WO2021179884A1 - 具有共轭二烯结构c环的齐墩果酸衍生物及其制备方法和应用 - Google Patents

具有共轭二烯结构c环的齐墩果酸衍生物及其制备方法和应用 Download PDF

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WO2021179884A1
WO2021179884A1 PCT/CN2021/076726 CN2021076726W WO2021179884A1 WO 2021179884 A1 WO2021179884 A1 WO 2021179884A1 CN 2021076726 W CN2021076726 W CN 2021076726W WO 2021179884 A1 WO2021179884 A1 WO 2021179884A1
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compound
pharmaceutically acceptable
alkyl
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substituted
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丁晔
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丁晔
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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
    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to an oleanolic acid derivative compound for preventing and treating inflammation-related diseases and tumor-related diseases, and a preparation method and application thereof.
  • Natural terpenoids and derivatives have unique chemical structures and biologically active properties, and are one of the important sources for the development of new small molecule drugs.
  • Paclitaxel from Bristol-Myers Squibb and Docetaxel from Sanofi although they have very similar taxane terpenoid structures, have their own characteristics in terms of pharmacokinetics, safety, and clinical application. Features [Practical Medicines and Clinics, 2010,4,281-283].
  • oleanane terpenoids such as oleanolic acid
  • have a wide range of biological activities such as antioxidant and anti-inflammatory.
  • Oleanolic acid is approved in my country for the adjuvant treatment of clinical liver diseases (such as hepatitis).
  • oleanolic acid has weak anti-inflammatory and other biological activities, high fat solubility and poor water solubility, and other shortcomings, which limit its clinical application.
  • the drug has not been approved for marketing by the United States, Europe and other drug regulatory authorities.
  • a series of derived oleanolic acid derivatives such as RTA401, RTA402 and RTA408, are compounds with strong biological activity [J.Med.Chem.2000,43,4233-4246]. Such compounds can be cytoprotective agents and inhibitors of cell inflammatory processes at low concentrations, and inhibit cell proliferation and promote apoptosis at high concentrations [Nat. Rev. Cancer, 2007, 7, 357-369]. Studies have shown that the mechanism of action of this type of compound is also very complicated, and there are many potential targets.
  • Keap1-NRF2 Multiple inflammation-related targets and signaling pathways including Keap1-NRF2 have been proven [Nat.Rev.Cancer,2007,7,357–369; J.Am.Soc.Nephrol.2012,1663-1673; PLOS ONE,2014, 9,e98896] participate in it.
  • the first type of compounds is mainly focused on the A ring, the preferred candidate compound RTA401 (also known as CDDO, Bardoxolone), namely 2-cyano-3,12-dioxole-1,9(11)-two Ene-28-acid; and RTA402 (also known as CDDO-Me, Bardoxolone methyl), which is 2-cyano-3,12-dioxole-1,9(11)-diene-28-methyl acid .
  • RTA401 also known as CDDO, Bardoxolone
  • RTA402 also known as CDDO-Me, Bardoxolone methyl
  • RTA402 is not a simple methyl ester prodrug of RTA401.
  • Reata Company advances both RTA401 and RTA402 into clinical trials and applies them in different indications. For example, in clinical trial NCT00322140, RTA401 was used to treat solid tumors and lymphomas, and in clinical trial NCT03749447, RTA402 was used to treat chronic kidney disease.
  • the C ring is also a key area that affects the activity and druggability of the compound.
  • the C ring of oleanane also has unique chemical properties.
  • the ethylenic bond of ring A of RTA402 can be reduced by catalytic hydrogenation at atmospheric pressure, while the ethylenic bond of ring C is very stable to catalytic hydrogenation.
  • Reata Company conducts special research on the C ring, such as saturating unsaturated ethylenic bonds [CN102164941B]. Although the safety of the compound is improved, the biological activity is reduced, and it has not been selected for clinical research so far.
  • the present invention provides an oleanane compound with a C ring having a conjugated diene rigid structure, its preparation method and its application.
  • the present invention provides a class of compounds of general formula (I) or pharmaceutically acceptable salts thereof:
  • Z -COOR 2 , -CONR 2 R 3 , or -NR 2 R 3 ;
  • R 1 is independently taken from hydrogen, or alkyl (C ⁇ 8), alkyl ether group, or halogen substituted form of at least one hydrogen atom of any one of these groups;
  • R 2 and R 3 are independently taken from hydrogen, or alkyl (C ⁇ 8), cycloalkyl (C ⁇ 8), spiroalkyl (C ⁇ 8), alkenyl (C ⁇ 8), alkynyl ( C ⁇ 8), aryl (C ⁇ 8), heteroaryl (C ⁇ 8, where at least one of the ring atoms is nitrogen, oxygen or sulfur), aralkyl (C ⁇ 8), alkyl ether group, or these At least one hydrogen atom of any one of the groups is substituted by halogen, hydroxyl, amino, or carboxyl.
  • the compound is further defined as a type of compound represented by the general formula (II) or a pharmaceutically acceptable salt thereof:
  • Z -COOR 1 , -CONR 1 R 2 , or -NR 1 R 2 ;
  • R 1 and R 2 are independently taken from hydrogen, or alkyl (C ⁇ 8), cycloalkyl (C ⁇ 8), spiroalkyl (C ⁇ 8), alkenyl (C ⁇ 8), alkynyl ( C ⁇ 8), aryl (C ⁇ 8), heteroaryl (C ⁇ 8, where at least one of the ring atoms is nitrogen, oxygen or sulfur), aralkyl (C ⁇ 8), alkyl ether group, or these At least one hydrogen atom of any one of the groups is substituted by halogen, hydroxyl, amino, or carboxyl.
  • the compound is further defined as a type of compound represented by the general formula (III) or a pharmaceutically acceptable salt thereof:
  • Z -COOR 1 , -CONR 1 R 2 , or -NR 1 R 2 ;
  • R 1 and R 2 are independently taken from hydrogen, or alkyl (C ⁇ 8), cycloalkyl (C ⁇ 8), spiroalkyl (C ⁇ 8), alkenyl (C ⁇ 8), alkynyl ( C ⁇ 8), aryl (C ⁇ 8), heteroaryl (C ⁇ 8, where at least one of the ring atoms is nitrogen, oxygen or sulfur), aralkyl (C ⁇ 8), alkyl ether group, or these At least one hydrogen atom of any one of the groups is substituted by halogen, hydroxyl, or amino.
  • the compound is further defined as a type of compound represented by the general formula (IV) or a pharmaceutically acceptable salt thereof:
  • Z -COOCH 3 , -CONHCH 2 CF 3 or -NHCOCF 2 CH 3 ;
  • R 1 is taken from alkyl (C ⁇ 8), cycloalkyl (C ⁇ 8), spiroalkyl (C ⁇ 8), alkenyl (C ⁇ 8), alkynyl (C ⁇ 8), aryl ( C ⁇ 8), heteroaryl (C ⁇ 8, wherein at least one of the ring atoms is nitrogen, oxygen or sulfur), aralkyl (C ⁇ 8), alkyl ether group, or at least one of any of these groups
  • the compound is further defined as a type of compound represented by the general formula (V) or a pharmaceutically acceptable salt thereof:
  • Z -COOCH 3 , -CONHCH 2 CF 3 or -NHCOCF 2 CH 3 ;
  • R 1 is taken from alkyl (C ⁇ 8), cycloalkyl (C ⁇ 8), alkenyl (C ⁇ 8), alkynyl (C ⁇ 8), aryl (C ⁇ 8), heteroaryl ( C ⁇ 8, where at least one of the ring atoms is nitrogen, oxygen or sulfur), aralkyl (C ⁇ 8), alkyl ether group, or halogen, hydroxyl, or amino substitution of at least one hydrogen atom of any of these groups form.
  • Non-limiting examples of the compounds provided in the present disclosure include compounds according to the chemical formula shown below, and pharmaceutically acceptable salts thereof:
  • some compounds can be selected by the following synthesis method (1) to construct a C-ring conjugated diene structure:
  • some compounds can also be selected to construct the C-ring conjugated diene structure by the following synthesis method (2):
  • some compounds can also be selected to construct the C-ring conjugated diene structure by the following synthesis method (3):
  • the above-mentioned compounds themselves, or optical isomers, and pharmaceutically acceptable salts possess anti-inflammatory activity at low concentration (nM) and anti-tumor activity at high concentration ( ⁇ M).
  • the above-mentioned compounds themselves, or optical isomers, and pharmaceutically acceptable salts have applications in the treatment of inflammatory-related diseases, tumors and other diseases.
  • the amount of the compound itself, or an optical isomer, or a pharmaceutically acceptable salt in the application is about 0.01 mg to about 1000 mg, about 0.1 mg to about 200 mg, about 1 mg to about 100 mg, and about 5 mg to about 5 mg. About 30 mg, about 10 mg to about 20 mg, about 12 mg to about 18 mg, or about 15 mg.
  • the present invention also provides a pharmaceutical composition.
  • the compound disclosed herein can be administered as a pure chemical, but is preferably administered as a pharmaceutical composition. Therefore, the present invention provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition may contain the compound or salt as the sole active agent, and may also contain at least one other active agent.
  • the compounds disclosed in the present invention can be injected, orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, by oral cavity, rectal, as an ophthalmic solution, or via a dosage unit formulation containing conventional pharmaceutical carriers.
  • the pharmaceutical composition can be formulated into any pharmaceutical form, such as: injection, aerosol, cream, gel, pill, capsule, tablet, syrup, transdermal patch, or ophthalmic solution.
  • Some dosage forms such as tablets and capsules can be subdivided into appropriate dosage unit dosage forms containing appropriate amounts of active ingredients such as effective amounts for the desired purpose.
  • Injection preparations preferably include injections, powder injections, etc., and are preferably injected intravenously, intramuscularly or locally.
  • Carriers include excipients and diluents, and must have sufficiently high purity and very low toxicity to make them suitable for administration to the patient to be treated.
  • the carrier may be inert or it may have medicinal benefits on its own.
  • the types of carriers include, but are not limited to: binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents Agent, and wetting agent.
  • Some carriers can be listed in more than one category, for example, vegetable oils can be used as lubricants in some formulations and as diluents in others.
  • Exemplary pharmaceutical carriers include sugar, starch, cellulose, malt, gelatin, talc and vegetable oils.
  • An optional active agent may be included in the pharmaceutical composition, which does not substantially affect the activity of the compound of the present invention.
  • Optical isomers are compounds that have the same chemical composition but differ in the arrangement of atoms or groups in space. It includes “diastereomers” and “enantiomers”.
  • Alkyl includes both branched and straight chain saturated aliphatic hydrocarbon groups, and has a specified number of carbon atoms, generally 1 to about 12 carbon atoms.
  • alkyl (C ⁇ 8) as used herein denotes an alkyl group having 1 to 8 carbon atoms. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, tert-butyl, n-pentyl, and sec-pentyl.
  • Cycloalkyl includes monocyclic, bicyclic or tricyclic alkyl groups, which usually have 3 to 12 carbon atoms. Cycloalkyl (C ⁇ 8) refers to having 3 to 8 carbon atoms, such as 3 to 8, or 3 to 4, 3 to 5, 3 to 6, or 3 to 7 carbon atoms. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Bicyclic and tricyclic ring systems include both bridged cycloalkyls and fused rings, such as, but not limited to, bicyclic [2.1.1] hexane and the like.
  • Cycloalkyl groups can be substituted or unsubstituted.
  • the substituted cycloalkyl group may be substituted one or more times with non-hydrogen and non-carbon groups as defined above.
  • substituted cycloalkyl groups may also include those rings substituted with straight or branched chain alkyl groups as defined above.
  • Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, for example, but not limited to, 2,2-, 2,3-, 2,4-, 2,5-, or 2,6- Disubstituted cyclohexyl, which may be substituted with substituents such as those listed above.
  • Alkenyl (C ⁇ 8) “alkenyl” refers to straight and branched hydrocarbon chains that include one or more unsaturated carbon-carbon bonds. The carbon-carbon bonds can occur at any stable point along the chain.
  • the alkenyl group (C ⁇ 8) described herein generally has 2 to 8 carbon atoms, such as C 2 -C 8 , C 2 -C 6 , and C 2 -C 4 alkenyl. In some embodiments, the alkenyl group includes one, two, or three carbon-carbon double bonds.
  • Alkenyl groups can be substituted or unsubstituted. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, for example, but not limited to, mono-, di- or tri-substituted alkenyl groups substituted with substituents such as those listed above.
  • Aryl is a cyclic aromatic hydrocarbon that does not contain heteroatoms.
  • the aryl group described herein includes monocyclic, bicyclic and tricyclic ring systems.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptyl, diphenyl, fluorenyl, phenanthryl, anthracenyl, indenyl, dihydroindenyl, pentacyclopentadienyl, and naphthyl.
  • the aryl group contains 6-14 carbon atoms in the ring portion of the group, and in other embodiments, it contains 6-12 carbon atoms or even 6-10 carbon atoms.
  • aryl groups are phenyl and naphthyl.
  • aryl includes those groups that contain fused rings, such as fused aryl-aliphatic ring systems (e.g., indenyl, tetrahydronaphthyl, etc.).
  • fused aryl-aliphatic ring systems e.g., indenyl, tetrahydronaphthyl, etc.
  • aryl also includes substituted aryl groups. Groups such as tolyl are called substituted aryl groups. Representative substituted aryl groups can be mono-substituted and substituted more than once.
  • monosubstituted aryl groups include, but are not limited to, 2-substituted, 3-substituted, 4-substituted, 5-substituted, or 6-substituted phenyl or naphthyl, which can be substituted by such as those listed above
  • the substituents and other substituents are substituted.
  • the aryl group is a phenyl group, which may be substituted or unsubstituted.
  • the substituted phenyl group has one or two substituents.
  • the substituted phenyl has one substituent.
  • Heteroaryl means a stable 5- or 6-membered monocyclic or polycyclic ring containing 1 to 4, or preferably 1 to 3 heteroatoms selected from N, O, and S, and the remaining ring atoms are carbon.
  • heteroaryl groups include, but are not limited to: pyridinyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, Azolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benzo[b]phenylthio, isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl , And 5,6,7,8-tetrahydroisoquinoline.
  • Alkynyl includes straight and branched chain alkyl groups as defined above, except that there is at least one triple bond between two carbon atoms.
  • the alkynyl group (C ⁇ 8) has 2-8 carbon atoms, and in some embodiments, 2-8, 2-6, or 2-4 carbon atoms.
  • the alkynyl group includes one, two, or three carbon-carbon triple bonds. Examples include but are not limited to -C ⁇ CH, -C ⁇ CCH 3 , -CH 2 C ⁇ CCH 3 , -C ⁇ CCH 2 CH(CH 2 CH 3 ) 2 and so on.
  • Alkynyl groups can be substituted or unsubstituted.
  • Representative substituted alkynyl groups may be mono-substituted or substituted more than once, for example, but not limited to, mono-, di- or tri-substituted alkynyl groups substituted with substituents such as those listed above .
  • Alkyl is an alkyl group as defined above, wherein the hydrogen or carbon bond of the alkyl group is replaced by a bond to the aryl group as defined above.
  • the aralkyl group (C ⁇ 8) has 7 to 8 carbon atoms.
  • Aralkyl groups can be substituted or unsubstituted.
  • the substituted aralkyl group can be substituted in the alkyl part, the aryl part, or both the alkyl and aryl parts of the group.
  • Representative aralkyl groups include, but are not limited to, benzyl and phenethyl.
  • Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above.
  • Alkyl ether group refers to any alkyl group as defined above in which at least one carbon-carbon bond is replaced with a carbon-oxygen bond.
  • the carbon-oxygen bond can be on the terminal (as in an alkoxy group) or the carbon-oxygen bond can be internal (ie, C-O-C).
  • Alkyl ethers contain at least one carbon-oxygen bond, but may include more than one carbon-oxygen bond.
  • polyethylene glycol (PEG) is included within the meaning of alkyl ether.
  • alkyl ether groups are optionally substituted.
  • the alkyl ether is substituted with an alcohol or phosphate ester.
  • spiroalkyl refers to a structure composed of two cycloalkyl moieties with exactly one atom in common. Examples include, but are not limited to, spiro[2.3]hexyl, spiro[2.5]octane and the like.
  • halogen refers to bromine, chlorine, fluorine, or iodine.
  • hydroxyl may refer to -OH or its ionized form -O - .
  • amino refers to a functional group having a nitrogen atom and 1 to 2 hydrogen atoms.
  • Amino groups are generally used herein to describe primary, secondary, or tertiary amines, and those skilled in the art can determine their identity in view of the context in which the term is used in this disclosure.
  • the term “amine” or “amine group” or “amino group” means a functional group containing a nitrogen atom derived from ammonia (NH 3 ).
  • the amine group may be a primary amine, which means that the nitrogen is bonded to two hydrogen atoms and a substituent that includes a substituted or unsubstituted alkyl or aryl group or an aliphatic or aromatic group.
  • the amine group may be a secondary amine, which means that the nitrogen is bonded to one hydrogen atom and two substituents, the substituents including substituted or unsubstituted alkyl or aryl groups or aliphatic or aromatic groups, This is defined as follows.
  • the amine group may be a tertiary amine, which means that the nitrogen is bonded to three substituents, which include substituted or unsubstituted alkyl or aryl groups or aliphatic or aromatic groups.
  • the amine group may also be a quaternary amine, which means that the designated amine group is bonded to the fourth group to form a positively charged ammonium group.
  • Carboxyl refers to the -C(O)OH group or its ionized form: -C(O)O - .
  • “Pharmaceutically acceptable salt” or “salt of a compound” is a derivative of the disclosed compound, wherein the parent compound is modified by preparing a non-toxic acid or base addition salt thereof, and also refers to these compounds and these salts
  • Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acids; etc., and include one or more Combinations of the above salts.
  • Pharmaceutically acceptable salts include non-toxic salts and quaternary ammonium salts such as parent compounds formed from non-toxic inorganic or organic acids.
  • non-toxic acid salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, Cesium salt, etc.; alkaline earth metal salt such as: calcium salt, magnesium salt, etc., and a combination including one or more of the above-mentioned salts.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.
  • other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, Cesium salt, etc.
  • alkaline earth metal salt such as: calcium salt, magnesium salt, etc., and a combination including one or more of the above-mentioned salts.
  • Organic salts include compounds such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, catabolic acid, maleic acid, hydroxymaleic acid, benzene Acetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid , Ethane disulfonic acid, oxalic acid, isethionic acid, HOOC-(CH 2 )n-COOH (where n is 0 to 4) and other organic acid salts; organic amine salts, such as: triethylamine salt, Pyridine
  • Figure 1 shows the co-crystal structure analysis and structural modification prediction of the BTB region protein of RTA401 and the target protein Keap1.
  • Fig. 2 is an MS spectrum of a representative compound 1 having a C ring of a conjugated diene structure.
  • Fig. 3 is a 1 H NMR spectrum of a representative compound 1 having a C ring of a conjugated diene structure.
  • Fig. 4 is an MS spectrum of the representative compound 7 with the C ring of the substituted conjugated diene structure.
  • Fig. 5 is a 1 H NMR chart of a representative compound 7 having a ring C of a conjugated diene structure with a substituent.
  • Fig. 6 is the rigid structure region (gray region) of the C ring core of the conjugated diene structure with substituents.
  • the embodiment of the present invention provides an oleanane compound with a C ring of a conjugated diene rigid structure, and a synthesis method and biological activity screening thereof.
  • the fine structure of the compound C ring has an extremely important influence on the activity of the compound.
  • the available data show that, without the enol-like conjugated structure, OA-26 reduces the anti-inflammatory activity of RTA401 compound by 1000 times [J.Med.Chem.2000,43,4233-4246].
  • the structure of the compound C ring is affected by the large steric hindrance of the surrounding multi-ring rigid structure, which makes its reactivity significantly passivated.
  • the C-cyclic ethylenic bond of oleanolic acid (OA) does not react at all under atmospheric catalytic hydrogenation conditions.
  • the A-ring ethylenic bond of OA-20 can be reduced quantitatively, but the C-ring ethylenic bond can not be reacted at all, and the final compound is OA-19. Therefore, it can be seen that the C ring possesses the unique chemical properties of natural terpenoid products.
  • Synthetic reagents and conditions a) hydrogen, palladium on carbon, tetrahydrofuran, normal pressure and normal temperature, 24 to 48 hours.
  • Synthetic reagents and conditions a) benzyl chloride, potassium carbonate, N,N-dimethylformamide, 55°C, 5 hours; b) acetic anhydride, pyridine, 0°C ⁇ room temperature, 4 hours; c) 30% hydrogen peroxide , Formic acid, dichloromethane, room temperature, 24 hours; d) bromine, hydrobromic acid, acetic acid, 50 °C ⁇ room temperature, 24 hours; e) potassium hydroxide, methanol, reflux, 54 minutes; f) Jones reagent, acetone, 0°C ⁇ room temperature, 20 minutes; g) ethyl formate, sodium methoxide, dichloromethane, 0°C ⁇ room temperature, 12 hours; h) hydroxylamine hydrochloride, ethanol, reflux, 1 hour; i) zinc amalgam reagent, acetone, Room temperature, 4 hours; j) Sodium methoxide, ether, methanol, 0°C ⁇ room
  • the white solid (2.1 g, 3.74 mmol) obtained in the previous step was dissolved in a mixed solution of methanol (60 mL) and ether (125 mL), sodium methoxide (6.38 g, 118 mmol) was added at 0°C, and the reaction was carried out at room temperature for 45 minutes.
  • the reaction solution was diluted with dichloromethane (50 mL), and the organic layer was washed 3 times with 5% dilute hydrochloric acid, saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a pale yellow solid OA-10 ( 2.0g, 95%).
  • Synthesis reagents and conditions a) hydrogen, palladium on carbon, tetrahydrofuran, normal pressure and room temperature, 1 to 2 hours; b) methyl iodide, potassium carbonate, acetone, room temperature, 4 hours; c) zinc amalgam reagent, acetone, room temperature, 4 Hours; d) sodium methoxide, ether, methanol, 0°C ⁇ room temperature, 1 hour; e) 2,3-dichloro-5,6-dicyanoquinone, benzene, reflux, 20 minutes.
  • OA-8 was used as the starting material to prepare OA-11, and the preparation method was completed according to the previous study [CN102070697A].
  • Compound OA-11 (1.05g, 2.1mmol) was dissolved in acetone (50mL), potassium carbonate (3.0g, 21mmol) was added and suspended in the solution, stirred vigorously for 2 hours, and slowly added dropwise methyl iodide (0.85g, 6mmol), react at room temperature for 5 hours.
  • the reaction solution was filtered, the filtrate was concentrated, and dichloromethane (50mL) was diluted.
  • Synthetic reagents and conditions a) zinc amalgam reagent, acetone, room temperature, 4 hours; b) sodium methoxide, ether, methanol, 0°C ⁇ room temperature, 1 hour; c) 2,3-dichloro-5,6-di Cyano-p-benzoquinone, benzene, reflux, 20 minutes; d)i. Oxalyl chloride, anhydrous dichloromethane, 0°C ⁇ room temperature, 12 hours; ii. NH 2 CH 2 CF 3 , triethylamine, anhydrous dichloride Methane, 0°C ⁇ room temperature, 4 hours.
  • Dissolve compound 3 (1.4g, 3.04mmol) in anhydrous dichloromethane (5mL), add oxalyl chloride (3.86g, 30.4mmol) at 0°C, stir, return to room temperature, react overnight, spin dry to obtain a pale yellow solid , Directly into the next reaction.
  • the solid obtained in the previous step was dissolved in anhydrous dichloromethane (5mL).
  • Trifluoroethylamine (0.36g, 3.64mmol) and triethylamine (0.92g, 9.12mmol) were added at 0°C, stirred, returned to room temperature, and reacted.
  • Synthesis reagents and conditions a) Sodium methoxide, ether, methanol, 0°C ⁇ room temperature, 1 hour; b) 2,3-Dichloro-5,6-dicyano-p-benzoquinone, benzene, reflux, 20 minutes; c) Acetic anhydride, concentrated sulfuric acid, p-toluenesulfonic acid, room temperature, 2 hours.
  • Synthesis reagents and conditions a) Sodium methoxide, ether, methanol, 0°C ⁇ room temperature, 1 hour; b) 2,3-Dichloro-5,6-dicyano-p-benzoquinone, benzene, reflux, 20 minutes; c) Acetic anhydride, concentrated sulfuric acid, p-toluenesulfonic acid, room temperature, 2 hours.
  • Synthesis reagents and conditions a) methyl iodide, potassium carbonate, acetone, room temperature, 4 hours; b) acetic anhydride, concentrated sulfuric acid, p-toluenesulfonic acid, room temperature, 2 hours.
  • the preparation of OA-21 from compound OA-20 was completed by referring to the method of preparing OA-12 from OA-11 in Example 2 of the present invention.
  • the preparation of compound 7 from compound OA-21 is completed by referring to the method of preparing compound 5 from OA-18 in Example 4 of the present invention.
  • MS[M+H] + of compound 7 calcd for C 34 H 46 NO 5 , 548.3; found 548.5; MS [M+NH 4 ] + : calcd for C 34 H 49 N 2 O 5 , 565.4; found 565.6.
  • Synthesis reagents and conditions a) i. Oxalyl chloride, anhydrous dichloromethane, 0°C ⁇ room temperature, 12 hours; ii.NH 2 CH 3 , triethylamine, anhydrous dichloromethane, 0°C ⁇ room temperature, 4 hours B) i. Oxalyl chloride, anhydrous dichloromethane, 0°C ⁇ room temperature, 12 hours; ii. NH 2 CH 2 CF 3 , triethylamine, anhydrous dichloromethane, 0°C ⁇ room temperature, 4 hours; c )i. Oxalyl chloride, anhydrous dichloromethane, 0°C ⁇ room temperature, 12 hours; ii.
  • the preparation of OA-22, OA-23 and OA-24 from compound OA-20 was completed by referring to the method of preparing compound 4 from compound 3 in Example 3 of the present invention.
  • the preparation of compounds 8, 9 and 10 from compounds OA-22, OA-23 and OA-24, respectively, was completed with reference to the method for preparing compound 5 from OA-18 in Example 4 of the present invention.
  • Synthesis reagents and conditions a) Acetic anhydride, concentrated sulfuric acid, p-toluenesulfonic acid, room temperature, 2 hours.
  • Compound OA-25 was prepared from OA-20 with reference to CN104395332B.
  • the preparation of compound 11 from compound OA-25 was completed by referring to the method of preparing compound 5 from OA-18 in Example 4 of the present invention.
  • MS[M+H] + of compound 11 calcd for C 35 H 47 F 2 N 2 O 4 , 597.4; found 597.4.
  • Synthesis reagents and conditions a) Anhydrous tetrahydrofuran, lithium diisopropylamine solution, chloroacetic anhydride, -78°C, 2-4 hours.
  • Synthetic reagents and conditions a) Anhydrous tetrahydrofuran, lithium diisopropylamide solution, isobutyryl chloride, -78°C, 2-4 hours.
  • compound 13 was prepared from compound OA-21 in a small amount. MS[M+H] + of compound 13: calcd for C 36 H 50 NO 5 , 576.3; found 576.4.
  • Synthetic reagents and conditions a) Anhydrous tetrahydrofuran, lithium diisopropylamide solution, isobutyryl chloride, -78°C, 2-4 hours.
  • compound 14 was prepared from compound OA-21. MS[M+H] + of compound 14: calcd for C 36 H 48 NO 6 ,590.3; found 590.3.
  • the results of the determination of the inhibition of IFN ⁇ -induced nitric oxide production by RAW264.7 mouse macrophages by several compounds of the present invention were carried out with reference to the document J. Med. Chem. 2000, 43, 4233-4246. 20,000 cells/well were seeded in RPMI1640 medium and 0.5% FBS in a 96-well plate, and incubated at 37° C. and 5% CO 2. DMSO or test compound and recombinant mouse IFN ⁇ were treated together for 48 hours. Griess reagent is used to react with nitric oxide in the medium and detected at 550 nM. In this way, the anti-inflammatory biological activity of the compound was initially screened.
  • a "-" means IC 50 is greater than 100nM
  • IC 50 is between 10 and 100nM
  • b OA-26 is a known compound, prepared according to the document J. Med. Chem. 2000, 43, 4233-4246.
  • Example 14 Based on the screening model in Example 14, before adding DMSO or test compound 7 or 12, add a known cell esterase inhibitor (for example, 1mmol/L Paraoxon) [J.Appl.Toxicol.2005,25,562–67] , And then also use Griess reagent to react with nitric oxide in the medium and detect at 550nM. In this way, the interaction between the esterase inhibitor and the test compound is tested.
  • the results show (see Table 2), which represents that the anti-inflammatory biological activity produced by compounds 7 and 12 is completely independent of the presence of esterase.
  • a "-" means IC 50 is greater than 100nM
  • IC 50 is between 10 and 100nM
  • Human pancreatic cancer PANC-1 cells were seeded in a 96-well plate at a density of 3000 cells/well with 100 ⁇ L of culture medium. Add the required drug concentration to the culture medium, shake and mix, and each drug concentration is at least three replicate holes. After 72 hours, discard the culture medium, add 60 ⁇ L of 50% TCA to each well, let stand for 60 minutes at 4°C, wash with deionized water, and dry naturally; add 60 ⁇ L of 0.4% SRB to each well, and keep it at room temperature away from light.
  • the cell proliferation inhibition rate (%) (1-OD experimental group /OD solvent group )*100%.
  • the experimental results are shown in Table 3 below.
  • Most of the compounds can inhibit the proliferation of human pancreatic cancer PANC-1 cells at the micromolar level.
  • the electron-withdrawing substituent side chain can significantly enhance the anti-tumor biological activity of the compound.
  • compound 11 And 12 compared with RTA402, the biological activity has a significant difference (P ⁇ 0.05).
  • a "-" means IC 50 is greater than 100 ⁇ M
  • IC 50 is between 10 and 100 ⁇ M
  • b OA-26 is a known compound, prepared according to the document J. Med. Chem. 2000, 43, 4233-4246.
  • the toxicity of compounds 7 and 12 at the IC 50 concentration level of anti-tumor and anti-inflammatory on human normal cells representative LO2 hepatocytes was investigated.
  • the logarithmic growth phase cells were seeded in a 96-well plate (100 ⁇ L/well) at a cell density of 8 ⁇ 10 3 cells per well, and placed in a cell incubator for culture.
  • different concentrations of test compounds were added to the culture medium, and each group had 4 multiple wells. Put the cells back into the CO 2 incubator for culture.
  • test compound After 24 hours, discard the cell culture medium, add 200 ⁇ L of medium containing 10% MTT (5mg/mL), continue incubating for 4 hours in the 37°C incubator, discard the medium containing MTT, and then add 150 ⁇ L of DMSO to each well , Shake for 8min to fully dissolve the formed formazan.
  • the results are shown in Table 4.
  • the test compound has little inhibitory activity against normal cells at the concentration level of anti-tumor (1-10 ⁇ M), and has good tumor cell/normal cell selectivity; while at the concentration of anti-inflammatory activity (Around 10 nM), no inhibitory activity was seen at all.
  • compounds 7 and 12 the toxicity to human normal cells is slightly reduced or equivalent, and there is no significant inhibitory effect on human normal cells at therapeutic concentrations.
  • a "-" means that the inhibition rate is less than or equal to 0%, and there is no cell proliferation inhibition at all.

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Abstract

本发明属于药物化学领域，具体涉及一种具有共轭二烯结构C环的齐墩果酸衍生物及其制备方法和应用。特别的，本发明还提供一种药物组合物，其包含有效量的所述衍生物或其药学上可接受的盐、以及药学上可接受的载体；并且，所述衍生物或其药学上可接受的盐能够用于治疗炎症相关疾病、以及具有抗肿瘤活性，同时化合物安全性有所改善或相当。

Description

具有共轭二烯结构C环的齐墩果酸衍生物及其制备方法和应用 技术领域
本发明属于药物化学领域，具体涉及一种用于预防和治疗炎症相关疾病和肿瘤相关疾病的齐墩果酸衍生物化合物、制备方法和应用。
背景技术
天然萜类化合物及衍生物具备独特的化学结构和生物活性性质，是开发小分子新药的重要来源之一。例如百时美施贵宝公司的紫杉醇(Paclitaxel)和赛诺菲公司的多西他赛(Docetaxel)虽然具备极为相似的紫衫烷萜类结构，却在药代、安全性和临床应用等方面各具特色[实用药物与临床,2010,4,281-283]。
类似地，齐墩果烷萜类化合物，例如齐墩果酸，具有抗氧化、抗炎等广泛的生物活性。齐墩果酸在我国批准用于临床肝病(例如肝炎)的辅助治疗。然而齐墩果酸抗炎等生物活性弱，高度脂溶性而水溶性差等缺点，限制其临床应用，该药物未被美国、欧洲等药品管理部门批准上市。
一系列衍生获得的齐墩果酸衍生物，例如RTA401、RTA402和RTA408等，是具有强烈生物活性的化合物[J.Med.Chem.2000,43,4233-4246]。该类化合物可在低浓度下是细胞保护剂和细胞炎性过程抑制剂，高浓度下则抑制细胞增殖，促进细胞凋亡[Nat.Rev.Cancer,2007,7,357–369]。研究表明，该类化合物作用机制也十分复杂，潜在的靶点众多。已证明包括Keap1-NRF2在内的多个炎症相关靶点和信号通路[Nat.Rev.Cancer,2007,7,357–369；J.Am.Soc.Nephrol.2012,1663-1673；PLOS ONE,2014,9,e98896]参与其中。
Figure PCTCN2021076726-appb-000001
研发过程发现，齐墩果酸的A环、C环和28位羧基是关键的活性改造位点。第一类化合物主要集中在A环上进行研究，优选的候选化合物RTA401(又名CDDO，Bardoxolone)，即2-氰基-3，12-二氧齐墩果-1，9(11)-二烯-28-酸；以及RTA402(又名CDDO-Me，Bardoxolone methyl)，即2-氰基-3，12-二氧齐墩果-1，9(11)-二烯-28-酸甲酯。需要指出的是，由于该天然产物的结构独特性，例如多环骈合形成的空间位阻效应，导致28位羧基甲酯不易水解。不同于药物化学常识，RTA402并不是RTA401的简单的甲酯化前药。Reata公司将RTA401和RTA402都推进至临床试验，应用在不同的适应症中，例如在临床试验NCT00322140中RTA401用于治疗实体瘤和淋巴瘤，而在临床试验NCT03749447中RTA402用于治疗慢性肾病。
后续研究主要针对28位羧基进行[CN102066398B；CN102083442B]，优选的候选化合物RTA408，即2-氰基-3，12-二氧齐墩果-1，9(11)-二烯-28-(2’,2’-二氟-丙酰基)胺。Reata公司又将RTA408推进至临床试验，用于治疗不同的炎症相关的病症，例如在临床试验NCT03902002中RTA408用于治疗肝损伤。
除A环和28位羧基以外，C环也是影响化合物活性和成药性的关键区域。此 外，齐墩果烷的C环在化学上也具备独特的性质。例如RTA402的A环的烯键可以通过常压催化氢化还原，而C环上的烯键对催化氢化却十分稳定。Reata公司对C环进行专门研究，例如对不饱和烯键进行饱和化[CN102164941B]，虽然改善了化合物安全性，但生物活性下降，至今未获选进入临床研究。
鉴于已知齐墩果酸衍生物独特的化学结构(例如钝化的28位羧基酯、C环等)和多重生物学活性性质，具有治疗多样性的慢性炎症、肿瘤等疾病的潜力；同时作为构效关系中关键的活性改造位点，C环的结构研究没有取得明显进展，因此有需要合成具备更多C环化学结构、生物活性多样性进一步扩展的不同结构齐墩果酸新化合物，用于***、慢性炎症相关疾病。
发明内容
为解决上述技术问题，本发明提供一种C环具有共轭二烯刚性结构的齐墩果烷化合物、其制备方法和其应用。
具体地，本发明提供了一类通式(Ⅰ)的化合物或其药学上可接受的盐：
Figure PCTCN2021076726-appb-000002
其中：
X＝-H(Y取代基不存在)或O；
Y＝-COR 1；
Z＝-COOR 2,-CONR 2R 3,或-NR 2R 3；
其中R 1分别独立取自氢、或烷基(C≤8)、烷醚基，或者这些基团的任一个的 至少一个氢原子的卤素取代形式；
其中R 2和R 3分别独立取自氢、或烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基、羧基取代形式。
在具体实施方案中，化合物进一步地定义为通式(Ⅱ)所示的一类化合物或其药学上可接受的盐：
Figure PCTCN2021076726-appb-000003
Z＝-COOR 1,-CONR 1R 2,或-NR 1R 2；
其中R 1和R 2分别独立取自氢、或烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基、羧基取代形式。
在具体实施方案中，化合物进一步地定义为通式(Ⅲ)所示的一类化合物或其药学上可接受的盐：
Figure PCTCN2021076726-appb-000004
X＝-O；
Y＝-COR 1；
Z＝-COOR 1,-CONR 1R 2,或-NR 1R 2；
其中R 1和R 2分别独立取自氢、或烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基取代形式。
在具体实施方案中，化合物进一步地定义为通式(Ⅳ)所示的一类化合物或其药学上可接受的盐：
Figure PCTCN2021076726-appb-000005
X＝-O；
Y＝-COR 1；
Z＝-COOCH 3、-CONHCH 2CF 3或-NHCOCF 2CH 3；
其中R 1取自烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基取代形式。
在具体实施方案中，化合物进一步地定义为通式(Ⅴ)所示的一类化合物或其药学上可接受的盐：
Figure PCTCN2021076726-appb-000006
X＝-O；
Y＝-COR 1；
Z＝-COOCH 3、-CONHCH 2CF 3或-NHCOCF 2CH 3；
其中R 1取自烷基(C≤8)、环烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基取代形式。
本公开所提供化合物的非限定性实例包括根据下面所示化学式的化合物，以及其可药用盐：
Figure PCTCN2021076726-appb-000007
Figure PCTCN2021076726-appb-000008
在具体实施方案中，部分化合物可选择由如下合成方法(一)构建C环共轭二烯结构：
Figure PCTCN2021076726-appb-000009
在具体实施方案中，部分化合物也可选择由如下合成方法(二)构建C环共轭二烯结构：
Figure PCTCN2021076726-appb-000010
在具体实施方案中，部分化合物也可选择由如下合成方法(三)构建C环共轭二烯结构：
Figure PCTCN2021076726-appb-000011
上述化合物自身、或光学异构体、药学上可接受的盐具备在低浓度(nM)下的抗炎活性、以及高浓度(μM)下的抗肿瘤活性。上述化合物自身、或光学异构体、药学上可接受的盐具备应用于治疗炎性相关疾病以及肿瘤等疾病的用途。具体的，所述化合物自身、或光学异构体、药学上可接受的盐在所述用途中的用量约0.01mg至约1000mg、约0.1mg至约200mg、约1mg至约100mg、 约5mg至约30mg、约10mg至约20mg、约12mg至约18mg、或约15mg。
本发明还提供了一种药物组合物，本文中公开的化合物可以以纯化学品给予，但优选作为药物组合物给予。因此，本发明提供了包括化合物或药用盐连同至少一种药用载体的药物组合物。药物组合物可以包含化合物或盐作为唯一的活性剂，也可以包含至少一种其他活性剂。
本发明公开的化合物可以以包含常规药用载体的剂量单位制剂注射、口服、局部、非肠道、通过吸入或喷雾、舌下、透皮、通过口腔给予、直肠、作为眼用溶液、或通过其它方式来给予。可以将药物组合物配制成任何药用形式，如：注射制剂、气雾剂、乳膏剂、凝胶剂、丸剂、胶囊剂、片剂、糖浆剂、透皮贴剂、或眼用溶液。诸如片剂和胶囊剂的一些剂型可以再分成包含诸如达到期望目的的有效量的适当量活性组分的适当剂量单位剂型。注射制剂优选地包括注射液、粉针剂等，优选地通过静脉、肌肉或局部注射。
载体包括赋形剂和稀释剂，并且必须具有足够高的纯度和十分低的毒性以使它们适于被给予待治疗的患者。载体可以是惰性的或其可以本身具有药用益处。载体的种类包括但不限于：粘合剂、缓冲剂、着色剂、稀释剂、崩解剂、乳化剂、调味剂、助流剂、滑润剂、防腐剂、稳定剂、表面活性剂、制片剂、以及润湿剂。一些载体可以列在多于一种的类别中，如：植物油可以在一些制剂中用作滑润剂并在其他制剂中用作稀释剂。示例性药用载体包括糖、淀粉、纤维素、麦芽、明胶、滑石和植物油。可选的活性剂可以包括在药物组合物中，其基本上不影响本发明的化合物的活性。
术语约定：
“光学异构体”是具有相同化学组成但原子或基团在空间中的排布不同的化合物。其包括“非对映异构体”和“对映异构体”。
“烷基”包括支链和直链饱和脂肪族烃基两者，并具有指定数量的碳原子数量，一般1至约12个碳原子。如在本文中使用的术语烷基(C≤8)表示具有1至8个碳原子的烷基。烷基的实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、3-甲基丁基、叔丁基、正戊基、和仲戊基。
“环烷基”包括单环、双环或三环烷基，其通常在具有3～12个碳原子。环烷基(C≤8)指具有3～8个碳原子，如3～8、或3～4、3～5、3～6、3～7个碳原子。示例性单环环烷基包括但不限于环丙基、环丁基、环戊基、环己基、环庚基、和环辛基。双环和三环环系既包括桥接环烷基也包括稠环，例如，但不限于，双环[2.1.1]己烷等等。环烷基可以是取代或未取代的。取代的环烷基可以被如上所定义的非氢和非碳基团取代一次或多次。然而，取代的环烷基还可以包括那些被如上所定义的直链或支链烷基取代的环。代表性的取代的环烷基可以是单取代的或者是取代一次以上的，例如但不限于，2,2-，2,3-，2,4-，2,5-，或2,6-二取代的环己基，其可以被诸如如上所列的取代基取代。
烯基(C≤8)、“烯基”指包括一个或多个不饱和的碳-碳键的直链和支链烃链，碳-碳键可以出现在沿着链的任一稳定点。本文中所述的烯基(C≤8)通常具有2至8个碳原子，如：C 2-C 8、C 2-C 6、和C 2-C 4烯基。在一些实施方式中，烯基包括一个、两个、或三个碳-碳双键。烯基的实例包括乙烯基、丙烯基、丁烯基、烯丙基、CH＝CH(CH 3)、-CH＝C(CH 3) 2、-C(CH 3)＝CH 2、-C(CH 3)＝CH(CH 3)、-C(CH 2CH 3)＝CH 2等等。烯基可以是取代或未取代的。代表性的取代的烯基可以是单-取代的或者是取代一次以上的，例如但不限于，被诸如如上所列的取代基取代的单-，二-或三-取代的烯基。
“芳基”为不含杂原子的环芳香烃。本文所述的芳基包括单环、双环和三环体系。因此，芳基包括但不限于，苯基、薁基、庚搭基、二苯基、芴基、菲 基、蒽基、茚基、二氢茚基、并环戊二烯基、和萘基。在一些实施方式中，芳基在基团的环部分含有6-14个碳原子，在其他实施方式中，含有6～12个碳原子或者甚至是6～10个碳原子。在一些实施方式中，芳基为苯基和萘基。“芳基”一词包括那些含有稠环的基团，例如稠合芳-脂族环体系(例如，二氢茚基、四氢萘基等等)。“芳基”一词还包括取代的芳基。诸如甲苯基等基团被称为取代的芳基。代表性的取代的芳基可以是单取代的和取代一次以上的。例如，单取代的芳基包括但不限于，2-取代的、3-取代的、4-取代的、5-取代的、或6-取代的苯基或萘基，其可以被诸如如上所列的取代基等取代基取代。在一些实施方式中，芳基是苯基，其可以是取代或未取代的。在一些实施方式中，取代的苯基具有一个或两个取代基。在一些实施方式中，取代的苯基具有一个取代基。
“杂芳基”表示包含选自N、O和S的1至4个、或优选1至3个杂原子并且剩余环原子为碳的稳定的5-或6-元单环或多环。杂芳基的实例包括但不限于：吡啶基、吲哚基、嘧啶基、哒嗪基、吡嗪基、咪唑基、噁唑基、呋喃基、苯硫基、噻唑基、***基、四唑基、异噁唑基、喹啉基、吡咯基、吡唑基、苯并[b]苯硫基、异喹啉基、喹唑啉基、喹喔啉基、噻吩基、异吲哚基、和5,6,7,8-四氢异喹啉。
“炔基”包括如上所定义的直链和支链烷基，不同在于两个碳原子之间具有至少一个三键。炔基(C≤8)具有2～8个碳原子，在一些实施方式中，具有2～8、2～6、或2～4个碳原子。在一些实施方式中，炔基包括一个、两个、或三个碳-碳三键。例子包括但不限于-C≡CH、-C≡CCH 3、-CH 2C≡CCH 3、-C≡CCH 2CH(CH 2CH 3) 2等等。炔基可以是取代或未取代的。代表性的取代的炔基可以是单-取代的或者是取代一次以上的，例如但不限于，被诸如如上所列的取代基等取代基取代的单-，二-或三-取代的炔基。
“芳烷基”是如上所定义的烷基，其中烷基的氢或碳键被连接到如上所定义的芳基的键取代。芳烷基(C≤8)具有7～8个碳原子。芳烷基可以是取代或未取代的。取代的芳烷基可以在基团的烷基部分、芳基部分或烷基及芳基两个部分进行取代。代表性的芳烷基包括但不限于，苄基和苯乙基。代表性的取代的芳烷基可以被诸如如上所列的取代基等取代基取代一次或多次。
“烷醚基”指如上文定义的任何烷基，其中至少一个碳-碳键用碳-氧键替换。碳-氧键可以在末端上(如在烷氧基中)或碳氧键可以是内部的(即，C-O-C)。烷基醚包含至少一个碳氧键，但是可以包括多于一个碳氧键。例如，在烷基醚的含义内包括聚乙二醇(PEG)。除非在说明书中另有明确说明，否则烷基醚基是任选取代的。例如，在一些实施方案中，烷基醚用醇或磷酸酯取代。
术语“螺烷基”是指由具有刚好一个共有原子的两个环烷基部分组成的结构。其例子包括但不限于螺[2.3]己基、螺[2.5]辛烷等。
术语“卤素”或“卤代”指的是溴、氯、氟、或碘。
术语“羟基”可以指的是–OH或其离子化形式–O –。
术语“氨基”指具有氮原子和1至2个氢原子的官能团。“氨基”通常在本文中可用于描述伯胺、仲胺或叔胺，且本领域技术人员鉴于本公开使用该术语的上下文而能够确定它们的身份。术语“胺”或“胺基团”或“氨基团”意指含有由氨(NH 3)衍生的氮原子的官能团。胺基团可为伯胺，其意指氮与两个氢原子和一个取代基键合，所述取代基包含经取代或未取代的烷基或芳基或脂族或芳族基团。胺基团可为仲胺，其意指氮与一个氢原子和两个取代基键合，所述取代基包含经取代或未经取代的烷基或芳基或脂族或芳族基团，这如下定义。胺基团可为叔胺，其意指氮与三个取代基键合，所述取代基包含经取代或未经取代的烷基或芳基或脂族或芳族基团。胺基团也可为季胺，其意指所指定的胺 基团与第四基团键合，形成带正电的铵基团。
术语“羧基”指的是–C(O)OH基或其离子化形式：–C(O)O –。
“药学上可接受的盐”或“化合物的盐”是所公开的化合物的衍生物，其中，母体化合物通过制备无毒的酸或其碱加成盐改性，并且还指这些化合物和这些盐的药用溶剂化物，包括水合物。药用盐的实例包括但不限于：碱性残基如胺类的无机或有机酸加成盐；酸性残基如羧酸的碱或有机加成盐；等等，以及包括一种或多种上述盐的组合。药用盐包括诸如从无毒无机或有机酸形成的母体化合物的无毒盐和季铵盐。例如，无毒酸性盐包括衍生自无机酸的那些，例如：盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等；其他可接受的无机盐包括金属盐如：钠盐、钾盐、铯盐等；碱土金属盐如：钙盐、镁盐等，以及包括一种或多种上述盐的组合。有机盐包括由诸如乙酸、三氟乙酸、丙酸、丁二酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、扑酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、甲磺酸、乙磺酸、苯磺酸、对氨基苯磺酸、2-乙酸基苯酸、富马酸、对甲苯磺酸、甲磺酸、乙烷二磺酸、草酸、羟乙磺酸、HOOC-(CH 2)n-COOH(其中n为0至4)等的有机酸制备的盐；有机胺盐，如：三乙胺盐、吡啶盐、甲基吡啶盐、乙醇胺盐、三乙醇胺盐、二环己基胺盐、N,N'-二苄基乙二胺盐等；和氨基酸盐，如：精氨酸盐、天冬氨酸盐、谷氨酸盐等，以及包括一种或多种上述盐的组合。
附图说明
图1是RTA401与靶蛋白Keap1的BTB区域蛋白共结晶结构分析与结构改造预测。
图2是具有共轭二烯结构C环的代表化合物1的MS图谱。
图3是具有共轭二烯结构C环的代表化合物1的 1H NMR图谱。
图4是具有取代基共轭二烯结构C环的代表化合物7的MS图谱。
图5是具有取代基共轭二烯结构C环的代表化合物7的 1H NMR图谱。
图6是具有取代基共轭二烯结构C环核心的刚性结构区域(灰色区域)。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案，但本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
本发明实施例提供具有共轭二烯刚性结构C环的齐墩果烷化合物、及其合成方法与生物活性筛选。
实施例1 C环的结构与活性
化合物C环的精细结构对化合物的活性具备极其重要影响。已有的数据显示，失去烯醇式共轭结构，OA-26比RTA401化合物抗炎活性降低1000倍[J.Med.Chem.2000,43,4233-4246]。
Figure PCTCN2021076726-appb-000012
采用数据库RSC PDB中公开的RTA401与靶蛋白Keap1的BTB区域蛋白共结晶(序列号PDB 4CXT)数据分析，结合状态下RTA401的C环具备明确的烯酮式结构，羰基面向结合口袋外的开放溶剂区，通过计算机辅助药物设计判断，此处(图1，箭头处)空间结构上具备较大改造潜力。
实施例2 C环的化学性质
化合物C环的结构受周围多环刚性结构的大空间位阻影响，使其反应性显著钝化，例如齐墩果酸(OA)C环烯键在常压催化氢化条件下完全不反应。同样的条件下，OA-20的A环烯键可以定量还原，而C环烯键也完全不能反应，最终得到化合物是OA-19。因此可见C环具备萜类天然产物独特的化学性质。
Figure PCTCN2021076726-appb-000013
合成试剂与条件：a)氢气，钯碳，四氢呋喃，常压常温，24至48小时。
将化合物OA-20(0.2g,0.41mmol)溶于四氢呋喃(5mL)中，钯碳(10％,0.02g)催化常压氢化反应24至48小时，过滤，滤液浓缩，快速硅胶柱层析得白色固体化合物得白色固体OA-19(0.16g,84％)。化合物OA-19的质谱[M+H] +:calcd for C 31H 44NO 4,494.3；found 494.3。化合物OA-19的氢谱 1HNMR(300M Hz,CDCl 3,25℃,TMS):δ8.01(s,1H),5.9(s,1H),3.03(m,1H),2.93(d,J＝4.5Hz,1H),2.41(d,J＝9.0Hz,1H),2.26(d,J＝9.0Hz,1H),1.26,1.21,1.24,1.20,1.16,1.00,0.91(s,each 3H)ppm。
实施例3化合物1的合成如下路线所示：
Figure PCTCN2021076726-appb-000014
Figure PCTCN2021076726-appb-000015
合成试剂与条件:a)苄基氯,碳酸钾,N,N-二甲基甲酰胺,55℃,5小时；b)乙酸酐，吡啶，0℃→室温，4小时；c)30％双氧水，甲酸，二氯甲烷，室温，24小时；d)溴,氢溴酸，乙酸，50℃→室温,24小时；e)氢氧化钾,甲醇，回流，54分钟；f)琼斯试剂，丙酮，0℃→室温,20分钟；g)甲酸乙酯,甲醇钠，二氯甲烷，0℃→室温,12小时；h)盐酸羟胺,乙醇，回流，1小时；i)锌汞齐试剂，丙酮，室温，4小时；j)甲醇钠，***，甲醇，0℃→室温,1小时；k)2,3-二氯-5,6-二氰对苯醌，苯,回流，20分钟。
以市售齐墩果酸为起始原料，其中中间体OA-8制备方法按照前期研究完成[CN102070697A]。将化合物OA-8(2.3g,3.94mmol)，溶于丙酮(75mL)，反应体系中加入过量的锌汞齐试剂，常温反应约4小时，反应结束，过滤，滤液浓缩得白色固体OA-9(2.1g,95％)。将上一步获得的白色固体(2.1g,3.74mmol)，溶于甲醇(60mL)和***(125mL)的混合溶液，0℃下加入甲醇钠(6.38g,118mmol)，室温反应45分钟。反应液用二氯甲烷(50mL)稀释，有机层用5％稀盐酸、饱和碳酸氢钠和饱和食盐水各洗涤3次，无水硫酸钠干燥，过滤，滤液浓缩得淡黄色固体OA-10(2.0g,95％)。将此固体(2.0g,3.51mmol)和2,3-二氯-5,6-二氰对苯醌(0.80g,3.51mmol)溶于无水苯(40mL)中，加热回流20分钟。反应结束后，过滤，滤液浓缩，快速硅胶柱层析得白色固体化合 物1(1.7g,88％)。化合物1的质谱[M+Na] +:calcd for C 38H 47NNaO 3,588.3；found 588.4；[M+K] +:calcd for C 38H 47NKO 3,604.9；found 604.4。化合物1的氢谱 1H NMR(300M Hz,CDCl 3,25℃,TMS):δ8.01(d,1H),7.34-7.27(m,5H),5.88-5.67(dd,2H),5.16-5.03(dd,2H),3.10-3.07(m,1H),2.00(m,1H),1.71-1.55(m,9H),1.50(m,5H),1.26,1.21,1.24,1.20,1.16,1.00,0.91(s,each 3H)ppm。
实施例4化合物2的合成如下路线所示：
Figure PCTCN2021076726-appb-000016
合成试剂与条件:a)氢气，钯碳,四氢呋喃,常压常温,1至2小时；b)碘甲烷，碳酸钾，丙酮，室温，4小时；c)锌汞齐试剂，丙酮，室温，4小时；d)甲醇钠，***，甲醇，0℃→室温,1小时；e)2,3-二氯-5,6-二氰对苯醌，苯,回流，20分钟。
以中间体OA-8为起始原料制备OA-11，其制备方法按照前期研究完成[CN102070697A]。将化合物OA-11(1.05g,2.1mmol)溶于丙酮(50mL)，加入碳酸钾(3.0g,21mmol)悬浮于溶液中，剧烈搅拌2小时，0℃下缓慢滴加碘甲烷(0.85g,6mmol)，室温反应5小时。反应液过滤，滤液浓缩，二氯甲烷(50mL)稀释，有机层用饱和碳酸氢钠溶液和饱和食盐水各洗涤3次，无水硫酸钠干燥，过滤，滤液浓缩后得淡黄色固体，经快速硅胶柱层析制得白色固体OA-12，直接投入后续反应。而由化合物OA-12制备化合物2参考本发明实施例1中OA-8制备化合物1方法完成。化合物2的质谱MS[M+Na] +:calcd for C 32H 43NNaO 3,512.3；found 512.3。
实施例5化合物3和4的合成如下路线所示：
Figure PCTCN2021076726-appb-000017
合成试剂与条件:a)锌汞齐试剂，丙酮，室温，4小时；b)甲醇钠，***，甲醇，0℃→室温,1小时；c)2,3-二氯-5,6-二氰对苯醌，苯,回流，20分钟；d)i.草酰氯，无水二氯甲烷，0℃→室温,12小时；ii.NH 2CH 2CF 3，三乙胺，无水二氯甲烷，0℃→室温,4小时。
而由化合物OA-11制备化合物3参考本发明实施例1中OA-8制备化合物1方法完成。化合物3的MS[M-H] -:calcd for C 31H 40NO 3,474.3；found 474.3。
将化合物3(1.4g,3.04mmol)溶于无水二氯甲烷(5mL)中，0℃下加入草酰氯(3.86g,30.4mmol),搅拌，恢复室温，反应过夜，旋干得淡黄色固体，直接投入下一步反应。上一步所得固体，溶于无水二氯甲烷(5mL)中，0℃下加入三氟乙胺(0.36g,3.64mmol)和三乙胺(0.92g,9.12mmol)，搅拌，恢复室温，反应4小时，加入二氯甲烷(50mL)稀释，有机层用稀盐酸、饱和碳酸氢钠溶液和饱和食盐水洗涤三次，干燥，旋干，快速柱层析得白色固体4(0.9g,53％)。化合物4的MS[M+H] +:calcd for C 33H 44F 3N 2O 2,557.3；found 557.4。
实施例6化合物5的合成如下路线所示：
Figure PCTCN2021076726-appb-000018
合成试剂与条件:a)甲醇钠，***，甲醇，0℃→室温,1小时；b)2,3-二氯-5,6-二氰对苯醌，苯,回流，20分钟；c)乙酸酐，浓硫酸，对甲苯磺酸，室温，2小时。
将化合物OA-8(2.3g,3.94mmol)，溶于甲醇(60mL)和***(125mL)的混合溶液，0℃下加入甲醇钠(6.38g,118mmol)，室温反应45分钟。反应液用二氯甲烷(50mL)稀释，有机层用5％稀盐酸、饱和碳酸氢钠和饱和食盐水各洗涤3次，无水硫酸钠干燥，过滤，滤液浓缩得淡黄色固体OA-17(2.2g,95％)。随后将OA-17(2.2g,3.77mmol)和2,3-二氯-5,6-二氰对苯醌(0.9g,3.77mmol)溶于无水甲苯，回流20分钟后停止，旋干反应液，加二氯甲烷和碳酸氢钠饱和溶液搅洗，取有机层，用碳酸氢钠溶液洗涤三次，无水硫酸钠干燥，过滤，滤液旋干，快速柱层析分离得白色固体OA-18(1.9g，86％)。取OA-18(1g，1.7mmol)溶于乙酸酐(5mL)，加入催化量的浓硫酸和对甲苯磺酸，室温反应2小时，反应结束，反应液倾入冰中，加入碳酸氢钠溶液和二氯甲烷，搅拌充分后，调节pH至近中性，取有机层，干燥，过滤，滤液旋干，快速柱层析得白色固体5(0.71g，67％)。化合物5的MS[M+H] +:calcd for C 40H 50NO 5,624.4；found 624.4；MS[M+NH 4] +:calcd for C 40H 53N 2O 5,641.4；found 641.4。
实施例7化合物6的合成如下路线所示：
Figure PCTCN2021076726-appb-000019
合成试剂与条件:a)甲醇钠，***，甲醇，0℃→室温,1小时；b)2,3-二氯-5,6-二氰对苯醌，苯,回流，20分钟；c)乙酸酐，浓硫酸，对甲苯磺酸，室温，2小时。
由化合物OA-11制备化合物6参考本发明实施例4中OA-8制备化合物5方法完成。化合物6的MS[M-H] -:calcd for C 33H 42NO 5,532.3；found 532.3。
实施例8化合物7的合成如下路线所示：
Figure PCTCN2021076726-appb-000020
合成试剂与条件:a)碘甲烷，碳酸钾，丙酮，室温，4小时；b)乙酸酐，浓硫酸，对甲苯磺酸，室温，2小时。
由化合物OA-20制备OA-21参考本发明实施例2中OA-11制备OA-12的方法完成。而由化合物OA-21制备化合物7参考本发明实施例4中OA-18制备化合物5方法完成。化合物7的MS[M+H] +:calcd for C 34H 46NO 5,548.3；found 548.5；MS[M+NH 4] +:calcd for C 34H 49N 2O 5,565.4；found 565.6。化合物7的氢谱 1HNMR(300M Hz,CDCl 3,25℃,TMS):δ8.01(s,1H),5.70(s,1H),3.63(s,3H),3.26(m,1H),2.19(s,3H),2.00(m,1H),1.71-1.55(m,9H),2.26(m,5H),1.26,1.21,1.24,1.20,1.16,1.00,0.91(s,each 3H)ppm。
实施例9化合物8、9和10的合成如下路线所示：
Figure PCTCN2021076726-appb-000021
合成试剂与条件:a)i.草酰氯，无水二氯甲烷，0℃→室温,12小时；ii.NH 2CH 3，三乙胺，无水二氯甲烷，0℃→室温,4小时；b)i.草酰氯，无水二氯甲烷，0℃→室温,12小时；ii.NH 2CH 2CF 3，三乙胺，无水二氯甲烷，0℃→室温,4小时；c)i.草酰氯，无水二氯甲烷，0℃→室温,12小时；ii.3-氯-4-氟苯胺，三乙胺，无水二氯甲烷，0℃→室温,4小时；d)乙酸酐，浓硫酸，对甲苯磺酸，室温，2小时。
由化合物OA-20制备OA-22、OA-23和OA-24参考本发明实施例3中化合物3制备化合物4的方法完成。而由化合物OA-22、OA-23和OA-24分别制备化合物8、9和10则参考本发明实施例4中OA-18制备化合物5方法完成。
化合物8的MS[M+H] +:calcd for C 34H 47N 2O 4,547.4；found 547.4。
化合物9的MS[M+H] +:calcd for C 35H 46F 3N 2O 4,615.3；found 615.3。
化合物10的MS[M+H] +:calcd for C 39H 47ClFN 2O 4,661.3；found 661.3。
实施例10化合物11的合成如下路线所示：
Figure PCTCN2021076726-appb-000022
合成试剂与条件:a)乙酸酐，浓硫酸，对甲苯磺酸，室温，2小时。
化合物OA-25参考CN104395332B由OA-20制备获得。由化合物OA-25 制备化合物11参考本发明实施例4中OA-18制备化合物5方法完成。化合物11的MS[M+H] +:calcd for C 35H 47F 2N 2O 4,597.4；found 597.4。
实施例11化合物12合成方法如下路线所示：
Figure PCTCN2021076726-appb-000023
合成试剂与条件:a)无水四氢呋喃,二异丙基胺基锂溶液,氯乙酸酐，-78℃，2-4小时。
干燥氮气保护下，将化合物OA-21(1.1g,2.18mmol)溶于无水四氢呋喃(30mL)，冷却至-78℃。将二异丙基胺基锂溶液(2.62mmol)控温缓慢滴入溶液中，-78℃下继续搅拌30至60分钟。随后将氯乙酸酐(0.75g,4.36mmol)的无水四氢呋喃溶液(10mL)控温缓慢滴加至反应溶液中，继续反应1至2小时。淬灭反应，并用二氯甲烷(50mL)萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液浓缩得淡黄色固体，快速柱层析得白色固体12(0.20g,16％)。化合物12的MS[M+H] +:calcd for C 34H 45ClNO 5,582.3；found 582.3。
实施例12化合物13的合成如下路线所示：
Figure PCTCN2021076726-appb-000024
合成试剂与条件:a)无水四氢呋喃,二异丙基胺基锂溶液,异丁酰氯，-78℃，2-4小时。
参考本发明实施例9中OA-21制备化合物12方法，由化合物OA-21微量制备化合物13。化合物13的MS[M+H] +:calcd for C 36H 50NO 5,576.3；found 576.4。
实施例13化合物14的合成如下路线所示：
Figure PCTCN2021076726-appb-000025
合成试剂与条件:a)无水四氢呋喃,二异丙基胺基锂溶液,异丁酰氯，-78℃，2-4小时。
参考本发明实施例9中OA-21制备化合物12方法，由化合物OA-21制备化合物14。化合物14的MS[M+H] +:calcd for C 36H 48NO 6,590.3；found 590.3。
实施例14化合物的抗炎生物学活性筛选
本发明的几种化合物对IFNγ诱导的RAW264.7小鼠巨噬细胞一氧化氮产生的抑制的测定结果。该抗炎生物活性模型参照文献J.Med.Chem.2000,43,4233-4246进行。将以20000个细胞/孔接种于96孔板中的RPMI1640培养基和0.5％FBS中，37℃、5％CO 2下孵育。DMSO或受试化合物，以及重组小鼠IFNγ一起处理48小时。使用Griess试剂与培养基中的一氧化氮反应并在550nM下检测。以此初步筛选化合物的抗炎生物学活性。多数化合物纳摩尔级别即可抑制炎症标记物一氧化氮的生成，其中具备吸电子性质取代基侧链的化合物12，相比RTA402具备更强的活性(见表1)，具有极显著性差异(P＜0.01)。
表1化合物抑制IFNγ诱导RAW264.7小鼠巨噬细胞释放一氧化氮生物活性
化合物 生物活性 a 化合物 生物活性 a
RTA402 ++ 8 ++
1 - 9 +++
2 + 10 ++
3 - 11 +++
4 ++ 12 ++++
5 ++ 13 +
6 ++ 14 +
7 +++ OA-26 b -
备注： a“-”表示IC 50大于100nM；
“+”表示IC 50介于10至100nM；
“++”表示IC 50介于1至10nM；
“+++”表示IC 50介于0.1至1nM；
“++++”表示IC 50小于0.1nM。
bOA-26是已知化合物，按照文献J.Med.Chem.2000,43,4233-4246制备获得。
实施例15酯酶抑制剂对化合物的抗炎生物学活性影响
在实施例14筛选模型基础上，在加入DMSO或受试化合物7或12前，加入已知的细胞酯酶抑制剂(例如1mmol/L Paraoxon)[J.Appl.Toxicol.2005,25,562–67]，随后同样使用Griess试剂与培养基中的一氧化氮反应并在550nM下检测。以此检验酯酶抑制剂与受试化合物的相互作用。结果显示(见表2)，代表化合物7和12所产生的抗炎生物活性完全不依赖酯酶的存在。
表2化合物抑制IFNγ诱导RAW264.7小鼠巨噬细胞释放一氧化氮生物活性
化合物 生物活性 a 化合物 生物活性 a
RTA402+Paraoxon ++ Paraoxon -
7+Paraoxon +++ 12+Paraoxon ++++
备注： a“-”表示IC 50大于100nM；
“+”表示IC 50介于10至100nM；
“++”表示IC 50介于1至10nM；
“+++”表示IC 50介于0.1至1nM；
“++++”表示IC 50小于0.1nM。
实施例16磺酰罗丹明B(SRB)法检测人胰腺癌PANC-1细胞增殖抑制实验
将人胰腺癌PANC-1细胞以3000cell/well密度接种于96孔板中，100μL培养基。将所需药物浓度加入培养基中，振荡混匀，每个药物浓度至少三个复孔。72小时后，弃去培养基，每孔加入60μL的50％的TCA，4℃静置固定60分钟，去离子水洗涤，自然晾干；每孔加60μL的0.4％的SRB，室温避光静置20分钟，1％的醋酸洗涤，自然晾干；每孔加10mM非缓冲Tris碱液150μL，振荡至染料完全溶解；在515nm波长下检测样本吸光度。根据吸光度计算，细胞增殖抑制率(％)＝(1-OD 实验组/OD 溶剂组)*100％。实验结果见下表3所示，多数化合物微摩尔级别即可抑制人人胰腺癌PANC-1细胞增殖，类似地具备吸电子性质取代基侧链可以显著加强化合物的抗肿瘤生物活性，其中化合物11和12，相比RTA402，生物活性具有显著性差异(P＜0.05)。
表3化合物抑制人胰腺癌PANC-1细胞增殖
化合物 生物活性 a 化合物 生物活性 a
RTA402 ++ 8 ++
1 - 9 ++
2 - 10 ++
3 - 11 +++
4 + 12 +++
5 + 13 +
6 + 14 +
7 ++ OA-26 b -
备注： a“-”表示IC 50大于100μM；
“+”表示IC 50介于10至100μM；
“++”表示IC 50介于1至10μM；
“+++”表示IC 50小于1μM。
bOA-26是已知化合物，按照文献J.Med.Chem.2000,43,4233-4246制备获得。
实施例17四甲基偶氮唑蓝(MTT)法检测人正常细胞LO2肝细胞毒实验
为考察代表化合物RTA402，化合物7和12在抗肿瘤和抗炎的IC 50浓度级别对人正常细胞代表LO2肝细胞的毒性考察。将对数生长期细胞以每孔8×10 3个的细胞密度接种于96孔板中(100μL/孔)，置于细胞培养箱中培养。实验组加入不同浓度的受试化合物加入培养基，每组设4个复孔。将细胞放回CO 2培养箱中培养。24小时之后，弃去细胞培养液，加入200μL的含有10％MTT(5mg/mL)的培养基，37℃培养箱中继续培养4小时后弃去含MTT的培养液，然后每孔加入150μL DMSO，震荡8min，使生成的甲臜充分溶解。使用酶标仪在490nm波长处测定其吸光值，计算细胞的抑制率。结果如表4所示，受试化合物在抗肿瘤(1-10μM)浓度级别下对正常细胞抑制活性均很小，具有很好的肿瘤细胞/正常细胞的选择性；而在抗炎活性浓度下(10nM附近)则完全未见抑制活性。总的来说相比RTA402、化合物7和12对人正常细胞的毒性小幅下降或相当，在治疗浓度下未见对人正常细胞的显著抑制作用。
表4化合物对人正常细胞LO2毒性考察
Figure PCTCN2021076726-appb-000026
Figure PCTCN2021076726-appb-000027
备注： a“-”表示抑制率小于等于0％，完全无细胞增殖抑制作用。
综合实施例1的药物与靶点结合信息、分析和预测，以及后续实施例的实验结果，我们发现通过计算机辅助药物设计判断的，C环烯醇的羰基，面向结合口袋外的开放溶剂区，此处(图1，箭头处)空间结构确实具备明确的优化潜力。具体来说，随着C环核心的刚性结构区域逐步扩大(图6，灰色区域代表分子平面刚性结构区域)，化合物的生物活性可以得到进一步提升，例如专利化合物6对比已有化合物OA-26，化合物7和12对比RTA402等在各项活性数据上有非常显著提高，R取代基具备一定吸电子作用时，化合物生物活性可得到加强，同时化合物安全性有所改善或相当。

Claims (10)

  1. 通式(Ⅰ)所示的化合物或其药学上可接受的盐：
    Figure PCTCN2021076726-appb-100001
    其中：
    X＝-H(Y取代基不存在)或O；
    Y＝-COR 1；
    Z＝-COOR 2,-CONR 2R 3,或-NR 2R 3；
    其中R 1分别独立取自氢、或烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素取代形式；
    其中R 2和R 3分别独立取自氢、或烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基、羧基取代形式。
  2. 通式(Ⅱ)所示的化合物或其药学上可接受的盐：
    Figure PCTCN2021076726-appb-100002
    Z＝-COOR 1,-CONR 1R 2,或-NR 1R 2；
    其中R 1和R 2分别独立取自氢、或烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、 烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基、羧基取代形式。
  3. 通式(Ⅲ)所示的化合物或其药学上可接受的盐：
    Figure PCTCN2021076726-appb-100003
    X＝-O；
    Y＝-COR 1；
    Z＝-COOR 1,-CONR 1R 2,或-NR 1R 2；
    其中R 1和R 2分别独立取自氢、或烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基取代形式。
  4. 通式(Ⅳ)所示的化合物或其药学上可接受的盐：
    Figure PCTCN2021076726-appb-100004
    X＝-O；
    Y＝-COR 1；
    Z＝-COOCH 3、-CONHCH 2CF 3或-NHCOCF 2CH 3；
    其中R 1取自烷基(C≤8)、环烷基(C≤8)、螺烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基取代形式。
  5. 通式(Ⅴ)所示的化合物或其药学上可接受的盐：
    Figure PCTCN2021076726-appb-100005
    X＝-O；
    Y＝-COR 1；
    Z＝-COOCH 3、-CONHCH 2CF 3或-NHCOCF 2CH 3；
    其中R 1取自烷基(C≤8)、环烷基(C≤8)、烯基(C≤8)、炔基(C≤8)、芳基(C≤8)、杂芳基(C≤8，其中至少环原子之一是氮、氧或硫)、芳烷基(C≤8)、烷醚基，或者这些基团的任一个的至少一个氢原子的卤素、羟基、氨基取代形式。
  6. 根据权利要求1-5任一所述的化合物或其药学上可接受的盐，其特征在于，所述化合物选自如下所示化学式的化合物：
    Figure PCTCN2021076726-appb-100006
    Figure PCTCN2021076726-appb-100007
    Figure PCTCN2021076726-appb-100008
  7. 药物组合物，其特征在于，包含有效量的权利要求1-6任一所述的化合物或其药学上可接受的盐、以及药学上可接受的载体。
  8. 根据权利要求1-6任一所述的化合物或其药学上可接受的盐的制备方法，其特征在于，选择由如下合成方法构建C环共轭二烯结构：
    Figure PCTCN2021076726-appb-100009
  9. 根据权利要求1-6任一所述的化合物或其药学上可接受的盐、根据权利要求7所述的药物组合物在制备治疗炎性相关疾病药物中的应用。
  10. 根据权利要求1-6任一所述的化合物或其药学上可接受的盐、根据权利要求7所述的药物组合物在制备治疗抗肿瘤活性药物中的应用。
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