THRβ receptor agonist compound and preparation method and use thereof

Abstract

The present application relates to a THRβ receptor agonist compound and its preparation method and use. The present invention discloses a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof. The compound improves the selectivity for THRα while maintaining good THRβ agonist activity, thereby improving the drug properties.

Description

[0001] This application is a divisional application of an invention patent application with an application date of December 12, 2019, application number 201980081797.3 and invention name “THRβ receptor agonist compounds, preparation methods and uses thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] 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. Technical Field

[0004] The present invention relates to the field of drug synthesis, and in particular to a compound that can be used as a novel agonist of THRβ receptor, and a preparation method and use thereof. Background Art

[0005] Thyroid hormone (TH) is synthesized in the thyroid gland in response to thyroid stimulating hormone (TSH) secreted by the pituitary gland. Thyroid hormone plays a very important role in regulating body growth, development, metabolism, and matrix balance. There are two main types of thyroid hormones, 3,5,3`-triiodo-L-thyroxine (T3) and thyroxine (T4). The human body mainly secretes T4. In peripheral organs, T4 is converted into more active T3 by deiodinase. T3 and T4 produced by the thyroid gland are under negative feedback control. Thyroid stimulating hormone (TSH) is responsible for normal thyroid function and thyroid hormone secretion. TSH is synthesized in the anterior lobe of the pituitary gland, and its secretion is controlled by thyroid releasing hormone (TRH) synthesized in the hypothalamus.

[0006] Thyroid hormones exert their functions by binding to thyroid hormone receptors (THRs). THRs belong to the large family of nuclear receptors and regulate the expression of target genes. There are two different subtypes of thyroid hormone receptors, THRα and THRβ. THRα is mainly distributed in heart tissue and plays an important regulatory role in heart function. The THRβ subtype is mainly expressed in the liver and pituitary gland, regulating cholesterol metabolism and regulating thyroid-stimulating hormone secretion.

[0007] At normal levels, thyroid hormones THs maintain body weight, metabolic rate, body temperature, mood and regulate serum cholesterol. People have tried to use thyroid hormones to regulate serum cholesterol. However, the possible side effects of taking natural thyroid hormones on the heart (such as tachycardia and arrhythmia, heart failure, and causing thyroid axis function, muscle metabolism and osteoporosis) make it unsuitable for the treatment of high cholesterol and obesity. Animal studies with selective knockout of the THR gene and the results of some studies on selective THR ligands have shown that these cardiac side effects caused by thyroid hormones can be attributed to THRα.

[0008] The thyroid hormone receptor pathway regulates lipid metabolism, including cholesterol, triglycerides, and lipoproteins. Clinically, it has been shown that lowering low-density cholesterol can reduce the incidence of cardiovascular and cerebrovascular diseases.

[0009] Non-alcoholic fatty liver disease (NAFLD) is also a type of metabolic disorder caused by excessive accumulation of triglycerides in the liver, which can further cause liver cell damage and inflammation, leading to non-alcoholic steatohepatitis (NASH). In addition to NASH, NASH patients are usually also accompanied by type 2 diabetes, high cholesterol, high blood lipids and obesity. NASH patients also have a higher probability of developing cirrhosis, liver failure, and eventually liver cancer. In this field, there is currently a lack of effective drugs for the treatment of NASH. The function of thyroid hormones in regulating lipid metabolism makes the thyroid receptor pathway a potential target for the treatment of NASH and NAFLD. It has been confirmed in animals that thyroid hormone analogs can significantly reduce the fat content of animal livers.

[0010] Selective THRβ agonists can be used to circumvent the cardiac side effects caused by conventional THR receptor agonists, and selectively activate only THRβ, improve cellular lipid metabolism, and play a role in lowering cholesterol and blood lipids. However, selective THRβ agonists may also inhibit the thyroid axis, leading to side effects such as depression, fatigue, and osteoporosis. Therefore, it is necessary to develop a selective THRβ agonist that activates THRβ but reduces the inhibitory effect on the thyroid axis, thereby circumventing the side effects associated with thyroid axis inhibition.

[0011] Patents such as WO03094845, WO2007009913, WO2010122980 and WO2011038207 disclose some THR receptor agonists. These agonist structures are almost all designed and developed based on the natural ligand T3 of THR receptor. Based on these backgrounds, there is still a need to develop selective THRβ receptor agonists that have the beneficial effects of thyroid hormones but avoid adverse cardiac side effects.

[0012]

[0013] The present invention is also based on the structural modification of the natural ligand T3 of the THR receptor. The inventor unexpectedly found that most of the modified compounds maintained good THRβ receptor agonist activity, and compared with the reference ("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 ReceptorβAgonist in Clinical Trials for the Treatment of Dyslipidemia" Martha et al., Journal of medicinal chemistry, 2014, 3912-3923) in the reference compound 53, some compounds also improved the selectivity for THRα. At the same time, some compounds of the present invention also show very good pharmacokinetic properties.

[0014] Some preferred compounds have significantly better pharmacokinetic properties than comparative compounds, thereby improving drugability.

[0015] Summary of the invention

[0016] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0017] According to one aspect of the present invention, the present invention provides a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof:

[0018]

[0019] in,

[0020] R 1Selected from hydrogen, cyano, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6 The group consisting of cycloalkyl, wherein the substituent is selected from halogen atoms, hydroxyl groups and C 1-6 A group consisting of alkoxy groups;

[0021] R 2 and R 3 are each independently selected from a halogen atom or a substituted or unsubstituted C 1-6 The substituent is selected from the group consisting of halogen atoms, hydroxyl groups and C 1-6 A group consisting of alkoxy groups;

[0022] Ring A is a substituted or unsubstituted saturated or unsaturated C 5-10 Aliphatic ring or substituted or unsubstituted C 5-10 Aromatic ring, the substituent is selected from hydrogen, halogen atoms, hydroxyl, -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 species of the group consisting of cycloalkyl, when containing two substituents, the two substituents can form a ring structure with the carbon to which they are attached; and

[0023] The halogen atom is selected from the group consisting of F, Cl or Br.

[0024] The compound according to the present invention has a structure shown in the following formula (II):

[0025]

[0026] in,

[0027] R 1 To R 3 Definitions are as described above in formula (I);

[0028] L is absent or selected from -CH 2 -and-CH 2 CH 2 - Groups formed;

[0029] R 4Selected from hydrogen, halogen atoms, hydroxyl groups, -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 The group consisting of cycloalkyl groups;

[0030] n is an integer from 1 to 4;

[0031] m is an integer from 1 to 4; and

[0032] When L is absent, the ring may have two or more substituents R 4 ;and

[0033] The halogen atom is selected from the group consisting of F, Cl or Br.

[0034] Preferably, in the structure shown in formula (II), R 4 Selected from hydrogen, halogen atoms, hydroxyl groups, C 1-3 Alkyl, C 1-3 Alkoxy or C 3-6 The group consisting of cycloalkyl groups;

[0035] L is absent or selected from -CH 2 -or-CH 2 CH 2 - Groups formed;

[0036] n is 1, 2 or 3; and

[0037] m is 1 or 2.

[0038] More preferably, in the structure shown in formula (II), R 4 Selected from hydrogen or C 1-3 The group consisting of alkyl groups;

[0039] L is -CH 2 -or-CH 2 CH 2 -;

[0040] n is 1, 2 or 3; and

[0041] m is 1 or 2.

[0042] More preferably, in the structure shown in formula (II), R 4 Selected from hydrogen or C 1-3 The group consisting of alkyl groups;

[0043] L does not exist;

[0044] n is 1, 2 or 3; and

[0045] m is 1 or 2.

[0046] The compound according to the present invention has a structure shown in the following formula (III):

[0047]

[0048] in,

[0049] R 1 To R 3 Definitions are as described above in formula (I);

[0050] R 4 Selected from hydrogen, halogen atoms, hydroxyl groups, -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 The group consisting of cycloalkyl groups;

[0051] m is an integer from 1 to 4; and

[0052] The halogen atom is selected from the group consisting of F, Cl or Br.

[0053] Preferably, in the structure shown in formula (III), R 4 Selected from hydrogen, halogen atoms, hydroxyl groups, -OCF 3 , C 1-6 Alkyl, C 1-6 Alkoxy or C 3-6 The group consisting of cycloalkyl; m is an integer from 1 to 3.

[0054] Preferably, in the structure shown in formula (III), R 4 Selected from the group consisting of hydrogen, halogen atoms or C 1-3 Alkyl; m is 1 or 2.

[0055] Preferably, in the above-mentioned compound having a structure as described in formula (I), (II) or (III) according to the present invention, R 1 Selected from hydrogen, cyano, substituted or unsubstituted C 1-6The group consisting of alkyl; further preferably, R 1 Selected from cyano or C 1-3 The group consisting of alkyl; More preferably, R 1 It's cyano.

[0056] Preferably, in the above-mentioned compound having a structure as described in formula (I), (II) or (III) according to the present invention, R 2 and R 3 Each is independently selected from the group consisting of F, Cl or Br, and more preferably, R 2 and R 3 They are all Cl.

[0057] Preferably, the compound according to the invention and its pharmaceutically acceptable salt is one of the following compounds:

[0058]

[0059] According to another aspect of the present invention, the present invention provides a method for preparing the compound, the preparation method comprising the following steps:

[0060]

[0061] 1) reacting an internal anhydride compound Ia with hydrazine hydrochloride to obtain a compound of formula Ib;

[0062] 2) heating the obtained compound of formula Ib in phosphorus oxychloride to generate a compound of formula Ic;

[0063] 3) The obtained compound of formula Ic is subjected to a high temperature coupling reaction with compound Id to obtain a compound of formula Ie, wherein the catalyst under such conditions is preferably cuprous iodide;

[0064] 4) reacting the obtained compound of formula Ie under acidic or alkaline conditions at high temperature to obtain a compound of formula If;

[0065] 5) The obtained compound of formula If is reacted with sodium nitrite in an acidic aqueous solution, and then compound Ig is added for further reaction, and the ring is closed at high temperature to obtain a compound of formula I. The acid under this condition is preferably hydrochloric acid.

[0066] The compound of general formula I of the present invention can also be prepared as follows:

[0067]

[0068] The compound of general formula IB-1 is hydrolyzed at high temperature in an acidic aqueous solution to obtain a compound of general formula IB-2, wherein the acid under such conditions is preferably hydrochloric acid; the obtained compound of general formula IB-2 is reacted at high temperature in the presence of thioglycolic acid to eliminate the carboxyl group to obtain a compound of general formula I.

[0069] The compound of general formula I of the present invention can also be prepared by the following method:

[0070]

[0071] The compound of the general formula If reacts with sodium nitrite under acidic conditions to generate a diazonium salt compound, and then a halide anion is added to obtain a compound of the general formula Ig; the obtained compound of the general formula Ig is coupled with the intermediate Ih under transition metal catalysis to obtain a compound of the general formula I.

[0072] According to another aspect of the present invention, the present invention provides use of the compound in preparing a drug for treating metabolism-related diseases.

[0073] According to another aspect of the present invention, the present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of the compound according to the present invention and a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient.

[0074] Preferably, the metabolism-related disease is selected from the group consisting of: obesity, hyperlipidemia, hypercholesterolemia, diabetes, and non-alcoholic fatty liver disease (NASH), hepatic steatosis, atherosclerosis, hypothyroidism and thyroid cancer; preferably, the metabolism-related disease is selected from the group consisting of: non-alcoholic fatty liver disease (NASH), hypothyroidism and thyroid cancer.

[0075] According to another aspect of the present invention, the present invention provides a method for treating metabolic-related diseases, the method comprising administering to a subject an effective amount of the compound according to the present invention or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable salt thereof as an active ingredient.

[0076] Preferably, according to the method for treating metabolism-related diseases, the metabolism-related diseases are selected from the group consisting of: obesity, hyperlipidemia, hypercholesterolemia, diabetes, and non-alcoholic fatty liver disease (NASH), hepatic steatosis, atherosclerosis, hypothyroidism and thyroid cancer; preferably, the metabolism-related diseases are selected from the group consisting of: non-alcoholic fatty liver disease (NASH), hypothyroidism and thyroid cancer. DETAILED DESCRIPTION

[0077] Below, the present invention will be described in detail. Before describing, it should be understood that the terms used in this specification and the attached claims should not be interpreted as being limited to the general meaning and dictionary meaning, but should be interpreted according to the meaning and concept corresponding to the technical aspects of the present invention on the basis of the principle that the inventor is allowed to appropriately define the terms for the best interpretation. Therefore, the descriptions presented here are only preferred examples for illustrative purposes and are not intended to limit the scope of the present invention. It should be understood that other equivalents or improvements can be obtained therefrom without departing from the spirit and scope of the present invention.

[0078] According to the present invention, if not otherwise specified, all terms cited herein have the same meanings as those understood by those skilled in the art.

[0079] As used herein, the term "salt" refers to a compound containing a cation and an anion, which can be generated by protonation of an acceptable proton site and / or deprotonation of a proton-donating site. It is worth noting that protonation of an acceptable proton site results in the formation of a cationic species, the charge of which is balanced by the presence of a physiological anion, while deprotonation of a proton-donating site results in the formation of an anionic species, the charge of which is balanced by the presence of a physiological cation.

[0080] The term "pharmaceutically acceptable salt" means that the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, 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, etc.; or with 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-p-toluenesulfonic acid, camphoric acid, dodecylsulfuric acid, gluconic acid, glutamic acid, salicylic acid and cis-hexanedioic acid, etc.; or (2) base addition salts, formed with the conjugate base of any of the above inorganic acids, wherein the conjugate base comprises a compound selected from the group consisting of Na + , K + Mg 2+ , Ca 2+ and NH x R 4-x + The cationic components in the group consisting of NH x R 4-x + (R is C 1-4alkyl, the subscript x is an integer selected from the group consisting of 0, 1, 2, 3 or 4) represents the cation in the quaternary ammonium salt. It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein of the same acid addition salt.

[0081] The term "C 1-M The term "alkyl" refers to an alkyl group containing 1 to M carbon atoms, for example, wherein M is an integer having the following 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, the term "C 1-6 "Alkyl" refers to an alkyl group containing 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, lower alkyl groups including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl and octyl.

[0082] The term "aromatic" refers to an aromatic system, which may be a single ring or multiple aromatic rings that are originally fused or linked together so that at least a portion of the fused or linked rings form a conjugated aromatic system. Aryl groups include, but are not limited to, phenyl, naphthyl, and tetrahydronaphthyl. Aryl groups may be optionally substituted, such as aryl or heterocyclic groups substituted with 1-4 groups selected from the group consisting of halogen, -CN, -OH, -NO 2 , amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, substituted alkoxy, alkylcarbonyl, alkylcarboxyl, alkylamino or arylthio.

[0083] The term "substituted" means that the referenced group may be substituted with one or more additional groups, wherein the additional groups are individually and independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, carbonyl, thiocarbonyl, nitro, haloalkyl, fluoroalkyl and amino, including mono- and di-substituted amino groups and protected derivatives thereof.

[0084] The compound represented by formula (I) or its pharmaceutically acceptable salt provided by the present invention, and the pharmaceutical composition comprising the compound can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in a suitable solid or liquid carrier or diluent and in a suitable sterile device for injection or instillation.

[0085] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. For example, the unit dose of the formulation contains 0.05-200 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof, preferably, the unit dose of the formulation contains 0.1 mg-100 mg of the compound of formula (I).

[0086] The compound or pharmaceutical composition represented by the general formula (I) provided by the present invention can be used clinically in mammals, including humans and animals, and can be administered by oral, nasal, skin, lung, or gastrointestinal routes. Oral administration is most preferred. The best preferred daily dose is 0.01-200 mg / kg body weight, taken at one time, or 0.01-100 mg / kg body weight taken in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Usually, it is started with a small dose and gradually increased until the most suitable dose is found.

[0087] In the present invention, the term "effective amount" may refer to an effective amount of the dosage and time period required to achieve the desired effect. This effective dose may vary due to certain factors, such as the type of disease, the condition of the disease during treatment, the structure of the specific target organ to be administered, the height and weight of the individual patient, or the severity of the disease or symptoms. A person with ordinary knowledge in the art can determine the effective amount of a specific compound empirically without undue experimentation.

[0088] A typical formulation is prepared by mixing the compound represented by the general formula (I) of the present invention and 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.

[0089] The specific carrier, diluent or excipient used will be determined according to the mode of use and purpose of the compound of the present invention. Solvents are generally selected based on solvents that are considered safe and effective for administration to mammals by those skilled in the art. Generally speaking, safe solvents are non-toxic aqueous solvents such as water, and other non-toxic solvents that are soluble in water or miscible with water. Suitable aqueous solvents include one or more of water, ethanol, propylene glycol, polyethylene glycol (such as PEG400 and PEG300), etc. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents, flavoring agents or other known additives to make the drug in an acceptable form for manufacture or use.

[0090] When the compound of formula (I) of the present invention is used in combination with at least one other drug, the two drugs or multiple 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 the subject separately or together in any known oral, intravenous, rectal, vaginal, transdermal, or other local or systemic administration forms.

[0091] These pharmaceutical compositions may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents, flavoring agents or other known additives so that the pharmaceutical compositions can be manufactured or used in an acceptable form.

[0092] The drug of the present invention is preferably administered orally. Solid dosage forms for oral administration may include capsules, tablets, powders or granular preparations. In the solid dosage form, 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, silicic acid, etc.; binders such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic, etc.; wetting agents such as glycerol, etc.; disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, specific complex silicates and sodium carbonate, etc.; solution retardants such as paraffin, etc.; absorption promoters such as quaternary ammonium compounds, etc.; adsorbents such as kaolin and bentonite, etc.; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol and sodium lauryl sulfate, etc. In the case of capsules and tablets, the dosage form may also include a buffer. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or other excipients such as high molecular weight polyethylene glycols.

[0093] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the compound of the present invention or its pharmaceutical composition, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents; solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide; oils (such as cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil); glycerol; tetrahydrofurfuryl alcohol; fatty acid esters of polyethylene glycol and dehydrated sorbitan; or a mixture of several of these substances, etc.

[0094] In addition to these inert diluents, the composition may also include excipients such as one or more of wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, and perfuming agents.

[0095] As for the suspension, in addition to the compound represented by general formula (I) of the present invention or its pharmaceutically acceptable salt or a pharmaceutical composition comprising the same, the suspension may further contain a carrier such as a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, or a mixture of several of these substances.

[0096] The compound represented by the general formula (I) of the present invention or its pharmaceutically acceptable salt or pharmaceutical composition comprising the same can be administered in other topical dosage forms, including creams, powders, sprays and inhalants. The drug can be mixed with a pharmaceutically acceptable excipient, diluent or carrier and any required preservative, buffer or propellant under sterile conditions. Ophthalmic formulations, ophthalmic ointments, powders and solutions are also intended to be included within the scope of the present invention.

[0097] In addition, the present disclosure also encompasses kits (e.g., pharmaceutical packaging). The kits provided may include a pharmaceutical composition or compound described herein and a container (e.g., a medicine bottle, an ampoule, a bottle, a syringe, and / or a subpackage or other suitable container). In some embodiments, the kit provided 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 composition or compound described herein disposed in the first container and the second container are combined to form a unit dosage form.

[0098] In certain embodiments, the kit described herein further includes instructions for use of the compound or pharmaceutical composition contained in the kit. The kit described herein may also include information required by an administrative agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and instructions for use provide for treating a proliferative disease in a subject in need thereof and / or preventing a proliferative disease in a subject in need thereof. The kit described herein may include one or more additional pharmaceutical preparations as separate compositions.

[0099] The present invention is further described in detail below in conjunction with specific examples, but the present invention is not limited to the following examples, which are intended to better illustrate certain specific embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way. The conditions not specified in the examples are normal conditions. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0100] The structures of the compounds in the following examples are determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). NMR was measured using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3) and deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0101] MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvance max).

[0102] Thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm-0.2mm, and the specification of silica gel plate used for thin layer chromatography to separate and purify products is 0.4mm-0.5mm.

[0103] Column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

[0104] Unless otherwise specified in the examples, the reaction temperature is room temperature, i.e., 20°C to 30°C.

[0105] The reaction progress in the examples was detected by thin layer chromatography (TLC). The developing solvent system used and the eluent system of column chromatography used for purifying the compounds included: 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, wherein the volume ratio of the solvent was adjusted according to the different polarities of the compounds.

[0106] Example 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-carbonitrile

[0107]

[0108]

[0109] Step 1: Preparation of 2,3,5,6,7,8-hexahydrophthalazine-1,4-dione (Compound 1b)

[0110] 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), sodium acetate (3.69 g, 45 mmol) and hydrazine hydrochloride (3.08 g, 45 mmol) were added in sequence. After the addition, the mixture was heated to 100°C and stirred for 3 h. The reaction was stopped and cooled to room temperature naturally. Solids were precipitated and filtered to obtain compound 1b (4.2 g), which was directly used in the next step.

[0111] 1 H NMR (400 MHz, DMSO-d 6 ):11.26(s,2H),2.36(s,4H),1.65(s,4H).

[0112] Step 2: Preparation of 1,4-dichloro-5,6,7,8-tetrahydrophthalazine (Compound 1c)

[0113] Dissolve compound 1b (1g, 6.02mmol) in phosphorus oxychloride (8ml). Replace the air in the system with nitrogen three times. Heat the system to 110°C and stir for 3h. Stop the reaction and cool naturally. Slowly drip the reaction solution into ice water. Adjust the mixture to pH=10 with 1N sodium hydroxide aqueous solution and extract it with ethyl acetate three times. Combine the organic phases, wash the organic phases with saturated brine, and concentrate the organic phase to obtain compound 1c (1.1g). The product is directly used in the next step reaction.

[0114] Step 3: Preparation of 3,5-dichloro-4-((4-chloro-5,6,7,8-tetrahydrophthalazin-1-yl)oxy)aniline (Compound 1d)

[0115] Add dimethyl sulfoxide (8 ml) 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). Replace the air in the system with nitrogen three times. Heat the system to 100 ° C and stir for 3 h. After stopping the reaction, cool it. First filter out the solid in the reaction solution and wash the filter residue repeatedly with ethyl acetate. Add 80 ml of water to the filtrate, and then extract the aqueous phase with ethyl acetate. Combine the organic phases and wash with saturated brine. Filter and concentrate the solvent to obtain compound 1d (450 mg) by column chromatography.

[0116] Step 4: Preparation of 4-(4-amino-2,6-dichlorophenoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (Compound 1e)

[0117] 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 h. After stopping the reaction, the solvent was removed under reduced pressure. 10 ml of water was added, and then 1N sodium hydroxide aqueous solution was added to adjust the pH to 8. It was extracted with ethyl acetate (10 ml * 3) and the organic phases were combined. The organic phases were washed with saturated sodium bicarbonate aqueous solution and saturated brine, respectively, and concentrated under reduced pressure to obtain a gray solid. 10 ml of methanol and 10 ml of 1N sodium hydroxide aqueous solution were added to the obtained solid, and the mixture was heated to 120 ° C and stirred for 12 h. After stopping the reaction, methanol was concentrated under reduced pressure. The remaining aqueous phase was repeatedly extracted with ethyl acetate 3 times, and the organic phases were combined. After the solvent was concentrated under reduced pressure, thin layer chromatography was used for separation and purification to obtain compound 1e (80 mg).

[0118] Step 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-carbonitrile (Compound 1)

[0119] 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 hydrochloric acid aqueous solution (1.25 mL), and stirred for 0.5 h under ice bath conditions until the solution was clear. At this temperature, a mixed solution of water (4.2 ml) and pyridine (1.3 ml) of N-cyanoacetylurea (32 mg) was added dropwise, and stirred overnight after the addition was complete. After stopping the reaction, the yellow solid was filtered out and washed with water and petroleum ether. Acetic acid (5 ml) and sodium acetate (160 mg, 2 mmol) were added to the obtained solid. The mixture was heated to 120 ° C and stirred for 6 h, then cooled to room temperature. 100 ml of water was added to precipitate a light yellow solid. Compound 1 (12.0 mg) was separated and purified by thin layer chromatography (DCM: MeOH = 8: 1).

[0120] 1 H NMR (400 MHz, DMSO-d 6 )δ12.03(s,1H),7.75(s,2H),2.68-2.63(m,2H),2.45-2.39(m,2H),1.80-1.67(m,4H).

[0121] MS m / z(ESI):447.4[M+1].

[0122] Example 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-carbonitrile (Compound 2b).

[0123]

[0124] The synthetic route of Example 1 was adopted, except that the first step raw material 3,4,5,6-tetrahydrophthalic anhydride 1a was replaced by bicyclo[2.2.2]oct-2-ene-2,3-dicarboxylic anhydride to obtain the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-o-ethylidenephthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 2).

[0125] 1 H NMR (400 MHz, DMSO-d 6 ):12.13(s,1H),7.77(s,2H),1.88-1.73(m,4H),1.39-1.17(m,6H).

[0126] MS m / z(ESI):473.2[M+1].

[0127] Example 3: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-methanophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 3).

[0128]

[0129] Step 1: Preparation of dimethyl-bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate

[0130]

[0131] Dimethyl butynedioate (compound 3b) (2.75 ml, 22.3 mmol) was slowly added dropwise to cyclopentadiene (compound 3a) (1.475 g, 22.3 mmol). After the addition, the mixture was stirred at room temperature for 2 hours. The reaction was stopped to obtain an oily product 3c (4.0 g). The crude product was used directly in the next step without purification.

[0132] Step 2: Preparation of dimethyl-bicyclo[2.2.1]hepta-2-ene-2,3-dicarboxylate:

[0133]

[0134] Compound 3c (1.0 g, 4.8 mmol), palladium on carbon (0.05 g) and 15 ml of acetone were placed in a reaction bottle and replaced three times under a hydrogen balloon. Stir at room temperature for 1 hour. Filter and concentrate the solvent to dryness under reduced pressure to obtain a light green liquid product 3d (0.9 g).

[0135] Step 3: Preparation of bicyclo[2.2.1]hepta-2-ene-2,3-diacid:

[0136]

[0137] 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 pH value of the solution was adjusted to 1 with 2N dilute hydrochloric acid, extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain white solid compound 3e (2.0 g).

[0138] Step 4: Preparation of 4,5,6,7-tetrahydro-4,7-isophenylfuran 1,3-dione:

[0139]

[0140] Acetic anhydride (10 mL) was added to compound 3e (200 mg, 1.1 mmol). The mixture was heated to 100 degrees Celsius and stirred for 2 hours. The solvent was removed by concentration under reduced pressure to obtain a crude solid compound 3f (200 mg).

[0141] Step 5: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-o-methylenephthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile:

[0142] The synthetic route of Example 1 was adopted, except that the first step raw material 4,5,6,7-tetrahydroisobenzofuran-1,3-dione (compound 1a) was replaced by 4,5,6,7-tetrahydro-4,7-isophenylfuran-1,3-dione (compound 3f), to obtain the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-isophenylphthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (compound 3).

[0143] 1H NMR (400 MHz, DMSO-d 6 )δ12.08(s,1H),7.77(s,2H),2.75-2.65(m,2H),1.80-1.72(m,4H),1.55-1.50(m,2H).

[0144] MS m / z(ESI):459.0[M+1].

[0145] Example 4: Preparation of 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopenta[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (Compound 4)

[0146]

[0147] Step 1: Preparation of methyl 3,3-dimethyl-2-oxocyclopentyl-1-carboxylate

[0148]

[0149] NAH (480 mg, 12 mmol) was added to a DMC (11 ml) solution of compound 2,2-dimethylcyclopentane-1-one (1.12 g, 10 mmol) and MeOH (0.5 ml). The mixture was heated to 82°C and stirred for 3 hours, then cooled. Methanol (0.5 ml) and acetic acid (1 ml) were added to the system in sequence, and the system was poured into ice water, extracted with dichloromethane, dried, and the solvent was removed under reduced pressure to obtain a colorless oil compound 4a (1.7 g), which was used directly in the next step.

[0150] Step 2: Preparation of methyl 3,3-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)cyclopent-1-ene-1-carboxylate

[0151]

[0152] 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 h. 50 ml of water was added, and the mixture was extracted with ethyl acetate. The solvent was removed under reduced pressure and purified by column chromatography (PE:EA=50:1) to obtain compound 4b (1.8 g) as a colorless oil.

[0153] Step 3: Preparation of 2-(methoxycarbonyl)-5,5-dimethylcyclopent-1-ene-1-carboxylic acid

[0154]

[0155] Under a nitrogen 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 diacetate (66.9 mg, 0.30 mmol) and lithium chloride (758 mg, 17.88 mmol). After the addition, the system was stirred at room temperature for 16 hours, and 300 ml of ethyl acetate was added to the system. The system was washed once with water and once with saturated brine. The solvent was removed under reduced pressure to obtain a colorless oil 4c (1.2 g).

[0156] Step 4: Preparation of 3,3-dimethylcyclopent-1-ene-1,2-dicarboxylic acid

[0157]

[0158] 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 pH was adjusted to 1 with concentrated hydrochloric acid, extracted with ethyl acetate, and the solvent was removed under reduced pressure to obtain white solid compound 4d (1.0 g).

[0159] Step 5: Preparation of 4,4-dimethyl-5,6-dihydro-1H-cyclopentyl[c]furan-1,3(4H)-dione

[0160]

[0161] Acetic anhydride (10 ml) was added to compound 4d (1.0 g, 5.43 mmol). The mixture was heated to 100 degrees Celsius and stirred for 3 hours, then cooled. Excess acetic anhydride was removed under reduced pressure to obtain a light brown liquid compound 4e (875 mg), which was directly used in the next step of synthesis.

[0162] Step 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-carbonitrile

[0163] The synthetic route of Example 1 was adopted, except that the first step raw material 3,4,5,6,-tetrahydrophthalic anhydride (compound 1a) was replaced by 4,4-dimethyl-5,6-dihydro-1H-cyclopentyl[c]furan-1,3(4H)-dione (compound 4e), to obtain the title product 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopenta[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (compound 4).

[0164] 1 H NMR (400 MHz, DMSO-d 6 )δ12.09(s,1H),7.79(s,2H),2.97-2.93(m,2H),2.01-1.98(m,2H),1.34-1.20(m,6H).

[0165] MS m / z(ESI):460.9[M+1].

[0166] Example 5: Preparation of 1-(3,5-dichloro-4-((5-methyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (Compound 5)

[0167]

[0168] Step 1: Preparation of methyl 3-methyl-2-oxocyclohexane-1-carboxylate 5b:

[0169]

[0170] To a solution of 2-methylcyclohexanone (compound 5a) (4.48 g, 40 mmol) in dichloromethane (50 ml) was added NaH (60% content, 1.92 g, 48 mmol). The mixture was heated to reflux, stirred for 3 hours, and cooled. The mixture was quenched with methanol (0.5 ml) and acetic acid (1 ml). The reaction system was added to an ice bath and extracted with dichloromethane. The solvent was removed under reduced pressure and purified by column chromatography to obtain an oil (compound 5b) (5.6 g).

[0171] Step 2: Preparation of methyl 3-methyl-2-(((trifluoromethyl)sulfonyl)oxy)cyclohexyl-1-ene-1-carboxylate

[0172]

[0173] Under ice bath conditions, NaH (60% content, 600 mg, 25 mmol) was added to compound 5b (0.85 g, 5 mmol) in ether (20 ml) and stirred for 0.5 h. Then trifluoromethanesulfonic anhydride (2.8 g, 10 mmol) was added dropwise to the system and stirred for 1 h at 0 °C. 2 O (50 ml) was added to quench the system, 1N HCl was added to adjust the pH to 1, and the mixture was extracted with dichloromethane. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (PE:EA=20:1) to give compound 5c (1.04 g) as a colorless oil.

[0174] Step 3: Preparation of 2-(methoxycarbonyl)-6-methylcyclohexyl-1-ene-1-carboxylic acid

[0175]

[0176] Under nitrogen atmosphere, diisopropylethylamine (0.714g, 7.00mmol) and acetic anhydride (0.903g, 7.00mmol) were added dropwise to a solution of compound 5c (0.714g, 10.5mmol) and sodium formate (0.714g, 10.50mmol) in N,N-dimethylformamide (15ml) in sequence, and stirred at room temperature for 1 hour. Palladium acetate (40mg, 0.18mmol) and lithium chloride (445mg, 10.50mmol) were then added, and the system was stirred at room temperature overnight. Ethyl acetate (30ml) was added, and the mixture was washed with water. The solvent was removed under reduced pressure to obtain a light yellow oil compound 5d (590mg).

[0177] Step 4: Preparation of 3-methylcyclohexyl-1-ene-1,2-dioic acid 5e

[0178]

[0179] 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 the mixture was stirred at room temperature for 3 h. Methanol was then removed under reduced pressure. 1N aqueous hydrochloric acid solution was added to adjust the pH of the system to 1, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to remove the solvent to obtain compound 5e (0.550 g) as an oil.

[0180] Step 5: Preparation of 4-methyl-4,5,6,7-tetrahydroisophenylfuran-1,3-dione 5f

[0181]

[0182] Acetic anhydride (6 ml) was added to compound 5e (0.550 g, 2.99 mmol). The mixture was heated to 100 degrees Celsius and stirred for 2 hours. The system was cooled and the solvent was removed under reduced pressure to obtain a brown oil compound 5f (0.34 g), which was directly used in the next step of synthesis.

[0183] Step 6: Preparation of 1-(3,5-dichloro-4-((5-methyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (Compound 5)

[0184] The synthetic route of Example 1 was adopted, except that the first step raw material 3,4,5,6,-tetrahydrophthalic anhydride (compound 1a) was replaced by 4-methyl-4,5,6,7-tetrahydroisophenylfuran-1,3-dione (compound 5f), to obtain the title product 1-(3,5-dichloro-4-((5-methyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (compound 5).

[0185] MS m / z(ESI):460.1[M+1]

[0186] Example 6: Preparation of 1-(3,5-dichloro-4-((5,5-dimethyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile

[0187]

[0188] The synthetic route of Example 5 was adopted, except that the first step raw material 2-methylcyclohexanone (compound 5a) was replaced by 2,2-dimethylcyclohexanone, to obtain the title product 1-(3,5-dichloro-4-((5,5-dimethyl-4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (compound 6).

[0189] MS m / z(ESI):474.1[M+1]

[0190] Example 7: Preparation of 1-(3,5-dichloro-4-((7-methyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (Compound 7).

[0191]

[0192] The synthetic route of Example 5 was adopted, except that the first step raw material 2-methylcyclohexanone (compound 5a) was replaced by 2-methylcyclopentanone, to obtain the title product 1-(3,5-dichloro-4-((7-methyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (compound 7).

[0193] MS m / z(ESI):446.0[M+1]

[0194] Example 8: Preparation of 1-(3,5-dichloro-4-((7-ethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopenta[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (Compound 8)

[0195]

[0196] The synthetic route of Example 5 was adopted, except that the first step raw material 2-methylcyclohexanone (compound 5a) was replaced by 2-ethylcyclopentanone, to obtain the title product 1-(3,5-dichloro-4-((7-ethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopenta[d]pyridazin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (compound 8).

[0197] MS m / z(ESI):460.1[M+1]

[0198] Example 9: Preparation of 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-carbonitrile (Compound 9)

[0199]

[0200] The synthetic route of Example 1 was adopted, except that the raw material 1,4-dichloro-5,6,7,8-tetrahydrophthalazine (Compound 1c) in the third step was replaced by 1,4-dichlorophthalazine, to obtain the title product 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-carbonitrile (Compound 9).

[0201] 1 H NMR (400 MHz, DMSO-d 6): 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).

[0202] MS m / z(ESI):443.0[M+1].

[0203] Example 10: Preparation of 2-(3,5-dichloro-4-((5-chloro-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 10):

[0204]

[0205] The synthetic route of Example 1 was adopted, except that the first step raw material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced by 3-chlorophthalic anhydride to obtain the title product 2-(3,5-dichloro-4-((5-chloro-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 10).

[0206] 1 H NMR (400 MHz, DMSO-d 6 ): 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).

[0207] MS m / z(ESI):477.0[M+1].

[0208] Example 11: Preparation of 2-(3,5-dichloro-4-((5-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 11):

[0209]

[0210] The synthetic route of Example 1 was adopted, except that the first step raw material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced by 3-methylphthalic anhydride to obtain the title product 2-(3,5-dichloro-4-((5-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 11).

[0211] MS m / z(ESI):456.9[M+1].

[0212] Example 12: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 12):

[0213]

[0214] Step 1: 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-carboxylic acid

[0215] Add acetic acid (4 ml) and concentrated hydrochloric acid (1 ml) to compound 2 (77 mg), heat to 120 degrees Celsius, stir for 5 hours, and cool. Dilute the reaction solution with water. The obtained solid is filtered, washed with water and petroleum ether to obtain solid compound 12a (35 mg), which is directly used in the next step.

[0216] Step 2: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydro-5,8-ethanophthalazin-1-yl)oxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione

[0217] Thioglycolic acid (2 ml) was added to compound 12a (35.0 mg), and the mixture was heated to 170 degrees Celsius and stirred for 2 hours. The mixture was cooled, the pH was adjusted to 8 with 1N sodium hydroxide aqueous solution, and extracted with ethyl acetate three times. The organic phases were combined. The solvent was removed under reduced pressure, and compound 12 (10.0 mg) was separated and purified by thin layer chromatography (DCM: MeOH = 8: 1).

[0218] 1 H NMR (400 MHz, DMSO-d 6 ):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).

[0219] MS m / z(ESI):448.0[M+1].

[0220] Example 13: Preparation of 2-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 13)

[0221]

[0222] The synthetic route of Example 12 was adopted, except that the first step 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-carbonitrile (Compound 2) was replaced by 1-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1,2,4-triazine-6-carbonitrile) The title product 2-(3,5-dichloro-4-((7,7-dimethyl-1-oxo-2,5,6,7-tetrahydro-1H-cyclopentyl[d]pyridazin-4-yl)oxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 13) was obtained.

[0223] 1 H NMR (400 MHz, DMSO-d 6 ):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).

[0224] MS m / z(ESI):436.0[M+1].

[0225] Example 14: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-1,2,4-triazine-3,5-(2H,4H)dione (Compound 14)

[0226]

[0227] The synthetic route of Example 12 was adopted, except that the first step 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-carbonitrile (Compound 2) was replaced by 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-carbonitrile (Compound 9) to obtain the title product 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-1,2,4-triazine-3,5-(2H,4H)dione (Compound 14).

[0228] 1 H NMR (400 MHz, DMSO-d 6 ):12.00(s,1H),8.30-8.23(m,2H),8.09-7.99(m,2H),7.18(s,1H).

[0229] MS m / z(ESI):418.0[M+1].

[0230] Example 15: Preparation of 2-(3,5-dichloro-4-((5-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 15)

[0231]

[0232] The synthetic route of Example 1 was adopted, except that the first step raw material 3,4,5,6-tetrahydrophthalic anhydride (Compound 1a) was replaced by 3-fluorophthalic anhydride to obtain the title product 2-(3,5-dichloro-4-((5-fluoro-4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (Compound 15).

[0233] MS m / z(ESI):461.0[M+1].

[0234] Example 16: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 16)

[0235]

[0236] Step 1: Preparation of 4-(2,6-dichloro-4-iodophenoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (Compound 16a)

[0237]

[0238] Under ice bath conditions, water (4 ml) and concentrated hydrochloric acid (2 ml) were added to 1e (100 mg) in sequence, and then a solution of sodium nitrite (30 mg) in water (2 ml) was added dropwise. The reaction was stirred for 1 hour while maintaining ice bath conditions. Potassium iodide (104 mg) was added dropwise, and the mixture was warmed to room temperature and stirred for 16 hours. The mixture was extracted with dichloromethane, and the solvent was removed under reduced pressure to obtain a yellow solid 16a (100 mg), which was directly used in the next step reaction.

[0239] Step 2: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 16)

[0240]

[0241] N,N-diformamide (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). The system was heated to 120 degrees and stirred for 12 hours. The system was cooled, filtered, and separated by adding ethyl acetate and water. The organic phase was decompressed to remove the solvent, and the compound 16 (5.0 mg) was separated and purified by thin layer chromatography (DCM: MeOH = 8: 1).

[0242] MS m / z(ESI):436.1[M+1].

[0243] Example 17: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-6-isopropyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 17)

[0244]

[0245] The synthetic route of Example 16 was adopted, except that the second step raw material 6-azathymine was replaced by 6-isopropyl-1,2,4-triazine-3,5(2H,4H)-dione (using the known method "Chemistry and Biodiversity, 2012, 9(3)536)

[0246] -556" to obtain the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-6-isopropyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 17).

[0247] MS m / z(ESI):464.1[M+1].

[0248] Example 18: Preparation of 6-cyclopropyl-2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (Compound 18)

[0249]

[0250] The synthetic route of Example 16 was adopted, except that the second step raw material 6-azathymine was replaced by 6-cyclopropyl-1,2,4-triazine-3,5(2H,4H)-dione (prepared by the known method "Collection of Czechoslovak Chemical Communications, 1975, 40, 1038-1041"), to obtain the title product 2-(3,5-dichloro-4-((4-oxo-3,4,5,6,7,8-hexahydrophthalazin-1-yl)oxy)phenyl)-6-cyclopropyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 18).

[0251] MS m / z(ESI):462.1[M+1].

[0252] Example 19: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 19)

[0253]

[0254] Step 1: Preparation of 3,5-dichloro-4-((4-chlorophthalazin-1-yl)oxy)aniline (Compound 19a)

[0255]

[0256] The synthetic route of the intermediate 1d in the third step of Example 1 was adopted, except that 1,4-dichloro-5,6,7,8-tetrahydrophthalazine (compound 1c) was replaced by 1,4-dichlorophthalazine to obtain the title product 3,5-dichloro-4-((4-chlorophthalazin-1-yl)oxy)aniline (compound 19a).

[0257] Step 2: Preparation of 4-(4-amino-2,6-dichlorophenoxy)phthalazin-1(2H)-one (Compound 19b)

[0258]

[0259] The synthetic route of intermediate 1e in the fourth step of Example 1 was adopted, except that 3,5-dichloro-4-((4-chloro-5,6,7,8-tetrahydrophthalazin-1-yl)oxy)aniline (compound 1d) was replaced by 3,5-dichloro-4-((4-chlorophthalazin-1-yl)oxy)aniline (compound 19a), to obtain the title product 4-(4-amino-2,6-dichlorophenoxy)phthalazin-1(2H)-one (compound 19b).

[0260] Step 3: Preparation of 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 19)

[0261] The synthetic route of Example 16 was adopted, except that 4-(2,6-dichloro-4-iodophenoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (Compound 16a) in the first step was replaced by 4-(4-amino-2,6-dichlorophenoxy)phthalazin-1(2H)-one (Compound 19b), to obtain the title product 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-6-methyl-1,2,4-triazine-3,5(2H,4H)-dione (Compound 19).

[0262] MS m / z(ESI):432.0[M+1].

[0263] Example 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-carbonitrile (Compound 20):

[0264]

[0265] The synthetic route of Example 1 was adopted, except that the first step raw material tetrahydrophthalic anhydride (1a) was replaced by 4-methylphthalic anhydride, to obtain 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-carbonitrile (Compound 20).

[0266] MS m / z(ESI):457.0[M+1].

[0267] Example 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-carbonitrile (Compound 21):

[0268]

[0269] The synthetic route of Example 1 was adopted, except that the first step raw material tetrahydrophthalic anhydride (1a) was replaced by 4-chlorophthalic anhydride to obtain 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-carbonitrile (Compound 21).

[0270] MS m / z(ESI):477.0[M+1].

[0271] Test Example 1: Test of compound binding to TRα:

[0272] 1. Main experimental materials and instruments:

[0273] Envision 2104 microplate reader;

[0274] Biotin-SRC2-2 coactivator peptide was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.;

[0275] TRαLBD, GST, purchased from Thermo Fisher (Cat. No. PV4762);

[0276] Europium-conjugated anti-glutathione antibody, purchased from Cisbio (Cat. No. 61GSTKLB); and

[0277] Streptavidin-D2, purchased from Cisbio (Cat. No. 610SADAB)

[0278] 2. Preparation and treatment of compounds

[0279] 2.1 Preparation of dimethyl sulfoxide stock solution containing compounds

[0280] All compounds were dissolved in dimethyl sulfoxide to prepare 10 mM stock solutions.

[0281] 2.2 Compound Storage

[0282] After the compound is dissolved in dimethyl sulfoxide, the solution can be stored in a desiccator at room temperature for three months. For long-term storage, the compound should be placed in a -20℃ refrigerator.

[0283] 3. Experimental Procedure

[0284] 3.1 Preparation of 1x reaction buffer

[0285] 3.2 Screening of compounds:

[0286] a) The positive drug triiodothyronine (T3) was diluted from 10 mM (100X) or the test compound was diluted from 1 mM (100X) in 1:3 ratio with 100% dimethyl sulfoxide, for a total of 10 concentrations.

[0287] b) Prepare 4x serial dilutions of compound in 1x reaction buffer.

[0288] c) Add 5 μl of 4x serially diluted compounds to a 384-well assay plate.

[0289] d) Prepare 4X TRαLBD and 4X RXRα in 1x reaction buffer.

[0290] e) Add 5 μl of 4x TRαLBD and 4X RXRα to a 384-well assay plate.

[0291] f) Prepare 2X biotin-SRC2-2, 2X europium-conjugated anti-glutathione antibody and 2X streptavidin-d2 in 1X reaction buffer.

[0292] g) Add 10 μl of 2X mixture (see step f) to a 384-well assay plate.

[0293] h) Centrifuge the 384-well assay plate at 1000 rpm for 1 minute.

[0294] i) Incubate at room temperature in the dark for 1 hour.

[0295] j) The fluorescence signal values ​​at 665 nm and 615 nm wavelengths of each well of the 384-well test plate were recorded using an Envision 2104 microplate reader, and the fluorescence ratio of 665 nm / 615 nm was calculated.

[0296] 4. Data Analysis

[0297] 4.1 Calculate the relative ratio (ratio) of each well 665nm / 615nm -ratio 空白 )

[0298] 4.2 The activity percentage is calculated as follows:

[0299]

[0300] Average value of relative ratio of the pores of the example compounds

[0301] The relative ratios of all positive control wells are averaged.

[0302] The relative ratios of all negative control wells are averaged.

[0303] 4.3 Curve drawing and EC50 calculation:

[0304] Graphpad 5.0 was used to calculate the EC50 by fitting the relationship between activity (%) and the logarithmic concentration of the compound using the nonlinear regression method.

[0305] Y = bottom + (top - bottom) / (1 + 10^((LogEC50-X) * slope))

[0306] X: Logarithmic concentration of compound Y: Percent activity

[0307] The specific test data is shown in Table 1 below.

[0308] Test Example 2: Evaluation of Compounds' Agonistic Activity on TRα

[0309] 1. Main experimental materials and instruments:

[0310] Envision 2104 Microplate Reader

[0311] HEK293T cell line, purchased from ATCC (Cat. No.: CRL-3216)

[0312] pGL4.35[luc2P / 9XGAL4 UAS / Hygro], purchased from Promega (Cat. No.: E1370)

[0313] pBIND-TRα plasmid was obtained from Pharmaron

[0314] pBIND-RXRα plasmid was obtained from Pharmaron

[0315] LipoLTX transfection reagent, purchased from Thermo Fisher (Cat. No.: 15338-100)

[0316] 2. Compound Preparation

[0317] 2.1 Compound Dissolution

[0318] Compound powders were prepared as 10 mM dimethyl sulfoxide stock solutions according to standard protocols.

[0319] 2.2 Compound Storage

[0320] All compounds dissolved in dimethyl sulfoxide were stored in a desiccator at room temperature for short term storage or at -20°C for long term storage.

[0321] 2.3 Preparation of experimental compounds

[0322] 2.3.1 All test compounds were diluted 3-fold with dimethyl sulfoxide, with a 10-step dilution gradient and a starting concentration of 10 μM.

[0323] 2.3.2 Positive control triiodothyronine (T3) was diluted 3-fold with dimethyl sulfoxide, with a 10-step dilution gradient and a starting concentration of 16.67 μM.

[0324] 2.3.3 Prepare 166.7× positive control (16.67 μM, triiodothyronine (T3)) and 166.7× negative control (100% dimethyl sulfoxide).

[0325] 2.4 Block the compound plate and shake for 5 minutes.

[0326] 3 Experimental procedures

[0327] 3.1 Preparation of cell suspension and plating

[0328] a) All cells were cultured according to ATCC standard procedures, and HEK293T cells were used for experiments in the exponential growth phase.

[0329] b) discarding the culture medium;

[0330] c) washing the cells twice with phosphate buffered saline;

[0331] d) adding trypsin digestion solution to digest the cells and stopping the digestion with complete medium;

[0332] e) Collect and count cells. Experiments can only be performed when the cell viability is greater than 90%;

[0333] f) 2.5*10 6 HEK293-LUC cells into a 60 mm cell culture dish; and

[0334] g) Place the culture dish with the cells in a 37°C and 5% CO 2 Incubate overnight.

[0335] 3.2 Cell transfection

[0336] a) Place the LipoLTX transfection reagent at room temperature for equilibrium;

[0337] b) Add 6 μl of Plus reagent and 6 μg of DNA to 250 μl of Opti-MEM TM medium, being careful not to touch the tube wall, pipette to mix and

[0338] Plasmids: 2.5 μg pBIND-TRα, 2.5 μg pBIND-RXRα, and 1 μg pGL4.35 plasmids were added respectively;

[0339] c) Add 12 μl Lipo LTX and 250 μl Opti-MEM TM The culture medium, being careful not to touch the tube wall, is mixed by pipetting and pipetting;

[0340] d) Add the mixed DNA Plus reagent (see step 3.2.b) to the diluted LipoLTX (see step 3.2.c) transfection reagent and let stand at room temperature for 15 minutes;

[0341] e) Add the transfection reagent mixed with DNA to a 60 mm cell culture dish (see step 3.1); and

[0342] f) Place the culture dish in a 37°C and 5% CO 2 Incubate in incubator for 5 hours.

[0343] 3.3 Compound treatment

[0344] a) Transfer 150 nl of the diluted compound (see step 2.3) to the cell culture plate using Echo550 (6007680-50, PE);

[0345] b) seed the cells (see step 3.2) into a 384 cell culture plate (6007680-50, PE) with 15,000 cells per well and 25 μl of medium containing 5% fetal bovine serum; and

[0346] c) The cells were cultured at 37°C and 5% CO 2 Incubate overnight in an incubator.

[0347] 3.4 Compound detection

[0348] a) Steady-Glo TM The test reagents were placed at room temperature;

[0349] b) Place the 384-cell plate (see step 3.3) at room temperature;

[0350] c) Add 25 μl Steady-Glo to each well TM Detection reagents in cell culture plates (see step 3.4b);

[0351] d) Place the plate on a shaker in the dark for 5 minutes; and

[0352] e) The chemiluminescence value was detected using Envision 2104 microplate reader.

[0353] 4. Data Analysis

[0354] 4.1 Calculation of activity (%):

[0355]

[0356] RLU: Fluorescence generated

[0357] Example Compound Pore Average Value

[0358] Positive control average

[0359] Negative control mean

[0360] 4.2 Calculate EC50 and draw the dose-effect curve of the compound:

[0361] The EC50 of the compounds was calculated by fitting the logarithmic values ​​of activity (%) and compound concentration using Graphad 5.0.

[0362] Y = bottom + (top - bottom) / (1 + 10^((LogIC50 - X) * slope))

[0363] X: Logarithmic concentration of compound Y: Percent inhibition rate

[0364] The specific test data is shown in Table 1 below.

[0365] Test Example 3: Test of Compound Binding Ability to TRβ

[0366] 1. Main experimental materials and instruments:

[0367] Envision 2104 microplate reader,

[0368] Biotin-SRC2-2 coactivator peptide was purchased from Shanghai Shenggong Biotechnology Co., Ltd.

[0369] TRβLBD, GST, purchased from Thermo Fisher (Cat. No. PV4762)

[0370] Europium-conjugated anti-glutathione antibody, purchased from Cisbio (Cat. No. 61GSTKLB)

[0371] Streptavidin-D2, purchased from Cisbio (Cat. No. 610SADAB)

[0372] 2. Preparation and treatment of compounds

[0373] 2.1 Preparation of dimethyl sulfoxide stock solution containing compounds

[0374] All compounds were dissolved in dimethyl sulfoxide to prepare 10 mM stock solutions.

[0375] 2.2 Compound Storage

[0376] After the compound is dissolved in dimethyl sulfoxide, the solution can be stored in a desiccator at room temperature for three months. For long-term storage, the compound should be placed in a -20℃ refrigerator.

[0377] 3. Experimental Procedure

[0378] 3.1 Preparation of 1x reaction buffer

[0379] 3.2 Screening of compounds:

[0380] a) Use 100% dimethyl sulfoxide to dilute the positive drug triiodothyronine (T3) from 10 micromolar (100X) or the test compound from 1 millimolar (100X) in a 1:3 ratio, for a total of 10 concentrations;

[0381] b) Prepare 4x concentration gradient dilutions of the compound using 1x reaction buffer;

[0382] c) Add 5 μl of 4x serially diluted compounds to a 384-well assay plate;

[0383] d) Prepare 4X TRβLBD and 4X RXRβ using 1x reaction buffer;

[0384] e) Add 5 μl of 4x TRβLBD and 4X RXRβ to a 384-well assay plate;

[0385] f) preparing 2X biotin-SRC2-2, 2X europium-conjugated anti-glutathione antibody and 2X streptavidin-d2 using 1X reaction buffer;

[0386] g) Add 10 μl of 2X mixed solution (see step f) to the 384-well assay plate;

[0387] h) Centrifuge the 384-well test plate at 1000 rpm for 1 minute;

[0388] i) incubate at room temperature in the dark for 1 hour; and

[0389] j) Use Envision 2104 microplate reader to record the fluorescence signal values ​​at 665 nm and 615 nm wavelengths in each well of the 384-well test plate, and calculate the fluorescence ratio of 665 nm / 615 nm.

[0390] 4. Data Analysis

[0391] 4.1 Calculate the relative ratio (ratio) of each well 665nm / 615nm -ratio 空白 )

[0392] 4.2 The activity percentage is calculated as follows:

[0393]

[0394] Average value of relative ratio of the pores of the example compounds

[0395] The average of the relative ratios of all positive control wells

[0396] The average of the relative ratios of all negative control wells

[0397] 4.3 Curve drawing and EC50 calculation:

[0398] Graphpad 5.0 was used to calculate the EC50 by fitting the relationship between activity (%) and the logarithmic concentration of the compound using the nonlinear regression method.

[0399] Y = bottom + (top - bottom) / (1 + 10^((LogEC50-X) * slope))

[0400] X: Logarithmic concentration of compound Y: Percent activity

[0401] The specific test data is shown in Table 1 below.

[0402] The binding activity of the compounds in Table 1 to thyroxine receptor β is as follows:

[0403]

[0404] Conclusion: Compared with the disclosed comparative compound 53, some compounds of the present invention unexpectedly showed very high THRβ activity (<0.2μM), and some compounds showed higher selectivity for THRα than comparative compound 53.

[0405] Test Example 4: Evaluation of the TRβ receptor agonist activity of compounds

[0406] Experimental Overview: TRβ-LBD and RXRα-LBD coding sequences were inserted into pBIND plasmid (Promega, E1581) respectively. The expression vector and the reporter vector (pGL4.35 carrying a stably integrated GAL4 promoter-driven luciferase reporter gene) were co-expressed in host cells. When the agonist binds to the corresponding chimeric receptor, the chimeric receptor binds to the GAL4 binding site on the reporter gene vector and stimulates the expression of the reporter gene. The agonist activity of the compound on the TRβ receptor is determined based on the strength of the chemiluminescent signal.

[0407] Experimental materials and instruments:

[0408] Envision 2104 Microplate Reader

[0409] HEK293T cell line, purchased from ATCC (Cat. No.: CRL-3216)

[0410] pGL4.35[luc2P / 9XGAL4 UAS / Hygro], purchased from Promega (Cat. No.: E1370)

[0411] pBIND-TRβ plasmid was obtained from Pharmaron

[0412] pBIND-RXRα plasmid was obtained from Pharmaron

[0413] LipoLTX transfection reagent, purchased from Thermo Fisher (Cat. No.: 15338-100)

[0414] Steady-Glo TM Luciferase assay kit, purchased from Promega (Cat. No.: E2520)

[0415] 2.3 Preparation of experimental compounds

[0416] All test compounds were diluted 3-fold with dimethyl sulfoxide, with a starting concentration of 10 mM.

[0417] The positive control triiodothyronine (T3) was diluted 3-fold with dimethyl sulfoxide, with a 10-step dilution gradient and a starting concentration of 16.67 μM.

[0418] A 166.7× positive control (16.67 μM triiodothyronine (T3)) and a 166.7× negative control (100% dimethyl sulfoxide) were prepared.

[0419] The compound plates were blocked and shaken for 5 minutes.

[0420] Experimental procedures

[0421] 3.1 Preparation of cell suspension and plating

[0422] a) All cells were cultured according to ATCC standard operations, and HEK293T was used for experiments in the exponential growth phase;

[0423] b) discarding the culture medium;

[0424] c) washing the cells twice with phosphate buffered saline;

[0425] d) adding trypsin digestion solution to digest the cells and stopping the digestion with complete medium;

[0426] e) Collect and count cells. Experiments can only be performed when the cell viability is greater than 90%;

[0427] f) 2.5×106 HEK293-LUC cells were placed in 60 mm cell culture dishes;

[0428] g) Place the culture dish with the cells in a 37°C and 5% CO 2 Incubate overnight.

[0429] 3.2 Cell transfection

[0430] a) Place the LipoLTX transfection reagent at room temperature for equilibrium;

[0431] b) Add 6 μl of Plus reagent and 6 μg of DNA to 250 μl of Opti-MEM TM Culture medium, be careful not to touch the tube wall, pipette to mix and use a pipette to transfer; Plasmid: 2.5 μg pBIND-TRβ, 2.5 μg pBIND-RXRα and 1 μg pGL4.35 plasmid

[0432] c) Add 12 μl Lipo LTX and 250 μl Opti-MEM TM The culture medium, being careful not to touch the tube wall, is mixed by pipetting and pipetting;

[0433] d) Add the mixed DNA Plus reagent (see step 3.2.b) to the diluted Lipo LTX (see step 3.2.c) transfection reagent and let stand at room temperature for 15 minutes;

[0434] e) Add the transfection reagent mixed with DNA to a 60 mm cell culture dish (see step 3.1); and

[0435] f) Place the culture dish in a 37°C and 5% CO 2 Incubate in incubator for 5 hours.

[0436] 3.3 Compound treatment

[0437] a) Transfer 150 nl of the diluted compound (see step 2.3) to the cell culture plate using Echo550 (6007680-50, PE);

[0438] b) seeding the cells (see step 3.2) into 384 cell culture plates (6007680-50, PE) with 15,000 cells per well and 25 μl of culture medium; and

[0439] c) The cells were cultured at 37°C and 5% CO 2 Incubate overnight in an incubator.

[0440] 3.4 Compound detection

[0441] a) Steady-Glo TMThe test reagents were placed at room temperature;

[0442] b) Place the 384-cell plate (see step 3.3) at room temperature;

[0443] c) Add 25 μl Steady-Glo to each well TM Detection reagents to cell culture plates (see step 3.4b);

[0444] d) Place the plate on a shaker in the dark for 5 minutes; and

[0445] e) The chemiluminescence value was detected using Envision 2104 microplate reader.

[0446] Data Analysis:

[0447] 4.1 Calculation of activity (%):

[0448]

[0449] RLU: Fluorescence generated

[0450] Example Compound Pore Average Value

[0451] Positive control average

[0452] Negative control mean

[0453] 4.2 Calculate EC50 and draw the dose-effect curve of the compound:

[0454] The EC50 of the compounds was calculated by fitting the logarithmic values ​​of activity (%) and compound concentration using Graphad 5.0.

[0455] Y = bottom + (top - bottom) / (1 + 10^((LogIC50 - X) * slope))

[0456] X: Logarithmic concentration of compound Y: Percent inhibition rate

[0457] Table 2: The agonist activity of the compounds of the present invention on thyroxine receptor β is as follows:

[0458]

[0459] Conclusion: The compounds of the present invention can activate the downstream signaling of thyroid hormone receptor β.

[0460] Test Example 5: Pharmacokinetic Evaluation:

[0461] Rats were used as test animals. The drug concentrations in plasma at different times after rats were gavaged with the compounds of Example 4 and Example 9 were tested. The pharmacokinetic behavior of the compounds of the present invention in rats was studied, and their drug metabolism characteristics were evaluated. Three SD male rats of similar body weight were selected for each group of examples, and the oral dose was 2 mg / kg, and the drug was administered once. Blood was collected at 15min, 30min, 1h, 2h, 4h, 6h, 10h and 24h after the animals were administered. The compound content in plasma was detected by LC-MS / MS analysis, and the quantitative lower limit of the method was 20ng / ml. The metabolic kinetic data analysis software WinNonlin 7.0 was used to perform statistics on VT088 and concentration data in plasma. The pharmacokinetic parameters were calculated using the non-compartmental model method (NCA), as shown in Table 2 below.

[0462] Experimental protocol:

[0463] Experimental drugs: compounds of Example 4 and Example 9.

[0464] Experimental Animals:

[0465] Six healthy SD male rats were equally divided into two groups, with three rats in each group. They were purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd., with animal production license number: SCXK (Shanghai) 2008-0016.

[0466] Drug preparation: Take a certain amount of drug and add 2% Klucel LF + 0.1% Tween 80 aqueous solution to prepare a clear solution or uniform suspension.

[0467] Administration: SD rats were fasted overnight and then given the drug by gavage. Each dose was 2 mg / kg and the administration volume was 10 mL / kg.

[0468] Procedure: Rats were intragastrically administered with the compounds of Example 4 and Example 9. At least 0.2 mL of blood was collected from the tail vein before administration and 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h and 24 h after administration, and placed in a heparinized sample tube. The plasma was separated by centrifugation at 4 degrees Celsius and 3500 rpm for 10 minutes. The heparinized sample tube was then stored at -20 degrees Celsius, and the rats were fed 2 hours after administration.

[0469] Determination of the content of the test compound in rat plasma after oral administration of different concentrations of drugs: After the plasma samples were thawed at room temperature, 50 μL was taken, 130 μL of internal standard working solution (1000 ng / mL, acetonitrile, tolbutamide) was added, and the mixture was vortexed for about 1 min, and then centrifuged at 4°C and 13000 rpm for 10 min. 50 μL of the supernatant was mixed with 100 μL of 50% acetonitrile water and then introduced into LC / MS / MS analysis.

[0470] The pharmacokinetic parameters are shown in Table 3.

[0471] Table 3: Drug metabolism data in rats

[0472]

[0473]

[0474] Conclusion: The compounds of the present invention have good pharmacokinetic absorption and have obvious pharmacokinetic advantages. Compared with the reported comparative compound 53, some compounds of the present invention unexpectedly showed higher Cmax values ​​and exposure amounts under the same dose and formulation. All the above PK results show that the compounds provided by the present invention have good PK properties and can be used as therapeutic drugs for metabolic-related diseases.

Claims

1. A compound of formula (III) or a pharmaceutically acceptable salt thereof, in R 1 is selected from the group consisting of hydrogen or cyano; R 2 and R 3 are each independently selected from halogen; R 4 Selected from hydrogen, halogen and C 1-6 The group consisting of alkyl groups; m is an integer ranging from 1 to 4; and The halogen is selected from the group consisting of F, Cl and Br. 2 . The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein m is an integer ranging from 1 to 3.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein R 4 Selected from hydrogen, halogen and C 1-3 The group consisting of alkyl; and m is 1 or 2.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is cyano.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 2 and R 3 Each is Cl.

6. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is:

7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

8. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

9. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

10. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

11. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

12. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

13. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is:

14. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

15. Use of a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 14, in the preparation of a medicament for treating a disease in a subject in need thereof, wherein the disease is selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, type 2 diabetes, non-alcoholic fatty liver disease (NASH), hepatic steatosis, atherosclerosis, hypothyroidism, and thyroid cancer.

16. The use according to claim 15, wherein the disease is selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, type 2 diabetes, NASH, hepatic steatosis, hypothyroidism and thyroid cancer.

17. The use according to claim 16, wherein the disease is selected from the group consisting of NASH, hypothyroidism and thyroid cancer.

18. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

20. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

21. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

22. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

23. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

24. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition comprises a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

25. The use according to claim 17, wherein the disease is NASH.

26. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

27. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

28. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

29. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

30. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

31. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

32. The use according to claim 25, wherein the compound is or a pharmaceutically acceptable salt thereof.

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