A Wnt pathway inhibitor compound

Abstract

The present invention relates to a Wnt pathway inhibitor compound represented by Formula 4 and a method for synthesizing the same, as well as a pharmaceutical composition comprising the compound, and the use of the compound of Formula 4 for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, or immune-mediated diseases. #imgabs0#

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number "202280013624.X". The filing date of the parent application is July 26, 2022 (PCT international filing date), the Chinese national application number is "202280013624.X" (PCT international application number is PCT / CN2022 / 107727), and the name of the invention is "A Wnt pathway inhibitor compound".

[0002] The parent application and this divisional application claim priority to Chinese patent application 202110847130.9, filed with the State Intellectual Property Office of China on July 26, 2021, entitled “A Wnt pathway inhibitor compound”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to a heterocyclic compound, in particular to a highly active Wnt pathway inhibitor and a preparation method and application thereof. Background Art

[0004] The Wnt / β-catenin signaling pathway is an evolutionarily conserved pathway. In normal somatic cells, β-catenin functions solely as a cytoskeletal protein, forming a complex with E-cadherin at the cell membrane to maintain homotypic cell adhesion and prevent cell migration. When the Wnt signaling pathway is inactive, β-catenin in the cytoplasm becomes phosphorylated and forms a β-catenin degradation complex with proteins such as APC, Axin, and GSK3β. This triggers the ubiquitin system to degrade β-catenin via the proteasome, maintaining low cytoplasmic β-catenin levels. When cells are stimulated by Wnt signals, Wnt proteins bind to specific membrane receptors called Frizzled proteins. The activated Frizzled receptors recruit intracellular Dishevelled proteins, inhibiting the degradation activity of the β-catenin degradation complex formed by proteins such as GSK3β, thereby stabilizing free β-catenin in the cytoplasm. After stably accumulating in the cytoplasm, β-catenin enters the nucleus and binds to the LEF / TCF transcription factor family, initiating the transcription of downstream target genes (such as c-myc, c-jun, and Cyclin D1). Overactivation of the Wnt / β-catenin signaling pathway is closely associated with the development of various cancers, including colon, gastric, and breast cancer. For example, aberrant activation of the canonical Wnt signaling pathway and nuclear accumulation of β-catenin protein are common in colorectal cancer. Inhibiting Wnt signaling can inhibit the proliferation of cancers such as colon cancer. Mutations in APC are found in over 85% of colorectal cancers. Mutated APC blocks the phosphorylation and degradation of β-catenin, inducing the development of colorectal cancer. Furthermore, mutations in Axin and β-catenin itself can also cause intracellular accumulation of β-catenin, activating the Wnt / β-catenin pathway.

[0005] Although it is known that inhibiting the Wnt signaling pathway can effectively prevent and / or treat cancer, tumors, inflammatory diseases, autoimmune diseases, and immune-mediated diseases, the current state of the art lacks satisfactory and effective Wnt pathway inhibitor compounds. Therefore, the development of effective Wnt pathway inhibitor compounds is a need in the art. Summary of the Invention

[0006] In one aspect, the present invention provides a compound for inhibiting Wnt pathway activity having a structure of Formula 4 or a pharmaceutically acceptable salt, isotopic derivative, or stereoisomer thereof:

[0007]

[0008] Furthermore, the present invention also provides a pharmaceutical composition comprising the compound of the present invention or its pharmaceutically acceptable salt, isotope derivative or stereoisomer.

[0009] Furthermore, the present invention also provides the use of the compound of the present invention or its pharmaceutically acceptable salt, isotopic derivative, stereoisomer, or the pharmaceutical composition of the present invention in the preparation of a medicament for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, or immune-mediated diseases. It is particularly noted that, herein, when referring to the "compound" of Formula 4, it generally also includes its stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives.

[0010] Accordingly, the present invention provides a method for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases, comprising administering to a subject a compound of the present invention or a pharmaceutically acceptable salt, isotopic derivative, stereoisomer thereof or a pharmaceutical composition of the present invention.

[0011] It is well known to those skilled in the art that salts, solvates and hydrates of a compound are alternative forms of existence of the compound, and they can all be converted into the compound under certain conditions. Therefore, it is particularly noted that when referring to the compound of Formula 4 herein, it generally also includes its pharmaceutically acceptable salts, and further includes its solvates and hydrates.

[0012] Similarly, reference herein to a compound generally also includes prodrugs, metabolites, and N-oxides thereof.

[0013] Pharmaceutically acceptable salts of the present invention may be formed using, for example, the following inorganic or organic acids: "pharmaceutically acceptable salts" refers to salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, or the like, within the scope of sound medical judgment, and at a reasonable benefit / risk ratio. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base or free acid with a suitable reagent, as outlined below. For example, the free base function may be reacted with a suitable acid. Examples of pharmaceutically acceptable inorganic acid addition salts are salts formed of an amino group with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or by using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium salts, quaternary ammonium salts, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0014] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example, by dissolving the compound of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol and acetonitrile), adding an excess of an organic acid or an aqueous inorganic acid solution thereto to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.

[0015] The precursors or metabolites described herein may be those known in the art, as long as the precursors or metabolites are converted to compounds through in vivo metabolism. For example, "prodrugs" refer to those prodrugs of the compounds of the present invention that, within the scope of reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and are considered to have a reasonable benefit / risk ratio and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly converted in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism or N-demethylation of the compounds of the present invention.

[0016] As used herein, "solvate" refers to a physical association of a compound of the invention with one or more solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In some cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of isolation. The solvent molecules in the solvate may exist in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0017] "Stereoisomerism" as used herein is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism (i.e., optical isomerism). Conformational isomerism refers to the stereoisomerism phenomenon in which the atoms or atomic groups of an organic molecule with a certain configuration have different spatial arrangements due to the rotation or distortion of carbon-carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair and boat conformations that occur in the structure of cyclohexane. "Stereoisomers" refer to compounds of the present invention that contain one or more asymmetric centers and can therefore exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers. The compounds of the present invention have asymmetric centers, and each asymmetric center can produce two optical isomers. The scope of the present invention includes all possible optical isomers and diastereomeric mixtures, as well as pure or partially purified compounds. The compounds of the present invention may exist as tautomers, which have different hydrogen attachment points due to the displacement of one or more double bonds. For example, a ketone and its enol form are keto-enol tautomers. Each tautomer and mixtures thereof are included in the compounds of the present invention. Enantiomers, diastereomers, racemates, mesomorphs, cis-trans isomers, tautomers, geometric isomers, epimers and mixtures thereof of all compounds of formula 4 are included within the scope of the present invention.

[0018] The "isotopic derivative" of the present invention refers to a molecule in which the compound is isotopically labeled. The isotopes commonly used as isotopic labels are: hydrogen isotopes, 2 H and 3 H; Carbon isotope: 11 C, 13 C and 14 C; Chlorine isotope: 35 Cl and 37 Cl; Fluorine isotope: 18 F; Iodine isotope: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. 3 H and carbon 13 C, because they are easy to label and detect, they are more widely used. Some heavy isotopes, such as deuterium ( 2H), substitution can enhance metabolic stability and prolong half-life, thereby achieving a reduction in dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques similar to those used for synthesizing non-isotope-labeled compounds.

[0019] The present invention also provides use of the compound of the present invention in preparing a medicament for preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

[0020] In addition, the present invention provides a pharmaceutical composition for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases, or immune-mediated diseases, comprising a compound of the present invention as an active ingredient. The pharmaceutical composition may optionally contain a pharmaceutically acceptable carrier.

[0021] Furthermore, the present invention provides a method for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases or immune-mediated diseases, which comprises administering a compound of the present invention to a mammal in need thereof.

[0022] When the compound of the present invention or a pharmaceutically acceptable salt thereof is administered in combination with another anticancer agent or immune checkpoint inhibitor for treating cancer or tumors, the compound of the present invention or a pharmaceutically acceptable salt thereof may provide enhanced anticancer effects.

[0023] When the compounds of the present invention, or pharmaceutically acceptable salts thereof, are administered in combination with another therapeutic agent for treating inflammatory diseases, autoimmune diseases, and immune-mediated diseases, the compounds of the present invention, or pharmaceutically acceptable salts thereof, may provide enhanced therapeutic effects.

[0024] The compound of the present invention or its pharmaceutically acceptable salt can be used as an active ingredient by oral or parenteral administration. The dosage of the active ingredient can be adjusted according to multiple relevant factors (such as the situation of the subject to be treated, the type of disease and severity, the rate of administration and the doctor's opinion). In some cases, the amount less than the above dosage may be suitable. If no harmful side effects are caused, the amount greater than the above dosage can be used and the amount can be applied in divided doses every day.

[0025] In addition, the present invention also provides a method for preventing and / or treating tumors, cancers, viral infections, organ transplant rejection, neurodegenerative diseases, attention-related diseases or autoimmune diseases, which comprises administering the compound of the present invention or the pharmaceutical composition of the present invention to a mammal in need thereof.

[0026] The pharmaceutical composition of the present invention can be formulated into dosage forms for oral administration or parenteral administration (including intramuscular, intravenous and subcutaneous routes, intratumor injection) according to any of the conventional methods, such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions or suspensions.

[0027] The pharmaceutical composition of the present invention for oral administration can be prepared by mixing the active ingredient with carriers such as cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactants, suspending agents, emulsifiers and diluents.

[0028] Examples of carriers used in the pharmaceutical compositions for injection administration of the present invention may include water, saline solutions, glucose solutions, glucose-like solutions, alcohols, glycols, ethers (e.g., polyethylene glycol 400), oils, fatty acids, fatty acid esters, glycerides, surfactants, suspending agents, and emulsifying agents.

[0029] Other features of the present invention will become apparent as the present invention describes exemplary embodiments, which are given to illustrate the present invention and are not intended to be limiting thereof. The following examples were prepared, isolated, and characterized using the methods disclosed herein.

[0030] The compounds of the present invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the following methods and synthetic methods known in the field of organic synthetic chemistry or variations thereof known to those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent or solvent mixture suitable for the kit materials used and for the desired transformation. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule is consistent with the proposed transformation. This sometimes requires judgment to change the order of the synthesis steps or the raw materials to obtain the desired compounds of the present invention. DETAILED DESCRIPTION

[0031] the term

[0032] Unless otherwise indicated, the terms used in this application, including the specification and claims, are defined as follows. Unless otherwise indicated, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology will be used. Throughout this application, unless otherwise indicated, the use of "or" or "and" means "and / or."

[0033] In the specification and claims, a given chemical formula or name shall encompass all stereoisomers and optical isomers thereof and racemates thereof in which such isomers exist. Unless otherwise indicated, all chiral (enantiomers and diastereoisomers) and racemic forms are within the scope of the present invention. Multiple geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc. may also exist in the compounds, and all such stable isomers are encompassed by the present invention. The present invention describes cis- and trans- (or E- and Z-) geometric isomers of the compounds of the present invention, and they can be separated into mixtures of isomers or separate isomeric forms. The compounds of the present invention can be isolated in optically active or racemic form. All methods for preparing the compounds of the present invention and the intermediates prepared therein are considered part of the present invention. When enantiomeric or diastereomeric products are prepared, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization). Depending on the process conditions, the final products of the present invention are obtained in free (neutral) or salt form. Both the free forms and salts of these final products are within the scope of the present invention. If desired, one form of the compound can be converted into another form. A free base or acid can be converted into a salt; a salt can be converted into a free compound or another salt; and a mixture of isomeric compounds of the present invention can be separated into its individual isomers. The compounds of the present invention, their free forms, and salts can exist in multiple tautomeric forms, in which hydrogen atoms are transposed to other parts of the molecule and the chemical bonds between the atoms of the molecule are rearranged. It should be understood that all tautomeric forms that may exist are included in the present invention.

[0034] The term "patient" as used herein refers to an organism to be treated by the method of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., mice, apes, monkeys, horses, cows, pigs, dogs, cats, etc.) and most preferably refer to humans.

[0035] As used herein, the term "effective amount" means an amount of a drug or pharmaceutical agent (i.e., a compound of the present invention) that will cause a biological or medical response in a tissue, system, animal, or human being, such as that sought by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the above amount. An effective amount can be given in one or more administrations, applications, or dosages and is not intended to be limited by a specific formulation or route of administration. The term also includes within its scope an effective amount that enhances normal physiological function.

[0036] As used herein, the term "treating" includes any effect that results in improvement of a condition, disease, disorder, etc., such as alleviation, reduction, modulation, improvement, or elimination, or amelioration of the symptoms thereof.

[0037] The term "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response and / or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0038] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutical substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating substance, which is involved in carrying or transporting the subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0039] The term "pharmaceutical composition" means a composition comprising a compound of the present invention and at least one other pharmaceutical carrier. "Pharmaceutical carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals (particularly mammals), including (i.e.) adjuvants, excipients or vehicles such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants, depending on the mode of administration and the nature of the dosage form.

[0040] The term "acceptable," as used herein, means that a prescribed ingredient or active ingredient has no undue adverse effect on health and well-being for the general purpose of treatment.

[0041] The term "cancer," as used herein, refers to an abnormal, uncontrolled growth of cells that can metastasize (spread) under certain conditions. This type of cancer includes, but is not limited to, solid tumors (such as those of the bladder, intestine, brain, chest, uterus, heart, kidney, lung, lymphoid tissue (lymphoma), ovary, pancreas or other endocrine organs (such as the thyroid), prostate, skin (melanoma), or blood tumors (such as non-leukemic leukemias).

[0042] The term "combination administration" or its like, as used herein, refers to the administration of several selected therapeutic agents to a single patient, using the same or different administration routes at the same or different times.

[0043] The terms "enhance" or "capable of enhancing," as used herein, refer to the ability to increase or prolong the potency or duration of a desired outcome. Thus, in the context of enhancing the therapeutic effect of a drug, the term "capable of enhancing" refers to the ability of the drug to increase or prolong the potency or duration of the drug in a system. "Potentiation," as used herein, refers to the ability of another therapeutic agent to maximize its effectiveness in an ideal system.

[0044] The term "immune disease" refers to a disease or condition that results from an adverse or deleterious response to an endogenous or exogenous antigen. The result is usually cellular dysfunction, or the resulting damage and malfunction of, or destruction of, organs or tissues that may be responsible for the immune condition.

[0045] The terms "kit" and "product packaging" are synonymous.

[0046] The term "subject" or "patient" includes both mammals and non-mammals. Mammals include, but are not limited to, mammals such as humans, non-human primates such as gorillas, apes, and monkeys; agricultural animals such as cattle, horses, goats, sheep, and pigs; livestock such as rabbits and dogs; and laboratory animals including rodents such as rats, mice, and guinea pigs. Non-mammals include, but are not limited to, birds and fish. In a preferred aspect, the selected mammal is a human.

[0047] The terms "treat," "treatment," or "therapy" as used herein include alleviating, inhibiting, or ameliorating the symptoms of a disease or condition; inhibiting the development of complications; ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or symptom, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing a regression of a disease or symptom; alleviating complications caused by a disease or symptom, or preventing and / or treating signs caused by a disease or symptom.

[0048] As used herein, a compound or pharmaceutical composition, when administered, can improve a disease, symptom, or condition, particularly by improving its severity, delaying its onset, slowing its progression, or reducing its duration, regardless of whether the administration is fixed or temporary, continuous or intermittent, and can be attributed to or related to the administration.

[0049] Route of administration

[0050] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, auricular, nasal, and topical administration. In addition, parenteral administration, by way of example only, includes intramuscular, subcutaneous, intravenous, intramedullary, intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0051] In one aspect, the compounds described herein are administered locally rather than systemically. In certain embodiments, the long-acting formulations are administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Additionally, in another embodiment, the drug is administered via a targeted drug delivery system. For example, liposomes coated with organ-specific antibodies. In such embodiments, the liposomes are selectively directed to specific organs and absorbed.

[0052] The present invention includes within its scope pharmaceutical compositions comprising (alone or in combination with a pharmaceutical carrier) a therapeutically effective amount of at least one compound of the present invention as an active ingredient. Optionally, the compounds of the present invention may be used alone, in combination with other compounds of the present invention, or in combination with one or more other therapeutic agents (e.g., anticancer agents or other pharmaceutically active substances).

[0053] Regardless of the route of administration selected, the compound of the present invention (which may be used in a suitable hydrated form) and / or the pharmaceutical composition of the present invention are formulated into pharmaceutical dosage forms by conventional methods known to those skilled in the art.

[0054] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0055] The selected dosage level will depend upon a variety of factors, including the activity of the specific compound of the present invention being employed, or its ester, salt or amide; the route of administration; the time of administration; the rate of excretion of the specific compound being employed; the rate and extent of absorption; the duration of the treatment; other drugs, compounds and / or substances used in combination with the specific compound being employed; and factors well known in the medical arts such as the age, sex, weight, condition, general health and prior medical history of the patient being treated.

[0056] While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[0057] Kit / Product Packaging

[0058] All features described in this specification (including any claims, abstract and drawings), and / or all steps involved in any method or process, may exist in any combination, unless certain features or steps are mutually exclusive in the same combination.

[0059] The features described above, or in the embodiments, may be combined in any combination. All features disclosed in this specification may be used in any combination, and each feature disclosed in this specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features.

[0060] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0061] The units used in the present invention for weight-volume percentages are well known to those skilled in the art, for example, referring to the weight (g) of solute in 100 ml of solution. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0062] Example

[0063] General Process

[0064] When the preparation route is not included, the raw materials and reagents used in the present invention are all known products, which can be synthesized according to methods known in the art, or can be obtained by purchasing commercial products. No further purification is required for the commercially available reagents used.

[0065] Room temperature refers to 20-30℃.

[0066] Unless otherwise specified in the reaction examples, all reactions were carried out under a nitrogen atmosphere, which means that the reaction flask was connected to a nitrogen balloon of approximately 1 L.

[0067] The hydrogenation reaction is usually carried out by evacuating the flask and filling it with hydrogen, and this operation is repeated three times. The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1L.

[0068] Microwave reaction use Initiator + microwave reactor.

[0069] The structures of the compounds of the present invention were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker Ascend TM Spectra were measured using a 500 nm NMR spectrometer. Solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard. The following abbreviations are used for NMR signal multiplicity: s = singlet, brs = broad, d = doublet, t = triplet, and m = multiplet. Coupling constants are listed as J values ​​and are measured in Hz.

[0070] LC-MS analysis was performed using a Thermo LC-MS / MS instrument (UltiMate 3000+MSQ PLUS). HPLC analysis was performed using a Thermo HPLC instrument (UltiMate 3000). Reverse-phase preparative chromatography was performed using a Thermo HPLC instrument (UltiMate 3000). Flash column chromatography was performed using an Agilent FS-9200T automatic column analyzer, and silica gel prepacked columns were performed using a Santai HPLC instrument. Pre-packed columns. Thin layer chromatography silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications used for thin layer chromatography separation and purification products are 0.4mm to 0.5mm.

[0071] Example 1

[0072] (S)-4,5-Dimethyl-2-((trans-3-(3,4,5-trifluorophenoxy)cyclobutyl)amino)-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pteridin-6-one

[0073]

[0074] Compound 1 was prepared by the following steps:

[0075]

[0076] Step 1: Dissolve cis-3-BOC-aminocyclobutanol 1a (250 mg, 1.34 mmol), methanesulfonic anhydride (465 mg, 2.67 mmol), and N,N-diisopropylethylamine (517 mg, 4.01 mmol) in dichloromethane (2 mL) and stir at room temperature overnight. Monitor the reaction by TLC. The reaction mixture is diluted with dichloromethane and washed sequentially with water and saturated brine. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated to yield 1b (300 mg, 84% yield) as a yellow solid. 1 H NMR (500MHz, DMSO-d6) δ7.23 (d, J = 8.3Hz, 1H), 4.69-4.64 (m, 1H), 3.63-3. 60(m,1H),3.13(s,3H),2.70-2.62(m,2H),2.16-2.09(m,2H),1.37(s,9H).

[0077] Step 2: Dissolve compound 1b (300 mg, 1.13 mmol), compound 1c (251 mg, 1.70 mmol), and cesium carbonate (737 mg, 2.26 mmol) in N,N-dimethylformamide (2 mL) and stir at 80°C overnight. LCMS monitored the reaction completion. The reaction solution was diluted with ethyl acetate, washed sequentially with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to afford 1d (280 mg, 78% yield) as a white solid. ESI-MS (m / z): 318.6 [M+H] + .

[0078] Step 3: Dissolve compound 1d (280 mg, 882 μmol) in dichloromethane (2 mL) and add 1,4-dioxane hydrochloride solution (4 M, 1.10 mL) dropwise. Stir overnight at room temperature. LCMS monitoring of the reaction completes. The reaction solution is concentrated to afford 1e (170 mg, 75% yield) as a white solid. ESI-MS (m / z): 218.4 [M+H] + .

[0079] Step 4: Dissolve 2,4-dichloropyrido[3,2-d]pyrimidine 1f (1.7 g, 8.50 mmol) and (S)-methyl 2-(methylamino)propanoate hydrochloride 1g (1.70 g, 11.05 mmol) in tetrahydrofuran (40 mL), add triethylamine (2.58 g, 25.50 mmol, 3.53 mL), and stir at room temperature overnight. The reaction was monitored by LCMS. The reaction solution was concentrated and the residue was purified by silica gel column chromatography to obtain a yellow oil 1h (1.1 g, 46% yield). ESI-MS (m / z): 281.2 [M+H] + .

[0080] Step 5: Compound 1h (1.1 g, 3.92 mmol) was dissolved in tetrahydrofuran (20 mL), and aqueous hydrochloric acid (6N, 0.65 mL) and platinum dioxide (88 mg, 0.39 mmol) were added. The reaction system was purged with a hydrogen balloon and stirred under hydrogen balloon pressure at room temperature for 48 hours. The reaction was monitored by LCMS. The reaction solution was diluted with methanol, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain 1i (900 mg, 90% yield) as a white solid. ESI-MS (m / z): 253.2 [M+H] + .

[0081] Step 6: Dissolve compound 1i (50 mg, 197 μmol), compound 1e (65 mg, 257 μmol), and p-toluenesulfonic acid monohydrate (3.7 mg, 19 μmol) in n-butanol (2 mL) and react under microwave conditions at 160°C for 2 hours. LCMS monitoring of the reaction completion was performed. The reaction solution was purified by reverse-phase preparative HPLC to obtain 1 (13 mg, 15% yield) as a white solid. ESI-MS (m / z): 434.3 [M+H] + ; 1H NMR(500MHz,DMSO-d6)δ6.90-6.82(m,3H),4.86-4.81(m,1H),4.42-4.36(m,1H),4.12(q,J=6.8Hz,1H),4.05-4.00(m,1H),3.30-3.24(m ,1H),2.94(s,3H),2.55-2.52(m,2H),2.48-2.38(m,2H),2.36-2.28(m,2H),1.97-1.88(m,1H),1.85-1.76(m,1H),1.23(d,J=6.8Hz,3H).

[0082] Example 2

[0083] (S)-4,5-Dimethyl-2-((trans-3-((6-(trifluoromethyl)pyridin-3-yl)oxy)cyclobutyl)amino)-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pteridin-6-one

[0084]

[0085] Compound 2 was prepared by the following steps:

[0086]

[0087] Step 1: Dissolve compound 1b (500 mg, 1.88 mmol), compound 2a (461 mg, 2.83 mmol), and cesium carbonate (1.23 g, 3.77 mmol) in N,N-dimethylformamide (2 mL) and stir at 80°C overnight. LCMS monitored the reaction. The reaction solution was diluted with ethyl acetate, washed sequentially with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to obtain 2b (500 mg, 79% yield) as a white solid. ESI-MS (m / z): 333.3 [M+H] + .

[0088] Step 2: Dissolve compound 2b (500 mg, 1.50 mmol) in dichloromethane (2 mL) and add 1,4-dioxane hydrochloride (4 M, 1.88 mL) dropwise. Stir at room temperature overnight. LCMS monitoring of the reaction completes. The reaction solution is concentrated to afford 2c (300 mg, 74% yield) as a white solid. ESI-MS (m / z): 233.5 [M+H] + .

[0089] Step 3: Compound 1i (50 mg, 197 μmol), compound 2c (68 mg, 256 μmol), and p-toluenesulfonic acid monohydrate (3.7 mg, 19 μmol) were dissolved in n-butanol (2 mL) and reacted in a microwave oven at 160°C for 2 hours. LCMS monitored the completion of the reaction. The reaction solution was purified by reverse-phase preparative HPLC to obtain 2 as a white solid (10.1 mg, 11% yield). ESI-MS (m / z): 449.2 [M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ8.38(d,J=2.8Hz,1H),7.83(d,J=8.7Hz,1H),7.46(dd ,J=8.7,2.9Hz,1H),6.91(d,J=6.9Hz,1H),5.05-5.00(m,1H),4.46-4.42(m,1H ),4.12(q,J=6.9Hz,1H),4.03-3.99(m,2H),3.28-3.25(m,1H),2.95(s,3H),2 .50-2.38(m,4H),1.97-1.87(m,1H),1.84-1.77(m,1H),1.23(d,J=6.7Hz,3H).

[0090] Example 3

[0091] (R)-4,6-dimethyl-N-(trans-3-(3,4,5-trifluorophenoxy)cyclobutyl)-5,6-dihydro-4H-pyrrolo[3,2,1-de]

[0092] Pteridin-2-amine

[0093]

[0094] Compound 3 was prepared by the following steps:

[0095]

[0096] Step 1: Dissolve compound 3a (500 mg, 2.66 mmol), compound 3b (946 mg, 3.99 mmol), potassium carbonate (1.47 g, 10.64 mmol), and 18-crown-6 (351 mg, 1.33 mmol) in 1,4-dioxane (10 mL) and stir at 80°C overnight. LCMS monitored the reaction. The reaction solution was diluted with ethyl acetate, washed sequentially with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to afford 3c (600 mg, 65% yield) as a yellow solid. ESI-MS (m / z): 345.3 [M+H]+ .

[0097] Step 2: Dissolve compound 3c (600 mg, 1.74 mmol) in dichloromethane (2 mL) and add 1,4-dioxane hydrochloride (4 M, 2.17 mL) dropwise. Stir overnight at room temperature. LCMS monitoring of the reaction revealed completion. The reaction solution was concentrated to afford 3d (400 mg, 81% yield) as a white solid. ESI-MS (m / z): 245.3 [M+H] + .

[0098] Step 3: Dissolve compound 3d (400 mg, 1.63 mmol) and N,N-diisopropylethylamine (632 mg, 4.90 mmol) in 1,4-dioxane (5 mL) and stir at 100°C overnight. The reaction was monitored by LCMS. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give a yellow solid 3e (150 mg, 44% yield). ESI-MS (m / z): 209.4 [M+H] + .

[0099] Step 4: Compound 3e (150 mg, 718 μmol), iodomethane (153 mg, 1.08 mmol), and cesium carbonate (468 mg, 1.44 mmol) were dissolved in N,N-dimethylformamide (2 mL) and stirred at 80°C overnight. LCMS monitored the reaction completion. The reaction solution was diluted with ethyl acetate, washed sequentially with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to afford 3f (100 mg, 62% yield) as a yellow solid. ESI-MS (m / z): 223.4 [M+H] + .

[0100] Step 5: Compound 3f (30 mg, 134 μmol), compound 1e (68 mg, 256 μmol), and trifluoroacetic acid (1.5 mg, 13 μmol) were dissolved in n-butanol (2 mL) and reacted in a microwave oven at 160°C for 2 hours. LCMS monitored the completion of the reaction. The reaction solution was purified by reverse-phase preparative HPLC to obtain 3 as a white solid (6.8 mg, 12% yield). ESI-MS (m / z): 404.2 [M+H] + ; 1H NMR(500MHz,DMSO-d6)δ8.19(s,1H),7.30(d,J=2.8Hz,1H),6.92-6.82(m,2H),6.04(d,J=2.8Hz,1H),4.93-4.81(m,1H),4.46-4.41(m,1H),4 .37-4.24(m,1H),3.64(dd,J=12.4,3.9Hz,1H),3.35-3.30(m,1H),3.05(s,3H),2.48-2.42(m,2H),2.38-2.33(m,2H),1.42(d,J=6.4Hz,3H).

[0101] Example 4

[0102] (R)-4,6-Dimethyl-N-(trans-3-((6-(trifluoromethyl)pyridin-3-yl)oxy)cyclobutyl)-5,6-dihydro-4H-pyrrolo[3,2,1-de]pteridin-2-amine

[0103]

[0104] Compound 4 was prepared by the following steps:

[0105]

[0106] Step 1: Compound 3f (30 mg, 134 μmol), compound 2b (40 mg, 175 μmol), and trifluoroacetic acid (1.5 mg, 13 μmol) were dissolved in n-butanol (2 mL) and reacted in a microwave oven at 160°C for 2 hours. LCMS monitored the completion of the reaction. The reaction solution was purified by reverse-phase preparative HPLC to afford 4 (9.3 mg, 16% yield) as a white solid. ESI-MS (m / z): 419.5 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=2.8Hz,1H),8.20(s,1H),7.84(d,J=8.7Hz,1H),7.47(dd,J=8.7,2.9Hz,1H),7.30(d,J=2.8Hz,1H),6.69(br s,1H),6.04(d,J=2.8Hz,1H),5.08-5.02(m,1H),4.53-4.45(m,1H),4.35-4.25(m,1H) ,3.64(dd,J=12.3,3.9Hz,2H),3.05(s,3H),2.50-2.40(m,4H),1.42(d,J=6.4Hz,3H).

[0107] Wnt pathway inhibitor biological screening and results

[0108] Experimental Example 1: Construction of Colo205-LUC-TCF / LEF-M1 reporter cell line

[0109] Colo205 cell lines (Cell Bank of the Chinese Academy of Sciences, Cat# TCHu102) were purchased from the Cell Bank of the Chinese Academy of Sciences. After expansion and subculture, cells were transfected with a luciferase reporter plasmid (Promega) carrying the TCF / LEF transcription factor-driven gene using lipofectamine 3000 during the exponential growth phase. This plasmid carries a resistance gene, allowing for resistance selection. Transfections were performed in 10 cm culture dishes using standard complete medium without resistance. After two days, the medium containing the resistance gene was replaced and culture continued. Thereafter, the resistance medium was replaced every two days, and the suspended cells were discarded. The original medium was centrifuged to remove cells and debris and retained as the adaptation medium. When cells had confluently grown in the culture dish, they were digested, counted, and passaged into 96-well plates, averaging 1.5 cells per well. Adaptation medium was used for each passage. The remaining cells were frozen. After passage, the cells were cultured for 4 hours to allow them to adhere, and the cell number in each well was observed under a microscope. Wells with only one cell per well are marked, which are monoclonal wells. Then culture normally, change the culture medium every 2 days, and observe. In the early stage, the wells where monoclonal cells continue to grow are marked twice and can be replaced with normal resistant culture medium. When the cells in the monoclonal wells are full of 96-well plate wells, they are digested and passaged to 24-well culture plates. After the 24-well plate is full, they are passaged to 1 96-well plate and 1 6-well plate, of which the 96-well plate cells are passaged to at least 6 wells, of which 3 wells are added with known Wnt inhibitors, and the other 3 wells are not treated. After 24h, fluorescent detection reagents are added to the 96-well plate cells to detect the fluorescence intensity. Select the cell lines that have fluorescence expression when not treated and whose fluorescence is reduced after inhibition and further culture. The Colo205-LUC-TCF / LEF-M1 cell line is one of the cell lines screened above. Its growth curve, cell morphology, and cell growth status are similar to those of the original Colo205 cells. In addition, the ratio of fluorescent signals between the treated and untreated cells with inhibitors is the largest among all cell lines, with the ratio being inhibited by 4-5 times within 4 hours, making it fully suitable for the subsequent screening of Wnt inhibitors.

[0110] Experimental Example 2: Detection of the inhibitory ability of compounds on Colo205-LUC-TCF / LEF M1 reporter cell line

[0111] The Colo205-LUC-TCF / LEF M1 cell line is a reporter cell line stably transfected with the pGL4.49-LUC2-TCF / LEF vector. Its β-catenin Wnt pathway is continuously activated. After the addition of the inhibitor, the Wnt pathway is inhibited, and the expression level of firefly luciferase regulated by the TCF / LEF cis-element on the vector decreases. After the subsequent addition of the detection substrate, the detected light signal decreases accordingly, thereby detecting the inhibitory effect of the compound.

[0112] Add 100 μL of compound with a maximum concentration of 20 μM to each well of a 96-well cell culture plate, and perform a 3-fold gradient dilution of the compound concentration. Then, inoculate 10,000 colo205 cells stably transfected with the reporter gene and 100 μL of culture medium into each well, and perform corresponding treatments as positive and negative control wells. Place the cells in a 5% CO2 cell culture incubator and culture at 37°C for 4 hours. After 4 hours, remove the culture medium, add 100 μL of a reagent containing the corresponding firefly luciferase substrate (Promega) to each well, and measure the activity of the luciferase reporter gene. Use SpectraMax to read the luminescence intensity in full wavelength mode. The light signal intensity of cells treated only with DMSO is the positive control, and the light signal intensity of the cell-free well is the negative control. Calculate the IC value of the compound. 50 The Colo 205 reporter gene assay data are summarized in Table 1 below.

[0113] Table 1 IC of compounds against Colo205-LUC-TCF / LEF reporter gene inhibition 50 value

[0114]

[0115]

[0116] Experimental Example 3: Proliferation inhibition test of compounds on Wnt mutant cell lines (Colo205, DU4475, NCI-H929 and HepG2) and non-Wnt mutant cell lines (Hela and RKO)

[0117] The cell lines used in the experiment were Colo205, DU4475, NCI-H929, and HepG2 cell lines, in which the Wnt pathway is continuously activated and whose proliferation is Wnt pathway-dependent. HELA and RKO cell lines, in which the Wnt pathway is normally inactivated and whose proliferation is not dependent on the Wnt pathway, were used as control cell lines to determine that the inhibitory effect of the compounds of the present invention on Wnt-dependent proliferation was not due to other non-specific toxicity.

[0118] Colo205, Du4475, NCI-H929, HepG2, HELA and RKO cell lines cultured in their respective culture media were treated during the logarithmic growth phase, and the cells were collected and prepared into a uniform cell suspension of known concentration. The cell suspension was then added to a 96-well cell culture plate so that each well contained 1,000 cells. The plate was placed in a 5% CO2 cell culture incubator and cultured at 37°C for 20-24 hours. The next day, completely dissolved, 3-fold gradient dilutions of the compound were added to each cell culture well so that the final maximum concentration in the cell culture well was 20 μM, and the culture was continued for 96 hours. This experiment was performed using Promega's cell activity detection test. The more the cells proliferated, the stronger the final signal intensity. The detection instrument was SpectraMax, full wavelength mode. The wells where only DMSO was added served as positive control wells, and the wells where no cells were inoculated served as negative control wells. The IC value of the compound for inhibiting the proliferation of cells that were continuously activated or proliferation-dependent on Wnt was calculated. 50 values, and IC values ​​for proliferation inhibition in Wnt-inactive or proliferation-independent cells 50 The inhibitory effect of the compound on the Wnt pathway and the cytotoxic effect on normal cells were evaluated. The results are shown in Table 2 below.

[0119] Table 2 IC values ​​of compounds for inhibition of proliferation of Wnt mutant cell lines 50 value

[0120]

[0121] The above results show that the compounds of the present invention have significant inhibitory activity against mutant cell lines Colo205, DU4475, NCI-H929 and HepG2, but have basically no significant inhibitory activity against Hela and RKO cell lines, which indicates that the compounds of the present invention have significant and selective Wnt pathway inhibitory effects.

Claims

1. A compound having the structure of Formula 4 or a pharmaceutically acceptable salt thereof:

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing and / or treating cancer or inflammatory diseases.

4. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing and / or treating tumors and autoimmune diseases.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing and / or treating immune-mediated diseases.

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