Chlolic acid derivatives and its preparation method and applicationTechnical field
The invention belongs to field of medicaments, are related to a kind of chlolic acid derivatives as FXR agonist and preparation method thereof and useOn the way.
Background technique
Farnesoid X receptor (farnesoid X receptor, FXR) is a member of nuclear receptor superfamily.Mainly in liverWith expression high in small intestine.Nucleus can be moved to, with view after the activation of the FXR native ligand such as bile acid by chenodeoxycholic acid in vivoYellow aldehyde X receptor (retinoid X receptor, RXR) combines and forms heterodimer, in the common work of a variety of co-activation factorsIn conjunction with the lower FXR response element on DNA, the expression of target gene is adjusted.FXR widely participates in bile acid, sugar, rouge generationIt thanks, the regulation of the processes such as inflammation, hepatic fibrosis-renal tubular ectasia syndrome.FXR can be inhibited indirectly the gene expression of CYP7A1 after excitement, to inhibitCholic acid synthesis, to treat primary biliary cirrhosis, inhibit hepatic fibrosis-renal tubular ectasia syndrome process, nonalcoholic fatty liver, diabetes, heightBlood lipid disease etc..
With social development, metabolism class disease incidence is higher and higher, and numerous diseases caused by glycolipid metabolism become harmThe high morbidity of health.FXR agonist is less as new drug listing number at present, and nearest OCA is as FXR agonist by FDA batchesQuasi- listing treatment primary biliary cirrhosis, while the indication of nonalcoholic fatty liver is also carrying out clinical research.ButReceiving OCA treatment patient in clinic will appear the ill symptoms such as serum cholesterol raising, insulin resistance.
It is still necessary to further be studied FXR agonist for this field.
Summary of the invention
The purpose of the present invention is to provide a kind of chlolic acid derivatives and its pharmaceutically acceptable salt and preparation method thereof andPurposes.
The first aspect of the present invention provides a kind of compound of formula I or its pharmaceutically acceptable salt:
In formula, A ring is 5-10 member aromatic ring or 5-10 member hetero-aromatic ring；
N is 1,2,3 or 4；
M is 0,1 or 2；
Each R1It is identical or different, and it is each independently hydrogen, hydroxyl, sulfydryl, cyano, halogen, C1-C6Alkyl, C1-C6AlkaneOxygroup, C1-C6Halogenated alkyl ,-COOR ' or-SO3R'；
R2And R3It is identical or different, and be each independently H ,-COR ', C1-C6Alkyl ,-COOR '；
Each R ' stands alone as hydrogen or C1-C6Alkyl；
For singly-bound or double bond；
X is CH2, NH, O, S or CH.
In another preferred example, the pharmaceutically acceptable salt refers to and the inorganic acids such as phosphoric acid, sulfuric acid, hydrochloric acid or vinegarThe acidic amino acids such as the organic acids such as acid, tartaric acid, citric acid, malic acid or aspartic acid, glutamic acid formed salt, or with nothingThe salt that machine alkali is formed, such as sodium, potassium, calcium, aluminium salt and ammonium salt.
In another preferred example, A ring be phenyl ring, how ring, pyridine ring, pyrimidine ring, pyrrole ring, thiphene ring, furan nucleus, pyrazolesRing, pyrazine or pyranoid ring.
In another preferred example, the compound of formula I has following one or more features:
(1) each R1It is each independently hydrogen, hydrogen, fluorine, chlorine, hydroxyl, sulfydryl, cyano, methyl, ethyl, n-propyl, positive fourthBase, isopropyl, isobutyl group, methoxyl group, ethyoxyl, propoxyl group, trifluoromethyl ,-COOH ,-COOCH3、-COOCH2CH3、-SO3HOr-SO3CH3；
(2)R2And R3It is identical or different, and it is each independently H or-COR '；
(3) each R ' stands alone as hydrogen, methyl, ethyl, n-propyl, normal-butyl, isopropyl or isobutyl group.
In another preferred example, compound shown in the Formulas I or its pharmaceutically acceptable salt are selected from the group:
The second aspect of the present invention, provides a kind of pharmaceutical composition, and described pharmaceutical composition includes described in first aspectCompound of formula I or its pharmaceutically acceptable salt；With
Pharmaceutically acceptable carrier.
The third aspect of the present invention provides compound of formula I described in first aspect or the system of its pharmaceutically acceptable saltPreparation Method, the preparation method comprise the steps of:
Wherein
A) with shellfish cholic acid (OCA) difficult to understand for starting material, 2 compound represented of general formula is obtained after hydroxy esterification；
B) 2 compound represented of general formula decarboxylation under the conditions of iodobenzene acetate or palladium acetate generates chemical combination shown in general formula 3Object；
C) 3 compound represented of general formula obtains 4 compound represented of general formula through oxidation；
D) 4 compound represented of general formula and amineReductive amination process occurs and generates Formulas I shown in general formula 5Object is closed,
Optionally, e) compound of formula I shown in general formula 5 is compound of formula I shown in general formula 6；
Wherein, n, m, R1It is identical as the definition in claim 1 with the definition of A ring；
R2And R3It is identical or different, and it is each independently-COR ', C1-C6Alkyl ,-COOR '；
Each R ' stands alone as hydrogen or C1-C6Alkyl.
The fourth aspect of the present invention provides compound of formula I described in first aspect or the system of its pharmaceutically acceptable saltPreparation Method, the preparation method comprise the steps of:
F) 4 compound represented of general formula obtains 7 compound represented of general formula through reduction；
G) 7 compound represented of general formula generates 8 compound represented of general formula with methane sulfonyl chloride in the presence of alkali；
H) 8 compound represented of general formula fortified phenol or hydroxy compounds in the presence of alkaliReaction lifeAt compound of formula I shown in general formula 9；
Optionally, i) compound of formula I shown in general formula 9 is hydrolyzed to compound of formula I shown in general formula 10；
Wherein, n, m, R1It is identical as the definition in claim 1 with the definition of A ring；
R2And R3It is identical or different, and it is each independently-COR ', C1-C6Alkyl ,-COOR '；
Each R ' stands alone as hydrogen or C1-C6Alkyl.
The fifth aspect of the present invention provides compound of formula I described in first aspect or the system of its pharmaceutically acceptable saltPreparation Method, the preparation method comprise the steps of:
J) 4 compound represented of general formula and phosphorus ylide reagentReaction generates Formulas I shown in general formula 11Close object；
Optionally, k) compound of formula I shown in general formula 11 through reduction obtains compound of formula I shown in general formula 12；
Optionally, l) compound of formula I shown in general formula 12 is hydrolyzed to compound of formula I shown in general formula 13,
Wherein, n, m, R1It is identical as the definition in claim 1 with the definition of A ring；R2And R3It is identical or different, and respectivelyIt independently is-COR ', C1-C6Alkyl ,-COOR '；Each R ' stands alone as hydrogen or C1-C6Alkyl.
The sixth aspect of the present invention provides compound of formula I described in first aspect or the use of its pharmaceutically acceptable saltOn the way, (1) is used to prepare FXR agonist；
(2) it is used to prepare prevention and/or treats the drug of FXR related disease；And/or
(3) prevention and/or treatment bile acid biosynthesis, glycometabolism, lipid metaboli, inflammation, hepatic fibrosis-renal tubular ectasia syndrome process are used to prepareThe drug of related disease.
In another preferred example, the FXR related disease is selected from: nonalcoholic fatty liver (NASH), primary biliaryCirrhosis (PBC), primary sclerotic cholangitis (PSC), gall stone, evaluation of non-alcoholic cirrhotic patients, cholestatic liver disease, high bloodRouge disease, hypercholesterolemia, diabetes.
The seventh aspect of the present invention, provide it is a kind of prevention and/or treatment FXR related disease method, including to have this needThe step of compound of formula I described in the object application first aspect wanted or its pharmaceutically acceptable salt.
Chlolic acid derivatives of the invention have FXR preferably as FXR agonist in molecular level and cellular levelFXR related disease can be prevented and/or be treated to exciting ability, can treat primary biliary cirrhosis, inhibit hepatic fibrosisThe metabolic diseases such as change process, nonalcoholic fatty liver, diabetes, hyperlipemia.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present inventionIt can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.Institute in specificationThe each feature disclosed can be replaced by any alternative characteristics for providing identical, impartial or similar purpose.As space is limited, existThis no longer tires out one by one states.
Specific embodiment
Present inventor develops a kind of chlolic acid derivatives for the first time, swashs as FXR by depth studying extensivelyDynamic agent, structure is shown in formula I, has preferable exciting ability to FXR in molecular level and cellular level.On this basis, completeAt the present invention.
Term
“C1-C6Alkyl " refers to the radical of saturated aliphatic alkyl of the linear chain or branched chain with 1 to 6 carbon atom.Including but not limited toMethyl, ethyl, propyl, isopropyl, normal-butyl, isobutyl group, tert-butyl, sec-butyl, n-pentyl, 1,1- dimethyl propyl, 1,2-Dimethyl propyl, 2,2- dimethyl propyl, 1- ethyl propyl, 2- methyl butyl, 3- methyl butyl, n-hexyl, 1- ethyl -2- firstBase propyl, 1,1,2- thmethylpropyl, 1,1- dimethylbutyl, 1,2- dimethylbutyl, 2,2- dimethylbutyl, 1,3- diformazanBase butyl, 2- ethyl-butyl, 2- methyl amyl, 3- methyl amyl, 4- methyl amyl, 2,3- dimethylbutyl, n-heptyl etc..AlkaneBase can be substituted or unsubstituted, and when substituted, substituent group can be substituted on any workable tie point, excellentBe selected as one or more following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio group, alkyl amino, halogen,It is mercaptan, hydroxyl, nitro, cyano, naphthenic base, heterocycle, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, miscellaneousCycloalkylthio, oxo base, amino, hydroxyalkyl, carboxyl or carboxylate.
“C1-C6Halogenated alkyl " refers to C1-C6Alkyl is replaced by 1,2,3,4 or 5 halogen atom (preferably fluorine atom), excellentSelect halogenated C1-3Alkyl.Such as a chloroethyl, dichloromethyl, 1,2- Dichloroethyl, a bromoethyl, a fluoro ethyl, a fluorine firstBase, difluoromethyl, trifluoromethyl etc..
“C1-C6Alkoxy " refers to (C1-C6Alkyl)-O-.Such as methoxyl group, ethyoxyl, propoxyl group, butoxy etc..
" 5-10 member aryl " is used interchangeably with " 5-10 member aromatic ring ", refers to the aryl radical with 6-10 carbon atom,Such as phenyl, naphthalene etc..
" halogen " refers to fluorine, chlorine, bromine or iodine.
" 5-10 member hetero-aromatic ring " is used interchangeably with " 5-10 unit's heteroaryl ", is referred to 5 to 10 annular atoms, preferably 5,6 or 8 annular atoms；6,10 or 14 pi-electrons are shared in ring array；And also there are 1 to 5 heteroatomic bases outside carbon atomGroup.Term " hetero atom " refers to nitrogen, oxygen or sulphur.
" pharmaceutically acceptable salt " includes pharmaceutically acceptable acid-addition salts and pharmaceutically acceptable base addition salts." pharmaceutically acceptable acid-addition salts " be refer to retain the biological effectiveness of free alkali and without other side effects, and it is inorganicAcid or organic acid are formed by salt.Inorganic acid salt includes but is not limited to hydrochloride, hydrobromate, sulfate, phosphate etc.；It is organicHydrochlorate includes but is not limited to formates, acetate, propionate, glycollate, gluconate, lactate, oxalates, maleic acidSalt, succinate, fumarate, tartrate, citrate, glutamate, aspartate, benzoate, mesylate,Tosilate and salicylate etc..These salt can be prepared by method known in the art." pharmaceutically acceptable alkali additionSalt ", the including but not limited to salt of inorganic base such as sodium salt, sylvite, calcium salt and magnesium salts etc..The including but not limited to salt of organic base, thanSuch as ammonium salt, triethylamine salt, lysine salt, arginine salt etc..These salt can be prepared by method known in the art.
Preparation method
Bile acid derivative of the invention or its pharmaceutically acceptable salt can pass through following three reaction process systemIt is standby.
Reaction stream formula 1
Reaction stream formula 2
Reaction stream formula 3
It a) is that starting material reacts hydroxyl in formic acid or acetic acid solution under strong acid catalyst with shellfish cholic acid (OCA) difficult to understand2 compound represented of general formula is obtained after esterification；
B) 2 compound represented of general formula decarboxylation under the conditions of iodobenzene acetate or palladium acetate generates alkene shown in general formula 3Compound；
C) 3 compound represented of general formula is then oxidized to aldehyde compound shown in general formula 4 under the action of ozone；
D) 4 compound represented of general formula and amineIt issues raw reductive amination process in reducing agent reduction and generates and lead to5 compound represented of formula,
E) 5 compound represented of general formula is hydrolyzed to 6 compound represented of general formula in the presence of alkali；
F) 4 compound represented of general formula is reduced to hydroxy compounds shown in general formula 7 under the action of reducing agent；
G) 7 compound represented of general formula generates 8 compound represented of general formula with methane sulfonyl chloride in the presence of alkali；
H) 8 compound represented of general formula fortified phenol or hydroxy compounds in the presence of alkaliReaction lifeAt 9 compound represented of general formula；
I) 9 compound represented of general formula is hydrolyzed to 10 compound represented of general formula in the presence of alkali；
J) 4 compound represented of general formula in the presence of alkali with phosphorus ylide reagentReaction generates general formula 11Compound represented；
K) 11 compound represented of general formula is reduced to 12 compound represented of general formula under palladium carbon or the hydrogenization of nickel；
L) 12 compound represented of general formula is hydrolyzed to 13 compound represented of general formula in the presence of alkali；
Wherein, n, m, R1、R2、R3It is as defined above with A ring.
Strong acid in step a) is selected from sulfuric acid, hydrochloric acid, periodic acid, perchloric acid, hyperbromic acid, methanesulfonic acid, trifluoromethanesulfonic acid；
Reducing agent in step d) is selected from sodium borohydride, sodium triacetoxy borohydride, sodium cyanoborohydride；
Step e) i) l) in alkali be selected from sodium carbonate, potassium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium methoxide,Sodium ethoxide, potassium ethoxide；
Reducing agent in step f) is selected from sodium borohydride, tetrahydrochysene lithium aluminium, borine；
Step g) h) in alkali be selected from triethylamine, diisopropylethylamine, pyridine, DBU, sodium carbonate, potassium carbonate, hydroxideLithium, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, potassium ethoxide；
Alkali in step j) is selected from potassium tert-butoxide, sodium tert-butoxide, butyl lithium, LDA, sodium methoxide, sodium ethoxide, potassium ethoxide.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical compositions, it includes the active constituent and medicine within the scope of safe and effective amountAcceptable carrier on.
" active constituent " of the present invention refers to compound of formula I of the present invention.
" active constituent " of the present invention and pharmaceutical composition are used to prevent and/or treat the medicine of FXR related diseaseObject.
" safe and effective amount " refers to: the amount of active constituent is enough to be obviously improved the state of an illness, and is unlikely to generate serious pairEffect.In general, pharmaceutical composition contains 1-2000mg active constituent/agent, more preferably, contain 10-200mg active constituent/agent.Compared withGoodly, it is described it is " one " be a tablet.
" pharmaceutically acceptable carrier " refers to: one or more biocompatible solids or liquid filler or gelatinous mass,They are suitable for people's use and it is necessary to have enough purity and sufficiently low toxicity." compatibility " referred to herein as compositionMiddle each component energy and active constituent of the invention and they between mutually admix, and significantly reduce the drug effect of active constituent.Pharmaceutically acceptable carrier part example have cellulose and its derivates (such as sodium carboxymethylcellulose, ethyl cellulose sodium,Cellulose ethanoate etc.), gelatin, talcum, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soya-bean oil,Sesame oil, peanut oil, olive oil etc.), polyalcohol (such as propylene glycol, glycerol, mannitol, sorbierite), emulsifier (such as), wetting agent (such as lauryl sodium sulfate), colorant, flavoring agent, stabilizer, antioxidant, preservative, apirogen waterDeng.
The method of application of active constituent or pharmaceutical composition of the invention is not particularly limited, representative method of application packetInclude (but being not limited to): in oral, tumor, rectum, parenteral (intravenous, intramuscular or subcutaneous) etc..
Solid dosage forms for oral administration includes capsule, tablet, pill, powder and granule.
Liquid formulation for oral administration includes pharmaceutically acceptable lotion, solution, suspension, syrup or tincture.
The compounds of this invention can be administered alone, or be administered in combination with other treatment drug (such as antimicrobial).
It is the mammal that the compounds of this invention of safe and effective amount is applicable to treatment when using pharmaceutical composition(such as people), wherein dosage is the effective dosage pharmaceutically thought when application, for the people of 60kg weight, day is to medicamentAmount is usually 1~2000mg, preferably 20~500mg.Certainly, specific dosage is also contemplated that administration route, patient health situation etc.Factor, within the scope of these are all skilled practitioners technical ability.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present inventionRather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional stripPart (such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in) or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage andNumber is weight percent and parts by weight.
Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the artJustice is identical.In addition, any method similar to or equal to what is recorded and material can be applied to the method for the present invention.Wen ZhongThe preferred implement methods and materials are for illustrative purposes only.
Prepare embodiment
Instrument and main experimental materials are as follows:
BrukerAM-400 type and Varian Mercury plus-400 type Nuclear Magnetic Resonance, 6230 type matter of AgilentSpectrometer and 200-300 mesh column chromatography silica gel (Haiyang Chemical Plant, Qingdao), HSGF254TLC plate (chemical research institute of Yantai City).
Route 1
Embodiment 1:
The formic acid of 5g Austria shellfish cholic acid (OCA), 20mL 88% mixes, and after stirring and dissolving, 4 drop HClO4 is added dropwise, are warming up to 60DEG C, after stirring 4 hours, stops heating, remove oil bath.When temperature is down to 40 DEG C, Ac2O is added dropwise, temperature is kept to be not higher than 40DEG C, until there is a large amount of bubbles to generate (about 10ml has been added dropwise).Above-mentioned system is poured into 200ml water, EA extraction, great Liang ShuiIt washes, dry, the crude product 1A of concentration, yield 95%.1H NMR(400MHz,CDCl3)δ8.16(s,1H),8.05(s,1H),5.20(s,1H),4.79–4.65(m,1H),2.40(m,1H),2.26(m,1H),0.98–0.89(m,9H),0.66(s,3H)；
In the toluene solution of 3g compound 1A (150mL), 370mg pyridine and 240mg copper acetate is added, stirs 10min.Afterwards, 9.4g PhI (OAc) 2 is added portionwise, is heated to reflux 12h.Water quenching reaction, ethyl acetate extraction, washing is added.EA:PE=1:10 column chromatography, obtains product 1B (58%).1H NMR(400MHz,CDCl3) δ 8.16 (s, 1H), 8.06 (d, J=0.6Hz,1H), 5.66 (ddd, J=17.1,10.1,8.5Hz, 1H), 5.21 (s, 1H), 4.92 (dd, J=17.1,1.2Hz, 1H), 4.84(dd, J=10.2,1.9Hz, 1H), 4.78-4.67 (m, 1H), 1.05 (d, J=6.6Hz, 3H), 0.98 (s, 3H), 0.92 (t, J=7.4Hz, 3H), 0.70 (s, 3H)；
1.5g compound 1B is dissolved in 20mL methylene chloride/methanol (10/1) solution, it is anti-that ozone is passed through under the conditions of -78 DEG CIt should continue logical ozone 0.5 hour to solution turned blue, with dimethyl sulphide quenching reaction, be concentrated after being warmed to room temperature stirring naturally 2 hoursSolvent is removed, column chromatographs to obtain product 1C, yield 80%.1H NMR(400MHz,CDCl3) δ 9.49 (d, J=3.2Hz, 1H), 8.08(s, 1H), 7.97 (s, 1H), 5.14 (s, 1H), 4.70-4.58 (m, 1H), 2.28 (m, 1H), 1.05 (d, J=6.8Hz, 4H),0.90 (s, 3H), 0.83 (t, J=7.4Hz, 3H), 0.64 (s, 3H)；
80mg compound 1C and 3- Methyl anthranilate is dissolved in 2mL methylene chloride, 20uL acetic acid, room temperature is added40mg sodium triacetoxy borohydride is added after stirring 0.5 hour, reaction converts completely to raw material, and add water methylene chloride to extract,Dry evaporating column chromatographs to obtain product 1D, yield 79%.1H NMR(400MHz,CDCl3)δ8.17(s,1H),8.06(s,1H),7.35 (d, J=7.7Hz, 1H), 7.27-7.20 (m, 2H), 6.77 (dd, J=8.1,1.6Hz, 1H), 5.22 (s, 1H), 4.82-4.69 (m, 1H), 3.91 (s, 3H), 3.24 (dd, J=12.3,3.0Hz, 1H), 2.84 (dd, J=12.2,8.3Hz, 1H),1.07 (d, J=6.6Hz, 3H), 0.98 (s, 3H), 0.93 (t, J=7.4Hz, 4H), 0.71 (s, 3H)；
50mg compound 1D is dissolved in 2.5mL methanol, in the mixed solution of 1.5mL tetrahydrofuran and 0.5mL water, is added200mg lithium hydroxide is reacted at room temperature to conversion completely, after concentration removes most of solvent, with 1M dilute hydrochloric acid tune PH to highly acid,Solid is precipitated, filtration drying obtains product 1, yield 91%.1H NMR(400MHz,CDCl3)δ7.61(m,2H),7.33(m,1H),7.21 (s, 1H), 3.35 (m, 3H), 3.62-3.22 (m, 1H), 1.09 (d, J=6.2Hz, 3H), 0.84 (m, 7H), 0.62 (s,3H).
Embodiment 2 is prepared to the operation for preparing reference implementation example 1 of embodiment 18 from intermediate 1C pass course 1It obtains, it is as a result as follows.
Route 2
Embodiment 19
1g intermediate 1C is dissolved in 20mL methylene chloride, acetic acid 0.4mL is added, sodium borohydride is added under ice-water bath0.2g, is warmed to room temperature stirring 1 hour, washing methylene chloride extraction, and drying is concentrated, the 0.7g product 19A of column chromatography, yield70%；1H NMR(400MHz,CDCl3)δ8.08(s,1H),7.98(s,1H),5.14(s,1H),4.73–4.59(m,1H),3.57 (dd, J=10.5,3.2Hz, 1H), 3.35-3.26 (m, 1H), 0.99 (d, J=6.6Hz, 3H), 0.90 (s, 3H), 0.84(t, J=7.4Hz, 3H), 0.62 (s, 3H)
0.5g compound 19A is dissolved in 10mL methylene chloride, 0.1mL is added dropwise after 0.3mL triethylamine is added under ice-water bathMethane sulfonyl chloride is warmed to room temperature reaction 2 hours after dripping off, add water quenching reaction, methylene chloride extraction, dry, concentration, column chromatographyObtain 0.52g product 19B, yield 88%；1H NMR(400MHz,CDCl3)δ8.17(s,1H),8.06(s,1H),5.23(s,1H),4.79-4.67 (m, 1H), 4.20 (dd, J=9.3,3.1Hz, 1H), 4.00 (dd, J=9.3,6.6Hz, 1H), 3.02 (s, 3H),1.12 (d, J=6.7Hz, 3H), 0.98 (s, 3H), 0.92 (t, J=7.4Hz, 3H), 0.70 (s, 3H)；
By 100mg compound 19B, 100mg potassium carbonate, 10mg tetrabutylammonium iodide and 30mg methyl p-hydroxybenzoateIt is dissolved in anhydrous acetonitrile, reacts 18 hours at 60 DEG C and converted completely to raw material, be cooled to room temperature ethyl acetate extraction, washing, doDry, concentration, column chromatograph to obtain 97mg compound 19C, yield 87%；1H NMR(400MHz,CDCl3)δ8.16(s,1H),8.05(s,1H), 7.98 (d, J=8.9Hz, 2H), 6.89 (d, J=8.9Hz, 2H), 5.21 (s, 1H), 4.78-4.67 (m, 1H), 3.95(dd, J=8.9,3.2Hz, 1H), 3.88 (s, 3H), 3.72 (dd, J=8.8,7.3Hz, 1H), 1.14 (d, J=6.6Hz, 3H),0.97 (s, 3H), 0.90 (d, J=7.4Hz, 3H), 0.72 (s, 3H)
50mg compound 19C is dissolved in 2.5mL methanol, in the mixed solution of 1.5mL tetrahydrofuran and 0.5mL water, is addedEnter 200mg lithium hydroxide to react at room temperature to conversion completely, after concentration removes most of solvent, with 1M dilute hydrochloric acid tune PH to strong acidProperty, solid is precipitated, filtration drying obtains product 19, yield 96%.1H NMR (400MHz, DMSO) δ 7.86 (d, J=8.6Hz,2H), 6.95 (d, J=8.6Hz, 2H), 3.97-3.95 (m, 1H), 3.75-3.71 (s, 1H), 3.14 (s, 1H), 1.08 (d, J=6.2Hz,3H),0.86–0.80(m,6H),0.65(s,3H).
Embodiment 20 is made to the operation for preparing reference implementation example 19 of embodiment 21 from intermediate 19B pass course 2It is standby to obtain, it is as a result as follows.
Route 3
Embodiment 22
By 300mg compound 1C, 330mgAnd 100mg potassium tert-butoxide is dissolved in 10mL methylene chloride, insteadIt should stay overnight, add water quenching reaction, methylene chloride extraction, washing, dry, concentration, column chromatograph to obtain compound 22A, yield 89%；1HNMR(400MHz,CDCl3) δ 8.13 (s, 1H), 8.04 (s, 1H), 7.99 (s, 1H), 7.84 (d, J=7.8Hz, 1H), 7.48(d, J=7.8Hz, 1H), 7.34 (t, J=7.8Hz, 1H), 6.32 (d, J=15.8Hz, 1H), 6.13 (dd, J=15.8,8.8Hz, 1H), 5.19 (s, 1H), 4.79-4.64 (m, 1H), 3.92 (s, 3H), 1.12 (d, J=6.6Hz, 3H), 0.97 (s,3H), 0.90 (t, J=7.4Hz, 4H), 0.72 (s, 3H)
50mg compound 22A is dissolved in 2.5mL methanol, in the mixed solution of 1.5mL tetrahydrofuran and 0.5mL water, is addedEnter 200mg lithium hydroxide to react at room temperature to conversion completely, after concentration removes most of solvent, with 1M dilute hydrochloric acid tune PH to strong acidProperty, solid is precipitated, filtration drying obtains product 22, yield 95%.1H NMR(400MHz,DMSO)δ7.91(s,1H),7.76(d,J=7.8Hz, 1H), 7.59 (d, J=7.8Hz, 1H), 7.41 (t, J=7.8Hz, 1H), 6.40 (d, J=15.8Hz, 1H), 6.21(dd, J=15.8,8.8Hz, 1H), 4.30 (s, 1H), 4.06 (d, J=4.8Hz, 1H), 3.50 (s, 1H), 3.14 (s, 1H),1.11 (d, J=6.6Hz, 3H), 0.87-0.80 (m, 6H), 0.68 (s, 3H)
Embodiment 23
50mg compound 22 is dissolved in 3mL methanol, 5mg palladium carbon is added under nitrogen protection, reaction 3 is small under hydrogen environmentWhen, raw material fully reacting, diatomite is filtered to remove palladium carbon, and solvent concentration obtains product 23, quantitative yield.1H NMR(400MHz,DMSO) δ 7.83-7.72 (m, 2H), 7.51-7.34 (m, 2H), 4.29 (s, 1H), 4.03 (d, J=5.2Hz, 1H), 3.50 (s,1H), 3.13 (s, 1H), 2.80-2.66 (m, 1H), 1.01 (d, J=6.3Hz, 3H), 0.83-0.81 (m, 6H), 0.60 (s,3H).
Embodiment 24
FXR molecular level activity test method
Using recombination GST-FXR fusion protein, measured by the AlphaScreen detection reagent of Perkin Elmer companyFXR activity.The reaction of this method is in 384 orifice plates, and reaction total volume is 15 μ L.Albumen, agonist, counselor work,Acceptor beads andThe mixed liquor of donor microballon is including Tris-HCl 50mM(pH7.4), it is reacted in the buffer of 50mM NaCl, BSA 0.1%, 1mM DTT, passes through fluorescence detector EnvisionDetect the fluorescence signal intensity reaction FXR activity under 570nm wavelength.EC50Value pass through software Graphpad Prism 5 countIt obtains.
FXR cellular level activity test method
By FXR expression plasmid and FXRE luciferase reporter plasmid with the ratio cotransfection to 293T cell of 1:9 after,With 5*105/ hole will be transfected cell inoculation in 96 hole flat bottom microtiter plate (ViewPlate-96, White96-wellMicroplate with Clear Bottom, PerkinElmer).Culture cell ensures plasmid expression for 24 hours, and FXR to be measured is addedReceptor stimulating agent；After untested compound acts on 18h, luciferase kit (steady-Glo Luciferase Assay is usedSystem fluorescence power is detected) to reflect compound to the activation efficiency of FXR receptor.
Wherein when primary dcreening operation, untested compound and positive compound Austria shellfish cholic acid (OCA) act on cell with 10 μM, survey respectivelyUntested compound is determined to relative activity (relative activity=(the untested compound signal strength-blank)/(positive of positive compoundCompound signal strength-blank) * 100%), the compound that relative activity is higher than positive compound 50% enters secondary screening, selectsSuitable concentration section calculates its dose-dependence, i.e. EC50Value.
Active testing result
Conclusion: test result shows that compound of the present invention has FXR preferably in molecular level and cellular levelExciting ability (wherein 16 activity of embodiment is substantially better than positive control) can be used as FXR agonist, prevention and/or treatment FXRRelated disease, can treat primary biliary cirrhosis, inhibit hepatic fibrosis-renal tubular ectasia syndrome process, nonalcoholic fatty liver, diabetes,The metabolic diseases such as hyperlipemia.
All references mentioned in the present invention is incorporated herein by reference, independent just as each documentIt is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art canTo make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claimsIt encloses.