THR b receptor agonist compounds, and preparation method and use thereof

Abstract

To provide a compound capable of functioning as a new agonist of a THRβ receptor, and to provide a method for producing the same and use thereof.SOLUTION: The present invention provides a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof. The compound improves THRα selectivity while maintaining good THRβ agonist activity, thereby improving the properties of the final drug. (I) SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese Patent Application No. 201811527414.4, filed on December 13, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Technical Field The present invention relates to the field of pharmaceutical synthesis, and in particular to compounds that can function as novel agonists of the THRβ receptor, and methods for their preparation and use. [Background technology]

[0003] Thyroid hormones (TH) are synthesized in the thyroid gland in response to thyroid-stimulating hormone (TSH) secreted by the pituitary gland. Thyroxine plays a crucial role in regulating growth, development, metabolism, and bodily balance. There are two main types of thyroid hormones: 3,5,3'-triiodo-L-thyroxine (T3) and thyroxine (T4). The human body primarily secretes T4. In peripheral organs, T4 is converted to the more active form, T3, by deiodinase. T3 and T4 produced by the thyroid gland are under negative feedback control. Thyroid-stimulating hormone (TSH) is involved in normal thyroid function and thyroid hormone secretion. Thyroid-stimulating hormone is synthesized in the anterior pituitary gland, and its secretion is controlled by thyroid-releasing hormone (TRH) synthesized in the hypothalamus.

[0004] Thyroid hormones function by binding to thyroid hormone receptors (THRs). Thyroid hormone receptors belong to the nuclear receptor family and regulate the expression of target genes. Thyroid hormone receptors include two distinct subtypes, THRα and THRβ. THRα is primarily distributed in cardiac tissue and plays an important role in regulating cardiac function. The THRβ subtype is primarily expressed in the liver and pituitary gland and regulates cholesterol metabolism and thyroid-stimulating hormone secretion.

[0005] At normal levels, thyroid hormones (THRα) maintain body weight, metabolic rate, body temperature, and mood, and are involved in regulating serum cholesterol. Attempts have been made to use thyroid hormones to regulate serum cholesterol. However, given the potential cardiac side effects of natural thyroid hormone intake (e.g., tachycardia and arrhythmia, heart failure, thyroid axis function, muscle metabolism, and osteoporosis), thyroid hormones are not suitable for treating high cholesterol or obesity. Research findings from studies of animals with selective THR gene knockouts and from studies of several selective THR ligands suggest that these cardiac side effects caused by thyroid hormones may be due to THRα.

[0006] The thyroid hormone receptor pathway regulates the metabolism of lipids, including cholesterol, triglycerides, and lipoproteins. Clinical evidence has shown that lowering low-density cholesterol can reduce the incidence of cardiovascular disease.

[0007] Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder caused by excessive accumulation of triglycerides in the liver, which can further damage and inflame liver cells, resulting in nonalcoholic steatohepatitis (NASH). Patients with NASH typically have type 2 diabetes, high cholesterol, hyperlipidemia, and obesity. Furthermore, they are more likely to develop cirrhosis, liver failure, and ultimately liver cancer. Currently, there are limited drugs available to effectively treat NASH. Considering the function of thyroid hormone in regulating lipid metabolism, the thyroid receptor pathway is a potential target for the treatment of NASH and NAFLD. Thyroid hormone analogs have been shown to significantly reduce liver fat levels in animals.

[0008] Selective THRβ agonists can be used to avoid the cardiac side effects of conventional THR receptor agonists and selectively activate THRβ, thereby improving cellular lipid metabolism and lowering cholesterol and blood lipids. However, selective THRβ agonists also inhibit the thyroid axis, which can cause depression, fatigue, osteoporosis, and other side effects. Therefore, to avoid the side effects associated with thyroid axis inhibition, it is desirable to develop selective THRβ agonists that activate THRβ while reducing thyroid axis inhibition.

[0009] Patents such as Patent Documents 1, 2, 3, and 4 disclose several THR receptor agonists. The structures of these agonists are almost all designed and developed based on the natural ligand of THR receptor, T3. Based on this background, it is still desirable to develop a selective THRβ receptor agonist that not only has the beneficial therapeutic effects of thyroid hormone but also avoids the side effects that affect the heart. [ka]

[0010] Structural modifications were also carried out in the present invention based on the natural ligand for the THR receptor, T3. The inventors unexpectedly discovered that most modified compounds maintained good agonistic activity against the THRβ receptor, and some compounds even exhibited improved selectivity for THRα compared with the reference compound 53 in the reference literature ("Discovery of 2-[3,5-dichloro-4-(5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yloxy)phenyl]-3,5-dioxo-2,3,4,5-tetrahydro[1,2,4]triazine-6-carbonitrile (MGL-3196), a highly selective thyroid hormone receptor β agonist in clinical trials for the treatment of dyslipidemia" (Non-Patent Document 1). At the same time, some compounds of the present invention also exhibit highly desirable pharmacokinetic properties. The pharmacokinetic properties of some preferred compounds are significantly superior to those of the reference compounds, thus improving the properties of the final drug. [ka] Comparative compound 53 [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO03 / 094845 publication [Patent Document 2] WO2007 / 009913 publication [Patent Document 3] WO2010 / 122980 publication [Patent Document 4] WO2011 / 038207 publication [Non-patent literature]

[0012] [Non-Patent Document 1] Martha et al., Journal of Medicinal Chemistry, 2014, 3912-3923 Summary of the Invention

[0013] In order to solve the above technical problems, the present invention uses the following technical solutions:

[0014] According to one aspect of the present invention, the present invention provides a compound of formula (I): [ka] (I) [In the formula, R1 is hydrogen, cyano, substituted or unsubstituted C 1-6 Alkyl or substituted or unsubstituted C 3-6 cycloalkyl, the substituents being selected from the group consisting of halogen atoms, hydroxy, and C 1-6 is selected from the group consisting of alkoxy; R2 and R3 each independently represent a halogen atom or a substituted or unsubstituted C 1-6 alkyl, the substituents being selected from the group consisting of halogen atoms, hydroxy, and C 1-6 selected from the group consisting of alkoxy; Ring A is a substituted or unsubstituted saturated or unsaturated C 5-10 Aliphatic ring or substituted or unsubstituted C 5-10 It is an aromatic ring, and the substituents are hydrogen, halogen atoms, hydroxy, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C 1-4 alkyl)2, -CONH2, -CONHC 1-4 Alkyl, -CON(C 1-4 alkyl)2, -NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-6 one or more groups selected from the group consisting of cycloalkyl, and when two substituents are included, the two substituents together with the carbons to which they are attached may form a ring structure; and The halogen atom is selected from the group consisting of F, Cl, or Br. and pharmaceutically acceptable salts thereof.

[0015] The compound according to the present invention has the following formula (II): [ka] (II) [In the formula, R1 to R3 are as defined above in formula (I); L is absent or selected from the group consisting of -CH2-, and -CH2CH2-; R4 is hydrogen, halogen atom, hydroxy, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C 1-4 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, or C 3-6 cycloalkyl; n is an integer ranging from 1 to 4; m is an integer ranging from 1 to 4; When L is absent, the ring may have two or more substituents R4; and The halogen atom is selected from the group consisting of F, Cl, or Br. It is a compound represented by the structure:

[0016] Preferably, in the structure represented by formula (II), R4 is hydrogen, a halogen atom, hydroxy, C 1-3 Alkyl, C 1-3 Alkoxy, or C 3-6 cycloalkyl; L is absent or selected from the group consisting of -CH2-, or -CH2CH2-; n is 1, 2, or 3; and m is 1 or 2.

[0017] More preferably, in the structure of formula (II), R4 is hydrogen or C 1-3 selected from the group consisting of alkyl; L is selected from the group consisting of -CH2-, or -CH2CH2-; n is 1, 2, or 3; and m is 1 or 2.

[0018] More preferably, in the structure of formula (II), R4 is hydrogen or C 1-3 selected from the group consisting of alkyl; L is absent; n is 1, 2, or 3; and m is 1 or 2.

[0019] The compound according to the present invention has the following formula (III): [ka] [In the formula, R1 to R3 are as defined above in formula (I); R4 is hydrogen, halogen atom, hydroxy, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C 1-4 alkyl)2, -CONH2, -CONHC 1-4 Alkyl, -CON(C 1-4 alkyl)2, -NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, or C 3-6 cycloalkyl; m is an integer ranging from 1 to 4; and The halogen atom is selected from the group consisting of F, Cl, or Br. It is a compound represented by the structure:

[0020] Preferably, in the structure represented by formula (III), R4 is hydrogen, a halogen atom, hydroxy, -OCF3, C 1-6 Alkyl, C 1-6 Alkoxy, or C 3-6 cycloalkyl; and m is an integer ranging from 1 to 3.

[0021] Preferably, in the structure represented by formula (III), R4 is hydrogen, a halogen atom, or C 1-3 alkyl; and m is 1 or 2.

[0022] Preferably, in the compound of the present invention having the structure of formula (I), formula (II), or formula (III), R is hydrogen, cyano, substituted or unsubstituted C 1-6 alkyl, and more preferably, R is selected from the group consisting of cyano, or C 1-3 Preferably, R1 is selected from the group consisting of alkyl, and even more preferably, R1 is cyano.

[0023] Preferably, in the compound according to the present invention having the structure of formula (I), formula (II), or formula (III), R2 and R3 are each independently selected from the group consisting of F, Cl, or Br, and more preferably, R2 and R3 are both Cl.

[0024] Preferably, the compound and its pharmaceutically acceptable salts are the following compounds: [ka] It is one of the following.

[0025] According to another aspect of the present invention, there is provided a method for preparing the compound, the method comprising the steps of: [ka] 1) reacting the intramolecular anhydride compound Ia with hydrazine hydrochloride to obtain a compound of general formula Ib; 2) heating the resulting compound of general formula Ib in phosphorus oxychloride to form a compound of general formula Ic; 3) coupling the obtained compound of general formula Ic with compound Id at high temperature to obtain a compound of general formula Ie (under these conditions, the catalyst is preferably cuprous iodide); 4) reacting the obtained compound of general formula Ie at high temperature in an acidic or alkaline environment to obtain a compound of general formula If; and 5) The obtained compound of general formula If is reacted with sodium nitrite in an acidic aqueous solution, and then compound Ig is added and further reacted, followed by ring closure at high temperature to obtain the compound of general formula I (under these conditions, the acid is preferably hydrochloric acid); Includes.

[0026] The compounds of general formula I according to the present invention are [ka] It may be produced as follows. The compound of general formula IB-1 is hydrolyzed in an acidic aqueous solution at high temperature to give the compound of general formula IB-2 (under these conditions, the acid is preferably hydrochloric acid); and the obtained compound of general formula IB-2 is then reacted at high temperature in the presence of mercaptoacetic acid to remove the carboxyl group to give the compound of general formula I.

[0027] The compounds of general formula I according to the present invention can be prepared by the following method: [ka] It may also be produced by The compound of general formula If is reacted with sodium nitrite under acidic conditions to form a diazonium salt compound, and then a halide anion is added to obtain a compound of general formula Ig; and the obtained compound of general formula Ig is coupled with intermediate Ih under transition metal catalysis to obtain a compound of general formula I.

[0028] In another aspect, the present invention provides use of the compound in the manufacture of a medicament for treating a metabolic-related disease.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable adjuvant.

[0030] Preferably, the metabolic-related disease is selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, diabetes, and non-alcoholic steatohepatitis (NASH), fatty liver, atherosclerosis, hypothyroidism, and thyroid cancer; preferably, the metabolic-related disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), hypothyroidism, and thyroid cancer.

[0031] In another aspect, the present invention provides a method for treating a metabolic-related disease, comprising administering to a subject an effective amount of a compound of the present invention or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0032] Preferably, in the method for treating a metabolic-related disease, the metabolic-related disease is selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, diabetes, non-alcoholic steatohepatitis (NASH), fatty liver, atherosclerosis, hypothyroidism, and thyroid cancer, and preferably, the metabolic-related disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), hypothyroidism, and thyroid cancer. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be described in detail below. Prior to the description, it should be understood that the terms used in this specification and the appended claims should not be interpreted as being limited to their general meanings and dictionary meanings, but should be interpreted according to the corresponding meanings and concepts in the technical aspects of the present invention based on the principle that the inventor is allowed to properly define terms for the best interpretation. Therefore, the description proposed here is merely a preferred example for illustrative purposes and is not intended to limit the scope of the present invention. Therefore, it should be understood that other equivalents or improvements can be obtained therefrom without departing from the spirit and scope of the present invention.

[0034] In accordance with the present invention, all terms recited herein, unless otherwise specified, have the same meaning as one of ordinary skill in the art would understand the present invention.

[0035] As used herein, the term "salt" refers to a compound containing a cation and an anion, which can be formed by protonation of a site capable of accepting a proton and / or proton abstraction of a site capable of donating a proton. Notably, protonation of a site capable of accepting a proton leads to the formation of a cationic substance, the charge of which is balanced by the presence of a physiological anion, whereas proton abstraction of a site capable of donating a proton leads to the formation of an anionic substance, the charge of which is balanced by the presence of a physiological cation.

[0036] The term "pharmaceutically acceptable salt" means that the salt is pharmaceutically acceptable. Pharmaceutically acceptable salts include, for example, but are not limited to, (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, or organic acids such as glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-paratoluenesulfonic acid, camphoric acid, dodecylsulfuric acid, gluconic acid, glutamic acid, salicylic acid, and cis-muconic acid; or (2) alkali addition salts formed with the conjugate bases of the above inorganic acids, where the conjugate base is Na + , K. + , Mg 2+ , Ca 2+ , and N.H. x R 4-x + wherein NH x R 4-x + (R is C1-4 where x is an alkyl group and the subscript x is an integer selected from the group consisting of 0, 1, 2, 3, or 4) represents the cation of a quaternary ammonium salt. It should be understood that all pharmaceutically acceptable salts involved include solvent addition forms (solvates) or crystalline forms (polymorphs) as defined herein for the same acid addition salt.

[0037] "C 1-M The term "alkyl" means an alkyl containing 1 to M carbon atoms, where M is an integer having the following numerical values: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. For example, "C 1-6 "Alkyl" means an alkyl containing 1 to 6 carbon atoms. Examples of alkyl include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl, or amyl, i-amyl, neopentyl, hexyl, heptyl, and octyl, but are not limited to lower alkyl.

[0038] The term "aromatic group" refers to an aromatic system, which may be a monocyclic ring or multiple aromatic rings originally fused or bonded together such that at least some of the fused or bonded rings form a conjugated aromatic system. Aryl groups include, but are not limited to, phenyl, naphthyl, and tetralyl. Aryl may be optionally substituted, for example, one to four aryl groups or heterocycles may be substituted with a group selected from the group consisting of halogen, -CN, -OH, -NO, amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, substituted alkoxy, alkylcarbonyl, alkylcarboxy, alkylamino, or arylthio.

[0039] The term "substituted" means that the referenced group can be substituted with one or more additional groups, where the additional groups are individually and independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic hydrocarbon, hydroxy, alkoxy, alkylthiol, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, cyano, halo, carbonyl, thiocarbonyl, nitro, haloalkyl, fluoroalkyl, and amino (including mono- and di-substituted amino groups and their protected derivatives).

[0040] The compound represented by formula (I) or a pharmaceutically acceptable salt thereof provided by the present invention, and pharmaceutical compositions containing the compound may be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions, and aerosols, and may be present in a suitable solid or liquid carrier or diluent, and in a sterile liquid suitable for injection or infusion.

[0041] Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional pharmaceutical manufacturing methods. For example, a unit dose of the preparation contains 0.05-200 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof, preferably 0.1-100 mg of the compound of formula (I).

[0042] The compounds or pharmaceutical compositions of the present invention represented by general formula (I) can be used clinically in mammals, including humans and animals, via oral, nasal, transdermal, pulmonary, or gastrointestinal routes. Oral administration is most preferred. The most preferred daily dose is 0.01 to 200 mg / kg body weight per single dose or 0.01 to 100 mg / kg body weight per divided dose. Regardless of the administration method, the optimal dose for each individual should be determined based on the specific treatment. Typically, administration begins at a low dose and is gradually increased until the optimal dose is found.

[0043] In the present invention, the term "effective amount" can refer to an effective amount for the dosage and duration required to achieve the desired effect. This effective amount can vary depending on certain factors such as the type of disease, the symptoms of the disease during treatment, the structure of the specific target organ to be administered, the individual height and weight of the patient, or the severity of the disease or symptoms. Those skilled in the art can determine the effective amount of a specific compound through experience without unnecessary experimentation.

[0044] A typical formulation is prepared by mixing the compound of the present invention represented by general formula (I) with a carrier, diluent or excipient. Suitable carriers, diluents or excipients are well known to those skilled in the art and include substances such as carbohydrates, waxes, water-soluble and / or swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, and water.

[0045] The specific carrier, diluent, or excipient used will be determined according to the use and purpose of the compound of the present invention. Generally, the solvent will be selected by those skilled in the art based on the solvent that can be safely and effectively administered to mammals. Generally, safe solvents are non-toxic aqueous solvents such as water, and other non-toxic solvents that are soluble in or miscible with water. Suitable aqueous solvents include one or more of water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400 and PEG300), etc. The formulation may also include one or more of buffering agents, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, light-shielding agents, flow aids, processing aids, colorants, sweeteners, flavors, flavoring agents, or other known additives, so that the drug can be manufactured or used in an acceptable form.

[0046] When the compound of formula (I) of the present invention is used in combination with at least one other drug, the two or more drugs can be used separately or in combination, preferably in the form of a pharmaceutical composition.The compound of formula (I) of the present invention or the pharmaceutical composition can be administered to a subject separately or together in a known oral, intravenous, rectal, vaginal, transdermal, or other local or systemic administration form.

[0047] These pharmaceutical compositions may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, light-screening agents, flow aids, processing aids, colorants, sweeteners, flavors, flavorings, or other known additives to enable the pharmaceutical compositions to be manufactured or used in an acceptable form.

[0048] Oral administration is preferred for the drug of the present invention. Solid dosage forms for oral administration can include capsules, tablets, powders, or granular preparations. In solid dosage forms, the compound or pharmaceutical composition of the present invention is mixed with at least one inert excipient, diluent, or carrier. Suitable excipients, diluents, or carriers include substances such as sodium citrate or dicalcium phosphate, or starch, lactose, sucrose, mannitol, and silicic acid; adhesives such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; humectants such as glycerol; disintegrants such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain complex silicates, and sodium carbonate; solution blocking agents such as paraffin; absorption enhancers such as quaternary ammonium compounds; adsorbents such as kaolin and bentonite; and lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate. In the case of capsules and tablets, the dosage form can also contain a buffer solution. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose and high molecular weight polyethylene glycols as excipients.

[0049] The liquid dosage form for oral administration includes pharmaceutically acceptable emulsion, solution, suspension, and syrup and elixir.In addition to the compound of the present invention or its pharmaceutical composition, liquid dosage form can comprise the inert diluent commonly used in this field, such as water or other solvent; solubilizer and emulsifier such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide; oil (for example, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil); glycerol; tetrahydrofurfuryl alcohol; fatty acid ester of polyethylene glycol and sorbitan; or the mixture of some of these substances.

[0050] Besides these inert diluents, compositions can also include excipients, such as one or more wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, and perfuming agents.

[0051] Regarding suspensions, in addition to the compound represented by general formula (I) of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, the suspension may further contain a carrier such as a suspending agent, for example, ethoxylated isostearol, polyoxyethylene sorbitol, sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum, or a mixture of any of these substances.

[0052] The compound of the present invention represented by general formula (I) or its pharmaceutically acceptable salt or pharmaceutical composition containing it can be administered using other topical dosage forms, including ointments, powders, sprays and inhalants.The drug can be mixed under sterile conditions with pharmaceutically acceptable excipients, diluents or carriers, and optionally with preservatives, buffers or propellants.Ophthalmic preparations, ophthalmic ointments, powders and solutions are also intended to be included in the scope of the present invention.

[0053] Additionally, the present disclosure further encompasses kits (e.g., pharmaceutical packages). The provided kits may include a pharmaceutical composition or compound described herein and a container (e.g., a drug bottle, an ampoule, a bottle, a syringe, and / or a subpackage, or other suitable container). In some embodiments, the provided kits may optionally further include a second container containing a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical compositions or compounds described herein provided in the first and second containers are combined to form a unit dosage form.

[0054] In some embodiments, the kits described herein further include instructions for use of the compounds or pharmaceutical compositions included in the kit. The kits described herein may include information required by regulatory authorities, such as the U.S. Food and Drug Administration (FDA). In some embodiments, the information included in the kit is formulation information. In some embodiments, the kit and instructions are provided for treating a proliferative disorder in a subject in need thereof and / or preventing a proliferative disorder in a subject in need thereof. The kits described herein may include one or more additional pharmaceutical formulations as separate compositions. [Example]

[0055] The present invention will be described in more detail below in combination with specific embodiments, but the present invention is not limited to the following embodiments, which are provided for the purpose of better illustrating specific embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way. Conditions not described in the embodiments are conventional conditions. Unless otherwise specified, all reagents and devices used in the following embodiments are commercially available products.

[0056] The structures of the compounds in the following embodiments are determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR displacements (δ) are given in units of 10 (ppm). NMR measurements are performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents used are deuterated dimethyl sulfoxide (DMSO-d), deuterated chloroform (CDCl), and deuterated methanol (CDOD), and the internal standard is tetramethylsilane (TMS).

[0057] For MS measurement, a FINNIGAN LCQAd(ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advance max) is used.

[0058] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used for thin-layer chromatography (TLC). The silica gel plate specifications are 0.15 mm to 0.2 mm, and the silica gel plate specifications for thin-layer chromatography (TLC) for product separation and purification are 0.4 mm to 0.5 mm.

[0059] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0060] Unless otherwise specified in the embodiments, the reaction temperature is room temperature, that is, 20°C to 30°C.

[0061] Thin layer chromatography (TLC) is used to detect the reaction process in the embodiment. The developing system used and the column chromatography eluent system used to purify the compound include: A: dichloromethane and methanol system; B: n-hexane and ethyl acetate system; C: petroleum ether and ethyl acetate system; D: acetone and petroleum ether system, and the volume ratio of the solvents is adjusted according to the different polarity of the compound.

[0062] Embodiment 1 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile [ka] [ka]

[0063] Process 1 Preparation of 2,3,5,6,7,8-hexahydrophthalazine-1,4-dione (compound 1b) Sodium acetate (3.69 g, 45 mmol) and hydrazine hydrochloride (3.08 g, 45 mmol) were added sequentially to a solution of compound 3,4,5,6-tetrahydrophthalic anhydride 1a (4.56 g, 30 mmol) in acetic acid (50 mL) and water (100 mL). After the addition, the mixture was heated to 100°C and stirred for 3 hours. The reaction was stopped and allowed to cool to room temperature. The solid was precipitated and filtered to obtain compound 1b (4.2 g), which was used directly in the next reaction. 1 H NMR (400 MHz, DMSO-d6): 11.26 (s, 2H), 2.36 (s,4H), 1.65(s, 4H).

[0064] Process 2 Preparation of 1,4-dichloro-5,6,7,8-tetrahydrophthalazine (compound 1c) Compound 1b (1 g, 6.02 mmol) was dissolved in phosphorus oxychloride (8 ml). The air in the system was replaced with nitrogen gas three times. The system was heated to 110°C and stirred for 3 hours. The reaction was stopped and then allowed to cool naturally. The reaction solution was slowly poured into ice water. The mixture was adjusted to pH 10 with 1N aqueous sodium hydroxide solution and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, concentrated, and dried to obtain compound 1c (1.1 g). The product was used directly in the next reaction.

[0065] Process 3 Preparation of 3,5-dichloro-4-((4-chloro-5,6,7,8-tetrahydrophthalazin-1-yl)oxy)aniline (Compound 1d) Dimethyl sulfoxide (8 ml) was added to a mixture of compound 1c (1.0 g, 5.0 mmol), 2,6-dichloro-4-aminophenol (0.93 g, 6 mmol), potassium carbonate (2.76 g, 20 mmol), and CuI (0.57 g, 3 mmol). The air in the system was replaced with nitrogen gas three times. The system was heated to 100 °C and stirred for 3 hours. The reaction was stopped and then cooled. The solids in the reaction solution were first filtered, and the filter residue was repeatedly washed with ethyl acetate. 80 ml of water was added to the filtrate, and then the aqueous phase was extracted with ethyl acetate. The organic phases were combined and washed with saturated brine. After filtration and solvent concentration, compound 1d (450 mg) was obtained by column chromatography.

[0066] Process 4 Preparation of 4-(4-amino-2,6-dichlorophenoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (Compound 1e) Compound 1d (100 mg, 0.3 mmol) was dissolved in acetic acid (4 ml), and sodium acetate (200 mg, 2.5 mmol) was added. The mixture was heated to 120°C and stirred for 12 hours. After the reaction was stopped, the solvent was removed under reduced pressure. 10 ml of water was added, followed by the addition of 1N aqueous sodium hydroxide solution to adjust the pH to 8. The mixture was then extracted with ethyl acetate (10 ml x 3), and the organic phases were combined. The organic phase was washed with saturated aqueous sodium bicarbonate solution and saturated brine, respectively, and concentrated under reduced pressure to obtain a gray solid. 10 ml of methanol and 10 ml of 1N aqueous sodium hydroxide solution were added to the resulting solid, and the mixture was heated to 120°C and stirred for 12 hours. After the reaction was stopped, the methanol was concentrated under reduced pressure. The remaining aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined. After the solvent was concentrated under reduced pressure, compound 1e (80 mg) was obtained by separation and purification by thin-layer chromatography.

[0067] Process 5 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 1) Sodium nitrite (16 mg) was dissolved in water (0.5 ml). Under ice bath conditions, this solution was slowly added dropwise to a mixture of compound 1e (60 mg, 0.185 mmol), water (2.5 ml), and saturated aqueous hydrochloric acid (1.25 ml). The mixture was stirred for 0.5 hours while maintaining ice bath conditions until the solution became clear. At this temperature, a mixture of N-cyanoacetylurethane (32 mg), water (4.2 ml), and pyridine (1.3 ml) was further added dropwise. After the addition, the mixture was stirred overnight. After quenching the reaction, the yellow solid was filtered and washed with water and petroleum ether. Acetic acid (5 ml) and sodium acetate (160 mg, 2 mmol) were added to the resulting solid. The mixture was heated to 120°C, stirred for 6 hours, and then cooled to room temperature. 100 ml of water was added, and a pale yellow solid precipitated. After separation and purification by thin layer chromatography (DCM:MeOH=8:1), compound 1 (12.0 mg) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 12.03 (s, 1H), 7.75 (s,2H), 2.68-2.63 (m, 2H), 2.45-2.39 (m, 2H), 1.80-1.67 (m, 4H). MS m / z (ESI): 447.4 [M+1].

[0068] Embodiment 2 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 2b) [ka] 2 The synthetic route of Example 1 was carried out to produce the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 2), except that in step 1 of Example 1, the raw material 3,4,5,6-tetrahydrophthalic anhydride 1a was replaced with bicyclo[2.2.2]oct-2-ene-2,3-dicarboxylic anhydride. 1 H NMR (400 MHz, DMSO-d6): 12.13 (s, 1H), 7.77 (s,2H), 1.88-1.73 (m, 4H), 1.39-1.17 (m, 6H). MS m / z (ESI): 473.2 [M+1].

[0069] Embodiment 3 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-methanophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 3) [ka] 3

[0070] Process 1 Preparation of dimethyl-bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate [ka] Dimethyl acetylenedicarboxylate (compound 3b) (2.75 ml, 22.3 mmol) was slowly added dropwise to cyclopentadiene (compound 3a) (1.475 g, 22.3 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction was stopped to give oily product 3c (4.0 g). The crude product was used directly in the next reaction without further purification.

[0071] Process 2 Preparation of dimethyl bicyclo[2.2.1]hept-2-ene-2,3-dicarboxylate [ka] Compound 3c (1.0 g, 4.8 mmol), palladium on carbon (0.05 g), and 15 ml of acetone were placed in a reaction flask and purged three times under a hydrogen balloon. The system was stirred at room temperature for 1 hour. After filtration, the solvent was concentrated and dried under reduced pressure to give product 3d (0.9 g) as a pale green liquid.

[0072] Process 3 Preparation of bicyclo[2.2.1]hept-2-ene-2,3-diacide [ka] Tetrahydrofuran (20 ml) and water (20 ml) were added to a mixture of compound 3d (3.0 g, 14.3 mmol) and lithium hydroxide monohydrate (1.54 g, 35.8 mmol) and stirred at room temperature for 2 hours. The solution was adjusted to pH 1 with 2N dilute hydrochloric acid, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 3e (2.0 g) as a white solid.

[0073] Process 4 Preparation of 4,5,6,7-tetrahydro-4,7-methanoisophenylfuran 1,3-dione [ka] Acetic anhydride (10 ml) was added to compound 3e (200 mg, 1.1 mmol). The mixture was heated to 100° C. and stirred for 2 hours. The solvent was removed by concentration under reduced pressure to obtain a crude product of compound 3f (200 mg) as a solid.

[0074] Process 5 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-methanophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile The synthetic route of Example 1 was carried out to prepare the title product, 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-methanophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 3), except that in step 1 of Example 1, the starting material 4,5,6,7-tetrahydroisobenzofuran-1,3-dione (Compound 1a) was replaced with 4,5,6,7-tetrahydro-4,7-methanoisophenylfuran-1,3-dione (Compound 3f). 1H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 7.77 (s,2H), 2.75-2.65 (m, 2H), 1.80-1.72 (m, 4H), 1.55-1.50 (m, 2H). MS m / z (ESI): 459.0 [M+1].

[0075] Embodiment 4 Preparation of 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentane[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 4) [ka] 4

[0076] Process 1 Preparation of methyl 3,3-dimethyl-2-oxocyclopentyl-1-carboxylate [ka] NAH (480 mg, 12 mmol) was added to a solution of 2,2-dimethylcyclopentan-1-one (1.12 g, 10 mmol) and MeOH (0.5 ml) in DMC (11 ml). The mixture was heated to 82 °C, stirred for 3 hours, and cooled. Methanol (0.5 ml) and acetic acid (1 ml) were added sequentially, and the mixture was poured into ice water. The mixture was extracted with dichloromethane, dried, and the solvent was removed under reduced pressure to obtain colorless oily compound 4a (1.7 g), which was used directly in the next step.

[0077] Process 2 Preparation of methyl 3,3-dimethyl-2-(((trifluoromethyl)sulfo)oxo)cyclopent-1-ene-1-carboxylate [ka] Trifluoroacetic anhydride (2 ml, 12 mmol) was added dropwise to a solution of compound 4a (1.7 g, 10 mmol) and diisopropylethylamine (8.2 ml, 50 mmol) in dichloromethane (17 ml) at -60°C. After the addition, the mixture was slowly warmed to room temperature and stirred for 16 hours. 50 ml of water was added, and the mixture was extracted with ethyl acetate. The solvent was removed under reduced pressure, and the extract was purified by column chromatography (PE:EA = 50:1) to give compound 4b (1.8 g) as a colorless oil.

[0078] Process 3 Preparation of 2-(methoxycarbonyl)-5,5-dimethylcyclopent-1-ene-1-carboxylic acid [ka] Under a nitrogen gas balloon atmosphere, DMF (25 mL) was added to a mixture of 4b (1.8 g, 5.96 mmol), diisopropylethylamine (1.97 mL, 11.92 mmol), acetic anhydride (1.13 mL, 11.92 mmol), sodium formate (1.22 g, 5.96 mmol), palladium acetate (66.9 mg, 0.30 mmol), and lithium chloride (758 mg, 17.88 mmol). After the addition, the mixture was stirred at room temperature for 16 hours, and then 300 mL of ethyl acetate was added. The mixture was washed once with water and once with saturated brine. The solvent was removed under reduced pressure to give 4c (1.2 g) as a colorless oil.

[0079] Process 4 Preparation of 3,3-dimethylcyclopent-1-ene-1,2-dicarboxylic acid [ka] Methanol (6 mL) and water (6 mL) were added to a mixture of 4c (1.2 g, 6 mmol) and lithium hydroxide monohydrate (756 mg, 18 mmol) and stirred at room temperature for 3 h. Methanol was removed under reduced pressure, and the solution was then adjusted to pH 1 with concentrated hydrochloric acid, extracted with ethyl acetate, and the solvent was removed under reduced pressure to give compound 4d (1.0 g) as a white solid.

[0080] Process 5 Preparation of 4,4-dimethyl-5,6-dihydro-1H-cyclopentyl[c]furan-1,3(4H)-dione [ka] Acetic anhydride (10 ml) was added to compound 4d (1.0 g, 5.43 mmol). The mixture was heated to 100° C., stirred for 3 hours, and cooled. Excess acetic anhydride was removed under reduced pressure to give compound 4e (875 mg) as a light brown liquid, which was used directly in the next step.

[0081] Process 6 Preparation of 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentane[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile The synthetic route of Example 1 was carried out to prepare the title product 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentane[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 4), except that in step 1 of Example 1, the starting material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced with 4,4-dimethyl-5,6-dihydro-1H-cyclopentyl[c]furan-1,3(4H)-dione (Compound 4e). 1 H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 7.79 (s,2H), 2.97-2.93 (m, 2H), 2.01-1.98 (m, 2H), 1.34-1.20 (m, 6H). MS m / z (ESI): 460.9 [M+1].

[0082] Embodiment 5 Preparation of 1-(3,5-dichloro-4-((5-methyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 5) [ka] 5

[0083] Process 1 Preparation of methyl 3-methyl-2-oxocyclohexane-1-carboxylate 5b [ka] NaH (60% content, 1.92 g, 48 mmol) was added to a solution of 2-methylcyclohexanone (compound 5a) (4.48 g, 40 mmol) in dichloromethane (50 ml). The mixture was heated to reflux, stirred for 3 hours, and cooled. The mixture was roughly quenched with methanol (0.5 ml) and acetic acid (1 ml). The reaction was added to an ice bath and extracted with dichloromethane. The solvent was removed under reduced pressure, and purified by column chromatography to give an oil (compound 5b) (5.6 g).

[0084] Process 2 Preparation of methyl 3-methyl-2-(((trifluoromethyl)sulfo)oxo)cyclohexyl-1-ene-1-carboxylate [ka] Under ice bath conditions, NaH (60% content, 600 mg, 25 mmol) was added to a solution of compound 5b (0.85 g, 5 mmol) in ethyl ether (20 mL) and stirred for 0.5 hours. Then, trifluoromethanesulfonic anhydride (2.8 g, 10 mmol) was added dropwise to the system, and the mixture was stirred at 0 °C for an additional 1 hour. The system was quenched by adding HO (50 mL), adjusted to pH 1 with 1N HCl, and extracted with dichloromethane. The solvent was removed under reduced pressure, and the product was purified by column chromatography (PE:EA = 20:1) to obtain compound 5c (1.04 g) as a colorless oil.

[0085] Process 3 Preparation of 2-(methoxycarbonyl)-6-methylcyclohexyl-1-ene-1-carboxylic acid [ka] Under a nitrogen atmosphere, diisopropylethylamine (0.714 g, 7.00 mmol) and acetic anhydride (0.903 g, 7.00 mmol) were added dropwise, successively, to a solution of compound 5c (0.714 g, 10.5 mmol) and sodium formate (0.714 g, 10.50 mmol) in N,N-dimethylformamide (15 mL), and the mixture was stirred at room temperature for 1 hour. Next, palladium acetate (40 mg, 0.18 mmol) and lithium chloride (445 mg, 10.50 mmol) were added, and the mixture was stirred at room temperature overnight. Ethyl acetate (30 mL) was added, and the mixture was washed with water. The solvent was removed under reduced pressure to give compound 5d (590 mg) as a pale yellow oil.

[0086] Process 4 Preparation of 3-methylcyclohexyl-1-ene-1,2-diacide 5e [ka] Lithium hydroxide monohydrate (0.375 g, 8.94 mmol) was added to a solution of compound 5d (0.590 g, 2.98 mmol) in methanol (3 ml) and water (3 ml) and stirred at room temperature for 3 hours. Methanol was then removed under reduced pressure. 1N aqueous hydrochloric acid was added to adjust the system to pH 1, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, concentrated under reduced pressure, and the solvent was removed to give compound 5e (0.550 g) as an oil.

[0087] Process 5 Preparation of 4-methyl-4,5,6,7-tetrahydroisophenylfuran-1,3-dione 5f [ka] Acetic anhydride (6 ml) was added to compound 5e (0.550 g, 2.99 mmol). The mixture was heated to 100° C. and stirred for 2 hours. The system was cooled, and the solvent was removed under reduced pressure to give compound 5f (0.34 g) as a brown oil, which was used directly in the next step.

[0088] Process 6 Preparation of 1-(3,5-dichloro-4-((5-methyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 5) The synthetic route of Example 1 was carried out to prepare the title product 1-(3,5-dichloro-4-((5-methyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 5), except that in step 1 of Example 1, the starting material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced with 4-methyl-4,5,6,7-tetrahydroisophenylfuran-1,3-dione (Compound 5f). MS m / z (ESI): 460.1 [M+1]

[0089] Embodiment 6 Preparation of 1-(3,5-dichloro-4-((5,5-dimethyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile [ka] 6 The synthetic route of Example 5 was carried out to prepare the title product 1-(3,5-dichloro-4-((5,5-dimethyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 6), except that in step 1 of Example 5, the raw material 2-methylcyclohexanone (Compound 5a) was replaced with 2,2-dimethylcyclohexanone. MS m / z (ESI): 474.1 [M+1]

[0090] Embodiment 7 Preparation of 1-(3,5-dichloro-4-((7-methyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 7) [ka] 7 The synthetic route of Example 5 was carried out to prepare the title product 1-(3,5-dichloro-4-((7-methyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 7), except that in step 1 of Example 5, the starting material 2-methylcyclohexanone (Compound 5a) was replaced with 2-methylcyclopentanone. MS m / z (ESI): 446.0 [M+1]

[0091] Embodiment 8 Preparation of 1-(3,5-dichloro-4-((7-ethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentane[d]pyridazin-4-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 8) [ka] 8 The synthetic route of Example 5 was carried out to prepare the title product 1-(3,5-dichloro-4-((7-ethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentane[d]pyridazin-4-yl)oxo)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-nitrile (Compound 8), except that in step 1 of Example 5, the raw material 2-methylcyclohexanone (Compound 5a) was replaced with 2-ethylcyclopentanone. MS m / z (ESI): 460.1 [M+1]

[0092] Embodiment 9 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 9) [ka] 9 The synthetic route of Example 1 was carried out to prepare the title product, 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 9), except that the starting material, 1,4-dichloro-5,6,7,8-tetrahydrophthalazine (Compound 1c) in Step 3 of Example 1, was replaced with 1,4-dichlorophthalazine. 1 H NMR (400 MHz, DMSO-d6): 12.00(s, 1H), 8.31(d, J=8.0Hz, 1H), 8.26 (d, J=8.0Hz, 1H), 8.09 (t, J=12.0Hz 1H), 8.03 (t, J=16.0Hz 1H), 7.81 (s, 2H). MS m / z (ESI): 443.0 [M+1].

[0093] Embodiment 10 Preparation of 2-(3,5-dichloro-4-((5-chloro-4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 10) [ka] 10 The synthetic route of Example 1 was carried out to prepare the title product 2-(3,5-dichloro-4-((5-chloro-4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 10), except that in step 1 of Example 1, the raw material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced with 3-chlorophthalic anhydride. 1 H NMR (400 MHz, DMSO-d6): 12.00(s, 1H), 8.30 (d, J=8.0Hz, 1H), 8.15 (d, J=8.0Hz, 1H), 7.95 (m, 1H), 7.80 (s, 2H). MS m / z (ESI): 477.0 [M+1].

[0094] Embodiment 11 Preparation of 2-(3,5-dichloro-4-((5-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 11) [ka] 11 The synthetic route of Example 1 was carried out to prepare the title product 2-(3,5-dichloro-4-((5-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 11), except that in step 1 of Example 1, the raw material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced with 3-methylphthalic anhydride. MS m / z (ESI): 456.9 [M+1].

[0095] Embodiment 12 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxo)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 12) [ka] 12 [ka]

[0096] Process 1 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid Acetic acid (4 ml) and concentrated hydrochloric acid (1 ml) were added to compound 2 (77 mg), heated to 120°C, stirred for 5 hours, and cooled. The reaction solution was diluted with water. The resulting solid was filtered, washed with water, and then washed with petroleum ether to obtain solid compound 12a (35 mg), which was used directly in the next reaction.

[0097] Process 2 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxo)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione Mercaptoacetic acid (2 ml) was added to compound 12a (35.0 mg), and the mixture was heated to 170° C. and stirred for 2 hours. The mixture was cooled, adjusted to pH 8 with 1N aqueous sodium hydroxide solution, and extracted three times with ethyl acetate. The organic phases were combined. The solvent was removed under reduced pressure, and the mixture was separated and purified by thin-layer chromatography (DCM:MeOH=8:1) to give compound 12 (10.0 mg). 1 H NMR (400 MHz, DMSO-d6): 12.10 (s, 1H), 7.77 (s, 2H), 7.33 (s, 1H), 2.01 (m, 2H), 1.93-1.75 (m, 4H), 1.39-1.27 (m, 4H). MS m / z (ESI): 448.0 [M+1].

[0098] Embodiment 13 Preparation of 2-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxo)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 13) [ka] 13 In step 1 of embodiment 12, the raw material 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (compound 2) is converted into 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentane[d]pyridazin-4-yl) The synthetic route of Example 12 was carried out, except for substituting 2-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxo)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 13). 1 H NMR (400 MHz, DMSO-d6): 12.02 (s, 1H), 7.77 (s, 2H), 7.35 (s, 1H), 2.94-2.90 (m, 2H), 1.99-1.95 (m, 2H), 1.32 (d, 6H). MS m / z (ESI): 436.0 [M+1].

[0099] Embodiment 14 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-1,2,4-triazine-3,5-(2H,4H)dione (Compound 14) [ka] 14 In step 1 of embodiment 12, the raw material 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (compound 2) is converted into 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile). The synthetic route of Example 12 was carried out, except for substituting 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-1,2,4-triazine-3,5-(2H,4H)dione (Compound 14), which is the title product. 1 H NMR (400 MHz, DMSO-d6): 12.00 (s, 1H), 8.30-8.23 (m, 2H), 8.09-7.99 (m, 2H), 7.18 (s, 1H). MS m / z (ESI): 418.0 [M+1].

[0100] Embodiment 15 Preparation of 2-(3,5-dichloro-4-((5-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 15) [ka] The synthetic route of Example 1 was carried out to produce the title product 2-(3,5-dichloro-4-((5-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 15), except that in step 1 of Example 1, the raw material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced with 3-fluorophthalic anhydride. MS m / z (ESI): 461.0 [M+1].

[0101] Embodiment 16 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 16) [ka]

[0102] Process 1 Preparation of 4-(2,6-dichloro-4-iodophenoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (compound 16a) [ka] Under ice-bath conditions, water (4 ml) and concentrated hydrochloric acid (2 ml) were added sequentially to 1e (100 mg), followed by the dropwise addition of a solution of sodium nitrite (30 mg) in water (2 ml). The reaction was stirred for 1 hour while maintaining the ice-bath conditions. Potassium iodide (104 mg) was added dropwise, and the mixture was allowed to warm to room temperature and stirred for 16 hours. The mixture was extracted with dichloromethane, and the solvent was removed under reduced pressure to give a yellow solid, 16a (100 mg), which was used directly in the next reaction.

[0103] Process 2 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 16) [ka] N,N-dicarbonamide (2.0 ml) was added to a mixture of compound 16a (20 mg), 6-azathymine (6.6 mg), cuprous iodide (8.9 mg), and potassium carbonate (30 mg), and the mixture was heated to 120°C and stirred for 12 hours. The mixture was cooled, filtered, and ethyl acetate was added to separate it from the water. The solvent was removed from the organic phase under reduced pressure, and the mixture was separated and purified by thin-layer chromatography (DCM:MeOH = 8:1) to give compound 16 (5.0 mg). MS m / z (ESI): 436.1 [M+1].

[0104] Embodiment 17 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-6-isopropyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 17) [ka] The synthetic route of Example 16 was carried out to produce the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-6-isopropyl-1,2,4-triazine-3,5(2H,4H)-dione (compound 17), except that in step 2 of Example 16, the starting material 6-azathymine was replaced with 6-isopropyl-1,2,4-triazine-3,5(2H,4H)-dione (prepared by carrying out the known method in "Chemistry and Biodiversity, 2012, 9(3)536-556"). MS m / z (ESI): 464.1 [M+1].

[0105] Embodiment 18 Preparation of 6-cyclopropyl-2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 18) [ka] The synthetic route of Example 16 was carried out to produce the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxo)phenyl)-6-cyclopropyl-1,2,4-triazine-3,5(2H,4H)-dione (compound 18), except that in step 2 of Example 16, the starting material 6-azathymine was replaced with 6-cyclopropyl-1,2,4-triazine-3,5(2H,4H)-dione (prepared by carrying out the known method in "Collection of Czechoslovak Chemical Communications, 1975, 40, 1038-1041"). MS m / z (ESI): 462.1 [M+1].

[0106] Embodiment 19 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 19) [ka]

[0107] Process 1 Preparation of 3,5-dichloro-4-((4-chlorophthalazin-1-yl)oxo)aniline (Compound 19a) [ka] The synthetic route of intermediate 1d in step 3 of embodiment 1 was carried out to prepare the title product 3,5-dichloro-4-((4-chlorophthalazin-1-yl)oxo)aniline (compound 19a), except that 1,4-dichloro-5,6,7,8-tetrahydrophthalazine (compound 1c) in embodiment 1 was replaced with 1,4-dichlorophthalazine.

[0108] Process 2 Preparation of 4-(4-amino-2,6-dichlorophenoxy)phthalazin-1(2H)-one (compound 19b) [ka] The synthetic route for intermediate 1e in step 4 of Example 1 was carried out to prepare the title product 4-(4-amino-2,6-dichlorophenoxy)phthalazin-1(2H)-one (Compound 19b), except that 3,5-dichloro-4-((4-chloro-5,6,7,8-tetrahydrophthalazin-1-yl)oxy)aniline (Compound 1d) in Example 1 was replaced with 3,5-dichloro-4-((4-chlorophthalazin-1-yl)oxo)aniline (Compound 19a).

[0109] Process 3 Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 19) The synthetic route of Example 16 was carried out to produce the title product, 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxo)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 19), except that in step 1 of Example 16, 4-(2,6-dichloro-4-iodophenoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (Compound 16a) was replaced with compound 4-(4-amino-2,6-dichlorophenoxy)phthalazin-1(2H)-one (Compound 19b). MS m / z (ESI): 432.0 [M+1].

[0110] Embodiment 20 Preparation of 2-(3,5-dichloro-4-((6-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 20) [ka] The synthetic route of Example 1 was carried out to produce the title product, 2-(3,5-dichloro-4-((6-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (compound 20), except that in step 1 of Example 1, the starting material tetrahydrophthalic anhydride (1a) was replaced with 4-methylphthalic anhydride. MS m / z (ESI): 457.0 [M+1].

[0111] Embodiment 21 Preparation of 2-(3,5-dichloro-4-((6-chloro-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 21) [ka] The synthetic route of Example 1 was carried out to produce the title product, 2-(3,5-dichloro-4-((6-chloro-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile (Compound 21), except that in step 1 of Example 1, the starting material tetrahydrophthalic anhydride (1a) was replaced with 4-chlorophthalic anhydride. MS m / z (ESI): 477.0 [M+1].

[0112] Test 1: Test of compound binding ability to TRα 1. Main experimental materials and equipment Envision2104 microplate reader; Biotin-SRC2-2 coactivator peptide (commercially available from Sangon Biotech (Shanghai) Co., Ltd.); TRα LBD, GST (commercially available from ThermoFisher) (Art. No. PV4762); Europium-conjugated anti-glutathione antibody (commercially available from Cisbio) (Art. No. 61GSTKLB); and Streptavidin-D2 (commercially available from Cisbio) (Art. No. 610SADAB)

[0113] 2. Compound Preparation and Treatment 2.1 Preparation of dimethyl sulfoxide stock solutions of compounds All compounds were dissolved in dimethyl sulfoxide to prepare 10 mmol stock solutions. 2.2 Compound storage After dissolving the compound in dimethyl sulfoxide, the solution can be stored in a desiccator at room temperature for up to 3 months. For long-term storage, place the compound in a -20°C freezer.

[0114] 3. Experimental Steps 3.1 Preparation of 1x reaction buffer 3.2 Compound screening a) The positive drug triiodothyronine (T3) was diluted from 10 mmol (100x) or the compound to be tested from 1 mmol (100x) in 100% dimethyl sulfoxide in an equal ratio of 1:3 for a total of 10 concentrations. b) A 4x gradient dilution of compounds was prepared in 1x reaction buffer. c) 5 μl of 4× gradient diluted compounds was added to a 384-well test plate. d) 4xTRαLBD and 4xRXRα were prepared in 1x reaction buffer. e) 5 μl of 4xTRαLBD and 4xRXRα were added to a 384-well test plate. f) 2x biotin-SRC2-2, 2x europium-conjugated anti-glutathione antibody, and 2x streptavidin-d2 were prepared in 1x reaction buffer. g) 10 μl of the 2x mixed solution (see step f) was added to a 384-well test plate. h) The 384-well test plate was centrifuged at 1000 rpm in a centrifuge. i) Incubation was carried out for 1 hour at room temperature, protected from light. j) The fluorescence signal values ​​of each well of the 384-well test plate at wavelengths of 665 nm and 615 nm were recorded using an Envision2104 microplate reader, and the 665 nm / 615 nm fluorescence ratio was calculated.

[0115] 4. Data Analysis 4.1 Relative ratio of each well (ratio 665nm / 615nm -ratio ブランク ) calculation 4.2 The activity rate was calculated as follows:

number

[0116] Specific test data is shown in Table 1 below.

[0117] Test 2: Evaluation of compound agonist activity against TRα 1. Main experimental materials and equipment Envision2104 Microplate Reader HEK293T cell line (commercially available from ATCC) (Art. No.: CRL-3216) pGL4.35 [luc2P / 9XGAL4 UAS / Hygro] (commercially available from Promega) (Art. No.: E1370) pBIND-TRα plasmid (from Pharmaron) pBIND-RXRα plasmid (from Pharmaron) LipoLTX transfection agent (commercially available from ThermoFisher) (Art. No.: 15338-100)

[0118] 2. Compound Preparation 2.1 Dissolution of compounds Compound powders were prepared into 10 mmol dimethyl sulfoxide stock solutions according to standard schemes. 2.2 Compound storage All compounds dissolved in dimethyl sulfoxide were stored in a desiccator at room temperature for short periods or at −20°C for long periods. 2.3 Preparation of experimental compounds 2.3.1 All compounds to be tested were diluted in dimethyl sulfoxide in a 3-fold gradient of 10 dilutions, starting from an initial concentration of 10 μmol. 2.3.2 The positive control, triiodothyronine (T3), was diluted in dimethyl sulfoxide over a 3-fold gradient of 10 dilutions, starting from an initial concentration of 16.67 μmol. 2.3.3 Prepare a 166.7x positive control (16.67 μmol triiodothyronine (T3)) and a 166.7x negative control (100% dimethyl sulfoxide). 2.4 The compound plate was closed and shaken for 5 minutes.

[0119] 3 Experimental process 3.1 Preparation of cell suspension and plating a) All cells were cultured according to ATCC standard procedures, and HEK293T were tested in exponential growth phase. b) The medium was discarded. c) Cells were washed twice with phosphate buffer. d) Trypsinization solution was added to treat the cells and the treatment was stopped with complete medium. e) Cells were collected and counted, and experiments could only be performed if cell viability was greater than 90%. f) 2.5 x 10 6 HEK293-LUC cells were seeded onto a 60 mm cell culture dish. g) The culture dish on which the cells were seeded was placed in an incubator at 37°C and 5% CO2, and cultured overnight. 3.2 Cell transfection a) LipoLTX transfection agent was placed at room temperature for equilibration. b) 6 μl of Plus reagent and 6 μg of DNA were added to 250 μl of Opti-MEM™ medium without contacting the tube wall, and mixed uniformly by blowing with a pipetting gun and pipetting. Plasmids: 2.5 μg of pBIND-TRα, 2.5 μg of pBIND-RXRα, and 1 μg of pGL4.35 plasmid were added, respectively. c) 12 μl of LipoLTX and 250 μl of Opti-MEM™ medium were added without contacting the walls of the tube, and mixed evenly by blowing with a pipetting gun and pipetting. d) The agent mixed with DNA Plus (see step 3.2.b) was added to the diluted LipoLTX (see step 3.2.c) transfection agent and left at room temperature for 15 minutes. e) The transfection agent mixed with DNA was added to a 60 mm cell culture dish (see step 3.1). f) The culture dish was placed in an incubator at 37°C and 5% CO2, and cultured for 5 hours. 3.3 Compound Treatment a) 150 nl of diluted compound (see step 2.3) was transferred to a cell culture plate (6007680-50, PE) by Echo550. b) Cells (see step 3.2) were seeded into 384 cell culture plates (6007680-50, PE) at 15,000 cells per well in 25 μl of medium (containing 5% fetal bovine serum). c) The cells were cultured overnight in an incubator at 37°C and 5% CO2. 3.4 Compound detection a) Steady-Glo™ detection agent was placed at room temperature. b) The 384 cell plate (see step 3.3) was placed at room temperature. c) 25 μl per well of Steady-Glo™ detection agent was added to the cell culture plate (see step 3.4b). d) The plate was placed on a shaker and shaken for 5 minutes while protected from light. e) Chemiluminescence values ​​were detected using an Envision2104 microplate reader.

[0120] 4. Data Analysis 4.1 Calculation of activity (%)

number

[0121] Specific test data is shown in Table 1 below.

[0122] Test 3: Test of compound binding ability to TRβ 1. Main experimental materials and equipment Envision2104 microplate reader; Biotin-SRC2-2 coactivator peptide (commercially available from Sangon Biotech (Shanghai) Co., Ltd.) TRβ LBD, GST (commercially available from ThermoFisher) (Art. No. PV4762) Europium-conjugated anti-glutathione antibody (commercially available from Cisbio) (Art. No. 61GSTKLB) Streptavidin-D2 (commercially available from Cisbio) (Art. No. 610SADAB)

[0123] 2. Compound Preparation and Treatment 2.1 Preparation of dimethyl sulfoxide stock solutions of compounds All compounds were dissolved in dimethyl sulfoxide to prepare 10 mmol stock solutions. 2.2 Compound storage After dissolving the compound in dimethyl sulfoxide, the solution can be stored in a desiccator at room temperature for up to 3 months. For long-term storage, place the compound in a -20°C freezer.

[0124] 3. Experimental Steps 3.1 Preparation of 1x reaction buffer 3.2 Compound screening a) The positive drug triiodothyronine (T3) was diluted from 10 μmol (100x) or the compound to be tested from 1 mmol (100x) in 100% dimethyl sulfoxide in an equal ratio of 1:3 for a total of 10 concentrations. b) A 4x gradient dilution of compounds was prepared in 1x reaction buffer. c) 5 μl of 4× gradient diluted compounds was added to a 384-well test plate. d) 4xTRβLBD and 4xRXRβ were prepared in 1x reaction buffer. e) 5 μl of 4xTRβLBD and 4xRXRβ were added to a 384-well test plate. f) 2x biotin-SRC2-2, 2x europium-conjugated anti-glutathione antibody, and 2x streptavidin-d2 were prepared in 1x reaction buffer. g) 10 μl of the 2x mixed solution (see step f) was added to a 384-well test plate. h) The 384-well test plate was centrifuged at 1000 rpm in a centrifuge. i) Incubation was carried out for 1 hour at room temperature, protected from light. j) The fluorescence signal values ​​of each well of the 384-well test plate at wavelengths of 665 nm and 615 nm were recorded using an Envision2104 microplate reader, and the 665 nm / 615 nm fluorescence ratio was calculated.

[0125] 4. Data Analysis 4.1 Relative ratio of each well (ratio 665nm / 615nm -ratio ブランク ) calculation 4.2 The activity rate was calculated as follows:

number

[0126] Specific test data is shown in Table 1 below.

[0127] Table 1 The binding activity of compounds to thyroxine receptor β is shown below. [Table 1]

[0128] conclusion Compared to the disclosed comparative compound 53, several compounds according to the present invention exhibited unexpectedly high THRβ activity (<0.2 μM), and several compounds exhibited higher selectivity for THRα than comparative compound 53.

[0129] Test 4: Evaluation of the agonist activity of compounds at TRβ receptors Experimental Summary The TRβ-LBD and RXRα-LBD coding sequences were inserted into the pBIND plasmid (Promega, E1581), respectively. The expression vector and reporter vector (pGL4.35, carrying a luciferase reporter gene driven by a stable integrated GAL4 promoter) were coexpressed in host cells. When an agonist binds to the corresponding chimeric receptor, the chimeric receptor binds to the GAL4 binding site of the reporter gene vector, stimulating reporter gene expression. The agonist activity of the compound against the TRβ receptor was determined according to the intensity of the chemiluminescent signal.

[0130] Experimental materials and equipment Envision2104 Microplate Reader HEK293T cell line (commercially available from ATCC) (Art. No.: CRL-3216) pGL4.35 [luc2P / 9XGAL4 UAS / Hygro] (commercially available from Promega (Art. No.: E1370) pBIND-TRβ plasmid (from Pharmaron) pBIND-RXRα plasmid (from Pharmaron) LipoLTX transfection agent (commercially available from ThermoFisher) (Art. No.: 15338-100) Steady-Glo™ Luciferase Detection Kit (commercially available from Promega) (Art. No.: E2520)

[0131] 2.3 Preparation of experimental compounds All compounds to be tested were diluted in dimethyl sulfoxide in a 3-fold gradient over 10 dilutions, starting from an initial concentration of 10 mmol. The positive control, triiodothyronine (T3), was diluted in dimethyl sulfoxide in a 3-fold gradient over 10 dilutions, starting from an initial concentration of 16.67 μmol. A 166.7x positive control (16.67 μmol, triiodothyronine (T3)) and a 166.7x negative control (100% dimethyl sulfoxide) were prepared. The compound plate was closed and shaken for 5 minutes.

[0132] Experimental Process 3.1 Preparation of cell suspension and plating a) All cells were cultured according to ATCC standard procedures, and HEK293T were tested in exponential growth phase. b) The medium was discarded. c) Cells were washed twice with phosphate buffer. d) Trypsinization solution was added to treat the cells and the treatment was stopped with complete medium. e) Cells were collected and counted, and experiments could only be performed if cell viability was greater than 90%. f) 2.5 x 10 6 HEK293-LUC cells were seeded onto a 60 mm cell culture dish. g) The culture dish on which the cells were seeded was placed in an incubator at 37°C and 5% CO2, and cultured overnight. 3.2 Cell transfection a) LipoLTX transfection agent was placed at room temperature for equilibration. b) 6 μl of Plus reagent and 6 μg of DNA were added to 250 μl of Opti-MEM™ medium without contacting the tube wall, and mixed evenly by blowing with a pipetting gun and pipetting. Plasmids: 2.5 μg of pBIND-TRβ, 2.5 μg of pBIND-RXRα, and 1 μg of pGL4.35 plasmid c) 12 μl of LipoLTX and 250 μl of Opti-MEM™ medium were added without contacting the walls of the tube, and mixed evenly by blowing with a pipetting gun and pipetting. d) The agent mixed with DNA plus (see step 3.2.b) was added to the diluted LipoLTX (see step 3.2.c) transfection agent and left to stand at room temperature for 15 min. e) The transfection agent mixed with DNA was added to a 60 mm cell culture dish (see step 3.1). f) The culture dish was placed in an incubator at 37°C and 5% CO2, and cultured for 5 hours. 3.3 Compound Treatment a) 150 nl of diluted compound (see step 2.3) was transferred to a cell culture plate (6007680-50, PE) by Echo550. b) Cells (see step 3.2) were seeded into 384 cell culture plates (6007680-50, PE) at 15,000 cells per well in 25 μl of medium. c) The cells were cultured overnight in an incubator at 37°C and 5% CO2. 3.4 Compound detection a) Steady-Glo™ detection agent was placed at room temperature. b) The 384 cell plate (see step 3.3) was placed at room temperature. c) 25 μl per well of Steady-Glo™ detection agent was added to the cell culture plate (see step 3.4b). d) The plate was placed on a shaker and shaken for 5 minutes while protected from light. e) Chemiluminescence values ​​were detected using an Envision2104 microplate reader.

[0133] Data analysis 4.1 Calculation of activity (%)

number

[0134] Table 2: Agonist activity of compounds of the present invention on thyroxine receptor beta is shown below. [Table 2]

[0135] conclusion The compounds of the present invention can activate downstream signals of thyroid hormone receptor beta.

[0136] Study 5: Pharmacokinetic evaluation Rats were used as test animals. After intragastric injection of the compounds of Example 4 and Example 9, the drug concentrations in the rats' plasma were measured at different times. The pharmacokinetic behavior of the compounds of the present invention in rats was tested and their drug metabolism characteristics were evaluated. Three male SD rats with similar body weights were selected for each group of the present invention, and a single oral dose of 2 mg / kg was administered. After the animals were administered the dose, blood samples were collected at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, and 24 hours. The content of the compound in plasma was detected using LC-MS / MS analysis, and the lower limit of quantification for this method was 20 ng / ml. Statistics for the VT088 and plasma concentration data were generated using metakinetic data analysis software WinNonlin 7.0. Pharmacokinetic parameters were calculated using non-compartmental modeling (NCA), particularly as shown in Table 2 below.

[0137] Experimental scheme Experimental drug: the compound of embodiment 4 and embodiment 9. experimental animals Six healthy male SD rats, commercially available from Shanghai Sippr-Bk Laboratory Animal Co., Ltd. with Animal Production License Number: SCXK (Shanghai) 2008-0016, were divided into two groups, with three rats in each group. Drug preparation A certain amount of drug was taken and added to a 2% Klucel LF+0.1% Tween 80 aqueous solution to prepare a clear solution or a uniform suspension. dosage SD rats were fasted overnight and given drugs by intragastric infusion at a dose of 2 mg / kg and a dose volume of 10 mL / kg, respectively. method The compounds of embodiment 4 and embodiment 9 are administered to rats by intragastric injection.At least 0.2mL of blood is collected from the tail vein before and after administration at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 10 hours and 24 hours.The blood is then placed into a heparinized sample tube and centrifuged at 4 ℃, 3500 rpm for 10 minutes to separate plasma.The heparinized sample tube is then stored at -20 ℃, and the rats are allowed to eat 2 hours after administration. Determination of the content of the tested compounds in the plasma of rats after intragastric infusion of different concentrations of the drug The plasma samples were thawed at room temperature, and 50 μL of each was taken and added to 130 μL of an internal standard solution (1000 ng / mL, acetonitrile, tolbutamide). The mixture was stirred for approximately 1 minute and then centrifuged at 13,000 rpm for 10 minutes at 4°C. 50 μL of the supernatant was taken and mixed with 100 μL of 50% acetonitrile water, followed by LC / MS / MS analysis.

[0138] Table 3 shows the results of the pharmacokinetic parameters. Table 3: Drug metabolism data in rats [Table 3]

[0139] conclusion The compounds of the present invention have good pharmacokinetic absorption and significant pharmacokinetic advantages. Compared with the reported comparative compound 53, some of the compounds of the present invention show unexpectedly high Cmax values ​​and exposure levels at the same dose and formulation. All of the above PK results indicate that the compounds provided in the present invention have good PK properties and can be used as therapeutic agents for metabolic diseases.

Claims

1. The following formula (I): 【Chemistry 1】 (I) [In the formula, R １ is hydrogen, cyano, substituted or unsubstituted C １－６ Alkyl or substituted or unsubstituted C ３－６ cycloalkyl, the substituents being selected from the group consisting of halogen atoms, hydroxy, and C １－６ selected from the group consisting of alkoxy; R ２ and R ３ are each independently a halogen atom or a substituted or unsubstituted C １－６ alkyl, the substituents being selected from the group consisting of halogen atoms, hydroxy, and C １－６ selected from the group consisting of alkoxy; Ring A is a substituted or unsubstituted saturated or unsaturated C ５－１０ an aliphatic ring or a substituted or unsubstituted C ５－１０ The aromatic ring is a ring having a substituent selected from the group consisting of hydrogen, halogen, hydroxy, and -OCF ３ , -NH ２ , -NHC １－４ Alkyl, —N(C １－４ alkyl) ２ , -CONH ２ , -CONHC １－４ Alkyl, -CON(C １－４ alkyl) ２ , -NHCOC １－４ Alkyl, C １－６ Alkyl, C １－６ Alkoxy or C ３－６ one or more groups selected from the group consisting of cycloalkyl, and when two substituents are included, the two substituents together with the carbons to which they are attached may form a ring structure; and The halogen atom is selected from the group consisting of F, Cl, or Br. and pharmaceutically acceptable salts thereof.

2. The following formula (II): 【Chemistry 2】 (II) [In the formula, R １ From R ３ is as defined in claim 1; L is absent or -CH ２ - and -CH ２ CH ２ - selected from the group consisting of; R ４ represents hydrogen, halogen atoms, hydroxy, -OCF ３ , -NH ２ , -NHC １－４ Alkyl, —N(C １－４ alkyl) ２ , C １－６ Alkyl, C １－６ Alkoxy, or C ３－６ cycloalkyl; n is an integer ranging from 1 to 4; m is an integer ranging from 1 to 4; When L is absent, the ring may contain two or more substituents R ４ and The halogen atom is selected from the group consisting of F, Cl, or Br.

2. The compound of claim 1, which is a compound represented by the structure: and a pharmaceutically acceptable salt thereof.

3. R ４ is hydrogen, halogen atom, hydroxy, C １－３ Alkyl, C １－３ Alkoxy, or C ３－６ cycloalkyl; L is absent or -CH ２ - or -CH ２ CH ２ - selected from the group consisting of; n is 1, 2, or 3; and m is 1 or 2; 3. The compound of claim 2, and pharmaceutically acceptable salts thereof.

4. R ４ is hydrogen, or C １－３ selected from the group consisting of alkyl; L is absent; n is 1, 2, or 3; and m is 1 or 2; 4. The compound of claim 3, and pharmaceutically acceptable salts thereof.

5. R ４ is hydrogen, or C １－３ selected from the group consisting of alkyl; L is -CH ２ - or -CH ２ CH ２ - selected from the group consisting of; n is 1, 2, or 3; and m is 1 or 2; 4. The compound of claim 3, and pharmaceutically acceptable salts thereof.

6. The following formula (III): 【Transformation 3】 [In the formula, R １ From R ３ is as defined in claim 1; R ４ represents hydrogen, halogen atoms, hydroxy, -OCF ３ , -NH ２ , -NHC １－４ Alkyl, —N(C １－４ alkyl) ２ , -CONH ２ , -CONHC １－４ Alkyl, -CON(C １－４ alkyl) ２ , -NHCOC １－４ Alkyl, C １－６ Alkyl, C １－６ Alkoxy, or C ３－６ cycloalkyl; m is an integer ranging from 1 to 4; and The halogen atom is selected from the group consisting of F, Cl, or Br.

2. The compound of claim 1, which is a compound represented by the structure: and a pharmaceutically acceptable salt thereof.

7. R ４ is hydrogen, halogen atom, hydroxy, -OCF ３ , C １－６ Alkyl, C １－６ Alkoxy, or C ３－６ cycloalkyl; and m is an integer ranging from 1 to 3; 7. The compound of claim 6, and pharmaceutically acceptable salts thereof.

8. R ４ is a hydrogen atom, a halogen atom, or C １－３ and m is 1 or 2, and pharmaceutically acceptable salts thereof.

9. R １ is hydrogen, cyano, and substituted or unsubstituted C １－６ alkyl, the substituents being selected from the group consisting of halogen atoms, hydroxy, and C １－６ alkoxy; and The halogen atom is selected from the group consisting of F, Cl, or Br; The compound according to any one of claims 1 to 8, and a pharmaceutically acceptable salt thereof.

10. R １ But cyano, or C １－３ 10. The compound of claim 9, wherein the compound is selected from the group consisting of alkyl, and pharmaceutically acceptable salts thereof.

11. R １ 11. The compound of claim 10, and pharmaceutically acceptable salts thereof, wherein is cyano.

12. R ２ and R ３ are each independently selected from the group consisting of F, Cl, or Br, and pharmaceutically acceptable salts thereof.

13. R ２ and R ３ and pharmaceutically acceptable salts thereof.

14. The compound and its pharmaceutically acceptable salts are the following compounds: 【Chemistry 4】 14. The compound according to any one of claims 1 to 13, which is any one of:

15. Use of the compound according to any one of claims 1 to 14 and its pharmaceutically acceptable salts in the manufacture of a medicament for treating a metabolic-related disease.

16. Use of the compound of claim 15 and its pharmaceutically acceptable salts in the manufacture of a medicament for treating a metabolic-related disease, wherein the metabolic-related disease is selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, diabetes, nonalcoholic steatohepatitis (NASH), fatty liver, atherosclerosis, hypothyroidism, and thyroid cancer.

17. 17. Use of the compound of claim 16 and its pharmaceutically acceptable salts in the manufacture of a medicament for treating a metabolic-related disease, wherein the metabolic-related disease is selected from the group consisting of nonalcoholic steatohepatitis (NASH), hypothyroidism, and thyroid cancer.

18. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 14 and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

19. A method for treating a metabolic-related disease, comprising administering to a subject an effective amount of the compound according to any one of claims 1 to 14, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

20. 20. The method for treating a metabolic-related disease according to claim 19, wherein the metabolic-related disease is selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, diabetes, nonalcoholic steatohepatitis (NASH), fatty liver, atherosclerosis, hypothyroidism, and thyroid cancer.

21. 21. The method of claim 20, wherein the metabolic-related disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), hypothyroidism, and thyroid cancer.

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