A wnt pathway inhibitor compound

Abstract

The present application relates to Wnt pathway inhibitor compounds represented by Formula 1 and methods for preparing the same, and pharmaceutical compositions comprising the same, and uses of the compounds of Formula 1 for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, or immune-mediated diseases.

Description

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

[0002] This application claims priority to Chinese Patent Application No. 202110847130.9, entitled “A Wnt Pathway Inhibitor Compound,” filed on July 26, 2021, with the China National Intellectual Property Administration, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to a heterocyclic compound, specifically to a highly active Wnt pathway inhibitor, its preparation method, and its uses. Background Technology

[0004] The Wnt / β-catenin signaling pathway is a conserved pathway throughout biological evolution. In normal somatic cells, β-catenin functions as a cytoskeletal protein, forming a complex with E-cadherin at the cell membrane to maintain homologous cell adhesion and prevent cell migration. When the Wnt signaling pathway is not activated, intracellular β-catenin is phosphorylated and forms a β-catenin degradation complex with APC, Axin, and GSK3β, thereby initiating the ubiquitin system to degrade β-catenin via the proteasome pathway, maintaining intracellular β-catenin at a low level. When cells are stimulated by Wnt signaling, Wnt protein binds to the specific receptor Frizzled protein on the cell membrane. The activated Frizzled receptor recruits intracellular Disheveled protein, inhibiting the degradation activity of the β-catenin degradation complex formed by proteins such as GSK3β, thus stabilizing the free β-catenin protein in the cytoplasm. β-catenin, stably accumulated in the cytoplasm, 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, Cyclin D1, etc.). Overactivation of the Wnt / β-catenin signaling pathway is closely related to the development of various cancers (including colon cancer, gastric cancer, and breast cancer). For example, abnormal activation of the classical Wnt signaling pathway and nuclear accumulation of β-catenin protein are widespread in colorectal cancer, while inhibiting Wnt signaling pathway activity can suppress the proliferation of cancers such as colon cancer. Mutations in APC are present in more than 85% of colorectal cancers; mutated APC blocks β-catenin phosphorylation and degradation, inducing colorectal cancer development. 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, there is currently a lack of satisfactory and effective Wnt pathway inhibitor compounds in the existing technology. Therefore, researching effective Wnt pathway inhibitor compounds is a necessity in the current technology. Summary of the Invention

[0006] In one aspect, the present invention provides a compound having the structure of Formula 1 that inhibits Wnt pathway activity, or a pharmaceutically acceptable salt, isotope derivative, or stereoisomer thereof:

[0007]

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

[0009] Furthermore, the present invention also provides the use of the compounds described herein, or pharmaceutically acceptable salts, isotope derivatives, stereoisomers, or pharmaceutical compositions described herein, in the preparation of medicaments for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases, or immune-mediated diseases. It is particularly noteworthy that, herein, when referring to a "compound" of Formula 1, this generally also encompasses its stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotope 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, isotope derivative, stereoisomer or pharmaceutical composition of the present invention.

[0011] As is known to those skilled in the art, the salts, solvates, and hydrates of a compound are alternative forms of the compound, and they can all be converted into the compound under certain conditions. Therefore, it is particularly noteworthy that when referring to a compound of Formula 1 herein, its pharmaceutically acceptable salt is generally also included, and further included, its solvates and hydrates.

[0012] Similarly, when referring to a compound in this article, its prodrug, metabolites, and nitrogen oxides are generally also included.

[0013] The pharmaceutically acceptable salts described in this invention can be formed using, for example, inorganic or organic acids: “Pharmaceutically acceptable salt” means a salt that, within a reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. The salts can be prepared in situ during the final separation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent, as outlined below. For example, the free base function can react with a suitable acid. Examples of pharmaceutically acceptable inorganic acid addition salts are salts formed by amino groups 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 salts formed using other methods 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 compounds of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol, and acetonitrile), adding an excess of an aqueous solution of an organic or inorganic acid to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid, and then separating the precipitated salt.

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

[0016] The term "solvate" as used in this invention refers to the physical association of the compound of this invention with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0017] The "stereoisomerism" described in this invention is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism. Conformational isomerism refers to the phenomenon where organic molecules with a certain configuration undergo different spatial arrangements of atoms or groups of atoms due to the rotation or twisting of carbon or carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair conformation and boat conformation in the cyclohexane structure. "Stereoisomers" refer to compounds of this invention containing one or more asymmetric centers, thus allowing them to exist as racemic mixtures and racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers. The compounds of this invention have asymmetric centers, each of which produces two optical isomers. The scope of this invention includes all possible optical isomers and diastereomer mixtures, as well as pure or partially pure compounds. The compounds of this invention can exist as tautomers, which have different hydrogen bonding sites through one or more double bond shifts. For example, ketones and their enol forms are ketone-enol tautomers. All tautomers and mixtures thereof are included in the compounds of this invention. All enantiomers, diastereomers, racemates, mesomates, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof of compounds of formulas (I) to (III) are included within the scope of this invention.

[0018] The term "isotope derivative" in this invention refers to molecules in which the compounds described herein are isotopically labeled. Commonly used isotopes for isotopic labeling are hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 11 C, 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 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 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially deuterium. 3 H and carbon 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2Substitution with H can enhance metabolic stability and prolong the half-life, thereby reducing the dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0019] The present invention also provides the use of the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases.

[0020] Furthermore, the present invention provides pharmaceutical compositions for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases, or immune-mediated diseases, comprising compounds of the present invention as active ingredients. The pharmaceutical compositions may optionally comprise 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, comprising administering the compound of the present invention to a mammal in need of such treatment.

[0022] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with other anticancer agents or immune checkpoint inhibitors used to treat cancer or tumors, the compounds of the present invention or their pharmaceutically acceptable salts may provide enhanced anticancer effects.

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

[0024] The compounds of the present invention, or their pharmaceutically acceptable salts, can be administered orally or parenterally as active ingredients. The dosage of the active ingredient can be adjusted based on several relevant factors, such as the condition of the patient, the type and severity of the disease, the rate of administration, and physician advice. In some cases, amounts less than the above dosages may be appropriate. Amounts greater than the above dosages may be used if no harmful side effects are caused, and this amount can be administered in divided doses daily.

[0025] In addition, the present invention provides a method for preventing and / or treating tumors, cancer, viral infections, organ transplant rejection, neurodegenerative diseases, attention-related diseases, or autoimmune diseases, comprising administering the compounds of the present invention or the pharmaceutical compositions of the present invention to mammals in need of such treatment.

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

[0027] The pharmaceutical compositions of the present invention for oral administration can be prepared by mixing the active ingredient with, for example, a carrier including: cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactant, suspending agent, emulsifier, and diluent.

[0028] Examples of carriers used in the injectable pharmaceutical compositions of the present invention may be water, salt solution, glucose solution, glucose-like solution, alcohol, glycol, ether (e.g., polyethylene glycol 400), oil, fatty acid, fatty acid ester, glycerol ester, surfactant, suspending agent, and emulsifier.

[0029] Other features of the invention will become apparent as the exemplary embodiments are described. The embodiments are given to illustrate the invention and are not intended to be limiting. The following examples use the methods disclosed in the invention to prepare, separate, and characterize.

[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. They can be synthesized using the methods described below, as well as synthetic methods known in the field of organic synthetic chemistry, or by variations thereof understood by 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 suitable for the transformation achieved. Those skilled in the art of organic synthesis will understand that the functionalities present on the molecule are consistent with the proposed transformation. This sometimes necessitates determining whether to change the order of synthetic steps or the starting materials to obtain the desired compound of the present invention. Detailed Implementation

[0031] the term

[0032] Unless otherwise specified, the terms used in this application, including those in the specification and claims, are defined as follows. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0033] In the specification and claims, the given chemical formula or name shall encompass all its stereoisomers and optical isomers, as well as racemic forms containing such isomers. Unless otherwise specified, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of this invention. Various geometric isomers of C=C double bonds, C=N double bonds, ring systems, etc., may also be present in the compounds, and all such stable isomers are covered within this invention. This invention describes the cis- and trans- (or E- and Z-) geometric isomers of the compounds of this invention, which can be separated into mixtures of isomers or separate isomeric forms. The compounds of this invention can be separated in optically active or racemic forms. All methods used to prepare the compounds of this invention and the intermediates prepared therein are considered part of this invention. In the preparation of enantiomers or diastereomers, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization). Depending on the method conditions, the final products of this invention are obtained in free (neutral) or salt form. The free forms of these end products and their salts are all within the scope of this invention. If desired, one form of the compound can be converted to another. A free base or acid can be converted to a salt; a salt can be converted to a free compound or another salt; a mixture of isomers of the present invention can be separated into individual isomers. The compounds of the present invention, their free forms, and their salts can exist in a variety of tautomeric forms, wherein hydrogen atoms are transposed to other parts of the molecule and thereby the chemical bonds between the atoms of the molecule are rearranged. It should be understood that all possible tautomeric forms are included within the scope of this invention.

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

[0035] As used herein, the term "effective amount" means the amount of a drug or agent (i.e., the compound of the present invention) that will elicit a biological or medical response in a tissue, system, animal, or human, as sought by, for example, a researcher or clinician. Furthermore, the term "therapeutic effective amount" means an amount that, compared to a corresponding subject who has not received the aforementioned amount, results in improved treatment, cure, prevention, or reduction of a disease, symptom, or side effect, or a slower rate of progression of a disease or symptom. Effective amounts may be administered, applied, or dosed in one or more ways and are not intended to be limited to a particular formulation or route of administration. The term also includes effective amounts within its scope that enhance normal physiological function.

[0036] The term “treatment” as used in this article includes any effect that results in improvement of a condition, disease, disorder, etc., such as reducing, decreasing, regulating, improving or eliminating, or improving its symptoms.

[0037] The term "pharmaceutical" as used herein refers to compounds, substances, compositions, and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, and / or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0038] As used herein, the phrase "pharmaceutical carrier" refers to a pharmaceutical substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulation substance, relating to the carrying or delivery of a subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient.

[0039] The term "pharmaceutical composition" means a composition comprising the compounds of the present invention and at least one other pharmaceutical carrier. "Pharmaceutical carrier" refers to a medium commonly accepted in the art for delivering a bioactive agent to an animal (specifically a mammal), including (i) adjuvants, excipients, or mediators such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, 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 prescription component or active ingredient does not have an excessively harmful effect on health for general therapeutic purposes.

[0041] The term "cancer," as used in this article, refers to an uncontrolled abnormal growth of cells that, under certain conditions, can metastasize (spread). This type of cancer includes, but is not limited to, solid tumors (such as those of the bladder, intestines, brain, chest, uterus, heart, kidneys, lungs, lymphoid tissue (lymphoma), ovaries, pancreas or other endocrine organs (such as the thyroid), prostate, skin (melanoma), or hematologic malignancies (such as nonleukemic leukemia).

[0042] The term “combined administration” or similar terms, as used herein, refers to the administration of several selected therapeutic agents to a patient in the same or different manners of administration at the same or different times.

[0043] The terms “enhancement” or “potential enhancement,” as used herein, refer to the expected increase or prolongation of either efficacy or duration of effect. Therefore, in the context of enhancing the therapeutic effect of a drug, the term “potential enhancement” refers to the ability of a drug in a system to increase or prolong its efficacy or duration. The term “synergistic value,” as used herein, refers to the ability of an ideal system to maximally enhance the efficacy of another therapeutic agent.

[0044] The term "immune disease" refers to a disease or symptom that results from an adverse or harmful reaction to endogenous or exogenous antigens. The result is often impaired cell function, or damage to cells leading to dysfunction, or damage to organs or tissues that may produce immune symptoms.

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

[0046] The terms "subject" or "patient" include both mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as orangutans, 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 “treatment,” “treatment process,” or “therapy” as used herein include alleviating, suppressing, or improving symptoms or conditions of a disease; suppressing the development of complications; improving or preventing underlying metabolic syndrome; suppressing the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; mitigating a disease or symptom; reducing complications arising from a disease or symptom, or preventing and / or treating signs arising from 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. This may be attributable to or related to the administration, whether the administration is fixed or intermittent, continuous or discontinuous.

[0049] route of administration

[0050] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, ocular, nasal, and topical administration. Furthermore, for illustrative purposes only, parenteral administration includes intramuscular, subcutaneous, intravenous, intramedullary, ventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0051] In one respect, the compounds described herein are administered via local rather than systemic routes of administration. In specific embodiments, long-acting formulations are administered via implantation (e.g., subcutaneous or intramuscular) or via intramuscular injection. Furthermore, in another specific embodiment, the drug is administered via a targeted drug delivery system, such as liposomes encapsulated by organ-specific antibodies. In this embodiment, the liposomes are selectively directed to a specific organ and absorbed.

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

[0053] Regardless of the chosen route of administration, the compounds of the present invention (which may be used in a suitable hydrated form) and / or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0054] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be altered to obtain an amount of active ingredient that is effective and non-toxic to patients in achieving the desired therapeutic response, composition, and administration mode for a particular patient.

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

[0056] Although the compounds of the present invention can be administered alone, they are preferably administered in the form of pharmaceutical formulations (compositions).

[0057] Reagent kit / product packaging

[0058] All features described in this specification (including any claims or abstracts) 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 mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0060] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

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

[0062] Example

[0063] General process

[0064] When the preparation method is not specified, all raw materials and reagents used in this invention are known products that can be synthesized according to methods known in the art, or can be obtained by purchasing commercially available products. None of the commercially available reagents used require further purification.

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

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

[0067] Hydrogenation reactions are typically carried out under vacuum, filled with hydrogen gas, and repeated three times. A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon of approximately 1L.

[0068] Microwave reaction use Initiator + Microwave Reactor.

[0069] The structure of the compounds of this invention was determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR shifts (δ) were expressed in terms of 10⁻¹⁰. -6 The measurements are given in units of (ppm). NMR determinations are performed using (Bruker Ascend) TM A Model 500 NMR spectrometer was used. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The following abbreviations are used for NMR signal multiplicity: s = singlet, brs = broad peak, d = doublet, t = triplet, m = multiplet. Coupling constants are listed in J values ​​and measured in Hz.

[0070] LC-MS was performed using a Thermo UltiMate 3000+MSQ PLUS system. HPLC was performed using a Thermo UltiMate 3000 high-performance liquid chromatograph. Reversed-phase preparative chromatography was performed using a Thermo UltiMate 3000 reversed-phase preparative chromatograph. Rapid column chromatography was performed using an Agilent FS-9200T automated column press, and pre-packed silica gel columns were obtained from Sante. Pre-packed column. Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The thickness used for thin-layer chromatography separation and purification of products is 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]pterodin-6-one

[0073]

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

[0075]

[0076] Step 1: 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) were dissolved in dichloromethane (2 mL) and stirred overnight at room temperature. The reaction was monitored by TLC until complete. The reaction solution was diluted with dichloromethane, washed successively with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solution was filtered and concentrated to obtain a yellow solid 1b (300 mg, 84% yield). 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: Compound 1b (300 mg, 1.13 mmol), compound 1c (251 mg, 1.70 mmol), and cesium carbonate (737 mg, 2.26 mmol) were dissolved in N,N-dimethylformamide (2 mL) and stirred overnight at 80 °C. The reaction was monitored by LCMS until completion. The reaction solution was diluted with ethyl acetate, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a white solid 1d (280 mg, 78% yield). ESI-MS (m / z): 318.6 [M+H] + .

[0078] Step 3: Compound 1d (280 mg, 882 μmol) was dissolved in dichloromethane (2 mL), and 1,4-dioxane hydrochloric acid solution (4 M, 1.10 mL) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was monitored by LCMS until completion. The reaction solution was concentrated to give a white solid 1e (170 mg, 75% yield). ESI-MS (m / z): 218.4 [M+H] + .

[0079] Step 4: 1f of 2,4-dichloropyrido[3,2-d]pyrimidine (1.7g, 8.50mmol) and 1g of (S)-2-(methylamino)propionate hydrochloride (1.70g, 11.05mmol) were dissolved in tetrahydrofuran (40mL), and triethylamine (2.58g, 25.50mmol, 3.53mL) was added. The mixture was stirred overnight at room temperature. The reaction was monitored by LCMS until completion. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to give a yellow oily substance (1h, 1.1g, yield 46%). 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 hydrochloric acid aqueous solution (6 N, 0.65 mL) and platinum dioxide (88 mg, 0.39 mmol) were added. The reaction system was purged with hydrogen gas using a hydrogen balloon, and stirred at room temperature under hydrogen balloon pressure for 48 hours. The reaction was monitored by LCMS until completion. The reaction solution was diluted with methanol, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give a white solid 1i (900 mg, 90% yield). ESI-MS (m / z): 253.2 [M+H] + .

[0081] Step 6: Compound 1i (50 mg, 197 μmol), compound 1e (65 mg, 257 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. The reaction was monitored by LCMS until completion. The reaction solution was purified by reverse-phase preparative HPLC to give a white solid 1 (13 mg, yield 15%). 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] Biological screening and results of Wnt pathway inhibitors

[0083] Experiment 1: Construction of the Colo205-LUC-TCF / LEF-M1 reporter cell line

[0084] The Colo205 cell line (Chinese Academy of Sciences Cell Bank, Cat#TCHu102) was purchased from the Chinese Academy of Sciences Cell Bank. After expansion and passage, during the exponential growth phase, the cells were transfected with a luciferase reporter plasmid (Promega) driven by the TCF / LEF transcription factor during the lipo3000 transfection method. This plasmid carries an antibiotic resistance gene, allowing for antibiotic selection. Transfection was performed in 10cm culture dishes using standard complete medium without antibiotics. After 2 days, the medium was replaced with antibiotic-containing medium, and culture continued. Thereafter, the antibiotic-containing medium was replaced every 2 days, and the suspended cells were discarded. The original medium was centrifuged to remove cells and debris and retained as adaptation medium. When the cells reached confluence, they were digested, counted, and passaged into 96-well plates, ensuring an average of 1.5 cells / well. Adaptation medium was used for passage. The remaining cells were cryopreserved. After passage, the cells were cultured for 4 hours to allow them to adhere, and then the cell count in each well was observed under a microscope. Each well containing only one cell was labeled as a monoclonal well. The cells were then cultured normally, with the medium changed every two days and observations performed. Wells showing continued monoclonal cell growth were labeled twice and then replaced with normal antibiotic-containing medium. When cells from monoclonal wells reached confluence in a 96-well plate, they were digested and passaged into 24-well plates. After confluence in the 24-well plates, the cells were passaged into one 96-well plate and one 6-well plate. In the 96-well plate, cells were passaged into at least six wells, with three wells treated with a known Wnt inhibitor and the other three untreated. After 24 hours, fluorescence detection reagent was added to the 96-well plate cells, and fluorescence intensity was measured. Cell lines showing fluorescence expression without treatment and exhibiting reduced fluorescence after inhibition were selected for further culture. The Colo205-LUC-TCF / LEF-M1 cell line is one of the cell lines selected above. Its growth curve, cell morphology, and cell growth status are similar to those of the original Colo205 cells. Moreover, the ratio of fluorescence signal with and without inhibitor treatment is relatively large among all cell lines. The ratio can reach 4-5 times the inhibition at 4h, which is fully suitable for the subsequent screening of Wnt inhibitors.

[0085] Experimental Example 2: Detection of the inhibitory effect of the compound on the Colo205-LUC-TCF / LEF M1 reporter cell line

[0086] 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 an inhibitor, the Wnt pathway is inhibited, and the expression level of firefly luciferase regulated by the TCF / LEF cis-elements on the vector decreases. Subsequently, after the addition of the detection substrate, the detected light signal decreases accordingly, thus detecting the inhibitory effect of the compound.

[0087] 100 μL of a compound at a maximum concentration of 20 μM was added to each well of a 96-well cell culture plate, with the compound concentration serially diluted 3-fold. Then, 10,000 stable colo205 cells transfected with the reporter gene and 100 μL of culture medium were seeded into each well. Appropriate treatments were performed as positive and negative control wells. Cells were placed in a 5% CO2 cell culture incubator at 37°C for 4 h. After 4 hours, the culture medium was removed, and 100 μL of reagent containing the corresponding firefly luciferase substrate (Promega) was added to each well. The activity of the luciferase reporter gene was measured. The luminescence intensity was read using SpectraMax in full-wavelength mode. The light signal intensity of cells treated only with DMSO served as a positive control, and the light signal intensity of wells without cells served as a negative control. The IC50 of compound 1 was calculated. 50 The concentration of Colo 205 reporter gene assay data is summarized in Table 1 below.

[0088] Table 1. IC50 of the compounds on the inhibition of the Colo205-LUC-TCF / LEF reporter gene. 50 value

[0089]

[0090] Experimental Example 3: Inhibition of proliferation of compounds on Wnt mutant cell lines (Colo205, DU4475, NCI-H929, and HepG2) and non-Wnt mutant cell lines (HeLa and RKO)

[0091] The cell lines used in the experiment were Colo205, DU4475, NCI-H929, and HepG2 cell lines, which had sustained activation of the Wnt pathway and whose proliferation was Wnt-dependent. The HELA and RKO cell lines, which do not have Wnt pathway activation under normal conditions and whose proliferation is not dependent on the Wnt pathway, were used as control cell lines to determine that the inhibitory effect of the compound of the present invention on Wnt-dependent proliferation was not due to other non-specific toxicity.

[0092] Colo205, Du4475, NCI-H929, HepG2, HELA, and RKO cell lines cultured in their respective media were treated during the logarithmic growth phase. Cells were collected and prepared into homogeneous cell suspensions of known concentrations. These suspensions were then added to 96-well cell culture plates, ensuring each well contained 1000 cells. The plates were incubated in a 5% CO2 incubator at 37°C for 20–24 h. The next day, completely dissolved, 3-fold serially diluted compounds were added to each well, bringing the final maximum concentration to 20 μM. The plates were then cultured for another 96 h. Promega's cell viability assay was used for detection; higher cell proliferation resulted in a stronger signal intensity. The assay was performed using a SpectraMax in full-wavelength mode. Wells containing only DMSO served as positive controls, and uninoculated wells served as negative controls. The IC50 of compound 1 for inhibiting the proliferation of Wnt-activated or proliferation-dependent cells was calculated. 50 Values, and IC50 values ​​for proliferation inhibition in Wnt-inactive or proliferation-independent cells. 50 The values ​​were used to evaluate the inhibitory effect of the compound on the Wnt pathway and its toxicity to normal cells. The results are shown in Table 2 below.

[0093] Table 2. IC50 of the compounds on the proliferation inhibition of Wnt mutant cell lines 50 value

[0094]

[0095] The above results indicate that the compounds of the present invention have significant inhibitory activity against mutant cell lines Colo205, DU4475, NCI-H929 and HepG2, but have little to no significant inhibitory activity against Hela and RKO cell lines. This suggests that the compounds of the present invention have significant and selective inhibitory effects on the Wnt pathway.

Claims

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

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

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

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

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

Images