WO2022022623A1 - Dérivé d'arénobufagine, son procédé de préparation, composition pharmaceutique et utilisation associées - Google Patents

Dérivé d'arénobufagine, son procédé de préparation, composition pharmaceutique et utilisation associées Download PDF

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WO2022022623A1
WO2022022623A1 PCT/CN2021/109182 CN2021109182W WO2022022623A1 WO 2022022623 A1 WO2022022623 A1 WO 2022022623A1 CN 2021109182 W CN2021109182 W CN 2021109182W WO 2022022623 A1 WO2022022623 A1 WO 2022022623A1
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acid
alkyl
compound
cycloalkyl
heterocyclyl
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Chinese (zh)
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罗川
高波
缪震元
吴岳林
庄春林
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安徽华润金蟾药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to a class of Xenopusin derivatives, a preparation method thereof, a pharmaceutical composition and application thereof.
  • Arenobufagin is a bufatolactone compound extracted from toadstools, and its chemical structure is as follows:
  • Xenopus has strong anti-tumor activity, and can inhibit the adhesion, migration, invasion and angiogenesis of liver cancer HepG2 cells (Chinese Pharmacological Bulletin, 2011, 27; 19-23), and can also significantly inhibit liver cancer.
  • SMMC-7721 mitochondrial apoptosis pathway induces apoptosis of tumor cells (China Emergencies of Traditional Chinese Medicine, 2013, 22: 1845-1883). Therefore, it is of great significance to synthesize Xenopusin derivatives, study their structure-activity relationship, and find high-efficiency and low-toxic Xenopusin derivatives.
  • WO201185641A1 discloses a class of bufalin derivatives and their treatment Cancer Uses.
  • CN102532235 reports a class of bufaxin derivatives and preparation methods thereof, and uses of compositions comprising the derivatives.
  • CN102656179, CN103980337, CN110483608 and CN103980338 disclose a class of bufalin derivatives, pharmaceutical compositions and uses thereof, mainly by esterifying the 3-position hydroxyl group to obtain a series of derivatives.
  • Xenopus can induce apoptosis of breast cancer cell line MCF-7 by regulating JNK-mediated phosphorylation of Yes-related proteins (Deng et al. Cancer Cell Int (2016) 18:209). Xenopus can inhibit the PI3K enzyme of Rapamycin signaling pathway. Induction of apoptosis and autophagy in pancreatic cancer cells (Xiaolu, et al. Pancreas (2020) 49:261). The study found that the effect of Xenopus venom on hepatocellular carcinoma is by inhibiting the production of Na/K ATPase.
  • Xenopus Although Xenopus has excellent anti-tumor activity, its toxicity is relatively large and its therapeutic index is relatively narrow. Therefore, it is necessary to carry out structural modification on this basis to enhance the activity of the compound and reduce the toxicity, and discover new anti-tumor drugs from it. .
  • the purpose of the present invention is to provide a new class of Xenobufacin derivatives and a preparation method thereof, and also to provide the application of the Xenopusin derivatives in the preparation of anti-tumor drugs.
  • the Xenopusin derivatives of the present invention not only have excellent antitumor activity, but also have low toxicity.
  • the first aspect of the present invention is to provide a new class of Xenobufacin derivatives, which is a compound represented by the following general formula (I) or (II) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from hydrogen, deuterium, and substituted or unsubstituted of the following: alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkane aryl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl;
  • R2, R3 are independently selected from hydrogen , deuterium, and the following substituted or unsubstituted: alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl , cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl; or R 2 , R 3 together with the N atom to which they are attached form a substituted or unsubstituted of heterocycles.
  • alkyl refers to a saturated straight or branched monovalent hydrocarbon group having one to twelve carbon atoms, wherein the alkyl group may be optionally independently substituted with one or more substituents.
  • alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2 -propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2 -Methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl , 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl
  • alkenyl refers to a linear or branched monovalent hydrocarbon group of two to twelve carbon atoms having at least one site of unsaturation, ie, a carbon-carbon sp double bond, wherein the alkenyl can be any is optionally substituted independently with one or more substituents, and includes groups having "cis” and “trans” orientations or "E” and “Z” orientations.
  • Examples include, but are not limited to, vinyl, allyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 5-hexenyl, 1 - cyclohex-2-enyl, 1-cyclohex-2-enyl and 1-cyclohex-3-enyl.
  • alkynyl refers to a linear or branched monovalent hydrocarbon group of two to twelve carbon atoms in at least one position of unsaturation, ie, a carbon-carbon sp triple bond, wherein the alkynyl group may optionally is independently substituted with one or more substituents. Examples include, but are not limited to, ethynyl and propynyl.
  • cycloalkyl refers to a monovalent non-aromatic saturated or partially saturated cyclic hydrocarbon radical having three to ten carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl , 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclohexyl Undecyl, cyclododecyl.
  • cycloalkyl also includes polycyclic (eg, bicyclic and tricyclic) cycloalkyl structures, wherein the polycyclic structure optionally includes cycloalkyl or heterocyclyl or aryl groups with saturated or partially unsaturated Saturated or partially unsaturated cycloalkyl fused to a radical or heteroaryl ring.
  • Bicyclic carbocycles having 7 to 12 atoms can be arranged as bicyclo[4,5], [5,5], [5,6] or [6,6] systems, for example, or as bridged systems such as bicyclo[2.2. 1] Heptane, bicyclo[2.2.2]heptoctane and bicyclo[3.2.2]nonane.
  • heteroalkyl refers to a saturated straight or branched monovalent hydrocarbon group having one to twelve carbon atoms, wherein at least one carbon atom is replaced by a heteroatom selected from nitrogen, oxygen and sulfur , and wherein the group may be a carbon group or a heteroatom group (ie, the heteroatom may be present in the middle or at the end of the group). Heteroalkyl groups can be optionally substituted independently with one or more substituents.
  • heteroalkyl includes alkoxy and heteroalkoxy.
  • heteroalkenyl used in the present invention refers to a linear or branched monovalent hydrocarbon group containing at least one double bond and two to twelve carbon atoms, examples of which include, but are not limited to, vinyl, propenyl, and the like, wherein at least One carbon atom is replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, and wherein the group may be a carbon group or a heteroatom group (ie, the heteroatom may be present in the middle or at the end of the group).
  • Heteroalkenyl groups are optionally substituted independently with one or more substituents, and include radicals having "cis” and "trans” orientations or "E” and "Z" orientations.
  • heteroalkynyl used in the present invention refers to a linear or branched monovalent hydrocarbon group containing at least one triple bond and two to twelve carbon atoms, examples of which include, but are not limited to, ethynyl, propynyl, and the like, wherein At least one carbon atom is replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, and wherein the group can be a carbon group or a heteroatom group (ie, the heteroatom can be present in the middle or at the end of the group).
  • a heteroalkynyl group can be optionally substituted independently with one or more substituents.
  • heterocyclyl and “heterocycle” are used interchangeably and refer to a saturated or partially unsaturated carbocyclic group having 3 to 8 ring atoms, at least one of which is Heteroatoms independently selected from nitrogen, oxygen and sulfur, the remaining ring atoms are carbon atoms, wherein one or more ring atoms may be optionally substituted independently with one or more substituents.
  • This group can be a carbon group or a heteroatom group.
  • heterocyclyl includes heterocycloalkoxy.
  • Heterocyclyl also includes groups in which a heterocyclyl is fused to a saturated, partially unsaturated, or fully unsaturated (ie, aromatic) carbocyclic or heterocyclic ring.
  • heterocyclyl rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, Linyl, 4-thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietane, homo piperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3- Pyrroline,
  • Heterocyclyl groups can be C-attached or N-attached as long as it is possible.
  • a group derived from pyrrole can be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached).
  • imidazole-derived groups can be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached).
  • Heterocyclyl groups in the present invention may be unsubstituted or substituted with various groups as indicated at one or more substitutable positions.
  • aryl refers to an optionally substituted monocyclic or polycyclic group or ring system containing at least one aromatic hydrocarbon ring, such as, but not limited to, phenyl, naphthyl, fluorenyl, azulenyl base, anthracenyl, phenanthrenyl, pyrenyl, biphenyl and terphenyl.
  • heteroaryl refers to an optionally substituted monocyclic or polycyclic group containing at least one aromatic ring having one or more heteroatoms independently selected from nitrogen, oxygen and sulfur or Ring systems, examples of monocyclic heteroaryl groups such as but not limited to furyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, Pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl and triazolyl.
  • bicyclic heteroaryl groups are, but are not limited to, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furanopyridyl, imidazopyridyl, imidazothiazolyl, indolazinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothiophene base, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridyl, phthalazinyl, pteridyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl , quinoxalinyl, quinazolinyl, thiadiazol
  • tricyclic heteroaryl groups such as, but not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, pyridyl, phenanthroline, phenidyl, phenazinyl, phenazine base, phenothiazinyl, phenoxazinyl and xanthyl.
  • arylalkyl refers to an alkyl moiety (as defined above) substituted with one or more aryl moieties (as defined above).
  • arylalkyl groups include aryl-Ci- 3 -alkyl groups such as, but not limited to, benzyl, phenylethyl, and the like.
  • heteroarylalkyl refers to an alkyl moiety (as defined above) substituted with a heteroaryl moiety (as defined above).
  • heteroarylalkyl groups include 5- or 6-membered heteroaryl-Ci- 3 -alkyl groups such as, but not limited to, oxazolylmethyl, pyridylethyl, and the like.
  • heterocyclylalkyl refers to an alkyl moiety (as defined above) substituted with a heterocyclyl moiety (as defined above).
  • heterocyclylalkyl groups include 5- or 6-membered heteroaryl-Ci- 3 -alkyl groups such as, but not limited to, tetrahydropyranylmethyl.
  • cycloalkylalkyl refers to an alkyl moiety (as defined above) substituted with a cycloalkyl moiety (as defined above).
  • examples of cycloalkylalkyl groups include 5- or 6-membered cycloalkyl-Ci- 3 -alkyl groups such as, but not limited to, cyclopropylmethyl.
  • substituted alkyl refers to an alkyl group in which one or more hydrogen atoms are each independently replaced by a substituent.
  • Alkenyl, alkynyl, allyl, cycloalkyl, heteroalkyl, heterocyclyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, cycloalkylalkyl, aryl as defined above and heteroaryl groups can be similarly substituted.
  • halogen as used herein includes fluorine, bromine, chlorine, iodine.
  • R 1 is selected from substituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl , cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl; the substituted alkyl, alkenyl, alkyne alkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl or heterocyclylalkane is optionally substituted with one or more groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
  • R 1 is preferably a C1-C8 alkyl group, a 3- to 8-membered cycloalkyl C1-C8 alkyl group, a 3- to 8-membered heterocyclic C1-C8 alkyl group, an ether bond-containing 3- to 8-membered alkyl group or 3- to 8-membered alkyl groups containing thioether bonds, the C1-C8 alkyl groups, 3- to 8-membered cycloalkyl C1-C8 alkyl groups, 3- to 8-membered heterocyclic groups C1-C8 alkyl groups, and ether bond-containing
  • R 2 and R 3 are independently selected from hydrogen, deuterium, and the following substituted or unsubstituted: alkyl, alkenyl, alkynyl , heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl ; the substituted alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl alkenyl, heteroarylalkyl, heterocyclylalkyl optionally substituted with one or more groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl , heteroalkyl, heteroalkenyl, heteroalky
  • R 2 , R 3 together with the N atom to which they are attached form a heterocycle which does not otherwise contain or optionally additionally contains one or more heteroatoms selected from the group consisting of N, O, S, SO, SO2 , NR6 , wherein the heterocycle is optionally substituted with one or more groups independently selected from the group consisting of: oxo, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, Heteroalkynyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, cyano, nitro, aryl, OR4 , NR4R5 , heteroaryl, arylalkyl , heteroarylalkyl or Heterocyclylalkyl wherein said alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, cyclyl
  • R 2 and R 3 are independently hydrogen, deuterium, C1-C8 alkyl, 3-8 membered cycloalkyl C1-C8 alkyl, 3-8 membered heterocyclic C1-C8 alkyl, ether-containing 3-8 membered alkyl or sulfide-containing ether bond, wherein the C1-C8 alkyl, 3-8 membered cycloalkyl C1-C8 alkyl, 3-8 membered heterocyclic C1-C8 alkyl, containing
  • R 4 , R 5 is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, or NR 4 R 5 forms a 4-6 membered substituted or unsubstituted heterocycle;
  • each R is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl ;
  • Each of the cycloalkyl and heterocyclyl groups described is saturated or partially unsaturated.
  • Xenopusin derivatives of the present invention are selected from the following compounds:
  • the pharmaceutically acceptable salts of the xenopusin derivatives of the present invention include the salts formed by the above-mentioned compounds and pharmaceutically acceptable inorganic and organic acids;
  • the preferred inorganic acids include: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid and sulfuric acid;
  • Preferred organic acids include: formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, succinic acid, carbenoxic acid, glycyrrhetic acid, oleanolic acid, maslinic acid, ursolic acid, Corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid , maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, niacin, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid and amino acids.
  • the second aspect of the present invention is to provide the preparation method of the Xenobufacin derivative, characterized in that it comprises the following steps of preparing the compound of general formula (I):
  • Compound II is obtained by reacting compound IV with a molar ratio of 1:(0.05-0.5):(2-4), a basic catalyst and an amine compound NHR 2 R 3 in a second solvent.
  • the acid compound R 1 OOH is a compound containing a carboxylic acid group, preferably N-Boc-D-aminobutyric acid, N-Boc-D-amino Propionic acid, N-Boc-D-valine, N-Boc-L-leucine, Boc-8-aminooctanoic acid, 8-chlorooctanoic acid, Boc-D-glutamine, N-Boc-L-methionine , S-acetamidomethyl-N-tert-butoxycarbonyl-L-cysteine, 1-(tert-butoxycarbonyl) azetidine-3-acetic acid, trifluoroacetic acid, acetic acid, etc.;
  • the first basic catalyst is a nitrogen-containing compound, preferably 4-dimethylaminopyridine, triethylamine, pyridine, etc.;
  • the condensing agent is a carbodiimide condensing agent, preferably 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC) ), diisopropylcarbodiimide (DIC), etc.;
  • EDCI 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • the solvent is selected from methylene chloride, benzene, toluene, tetrahydrofuran, etc.;
  • the reaction is carried out at room temperature.
  • the second basic catalyst is a nitrogen-containing compound, preferably 4-dimethylaminopyridine, triethylamine, pyridine, etc.;
  • the second solvent is selected from methylene chloride, benzene, toluene, tetrahydrofuran, etc.;
  • the amine compound NHR 2 R 3 is selected from morpholine, N-(2-aminoethyl)morpholine, N-aminopropylmorpholine, piperidine, pyrrolidine, piperazine, diethylamine, N-methyl ylpiperazine, cyclohexylamine, 1-(2-aminoethyl)pyrrolidine, L-alanine ethyl ester hydrochloride, cyclopentylamine, 3,3-difluoropiperidine hydrochloride, 3-fluoro Piperidine hydrochloride, cis-1-Boc-3-amino-4-fluoropyrrolidine, 4,4-difluoropiperidine hydrochloride, 3,3-difluoropyrrolidine hydrochloride, 4-fluoromethane Piperidine hydrochloride, 4-difluoromethylpiperidine hydrochloride, 3-fluoroazetidine hydrochloride, 4,4-difluorocycl
  • the reaction is carried out at normal temperature
  • compound IV can be prepared by the following steps:
  • compound III with a molar ratio of 1:(8-20):(8-20) is dissolved in a third solvent to react with p-nitrophenyl chloroformate and a third basic catalyst to obtain compound IV;
  • the third basic catalyst is a nitrogen-containing compound, preferably 4-dimethylaminopyridine, triethylamine, pyridine, etc.;
  • the third solvent is selected from methylene chloride, benzene, toluene, tetrahydrofuran, etc.;
  • the reaction is carried out at room temperature.
  • the third aspect of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising the Xenobufacin derivative, which also includes one or more of pharmaceutically acceptable carriers, excipients and adjuvants.
  • the dosage forms of the pharmaceutical composition are injections (including small-volume injections, medium-volume injections, and large-volume injections), powder injections, emulsions for injection, tablets, pills, capsules, ointments, creams, Patches, liniments, powders, sprays, implants, drops, suppositories, ointments.
  • the pharmaceutical composition can be various nano-formulations, such as liposomes.
  • the fourth aspect of the present invention is to provide the application of the Xenobufinoid derivatives in the preparation of antitumor drugs, drugs for treating cardiovascular and cerebrovascular diseases or drugs for treating nervous system diseases.
  • the Xenopusin derivatives of the present invention have anti-tumor activity and can be used to treat tumors, including esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, Cancers in the testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system, as well as thyroid cancer, leukemia, Hocking's disease, lymphoma and myeloma.
  • tumors including esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, Cancers in the testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system, as well as thyroid cancer, leukemia, Hocking's disease, lymph
  • the pharmacological activity of the compounds of the present invention enables them to be used for the preparation of anti-tumor, cardiovascular and anti-nervous system drugs, so the present invention also includes pharmaceutical compositions using these compounds and their pharmaceutically acceptable salts as active ingredients.
  • the pharmaceutical composition can be in solid form or liquid form.
  • the compounds of the present invention can also be used in the preparation of the following medicines: therapeutic medicines for cardiovascular and cerebrovascular diseases and nervous system diseases, in addition to their application in the preparation of antitumor drugs.
  • the compound of the present invention Compared with Xenopus, the compound of the present invention has reduced toxicity due to the introduction of diester groups, and has less toxic and side effects when used for preparing antitumor drugs.
  • N-Boc-D-aminopropionic acid was used instead of N-Boc-D-aminobutyric acid to obtain a white solid with a yield of 44.3%.
  • 1 H NMR 500MHz, CDCl 3 ) ⁇ : 7.72(d,1H), 7.38(s,1H), 6.28(d,1H), 5.43(d,1H), 5.00-5.16(m,3H), 4.03- 4.32(m,3H),1.72–2.28(m,12H),1.26–1.57(m,28H),1.15(s,3H),0.99(s,3H).
  • ESI-HRMS calcd C40H58N2O12[M+Na] + m/z, 781.3887; found 781.3886.
  • the compound of the present invention was subjected to tumor cell proliferation inhibition test, and the test method adopted conventional MTT method (eg, "New Pharmacology Research Methods” edited by Lv Qiujun, 2007: 242-243).
  • the cell lines were selected from A549 (human lung adenocarcinoma cells), HCT116 (human intestinal cancer cells), MDA-MB-231 (human breast cancer cells), Bel7404 (human liver cancer cells), and were cryopreserved by the Pharmacology Laboratory of Shanghai Institute of Pharmaceutical Industry. and passage.
  • the culture medium is DMEM+10%FBS+double antibody.
  • In vitro activity test 100 ⁇ L of cell suspension with a concentration of 4-5 ⁇ 10 4 cells/mL was added to each well of a 96-well plate, and placed in a 37° C., 5% CO 2 incubator. After 24h, add sample solution, 10 ⁇ L/well, set up double wells, 37°C, 5% CO 2 for 72h. Add 20 ⁇ L of 5 mg/ml MTT solution to each well, add 100 ⁇ L/well of dissolving solution after 4 h, put it in an incubator, and measure the OD value at 570 nm with a full-wavelength multi-function microplate reader after dissolving. The inhibition rate (IR) and the median inhibitory concentration (IC 50 ) were calculated with Excel software.
  • test results are shown in Table 1, wherein the sample refers to the Xenopusin derivatives prepared in the corresponding examples.
  • the above experimental results show that the compound of the present invention has good anti-tumor activity, and exhibits excellent activity against lung cancer, breast cancer, colon cancer and liver cancer cell lines, so the compound of the present invention and its salts can be used for the preparation of anti-tumor drug.
  • the compounds IIb, IIe, IIf, IIp and IIq of the present invention were subjected to tumor cell proliferation inhibition test using conventional MTT method (eg, "New Pharmacological Research Methods” edited by Lv Qiujun, 2007: 242-243).
  • Cell lines were selected from KYSE-150 (human esophageal cancer cells), MGC803 (human gastric cancer cells), and SHG44 (human glioma cells), which were cryopreserved and passaged by the Pharmacology Laboratory of Shanghai Institute of Pharmaceutical Industry.
  • the culture medium is DMEM+10%FBS+double antibody.
  • In vitro activity test 100 ⁇ L of cell suspension with a concentration of 4-5 ⁇ 10 4 cells/mL was added to each well of a 96-well plate, and placed in a 37° C., 5% CO 2 incubator. After 24h, add sample solution, 10 ⁇ L/well, set up double wells, 37°C, 5% CO 2 for 72h. Add 20 ⁇ L of 5 mg/ml MTT solution to each well, add 100 ⁇ L/well of dissolving solution after 4 h, put it in an incubator, and measure the OD value at 570 nm with a full-wavelength multi-function microplate reader after dissolving. The inhibition rate (IR) and the median inhibitory concentration (IC 50 ) were calculated with Excel software.
  • test results are shown in Table 2, wherein the sample refers to the Xenopusin derivatives prepared in the corresponding examples.
  • Example 40 In vivo antitumor activity test of the compound Ij of the present invention against lung cancer A549
  • mice BALB/C nude mice (SPF grade), male, 18-20 grams.
  • Human lung adenocarcinoma cells A549 were cultured in DMEM medium containing 10% FBS. After the cells grew to exponential growth phase, they were digested with 0.25% trypsin and then centrifuged at 1200 r ⁇ min -1 for 5 min. The cell pellet was washed twice with PBS. After removing the serum, the cells were resuspended in PBS, the cell concentration was adjusted to 1 ⁇ 10 7 cells/ml, and 200 ⁇ L of each Balb/c nude mouse was inoculated into the fat pad of the left rib near the axilla. After 9 days, when the tumor volume grew to 100-130 mm 3 , the patients were administered into groups.
  • the compound was dissolved in 97% CMC-Na glucose sodium chloride solution (CMC-Na 0.05%) + 3% Tween 80, and ultrasonically dissolved into a uniform dispersion liquid.
  • Compound Ij was administered at a dose of 10 mg/Kg; cisplatin was administered at 2 mg/kg.
  • mice with successful modeling were selected and randomly divided into model groups with 6 mice in each group.
  • Cisplatin 0.4ml/only (0.1mg/mL, about 2mg/kg).
  • Each group was given the corresponding drugs by intraperitoneal injection for a total of 2 weeks (5 consecutive days per week and 2 days of drug withdrawal), and the cisplatin group was intraperitoneally administered for 5 consecutive days.
  • 24h after the last administration 6 mice in each group were randomly sacrificed by cervical vertebrae, weighed, and the tumor tissue was stripped and weighed, and the tumor growth inhibition rate (tumor inhibition rate) was calculated.
  • Tumor weight inhibition rate (%) (average tumor weight of control group ⁇ average tumor weight of administration group)/average tumor weight of control group ⁇ 100%.
  • Example 41 In vivo antitumor activity test of the compounds Il, IIb, IIe, IIf, IIp and IIq of the present invention against lung cancer A549
  • a well-grown lung cancer A549 tumor was taken, cut into uniform small pieces of about 3 mm in size under sterile conditions, and one piece was inoculated subcutaneously in the right armpit of each mouse with a trocar.
  • the average tumor mass volume was about 130mm 3 .
  • the animals were regrouped according to the size of the tumor. Animals with too large and too small tumors were eliminated.
  • the average tumor volume in each group was basically the same. /20g body weight. From the 9th day of inoculation, the long diameter of the tumor a (mm) and the vertical short diameter b (mm) of the tumor were measured twice a week with a digital electronic caliper.
  • Example 42 In vivo antitumor activity test of the compounds IIp and IIq of the present invention in gastric cancer MGC803
  • a well-grown gastric cancer MGC803 tumor was taken, cut into uniform pieces of about 3 mm in size under sterile conditions, and one piece was inoculated subcutaneously in the right armpit of each mouse with a trocar.
  • the average tumor mass volume was about 130mm 3 .
  • the animals were regrouped according to the size of the tumor. Animals with too large and too small tumors were eliminated.
  • the average tumor volume in each group was basically the same. /20g body weight. From the 9th day of inoculation, the long diameter of the tumor a (mm) and the vertical short diameter b (mm) of the tumor were measured twice a week with a digital electronic caliper.
  • Example 43 In vivo antitumor activity test of brain glioma SHG44 of the compounds IIp and IIq of the present invention
  • a well-grown glioma SHG44 tumor block was taken, cut into uniform small pieces of about 3 mm in size under sterile conditions, and one piece was inoculated subcutaneously in the right armpit of each mouse with a trocar.
  • the average tumor mass volume was about 130mm 3 .
  • the animals were regrouped according to the size of the tumor. Animals with too large and too small tumors were eliminated.
  • the average tumor volume in each group was basically the same. /20g body weight. From the 9th day of inoculation, the long diameter of the tumor a (mm) and the vertical short diameter b (mm) of the tumor were measured twice a week with a digital electronic caliper.
  • a well-grown colon cancer HCT116 tumor was taken, cut into uniform pieces of about 3 mm in size under sterile conditions, and one piece was inoculated subcutaneously in the right armpit of each mouse with a trocar.
  • the average tumor mass volume was about 130mm 3 .
  • the animals were regrouped according to the size of the tumor. Animals with too large and too small tumors were eliminated.
  • the average tumor volume in each group was basically the same. /20g body weight. From the 9th day of inoculation, the long diameter of the tumor a (mm) and the vertical short diameter b (mm) of the tumor were measured twice a week with a digital electronic caliper.
  • Example 45 Test of the effect of the compound of the present invention on hERG potassium channel
  • CHO-hERG cells were cultured in a 175cm 2 culture flask. When the cell density reached 60-80%, the culture medium was removed, washed with 7mL of PBS (Phosphate Buffered Saline phosphate buffered saline), and then digested with 3mL of Detachin. After the digestion is complete, add 7 mL of culture medium to neutralize, then centrifuge, remove the supernatant, and add 5 mL of culture medium to resuspend to ensure that the cell density is 2-5 ⁇ 10 6 /mL.
  • PBS Phosphate Buffered Saline phosphate buffered saline
  • Single-cell high-impedance sealing and whole-cell pattern formation are all performed automatically by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cell is clamped at -80 mV, before a 5-second +40 mV depolarizing stimulus is given. , given a pre-voltage of -50 mV for 50 ms, then repolarized to -50 mV for 5 seconds, and then returned to -80 mV. This voltage stimulus was applied every 15 seconds, and the extracellular fluid was recorded for 2 minutes and then recorded for 5 minutes. Then the dosing process was started. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2.5 minutes. After all concentrations were continuously administered, the administration Positive control compound 3 ⁇ M Cisapride. At least 3 cells were tested at each concentration (n ⁇ 3).
  • the 20 mM compound stock solution was diluted with DMSO, and 10 ⁇ L of 20 mM compound stock solution was added to 20 ⁇ L DMSO solution, and serially diluted 3 times to 6 DMSO concentrations. Take 4 ⁇ L of 6 compounds with 6 DMSO concentrations, add them to 396 ⁇ L of extracellular fluid, dilute 100 times to 6 intermediate concentrations, and then take 80 ⁇ L of 6 intermediate concentrations of compounds and add them to 320 ⁇ L of extracellular fluid, 5 times Dilute to final concentration to be tested. The highest tested concentration was 40 ⁇ M, followed by 40, 13.33, 4.44, 1.48, 0.49, and 0.16 ⁇ M in a total of 6 concentrations.
  • the DMSO content in the final test concentration did not exceed 0.2%, and this concentration of DMSO had no effect on the hERG potassium channel.
  • Compound preparation is performed by the Bravo instrument through the entire dilution process. Experimental data were analyzed by GraphPad Prism 5.0 software.
  • HEK293 cells stably expressing hCav1.2 were cultured in a cell culture dish with a diameter of 35mm, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:4 every 48 hours.
  • the cell culture medium was aspirated, rinsed with extracellular fluid, and then 0.25% Trypsin-EDTA (Invitrogen) solution was added, and the cells were digested at room temperature for 3-5 minutes. Aspirate the digestion solution, resuspend the cells with extracellular fluid, and transfer the cells to the experimental dish for electrophysiological recording.
  • Trypsin-EDTA Invitrogen
  • the compound stock solution was diluted with 100% DMSO to an intermediate concentration, and then 10 ⁇ L of the intermediate concentration of the compound was added to 4990 ⁇ L of extracellular fluid, and diluted 500 times to obtain the final concentration to be tested, 10 ⁇ M.
  • Preparation of positive control compound nifedipine 10 ⁇ L of 150 ⁇ M nifedipine stock solution was added to 4990 ⁇ L of extracellular fluid, and diluted 500 times to obtain the final concentration to be tested, 300 nM.
  • the content of DMSO in the final test concentration did not exceed 0.2%, and this concentration of DMSO had no effect on the Cav1.2 calcium channel.
  • Cav1.2 channel currents were recorded by whole-cell patch-clamp technique at room temperature.
  • the glass microelectrode is drawn from the glass electrode blank (BF150-86-10, Sutter) by a drawing machine.
  • the tip resistance after filling the electrode liquid is about 2-5M ⁇ . Insert the glass microelectrode into the amplifier probe to connect to the patch clamp amplifier. Clamp voltage and data recording were controlled and recorded by a computer via pClamp 10 software with a sampling frequency of 10 kHz and a filter frequency of 2 kHz.
  • the cells were clamped at -80mV and the step voltage evoking Cav1.2 currents was given a 500ms depolarization voltage from -80mV to +20mV and back to -80mV. This voltage stimulation was given every 10 s, and the administration process was started after the current was determined to be stable (1 minute). Each test concentration was given for at least 1 minute. At least 3 cells were tested per concentration (n > 3), and positive compounds were tested at least 2 cells per concentration (n > 2).
  • Inhibition% represents the percentage of inhibition of Cav1.2 current by the compound
  • I and Io represent the amplitude of Cav1.2 current after dosing and before dosing, respectively.

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Abstract

La présente invention se rapporte au domaine technique de la médecine, et concerne en particulier un dérivé d'arénobufagine, son procédé de préparation, une composition pharmaceutique et une utilisation associées. Le dérivé d'arénobufagine selon la présente invention a une activité antitumorale. L'activité pharmacologique du composé lui permet d'être utilisé dans la préparation d'un médicament ayant des effets antitumoraux, cardiovasculaires et anti-système nerveux. La présente invention concerne un composé tête de série pour le développement de nouveaux médicaments antitumoraux, et ouvre un nouvelle voie pour la recherche en profondeur et le développement de nouveaux médicaments antitumoraux.
PCT/CN2021/109182 2020-07-31 2021-07-29 Dérivé d'arénobufagine, son procédé de préparation, composition pharmaceutique et utilisation associées WO2022022623A1 (fr)

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