WO2021078150A1 - 五环三萜类tgr5受体激动剂、其制备方法和用途 - Google Patents

五环三萜类tgr5受体激动剂、其制备方法和用途 Download PDF

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WO2021078150A1
WO2021078150A1 PCT/CN2020/122472 CN2020122472W WO2021078150A1 WO 2021078150 A1 WO2021078150 A1 WO 2021078150A1 CN 2020122472 W CN2020122472 W CN 2020122472W WO 2021078150 A1 WO2021078150 A1 WO 2021078150A1
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alkyl
substituted
hydrogen
group
unsubstituted
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PCT/CN2020/122472
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English (en)
French (fr)
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南发俊
谢欣
张晨露
郭世猛
卓宁
梁晓影
张仰明
贠盈
刘桦楠
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中国科学院上海药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms

Definitions

  • the present invention relates to a class of TGR5 (bile acid G protein coupled receptor) agonists, in particular to a class of pentacyclic triterpenoids, and a preparation method thereof, and this class of TGR5 receptor agonists or their pharmacological agents Use of acceptable salts or pharmaceutical compositions containing any of them in the preparation of drugs for treating diabetes, obesity, hyperlipidemia, liver injury and inflammatory diseases.
  • TGR5 bile acid G protein coupled receptor
  • TGR5 is a G protein-coupled receptor that can be activated by bile acids and is named Membrane Bile Acid Receptor (M-BAR) or TGR5.
  • M-BAR Membrane Bile Acid Receptor
  • TGR5 is highly expressed in the gallbladder, bile duct epithelial cells, brown adipose tissue, muscle, intestine, kidney, placenta and brain. When the ligand binds to TGR5, it activates adenylate cyclase, which increases the level of intracellular cAMP, showing different physiological effects in different tissues.
  • cAMP levels can promote the secretion of GLP-1, thereby promoting insulin secretion and improving the body's sensitivity to insulin; in liver endothelial cells, it can promote the expression of endothelial NO synthase and promote the release of NO, thereby Protect endothelial cells from excessive oxidation of bile acids and lipids; in muscle and brown adipose tissue, the inactive thyroxine T4 can be converted into the active form of T3 by activating cAMP-dependent deiodinase 2 (DIO2), thereby Increase energy and oxygen consumption.
  • DIO2 cAMP-dependent deiodinase 2
  • TGR5 also has potential in the treatment of inflammatory diseases such as atherosclerosis, colitis and non-alcoholic fatty liver disease.
  • inflammatory diseases such as atherosclerosis, colitis and non-alcoholic fatty liver disease.
  • TGR5 activation can also promote mitochondrial division and white adipose tissue remodeling. Therefore, TGR5 receptor agonists are expected to be developed as drugs for the treatment of diabetes, obesity, hyperlipidemia, liver damage and inflammatory diseases.
  • TGR5 receptor agonists currently under study are mainly divided into the following three types according to their structural types: steroidal TGR5 receptor agonists, triterpenoid TGR5 receptor agonists and synthetic small molecule TGR5 receptor agonists.
  • Synthetic small molecule TGR5 receptor agonists are mostly active, but these compounds can strongly activate the gallbladder TGR5 receptor, leading to smooth muscle relaxation and promoting gallbladder filling.
  • this type of TGR5 receptor agonist can not only in the central nervous system Promote the release of neuropeptides in the spinal cord to cause itching, and activation of TGR5 receptors in the cardiovascular system can also lead to systemic vasodilation, which leads to side effects such as increased heart rate and increased cardiac output. Therefore, the development of intestinal selective TGR5 receptors Agonists are the main strategy to avoid toxic side effects.
  • INT777 a typical representative of steroidal TGR5 agonists, is currently in phase I clinical phase. Its agonistic activity on TGR5 receptors has an EC 50 of 0.82 ⁇ M. At a dose of 30 mg/kg/d, it can significantly improve the performance of obese C57BL/6 mice. Energy expenditure, relieve liver fatty disease and increase its glucose tolerance and insulin sensitivity.
  • the compound INT777 has the disadvantage of difficulty in preparation. It takes cholic acid as the starting point and has as many as twelve steps in the synthesis route. The multi-step reaction conditions are extremely harsh. Recent literature also shows that INT777 also has obvious gallbladder filling phenomenon (Finn, PD, etc., Am J Physiol Gastrointest Liver Physiol, 2019, 316, 412–424).
  • the pentacyclic triterpenoid natural product oleanolic acid can stimulate TGR5 receptors.
  • Oral 100mg/kg oleanolic acid can significantly reduce the body weight and blood sugar levels of mice fed high-fat diet, but due to its low TGR5 receptor agonistic activity Therefore, it cannot be ruled out that the drug effect is caused by off-target effects (inhibition of the PTP1B receptor) (Biochem. Biophys. Res. Commun 2007, 362, 793-8).
  • the betulinic acid which also has a pentacyclic triterpene skeleton, also has good TGR5 receptor agonistic activity (Journal of Medicinal Chemistry.2008,51,4849-4849; Journal of Medicinal Chemistry 2010,53,178-90), of which the activity is the best
  • the greater fat solubility of the fatty acid nucleus results in poor physical and chemical properties such as solubility, permeability, and low bioavailability.
  • WO2015135449 a class of pentacyclic triterpenoid TGR5 receptor agonists with the following structure:
  • EC 50 237.8-455.7nM
  • the object of the present invention is to provide a pentacyclic triterpenoid compound as a TGR5 receptor agonist.
  • R 1 is hydrogen, hydroxy, halogen or C1-C6 alkyl
  • R 3 and R 4 are each independently an unsubstituted or substituted C1-C6 alkyl group; or R 3 and C at position 3 form an unsubstituted or substituted C3-C6 cycloalkyl group, and R 4 is hydrogen, or unsubstituted or substituted ⁇ C1-C6 alkyl;
  • R 5 is hydrogen, hydroxyl, hydroxymethyl, formyl or carboxy
  • R 6 and R 7 are each independently hydrogen, hydroxy or C1-C6 alkyl
  • R 8 and R 9 are each independently hydrogen, hydroxyl, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-6 membered oxygen-containing heterocyclic ring,
  • R c is hydrogen, hydroxyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-7 membered heterocyclic group, substituted or unsubstituted 4-8 membered heteroaryl Group, Rd NH-, Rd O(CH 2 ) s NH-, or Rd O(CH 2 ) s -; s is 0, 1, 2, 3 or 4, each Rd is independently hydrogen, substituted or Unsub
  • Z is -(CH 2 )n-, n is 1, 2 or 3;
  • Each * independently represents R configuration, S configuration or racemization
  • R 1 is hydrogen, hydroxy or C1-C4 alkyl. In another preferred embodiment, R 1 is hydrogen.
  • n 1 or 2;
  • R is a substituted or unsubstituted following groups: C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, 5-7 membered heteroaryl, 5-7 membered saturated heterocyclic ring, R a N ( C1-C4 alkyl) -, R a NH- or R a 0-; wherein each R a is independently a substituted or unsubstituted following groups: hydrogen, C1-C8 alkyl, C3-C10 cycloalkyl, C6 -C10 aryl, 5-7 membered heteroaryl or 5-7 membered saturated heterocyclic group;
  • X is NH, O or S
  • Y is C, S or P
  • substitution means that the hydrogen on the group is substituted by one or more substituents selected from the group consisting of hydroxyl, methoxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, carboxyl (-COOH ), sulfonic acid group (-SO 2 OH), glucosamine group, morpholinyl group, N,N-dimethyl.
  • the substitution means that the hydrogen on the group is substituted by one or more substituents selected from the group consisting of hydroxyl, halogen, C1-C6 alkoxy, carboxy (- COOH), sulfonic acid group (-SO 2 OH), morpholino group, N,N-dimethyl.
  • R 2 is a carboxyl group, RCOO-, RCSO- or RSO 2 O-, and the definition of R is the same as described above.
  • R is R a NH-, R a N ( C1-C4 alkyl) -, R a O, C1 -C6 alkyl, C1-C6-alkoxy -C1-C6 alkylene or hydroxy -C1-C6 alkylene group; wherein each R a is independently hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, phenyl, halophenyl, C1-C4 alkoxy-substituted phenyl group, Imidazolyl, morpholino, carboxy-C1-C6 alkylene, sulfonic acid-C1-C6 alkylene, hydroxy-substituted C1-C6 alkyl, morpholino-C1-C6 alkylene
  • R 2 is connected to the ring in an ⁇ configuration.
  • R 3 and R 4 are each independently a C1-C4 alkyl group
  • R 3 and the 3 position C form a C3-C6 cycloalkyl substituted with a C1-C6 alkyl group, and R 4 is hydrogen.
  • R 3 and R 4 are each independently a C1-C3 alkyl group. In another preferred example, R 3 and R 4 are both methyl groups.
  • R 5 is a hydroxymethyl group, a formyl group or a carboxyl group. In another preferred embodiment, R 5 is a carboxyl group.
  • R 6 and R 7 are each independently hydrogen, methyl or ethyl.
  • R 8 and R 9 are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl,
  • R 12 , R 13 and R 14 are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, R c SO 2 -, or R c CO-, where R c is hydrogen, hydroxyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-7 membered nitrogen-containing heterocyclic group (such as morpholine substituted piperidinyl), Rd NH-, Rd O(CH 2 ) s NH-, Rd O(CH 2 ) s -; s is 0, 1, 2, 3 or 4; each R d is taken from hydrogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted 5-6 membered nitrogen-containing hetero Aryl (such as pyridyl);
  • B is NH or O
  • R 15 is hydrogen, substituted or unsubstituted C1-C6 alkyl, and 5-7 membered saturated heterocyclic ring. The definition of each substitution is the same as described above.
  • R c is hydroxyl, morpholine substituted C1-C4 alkyl, carboxyl substituted C1-C4 alkyl, piperidine substituted C1-C4 alkyl,
  • R d is C1-C4 alkyl substituted with morpholine, C1-C4 alkyl substituted with pyrrolidine, C1-C4 alkyl substituted with piperidine, C1-C4 substituted with 4-methylpiperazinyl C4 alkyl, pyridine-substituted C1-C4 alkyl, amino-substituted C1-C4 alkyl, hydroxy-substituted C1-C6 alkyl, pyridyl, imidazolyl, amino-substituted imidazolyl, carboxy-substituted C1-C4 alkane base.
  • R 15 is a C1-C4 alkyl group substituted with a carboxyl group, a morpholino group, a sulfonic acid group-C1-C6 alkylene group, N,N-dimethyl-C1-C4 alkylene group, Alkyl-C1-C4 alkylene group,
  • the compound is any one of T1-T110.
  • the compound of the present invention has an asymmetric center, a chiral axis, and a chiral plane, and can exist in the form of racemate, R-isomer, or S-isomer. Those skilled in the art can use conventional technical means to obtain R-isomer and/or S-isomer from racemate resolution.
  • the second aspect of the present invention provides a method for preparing the compound of the first aspect or a pharmaceutically acceptable salt or dimer thereof, wherein the method is As a raw material, through the introduction of ester groups, carbamates, sulfamates, and carbonates at the C3 hydroxyl group, diversified modification of the C20 double bond, such as the introduction of derivatizable groups such as hydroxyl and carboxyl groups, and then proceed to ester formation , Amide, carbamate, carbonate, etc., and finally hydrogenation to remove the benzyl protecting group on the C17 carboxyl group to obtain the compound described in the first aspect, and the definition of each substituent is as described in the first aspect.
  • the third aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising the compound described in the first aspect, a pharmaceutically acceptable salt or dimer thereof;
  • a pharmaceutically acceptable carrier or excipient is provided.
  • the present invention provides novel compounds, which can be used alone or mixed with pharmaceutically acceptable excipients (such as excipients, diluents, etc.) to prepare tablets, capsules, granules or syrups for oral administration, etc. .
  • the pharmaceutical composition can be prepared according to conventional methods in pharmacy.
  • the fourth aspect of the present invention provides the use of the compound of the first aspect or the pharmaceutical composition of the third aspect, (i) for preparing a bile acid G protein coupled receptor (TGR5) agonist; or
  • the metabolic disease is selected from the group consisting of diabetes, obesity, hyperlipidemia, liver damage and inflammatory diseases.
  • the present invention also provides a method for treating metabolic diseases by administering the compound of the first aspect or the pharmaceutical composition of the third aspect to a subject in need.
  • novel pentacyclic triterpenoids of the present invention can effectively agonize TGR5 receptors, and can agonize TGR5 receptors at nanomolar concentrations. Compared with some existing natural source TGR5 agonists, it has more excellent TGR5 agonistic activity , More abundant natural sources and simpler synthetic methods.
  • the inventor of the present application introduced large polar groups (mainly the C3 hydroxyl group inverted and passed the C3 hydroxyl and C29 hydroxyl groups at the sites that are not sensitive to the activity of pentacyclic triterpenoids).
  • the introduction of ester bonds, carbamates and carbonates and other polar groups not only significantly improves the physical and chemical properties of pentacyclic triterpenoids, such as increased solubility and permeability, thereby overcoming pentacyclic triterpenoids It has a congenital deficiency due to excessive fat solubility, and it was unexpectedly found to significantly increase the agonistic activity of TGR5 receptors.
  • the agonistic activity of human TGR5 of compound T75 is about 100 times that of compound C101 (WO2015135449) in the previous invention, and its solubility is obvious compared with C101. It increases and can penetrate the Caco-2 monolayer cells, which can ensure the in vivo efficacy of this type of compound after oral administration.
  • this type of TGR5 receptor agonist not only has significantly improved physical and chemical properties, but also has greatly improved TGR5 agonistic activity. It is expected to be further developed as a drug for the treatment of metabolic diseases represented by diabetes. On this basis, the present invention has been completed.
  • the halogen is F, Cl, Br or I.
  • C 1 -C 6 means having 1, 2, 3, 4, 5 or 6 carbon atoms
  • C 1 -C 8 means having 1, 2, 3, 4, 5 , 6, 7, or 8 carbon atoms, and so on.
  • 3-10 membered means having 3-10 ring atoms, and so on.
  • alkyl means a saturated linear or branched hydrocarbon moiety
  • C 1 -C 8 alkyl means having 1, 2, 3, 4, 5, 6 Straight or branched chain alkyl groups of 1, 7, or 8 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert Butyl, pentyl, hexyl, etc.; preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
  • alkoxy means -O-alkyl.
  • C 1 -C 6 alkoxy refers to a linear or branched alkoxy group having 1 to 6 carbon atoms, including non-limiting methoxy, ethoxy, n-propoxy, iso Propoxy and butoxy, etc.
  • alkenyl means a straight or branched chain hydrocarbon moiety containing at least one double bond.
  • C 2 -C 6 alkenyl means a double bond with 2 to 6 carbon atoms.
  • the straight-chain or branched alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl and the like.
  • C 2 -C 6 alkynyl refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms and containing a triple bond, and includes, without limitation, ethynyl, propynyl, Butynyl, isobutynyl, pentynyl and hexynyl, etc.
  • cycloalkyl means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon moiety.
  • C 3 -C 10 cycloalkyl means having 3 to 10 carbons in the ring.
  • Cyclic alkyl groups of atoms include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and the like.
  • C 3 -C 8 cycloalkyl C 3 -C 7 cycloalkyl
  • C 3 -C 6 cycloalkyl have similar meanings.
  • Polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyls.
  • cycloalkoxy refers to -O-(cycloalkyl), where cycloalkyl is as defined above.
  • heterocyclic group means a cyclic group containing at least one ring heteroatom (for example, N, O or S).
  • heteroatom for example, N, O or S.
  • heterocyclic group refers to a saturated or unsaturated 3-12 membered ring group containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen in the ring, such as a dioxane Pentyl, tetrahydropyridyl, dihydropyridyl, dihydrofuranyl, dihydrothienyl, etc.
  • the term “3-7 membered heterocyclyl” has a similar meaning.
  • 3-7 membered oxygen-containing heterocyclic ring refers to a cycloalkyl ring having 3-7 ring atoms and containing 1, 2 or 3 O atoms, including but not limited to propylene oxide ring , Butylene oxide ring, heptane oxide ring, etc.
  • C 3 -C 10 cycloalkenyl refers to a cyclic alkenyl group having 3 to 10 carbon atoms in the ring, and includes, without limitation, cyclopropenyl, cyclobutenyl, and cyclopentyl Alkenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl and cyclodecylene, etc.
  • C 3 -C 7 cycloalkenyl has a similar meaning.
  • aryl means a hydrocarbyl moiety containing one or more aromatic rings and having a conjugated ⁇ -electron system.
  • C 6 -C 12 aryl group refers to an aromatic ring group having 6 to 12 carbon atoms that does not contain heteroatoms in the ring, such as phenyl, naphthyl, and the like.
  • C 6 -C 10 aryl has a similar meaning. Examples of aryl groups include, but are not limited to, phenyl (Ph), naphthyl, pyrenyl, anthracenyl, and phenanthryl.
  • heteroaryl means a part of an aromatic ring containing one or more heteroatoms (such as N, O or S).
  • heteroaryl groups include furyl, fluorenyl, pyrrolyl, and thienyl. , Oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrrolinyl, pyrimidinyl, quinazolinyl, quinolinyl, pyranyl, isoquinolinyl and indolyl.
  • the aryl ring may be condensed with a heterocyclic group, a heteroaryl group or a cycloalkyl ring, non-limiting examples include benzimidazole, benzothiazole, benzoxazole, benzisoxazole, benzopyrazole , Quinoline, benzindole, benzodihydrofuran.
  • the heteroaryl group can be fused to an aryl group, a heterocyclic group or a cycloalkyl ring, and the ring connected to the parent structure is a heteroaryl ring.
  • alkyl, alkoxy, cycloalkyl, heterocyclyl, and aryl groups described herein are substituted and unsubstituted groups.
  • Possible substituents on alkyl, alkoxy, cycloalkyl, heterocyclyl and aryl groups include, but are not limited to: hydroxyl, amino, nitro, nitrile, halogen, C1-C6 alkyl, C2-C10 alkene Group, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C6 alkoxy, aryl, heteroaryl Group, heteroaryloxy, C1-C10 alkylamino, C1-C20 dialkylamino, arylamino, diarylamino, C1-C10 alkylsulfamoyl, aryls
  • the substitution is mono-substitution or poly-substitution
  • the poly-substitution is di-substitution, tri-substitution, tetra-substitution or penta-substitution.
  • the di-substitution refers to having two substituents, and so on.
  • the structural formula described in the present invention is intended to include all tautomers, optical isomers and stereoisomeric forms (such as enantiomers, diastereomers, geometric isomers or conformational isomers).
  • Conformers For example, R and S configurations containing asymmetric centers, (Z), (E) isomers and (Z), (E) conformers of double bonds. Therefore, individual stereochemical isomers, tautomers or enantiomers, diastereomers or geometric isomers or conformational isomers or mixtures of tautomers of the compounds of the present invention All belong to the scope of the present invention.
  • tautomers means that structural isomers with different energies can exceed the low energy barrier to convert into each other.
  • proton tautomers ie, proton transfer
  • interconversion through proton transfer such as 1H-indazole and 2H-indazole, 1H-benzo[d]imidazole and 3H-benzo[d]imidazole
  • Valence tautomers include interconversion through some bond-forming electron recombination.
  • the pharmaceutically acceptable salts are not particularly limited, and preferably include: inorganic acid salts, organic acid salts, alkyl sulfonates and aryl sulfonates;
  • the inorganic acid salts include hydrochlorides , Hydrobromide, nitrate, sulfate, phosphate, etc.;
  • the organic acid salt includes formate, acetate, propionate, benzoate, maleate, fumarate, succinate Acid salt, tartrate, citrate, etc.;
  • the alkyl sulfonate includes methane sulfonate, ethyl sulfonate, etc.;
  • the aryl sulfonate includes benzene sulfonate, p-toluene sulfonate Wait.
  • the pentacyclic triterpenoids of the present invention can be prepared by the following route.
  • the present invention also provides a pharmaceutical composition, which contains active ingredients in a safe and effective amount, and a pharmaceutically acceptable carrier.
  • the “active ingredient” in the present invention refers to the compound of formula I in the present invention.
  • the "active ingredients” and pharmaceutical compositions of the present invention are used to prepare drugs for the treatment of metabolic diseases.
  • the “active ingredients” and pharmaceutical compositions of the present invention can be used as TGR5 receptor agonists. In another preferred embodiment, it is used to prepare medicines for preventing and/treating diseases regulated by TGR5 agonists.
  • Safe and effective amount refers to: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of active ingredient/dose, more preferably, 10-200 mg of active ingredient/dose.
  • the "one dose” is a tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components in the composition can be blended with the active ingredients of the present invention and between them without significantly reducing the efficacy of the active ingredients.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid).
  • Magnesium stearate calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • the method of administration of the active ingredient or the pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), etc.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the compounds of the present invention can be administered alone or in combination with other therapeutic drugs.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment.
  • the administered dose is usually 1 to 2000 mg, preferably 20 to 500 mg.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • NMR NMR was measured with a Mercury-Vx 300M instrument produced by Varian, and NMR calibration was: ⁇ H 7.26ppm (CDCl 3 ), 2.50ppm (DMSO-d 6 ); Agilent 1200 Quadrupole LC/MS was used for mass spectrometry.
  • Example 2 Using a similar method in Example 2, the following compounds can be obtained by reacting intermediate S7 with different amines.
  • the compound T109 can be obtained through a synthetic process similar to that of the compound T31.
  • 1 H NMR 400MHz, d 6 -DMSO
  • 4.38 (dd, J 11.6, 4.8 Hz, 1H)
  • 4.24 (t, 1H, J 5.2 Hz)
  • 1.07(d,3H,J 11.2Hz)
  • the compound T110 can be obtained through a synthetic process similar to that of the compound T49.
  • 1 H NMR 400MHz, d 6 -DMSO
  • ⁇ 11.97 (br s, 1H), 7.14 (br s, 1H), 4.25-4.18 (m, 2H), 3.58-3.53 (m, 1H), 3.17-3.10 (m, 1H), 2.44 (s, 1H), 2.25-2.08 (m, 3H), 1.70-1.23 (m, other aliphatic ring protons), 1.07 (d, 3H, J 11.6 Hz), 0.93 (s, 3H), 0.87-0.76 (m, 15H), 0.55-0.52 (m, 2H), 0.37-0.36 (m, 2H).
  • HEK293 cells transiently transduced with TGR5 were stimulated with compounds, and then homogeneous time-resolved fluorescence (HTRF) was used to detect whether these compounds can stimulate TGR5.
  • HTRF homogeneous time-resolved fluorescence
  • TGR5 is a bile acid membrane receptor and a member of the GPCR family. It has a regulatory effect on the metabolism of bile acids, lipids and carbohydrates. TGR5 is coupled to the Gs protein, and after activation, it further activates adenylate cyclase to produce the second messenger cAMP.
  • HTRF is a method for detecting the content of cAMP, which combines two technologies: Fluorescence Resonance Energy Transfer (FRET) and Time Resolved Fluorescence (TRF).
  • FRET Fluorescence Resonance Energy Transfer
  • TRF Time Resolved Fluorescence
  • the cryptate containing Eu is used as a fluorescent donor, and its emission spectrum overlaps with the excitation spectrum of the fluorescent acceptor. FRET induces the acceptor to produce fluorescence, while Eu has a long fluorescence lifetime.
  • TRF can distinguish the acceptor emission from the fluorescent background. ⁇ Fluorescence signal. Combine the fluorescent donor with the cAMP specific antibody, and at the same time label cAMP with the fluorescent acceptor. The two approach each other through the antigen-antibody specific recognition reaction to produce FRET, and the cAMP produced by the cell competes with the labeled cAMP for the antibody binding site, resulting in The fluorescence intensity decreases.
  • the TGR5 agonist INT777 was used as a positive control to explore the effect of the compound on TGR5.
  • the compound was dissolved in DMSO to prepare the mother liquor, and then diluted with the culture solution to the required concentration during use.
  • the compound to be tested is made up to 2 times the final concentration with 1xPBS.
  • the final concentration is 100 ⁇ M, 10 ⁇ M, 1 ⁇ M, 100nM, 10nM, 1nM, 0.1nM, DMSO (each well contains 1% DMSO).
  • cAMP-d2 is diluted to 20 ⁇ L with cAMP&cGMP conjugates&lysis buffer.
  • EC 50 is the evaluation of the agonistic activity of the sample drug on TGR5, half of the effective concentration is 50%. 1-10 ⁇ M: “*”, 0.1-1 ⁇ M: “**”, 100-1nM: "***"
  • Caco-2 cells are cultured in high-sugar DMEM medium at 37°C, 5% CO 2 , and 90% air relative humidity.
  • the medium is supplemented with 10% fetal bovine serum, 10mmol/L HEPES, 1mmol/L Sodium pyruvate, 1% glutamine, 1% non-essential amino acids, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin. It is passed from one generation to the next every 7 days, and the generation ratio is 1:10.
  • the experiment uses cells between 40 and 60 passages.
  • Caco-2 cells were inoculated into a Millicell-24 well plate at a concentration of 2 ⁇ 10 5 /mL, 400 ⁇ L per well, 800 ⁇ L of culture medium was added to the basal side, and cultured in an incubator at 37° C. and 5% CO 2. After 72 hours of cell inoculation, the medium was changed, and then the medium was changed every other day, and cultured for 21 days.
  • the TEER value is used to detect the compactness of the cell monolayer, generally greater than 200 ⁇ cm 2 can be regarded as the monolayer compact and complete.
  • Caco-2 cells were cultured in a Millicell culture plate, and the transmembrane resistance value was measured with a resistance meter Millicell-ERS II at 21 days.
  • the drug transport from the top layer of the cell (A side) to the basal layer (B side) and from the B side to the A side was investigated.
  • the test method is as follows: After washing the cells three times with HBSS, the compounds of the corresponding concentration are added to the corresponding cell wells (pH 6.8 on the A side and pH 7.4 on the B side). Incubate in a 37°C incubator for 95 minutes, take samples from the administration side at 5 minutes and 95 minutes, and take samples from the receiving side at 35 minutes and 95 minutes. The concentration of the sample was detected by LC-MS/MS. To verify the cells, a standard curve was fitted using the expected Fabs and Papp values of several positive control compounds. Determine the Papp value of the test compound and calculate its Fabs value from the standard curve.
  • T36, T38, T39, and T64 have similar permeation rates on both sides of the membrane, while T66 and T75 have significant differences in permeation rates on both sides of the membrane, indicating that T66 and T75 may be substrates of Pgp protein.

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Abstract

一种五环三萜类TGR5受体激动剂、其制备方法和用途。五环三萜类TGR5受体激动剂结构如式I所示，各取代基的定义如说明书和权利要求书所述。五环三萜类化合物，溶解度增加，渗透性提高，且显著提升TGR5受体激动活性，可以穿透Caco-2单层细胞，保证该类化合物口服给药后的体内药效发挥。该类TGR5受体激动剂有望进一步开发成为治疗以糖尿病为代表的代谢性疾病的药物。

Description

五环三萜类TGR5受体激动剂、其制备方法和用途 技术领域
本发明涉及一类TGR5(胆汁酸G蛋白偶联受体)激动剂，具体而言，涉及一类五环三萜类化合物，及其制备方法，以及该类TGR5受体激动剂或其药学上可接受的盐或含有它们中任何一种的药物组合物在制备治疗糖尿病、肥胖、高血脂、肝损伤和炎症性疾病药物方面的用途。
背景技术
TGR5是一种可被胆汁酸激活的G蛋白偶联受体，并命名为膜胆汁酸受体(M-BAR)或TGR5。TGR5高表达于胆囊、胆管上皮细胞、棕色脂肪组织、肌肉、肠道、肾脏、胎盘以及大脑中。当配体与TGR5结合后就会激活腺苷酸环化酶，使细胞内cAMP水平升高，在不同的组织中表现出不同的生理学效应。在小肠中，cAMP水平升高可促进GLP-1的分泌，从而促进胰岛素分泌，提高机体对胰岛素的敏感性；在肝脏内皮细胞中，它可以促进内皮NO合成酶的表达，促进NO释放，从而保护内皮细胞免受胆汁酸和脂质过度氧化的损伤；在肌肉和棕色脂肪组织中，可通过激活cAMP依赖的脱碘酶2(DIO2)将非活化的甲状腺素T4转换为活性形式T3，从而增加能量和氧气消耗。同时，激动TGR5在治疗炎症性疾病如动脉粥样硬化，结肠炎和非酒精性脂肪肝病等疾病方面也有潜力。最近有研究表明，TGR5激活还可以促进线粒体分裂和白色脂肪组织重塑。故TGR5受体激动剂有望开发成为治疗糖尿病、肥胖、高血脂、肝损伤和炎症性疾病等的药物。
目前在研的TGR5受体激动剂根据结构类型的不同主要分为以下三种：甾体类TGR5受体激动剂，三萜类TGR5受体激动剂以及合成类小分子TGR5受体激动剂。
合成类小分子TGR5受体激动剂活性大都很强，但此类化合物可强烈激活胆囊TGR5受体，从而导致平滑肌松弛，促进胆囊充盈，同时该类TGR5受体激动剂不仅可在中枢神经系统中促进脊髓中神经肽的释放从而引起瘙痒，而且在心血管系统的TGR5受体被激活还可导致全身血管舒张，进而导致心率加快和心输出量增加等副作用，所以目前开发肠道选择性TGR5受体激动剂是避免毒副作用的主要策略。其中Ardelyx公司报道的化合物12(J.Med.Chem.,2018,61,7589–7613)以及未披露结构的RDX-8940(Am J Physiol Gastrointest Liver Physiol,2019,316,412–424)通过降低分子渗透性初步实现了短期给药胆囊毒性的规避。
甾体类TGR5激动剂的典型代表INT777目前处于I期临床阶段，它对TGR5受体的激动活性EC 50为0.82μM，在30mg/kg/d的剂量下可明显提升肥胖C57BL/6小鼠的能量消耗，缓解肝脏脂肪病变以及增加其糖耐量和胰岛素敏感度。但化合物INT777具有制备困难的缺点，以胆酸为起点，合成路线多达十二步，多步反应条件极为苛刻，最近的文献也表明INT777同样存在明显的胆囊充盈现象(Finn,PD等，Am J Physiol Gastrointest Liver Physiol,2019,316,412–424)。
五环三萜类天然产物齐墩果酸可激动TGR5受体，口服100mg/kg齐墩果酸可明显降 低高脂饮食喂养小鼠的体重和血糖水平，但由于其TGR5受体激动活性较低，所以并不能排除药效为脱靶效应(抑制PTP1B受体)所致(Biochem.Biophys.Res.Commun 2007,362,793-8)。同样具有五环三萜骨架的白桦脂酸也具有较好的TGR5受体激动活性(Journal of Medicinal Chemistry.2008,51,4849-4849；Journal of Medicinal Chemistry 2010,53,178-90)，其中活性最好的化合物18 dia 2(EC 50＝0.12μM)(Journal of Medicinal Chemistry 2010,53,178-90)在高脂饲养小鼠上并未表现出明显的降低体重以及降血糖的药效，作者推测是由白桦脂酸母核较大的脂溶性导致溶解度渗透性等理化性质不佳和生物利用度较低造成的。
发明人于WO2015135449公开了一类如下结构的五环三萜类TGR5受体激动剂：
Figure PCTCN2020122472-appb-000001
其中当R 2为α构型的丁酰氧基时的化合物(C40，C99，C101，C103)人源TGR5激动活性(EC 50＝237.8-455.7nM)与INT777(EC 50＝395.1nM)相当。后续的深入研究发现，上述化合物虽然在体外可明显促进NCI-H716细胞GLP-1的分泌，但因为脂溶性过高导致其溶解度极差，在肠道中无法溶出，因此口服给药后无法在体内起效。Caco-2单层细胞渗透性实验表明，该化合物无法透过细胞膜，而TGR5受体位于肠道表皮内分泌L细胞基底膜侧，渗透性不足也决定了化合物无法透过无法肠道表皮内分泌L细胞基底膜来发挥药效。
发明内容
本发明的目的在于提供一种用作TGR5受体激动剂的五环三萜类化合物。
本发明的第一方面，提供一种式I化合物、其药学上可接受的盐或其二聚体：
Figure PCTCN2020122472-appb-000002
R 1为氢、羟基、卤素或C1-C6烷基；
R 2为氢、羟基、羧基、卤素、C1-C6烷氧基、3至10元环烷氧基、＝O、＝N-OH或R-Y(＝X) m-O-；其中，m为1或2；R为取代或未取代的以下基团：C1-C8烷基、C3-C10环烷基、C6-C10芳基、5-7元杂芳基、5-7元饱和杂环、R aN(C1-C6烷基)-、R aNH-或R aO-；其中，各R a独立选自取代或未取代的以下基团：氢，C1-C8烷基、C3-C10环烷基、C6-C10 芳基、5-7元杂芳基或5-7元饱和杂环基；X为NH，O或S，Y为C，S或者P；
R 3和R 4各自独立地为未取代或取代的C1-C6烷基；或者R 3和3位C形成未取代或取代的C3-C6环烷基，R 4为氢、或未取代或取代的C1-C6烷基；
R 5为氢、羟基、羟甲基、甲酰基或羧基；
R 6和R 7各自独立地为氢、羟基或C1-C6烷基；
R 8和R 9各自独立地为氢、羟基、卤素、取代或未取代的C1-C6烷基、取代或未取代的3-6元含氧杂环、
Figure PCTCN2020122472-appb-000003
其中R 12、R 13和R 14为氢、取代或未取代的C1-C6烷基或R c-M(＝R’) r-；其中，r为1或2；M为C，S或P，R’为O或S；R c为氢、羟基、取代或未取代的C1-C6烷基、取代或未取代的3-7元杂环基、取代或未取代的4-8元杂芳基、R dNH-、R dO(CH 2) sNH-、或R dO(CH 2) s-；s为0、1、2、3或4，各R d独立为氢、取代或未取代的C1-C6烷基、或取代或未取代的4-8元杂芳基；A为O或S，B为NH或O，R 15为氢、取代或未取代的C1-C6烷基、C3-C8环烷基或5-7元饱和杂环；
R 10为氢、羟基、氧代基团(＝O)或C1-C6烷基；
R 11为氢、羟基、氧代基团(＝O)或C1-C6烷基；
Z为-(CH 2)n-，n为1、2或3；
Figure PCTCN2020122472-appb-000004
表示单键或双键；
各*独立地表示R构型、S构型或消旋；
所述取代是指被基团上的氢被选自下组的一个或多个取代基取代：羟基、氨基、甲氧基、卤素、C1-C6烷基、C1-C6烷氧基、氧代基团(＝O)、环氧基团、肟(＝N-OH)、羧基(-COOH)、磺酸基(-SO 2OH)、葡糖胺基、吗啉基、N,N-二甲基、4-甲基哌嗪基、吡啶基、哌啶基、吡咯烷基。
在另一优选例中，R 1为氢、羟基或C1-C4烷基。在另一优选例中，R 1为氢。
在另一优选例中，R 2为氢、羟基、羧基、卤素、C1-C4烷氧基、3至6元环烷氧基、＝O、＝N-OH或R-Y(＝X) m-O-；
其中，m为1或2；
R为取代或未取代的以下基团：C1-C6烷基、C3-C8环烷基、C6-C10芳基、5-7元杂芳基、5-7元饱和杂环、R aN(C1-C4烷基)-、R aNH-或R aO-；其中，各R a独立为取代或未取代的以下基团：氢，C1-C8烷基、C3-C10环烷基、C6-C10芳基、5-7元杂芳基或5-7元饱和杂环基；
X为NH，O或S；
Y为C，S或者P；
所述取代是指被基团上的氢被选自下组的一个或多个取代基取代：羟基、甲氧基、卤素、C1-C6烷基、C1-C6烷氧基、羧基(-COOH)、磺酸基(-SO 2OH)、葡糖胺基、吗啉基、N,N-二甲基。
在另一优选例中，对于R 2，所述取代是指被基团上的氢被选自下组的一个或多个取代基取代：羟基、卤素、C1-C6烷氧基、羧基(-COOH)、磺酸基(-SO 2OH)、吗啉基、N,N-二甲基。
在另一优选例中，R 2为羧基、RCOO-、RCSO-或RSO 2O-，R的定义同前所述。在另一优选例中，R为R aNH-、R aN(C1-C4烷基)-、R aO、C1-C6烷基、C1-C6烷氧基-C1-C6亚烷基或羟基-C1-C6亚烷基；其中，各R a独立为氢、C1-C4烷基、C3-C6环烷基、苯基、卤代苯基、C1-C4烷氧基取代的苯基、咪唑基、吗啉基、羧基-C1-C6亚烷基、磺酸基-C1-C6亚烷基、羟基取代的C1-C6烷基、吗啉基-C1-C6亚烷基或N,N-二甲基-C1-C6亚烷基。
在另一优选例中，R 2以α构型与环连接。
在另一优选例中，R 3和R 4各自独立地为C1-C4烷基；
或者R 3和3位C形成C1-C6烷基取代的C3-C6环烷基，R 4为氢。
在另一优选例中，R 3和R 4各自独立地为C1-C3烷基。在另一优选例中，R 3和R 4同时为甲基。
在另一优选例中，R 5为羟甲基、甲酰基或羧基。在另一优选例中，R 5为羧基。
在另一优选例中，R 6和R 7各自独立地为氢、甲基或乙基。
在另一优选例中，R 8和R 9各自独立地为氢、甲基、乙基、正丙基、异丙基、
Figure PCTCN2020122472-appb-000005
Figure PCTCN2020122472-appb-000006
其中R 12、R 13和R 14各自独立为氢、取代或未取代的C1-C6烷基、R cSO 2-、或R cCO-，其中，R c为氢、羟基、取代或未取代的C1-C6烷基、取代或未取代的3-7元含氮杂环基(如吗啉取代的哌啶基)、R dNH-、R dO(CH 2) sNH-、R dO(CH 2) s-；s为0、1、2、3或4；各R d取自氢、取代或未取代的C1-C6烷基或取代或未取代的5-6元含氮杂芳基(如吡啶基)；
B为NH或O，
R 15为氢、取代或未取代的C1-C6烷基、5-7元饱和杂环。各取代的定义同前所述。
在另一优选例中，R c为羟基、吗啉取代的C1-C4烷基、羧基取代的C1-C4烷基、哌啶取代的C1-C4烷基、
在另一优选例中，R d为吗啉取代的C1-C4烷基、吡咯烷的C1-C4烷基、哌啶取代的C1-C4烷基、4-甲基哌嗪基取代的C1-C4烷基、吡啶取代的C1-C4烷基、氨基取代的C1-C4烷基、羟基取代的C1-C6烷基、吡啶基、咪唑基、氨基取代的咪唑基、羧基取代的C1-C4烷基。
在另一优选例中，R 15为羧基取代的C1-C4烷基、吗啉基、磺酸基-C1-C6亚烷基、N,N-二甲基-C1-C4亚烷基、吗啉基-C1-C4亚烷基、
在另一优选例中，R 10为氢、羟基或氧代基团(＝O)。
在另一优选例中，R 11为氢、羟基或氧代基团(＝O)。
在另一优选例中，所述化合物为：T1-T110中的任一化合物。
本发明的化合物具有不对称中心、手性轴和手性平面，并且可以以外消旋体、R-异构体或S-异构体的形式存在。本领域技术人员能够采用常规技术手段由外消旋体拆分获得R-异构体和/或S-异构体。
本发明的第二方面，提供一种第一方面所述的化合物或其药学上可接受的盐或其二聚体的制备方法，所述方法以
Figure PCTCN2020122472-appb-000007
为原料，通过在C3位羟基引入酯基、氨基甲酸酯、氨基磺酸酯、碳酸酯，在C20位双键多样化改造，如引入羟基和羧基等可衍生化基团，再进行成酯、酰胺、氨基甲酸酯、碳酸酯等改造，最后氢化脱去C17位羧基上的苄基保护基，获得第一方面所述的化合物，各取代基的定义如第一方面所述。
本发明的第三方面，提供一种药物组合物，所述药物组合物包含第一方面所述的化合物、其药学上可接受的盐或二聚体；以及
药学上可接受的载体或赋形剂。
本发明提供新型的化合物，可以单独使用，或者将其与可药用的辅料(例如赋形剂、稀释剂等)混合，配制成口服给药的片剂、胶囊剂、颗粒剂或糖浆剂等。该药物组合物可以按照制药学上常规方法制得。
本发明的第四方面，提供第一方面所述的化合物或第三方面所述的药物组合物的用途，(i)用于制备胆汁酸G蛋白偶联受体(TGR5)激动剂；或
(ii)用于制备治疗代谢性疾病的药物。
在另一优选例中，所述代谢性疾病选自：糖尿病、肥胖、高血脂、肝损伤和炎症性疾病。
本发明还提供了一种治疗代谢性疾病的方法，向有需要的对象施用第一方面所述的化合物或第三方面所述的药物组合物。
本发明新型的五环三萜类化合物，能有效激动TGR5受体，可在纳摩尔浓度下激动TGR5受体，与现有的一些天然来源的TGR5激动剂相比，具有更优异的TGR5激动活性，更丰富的天然来源和更简便的合成方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所 揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一累述。
具体实施方式
本申请的发明人经过广泛而深入地研究，在五环三萜类化合物活性不敏感位点处引入大的极性基团(主要是C3位羟基反转并通过在其C3位羟基和C29位处引入酯键、氨基甲酸酯和碳酸酯等极性基团)，不仅显著改善了五环三萜类化合物的理化性质，如溶解度增加，渗透性提高，从而克服了五环三萜类化合物本身脂溶性过大的先天不足，而且意外发现显著提升TGR5受体激动活性，其中化合物T75人源TGR5激动活性为前一发明中化合物C101(WO2015135449)的100倍左右，且其溶解度与C101相比明显增加，并可以穿透Caco-2单层细胞，可保证该类化合物口服给药后的体内药效发挥。该类TGR5受体激动剂与INT777相比不仅理化性质明显改善，而且TGR5激动活性大幅提高，有望进一步开发成为治疗以糖尿病为代表的代谢性疾病的药物。在此基础上，完成了本发明。
术语
在本发明中，所述卤素为F、Cl、Br或I。
在本发明中，除非特别指出，所用术语具有本领域技术人员公知的一般含义。
在本发明中，术语“C 1-C 6”是指具有1、2、3、4、5或6个碳原子，“C 1-C 8”是指具有1、2、3、4、5、6、7或8个碳原子，依此类推。“3-10元”是指具有3-10个环原子，依此类推。
在本发明中，术语“烷基”表示饱和的线性或支链烃部分，例如术语“C 1-C 8烷基”是指具有1个、2个、3个、4个、5个、6个、7个或8个碳原子的直链或支链烷基，非限制性地包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、戊基和已基等；优选乙基、正丙基、异丙基、正丁基、异丁基、仲丁基和叔丁基。
在本发明中，术语“烷氧基”表示-O-烷基。例如术语“C 1-C 6烷氧基”是指具有1至6个碳原子的直链或支链烷氧基，非限制性地包括甲氧基、乙氧基、正丙氧基、异丙氧基和丁氧基等。
在本发明中，术语“烯基”表示包含至少一个双键的直链或支链烃基部分，例如术语“C 2-C 6烯基”是指具有2至6个碳原子的含有一个双键的直链或支链烯基，非限制性地包括乙烯基、丙烯基、丁烯基、异丁烯基、戊烯基和己烯基等。
在本发明中，术语“C 2-C 6炔基”是指具有2至6个碳原子的含有一个三键的直链或支链炔基，非限制性地包括乙炔基、丙炔基、丁炔基、异丁炔基、戊炔基和己炔基等。
在本发明中，术语“环烷基”表示饱和或者部分不饱和单环或多环环状烃基部分，例如术语“C 3-C 10环烷基”是指在环上具有3至10个碳原子的环状烷基，非限制性地包括环丙基、环丁基、环戊基、环戊烯基、环己基、环庚基、环辛基和环癸基等。术语“C 3-C 8环烷基”、“C 3-C 7环烷基”、和“C 3-C 6环烷基”具有类似的含义。多环环烷基包括螺环、稠环和桥环的环烷基。
术语“环烷氧基”是指-O-(环烷基)，其中环烷基的定义如上所述。
在本发明中，术语“杂环基”表示包含至少一个环杂原子(例如N,O或S)的环状基团。例 如术语“3-12元杂环基”是指在环上含有1～3个选自氧、硫和氮中的杂原子的饱和或不饱和的3-12元环基，例如二氧杂环戊基、四氢吡啶基、二氢吡啶基、二氢呋喃基、二氢噻吩基等。术语“3-7元杂环基”具有类似的含义。
在本发明中，术语“3-7元含氧杂环”是指具有3-7个环原子且包含1、2或3个O原子的环烷基环，非限制性地包括环氧丙烷环、环氧丁烷环、环氧庚烷环等。
在本发明中，术语“C 3-C 10环烯基”是指在环上具有3至10个碳原子的环状烯基，非限制性地包括环丙烯基、环丁烯基、环戊烯基、环己烯基、环庚烯基、环辛烯基和环癸基烯等。术语“C 3-C 7环烯基”具有类似的含义。
在本发明中，术语“芳基”表示包含一个或多个芳环的烃基部分，具有共轭的π电子体系。例如术语“C 6-C 12芳基”是指在环上不含杂原子的具有6至12个碳原子的芳香族环基，如苯基、萘基等。术语“C 6-C 10芳基”具有类似的含义。芳基的例子包括但不限于苯基(Ph)、萘基、芘基、蒽基和菲基。
在本发明中，术语“杂芳基”表示包含一个或多个杂原子(例如N,O或S)的芳环的部分，杂芳基的例子包括呋喃基、芴基、吡咯基、噻吩基、噁唑基、咪唑基、噻唑基、吡啶基、吡咯啉基、嘧啶基、喹唑啉基、喹啉基、吡喃基、异喹啉基和吲哚基。
所述芳基环可以与杂环基、杂芳基或环烷基环稠合，非限制性实例含苯并咪唑、苯并噻唑、苯并恶唑、苯并异恶唑、苯并吡唑、喹啉、苯并吲哚、苯并二氢呋喃。
所述的杂芳基可以稠合于芳基、杂环基或者环烷基环上，其中与母体结构连接在一起的环为杂芳基环。
在本发明中除非另外指出，
Figure PCTCN2020122472-appb-000008
表示连接位点。
除非另外说明，本文所述的烷基、烷氧基、环烷基、杂环基和芳基为取代的和未取代的基团。烷基、烷氧基、环烷基、杂环基和芳基上可能的取代基包括，但不限于：羟基、氨基、硝基、腈基、卤素、C1-C6烷基、C2-C10烯基、C2-C10炔基、C3-C20环烷基、C3-C20环烯基、C1-C20杂环烷基、C1-C20杂环烯基、C1-C6烷氧基、芳基、杂芳基、杂芳氧基、C1-C10烷基氨基、C1-C20二烷基氨基、芳基氨基、二芳基氨基、C1-C10烷基氨磺酰基、芳基氨磺酰基、C1-C10烷基亚氨基、C1-C10烷基磺基亚氨基、芳基磺基亚氨基、巯基、C1-C10烷硫基、C1-C10烷基磺酰基、芳基磺酰基、酰基氨基、氨酰基、氨基硫代酰基、胍基、脲基、氰基、酰基、硫代酰基、酰氧基、羧基和羧酸酯基。另一方面，环烷基、杂环烷基、杂环烯基、芳基和杂芳基也可互相稠合。
本发明中，所述取代为单取代或多取代，所述多取代为二取代、三取代、四取代、或五取代。所述二取代就是指具有两个取代基，依此类推。
除非特别说明，本发明所描述的结构式意在包括所有的互变异构、光学异构和立体异构形式(如对映异构体、非对映异构体，几何异构体或构象异构体)：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体和(Z)、(E)的构象异构体。因此本发明的化合物的单个立体化学异构体、互变异构体或其对映异构体、非对映异构体或几何异构体或构象异构体或互变异构体的混合物都属于本发明的范围。
术语“互变异构体”表示具有不同能量的结构同分异构体可以超过低能垒，从而互相转化。比如，质子互变异构体(即质子移变)包括通过质子迁移进行互变，如1H-吲唑与2H-吲唑、1H-苯并[d]咪唑与3H-苯并[d]咪唑，化合价互变异构体包括通过一些成键电子重组而进行互变。
在本文中，所述的药学上可接受的盐没有特别的限制，优选包括：无机酸盐、有机酸盐、烷基磺酸盐和芳基磺酸盐；所述无机酸盐包括盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐等；所述有机酸盐包括甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐等；所述烷基磺酸盐包括甲基磺酸盐、乙基磺酸盐等；所述芳基磺酸盐包括苯磺酸盐、对甲苯磺酸盐等。
制备方法
本发明的五环三萜类化合物，能够采用以下路线进行制备。
路线1
Figure PCTCN2020122472-appb-000009
路线2
Figure PCTCN2020122472-appb-000010
路线3
Figure PCTCN2020122472-appb-000011
路线4
Figure PCTCN2020122472-appb-000012
路线5
Figure PCTCN2020122472-appb-000013
Figure PCTCN2020122472-appb-000014
路线6
Figure PCTCN2020122472-appb-000015
路线7
Figure PCTCN2020122472-appb-000016
Figure PCTCN2020122472-appb-000017
路线8
Figure PCTCN2020122472-appb-000018
路线9
Figure PCTCN2020122472-appb-000019
路线10
Figure PCTCN2020122472-appb-000020
路线11
Figure PCTCN2020122472-appb-000021
路线12
Figure PCTCN2020122472-appb-000022
路线13
Figure PCTCN2020122472-appb-000023
路线14
Figure PCTCN2020122472-appb-000024
路线15
Figure PCTCN2020122472-appb-000025
路线16
Figure PCTCN2020122472-appb-000026
路线17
Figure PCTCN2020122472-appb-000027
路线18
Figure PCTCN2020122472-appb-000028
路线19
Figure PCTCN2020122472-appb-000029
路线20
Figure PCTCN2020122472-appb-000030
路线21
Figure PCTCN2020122472-appb-000031
药物组合物
本发明还提供了一种药物组合物，它包含安全有效量范围内的活性成分，以及药学上可接受的载体。
本发明所述的“活性成分”是指本发明所述的式I化合物。
本发明所述的“活性成分”和药物组合物用于制备治疗代谢性疾病的药物。本发明所述的“活性成分”和药物组合物可用作TGR5受体激动剂。在另一优选例中，用于制备预防和/治疗受TGR5激动剂调节的疾病的药物。
“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg活性成分/剂，更佳地，含有10-200mg活性成分/剂。较佳地，所述的“一剂”为一个药片。
“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂
Figure PCTCN2020122472-appb-000032
润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明的活性成分或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)等。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物可以单独给药，或者与其他治疗药物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选20～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件(如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件)或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文 中所述的较佳实施方法与材料仅作示范之用。
下述制备实施例中，NMR用Varian生产的Mercury-Vx 300M仪器测定，NMR定标：δH 7.26ppm(CDCl 3)，2.50ppm(DMSO-d 6)；质谱用Agilent 1200 Quadrupole LC/MS液质联用仪或SHIMADZU GCMS-QP5050A测定；试剂主要由上海化学试剂公司提供；TLC薄层层析硅胶板由山东烟台会友硅胶开发有限公司生产，型号HSGF 254；化合物纯化使用的正相柱层析硅胶为山东青岛海洋化工厂分厂生产，型号zcx-11，200-300目。
本文缩写所对应的的中文如下：DMAP:4-二甲氨基吡啶；DCM:二氯甲烷；DMF：N,N-二甲基甲酰胺；THF：四氢呋喃。
实施例1
Figure PCTCN2020122472-appb-000033
(1)室温下将原料白桦脂酸S1(1.2g,2.63mmol)溶于甲醇(50mL)中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温下搅拌。24小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到化合物S2 1.04g(2.27mmol)，为白色固体，收率：86％。 1H NMR(300MHz,CDCl 3)δ3.13(t,1H,J＝9.0,6.9Hz),2.28–2.16(m,2H),1.98–1.78(m,4H),1.64–0.96(m,其它脂肪环质子),0.96(s,3H),0.93(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.78(s,3H).
(2)室温下将上步产物S2(1.04g,2.27mmol)溶解在DMF(20mL)中，加入无水碳酸钾(0.626g,4.54mmol)，搅拌下慢慢滴加氯化苄(0.313mL,2.72mmol)。滴加完毕后将反应液移至50℃搅拌3h，TLC监测显示反应进行完全。将混合物冷却至室温，加入去离子水50mL稀释，用乙酸乙酯(2×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥后并减压蒸馏得到所需白色固体化合物S3 1.21g(2.20mmol)，直接用于下一步反应，收率：97％。
(3)冰水浴下将S3(1.21g，2.20mmol)溶于二氯甲烷(30mL),分批加入Dess-Martin氧化剂(1.87g,4.40mmol),慢慢升至室温并搅拌1小时。将反应混合物过滤后旋干，用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到化合物S4 0.983g(1.80mmol)，为白色固体，收率：82％。 1H NMR(300MHz,CDCl 3)δ7.38-7.30(m,5H),5.16-5.05(m,2H),2.55-2.34(m,2H),2.31–2.18(m,3H),1.95–1.12(m,其它脂肪环质子),1.06(s,3H),1.01(s,3H),0.93(s,3H),0.91(s,3H),0.86(d,3H,J＝6.9Hz),0.79-0.73(m,6H).
(4)上步产物S4(0.983g,1.80mmol)和S-(-)-2-甲基恶唑硼烷(100mg,0.36mmol)与100mL烘干的圆底烧瓶，加入新鲜钠丝处理过的THF(70mL)。室温下慢慢滴加10M的硼烷-四氢呋喃溶液(0.32mL)，控制滴加速度,在十分钟内加完，室温下搅拌十分钟， TLC监测显示反应已经进行完。将反应瓶移至冰水浴，慢慢滴加甲醇淬灭反应，待不再有气泡生成后旋干溶剂，用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到化合物S5 827mg(1.51mmol)，为白色固体，收率：84％。 1H NMR(300MHz,CDCl 3)δ7.37-7.30(m,5H),5.15-5.06(m,2H),3.38(t,1H,J＝3.0Hz),2.55-2.34(m,2H),2.31–2.18(m,3H),1.95–1.12(m,其它脂肪环质子),0.94(s,3H),0.93(s,3H),0.91(s,3H),0.85-0.82(m,9H),0.75-0.73(m,6H).
(5)将中间体S5(100mg,0.182mmol)溶于干燥DCM(5mL)中，0℃下滴加三氯乙酰异氰酸酯(25.9μL,0.218mmol)，保持该温度反应1小时，旋出DCM，加甲醇(5mL)溶解，然后室温下加入碳酸钾(50.2mg,0.364mmol)并搅拌2小时，TLC显示反应完全。加入去离子水淬灭，旋出甲醇，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S6 103mg(0.174mmol)，收率：95.5％。 1H NMR(300MHz,CDCl 3)δ7.37-7.29(m,5H),5.15-5.06(m,2H),4.59(br s,2H),4.49(t,1H,J＝3.0Hz),2.30-2.15(m,3H),1.92-0.99(m,其它脂肪环质子),0.97(s,3H),0.87-0.83(m,12H),0.75-0.73(m,6H).
(6)化合物T1的制备：室温下将中间体S6(103mg,0.174mmol)溶于甲醇(20mL)中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温下搅拌。2小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯的洗脱剂体系进行柱层析分离，得到化合物T1 79.0mg，为白色固体，收率：90.5％。 1H NMR(500MHz,CDCl 3)δ4.86(br s,2H),4.50(t,1H,J＝3.0Hz),2.28-2.21(m,3H),1.90-1.09(m,其它脂肪环质子),1.00(s,3H),0.94(s,3H),0.88-0.85(m,12H),0.76(d,3H,J＝6.5Hz).
实施例2
Figure PCTCN2020122472-appb-000034
(1)将S5(1.00g,1.82mmol)溶于干燥DCM(10mL)中，0℃下滴加吡啶(732μL,9.10mmol)，0.5小时后滴加对硝基苯基氯甲酸酯(1.10g,5.46mmol)的干燥DCM溶液(10mL)，恢复至室温后搅拌5小时，TLC显示反应完全。旋出DCM，用乙酸乙酯(3×100mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S7 1.20g(1.69mmol)，收率：92.2％。 1H NMR(300MHz,CDCl 3)δ8.30-8.27(m,2H),7.40-7.35(m,7H),5.15-5.06(m,2H),4.60(t,1H,J＝3.0Hz),2.30-2.15(m,5H),2.04-1.05(m,其它脂肪环质子),0.96(s,3H),0.95(s,3H),0.92(s,3H),0.86-0.84(m,6H),0.76-0.74(m,6H).
(2)将中间体S-7(100mg,0.182mmol),DMAP(66.7mg,0.546mmol)和三乙胺(75.7μL,0.546mmol)溶于干燥DCM(2mL)中,滴加环丙胺(63.0μL,0.910mmol)，室温下搅拌过夜，次日TLC显示反应完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的 有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物81mg(0.128mmol)。 1H NMR(300MHz,CDCl 3)δ7.38-7.30(m,5H),5.15-5.06(m,2H),4.82(br s,1H),4.51(t,1H,J＝2.7Hz),2.61-2.57(m,1H),2.30-2.15(m,3H),1.92-0.98(m,其它脂肪环质子),0.95(s,3H),0.86-0.83(m,12H),0.75-0.68(m,8H),0.55-0.50(m,2H).
(3)经类似的脱苄基过程可得化合物T6 53.5mg,产率77.0％。 1H NMR(300MHz,CDCl 3)δ4.51(t,1H,J＝3.0Hz),2.60-2.55(m,1H),2.29-2.17(m,3H),1.91-1.07(m,其它脂肪环质子),0.97(s,3H),0.93(s,3H),0.87-0.84(m,12H),0.76-0.68(m,5H),0.56-0.51(m,2H).
用实施例2中类似的方法，中间体S7与不同的胺反应可得到以下化合物。
Figure PCTCN2020122472-appb-000035
Figure PCTCN2020122472-appb-000036
Figure PCTCN2020122472-appb-000037
实施例3
Figure PCTCN2020122472-appb-000038
(1)中间体S8的制备：氢化钠(9.84mg,0.246mmol,60％)悬浮于干燥DMF(1mL)中，0℃下滴入中间体S8(76.0mg，0.123mmol)的干燥DMF溶液2mL，然后保持0℃滴入碘甲烷(38.3μL,0.615mmol)，10分钟后恢复至室温，搅拌过夜。次日TLC监测显示反应进行完全。加入去离子水淬灭，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S9 59.0mg(0.093mmol)，收率：76.0％。 1H NMR(300MHz,CDCl 3)δ7.37-7.30(m,5H),5.15-5.06(m,2H),4.49(t,1H,J＝2.7Hz),3.35-3.28(m,2H),2.90(s,3H),2.30-2.15(m,3H),1.91-0.96(m,其它脂肪环质子),0.93(s,3H),0.88-0.84(m,12H),0.76-0.73(m,6H).
(2)中间体S9经类似的脱苄基过程可得化合物T20(32.0mg,63.2％)。 1H NMR(300MHz,CDCl 3)δ4.50(t,1H,J＝2.7Hz),3.36-3.29(m,2H),2.91(s,3H),2.29-2.17(m,3H),1.92-1.08(m,其它脂肪环质子),0.97(s,3H),0.94(s,3H),0.88-0.85(m,12H),0.76(d,3H,J＝6.9Hz).
实施例4
Figure PCTCN2020122472-appb-000039
(1)中间体S10的制备：将化合物S5(130mg,0.237mmol)溶于3mL吡啶中，-20℃下加入氯甲酸乙酯(113μL,1.19mmol)，10分钟后回至室温搅拌，2小时后TLC检测反应完全。加入去离子水30mL稀释，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用稀盐酸，去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩后进行柱层析分离，得产物 S10 97.0mg(0.156mmol)，收率：66.0％。 1H NMR(300MHz,CDCl 3)δ7.38-7.30(m,5H),5.15-5.06(m,2H),4.46(t,1H,J＝3.0Hz),4.19(q,2H,J＝7.2Hz),2.30-2.14(m,3H),1.97-0.98(m,其它脂肪环质子),0.95(s,3H),0.90-0.85(m,9H),0.83(s,3H),0.75-0.73(m,6H).
(2)化合物T21的制备：将中间体S10(97.0mg,0.156mmol)溶于乙酸乙酯和叔丁醇(1:1,10mL)中，换氮气后，迅速加入10％的Pd(OH) 2/C,再换氮气后换氢气，室温下搅拌。1小时后TLC检测反应完全。换氮气后滤去Pd(OH) 2/C，反应液旋干后进行柱层析分离得化合物T21 48.2mg(0.091mmol)，收率：58.1％。 1H NMR(300MHz,CDCl 3)δ4.47(t,1H,J＝3.0Hz),4.20(q,2H,J＝7.2Hz),2.28-2.14(m,3H),1.96-1.14(m,其它脂肪环质子),0.98(s,3H),0.93(s,3H),0.90(s,3H),0.88-0.84(m,9H),0.75(d,3H,J＝6.9Hz).
实施例5
Figure PCTCN2020122472-appb-000040
(1)中间体S5(50.0mg，0.091mmol)溶于吡啶溶液1mL，然后保持0℃滴入甲氨基磺酰氯(39.3μL,0.455mmol)，10分钟后恢复至室温，搅拌过夜，次日TLC显示反应完全。旋出吡啶，加入去离子水，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物51.0mg(0.080mmol)，收率：87.2％。 1H NMR(300MHz,CDCl 3)δ7.38-7.30(m,5H),5.15-5.06(m,2H),4.33-4.28(m,2H),2.82(d,3H,J＝5.1Hz),2.30-2.15(m,3H),1.95-1.10(m,其它脂肪环质子),1.00(s,3H),0.95(s,3H),0.88(s,3H),0.86-0.83(m,6H),0.74(t,6H,J＝3.3Hz).
(2)经与化合物T1制备中类似的脱苄基过程可得化合物T22(28.0mg,90.5％)。 1H NMR(300MHz,CDCl 3)δ4.40-4.33(m,1H),4.30(t,1H,J＝2.7Hz),2.83(d,3H,J＝5.4Hz),2.28-2.16(m,4H),1.96-1.14(m,其它脂肪环质子),1.01(s,3H),0.99(s,3H),0.93(s,3H),0.89-0.84(m,9H),0.76(d,3H,J＝6.6Hz).
用实施例5中类似的方法，中间体S5与酰氯、磺酰氯和氨基磺酰氯反应可得到化合物T23-T26。
Figure PCTCN2020122472-appb-000041
Figure PCTCN2020122472-appb-000042
实施例6
Figure PCTCN2020122472-appb-000043
(1)中间体S5经类似的脱苄基过程可得化合物S11(35.0mg,87.5％)。 1H NMR(300MHz,CDCl 3)δ3.39(s,1H),2.28-2.16(m,2H),1.98-1.78(m,4H),1.64-0.96(m,其它脂肪环质子),0.96(s,3H),0.93(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.78(s,3H).
(2)氢化钠(15.3mg,0.382mmol)悬浮于干燥THF 1mL中，0℃下搅拌0.5h，然后加入乙基硫代异氰酸酯(100μL,3.82mmol)，回至室温搅拌过夜，次日TLC显示反应完全。加入去离子水淬灭，旋出THF，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物T27 26.0mg(0.048mmol)，收率：62.4％。 1H NMR(400MHz,CDCl 3)δ6.71(t,0.5H,J＝5.6Hz),6.24(t,0.5H,J＝5.6Hz),5.20(t,1H,J＝2.8Hz),3.64-3.57(m,1H),3.37-3.31(m,1H),2.25-2.20(m,4H),1.91-1.05(m,其它脂肪环质子),0.97-0.91(m,9H),0.89-0.82(m,12H),0.75(d,3H,J＝6.4Hz).
实施例7
Figure PCTCN2020122472-appb-000044
(1)白桦脂酸苄基保护之后再经翻转羟基得到中间体S12。 1H NMR(300MHz,CDCl 3)δ7.37-7.31(m,5H),5.17-5.06(m,2H),4.72(s,1H),4.60-4.58(m,1H),3.39-3.36(m,1H),3.06-2.98(m,1H),2.29-2.13(m,3H),1.98-1.02(m,其它脂肪环质子),0.96(s,3H),0.92(s,3H),0.81(s,6H),0.76(s,3H).
(2)将S12(1.96g,3.59mmol)和DMAP(43.9mg,0.359mmol)溶于吡啶(20mL)中，0℃下滴加乙酸酐(1.70mL,18.0mmol)，10分钟后恢复至室温后搅拌过夜，次日TLC监测显示反应进行完全。旋出吡啶，用乙酸乙酯(3×100mL)萃取，将合并的有机层分别用去离子水，1N稀盐酸和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S132.07g(3.52mmol)，收率：98.1％。 1H NMR(300MHz,CDCl 3)δ7.39-7.31(m,5H),5.17-5.07(m,2H),4.72(s,1H),4.62-4.58(m,2H),3.07-2.98(m,1H),2.31-2.14(m,3H),2.06(s,3H),1.94-1.03(m,其它脂肪环质子),1.68(s,3H),1.00(s,3H),0.86(s,3H),0.82(s,6H),0.77(s,3H).
(3)中间体S13(60.0mg,0.102mmol)溶于MeOH/DCM(1:1,10mL)中，在-78℃下通入臭氧，1分钟后TLC显示反应完全。停止通入臭氧后用氧气排尽残余的臭氧，加入二甲硫醚25μL淬灭反应，升至室温搅拌过夜。次日，反应液旋干后柱层析分离得化合物S14 44.0mg(0.075mmol)，收率：73.1％。 1H NMR(300MHz,CDCl 3)δ7.37-7.31(m,5H),5.17-5.07(m,2H),4.60(t,1H,J＝3.0Hz),3.29-3.22(m,1H),2.33-2.27(m,1H),2.16(s,3H),2.07(s,3H),2.04-1.06(m,其它脂肪环质子),1.02(s,3H),0.85(s,3H),0.81(s,6H),0.74(s,3H).
(4)中间体S14经类似的脱苄基过程可得化合物T28(35.5mg,95.2％)。 1H NMR(300MHz,CDCl 3)δ4.61(s,1H),3.29-3.20(m,1H),2.31-2.25(m,1H),2.18(s,3H),2.08(s,3H),2.04-1.09(m,其它脂肪环质子),1.06(s,3H),0.93(s,3H),0.86(s,3H),0.84(s,3H),0.83(s,3H).
实施例8
Figure PCTCN2020122472-appb-000045
(1)将中间体S13(70.0mg,0.119mmol)溶于DCM(3mL)中，0℃下加入m-CPBA(51.3mg,0.238mmol,80％)，10分钟后恢复至室温后搅拌2小时，TLC监测显示反应进行 完全。加饱和亚硫酸钠水溶液淬灭，旋出DCM后用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，饱和碳酸氢钠水溶液和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S15 58.0mg(0.096mmol)，收率：80.7％。 1H NMR(300MHz,CDCl 3)δ7.35-7.32(m,5H),5.11-5.08(m,2H),4.61(t,1H,J＝3.0Hz),2.65-2.54(m,2H),2.32-2.27(m,1H),2.17-2.10(m,2H),2.07(s,3H),1.97-1.02(m,其它脂肪环质子),1.23(s,3H),0.99(s,3H),0.86(s,3H),0.82(s,6H),0.76(s,3H).
(2)中间体S15经类似的脱苄基过程可得化合物T29(27.2mg,55.1％)。 1H NMR(300MHz,CDCl 3)δ4.62(t,1H,J＝2.4Hz),2.66-2.63(m,2H),2.30-2.27(m,1H),2.18-2.11(m,2H),2.08(s,3H),2.04-1.05(m,其它脂肪环质子),1.25(s,3H),1.02(s,3H),0.94(s,3H),0.87(s,3H),0.86(s,3H),0.84(s,3H).
实施例9
Figure PCTCN2020122472-appb-000046
(1)将中间体S14(25.0mg,0.043mmol)溶于吡啶(1mL)中，0℃下加入羟胺盐酸盐(34.7mg,0.500mmol)，10分钟后恢复至室温后搅拌过夜，次日TLC显示反应完全。旋出大部分吡啶，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，1N稀盐酸和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S16 16.0mg(0.026mmol)，收率：62.2％。 1H NMR(300MHz,CDCl 3)δ7.38-7.30(m,5H),5.17-5.08(m,2H),4.61(t,1H,J＝2.7Hz),3.21-3.13(m,1H),2.33-2.28(m,1H),2.21-2.11(m,2H),2.06(s,3H),2.04-1.00(m,其它脂肪环质子),1.81(s,3H),0.98(s,3H),0.86(s,3H),0.82(s,6H),0.75(s,3H).
(2)中间体S16经类似的脱苄基过程可得化合物T30(6.00mg,44.1％)。 1H NMR(500MHz,CDCl 3)δ4.60(t,1H,J＝3.0Hz),3.16(t,1H,J＝11.0Hz),2.38-2.36(m,1H),2.24-2.20(m,1H),2.11-2.03(m,5H),1.90(s,3H),1.86-1.75(m,3H),1.61-1.03(m,其它脂肪环质子),1.00(s,3H),0.92(s,3H),0.84(s,3H),0.82(s,3H),0.81(s,3H).
实施例10
Figure PCTCN2020122472-appb-000047
(1)将S13(53.0mg,0.090mmol)溶于干燥THF(1mL)中，0℃下滴入10M的BH 3-Me 2S(27.0μL,0.270mmol)，搅拌1小时后，TLC检测基本反应完全。向反应液中依 次加入乙醇(100μL)，饱和碳酸钠溶液(80μL)和30％的过氧化氢溶液(100μL)，室温下搅拌2小时，TLC检测反应完全。加饱和亚硫酸钠溶液中和剩余的过氧化氢，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，1N稀盐酸和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得到化合物S17 39.0mg(0.065mmol)，收率：71.6％。 1H NMR(300MHz,CDCl 3)δ7.36-7.30(m,5H),5.15-5.05(m,2H),4.61(t,1H,J＝3.0Hz),3.77(dd,1H,J＝10.5Hz,4.8Hz),3.41(dd,1H,J＝10.5Hz,7.8Hz),2.35-2.16(m,3H),2.07(s,3H),1.90-1.80(m,3H),1.69-1.03(m,其它脂肪环质子),0.98(s,3H),0.95(d,3H,J＝6.9Hz),0.86(s,3H),0.82(s,6H),0.75(s,3H).
(2)中间体S17经类似的脱苄基过程可得化合物T31(8.00mg,55.3％)。 1H NMR(500MHz,CDCl 3)δ4.62(t,1H,J＝3.0Hz),3.79(dd,1H,J＝10.5,4.5Hz),3.44(dd,1H,J＝10.5,8.0Hz),2.35-2.30(m,1H),2.28-2.19(m,2H),2.08(s,3H),1.91-1.84(m,3H),1.75-1.08(m,其它脂肪环质子),1.01(s,3H),0.97(d,3H,J＝6.5Hz),0.94(s,3H),0.87(s,3H),0.86(s,3H),0.83(s,3H).
实施例11
Figure PCTCN2020122472-appb-000048
(1)将SeO 2(11.3mg,0.102mmol)和TBHP(39.1μL,0.408mmol)溶于干燥DCM(1mL)中，0℃下滴入乙酸(1.14μL,0.020mmol)，搅拌10分钟后，加入中间体S13(120mg,0.204mmol)的干燥DCM溶液(3mL)，室温下搅拌过夜，次日TLC检测反应完全。加饱和亚硫酸钠溶液中和剩余的TBHP，旋出DCM，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，饱和Na 2SO 3溶液和饱和食盐水洗涤，经硫酸钠干燥后浓缩，然后加如甲醇(5mL)溶解，加入NaBH 4(15.4mg,0.408mmol)反应1小时，TLC检测反应完全。加丙酮淬灭，直接旋干后经柱层析分离得化合物S18 85.4mg(0.141mmol)，收率：69.3％。 1H NMR(300MHz,CDCl 3)δ7.37-7.32(m,5H),5.17-5.07(m,2H),4.95(s,1H),4.91(s,1H),4.61(t,1H,J＝3.0Hz),4.13-4.10(m,2H),2.93-2.85(m,1H),2.34-2.28(m,1H),2.21-2.12(m,2H),2.06(s,3H),1.98-1.03(m,其它脂肪环质子),1.00(s,3H),0.86(s,3H),0.82(s,6H),0.76(s,3H).
(2)中间体S18(45.0mg,0.075mmol)溶于MeOH/DCM(1:1,10mL)中，在-78℃下通入臭氧，1分钟后TLC显示反应完全。停止通入臭氧后用氧气排尽残余的臭氧，加入二甲硫醚25μL淬灭反应，升至室温搅拌过夜。次日，反应液旋干后硅胶柱层析得化合物S19 31.0mg(0.051mmol)，收率：68.7％。 1H NMR(300MHz,CDCl 3)δ7.37-7.32(m,5H),5.18-5.07(m,2H),4.61(t,1H,J＝3.0Hz),4.27(d,2H,J＝4.5Hz),3.28-3.19(m,1H),3.11(t,1H,J＝4.8Hz),2.35-2.20(m,2H),2.07(s,3H),2.04-1.05(m,其它脂肪环质子),1.01(s, 3H),0.86(s,3H),0.81(s,6H),0.74(s,3H).
(3)化合物T32的制备：中间体S19经类似的脱苄基过程可得化合物T32(21.1mg,79.9％)。 1H NMR(300MHz,CDCl 3)δ4.62(s,1H),4.30(s,2H),3.27-3.18(m,2H),2.33-2.25(m,2H),2.08(s,3H),2.05-1.08(m,其它脂肪环质子),1.05(s,3H),0.92(s,3H),0.86(s,3H),0.84(s,3H),0.83(s,3H).
实施例12
Figure PCTCN2020122472-appb-000049
(1)将S18(40.4mg,0.067mmol)溶于干燥THF(1mL)中，0℃下滴入10M的BH 3-Me 2S(20.1μL,0.201mmol)，搅拌1小时后，TLC检测基本反应完全。向反应液中依次加入乙醇(100μL)，饱和碳酸钠溶液(80μL)和30％的过氧化氢溶液(100μL)，室温下搅拌2小时，TLC检测反应完全。加饱和亚硫酸钠溶液中和剩余的过氧化氢，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，1N稀盐酸和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得到化合物S20 23.0mg(0.037mmol)，收率：55.3％。 1H NMR(300MHz,CDCl 3)δ7.36-7.30(m,5H),5.14-5.04(m,2H),4.61(t,1H,J＝2.7Hz),3.91-3.87(m,1H),3.80-3.67(m,3H),2.34-2.17(m,4H),2.08(s,3H),2.04-1.03(m,其它脂肪环质子),0.99(s,3H),0.86(s,3H),0.82(s,6H),0.75(s,3H).
(2)中间体S20经类似的脱苄基过程可得化合物T33(15.5mg,78.8％)。 1H NMR(500MHz,CD 3OD)δ4.61(t,1H,J＝2.5Hz),3.74(dd,1H,J＝11.0,4.5Hz),3.66(dd,1H,J＝11.0,6.5Hz),3.55(dd,1H,J＝11.0,7.5Hz),3.46(dd,1H,J＝11.0,7.5Hz),2.58-2.52(m,1H),2.40-2.35(m,1H),2.26-2.22(m,1H),2.06(s,3H),1.98-1.89(m,2H),1.84-1.80(m,1H),1.76-1.74(m,1H),1.68-1.16(m,其它脂肪环质子),1.05(s,3H),0.99(s,3H),0.92(s,3H),0.91(s,3H),0.85(s,3H).
实施例13
Figure PCTCN2020122472-appb-000050
(1)将中间体S17(201mg,0.332mmol)溶于DCM(5mL)中，0℃下加入DMP(211mg,0.498mmol)，保持该温度反应15分钟，TLC监测显示反应进行完全。硅藻土过滤，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，饱和Na 2SO 3水溶液 和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S21 56.0mg(0.093mmol)，摩尔收率：28.0％。中间体S21不稳定，直接投下一步。
(2)将中间体S21(56.0mg,0.093mmol)和过量的2-甲基-2-丁烯(0.2mL)溶于叔丁醇(2mL)中，室温下下加入NaClO 2(29.5mg,0.326mmol)和NaH 2PO 4(55.8mg,0.465mmol)的混合水溶液(2mL)，反应4小时，TLC显示反应完全。用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水，饱和NH 4Cl水溶液和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S22 56.2mg(0.093mmol)，摩尔收率：97.5％。 1H NMR(300MHz,CDCl 3)δ7.37-7.31(m,5H),5.16-5.05(m,2H),4.62(s,1H),2.80-2.71(m,1H),2.45-2.38(m,1H),2.30-2.12(m,2H),2.07(s,3H),1.92-1.07(m,其它脂肪环质子),1.16(d,3H,J＝7.2Hz),0.97(s,3H),0.87(s,3H),0.83(s,6H),0.75(s,3H).ESI-MS(m/z):643.4(M+Na) +.
(3)中间体S22经类似的脱苄基过程可得化合物T34(12.4mg,72.5％)。 1H NMR(300MHz,d 6-DMSO)δ12.02(br s,1H),4.50(s,1H),2.24-2.07(m,3H),2.02(s,3H),1.99-1.07(m,其它脂肪环质子),1.02(d,3H,J＝6.9Hz),0.93(s,3H),0.89(s,3H),0.84-0.83(m,6H),0.78(s,3H).
实施例14
Figure PCTCN2020122472-appb-000051
(1)将中间体S17(50.0mg,0.083mmol)溶于干燥DMF(3mL)中，室温下加入三氧化硫三乙胺盐(150mg,0.830mmol)，加热至95℃反应24小时，TLC监测显示反应进行完全。加水稀释，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S23 21.5mg(0.031mmol)，收率：37.9％。 1H NMR(300MHz,CD 3OD)δ7.40-7.29(m,5H),5.17-5.05(m,2H),4.59(t,1H,J＝2.7Hz),4.16-4.11(m,1H),3.86-3.80(m,1H),2.34-2.14(m,3H),2.05(s,3H),1.99-1.06(m,其它脂肪环质子),1.03(s,3H),0.97(d,3H,J＝6.9Hz),0.89(s,3H),0.87(s,3H),0.84(s,3H),0.74(s,3H).
(2)中间体S23经类似的脱苄基过程可得化合物T35(7.10mg,70.4％)。 1H NMR(300MHz,CD 3OD)δ4.60(t,1H,J＝2.4Hz),4.19-4.14(m,1H),3.87-3.81(m,1H),2.37-2.31(m,2H),2.22(d,1H,J＝11.7Hz),2.06(s,3H),2.02-1.14(m,其它脂肪环质子),1.06(s,3H),1.00-0.98(m,6H),0.91(s,6H),0.85(s,3H).
实施例15
Figure PCTCN2020122472-appb-000052
(1)将中间体S17(30.0mg,0.050mmol),DMAP(1.22mg,0.010mmol)，EDCI(19.1mg,0.100mmol)和4-吗啉乙酸(14.5mg,0.100mmol)溶于干燥DCM(2mL)中，室温下搅拌过夜，次日TLC显示反应完全。旋出DCM，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物33.5mg(0.046mmol)，收率：92.0％。 1H NMR(300MHz,CDCl 3)δ7.37-7.31(m,5H),5.15-5.04(m,2H),4.61(t,1H,J＝3.0Hz),4.30-4.25(m,1H),3.90-3.84(m,1H),3.78-3.73(m,4H),3.21(s,3H),2.60-2.57(m,4H),2.40-2.16(m,3H),2.09-2.02(m,4H),1.87-1.80(m,2H),1.67-1.07(m,其它脂肪环质子),0.98(s,3H),0.94(d,3H,J＝6.9Hz),0.93(s,3H),0.86(s,3H),0.82(s,3H),0.74(s,3H).
(2)经类似的脱苄基过程可得化合物T36(22.0mg,74.9％)。 1H NMR(500MHz,CDCl 3)δ4.62(t,1H,J＝3.0Hz),4.28(dd,1H,J＝10.5,4.5Hz),3.89(dd,1H,J＝10.5,8.0Hz),3.76(t,4H,J＝4.5Hz),3.23(s,2H),2.62-2.60(m,4H),2.37-2.31(m,1H),2.28-2.18(m,2H),2.09(s,3H),2.07-2.00(m,1H),1.91-1.83(m,2H),1.77-1.09(m,其它脂肪环质子),1.02(s,3H),0.95(d,1H,J＝7.0Hz),0.93(s,3H),0.86(s,3H),0.85(s,3H),0.83(s,3H).
用实施例15中类似的方法，中间体S17与丁二酸缩合可得到化合物T37。
Figure PCTCN2020122472-appb-000053
实施例16
Figure PCTCN2020122472-appb-000054
(1)将中间体S17(150mg,0.248mmol)溶于干燥DCM(2mL)中，0℃下滴加吡啶 (99.9μL,1.24mmol)，0.5小时后滴加对硝基苯基氯甲酸酯(150mg,0.744mmol)的干燥DCM溶液(3mL)，恢复至室温后搅拌5小时，TLC显示反应完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S24 153mg(0.198mmol)，收率：79.9％。 1H NMR(300MHz,CDCl 3)δ8.31-8.26(m,2H),7.41-7.31(m,5H+2H),5.16-5.06(m,2H),4.62(t,1H,J＝2.7Hz),4.40(dd,1H,J＝10.5,4.8Hz),4.07(dd,1H,J＝10.5,7.8Hz),2.46-2.19(m,6H),2.06(s,3H),1.91-1.08(m,其它脂肪环质子),1.03-1.00(m,6H),0.86(s,3H),0.83(s,3H),0.82(s,3H),0.75(s,3H).
(2)将中间体S24(40.0mg,0.052mmol),DMAP(19.1mg,0.156mmol)和三乙胺(21.6μL,0.156mmol)溶于干燥DCM(2mL)中,加入N-(2-氨基乙基)吗啉(34.0μL,0.260mmol)，室温下搅拌过夜，次日TLC显示反应完全。加水稀释，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物35.0mg(0.046mmol)，收率：88.5％。 1H NMR(300MHz,CDCl 3)δ7.37-7.30(m,5H),5.15-5.05(m,2H+1H),4.61(t,1H,J＝2.7Hz),4.18-4.16(m,1H),3.87-3.81(m,1H),3.73-3.70(m,4H),3.32-3.27(m,2H),2.50-2.46(m,6H),2.35-2.13(m,6H),2.08(s,3H),1.92-1.03(m,其它脂肪环质子),0.98(s,3H),0.93(d,3H,J＝6.6Hz),0.86(s,3H),0.82(s,3H),0.74(s,3H).
(3)经类似的脱苄基过程可得化合物T38(24.3mg,78.7％)。 1H NMR(300MHz,CDCl 3)δ5.33(s,1H),4.62(s,1H),4.26-4.21(m,1H),3.82-3.73(m,5H),3.38-3.28(m,2H),3.32-3.27(m,2H),2.52-2.50(m,6H),2.37-2.17(m,6H),2.09(s,3H),2.05-1.10(m,其它脂肪环质子),1.02(s,3H),0.98-0.93(m,6H),0.86(s,3H),0.85(s,3H),0.83(s,3H).
用实施例16中类似的方法，中间体S24与2-吗啉乙醇缩合可得到化合物T39。
Figure PCTCN2020122472-appb-000055
实施例17
Figure PCTCN2020122472-appb-000056
(1)将中间体S22(28.0mg,0.045mmol),甘氨酸苄酯盐酸盐(10.9mg,0.054mmol)，EDCI(13.0mg,0.068mmol)，HOBt(6.08mg,0.045mmol)和4-甲基吗啉(14.8μL, 0.135mmol)溶于干燥DMF(2mL)中，室温下搅拌过夜，次日TLC监测显示反应进行完全。加水稀释，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物27.0mg(0.035mmol)，收率：77.9％。 1H NMR(300MHz,CDCl 3)δ7.39-7.30(m,10H),5.83(t,1H,J＝5.4Hz),5.19(s,2H),5.15-5.04(m,2H),4.62(t,1H,J＝3.0Hz),4.08(d,2H,J＝5.1Hz),2.51-2.17(m,3H),2.07(s,3H),1.94-1.05(m,其它脂肪环质子),1.16(d,3H,J＝6.9Hz),0.95(s,3H),0.87(s,3H),0.83(s,6H),0.74(s,3H).
(2)经类似的脱苄基过程可得化合物T40(10.7mg,51.8％)。 1H NMR(300MHz,CD 3OD)δ4.61(s,1H),3.90-3.76(m,2H),2.58-2.54(m,1H),2.35-2.16(m,4H),2.06(s,3H),2.02-1.91(m,1H),1.72-1.12(m,其它脂肪环质子),1.15(d,3H,J＝6.9Hz),1.03(s,3H),0.98(s,3H),0.92(s,3H),0.91(s,3H),0.85(s,3H).
用实施例17中类似的方法，中间体S22与不同的胺缩合可得到以下化合物。
Figure PCTCN2020122472-appb-000057
Figure PCTCN2020122472-appb-000058
实施例18
Figure PCTCN2020122472-appb-000059
(1)将中间体S22(81.5mg,0.131mmol),DMAP(1.22mg,0.010mmol)，EDCI(37.8mg,0.197mmol)和N-(2-羟基乙基)吗啉(159μL,1.31mmol)溶于干燥DCM(2mL)中，室温下搅拌过夜，次日TLC监测显示反应进行完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物70.2mg(0.096mmol)，收率：72.9％。 1H NMR(300MHz,CDCl 3)δ7.37-7.29(m,5H),5.15-5.05(m,2H),4.62(t,1H,J＝2.7Hz),4.20(t,2H,J＝5.7Hz),3.71(t,4H,J＝4.5Hz),2.72-2.60(m,4H),2.51(t,4H,J＝4.5Hz),2.39-2.12(m,5H),2.08(s,3H),2.04-1.02(m,其它脂肪环质子),1.13(d,3H,J＝7.2Hz),0.98(s,3H),0.87(s,3H),0.83(s,6H),0.75(s,3H).
(2)经类似的脱苄基过程可得化合物T46(59.3mg,96.3％)。 1H NMR(500MHz,CDCl 3)δ4.62(t,1H,J＝3.0Hz),4.23-4.21(m,2H),3.72(t,4H,J＝4.5Hz),2.74-2.69(m,1H),2.65(t,2H,J＝6.0Hz),2.54(t,4H,J＝4.5Hz),2.35-2.31(m,1H),2.27-2.16(m,2H),2.08(s,3H),1.91-1.17(m,其它脂肪环质子),1.14(d,3H,J＝7.0Hz),1.01(s,3H),0.94(s,3H),0.87(s,3H),0.86(s,3H),0.84(s,3H).
实施例19
Figure PCTCN2020122472-appb-000060
(1)将中间体S12(1.50g,2.75mmol)溶于干燥DCM(20mL)中，0℃下滴加吡啶(665μL,8.25mmol)，0.5小时后滴加对硝基苯基氯甲酸酯(2.77g,13.8mmol)的干燥DCM溶液(10mL)，恢复至室温后搅拌5小时，TLC监测显示反应进行完全。旋出DCM，用乙酸乙酯(3×100mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫 酸钠干燥浓缩，柱层析分离得产物S25 1.74g(2.45mmol)，收率：89.1％。 1H NMR(300MHz,CDCl 3)δ8.31-8.27(m,2H),7.41-7.33(m,7H),5.17-5.07(m,2H),4.73(d,1H,J＝2.4Hz),4.60-4.59(m,1H),3.07-2.98(m,1H),2.31-2.13(m,2H),2.02-1.01(m,其它脂肪环质子),0.96(s,3H),0.95(s,3H),0.91(s,3H),0.84(s,3H),0.76(s,3H).
(2)将中间体S25(100mg,0.141mmol),DMAP(51.7mg,0.423mmol)和三乙胺(58.6μL,0.423mmol)溶于干燥DCM(2mL)中,滴加环丙胺(58.6μL,0.910mmol)，室温下搅拌过夜，次日TLC显示反应完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物78mg(0.126mmol)。 1H NMR(300MHz,CDCl 3)δ7.39-7.33(m,5H),5.18-5.07(m,2H),4.76-4.71(m,2H),4.59(s,1H),4.49(s,1H),3.07-2.98(m,1H),2.81(d,3H,J＝3.3Hz),2.32-2.13(m,2H),1.94-1.03(m,其它脂肪环质子),0.97(s,3H),0.85(s,6H),0.81(s,3H),0.76(s,3H).
(3)经与中间体S17类似的方法制备得到化合物22.4mg(55.3％)。 1H NMR(300MHz,CDCl 3)δ7.39-7.30(m,5H),5.15-5.05(m,2H),4.64(br s,1H),4.49(s,1H),3.77(dd,1H,J＝10.5,4.5Hz),3.42(dd,1H,J＝10.5,7.8Hz),2.80(d,3H,J＝4.8Hz),2.34-2.16(m,4H),1.92-1.07(m,其它脂肪环质子),0.97-0.94(m,6H),0.86-0.82(m,9H),0.74(s,3H).
(4)经类似的脱苄基过程可得化合物T47(5.80mg,96.8％)。 1H NMR(300MHz,CDCl 3)δ4.68-4.66(m,1H),4.49(s,1H),3.79(dd,1H,J＝10.5,4.8Hz),3.43(dd,1H,J＝10.5,8.1Hz),2.80(d,3H,J＝4.8Hz),2.35-2.17(m,5H),1.92-1.09(m,其它脂肪环质子),0.98-0.96(m,6H),0.93(s,3H),0.86-0.84(m,9H).
用实施例19中类似的方法，可得到化合物T48-T50。
Figure PCTCN2020122472-appb-000061
实施例20
Figure PCTCN2020122472-appb-000062
中间体S26按照化合物T28类似的方法得产物T51(30.7mg,79.5％)。 1H NMR(400MHz,CDCl 3)δ4.94(br s,1H),4.51(s,1H),3.28-3.22(m,1H),2.58(s,1H),2.28(d,1H,J＝10.0Hz),2.15-1.06(m,其它脂肪环质子),1.02(s,3H),0.92(s,3H),0.86-0.84(m,9H),0.74-0.69(m,2H),0.56-0.51(m,2H).
实施例21
Figure PCTCN2020122472-appb-000063
中间体S26按照化合物T29类似的方法得产物T52(42.0mg,84.2％)。 1H NMR(400MHz,CDCl 3)δ4.92(br s,1H),4.51(s,1H),2.67-2.57(m,3H),2.28(d,1H,J＝12.4Hz),2.19-1.02(m,其它脂肪环质子),0.99(s,3H),0.93(s,3H),0.87-0.86(m,9H),0.72-0.71(m,2H),0.54(s,2H).
实施例22
Figure PCTCN2020122472-appb-000064
中间体S26按照化合物T30类似的方法得产物T53(53.0mg,76.5％)。 1H NMR(400MHz,CDCl 3)δ4.96(br s,1H),4.50(s,1H),3.14(t,1H,J＝8.4Hz),2.58(s,1H),2.37(d,1H,J＝12.0Hz),2.26-1.16(m,其它脂肪环质子),0.96(s,3H),0.91(s,3H),0.84(s,6H),0.80(s,3H),0.71-0.70(m,2H),0.54(s,2H).
实施例23
Figure PCTCN2020122472-appb-000065
中间体S26按照化合物T32类似的方法得产物T54(28.2mg,55.6％)。 1H NMR(400MHz,CDCl 3)δ4.50(s,1H),4.34-4.25(m,2H),3.25-3.18(m,1H),2.57(s,1H),2.32-2.22(m,2H),2.10-1.19(m,其它脂肪环质子),1.00(s,3H),0.90(s,3H),0.85-0.83(m,9H),0.71-0.70(m,2H),0.53(s,2H).
实施例24
Figure PCTCN2020122472-appb-000066
中间体S26按照化合物T33类似的方法得产物T55(7.6mg,22.4％)。 1H NMR(400MHz,CD 3OD)δ4.41(s,1H),3.70-3.68(m,1H),3.63-3.59(m,1H),3.53-3.48(m,1H),3.44-3.37(m,1H),2.53-2.47(m,2H),2.33-2.32(m,1H),2.21-2.18(m,1H),1.94-1.11(m,其它脂肪环质子),0.99-0.95(m,6H),0.86-0.84(m,9H),0.62(s,2H),0.43(s,2H).
实施例25
Figure PCTCN2020122472-appb-000067
中间体S26按照化合物T34类似的方法得产物T56(10.5mg,57.3％)。 1H NMR(300MHz,CD 3OD)δ4.45(s,1H),2.68-2.65(m,1H),2.51(s,1H),2.38-1.16(m,其它脂肪环质子),1.11(d,3H,J＝6.9Hz),0.98(s,6H),0.90-0.87(m,9H),0.67-0.64(m,2H),0.49-0.44(m,2H).
实施例26
Figure PCTCN2020122472-appb-000068
(1)中间体S27(180mg,0.285mmol)溶于MeOH 5mL中，0℃下加入NaBH 4(53.9mg,1.425mmol),保持该温度反应5h，TLC显示反应完全。加丙酮淬灭反应，旋出溶剂，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水，饱和Na 2SO 3水溶液和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S28和S29，分别85.7mg(0.135mmol)和81.9mg(0.129mmol)，收率：92.8％。S28: 1H NMR(400MHz,CDCl 3)δ7.36-7.30(m,5H),5.14-5.07(m,2H),4.88(br s,1H),4.50(s,1H),3.89(s,1H),2.58(s,1H),2.30-2.17 (m,3H),1.90-1.81(m,3H),1.70-1.08(m,其它脂肪环质子),1.13(d,3H,J＝6.4Hz),0.96(s,6H),0.82(s,3H),0.75(s,3H),0.72-0.70(m,2H),0.53(s,2H).S29: 1H NMR(400MHz,CDCl 3)δ7.36-7.31(m,5H),5.11(s,2H),4.84(br s,1H),4.50(s,1H),4.02(s,1H),2.59-2.53(m,2H),2.32-2.29(m,1H),2.22-2.16(m,1H),1.91-1.83(m,2H),1.73-1.10(m,其它脂肪环质子),1.06(d,3H,J＝6.0Hz),0.92(s,3H),0.86(s,6H),0.82(s,3H),0.74(s,3H),0.72-0.70(m,2H),0.53(s,2H).
(2)中间体S28和S29经类似的脱苄基过程可得化合物T57(30.2mg,79.5％)和T58(32.5mg,％)。T57: 1H NMR(400MHz,d 6-DMSO)δ11.91(s,1H),7.15(br s,1H),4.32(s,1H),4.15(d,1H,J＝4.8Hz),4.09(q,1H,J＝5.2Hz),3.65-3.62(m,1H),2.44(s,1H),2.25-2.18(m,1H),2.10(d,1H,J＝12.0Hz),2.05-1.99(m,1H),1.88-1.81(m,1H),1.70-1.64(m,1H),1.56-1.06(m,其它脂肪环质子),0.98(d,3H,J＝6.0Hz),0.92(s,3H),0.88(s,3H),0.82(s,9H),0.55(s,2H),0.40(s,2H).T58: 1H NMR(400MHz,CDCl 3)δ4.93(br s,1H),4.51(s,1H),4.05(t,1H,J＝5.6Hz),2.59-2.54(m,2H),2.29-2.20(m,2H),1.97-1.13(m,其它脂肪环质子),1.08(d,3H,J＝6.4Hz),0.94(s,6H),0.87-0.86(m,9H),0.72-0.71(m,2H),0.54(s,2H).
实施例27
Figure PCTCN2020122472-appb-000069
(1)将中间体S30(300mg,0.464mmol)溶于DCM(5mL)中，0℃下加入DMP(295mg,0.696mmol)，保持该温度反应15分钟，TLC显示反应完全。硅藻土过滤，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水，饱和Na 2SO 3水溶液和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S31 210mg(0.325mmol)，收率：70.1％。S31不稳定，直接投下一步。
(2)将中间体S31(240mg,0.372mmol)溶于干燥THF(5mL)中，-78℃下加入甲基溴化镁(186μL,0.558mmol)，保持该温度反应15分钟，恢复至室温反应过夜，TLC显示反应完全。硅藻土过滤，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水，饱和Na 2SO 3水溶液和饱和食盐水洗涤，经硫酸钠干燥浓缩的中间体S32，直接投下一步。S32经与S31类似的氧化方法可得中间体S33,收率：81.9％。 1H NMR(400MHz,CDCl 3)δ7.35-7.30(m,5H),5.13-5.05(m,2H),4.90(s,1H),4.50(s,1H),2.87-2.81(m,1H),2.58(s,1H),2.36-2.15(m,3H),2.13(s,3H),2.09(s,1H),2.04(s,1H),1.90-1.06(m,其它脂肪环质子),1.13(d,3H,J＝6.8Hz),0.91(s,3H),0.86-0.82(m,9H),0.74(s,3H),0.72-0.70(m,2H),0.53(s,2H).
(4)中间体S33经类似的脱苄基过程可得化合物T59(40.0mg,84.2％)。 1H NMR(400MHz,CDCl 3)δ4.94(s,1H),4.51(s,1H),2.88-2.81(m,1H),2.58(s,1H),2.34-2.19(m,3H),2.14(s,3H),1.90-1.17(m,其它脂肪环质子),1.14(d,3H,J＝6.8Hz),0.94(s,3H),0.92(s, 3H),0.86-0.85(m,9H),0.72-0.70(m,2H),0.53(s,2H).
实施例28
Figure PCTCN2020122472-appb-000070
(1)中间体S34酯化后得到中间体S35(11.0mg,31.3％)。 1H NMR(300MHz,CDCl 3)δ7.36-7.29(m,5H),5.15-5.04(m,2H),4.53(t,1H,J＝2.7Hz),4.21-4.19(m,2H),3.71(t,4H,J＝4.8Hz),2.72-2.55(m,5H),2.51(t,4H,J＝4.5Hz),2.41-0.99(m,其它脂肪环质子),1.13(d,3H,J＝6.9Hz),0.94(s,3H),0.86(s,6H),0.83(s,3H),0.74(s,3H),0.71-0.67(m,2H),0.54-0.49(m,2H).
(2)中间体S35经类似的脱苄基过程可得化合物T60(7.10mg,73.0％)。 1H NMR(300MHz,CDCl 3)δ5.22(br s,1H),4.54(s,1H),4.23-4.19(m,2H),3.73(t,4H,J＝4.8Hz),2.74-1.04(m,其它脂肪环质子),1.14(d,3H,J＝6.9Hz),0.97(s,3H),0.94(s,3H),0.87-0.86(m,9H),0.74-0.68(m,2H),0.54-0.51(m,2H).
实施例29
Figure PCTCN2020122472-appb-000071
(1)中间体S36与吗啉乙酸反应得缩合产物28.0mg,产率：75.9％。 1H NMR(300MHz,CDCl 3)δ7.37-7.30(m,5H),5.14-5.04(m,2H),4.20(dd,1H,J＝10.8,5.1Hz),3.91(dd,1H,J＝10.8,7.8Hz),3.77-3.74(m,1H),3.38(t,1H,J＝2.7Hz),3.21(s,2H),2.61-2.58(m,4H),2.38-1.05(m,其它脂肪环质子),0.94-0.91(m,9H),0.81(s,6H),0.74(s,3H).
(2)经与中间体S7类似的方法制备得到活性碳酸酯产物95.0mg,产率：74.5％。 1H NMR(300MHz,CDCl 3)δ8.31-8.26(m,2H),7.42-7.31(m,5H+2H),5.15-5.05(m,2H),4.59(s,1H),4.26(dd,1H,J＝10.8,4.5Hz),3.88(dd,1H,J＝10.8,8.1Hz),3.75(t,4H,J＝4.8Hz),3.22(s,2H),2.60-2.57(m,4H),2.36-2.17(m,3H),2.08-1.05(m,其它脂肪环质子),0.96-0.92(m,12H),0.85(s,3H),0.75(s,3H).
(3)活性碳酸酯与环丙胺反应得产物48.0mg,产率98.3％。 1H NMR(300MHz,CDCl 3)δ7.36-7.31(m,5H),5.15-5.05(m,2H),4.51(t,1H,J＝2.7Hz),4.27(dd,1H,J＝11.1,4.8Hz),3.87(dd,1H,J＝10.5,8.4Hz),3.76(t,4H,J＝4.8Hz),3.21(s,2H),2.61-2.57(m,5H),2.41-1.06(m,其它脂肪环质子),0.95-0.93(m,6H),0.86(s,6H),0.82(s,3H), 0.74-0.69(m,3H+2H),0.55-0.53(m,2H).
(4)经类似的脱苄基过程可得化合物T66(33.8mg,79.7％)。 1H NMR(500MHz,CDCl 3)δ5.01(br s,1H),4.52(t,1H,J＝3.0Hz),4.29-4.23(m,1H),3.89(t,1H,J＝9.5Hz),3.76(t,4H,J＝4.5Hz),3.22(s,2H),2.61-2.59(m,4H),2.37-2.32(m,1H),2.28-2.19(m,2H),2.05-2.01(m,1H),1.92-1.07(m,其它脂肪环质子),0.99(s,3H),0.95(d,3H,J＝6.9Hz),0.93(s,3H),0.86-0.85(m,9H),0.73-0.71(m,2H),0.56-0.53(m,2H).
用实施例29中类似的方法，可得到以下化合物。
Figure PCTCN2020122472-appb-000072
实施例30
Figure PCTCN2020122472-appb-000073
(1)将中间体S30(300mg,0.464mmol)溶于干燥DCM(5mL)中，0℃下滴加吡啶(187μL,2.32mmol)，0.5小时后滴加对硝基苯基氯甲酸酯(280mg,1.39mmol)的干燥DCM溶液(5mL)，恢复至室温后搅拌5小时，TLC监测显示反应进行完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S37 374mg(0.461mmol)，收率：99.5％。 1H NMR(300MHz,CDCl 3)δ8.32-8.26(m,2H),7.40-7.30(m,7H),5.16-5.06(m,2H),4.85(br s,1H),4.50(s,1H),4.42-4.37(m,1H),4.11-4.04(m,1H),2.56(s,1H),2.46-2.39(m,1H),2.32-2.16(m,3H),1.91-1.07(m,其它脂肪环质子),1.02(d,3H,J＝6.9Hz),0.96(s,3H),0.86-0.83(m,9H),0.75(s,3H),0.71-0.65(m,2H),0.53-0.48(m,2H).
(2)中间体S37与N-(3-氨丙基)吗啉缩合得到产物14.5mg,产率48.0％。 1H NMR(400MHz,CDCl 3)δ7.37-7.31(m,5H),5.56(br s,1H),5.23(br s,1H),5.14-5.05(m,2H),4.51(t,1H,J＝2.8Hz),4.19-4.17(m,1H),3.81-3.71(m,5H),3.30-3.25(m,2H),2.59(s,1H),2.45-2.42(m,6H),2.37-1.06(m,其它脂肪环质子),0.95-0.92(m,6H),0.86-0.79(m,9H),0.73-0.70(m,3H+2H),0.54-0.50(m,2H).
(3)经类似的脱苄基过程可得化合物T75(10.1mg,78.3％)。 1H NMR(400MHz,CDCl 3)δ5.49(br s,1H),5.21(s,1H),4.51(s,1H),4.19(s,1H),3.82-3.73(m,5H),3.27-3.24(m,2H),2.59(s,1H),2.49-2.45(m,6H),2.35-2.30(m,1H),2.26-2.20(m,2H),1.99(s,1H),1.89-1.84(m,2H),1.72-1.09(m,其它脂肪环质子),0.98(s,3H),0.94-0.92(m,6H),0.86-0.84(m,9H),0.72-0.69(m,2H),0.56-0.51(m,2H).
用实施例30中类似的方法，可得到以下化合物。
Figure PCTCN2020122472-appb-000074
Figure PCTCN2020122472-appb-000075
Figure PCTCN2020122472-appb-000076
Figure PCTCN2020122472-appb-000077
实施例31
Figure PCTCN2020122472-appb-000078
(1)中间体S7(100mg,0.140mmol)与2,2’-(乙烯二氧)双(乙胺)(10.4mg,0.070mmol)，三乙胺(19.4μL,0.140mmol)和DMAP(1.71mg,0.014mmol)溶于DCM 2mL中，室温下搅拌过夜，TLC监测显示反应进行完全。旋出DCM，加入去离子水30mL稀释，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S38 51.6mg(0.040mmol)，收率：28.6％。 1H NMR(300MHz,CDCl 3)δ7.38-7.30(m,10H),5.21(br s,2H),5.15-5.06(m,4H),4.49(s,2H),3.65(s,4H),3.61-3.57(m,4H),3.40(s,4H),2.28-1.03(m,其它脂肪环质子),0.95(s,6H),0.85-0.81(m,24H),0.75-0.72(m,12H).
(2)经类似的脱苄基过程可得化合物T89(15.1mg,34.0％)。 1H NMR(300MHz,CDCl 3)δ5.18(br s,2H),4.51(s,2H),3.66(s,4H),3.62-3.58(m,4H),3.40(s,4H),2.28-1.07(m,其它脂肪环质子),1.00(s,6H),0.93(s,6H),0.86-0.85(m,24H),0.77-0.74(m,6H).
实施例32
Figure PCTCN2020122472-appb-000079
(1)中间体S37(100mg,0.123mmol)与2,2’-(乙烯二氧)双(乙胺)(9.2mg,0.062mmol)，三乙胺(17.1μL,0.123mmol)和DMAP(1.47mg,0.012mmol)溶于DCM 2mL中，室温下搅拌过夜，TLC监测显示反应进行完全。旋出DCM，加入去离子水30mL稀释，用乙酸乙酯(3×30mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S39 66.7mg(0.045mmol)，收率：36.6％。 1H NMR(300MHz,CDCl 3)δ7.36-7.32(m,10H),5.15-5.05(m,4H),4.51(s,2H),3.63(s,4H),3.57(t,4H,J＝5.1Hz),3.39(s,4H),2.39-1.04(m,其它脂肪环质子),0.93(s,12H),0.86(s,12H),0.82(s,6H),0.73-0.70(m,10H),0.55-0.54(m,4H).
(2)经类似的脱苄基过程可得化合物T90(32.4mg,55.2％)。 1H NMR(300MHz,CDCl 3)δ4.50(s,2H),3.63(s,4H),3.57(t,4H,J＝5.1Hz),3.40-3.38(m,4H),2.60(s,2H),2.38-1.13(m,其它脂肪环质子),0.97-0.92(m,18H),0.86-0.84(m,18H),0.74-0.68(m,4H),0.56-0.51(m,4H).
实施例33
Figure PCTCN2020122472-appb-000080
(1)中间体S29与吗啉乙酸反应得缩合产物19.2mg,产率：45.6％。 1H NMR(400MHz,CDCl 3)δ7.34-7.30(m,5H),5.18-5.06(m,3H),4.85(br s,1H),4.50(s,1H),3.73-3.61(m,3H),3.14(s,1H),2.65-2.57(m,5H),2.31-1.05(m,其它脂肪环质子),0.91(s,3H),0.86(s,6H),0.82(s,3H),0.72-0.69(m,5H),0.53(s,2H).
(2)经类似的脱苄基过程可得化合物T91(13.2mg,77.9％)。 1H NMR(400MHz,CDCl 3)δ5.14(t,1H,J＝4.8Hz),4.41(s,1H),3.66(s,4H),3.16(s,2H),2.63-2.47(m,6H),2.31-1.18(m,其它脂肪环质子),1.13(d,3H,J＝6.4Hz),0.94(s,6H),0.88-0.84(m,9H),0.62(s,2H),0.42(s,2H).
实施例34
Figure PCTCN2020122472-appb-000081
(1)将中间体S29(100mg,0.158mmol)和DMAP(193mg,1.58mmol)溶于干燥DCM(2mL)中，0.5小时后滴加对硝基苯基氯甲酸酯(63.7mg,0.316mmol)的干燥DCM溶液(2mL)，恢复至室温后搅拌5小时，TLC监测显示反应进行完全，然后滴加N-(3-氨丙基)吗啉(231μL,1.58mmol),室温下反应过夜。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物91.2mg(0.128mmol)，收率：81.0％。 1H NMR(400MHz,CDCl 3)δ7.35-7.30(m,5H),5.58(s,1H),5.14-5.06(m,2H),4.99(br s,1H),4.86(br s,1H),4.51(s,1H),3.71(s,4H),3.25-3.23(m,2H),2.64-2.58(m,2H),2.45(s,6H),2.31-2.28(m,1H),2.20-2.14(m,1H),1.89-0.96(m,其它脂肪环质子),0.91(s,3H),0.86(s,6H),0.81(s,3H),0.72-0.70(m,5H),0.53(s,2H).
(2)经类似的脱苄基过程可得化合物T94(50.2mg,55.0％)。 1H NMR(400MHz,CD 3OD)δ4.96(s,1H),4.45(s,1H),3.71(s,4H),3.13(s,2H),2.66-2.61(m,1H),2.55-2.45(m,7H),2.37-2.31(m,1H),2.25(d,1H,J＝12.4Hz),1.97-1.12(m,其它脂肪环质子),0.97(s,6H),0.91-0.88(m,9H),0.65(s,2H),0.47(s,2H).
用实施例34中类似的方法，可得到以下化合物。
Figure PCTCN2020122472-appb-000082
Figure PCTCN2020122472-appb-000083
实施例35
Figure PCTCN2020122472-appb-000084
(1)将中间体S30(50mg,0.0731mmol)与三苯基膦(48mg，0.183mmol)溶于干燥THF(2mL)中，0℃下滴加偶氮二甲酸二异丙酯(75μL,0.381mmol)，15分钟后滴加叠氮磷酸二苯酯(75mg,0.272mmol)，恢复至室温后搅拌过夜，TLC监测显示反应进行完全，加入三苯基膦(200mg，0.762mmol)的THF溶液(2mL)，搅拌过夜，TLC监测显示反应进行完全，加入水(2mL)，搅拌4小时，TLC监测显示反应进行完全。旋出THF，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S40 29.2mg(0.0452mmol)，收率：61.8％。 1H NMR(400MHz,CDCl 3)δ7.35-7.30(m,5H),5.09(q,2H,J＝12.0Hz),4.92(s,1H),4.50(s,1H),2.91(d,4H,J＝12.0Hz),2.58(s,1H),2.42(t,1H,J＝11.2Hz),2.34(t,1H,J＝10.8Hz),2.30-2.26(m,1H),2.22(d,1H,J＝12.0Hz),2.17(s,1H),1.86–1.20(m,其它脂肪环质子),1.08(d,3H,J＝10.8Hz),0.98(d,3H,J＝6.8Hz),0.94(s,3H)，0.86(s,8H)，0.81(s,4H),0.71(d,2H,J＝6.8Hz),0.53(s,2H).
(2)经类似的脱苄基过程可得化合物T96(16.7mg,66.4％)。 1H NMR(400MHz,CD 3OD)δ4.45(s,1H),3.11(d,1H,J＝11.2Hz),2.69(t,1H,J＝12.0Hz),2.52-2.46(m,3H),2.27(d,1H,J＝12.8Hz),2.05-1.16(m,其它脂肪环质子),1.06(d,3H,J＝6.8Hz),1.03-0.99(m,8H),0.98(s,2H)，0.92(s,7H),0.89(s,3H),0.67(s,2H),0.48(s,2H).
实施例36
Figure PCTCN2020122472-appb-000085
(1)将中间体S40(50mg,0.0773mmol)溶于干燥DCM(2mL)中，加入4-二甲氨基吡啶(9.44mg,0.0773mmol)和三乙胺(31.49μL,0.386mmol)，5分钟后滴加对硝基苯基氯甲酸酯(46.73mg,0.232mmol)的干燥DCM溶液(1mL)，室温搅拌5小时，TLC监测显示反应进行完全后加入3-(4-吗啉)-1-丙醇(50.00μL，0.386mmol)，室温搅拌过夜。TLC监测显示反应进行完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S41 38.1mg(0.0524mmol)，收率：60.3％。 1H NMR(400MHz,CDCl 3)δ7.35-7.31(m,5H),5.09(q,2H,J＝12.0Hz),4.51(s,1H),4.21(s,1H),4.12-4.08(m,1H),3.73(t,3H,J＝4.8Hz),3.39(s,1H),2.76(s,1H),2.60(d,1H,J＝10.4Hz),2.47-2.42(m,4H),2.35(d,1H,J＝11.2Hz),2.29-2.26(m,1H),2.23–2.16(m,1H),1.85-1.25(m,其它脂肪环质子),1.08(d,3H,J＝12.4Hz),0.96(s,2H),0.91(d,3H,J＝7.2Hz),0.83(d,12H,J＝15.6Hz),0.73(s,3H),0.71(d,2H,J＝6.8Hz),0.53(s,2H).
(2)经类似的脱苄基过程可得化合物T97(27.6mg,72.4％)。 1H NMR(400MHz,CDCl 3)δ4.52(s,1H),4.09(d,1H,J＝6.4Hz),3.75(t,3H,J＝4.8Hz),3.40(s,1H),2.78(s,1H),2.60(s,1H),2.50-2.46(m,5H),2.32-2.20(m,4H),2.07-2.00(m,2H),1.86-1.08(m,其它脂肪环质子),0.99(d,6H,J＝6.8Hz),0.92(d,7H,J＝6.0Hz),0.87-0.84(m,13H),0.72(d,2H,J＝6.8Hz),0.54(s,2H).
用实施例36中类似的方法，可得到化合物T98。
Figure PCTCN2020122472-appb-000086
实施例37
Figure PCTCN2020122472-appb-000087
(1)将齐墩果酸(200mg,0.439mmol)溶解在DMF(5mL)中，加入无水碳酸钾(121mg,0.878mmol)，室温搅拌下慢慢滴加氯化苄(60.7μL,0.527mmol)。滴加完毕后将反应液移至50℃搅拌3h，TLC监测显示反应进行完全。将混合物冷却至室温，加入去离子水50mL稀释，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S43 208mg(0.381mmol)，收率：86.9％。 1H NMR(300MHz,CDCl 3)δ7.38-7.28(m,5H),5.29(t,1H,J＝3.9Hz),5.12-5.02(m,2H),3.23-3.18(m,1H),2.94-2.88(m,1H),2.04-1.93(m,1H),1.89-1.83(m,1H),1.72-1.22(m,其它脂肪环质子),1.13(s,3H),0.98(s,3H),0.92(s,3H),0.90(s,3H),0.88(s,3H),0.78(s,3H),0.61(s,3H).
(2)将S43(208mg,0.381mmol)溶于DCM(5mL)中，0℃下缓慢加入Dess-Martin氧化剂(323mg,0.762mmol),慢慢升至室温并搅拌1小时，TLC监测显示反应进行完全。将反应混合物过滤后旋干，经柱层析分离得到化合物S44 190mg(0.349mmol)，收率：91.7％。 1H NMR(300MHz,CDCl 3)δ7.37-7.28(m,5H),5.31(t,1H,J＝3.6Hz),5.13-5.03(m,2H),2.92(dd,1H,J＝13.8,4.5Hz),2.60-2.48(m,1H),2.40-2.31(m,1H),2.04-1.17(m,其它脂肪环质子),1.13(s,3H),1.08(s,3H),1.04(s,3H),1.02(s,3H),0.92(s,3H),0.90(s,3H),0.66(s,3H).
(3)将上步产物S44(190mg,0.349mmol)和S-(-)-2-甲基恶唑硼烷(19.4mg,0.070mmol)溶于干燥的THF(10mL)中。室温下慢慢滴加10M的BH 3-Me 2S(41.9μL,0.419mmol)，搅拌10分钟，TLC监测显示反应已经进行完。将反应瓶移至冰水浴，慢慢滴加甲醇淬灭反应，待不再有气泡生成后旋干溶剂，柱层析分离得到化合物S45 40.1mg(0.073mmol)，收率：21.0％。 1H NMR(300MHz,CDCl 3)δ7.37-7.32(m,5H),5.29(t,1H,J＝3.6Hz),5.12-5.02(m,2H),3.40(t,1H,J＝3.0Hz),2.94-2.88(m,1H),1.99-1.84(m,3H),1.72-1.17(m,其它脂肪环质子),1.14(s,3H),0.95(s,3H),0.92(s,3H),0.90-0.89(m,6H),0.84(s,3H),0.62(s,3H).
(4)将S45(153mg,0.280mmol)和DMAP(2.44mg,0.02mmol)溶于吡啶(5mL)中，0℃下滴加乙酸酐(132μL,1.40mmol)，10分钟后恢复至室温后搅拌过夜，次日TLC显示反应完全。旋出吡啶，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水，1N稀盐酸和饱和食盐水洗涤，经硫酸钠干燥浓缩，柱层析分离得产物S46 163mg(0.277mmol)，收率：98.9％。 1H NMR(300MHz,CDCl 3)δ7.35-7.30(m,5H),5.30(t,1H,J＝3.6Hz),5.12-5.02(m,2H),4.63(t,1H,J＝3.0Hz),2.91(dd,1H,J＝13.8,4.5Hz),2.06(s,3H),2.00-0.96(m,其它脂肪环质子),1.18(s,3H),0.92(s,3H),0.90(s,6H),0.88(s,3H),0.84(s,3H),0.62(s,3H).
(5)中间体S46经类似的脱苄基过程可得化合物T99(129mg,93.4％)。 1H NMR(300MHz,CDCl 3)5.30(t,1H,J＝3.6Hz),4.63(t,1H,J＝3.0Hz),2.83(dd,1H,J＝14.1,4.5Hz),2.07(s,3H),2.04-1.07(m,其它脂肪环质子),1.19(s,3H),0.93(s,6H),0.91(s,3H),0.88(s,3H),0.85(s,3H),0.77(s,3H).
用实施例37中类似的方法，可得到以下化合物。
Figure PCTCN2020122472-appb-000088
实施例38
Figure PCTCN2020122472-appb-000089
(1)三氧化铬(13.6mg,0.136mmol)溶于干燥的DCM(1.0mL)中，0℃下滴加吡啶(21.9μL,0.272mmol)搅拌1小时，然后加入中间体S47(40mg,0.068mmol)的干燥DCM溶液(1.0mL)，室温下搅拌过夜，次日约一半原料剩余。过滤，直接浓缩，柱层析分离得产物S48 8.7mg，回收原料29mg，产率：77.3％。 1H NMR(300MHz,CDCl 3)δ7.35-7.29(m,5H),5.59(s,1H),5.04(s,2H),4.63(t,1H,J＝3.0Hz),2.58-2.51(m,1H),2.44(d,1H,J＝11.1Hz),2.37(s,1H),2.06(s,3H),2.15-1.06(m,其它脂肪环质子),1.33(s,3H),1.10(s,3H),0.97-0.84(m,12H),0.76(s,3H).
(2)中间体S48经类似的脱苄基过程可得化合物T101(5.6mg,75.7％)。 1H NMR(300MHz,CDCl 3)δ5.59(s,1H),4.63(t,1H,J＝3.0Hz),2.58-2.53(m,1H),2.40(s,1H),2.36(s,1H),2.06(s,3H),2.13-1.09(m,其它脂肪环质子),1.35(s,3H),1.14(s,3H),0.98-0.91(m,6H),0.88-0.86(m,6H),0.84(s,3H).
实施例39
Figure PCTCN2020122472-appb-000090
(1)中间体S47(59.8mg,0.102mmol)溶于DCM(1.0mL)中，室温下加入过量的甲酸(100μL)，然后滴加过氧化氢(30％,52μL,0.408mmol)，室温下搅拌过夜，次日点板仍有一半原料剩余。加入无水亚硫酸钠淬灭，旋出DCM，乙酸乙酯萃取，水洗，有机 相浓缩，柱层析分离得产物S49 21.1mg(0.035mmol)，回收原料37.2mg,产率：91.4％。 1H NMR(300MHz,CDCl 3)δ7.38-7.31(m,5H),5.22-5.09(m,2H),4.64(t,1H,J＝2.7Hz),2.85-2.78(m,1H),2.64-2.54(m,1H),2.43-2.36(m,1H),2.06(s,3H),1.93-1.00(m,其它脂肪环质子),1.33(s,3H),0.96-0.91(m,9H),0.85(s,3H),0.70-0.66(m,6H).
(2)中间体S49经类似的脱苄基过程可得化合物T102(17.1mg,95.2％)。 1H NMR(300MHz,CDCl 3)δ4.65(t,1H,J＝2.7Hz),2.76-2.70(m,1H),2.64-2.54(m,1H),2.43-2.37(m,1H),2.07(s,3H),1.93-1.08(m,其它脂肪环质子),1.31(s,3H),1.02(s,3H),0.95(s,3H),0.91(s,3H),0.86(s,3H),0.81(s,3H),0.73(d,3H,J＝5.4Hz).
实施例40
Figure PCTCN2020122472-appb-000091
(1)将中间体S45(89.0mg,0.163mmol)溶于干燥DCM(1mL)中，0℃下滴加吡啶(65.7μL,0.815mmol)，0.5小时后滴加对硝基苯基氯甲酸酯(98.6mg,0.489mmol)的干燥DCM溶液(1mL)，恢复至室温后搅拌5小时，TLC显示反应完全。旋出DCM，用乙酸乙酯(3×50mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经无水硫酸钠干燥浓缩，柱层析分离得产物S50 102mg(0.143mmol)，产率：88.0％。 1H NMR(300MHz,CDCl 3)δ8.29-8.23(m,2H),7.39-7.31(m,5H+2H),5.29(t,1H,J＝3.6Hz),5.12-5.02(m,2H),4.60(t,1H,J＝3.0Hz),2.91(dd,1H,J＝13.8,4.5Hz),2.03-1.02(m,其它脂肪环质子),1.14(s,3H),0.97(s,3H),0.93-0.91(m,12H),0.61(s,3H).
(2)中间体S50与环丙胺缩合得到产物14.0mg,产率：69.8％。 1H NMR(300MHz,CDCl 3)δ7.36-7.29(m,5H),5.30(t,1H,J＝3.6Hz),5.12-5.02(m,2H),4.82(br s,1H),4.52(t,1H,J＝2.7Hz),2.91(dd,1H,J＝14.1,4.5Hz),2.57(s,1H),2.04-1.02(m,其它脂肪环质子),1.15(s,3H),0.92-0.88(m,15H),0.73-0.69(m,2H),0.62(s,3H),0.55-0.50(m,2H).
(3)经类似的脱苄基过程可得化合物T105(7.20mg,60.0％)。 1H NMR(300MHz,CDCl 3)5.29(t,1H,J＝3.3Hz),4.95(br s,1H),4.52(t,1H,J＝2.7Hz),2.83(dd,1H,J＝13.8,4.5Hz),2.57(s,1H),2.03-1.05(m,其它脂肪环质子),1.15(s,3H),0.93-0.88(m,15H),0.76(s,3H),0.71-0.69(m,2H),0.55-0.50(m,2H).
用实施例40中类似的方法，可得到以下化合物。
Figure PCTCN2020122472-appb-000092
Figure PCTCN2020122472-appb-000093
实施例41
Figure PCTCN2020122472-appb-000094
由中间体S51出发，经与化合物T31相似的合成过程可得化合物T109。 1H NMR(400MHz,d 6-DMSO)δ11.98(br s,1H),4.38(dd,J＝11.6,4.8Hz,1H),4.24(t,1H,J＝5.2Hz),3.58-3.53(m,1H),3.17-3.10(m,1H),2.25-2.08(m,3H),2.00(s,3H),1.70-1.21(m,其它脂肪环质子),1.07(d,3H,J＝11.2Hz),0.93(s,3H),0.87-0.79(m,15H).
实施例42
Figure PCTCN2020122472-appb-000095
由中间体S51出发，经与化合物T49相似的合成过程可得化合物T110。 1H NMR(400MHz,d 6-DMSO)δ11.97(br s,1H),7.14(br s,1H),4.25-4.18(m,2H),3.58-3.53(m,1H), 3.17-3.10(m,1H),2.44(s,1H),2.25-2.08(m,3H),1.70-1.23(m,其它脂肪环质子),1.07(d,3H,J＝11.6Hz),0.93(s,3H),0.87-0.76(m,15H),0.55-0.52(m,2H),0.37-0.36(m,2H).
实施例43TGR5受体激动试验
1.实验目的
利用瞬转TGR5的HEK293细胞用化合物进行刺激，然后用均相时间分辨荧光(Homogeneous Time-Resolved Fluorescence,HTRF)检测这些化合物是否可以激动TGR5。
2.实验原理
TGR5是胆汁酸膜受体，也是GPCR家族的一员，对胆汁酸、脂类及糖类的代谢具有调控作用。TGR5与Gs蛋白偶联，活化后进一步激活腺苷酸环化酶，产生第二信使cAMP。HTRF是一种检测cAMP含量的方法，它结合了荧光共振能量转移(FRET,Fluorescence Resonance Energy Transfer)和时间分辨荧光(TRF,Time Resolved Fluorescence)两种技术。含Eu的穴状化合物作为荧光供体，其发射光谱与荧光受体激发光谱有一定重叠，通过FRET诱导受体产生荧光，而Eu的荧光寿命长，通过TRF可以从荧光背景中区分受体发出的荧光信号。将荧光供体与cAMP特异性抗体结合，同时以荧光受体标记cAMP，二者通过抗原抗体特异性识别反应相互靠近，产生FRET，而细胞产生的cAMP与标记的cAMP竞争抗体结合位点，导致荧光强度降低。本实验以TGR5激动剂INT777为阳性对照，探究化合物对TGR5的作用。
3.实验样品
试验前将化合物溶于DMSO，配制母液，使用时用培养液稀释至所需浓度。
4.实验方法
4.1将待测化合物用1xPBS配成终浓度的2倍.其中终浓度为100μM、10μM、1μM、100nM、10nM、1nM、0.1nM、DMSO(每个孔都含有1％的DMSO)。
4.2细胞处理:
(1)用胰酶消化细胞，然后用无血清培液悬浮。
(2)定细胞密度，并同时在无血清培液中加IBMX(终浓度为500μM)，细胞数为2000/5μl/孔。
(3)加入5μL待测化合物&5μL含IBMX的细胞悬液混合，锡箔纸将384孔板封闭好，室温避光反应不超过30分钟。
4.3检测底物配置
(1)1μL cAMP-d2用cAMP&cGMP conjugates&lysis buffer稀释到20μL。
(2)1μL anti-cAMP-Cryptate用cAMP&cGMP conjugates&lysis buffer稀释至20μL。
(3)30分钟后，加入5μL(1.3.1)+5μL(1.3.2)，锡箔纸将384孔板封闭好，室温避光反应30分钟。
4.4 60分钟后，Envision2101多功能微孔板酶标仪(PerkinElmer)读数。
5.实验结果(以T2等40个化合物为例，但不局限于这些化合物)
表1化合物TGR5激动活性测试试验
化合物编号 hTGR5EC 50(nM) Max Response(％)
INT777 ** 100
T2 *** 100
T3 *** 100
T6 *** 100
T10 ** 100
T18 *** 100
T22 *** 100
T25 *** 100
T26 *** 100
T27 *** 100
T28 *** 100
T29 *** 100
T31 *** 100
T33 *** 100
T36 *** 100
T38 *** 100
T39 *** 100
T46 *** 100
T49 *** 100
T51 *** 100
T52 *** 100
T53 *** 100
T58 *** 100
T59 *** 100
T66 *** 100
T68 *** 100
T69 *** 100
T75 *** 100
T77 *** 100
T78 *** 100
T79 *** 100
T81 *** 100
T84 *** 100
T90 ** 100
T91 *** 100
T93 *** 100
T97 ** 100
T101 *** 100
T108 *** 100
T109 ** 100
T110 * 100
注：EC 50为样品药物对TGR5激动活性的评价，半数50％有效浓度。1-10μM:“*”，0.1-1μM：“**”，100-1nM：“***”
6.结果与讨论：
由表1中TGR5受体激动活性测试结果可以看出，本发明所公开大部分化合物人源TGR5受体激动活性显著高于INT777及WO2015135449所公开的化合物，由T109和T110的人源TGR5激动活性结果可以看出，α构型化合物显著优于β构型化合物。
实施例44化合物动力学溶解度测试
1.实验原理
将化合物沉淀作为化合物浓度限制的指示剂。由于颗粒阻挡光线到达检测器，使得光线强度减弱，通过溶液对紫外光吸附的增加来间接检测化合物沉淀的出现。
2.实验步骤
将化合物固体粉末制成DMSO溶液。将少量化合物DMSO溶液分别加入pH＝2.0和pH＝7.4的DPBS缓冲液中，直至到达浓度上限。通过光学方法检测到达浓度上限时出现的沉淀，沉淀出现是的溶解度即动力学溶解度值。
3.实验结果
表2化合物动力学溶解度
Figure PCTCN2020122472-appb-000096
4.结果与讨论
动力学溶解度结果如表2所示：化合物T36，T38，T39，T64，T66的动力学溶解度较C101均有所提升。
实施例45化合物Caco-2单层细胞渗透性试验
1.实验操作
1.1 Caco-2细胞培养
Caco-2细胞用高糖DMEM培养基，置于37℃、5％CO 2、空气相对湿度90％的培养箱中培养，培养基中添加10％胎牛血清、10mmol/L HEPES、1mmol/L丙酮酸钠、1％谷氨酰胺、1％非必需氨基酸、100U/mL青霉素和100μg/mL链霉素。每隔7天传一代，传代比率1:10。实验使用40至60代之间的细胞。
1.2 Caco-2细胞单层模型建立
将Caco-2细胞按2×10 5/mL的浓度接种至Millicell-24孔板，每孔400μL，基底侧加入培养液800μL，在37℃，5％CO 2的培养箱中培养。细胞接种72小时后换液，以后隔天换液，培养21天。
1.3 Caco-2细胞单层模型验证
培养21天以后，用TEER值检测细胞单层的紧密程度，一般大于200Ω·cm 2即可视为单层致密完整。TEER值越高，表示单层越致密，一般不超过1000Ω·cm 2。本实验中将Caco-2细胞培养于Millicell培养板中，21天时用电阻仪Millicell-ERS II测量跨膜电阻值。
1.4受试化合物双侧转运试验
考察从细胞顶层(A侧)到基底层(B侧)和B侧到A侧的药物转运。试验方法如下：用HBSS洗细胞三次以后，相应浓度的化合物分别加到对应的细胞孔中(A侧pH6.8,B侧pH7.4)。在37℃培养箱中培养95分钟，分别在5分钟和95分钟时给药侧取样，在35分钟和95分钟时接收侧取样。用LC-MS/MS检测样品的浓度。为了验证细胞，使用几个阳性对照化合物的预期Fabs值和Papp值拟合一条标准曲线。测定被试化合物的Papp值并通过该标准曲线计算其Fabs值。
2.实验结果
表3化合物Caco-2单层细胞渗透性实验结果
Figure PCTCN2020122472-appb-000097
Figure PCTCN2020122472-appb-000098
a：P app<2×10 -6cm/s:低渗透性；2×10 -6<P app<20×10 -6cm/s:中等渗透性；P app>20×10 -6cm/s:高渗透性
3.结果与讨论
结果如表3所示：化合物C101(WO2015135449，
Figure PCTCN2020122472-appb-000099
)，无论是在Caco-2单层细胞的顶端膜侧还是基底膜侧，都完全检测不到化合物的存在，这说明化合物并不能透过细胞膜，化合物可能由于脂溶性太高而直接粘附于细胞膜上，或者进入细胞内无法渗出；在分子中引入大极性基团的化合物T36，T38，T39，T64，T66，T75渗透性都明显改善。其中化合物T36，T38，T39和T64在膜两侧渗透速率相近，而T66和T75在膜两侧渗透速率存在明显差异，这说明T66和T75可能是Pgp蛋白的底物。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种式I化合物、其药学上可接受的盐或其二聚体：
    Figure PCTCN2020122472-appb-100001
    R 1为氢、羟基、卤素或C1-C6烷基；
    R 2为氢、羟基、羧基、卤素、C1-C6烷氧基、3至10元环烷氧基、＝O、＝N-OH或R-Y(＝X) m-O-；其中，m为1或2；R为取代或未取代的以下基团：C1-C8烷基、C3-C10环烷基、C6-C10芳基、5-7元杂芳基、5-7元饱和杂环、R aN(C1-C6烷基)-、R aNH-或R aO-；其中，各R a独立选自取代或未取代的以下基团：氢，C1-C8烷基、C3-C10环烷基、C6-C10芳基、5-7元杂芳基或5-7元饱和杂环基；X为NH，O或S，Y为C，S或者P；
    R 3和R 4各自独立地为未取代或取代的C1-C6烷基；或者R 3和3位C形成未取代或取代的C3-C6环烷基，R 4为氢、或未取代或取代的C1-C6烷基；
    R 5为氢、羟基、羟甲基、甲酰基或羧基；
    R 6和R 7各自独立地为氢、羟基或C1-C6烷基；
    R 8和R 9各自独立地为氢、羟基、卤素、取代或未取代的C1-C6烷基、取代或未取代的3-6元含氧杂环、
    Figure PCTCN2020122472-appb-100002
    其中R 12、R 13和R 14为氢、取代或未取代的C1-C6烷基或R c-M(＝R’) r-；其中，r为1或2；M为C，S或P，R’为O或S；R c为氢、羟基、取代或未取代的C1-C6烷基、取代或未取代的3-7元杂环基、取代或未取代的4-8元杂芳基、R dNH-、R dO(CH 2) sNH-、或R dO(CH 2) s-；s为0、1、2、3或4，各R d独立为氢、取代或未取代的C1-C6烷基、或取代或未取代的4-8元杂芳基；A为O或S，B为NH或O，R 15为氢、取代或未取代的C1-C6烷基、C3-C8环烷基或5-7元饱和杂环；
    R 10为氢、羟基、氧代基团(＝O)或C1-C6烷基；
    R 11为氢、羟基、氧代基团(＝O)或C1-C6烷基；
    Z为-(CH 2)n-，n为1、2或3；
    Figure PCTCN2020122472-appb-100003
    表示单键或双键；
    各*独立地表示R构型、S构型或消旋；
    所述取代是指被基团上的氢被选自下组的一个或多个取代基取代：羟基、氨基、甲氧基、卤素、C1-C6烷基、C1-C6烷氧基、氧代基团、环氧基团、肟、羧基、磺酸基、葡糖胺基、吗啉基、N,N-二甲基、4-甲基哌嗪基、吡啶基、哌啶基、吡咯烷基。
  2. 如权利要求1所述的化合物，其特征在于，R 1为氢、羟基或C1-C4烷基。
  3. 如权利要求1所述的化合物，其特征在于，R 2为氢、羟基、羧基、卤素、C1-C4烷氧基、3至6元环烷氧基、＝O、＝N-OH或R-Y(＝X) m-O-；
    其中，m为1或2；
    R为取代或未取代的以下基团：C1-C6烷基、C3-C8环烷基、C6-C10芳基、5-7元杂芳基、5-7元饱和杂环、R aN(C1-C4烷基)-、R aNH-或R aO-；其中，各R a独立为取代或未取代的以下基团：氢，C1-C8烷基、C3-C10环烷基、C6-C10芳基、5-7元杂芳基或5-7元饱和杂环基；
    X为NH，O或S；
    Y为C，S或者P；
    所述取代是指被基团上的氢被选自下组的一个或多个取代基取代：羟基、甲氧基、卤素、C1-C6烷基、C1-C6烷氧基、羧基、磺酸基、葡糖胺基、吗啉基、N,N-二甲基。
  4. 如权利要求1所述的化合物，其特征在于，R 3和R 4各自独立地为C1-C4烷基；
    或者R 3和3位C形成C1-C6烷基取代的C3-C6环烷基，R 4为氢。
  5. 如权利要求1所述的化合物，其特征在于，R 6和R 7各自独立地为氢、甲基或乙基。
  6. 如权利要求1所述的化合物，其特征在于，R 8和R 9各自独立地为氢、甲基、乙基、正丙基、异丙基、
    Figure PCTCN2020122472-appb-100004
    Figure PCTCN2020122472-appb-100005
    其中R 12、R 13和R 14各自独立为氢、取代或未取代的C1-C6烷基、R cSO 2-、或R cCO-，其中，R c为氢、羟基、取代或未取代的C1-C6烷基、取代或未取代的3-7元含氮杂环基、R dNH-、R dO(CH 2) sNH-、或R dO(CH 2) s-；s为0、1、2、3或4，各R d取自氢、取代或未取代的C1-C6烷基或取代或未取代的5-6元含氮杂芳基；
    B为NH或O，
    R 15为氢、取代或未取代的C1-C6烷基、5-7元饱和杂环。
  7. 如权利要求1所述的化合物，其特征在于，所述化合物为：
    Figure PCTCN2020122472-appb-100006
    Figure PCTCN2020122472-appb-100007
    Figure PCTCN2020122472-appb-100008
    Figure PCTCN2020122472-appb-100009
    Figure PCTCN2020122472-appb-100010
    Figure PCTCN2020122472-appb-100011
    Figure PCTCN2020122472-appb-100012
    Figure PCTCN2020122472-appb-100013
    Figure PCTCN2020122472-appb-100014
  8. 一种药物组合物，其特征在于，包含：
    如权利要求1所述的化合物、其药学上可接受的盐或其二聚体；和
    药学上可接受的载体。
  9. 如权利要求1所述的化合物或权利要求8所述的药物组合物用途，其特征在于，
    (i)用于制备胆汁酸G蛋白偶联受体TGR5激动剂；或
    (ii)用于制备治疗代谢性疾病的药物。
  10. 如权利要求9所述的用途，其特征在于，所述代谢性疾病选自：糖尿病、肥胖、高血脂、肝损伤和炎症性疾病。
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