CN114213331A - URAT1 inhibitor and application thereof - Google Patents
URAT1 inhibitor and application thereof Download PDFInfo
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- CN114213331A CN114213331A CN202210131754.5A CN202210131754A CN114213331A CN 114213331 A CN114213331 A CN 114213331A CN 202210131754 A CN202210131754 A CN 202210131754A CN 114213331 A CN114213331 A CN 114213331A
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
- C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
Abstract
The invention belongs to the field of medicinal chemistry, and particularly relates to a URAT1 inhibitor and application thereof, wherein the URAT1 inhibitor is a compound with a structure shown in a formula (I) or a formula (II) or a pharmaceutically acceptable salt thereof. Experiments show that the compound provided by the invention has a very good inhibitory effect on uric acid transfer of hURAT1 in HEK293 transfected cells, and shows that the compound has a good application prospect in the aspect of treating hyperuricemia or gout.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to URAT1 inhibitor compounds and application of the compounds.
Background
Gout is a metabolic disease caused by hyperuricemia. When purine metabolism disorder or excessive high-purine food is taken in a human body, the production of uric acid is increased, and the kidney is unsmooth in the excretion of uric acid, so that the blood uric acid concentration exceeds the normal level, saturation is achieved, the urate is accumulated in a large amount and crystallized and deposited on joints, tendons, kidneys and the like, and finally repeated pain caused by arthritic pain is caused (Richette P, Bardin T.Gout.Lancet.2010,375(9711): 318-. Hyperuricemia is clinically defined when the uric acid concentration in the blood of men is greater than 7mg/dL or the uric acid concentration in the blood of women is greater than 6 mg/dL. The cause of approximately 80-85% of patients with hyperuricemia is poor excretion of uric acid by the kidneys (Cheeseman C. solution carrier family 2, member 9 and uric acid hoscostasis. Current Opinion in physiology and Hypertension,2009,18(5): 428-432).
Gout has a very serious hazard. The onset of which can cause severe pain to the patient. If a large amount of sodium urate crystals are deposited subcutaneously in the human body, tophus may be formed, which may deform joints, lose function and rupture epidermal cells. Tophus formed in the kidney can cause uric acid lithiasis and nephritis, and even cause renal injury. Gout may also have certain interactions with hypertension, metabolic syndrome, hyperlipidemia, diabetes, insulin resistance, etc., and exacerbate the risk factors of the above diseases. (Rho YH, Woo JH, Choi SJ, et al.Association between serum uric acid and the adult waste model III-defined metabolic syndrome: results from a single genetic database.2008, 57 (1) 71-76).
With the increasing of human living standard and the change of diet and living habits, the incidence rate of gout is on the rise year by year, and gout has become the most common chronic disease and is listed as one of the 21 st century 20 stubborn diseases by united nations (Grobner W, Walter SI. treatment of hyperuricemia and gout. Med Monatsschr Pharm,2005,28: 159-. At present, nearly 1 hundred million gout patients exist in the world, and the market is huge. The incidence of gout in Europe is about 1-2%, with the main population being middle-aged and elderly men (Michael Doherty, Tim L Jansen, George Nuki, et al. Gout: whhy is this secure disease so selected dom cured. Annals of the theoretical diseases.2012,71(11): 1765-); the number of gout patients in the United states is 830 ten thousand; the number of hyperuricemia patients in China is about 1.2 hundred million, wherein the number of gout patients exceeds 5000 million, and the incidence rate of gout and hyperuricemia in developed areas (such as Qingdao, hong Kong, Taiwan, and the like) in coastal areas is high. According to the statistics of Taiwan hospital in China in 2007 in 2000-, the prevalence rate of hyperuricemia is as high as 22.8%.
A standardized treatment regimen for gout comprises urate-lowering therapy (ULT). It can make the concentration of blood uric acid in body lower than saturated concentration, and can dissolve urate crystal in focus position. Gout is no longer formed after the urate crystals disappear in vivo. The purpose of ULT is to reduce the patient's blood uric acid concentration to below 6 mg/dL. For patients with tophus, the target blood uric acid concentration must be reduced to 5mg/dL, which is effective in reducing urate precipitation in patients within a certain time frame (Puja P Khanna, George Nuki, Thomas Bardin, et al. Tophi and front gout flares areas associated with interference of Life, and associated with capillary resource use: resources from cross-section and Quality of Life issues.2012, 10(117): 117; Richette P, Frazier A, Bardin T. biochemical associations for food waste. expression Opt. 20147. Tophic et 7. 20147). ULT drugs are classified into uric acid production inhibitors, uricosuric agents and uricases. Allopurinol and febuxostat are xanthine oxidase inhibitors which are main uric acid generation inhibitors, but allopurinol is large in clinical application dose and has various adverse reactions, such as fatal rash; febuxostat has serious side effects of cardiovascular and gastrointestinal discomfort and also has hepatotoxicity. And about 40-80% of patients are clinically unable to achieve control of uric acid levels by uric acid production inhibitors (Edwards NL. Febuxostat: a new treatment for hyperuricemia in gout. Rheumatology (Oxford) 2009,48(2): 15-19). Uricase drugs such as polyethylene glycol recombinant uricase (pegloticase) have remarkable curative effect, but can only be used for adult gout patients who have no effect or can not tolerate the conventional treatment due to serious adverse reactions such as cardiovascular events, transfusion reaction and immunogenicity reaction, so that the uricase drugs are rarely used clinically. (Lipsky PE, Calabrese LH, Kavanaugh A, et al. Pegliotic immunity: the correlation between sensitivity and sensitivity with sensitivity in strategies and strategies for reactivity chlorine. arthritis Research therapy 2014,16(2):60.)
Another ULT class of drugs is drugs that promote uric acid excretion. The action mechanism is that the transport function of human urate anion transport protein 1(hURAT1) of the epithelial cells of the proximal convoluted tubule of the kidney on uric acid is inhibited, and the reabsorption of uric acid on the proximal convoluted tubule of the kidney is reduced, so that the excretion function of the kidney on uric acid is promoted. hURAT1 is specifically expressed on brush border membrane of epithelial cells of proximal convoluted tubule of human kidney, is the most important uric acid reabsorption protein in human body, and controls reabsorption of uric acid after filtering of glomeruli by more than about 90% (Michael FW, Juutabha P, quad B. developing patent human uric acid transporter 1(hURAT1) inhibition. journal of Medicinal chemistry.2011,54: 2701-.
Uricosuric URAT1 inhibitors currently used primarily clinically for the treatment of gout include Benzbromarone (benzebramarone), Zurampic, probenecid, and tribenuron-methyl. The Zurampic of Aslicon was approved by the United states and Europe at a dose of 200 mg/day for use with allopurinol at 12 months 2015 and 2016 2 months, and was far less effective than benzbromarone. Clinical trials show that Lesinurad has various toxic and side effects: (1) the medicine may cause serious cardiovascular adverse reactions such as fatal cardiovascular diseases, non-fatal myocardial infarction or cerebral pruritus of patients. (2) Lesinurad can cause adverse reactions related to renal function immediately after treatment, and when 400mg is taken alone, the incidence rate of serious adverse events is highest, so that the single use of high dose is forbidden clinically, and the renal function needs to be detected regularly before and after treatment. (3) The medicine can cause mild and moderate liver injury. Thus, the FDA requires Lesinurad to have its severe nephrotoxicity indicated in the specification by a black box. The old medicines of probenecid and metconazole have poor curative effect, large using dose and great side effect.
Benzbromarone, a typical selective inhibitor of URAT1, is currently the most effective uricosuric agent on the market, was developed by the company Snaofi-synthelobo, france, and was marketed in 1976. It has been reported that 92% of gout patients who have failed allopurinol therapy can have their blood uric acid value reduced to 5mg/dl after 2 months of continuous benzbromarone use (Halevy S, Ghislain PD, Mockenhaupt M, et al, allopurinol is the most common mouse of people of Stevens-Johnson syndrome and toxin epidemic neurology in Europe and Israel. journal of the American Academy of Dermatology.2008,58(1): 25-32). Benzbromarone is easily oxidized and metabolized into 6-hydroxybenzbromarone by CYP2C9, and further metabolized into o-bisquinone products at the 6, 7-position or 5, 6-position (Matthew G.McDonald, Retentie AE.sequential metabolism and bioactivation of the hepatotoxin benzebra: formation of glutathione adproducts from a hydrocarbon interlayer. chemical Research in biology.2007, 20(12): 1833-; benzbromarone contains phenolic hydroxyl, and is oxidized by CYP2C9 through an ipso-substitution reaction to generate 2, 6-dibromohydroquinone, which is further metabolized into a hydroquinone product (Yuminia Kitagawa, Tomoyuki Ohe, Kumiko Tachibana. novel biological activity Pathway of Benzbromoarone Mediated by Cytochrome P450.ASPET journal.2015). The chemical activity of these three biquinone products can lead to hepatotoxicity through conjugate addition with thiol groups on cysteine residues of proteins or polypeptides. In addition, benzbromarone has strong inhibition on CYP2C 9. This drug failed to enter the US market and was also released from some European countries in 2003 (Jansen TL, Reinders MK, van Roon EN, et al, Benzbrolone with draw from the European market: the other case of "absence of evidence of absence". Clinic Experimental Rheumatology,2004,22(5): 651). However, due to the lack of good anti-gout drugs in the market, more than 20 countries such as china, germany, japan, brazil, new zealand, etc. are still in wide use.
Disclosure of Invention
The invention aims to provide a series of novel compounds based on the prior art, and aims to obtain a URAT1 inhibitor with low toxicity and better drug effect for treating hyperuricemia or gout diseases.
The object of the invention can be achieved by the following measures:
a compound with a structure shown in a formula (I) or a formula (II) or a pharmaceutically acceptable salt thereof,
wherein the content of the first and second substances,
A1、A2、A3or A4Is CH or N;
g is carbonyl, sulfur, sulfuryl, sulfoxyl, optionally substituted methylene or imino;
R1selected from hydrogen,Deuterium, halogen, cyano, hydroxyl, nitro, amino, carboxyl, substituted amino or one or more of the following substituted or unsubstituted groups: c1-5Alkyl radical, C1-5Alkoxy or C1-5An alkylthio group;
R2selected from hydrogen, deuterium, halogen, cyano, hydroxy, nitro, substituted amino, C2-3Alkenyl radical, C2-3Alkynyl or one or more of the following substituted or unsubstituted groups: c1-4Alkyl radical, C1-5Alkoxy or C1-5An alkylthio group;
R3selected from the following substituted or unsubstituted groups: c1-4Alkyl or C3-4Cycloalkyl with substituents selected from deuterium, halogen, C1-2Alkyl or C3-4A cycloalkyl group;
m is an integer of 0 to 3;
n is an integer of 1 to 3;
the substituents in the group G are selected from hydroxy, cyano, nitro, amino, carboxyl or C1-3Alkoxy radical, R1Or R2Wherein the substituent is selected from deuterium, halogen, cyano, hydroxy, nitro, amino, C1-3Alkyl radical, C3-4Cycloalkyl or C1-3An alkoxy group.
When m is 2 or 3 in the invention, it means that the compound contains two R1A group, and the two R1The radicals may be identical or may each be R in the present application1Different groups as defined. When R is1Group and A1、A2、A3And A4When one or more ring atoms of (a) are attached, and when the ring atom is CH, the group at the ring atom is C-R1。
When n in the present invention is 2 or 3, it means that the compound contains two R2A group, and the two R2The radicals may be identical or may each be R in the present application2Different groups as defined.
Containing A1、A2、A3、A4The six-membered ring of (A) constitutes an aromatic ring, preferably, the aromatic ring is a benzene ring or a pyridazine ringA pyrimidine ring, a pyrazine ring or a pyridine ring.
In a preferred embodiment, A1Is CH, A2Is CH or N, A3Or A4Is CH.
In another preferred embodiment, A1And A4Is CH, A2And A3Each independently CH or N.
In one embodiment, R1Selected from hydrogen, deuterium, fluorine, chlorine, bromine, cyano, hydroxy, C1-3Alkyl radical, C1-3Haloalkyl, C1-3Deuterated alkyl, C1-3Alkoxy or C1-3One or more of deuterated alkoxy; m is 0, 1, 2 or 3.
In a preferred embodiment, R1One or more selected from hydrogen, deuterium, fluorine, chlorine, bromine, cyano, methyl, ethyl, methoxy, deuterated methoxy and ethoxy; m is 0, 1, 2 or 3.
In one embodiment, R2Selected from hydrogen, deuterium, halogen, cyano, nitro, ethenyl, ethynyl, C1-2Alkyl, substituted C1-2Alkyl radical, C1-2Alkoxy, substituted C1-2Alkoxy radical, C1-2Alkylthio, substituted C1-2One or more alkylthio groups; the substituents are selected from deuterium, halogen, C1-2Alkyl radical, C3-4Cycloalkyl or C1-3An alkoxy group; n is 1 or 2.
In a preferred embodiment, R2Selected from hydrogen, deuterium, halogen, cyano, C1-2Alkyl radical, C1-2Haloalkyl, C1-2Alkoxy or C1-2One or more alkylthio groups; n is 1 or 2.
In a more preferred embodiment, R2One or more selected from bromine, chlorine and cyano; n is 1 or 2.
In one embodiment, R3Selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl or cyclobutyl.
In a more preferred embodiment, the compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:
the preparation method of the compound with the structure shown in the formula (I) or the formula (II) comprises the following steps:
reaction of a substituted indazole (or pyridopyrazole) with a halide affords the corresponding 1-substituted compound (I-A) or 2-substituted compound (II-A). And (3) carrying out oxidation reaction and hydroxyl deprotection reaction on the product obtained after the reaction of the I-A and aryl aldehyde to obtain a corresponding hydroxyl compound, wherein the compound can be a final product, and can also be used for obtaining a corresponding target product (I) through halogenation reaction, reduction reaction or other reactions. Reacting the II-A with acyl chloride, and then carrying out hydroxyl deprotection reaction to obtain a corresponding hydroxyl compound which can be a final product, or can obtain a corresponding target product (II) through halogenation reaction, reduction reaction or other reactions. A1, A2, A3, A4, R1、R2And R3The definitions of (A) and (B) are as described in the claims and the general formula in the summary of the invention.
Unless otherwise indicated, the following terms used in the claims and specification have the following meanings:
"six-membered aromatic ring" refers to a fused ring group having a conjugated planar ring structure composed of six ring atoms, which has aromatic properties and the ring atoms may be other than carbon atoms, i.e., heteroatoms. When a heteroatom is contained in the six-membered aromatic ring, the heteroatom may be N, S or O, and the number of heteroatoms is not limited to one, and may be two, three, or the like. The six-membered aromatic ring containing a heteroatom in the present invention includes, but is not limited to, a pyridine ring, a pyrimidine ring, a pyrazine ring and the like.
"Hydrogen" means protium (1H), which is the predominant stable isotope of hydrogen.
"deuterium", which is a stable isotope of hydrogen, also known as deuterium, has the elemental symbol D.
"halogen" means a fluorine atom, chlorine atom, bromine atom or iodine atom.
"alkyl" means a saturated aliphatic radical of 1 to 20 carbon atoms, including straight and branched chain radicals (a numerical range referred to herein, e.g., "1 to 20", means that the radical, in this case alkyl, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). Alkyl groups having 1 to 4 carbon atoms are referred to as lower alkyl groups. When a lower alkyl group has no substituent, it is referred to as unsubstituted lower alkyl. More preferably, the alkyl group is a medium size alkyl group having 2 to 5 carbon atoms. Examples of the alkyl group in the present invention include methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl and the like. Preferably, the alkyl group is a lower alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, or the like. Alkyl groups may be substituted or unsubstituted.
"alkoxy" denotes the group-O- (unsubstituted alkyl) and-O- (unsubstituted cycloalkyl), which further denotes the group-O- (unsubstituted alkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.
"carbonyl" refers to a C ═ O group.
"sulfone group", represents-S (O)2-a group.
"sulfoxide group" means an-S (O) -group.
"methylene" means-CH2-a group.
"imino", represents an-NH-group.
"hydroxy" represents an-OH group.
"nitro" means-NO2A group.
"amino" means-NH2A group.
"carboxyl" represents a-COOH group.
"cyano" refers to the group-CN.
"pharmaceutically acceptable salts" are salts comprising a compound of formula (I) with an organic or inorganic acid, and refer to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:
(1) salts with acids are formed by reaction of the free base of the parent compound with inorganic acids such as, but not limited to, hydrochloric, hydrobromic, nitric, phosphoric, metaphosphoric, sulfuric, sulfurous, and perchloric acids or organic acids such as, but not limited to, acetic, propionic, acrylic, oxalic, (D) or (L) malic, fumaric, maleic, hydroxybenzoic, γ -hydroxybutyric, methoxybenzoic, phthalic, methanesulfonic, ethanesulfonic, naphthalene-1-sulfonic, naphthalene-2-sulfonic, p-toluenesulfonic, salicylic, tartaric, citric, lactic, mandelic, succinic, or malonic acids, and the like.
(2) The acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or is complexed with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, or the like.
"pharmaceutical composition" refers to a mixture of one or more compounds described herein, or their pharmaceutically acceptable salts and prodrugs, with other chemical components, such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.
The invention comprises a pharmaceutical composition, which comprises any one of the compounds, pharmaceutically acceptable salts thereof or easily hydrolyzed prodrug esters thereof as an active ingredient, or further comprises other compounds with pharmacodynamic activity as one of the active ingredients, and pharmaceutically acceptable auxiliary materials.
The compounds or the pharmaceutically acceptable salts thereof can be applied to the preparation of uric acid excretion promoting medicines, in particular to the preparation of medicines for treating or preventing hyperuricemia, nephropathy or gout. Experiments show that the compound provided by the invention has a very good inhibitory effect on uric acid transfer of hURAT1 in HEK293 transfected cells, and shows that the compound has a good application prospect in the aspect of treating hyperuricemia or gout.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the scope of the present invention is not limited to the following examples.
Example 1: synthesis of (3, 5-dibromo-4-hydroxyphenyl) (2-ethyl-2H-indazol-3-yl) methanone (6)
Step A: a mixture containing indazole (5.00g, 42.3mmol), iodoethane (13.2g, 90.3mmol), potassium hydroxide (5.50g, 98.0mmol) and ethanol (60mL) was stirred at 65 ℃ for 5 hours. Cooled to room temperature and filtered to remove insoluble matter. The solvent was distilled off under reduced pressure, followed by addition of water (30mL), extraction with methylene chloride (20 mL. times.3) and drying over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether ═ 1: 20-1: 5 elution) to give 2-ethyl-2H-indazole (1) (1.56g) and 1-ethyl-1H-indazole (2) (3.06 g). The yields were 25.2% and 49.5%, respectively. Compound 1:1H NMR(DMSO-d6400MHz) δ 8.37(s, 1H), 7.69(d, J ═ 8.4Hz, 1H), 7.60(d, J ═ 8.4Hz, 1H), 7.24-7.20(m, 1H), 7.05-7.01(m, 1H), 4.46(q, J ═ 7.2Hz, 2H), 1.51(t, J ═ 7.2Hz, 3H). Compound 2:1H NMR(DMSO-d6，400MHz)δ8.05(s,1H)，7.76(d，J＝8.0Hz，1H)，7.67(dd，J＝0.8，8.4Hz，1H)，7.41-7.36(m，1H)，7.15-7.11(m，1H)，4.45(q，J＝7.2Hz，2H)，1.40(t，J＝7.2Hz，3H)。
and B: to a solution of the compound 1(480mg, 3.28mmol) and diisopropylamine (432mg, 4.27mmol) in anhydrous THF (10mL) was added dropwise a 2.5M n-butyllithium n-hexane solution (1.7mL, 4.25mmol) at-70 to-80 ℃. After the addition is finished, stirring is continued for 10 minutes at the temperature, then the temperature is slowly raised to-10 to-20 ℃ after about 20 minutes, and stirring is continued for 15 minutes at the temperature. Cooled again to-70-80 ℃ and 4-methoxybenzaldehyde (514mg, 3.77mmol) was added via syringe. After stirring for about 15 minutes, the temperature was allowed to rise to room temperature naturally and stirring was continued overnight. Water (25mL) was added, extraction was performed with ethyl acetate (20 mL. times.3), and the combined organic phases were washed with saturated brine (15mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether ═ 1: 20-1: 3 elution) to give (2-ethyl-2H-indazol-3-yl) (4-methoxyphenyl) methanol (3) (536 mg). The yield thereof was found to be 57.9%.1H NMR(DMSO-d6，400MHz)δ7.52(d，J＝8.4Hz，1H)，7.42(d，J＝8.4Hz，1H)，7.30(d，J＝8.4Hz，2H)，7.19-7.15(m，1H)，6.93-6.89(m，3H)，6.35(d，J＝4.4Hz，1H)，6.30(d，J＝4.4Hz，1H)，4.40(q，J＝7.2Hz，2H)，3.73(s，3H)，1.30(t，J＝7.2Hz，3H)。
And C: to a solution of compound 3(320mg, 1.13mmol) in DMSO (10mL) was added 2-iodoxybenzoic acid (416mg, 1.49mmol), and the resulting mixture was stirred at room temperature overnight. Water (40mL) was added, extraction was performed with ethyl acetate (30 mL. times.3), and the combined organic phases were washed successively with water (20 mL. times.2) and saturated brine (15mL), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give (2-ethyl-2H-indazol-3-yl) (4-methoxyphenyl) methanone (4) (275 mg). The yield thereof was found to be 86.8%.
Step D: a mixture containing compound 4(267mg, 0.952mmol), 1M boron tribromide in toluene (2.9mL), and dichloromethane (15mL) was stirred at room temperature overnight. The reaction was slowly poured into crushed ice (40g), the pH was adjusted to 6-7 with 2M aqueous sodium hydroxide solution, extracted with dichloromethane (20 mL. times.3), and the combined organic phases were washed successively with saturated aqueous sodium bicarbonate solution (10mL) and saturated brine (10mL), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give (2-ethyl-2H-indazol-3-yl) (4-hydroxyphenyl) methanone (5) (200 mg). The yield thereof was found to be 78.9%.
Step E: a solution of bromine (257mg, 1.61mmol) in acetic acid (5mL) was added dropwise to a solution of compound 5(195mg, 0.732mmol) and sodium acetate (180mg, 2.19mmol) in acetic acid (10mL), and after the addition was complete, the resulting mixture was stirred at room temperature for 1 hour. The reaction was quenched with aqueous sodium bisulfite solution and most of the solvent was then distilled off under reduced pressure. Adding water (20mL), separating insoluble substances, filtering, and adding petroleum ether/acetic acid to filter cakeThe ethyl ester was recrystallized to give (3, 5-dibromo-4-hydroxyphenyl) (2-ethyl-2H-indazol-3-yl) methanone (6) (236 mg). The yield thereof was found to be 76.0%.1H NMR(DMSO-d6，400MHz)δ7.95(s，2H)，7.82(d，J＝8.8Hz，1H)，7.38-7.34(m，1H)，7.22-7.18(m，1H)，7.12(d，J＝8.4Hz，1H)，4.69(q，J＝7.2Hz，2H)，1.54(t，J＝7.2Hz，3H)。MS(EI，m/z)：422.9[M-H]-。
Example 2: synthesis of 2, 6-dibromo-4- [ (2-ethyl-2H-indazol-3-yl) hydroxymethyl ] phenol (7)
To a solution of compound 6(100mg, 0.236mmol) in methanol (10mL) was added sodium borohydride (90mg, 2.38mmol), and after the addition was complete, the resulting mixture was stirred at reflux for 30 minutes, then sodium borohydride (90mg, 2.38mmol) was added, and stirring continued at reflux for 1 hour. Water (20mL) was added, the pH was adjusted to 6-7 with 2M aqueous citric acid, extracted with ethyl acetate (20 mL. times.3), and the combined organic phases were washed with water (20mL) and dried over anhydrous sodium sulfate. The solvent is removed by reduced pressure evaporation, and the product is purified by column chromatography (200-300 mesh silica gel, ethyl acetate and petroleum ether are eluted at the ratio of 1: 20-1: 3) to obtain 2, 6-dibromo-4- [ (2-ethyl-2H-indazole-3-yl) hydroxymethyl group]Phenol (7).1H NMR(DMSO-d6，400MHz)δ9.99(s，1H)，7.55(d，J＝8.8Hz，1H)，7.51(s，2H)，7.38(d，J＝8.8Hz，1H)，7.21-7.17(m，1H)，6.97-6.93(m，1H)，6.51(d，J＝4.4Hz，1H)，6.35(d，J＝4.4Hz，1H)，4.43(q，J＝7.2Hz，2H)，1.35(t，J＝7.2Hz，3H)。MS(EI，m/z)：427.0[M+H]+。
Example 3: synthesis of (3, 5-dibromo-4-hydroxyphenyl) (1-ethyl-1H-indazol-3-yl) methanone (10)
Step A: comprises compound 2(2.59g, 17.7mmol), p-methoxybenzoyl chloride (3.02g, 17.7mmol) and anhydrous aluminium trichloride(3.54g, 26.6mmol) of the mixture was stirred at 105 ℃ overnight. After cooling to room temperature, water (30mL) and ethyl acetate (30mL) were added and stirred for about 5 minutes. The layers were separated, the aqueous layer was extracted with ethyl acetate (10 mL. times.3), and the combined organic phases were washed with water (15mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether ═ 1:25 to 1:15 elution) to give (1-ethyl-1H-indazol-3-yl) (4-methoxyphenyl) methanone (8) (990 mg). The yield thereof was found to be 21.1%.1H NMR(DMSO-d6，400MHz)δ8.34-8.29(m，3H)，7.88(d，J＝8.4Hz，1H)，7.56-7.52(m，1H)，7.41-7.37(m，1H)，7.15-7.11(m，2H)，4.63(q，J＝7.2Hz，2H)，3.89(s，3H)，1.51(t，J＝7.2Hz，3H)。
And B: a mixture containing compound 8(200mg, 0.714mmol), 1M boron tribromide in toluene (2.3mL), and dichloromethane (5mL) was stirred at room temperature overnight. The reaction was slowly poured into crushed ice (40g), the pH was adjusted to 6-7 with 2M aqueous sodium hydroxide solution, extracted with dichloromethane (20 mL. times.3), and the combined organic phases were washed successively with saturated aqueous sodium bicarbonate solution (10mL) and saturated brine (10mL), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give (1-ethyl-1H-indazol-3-yl) (4-hydroxyphenyl) methanone (9) (131 mg). The yield thereof was found to be 68.9%.
And C: a solution of bromine (236mg, 1.48mmol) in acetic acid (2mL) was added dropwise to a solution of compound 9(106mg, 0.398mmol) and sodium acetate (109mg, 1.33mmol) in acetic acid (3mL), and the resulting mixture was stirred at room temperature overnight. The reaction was quenched with aqueous sodium bisulfite solution and most of the solvent was then distilled off under reduced pressure. Water (20mL) was added, the pH was adjusted to 7-8 with saturated aqueous sodium bicarbonate, extracted with ethyl acetate (20 mL. times.2), and the combined organic phases were washed with water (10mL) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give (3, 5-dibromo-4-hydroxyphenyl) (1-ethyl-1H-indazol-3-yl) methanone (10) (70 mg). The yield thereof was found to be 41.4%.1H NMR(DMSO-d6，400MHz)δ8.53(s,2H)，8.28(d，J＝8.4Hz，1H)，7.89(d,J＝8.4Hz，1H)，7.54(d，J＝7.6Hz，1H)，7.39(d，J＝7.6Hz，1H)，4.63(q，J＝7.2Hz，2H)，1.52(t，J＝7.2Hz，3H)。MS(EI，m/z)：422.9[M-H]-。
Example 4: synthesis of 3- (3, 5-dibromo-4-hydroxybenzoyl) -1-ethyl-1H-indazole-5-carbonitrile (14)
Step A: selective fluorine (695mg, 1.96mmol) was added to a mixture containing compound 8(550mg, 1.96mmol), iodine (259mg, 1.02mmol) and acetonitrile (10mL) in an ice-water bath, and after the addition was complete, the resulting mixture was stirred at room temperature overnight. Water (30mL) was added and a 2M aqueous solution of sodium thiosulfate was added dropwise until the color disappeared. The mixture was extracted with ethyl acetate (20 mL. times.3), and the combined organic phases were washed successively with water (20mL) and saturated brine (20mL), and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, dichloromethane: petroleum ether ═ 1:3 elution) to give (1-ethyl-5-iodo-1H-indazol-3-yl) (4-methoxyphenyl) methanone (11) (290 mg). The yield thereof was found to be 36.4%.1H NMR(DMSO-d6，400MHz)δ8.67(s,1H)，8.35(d，J＝8.8Hz，2H)，7.82-7.76(m，2H)，7.13(d，J＝8.8Hz，2H)，4.61(q，J＝7.2Hz，2H)，3.89(s，3H)，1.49(t，J＝7.2Hz，3H)。
And B: a mixture containing Compound 11(190mg, 0.468mmol), cuprous cyanide (187mg, 2.09mmol) and DMF (4mL) was stirred at 130 ℃ overnight. After cooling to room temperature, ethyl acetate (20mL) was added, and insoluble materials were removed by filtration through Celite. Water (20mL) was added, the layers were separated, the aqueous layer was extracted with ethyl acetate (20 mL. times.3), the combined organic layers were washed successively with water (20 mL. times.2) and saturated brine (20mL), and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give crude 1-ethyl-3- (4-methoxybenzoyl) -1H-indazole-5-carbonitrile (12) (150 mg). This compound was used in the next reaction without purification.1H NMR(DMSO-d6，400MHz)δ8.72(s,1H)，8.37(d，J＝8.8Hz，2H)，8.13(d，J＝8.8Hz，1H)，7.90(d，J＝8.8Hz，1H)，7.15(d，J＝8.8Hz，2H)，4.67(q，J＝7.2Hz，2H)，3.90(s，3H)，1.51(t，J＝7.2Hz，3H)。
And C: A1M toluene solution of boron tribromide (2mL) was added dropwise to a crude solution of Compound 12(150mg) in dichloromethane (5mL) in an ice-water bath, and after the addition was complete, the resulting mixture was stirred at room temperature overnight. The reaction was slowly poured into ice water (20mL), the pH was adjusted to 6-7 with 2M aqueous sodium hydroxide, extracted with dichloromethane (20 mL. times.3), and the combined organic phases were washed successively with saturated aqueous sodium bicarbonate (10mL) and saturated brine (10mL), and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether ═ 1: 20-1: 3 elution) to give 1-ethyl-3- (4-hydroxybenzoyl) -1H-indazole-5-carbonitrile (13) (72 mg). The total yield of the two reactions of the steps B and C is 52.5%.
Step D: NBS (97mg, 0.545mmol) was added to a solution of compound 13(72mg, 0.247mmol) in DMF (3mL), and the resulting mixture was stirred at room temperature for 1.5 h. Water (15mL) was added, filtered, the filter cake rinsed with a small amount of water, and the resulting solid was recrystallized from ethyl acetate to give 3- (3, 5-dibromo-4-hydroxybenzoyl) -1-ethyl-1H-indazole-5-carbonitrile (14).1H NMR(DMSO-d6，400MHz)δ8.71(s,1H)，8.54(s，2H)，8.15(d，J＝8.8Hz，1H)，7.92(d，J＝8.8Hz，1H)，4.69(q，J＝7.2Hz，2H)，1.53(t，J＝7.2Hz，3H)。MS(EI，m/z)：447.9[M-H]-。
Example 5: synthesis of 2, 6-dibromo-4- [ (2-ethyl-2H-indazol-3-yl) hydroxymethyl ] phenol (15)
A mixture containing compound 10(150mg, 0.354mmol), sodium borohydride (120mg, 3.17mmol) and THF (5mL) was stirred at room temperature overnight. Water (20mL) was added, extraction was performed with ethyl acetate (20 mL. times.2), and the combined organic phases were washed with water (10mL) and dried over anhydrous sodium sulfate. The solvent is removed by evaporation under reduced pressure to obtain 2, 6-dibromo-4- [ (2-ethyl-2H-indazol-3-yl) hydroxymethyl]Phenol (15) (119 mg). The yield thereof was found to be 78.9%.1H NMR(DMSO-d6，400MHz)δ9.84(s，1H)，7.71(d，J＝8.4Hz，1H)，7.61(d，J＝8.4Hz，1H)，7.56(s，2H)，7.37-7.33(m，1H)，7.10-7.06(m，1H)，6.23(d，J＝4.4Hz，1H)，6.02(d，J＝4.4Hz，1H)，4.40(q，J＝7.2Hz，2H)，1.37(t，J＝7.2Hz，3H)。MS(EI，m/z)：426.9[M+H]+。
Example 6: synthesis of (3-bromo-4-hydroxy-5-methylphenyl) (2-isopropyl-2H-indazol-3-yl) methanone (21)
Experimental procedure for Steps A, B, C and D the procedure was followed for the preparation of Steps A, B, C and D of example 1 wherein iodoethane in step A of example 1 was replaced with bromoisopropane and 4-methoxybenzaldehyde in step B of example 1 was replaced with 3-methyl-4-methoxybenzaldehyde. Compound 20:1H NMR(DMSO-d6，400MHz)δ10.57(s，1H)，7.81-7.79(m，1H)，7.64(s，1H)，7.56-7.53(m，1H)，7.34-7.30(m，1H)，7.14-7.08(m，2H)，6.95-6.93(m，1H)，5.30-5.26(m，1H)，2.17(s，3H)，1.58(d，J＝6.4Hz，6H)。
step E: NBS (69mg, 0.388mmol) was added to a solution of compound 20(95mg, 0.323mmol) in DMF (10mL) and after the addition was complete, the resulting mixture was stirred at room temperature for 1 hour. Water (40mL) was added, extraction was performed with ethyl acetate (20 mL. times.3), and the combined organic phases were washed successively with water (10 mL. times.2) and saturated brine (10mL), and dried over anhydrous sodium sulfate. The solvent is evaporated under reduced pressure, and the product is purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether ═ 1:5 elution) to obtain (3-bromo-4-hydroxy-5-methylphenyl) (2-isopropyl-2H-indazol-3-yl) methanone (21).1H NMR(DMSO-d6，400MHz)δ10.26(s，1H)，7.84-7.80(m，2H)，7.63(s，1H)，7.35-7.32(m，1H)，7.17-7.14(m，1H)，7.08-7.06(m，1H)，5.34-5.28(m，1H)，2.29(s，3H)，1.59(d，J＝6.4Hz，6H)。MS(EI，m/z)：373.1[M+H]+。
Example 7: synthesis of 2-bromo-4- [ hydroxy (2-isopropyl-2H-indazol-3-yl) methyl ] -6-methylphenol (22)
Synthesis of compound 22 using compound 21 as a starting material was carried out in accordance with the procedure of example 2.1H NMR(DMSO-d6，400MHz)δ9.05(s，1H)，7.56(d，J＝8.4Hz，1H)，7.48(d，J＝8.4Hz，1H)，7.30(s，1H)，7.20-7.16(m，1H)，7.03(s，1H)，6.96-6.92(m，1H)，6.38-6.33(m，2H)，5.01-4.96(m，1H)，2.17(s，3H)，1.43(d，J＝6.4Hz，3H)，1.31(d，J＝6.4Hz，3H)。MS(EI，m/z)：375.1[M+H]+。
Example 8: synthesis of 3-bromo-5- (2-ethyl-2H-indazole-3-carbonyl) -2-hydroxybenzonitrile (28)
Step A: a mixture containing 2-bromo-4-hydroxybenzaldehyde (3.0g, 14.9mmol), cuprous cyanide (1.74g, 19.4mmol) and N-methylpyrrolidone (15mL) was stirred at 180 ℃ for 5 hours. Cooled to room temperature, ethyl acetate (50mL) and water (50mL) were added. After filtration through celite, the layers were separated, the aqueous phase was extracted with ethyl acetate (40 mL. times.4), and the combined organic phases were washed with saturated brine (30mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: dichloromethane ═ 1:15 elution) to give 5-formyl-2-hydroxybenzonitrile (23) and a small amount of residual N-methylpyrrolidone, the total weight of the mixture being 2.40g, which was then used directly in the next reaction. MS (EI, m/z): 146.0[ M-H]-。
And B: chloromethyl methyl ether (1.26g, 15.7mmol) was added to a solution of crude compound 23(2.30g) and diisopropylethylamine (2.60g, 20.1mmol) in dichloromethane (25mL) in an ice-water bath, and after addition, the resulting mixture was stirred at room temperature overnight. Water (40mL) was added, the mixture was extracted with dichloromethane (40 mL. times.3), and the combined organic phases were washed with saturated brine (30mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, dichloromethane: petroleum ether ═ 1: 10-20: 1 elution) to give 5-formyl-2- (methoxymethoxy) benzonitrile (24) (673 mg). The total yield of the two reactions of the step A and the step B is 24.7 percent.
Experimental procedure for steps C and D following the procedure of example 1, steps B and C, 5- (2-ethyl-2H-indazol-3-yl) hydroxymethyl-2- (methoxymethoxy) benzonitrile (26) was obtained.
Step E: to a solution of compound 26(285mg, 0.845mmol) in dichloromethane (5mL) was added trifluoroacetic acid (1mL), and the resulting mixture was stirred at room temperature overnight. Water (30mL) was added, the pH was adjusted to 7-8 with a saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate (30 mL. times.3), and the combined organic phases were washed with a saturated brine (20mL) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give 5- (2-ethyl-2H-indazole-3-carbonyl) -2-hydroxybenzonitrile (27) (230 mg). The yield thereof was found to be 93.4%.
Experimental procedure for step F3-bromo-5- (2-ethyl-2H-indazole-3-carbonyl) -2-hydroxybenzonitrile (28) was obtained according to the preparation method of example 6, step E.1H NMR(DMSO-d6，400MHz)δ8.00(d，J＝2.4Hz，1H)，7.75(d，J＝8.8Hz，1H)，7.68(d，J＝2.4Hz，1H)，7.34-7.31(m，2H)，7.19-7.15(m，1H)，4.59(q，J＝7.2Hz，2H)，1.49(t，J＝7.2Hz，3H)。MS(EI，m/z)：368.0[M-H]-。
Example 9: synthesis of (3, 5-dibromo-4-hydroxyphenyl) (2-ethyl-2H-pyrazolo [3,4-c ] pyridin-3-yl) methanone (29)
Synthesis of compound 29 was prepared according to the method of example 1, wherein the indazole of example 1, step A was substituted with 1H-pyrazolo [3,4-c ]]Pyridine is substituted.1H NMR(DMSO-d6，400MHz)δ9.39(s，1H)，8.20(d，J＝5.6Hz，1H)，7.96(s，2H)，7.10(d，J＝5.6Hz，1H)，4.77(q，J＝7.2Hz，2H)，1.57(t，J＝7.2Hz，3H)。MS(EI，m/z)：424.0[M-H]-。
Example 10: synthesis of 2, 6-dibromo-4- { deuterium (2-ethyl-2H-pyrazolo [3,4-c ] pyridin-3-yl) hydroxymethyl } phenol (30)
To a solution of compound 29(65mg, 0.154mmol) in THF (6mL) was added sodium borodeuteride (32mg, 0.765mmol), and the resulting mixture was stirred at room temperature overnight. Water (20mL) was added, the pH was adjusted to 7-8 with 2M citric acid solution, and the mixture was extracted with ethyl acetate (20 mL. times.3), and the combined organic phases were washed with saturated brine (15mL) and dried over anhydrous sodium sulfate. The solvent is removed by reduced pressure evaporation, and the product is purified by column chromatography (200-300 mesh silica gel, ethyl acetate and petroleum ether are eluted at the ratio of 1: 3-2: 1) to obtain 2, 6-dibromo-4- { deuterium (2-ethyl-2H-pyrazolo [3, 4-c)]Pyridin-3-yl) hydroxymethyl } phenol (30).1H NMR(DMSO-d6，400MHz)δ10.07(s，1H)，9.24(s，1H)，7.88(d，J＝5.6Hz，1H)，7.57-7.55(m，3H)，6.74(s，1H)，4.61-4.55(m，2H)，1.40(t，J＝7.2Hz，3H)。MS(EI，m/z)：426.0[M-H]-。
Example 11: synthesis of (7-bromo-2-ethyl-6-methoxy-2H-indazol-3-yl) (3, 5-dibromo-4-hydroxyphenyl) methanone (37)
Step A: 2-fluoro-4-methoxybenzaldehyde (6.5g, 42.2mmol) and 85% hydrazine hydrate (50mL) were stirred at 120 ℃ for 30 hours with a sealed tube. Water (50mL) was added, extraction was performed with ethyl acetate (50 mL. times.3), and the combined organic phases were washed with water (20 mL. times.2). And then adding water (100mL) into the organic phase, adjusting the pH value to 1-2 by using 2M hydrochloric acid, and layering to obtain a product in the water phase. Adjusting the pH value of the water phase to 8-9 by using 2M sodium hydroxide solution, extracting by using ethyl acetate (50mL multiplied by 3), and drying by using anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was recrystallized from methyl t-butyl ether/petroleum ether to give 6-methoxy-1H-indazole (31) (3.47 g). The yield thereof was found to be 55.5%.
And B: a mixture containing compound (3.43g, 23.2mmol), iodoethane (9.06g, 58.1mmol), potassium hydroxide (3.41g, 60.8mmol) and ethanol (25mL) was stirred at reflux overnight. Most of the solvent was distilled off under reduced pressure, water (40mL) was added, extraction was performed with methylene chloride (40 mL. times.5), and drying was performed over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether ═ 1: 20-1: 5 elution) to give 2-ethyl-6-methoxy-2H-indazole (32) (1.48g) and 1-ethyl-6-methoxy-1H-indazole (33) (2.44 g). The yields were 36.2% and 59.7%, respectively.
Experimental procedure for steps C, D and E the procedure of example 8, steps C, D and E was followed, wherein compound 24 in step C of example 8 was replaced with 4-methoxymethylbenzaldehyde, to give (2-ethyl-6-methoxy-2H-indazol-3-yl) (4-hydroxyphenyl) methanone (36).1H NMR(DMSO-d6，400MHz)δ10.58(s,1H)，7.72-7.68(m，2H)，7.11(d，J＝4.0Hz，1H)，6.98-6.90(m，3H)，6.82-6.78(m，1H)，4.58(q，J＝6.8Hz，2H)，3.81(s，3H)，1.48(t，J＝6.8Hz，3H)。
Experimental procedure for step F using compound 36 as a starting material according to the preparation method of example 1, step E, (7-bromo-2-ethyl-6-methoxy-2H-indazol-3-yl) (3, 5-dibromo-4-hydroxyphenyl) methanone (37) was obtained.1H NMR(DMSO-d6，400MHz)δ7.96(s,2H)，7.23(d，J＝9.2Hz，1H)，7.11(d，J＝9.2Hz，1H)，4.64(q，J＝6.8Hz，2H)，3.92(s，3H)，1.52(t，J＝6.8Hz，3H)。MS(EI，m/z)：530.8[M-H]-。
Example 12: synthesis of (7-bromo-2-ethyl-6-trideuteromethoxy-2H-indazol-3-yl) (3, 5-dibromo-4-hydroxyphenyl) methanone (43)
Step A: a mixture containing compound 32(800mg, 4.54mmol), 1M boron tribromide in toluene (11mL), and dichloromethane (20mL) was stirred at room temperature overnight. The reaction was slowly poured into crushed ice (60g), the pH was adjusted to 6-7 with 2M aqueous sodium hydroxide solution, extracted with dichloromethane (30 mL. times.4), and the combined organic phases were washed successively with saturated aqueous sodium bicarbonate solution (25mL) and saturated brine (20mL), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give 2-ethyl-2H-indazol-6-ol (38) (710 mg). The yield thereof was found to be 96.4%.
And B: a mixture containing compound 38(500mg, 3.08mmol), potassium carbonate (852mg, 6.17mmol), deuterated iodomethane (581mg, 4.01mmol), and DMF (10mL) was stirred at 30 ℃ overnight. Water (40mL) was added, and the mixture was extracted with methylene chloride (40 mL. times.3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether: 1: 15-1: 1 elution) to give 2-ethyl-6-trideuteromethoxy-2H-indazole (39) (410 mg). The yield thereof was found to be 81.6%.
Experimental procedure for steps C, D, E and F (7-bromo-2-ethyl-6-trideuteromethoxy-2H-indazol-3-yl) (3, 5-dibromo-4-hydroxyphenyl) methanone (43) was obtained according to the preparation method of example 8, step C, D, E and F.1H NMR(DMSO-d6，400MHz)δ7.97(s,2H)，7.22(d，J＝9.2Hz，1H)，7.11(d，J＝9.2Hz，1H)，4.65(q，J＝7.2Hz，2H)，1.52(t，J＝7.2Hz，3H)。MS(EI，m/z)：533.9[M-H]-。
Example 13: synthesis of (3, 5-dibromo-4-hydroxyphenyl) (2-ethyl-5-methyl-2H-indazol-3-yl) methanone (44)
Synthesis of compound 44 was prepared according to the method of example 1, wherein the indazole of example 1, step A, was replaced with 5-methylindazole.1H NMR(DMSO-d6，400MHz)δ7.95(s,2H)，7.73(d，J＝8.8Hz，1H)，7.21(d，J＝8.8Hz，1H)，6.90(s，1H)，4.66(q，J＝7.2Hz，2H)，2.30(s，3H)，1.51(t，J＝7.2Hz，3H)。MS(EI，m/z)：436.9[M-H]-。
Example 13: synthesis of (3, 5-dibromo-4-hydroxyphenyl) (2-ethyl-6-fluoro-2H-indazol-3-yl) methanone (45)
Synthesis of Compound 45 was prepared according to the method of example 1, wherein the indazole of example 1, step A was replaced with 6-fluoro indazoleAnd (4) generation.1H NMR(DMSO-d6，400MHz)δ7.92(s,2H)，7.59(d，J＝9.6Hz，1H)，7.22-7.19(m，1H)，7.14-7.10(m，1H)，4.65(q，J＝7.2Hz，2H)，1.52(t，J＝7.2Hz，3H)。MS(ESI，m/z)：440.9[M-H]-。
Example 14 inhibition of transport of uric acid by the Compounds in HEK293 transfected cell line hURAT1
First, reagent name and source:
zurampic is available from Oncomen super medicine science and technology, Inc.; plasmid pCMV6-hURAT1 was purchased from origin Technologies, Inc; g418 was purchased from Biotechnology, Inc.; the HEK293 cell strain is purchased from the cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences; polylysine was purchased from Sigma-Aldrich co.llc;14c-uric acid is available from American radio laboratory Chemicals, Inc; sodium gluconate, potassium gluconate, calcium gluconate, KH2PO4、MgSO4Glucose and HEPES were purchased from national drug group chemicals, ltd; DMEM medium, fetal bovine serum was purchased from Thermo Fisher Scientific Inc;
II, test method and result:
1. constructing HEK293 stable transfer cell strain with high expression of hURAT 1: plasmid pCMV6-hURAT1 is transfected into HEK293 cells, and then G418 (final concentration 500 mug/mL) resistance screening is carried out to obtain a stable cell strain which highly expresses hURAT1 transport membrane protein and can be used for an inhibition test for in vitro hURAT1 transport of uric acid (Weaver YM, Ehresman DJ, Butenhoff JL, et al. circles of rate crude organic transport in transporting fluorinated carbohydrates with differential chain length hs. toxicolial Sciences,2009,113(2):305 and 314).
2. Coating 24-hole plate: polylysine was added at 0.1mg/mL per well at 200. mu.l/well and allowed to stand overnight. The polylysine was removed, washed with sterile water and dried thoroughly for future use.
3. The HEK293-hURAT1 cells were stably transfected at 2X 105The cells/well were plated in coated 24-well plates at 37 ℃ with 5% CO2Cultured for 3 days.
Preparation of HBSS: according to the weight ratio of 125mM sodium gluconate and 4.8mM glucosePotassium, 1.3mM calcium gluconate, 1.2mM KH2PO4、1.2mM MgSO4Weighing each reagent according to the final concentration of 5.6mM glucose and 25mM HEPES, adding deionized water to a constant volume to a corresponding volume, fully and uniformly mixing to obtain HBSS solution with pH of 7.4, and storing in a refrigerator at-20 ℃.
5. On the day of the experiment, the HBSS was removed from the refrigerator and heated in a water bath to 37 ℃. Taking out the 24-hole cell culture plate, washing 2 times of cells by HBSS, completely sucking, adding HBSS according to 160 mul/hole, and adding test compound with final concentration of 500nM according to 20 mul/hole to serve as test compound hole; HBSS was added at 180. mu.l/well without test compound and used as a blank control well. Standing at room temperature for 10 min.
6. Add 20. mu.l/well of 50. mu.M final14And C, placing the mixture at room temperature for 20 min.
7. The solution was pipetted off each well and the cells were washed with pre-cooled HBSS and pipetted off. Finally, 0.2M NaOH is added to dissolve cells, cell debris is collected and a proper amount of scintillation fluid is added, and then the cell debris is placed on a PerkinElmer Microbeta Trilux 1450 liquid scintillation analyzer to detect isotopes14C radioactivity in uric acid (CPM value).
8. The inhibition rate of the compound on transport of uric acid by hURAT1 in HEK293 transfected cell line is calculated as follows, and CPM value of the test compound is expressed as CPM(test Compound)Represents; CPM value of blank control with CPM(blank control)And (4) showing. The test compounds were repeated three times, the test results were averaged, and the standard deviation SD was calculated. The test results are shown in Table 1.
Third, test results
Compared with Zurampic, the compounds of the invention (especially compounds 6,7, 10, 15, 28, 29, 30 and 44) had very good inhibitory effect on transport of uric acid by hURAT1 in HEK293 transfected cells at a concentration of 500 nM.
TABLE 1 inhibition of transport of uric acid by hURAT1 in HEK293 transfected cell lines by test Compounds and Zurampic
Claims (10)
1. A compound having a structure represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof,
wherein the content of the first and second substances,
A1、A2、A3or A4Is CH or N;
g is carbonyl, sulfur, sulfuryl, sulfoxyl, optionally substituted methylene or imino;
R1selected from one or more of hydrogen, deuterium, halogen, cyano, hydroxyl, nitro, amino, carboxyl, substituted amino or substituted or unsubstituted following groups: c1-5Alkyl radical, C1-5Alkoxy or C1-5An alkylthio group;
R2selected from hydrogen, deuterium, halogen, cyano, hydroxy, nitro, substituted amino, C2-3Alkenyl radical, C2-3Alkynyl or one or more of the following substituted or unsubstituted groups: c1-4Alkyl radical, C1-5Alkoxy or C1-5An alkylthio group;
R3selected from the following substituted or unsubstituted groups: c1-4Alkyl or C3-4Cycloalkyl with substituents selected from deuterium, halogen, C1-2Alkyl or C3-4A cycloalkyl group;
m is an integer of 0 to 3;
n is an integer of 1 to 3;
the substituents in the group G are selected from hydroxy, cyano, nitro, amino, carboxyl or C1-3Alkoxy radical, R1Or R2Wherein the substituent is selected from deuterium, halogen, cyano, hydroxy, nitro, amino, C1-3Alkyl radical, C3-4CycloalkanesRadical or C1-3An alkoxy group.
2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1Selected from hydrogen, deuterium, fluorine, chlorine, bromine, cyano, hydroxy, C1-3Alkyl radical, C1-3Haloalkyl, C1-3Deuterated alkyl, C1-3Alkoxy or C1-3One or more of deuterated alkoxy; m is 0, 1, 2 or 3.
3. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R2Selected from hydrogen, deuterium, halogen, cyano, nitro, ethenyl, ethynyl, C1-2Alkyl, substituted C1-2Alkyl radical, C1-2Alkoxy, substituted C1-2Alkoxy radical, C1-2Alkylthio, substituted C1-2One or more alkylthio groups; the substituents are selected from deuterium, halogen, C1-2Alkyl radical, C3-4Cycloalkyl or C1-3An alkoxy group; n is 1 or 2.
4. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R2Selected from hydrogen, deuterium, halogen, cyano, C1-2Alkyl radical, C1-2Haloalkyl, C1-2Alkoxy or C1-2One or more alkylthio groups; n is 1 or 2.
5. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R3Selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl or cyclobutyl.
6. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein a1And A4Is CH, A2And A3Each independently CH or N.
7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:
8. a pharmaceutical composition, which comprises the compound or the pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 7 as an active ingredient or a main active ingredient, and pharmaceutically acceptable auxiliary materials.
9. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for promoting uricosuric acid.
10. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of hyperuricemia, nephropathy or gout.
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| KR20200112908A (en) * | 2018-01-19 | 2020-10-05 | 쑤저우 시노벤트 파마슈티칼즈 씨오., 엘티디. | Heterocyclic compounds, methods of manufacture and their use in pharmaceuticals |
| CN113527236B (en) * | 2021-08-20 | 2022-12-23 | 苏州弘森药业股份有限公司 | Method for preparing amiodarone hydrochloride |
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