CN108516971A - A kind of benzo [b] thiophenes and its application in fat and diabetes - Google Patents

Abstract

Application the invention discloses a kind of benzo [b] thiophenes and its in fat and diabetes,Wherein：R₁、R₂、R₃It is independently selected from H, F or CH₃.Tested by pharmacological activity when confirming 10 μM of the compounds of this invention has preferable inhibitory activity to GOAT, GOAT inhibitor can be used as to carry out more extensive pharmacological effect experiment in the disease animal models such as fat and diabetes, to obtain the fat and new medicine of diabetes.

Description

A kind of benzo [b] thiophenes and its application in fat and diabetes

Technical field

The invention belongs to chemical medicine, it is related to a kind of benzo [b] thiophenes and its in fat and diabetes In application.

Background technology

Today's society, obesity patient's quantity increase severely in the world, and show the apparent trend that becomes younger, overweight With fat so that ascendant trend is presented in the incidence of other Chronic Non-Communicable Diseases of China, it is strong that obesity has become influence resident One of public safety problem of health.Obesity needs to obtain long-term treatment as a kind of chronic metabolic class disease.It is moving With lifestyle change, diet control it is invalid in the case of, drug therapy is particularly important.Currently, anti-obesity drug Developing history is relatively short, requires higher to Drug safety and validity, in recent years, anti-obesity medicament research and development causes Researchers pay close attention to, but clinically bariatrician is very few.Meanwhile some marketed drugs because drug safety with Side-effect problem and be forced to remove city, be currently available that anti-obesity drug is very few.

Diabetes are common disease in crowd, high morbidity, are with high caused by hypoinsulinism or insulin resistance The incretion metabolism disease that blood glucose is characterized has become the third-largest harm human health for being only second to angiocardiopathy and cancer Disease.Diabetes are the impaired diseases of Patients' rights blood glucose ability, and patient has lost to some extent makees insulin With the ability for making appropriate reaction.It is largely type II diabetes (i.e. Non-Insulin Dependent Diabetes Mellitus) in diabetes, accounts for about 80%-90%, the study found that the insulin resistance of peripheral tissues' (including skeletal muscle, liver and adipose tissue etc.) is in II type sugar It urinates in sick generation, development and plays particularly important effect.

Ghrelin O- acyltransferases (ghrelin O-acyltransferase, GOAT) belong to film combination O- acyltransferases (MBOAT) enzyme family.Aliphatic acid by being transferred to the Ser3 residues of acyl group ghrelin by it Acyl group-ghrelin (also referred to as 1-28 UAG or UAG) will be gone to be converted to biologically active form acyl group-life Long element release peptide (AG).Acyl group-ghrelin in the mankind and rodent has been displayed to increase food intake and increase fertilizer It is fat.Also it has been displayed and is transfused the insulin secretion that AG inhibits glucose induction in people.Disappearing for ghrelin gene has been displayed The glucose intolerance in ob/ob mouse except enhancing insulin releasing to prevent or improve feeding high fat diet.

However, fat and diabetes prevalences and obesity are with the different validity of the Current therapeutic of diabetes and to treatment Reaction make patient it is necessary to obtain more therapeutic choices.GOAT inhibitor as diet and/or movement, be designed to subtract The auxiliary of few weight gain or the fat other healing potions or program for the treatment of, it can also be used to reduce weight gain or weight is anti- Bullet.GOAT inhibitor can combine individually or with the other therapies of diabetes B and be used to treat diabetes B.

Invention content

The invention discloses a kind of benzo [b] thiophenes, general structure is formula I

Wherein：R₁、R₂、R₃It is independently selected from H, F or CH₃。 The invention further relates to the pharmaceutically acceptable salt of the Formula I or solvates.

Further, benzo [b] thiophenes formula described in some preferred schemes I are

The synthetic route that another object of the present invention discloses benzo [b] the thiophenes formula I is：

Specifically synthetic method is：Its synthesis step is as follows：

1) hydroxyl position (- OH) and 5- methylpyrazine -2- carbonyls chlorine (compound 2) are sent out in compound 1 in a suitable solvent Raw condensation reaction, generates compound 3；

2) halogenation generation compound 4 occurs for compound 3 and NBS；

3) under cryogenic conditions, the sulphur in benzothiophene ring in compound 4 is oxidized to sulfinyl and generates compound 5；

4) compound 5 is undergone coupling reaction to produce with corresponding alcohol under the action of solvent DMF and inorganic base corresponding Sulfinyl derivative；

5) sulfinyl in previous step product is in H₂Hydrogenization and Pd/C catalytic action under be reduced to sulphur, it is raw At final product benzo [b] thiophene derivants.

Wherein, the solvent in the step 1) can be dichloromethane, 1,2- dichloroethanes, second eyeball, N, N- dimethyl methyls Amide (DMF), tetrahydrofuran, ethyl acetate, toluene, dimethylbenzene or other suitable solvents, preferably tetrahydrofuran.

Wherein, the temperature range of sub-cooled is -15~-10 DEG C, preferably -10 DEG C in the step 3).

Wherein, the inorganic base in the step 4) can be potassium ethoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide etc., preferably Potassium tert-butoxide.

Another object of the present invention provides the benzo [b] thiophenes and turns as ghrelin O- acyl groups Move the application of enzyme (GOAT) inhibitor.

Another object of the present invention provides the benzo [b] thiophenes in prevention or obesity treating medicine Application.

Another object of the present invention provides the benzo [b] thiophenes in preventing or treating diabetes medicament Application.Further, the diabetes are type II diabetes and/or obese diabetic.

Related disease further includes with ghrelin (ghrelin)：Obesity, interior fat syndrome, non-alcohol Property fatty liver, nonalcoholic steatohepatitis, metabolic syndrome, diabetes are (for example, type-1 diabetes mellitus, type-2 diabetes mellitus, the gestational period Diabetes, obese diabetes), post prandial hyperglycemia, hyperlipidemia (for example, hypertriglyceridemia, hypercholesterolemia, High LDL- cholesteremias, low HDL-cholesteremia, postprandial hyperlipemia), the complication of diabetes (for example, neuropathy, nephrosis, Retinopathy, cardiomyopathy, huge angiosis, sclerotin reduction, hyperosmolar coma, gangrene, xerostomia, hypacusia, cerebrovascular condition, Peripheral blood circulatory disorders, infection are (for example, respiratory tract infection, urinary tract infections, gastrointestinal infection, epidermis soft tissue infection, lower limb sense Dye), diabetic gangrene), digestive disease (for example, irritable bowel syndrome, acute or chronic diarrhea, functional gastrointestinal disease, Ulcerative colitis, acute corrosive esophagitis, acute corrosive gastritis, Crohn disease, acute pancreatitis, chronic pancreatitis), kidney Dirty disease is (for example, chronic nephritis, nephrosis, glomerulonephritis, glomerulosclerosis, kidney failure, End-stage renal disease Disease, hypertensive nephrosclerosis, pyelonephritis, water nephrosis), circulation system disease is (for example, ischemic heart disease (myocardial infarction, the heart Angina), cardiomegaly, cardiomyopathy, hypertension, artery sclerosis, arrhythmia cordis, heart failure, coronary heart disease, endocarditis, artery Tumor, mitral valve prolapse, venous thromboembolism, valvular inadequacy), autoimmune disease is (for example, rheumatoid arthritis, more Hair property hardening, psoriasis, systemic loupus erythematosus, Sjogren's syndromes) etc. and growth disorder (for example, acra is fertile Big disease, giantism), Chronic Obstructive Pulmonary Disease, pneumonia, spill smart dependence, excitant dependence, narcotic dependence, tobacco Dependence, gambling dependence, eating disorder are (for example, bulimia, neuropath's bulimia, mad food illness, Prader- Willi syndromes), prostate cancer, breast cancer, hypophysis tumor, ovarian neoplasm, carcinoma of uterine body, neck cancer, etc..

Pharmaceutically acceptable salt of the present invention refers to the organic salt and inorganic salts of the compounds of this invention.Pharmaceutically may be used The acid-addition salts of receiving can be formed with inorganic acid and organic acid, including but not limited to, acetate, aspartate, benzene first Hydrochlorate, benzene sulfonate, bromide/hydrobromate, bicarbonate/carbonate, disulfate/sulfate, camsilate (camphorsulfornate), chloride/hydrochloride, chloro theophylline salt (chlortheophyllonate), citrate, second two Sulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydriodate/iodide, hydroxyl second Base sulfonate, lactate, Lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, Mesylate, Methylsulfate, naphthoate, naphthalene sulfonate, nicotinate, nitrate, octadecane hydrochlorate, oleate, oxalates, Palmitate, embonate, phosphate/phosphor acid hydrogen salt/dihydric phosphate, Polygalacturonate, propionate, stearic acid Salt, succinate, sulfosalicylate, tartrate, toluene fulfonate and trifluoroacetate.Can the nothing of salt be obtained by its derivative Machine acid includes such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid.The organic acid that salt can be obtained by its derivative includes such as second Acid, propionic acid, glycolic, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, Methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid etc..

Solvate of the present invention refers to that one or more solvent molecules are formed by with the compound of the present invention and form Close object.The solvent for forming solvate includes, but is not limited to, water, isopropanol, ethyl alcohol, methanol, dimethyl sulfoxide, ethyl acetate, Acetic acid and ethylaminoethanol.

Specific embodiment

Embodiment 1：The synthesis of 2- (furans -2- oxygroups) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates

The synthesis of 1-1, benzo [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters：

In a nitrogen atmosphere, by benzo [b] thiophene -6- alcohol (compound 1) (5.86g, 39.01mmol), 4- dimethylaminos After pyridine DMAP (473mg, 3.87mmol) is dissolved in together in THF (100mL), add triethylamine (4.15g, 41.03mmol) and 5- methylpyrazine -2- carbonyls chlorine (compound 2) (6.42g, 40.99mmol), being heated to reflux mixture, to hold 7 small When, then the reaction is cooled to 50 DEG C.Add the mixture of water (20mL) and acetic acid (3.67mL), the solution being layered. Separation organic layer simultaneously gives up water layer, you can obtains benzo [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters (compound 3) The solution is directly used in the synthesis of next step by THF solution, 10.54g.¹H-NMR(400MHz,CDCl₃)δ:2.59(s, 3H),7.32(d,2H),7.42(d,1H),7.58(s,1H),7.75(d,1H),8.88(s,1H),9.16(s,1H).¹³C-NMR (125MHz,CDCl₃)δ:21.51,113.16,118.59,122.13,122.15,124.54,135.42,139.92, 140.36,141.12,148.29,151.16,156.17,162.82.LC-MS(ESI,pos,ion)m/z:271[M+H].

The synthesis of 1-2,2- bromobenzene simultaneously [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters：

NBS (7.30g, 40.99mmol) is added in the solution that synthesis step 1-1 is obtained at room temperature, is stirred Object 30 minutes.Then acquired solution is distilled to 50mL, methanol (25mL) is added and solution is cooled to 50 DEG C, then to this Crystal seed 2- bromobenzenes simultaneously [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters (331mg, 0.8mmol) are added in cooling solution, Then stirring mixture 20 minutes.It is slowly added to water (45mL) so that mixture is cooled to room temperature and maintains 1 hour.By true Product is collected by filtration in sky, and aqueous solution (30mL, the water of THF is used in combination：THF=2:1) it is washed, and dry in vacuum drying oven, Obtaining the 2- bromobenzenes of 12.05g, simultaneously [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters (compound 4), yield are 88.5%.¹H-NMR(400MHz,CDCl₃)δ:2.59(s,3H),7.32(d,1H),7.38(s,1H),7.58(s,1H),7.75(d,1H), 8.88(s,1H),9.16(s,1H).¹³C-NMR(125MHz,CDCl₃)δ:21.51,110.13,112.50,117.65, 122.18,122.42,134.51,139.23,139.92,141.12,148.91,151.16,156.17,162.82.LC-MS (ESI,pos,ion)m/z:349[M+H].

The synthesis of bromo- 1- oxos benzo [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters of 1-3,2-：

At -10 DEG C, the compound 4 (12.05g, 34.51mmol) that synthesis step 1-2 is obtained is added to sulfolane In the solution of (110mL) and sulfuric acid (55mL), 30% hydrogen peroxide (20.25mL) is then added dropwise, is dripped within 3 hours It adds into, and keeps temperature always close to -10 DEG C.After being added dropwise to complete, stirring mixture 1 hour takes sample and measures and reacted At.After the completion of reaction once, any possible remaining oxidant is quenched with 5% aqueous solution of sodium bisulfite (85mL).Then Acetic acid (27.5mL) is added into this mixture and adjusts temperature to 20~25 DEG C.The bromo- 1- oxos of 2- are added into this mixture Benzo [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters (compound 5) (2.5g, 6.85mmol) are to grow crystal, then 2 hours are stirred to promote crystal growth.After 2 hours, slowly add water (150mL) to complete to crystallize within 3 hours.Then it stirs Slurries filter after at least 2 hours.Then water (85mL), ammonium hydroxide (14%, 85mL) and water (85mL) is used to wash filter cake successively. Gained wet cake is dissolved at 65 DEG C in the two phase liquid of ethyl acetate (140mL) and water (127.5mL), then stirring and dissolving Product at least 30 minutes, then remove following water layer.Then compound 5 is added to 60~65 DEG C in cooling solution temperature (2.5g, 6.85mmol) is to grow crystal.Slurries are cooled to 0-5 DEG C by growing the grain 30~60 minutes in 2-3 hours.Filtering institute Product, be used in combination cold (0~5 DEG C) ethyl acetate solution to be washed, then the dry products at 50~60 DEG C of vacuum, you can Bromo- 1- oxos benzo [b] thiophene -6- base 5-Methylpyrazine-2-carboxylic acid ethyl esters (compound 5) of 2- of 8.81g are obtained, yield is 69.9%.¹H-NMR(400MHz,CDCl₃)δ:2.59(s,3H),7.41-7.58(m,4H),8.86(s,1H),9.15(s,1H) .¹³C-NMR(125MHz,CDCl₃)δ:21.51,117.34,127.18,135.09,136.69,139.92,141.12, 141.24,151.08,151.16,156.17,162.82.LC-MS(ESI,pos,ion)m/z:365[M+H].

The synthesis of 1-4,2- furans -2- Oxy-1s-oxo benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates：

Furans -2- alcohol (5.08mmol) is added into container, potassium tert-butoxide (25.03mmol) is then added into this container THF solution, be then rinsed using the DMF less than 100mL, agitating solution about 15 minutes is to ensure that furans -2- alcohol is complete Deprotonation.The compound 5 (8.81g, 24.12mmol) that synthesis step 1-3 is obtained is added in supplemental tank, with no more than 100- The DMF of 500mL transfers the material into reaction vessel (furans -2- alcohol for including deprotonation), is then transferred to reaction In container.The solution of eluant container is also transferred in reactor using DMF.Reactor is heated to 50 DEG C, and at 50 DEG C Aging 4 hours, or until reaction assay terminate, be displayed without more compounds 5 and be converted into 2- furans -2- Oxy-1s-oxygen For benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates, solution is directly used in next step.¹H-NMR(400MHz, CDCl₃)δ:2.59(s,3H),5.75(dd,1H),6.35(t,1H),6.86(s,1H),7.44-7.51(m,4H),8.87(s, 1H),9.16(s,1H).¹³C-NMR(125MHz,CDCl₃)δ:21.51,88.64,109.16,119.73,124.3,130.29, 132.04,133.65,139.33,139.92,141.12,150.6,151.16,151.74,156.17,162.82.LC-MS (ESI,pos,ion)m/z:369[M+H].

The synthesis of 1-5,2- (furans -2- oxygroups) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates：

75% phosphoric acid (about 140mL) is added in the organic layer (21.75mmol) obtained to synthesis step 1-4, until pH Less than 2.0, then the solution of acidification is transferred in hydrogenator, and 10%Pd/C catalyst (4.3g) is added.With 0.5L/kg's Deionized water (225ml) rinses catalyst and is directly entered dehydrogenator.It is hydrogenated under the Hydrogen Vapor Pressure of 65psi at 110 DEG C.Then The the suspended of -6- base -5- methylpyrazine -2- methyl formates Han 2- (furans -2- oxygroups) benzo [b] thiophene is taken out from hydrogenator Liquid is used in combination filter aid catalyst, vacuum to shift solvent, and flash column chromatography separation obtains 7.05g yellow powders, two steps are total Yield 83%.¹H-NMR(400MHz,CDCl₃)δ:2.53(s,3H),5.79(dd,1H),6.35(t,1H),6.61(d,1H), 7.32(dd,1H),7.44(dd,1H),7.78(d,1H),7.75(m,1H),8.89(s,1H),9.10(s,1H).¹³C-NMR (125MHz,CDCl₃)δ:21.51,88.64,109.03,109.16,114.05,116.48,123.44,129.24,139.33, 139.92,141.12,147.48,147.97,151.16,151.74,156.17,160.14,162.82.LC-MS(ESI,pos, ion)m/z:353[M+H]。

Embodiment 2：2- (the fluoro- furans -2- oxygroups of 5-) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates Synthesis

2-1,2- (5- fluoro- furans-2) Oxy-1-oxo benzo [b] thiophene-6- base-5- methylpyrazine-2- methyl formates Synthesis：

Fluoro- furans -2- the alcohol (25.08mmol) of 5- are added into container, potassium tert-butoxide then is added into this container The THF solution of (25.03mmol) is then rinsed using the DMF less than 100mL, and agitating solution about 15 minutes is to ensure 5- The fluoro- complete deprotonation of furans -2- alcohol.Compound 5 (8.81g, 24.12mmol) is added in supplemental tank, with no more than 100- The DMF of 500mL transfers the material into reaction vessel (the fluoro- furans -2- alcohol of 5- for including deprotonation), is then transferred to In reaction vessel.The solution of eluant container is also transferred in reactor using DMF.Reactor is heated to 50 DEG C, and 50 Aging 4 hours at DEG C, or until reaction assay terminate, be displayed without more compounds 5 and be converted into 2- (the fluoro- isoquinolin-of 6- 8) Oxy-1-oxo benzo [b] thiophene-6- base-5- methylpyrazine-2- methyl formates, solution are directly used in next step.LC-MS (ESI,pos,ion)m/z:387[M+H].

The synthesis of 2-2,2- (the fluoro- furans -2- oxygroups of 5-) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates：

In the organic layer obtained to synthesis step 2-1 be added 75% phosphoric acid (about 140mL), until pH be less than 2.0, then The solution of acidification is transferred in hydrogenator, and 10%Pd/C catalyst (4.3g) is added.With the deionized water of 0.5L/kg (225ml) rinses catalyst and is directly entered dehydrogenator.It is hydrogenated under the Hydrogen Vapor Pressure of 65psi at 110 DEG C.Then from hydrogenator The middle suspension for taking out 2- (the fluoro- furans -2- oxygroups of 5-) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates, is used in combination Filter aid catalyst, vacuum shift solvent, and flash column chromatography separation obtains 7.76g yellow powders, two step gross production rates 87%.LC-MS(ESI,pos,ion)m/z:371[M+H].

Embodiment 3：2- (5- methyl-ribofuranosyl -2- oxygroups) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates Synthesis

3-1,2- (5- methyl-ribofuranosyls-2) Oxy-1-oxo benzo [b] thiophene-6- base-5- methylpyrazine-2- formic acid first The synthesis of ester：

5- methyl-ribofuranosyl -2- alcohol (25.08mmol) is added into container, potassium tert-butoxide then is added into this container The THF solution of (25.03mmol) is then rinsed using the DMF less than 100mL, and agitating solution about 15 minutes is to ensure 5- The complete deprotonation of methyl-ribofuranosyl -2- alcohol.By compound 5 (8.81g, 24.12mmol) be added supplemental tank in, with no more than The DMF of 100-500mL transfers the material into reaction vessel (the 5- methyl-ribofuranosyl -2- alcohol for including deprotonation), then by it It is transferred in reaction vessel.The solution of eluant container is also transferred in reactor using DMF.Reactor is heated to 50 DEG C, And aging 4 hours at 50 DEG C, or until reaction assay terminate, be displayed without more compounds 5 and be converted into 2- (5- first Base-furans-2) Oxy-1-oxo benzo [b] thiophene-6- base-5- methylpyrazine-2- methyl formates, solution is directly used in next Step.LC-MS(ESI,pos,ion)m/z:3383[M+H].

The conjunction of 3-2,2- (5- methyl-ribofuranosyl -2- oxygroups) benzo [b] thiophene -6- base -5- methylpyrazine -2- methyl formates At：

In the organic layer obtained to synthesis step 3-1 be added 75% phosphoric acid (about 140mL), until pH be less than 2.0, then The solution of acidification is transferred in hydrogenator, and 10%Pd/C catalyst (4.3g) is added.With the deionized water of 0.5L/kg (225ml) rinses catalyst and is directly entered dehydrogenator.It is hydrogenated under the Hydrogen Vapor Pressure of 65psi at 110 DEG C.Then from hydrogenator The middle suspension for taking out -6- base -5- methylpyrazine -2- methyl formates Han 2- (5- methyl-ribofuranosyl -2- oxygroups) benzo [b] thiophene, It is used in combination filter aid catalyst, vacuum to shift solvent, flash column chromatography separation obtains 6.97g yellow powders, two step gross production rates 79%.LC-MS(ESI,pos,ion)m/z:367[M+H].

Test example：External inhibitory activity of the compounds of this invention to GOAT

One, the structure of GOAT expression plasmids

With PCR amplification people GOAT (registration number EU518495) cDNA, including following reaction：(1) 98 DEG C, 1 minute, (2) 35 98 DEG C of secondary repetition, 10 seconds 65 DEG C, 15 seconds 72 DEG C, 80 seconds, and (3) 72 DEG C, 5 minutes, people's stomach cDNA used as template Library (Takara Bio) and Pyrobest archaeal dna polymerases (Takara Bio).By amplified fragments restriction enzyme treatment, make With Ligation High (TOYOBO), the Stu I/Not I sites of pFastBac1 (Invitrogen) are inserted it into, and turn It contaminates in ECOS JM109 (Nippon Gene), builds pFB/hGOAT.

Two, virus amplification and people's GOAT expression

In order to prepare baculoviral (baculovirus) by pFB/hGOAT, BacToBac baculovirus expression systems are used (Invitrogen).Using SYBR Green (Takara Bio), virus titer is measured by real-time PCR.Make Sf9 cell senses Obtained baculoviral, multiple infections (MOI)=0.2 are caught, and are cultivated 70 hours.Sf9 cells are recycled, suspension is suspended in In buffer solution (TBS (0.3M NaCl), 1mM DTT, 1mM EDTA, Complete (Roche) (1tablet/50mL)), and make With polytron homogenizers, on ice, at 20000rpm by the cell fragmentation 30 seconds.At 4 DEG C, smudge cells solution is existed Centrifuged 10 minutes under 1000rpm, by supernatant at 4 DEG C, at 40000rpm further ultracentrifugation 30 minutes, and recycle precipitation. The precipitation of acquisition is suspended in the buffer suspension liquid being used above, obtains people's GOAT expression microsome fractions.Use BCA albumen Testing reagent (PIERCE) measures protein level.

Three, enzymatic activity is measured using HTRF methods

It will be diluted and be reached with experiment buffer solution (containing 50mM Tris-HCl (pH7.5), 0.5%TWEEN20 and Complete) 20 points are cultivated at room temperature to the compound solution (2 μ L) of 15%DMSO and 90 μ g/mL people GOAT expression microsome fractions (2 μ L) Clock then will contain 30 μM of caprylyl CoA (CHEM-IMPLEX) and 3 μM of ghrelin (Ghrelin)-biotins (biotin)(Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu- Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-epsilon aminocaproic acids-biotin (Greiner Coutom synthesis)) substrate mixed solution be added in the white small size plate (Greiner) in 384 (holes).20 points of reaction After clock, citrate buffer (pH3) (0.1M citric acids (39.8mL) and the 0.2M Na of 2 μ L is added₂HPO₄(10.2mL's) Mixed solution), the Eu (K)-of 25nM resistant activities ghrelin (ghrelin) antibody (being generated in mouse) of 2 μ L, 5 μ L The streptavidin-of anti-mouse IgG Pab (CisBio) (diluting 100 times with HTRF detection buffer solutions (Cis Bio)) and 5 μ L Xlent (Cis Bio) (dilutes 66.6 times) with HTRF detection buffer solutions (CisBio).When cultivating 1 hour or longer at room temperature Between after, utilize Envision (PerkinElmer) measure 620nm and 665nm under fluorescent value.Calculating data, [(665nm believes Number/620nm signals) × 10⁴], it is indicated by data for the inhibitory activity of people GOAT, wherein 0% reference material is free from chemical combination Object, 100% reference material are free from enzyme.

Four, to the inhibiting rate of GOAT when 10 μM of compound

Illustrate that there is preferable inhibitory activity to GOAT when 10 μM of the compounds of this invention, can exist as GOAT inhibitor More extensive pharmacological effect experiment is carried out in the disease animal models such as fat and diabetes, it is new to obtain fat and diabetes Medicine.

Claims (7)

1. a kind of benzo [b] thiophenes, general structure is formula I,

Wherein：R₁、R₂、R₃It is independently selected from H, F or CH₃。

2. the pharmaceutically acceptable salt or solvate of benzo [b] thiophenes as described in claim 1.

3. benzo [b] thiophenes as described in claim 1, characterized in that be selected from following compound：

4. benzo [b] thiophenes as claimed in claim 1 or 2 inhibit as ghrelin O- acyltransferases The application of agent.

5. application of benzo [b] thiophenes as claimed in claim 1 or 2 in prevention or obesity treating medicine.

6. application of benzo [b] thiophenes as claimed in claim 1 or 2 in preventing or treating diabetes medicament.

7. application as claimed in claim 6, characterized in that the diabetes are type II diabetes and/or obese diabetic.