WO2023134692A1 - 多环类化合物及其用途 - Google Patents

多环类化合物及其用途 Download PDF

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WO2023134692A1
WO2023134692A1 PCT/CN2023/071705 CN2023071705W WO2023134692A1 WO 2023134692 A1 WO2023134692 A1 WO 2023134692A1 CN 2023071705 W CN2023071705 W CN 2023071705W WO 2023134692 A1 WO2023134692 A1 WO 2023134692A1
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group
compound
substituted
yap
ring
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PCT/CN2023/071705
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English (en)
French (fr)
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梁阿朋
李钧
吴豫生
刘广斌
董胜利
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浙江同源康医药股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the technical field of medicine, in particular to a polycyclic compound used for regulating the Hippo pathway and its preparation method and use, mainly used for the treatment or prevention of proliferative diseases (such as cancer), especially for the regulation and treatment of YAP/ Related diseases caused by abnormal activity of TEAD.
  • proliferative diseases such as cancer
  • the Hippo pathway essentially consists of a core kinase cascade comprising the Ste-20 family of protein kinases MST1-2, the scaffolding protein Salvador and the large tumor suppressor kinase LATS1-2, and the repressive transcriptional coactivator YAP (Yes1 associated protein) and TAZ (transcriptional coactivator with PDZ binding motif).
  • YAP and TAZ are the main effectors of the Hippo signaling pathway, and together with TEAD (transcriptional enhancer association domain) in the nucleus, they act as transcription factors to increase the expression of target genes such as CTGF (connective tissue growth factor), CYR61, etc.
  • CTGF connective tissue growth factor
  • CYR61 CYR61
  • TEAD transcription factors lie at the heart of the Hippo pathway and are critical for regulating organ growth and wound repair.
  • Dysregulation of TEAD and its regulatory cofactor Yes-associated protein (YAP) have been implicated in many human cancers and hyperproliferative pathological processes, and dysregulation of this pathway is frequently detected in human cancers.
  • YAP and TAZ activation have been identified in many human tumors and are critical for tumor initiation, progression, and metastasis, as seen in patients with breast, ovarian, colon, liver, and pancreatic cancers. There was elevated expression of YAP and it was associated with reduced survival.
  • YAP or TAZ enhances TEAD-dependent gene expression (eg, CCN1, CTGF, ITGB2 and Birc5/survivin) and promotes cell proliferation and migration in many cell types.
  • TEAD-dependent gene expression eg, CCN1, CTGF, ITGB2 and Birc5/survivin
  • blocking signaling or interfering with YAP/TAZ-TEAD complex formation to prevent the expression of many mitogenic TEAD target genes significantly reduced cell proliferation and oncogenic transformation activity.
  • the Hippo pathway also cross-talks with other signaling pathways such as Wnt, Notch, Hedgehog, and MAPK, thereby affecting a variety of biological functions, and its dysfunction may not only be involved in cancer, but also be involved in in many human diseases. Therefore, the YAP-TEAD complex is a promising therapeutic target.
  • the object of the present invention is to provide a compound represented by formula I, its preparation method and its use in regulating and treating diseases related to the abnormal activity of YAP/TEAD.
  • the first aspect of the present invention provides a compound represented by formula I, or a pharmaceutically acceptable salt, solvate or prodrug thereof,
  • Ring A is selected from the group consisting of:
  • Ring B is a substituted or unsubstituted 5-10 membered heteroaryl group containing 1-3 heteroatoms selected from N, O or S, wherein a C atom on the ring is connected to R 1 , and the substitution means being replaced by 1 -3 substituents R are substituted;
  • Ring C is selected from the group consisting of substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 6-10 membered heteroaryl containing 1-3 heteroatoms selected from N, O or S, the substituted means substituted by 1-3 substituents R;
  • R 1 is selected from the group consisting of:
  • R 2 , R 3 , R 4 and R 5 are each independently selected from the following substituted or unsubstituted groups: H, halogen, CN, NH 2 , ester group, urea group, carbamate group, amide group, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 cycloalkoxy, C6- C10 aryl, 6-10 membered heteroaryl containing 1-3 heteroatoms selected from N, O or S, wherein the substitution refers to being substituted by 1-3 substituents R;
  • R 4 , R 5 and adjacent N form a 4-6 membered heterocyclic group containing 1 N;
  • R 6 and R 7 are each independently selected from the following group: C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, 6-6 containing 1-3 heteroatoms selected from N, O or S 10-membered heteroaryl, wherein the substitution refers to being substituted by 1-3 substituents R;
  • Each R is independently selected from the group consisting of halogen, CN, OH, NH 2 , ester group, urea group, carbamate group, amido group, C1-C6 alkyl group, C1-C6 alkoxy group, C3-C6 ring Alkyl, C3-C6 cycloalkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 aryl, 6-10 membered heteroatoms containing 1-3 heteroatoms selected from N, O or S Aryl;
  • n is selected from the following group: 0, 1, 2, 3;
  • n is selected from the group: 0,1,2,3.
  • ring B is selected from the following group:
  • Ring C is selected from the group consisting of:
  • R is selected from the following group:
  • R 2 is selected from the group consisting of trifluoromethyl, fluorine, chlorine, bromine, iodine, cyclopentyl, cyclohexyl.
  • the compound is selected from the following group:
  • the second aspect of the present invention provides a pharmaceutical composition, comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof , solvate or prodrug.
  • the third aspect of the present invention provides the use of the pharmaceutical composition described in the second aspect of the present invention for the preparation of drugs for treating related diseases caused by Hippo pathway disorders.
  • the fourth aspect of the present invention provides a use of the pharmaceutical composition described in the second aspect of the present invention, which is used for the preparation and treatment of disorders caused by YAP or TAZ or YAP/TAZ or YAP/TEAD or YAP/TAZ/TEAD Drugs that cause related diseases.
  • the fifth aspect of the present invention provides a use of the pharmaceutical composition described in the second aspect of the present invention for the preparation of medicines for treating related diseases caused by YAP/TEAD or YAP/TAZ/TEAD imbalance.
  • the disease is cancer.
  • the sixth aspect of the present invention provides a use of the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a second active ingredient for the preparation of a drug for treating cancer ;
  • the second active ingredient is selected from the group consisting of ERK inhibitors, MEK inhibitors, KRAS inhibitors, BRAF inhibitors, EGFR inhibitors, Wnt inhibitors, and PD-1 inhibitors.
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, liver cancer, and pancreatic cancer.
  • Figure 1 is the activity test results of the present invention, wherein TY-2400 corresponds to T-1 of the present invention, TY-2401 corresponds to compound FA of the present invention, TY-2407b corresponds to compound T-2 of the present invention, TY-2410 corresponds to compound T-4 of the present invention, TY-2412 corresponds to compound T-8 of the present invention.
  • Figure 2 is the structural formula of TY-2401 corresponding compound FA.
  • halogen refers to F, Cl, Br or I.
  • C1-C6 alkyl refers to a linear or branched alkyl group including 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl group, tert-butyl group, neopentyl group, tertyl group, or similar groups.
  • C2-C6 alkenyl refers to a straight chain or branched alkenyl group with 2-6 carbon atoms containing a double bond, including non-limiting ethenyl, propenyl, butenyl , Isobutenyl, Pentenyl and Hexenyl etc.
  • C2-C6 alkynyl refers to a straight-chain or branched-chain alkynyl group with 2-6 carbon atoms containing a triple bond, including without limitation ethynyl, propynyl, butynyl, group, isobutynyl, pentynyl and hexynyl, etc.
  • C3-C8 cycloalkyl refers to a cyclic alkyl group having 3-8 carbon atoms in the ring, including without limitation cyclopropyl, cyclobutyl, cyclopentyl, cyclo Hexyl, cycloheptyl, cyclooctyl, etc.
  • C3-C6 cycloalkyl has a similar meaning.
  • C1-C6 alkoxy refers to a straight-chain or branched alkoxy group with 1-6 carbon atoms, including without limitation methoxy, ethoxy, propoxy, Isopropoxy and butoxy, etc. Preference is given to C1-C4 alkoxy.
  • heterocyclic group is a 4-8 membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O, S, including (but not limited to) the following groups:
  • aromatic ring or “aryl” has the same meaning, preferably “C6-C10 aryl”.
  • C6-C10 aryl refers to an aromatic ring group having 6-10 carbon atoms without heteroatoms in the ring, such as phenyl, naphthyl and the like.
  • heteroaryl has the same meaning and refers to a heteroaromatic group containing one to more heteroatoms.
  • C3-C10 heteroaryl refers to an aromatic heterocyclic ring containing 1-4 heteroatoms selected from oxygen, sulfur and nitrogen and 3-10 carbon atoms.
  • Non-limiting examples include: furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring.
  • Heteroaryl groups can be optionally substituted or unsubstituted.
  • halo refers to substitution by halogen.
  • substituted means that one or more hydrogen atoms on a specific group are replaced by a specific substituent.
  • the specific substituents are the corresponding substituents described above, or the substituents appearing in each embodiment.
  • a substituted group may have a substituent selected from a specific group at any substitutable position of the group, and the substituents may be the same or different at each position.
  • substituents contemplated by this invention are those that are stable or chemically feasible.
  • the substituents are for example (but not limited to): halogen, hydroxyl, carboxyl (-COOH), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 3- to 12-membered heterocyclic group, aryl group, heteroaryl group, C1-C8 aldehyde group, C2-C10 acyl group, C2-C10 ester group, amino group, C1-C6 alkoxy group, C1-C10 sulfonyl group, etc.
  • the term 1-6 means 1, 2, 3, 4, 5 or 6. Other similar terms have similar meanings.
  • ester group has the structure -C(O)-O-R' or R'-C(O)-O-, wherein R' independently represents hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl , C6-C10 aryl, heteroaryl, heterocyclyl, as defined above.
  • ureido has Structure, wherein Ra, Rb are each independently selected from H, C1-C6 alkyl, halogenated C1-C6 alkyl, C6-C10 aryl.
  • urethane group has Structure, wherein Ra, Rb are each independently selected from H, C1-C6 alkyl, halogenated C1-C6 alkyl, C6-C10 aryl.
  • Amido refers to a group with the structure -CONRR', wherein R and R' can independently represent hydrogen, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, aryl or Substituted aryl, heterocycle or substituted heterocycle, as defined above. R and R' can be the same or different in the dialkylamine moiety.
  • the present invention provides a compound represented by formula I, or a pharmaceutically acceptable salt, solvate or prodrug thereof,
  • each group is as defined above.
  • any one of ring A, ring B, ring C, R 1 , R 2 and m is the corresponding group in the specific compound described in this application.
  • the compound is preferably the compound prepared in the examples.
  • the term "pharmaceutically acceptable salt” refers to a salt of a compound of the present invention with an acid or a base which is suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and other organic acids; amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid
  • salts of the compounds of the present invention with bases such as alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. magnesium or calcium salts), ammonium salts (e.g.
  • lower alkanolammonium salts and other pharmaceutically acceptable amine salts such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butyl amine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.
  • methylamine salts such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butyl amine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salt
  • solvate refers to a complex in which a compound of the present invention coordinates with solvent molecules to form a specific ratio.
  • prodrug includes itself may be biologically active or inactive, when administered in an appropriate manner, it undergoes metabolism or chemical reactions in the human body to convert into a class of compounds of formula I, or formula A A salt or solution of a compound.
  • the prodrugs include (but are not limited to) carboxylates, carbonates, phosphates, nitrates, sulfates, sulfone esters, sulfoxide esters, amino compounds, carbamates, azo compounds of the compounds , phosphoramide, glucoside, ether, acetal and other forms.
  • the preparation method of the compound of formula I of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention.
  • the compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in the specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention belongs.
  • Example 1 the preparation process of the compound of the present invention is shown in Example 1, wherein the raw materials and reagents used can be purchased through commercial channels unless otherwise specified.
  • compositions and methods of administration are provided.
  • the compound of the present invention has excellent antitumor activity, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and drugs containing the compound of the present invention as the main active ingredient
  • the composition can be used for the treatment, prevention and alleviation of tumor-related diseases.
  • the pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier.
  • safe and effective dose refers to: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-1000 mg of the compound of the present invention per dose.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low enough toxicity. "Compatibility” herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound.
  • Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and derivatives thereof such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid , magnesium stearate
  • the pharmaceutical composition is injection, capsule, tablet, pill, powder or granule.
  • the mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostea, or
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner.
  • coatings and shell materials such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner.
  • Examples of usable embedding components are polymeric substances and waxy substances.
  • the active compounds can also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.
  • inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and
  • compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required, if necessary.
  • the compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as antineoplastic drugs).
  • other pharmaceutically acceptable compounds such as antineoplastic drugs.
  • the treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily
  • the dosage is usually 1-2000 mg, preferably 50-1000 mg.
  • factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the present invention has the following main advantages:
  • the compound of the present invention has very good inhibitory activity on YAP/TEAD.
  • the compound of the present invention has good pharmacology, pharmacokinetics and pharmacodynamic effects.
  • the synthetic route is as follows:
  • DMSO DMEM medium containing 1% DMSO
  • Dual-Lumi II Renilla luciferase detection reagent According to the amount of 50 ⁇ L Dual-Lumi II Renilla luciferase detection reagent required for each sample, prepare an appropriate amount of Dual-Lumi II Renilla luciferase detection reagent. Mix an appropriate amount of Dual-Lumi II Renilla luciferase detection substrate and Dual-Lumi II Renilla luciferase detection buffer at a ratio of 1:100 to prepare the Dual-Lumi II Renilla luciferase detection reagent.
  • Renilla luciferase Using Renilla luciferase as an internal reference, the RLU value obtained by firefly luciferase was divided by the RLU value obtained by Renilla luciferase to obtain the relative fluorescence signal intensity of each well.
  • the average signal intensity of the DMSO group was regarded as 100%, and the percentage of signal intensity at each concentration was calculated respectively.
  • Tumor cells were cultured in RPMI-1640 (containing 10% FBS and 100 U/mL penicillin-G/streptomycin) at 37°C, 5% CO2 and saturated humidity to a density of 80-90%.
  • the cell suspension was added to a 96-well plate with a volume of 100 ⁇ L per well, and cultured overnight in a 37° C., 5% CO2 incubator.
  • test compound gradient dilution solution the test compound was prepared as 1 mM stock solution. Then, 1.5 ⁇ l of the stock solution was dissolved in 1.5 ml of DMSO-free culture solution, and then 3-fold serial dilution was performed with 0.1% DMSO culture solution, with a total of 9 concentrations.
  • concentration of the compound after dilution was as follows:
  • Cell viability The cell proliferation activity or cytotoxicity activity is obtained by curve fitting with GraphPad Prism 8 software to obtain the IC50 value.
  • Test example 2 Nano Luciferase method to detect the activity of YAP-TEAD inhibitors
  • Vi-cell to measure the cell concentration and dilute to a suspension of 5*10 ⁇ 5 cells per ml. After the cell suspension is prepared, mix gently and add 10ml of liquid to a 10cm culture dish. In this way, the number of cells in each 10cm dish is 5*10 ⁇ 6. Incubate at 37°C for 1 day in a 5% CO2 incubator.
  • the above mixture was evenly added dropwise to a 10 cm Petri dish, and replaced with fresh complete medium after 6 hours of cultivation.
  • Vi-cell to measure the cell concentration and dilute to a suspension of 20,000 cells per milliliter.
  • the 10 mM compound stock solution was diluted to 50 ⁇ M with the culture medium, and the 50 ⁇ M compound solution was added to the third column of the deep well plate in sequence, and 216 ⁇ L of the medium containing 0.5% DMSO was added to the fourth to eleventh columns.
  • the 96-well plate was incubated in a 5% CO2 incubator at 37° C. for 48 hours, then taken out and equilibrated at room temperature for 10 minutes.

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Abstract

本发明涉及多环类化合物及其用途。具体地，本发明化合物具有式I所示结构，其中各基团和取代基的定义如说明书中所述；本发明还公开了所述化合物的制备方法及其在调节和治疗与YAP/TEAD的异常活性所导致的相关疾病方面的用途。

Description

多环类化合物及其用途 技术领域
本发明涉及医药技术领域，具体涉及用作调节Hippo通路的多环化合物及其制备方法和用途，主要用于增殖性疾病(如癌症)的治疗或预防，尤其是用于调节和治疗与YAP/TEAD的异常活性所导致的相关疾病。
背景技术
Hippo通路基本上由一个核心激酶级联反应组成，该级联反应包含蛋白激酶MST1-2的Ste-20家族，支架蛋白Salvador和大肿瘤抑制激酶LATS1-2，以及抑制转录共激活因子YAP(Yes1相关蛋白)和TAZ(具有PDZ结合基序的转录共激活子)。YAP和TAZ是Hippo信号通路的主要效应因子，它们与核内的TEAD(转录增强的缔合结构域)一起充当转录因子，从而增加诸如CTGF(结缔组织生长因子)、CYR61等靶基因的表达。Hippo途径是细胞生长、增殖和迁移的重要调节因子。TEAD转录因子位于Hippo途径的核心，对于调节器官生长和伤口修复至关重要。TEAD的失调和其调节辅因子Yes-相关蛋白(YAP)已涉及许多人类癌症和过度增殖病理过程，在人类癌症中经常检测到该通路的失调。与TEAD蛋白一样，YAP和TAZ激活已在许多人类肿瘤中被鉴定，并且对肿瘤的起始、进展和转移至关重要，如在乳腺癌、卵巢癌、结肠癌、肝癌和胰腺癌患者中均存在YAP表达升高，且与生存率降低有关。与此一致，YAP或TAZ的激活或过表达增强TEAD依赖性基因表达(例如，CCN1，CTGF，ITGB2和Birc5/存活蛋白)并促进许多细胞类型中的细胞增殖和迁移。相反，阻断YAP/TAZ-TEAD复合物形成的信号或干预阻止许多促有丝分裂TEAD靶基因的表达，能显著降低细胞增殖和致癌转化活性。此外，Hippo通路还与其他信号传导通路诸如Wnt、Notch、Hedgehog和MAPK等通路发生串扰(cross-talk)，从而影响多种生物功能，其功能失调可能除了参与到癌症中之外，还参与到很多人类疾病中。因此YAP-TEAD复合物是一种很有前景的治疗靶点。
发明内容
本发明的目的在于提供一种式I所示化合物及其制备方法和其在调节和治疗与YAP/TEAD的异常活性所导致的相关疾病方面的用途。
本发明的第一方面，提供了式Ⅰ所示的化合物，或其药学上可接受的盐、溶剂化物或前药，
Figure PCTCN2023071705-appb-000001
其中，
环A选自下组：
Figure PCTCN2023071705-appb-000002
环B为取代或未取代的含1-3个选自N、O或S的杂原子的5-10元杂芳基,其中环上的一个C原子与R 1相连，所述取代指被1-3个取代基R取代；
环C选自下组：取代或未取代的C6-C10芳基、取代或未取代的含1-3个选自N、O或S的杂原子的6-10元杂芳基，所述取代指被1-3个取代基R取代；
R 1选自下组：
Figure PCTCN2023071705-appb-000003
R 2、R 3、R 4和R 5各自独立地选自取代或未取代的下组基团：H、卤素、CN、NH 2、酯基、脲基、氨基甲酸酯基、酰胺基、C1-C6烷基、C2-C6烯基、C2-C6炔基、卤代C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷氧基、C6-C10芳基、含1-3个选自N、O或S的杂原子的6-10元杂芳基，其中，所述取代指被1-3个取代基R取代；
或者R 4、R 5与相邻的N形成含1个N的4-6元杂环基；
R 6和R 7各自独立地选自下组：C1-C6烷基、C3-C6环烷基、C6-C10芳基、含1-3个选自N、O或S的杂原子的6-10元杂芳基，其中，所述取代指被1-3个取代基R取代；
各R独立地选自下组：卤素、CN、OH、NH 2、酯基、脲基、氨基甲酸酯基、酰胺基、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷氧基、C2-C6烯基、C2-C6炔基、C6-C10芳基、含1-3个选自N、O或S的杂原子的6-10元杂芳基；
m选自下组：0、1、2、3；
n选自下组：0、1、2、3。
在另一优选例中，环B选自下组：
Figure PCTCN2023071705-appb-000004
Figure PCTCN2023071705-appb-000005
环C选自下组：
Figure PCTCN2023071705-appb-000006
在另一优选例中，R 1选自下组：
Figure PCTCN2023071705-appb-000007
R 2选自下组：三氟甲基、氟、氯、溴、碘、环戊基、环己基。
在另一优选例中，所述化合物选自下组：
Figure PCTCN2023071705-appb-000008
本发明的第二方面，提供了一种药物组合物，包含药学上可接受的载体和一种或多种安全有效量的本发明第一方面所述的化合物，或其药学上可接受的盐、溶剂化物或前药。
本发明的第三方面，提供了一种本发明第二方面所述的药物组合物的用途，用于制备用于治疗由Hippo通路失调导致的相关疾病的药物。
本发明的第四方面，提供了一种本发明第二方面所述的药物组合物的用途，用于制备用于治疗由YAP或TAZ或YAP/TAZ或YAP/TEAD或YAP/TAZ/TEAD失调导 致的相关疾病的药物。
本发明的第五方面，提供了一种本发明第二方面所述的药物组合物的用途，用于制备用于治疗由YAP/TEAD或YAP/TAZ/TEAD失调导致的相关疾病的药物。
在另一优选例中，所述疾病为癌症。
本发明的第六方面，提供了一种本发明第一方面所述的化合物，或其药学上可接受的盐、溶剂化物或前药与第二活性成分的用途，用于制备治疗癌症的药物；
所述第二活性成分选自下组：ERK抑制剂、MEK抑制剂、KRAS抑制剂、BRAF抑制剂、EGFR抑制剂、Wnt抑制剂、PD-1抑制剂。
在另一优选例中，所述癌症选自下组：乳腺癌、卵巢癌、结肠癌、肝癌、胰腺癌。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为本发明活性测试结果，其中，TY-2400对应本发明T-1、TY-2401对应化合物FA、TY-2407b对应本发明化合物T-2、TY-2410对应本发明化合物T-4、TY-2412对应本发明化合物T-8。
图2为TY-2401对应化合物FA的结构式。
具体实施方式
本发明人经过长期而深入的研究，意外地制备了一种对于与YAP/TEAD的异常活性所导致的相关疾病具有优异调节和治疗作用的式I所示化合物。在此基础上，发明人完成了本发明。
术语
在本发明中，除非特别指出，所用术语具有本领域技术人员公知的一般含义。
在本发明中，术语“卤素”指F、Cl、Br或I。
在本发明中，“C1-C6烷基”是指包括1-6个碳原子的直链或支链的烷基，例如 甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、新戊基、特戊基、或类似基团。
在本发明中，术语“C2-C6烯基”是指具有2-6个碳原子的含有一个双键的直链或支链烯基，非限制性地包括乙烯基、丙烯基、丁烯基、异丁烯基、戊烯基和己烯基等。
在本发明中，术语“C2-C6炔基”是指具有2-6个碳原子的含有一个三键的直链或支链炔基，非限制性地包括乙炔基、丙炔基、丁炔基、异丁炔基、戊炔基和己炔基等。
在本发明中，术语“C3-C8环烷基”是指在环上具有3-8个碳原子的环状烷基，非限制性地包括环丙基、环丁基、环戊基、环己基、环庚基、环辛基等。术语“C3-C6环烷基”具有类似含义。
在本发明中，术语“C1-C6烷氧基”是指具有1-6个碳原子的直链或支链烷氧基，非限制性地包括甲氧基、乙氧基、丙氧基、异丙氧基和丁氧基等。优选为C1-C4烷氧基。
在本发明中，术语“杂环基”为含1、2或3个选自N、O、S的杂原子的4-8元杂环基，包括(但并不限于)如下基团：
Figure PCTCN2023071705-appb-000009
在本发明中，术语“芳环”或“芳基”具有相同的含义，优选为“C6-C10芳基”。术语“C6-C10芳基”是指在环上不含杂原子的具有6-10个碳原子的芳香族环基，如苯基、萘基等。
在本发明中，术语“芳香杂环”或“杂芳基”具有相同的含义，指包含一个到多个杂原子的杂芳族基团。例如“C3-C10杂芳基”是指含有1～4个选自氧、硫和氮中的杂原子以及3-10个碳原子的芳香杂环。非限制性例子包括：呋喃基、噻吩基、吡啶基、吡唑基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。杂芳基可以是任选取代的或未取代的。
在本发明中，术语“卤代”是指被卤素取代。
在本发明中，术语“取代”指特定的基团上的一个或多个氢原子被特定的取代基所取代。特定的取代基为在前文中相应描述的取代基，或各实施例中所出现的取代基。除非特别说明，某个取代的基团可以在该基团的任何可取代的位点上具有一个选自特定组的取代基，所述的取代基在各个位置上可以是相同或不同的。本领域技术人员应理解，本发明所预期的取代基的组合是那些稳定的或化学上可实现的组合。所述取代基例如(但并不限于)：卤素、羟基、羧基(-COOH)、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、3-至12元杂环基、芳基、杂芳基、C1-C8醛基、C2-C10酰基、C2-C10酯基、氨基、C1-C6烷氧基、C1-C10磺酰基 等。
在本发明中，术语1-6个指1、2、3、4、5或6个。其他类似术语具有类似含义。
术语“酯基”具有-C(O)-O-R’或R’-C(O)-O-结构，其中，R’独立地代表氢、C1-C6烷基、C3-C6环烷基、C6-C10芳基、杂芳基、杂环基，如上文所定义。
术语“脲基”具有
Figure PCTCN2023071705-appb-000010
结构，其中Ra、Rb各自独立地选自H、C1-C6烷基、卤代C1-C6烷基、C6-C10芳基。
术语“氨基甲酸酯基”具有
Figure PCTCN2023071705-appb-000011
结构，其中Ra、Rb各自独立地选自H、C1-C6烷基、卤代C1-C6烷基、C6-C10芳基。
术语“酰胺基”是指带有结构-CONRR'的基团，其中，R和R'可以独立的代表氢、烷基或取代的烷基、环烷基或取代的环烷基、芳基或取代的芳基、杂环或取代的杂环，如上文所定义。R和R'在二烷基胺片段中可以相同或不同。
化合物
本发明提供了式Ⅰ所示的化合物，或其药学上可接受的盐、溶剂化物或前药，
Figure PCTCN2023071705-appb-000012
其中，各基团如上文所定义。
在另一优选例中，所述的化合物中，环A、环B、环C、R 1、R 2和m中任一个分别为本申请所述具体化合物中所对应的基团。
在另一优选例中，所述化合物优选为实施例中所制备的化合物。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸；甲酸、乙酸、三氟乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、苯甲酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘磺酸等有机酸；以及脯氨酸、苯丙氨酸、 天冬氨酸、谷氨酸等氨基酸。
另一类优选的盐是本发明化合物与碱形成的盐，例如碱金属盐(例如钠盐或钾盐)、碱土金属盐(例如镁盐或钙盐)、铵盐(如低级的烷醇铵盐以及其它药学上可接受的胺盐)，例如甲胺盐、乙胺盐、丙胺盐、二甲基胺盐、三甲基胺盐、二乙基胺盐、三乙基胺盐、叔丁基胺盐、乙二胺盐、羟乙胺盐、二羟乙胺盐、三羟乙胺盐，以及分别由吗啉、哌嗪、赖氨酸形成的胺盐。
术语“溶剂化物”指本发明化合物与溶剂分子配位形成特定比例的配合物。
术语“前药”包括其本身可以是具有生物学活性的或非活性的，当用适当的方法服用后，其在人体内进行代谢或化学反应而转变成式I的一类化合物，或式A的一个化合物所组成的盐或溶液。所述的前药包括(但不局限于)所述化合物的羧酸酯、碳酸酯、磷酸酯、硝酸酯、硫酸酯、砜酯、亚砜酯、氨基化合物、氨基甲酸盐、偶氮化合物、磷酰胺、葡萄糖苷、醚、乙缩醛等形式。
制备方法
下面更具体地描述本发明式I结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。
典型地，本发明化合物的制备工艺流程如实施例1所示，其中所用原料和试剂如无特殊说明，均可通过商业途径购买。
药物组合物和施用方法
由于本发明化合物具有优异的抗肿瘤活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解与肿瘤相关的疾病。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-1000mg本发明化合物/剂。较佳地， 所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2023071705-appb-000013
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
所述的药物组合物为注射剂、囊剂、片剂、丸剂、散剂或颗粒剂。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙 二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的其他化合物(如抗肿瘤药物)联合给药。
本发明治疗方法可以单独施用，或者与其它治疗手段或者治疗药物联用。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选50～1000mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
与现有技术相比，本发明具有以下主要优点：
(1)本发明化合物对YAP/TEAD有非常好的抑制活性。
(2)本发明化合物有很好的药理、药代和药效效果。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明， 否则百分比和份数按重量计算。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
本发明合成的化合物：
Figure PCTCN2023071705-appb-000014
合成路线如下：
Figure PCTCN2023071705-appb-000015
实验过程如下：
(1)化合物3的合成
在250mL的三口瓶中加入化合物1(10g,64.4mmol),化合物2(20g,74.1mmol),醋酸(100mL),130度反应16h。反应完毕，加入200mL水，加碳酸氢钠调pH＝8左右，乙酸乙酯萃取，有大量悬浮固体，为产品，萃取至水相无产物，有机相和固体加甲醇溶解，干燥过柱，分离4.0g纯品。
(2)化合物5的合成
500mL的单口瓶中依次加入化合物3(3.9g,0.011mol,1.0eq)，水/1，4-二氧六环(1/5)(240mL)，Pd(dppf)Cl 2(0.48g,0.655mmol,0.11eq)，碳酸钠(3.43g,0.032mol,3.0eq)，化合物4(2.69g,0.013mol,1,2eq)，氮气置换三次后放入油浴锅中，升至100度18h,反应结束后降至室温后过滤，乙酸乙酯洗，滤液旋干过柱子得到产品2.3g。
(3)化合物6的合成
将化合物5(2.3g,6.33mmol,1.0eq)溶于乙醇(50mL)中，加入Pd/C(0.07g,10％)，氢气环境下反应1.5天至原料消失，直接过滤旋干得到粗品2.0g.
(4)化合物7的合成
将化合物6(1.6g,4.38mol)加入乙醇中，再加入水合肼9.6g，与95度下回流3h，冷却后直接旋干得到粗品1.5g。
(5)化合物9的合成
将化合物7(1.5g,4.27mmol,1.0eq)溶于DCM/DMF(30mL/30mL)中，加入三乙胺(863mg,8.54mmol,2.0eq)，将反应液降温至0度，滴加化合物8(1.04mg,8.54mmol,2.0eq)，后升至室温反应4h，反应完毕直接旋干过柱子得2.145g。
(6)化合物10的合成
将化合物9(2.145g,4.91mmol,1.0eq)溶于DCM/DMF(40mL/40mL)中，后加入对甲苯磺酰氯(2.79g,14.6mmol,3.0eq)，三乙胺(1.49g,14.7mmol,3.0eq)，室温反应5h，反应完成后，加水，EA萃取后，干燥过滤旋干，过柱子纯化得到黄色固体488mg.
(7)化合物T-1的合成
将化合物10(35mL,0.083mmol,1.0eq)，加入氨水(3mL)，室温搅拌3h,固体直接过滤得9mg产品。LC-MS[M+1]：405.1。
参照实施例1的合成方法，合成如下化合物：
Figure PCTCN2023071705-appb-000016
Figure PCTCN2023071705-appb-000017
Figure PCTCN2023071705-appb-000018
(一)生物活性测试实验过程如下：
(1)转染
a)细胞培养：在转染前一天将2*10^6 293T细胞接种到10cm培养皿内进行培养，第二天细胞密度约70％。
b)在进行转染之前，把培养有细胞的培养皿换成10mL新鲜DMEM培养基(含10％血清，不含双抗)。
c)取两个无菌离心管，分别加入750μL不含抗生素和血清的DMEM，然后于一管加入10μg 3xSDYAP-luci和3μg Renilla质粒，并用移液器轻轻吹打混匀；另一管加入30μL Lipo6000转染试剂，用移液器轻轻吹打混匀。室温静置5分钟，将含有DNA的培养液用移液器轻轻加入含Lipo6000转染试剂的培养液中，轻轻颠倒离心管混匀，室温静置5分钟。
d)将混匀的Lipo6000转染试剂-DNA混合物均匀的滴加到培养皿内，随后轻轻混匀。
e)培养6小时后更换为新鲜的完全培养液。
f)继续培养24小时后，trypsin处理并计数细胞，按照每孔6000细胞加入96孔培养板。
(2)药物处理
药物浓度：
Figure PCTCN2023071705-appb-000019
Blank:DMEM培养基
DMSO：含1％DMSO的DMEM培养基
处理48小时后检测荧光信号
(3)荧光信号的检测
a)检测试剂的准备
i.融解Dual-Lumi II荧火虫荧光素酶检测试剂和Dual-Lumi II海肾荧光素酶检测缓冲液，并达到室温。
ii.按照检测每个样品需50μL Dual-Lumi II海肾荧光素酶检测试剂的量，配置适 量Dual-Lumi II海肾荧光素酶检测试剂。按照1:100比例混合适量的Dual-Lumi II海肾荧光素检测底物和Dual-Lumi II海肾荧光素酶检测缓冲液，配制成Dual-Lumi II海肾荧光素酶检测试剂。
b)荧光素酶的检测
萤火虫荧光素酶的检测
i.取出细胞培养板在室温平衡10分钟。
ii.96孔板，吸去培养液，每孔加入50μL Dual-Lumi II萤火虫荧光素酶检测试剂，混匀。
iii.室温孵育10分钟，使发光信号稳定。
iv.使用酶标仪进行化学发光检测，每个孔检测时间为1秒。
海肾荧光素酶的检测
i.96孔板每孔加入50μL Dual-Lumi II海肾荧光素酶检测试剂，混匀。
ii.室温孵育10分钟，使发光数据稳定。
iii.使用酶标仪进行化学发光检测，每个孔检测时间为1秒
c)数据计算
以海肾荧光素酶为内参，用萤火虫荧光素酶测定得到的RLU值除以海肾荧光素酶测定得到的RLU值得到每个孔的相对荧光信号强度。将DMSO组的平均信号强度视为100％，分别计算各个浓度下信号强度的百分比。
(4)实验结果如图1所示：其中化合物的浓度为2.5μM,化合物处理时间为72h。
Luciferase activity of DMSO vs drug treatment
*,ρ<0.05
**,ρ<0.01
***,ρ<0.001
(二)生物活性测试实验过程如下：
试验例1细胞抗增殖实验
YAP-TEAD抑制剂化合物对人胸膜间皮瘤细胞NCI-H226的增殖抑制测定
实验材料和设备：人胸膜间皮瘤细胞NCI-H226购于南京科佰生物科技有限公司。RPMI-1640培养基(Bio-Channel)，DMSO(二甲基亚砜),CCK8(WST-8)细胞分析试剂盒(碧云天)，0.25％EDTA-Tripsin(胰酶消化液),1xPBS(磷酸盐缓冲液，PH7.2)，96孔板(Corning),胎牛血清(FBS)，10,000U/mL青霉素-G/链霉素,高速冷冻离心机(EPPENDORF 5810R)，酶联免疫检测仪(Tecan Spark)。
实验准备：
1、细胞铺板
A)肿瘤细胞在37℃，5％CO2及饱和湿度的条件下，在RPMI-1640(含10％FBS和100U/mL青霉素-G/链霉素)中培养至80-90％密集度。
B)去除10cm培养皿中的培养基；
C)用10ml 1xPBS润洗细胞一遍；
D)加4ml 0.25％EDTA-Tripsin放入37℃，5％CO2培养箱胰酶消化5分钟，转移到15ml离心管，200g离心5分钟，弃上清得到细胞沉淀；
E)用4ml DMEM培养基重悬，计数并调整到10,000细胞/ml。
F)将细胞悬液加入96孔板每孔体积100μL，在37℃,5％CO2培养箱中培养过夜。
2、化合物处理
化合物稀释
A)配制受测化合物梯度稀释溶液：将测试化合物配置成1mM储液。然后用1.5μl储液溶解于1.5ml无DMSO培养液中，再以0.1％DMSO培养液进行3倍连续梯度稀释，共9个浓度，稀释后化合物浓度如下：
333.33nM，111.11nM，37.03nM，12.35nM，4.15nM，1.37nM，0.46nM，0.15nM
B)充分混匀后分别取100μL培养化合物溶液替代细胞培养板中的培养液，每个浓度4个复孔；
C)将细胞转移至培养箱孵育3天。
3、CCK8(WST-8)细胞分析检测
A)取出细胞培养板，在生物安全柜中每孔加入10微升CCK-8(WST-8)溶液；
B)把细胞培养板放回培养箱继续孵育3小时；
C)在TECAN酶联免疫检测仪上选择450nm波长测定吸光度值。
4、数据分析
使用如下公式计算细胞活力(％Cell Viability):
细胞活力(％)＝[A(加药)－A(空白)]/[A(0加药)－A(空白)]×100
A(加药)：具有细胞、CCK8溶液和药物溶液的孔的吸光度
A(空白)：具有培养基和CCK8溶液而没有细胞的孔的吸光度
A(0加药)：具有细胞、CCK8溶液而没有药物溶液的孔的吸光度
细胞活力：细胞增殖活力或细胞毒性活力通过GraphPad Prism 8软件进行曲线拟合得到IC50数值。
试验例2、Nano Luciferase方法检测YAP-TEAD抑制剂活性
(1)实验材料和设备：293T细胞购于南京科佰生物科技有限公司。DMEM培养基(高糖,无酚红，Bio-Channel)，DMSO(二甲基亚砜),Lipo6000 TM转染试剂(碧云天)，pGL3B-8xGTiiC-nLuc-CMV-fLuc质粒，0.25％EDTA-Tripsin(胰酶消化液),1xPBS(磷酸盐缓冲液，PH7.2)，96孔白色细胞培养板(PerkinElmer),胎牛血清(FBS)，10,000U/mL青霉素-G/链霉素,高速冷冻离心机(EPPENDORF5810R)，37℃CO2培养箱，
Figure PCTCN2023071705-appb-000020
细胞计数仪，Envision酶标仪(PerkinElmer)。
试剂
Figure PCTCN2023071705-appb-000021
Figure PCTCN2023071705-appb-000022
(2)293T细胞瞬时转染
将复苏的293T细胞接种到10cm培养皿，置于5％的CO2培养箱中，37℃恒温培养，为确保转染效率，应当使用处于对数期(细胞密度约50％-70％)的细胞。
在转染前一天，用Trypsin-EDTA消化对数期细胞，加入培养基终止反应，用移液器吹打混匀制成细胞悬液。
使用Vi-cell测定细胞浓度，稀释成每毫升5*10^5个细胞的悬液。细胞悬液制备完成后，轻轻混匀，在10cm培养皿中加入10ml液体。这样每个10cm培养皿中的细胞数量为5*10^6。在5％的CO2培养箱中，37℃恒温培养1天。
取两个洁净无菌离心管，分别加入750μL不含抗生素和血清的opti-MEM Medium，然后于其中一管加入15μg质粒(pGL3B-8xGTiiC-nLuc-CMV-fLuc)，并用移液器轻轻吹打混匀；另一管加入Lipo6000转染试剂，用移液器轻轻吹打混匀。室温静置5分钟后，将含有DNA的培养液轻轻加入含Lipo6000转染试剂的培养液中，轻轻颠倒离心管混匀，室温静置5分钟。
将上述的混合物均匀的滴加到10cm培养皿中，并在培养6小时后更换为新鲜的完全培养基。
(3)96孔板铺板
转染1天后，Trypsin EDTA消化细胞，加入培养基终止反应，用移液器吹打混匀制成细胞悬液。
使用Vi-cell测定细胞浓度，稀释成每毫升20000个细胞的悬液。
细胞悬液制备完成后，轻轻混匀，在96孔板中，每孔加入100μL，这样待测的细胞的密度为2000个每孔。
(4)加入化合物
将接种好的细胞培养板放入培养箱中培养，约24小时后加入浓度梯度的化合物。
将10mM的化合物储液用培养基稀释到50μM，依次在深孔板的第三列加入50μM的化合物溶液，第四到十一列加入216μL含0.5％DMSO的培养基。
梯度稀释：从第三列吸取100μL溶液加入第四列，混匀以后；再从第四列吸取100μL溶液加入第五列，重复该操作直至第十一列。
使用多通道移液器，从深孔板中吸取25μL化合物溶液加入96孔培养板，每个化合物在96孔板上重复四次。最终在96孔板上形成最高浓度为10000nM，1:3.16的浓度梯度。
(5)加入Nano-luciferase检测试剂，读数
96孔板在5％的CO2培养箱中，37℃恒温培养48小时后，取出于室温平衡10分钟。
每孔加入100μL检测试剂，置于水平摇床低速震荡10分钟，充分裂解细胞。使用PerkinElmer Envision酶标仪检测每个孔的荧光值。
(6)结果计算
以0nM作为对照，将每个孔的数值转换成百分比，使用GrahpPad prism软件中的[Inhibitor]vs.response(three parameters)进行非线性拟合计算IC 50。
如表1所示，其中A≤1μM；1μM<B<5μM；C≥5μM
表1
Figure PCTCN2023071705-appb-000023
Figure PCTCN2023071705-appb-000024
Figure PCTCN2023071705-appb-000025
Figure PCTCN2023071705-appb-000026
Figure PCTCN2023071705-appb-000027
Figure PCTCN2023071705-appb-000028
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 式Ⅰ所示的化合物，或其药学上可接受的盐、溶剂化物或前药，
    Figure PCTCN2023071705-appb-100001
    其中，
    环A选自下组：
    Figure PCTCN2023071705-appb-100002
    环B为取代或未取代的含1-3个选自N、O或S的杂原子的5-10元杂芳基,其中环上的一个C原子与R 1相连，所述取代指被1-3个取代基R取代；
    环C选自下组：取代或未取代的C6-C10芳基、取代或未取代的含1-3个选自N、O或S的杂原子的6-10元杂芳基，所述取代指被1-3个取代基R取代；
    R 1选自下组：
    Figure PCTCN2023071705-appb-100003
    R 2、R 3、R 4和R 5各自独立地选自取代或未取代的下组基团：H、卤素、CN、NH 2、酯基、脲基、氨基甲酸酯基、酰胺基、C1-C6烷基、C2-C6烯基、C2-C6炔基、卤代C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷氧基、C6-C10芳基、含1-3个选自N、O或S的杂原子的6-10元杂芳基，其中，所述取代指被1-3个取代基R取代；
    或者R 4、R 5与相邻的N形成含1个N的4-6元杂环基；
    R 6和R 7各自独立地选自下组：C1-C6烷基、C3-C6环烷基、C6-C10芳基、含1-3个选自N、O或S的杂原子的6-10元杂芳基，其中，所述取代指被1-3个取代基R取代；
    各R独立地选自下组：卤素、CN、OH、NH 2、酯基、脲基、氨基甲酸酯基、酰胺基、C1-C6烷基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烷氧基、C2-C6烯基、C2-C6炔基、C6-C10芳基、含1-3个选自N、O或S的杂原子的6-10元杂芳基；
    m选自下组：0、1、2、3；
    n选自下组：0、1、2、3。
  2. 如权利要求1所述的化合物，其特征在于，环B选自下组：
    Figure PCTCN2023071705-appb-100004
    Figure PCTCN2023071705-appb-100005
    环C选自下组：
    Figure PCTCN2023071705-appb-100006
  3. 如权利要求1所述的化合物，其特征在于，R 1选自下组：
    Figure PCTCN2023071705-appb-100007
    Figure PCTCN2023071705-appb-100008
    R 2选自下组：三氟甲基、氟、氯、溴、碘、环戊基、环己基。
  4. 如权利要求1所述的化合物，其特征在于，所述化合物选自下组：
    Figure PCTCN2023071705-appb-100009
  5. 一种药物组合物，其特征在于，包含药学上可接受的载体和一种或多种安全有效量的权利要求1所述的化合物，或其药学上可接受的盐、溶剂化物或前药。
  6. 一种权利要求5所述的药物组合物的用途，其特征在于，用于制备用于治疗由Hippo通路失调导致的相关疾病的药物。
  7. 一种权利要求5所述的药物组合物的用途，其特征在于，用于制备用于治疗由YAP或TAZ或YAP/TAZ或YAP/TEAD或YAP/TAZ/TEAD失调导致的相关疾病的药物。
  8. 一种权利要求5所述的药物组合物的用途，其特征在于，用于制备用于治疗由YAP/TEAD或YAP/TAZ/TEAD失调导致的相关疾病的药物。
  9. 如权利要求6-8任一所述的用途，其特征在于，所述疾病为癌症。
  10. 一种权利要求1所述的化合物，或其药学上可接受的盐、溶剂化物或前药与第二活性成分的用途，其特征在于，用于制备治疗癌症的药物；
    所述第二活性成分选自下组：ERK抑制剂、MEK抑制剂、KRAS抑制剂、BRAF抑制剂、EGFR抑制剂、Wnt抑制剂、PD-1抑制剂。
PCT/CN2023/071705 2022-01-13 2023-01-10 多环类化合物及其用途 WO2023134692A1 (zh)

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