A wnt pathway inhibitor compound

Abstract

The present application relates to Wnt pathway inhibitor compounds represented by Formula 25 and a method for synthesizing the same, and a pharmaceutical composition comprising the same, and use of the compound of Formula 25 for preventing and / or treating cancer, a tumor, an inflammatory disease, an autoimmune disease, or an immune-mediated disease.

Description

[0001] This application is a divisional application of Chinese patent application No. 202280013624.X. The parent application has a filing date of July 26, 2022 (PCT international filing date), a Chinese national application number of “202280013624.X” (a PCT international application number of PCT / CN2022 / 107727), and an invention title of “A Wnt pathway inhibitor compound”.

[0002] The parent application and the present divisional application claim priority to Chinese patent application No. 202110847130.9, filed on July 26, 2021, and entitled “A Wnt pathway inhibitor compound”, the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD

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

[0004] The Wnt / β-catenin signaling pathway is a conserved pathway in biological evolution. In normal somatic cells, β-catenin only plays a role in maintaining the adhesion of homotypic cells and preventing cell movement as a cytoskeletal protein complexed with E-cadherin at the cell membrane. When the Wnt signaling pathway is not activated, β-catenin in the cytoplasm is phosphorylated and forms a β-catenin degradation complex with APC, Axin, GSK3β, and the like, thereby initiating the degradation of β-catenin by the ubiquitin system through the proteasome pathway, so that the β-catenin in the cytoplasm is maintained at a low level. When the cell is stimulated by Wnt signal, Wnt protein binds to the specific receptor Frizzled protein on the cell membrane, and the activated Frizzled receptor recruits intracellular Dishevelled protein to inhibit the degradation activity of the β-catenin degradation complex formed by GSK3β and the like, and stabilize the free state of β-catenin protein in the cytoplasm. The stable accumulation of β-catenin in the cytoplasm enters the nucleus to bind to the LEF / TCF transcription factor family, and initiates 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 occurrence of various cancers (including colon cancer, gastric cancer, breast cancer, etc.). For example, abnormal activation of the Wnt classic signaling pathway and nuclear accumulation of β-catenin protein are widespread in colorectal cancer, and inhibition of Wnt signaling pathway activity can inhibit the proliferation of cancers such as colon cancer. More than 85% of colorectal cancers have mutations in APC, and the mutated APC blocks the phosphorylation and degradation of β-catenin, inducing the occurrence of colorectal cancer. In addition, Axin mutation and β-catenin mutation itself can also cause intracellular aggregation of β-catenin and activation of the Wnt / β-catenin pathway.

[0005] Although it is known that inhibition of the Wnt signaling pathway can effectively prevent and / or treat cancer, tumor, inflammatory disease, autoimmune disease, and immune-mediated disease, there is still a lack of satisfactory effective Wnt pathway inhibitor compounds in the prior art. Therefore, it is necessary to study effective Wnt pathway inhibitor compounds in the prior art. SUMMARY

[0006] In one aspect, the present application provides a compound having the structure of formula 25 or a pharmaceutically acceptable salt, isotope derivative, stereoisomer thereof, which inhibits the activity of the Wnt pathway:

[0007]

[0008] Further, the present application also provides a pharmaceutical composition comprising the compound of the present application or a pharmaceutically acceptable salt, isotope derivative or stereoisomer thereof.

[0009] Further, the present application also provides the use of the compound of the present application or a pharmaceutically acceptable salt, isotope derivative, stereoisomer thereof or the pharmaceutical composition of the present application in the manufacture of a medicament for preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

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

[0011] It is well known to those skilled in the art that a salt, solvate, hydrate of a compound is an alternative form of the compound, which can be converted into the compound under certain conditions, therefore, it is particularly noted that herein when referring to a compound of the structure of Formula 25, it generally also includes its pharmaceutically acceptable salt, and further includes its solvate and hydrate.

[0012] Similarly, herein when referring to a compound, it generally also includes its prodrug, metabolite and nitroxide.

[0013] The pharmaceutically acceptable salts of the present application can be formed, for example, using the following inorganic or organic acids: "Pharmaceutically acceptable salt" refers to salts of acidic groups which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. The salts can be prepared in situ during the final isolation and purification of the compounds of the application, or separately by reacting the free base or free acid with a suitable reagent, as outlined below. For example, the free base functionality can be reacted with the appropriate acid. Examples of pharmaceutically acceptable inorganic salts are salts of ammonium, of alkali metals such as potassium and sodium, and of alkaline earth metal such as calcium and magnesium formed, for example, as bicarbonates, borates, carbonates, sulfates, phosphates, hydroxides, and perchlorates, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed by counterion with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or by using other methods known in the art such as ion exchange.

[0014] The pharmaceutically acceptable salts of the present application can be prepared by conventional methods, e.g., by dissolving the compound of the present application in an organic solvent which is miscible with water, such as acetone, methanol, ethanol and acetonitrile, adding thereto an excess of an aqueous solution of an organic or inorganic acid, so that the salt precipitates from the resulting mixture, removing the solvent and the excess free acid therefrom, and isolating the precipitated salt.

[0015] The precursors or metabolites described herein can be those known in the art, so long as the precursors or metabolites are converted by in vivo metabolism to form the compounds. For example, "prodrugs" refers to those compounds of the present application which are useful for contacting the tissues of humans and lower animals with reasonable safety, without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, within the scope of sound medical judgment. The term "prodrug" refers to compounds which rapidly undergo chemical conversion by in vivo metaboliεm, or N-demethylation of the compounds of the present application, to form the parent compound of the above formula.

[0016] The term "solvate" as used herein means a physical association between one or more solvent molecules (whether organic or inorganic) and one or more of the compounds of the present application. This physical association can, for example, be a hydrogen bond. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The solvent molecules can be present in a defined and constant ratio. Solvates include hydrates. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods for solvation are known in the art.

[0017] The "stereoisomers" of the present application are divided into conformational isomers and configurational isomers, and the configurational isomers are further divided into cis-trans isomers and optical isomers (i.e. optical isomers). Conformational isomers refer to a kind of stereoisomerism phenomenon of organic molecules with certain configuration, in which the spatial arrangement of atoms or atomic groups is different due to the rotation or distortion of carbon-carbon single bond. Common examples include the chair conformation and boat conformation of cyclohexane structure. "Stereoisomers" refer to the compounds of the present application containing one or more asymmetric centers, which can exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and single diastereomers. The compounds of the present application have asymmetric centers, and each asymmetric center will produce two optical isomers. The scope of the present application includes all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds. The compounds of the present application can exist in the form of tautomers, which have different hydrogen bonding sites by shifting one or more double bonds. For example, ketone and its enol form are ketone-enol tautomers. Each tautomer and its mixture is included in the compounds of the present application. All enantiomers, diastereomers, racemates, meso isomers, cis-trans isomers, tautomers, geometric isomers, epimers and mixtures thereof of the compounds of formula 25 are included in the scope of the present application.

[0018] The "isotopic derivatives" of the present application refer to the molecules in which the compounds are isotopically labeled. The isotopes commonly used for isotopic labeling are: 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 isotopically labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. In particular, deuterium 3 H and carbon 13 C are more widely used because they are easy to label and convenient to detect. Certain heavy isotopes, such as heavy hydrogen 2Substitution of the compound of Formula (I) with an isotope of a naturally occurring element, such as deuterium, can enhance metabolic stability, prolong half-life, and thereby provide a therapeutic advantage by reducing dosage amounts and providing a therapeutic advantage. Isotopically labeled compounds generally are prepared from isotopically labeled reagents by using the same synthetic techniques as those used to prepare the non-isotopically labeled compounds.

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

[0020] In addition, the present application provides a pharmaceutical composition for preventing and / or treating cancer, a tumor, an inflammatory disease, an autoimmune disease, a neurodegenerative disease, an attention-related disease, or an immune-mediated disease, which comprises the compound of the present application as an active ingredient. The pharmaceutical composition can optionally comprise a pharmaceutically acceptable carrier.

[0021] In addition, the present application provides a method of preventing and / or treating cancer, a tumor, an inflammatory disease, an autoimmune disease, a neurodegenerative disease, an attention-related disease, or an immune-mediated disease, which comprises administering the compound of the present application to a mammal in need thereof.

[0022] When the compound of the present application or a pharmaceutically acceptable salt thereof is administered in combination with another anti-cancer agent or an immune checkpoint inhibitor for treating cancer or a tumor, the compound of the present application or a pharmaceutically acceptable salt thereof can provide an enhanced anti-cancer effect.

[0023] When the compound of the present application or a pharmaceutically acceptable salt thereof is administered in combination with another therapeutic agent for treating an inflammatory disease, an autoimmune disease, and an immune-mediated disease, the compound of the present application or a pharmaceutically acceptable salt thereof can provide an enhanced therapeutic effect.

[0024] The compound of the present application or a pharmaceutically acceptable salt thereof can be administered orally or parenterally as an active ingredient. The dose of the active ingredient can be adjusted according to various relevant factors (e.g., the condition of the subject to be treated, the type and severity of the disease, the rate of administration, and the physician's opinion). In some cases, an amount less than the above dose can be appropriate. An amount greater than the above dose can be used if it does not cause harmful side effects and can be administered in divided doses per day.

[0025] In addition, the present application also provides a method of preventing and / or treating a tumor, cancer, a viral infection, organ transplant rejection, a neurodegenerative disease, an attention-related disease, or an autoimmune disease, which comprises administering the compound of the present application or the pharmaceutical composition of the present application to a mammal in need thereof.

[0026] The pharmaceutical compositions of the present application can be formulated according to any of the methods known in the art for manufacture of pharmaceuticals, comprising conventional methods for the formulation of pharmaceutical compositions for oral administration or parenteral administration (including intramuscular, intravenous, and subcutaneous routes, intratumoral injection) such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions, or suspensions.

[0027] The pharmaceutical compositions of the present application for oral administration can be prepared by mixing the active ingredient with a carrier, for example, 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 the carriers employed in the pharmaceutical compositions of the present application for injection administration can be water, salt solution, glucose solution, glucose-like solution, alcohol, glycol, ether (e.g., polyethylene glycol 400), oil, fatty acid, fatty acid ester, glyceride, surfactant, suspending agent, and emulsifier.

[0029] Other features of the present application will become apparent in the course of the procedures described in the examples of exemplary embodiments, which are given for illustration of the application 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 application can be prepared in a number of ways known to one skilled in the art of organic synthesis, using the methods described below as well as synthetic methods known to those skilled in the art of synthetic organic chemistry or by variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reactions are performed in solvents appropriate to the reagents and materials employed and appropriate to the solvent used. One skilled in the art of organic synthesis will recognize that the order of synthetic steps can be varied to facilitate the reaction process. It will also be appreciated by one skilled in the art that the functionality present on the molecule should be compatible with the reagents and conditions employed in the subsequent transformations. DETAILED DESCRIPTION

[0031] Terminology

[0032] The terms used in the present application, including the specification and claims, are defined as follows, if not otherwise indicated. The conventional methods of mass spectrometry, nuclear magnetic, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used, if not otherwise indicated. In the present application, "or" or "and" means "and / or", if not otherwise indicated.

[0033] In the description and claims, a given chemical formula or name shall encompass all stereoisomers and optical isomers of the substance; racemic mixtures as well as mixtures of the separating individual isomers, where the mentioned isomers exist. Unless otherwise specified, all chiral (enantiomeric and diastereomeric) and racemic forms are covered by the application. Numerous geometric isomers can exist for compounds described herein; all such isomers are intended to be included. The present application describes the cis- and trans- (or E- and Z-) geometric isomers of the compounds of the present application and they can be isolated in, or converted into, mixtures of isomers. The compounds of the present application can be isolated in, or converted into, racemic mixtures or enantiomeric mixtures. All methods for preparing the compounds of the present application and intermediates used in preparing them are considered to be part of the present application. When preparing enantiomeric or diastereomeric products, they can be separated by conventional methods, e.g., by chromatography or fractional crystallization. The end products of the present application are obtained in either free (neutral) or salt form. Both the free form and the salts of these end products are within the scope of the present application. If desired, one form of a compound can be converted into another form. A free base or acid can be converted into a salt; a salt can be converted into the free compound or another salt; a mixture of isomers of a compound of the present application can be separated into the individual isomers. The compounds of the present application, free forms and salts, can exist in a variety of tautomeric forms; all such forms are intended to be included. For example, the compounds of the present application, free forms and salts, can exist in amomeric forms. All such forms are intended to be included.

[0034] The term "patient" as used herein refers to an organism to be treated by the methods of the present application. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, monkeys, equines, bovines, porcines, canines, felines, etc.) and most preferably refers to humans.

[0035] The term "effective amount" as used herein means that amount of a drug or pharmaceutical agent (i.e., a compound of the present application), which will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount of a compound to result in improved treatment, healing, prevention, or lessening of disease, condition, or side effect, or decrease in the rate of advancement of a disease or condition, as compared to a corresponding subject that does not receive the amount. An effective amount can be given in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. The term also includes within its scope effective amounts that enhance normal physiological function.

[0036] The term "treatment" as used herein includes any effect that relieves, reduces, modulates, ameliorates, or eliminates a condition, disease, disorder, etc., or its symptoms.

[0037] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which 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 problem or complication commensurate with a reasonable benefit / risk ratio.

[0038] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, 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 steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which have been used to form pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylate, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, sodium lauryl sulfate, and cetyl alcohol.

[0039] The term "pharmaceutical composition" means a composition comprising a compound of the present application in combination with at least one other pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for the delivery of a biologically active agent to an animal, particularly a mammal, and includes, i.e., adjuvants, excipients or vehicles such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricants and dispersing agents, depending on the nature of the mode of administration and the nature of the dosage form.

[0040] The term "acceptable", as used herein, means no excessive deleterious effect on the health of the subject of the general treatment goal.

[0041] The term "cancer", as used herein, refers to an uncontrolled abnormal growth of cells and, under certain conditions, the ability to metastasize (spread). This type of cancer includes, but is not limited to, solid tumors (e.g., bladder, bowel, brain, breast, uterine, cardiac, kidney, lung, lymphatic tissue (lymphoma), ovarian, pancreatic or other endocrine organ (e.g., thyroid), prostate, skin (melanoma) or blood tumors (e.g., non-leukemic leukemia).

[0042] The term "co-administration" or its grammatical equivalents, as used herein, means the administration of two or more selected therapeutic agents to a single patient at the same time by the same or different routes of administration.

[0043] The term "enhance" or "enhancing", as used herein, means an intended result that can be an increase or prolongation in potency or duration. Thus, in the context of enhancing the therapeutic effect of a drug, the term "enhancing" means the ability of a drug to have an increased or prolonged potency or duration in the system. "Enhancing value", as used herein, means the ability to maximize the enhancement of another therapeutic agent in an ideal system.

[0044] The term "immune disease" refers to a disease or condition resulting from an adverse or deleterious reaction to an endogenous or exogenous antigen. The result is usually a dysfunction of cells, or destruction and dysfunction as a result, or destruction of organs or tissues that can produce the immune condition.

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

[0046] The terms "subject" or "patient" include mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and house cats; laboratory animals such as rats, mice, and guinea pigs; and the like. Non-mammalian animals include, but are not limited to, birds, fish, and the like. In a preferred aspect, the mammal is human.

[0047] The terms "treatment," "treatment regime," or "therapy" as used herein include alleviating, inhibiting or ameliorating a disease symptom or condition; inhibiting the onset of complications; ameliorating or preventing underlying metabolic syndrome; inhibiting the onset of a disease or symptom, such as controlling the development of a disease or condition; reducing a disease or symptom; causing regression of a disease or symptom; reducing complications resulting from a disease or symptom, or preventing and / or treating an indication resulting from a disease or symptom.

[0048] As used herein, an improvement in a disease, symptom, or condition, particularly an improvement in the severity thereof, a delay in the onset thereof, a slowing of the progression thereof, or a reduction in the duration thereof, can result from administration of a compound or pharmaceutical composition, regardless of whether the administration is fixed or intermittent, continuous or discontinuous.

[0049] Routes of administration

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

[0051] In one aspect, the compounds described herein are administered in a manner that is local rather than systemic. In a particular embodiment, the long-acting formulation is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Further, in another embodiment, the drug is administered by a targeted drug delivery system. For example, a liposome encapsulated with an organ-specific antibody. In this embodiment, the liposome is selectively directed to a particular organ and is taken up.

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

[0053] Regardless of the route of administration selected, the compounds of the present application, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present application are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

[0054] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application can be varied so as to obtain an amount of the active ingredient which 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 on a variety of factors including the activity of the particular compound of the present application employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and pre-existing medical conditions of the patient being treated, and like factors well known in the medical arts.

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

[0057] Kit / product package

[0058] All of the features described in this specification (including any accompanying claims, abstract and / or drawings), and / or all of the steps of any method or process described in this specification, can be combined in any combination, unless the context explicitly indicates that some features, combinations only or some steps, combinations only are excluded.

[0059] The above features mentioned in the summary of the application, or features mentioned in the examples, can be combined in any combination. All features disclosed in the specification may

[0060] The present application is further illustrated by the following examples. It is to be understood that these examples are merely illustrative of the present application and do not in any way limit the scope of the application. Unless otherwise indicated, the methods of the following examples were carried out in accordance with conventional procedures or as otherwise described in the specification. Unless otherwise indicated, all percentages, ratios, proportions, or parts are by weight.

[0061] The units in the weight volume percentages in the present application are well known to those skilled in the art, for example, it means the weight (g) of solute in 100 ml of solution. Unless otherwise defined, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. In addition, methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are illustrative only and not intended to be limiting.

[0062] Examples

[0063] General procedures

[0064] When not included in the preparation route, the starting materials and reagents used in the present application are known products, which can be synthesized according to the methods known in the art, or can be obtained by purchasing commercially available products. The commercially available reagents used do not need further purification.

[0065] Room temperature means 20-30 °C.

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

[0067] The hydrogenation reaction is usually vacuumed and filled with hydrogen, and the operation is repeated 3 times. The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L.

[0068] Microwave reaction uses Initiator + microwave reactor.

[0069] The structure of the compounds of the present application is determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR shift (δ) is given in units of 10 -6 (ppm). The NMR was measured by a (Bruker Ascend TM 500 type) nuclear magnetic instrument, and the measuring solvent was deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS). The following abbreviations are used for the multiplicity of NMR signals: s = singlet, br s = broad singlet, d = doublet, t = triplet, m = multiplet. The coupling constant is listed as J value, measured in Hz.

[0070] The measurement of LC-MS uses Thermo liquid chromatograph-mass spectrometer (UltiMate 3000+MSQ PLUS). The measurement of HPLC uses Thermo high pressure liquid chromatograph (UltiMate 3000). The reverse phase preparation chromatography uses Thermo (UltiMate 3000) reverse phase preparation chromatograph. The flash column chromatography uses the automatic column machine of Ajler (FS-9200T), and the silica gel pre-packed column uses Sansheng Pre-packed column. TLC silica gel plate is Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate, and the specification for TLC separation and purification of the product is 0.4 mm to 0.5 mm.

[0071] Example 1

[0072] (S)-4,5-dimethyl-2-((trans-3-(3,4,5-trifluorophenoxy)cyclobutyl)amino)-4,5,9,10- tetrahydro

[0073] -6H,8H-pyrido[3,2,1-de]pteridin-6-one

[0074]

[0075] Compound 1 was prepared by the following steps:

[0076]

[0077] First step: cis-3-BOC-amino cyclobutanol la (250 mg, 1.34 mmol), methyl sulfonic anhydride (465 mg, 2.67 mmol) and N,N-diisopropyl ethylamine (517 mg, 4.01 mmol) were dissolved in dichloromethane (2 mL) and stirred at room temperature overnight. TLC monitored the end of the reaction, the reaction liquid was diluted with dichloromethane, washed with water and saturated brine in turn, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain yellow solid lb (300 mg, yield 84%). 1 H NMR (500 MHz, DMSO-d6) δ 7.23 (d, J = 8.3 Hz, 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).

[0078] Second step: compound lb (300 mg, 1.13 mmol), compound lc (251 mg, 1.70 mmol) and cesium carbonate (737 mg, 2.26 mmol) were dissolved in N,N-dimethylformamide (2 mL) and stirred at 80 °C overnight. LCMS monitored the end of the reaction, the reaction liquid was diluted with ethyl acetate, washed with water and saturated brine in turn, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to obtain white solid Id (280 mg, yield 78%). ESI-MS (m / z): 318.6 [M+H] + .

[0079] Step 3: Compound 1d (280 mg, 882 umol) was dissolved in dichloromethane (2 mL), hydrochloric acid 1,4-dioxane solution (4 M, 1.10 mL) was added dropwise, and the reaction was stirred at room temperature overnight. LCMS monitoring showed that the reaction was completed. The reaction solution was concentrated to obtain white solid 1e (170 mg, yield 75%). ESI-MS (m / z): 218.4 [M+H] + .

[0080] Step 4: 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) were dissolved in tetrahydrofuran (40 mL), triethylamine (2.58 g, 25.50 mmol, 3.53 mL) was added, and the reaction was stirred at room temperature overnight. LCMS monitoring showed that the reaction was completed. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain yellow oil 1h (1.1 g, yield 46%). ESI-MS (m / z): 281.2 [M+H] + .

[0081] Step 5: Compound 1h (1.1 g, 3.92 mmol) was dissolved in tetrahydrofuran (20 mL), aqueous hydrochloric acid (6 N, 0.65 mL) and platinum dioxide (88 mg, 0.39 mmol) were added, and the reaction system was replaced with hydrogen gas using a hydrogen balloon. The reaction was stirred at room temperature under the pressure of the hydrogen balloon for 48 hours, and LCMS monitoring showed that the reaction was completed. The reaction solution was diluted with methanol, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain white solid 1i (900 mg, yield 90%). ESI-MS (m / z): 253.2 [M+H] + .

[0082] Step 6: Compound 1i (50 mg, 197 umol), compound 1e (65 mg, 257 umol) and p-toluenesulfonic acid monohydrate (3.7 mg, 19 umol) were dissolved in n-butanol (2 mL), and the reaction was carried out at 160°C in a microwave for 2 hours. LCMS monitoring showed that the reaction was completed. The reaction solution was purified by reverse phase preparative HPLC to obtain white solid 1 (13 mg, yield 15%). ESI-MS (m / z): 434.3 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 6.90-6.82 (m, 3H), 4.86-4.81 (m, 1H), 4.42-4.36 (m, 1H), 4.12 (q, J = 6.8 Hz, 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 = 6.8 Hz, 3H).

[0083] Example 2

[0084] (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

[0085]

[0086] Compound 2 was prepared from the following steps:

[0087]

[0088] First step: Cis-3-BOC-amino cyclobutanol la (250 mg, 1.34 mmol), methyl sulfonic anhydride (465 mg, 2.67 mmol) and N,N-diisopropyl ethylamine (517 mg, 4.01 mmol) were dissolved in dichloromethane (2 mL) and stirred at room temperature overnight. The reaction was monitored by TLC and upon completion, the reaction was diluted with dichloromethane and washed with water and saturated brine successively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give yellow solid lb (300 mg, yield 84%). 1 H NMR (500 MHz, DMSO-d6) δ 7.23 (d, J = 8.3 Hz, 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).

[0089] Second step: Compound 1b (500 mg, 1.88 mmol), compound 2a (461 mg, 2.83 mmol) and cesium carbonate (1.23 g, 3.77 mmol) were dissolved in N,N-dimethylformamide (2 mL) and stirred at 80 °C overnight. LCMS monitored the reaction to be completed. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine successively. 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 white solid 2b (500 mg, yield 79%). ESI-MS (m / z): 333.3 [M+H] + .

[0090] Third step: Compound 2b (500 mg, 1.50 mmol) was dissolved in dichloromethane (2 mL) and hydrochloric acid 1,4-dioxane solution (4 M, 1.88 mL) was added dropwise. The reaction was stirred at room temperature overnight. LCMS monitored the reaction to be completed. The reaction solution was concentrated to obtain white solid 2c (300 mg, yield 74%). ESI-MS (m / z): 233.5 [M+H] + .

[0091] Fourth step: Compound 1i (50 mg, 197 umol), compound 2c (68 mg, 256 umol) and p-toluenesulfonic acid monohydrate (3.7 mg, 19 umol) were dissolved in n-butanol (2 mL) and reacted at 160 °C for 2 hours under microwave. LCMS monitored the reaction to be completed. The reaction solution was purified by reverse phase preparative HPLC to obtain white solid 2 (10.1 mg, yield 11%). ESI-MS (m / z): 449.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.38 (d, J = 2.8 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.46 (dd, J = 8.7, 2.9 Hz, 1H), 6.91 (d, J = 6.9 Hz, 1H), 5.05-5.00 (m, 1H), 4.46-4.42 (m, 1H), 4.12 (q, J = 6.9 Hz, 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 = 6.7 Hz, 3H).

[0092] Example 11

[0093] (S)-4,5-dimethyl-2-(((1s,3R)-3-(((6-(trifluoromethyl)pyridin-3-yl)oxy)methyl)cyclobutyl)amino)- 4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pteridin-6-one

[0094]

[0095] Compound 11 was prepared from the following steps:

[0096]

[0097] First step: Dissolve cis-tert-butyl 3-hydroxymethylcyclobutylcarbamate 11a (150 mg, 0.745 mmol), methanesulfonic anhydride (259 mg, 1.49 mmol) and N,N-diisopropylethylamine (385 mg, 2.98 mmol) in dichloromethane (5 mL) and stir at room temperature overnight. Monitor the reaction by TLC, dilute the reaction with dichloromethane, wash with water and saturated brine successively, dry the organic phase over anhydrous sodium sulfate, filter and concentrate to give yellow solid 11b (204 mg, yield 98%). 1 HNMR (500 MHz, DMSO-d6) δ 7.10 (d, J = 8.1 Hz, 1H), 4.11 (d, J = 5.2 Hz, 2H), 3.19 (d, J = 1.6 Hz, 1H), 3.16 (d, J = 1.6 Hz, 3H), 2.26 (d, J = 5.9 Hz, 1H), 1.68 (d, J = 8.7 Hz, 2H), 1.57-1.47 (m, 2H), 1.37 (s, 9H).

[0098] Second step: Dissolve compound 11b (204 mg, 731 umol), compound 2a (131 mg, 0.8 mmol) and cesium carbonate (485 mg, 1.49 mmol) in N,N-dimethylformamide (5 mL) and stir at 90 °C overnight. Monitor the reaction by TLC, dilute the reaction with water, extract with ethyl acetate, wash with saturated brine, dry the organic phase over anhydrous sodium sulfate, filter and concentrate, purify the residue by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to give yellow solid 11c (185 mg, yield 72%). ESI-MS (m / z): 347.2 [M+H] + .

[0099] Step 3: Compound 11c (120 mg, 346 umol) was dissolved in dichloromethane (5 mL), 4M hydrochloric acid in dioxane (0.87 mL) was added at 0 °C and stirred overnight. TLC monitored the reaction was finished, directly spin dry to get white solid 11d (85 mg, yield 99%). ESI-MS (m / z): 247.4 [M+H] + .

[0100] Step 4: Compound 11d (82 mg, 336 umol), compound 1i (85 mg, 336 umol) and p-toluenesulfonic acid monohydrate (5.7 mg, 33.6 umol) were dissolved in n-butanol (3 mL) and reacted at 160 °C for 3 hours under microwave. LCMS monitored the reaction was finished. The reaction solution was purified by reverse phase preparative HPLC to get white solid 11 (25 mg, yield 16%). ESI-MS (m / z): 463.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) d 8.47 (d, J = 3.0 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.64 (dd, J = 9.0, 3.0 Hz, 1H), 6.81 (br s, 1H), 4.32 - 4.23 (m, 1H), 4.16 - 4.09 (m, 3H), 4.08 - 4.01 (m, 1H), 3.30 - 3.24 (m, 2H), 2.95 (s, 3H), 2.48 - 2.38 (m, 3H), 1.97 - 1.88 (m, 1H), 1.86 - 1.73 (m, 3H), 1.23 (d, J = 7.0 Hz, 3H).

[0101] Example 12

[0102] (S)-2-(((1s,3R)-3-(((1-cyclopropyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)oxy)methyl)cyclobutyl)amino)-4,5-dimethyl-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pteridin-6-one

[0103]

[0104] Compound 12 was prepared from the following steps:

[0105]

[0106] First step: Compound 12a (1.7 g, 9.21 mmol) and compound 12b (1.0 g, 9.21 mmol) were dissolved in 20 mL of ethanol and stirred at 80 °C overnight. The reaction was concentrated and the residue was slurried with petroleum ether to give compound 12c (800 mg, yield 45%) as a brown solid. ESI-MS (m / z): 193.2 [M+H] + .

[0107] Second step: Compound 11b (872 mg, 3.12 mmol), compound 12c (500 mg, 2.61 mmol) and cesium carbonate (1.70 g, 5.22 mmol) were dissolved in N,N-dimethylformamide (20 mL) and stirred at 90 °C overnight. The reaction was monitored by TLC and the reaction was diluted with water and extracted with ethyl acetate, then washed with saturated brine. 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 = 3 / 1) to give compound 12d (495 mg, yield 52%) as a yellow solid. ESI-MS (m / z): 362.6 [M+H] + .

[0108] Third step: Compound 12d (495 mg, 1.1 mmol) was dissolved in dichloromethane (30 mL) and 4 M hydrochloric acid in dioxane (1.37 mL) was added at 0 °C and stirred overnight. The reaction was monitored by TLC and directly rotary evaporated to give compound 12e (231 mg, yield 80%) as a white solid. ESI-MS (m / z): 262.6 [M+H] + Fourth step: Compound 12e (26 mg, 95 umol), compound 1i (20 mg, 79 umol) and p-toluenesulfonic acid monohydrate (0.4 mg, 7.9 umol) were dissolved in n-butanol (3 mL) and reacted at 160 °C for 3 hours under microwave. The reaction was monitored by LCMS and the reaction was purified by reverse phase preparative HPLC to give compound 12 (13 mg, yield 35%) as a white solid. ESI-MS (m / z): 492.4 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 6.67 (br s, 1H), 6.17 (s, 1H), 4.26-4.18 (m, 1H), 4.15-4.06 (m, 2H), 4.05-3.97 (m, 1H), 3.54-3.21 (m, 5H), 2.94 (s, 3H), 2.60-2.50 (m, 2H), 2.47-2.35 (m, 3H), 1.96-1.86 (m, 1H), 1.85-1.70 (m, 3H), 1.21 (d, J = 6.8 Hz, 3H), 1.05-0.90 (m, 4H).

[0109] Example 13

[0110] (S)-2-(((1s,3R)-3-(((1-cyclopropyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)oxy)methyl)cyclobutyl)amino)-5-(hydroxymethyl)-4,5-dimethyl-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-

[0111] de]pyrido[3,2-d]pyrimidin-6-one Compound 13 was prepared from the following steps:

[0112]

[0113] First step: 2,4-dichloropyrido[3,2-d]pyrimidine 1f (4.0 g, 20.0 mmol) and 2-methyl-L-serine methyl ester hydrochloride 13a (4.07 g, 24.0 mmol) were dissolved in dichloromethane (30 mL), N,N-diisopropylethylamine (7.75 g, 59.99 mmol, 10.45 mL) was added, and the mixture was stirred at room temperature overnight. The reaction was monitored by LCMS, and the reaction solution was diluted with dichloromethane and washed with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give white solid 13b (5.0 g, yield 84%). ESI-MS (m / z): 297.3 [M+H] + .

[0114] Second step: Compound 13b (5.0 g, 16.85 mmol) was dissolved in tetrahydrofuran (50 mL), and hydrochloric acid aqueous solution (6 M, 5.62 mL) and platinum dioxide (382 mg, 1.69 mmol) were added. The reaction system was replaced with hydrogen gas by a hydrogen balloon, and the mixture was stirred at room temperature under the pressure of the hydrogen balloon for 48 hours. The reaction was monitored by LCMS, and the reaction solution was diluted with methanol, filtered, and concentrated to give white solid 13c (4.0 g, yield 88%). ESI-MS (m / z): 269.3 [M+H] + .

[0115] Step 3: Compound 13c (1.5 g, 5.58 mmol) and iodomethane (1.58 g, 11.16 mmol) were dissolved in acetonitrile (5 mL), and cesium carbonate (3.64 g, 11.16 mmol) was added. The reaction mixture was stirred at room temperature for 48 hours. The reaction was monitored by LCMS until completion. The reaction solution was diluted with ethyl acetate, filtered, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give a yellow solid 13d (1.1 g, 69% yield). ESI-MS (m / z): 283.3 [M+H] + .

[0116] Step 4: Compound 12e (25 mg, 84 μmol), compound 13d (20 mg, 70.7 μmol), and p-toluenesulfonic acid monohydrate (0.4 mg, 7.07 μmol) were dissolved in n-butanol (3 mL), and reacted in a microwave oven at 160 °C for 3 hours. The reaction was monitored by LCMS until completion. The reaction solution was purified by reverse-phase preparative HPLC to give a white solid 13 (13 mg, yield 35%). ESI-MS (m / z): 522.3 [M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ6.54(br s,1H),6.17(s,1H),5.06(t,J=5.5Hz,1H),4.29-4.18(m,1H),4.10(d,J=4.9Hz,2H),3.76-3.67(m,2H),3.65-3.58(m,1H),3 .57-3.49(m,2H),2.97(s,3H),2.55-2.45(m,2H),2.44-2.32(m,3H),1.88-1.72(m,4H),1.33(s,3H),0.98(d,J=7.8Hz,4H).

[0117] Example 14

[0118] (S)-5-(hydroxymethyl)-4,5-dimethyl-2-(((1s,3R)-3-(((2-methyl-6-(trifluoromethyl)pyridin-3-yl)oxo)methyl)cyclobutyl)amino)-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pteroidine

[0119] -6-keto

[0120] Compound 14 was prepared by the following steps:

[0121]

[0122] Step 1: Compound 2a (2.0 g, 12.26 mmol), Na2CO3(2.6 g, 24.52 mmol) were added to water (60 mL) and stirred to dissolve. Elemental iodine (3.11 g, 12.26 mmol) was added to the reaction solution, which was stirred at room temperature for 3 hours. LCMS was used to monitor the disappearance of the starting material. The reaction solution was adjusted to pH 5-6 with dilute hydrochloric acid, and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-30% gradient elution) to obtain white solid 14a (1.5 g, yield 42%). ESI-MS (m / z): 290.2 [M+H] + .

[0123] Step 2: Compound 14a (0.5 g, 1.73 mmol), benzyl bromide (337 mg, 1.98 mmol) were dissolved in DMF (5 mL), and K2CO3(364 mg, 2.64 mmol) was added. The reaction solution was stirred at 50°C for 2 hours, and LCMS was used to monitor the end of translation. The reaction solution was diluted with water, and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-15% gradient elution) to obtain white solid 14b (0.5 g, yield 76%). ESI-MS (m / z): 380.2 [M+H] + .

[0124] Step 3: Compound 14b (412 mg, 1.09 mmol), palladium acetate (24 mg, 109 umol), trimethylboroxine (682 mg, 5.43 mmol), tricyclohexylphosphine (152 mg, 523 umol), potassium phosphate tribasic (922 mg, 4.35 mmol), water (3 mL), 1,4-dioxane (30 mL) were added to a 100 mL two-necked flask, which was replaced with nitrogen and stirred at 90°C overnight. TLC was used to monitor the end of the reaction. The reaction solution was diluted with water, filtered with diatomite, extracted with ethyl acetate, and finally washed with saturated brine. 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 = 10 / 1) to obtain yellow liquid 14c (198 mg, yield 68%). ESI-MS (m / z): 268.2 [M+H]+.

[0125] Fourth step: Compound 14c (198 mg, 741 umol) was dissolved in dichloromethane (10 mL), and boron tribromide (928 mg, 3.71 umol) was added dropwise at -78 °C and stirred for 1 hour. TLC monitoring showed that the reaction was completed. The reaction solution was diluted with water and extracted with dichloromethane. 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 obtain yellow liquid 14d (143 mg, yield 91%). ESI-MS (m / z): 178.4 [M+H]+.

[0126] Fifth step: Compound 14d (120 mg, 677 umol), compound 11b (208 mg, 745 umol) and cesium carbonate (441 mg, 1.35 mmol) were dissolved in N,N-dimethylformamide (5 mL) and stirred at 90 °C overnight. TLC monitoring showed that the reaction was completed. The reaction solution was diluted with water, extracted with ethyl acetate, washed with 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 = 3 / 1) to obtain yellow solid 14e (196 mg, yield 80%). ESI-MS (m / z): 361.4 [M+H]+. + .

[0127] Sixth step: Compound 14e (196 mg, 543 umol) was dissolved in dichloromethane (10 mL), and 4M hydrochloric acid (0.952 mL) was added at 0 °C and stirred overnight. TLC monitoring showed that the reaction was completed. Directly rotary evaporation to obtain white solid 14f (112 mg, yield 79%). ESI-MS (m / z): 261.3 [M+H]+. + .

[0128] Seventh step: Compound 14f (27 mg, 106 umol), compound 13d (20 mg, 70.7 umol) and p-toluenesulfonic acid monohydrate (0.4 mg, 7.07 umol) were dissolved in n-butanol (3 mL) and reacted at 160 °C under microwave for 3 hours. LCMS monitoring showed that the reaction was completed. The reaction solution was purified by reverse phase preparative HPLC to obtain white solid 14 (19 mg, yield 52%). ESI-MS (m / z): 507.3 [M+H]+. + ; 1HNMR (500 MHz, DMSO-d6) δ 7.67 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 6.54 (br s, 1H), 5.06 (t, J = 5.5 Hz, 1H), 4.29-4.19 (m, 1H), 4.05 (d, J = 4.8 Hz, 2H), 3.77-3.66 (m, 2H), 3.65-3.60 (m, 1H), 3.58-3.53 (m, 1H), 2.98 (s, 3H), 2.48-2.35 (m, 8H), 1.90-1.74 (m, 4H), 1.33 (s, 3H).

[0129] Example 25

[0130] (S)-4,5-Dimethyl-2-(((1r,3S)-3-((2-methyl-6-(trifluoromethyl)pyridin-3- yl)oxy)cyclobutyl)amino)-4,5,9,10-tetrahydro-6H,8H-pyrido[3,2,1-de]pteridin-6- one

[0131]

[0132] Using intermediate 14d instead of 2a in the first step of Example 2, compound 25 was obtained in a similar manner and procedure. Details as follows:

[0133]

[0134] First step: Compound 1b, compound 14d and cesium carbonate were dissolved in N,N-dimethylformamide and stirred at 80 °C overnight. LCMS monitored the reaction was completed, the reaction was diluted with ethyl acetate, washed with water and saturated brine successively, the organic phase was dried over anhydrous sodium sulfate, the reaction was concentrated, the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give white solid 25b.

[0135] Second step: Compound 25b was dissolved in dichloromethane, hydrochloric acid 1,4-dioxane solution was added dropwise, and stirred at room temperature overnight. LCMS monitored the reaction was completed, the reaction was concentrated to give white solid 25c.

[0136] Third step: Compound 1i, compound 25c and p-toluenesulfonic acid monohydrate were dissolved in n-butanol, and reacted at 160 °C for 2 hours under microwave. LCMS monitored the reaction was completed. The reaction was purified by reverse phase preparative HPLC to give white solid 25. ESI-MS (m / z): 463.5 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 7.66 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 6.8 Hz, 1H), 4.97 (br s, 1H), 4.50 - 4.36 (m, 1H), 4.11 (q, J = 6.8 Hz, 1H), 4.04 - 3.98 (m, 1H), 3.35 - 3.29 (m, 2H), 2.94 (s, 3H), 2.60 - 2.30 (m, 9H), 2.00 - 1.88 (m, 1H), 1.85 - 1.75 (m, 1H), 1.23 (d, J = 6.6 Hz, 3H).

[0137] Wnt pathway inhibitor biological screening and results

[0138] Test Example 1: Construction of Colo205-LUC-TCF / LEF-Ml reporter cell line

[0139] Colo205 cell line (Chinese Academy of Sciences Cell Bank, Cat#TCHu102) was purchased from Chinese Academy of Sciences Cell Bank, after expansion and subculture, the cells were in the exponential growth phase, and then transfected with TCF / LEF transcription factor driven luciferase reporter plasmid (Promega) by lipo3000 liposome transfection method. The plasmid contains a resistance gene, which can be used for resistance screening. Transfection was performed in a 10 cm culture dish using regular complete medium without antibiotics. After 2 days, replace the medium with antibiotic-containing medium and continue to culture. Then replace the antibiotic-containing medium every 2 days, discard the suspended cells, and retain the cells and debris removed by centrifugation of the original medium as an adaptive medium. When the cells grow to cover the culture dish, the cells are digested, counted, and subcultured into a 96-well plate, with an average of 1.5 cells per well. The adaptive medium is used during subculture. The remaining cells are frozen. After subculture, the cells are cultured for 4 hours to allow them to adhere, and then observed under a microscope to count the number of cells in each well. Wells with only one cell per well are labeled as single clone wells. Then, normal culture is carried out, and the medium is replaced every 2 days and observed. The single clone cells that continue to grow are labeled twice, and the normal antibiotic-containing medium is replaced. When the cells in the single clone wells grow to cover the 96-well plate, they are digested and subcultured into a 24-well plate. When the 24-well plate is full, it is subcultured into a 96-well plate and a 6-well plate. The cells in the 96-well plate are subcultured into at least 6 wells, and 3 of them are treated with a known Wnt inhibitor, and the other 3 are not treated. After 24 hours, the cells in the 96-well plate are added with a luciferase detection reagent to detect the fluorescence intensity. The cell line with fluorescence expression without treatment and reduced fluorescence after inhibition is selected for further culture. Colo205-LUC-TCF / LEF-M1 cell line is one of the cell lines selected by the above screening method. Its growth curve, cell morphology, and cell growth state are similar to those of the original Colo205 cells. The ratio of fluorescence signals with and without inhibitor treatment is relatively large among all cell lines, and the ratio can reach 4-5 times after 4 hours of inhibition, which is completely suitable for the later screening of Wnt inhibitors.

[0140] Test Example 2: Detection of the inhibitory ability of the compound on Colo205-LUC-TCF / LEF M1 reporter cell line

[0141] Colo205-LUC-TCF / LEF M1 cell line is a stable transfection pGL4.49-LUC2-TCF / LEF vector reporter tool cell, whose β-catenin Wnt pathway is continuously activated. After adding an inhibitor, the Wnt pathway is inhibited, the expression of firefly luciferase regulated by TCF / LEF cis-element on the vector decreases, and after adding a detection substrate, the detected light signal decreases accordingly, thereby detecting the inhibitory effect of the compound.

[0142] To each well of a 96-well cell culture plate, 100 μL of a compound was added at a maximum concentration of 20 μM, and the compound concentration was diluted by 3-fold gradient. Then, 10000 stable transfection reporter gene colo205 cells and 100 μL of culture medium were inoculated into each well, and the corresponding treatment was performed as positive and negative control wells. The cells were placed in a 5% CO2 cell incubator at 37°C for 4 h, and after 4 h, the culture medium was removed, and 100 μL of a reagent (Promega) containing the corresponding luciferase substrate was added to each well to determine the luciferase reporter gene activity. The luminescence intensity was read by SpectraMax in full wavelength mode. The light signal intensity of the cells treated only by DMSO was the positive control, and the light signal intensity of the cell-free wells was the negative control, and the IC 50 value of the compound was calculated. The colo 205 reporter gene detection data are summarized in Table 1 below.

[0143] Table 1 IC value of the compound for inhibition of Colo205-LUC-TCF / LEF reporter gene 50

[0144]

[0145] Test Example 3: Proliferation inhibition test of the compound on Wnt mutant cell lines (Colo205, DU4475, NCI-H929 and HepG2) and non-Wnt mutant cell lines (Hela and RKO)

[0146] The cell lines used in the test are Colo205, DU4475, NCI-H929 and HepG2 cell lines whose Wnt pathway is continuously activated and whose proliferation is Wnt pathway dependent; and HE LA and RKO cell lines which are not activated under normal circumstances and whose proliferation is not dependent on the Wnt pathway as control cell lines, so as to determine whether the inhibition of the compound of the present application on Wnt-dependent proliferation is caused by other non-specific toxicity.

[0147] ​Colo205, Du4475, NCI-H929, HepG2, HELA and RKO cell lines cultured in respective culture medium were treated in logarithmic growth phase, and after the cells were collected, a uniform cell suspension of known concentration was prepared, and then the cell suspension was added to a 96-well cell culture plate so that each well contained 1000 cells. It was placed in a 5% CO2 cell incubator at 37°C for 20-24h. The next day, the completely dissolved 3-fold gradient diluted compound was added to each cell culture well, and the final highest concentration in the cell culture well was 20μM, and it was continued to be cultured for 96h. Promega's cell viability detection test was used for detection, and the more the cell proliferation, the stronger the final signal strength. The detection instrument was SpectraMax, full wavelength mode. Only the DMSO added well was used as the positive control well, and the well without inoculating cells was used as the negative control well, and the IC 50 value of the compound for the proliferation inhibition of the Wnt continuously activated or proliferation dependent cells, and the IC 50 value of the compound for the proliferation inhibition of the Wnt non-activated or proliferation independent cells were calculated to evaluate the inhibition effect of the compound on the Wnt pathway and the toxic effect on normal cells. The results are shown in Table 2 below.

[0148] Table 2 IC 50 value of the compound for the proliferation inhibition of the Wnt mutant cell lines

[0149]

[0150]

[0151] The above results show that the compound of the present application has significant inhibitory activity on mutant cell lines Colo205, DU4475, NCI-H929 and HepG2, and basically has no significant inhibitory activity on Hela and RKO cell lines, which shows that the compound of the present application has significant and selective Wnt pathway inhibition effect.

Claims

1. A compound having the structure of Formula 25: ###0001### 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 manufacture of a medicament for the prevention and / or treatment of cancer, an inflammatory disease.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of claim 2 in the manufacture of a medicament for the prevention and / or treatment of a tumor, an autoimmune disease.

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

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