WO2024140450A1 - Antibody-conjugated drug of n-alkoxyalkyl substituted camptothecin derivative - Google Patents
Antibody-conjugated drug of n-alkoxyalkyl substituted camptothecin derivative Download PDFInfo
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- WO2024140450A1 WO2024140450A1 PCT/CN2023/140881 CN2023140881W WO2024140450A1 WO 2024140450 A1 WO2024140450 A1 WO 2024140450A1 CN 2023140881 W CN2023140881 W CN 2023140881W WO 2024140450 A1 WO2024140450 A1 WO 2024140450A1
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Classifications
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- C07K—PEPTIDES
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
Definitions
- the classic ADC drug Kadcyla which treats HER2+ breast cancer that is resistant to naked anti-Herceptin, can further extend the overall survival by 6 months compared with standard therapy.
- Kadcyla Another clinical study showed that the incidence of grade 3 adverse reactions in the Kadcyla treatment group was reduced by 50% compared with the standard chemotherapy drug treatment group.
- ADC has greatly improved the therapeutic index compared with conventional chemotherapy drugs by reducing toxicity and increasing efficacy.
- ADC is expected to make breakthroughs in the treatment of tumor diseases.
- R 1 and R 2 are each independently selected from hydrogen, deuterium, and C 1 -C 6 alkyl;
- the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).
- Lp is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe-, and -Gly-Gly-Phe-Gly-.
- the tumor-associated antigen antibody is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody and anti-ROR1 antibody.
- R 1 , R 2 , n 1 , L p , Z and Ab have the same definitions as those of the compound of formula (I), and n' has the same definition as above.
- the present invention provides the following antibody-drug conjugates:
- Ab is selected from tumor-associated antigen antibodies, and is further selected from anti-Her2 antibodies, anti-Trop2 antibodies, anti-B7H3 antibodies, anti-5T4, anti-Nectin-4 antibodies, anti-CD20 antibodies, and anti-ROR1 antibodies.
- the present invention provides the following antibody-drug conjugates:
- n is selected from 6.5-6.8.
- n is selected from 7.3-7.6.
- n is selected from 7.1-7.2.
- n is selected from 7.3-7.5.
- n is selected from 7.3-7.4.
- n is selected from 2.1-2.3.
- n is selected from 4.3-4.6.
- Another aspect of the present invention provides a method for preparing the above-mentioned antibody-drug conjugate, which comprises the following steps:
- the compound of formula (a) is subjected to a condensation reaction with HO- Lz - Lj ' to obtain the compound of formula (b), and the compound of formula (b) is linked to an antibody to obtain the compound of formula (I).
- Another aspect of the present invention provides a pharmaceutical composition, which comprises the aforementioned antibody-drug conjugate or the antibody-drug conjugate prepared by the above method and a pharmaceutically acceptable carrier.
- Another aspect of the present invention provides a method for inhibiting or treating a tumor disease in a patient in need thereof, comprising administering the above-mentioned antibody-drug conjugate or pharmaceutical composition to the patient in need thereof.
- FIG3 is a graph showing the tumor growth curve of the KYSE-150 human esophageal squamous cell carcinoma CDX model in Test Example 2.
- FIG. 4 is a tumor photograph of the KYSE-150 human esophageal squamous cell carcinoma CDX model after dissection in Test Example 2.
- FIG6 is a photo of the tumor after dissection of the ES-2 human ovarian cancer CDX model in Test Example 2.
- the compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers.
- the stereoisomers include geometric isomers (such as cis and trans structures) and optical isomers (such as enantiomers), and therapeutic substances composed of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof.
- the compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be separated from racemic mixtures or by using hand
- the invention relates to the synthesis of a novel compound using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all within the scope of the present disclosure.
- C 1 -C 6 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.
- any variable e.g., Rn
- its definition at each occurrence is independent.
- the group may be optionally substituted with up to 5 Rs, and each occurrence of R is an independent choice.
- substituents and/or variants thereof are permitted only if such combinations result in stable compounds.
- the "-" between the drug-linker fragment and the antibody is intended to indicate the connection relationship between the antibody and the fragment, and is not intended to be limited to one antibody connected to one drug-linker fragment. It is well known in the art that an antibody can be connected to one or more drugs due to the presence of multiple inter-linking disulfide bonds.
- treating includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.
- patient refers to an animal, preferably a mammal, more preferably a human.
- an effective amount refers to a sufficient amount of a non-toxic drug or medicament that can achieve the desired effect.
- the amount of a given drug depends on many factors, such as a specific dosing regimen, a disease or condition type and its severity, the uniqueness of the subject or host to be treated (e.g., body weight), but, according to specific surrounding conditions, including, for example, a specific drug, route of administration, condition to be treated, and the subject or host to be treated, the dosage can be routinely determined by methods known in the art.
- antibody-drug conjugate refers to a small molecule drug with biological activity connected to a monoclonal antibody through a chemical link.
- the monoclonal antibody acts as a carrier to transport the small molecule drug into the target cells.
- the "antibody-drug conjugate” mentioned in this article has the same meaning as antibody-drug conjugate.
- Fmoc 9-fluorenylmethoxycarbonyl.
- the amino acid constituting the peptide residue LP Val: valine, has the structural formula Ala: Alanine, its structural formula is Gly: glycine, its structural formula is; Phe: phenylalanine, its structural formula is Leu: Leucine, its structural formula is Lys: lysine, its structural formula is
- LP can be obtained by an amino acid condensation method known in the art.
- the heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO: 3):
- the light chain amino acid sequence is as follows (SEQ ID NO: 4):
- the raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
- Example 1 7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (1)
- HX-9d (2.5g, 3.4mmol) was dissolved in 60ml methanol and 30ml DCM mixed solvent, 10% palladium carbon (1200mg) was added, and the mixture was replaced with hydrogen and reacted at room temperature and normal pressure for 2h. After the reaction was completed, the product HX-9e (2.01g, yield 91%) was obtained by filtration and concentration; LCMS: (M+1) + 645.42 (theoretical value: 645.67).
- HX-11a (2.66g, 3.4mmol) was dissolved in 50ml methanol and 25ml DCM mixed solvent, 1g of 10% palladium carbon was added, and after hydrogen substitution, the reaction was carried out at room temperature and normal pressure for 2h, and the product HX-11b (2.1g, 90%) was obtained by filtration and concentration; LCMS: (M+1) + 612.25 (theoretical value: 611.26).
- Example 7 7-[N-(HS627-Ac-PEG 2 -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (7)
- IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol)
- adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 ⁇ L)
- HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 ⁇ L) was added, and the prepared TCEP ⁇ HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
- TCEP ⁇ HCl tris(2-carboxyethyl)phosphine hydrochloride
- HX-11c (121 mg, 0.09 mmol) was dissolved in 0.2 mL of DMF, and then piperidine (71 mg, 0.9 mmol) was added. The reaction was stirred at room temperature for 1 h, and 10 mL of methyl tert-butyl ether was added. The mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-34d (82 mg, yield 81%), which was directly used in the next step reaction; LCMS: (M+1) + 1136.27 (theoretical value: 1135.50).
- reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min) and freeze-dried to obtain a yellow solid HX-34 (43 mg, yield 51%); LCMS: (M+1) + 1173.36 (theoretical value: 1172.40).
- Example 14 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (14)
- the ADC to be tested was diluted to 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM, 0.128nM, and 0.026nM using culture medium, and the diluted ADC was added to a 96-well cell culture plate at 100 ⁇ L per well, with 3 replicates for each concentration, and 100 ⁇ L of culture medium was added to each well of the negative control and blank control groups without ADC. After the addition of the sample, it was returned to the incubator at 37°C and 5% CO2 for further incubation for 6 days.
- Ab is IP140B antibody.
- the ADC compounds of the embodiments of the present invention have good inhibitory activity against NCI-H1975, MDA-MB-231 and OE-33 cells.
- the IC 50 of the inhibitory activity of some ADC compounds such as ADC10 and 12 against these cancer cells is even lower than 50 nM.
- Test Example 2 In vivo tumor growth inhibition activity of ADC
- ADC in vitro inhibitory activity test method Human non-small cell lung cancer Calu-6, human esophageal squamous cell carcinoma cell KYSE-150 and human ovarian cancer cell ES-2 were cultured in vitro monolayers. When the cell saturation was 80%-90%, they were digested with trypsin-EDTA, centrifuged and discarded the supernatant, and the cells were resuspended in PBS. The cell suspension was adjusted to an appropriate concentration. Calu-6 and KYSE-150 cells (2-10 ⁇ 10 6 cells/0.1ml) were subcutaneously inoculated into BALB/c nude mice, and ES-2 cells were subcutaneously inoculated into NOD-SCID mice. The animals and transplanted tumor growth were observed regularly.
- the tumor volume When the tumor volume grew to about 100-200mm 3 , they were randomly divided into vehicle control group (normal saline) and ADC administration group (dissolved in normal saline), with 6 animals in each group.
- the drugs were administered by intravenous injection, and the administration frequency was Q4D, with a total of 2 administrations (the time of the first administration was recorded as Day 1, and the second administration was recorded on Day 5).
- the experimental grouping and administration settings are shown in Table 2.
- a growth curve was drawn, and the tumor was finally removed and weighed.
- the tumor inhibition results were obtained by analyzing the data of tumor volume and weight of tumor-bearing mice at the end of the experiment using GraphPad Prism software.
- the control group " ⁇ " in the figure means that there is no result in this group and the tumor condition was not measured. There is no corresponding tumor in the experimental group, and "tumor reduction to 0" means that no tumor residue was found in the corresponding dissected animal.
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Abstract
An N-alkoxyalkyl-substituted camptothecin derivative is used as an antibody-drug conjugate represented by formula (I) and as a cytotoxic drug moiety, and the antibody-drug conjugate has a significant anti-tumor effect.
Description
本发明涉及医药领域。具体地说,本发明提供一系列N-烷氧烷基取代的喜树碱衍生物抗体偶联药物、其制备方法及其在抗肿瘤领域中的应用。The present invention relates to the field of medicine and specifically provides a series of N-alkoxyalkyl substituted camptothecin derivative antibody-drug conjugates, preparation methods thereof and applications thereof in the field of anti-tumor.
抗体-药物偶联物(antibody-drug conjugate,ADC)实现了单克隆抗体药物与小分子细胞毒素优势的联合,其在结构上包括抗体(antibody)、细胞毒素(cytotoxin)和连接子(linker)三个组成部分,它是通过抗体实现对肿瘤的特异性靶向,然后释放出细胞毒素进一步杀灭肿瘤细胞。ADC在一定程度上克服了单抗类药物的耐药问题。由于ADC的起效主要依赖于抗体所携带的细胞毒素,而抗体介导的生物效应则是非必须的,即使抗原发生一定程度的突变也并不会影响ADC发挥效力。例如经典的ADC药物Kadcyla,治疗对裸抗Herceptin产生耐药的HER2+乳腺癌,较标准疗法可将总生存期进一步延长6个月。另一项临床研究显示,Kadcyla治疗组较标准疗法化疗药治疗组的3级不良反应的发生率降低了50%。ADC通过减毒增效,与常规化疗药相比治疗指数得到大幅提高。作为一种新型的抗肿瘤“武器”,ADC有望在肿瘤疾病的治疗领域做出突破。Antibody-drug conjugate (ADC) combines the advantages of monoclonal antibody drugs with small molecule cytotoxins. Its structure includes three components: antibody, cytotoxin and linker. It achieves specific targeting of tumors through antibodies, and then releases cytotoxins to further kill tumor cells. ADC has overcome the drug resistance problem of monoclonal antibody drugs to a certain extent. Since the effectiveness of ADC mainly depends on the cytotoxins carried by antibodies, and the biological effects mediated by antibodies are not necessary, even if the antigen undergoes a certain degree of mutation, it will not affect the effectiveness of ADC. For example, the classic ADC drug Kadcyla, which treats HER2+ breast cancer that is resistant to naked anti-Herceptin, can further extend the overall survival by 6 months compared with standard therapy. Another clinical study showed that the incidence of grade 3 adverse reactions in the Kadcyla treatment group was reduced by 50% compared with the standard chemotherapy drug treatment group. ADC has greatly improved the therapeutic index compared with conventional chemotherapy drugs by reducing toxicity and increasing efficacy. As a new type of anti-tumor "weapon", ADC is expected to make breakthroughs in the treatment of tumor diseases.
一个ADC的成功,不仅依赖于对每个组成部分的优化,同时也依赖于各个组成部分的合理搭配与整合。ADC的三个组成部分所起的作用各不相同,因而ADC对其三个组成部分也分别具有不同的要求:ADC的抗体需要满足可以特异性靶向肿瘤,并具有适宜的亲和力和可内化性能;ADC的毒素则需要满足毒性高、作用机制明确、可被偶联等要求。喜树碱衍生物是一种光谱的抗肿瘤药物,被广泛用做ADC的毒素,但目前现有技术少有对烷氧烷基修饰的喜树碱衍生物作为毒素的结构设计或其对肿瘤抑制活性等相关内容的研究,这一课题的研究和推进对于喜树碱衍生物ADC技术的发展具有重要的价值。The success of an ADC depends not only on the optimization of each component, but also on the reasonable combination and integration of the components. The three components of ADC play different roles, so ADC also has different requirements for its three components: the antibody of ADC needs to meet the requirements of specific tumor targeting, suitable affinity and internalization performance; the toxin of ADC needs to meet the requirements of high toxicity, clear mechanism of action, and conjugation. Camptothecin derivatives are a kind of broad spectrum anti-tumor drugs, which are widely used as toxins of ADC. However, there are few existing technologies on the structural design of alkoxyalkyl-modified camptothecin derivatives as toxins or their tumor inhibitory activity. The research and promotion of this topic is of great value to the development of camptothecin derivative ADC technology.
发明内容Summary of the invention
本发明的目的在于提供一种以烷氧烷基取代的喜树碱衍生物作为药物毒性部分的抗体偶联药物,该些抗体-药物偶联物具有显著的抗肿瘤效果。The object of the present invention is to provide an antibody-drug conjugate with an alkoxyalkyl-substituted camptothecin derivative as the drug toxic part, and these antibody-drug conjugates have significant anti-tumor effects.
本发明的一方面,提供一种式(I)所示的抗体-药物偶联物,
In one aspect of the present invention, there is provided an antibody-drug conjugate represented by formula (I),
In one aspect of the present invention, there is provided an antibody-drug conjugate represented by formula (I),
式中,In the formula,
R1、R2各自独立地选自氢、氘、C1-C6烷基;R 1 and R 2 are each independently selected from hydrogen, deuterium, and C 1 -C 6 alkyl;
L1选自-L11-L12-L13-,其中,L11、L12、L13各自独立地选自-O-、C1-C3亚烷基和苯基;L 1 is selected from -L 11 -L 12 -L 13 -, wherein L 11 , L 12 , and L 13 are each independently selected from -O-, C 1 -C 3 alkylene, and phenyl;
LP为由2-7个氨基酸构成的肽残基;L P is a peptide residue consisting of 2-7 amino acids;
Z选自-Lz-Lj-,其中,Lz选自-C(=O)-C1-C8亚烷基和-C(=O)-(CH2CH2O)2-6-CH2CH2NH-,Lj为可偶联抗体的接头;Z is selected from -Lz - Lj- , wherein Lz is selected from -C(=O) -C1 - C8 alkylene and -C(=O)-( CH2CH2O ) 2-6 - CH2CH2NH- , and Lj is a linker that can be coupled to an antibody;
n1选自0、1、2或3;n 1 is selected from 0, 1, 2 or 3;
Ab为抗体。Ab is an antibody.
式(I)所示的抗体-药物偶联物中,其药物抗体比(DAR)选自2-8,进一步可以为2.1、2.3、4.3、4.6、6.2、
6.3、6.5、6.6、6.8、7.0、7.1、7.2、7.3、7.4或7.5。In the antibody-drug conjugate of formula (I), the drug-antibody ratio (DAR) is selected from 2-8, and can further be 2.1, 2.3, 4.3, 4.6, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.
所述式(I)所示的抗体-药物偶联物为式(I’)所示的抗体-药物偶联物,
The antibody-drug conjugate represented by formula (I) is the antibody-drug conjugate represented by formula (I'),
The antibody-drug conjugate represented by formula (I) is the antibody-drug conjugate represented by formula (I'),
n’选自2-8,进一步可以为2-4、2-6或4-6,例如可以为2.1、2.3、4.3、4.6、6.2、6.3、6.5、6.6、6.8、7.0、7.1、7.2、7.3、7.4或7.5。n' is selected from 2-8, and can further be 2-4, 2-6 or 4-6, for example, it can be 2.1, 2.3, 4.3, 4.6, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.
在一些具体实施方式中,R1、R2各自独立地选自氢、氘和C1-C3烷基。In some embodiments, R 1 and R 2 are each independently selected from hydrogen, deuterium and C 1 -C 3 alkyl.
在一些具体实施方式中,R1、R2各自独立地选自氢、氘、甲基、乙基和异丙基。In some embodiments, R 1 and R 2 are each independently selected from hydrogen, deuterium, methyl, ethyl and isopropyl.
在一些具体实施方式中,R1选自氢、氘、甲基、乙基和异丙基,R2选自氢、氘、甲基和乙基。In some embodiments, R 1 is selected from hydrogen, deuterium, methyl, ethyl and isopropyl, and R 2 is selected from hydrogen, deuterium, methyl and ethyl.
在一些具体实施方式中,R1为甲基,R2为氢或甲基。In some embodiments, R 1 is methyl, and R 2 is hydrogen or methyl.
在一些具体实施方式中,L11、L12、L13各自独立地选自-O-、亚甲基和苯基。In some specific embodiments, L 11 , L 12 , and L 13 are each independently selected from -O-, methylene, and phenyl.
在一些具体实施方式中,L11-L12-L13选自-O-CH2-苯基-和-CH2-O-CH2-。In some specific embodiments, L 11 -L 12 -L 13 are selected from -O-CH 2 -phenyl- and -CH 2 -O-CH 2 -.
在一些具体实施方式中,所述氨基酸选自苯丙氨酸(Phe)、甘氨酸(Gly)、缬氨酸(Val)、丙氨酸(Ala)、亮氨酸(Leu)、赖氨酸(Lys)、瓜氨酸(Cit)和天冬氨酸(Asn)。In some specific embodiments, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).
在一些具体实施方式中,Lp选自由2-5个选自苯丙氨酸(Phe)、甘氨酸(Gly)、缬氨酸(Val)、丙氨酸(Ala)、亮氨酸(Leu)、赖氨酸(Lys)、瓜氨酸(Cit)和天冬氨酸(Asn)的氨基酸构成的肽残基。In some specific embodiments, Lp is selected from a peptide residue consisting of 2-5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).
在一些具体实施方式中,Lp选自由2-4个选自苯丙氨酸(Phe)、甘氨酸(Gly)、缬氨酸(Val)、丙氨酸(Ala)、和亮氨酸(Leu)、赖氨酸(Lys)的氨基酸构成的肽残基。In some embodiments, Lp is selected from a peptide residue consisting of 2-4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), and lysine (Lys).
在一些具体实施方式中,Lp选自-Val-Cit-、-Val-Ala-、-Gly-Val-Ala-、-Gly-Val-Ala-Gly-、-Gly-Lys-、-Gly-Gly-Lys-、-Gly-Gly-Lys-Gly-、-Val-Ala-、-Ala-Ala-Asn-、-Gly-Leu-、-Gly-Gly-Leu-、-Gly-Gly-Leu-Gly-、-Gly-Phe-、-Gly-Gly-Phe-和-Gly-Gly-Phe-Gly-。In some specific embodiments, Lp is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe-, and -Gly-Gly-Phe-Gly-.
在一些具体实施方式中,Lp选自
其羰基端连接-NH-,另一端连接Z。In some embodiments, L p is selected from Its carbonyl end is connected to -NH- and the other end is connected to Z.
在一些具体实施方式中,Lp选自
其羰基端连接-NH-,另一端连接Z。
In some embodiments, L p is selected from Its carbonyl end is connected to -NH- and the other end is connected to Z.
在一些具体实施方式中,Lj选自
所示位置表示与抗体相连,所示位置表示与LZ基团相连。In some embodiments, L is selected from The positions shown indicate attachment to antibodies. The positions shown indicate attachment to the L Z group.
在一些具体实施方式中,Lz选自-C(=O)-C1-C8亚烷基和-C(=O)-(CH2CH2O)2-6-CH2CH2NH-。In some specific embodiments, L z is selected from —C(═O)—C 1 -C 8 alkylene and —C(═O)—(CH 2 CH 2 O) 2-6 —CH 2 CH 2 NH—.
在一些具体实施方式中,Lz选自-C(=O)-C1-C6亚烷基和-C(=O)-(CH2CH2O)2-4-CH2CH2NH-。In some specific embodiments, Lz is selected from -C( = O) -C1 -C6 alkylene and -C(=O)-( CH2CH2O ) 2-4 - CH2CH2NH- .
在一些具体实施方式中,Lz选自-C(=O)-(CH2)5-和-C(=O)-(CH2CH2O)2-CH2CH2NH-。In some specific embodiments, Lz is selected from -C(=O) - ( CH2 ) 5- and -C(=O)-( CH2CH2O ) 2 - CH2CH2NH- .
在一些具体实施方式中,Z选自
所示位置表示与抗体相连。所示位置表示与Lp基团相连。In some embodiments, Z is selected from The positions shown indicate attachment to antibodies. The positions shown indicate attachment to the L p group.
在一些具体实施方式中,所述抗体为肿瘤相关抗原抗体。In some embodiments, the antibody is an antibody against a tumor-associated antigen.
在一些具体实施方式中,所述肿瘤相关抗原抗体选自抗Her2抗体、抗Trop2抗体、抗B7H3抗体、抗5T4、抗Nectin-4抗体、抗CD20抗体和抗ROR1抗体。In some specific embodiments, the tumor-associated antigen antibody is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody and anti-ROR1 antibody.
在一些具体实施方式中,式(I)所示的抗体-药物偶联物具有如式(I-1)、式(I-1’)、式(I-2’)、或式(I-2)所示的结构,
In some embodiments, the antibody-drug conjugate of formula (I) has a structure as shown in formula (I-1), formula (I-1'), formula (I-2'), or formula (I-2),
In some embodiments, the antibody-drug conjugate of formula (I) has a structure as shown in formula (I-1), formula (I-1'), formula (I-2'), or formula (I-2),
式中,R1、R2、n1、Lp、Z、Ab各自定义同式(I)化合物,n’定义同前。In the formula, R 1 , R 2 , n 1 , L p , Z and Ab have the same definitions as those of the compound of formula (I), and n' has the same definition as above.
本发明提供以下抗体-药物偶联物:
The present invention provides the following antibody-drug conjugates:
The present invention provides the following antibody-drug conjugates:
其中,Ab定义同式(I)化合物。wherein Ab is defined as the compound of formula (I).
在一些具体实施方式中,Ab选自肿瘤相关抗原抗体,进一步选自抗Her2抗体、抗Trop2抗体、抗B7H3抗体、抗5T4、抗Nectin-4抗体、抗CD20抗体和抗ROR1抗体。In some specific embodiments, Ab is selected from tumor-associated antigen antibodies, and is further selected from anti-Her2 antibodies, anti-Trop2 antibodies, anti-B7H3 antibodies, anti-5T4, anti-Nectin-4 antibodies, anti-CD20 antibodies, and anti-ROR1 antibodies.
n选自2-8,进一步可以为2-4、2-6或4-6,例如可以为2.1、2.3、4.3、4.6、6.2、6.3、6.5、6.6、6.8、7.0、7.1、7.2、7.3、7.4或7.5。n is selected from 2-8, and may further be 2-4, 2-6 or 4-6, for example, may be 2.1, 2.3, 4.3, 4.6, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.
进一步地,本发明提供以下抗体-药物偶联物:
Furthermore, the present invention provides the following antibody-drug conjugates:
n选自6.5-6.8。 n is selected from 6.5-6.8.
n选自7.3-7.6。 n is selected from 7.3-7.6.
n选自7.1-7.2。 n is selected from 7.1-7.2.
n选自7.3-7.5。 n is selected from 7.3-7.5.
n选自7.3-7.4。 n is selected from 7.3-7.4.
n选自2.1-2.3。 n is selected from 2.1-2.3.
n选自4.3-4.6。 n is selected from 4.3-4.6.
其中,Ab定义同式(I)化合物。wherein Ab is defined as the compound of formula (I).
在一些具体实施方式中,Ab为HS627抗体或IP140B抗体,所述HS627的重链氨基酸序列如SEQ ID NO:1
所示,其轻链氨基酸序列如SEQ ID NO:2所示;所述IP140B抗体的重链氨基酸序列如SEQ ID NO:3所示,其轻链氨基酸序列如SEQ ID NO:4所示。In some embodiments, Ab is HS627 antibody or IP140B antibody, and the heavy chain amino acid sequence of HS627 is as shown in SEQ ID NO: 1 The amino acid sequence of its light chain is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain of the IP140B antibody is shown in SEQ ID NO:3, and the amino acid sequence of its light chain is shown in SEQ ID NO:4.
本发明的另一方面,提供上述抗体-药物偶联物的制备方法,其包括以下步骤:Another aspect of the present invention provides a method for preparing the above-mentioned antibody-drug conjugate, which comprises the following steps:
式(a)化合物与HO-Lz-Lj’缩合反应得到式(b)化合物,式(b)化合物与抗体连接得到式(I)化合物。
The compound of formula (a) is subjected to a condensation reaction with HO- Lz - Lj ' to obtain the compound of formula (b), and the compound of formula (b) is linked to an antibody to obtain the compound of formula (I).
The compound of formula (a) is subjected to a condensation reaction with HO- Lz - Lj ' to obtain the compound of formula (b), and the compound of formula (b) is linked to an antibody to obtain the compound of formula (I).
其中,n1、R1、R2、L1、Lp、Lz、Ab各自定义同式(I)化合物。wherein n 1 , R 1 , R 2 , L 1 , L p , L z , and Ab each have the same meaning as the compound of formula (I).
Lj’选自
L j 'selected
本发明的另一方面,提供一种药物组合物,其包括前述抗体-药物偶联物或上述方法制备得到的抗体-药物偶联物与药学上可接受的载体。Another aspect of the present invention provides a pharmaceutical composition, which comprises the aforementioned antibody-drug conjugate or the antibody-drug conjugate prepared by the above method and a pharmaceutically acceptable carrier.
本发明的另一方面,提供前述抗体-药物偶联物、上述方法制备得到的抗体-药物偶联物或上述药物组合物在制备抗肿瘤药物中的用途。Another aspect of the present invention provides use of the aforementioned antibody-drug conjugate, the antibody-drug conjugate prepared by the aforementioned method, or the aforementioned pharmaceutical composition in the preparation of anti-tumor drugs.
本发明的另一方面,提供一种抑制或治疗有需要患者肿瘤疾病的方法,其包括向有需要的患者施用上述抗体-药物偶联物或药物组合物。Another aspect of the present invention provides a method for inhibiting or treating a tumor disease in a patient in need thereof, comprising administering the above-mentioned antibody-drug conjugate or pharmaceutical composition to the patient in need thereof.
在一些具体实施方式中,上述抗体-药物偶联物或药物组合物的施用量为治疗有效量。In some embodiments, the amount of the antibody-drug conjugate or pharmaceutical composition administered is a therapeutically effective amount.
在一些具体实施方式中,所述肿瘤选自实体瘤。In some embodiments, the tumor is selected from a solid tumor.
在一些具体实施方式中,所述肿瘤选自肺癌、食管癌、乳腺癌、非小细胞肺癌、食管鳞癌或卵巢癌。In some embodiments, the tumor is selected from lung cancer, esophageal cancer, breast cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, or ovarian cancer.
在一些具体实施方式中,所述肺癌为非小细胞肺癌。In some embodiments, the lung cancer is non-small cell lung cancer.
图1为测试例2中Calu-6人非小细胞肺癌CDX模型肿瘤生长曲线图。FIG1 is a graph showing the tumor growth curve of the Calu-6 human non-small cell lung cancer CDX model in Test Example 2.
图2为测试例2中Calu-6人非小细胞肺癌CDX模型解剖后肿瘤照片。FIG2 is a photo of the tumor after dissection of the Calu-6 human non-small cell lung cancer CDX model in Test Example 2.
图3为测试例2中KYSE-150人食管鳞癌CDX模型肿瘤生长曲线图。FIG3 is a graph showing the tumor growth curve of the KYSE-150 human esophageal squamous cell carcinoma CDX model in Test Example 2.
图4为测试例2中KYSE-150人食管鳞癌CDX模型解剖后肿瘤照片。FIG. 4 is a tumor photograph of the KYSE-150 human esophageal squamous cell carcinoma CDX model after dissection in Test Example 2.
图5为测试例2中ES-2人卵巢癌CDX模型肿瘤生长曲线图。FIG5 is a graph showing the tumor growth curve of the ES-2 human ovarian cancer CDX model in Test Example 2.
图6为测试例2中ES-2人卵巢癌CDX模型解剖后肿瘤照片。FIG6 is a photo of the tumor after dissection of the ES-2 human ovarian cancer CDX model in Test Example 2.
I定义:IDefinition:
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.
如本文使用的和除非另作说明,术语“包含”,“包括”,“具有”,“含有”,包括其语法上的等同形式,通常应当理解为开放式且非限制性的,例如,不排除其他未列举的要素或步骤。As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.
本公开化合物可以是不对称的,例如,具有一个或多个立体异构体。除非另有说明,所有立体异构体都包括,如对映异构体和非对映异构体。所述的立体异构体包括几何异构(如顺式、反式结构)和光学异构(如对映异构体),以单体、消旋体、外消旋混合物及其药学上可接受的盐组成的治疗物。本公开的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分,或通过使用手
性原料或手性试剂合成。外消旋体、非对映异构体、对映异构体都包括在本公开的范围之内。The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The stereoisomers include geometric isomers (such as cis and trans structures) and optical isomers (such as enantiomers), and therapeutic substances composed of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be separated from racemic mixtures or by using hand The invention relates to the synthesis of a novel compound using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all within the scope of the present disclosure.
本文中的数字范围,是指给定范围中的各个整数。例如,“C1-C6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。Numeric ranges herein refer to each integer in the given range. For example, "C 1 -C 6 " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.
当任何变量(例如Rn)在化合物的组成或结构中出现一次以上时,其在每一种情况下的定义都是独立的。因此,例如,如果一个基团被1-5个R所取代,则所述基团可以任选地至多被5个R所取代,并且每种情况下的R都有独立的选项。此外,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。When any variable (e.g., Rn) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 Rs, the group may be optionally substituted with up to 5 Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and/or variants thereof are permitted only if such combinations result in stable compounds.
术语“C1-C6烷基”指具有1到6个碳原子的直链或支链烷基。烷基具体实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基,及其各种支链异构体等。The term "C 1- C 6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms. Specific examples of alkyl groups include, but are not limited to, 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, and various branched isomers thereof.
是指化学键连接处。需要说明的是,本发发明所记载的结构片段(例如-L11-L12-L12-或-Lz-Lj-),在无特别说明的时候,代表由左到右的顺序连接相应基团,例如当-L12-为C1-C2亚烷基-O-时,其表示C1-C2亚烷基-O-的左侧连接-L11-,右端连接-L13-。 Refers to the chemical bond connection. It should be noted that the structural fragments described in the present invention (e.g., -L 11 -L 12 -L 12 - or -L z -L j -), unless otherwise specified, represent the corresponding groups connected in sequence from left to right. For example, when -L 12 - is C 1 -C 2 alkylene-O-, it means that the left side of C 1 -C 2 alkylene-O- is connected to -L 11 -, and the right end is connected to -L 13 -.
可以理解地,本申请的抗体-药物偶联物中,例如式(I)、(I-1)、(I-2)中,药物-连接子片段与抗体之间的“-”意欲表示抗体与该片段的连接关系,不欲限定为一个抗体连接一个药物-连接子片段。本领域公知,一个抗体上由于存在多个连间二硫键,因此一个抗体可以连接一个或多个药物。It can be understood that in the antibody-drug conjugates of the present application, for example, in formula (I), (I-1), (I-2), the "-" between the drug-linker fragment and the antibody is intended to indicate the connection relationship between the antibody and the fragment, and is not intended to be limited to one antibody connected to one drug-linker fragment. It is well known in the art that an antibody can be connected to one or more drugs due to the presence of multiple inter-linking disulfide bonds.
药物或药物组合物Drug or drug composition
本公开的药物或药物组合物可以经口地、局部地、肠胃外地或粘膜地(例如,含服地、通过吸入或直肠地)以包含常规的非-毒性药学可接受的载体的剂量单位配制剂施用。通常希望使用口服途径。所述活性试剂可以经口地以胶囊、片剂等形式施用。The drugs or pharmaceutical compositions of the present disclosure can be administered orally, topically, parenterally or mucosally (e.g., buccally, by inhalation or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is generally desirable to use an oral route. The active agent can be administered orally in the form of capsules, tablets, etc.
术语“治疗”包括抑制、缓解、预防或消除与所治疗的疾病、病症或失调相关的一种或多种症状或副作用。The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.
术语“抑制”的使用是相对于对照的。本领域技术人员将容易地确定用于每个实验的适当对照。例如,将用化合物处理的受试者或细胞中的降低了的反应与未用化合物处理的受试者或细胞中的反应进行比较。The term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.
术语“患者”是指一种动物,最好为哺乳动物,更好的为人。The term "patient" refers to an animal, preferably a mammal, more preferably a human.
术语“药物组合物”意指包含本公开所述化合物或其药学上可接受的盐,以及依施用方式和剂型的性质而定的至少一种选自以下药学上可接受的成分的组合物,包括但不限于:载体、稀释剂、佐剂、赋形剂、防腐剂、填充剂、崩解剂、润湿剂、乳化剂、悬浮剂、甜味剂、矫味剂、香味剂、抗菌剂、抗真菌剂、润滑剂、分散剂、温敏材料、温度调节剂、黏附剂、稳定剂、助悬剂等。The term "pharmaceutical composition" means a composition comprising the compound described in the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.
术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。在本发明的实施方式中,在根据本发明对患者进行治疗时,给定药物的量取决于诸多因素,如具体的给药方案、疾病或病症类型及其严重性、需要治疗的受治疗者或宿主的独特性(例如体重),但是,根据特定的周围情况,包括例如已采用的具体药物、给药途径、治疗的病症、以及治疗的受治疗者或宿主,施用剂量可由本领域已知的方法常规决定。通常,就成人治疗使用的剂量而言,施用剂量典型地在0.02-5000mg/天,例如约1-1500mg/天的范围。该所需剂量可以方便地被表现为一剂、或同时给药的(或在短时间内)或在适当的间隔的分剂量,例如每天二、三、四剂或更多分剂。本领域技术人员可以理解的是,尽管给出了上述剂量范围,但具体的有效量可根据患者的情况并结合医师诊断而适当调节。The term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a non-toxic drug or medicament that can achieve the desired effect. In embodiments of the present invention, when treating a patient according to the present invention, the amount of a given drug depends on many factors, such as a specific dosing regimen, a disease or condition type and its severity, the uniqueness of the subject or host to be treated (e.g., body weight), but, according to specific surrounding conditions, including, for example, a specific drug, route of administration, condition to be treated, and the subject or host to be treated, the dosage can be routinely determined by methods known in the art. Usually, with respect to the dosage used for adult treatment, the dosage is typically in the range of 0.02-5000 mg/day, such as about 1-1500 mg/day. The desired dosage can be conveniently expressed as a single dose, or a dose administered simultaneously (or in a short period of time) or in a divided dose at appropriate intervals, such as two, three, four, or more divided doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range is given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.
术语“抗体-药物偶联物(antibody-drug conjugate,ADC)”是通过一个化学链接将具有生物活性的小分子药物连接到单抗上,单抗作为载体将小分子药物靶向运输到目标细胞中。本文所述“抗体偶联药物”含义同抗体-药物偶联物。The term "antibody-drug conjugate (ADC)" refers to a small molecule drug with biological activity connected to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into the target cells. The "antibody-drug conjugate" mentioned in this article has the same meaning as antibody-drug conjugate.
本文所述“抗体偶联药物”含义同抗体-药物偶联物(antibody-drug conjugate,ADC)。The “antibody-drug conjugate” mentioned in this article has the same meaning as antibody-drug conjugate (ADC).
术语“药物抗体比(DAR)”是指附着在单个单抗上的有效载荷分子(抗肿瘤化合物或药物)的平均数量。The term “drug-to-antibody ratio (DAR)” refers to the average number of payload molecules (anti-tumor compounds or drugs) attached to a single mAb.
缩写:abbreviation:
Fmoc:9-芴甲氧羰基。
Fmoc: 9-fluorenylmethoxycarbonyl.
构成所述肽残基LP的氨基酸,Val:缬氨酸,其结构式为Ala:丙氨酸,其结构式为Gly:甘氨酸,其结构式为;Phe:苯丙氨酸,其结构式为Leu:亮氨酸,其结构式为Lys:赖氨酸,其结构式为
The amino acid constituting the peptide residue LP , Val: valine, has the structural formula Ala: Alanine, its structural formula is Gly: glycine, its structural formula is; Phe: phenylalanine, its structural formula is Leu: Leucine, its structural formula is Lys: lysine, its structural formula is
LP可通过本领公知的氨基酸缩合方法得到。 LP can be obtained by an amino acid condensation method known in the art.
PABC:ha:
PABC: haha:
本发明中,抗体和用于连接抗体的接头Lj可以通过本领域公知的方法连接得到。例如,当Lj结构为(或)时,其可以是通过(或)与抗体上的硫醇等可反应基团反应连接得到,本发明对此不做特别限定。In the present invention, the antibody and the linker Lj used to connect the antibody can be connected by methods known in the art. For example, when the structure of Lj is (or ), which can be through (or ) is obtained by reacting with a thiol or other reactive group on an antibody, and the present invention is not particularly limited to this.
II实施例:II Embodiment:
本实施例中制备ADC使用但不限于帕妥珠单抗和IP140B抗体,帕妥珠单抗(HS627,PERTUZUMAB)重链氨基酸序列如下(SEQ ID NO:1):
In this embodiment, ADCs prepared using, but not limited to, Pertuzumab and IP140B antibodies, Pertuzumab (HS627, PERTUZUMAB) heavy chain amino acid sequence is as follows (SEQ ID NO: 1):
In this embodiment, ADCs prepared using, but not limited to, Pertuzumab and IP140B antibodies, Pertuzumab (HS627, PERTUZUMAB) heavy chain amino acid sequence is as follows (SEQ ID NO: 1):
轻链氨基酸序列如下(SEQ ID NO:2):
The light chain amino acid sequence is as follows (SEQ ID NO: 2):
The light chain amino acid sequence is as follows (SEQ ID NO: 2):
IP140B抗体的重链氨基酸序列如下(SEQ ID NO:3):
The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO: 3):
The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO: 3):
轻链氨基酸序列如下(SEQ ID NO:4):
The light chain amino acid sequence is as follows (SEQ ID NO: 4):
The light chain amino acid sequence is as follows (SEQ ID NO: 4):
如无特别说明,本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
中间体HX-10a:N-(S-)甲氧基异丙胺乙基喜树碱
Intermediate HX-10a: N-(S-)methoxyisopropylamine ethyl camptothecin
Intermediate HX-10a: N-(S-)methoxyisopropylamine ethyl camptothecin
向反应瓶中加入(S)-1-甲氧基2-丙胺(100mg,1.12mmol),盐酸(0.7mL,0.8mmol),DMSO(3mL)和7-甲基-10,11-亚甲基二氧喜树碱(50mg,0.12mmol),搅拌下升温至120°反应50分钟,冷却至室温,加入甲基叔丁基醚,过滤析出固体,用硅胶柱层析纯化得到产物标题化合物(36mg,产率61%,HPLC 99%);1H NMR(500MHz,DMSO-d6)δ8.64(s,2H),7.69(s,1H),7.57(s,1H),7.27(s,1H),6.53(s,1H),6.33(s,2H),5.44(s,1H),5.33(d,J=4.7Hz,2H),3.71-3.54(m,2H),3.47(dd,J=10.1,5.8Hz,6H),3.25(d,J=13.6Hz,2H),1.88(dt,J=14.2,9.2Hz,2H),1.24(d,J=6.4Hz,3H),0.88(dd,J=9.4,5.3Hz,3H);LC-MS(M+H)+508.37(理论值507.20)。(S)-1-methoxy-2-propylamine (100 mg, 1.12 mmol), hydrochloric acid (0.7 mL, 0.8 mmol), DMSO (3 mL) and 7-methyl-10,11-methylenedioxycamptothecin (50 mg, 0.12 mmol) were added to the reaction flask, the temperature was raised to 120° for reaction for 50 minutes with stirring, and the mixture was cooled to room temperature. Methyl tert-butyl ether was added, the precipitated solid was filtered out, and the title compound was purified by silica gel column chromatography to obtain the product (36 mg, yield 61%, HPLC 99%); 1 H NMR (500 MHz, DMSO-d 6 )δ8.64(s,2H),7.69(s,1H),7.57(s,1H),7.27(s,1H),6.53(s,1H),6.33(s,2H),5.44(s,1H),5.33(d,J=4.7Hz,2H),3.71-3.54(m,2H),3.47(dd,J=10.1,5.8Hz,6H),3.25(d,J=13.6Hz,2H),1.88(dt,J=14.2,9.2Hz,2H),1.24(d,J=6.4Hz,3H),0.88(dd,J=9.4,5.3Hz,3H); LC-MS(M+H)+508.37(theoretical value 507.20).
中间体HX-14a:N-甲氧乙胺乙基喜树碱
Intermediate HX-14a: N-methoxyethylaminoethyl camptothecin
Intermediate HX-14a: N-methoxyethylaminoethyl camptothecin
将2-甲氧基乙胺(2.5g,0.03mol)和盐酸(4mL,0.05mol)溶解于DMSO中(10mL),搅拌下加热至110℃,加入7-甲基-10,11-亚甲基二氧喜树碱(1.8g,4.4mmol),继续升温至120-130℃反应1h,冷却至室温,加入甲基叔丁基醚,过滤,硅胶柱层析纯化得到标题化合物(739mg,产率34%,HPLC 97%);1H NMR(500MHz,DMSO-d6)δ7.55(s,1H),7.44(s,1H),7.20(s,1H),6.51(s,1H),6.27(d,J=3.6Hz,2H),5.41(d,J=4.6Hz,2H),5.13(q,J=18.6Hz,2H),3.31-3.18(m,7H),2.94(t,J=7.5Hz,2H),2.85(d,J=4.9Hz,2H),1.86(dt,J=19.4,7.0Hz,2H),0.88(t,J=7.3Hz,3H);LC-MS(M+H)+494.12(理论值493.18)。2-Methoxyethylamine (2.5 g, 0.03 mol) and hydrochloric acid (4 mL, 0.05 mol) were dissolved in DMSO (10 mL), heated to 110°C with stirring, 7-methyl-10,11-methylenedioxycamptothecin (1.8 g, 4.4 mmol) was added, and the temperature was continued to rise to 120-130°C for 1 h, cooled to room temperature, methyl tert-butyl ether was added, filtered, and purified by silica gel column chromatography to obtain the title compound (739 mg, yield 34%, HPLC 97%); 1 H NMR (500 MHz, DMSO-d 6 )δ7.55(s,1H),7.44(s,1H),7.20(s,1H),6.51(s,1H),6.27(d,J=3.6Hz,2H),5.41(d,J=4.6Hz,2H),5.13(q,J=18.6Hz,2H),3.31-3.18(m,7H),2.94(t,J=7.5Hz,2H),2.85(d,J=4.9Hz,2H),1.86(dt,J=19.4,7.0Hz,2H),0.88(t,J=7.3Hz,3H); LC-MS(M+H)+494.12(theoretical value 493.18).
实施例1:7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(1)
Example 1: 7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (1)
Example 1: 7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (1)
在250ml单口瓶中,将Fmoc-Gly-Gly-OH(5g,14.11mmol)溶于DMF(50mL)中,搅拌溶解,依次加入醋酸铜(768mg,4.23mmol),醋酸(1.69g,28.22mmol),四乙酸铅(6.872g,15.5mmol),升温至60℃,反应20min,将反应液倒入冰水中,乙酸乙酯萃取,得到类白色固体产品HX-9a(3.2g,产率66%);LCMS:(M+1)+369.21(理论值:368.14)。In a 250 ml single-necked bottle, Fmoc-Gly-Gly-OH (5 g, 14.11 mmol) was dissolved in DMF (50 mL), stirred to dissolve, copper acetate (768 mg, 4.23 mmol), acetic acid (1.69 g, 28.22 mmol), lead tetraacetate (6.872 g, 15.5 mmol) were added in sequence, the temperature was raised to 60 ° C, the reaction was carried out for 20 min, the reaction solution was poured into ice water, and extracted with ethyl acetate to obtain an off-white solid product HX-9a (3.2 g, yield 66%); LCMS: (M+1) + 369.21 (theoretical value: 368.14).
将HX-9a(3.2g,9.3mmol)溶于无水DCM(30mL)中,加入乙醇酸苄酯(8.03g,93mmol),然后加入PPTS(0.24g,0.93mmol),反应液在50℃回流过夜,浓缩后用EtOAc(200ml)稀释反应混合物,用水(3x200ml)洗涤,无水MgSO4干燥,过滤并真空浓缩,粗残余物通过柱色谱法纯化,得到白色粉末产物HX-9b(4.05g,产率97%);LCMS:(M+1)+475.03(理论值:474.18)。HX-9a (3.2 g, 9.3 mmol) was dissolved in anhydrous DCM (30 mL), benzyl glycolate (8.03 g, 93 mmol) was added, and then PPTS (0.24 g, 0.93 mmol) was added. The reaction solution was refluxed at 50 ° C overnight. After concentration, the reaction mixture was diluted with EtOAc (200 ml), washed with water (3x200 ml), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo, and the crude residue was purified by column chromatography to give a white powder product HX-9b (4.05 g, yield 97%); LCMS: (M+1) + 475.03 (theoretical value: 474.18).
取HX-9b(3g,6.32mmol),加入100mL乙腈,再加入DBU(0.48g,3.16mmol),室温反应2h,依次加入PPTS(0.79g,3.16mmol)、HOBT(0.85g,6.32mmol)、Fmoc-Gly-Gly-Phe-OH(2.69g,5.36mmol)和EDCI(1.21g,6.32mmol),室温反应15h,旋干,加入100mL DCM和10mL异丙醇溶清,以100mL*3(0.5M)HCl、100mL*3饱和碳酸氢钠溶液、100mL饱和氯化钠溶液洗涤,20g无水硫酸钠干燥。过滤,滤液旋干,硅胶柱层析纯化,得白色粉末状固体产物HX-9d(2.5g,产率63%);LCMS:(M+1)+736.13(理论值:735.29)。Take HX-9b (3 g, 6.32 mmol), add 100 mL of acetonitrile, then add DBU (0.48 g, 3.16 mmol), react at room temperature for 2 h, add PPTS (0.79 g, 3.16 mmol), HOBT (0.85 g, 6.32 mmol), Fmoc-Gly-Gly-Phe-OH (2.69 g, 5.36 mmol) and EDCI (1.21 g, 6.32 mmol) in sequence, react at room temperature for 15 h, spin dry, add 100 mL of DCM and 10 mL of isopropanol to dissolve, wash with 100 mL*3 (0.5 M) HCl, 100 mL*3 saturated sodium bicarbonate solution, 100 mL of saturated sodium chloride solution, and dry over 20 g of anhydrous sodium sulfate. The filtrate was filtered and dried in a rotary evaporator. The product was purified by silica gel column chromatography to obtain a white powdery solid product HX-9d (2.5 g, yield 63%); LCMS: (M+1) + 736.13 (theoretical value: 735.29).
在500ml的单口瓶中,将HX-9d(2.5g,3.4mmol)溶于60ml甲醇和30ml的DCM混合溶剂中,加入10%的钯碳(1200mg),氢气置换后,室温常压反应2h,反应完,过滤浓缩拿到产品HX-9e(2.01g,产率91%);LCMS:(M+1)+645.42(理论值:645.67)。In a 500ml single-necked bottle, HX-9d (2.5g, 3.4mmol) was dissolved in 60ml methanol and 30ml DCM mixed solvent, 10% palladium carbon (1200mg) was added, and the mixture was replaced with hydrogen and reacted at room temperature and normal pressure for 2h. After the reaction was completed, the product HX-9e (2.01g, yield 91%) was obtained by filtration and concentration; LCMS: (M+1) + 645.42 (theoretical value: 645.67).
取HX-9e(0.2g,0.31mmol),加入2mL DMF,HATU(1.17g,0.31mmol),DIPEA(0.8g.0.62mmol)和N-(S-)甲氧基异丙胺乙基喜树碱(156mg,0.31mmol),室温反应15h,加入200mL DCM稀释,饱和氯化钠溶液洗涤,无水硫酸钠干燥,硅胶柱层析纯化得黄色固体产物HX-9f(201mg,产率56%);LCMS:(M+1)+1135.36(理论值:1134.43)。Take HX-9e (0.2 g, 0.31 mmol), add 2 mL DMF, HATU (1.17 g, 0.31 mmol), DIPEA (0.8 g. 0.62 mmol) and N-(S-)methoxyisopropylamineethylcamptothecin (156 mg, 0.31 mmol), react at room temperature for 15 h, add 200 mL DCM to dilute, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a yellow solid product HX-9f (201 mg, yield 56%); LCMS: (M+1) + 1135.36 (theoretical value: 1134.43).
将HX-9f(201mg,0.177mmol)溶于0.5ml的DMF中,然后加入哌啶(138mg,1.77mmol),室温搅拌反应1h,在反应液中加入30ml甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到HX-9g固体(121mg,产率74%)直接用于下一步反应;LCMS:(M+1)+913.36(理论值:912.37)。HX-9f (201 mg, 0.177 mmol) was dissolved in 0.5 ml of DMF, and then piperidine (138 mg, 1.77 mmol) was added. The reaction was stirred at room temperature for 1 h. 30 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain HX-9g solid (121 mg, yield 74%), which was directly used in the next step reaction; LCMS: (M+1) + 913.36 (theoretical value: 912.37).
将HX-9g(121mg,0.131mmol)溶解于1ml无水DMF中,依次加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(40mg,0.131mmol),DIPEA(17mg,0.131mmol),室温反应30min,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:15%至60%乙腈在水中,时间50min)洗脱,冻干,得到黄色固体产物HX-9(35mg,产率24%);LCMS:(M+1)+1106.16(理论值:1105.44)。HX-9g (121 mg, 0.131 mmol) was dissolved in 1 ml of anhydrous DMF, and 6-(maleimido)hexanoic acid succinimidyl ester (40 mg, 0.131 mmol) and DIPEA (17 mg, 0.131 mmol) were added in sequence. The reaction was allowed to react at room temperature for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium pressure reverse phase C18 chromatography (gradient: 15% to 60% acetonitrile in water, time 50 min), and freeze-dried to obtain a yellow solid product HX-9 (35 mg, yield 24%); LCMS: (M+1) + 1106.16 (theoretical value: 1105.44).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
将化合物HX-9(0.89mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 1(3.3mg/ml,2ml)。Compound HX-9 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 1 (3.3 mg/ml, 2 ml).
UV-HPLC计算平均值:n=6.8。UV-HPLC calculated average value: n=6.8.
实施例2:7-[N-(IP140B-mc-Gly-Gly-Phe-Gly-ha-Ac),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(2)
Example 2: 7-[N-(IP140B-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (2)
Example 2: 7-[N-(IP140B-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (2)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-9(0.89mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 2(3.2mg/ml,2ml)。Compound HX-9 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 2 (3.2 mg/ml, 2 ml).
UV-HPLC计算平均值:n=6.5。UV-HPLC calculated average value: n=6.5.
实施例3:7-[N-(HS627-Ac-PEG2-Val-Ala-PABC),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(3)
Example 3: 7-[N-(HS627-Ac-PEG 2 -Val-Ala-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (3)
Example 3: 7-[N-(HS627-Ac-PEG 2 -Val-Ala-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (3)
在10ml单口瓶中,依次加入N-(S-)甲氧基异丙胺乙基喜树碱(50mg,0.098mmol),0.5ml NMP,DIPEA(63mg,0.49mmol),Fmoc-VA-PAB-PNP(67mg,0.098mmol),HOBt(13.3mg,0.098mmol),室温搅拌反应0.5h,得到HX-10b直接投入至下一步;LCMS:(M+1)+1049.15(理论值:1048.42)。In a 10 ml single-necked bottle, N-(S-)methoxyisopropylamineethylcamptothecin (50 mg, 0.098 mmol), 0.5 ml NMP, DIPEA (63 mg, 0.49 mmol), Fmoc-VA-PAB-PNP (67 mg, 0.098 mmol), and HOBt (13.3 mg, 0.098 mmol) were added in sequence. The reaction was stirred at room temperature for 0.5 h to obtain HX-10b, which was directly used for the next step; LCMS: (M+1) + 1049.15 (theoretical value: 1048.42).
在上述HX-10b反应液中,加入0.5ml的哌啶(V:V=10%),室温反应30min,在反应液中加入20ml甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到固体产物HX-10c(65mg,产率80%)直接用于下一步反应;LCMS:(M+1)+826.35(理论值:825.35)。To the above HX-10b reaction solution, add 0.5 ml of piperidine (V:V = 10%), react at room temperature for 30 min, add 20 ml of methyl tert-butyl ether to the reaction solution, centrifuge, remove the supernatant, and remove the solvent under reduced pressure to obtain a solid product HX-10c (65 mg, yield 80%), which is directly used for the next step reaction; LCMS: (M+1) + 826.35 (theoretical value: 825.35).
在10ml的单口瓶中,依次加入DCM(1mL),HX-10c(65mg,0.078mmol),溴乙酰氨乙氧基乙氧基丙酸(23mg,0.078mmol)和DIC(10mg,0.078mmol),室温搅拌反应90min后,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:5%至40%乙腈在水中,时间30min)洗脱,冻干,得到黄色固体产物HX-10(24mg,产率27%);LCMS:(M+1)+1106.02(理论值:1105.36)。In a 10 ml single-mouth bottle, DCM (1 mL), HX-10c (65 mg, 0.078 mmol), bromoacetylaminoethoxyethoxypropionic acid (23 mg, 0.078 mmol) and DIC (10 mg, 0.078 mmol) were added in sequence. After stirring at room temperature for 90 min, the reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile and then balanced with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid product HX-10 (24 mg, yield 27%); LCMS: (M+1) + 1106.02 (theoretical value: 1105.36).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
将化合物HX-10(0.89mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM
蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 3(3.1mg/ml,2ml)。Compound HX-10 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM Sucrose, 0.2 g/L polysorbate 20, to obtain antibody-drug conjugate ADC 3 (3.1 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.3。UV-HPLC calculated average value: n=7.3.
实施例4:7-[N-(IP140B-Ac-PEG2-Val-Ala-PABC),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(4)
Example 4: 7-[N-(IP140B-Ac-PEG 2 -Val-Ala-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (4)
Example 4: 7-[N-(IP140B-Ac-PEG 2 -Val-Ala-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (4)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-10(0.89mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 4(2.9mg/ml,2ml)。Compound HX-10 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 4 (2.9 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.6。UV-HPLC calculated average value: n=7.6.
实施例5:7-[N-(HS627-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(5)
Example 5: 7-[N-(HS627-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (5)
Example 5: 7-[N-(HS627-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (5)
取HX-9b(3g,6.32mmol),加入100mL乙腈,再加入DBU(0.48g,3.16mmol),室温反应2h,加入PPTS(0.79g,3.16mmol)、HOBT(0.85g,6.32mmol)、Fmoc-Gly-Gly-Leu-OH(2.50g,5.36mmol)和EDCI(1.21g,6.32mmol),加毕,室温反应15h,反应液旋干,加入100mL DCM和10mL异丙醇溶清,以100mL*3(0.5M)HCl、100mL*3饱和碳酸氢钠溶液、100mL饱和氯化钠溶液洗涤,20g无水硫酸钠干燥。过滤,滤液旋干,硅胶柱层析纯化,得白色粉末状固体产物HX-11a(2.66g,产率61%);LCMS:(M+1)+702.23(理论值:701.31)。Take HX-9b (3 g, 6.32 mmol), add 100 mL of acetonitrile, then add DBU (0.48 g, 3.16 mmol), react at room temperature for 2 h, add PPTS (0.79 g, 3.16 mmol), HOBT (0.85 g, 6.32 mmol), Fmoc-Gly-Gly-Leu-OH (2.50 g, 5.36 mmol) and EDCI (1.21 g, 6.32 mmol), after the addition, react at room temperature for 15 h, spin dry the reaction solution, add 100 mL of DCM and 10 mL of isopropanol to dissolve, wash with 100 mL*3 (0.5 M) HCl, 100 mL*3 saturated sodium bicarbonate solution, 100 mL of saturated sodium chloride solution, and dry over 20 g of anhydrous sodium sulfate. The filtrate was filtered and dried in a rotary evaporator. The product was purified by silica gel column chromatography to obtain a white powdery solid product HX-11a (2.66 g, yield 61%); LCMS: (M+1) + 702.23 (theoretical value: 701.31).
在500ml的单口瓶中,将HX-11a(2.66g,3.4mmol)溶于50ml甲醇和25ml的DCM混合溶剂中,加入10%的钯碳1g,氢气置换后,室温常压反应2h,过滤浓缩得到产品HX-11b(2.1g,90%);LCMS:(M+1)+612.25(理论值:611.26)。In a 500ml single-necked bottle, HX-11a (2.66g, 3.4mmol) was dissolved in 50ml methanol and 25ml DCM mixed solvent, 1g of 10% palladium carbon was added, and after hydrogen substitution, the reaction was carried out at room temperature and normal pressure for 2h, and the product HX-11b (2.1g, 90%) was obtained by filtration and concentration; LCMS: (M+1) + 612.25 (theoretical value: 611.26).
取N-(S-)甲氧基异丙胺乙基喜树碱(0.16g,0.31mmol),加入2mL DMF,HATU(1.17g,0.31mmol),DIPEA(0.8g.0.62mmol)和HX-11b(189mg,0.31mmol),室温反应15h,加入200mL DCM稀释,饱和氯化钠溶液洗涤,无水硫酸钠干燥,硅胶柱层析纯化得黄色固体产物HX-11c(254mg,产率74%);LCMS:(M+1)+1101.33(理论值:
1100.45)。Take N-(S-)methoxyisopropylamineethyl camptothecin (0.16 g, 0.31 mmol), add 2 mL DMF, HATU (1.17 g, 0.31 mmol), DIPEA (0.8 g. 0.62 mmol) and HX-11b (189 mg, 0.31 mmol), react at room temperature for 15 h, add 200 mL DCM to dilute, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a yellow solid product HX-11c (254 mg, yield 74%); LCMS: (M+1) + 1101.33 (theoretical value: 1100.45).
将HX-11c(254mg,0.23mmol)溶于0.5ml的DMF中,然后加入哌啶(179mg,2.3mmol),室温搅拌反应1h,在反应液中加入30ml甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到固体产物HX-11d(150mg,产率73.4%)直接用于下一步反应;LCMS:(M+1)+879.36(计算值:878.38)。HX-11c (254 mg, 0.23 mmol) was dissolved in 0.5 ml of DMF, and then piperidine (179 mg, 2.3 mmol) was added. The reaction was stirred at room temperature for 1 h. 30 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-11d (150 mg, yield 73.4%), which was directly used in the next step reaction; LCMS: (M+1) + 879.36 (calculated value: 878.38).
在10ml的单口瓶中,依次加入DCM(3mL),HX-11d(150mg,0.17mmol),HX-3b(49mg,0.17mmol)和DIC(21mg,0.17mmol),室温搅拌反应90min,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:5%至40%乙腈在水中,时间30min)洗脱,冻干,得到黄色固体产物HX-11(50mg,产率25%);LCMS:(M+1)+1158.22(理论值:1157.39)。In a 10 ml single-necked bottle, DCM (3 mL), HX-11d (150 mg, 0.17 mmol), HX-3b (49 mg, 0.17 mmol) and DIC (21 mg, 0.17 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile and then balanced with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid product HX-11 (50 mg, yield 25%); LCMS: (M+1) + 1158.22 (theoretical value: 1157.39).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
将化合物HX-11(0.93mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 5(3.1mg/ml,2ml)。Compound HX-11 (0.93 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 5 (3.1 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.1。UV-HPLC calculated average value: n=7.1.
实施例6:7-[N-(IP140B-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(6)
Example 6: 7-[N-(IP140B-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (6)
Example 6: 7-[N-(IP140B-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (6)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-11(0.93mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 6(3.3mg/ml,2ml)。Compound HX-11 (0.93 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 6 (3.3 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.2。UV-HPLC calculated average value: n=7.2.
实施例7:7-[N-(HS627-Ac-PEG2-Val-Ala-PABC),N-甲氧乙基]胺乙基-10,11-亚甲基二氧喜树碱(7)
Example 7: 7-[N-(HS627-Ac-PEG 2 -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (7)
Example 7: 7-[N-(HS627-Ac-PEG 2 -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (7)
在10ml单口瓶中,依次加入N-甲氧乙胺乙基喜树碱(50mg,0.10mmol),0.5ml NMP,DIPEA(65mg,0.50mmol),Fmoc-VA-PAB-PNP(69mg,0.10mmol),HOBt(13.7mg,0.10mmol),室温搅拌反应0.5h,得到HX-14b反应液直接投入至下一步;LCMS:(M+1)+1035.12(理论值:1034.41)。In a 10 ml single-necked bottle, N-methoxyethylamineethylcamptothecin (50 mg, 0.10 mmol), 0.5 ml NMP, DIPEA (65 mg, 0.50 mmol), Fmoc-VA-PAB-PNP (69 mg, 0.10 mmol), and HOBt (13.7 mg, 0.10 mmol) were added in sequence, and the reaction was stirred at room temperature for 0.5 h to obtain HX-14b. The reaction solution was directly put into the next step; LCMS: (M+1) + 1035.12 (theoretical value: 1034.41).
在上述HX-14b反应液中,加入0.5ml的哌啶(V:V=10%),室温反应30min,在反应液中加入20ml甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到固体产物HX-14c(74mg,产率71%)直接用于下一步反应;LCMS:(M+1)+813.25(理论值:812.34)。To the above HX-14b reaction solution, add 0.5 ml of piperidine (V:V = 10%), react at room temperature for 30 min, add 20 ml of methyl tert-butyl ether to the reaction solution, centrifuge, remove the supernatant, and remove the solvent under reduced pressure to obtain a solid product HX-14c (74 mg, yield 71%), which is directly used for the next step reaction; LCMS: (M+1) + 813.25 (theoretical value: 812.34).
在10ml的单口瓶中,依次加入DCM(1mL),HX-14c(74mg,0.091mmol),HX-3b(26mg,0.091mmol)和DIC(11.48mg,0.091mmol),室温搅拌反应90min,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:5%至40%乙腈在水中,时间30min)洗脱,冻干,得到黄色固体产物HX-14(36mg,产率35%);LCMS:(M+1)+1092.17(理论值:1091.35)。In a 10 ml single-necked bottle, DCM (1 mL), HX-14c (74 mg, 0.091 mmol), HX-3b (26 mg, 0.091 mmol) and DIC (11.48 mg, 0.091 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile and then balanced with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid product HX-14 (36 mg, yield 35%); LCMS: (M+1) + 1092.17 (theoretical value: 1091.35).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
将化合物HX-14(0.88mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 7(3.0mg/ml,2ml)。Compound HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 7 (3.0 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.3。UV-HPLC calculated average value: n=7.3.
实施例8:7-[N-(IP140B-Ac-PEG2-Val-Ala-PABC),N-甲氧乙基]胺乙基-10,11-亚甲基二氧喜树碱(8)
Example 8: 7-[N-(IP140B-Ac-PEG 2 -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (8)
Example 8: 7-[N-(IP140B-Ac-PEG 2 -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (8)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-14(0.88mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 8(3.3mg/ml,2ml)。Compound HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 8 (3.3 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.5。UV-HPLC calculated average value: n=7.5.
实施例9:7-[N-(HS627-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac),N-甲氧乙基]胺乙基-10,11-亚甲基二氧喜树碱(9)
Example 9: 7-[N-(HS627-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (9)
Example 9: 7-[N-(HS627-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (9)
取N-甲氧乙胺乙基喜树碱(100mg,0.20mmol),加入2mL DMF,HATU(77mg,0.20mmol),DIPEA(52mg.0.4mmol)和HX-11b(123mg,0.20mmol),室温反应15h,加入200mL DCM稀释,饱和氯化钠溶液洗涤,无水硫酸钠干燥,硅胶柱层析纯化得黄色固体HX-15a(180mg,产率81%);LCMS:(M+1)+1087.15(理论值:1086.43)。Take N-methoxyethylaminoethyl camptothecin (100 mg, 0.20 mmol), add 2 mL DMF, HATU (77 mg, 0.20 mmol), DIPEA (52 mg. 0.4 mmol) and HX-11b (123 mg, 0.20 mmol), react at room temperature for 15 h, add 200 mL DCM to dilute, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a yellow solid HX-15a (180 mg, yield 81%); LCMS: (M+1) + 1087.15 (theoretical value: 1086.43).
将HX-15a(180mg,0.17mmol)溶于0.5ml的DMF中,然后加入哌啶(179mg,2.3mmol),室温搅拌反应1h,在反应液中加入30ml甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到固体产物HX-15b(114mg,产率77%)直接用于下一步反应;LCMS:(M+1)+865.35(理论值:864.37)。HX-15a (180 mg, 0.17 mmol) was dissolved in 0.5 ml of DMF, and then piperidine (179 mg, 2.3 mmol) was added. The reaction was stirred at room temperature for 1 h. 30 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-15b (114 mg, yield 77%), which was directly used in the next step reaction; LCMS: (M+1) + 865.35 (theoretical value: 864.37).
在10ml的单口瓶中,依次加入DCM(3mL),HX-11d(114mg,0.13mmol),HX-3b(38mg,0.13mmol)和DIC(16.5mg,0.13mmol),室温搅拌反应90min,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:5%至40%乙腈在水中,时间30min)洗脱,冻干,得到黄色固体HX-15(48mg,产率32%);LCMS:(M+1)+1145.03(理论值:1143.38)。In a 10 ml single-necked bottle, DCM (3 mL), HX-11d (114 mg, 0.13 mmol), HX-3b (38 mg, 0.13 mmol) and DIC (16.5 mg, 0.13 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid HX-15 (48 mg, yield 32%); LCMS: (M+1) + 1145.03 (theoretical value: 1143.38).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
将化合物HX-15(0.92mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 9(3.0mg/ml,2ml)。Compound HX-15 (0.92 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 9 (3.0 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.4。UV-HPLC calculated average value: n=7.4.
实施例10:7-[N-(IP140B-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac),N-甲氧乙基]胺乙基-10,11-亚甲基二氧喜树碱(10)
Example 10: 7-[N-(IP140B-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (10)
Example 10: 7-[N-(IP140B-Ac-PEG 2 -Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (10)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-15(0.92mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转
盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 10(3.2mg/ml,2ml)。Compound HX-15 (0.92 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and rotated at room temperature. The reaction was allowed to proceed for 2 h. After the reaction was completed, the buffer was replaced with a 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 10 (3.2 mg/ml, 2 ml).
UV-HPLC计算平均值:n=7.3。UV-HPLC calculated average value: n=7.3.
实施例11:7-[N-(HS627-Ac-PEG2-Gly-Gly-Lys-Gly-ha-Ac),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(11)
Example 11: 7-[N-(HS627-Ac-PEG 2 -Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (11)
Example 11: 7-[N-(HS627-Ac-PEG 2 -Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (11)
取HX-9b(2g,4.21mmol)溶解于60mL乙腈,加入DBU(0.31g,2.08mmol),室温反应2h,依次加入PPTS(0.52g,2.08mmol)、HOBT(0.56g,4.2mmol)、Fmoc-Gly-Gly-Lys-OH(3.29g,4.2mmol)和EDCI(0.85g,4.2mmol),加毕,室温反应15h,减压浓缩反应液,加入100mL DCM和10mL异丙醇溶清,用烯HCl、饱和碳酸氢钠溶液、饱和氯化钠溶液洗涤,无水硫酸钠干燥。过滤,滤液旋干,硅胶层析纯化,得白色粉末状固体HX-34a(2.5g,产率60%);LCMS:(M+1)+959.11(理论值:958.43)。Take HX-9b (2g, 4.21mmol) and dissolve it in 60mL acetonitrile, add DBU (0.31g, 2.08mmol), react at room temperature for 2h, add PPTS (0.52g, 2.08mmol), HOBT (0.56g, 4.2mmol), Fmoc-Gly-Gly-Lys-OH (3.29g, 4.2mmol) and EDCI (0.85g, 4.2mmol) in sequence, react at room temperature for 15h, concentrate the reaction solution under reduced pressure, add 100mL DCM and 10mL isopropanol to dissolve, wash with dilute HCl, saturated sodium bicarbonate solution, saturated sodium chloride solution, and dry over anhydrous sodium sulfate. Filter, spin dry the filtrate, and purify it by silica gel chromatography to obtain a white powder solid HX-34a (2.5g, yield 60%); LCMS: (M+1) + 959.11 (theoretical value: 958.43).
在500ml的单口瓶中,将HX-34a(2.5g,2.6mmol)溶于40ml甲醇和20ml的DCM混合溶剂中,加入10%的钯碳(1.0g),氢气置换后,室温常压反应2h,过滤浓缩,得到固体产品HX-34b(2.2g,产率97%);LCMS:(M+1)+869.12(计算值:868.38)。In a 500 ml single-necked bottle, HX-34a (2.5 g, 2.6 mmol) was dissolved in 40 ml of methanol and 20 ml of DCM mixed solvent, 10% palladium carbon (1.0 g) was added, and after hydrogen replacement, the reaction was carried out at room temperature and normal pressure for 2 h, and the solid product HX-34b (2.2 g, yield 97%) was obtained; LCMS: (M+1) + 869.12 (calculated value: 868.38).
取HX-10a(0.1g,0.19mmol)溶解于2mL DMF,加入HATU(0.15g,0.39mmol),DIPEA(0.05g.0.39mmol)和HX-34b(0.34mg,0.39mmol),室温反应15h,加入200mL DCM稀释,饱和氯化钠溶液洗涤,无水硫酸钠干燥,硅胶柱层析纯化得黄色固体产物HX-34c(121mg,产率76%);LCMS:(M+1)+1358.52(理论值:1357.57)。HX-10a (0.1 g, 0.19 mmol) was dissolved in 2 mL DMF, HATU (0.15 g, 0.39 mmol), DIPEA (0.05 g. 0.39 mmol) and HX-34b (0.34 mg, 0.39 mmol) were added, and the mixture was reacted at room temperature for 15 h. 200 mL DCM was added to dilute the mixture, and the mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a yellow solid product HX-34c (121 mg, yield 76%); LCMS: (M+1) + 1358.52 (theoretical value: 1357.57).
将HX-11c(121mg,0.09mmol)溶于0.2mL的DMF中,然后加入哌啶(71mg,0.9mmol),室温搅拌反应1h,加入10mL甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到固体产物HX-34d(82mg,产率81%)直接用于下一步反应;LCMS:(M+1)+1136.27(理论值:1135.50)。HX-11c (121 mg, 0.09 mmol) was dissolved in 0.2 mL of DMF, and then piperidine (71 mg, 0.9 mmol) was added. The reaction was stirred at room temperature for 1 h, and 10 mL of methyl tert-butyl ether was added. The mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-34d (82 mg, yield 81%), which was directly used in the next step reaction; LCMS: (M+1) + 1136.27 (theoretical value: 1135.50).
在10mL的单口瓶中,依次加入DCM(3mL),HX-34d(82mg,0.07mmol),溴乙酰胺乙氧基乙氧基丙酸(21.5mg,0.07mmol)和DIC(9.1mg,0.07mmol),室温搅拌反应90min,加入0.1ml的二氯乙酸,继续反应30min,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:5%至40%乙腈在水中,时间30min)洗脱,冻干,得到黄色固体HX-34(43mg,产率51%);LCMS:(M+1)+1173.36(理论值:1172.40)。In a 10 mL single-necked bottle, DCM (3 mL), HX-34d (82 mg, 0.07 mmol), bromoacetamide ethoxyethoxypropionic acid (21.5 mg, 0.07 mmol) and DIC (9.1 mg, 0.07 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. 0.1 ml of dichloroacetic acid was added and the reaction was continued for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min) and freeze-dried to obtain a yellow solid HX-34 (43 mg, yield 51%); LCMS: (M+1) + 1173.36 (theoretical value: 1172.40).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室
温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the mixture was stirred at room temperature. The reaction was carried out at 25°C on a rotating turntable for 90 min.
将化合物HX-34(0.94mg,0.8mmol)溶于0.09mL的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 11(3.3mg/ml,2ml)。Compound HX-34 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 11 (3.3 mg/ml, 2 ml).
UV-HPLC计算平均值:n=2.3。UV-HPLC calculated average value: n=2.3.
实施例12:7-[N-(IP140B-Ac-PEG2-Gly-Gly-Lys-Gly-ha-Ac),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(12)
Example 12: 7-[N-(IP140B-Ac-PEG 2 -Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (12)
Example 12: 7-[N-(IP140B-Ac-PEG 2 -Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (12)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-34(0.94mg,0.8mmol)溶于0.09mL的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 12(3.1mg/ml,2ml)。Compound HX-34 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 12 (3.1 mg/ml, 2 ml).
UV-HPLC计算平均值:n=2.1。UV-HPLC calculated average value: n=2.1.
实施例13:7-[N-(HS627-Ac-PEG2-Gly-Lys-PABC),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(13)
Example 13: 7-[N-(HS627-Ac-PEG 2 -Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (13)
Example 13: 7-[N-(HS627-Ac-PEG 2 -Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (13)
在10ml单口瓶中,依次加入HX-10a(101.5mg,0.2mmol),1ml NMP,DIPEA(129mg,1mmol),Fmoc-GK-PAB-PNP(187mg,0.2mmol),HOBt(27mg,0.2mmol),室温搅拌反应4h,得到HX-35a反应液直接投入至下一步;LCMS:(M+1)+1306.48(理论值:1305.54)。In a 10 ml single-necked bottle, HX-10a (101.5 mg, 0.2 mmol), 1 ml NMP, DIPEA (129 mg, 1 mmol), Fmoc-GK-PAB-PNP (187 mg, 0.2 mmol), and HOBt (27 mg, 0.2 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 4 h to obtain HX-35a. The reaction solution was directly used in the next step; LCMS: (M+1) + 1306.48 (theoretical value: 1305.54).
在上述HX-32a反应液中,加入0.1ml的哌啶(V:V=10%),室温反应0.5h,在反应液中加入20ml甲基叔丁基醚,离心,去除上清液,减压下去除溶剂,得到固体产物HX-35b(130mg,产率50%)直接用于下一步反应;LCMS:(M+1)+1076.39(理论值:1075.43)。To the above HX-32a reaction solution, add 0.1 ml of piperidine (V:V = 10%), react at room temperature for 0.5 h, add 20 ml of methyl tert-butyl ether to the reaction solution, centrifuge, remove the supernatant, and remove the solvent under reduced pressure to obtain a solid product HX-35b (130 mg, yield 50%), which is directly used for the next step reaction; LCMS: (M+1) + 1076.39 (theoretical value: 1075.43).
在10ml的单口瓶中,依次加入DCM(2mL),HX-35b(130mg,0.12mmol),HX-3b(35mg,0.12mmol)和DIC(15mg,0.12mmol),室温搅拌反应90min,中加入0.1ml的TFA,继续反应30min,将反应液注射入25g C18预柱(先用乙腈平衡,然后用水平衡,水相含0.1%TFA),然后通过中压反相C18色谱法(梯度:5%至40%乙腈在水中,时间30min)洗脱,冻干,得到黄色固体产物HX-35(41mg,产率31%);LCMS:(M+1)+1121.04(理论值:
1120.38)。In a 10 ml single-mouth bottle, DCM (2 mL), HX-35b (130 mg, 0.12 mmol), HX-3b (35 mg, 0.12 mmol) and DIC (15 mg, 0.12 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. 0.1 ml of TFA was added and the reaction was continued for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min) and freeze-dried to obtain a yellow solid product HX-35 (41 mg, yield 31%); LCMS: (M+1) + 1121.04 (theoretical value: 1120.38).
取HS627抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na 2 HPO 4 solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.
将化合物HX-35(0.90mg,0.8mmol)溶于0.09mL的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM L-组氨酸醋酸盐缓冲液,pH6.0,120mM蔗糖,0.2g/L聚山梨酯20,得到抗体偶联药物ADC 14(3.4mg/ml,2ml)。Compound HX-35 (0.90 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 14 (3.4 mg/ml, 2 ml).
UV-HPLC计算平均值:n=4.3。UV-HPLC calculated average value: n=4.3.
实施例14:7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC),N-(S)-甲氧基异丙基]胺乙基-10,11-亚甲基二氧喜树碱(14)
Example 14: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (14)
Example 14: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (14)
取IP140B抗体(20.0mg/mL,10mg,0.066mmol),用1M Na2HPO4溶液调pH至7.2,然后再加入0.1M乙二胺四乙酸二钠的溶液(25μL),加入配置好的TCEP·HCl(三(2-羧乙基)膦盐酸盐)溶液(10mM,0.04ml),室温25℃旋转转盘反应90min。Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na 2 HPO 4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.
将化合物HX-35(0.90mg,0.8mmol)溶于0.09ml的DMA中,加入到上述溶液体系中,混匀,室温旋转转盘反应2h,反应完毕后用NAP-5凝胶柱(Cytiva)将缓冲液置换为20mM组氨酸溶液,250mM山梨醇,0.02%吐温80,pH5.7,得到抗体偶联药物ADC 14(3.2mg/ml,2ml)。Compound HX-35 (0.90 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 14 (3.2 mg/ml, 2 ml).
UV-HPLC计算平均值:n=4.6。UV-HPLC calculated average value: n=4.6.
测试例1:ADC体外抑制肿瘤细胞生长活性Test Example 1: In vitro inhibition of tumor cell growth activity by ADC
ADC体外抑制活性测试方法:将作为活性检测的人食管癌细胞OE-33、肺癌细胞NCI-H1975和乳腺癌细胞MDA-MB-231细胞分别在含有10%胎牛血清(Cellmax)的RPMI1640(Cellmax)、RPMI1640(Cellmax)和DMEM(Cellmax)培养基中培养至指数增长期,胰酶消化后,离心弃上清,用培养基稀释至分别稀释至3×104cells/mL、0.5×104cells/mL和1.5×104cells/mL,以每孔100μL加入到96孔细胞培养板中,放回37℃,5%CO2的培养箱中过夜培养。第二天,使用培养基将待测ADC稀释至2000nM、400nM、80nM、16nM、3.2nM、0.64nM、0.128nM、0.026nM,并以每孔100μL将稀释后的ADC加入到96孔细胞培养板中,每个浓度设置3个复孔,未添加ADC的阴性对照和空白对照组每孔加入100μL的培养基。加样完成后,放回37℃,5%CO2的培养箱中继续孵育6天。孵育完成后,取出细胞培养板,用移液器将培养板中的培养基吸弃,每孔加入100μL含有10%CCK-8的培养基,37℃孵育3h。孵育完成后,取出培养板,避光,置于酶标板中,选择630nm为参比波长,450nm为测定波长测定吸光度。根据吸光值,使用GraphPad中四参数回归计算IC50(表2)。以Dxd的相应ADC药物为阳性对照药物,其具有以下结构:ADC in vitro inhibitory activity test method: Human esophageal cancer cell OE-33, lung cancer cell NCI-H1975 and breast cancer cell MDA-MB-231 cells used for activity detection were cultured in RPMI1640 (Cellmax), RPMI1640 (Cellmax) and DMEM (Cellmax) culture medium containing 10% fetal bovine serum (Cellmax) to the exponential growth phase, and after trypsin digestion, the supernatant was discarded by centrifugation and diluted with culture medium to 3×10 4 cells/mL, 0.5×10 4 cells/mL and 1.5×10 4 cells/mL, respectively, and 100 μL per well was added to a 96-well cell culture plate and returned to a 37°C, 5% CO 2 incubator for overnight culture. On the second day, the ADC to be tested was diluted to 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM, 0.128nM, and 0.026nM using culture medium, and the diluted ADC was added to a 96-well cell culture plate at 100μL per well, with 3 replicates for each concentration, and 100μL of culture medium was added to each well of the negative control and blank control groups without ADC. After the addition of the sample, it was returned to the incubator at 37°C and 5% CO2 for further incubation for 6 days. After the incubation was completed, the cell culture plate was removed, the culture medium in the culture plate was discarded with a pipette, 100μL of culture medium containing 10% CCK-8 was added to each well, and incubated at 37°C for 3h. After the incubation was completed, the culture plate was removed, protected from light, placed in an ELISA plate, and 630nm was selected as the reference wavelength and 450nm was selected as the measurement wavelength to measure the absorbance. According to the absorbance value, IC 50 was calculated using four-parameter regression in GraphPad (Table 2). The corresponding ADC drug of Dxd was used as the positive control drug, which has the following structure:
其中,Ab为IP140B抗体。 Among them, Ab is IP140B antibody.
表1:ADC药物对NCI-H1975、MDA-MB-231和OE-33细胞的抑制活性IC50(nM)
Table 1: IC 50 (nM) of ADC drugs against NCI-H1975, MDA-MB-231 and OE-33 cells
Table 1: IC 50 (nM) of ADC drugs against NCI-H1975, MDA-MB-231 and OE-33 cells
对于IC50值,其中“++++”表示IC50≤50nM;“+++”表示50nM<IC50≤200nM之间;“++”表示100<IC50≤500nM之间;“+”表示IC50>500nM。For IC 50 values, "++++" indicates IC 50 ≤ 50 nM; "+++" indicates 50 nM < IC 50 ≤ 200 nM; "++" indicates 100 < IC 50 ≤ 500 nM; and "+" indicates IC 50 > 500 nM.
本发明的实施例ADC化合物,对NCI-H1975、MDA-MB-231和OE-33细胞均具有较好的抑制活性,部分ADC化合物例如ADC10、12对该些癌细胞的抑制活性IC50甚均低于50nM。The ADC compounds of the embodiments of the present invention have good inhibitory activity against NCI-H1975, MDA-MB-231 and OE-33 cells. The IC 50 of the inhibitory activity of some ADC compounds such as ADC10 and 12 against these cancer cells is even lower than 50 nM.
测试例2:ADC体内抑制肿瘤生长活性Test Example 2: In vivo tumor growth inhibition activity of ADC
ADC体外抑制活性测试方法:人非小细胞肺癌Calu-6、人食管鳞癌细胞KYSE-150和人卵巢癌细胞ES-2于体外单层培养,细胞饱和度为80%-90%时,用胰酶-EDTA消化,离心弃上清,PBS重悬细胞,将细胞悬液调整至合适浓度,将Calu-6、KYSE-150细胞(2-10×106cells/0.1ml)皮下接种BALB/c裸小鼠,ES-2细胞皮下接种于NOD-SCID小鼠,定期观察动物及移植瘤生长情况,待瘤体积长到100-200mm3左右,根据瘤体积和体重进行随机分组,即溶媒对照组(生理盐水)和ADC给药组(溶于生理盐水),每组6只动物。静脉注射给药,给药频率为Q4D,共给药2次(第一次给药的时间记为Day 1,Day 5第二次给药),实验分组及给药设置见表2。每周2次用游标卡尺测量肿瘤长径a(mm)、短径b(mm)和小鼠体重,根据以下公式计算肿瘤体积(tumor volume,V):V=1/2×a×b2(mm3),其中a和b分别表示肿瘤长和宽,并绘制生长曲线,最后剥取肿瘤并称重。统计分析基于试验结束时肿瘤体积和荷瘤鼠体重的数据运用GraphPad Prism软件进行分析得到抑瘤结果,图中对照组“\”表示该组无结果,未测肿瘤状况,实验组无对应肿瘤、“肿瘤缩小为0”表示在相应解剖动物体内没有发现肿瘤残留。ADC in vitro inhibitory activity test method: Human non-small cell lung cancer Calu-6, human esophageal squamous cell carcinoma cell KYSE-150 and human ovarian cancer cell ES-2 were cultured in vitro monolayers. When the cell saturation was 80%-90%, they were digested with trypsin-EDTA, centrifuged and discarded the supernatant, and the cells were resuspended in PBS. The cell suspension was adjusted to an appropriate concentration. Calu-6 and KYSE-150 cells (2-10×10 6 cells/0.1ml) were subcutaneously inoculated into BALB/c nude mice, and ES-2 cells were subcutaneously inoculated into NOD-SCID mice. The animals and transplanted tumor growth were observed regularly. When the tumor volume grew to about 100-200mm 3 , they were randomly divided into vehicle control group (normal saline) and ADC administration group (dissolved in normal saline), with 6 animals in each group. The drugs were administered by intravenous injection, and the administration frequency was Q4D, with a total of 2 administrations (the time of the first administration was recorded as Day 1, and the second administration was recorded on Day 5). The experimental grouping and administration settings are shown in Table 2. The long diameter a (mm), short diameter b (mm) of the tumor and the weight of the mice were measured with a vernier caliper twice a week, and the tumor volume (V) was calculated according to the following formula: V = 1/2 × a × b 2 (mm 3 ), where a and b represent the length and width of the tumor, respectively. A growth curve was drawn, and the tumor was finally removed and weighed. Statistical analysis The tumor inhibition results were obtained by analyzing the data of tumor volume and weight of tumor-bearing mice at the end of the experiment using GraphPad Prism software. The control group "\" in the figure means that there is no result in this group and the tumor condition was not measured. There is no corresponding tumor in the experimental group, and "tumor reduction to 0" means that no tumor residue was found in the corresponding dissected animal.
表2:小鼠分组及给药设置
Table 2: Mouse grouping and drug administration settings
Table 2: Mouse grouping and drug administration settings
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.
Claims (11)
- 式(I)所示的抗体-药物偶联物,
The antibody-drug conjugate represented by formula (I)
式中,In the formula,R1、R2各自独立地选自氢、氘、C1-C6烷基;R 1 and R 2 are each independently selected from hydrogen, deuterium, and C 1 -C 6 alkyl;L1选自-L11-L12-L13-,其中,L11、L12、L13各自独立地选自-O-、C1-C3亚烷基和苯基;L 1 is selected from -L 11 -L 12 -L 13 -, wherein L 11 , L 12 , and L 13 are each independently selected from -O-, C 1 -C 3 alkylene, and phenyl;LP为由2-7个氨基酸构成的肽残基;L P is a peptide residue consisting of 2-7 amino acids;Z选自-Lz-Lj-,其中,Lz选自-C(=O)-C1-C8亚烷基和-C(=O)-(CH2CH2O)2-6-CH2CH2NH-,Lj为可偶联抗体的接头;Z is selected from -Lz - Lj- , wherein Lz is selected from -C(=O) -C1 - C8 alkylene and -C(=O)-( CH2CH2O ) 2-6 - CH2CH2NH- , and Lj is a linker that can be coupled to an antibody;n1选自0-3的整数;n 1 is an integer selected from 0-3;Ab为抗体。Ab is an antibody. - 根据权利要求1所述的抗体-药物偶联物,其中,R1、R2各自独立地选自氢、氘和C1-C3烷基;The antibody-drug conjugate according to claim 1, wherein R 1 and R 2 are each independently selected from hydrogen, deuterium and C 1 -C 3 alkyl;优选地,R1、R2各自独立地选自氢、氘、甲基、乙基和异丙基;Preferably, R 1 and R 2 are each independently selected from hydrogen, deuterium, methyl, ethyl and isopropyl;优选地,R1选自氢、氘、甲基、乙基和异丙基,R2选自氢、氘、甲基和乙基;Preferably, R 1 is selected from hydrogen, deuterium, methyl, ethyl and isopropyl, and R 2 is selected from hydrogen, deuterium, methyl and ethyl;优选地,R1为甲基,R2为氢或甲基。Preferably, R1 is methyl and R2 is hydrogen or methyl.
- 根据权利要求1或2所述的抗体-药物偶联物,其中,L11、L12、L13各自独立地选自-O-、亚甲基和苯基;The antibody-drug conjugate according to claim 1 or 2, wherein L 11 , L 12 , and L 13 are each independently selected from -O-, methylene, and phenyl;优选地,L11-L12-L13选自-O-CH2-苯基-和-CH2-O-CH2-。Preferably, L 11 -L 12 -L 13 are selected from -O-CH 2 -phenyl- and -CH 2 -O-CH 2 -.
- 根据权利要求1-3任一项所述的抗体-药物偶联物,其中,所述氨基酸选自苯丙氨酸(Phe)、甘氨酸(Gly)、缬氨酸(Val)、丙氨酸(Ala)、亮氨酸(Leu)、赖氨酸(Lys)、瓜氨酸(Cit)和天冬氨酸(Asn);The antibody-drug conjugate according to any one of claims 1 to 3, wherein the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn);优选地,Lp选自由2-5个选自苯丙氨酸(Phe)、甘氨酸(Gly)、缬氨酸(Val)、丙氨酸(Ala)、亮氨酸(Leu)、赖氨酸(Lys)、瓜氨酸(Cit)和天冬氨酸(Asn)的氨基酸构成的肽残基;Preferably, Lp is selected from a peptide residue consisting of 2-5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn);优选地,Lp选自由2-4个选自苯丙氨酸(Phe)、甘氨酸(Gly)、缬氨酸(Val)、丙氨酸(Ala)、和亮氨酸(Leu)、赖氨酸(Lys)的氨基酸构成的肽残基;Preferably, L p is selected from a peptide residue consisting of 2-4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys);优选地,Lp选自-Val-Cit-、-Val-Ala-、-Gly-Val-Ala-、-Gly-Val-Ala-Gly-、-Gly-Lys-、-Gly-Gly-Lys-、-Gly-Gly-Lys-Gly-、-Val-Ala-、-Ala-Ala-Asn-、-Gly-Leu-、-Gly-Gly-Leu-、-Gly-Gly-Leu-Gly-、-Gly-Phe-、-Gly-Gly-Phe-和-Gly-Gly-Phe-Gly-;Preferably, Lp is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe- and -Gly-Gly-Phe-Gly-;优选地,Lp选自 其羰基端连接-NH-,另一端连接Z。 Preferably, L p is selected from Its carbonyl end is connected to -NH- and the other end is connected to Z.
- 根据权利要求1-4任一项所述的抗体-药物偶联物,其中,Lj选自 所示位置表示与抗体相连,所示位置表示与LZ基团相连;The antibody-drug conjugate according to any one of claims 1 to 4, wherein L is selected from The positions shown indicate attachment to antibodies. The positions shown indicate attachment to the L Z group;优选地,Lz选自-C(=O)-C1-C8亚烷基和-C(=O)-(CH2CH2O)2-6-CH2CH2NH-;Preferably, L z is selected from —C(═O)—C 1 -C 8 alkylene and —C(═O)—(CH 2 CH 2 O) 2-6 -CH 2 CH 2 NH—;优选地,Lz选自-C(=O)-C1-C6亚烷基和-C(=O)-(CH2CH2O)2-4-CH2CH2NH-;Preferably, L z is selected from —C(═O)—C 1 -C 6 alkylene and —C(═O)—(CH 2 CH 2 O) 2-4 —CH 2 CH 2 NH—;优选地,Lz选自-C(=O)-(CH2)5-和-C(=O)-(CH2CH2O)2-CH2CH2NH-;Preferably, L z is selected from -C(=O)-(CH 2 ) 5 - and -C(=O)-(CH 2 CH 2 O) 2 -CH 2 CH 2 NH-;优选地,Z选自所示位置表示与抗体相连;所示位置表示与Lp基团相连。Preferably, Z is selected from The positions shown indicate attachment to antibodies; The positions shown indicate attachment to the L p group.
- 根据权利要求1-5任一项所述的抗体-药物偶联物,其中,所述抗体为肿瘤相关抗原抗体;The antibody-drug conjugate according to any one of claims 1 to 5, wherein the antibody is a tumor-associated antigen antibody;优选地,所述肿瘤相关抗原抗体选自抗Her2抗体、抗Trop2抗体、抗B7H3抗体、抗5T4、抗Nectin-4抗体、抗CD20抗体和抗ROR1抗体。Preferably, the tumor-associated antigen antibody is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody and anti-ROR1 antibody.
- 根据权利要求1-6任一项所述的抗体-药物偶联物,式(I)所示的抗体-药物偶联物具有如式(I-1)或式(I-2)所示的结构,
The antibody-drug conjugate according to any one of claims 1 to 6, wherein the antibody-drug conjugate represented by formula (I) has a structure as represented by formula (I-1) or formula (I-2),
式中,R1、R2、n1、Lp、Z、Ab各自定义同式(I)化合物。In the formula, R 1 , R 2 , n 1 , L p , Z and Ab have the same meanings as the compound of formula (I). - 以下抗体-药物偶联物:
The following antibody-drug conjugates:
其中,Ab定义同式(I)化合物;wherein Ab is defined as the compound of formula (I);优选地,Ab为HS627抗体或IP140B抗体,所述HS627的重链氨基酸序列如SEQ ID NO:1所示,其轻链氨基酸序列如SEQ ID NO:2所示;所述IP140B抗体的重链氨基酸序列如SEQ ID NO:3所示,其轻链氨基酸序列如SEQ ID NO:4所示。Preferably, Ab is HS627 antibody or IP140B antibody, the heavy chain amino acid sequence of HS627 is shown in SEQ ID NO:1, and the light chain amino acid sequence thereof is shown in SEQ ID NO:2; the heavy chain amino acid sequence of IP140B antibody is shown in SEQ ID NO:3, and the light chain amino acid sequence thereof is shown in SEQ ID NO:4. - 药物组合物,其包括权利要求1-8任一项所述的抗体-药物偶联与药学上可接受的载体。A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
- 权利要求1-8任一项所述的抗体-药物偶联物或权利要求9所述的药物组合物在制备抗肿瘤药物中的用途;Use of the antibody-drug conjugate according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of an anti-tumor drug;优选地,所述肿瘤选自实体瘤,更优选自肺癌、食管癌、乳腺癌、非小细胞肺癌、食管鳞癌或卵巢癌。Preferably, the tumor is selected from solid tumors, more preferably lung cancer, esophageal cancer, breast cancer, non-small cell lung cancer, esophageal squamous cell carcinoma or ovarian cancer.
- 治疗肿瘤疾病的方法,其包括向有需要的患者施用权利要求1-8任一项所述的抗体-药物偶联物或权利要求9所述的药物组合物的步骤。 A method for treating tumor diseases, comprising the step of administering the antibody-drug conjugate according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 to a patient in need thereof.
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