WO2013079018A1 - 2-位取代的齐墩果酸衍生物、及其制备方法和应用 - Google Patents

2-位取代的齐墩果酸衍生物、及其制备方法和应用 Download PDF

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WO2013079018A1
WO2013079018A1 PCT/CN2012/085652 CN2012085652W WO2013079018A1 WO 2013079018 A1 WO2013079018 A1 WO 2013079018A1 CN 2012085652 W CN2012085652 W CN 2012085652W WO 2013079018 A1 WO2013079018 A1 WO 2013079018A1
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group
alkyl
oleanolic acid
substituted
acid derivative
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PCT/CN2012/085652
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English (en)
French (fr)
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徐荣臻
荣风光
赖洪喜
谢福文
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杭州本生药业有限公司
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Priority to EP12852723.1A priority Critical patent/EP2786757B1/en
Priority to JP2014543761A priority patent/JP6211527B2/ja
Priority to DK12852723.1T priority patent/DK2786757T3/da
Priority to US14/362,017 priority patent/US9156801B2/en
Priority to NO12852723A priority patent/NO2786757T3/no
Priority to CN201280056729.XA priority patent/CN103945852B/zh
Publication of WO2013079018A1 publication Critical patent/WO2013079018A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/84—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/40—Acylated substituent nitrogen atom
    • 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
    • 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

Definitions

  • the invention belongs to the field of natural medicine and medicinal chemistry and relates to novel oleanolic acid derivatives, in particular to 2-substituted oleanolic acid derivatives, and a method for preparing the same, a composition comprising the same, and a preparation thereof Use in anti-tumor drugs.
  • Background technique
  • Oleanolic acid also known as earthworm, is a pentacyclic triterpenoid. It is extracted from the leaves and fruits of plants such as Hibiscus, Gentianaceae, Rubiaceae, and Polygonaceae, mainly in free form and/or in combination with sugar. Many scientists at home and abroad have done extensive research on pentacyclic triterpenoids. According to reports in the literature, oleanolic acid and its derivatives, analogs have a variety of biological activities, such as anti-inflammatory, anti-tumor, anti-virus, regulate immune function, inhibit platelet aggregation, reduce blood fat and protect liver, protect kidney, anti-HIV Wait. Li Yingxia et al .
  • Oleanolic acid and its derivatives and analogues Malignant tumors are the most serious diseases that threaten human health at present.
  • the development of anticancer drugs is a major research topic in the medical field. Finding high-efficiency and low-toxic drugs from traditional Chinese medicine plants has become a hot spot in anti-cancer research at home and abroad.
  • Oleanolic acid is a very promising anti-tumor drug with low toxicity and abundant Chinese medicine resources.
  • oleanolic acid has an anti-human lung cancer cell proliferation, invasion and induction of apoptosis.
  • the researchers examined the effects of oleanolic acid on the invasion of PGCL3 cells by cell proliferation inhibition assay and soft agar colony formation assay. The results showed that oleanolic acid can decrease the proliferation of PGCL3 cells in a dose-dependent manner, with anti-PGCL3.
  • Human lung cancer cells proliferate and invade, and induce apoptosis of PGCL3 cells. Its anti-invasive mechanism is not to block a certain part of the invasion, but to inhibit the basic links of the invasion.
  • oleanolic acid has a concentration-dependent effect on apoptosis of human lung adenocarcinoma cells.
  • Lin Xiukun et al reported that oleanolic acid and its pharmaceutical preparations have excellent anti-pancreatic cancer effects, which have obvious inhibitory activity against human pancreatic cancer cells in vitro, and have significant effects on tumor cells of tumor cells. Antitumor activity.
  • Lin Xiukun et al also studied the inhibitory effect of oleanolic acid on cervical cancer. The results showed that oleanolic acid and its pharmaceutical preparations have obvious inhibitory activity against human cervical cancer cells in vitro, and have obvious antitumor activity against transplanted tumor cells of tumor cells.
  • Lin Xiukun et al anti-pancreatic cancer effect of oleanolic acid and its pharmaceutical preparations.
  • CN 102151275 A.2011 Lin Xiukun et al, anti-cervical cancer effect of oleanolic acid and its pharmaceutical preparations.
  • CN 102133219 A .2011 Lin Xiukun et al, anti-cervical cancer effect of
  • Oleanolic acid is widely used in clinical practice due to its various pharmacological activities and low toxicity, but the bioavailability of such drugs in humans is low, so the development of high-efficiency and low-toxic oleanolic acid derivatives is good.
  • the present invention aligns the 2-position of the terminal acid with a substitution modification, and introduces a functional group to enhance its biological activity and availability.
  • the method of the present invention and 2-position substituted oleanolic acid have not been reported in the literature. Summary of the invention
  • One of the objects of the present invention is to provide a 2-substituted oleanolic acid derivative of the formula (I) or a pharmaceutically acceptable salt thereof,
  • optionally substituted ( ⁇ -( 18 alkyl, optionally substituted C 2 -C 18 alkenyl or alkynyl, optionally substituted C 3 -C 7 cycloalkyl or cycloalkenyl)
  • the group is also optionally substituted by CC 6 alkyl, or is a CC 6 alkane substituted by a group selected from the C 3 -C 7 cycloalkyl
  • W is selected from the group consisting of a hydroxyl group, an amino group, a dC 6 alkylamino group, a bis(dC 6 alkyl)amino group, a dc 6 alkoxy group, and a cc 6 alkyl fluorenyl group.
  • Another object of the present invention is to provide a process for the preparation of a 2-substituted oleanolic acid derivative of the formula (I) of the present invention:
  • Oleic acid (OA) is oxidized to form a ketone intermediate (OA-1) of oleanolic acid; this intermediate is methylated to form a (keto)methyl ester intermediate of oleanolic acid (OA-2)
  • This intermediate reacts with the Stiller reagent to introduce a methoxyl group to form a diester intermediate of oleanolic acid (OA-3) in the ortho position of the ketone group; this diester intermediate is hydrolyzed to obtain a mono-carboxy intermediate of oleic acid (OA-4); a mono-carboxy intermediate of oleanolic acid (OA-4) can also be obtained by one-pot reaction by introducing a methoxy group and a hydrolysis reaction; The organic amine is reacted by an amide bond to form a 2-substituted oleanolic acid derivative (I); wherein W is the same as defined in the above formula (I).
  • a third object of the present invention is to provide a pharmaceutical composition comprising a compound of the present invention, the pharmaceutical composition comprising at least one compound of the present invention, and optionally a pharmaceutically acceptable excipient.
  • a fourth object of the present invention is to provide a use of a compound of the present invention or a pharmaceutical composition comprising the same for the preparation of a medicament, particularly an antitumor medicament. Accordingly, the invention provides a method of treating a tumor patient comprising administering to a patient in need of treatment a therapeutically effective amount of at least one compound of the invention.
  • the tumor is particularly selected from the group consisting of leukemia, multiple myeloma, lymphoma, liver cancer, gastric cancer, breast cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, melanoma, human cervical cancer, glioma, Nasopharyngeal cancer, laryngeal cancer, esophageal cancer, middle ear tumor, prostate cancer, etc.
  • the invention also relates to compounds of the invention for use in the treatment of tumors. detailed description
  • the present invention relates to a novel 2-oleically substituted oleanolic acid derivative of the formula (I) or a pharmaceutically acceptable salt thereof,
  • optionally substituted ( ⁇ -( 18 alkyl, optionally substituted C 2 -C 18 alkene or alkyne, optionally substituted C 3 -C 7 cycloalkyl or cycloalkenyl)
  • W is selected from the group consisting of a hydroxyl group, an amino group, a dC 6 alkylamino group, a bis(dC 6 alkyl)amino group, a dc 6 alkoxy group, and a cc 6 alkyl fluorenyl group.
  • - dc 6 alkyl heterocyclyl-dC 6 alkyl, ( ⁇ - ⁇ alkylamino- ⁇ - ⁇ alkyl, bis(dC 6 alkyl)-dC 6 alkyl, heteroaryl-dC 6 alkyl , dC 6 alkoxy -dC 6 alkyl, said cycloalkyl, aryl, heterocyclyl and heteroaryl optionally substituted with halogen, hydroxy, dC 6 alkoxy, Ci-C 6 alkyl, amino, dC 6 Substituted by an alkylamino group or a di(dC 6 alkyl)amino group.
  • the heterocyclic group is a saturated heterocyclic group.
  • the heterocyclic ring contains a nitrogen atom It is a ring atom and serves as a connection point.
  • heterocyclic group is pyrrolidine
  • W is hydroxy or c r c 6 alkoxy.
  • the heteroaryl group is a pyridyl group, a furyl group, a thyryl group, a pyrrolyl group, a pyranyl group, an imidazolyl group.
  • the invention particularly preferably exemplifies the following compounds of the formula (I):
  • the 2-substituted oleanolic acid derivative of the present invention has anticancer activity.
  • the anticancer activity of the 2-substituted oleanolic acid derivative of the present invention is improved as compared with the oleanolic acid itself, for example, several times or even tens of times.
  • alkyl refers to a straight or branched chain containing the specified number of carbon atoms. Alkyl.
  • the alkyl group may contain from 1 to 18 carbon atoms, for example from 1 to 12, from 1 to 10,
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, t-butyl, n-pentyl, n-hexyl and n-octadecyl.
  • alkenyl refers to a straight or branched alkenyl group containing the specified number of carbon atoms.
  • the alkenyl group may contain 2 to 18 carbon atoms, for example 2 to 12, 2 to 10, 2 to 8 and 2 to 6
  • alkenyl groups include, but are not limited to, vinyl, allyl, and octadecyl.
  • alkynyl refers to a straight or branched alkynyl group containing the specified number of carbon atoms.
  • the alkynyl group may contain 2 to 18 carbon atoms, for example 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • Examples of alkynyl groups include, but are not limited to, ethynyl and propynyl.
  • C 3 -C 7 cycloalkyl or cycloalkenyl refers to a hydrocarbyl group of a 3-7 membered monocyclic ring system having a saturated or unsaturated ring.
  • the C 3 -C 7 cycloalkyl or cycloalkenyl group may be a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclopropenyl group, and a cyclohexenyl group.
  • aryl refers to a monocarbocyclic aryl group containing 6 to 14 (eg, 6 to 12, 6 to 20) carbon atoms or a fused or non-fused polycarbocyclic aryl group, in a multicarbocyclic ring. In the case, as long as one carbon ring is aromatic.
  • the aryl group also includes an aryl group fused to a heterocyclic group. Examples of the aryl group are a phenyl group, a biphenyl group, a naphthyl group, a 5,6,7,8-tetrahydronaphthyl group, a 2.3-dihydrobenzofuranyl group and the like.
  • heteroaryl refers to an aromatic ring group containing from 1 to 4 heteroatoms (e.g. 1, 2, 3 or 4 heteroatoms) as a ring member. Heteroatoms refer to nitrogen, oxygen or sulfur.
  • the heteroaryl group may be a monocyclic heteroaryl group having 5 to 7 ring atoms or a bicyclic heteroaryl group having 7 to 11 ring atoms. As long as one ring of the bicyclic aryl group is an aromatic heterocyclic ring, the other may be aromatic or non-aromatic, hetero atom-containing or hetero atom-free.
  • heteroaryl group examples include, for example, pyrrolyl group, pyrazolyl group, imidazolyl group, oxazolyl group, pyridyl group, pyrimidinyl group, furyl group, thienyl group, isoxazolyl group, fluorenyl group and the like.
  • heterocyclyl refers to a non-aromatic ring group containing from 1 to 4 heteroatoms (eg 1, 2, 3 or 4 heteroatoms) as ring members. Heteroatoms refer to nitrogen, oxygen or sulfur. Heterocyclic group It is a monocyclic heterocyclic group having 4 to 8 ring atoms (for example, a 4-7 membered ring, a 5-7 membered ring, a 5-6 membered ring), or a bicyclic heterocyclic group having 7 to 11 ring atoms. Heterocyclyl groups can be aromatic or non-aromatic.
  • heterocyclic groups are azetidinyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, dihydrofuranyl, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, tetrahydropyran Base, tetrahydrothiol and the like.
  • halogen means fluoro, chloro, bromo or iodo.
  • alkylamino refers to an amino group substituted by one or two alkyl groups as defined above.
  • alkoxy refers to an alkyl-O- group wherein the alkyl group is as defined above.
  • alkylthio refers to an alkyl-S- group wherein the alkyl group is as defined above.
  • the term "pharmaceutically acceptable salts of the compounds of formula (I)" is an organic acid salt formed from an organic acid which forms a pharmaceutically acceptable anion; these organic acid salts include, but are not limited to, toluene Acid salt, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate, lactic acid Salts and (X-glycerol phosphates; suitable inorganic salts are also formed; these include, but are not limited to, hydrochlorides, sulfates, nitrates, bicarbonates and carbonates, phosphates, hydrobromides , hydroiodide, etc.
  • Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art. For example, it is produced by administering a sufficient amount of a basic compound and a suitable pharmaceutically acceptable anion.
  • the eight chiral centers in the main structure of the 2-position-substituted oleanolic acid derivative of the present invention have a stereochemical structure represented by the formula (I).
  • treating generally refers to obtaining the desired pharmacological and/or physiological effects.
  • the effect # may completely or partially prevent the disease or its symptoms and may be preventive; and / Or it may be therapeutic based on partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • treatment covers any treatment for a patient's condition, including:
  • the 2-position-substituted oleanolic acid derivative of the formula (I) can be oxidized by the oleanolic acid (OA) extracted and extracted from the natural medicinal material to form a ketone intermediate (OA-1) of oleanolic acid.
  • OA oleanolic acid
  • oxidizing agents which can be used for oxidizing a hydroxyl group to a carbonyl group, for example, pyridinium chlorochromate (PCC), permanganic acid, and the like.
  • the ketone intermediate (OA-1) of oleanolic acid is methylated to form the (keto)methyl ester intermediate (OA-2) of oleanolic acid; many methylation reactions can be used for methyl ester formation. reaction.
  • the (keto)methyl ester intermediate (OA-2) of oleanolic acid can be formed by using methyl iodide in the presence of a base in a polar solvent.
  • the keto-acid (keto)methyl ester intermediate (OA-2) reacts with the Stiller reagent to introduce a methoxyl group to form a diester intermediate of oleanolic acid (OA-3) in the ortho position of the ketone group. ).
  • OA-2 keto-acid
  • OA-3 diester intermediate of oleanolic acid
  • a methoxy group can be introduced in the ortho position of the ketone.
  • the use of methoxymagnesium methyl carbonate in a polar solvent, in the ortho position of the ketone can be introduced into the methoxy group in a good yield to form a bisphenol intermediate of oleanolic acid (OA) -3 ).
  • the monocarboxylic intermediate (OA-4) of oleanolic acid is obtained by hydrolyzing a diester intermediate (OA-3) of oleanolic acid with sodium hydroxide in a polar solvent.
  • a single intermediate (OA-4) of oleanolic acid can also be obtained using other organic or inorganic bases.
  • oleanolic acid diester intermediate (OA-3) after introduction of the methoxyhistyl group can be directly hydrolyzed to obtain a monocarboxylic intermediate (OA-4) of oleanolic acid without isolation.
  • the monocarboxylic intermediate (OA-4) of oleanolic acid is reacted with an organic amine via an amide bond to form a 2-substituted oleanolic acid derivative (I).
  • the amidation reaction is generally carried out in the presence of a condensing agent.
  • the condensing agent here may be, but not limited to, an organic condensing agent.
  • the amidation reaction is generally carried out in the presence of a base.
  • the base here may be, but not limited to, an organic base.
  • DIPEA hydrazine, hydrazine-diisopropylethylamine
  • TEA triethylamine
  • pyridine 4-dimethylaminopyridine (DMAP).
  • the amidation reaction is generally carried out in a solvent or in the absence of a solvent.
  • Solvents used include, but are not limited to, organic polar solvents. For example: dichloromethane (DCM), tetrahydrofuran (THF), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like.
  • Protecting groups are those which, once attached to an active moiety (eg, a hydroxyl or amino group), prevent such protecting groups include, but are not limited to, alkyl, benzyl, allyl, trityl (ie, triphenylmethyl)
  • An acyl group for example, benzoyl, acetyl or HOOC-X"-CO-, X" is an alkylene, alkenylene, cycloalkylene or arylene group
  • a silyl group for example, Trimethylsilyl, triethylsilyl and tert-butyldimethylsilyl
  • alkoxycarbonyl aminocarbonyl (for example, dimethylaminocarbonyl, methylethylaminocarbonyl and phenylaminocarbonyl), Alkoxymethyl, benzyloxymethyl and alkyl fluorenylmethyl.
  • amino protecting group examples include, but are not limited to, an alkoxycarbonyl group, an alkanoyl group, an aryloxycarbonyl group, an aryl-substituted alkyl group, and the like. Hydroxy and amino protecting groups have been discussed in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons (1991). Both the 3 ⁇ 4 group and the amino protecting group can be removed by a conventional method after the reaction.
  • BS-OA-105, BS-OA-106, BS-OA-107, BS-OA-108, BS-OA-109 and BS-OA-110 first protect the hydroxyl group at position 28 via the Bn protecting group, and then After the reaction, the protective group is removed by a conventional method to prepare.
  • the invention also provides a pharmaceutical composition comprising a compound of formula (I) of the invention.
  • the present invention provides a pharmaceutical composition comprising at least one of the compounds of formula (I) of the present invention as described above, and optionally a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising at least one of the compounds of formula (I) of the present invention as described above, and optionally a pharmaceutically acceptable excipient.
  • Methods of preparing the pharmaceutical compositions include the incorporation of suitable pharmaceutical excipients, carriers, diluents and the like.
  • the pharmaceutical preparation of the present invention is produced by a known method, including a conventional mixing, dissolving or lyophilizing method.
  • the compounds of the present invention can be formulated into pharmaceutical compositions and administered to a patient in a variety of ways suitable for the chosen mode of administration, e.g., oral, gastrointestinal perfusion, intravenous injection or intramuscular and subcutaneous injection.
  • the compounds of the invention may be administered systemically, e.g., orally, in combination with a pharmaceutically acceptable carrier such as an inert diluent or an edible carrier. They can be enclosed in hard or soft gelatin capsules and can be compressed into tablets.
  • a pharmaceutically acceptable carrier such as an inert diluent or an edible carrier.
  • the active compound may be combined with one or more excipients and in the form of swallowable tablets, buccal tablets, tablets, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the ratio of such compositions and formulations may of course vary and may range from about 1% to about 99% by weight of a given unit dosage form.
  • the amount of active compound is such that an effective dosage level can be obtained.
  • Tablets, lozenges, pills, capsules, and the like may also contain: a binder such as tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch , potato starch, alginic acid, etc.; a lubricant such as magnesium stearate; and a sweetener such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as mint, wintergreen or cherry.
  • a binder such as tragacanth, acacia, corn starch or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrant such as corn starch , potato starch, alginic acid, etc.
  • a lubricant such as magnesium stearate
  • a sweetener such as sucrose, fructose, lactose or aspartame
  • a flavoring agent such as mint, winter
  • any material used to prepare any unit dosage form The material should be pharmaceutically acceptable and substantially non-toxic in the amount applied.
  • the active compound can be incorporated into sustained release formulations and sustained release devices.
  • the active compound can also be administered intravenously or intraperitoneally by infusion or injection.
  • An aqueous solution of the active compound or a salt thereof, optionally a miscible non-toxic surfactant can be prepared.
  • Dispersing agents in glycerin, liquid polyethylene glycols, triacetin and mixtures thereof, and oils can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent microbial growth.
  • the pharmaceutical dosage form suitable for injection or infusion may comprise a sterile aqueous solution or dispersion of the active ingredient (optionally encapsulated in a liposome) comprising a ready-to-use preparation suitable for sterile injectable or infusible solutions or dispersions. Or sterile powder.
  • the final dosage form must be sterile, liquid, and stable under the conditions of manufacture and storage.
  • the liquid carrier can be a solvent or liquid dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof.
  • Appropriate fluidity can be maintained, for example, by liposome formation, by maintaining the desired particle size in the case of dispersing agents, or by the use of surfactants.
  • Microbial action can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).
  • isotonic agents such as sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use of compositions that delay the absorption (e.g., aluminum monostearate and gelatin).
  • Sterile injectable solutions are prepared by combining the required active compound in a suitable solvent with the various other ingredients listed above, followed by filter sterilization.
  • the preferred preparation methods are vacuum drying and lyophilization techniques which result in a powder of the active ingredient plus any additional ingredients present in the previously sterile filtration solution. .
  • Useful solid carriers include 4 divided solids (e.g., talc, clay, ⁇ crystalline cellulose, silica, alumina, etc.).
  • Useful liquid carriers include water, ethanol or ethylene glycol or water-ethanol
  • the I glycol mixture, the compound of the present invention may be dissolved or dispersed in an effective amount, optionally with the aid of a non-toxic surfactant.
  • Adjuvants such as fragrances
  • additional antimicrobial agents can be added to optimize the properties for a given use.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified inorganic materials
  • Thickeners can also be used with liquid carriers to form coatable pastes, gels, ointments. , soap, etc., used directly on the user's skin.
  • the therapeutic requirements of a compound or an active salt or derivative thereof depend not only on the particular salt selected, but also on the mode of administration, the nature of the disease to be treated, and the age and condition of the patient, ultimately depending on the attending physician or clinician decision.
  • unit dosage form is a unit dosage unit containing a physical dispersion unit suitable for administration to humans and other mammalian bodies.
  • the unit dosage form can be a capsule or tablet, or a lot of capsules or tablets.
  • the amount of unit dose of the active ingredient can vary or be adjusted from about 0.1 to about 1000 grams or more.
  • the invention further provides the use of a compound of the invention or a composition comprising the compound for the preparation of a medicament, in particular an antitumor medicament. Accordingly, the invention provides a method of treating a tumor patient comprising administering to a patient in need of treatment a therapeutically effective amount of at least one compound of the invention.
  • the 2-position-substituted oleanolic acid derivative of the present invention or a pharmaceutically acceptable salt thereof can be used, for example, for treating leukemia, multiple myeloma, lymphoma, liver cancer, gastric cancer, breast cancer, cholangiocarcinoma, pancreatic cancer, Lung cancer, colorectal cancer, osteosarcoma, melanoma, human cervical cancer, glioma, nasopharyngeal carcinoma, laryngeal cancer, esophageal cancer, middle ear tumor, prostate cancer and other tumors.
  • Stile's Reagent Magnesium methoxycarbonate, 2M dissolved in hydrazine, hydrazine-dimethylformamide
  • T 3 P tripropyl brick anhydride
  • the compound BS-OA- 105-Bn (400 mg, 0.58 mmol) was dissolved in methanol (20 mL), Pd/C (80 mg, cat.) was added, and the reaction temperature was controlled under hydrogen (1 atm). The reaction mixture was stirred at 30 ° C for 2 hours. After the reaction was completed, the reaction mixture was filtered, and the obtained crude product was purified by preparative thin layer chromatography to afford white solid compound BS-OA- 105 (36.7 mg, 10.6 %).
  • ZS9S80/ZT0ZN3/X3d 8 ⁇ 06.0/ ⁇ OAV BS-OA-109 was prepared by reacting compound OA-8 with hydrazine, hydrazine-dimethylpropanediamine according to the method of BS-OA-105 using the same reagent as above:
  • Example 3 Determination of anti-leukemia activity of 2-substituted oleanolic acid derivatives of the present invention
  • Leukemia cell line leukemia cell line: K562/adr (resistant chronic myeloid leukemia, CML), NB4 (acute promyelocytic leukemia, AML), Kasumi-1 (acute myeloid leukemia M2, AML-M2), Jurkat (acute lymphocytic leukemia, ALL), the above cell lines were all donated to the Institute of Cancer Research, Zhejiang University; H9 (acute lymphocytic leukemia, ALL), purchased from the China Center for Type Culture Collection.
  • Oleanolic acid (OA) standard was purchased from Shifang City, Sichuan province, Huakang Pharmaceutical Raw Material Factory.
  • the 2-position substituted oleanolic acid derivative of the present invention was purchased from Shifang City, Sichuan province, Huakang Pharmaceutical Raw Material Factory.
  • 6000 well-grown leukemia cells were inoculated into the wells of a 96-well cell culture plate.
  • the culture broth was a 1640 cell culture medium containing 10% fetal bovine serum.
  • Different concentrations of 2-substituted oleanolic acid derivatives were added, mixed, and placed in a carbon dioxide (5% CO 2 ) cell incubator 37.
  • C was cultured for 72 hours.
  • the relative number of viable cells was then determined by the MTT method.
  • the cell proliferation inhibition rate was set to 0% in the control group (without compound treatment), and the half-growth inhibition concentration of leukemia cells (72 hours IC 5 o value, g/mL) was calculated according to the relative number of living cells. Inhibition rate of leukemia cells inhibited by IR and 16 h/mL of compound for 72 hours.
  • Table 1 shows that the 2-position-substituted oleanolic acid derivative of the present invention can induce cell death of human chronic myeloid leukemia, acute myeloid leukemia and acute lymphocytic leukemia and inhibit the growth of these leukemia cells, and the oleanolic acid itself Ratio, wherein the 2-position substituted oleanolic acid derivative of the present invention BS-OA-005, BS-OA-012, BS-OA-016, BS-OA-035, BS-OA-042, BS-OA- 078 increased the inhibition rate of K562/adr cell line by more than 3 times, and increased the inhibition rate of Jurkat cell line by more than 10 times; BS-OA-005, BS-OA-012, BS-OA-016, BS-OA-035 to Kasumi -1 cell strain inhibition rate increased more than 5 times; BS-OA-004, BS-OA-005, BS-OA-012, BS-OA-016, BS-OA-035, BS-OA-04
  • BS-OA-052 >16 38.0% >16 -5.0%
  • Example 4 The activity of the 2-position-substituted oleanolic acid derivative of the present invention against human multiple myeloma cells
  • Myeloma cell line RPMI8226 (multiple myeloma), purchased from Shanghai Fuxiang Biotechnology Co., Ltd.
  • Table 2 shows that the 2-substituted oleanolic acid derivatives of the present invention are capable of inducing death of human myeloma cells and inhibiting the growth of these tumor cells.
  • the oleanolic acid derivative of the present invention is 2-position substituted BS-OA-005, BS-OA-012, BS-OA-035, BS-OA-042, BS-OA -078 increased the inhibition rate of RPMI8226 cell line by more than 44 times.
  • Example 5 Determination of anti-human solid tumor action of 2-position-substituted oleanolic acid derivative of the present invention
  • Hep-2 (larynx cancer), A549 (human lung cancer), CaES-17 (esophage cancer cells), PC-3 (prostate cancer), CNE (nasopharyngeal carcinoma cells), SK-OV-3 (ovarian cancer cells), All purchased from China National Culture Collection; RKO (human colon adenocarcinoma), MGC 803 (human gastric cancer cells), MG63 (osteosarcoma), U87 MG (malignant glioma cells), all purchased from Shanghai Fuxiang Biotechnology Co., Ltd.; PANC-1 (pancreatic cancer), Hep G2 (human liver cancer cells), Becap37 (human breast cancer cells), Hela (human cervical cancer cells), were all donated to the Institute of Cancer Research, Zhejiang University.
  • the culture solution was a DMEM high glucose cell culture medium containing 10% fetal calf serum. Place in a carbon dioxide (5% C0 2 ) cell culture incubator 37. C was cultured for 24 hours, and then, different concentrations of 2-substituted oleanolic acid derivatives were added, and after mixing, carbon dioxide (5% CO 2 ) cell incubator 37 was further placed. C was cultured for 72 hours. The relative number of viable cells was then determined by the MTT method.
  • the control group (without compound treatment), the cell proliferation inhibition rate was set to 0%, and the relative number of living cells was calculated to determine the half-length growth inhibitory concentration of leukemia cells (72 hours IC 5 o value, g/mL) and Inhibition rate IR of solid tumor cells treated with 16 g/mL compound for 72 hours.
  • Table 2 shows that the 2-position-substituted oleanolic acid derivatives of the present invention are capable of inducing death of human solid tumor cells and inhibiting the growth of these tumor cells.
  • the 2-position-substituted oleanolic acid derivatives BS-OA-005, BS-OA-016, BS-OA-035 and BS-OA-042 of the present invention are related to A549 and RKO cells.
  • the inhibition rate of the strain was increased by 19 times and 6 times or more; BS-OA-005, BS-OA-012, BS-OA-016 and BS-OA-035 increased the inhibition rate of PANC-1 cell line by more than 11 times; BS- OA-012 and BS-OA-042 increased the inhibition rate of Becap37 cell line by more than 4 times; BS-OA-005, BS-OA-012, BS-OA-035 and BS-OA-042 to MG-63 and CNE cells The inhibition rate of the strain was increased by 3 times and more than 2 times respectively; BS-OA-016, BS-OA-035 and BS-OA-042 increased the inhibition rate of Hela cell strain by more than 4 times; BS-OA-005,
  • BS-OA-012, BS-OA-016, BS-OA-035 and BS-OA-042 increased the inhibition rates of U87 MG, PC-3, MGC 803 and Hep-2 cell lines by 4, 3 and 5 respectively. Times and 9 times or more; BS-OA-012, BS-OA-035 and BS-OA-042 increased the inhibition rate of CaES-17 and SK-OV-3 cell lines by 3 times and 11 times respectively; for Hep G2 cells
  • the strain, oleanolic acid itself did not show significant proliferation inhibition effect on this cell line, and the oleanolic acid derivatives of the present invention all showed good inhibition of cell proliferation, among which BS-OA-005, BS- OA-012, BS-OA-016, BS-OA-035 and BS-OA-042 are particularly obvious, and the inhibition rates are all above 96%.
  • Table 2 Determination of growth inhibitory concentration of 2-position substituted oleanolic acid derivatives on multiple myeloma and human solid tumor cells (72 hours,
  • BS-OA-008 >16 30.0% >16 3.4% >16 -4.6%
  • BS-OA-032 >16 30.3% >16 -6.6% >16 14.5%

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Abstract

本发明属于天然药物及药物化学领域，具体涉及通式I的新型2-位取代的齐墩果酸衍生物或其药学上可接受的盐，制备这些化合物的方法、包含该化合物的药物组合物及其在制备抗肿瘤药物中的用途。

Description

2-位取代的齐教果酸衍生物、 及其制备方法和应用 技术领域
本发明属于天然药物及药物化学领域并涉及新型齐墩果酸衍生 物， 特别是 2-位取代的齐墩果酸衍生物， 及制备这些化合物的方法、 包 含该化合物的组合物及其在制备抗肿瘤药物中的用途。 背景技术
齐墩果酸 (Oleanolic acid, OA), 别名土当归酸， 是一种五环三萜类化 合物。 从木樨科， 龙胆科， 茜草科， 苋科等植物的叶和果实中提取， 主 要以游离形式和（或） 与糖结合形式存在。 国内外许多科学家对五环三 萜类化合物做了广泛的研究。 据文献报道， 齐墩果酸及其衍生物， 类似 物具有多种生物活性， 如抗炎， 抗肿瘤， 抗病毒， 调节免疫功能， 抑制 血小板凝聚， 降血脂保肝， 护肾， 抗艾滋病毒等。 （李英霞等， 一种齐墩 果酸乳糖缀合物及其制备方法和用途 [P] CN 1414012 A. 2003; 张奕华等: 一种齐墩果酸衍生物，其制备方法及用途 [P] CN 102070697 A. 2011; Lin, Z. H.; Zhang, Y.; Zhang, Y. N.; Shen, H.; Hu, L. H.; Jiang, H. L.; Shen, X. Oleanolic acid derivative NPLG441 potently stimulates glucose transport in 3T3-L1 adipocytes via a multi-target mechanism. Biochemical Pharmacology. 2008, 76, 1251-1262; Chen, J.; Gong, Y. C; Liu, J.; Hua, W. Y.; Zhang, L. Y.; Sun, H. B. Synthesis and biological Evaluation of novel pyrazolo[4,3-b]oleanane derivatives as inhibitors of glycogen phosphorylase. Chemistry & Biodiversity. 2008, 5, 1304-1312)。
Figure imgf000002_0001
齐墩果酸 熊果酸 山楂酸
Figure imgf000003_0001
甘草次酸 3-邻甲酸苯甲酰氧基-齐墩果酸 吡唑 -[4,3b]-齐墩果酸
Figure imgf000003_0002
科罗索酸 2-氰基 -3, 12-二氧代齐墩果酸
齐墩果酸及其衍生物和类似物 恶性肿瘤是目前最严重的威胁人类健康的常见病， 抗癌类药物的开 发是现今医学界的重大研究课题。 从中药植物中寻找高效低毒的药物， 已成为国内外抗癌研究的热点。 齐墩果酸毒性小， 中药资源丰富， 是一 类非常有前途的抗肿瘤药物。
近年来， 有文献报道了齐墩果酸抗人肺癌细胞增殖， 侵袭和诱导细 胞凋亡的作用。 研究者通过细胞增殖抑制试验、 软琼脂集落形成试验等 测定观察齐墩果酸对 PGCL3细胞侵袭能力的影响， 结果表明， 齐墩果酸 可降低 PGCL3细胞增殖能力并呈剂量依赖性,具有抗 PGCL3人肺癌细胞 增殖和侵袭作用,并有诱导 PGCL3细胞凋亡的作用。其抗侵袭机理不是对 侵袭的某一环节的阻断,而是对侵袭各个基本环节都有抑制作用。 此外， 还有研究者探索了齐墩果酸对 A549细胞的作用及其可能的机制，研究结 果表明， 齐墩果酸具有浓度依赖性诱导人肺腺癌细胞凋亡的作用。 （张东 方等,齐墩果酸抗人肺癌细胞增殖、 侵袭和诱导细胞凋亡的研究.肿瘤防 治研究， 2003, 30(3) , 081-381; 卫小红等,齐墩果酸诱导人肺腺癌细胞 A549凋亡及其与细胞内钙离子的关系.同济大学学报（医学版）， 2009, 30(5), 19-23)。 有文献报道了研究者通过细胞增殖抑制试验， MTT法检测肿瘤细胞 的活性， 研究了齐墩果酸对卵巢癌细胞株 IGROV1和人乳腺癌细胞株 MDA-MB-231的抑制作用。 结果显示， 齐墩果酸可降低 IGROV1和 MDA-MB-231的细胞增殖能力并呈剂量依赖性。 表明齐墩果酸对这两株 恶性肿瘤细胞具有抑制活性。（吴林蔚等,齐墩果酸对卵巢癌细胞 IGROV1 和乳腺癌细胞 MDA-MB-231生长的抑制作用. 应用与环境生物学报, 2010, 16(2), 202-204)„
近期， 林秀坤等人报道了齐墩果酸及其药物制剂具有很好的抗胰腺 癌作用， 表现在对体外人胰腺癌细胞具有明显的抑制活性， 并对此肿瘤 细胞的棵鼠移植瘤具有显著的抑瘤活性。 此外， 林秀坤等人还研究了齐 墩果酸对宫颈癌的抑制作用。 研究结果表明， 齐墩果酸及其药物制剂对 体外人宫颈癌细胞具有明显的抑制活性， 并对此肿瘤细胞的棵鼠移植瘤 具有明显的抑瘤活性。 （林秀坤等， 齐墩果酸的抗胰腺癌作用及其药物制 剂. [P] CN 102151275 A.2011 ; 林秀坤等， 齐墩果酸的抗宫颈癌作用及其 药物制剂. [P] CN 102133219 A.2011)。
齐墩果酸由于其多种药理学活性且毒性较低被广泛应用于临床上， 但是该类药物在人体内的生物利用度较低， 因此开发高效低毒的齐墩果 酸衍生物具有良好的前景。 本发明对齐墩果酸的 2-位进行取代修饰， 引 入功能性基团， 以提高其生物活性及利用度。 本发明的方法以及 2-位取 代的齐墩果酸尚未见文献报道。 发明内容
本发明的目的之一是提供通式 (I)的 2-位取代的齐墩果酸衍生物或 其药学上可接受的盐，
Figure imgf000005_0001
式中 选自 H、 任选取代的（^-( 18的烷基、 任选取代的 C2-C18烯 基或炔基、 任选取代的 C3-C7环烷基或环烯基、 任选取代的芳基、 任选 取代的杂环基或杂芳基， 所述任选取代的取代基选自 |¾素、硝基、氰基、 氨基、羟基、巯基、 、 d-C6烷基氨基、二（d-C6烷基）氨基、 Ci-C6 烷氧基、 d-C6烷硫基， 所述 c3-c7环烷基或环烯基、 芳基、 杂环基或杂 芳基还任选地被 C C6烷基取代，或者 为由选自所述 C3-C7环烷基或 环烯基、 芳基、 杂环基或杂芳基的基团取代的 C C6烷基； 以及
式中 W选自羟基、 氨基、 d-C6烷基氨基、 二（d-C6烷基）氨基、 d-c6烷氧基和 c c6烷石克基。
本发明的目的之二是提供制备本发明通式（I ) 的 2-位取代的齐墩 果酸衍生物的方法：
Figure imgf000005_0002
Oleanolic Acid OA-1 OA-2
Figure imgf000005_0003
齐墩果酸（OA ) 经氧化， 生成齐墩果酸的酮中间体（OA-1 ) ； 此 中间体经甲酯化， 生成齐墩果酸的（酮）甲酯中间体（OA-2 ) ； 此中间体 与斯迪勒试剂反应， 在酮叛基的邻位引入甲氧叛基生成齐墩果酸的双酯 中间体（OA-3 ) ; 此双酯中间体经水解， 得齐墩果酸的单羧基中间体 ( OA-4 )； 引入甲氧叛基和水解反应也可一锅反应完成得齐墩果酸的单 羧基中间体（ OA-4 )； 此单羧基中间体与有机胺经酰胺键形成反应， 生 成 2-位取代的齐墩果酸衍生物（I ) ； 式中 和 W与上文通式（I ) 中 的定义相同。
本发明的目的之三是提供包含本发明化合物的药物组合物， 所述 药物组合物包括至少一种本发明化合物， 和任选的药学上可以接受的 赋形剂。
本发明的目的之四是提供本发明化合物或包含该化合物的药物组 合物在制备药物、 特别是抗肿瘤药物中的用途。 相应地， 本发明提供 一种治疗肿瘤患者的方法， 包括给予需要治疗的患者治疗有效量的至 少一种本发明的化合物。 所述肿瘤特别选自白血病、 多发性骨髓瘤、 淋巴瘤、 肝癌、 胃癌、 乳腺癌、 胆管细胞癌、 胰腺癌、 肺癌、 大肠癌、 骨肉瘤、 黑色素瘤、 人宫颈癌、 神经胶质瘤、 鼻咽癌、 喉癌、 食管癌、 中耳肿瘤、 前列腺癌等。
本发明还涉及用于治疗肿瘤的本发明的化合物。 具体实施方式
本发明涉及通式 (I)的 2-位取代的新型齐墩果酸衍生物或其药学上 可接受的盐，
Figure imgf000007_0001
式中 选自 H、 任选取代的（^-( 18的烷基、 任选取代的 C2-C18烯 烃基或炔烃基、任选取代的 C3-C7环烷基或环烯烃基、任选取代的芳基、 任选取代的杂环基或杂芳基， 所述任选取代的取代基选自鹵素、 硝基、 tt、 氨基、 羟基、 巯基、 、 d-C6烷基氨基、 二（d-C6烷基）氨 基、 d-C6烷氧基、 d-C6烷硫基， 所述 C3-C7环烷基或环烯烃基、 芳基、 杂环基或杂芳基还任选地被 C C6烷基取代， 或者 为由选自所述 C3-C7环烷基或环烯烃基、 芳基、 杂环基或杂芳基的基团取代的 d-C6 烷基； 以及
式中 W选自羟基、 氨基、 d-C6烷基氨基、 二（d-C6烷基）氨基、 d-c6烷氧基和 c c6烷石克基。
根据本发明一个优选的实施方式， 其中 为 c4-c8烷基、 环烷基
- d-c6烷基、杂环基 - d-C6烷基、（^-^烷基氨基- ^-^烷基、二（d-C6 烷基） - d-C6烷基、 杂芳基- d-C6烷基、 d-C6烷氧基 -d-C6烷基， 所 述环烷基、 杂环基和杂芳基任选地被鹵素、 羟基、 d-C6烷氧基、 Ci-C6 烷基、 氨基、 d-C6烷基氨基、 二（d-C6烷基）氨基所取代。
根据本发明另一个优选的实施方式， 其中 为 c4-c6烷基、 环烷 基- d-C4烷基、杂环基- d-C4烷基、 d-C6烷基氨基- d-C4烷基、二（ Ci-C6 烷基） - d-c4烷基、 杂芳基 - d-C4烷基、 d-C6烷氧基 -d-C4烷基， 所述 环烷基、 杂环基和杂芳基任选地被鹵素、 羟基、 d-C6烷氧基、 C C6烷 基、 氨基、 d-C6烷基氨基、 二（d-C6烷基）氨基所取代。
根据本发明一个优选的实施方式， 其中所述杂环基为饱和杂环基。 根据本发明另一个优选的实施方式， 其中所述杂环含有氮原子作 为环原子并作为连接点。
根据本发明一个特别优选的实施方式， 其中所述杂环基为吡咯烷
-1-基、 哌啶子基、 哌嗪 -1 -基、 4-甲基哌嗪小基、 吗啉代基、 噁唑烷基、 咪唑烷基、 异噁唑烷基。
根据本发明另一个特别优选的实施方式， 其中 W为羟基或 crc6 烷氧基。
根据本发明一个优选的实施方式， 其中所述杂芳基为吡啶基、 呋 喃基、 噻喻基、 吡咯基、 吡喃基、 咪唑基。
本发明的部分优选的 2-位取代的新型齐墩果酸衍生物如下所示。这 些实施例举只对本发明做进一步说明， 并不对本发明的范围构成任何限 制。
Figure imgf000008_0001
BS-OA-016 BS-OA-017 BS-OA-021
Figure imgf000009_0001
v9sgv〇sg s 88---
Figure imgf000010_0001
v〇sgv〇sg ozo 890----
Figure imgf000011_0001
BS-OA-075 BS-OA-078 BS-OA-082
Figure imgf000011_0002
BS-OA-105 BS-OA-106 BS-OA-107
Figure imgf000011_0003
BS-OA-108 BS-OA-109 BS-OA-110 上面所列化合物的部分数据如下表所示:
Figure imgf000011_0004
3/: O is9s8l£6/-oiAV
Figure imgf000012_0001
BS-OA-068 C35H55N04 553.8 白色 固体 15.6
BS-OA-070 C37H59N04 581.9 白色 固体 16
BS-OA-075 C36H57N05 583.84 白色 固体 14.9
BS-OA-078 C40H65N3O4 652 白色 固体 16.9
BS-OA-082 C37H53N05 591.82 黄色 固体 16.4
BS-OA-085 C38H55N05 605.9 白色 固体 13.5
BS-OA-086 C39H55N05 617.86 白色 固体 13.1
BS-OA-088 C38H55N04S 621.9 白色 油状 13.7
BS-OA-105 C37H58N2O4 594.87 白色 固体 3.63
BS-OA-106 C37H60N2O4 596.88 白色 固体 2.89
BS-OA-107 C38H60N2O4 608.89 白色 固体 2.23
BS-OA-108 C38H62N2O4 610.91 白色 固体 2.93
BS-OA-109 C36H58N204 582.86 白色 固体 2.24
BS-OA-110 C39H63N3O4 637.94 白色 固体 2.14
在另一种实施方式中 本发明特别优选如下的通式（I )化合物:
Figure imgf000013_0001
BS-OA-005
1-氧代 -2-(N- (吡咯烷基乙基))胺叛基齐墩果酸甲酯
Figure imgf000013_0002
BS-OA-012
1-氧代 -2-(N- (二乙基胺乙基))胺叛基齐墩果酸甲酯
Figure imgf000014_0001
BS-OA-035
-氧代 -2-(N- (二乙基胺丙基))胺叛基齐墩果酸甲酯
Figure imgf000015_0001
Figure imgf000015_0002
BS-OA-042
1 -氧代 -2-(N- (二甲基胺丙基))胺叛基齐墩果酸甲酯
Figure imgf000015_0003
BS-OA-078
1-氧代 -2-(N-(N-甲基哌嗪基丙基))胺羰基齐墩果酸甲酯 本发明的 2-位取代的齐墩果酸衍生物具有抗癌活性。 与齐墩果酸本 身比较， 本发明的 2-位取代的齐墩果酸衍生物的抗癌活性提高， 例如提 高数倍甚至数十倍。
如本文所使用， 术语"烷基"是指含有指定碳原子数的直链或支链 的烷基。 所述烷基可以包含 1- 18个碳原子， 例如 1- 12个、 1 - 10个、
1- 8个、 1-6个、 1 -5个、 1-4个或 1-3个碳原子。 烷基的例子包括但不 限于甲基、 乙基、 正丙基、 异丙基、 叔丁基、 正戊基、 正己基和正十 八烷基。
术语"烯基"是指含有指定碳原子数的直链或支链的烯基。 所述烯 基可以包含 2- 18个碳原子， 例如 2- 12个、 2- 10个、 2-8个、 2-6个、
2- 5个、 2-4个或 2-3个碳原子。 烯基的例子包括但不限于乙烯基、 烯 丙基、 和十八烯基。
术语"炔基"是指含有指定碳原子数的直链或支链的炔基。 所述炔 基可以包含 2- 18个碳原子， 例如 2- 12个、 2- 10个、 2-8个、 2-6个、 2-5个、 2-4个或 2-3个碳原子。 炔基的例子包括但不限于乙炔基和丙 炔基。
术语" C3-C7环烷基或环烯基"是指具有饱和或不饱和环的 3-7 元 单环系统的烃基。 C3-C7环烷基或环烯基可以为环丙基、 环丁基、 环戊 基、 环己基、 环庚基、 环丙烯基和环己烯基。
术语"芳基"是指含有 6- 14个 （例如 6- 12个、 6-20个）碳原子的 单碳环芳香基或稠合或非稠合的多碳环芳香基， 在多碳环的情况下， 只要一个碳环是芳香的即可。 芳基也包括与杂环基稠合的芳基。 所述 芳基的例子有苯基、 联苯基、 萘基、 5,6,7,8-四氢萘基、 2.3-二氢苯并 呋喃基等。
术语"杂芳基 "是指在环中含有 1 -4个杂原子 （例如 1、 2、 3或 4 个杂原子）作为环成员的芳香环基团。 杂原子是指氮、 氧或硫。 杂芳 基可以是具有 5-7个环原子的单环杂芳基， 或者具有 7-11个环原子的双 环杂芳基。 所述双环芳基中只要一个环是芳香杂环即可， 另一个可以是 芳香的或非芳香的、 含杂原子的或不含杂原子的。 杂芳基的例子有例如 吡咯基、 吡唑基、 咪唑基、 噁唑基、 吡啶基、 嘧啶基、 呋喃基、 噻吩基、 异噁唑基、 吲哚基等。
术语"杂环基 "是指含有 1-4个杂原子 （例如 1、 2、 3或 4个杂原 子）作为环成员的非芳香环基团。 杂原子是指氮、 氧或硫。 杂环基可 以是具有 4-8个环原子的单环杂环基（例如 4-7元环、 5-7元环、 5-6 元环） ， 或者具有 7- 11个环原子的双环杂环基。 杂环基可以是芳香 或非芳香的。 杂环基的例子有氮杂环丁基、 吡咯烷基、 吡咯啉基、 四 氢呋喃基、 二氢呋喃基、 哌嗪基、 哌啶基、 吗啉基、 硫代吗啉基、 四 氢吡喃基、 四氢噻喻基等。
术语"鹵素"是指氟、 氯、 溴或碘。
术语"烷基氨基"是指被一个或两个如上所定义的烷基取代的氨 基。
术语"烷氧基 "是指烷基 -0-基团， 其中所述烷基如上所定义。
术语"烷硫基 "是指烷基 -S-基团， 其中所述烷基如上所定义。
如本文所使用， 术语"式 (I)化合物的药学上可以接受的盐"的例子是 由形成药学上可以接受的阴离子的有机酸形成的有机酸盐； 这些有机酸 盐包括但不限于甲苯磺酸盐、 甲磺酸盐、 苹果酸盐、 醋酸盐、柠檬酸盐、 丙二酸盐、 酒石酸盐、 琥珀酸盐、 苯甲酸盐、 抗坏血酸盐、 α-酮戊二酸 盐、 乳酸盐和（X-甘油磷酸盐； 也可形成合适的无机盐； 这些无机酸盐包 括但不限于盐酸盐、 硫酸盐、 硝酸盐、 碳酸氢盐和碳酸盐、 磷酸盐、 氢 溴酸盐、 氢碘酸盐等。
药学上可以接受的盐可使用本领域熟知的标准程序获得。 例如， 通 过将足量的碱性化合物和提供药学上可以接受的阴离子的合适的^ 应生成。
本发明的 2-位取代的齐墩果酸衍生物主结构中的八个手性中心具 有式 （I ) 结构式所显示的立体化学结构。 本文使用的立体化学的定 义和约定一般遵循 MCGRAW-HILL DICTIONARY OF CHEMICAL TERMS (S. P. Parker, Ed., McGraw-Hill Book Company, New York, 1984); ELIEL, E. 和 WILEN, S.， STEREOCHEMISTRY OF ORGANIC COMPOUNDS (John Wiley & Sons, Inc., New York, 1994)„ 许多有机化合物以光学活性形式存在， 即 它们具有旋转平面偏光的平面的能力。
本文使用的术语"治疗"一般是指获得需要的药理和 /或生理效应。 该效应 #居完全或部分地预防疾病或其症状， 可以是预防性的； 和 / 或根据部分或完全稳定或治愈疾病和 /或由于疾病产生的副作用，可以 是治疗性的。本文使用的"治疗"涵盖了对患者疾病的任何治疗， 包括：
( a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或 症状； （b)抑制疾病的症状， 即阻止其发展； 或（c)緩解疾病的症状，
Figure imgf000018_0001
OA-3 OA -4
式 (I)的 2-位取代的齐墩果酸衍生物可由天然药材提取分离的齐墩果 酸（OA ) 经氧化， 生成齐墩果酸的酮中间体（OA-1 ) 。 氧化羟基至羰 基有很多氧化剂可以使用， 例如： 氯铬酸吡啶盐（PCC )， 高锰酸 4甲等。
齐墩果酸的酮中间体（OA-1 )经甲酯化， 生成齐墩果酸的（酮）甲酯 中间体（OA-2 ) ; 有很多甲基化反应可以用于甲酯的形成反应。 例如： 用碘甲烷在碱的存在下在极性溶剂中， 即可形成齐墩果酸的（酮）甲酯中 间体 ( OA-2 ) 。
齐墩果酸的（酮）甲酯中间体（OA-2 ) 与斯迪勒试剂反应， 在酮叛基 的邻位引入甲氧叛基生成齐墩果酸的双酯中间体（OA-3 )。 有很多试剂 可在酮叛基的邻位引入甲氧叛基。 例如： 在极性溶剂中使用甲氧基镁甲 基碳酸酯， 可在酮叛基的邻位， 以较好的收率引入甲氧叛基， 生成齐墩 果酸的双酯中间体（OA-3 ) 。
用氢氧化钠与齐墩果酸的双酯中间体（OA-3 )， 在极性溶剂中经水 解， 得到齐墩果酸的单羧基中间体（ OA-4 )。 使用其他的有机碱或无机 碱， 也可得到齐墩果酸的单 中间体（OA-4 ) 。
引入甲氧叛基后的齐墩果酸双酯中间体（OA-3 )可以不用分离， 直 接水解得到齐墩果酸的单羧基中间体（OA-4 ) 。
齐墩果酸的单羧基中间体（OA-4 )与有机胺经酰胺键形成反应， 生 成 2-位取代的齐墩果酸衍生物（ I ) 。
用于酰胺化反应的有机胺全部可以在市场上购买获得。
酰胺化反应一般在有缩合剂存在下进行。这里缩合剂可以是但不限 于有机缩合剂。 例如： 2-(7-偶氮苯并三氮唑) -Ν,Ν,Ν',Ν'-四甲基脲六氟磚 酸酯（HATU ) , 苯并三氮唑 -Ν,Ν,Ν',Ν'-四甲基脲六氟硼酸盐（ HBTU ) , 苯并三氮唑 -1 -基氧基三 (二甲基氨基)磷鑰六氟磚酸盐 （ΒΟΡ ) ， 苯并三 氮唑 -Ν,Ν,Ν',Ν'-四甲基脲六氟硼酸盐（TBTU ) ， 三丙基磚酸酐（Τ3Ρ ) 。
酰胺化反应一般在有碱存在下进行。 这里碱可以是但不限于有机 碱。 例如： Ν,Ν-二异丙基乙基胺（DIPEA )， 三乙基胺 ( TEA )， 吡啶， 4-二甲胺基吡啶（DMAP ) 。
酰胺化反应一般在溶剂中进行， 也可在无溶剂存在下进行。 使用的 溶剂包括但不限于有机极性溶剂。 例如： 二氯甲烷（DCM ) ， 四氢呋喃 ( THF ) ， N， N-二甲基甲酰胺（DMF ) ， 二甲亚砜（ DMSO ) 等。
常规的化学转换可用于实施本发明。 本领域的技术人员可以决定 用于这些化学转换的适当的化学试剂、 溶剂、 保护基和反应条件。 相 关信息描述于 R. Larock, Comprehensive Organic Transformations , VCH Publishers ( 1989); T. W. Greene and P.G.M. Wuts , Protective Groups in Organic Synthesis , 3rd Ed.， John Wiley and Sons ( 1999); L. Fieser and M. Fieser , Fieser and Fieser's Reagents for Organic Synthesis ， John Wiley and Sons( 1994)； L. A. Paquetteeditor, Encyclopedia of Reagents for Organic Synthesis， John Wiley and Sons(1995)及其后来的版本。
保护基指那些一旦连接活性部分 (例如， 羟基或氨基)， 防止这些 基保护基的例子包括但不限于，烷基、苯甲基、烯丙基、三苯甲基 (即， 三苯基甲基）、 酰基 (例如， 苯甲酰基、 乙酰基或 HOOC-X"-CO-， X" 为亚烷基、 亚链烯基、 亚环烷基或亚芳基)、 甲硅烷基 (例如， 三甲基 硅烷基、 三乙基硅烷基和叔丁基二甲基硅烷基)、 烷氧基羰基、 氨基 羰基 (例如， 二甲基氨基羰基、 甲基乙氨基羰基和苯基氨基羰基)、 烷 氧甲基、苯甲氧甲基和烷基巯甲基。氨基保护基的例子包括但不限于， 烷氧基羰基、 烷酰基、 芳氧基羰基、 芳基取代的烷基等。 羟基和氨基 保护基已在 T.W. Greene and P.G. M. Wuts, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons( 1991)中讨论。 ¾ 基和氨基保护基都可在反应后通过常规的方法去除。
具体而言，优选的本发明式 (I)化合物中， BS-OA-067, BS-OA-003,
BS-OA-004, BS-OA-005. BS-OA-008. BS-OA-01 BS-OA-012, BS-OA-016. BS-OA-017. BS-OA-02 BS-OA-024, BS-OA-027. BS-OA-028, BS-OA-03 BS-OA-032, BS-OA-033, BS-OA-034, BS-OA-035, BS-OA-037, BS-OA-038, BS-OA-042, BS-OA-044, BS-OA-046, BS-OA-048, BS-OA-052, BS-OA-053, BS-OA-054, BS-OA-058, BS-OA-059, BS-OA-062, BS-OA-064, BS-OA-068, BS-OA-070, BS-OA-075, BS-OA-078, BS-OA-082, BS-OA-085, BS-OA-086和 BS-OA-088直接按照以上反应流程来制备。
BS-OA-105, BS-OA-106, BS-OA-107, BS-OA-108, BS-OA-109 和 BS-OA-110则首先经 Bn保护基保护 28位上的羟基， 然后在反应 后通过常规的方法去除保护基来制备。
本发明还提供了包含本发明式 (I)化合物的药物组合物。
本发明提供了这样的药物组合物， 其中包含至少一种如上所述的 本发明的式 (I)化合物， 和任选的药学上可以接受的赋形剂。 制备各种含有一定量的活性成分的药物组合物的方法是已知的，
REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, E.W., ed" Mack Publishing Company, 19th ed. (1995)所述。制备所述药物组合物的方法 包括掺入适当的药学赋形剂、 载体、 稀释剂等。
以已知的方法制造本发明的药物制剂， 包括常规的混合、 溶解或 冻干方法。
本发明的化合物可以制成药物组合物， 并向患者以适于选定的施用 方式的各种途径施用， 例如： 口服， 肠胃灌注， 静脉内注射或肌内与皮 下注射。
因此， 本发明的化合物结合药学上可以接受的载体（如惰性稀释剂 或可食用的载体）可以全身施用， 例如， 口服。 它们可以封闭在硬或软 壳的明胶胶嚢中， 可以压为片剂。 对于口服治疗施用， 活性化合物可以 结合一种或多种赋形剂， 并以可吞咽的片剂、 颊含片剂、 含片、 胶嚢剂、 酏剂、 悬浮剂、 糖浆、 圆片等的形式使用。 这种组合物和制剂应该包含 至少 0.1%的活性化合物。 这种组合物和制剂的比例当然可以变化， 可以 占给定的单位剂型重量的大约 1%至大约 99%。 在这种治疗有用的组合 物中， 活性化合物的量使得能够获得有效剂量水平。
片剂、 含片、 丸剂、 胶嚢剂等也可以包含： 粘合剂， 如黄蓍胶、 阿 拉伯胶、 玉米淀粉或明胶； 赋形剂， 如磷酸氢二钙； 崩解剂， 如玉米淀 粉、 马铃薯淀粉、 藻酸等； 润滑剂， 如硬脂酸镁； 和甜味剂， 如蔗糖、 果糖、 乳糖或阿司帕坦； 或调味剂， 如薄荷、 冬青油或樱桃香味。 当单 位剂型是胶嚢时， 除了上面类型的材料， 它还可以包含液体载体， 如植 物油或聚乙二醇。 各种其他材料可以存在， 作为包衣， 或以其他方式改 变固体单位剂型的物理形式。 例如， 片剂、 丸剂或胶嚢剂可以用明胶、 蜡、 虫胶或糖等包衣。 糖浆或酏剂可以包含活性化合物， 蔗糖或果糖作 为甜味剂， 对羟苯甲酸甲酯或对羟苯甲酸丙酯作为防腐剂， 染料和调味 剂 （如樱桃香料或桔子香料） 。 当然， 用于制备任何单位剂型的任何材 料应该是药学上可以接受的且以应用的量基本上无毒。 此外， 活性化合 物可以掺入緩释制剂和緩释装置中。
活性化合物也可以通过输注或注射到静脉内或腹膜内施用。 可以制 备活性化合物或其盐的水溶液， 任选的可混和的无毒的表面活性剂。 也 可以制备在甘油、 液体聚乙二醇、 甘油三乙酸酯及其混合物以及油中的 分散剂。 在普通的储存和使用条件下， 这些制剂包含防腐剂以防止微生 物生长。
适于注射或输注的药物剂型可以包括包含适于无菌的可注射或可 输注的溶液或分散剂的即时制剂的活性成分（任选封装在脂质体中） 的 无菌水溶液或分散剂或无菌粉末。 在所有情况下， 最终的剂型在生产和 储存条件下必须是无菌的、 液体的和稳定的。 液体载体可以是溶剂或液 体分散介质， 包括， 例如水、 乙醇、 多元醇（例如， 甘油、 丙二醇、 液 体聚乙二醇等） 、 植物油、 无毒的甘油酯及其合适的混合物。 可以维持 合适的流动性， 例如， 通过脂质体的形成， 通过在分散剂的情况下维持 所需的粒子大小， 或通过表面活性剂的使用。 可以通过各种抗细菌剂和 抗真菌剂 （如对羟苯甲酸酯、 氯丁醇、 苯酚、 山梨酸、 硫柳汞等）产生 预防微生物的作用。 在许多情况下， 优选包括等渗剂， 如糖、 緩冲剂或 氯化钠。 通过使用延緩吸收剂的组合物（例如， 单硬脂酸铝和明胶）可 以产生可注射的组合物的延长吸收。
通过将合适的溶剂中的需要量的活性化合物与需要的上面列举的 各种其他成分结合， 然后进行过滤灭菌， 制备无菌可注射溶液。 在用于 制备无菌注射溶液的无菌粉末的情况下， 优选的制备方法是真空干燥和 冷冻干燥技术， 这会产生活性成分加上任何另外需要的以前无菌过滤溶 液中存在的成分的粉末。
有用的固体载体包括 4分碎的固体（如滑石、 粘土、 ^敖晶纤维素、 二 氧化硅、 氧化铝等）。 有用的液体载体包括水、 乙醇或乙二醇或水 -乙醇
I乙二醇混合物，本发明的化合物可以任选在无毒的表面活性剂的帮助下 以有效含量溶解或分散在其中。 可以加入佐剂 （如香味）和另外的抗微 生物剂来优化对于给定用途的性质。 增稠剂 （如合成的聚合物、 脂肪酸、 脂肪酸盐和酯、 脂肪醇、 改性 纤维素或改性无机材料）也可和液体载体用于形成可涂覆的糊剂、凝胶、 软膏、 肥皂等， 直接用于使用者的皮肤上。
化合物或其活性盐或衍生物的治疗需要量， 不仅取决于选择的特定 的盐， 而且取决于施药方式、待治疗的疾病的本质和患者的年龄和状态， 最终取决于在场医师或临床医生的决定。
上述制剂可以以单位剂型存在， 该单位剂型是含有单位剂量的物 理分散单元， 适于向人体和其它哺乳动物体给药。 单位剂型可以是胶 嚢或片剂， 或是很多胶嚢或片剂。 根据所涉及的具体治疗， 活性成分 的单位剂量的量可以在大约 0.1到大约 1000亳克或更多之间进行变化 或调整。
本发明还提供本发明的化合物或包含该化合物的组合物在制备药 物、 特别是抗肿瘤药物中的用途。 相应地， 本发明提供一种治疗肿瘤 患者的方法， 包括给予需要治疗的患者治疗有效量的至少一种本发明 的化合物。 本发明的 2-位取代的齐墩果酸衍生物或其药学上可接受的 盐例如可用于治疗白血病、 多发性骨髓瘤、 淋巴瘤、 肝癌、 胃癌、 乳 腺癌、 胆管细胞癌、 胰腺癌、 肺癌、 大肠癌、 骨肉瘤、 黑色素瘤、 人 宫颈癌、 神经胶质瘤、 鼻咽癌、 喉癌、 食管癌、 中耳肿瘤、 前列腺癌 等肿瘤。
在下列实施例中， 将更加具体地解释本发明。 但应理解， 下列实 施例旨在说明本发明而不对本发明的范围构成任何限制。 的合成方法制取。 实施例 1: 化合物（BS-OA-067)的合成
Figure imgf000024_0001
Oleanolic acid OA-1 式中， PCC: 氯铬酸吡啶盐
向二氯甲烷（1500 mL)中加入齐墩果酸（147 g, 0.32 mol) , 氯铬酸 吡啶盐（81.9 g, 0.38 mol)， 反应液室温下搅拌过夜。 反应结束后， 将反 应液过滤， 滤液浓缩后得到的粗产品经硅胶柱分离纯化后得到白色固 体化合物 OA-1 (123 g, 84.66%)。
Figure imgf000024_0002
OA-1 OA-2 向 Ν,Ν-二甲基甲酰胺 (800 mL)中加入化合物 OA-1 (63 g, 0.14 mol), 碳酸钾 (23.18 g, 0.168 mol) , 随后一次性加入碘甲烷 (23.86 g, 0.168 mol), 反应液室温下搅拌 5小时。 反应结束后， 将反应液浓缩， 向浓缩得到的粗产物中加入水， 用二氯甲烷萃取， 有机相用无水硫酸 钠干燥， 有机相浓缩得到的粗产物经硅胶柱分离纯化后得到白色固体 化合物 OA-2 (41.5 g, 63.34%)。 COOCH3 stile's Reagent
Figure imgf000025_0001
OA-2 OA-4
式中， Stile's Reagent (斯迪勒试剂）： 甲氧基碳酸镁， 2M溶于 Ν,Ν-二甲 基甲酰胺
在氮气保护下，将化合物 ΟΑ-2 (7.15 g, 15.28 mmol) ,斯迪勒试剂（73 mL)的混合液加热升温至 110 °C , 反应液搅拌 1.5小时后， 向反应液中 注入 5%的盐酸，反应液用乙酸乙酯（100 mL * 3)萃取，有机相经水洗 (100 mL*3) , 无水硫酸钠干燥， 浓缩后得到白色固体 OA-4 (4.68 g, 59.85%)。
Figure imgf000025_0002
式中，T3P: 三丙基磚酸酐
向二氯甲烷 (2 mL)中加入化合物 OA-4 (130 mg, 0.253 mmol)， 苯甲 胺 (27 mg, 0.253 mmol) , 随后加入三丙基碑酸酐 (80.4 mg, 0.253 mmol)。 反应液在 30 °C条件下震荡 16小时。 反应结束后， 反应液直接用制备薄 层色谱分离纯化得到白色油状化合物 BS-OA-067 (21.1 mg, 13.8%)。
LC-MS: 保留时间： 2.402 min (8.890%, isomer), 2.826 min (89.645%), m/z: 602.4 (M+H).
lR NMR (CDCI3, 400 MHz): δ 7.375-7.253 (m, 5H), 5.615-5.603 (d, J= 4.8 Hz, IH), 5.292 (s, IH), 4.576-4.525 (m, IH), 4.450-4.403 (m, IH), 3.624 (s, 3H), 2.875-2.851 (d, J= 9.6 Hz, IH ), 2.003- 1.950 (m, 3H), 1.874-1.733 (m, 2H), 1.628- 1.597 (m, 5H), 1.536- 1.450 (m, 4H), 1.351 -1.326 (m, 2H), 1.197- 1.104 (m, 13H), 1.088-0.951 (m, 9H), 0.915 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 3,4-二氟苯甲胺反应， 制备了 BS-OA-003 :
LC-MS: 保留时间： 2.52 min (8.16%, isomer), 2.93 min (82.50%), m/z: 638.3 (M+H).
ιΥί NMR (400 MHz, CDC13): δ 7.147 (m, 2H), 7.001 (s, IH), 5.292 (t,
IH), 3.620 (s, 3H), 2.842 (m, IH), 2.055- 1.826 (m, 4H), 1.785 (d, IH), 1.684- 1.594 (m, 4H), 1.536- 1.415 (m, 4H), 1.382- 1.245 (m, 3H), 1.175- 1.097 (m, 13H), 0.950-0.884 (m, 9H), 0.769 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 1 -哌啶乙胺反应， 制备了 BS-OA-004:
LC-MS: 保留时间： 1.81 min (92.73%), m/z: 623.4 (M+H).
ln NMR (400 MHz, CDC13): δ 5.360 (t, IH), 3.648 (s, 3H), 3.408 (s, 2H), 2.927 (m, IH), 2.583 (m, 5H), 2.044- 1.958 (m, 4H), 1.790- 1.634 (m, 1 1H), 1.555-1.442 (m, 6H), 1.394- 1.297 (m, 3H), 1.233- 1.070 (m, 12H), 0.956-0.924 (m, 9H), 0.792 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 1 -吡咯烷乙胺反应， 制备了 BS-OA-005 :
LC-MS: 保留时间： 1.77 min (93.89%), m/z: 609.4 (M+H).
ln NMR (400 MHz, CDC13): δ 6.106 (s, IH), 5.350 (t, IH), 3.629 (s, 3H), 3.517-3.290 (m, 2H), 2.912-2.869 (m, IH), 2.681 -2.574 (m, 6H), 2.074- 1.912 (m, 4H), 1.891 -1.61 1 (m, 10H), 1.547- 1.453 (m, 3H), 1.428- 1.289 (m, 3H), 1.232- 1.050 (m, 13H), 0.989-0.898 (m, 9H), 0.776 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 2-甲基丁胺反应， 制备了 BS-OA-008:
LC-MS: 保留时间： 2.71 min (50.06%, isomer), 2.98 min (44.45%), m/z: 582.3 (M+H), 604.3 (M+Na).
lll NMR (400 MHz, CDC13): δ 5.333 (m, IH), 3.620 (s, 3H), 3.157 (m, IH), 2.875 (m, IH), 2.577 (m, IH), 2.370 (m, IH), 1.998-1.839 (m, 5H), 1.763-1.603 (m, 7H), 1.476-1.385 (m, 3H), 1.370-1.296 (m, 4H), 1.193-1.032 (m, 15H), 0.945-0.890 (m, 9H), 0.769 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与异丁胺反应， 制备了 BS-OA-01 1 :
LC-MS: 保留时间： 2.72 min (17.02%, isomer), 2.89 min (79.56%), m/z: 568.3 (M+H), 590.4 (M+Na).
¾ NMR: (400 MHz, CDC13): δ 5.329 (t, IH), 3.626 (s, 3H), 3.314 (m, IH), 2.874 (m, IH), 2.525-2.314 (m, IH), 2.000-1.842 (m, 4H), 1.743-1.535 (m, 6H), 1.528-1.387 (m, 3H), 1.408-1.244 (m, 3H), 1.198-1.029 (m, 15H), 0.934-0.891 (m, 9H), 0.766 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 Ν,Ν-二乙基乙二胺反应， 制备了 BS-OA-012:
LC-MS: 保留时间： 1.82 min (94.04%), m/z: 611.4 (M+H).
IH NMR: (400 MHz, CDC13):. δ 6.265 (s, IH), 5.382-5.297 (m, IH), 3.629 (s, 3H), 3.326 (m, 2H), 2.913-2.869 (m, IH), 2.560 (m, 6H), 2.062-1.866 (m, 4H), 1.745-1.617 (m, 6H), 1.548-1.424 (m, 4H), 1.390-1.303 (m, 3H), 1.215-1.106 (m, 9H), 1.099-1.021 (m, 9H), 0.989-0.886 (m, 9H), 0.786 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 1-吡咯烷丙胺反应， 制备了 BS-OA-016:
LC-MS: 保留时间： 1.79 min (93.26%), m/z: 623.4 (M+H).
¾ NMR: (400 MHz, CDC13): δ 7.143 (s, IH), 5.320-5.303 (m, IH), 3.623 (s, 3H), 3.567-3.513 (m, IH), 3.492-3.302 (m, IH), 2.892-2.653 (m, 7H), 2.140-1.925 (m, 4H), 1.820-1.558 (m, 12H), 1.543-1.422 (m, 3H), 1.415-1.265 (m, 3H), 1.223-1.034 (m, 13H), 0.977-0.856 (m, 9H), 0.783 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与对氟苯甲胺反应， 制备了 BS-OA-017:
LC-MS: 保留时间： 2.40 min (7.08%, isomer), 2.81 min (86.17%), m/z: 620.3 (M+H), 642.3 (M+Na).
lll NMR: (400 MHz, CDC13): δ 7.305 (t, 2H), 7.072 (t, 2H), 5.323 (m, IH), 3.644 (s, 3H), 3.314 (m, IH), 2.864 (m, IH), 2.064-1.894 (m, 3H), 1.651-1.637 (m, 5H), 1.545-1.455 (m, 3H), 1.404-1.316 (m, 3H), 1.232-1.072 (m, 15H), 0.970-0.906 (m, 9H), 0.792 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 3-氟苯甲胺反应， 制备了 BS-OA-021 :
LC-MS: 保留时间： 2.40 min (7.28%, isomer), 2.82 min (90.96%), m/z: 620.3 (M+H), 642.3 (M+Na).
¾ NMR: (400 MHz, CDC13): δ 7.099 (d, IH), 7.032 (m, 2H), 5.329 (m, IH), 3.646 (s, 3H), 2.913 (m, IH), 2.066-1.932 (m, 3H), 1.87 (d, IH), 1.204-1.125 (m, 12H), 0.979-0.907 (m, 9H), 0.793 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 2,4-二氟苯甲胺反应， 制备了 BS-OA-024:
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ZS9S80/ZT0ZN3/X3d 8ΐ06.0/εΐΟΖ OAV LC-MS: 保留时间： 2.76 min (96.04%), m/z: 584.4 (M+H), 606.3 (M+Na).
¾ NMR: (400 MHz, CDC13): δ 5.318 (m, 1H), 3.627 (s, 3H), 3.523 (m, 2H), 3.418-3.361 (m, 5H), 2.891 (m, 1H), 2.075-2.001 (m, 1H), 1.965-1.875 (m, 2H), 1.807 (m, 2H), 1.728-1.682 (m, 2H), 1.644-1.542 (m, 4H), 1.503-1.417 (m, 3H), 1.341 (m, 2H), 1.203-1.081 (m, 12H), 0.941-0.898 (m, 9H), 0.771 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与乙氧基乙胺反应， 制备了 BS-OA-053:
LC-MS: 保留时间： 2.77 min (94.49%), m/z: 584.4 (M+H), 606.2 (M+Na).
lll NMR: (400 MHz, CDC13): . δ 5.347 (m, 1H), 3.633 (s, 3H), 3.553-3.454 (m, 6H), 2.068-1.863 (m, 4H), 1.774-1.595 (m, 6H), 1.549-1.270 (m, 7H), 1.239-1.091 (m, 15H), 0.946-0.905 (m, 9H), 0.778 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与甲氧基乙胺反应， 制备了 BS-OA-054:
LC-MS: 保留时间： 2.78 min (90.27%), m/z: 570.3 (M+H).
lll NMR: (400 MHz, CDC13): δ 5.340 (m, 1H), 3.628 (s, 3H), 3.583-3.357 (m, 7H), 2.899 (m, 1H), 2.073-1.920 (m, 4H), 1.544-1.467 (m, 3H), 1.414-1.306 (m, 2H), 1.200-1.085 (m, 12H), 0.941-0.899 (m, 9H), 0.777 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 2-异丙氧基乙胺反应， 制备了 BS-OA-058:
LC-MS: 保留时间： 2.86 min (94.21%), m/z: 598.4 (M+H).
¾ NMR: (400 MHz, CDC13): δ 5.358 (m, 1H), 3.649 (s, 3H), 2.908 ((()(£ 0ζ£ §9寸∞∞§mHHH卜--- ()()()()£ so6ζ6Όζ960£ 60二 99Π 6ΓΙHrH ςHΗ卜sSsS-- .. # ^¥^τ^-ί寸v〇 90v9s卜a -- ,·
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LC-MS: 保留时间： 2.82 min (94.76%), m/z: 554.4 (M+H).
¾ NMR: (400 MHz, CDC13): δ 5.364 (m, IH), 3.635 (s, 3H), 2.868 (m, IH), 2.082-1.886 (m, 3H), 1.768-1.656 (m, 6H), 1.383-1.335 (m, 3H) 1.168-1.091 (m, 1 IH), 0.960-0.902 (m, 1 1H), 0.785 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 3-戊胺反应， 制备了 BS-OA-070:
LC-MS: 2.72 min (25.86%, isomer), 2.95 min (70.07%), m/z: 582.4 (M+H), 604.3 (M+Na).
lll NMR: (400 MHz, CDC13): δ 5.343 (m, IH), 3.625 (s, 3H), 3.475 (m, IH), 2.879 (m, IH), 2.001-1.876 (m, 4H), 1.238-1.031 (m, 14H), 0.952-0.893 (m, 9H), 0.766 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 1-甲氧基 -2-丙胺反应， 制备了 BS-OA-075:
LC-MS: 保留时间： 4.14 min (13.50%, isomer), 4.62 min (86.00% ) m/z: 584.4 (M+H).
¾ NMR: (400 MHz, CDC13): δ 5.347 (m, IH), 3.630 (s, 3H), 3.372 (m, 5H), 2.870 (m, IH), 2.075-1.916 (m, 4H), 1.547-1.451 (m, 3H), 1.411-1.325 (m, 3H), 1.204-1.083 (m, 12H), 0.943-0.895 (m, 9H), 0.779 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 1-(3-氨丙基 )-4-甲基哌嗪反应， 制备了 BS-OA-078:
LC-MS: 保留时间： 2.30 min (90.63%), m/z: 652.5 (M+H).
¾ NMR: (400 MHz, CDC13): δ 6.447-6.443 (m, IH), 5.328-5.295 (m, IH), 3.630 (s, 3H), 3.565-3.517 (m, IH), 3.271-3.236 (m, IH), 2.907-2.863 (m, IH), 2.592-2.400 (m, 7H), 2.271 (s, 4H), 2.069-1.923 (m: 4H), 1.767-1.589 (m, 8H), 1.545-1.408 (m, 4H), 1.365-1.297 (m, 3H), 1.207-1.043 (m, 14H), 0.946 (s, 3H), 0.919 (s, 3H), 0.878 (s, 3H), 0.795 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与呋喃 -2-甲胺反应， 制备了 BS-OA-082:
LC-MS: 保留时间： 2.72 min (91.18%), m/z: 592.3 (M+H), 614.3 (M+Na).
¾ NMR: (400 MHz, CDC13): δ 7.401 (m, IH), 6.362-6.268 (m, 2H), 5.350 (m, IH), 3.648 (s, 3H), 2.921 (m, IH), 2.059-1.920 (m, 3H), 1.541-1.471 (m, 3H), 1.407-1.349 (m, 3H), 1.185-1.1 12 (m, 12H), 0.963-0.916 (m, 9H), 0.795 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 5-甲基糠胺反应， 制备了 BS-OA-085:
LC-MS: 保留时间： 2.80 min (90.36%), m/z: 606.4 (M+H).
¾ NMR: (400 MHz, CDC13): δ 6.122 (s, IH), 5.906 (s, IH), 5.322 (s, IH), 3.629 (s, 3H), 2.891 (m, IH), 2.285 (s, 3H), 2.064-1.873 (m, 4H), 1.765 (d, IH), 1.502-1.448 (m, 3H), 1.410-1.327 (m, 3H), 1.161-1.054 (m, 14H), 0.941-0.895 (m, 9H), 0.773 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 3-甲氧基苯胺反应， 制备了 BS-OA-086:
LC-MS: 保留时间： 4.40 min (28.43%, isomer), 4.86 min (66.82%), m/z: 618.5 (M+H).
lll NMR: (400 MHz, CDC13): δ 7.219 (m, IH), 6.971 (m, 2H), 6.708 (m, IH), 5.390 (m, IH), 3.829 (s, 3H), 3.658 (s, 3H), 2.936 (m, IH), 2.183 (d, IH), 1.732-1.642 (m, 5H), 1.595-1.500 (m, 4H), 1.464-1.362 (m, 3H), 1.219-1.131 (m, 12H), 0.960-0.927 (m, 9H), 0.823 (s, 3H). 按照 BS-OA-067的方法， 使用上述同样的试剂， 将化合物 OA-4 与 2-噻吩乙胺反应， 制备了 BS-OA-088:
LC-MS: 保留时间： 2.85 min (91.50%), m/z :622.4 (M+H).
¾ NMR: (400 MHz, CDC13): δ 7.190 (dd, J=5.2 Hz, IH), 6.974 (dd, J=52 Hz, IH), 6.841(m, IH), 5.311 (m, IH), 3.628 (s, 3H), 2.862 (m, IH), 2.031-1.929 (m, 2H), 1.849 (d, IH), 1.733-1.590 (m, 8H), 1.500-1.445 (m, 3H), 1.410-1.331 (m, 3H), 1.163-1.088 (m, 12H), 0.935-0.903 (m, 9H), 0.769 (s, 3H). 实施例 2: 化合物（BS-OA-105)的合成
Figure imgf000037_0001
Oleanolic acid OA-6 式中， BnCl: 氯苄
向 Ν,Ν-二甲基甲酰胺 (300 mL)中加入齐墩果酸 (20 g, 43.8 mmol), 碳酸钟 (6.7 g, 48.2 mmol), 随后一次性加入氯苯 (6.1 g, 48.2 mmol), 反 应液加热升温至 100°C：， 搅拌过夜。 反应结束后， 将溶剂旋干后加入 水 （100 mL)， 用二氯甲烷 (200mL*3)萃取， 合并有机相， 经无水硫酸 钠干燥， 旋干后得到固体粗产物 OA-6 (21.5 g, 90%)。
Figure imgf000038_0001
OA-6 OA-7 向二氯甲烷 (500 mL)中加入 OA-6 (21.5 g, 39.38 mmol),氯铬酸吡 啶盐（10.18 g, 47.25 mol)， 反应室温搅拌过夜。 反应接受， 将反应液过 滤， 得到的滤液旋干得到的粗产品经硅胶柱石油醚： 乙酸乙酯： 二氯 甲烷 （100:1:1〜20:1:1〜10:1:1)分离纯化后得到白色固体化合物 OA-7 (17 g, 79.36%)。
Figure imgf000038_0002
OA-7 OA-8 在氮气保护下， 将化合物 OA-7 (15 g, 27.57 mmol)， 斯迪勒试剂 (117.18 mL)的混合液加热升温至 110 °C, 反应液搅拌 1.5小时后， 向 反应液中注入 5%的盐酸， 反应液用乙酸乙酯（500 mL * 3)萃取， 有机 相经无水硫酸钠干燥， 浓缩后得到的粗产品经硅胶柱石油醚： 乙酸乙 酯 （1:0〜100:1〜80:1)分离纯化后得到白色固体化合物 OA-8 (12 g, 74.02%)„
Figure imgf000039_0001
OA-8 BS-OA-105-Bn 向二氯甲烷（10 mL)中加入化合物 OA-8 ( 1000 mg, 1.70 mmol) ,吡 咯烷乙胺 （194 mg, 1.70 mmol) , 随后加入三丙基磚酸酐（80.4 mg, 0.253 mmol) o 反应液在 3(TC条件下震荡 16小时。 反应结束后， 将反 应液浓缩， 得到的油状化合物经硅胶柱石油醚： 乙酸乙酯 10: 1 -1 : 1 分离纯化后得到黄色固体化合物 BS-OA- 105-Bn (400 mg, 34.42%)„
Figure imgf000039_0002
将化合物 BS-OA- 105-Bn (400 mg, 0.58 mmol) 溶于甲醇 (20 mL) 中， 加入 Pd/C (80 mg, cat.), 在氢气（1 atm)条件下， 控制反应温度为 30 °C , 搅拌反应 2小时， 反应结束后， 将反应液过滤， 得到的滤液旋 转浓缩后得到的粗产品经制备薄层色谱分离纯化后得到白色固体化 合物 BS-OA- 105 (36.7 mg, 10.6%)。
LC-MS: 保留时间： 2.92 min (99.23%), m/z: 595.4 (M+H).
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ZS9S80/ZT0ZN3/X3d 8ΐ06.0/εΐΟΖ OAV 按照 BS-OA-105的方法， 使用上述同样的试剂， 将化合物 OA-8 与 Ν,Ν-二甲基丙二胺反应， 制备了 BS-OA-109:
LC-MS: 保留时间： 2.87 min (99.20%), m/z: 583.4 (M+H).
lll NMR: (400 MHz, CDC13): δ 8.407-8.240 (m, 1H), 6.867 (m, 1H),
5.263-5.231 (m, 1H), 3.318-3.233 (m, 3H), 2.833-2.803 (m, 1H), 2.594-2.513 (m, 2H), 2.447-2.335 (m, 6H), 1.958-1.870 (m, 4H), 1.837-1.669 (m, 5H), 1.624-1.561 (m, 3H), 1.533-1.487 (m, 3H), 1.456-1.295 (m, 3H), 1.095-1.014 (m, 13H), 0.894-0.862 (m, 9H), 0.765 (s, 3H). 按照 BS-OA-105的方法， 使用上述同样的试剂， 将化合物 OA-8 与 1-(3-氨丙基 )-4-甲基哌嗪反应， 制备了 BS-OA-110:
LC-MS: 保留时间： 2.89 min (98.04%), m/z: 638.5 (M+H).
¾ NMR: (400 MHz, CDC13): δ 9.023-8.885 (m, 1H), 6.242 (m,
1H), 5.319 (m, 1H), 3.469-3.434 (m, 1H), 3.264-3.237 (m, 1H), 2.883-2.858 (m, 1H), 2.591-2.494 (m, 9H), 2.011-1.898 (m, 4H), 1.758-1.727 (m, 5H), 1.661-1.532 (m, 4H), 1.493-1.466 (m, 3H), 1.365-1.306 (m, 3H), 1.158-1.074 (m, 13H), 0.927-0.887 (m, 9H), 0.833 (s, 3H). 实施例 3: 本发明的 2-位取代的齐墩果酸衍生物抗白血病的活性测定
(1) 实验材料
白血病细胞株： 白血病细胞株： K562/adr (耐药慢性髓系白血病， CML)、 NB4 (急性早幼粒细胞白血病， AML)、 Kasumi-1 (急性髓系白血 病 M2型， AML-M2)、 Jurkat (急性淋巴细胞白血病， ALL)， 以上细胞系 均受赠于浙江大学肿瘤研究所； H9 (急性淋巴细胞白血病， ALL)， 购自 中国典型培养物保藏中心。
试剂： 齐墩果酸（OA )标准品购自四川省什邡市华康药物原料厂， 本发明的 2-位取代的齐墩果酸衍生物。
主要仪器： 细胞培养箱 （型号： Thermo Scientific 3111 ) ， 酶标仪 (型号： Bio-Rad iMark ) 。
(2) 实验方法
取生长良好的白血病细胞 6000个， 接种到 96孔细胞培养板孔内。 培养液为含 10%胎牛血清的 1640细胞培养液。 加入不同浓度的 2-位取 代的齐墩果酸衍生物， 混匀后， 置于二氧化碳（5%C02 )细胞培养箱 37 。C培养 72小时。 然后用 MTT法测定活细胞相对数。在本实验中对照组 (不加化合物处理） 细胞增殖抑制率设为 0%， ^^据活细胞相对数计算 出 72 小时白血病细胞半数生长抑制浓度 (72 小时 IC5o值， g/mL)和 16 g/mL化合物 72 小时作用的白血病细胞增殖抑制率 (Inhibition Rate) IR。
(3) 实验结果
实验结果见表 1。
表 1显示本发明的 2-位取代的齐墩果酸衍生物能诱导人慢性髓系白 血病、 急性髓系白血病和急性淋巴细胞白血病细胞死亡和抑制这些白血 病细胞生长， 与齐墩果酸本身相比， 其中本发明 2-位取代的齐墩果酸衍 生物 BS-OA-005， BS-OA-012 , BS-OA-016, BS-OA-035 , BS-OA-042, BS-OA-078对 K562/adr细胞株抑制率提高 3倍以上，对 Jurkat细胞株抑 制率提高 10倍以上； BS-OA-005 , BS-OA-012 , BS-OA-016, BS-OA-035 对 Kasumi-1 细胞株抑制率提高 5倍以上； BS-OA-004， BS-OA-005 , BS-OA-012 , BS-OA-016, BS-OA-035 , BS-OA-042对 NB4细胞株抑制 率提高 5倍以上； BS-OA-016， BS-OA-035对 H9细胞株抑制率提高 4 倍以上。
小时， IC5。值和 IR值， g/mL )
Figure imgf000042_0001
Figure imgf000043_0003
Figure imgf000043_0001
Figure imgf000043_0002
BS-OA-035 2.67 97.5% 1.67 96.6%
BS-OA-037 8.73 95.8% 11.46 80.4%
BS-OA-042 3.12 95.9% 2.43 99.0%
BS-OA-048 >16 28.9% >16 -21.2%
BS-OA-052 >16 38.0% >16 -5.0%
BS-OA-053 15.53 54.6% >16 13.0%
BS-OA-059 12.73 67.7% >16 23.8%
BS-OA-078 4.33 97.5% 2.5 98.3%
BS-OA-105 >16 31.5% >16 37.5%
BS-OA-106 >16 44.6% >16 43.2%
BS-OA-108 15.3 57.7% 12.61 66.5%
BS-OA-109 >16 39.4% >16 32.3%
BS-OA-110 >16 37.9% 18.36 45.5%
实施例 4: 本发明的 2-位取代的齐墩果酸衍生物抗人多发性骨髓瘤细胞 活性测定
(1) 实验材料
骨髓瘤细胞株： RPMI8226(多发性骨髓瘤)，购自上海复祥生物科技 有限公司。
试剂： 同实施例 3.
主要仪器： 细胞培养箱 （型号： Thermo Scientific 3111 ) ， 酶标仪 (型号： Bio-Rad iMark ) 。
(2) 实险方法
取生长良好的上述肿瘤细胞 6000个，接种到 96孔细胞培养板孔内。 培养液为含 10%胎牛血清的 1640细胞培养液。 加入不同浓度的 2-位取 代的齐墩果酸衍生物， 混匀后， 置于二氧化碳（5%C02 )细胞培养箱 37 。C培养 72小时。 然后用 MTT法测定活细胞相对数。在本实验中对照组 (不加化合物处理） 细胞增殖抑制率设为 0%， ^^据活细胞相对数计算 出 72小时白血病细胞半数生长抑制浓度 (72小时 IC5o值， g/mL)和 16 g/mL化合物 72小时作用的骨髓瘤细胞增殖抑制率 (Inhibition Rate) IR。 (3) 实验结果： 见表 2。
表 2显示本发明的 2-位取代的齐墩果酸衍生物能诱导人骨髓瘤细胞 死亡和抑制这些肿瘤细胞生长。 与齐墩果酸本身相比， 其中本发明 2-位 取代的齐墩果酸衍生物 BS-OA-005， BS-OA-012， BS-OA-035， BS-OA-042 , BS-OA-078对 RPMI8226细胞株抑制率提高 44倍以上。 实施例 5: 本发明的 2-位取代的齐墩果酸衍生物抗人实体瘤作用测定
(1) 实验材料
人实体瘤细胞株：
Hep-2(喉癌）、 A549(人肺癌）、 CaES-17(食道癌细胞)、 PC-3(前列腺 癌）、 CNE (鼻咽癌细胞)、 SK-OV-3(卵巢癌细胞)， 均购自中国典型培养 物保藏中心； RKO(人结肠腺癌细胞)、 MGC 803(人胃癌细胞)、 MG63(骨 肉瘤）、 U87 MG (恶性脑胶质瘤细胞)， 均购自上海复祥生物科技有限公 司； PANC-1(胰腺癌）、 Hep G2(人肝癌细胞)、 Becap37(人乳腺癌细胞)、 Hela (人宫颈癌细胞)， 均受赠于浙江大学肿瘤研究所。
试剂： 同实施例 4.
主要仪器： 细胞培养箱 （型号： Thermo Scientific 3111 ) ， 酶标仪 (型号： Bio-Rad iMark ) 。
(2) 实验方法
取生长良好的人实体瘤细胞 4000个，接种到 96孔细胞培养板孔内。 培养液为含 10%胎牛血清的 DMEM高糖细胞培养液。置于二氧化碳（ 5% C02 ) 细胞培养箱 37。C培养 24小时， 然后， 加入不同浓度的 2-位取代 的齐墩果酸衍生物， 混匀后， 继续置二氧化碳（5%C02 )细胞培养箱 37 。C培养 72小时。 然后用 MTT法测定活细胞相对数。在本实验中对照组 (不加化合物处理） 细胞增殖抑制率设为 0%， #居活细胞相对数计算 出 72小时白血病细胞半数生长抑制浓度 (72小时 IC5o值， g/mL)和 16 g/mL化合物 72小时作用的实体瘤细胞增殖抑制率 (Inhibition Rate) IR。
(3) 实验结果见表 2。 表 2显示本发明的 2-位取代的齐墩果酸衍生物能诱导人实体瘤细胞 死亡和抑制这些肿瘤细胞生长。 与齐墩果酸相比， 本发明的 2-位取代的 齐墩果酸衍生物 BS-OA-005， BS-OA-016, BS-OA-035和 BS-OA-042对 A549及 RKO细胞株抑制率分别提高 19倍， 6倍以上； BS-OA-005， BS-OA-012 , BS-OA-016和 BS-OA-035对 PANC-1细胞株抑制率提高 11倍以上； BS-OA-012和 BS-OA-042对 Becap37细胞株抑制率提高 4 倍以上； BS-OA-005， BS-OA-012, BS-OA-035和 BS-OA-042对 MG-63 及 CNE细胞株抑制率分别提高 3倍， 2倍以上； BS-OA-016， BS-OA-035 和 BS-OA-042对 Hela细胞株抑制率提高 4倍以上； BS-OA-005，
BS-OA-012 , BS-OA-016, BS-OA-035和 BS-OA-042对 U87 MG、 PC-3、 MGC 803及 Hep-2细胞株抑制率分别提高 4倍、 3倍、 5倍和 9倍以上； BS-OA-012 , BS-OA-035和 BS-OA-042对 CaES-17及 SK-OV-3细胞株 抑制率分别提高 3倍， 11倍以上； 对于 Hep G2细胞株， 齐墩果酸本身 未显示对此细胞株有明显的增殖抑制作用， 而本发明的齐墩果酸衍生物 均显示了较好的细胞增殖抑制作用， 其中 BS-OA-005， BS-OA-012 , BS-OA-016, BS-OA-035和 BS-OA-042尤为明显，抑制率均达 96%以上。 表 2: 2-位取代的齐墩果酸衍生物对多发性骨髓瘤和人实体瘤细胞 生长抑制浓度测定（72小时， IC5Q值和 IR值， g/mL ) 。
RPMI8226 A549 PANC-1
化合物 ic50 IR ic50 IR ic50 IR
OA >16 2.2% >16 5.0% >16 8.5%
BS-OA-004 2.08 98.3% 7.7 92.0% 8.77 94.1%
BS-OA-005 0.69 99.2% 2.19 96.8% 4.54 97.8%
BS-OA-008 >16 30.0% >16 3.4% >16 -4.6%
BS-OA-012 0.74 99.0% 2.83 97.4% 4.73 100.0%
BS-OA-016 1.15 99.0% 2.45 96.8% 4.38 98.5%
BS-OA-027 6.1 86.5% >16 45.6% >16 36.9%
BS-OA-031 >16 4.4% >16 -6.3% >16 -1.4%
BS-OA-032 >16 30.3% >16 -6.6% >16 14.5%
BS-OA-034 >16 15.5% >16 5.3% >16 -9.9%
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Figure imgf000047_0001
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Claims

权 利 要 求 书
1. 通式 (I)的 2-位取代的齐墩果酸衍生物或其药学上可接受的盐
Figure imgf000052_0001
式中 选自 H、 任选取代的（^-( 18的烷基、 任选取代的 C2-C18烯基 或炔基、 任选取代的 C3-C7环烷基或环烯基、 任选取代的芳基、 任选取 代的杂环基或杂芳基， 所述任选取代的取代基选自 1¾素、 硝基、 氰基、 氨基、羟基、巯基、 、 d-C6烷基氨基、二（d-C6烷基）氨基、 Ci-C6 烷氧基、 d-C6烷硫基， 所述 C3-C7环烷基或环烯基、 芳基、 杂环基或杂 芳基还任选地被 C C6烷基取代，或者 为由选自所述 C3-C7环烷基或 环烯基、 芳基、 杂环基或杂芳基的基团取代的 C C6烷基； 以及
式中 W选自羟基、 氨基、 d-C6烷基氨基、 二（d-C6烷基）氨基、 d-C6烷氧基和 C C6烷石克基。
2. 根据权利要求 1的 2-位取代的齐墩果酸衍生物或其药学上可接受 的盐， 其中 为 C4-C8烷基、 环烷基 - d-C6烷基、 杂环基 - d-C6烷基、 (^-^烷基氨基- ^-^烷基、二（d-C6烷基） - d-C6烷基、杂芳基 - d-C6 烷基、 d-C6烷氧基 -d-C6烷基， 所述环烷基、 杂环基和杂芳基任选地被 鹵素、羟基、 d-C6烷氧基、 d-C6烷基、氨基、 d-C6烷基氨基、二（d-C6 烷基）氨基所取代。
3. 根据权利要求 1的 2-位取代的齐墩果酸衍生物或其药学上可接受 的盐， 其中 为 C4-C6烷基、 环烷基 - d-C4烷基、 杂环基 - d-C4烷基、 C C6烷基氨基 - d-C4烷基、二（d-C6烷基） - d-C4烷基、杂芳基 - d-C4 烷基、 d-C6烷氧基 -d-C4烷基， 所述环烷基、 杂环基和杂芳基任选地被 鹵素、羟基、 d-C6烷氧基、 d-C6烷基、氨基、 d-C6烷基氨基、二（d-C6 烷基）氨基所取代。
4. 根据权利要求 1的 2-位取代的齐墩果酸衍生物或其药学上可接受 的盐， 其中所述杂环基为饱和杂环基。
5. 根据权利要求 1-4任一项的 2-位取代的齐墩果酸衍生物或其药学 上可接受的盐， 其中所述杂环含有氮原子作为环原子并作为连接点。
6. 根据权利要求 5的 2-位取代的齐墩果酸衍生物或其药学上可接受 的盐， 其中所述杂环基为吡咯烷 -1-基、 哌啶子基、 哌嗪 -1-基、 4-甲基哌 嗪 -1-基、 吗啉代基、 噁唑烷基、 咪唑烷基、 异噁唑烷基。
7. 根据权利要求 1-4任一项的 2-位取代的齐墩果酸衍生物或其药学 上可接受的盐， 其中 W为羟基或 C C6烷氧基。
8. 根据权利要求 1-4任一项的 2-位取代的齐墩果酸衍生物或其药学 上可接受的盐， 其中所述杂芳基为吡啶基、 呋喃基、 噻吩基、 吡咯基、 吡喃基、 咪唑基。
9. 根据权利要求 1的 2-位取代的齐墩果酸衍生物或其药学上可接 受的盐， 选自下述化合物：
Figure imgf000053_0001
BS-OA-005
1-氧代 -2-(N- (吡咯烷基乙基))胺叛基齐墩果酸甲酉
Figure imgf000054_0001
BS-OA-012
-氧代 -2-(N- ( 果酸甲酯
Figure imgf000054_0002
BS-OA-016
-氧代 -2-(N- (吡咯烷基丙基))胺叛基齐墩果酸甲酯
Figure imgf000054_0003
BS-OA-035
-氧代 -2-(N- (二乙基胺丙基))胺叛基齐墩果酸甲酯
Figure imgf000055_0001
BS-OA-037
1-氧代 -2-(N- (吗啡啉基丙基))胺叛基齐墩果酸甲酯
Figure imgf000055_0002
BS-OA-042
1 -氧代 -2-(N- (二甲基胺丙基))胺叛基齐墩果酸甲酯
Figure imgf000055_0003
BS-OA-078
1-氧代 -2-(N-(N-甲基哌嗪基丙基))胺羰基齐墩果酸甲酯
10. 一种制备式 I化合物的方法
Figure imgf000056_0001
Figure imgf000056_0002
OA-3 OA-4 齐墩果酸（OA ) 经氧化， 生成齐墩果酸的酮中间体（OA-1 ) ； 此 中间体经甲酯化， 生成齐墩果酸的（酮）甲酯中间体（OA-2 ) ； 此中间体 与斯迪勒试剂反应， 在酮叛基的邻位引入甲氧叛基生成齐墩果酸的双酯 中间体（OA-3 ) ； 此双酯中间体经水解， 得齐墩果酸的单羧基中间体 ( OA-4 )； 引入甲氧叛基和水解反应也可一锅反应完成得齐墩果酸的单 ^^中间体（OA-4 ) ; 此单^ ^中间体与有机胺经酰胺键形成反应， 生 成 2-位取代的齐墩果酸衍生物（ I )； 式中 和 W与权利要求 1-9中通 式（I ) 中的定义相同。
11. 一种药物组合物， 其中包含权利要求 1-9中任一项的 2-位取代 的齐墩果酸衍生物或其药学上可接受的盐和任选的药学上可以接受的 赋形剂。
12. 权利要求 1-10 中的任一项的 2-位取代的齐墩果酸衍生物或其 药学上可接受的盐在制备抗肿瘤药物中的用途。
13. 一种治疗肿瘤患者的方法， 包括给予需要治疗的患者治疗有 效量的根据权利要求 1-10任一项的 2-位取代的齐墩果酸衍生物或其药 学上可接受的 o
14.作为抗肿瘤治疗剂的权利要求 1-10任一项的 2-位取代的齐墩 果酸衍生物或其药学上可接受的盐。
15. #居权利要求 12, 13或 14的用途、方法或 2-位取代的齐墩果 酸衍生物， 其中， 所述肿瘤选自白血病、 多发性骨髓瘤、 淋巴瘤、 肝 癌、 胃癌、 乳腺癌、 胆管细胞癌、 胰腺癌、 肺癌、 大肠癌、 骨肉瘤、 人宫颈癌、 神经胶质瘤、 鼻咽癌、 喉癌、 食管癌、 中耳肿瘤、 黑色素 瘤和前列腺癌。
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US20140343064A1 (en) 2014-11-20
DK2786757T3 (da) 2017-11-20
JP6211527B2 (ja) 2017-10-11
EP2786757A1 (en) 2014-10-08
NO2786757T3 (zh) 2018-01-13
EP2786757B1 (en) 2017-08-16
JP2014534268A (ja) 2014-12-18

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