WO2023077977A1 - 秋水仙碱衍生物的制备方法及其用途 - Google Patents

秋水仙碱衍生物的制备方法及其用途 Download PDF

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WO2023077977A1
WO2023077977A1 PCT/CN2022/119478 CN2022119478W WO2023077977A1 WO 2023077977 A1 WO2023077977 A1 WO 2023077977A1 CN 2022119478 W CN2022119478 W CN 2022119478W WO 2023077977 A1 WO2023077977 A1 WO 2023077977A1
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compound
formula
group
methyl
contacted
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PCT/CN2022/119478
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French (fr)
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姚晨
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天津市昕晨投资发展有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/30—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
    • C07C233/32—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a ring other than a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/41—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a ring other than a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • 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 present application relates to organic compounds and their biological uses, more specifically to colchicine derivatives, their preparation methods, and their use in the preparation of medicines, such as the preparation of anti-inflammatory and analgesic medicines.
  • Colchicine is a plant alkaloid, originally extracted from the seeds and bulbs of the Liliaceae plant Colchicine. Its appearance is generally white or light yellow powder or needle-like crystals, which is highly toxic. Colchicine is currently mainly used to treat acute attacks of gout.
  • colchicine in the treatment of gout is that during the acute attack of gout, urate crystals in the joint cavity will cause leukocyte chemotaxis and enrichment, but after the leukocyte phagocytizes urate, there is no suitable enzyme to process it, and instead it is absorbed by uric acid. Salt crystals damage cell membranes and quickly release a large number of inflammatory factors, which will enrich more white blood cells, resulting in the release of more inflammatory factors, which can cause severe joint pain. Colchicine can effectively inhibit the chemotaxis and phagocytosis of leukocytes, and achieve the purpose of rapid analgesia and anti-inflammation.
  • colchicine has been widely used, its use is limited because of its side effects such as nausea, vomiting, and diarrhea, and its high cytotoxicity. . Therefore, those skilled in the art are still researching to find alternative drugs, even similar drugs with better effects.
  • the application discloses a compound of formula (A) or a pharmaceutically acceptable salt thereof,
  • the application discloses a compound of formula (A) or a pharmaceutically acceptable salt thereof, wherein said R 1 and R 2 are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl Group, and the R 3 is selected from the group consisting of H and methyl.
  • the application discloses a compound or a pharmaceutically acceptable salt thereof, for example, a preparation method of a compound of formula (A) or a pharmaceutically acceptable salt thereof, comprising combining a compound of formula (B) with a compound of formula (C) Compound or formula (C) in R 1 and R 2 at least one of the compound protected by a protecting group is contacted,
  • the application discloses a method for preparing a compound or a pharmaceutically acceptable salt thereof, such as a compound of formula (A) or a pharmaceutically acceptable salt thereof, comprising combining a compound of formula (B) with a compound of formula (C) or a compound of formula (C) In R1 and R2 , at least one compound protected by a protecting group is contacted, wherein R1 and R2 are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl R3 ' is selected from the group consisting of H and methyl; and M is selected from the group consisting of H, Li, Na, K, Rb, and Cs.
  • the application discloses a method of preparing a pharmaceutically acceptable salt of a compound of formula (A), contacting the compound of formula (A) with an acid.
  • the present application discloses the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating cancer, analgesia and/or anti-inflammation.
  • the pharmaceutically acceptable salt of the compound of formula (A) provided can achieve technical effects no less than or even better than colchicine on the one hand. More diverse or even superior alternative technical solutions are provided.
  • Figure 1 shows the compound of formula (A).
  • Fig. 2 is a synthetic pathway for the synthesis of N-glycyl deacetylcolchicine in some embodiments of the present application.
  • Fig. 3 is the 1 H NMR spectrum of N-tert-butoxycarbonylcolchicine synthesized in some examples of the present application.
  • Fig. 4 is the 1 H NMR spectrum of deacetylcolchicine synthesized in some embodiments of the present application.
  • Fig. 5 is a 1 H NMR spectrum of N-(N-(tert-butoxycarbonyl)glycyl)deacetylcolchicine synthesized in some embodiments of the present application.
  • 6A-6C are respectively the 1 H NMR spectrum, mass spectrometry detection results, and high performance liquid chromatography determination results of N-glycyl deacetylcolchicine synthesized in some embodiments of the present application.
  • Fig. 7 is a synthetic pathway of N-(N,N-diethyl)glycyl deacetylcolchicine and its hydrochloride in some embodiments of the present application.
  • Figures 8A-8B are the high-performance liquid chromatography and mass spectrometry results of N-(N,N-diethyl)glycyldeacetylcolchicine synthesized in some embodiments of the present application, respectively.
  • Figures 8C-8E are the 1 H NMR spectrum and high performance liquid chromatography of N-(N,N-diethyl)glycyl deacetylcolchicine hydrochloride synthesized in some embodiments of the present application, respectively. Determination results, and mass spectrometry detection results.
  • Fig. 9 is a 1 H NMR spectrum of N-(N,N-dimethyl)glycyl deacetylcolchicine synthesized in some embodiments of the present application.
  • Fig. 10 is the 1 H NMR spectrum of N-(N,N-di-n-propyl)glycyl deacetylcolchicine synthesized in some embodiments of the present application.
  • Figures 11A-11B are the 1 H NMR spectrum and high-performance liquid chromatography determination results of N-(N,N-diisopropyl)glycyl deacetylcolchicine synthesized in some embodiments of the present application, respectively .
  • Fig. 12 is a synthetic pathway of N-glycyldeacetyl-10-demethylcolchicine from N-glycyldeacetylcolchicine in some embodiments of the present application.
  • Figures 13A-13C are the 1 H NMR spectrum, ESI-MS positive ion mode spectrum, and ESI of N-glycyl deacetyl-10-demethylcolchicine synthesized in some embodiments of the present application, respectively.
  • NMR spectrum is the 1 H NMR spectra and 13 C of N-(N,N-diethyl)glycyl deacetyl-10-demethylcolchicine synthesized in some examples of the present application, respectively.
  • NMR spectrum heteronuclear single quantum correlation (HSQC) NMR spectrum, heteronuclear multiple bond correlation (HMBC) NMR spectrum, ESI-MS positive ion mode spectrum, and ESI-MS negative ion mode spectrum.
  • HSQC heteronuclear single quantum correlation
  • HMBC heteronuclear multiple bond correlation
  • Fig. 15 is the 1 H NMR spectrum of N-(N,N-dimethyl)glycyl deacetyl-10-demethylcolchicine synthesized in some embodiments of the present application.
  • Fig. 16 is a 1 H NMR spectrum of N-(N,N-di-n-propyl)glycyl deacetyl-10-demethylcolchicine synthesized in some embodiments of the present application.
  • Fig. 17 is a 1 H NMR spectrum of N-(N,N-diisopropyl)glycyl deacetyl-10-demethylcolchicine synthesized in some embodiments of the present application.
  • Fig. 18 is a diagram showing the change of 340nm light absorbance of various groups of tubulin polymerization reactions with time in the tubulin polymerization detection test of some embodiments of the present application.
  • Figure 19 and Figures 20A-20F are diagrams of the transdermal absorption rate of transdermal administration in the study of the absorption efficiency of transdermal administration in some embodiments of the present application.
  • Fig. 21 is a graph showing the number of cells evaluated by CCK8 method and the reading of OD 450 nm measured by ultraviolet spectrophotometer in the neutrophil chemotaxis experiment of some embodiments of the present application.
  • FIG. 22 is a comparison chart of the results of evaluating the number of migrated cells in the model cell migration experiments of some embodiments of the present application.
  • Fig. 23 is a micrograph after staining with crystal violet in the model cell migration experiment of some embodiments of the present application.
  • 24A-24D are comparison charts of the body weight, paw volume, arthritis index, and serum IL-6 level of experimental rats in each group in the in vivo drug efficacy test of some embodiments of the present application.
  • colchicine derivative in this application at least includes the compound of formula (A) disclosed in this application (the compound shown in Figure 1) or a pharmaceutically acceptable salt thereof, but is not limited thereto.
  • the application discloses a compound of formula (A) or a pharmaceutically acceptable salt thereof,
  • R is selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl. In some embodiments, R is selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl. In some embodiments, R 3 is selected from the group consisting of H and methyl. In some embodiments, R 1 is H. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is n-propyl. In some embodiments, R 1 is isopropyl. In some embodiments, R is H. In some embodiments, R 2 is methyl.
  • R 2 is ethyl. In some embodiments, R 2 is n-propyl. In some embodiments, R 2 is isopropyl. In some embodiments, R 3 is H. In some embodiments, R 3 is methyl. In some embodiments, R is selected from the group consisting of H and ethyl. In some embodiments, R is selected from the group consisting of H and ethyl. In some embodiments, R 1 and R 2 are both H, or R 1 and R 2 are both ethyl. In some embodiments, both R and R are H. In some embodiments, both R and R are methyl. In some embodiments, R 1 and R 2 are both ethyl. In some embodiments, both R and R are n-propyl. In some embodiments, both R and R are isopropyl.
  • the R and R are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl, and the R is selected from the group consisting of H and methyl composed of groups.
  • R 1 and R 2 are the same.
  • R and R are the same and are selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl.
  • R 1 and R 2 are the same.
  • R and R are the same and are selected from the group consisting of methyl, ethyl, n-propyl, and isopropyl.
  • the compound of formula (A) is selected from the group consisting of compounds of formula (A0) to compound of formula (A9),
  • the compound of formula (A) is selected from the compound of formula (A0), the compound of formula (A1), the compound of formula (A2), the compound of formula (A3), the compound of formula (A4), the compound of formula (A6), the compound of formula (A7) compound, formula (A8) compound, and the group formed by formula (A9) compound,
  • the pharmaceutically acceptable salt of the compound of formula (A) of the present application is selected from the group consisting of methanesulfonate, sulfate, p-toluenesulfonate, hydrochloride, succinate, maleate , the group consisting of fumarate.
  • the pharmaceutically acceptable salt of the compound of formula (A) of the present application is the mesylate salt of the compound of formula (A).
  • the pharmaceutically acceptable salt of the compound of formula (A) of the present application is the sulfate salt of the compound of formula (A).
  • the pharmaceutically acceptable salt of the compound of formula (A) of the present application is p-toluenesulfonate of the compound of formula (A). In some embodiments, the pharmaceutically acceptable salt of the compound of formula (A) of the present application is the compound of formula (A) In some embodiments, the pharmaceutically acceptable salt of the compound of formula (A) of the present application , is the succinate salt of the compound of formula (A). In some embodiments, the pharmaceutically acceptable salt of the compound of formula (A) of the present application is the maleate salt of the compound of formula (A). In some embodiments, the pharmaceutically acceptable salt of the compound of formula (A) of the present application is a fumarate salt of the compound of formula (A).
  • the application discloses a compound or a pharmaceutically acceptable salt thereof, for example, a preparation method of a compound of formula (A) or a pharmaceutically acceptable salt thereof, comprising combining a compound of formula (B) with formula ( C) the compound or formula (C) in R 1 and R 2 at least one of the compound protected by a protecting group is contacted,
  • R 3 ' is selected from the group consisting of H and methyl.
  • M is selected from the group consisting of H, Li, Na, K, Rb, and Cs.
  • R3 ' is methyl.
  • R3 is selected from the group consisting of H and methyl, and R3 ' is methyl.
  • R3 is H and R3 ' is methyl.
  • R3 is the same as R3 '.
  • both R3 and R3 ' are methyl.
  • M is H.
  • M is selected from the group consisting of Li, Na, K, Rb, and Cs.
  • M is selected from the group consisting of Li, Na, K, Rb, and Cs.
  • M is Li.
  • M is Na.
  • M is K.
  • M is Rb.
  • M is Cs.
  • the compound in which at least one of R 1 and R 2 in formula (C) is protected by a protecting group is at least one of H in R 1 and R 2 in formula (C) is protected by a protecting group.
  • the compound in which at least one of R 1 and R 2 is protected by a protecting group in formula (C) is that one of R 1 and R 2 or one of the two Hs in formula (C) is protected by Protective base protection.
  • M is H.
  • the protecting group is selected from the group consisting of tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, and p-methoxyphenyl.
  • the protecting group is t-butoxycarbonyl.
  • the protecting group is benzyloxycarbonyl. In some embodiments, the protecting group is 9-fluorenylmethoxycarbonyl. In some embodiments, the protecting group is benzyl. In some embodiments, the protecting group is p-methoxyphenyl.
  • R 1 and R 2 are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl; the protecting group is selected from tert-butoxycarbonyl, benzyloxy The group consisting of carbonyl, 9-fluorenylmethoxycarbonyl, benzyl, and p-methoxyphenyl; R 3 ' is selected from the group consisting of H and methyl; and M is selected from the group consisting of H, Li, Na, K, The group consisting of Rb, and Cs.
  • R 1 and R 2 are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl; R 3 ′ is selected from is selected from the group consisting of H and methyl; and M is selected from the group consisting of Li, Na, K, Rb, and Cs.
  • R 1 and R 2 are each independently selected from H, methyl, ethyl, n-propyl The group consisting of base, and isopropyl; the protecting group is selected from the group consisting of tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, and p-methoxyphenyl; R 3 'is selected from the group consisting of H and methyl; and M is H.
  • R 1 and R 2 are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl; the protecting group is selected from tert-butoxycarbonyl, benzyloxy The group consisting of carbonyl, 9-fluorenylmethoxycarbonyl, benzyl, and p-methoxyphenyl; R 3 ' is selected from the group consisting of H and methyl; and M is selected from the group consisting of H, Li, Na, K, The group consisting of Rb, and Cs.
  • the compound of formula (B) is contacted with the compound of formula (C), and in the compound of formula (C), R and R are each independently selected from H, methyl, ethyl, n-propyl R3 ' is selected from the group consisting of H and methyl; and M is selected from the group consisting of Li, Na, K, Rb, and Cs.
  • the compound of formula (B) is contacted with at least one of R 1 and R 2 protected by a protecting group in formula (C), at least one of R 1 and R 2 in formula (C)
  • R 1 and R 2 are each independently selected from the group consisting of H, methyl, ethyl, n-propyl, and isopropyl
  • the protecting group is selected from the group consisting of tert-butoxycarbonyl, the group consisting of benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, and p-methoxyphenyl
  • R 3 ' is selected from the group consisting of H and methyl
  • M is H.
  • contacting a compound of formula (B) with a compound of formula (C) or at least one of R and R in formula (C) protected by a protecting group comprises combining a compound of formula (B), and a compound of formula (C) compound or formula (C) in R 1 and R 2 at least one protected by a protecting group compound, and selected from 2-(7-azabenzotriazole)-N,N,N',N At least one member of the group consisting of '-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine is contacted.
  • M is H
  • contacting a compound of formula (B) with a compound of formula (C) or at least one of R and R in formula (C) is protected by a protecting group comprises contacting a compound of formula (B ) compound, and formula (C) compound or at least one protected compound of R 1 and R 2 in formula (C), and selected from 2-(7-azabenzotriazole)-N,N , N′,N′-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, and at least one of the group consisting of 4-dimethylaminopyridine, especially with 2-(7-aza Benzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate contact.
  • a compound of formula (B) is contacted with a compound of formula (C).
  • the compound of formula (B) is contacted with a compound of formula (C) in which at least one of R and R is protected by a protecting group.
  • the compound of formula (B) is contacted with the compound of formula (C) under an alkaline environment.
  • the compound of formula (B) is contacted with the compound of formula (C) in the presence of N,N-diisopropylethylamine, triethylamine or 2,2,6,6-tetramethyl In the presence of piperidines, especially N,N-diisopropylethylamine.
  • the method for preparing the compound of formula (A) of the present application also includes the compound of formula ( B) the product of a compound contacted with at least one of R and R in formula (C) protected by a protecting group, contacted with at least one selected from the group consisting of trifluoroacetic acid, hydrochloric acid, and hydrobromic acid, Especially in contact with trifluoroacetic acid.
  • the compound of formula (B) is contacted with at least one compound of R and R in formula (C) protected by a protecting group
  • the method for preparing the compound of formula (A) of the present application also includes the compound of formula ( B) the product of a compound contacted with at least one of R and R in formula (C) protected by a protecting group, contacted with at least one selected from the group consisting of trifluoroacetic acid, hydrochloric acid, and hydrobromic acid, Especially in contact with trifluoroacetic acid.
  • the method for preparing the compound of formula (A) of the present application further includes combining the compound of formula (B) with the compound of formula (C) or the compound of formula (C)
  • the product of contacting at least one of R1 and R2 with a compound protected by a protecting group is contacted with hydrochloric acid, especially hydrochloric acid and acetic acid.
  • R 3 ' is methyl
  • R 3 is H
  • the method for preparing the compound of formula (A) of the present application also includes combining the compound of formula (B) with the compound of formula (C) or the compound of formula (C)
  • the product of contacting at least one of R1 and R2 with a compound protected by a protecting group is contacted with hydrochloric acid, especially hydrochloric acid and acetic acid.
  • the method for preparing the compound of formula (A) of the present application also includes the compound of formula (B1) and a compound selected from di-tert-butyl dicarbonate, benzyl chloroformate, and 9-fluorenylmethyl chloroformate. At least one contact of the group consisting of groups yields a compound of formula (B2),
  • the L is selected from the group consisting of tert-butoxycarbonyl, benzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl, especially tert-butoxycarbonyl.
  • the method for preparing the compound of formula (A) of the present application comprises contacting the compound of formula (B1) with di-tert-butyl dicarbonate, wherein L is tert-butoxycarbonyl.
  • the method for preparing the compound of formula (A) of the present application comprises contacting the compound of formula (B1) with benzyl chloroformate, wherein L is benzyloxycarbonyl.
  • the method for preparing the compound of formula (A) of the present application comprises contacting the compound of formula (B1) with 9-fluorenylmethyl chloroformate, wherein L is 9-fluorenylmethoxycarbonyl.
  • the method for preparing the compound of formula (A) of the present application further includes contacting the compound of formula (B2) with at least one member selected from the group consisting of R'OM', hydrazine hydrate, and sodium borohydride, especially with R'OM' is contacted to obtain the compound of formula (B3),
  • R' is selected from the group consisting of methyl, ethyl, n-propyl, and isopropyl. In some embodiments, R' is ethyl. In some embodiments, M' is selected from the group consisting of Li, Na, K, Rb, and Cs. In some embodiments, M' is sodium. In some embodiments, R'OM' is sodium ethoxide.
  • the method for preparing the compound of formula (A) of the present application further comprises contacting the compound of formula (B3) with at least one selected from the group consisting of trifluoroacetic acid, hydrochloric acid, and hydrobromic acid, especially with Contacting with trifluoroacetic acid affords the compound of formula (B).
  • the concentration of hydrochloric acid is greater than or equal to 1M.
  • the concentration of hydrobromic acid is greater than or equal to 1M.
  • the concentration of hydrochloric acid is 1M to 2M.
  • the concentration of hydrobromic acid is 1M to 2M.
  • the present application discloses a method of preparing a pharmaceutically acceptable salt of a compound of formula (A), contacting the compound of formula (A) with an acid.
  • the method for preparing a pharmaceutically acceptable salt of the compound of formula (A) in the present application further includes preparing the compound of formula (A).
  • the method for preparing a pharmaceutically acceptable salt of the compound of formula (A) in the present application further includes preparing the compound of formula (A) according to the method for preparing the compound of formula (A) in the present application.
  • the present application discloses a method of treating a disease and/or condition comprising administering a compound of formula (A) or a pharmaceutically acceptable salt thereof to an individual in need thereof.
  • the present application discloses a medicament for treating diseases and/or symptoms, which comprises the compound of formula (A) or a pharmaceutically acceptable salt thereof.
  • the present application discloses the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases and/or symptoms.
  • the present application discloses the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of diseases and/or symptoms.
  • the treatment of diseases and/or symptoms is selected from at least one of treatment of cancer, analgesia and anti-inflammation. In some embodiments, the treatment of diseases and/or symptoms is at least one of anti-inflammatory and analgesic. In some embodiments, the disease and/or condition is at least one selected from the group consisting of cancer and arthritis. In some embodiments, the disease and/or condition is cancer. In some embodiments, the disease and/or condition is inflammation. In some embodiments, the disease and/or condition is arthritis.
  • the disease and/or condition is selected from ovarian cancer, breast cancer, lung cancer, Kaposi's sarcoma, cervical cancer, pancreatic cancer, rheumatism, acute arthritis, and acute gouty arthritis at least one of .
  • the disease and/or symptom is selected from at least one of ovarian cancer, breast cancer, lung cancer, Kaposi's sarcoma, cervical cancer, and pancreatic cancer.
  • the disease and/or symptom is selected from at least one of rheumatism, acute arthritis, and acute gouty arthritis.
  • the synthesis pathway of N-glycyldeacetylcolchicine from colchicine is shown in FIG. 2 . Specifically:
  • the synthetic pathway of N-glycyl deacetylcolchicine from colchicine can also be synthesized from N-tert-butoxycarbonylcolchicine (compound of formula (II)), N-tert-butoxy Carbonyl deacetylcolchicine (compound of formula (III)), deacetylcolchicine (compound of formula (IV)), or N-(N-tert-butoxycarbonyl) glycyl deacetylcolchicine (compound of formula (V) ) compound) start.
  • Colchicine introduces a tert-butoxycarbonyl (Boc) protecting group to obtain N-tert-butoxycarbonylcolchicine
  • Rotational concentration removes acetonitrile in the system.
  • the remaining material was diluted with dichloromethane and washed once with 10% aqueous citric acid and once with saturated brine.
  • the obtained organic phase was dried by adding anhydrous sodium sulfate, and spin-dried.
  • N-tert-butoxycarbonylcolchicine removes the acetyl group to obtain N-tert-butoxycarbonyl deacetylcolchicine
  • the system was diluted with dichloromethane, and the pH value was adjusted to 10 by adding saturated aqueous sodium carbonate solution.
  • the obtained system was separated into liquids, wherein the aqueous phase was extracted by adding dichloromethane for several times, and the organic phases obtained by extraction were combined with the organic phases obtained by liquid separation.
  • the system was diluted with dichloromethane, washed once with 10% citric acid aqueous solution and once with saturated brine.
  • the organic phase was dried by adding anhydrous sodium sulfate, and spin-dried.
  • the system was diluted with dichloromethane, and the pH value was adjusted to 10 by adding saturated aqueous sodium carbonate solution.
  • the obtained system was separated into liquids, wherein the water phase was extracted three times by adding dichloromethane, and the three organic phases obtained by extraction were combined with the organic phase obtained by liquid separation.
  • the synthesis pathway of N-(N,N-dialkyl)glycyldeacetylcolchicine and its hydrochloride salt from colchicine is shown in FIG. 7 .
  • the "alkyl” mentioned here, that is, R 1 and R 2 in FIG. 7 can be, for example, H, methyl, ethyl, n-propyl or isopropyl, but is not limited thereto. Specifically:
  • N-(N,N-dialkyl)glycyl deacetylcolchicine can also be converted to salt to obtain N-(N,N-dialkyl)glycyl deacetylcolchicine hydrochloride .
  • Example 1 and Example 2 can be replaced by each other, or their equivalent replacements, without resulting in failure to obtain the subsequent corresponding embodiments. compound of.
  • the alkyl group is H, methyl, n-propyl, or isopropyl, and can also be reacted by the corresponding method to obtain the corresponding N-(N,N-dialkyl) Glycyl deacetylcolchicine and its hydrochloride, such as N-(N,N-dimethyl)glycyl deacetylcolchicine and its hydrochloride, N-(N,N-di-n- Propyl)glycyldeacetylcolchicine and its hydrochloride, and N-(N,N-diisopropyl)glycyldeacetylcolchicine and its hydrochloride.
  • N-(N,N-dialkyl) Glycyl deacetylcolchicine and its hydrochloride such as N-(N,N-dimethyl)glycyl deacetylcolchicine and its hydrochloride, N-(N,N-di-n
  • Colchicine introduces a tert-butoxycarbonyl (Boc) protecting group to obtain N-tert-butoxycarbonylcolchicine
  • the remaining material was diluted with dichloromethane and washed once with 10% aqueous citric acid and once with saturated brine.
  • the obtained organic phase was dried by adding anhydrous sodium sulfate, and spin-dried.
  • the final product N-tert-butoxycarbonylcolchicine (compound of formula (II)) was about 32.5 g, and 6.2 g of raw materials were recovered.
  • N-tert-butoxycarbonylcolchicine removes the acetyl group to obtain N-tert-butoxycarbonyl deacetylcolchicine
  • the system was diluted with dichloromethane, and the pH value was adjusted to 10 by adding saturated aqueous sodium carbonate solution.
  • the obtained system was separated into liquids, wherein the aqueous phase was extracted three times by adding dichloromethane, and the organic phases obtained by extraction were combined with the organic phases obtained by liquid separation.
  • the system was vortex concentrated to remove DMF, and purified by column to obtain the product point.
  • Aqueous HCl was added to form a salt.
  • Ethyl acetate was added to extract impurities, the aqueous phase was taken, and saturated aqueous sodium carbonate solution was added to adjust the pH value to about 8.
  • the product is N-(N,N-diethyl)glycyl deacetylcolchicine hydrochloride.
  • the 1 H NMR spectrum is shown in Figure 8C.
  • LC-MS liquid chromatography-mass spectrometry
  • Colchicine introduces a tert-butoxycarbonyl (Boc) protecting group to obtain N-tert-butoxycarbonylcolchicine
  • Colchicine (compound of formula (I)) 1 equivalent, 4-dimethylaminopyridine 2 equivalents) is dissolved in acetonitrile, then add di-tert-butyl dicarbonate 3.5 equivalents and triethylamine 1 equivalent), react at 100 ° C 1.5 hours.
  • N-tert-butoxycarbonylcolchicine removes the acetyl group to obtain N-tert-butoxycarbonyl deacetylcolchicine
  • the aqueous solution was extracted with dichloromethane and the organic phase was collected.
  • N-tert-butoxycarbonyl deacetylcolchicine compound of formula (III)
  • the pH was adjusted to 8 by adding saturated aqueous sodium carbonate.
  • the resulting system was separated into liquids, and the aqueous phase was extracted three times by adding dichloromethane.
  • the reaction was checked using thin layer chromatography.
  • N-(N,N-dialkyl)glycyldeacetyl-10-desmethylcolchicine is synthesized from N-(N,N-dialkyl)glycyldeacetyl-10-demethylcolchicine
  • the synthetic pathway of Narcisine is shown in Figure 12.
  • the "alkyl” mentioned here, that is, R 1 and R 2 in FIG. 12 can be, for example, H, methyl, ethyl, n-propyl or isopropyl, but is not limited thereto. Specifically:
  • the alkyl group is H as an example. Specifically:
  • Electrospray ionization source mass spectrometry (m/z): 401[M+H] + , 423[M+Na] + , 399[MH] - .
  • the 1 H NMR spectrum is shown in Figure 13A.
  • the ESI-MS positive ion mode spectrum is shown in Figure 13B, and the ESI-MS negative ion mode spectrum is shown in Figure 13C.
  • the product is N-(N,N-diethyl)glycyldeacetyl-10-demethylcolchicine (compound of formula (A7)).
  • the 1 H NMR spectrum is shown in Figure 14A
  • the 13 C NMR spectrum is shown in Figure 14B
  • the heteronuclear single quantum correlation (HSQC) NMR spectrum is shown in Figure 14C
  • the heteronuclear multiple bond correlation (HMBC) NMR spectrum is shown in Figure 14D.
  • the ESI-MS positive ion mode spectrum is shown in Figure 14E
  • the ESI-MS negative ion mode spectrum is shown in Figure 14F.
  • alkyl group is methyl, n-propyl, and isopropyl as examples. Specifically:
  • reaction was monitored using thin layer chromatography until disappearance of reactant.
  • the dilute hydrochloric acid was evaporated to dryness under reduced pressure and purified by column chromatography to obtain N-(N,N-dialkyl)glycyl deacetyl-10-demethylcolchicine.
  • the compound of formula (A2) can form salts with common acids.
  • the amount of salt prepared in the examples is small, so the salt was not observed to crystallize directly from the solvent, but was obtained after concentration under reduced pressure.
  • Solvents such as acetone, dichloromethane, methanol, isopropanol or water can dissolve the salt, but the amount of salt used in the examples is less, and solids cannot be crystallized after direct cooling or dropwise addition of a reversed-phase solvent. If the reverse phase solvent is added dropwise in a slight excess, the system immediately becomes viscous.
  • the amount of water added was 0.1 mL, and the sample was dissolved, indicating that the solubility of the salt was >1 g/ml, which was very easy to dissolve.
  • N-glycyl deacetylcolchicine produced in Example 1 paclitaxel, vincristine and colchicine were selected as control groups, plus As blank control, tubulin polymerization test was carried out.
  • the experimental steps refer to the tubulin polymerization test kit BK006P from Cytoskeleton, as follows:
  • Tubulin glycerol buffer (tubulin glycerol buffer): a universal tubulin buffer with a final concentration of 15% glycerol. Store at 4°C.
  • Guanosine triphosphate stock solution 100mM solution of guanosine triphosphate. 10 ⁇ L per tube was divided into 10 aliquots and stored at -70°C.
  • Tubulin tubulin protein: add 10 ⁇ L guanosine triphosphate stock solution and 1.1 mL pre-cooled universal tubulin buffer, mix well, place on ice, 200 ⁇ L each tube is divided into 5 parts and stored at -70°C .
  • Paclitaxel mother solution 100 ⁇ L of 200 ⁇ M paclitaxel DMSO solution was prepared as the paclitaxel mother solution.
  • Vincristine mother solution 100 ⁇ L of 200 ⁇ M vincristine DMSO solution was prepared as the vincristine mother solution.
  • Colchicine mother solution 100 ⁇ L of 200 ⁇ M colchicine DMSO solution was prepared as colchicine mother solution.
  • N-glycyl deacetylcolchicine mother solution prepare 1mL of 200mM N-glycyl deacetylcolchicine DMSO solution, take 10 ⁇ L of 200mM solution and add it to 990 ⁇ L DMSO to prepare a 2mM solution; take 10 ⁇ L of 2mM The solution was added to 90 ⁇ L of DMSO to prepare a 200 ⁇ M solution; 10 ⁇ L of the 200 ⁇ M stock solution was added to 90 ⁇ L of DMSO to prepare a 20 ⁇ M solution.
  • tubulin polymerization buffer (tubulin polymerization buffer): 750 ⁇ L universal tubulin buffer plus 250 ⁇ L tubulin glycerol buffer and 10 ⁇ L guanosine triphosphate stock solution were mixed together, placed on ice superior.
  • step (6) Add 420 ⁇ L of cold tubulin polymerization buffer to 200 ⁇ L of tubulin in step (6) and mix to obtain a final concentration of: piperazine-1,4-diethanesulfonic acid (PIPES) 80 mM, magnesium chloride 2 mM, Ethylene glycol-bis-(2-aminoethyl ether) tetraacetic acid (EGTA) 0.5 mM, guanosine triphosphate 1 mM, glycerol 10.2%, 3 mg/mL tubulin.
  • PPES piperazine-1,4-diethanesulfonic acid
  • EGTA Ethylene glycol-bis-(2-aminoethyl ether) tetraacetic acid
  • Figure 18 shows the change of 340nm light absorbance with time in each group of tubulin polymerization reactions.
  • paclitaxel can promote tubulin polymerization, which belongs to tubulin polymerization agent; vincristine and colchicine can inhibit tubulin polymerization, and belongs to tubulin inhibitor.
  • the N-glycyldeacetylcolchicine of the embodiment of the present application can promote tubulin polymerization, and the effect is obviously stronger than that of paclitaxel at the same usage amount (for example, the same concentration is 10 ⁇ M). It can be seen that when the concentration of N-glycyldeacetylcolchicine is increased, for example, when the concentration is 100 ⁇ M, the obtained tubulin polymerization effect is better.
  • This embodiment discloses the pharmacokinetic characteristics of N-(N,N-diethyl)glycyl deacetylcolchicine (formula (A2) compound) single intravenous/transdermal administration in SD rats, and The administration methods of transdermal administration and intravenous injection in SD rats were compared to calculate the bioavailability, and at the same time calculate the drug content in the skin and joints of the administration site.
  • A2 compound N-(N,N-diethyl)glycyl deacetylcolchicine
  • test animals were 20 SD rats, half male and half male, and the body weight range was 229-285g for males and 185-217g for females at the time of administration.
  • Two male and female rats were administered by intravenous injection, and eight rats were administered by transdermal application.
  • the rats in the intravenous administration group were injected with the corresponding dose of the test product (select 1 mL sterile syringe) by tail vein injection, and the dose was calculated according to the body weight obtained recently.
  • the skin of the right ankle joint of the rats in the transdermal administration group was evenly smeared with the test product.
  • the technician grabbed the rats, fixed the administration site, and left it in the cold for about 5 minutes. Cover the administration site with 2 to 4 layers of gauze, and fix it with non-irritating adhesive tape. From the fixed time until the last time point sample is collected, observe the fixed site several times after administration to avoid animal friction.
  • Blood sample collection the time points of blood collection in the intravenous administration group (two male and female) were before administration, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, and 24 hours after administration .
  • the time points of blood collection in the transdermal administration group (2 males and females each) were before administration, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 12 hours and 24 hours after administration.
  • Skin tissue collection Animals were euthanized 3 hours, 8 hours, and 24 hours after administration, and skin tissues at the administration site were collected, 4 rats (2 males and 2 males) at each time point. All blank EP tubes were weighed in advance and placed in crushed ice. After adding the collected tissue, it was stored in an ultra-low temperature refrigerator. Add 9 times the volume of normal saline and 4–6 magnetic beads to the weighed tissue, homogenize on a homogenizer, check whether there are unbroken samples, and repeat the operation until the samples are evenly broken by visual inspection. The homogenate was stored in an ultra-low temperature refrigerator until analysis.
  • Skeletal tissue collection Animals were euthanized 3 hours, 8 hours, and 24 hours after administration. After collecting the skin tissue at the administration site, the bone tissue of the right ankle joint was collected, and 4 rats (2 males and 2 males) were collected at each time point. All blank EP tubes were weighed in advance and placed in crushed ice. After adding the collected tissue, it was stored in an ultra-low temperature refrigerator. Add 4 times the volume of normal saline and 4–6 magnetic beads to the weighed tissue, homogenize on a homogenizer, check whether there are unbroken samples, and repeat the operation until the samples are evenly broken by visual inspection. The homogenate was stored in an ultra-low temperature refrigerator until analysis.
  • the content of the compound of formula (A2) in plasma was detected by LC-MS/MS.
  • the lower limit of quantification of plasma method is 1ng/mL; the lower limit of quantification of skin tissue is 1000ng/g; the lower limit of quantification of bone tissue is 50ng/g.
  • the compound of formula (A2) was administered to SD rats by intravenous injection at a dose of 0.5mg/kg, the average elimination half-life t 1/2 was 3.26h, the extrapolated initial concentration was 249.46ng/mL, and the AUC (0-24h) was 172.28 h ⁇ ng/mL.
  • the blood concentration was low, and the average systemic exposure levels Cmax and AUC (0-24h) were 3.56ng/mL and 25.50h*ng/mL, respectively, and the absolute bioavailability The degree is 0.44%. It shows that a small amount of drug enters the blood circulation through the skin.
  • the average concentration of the compound of formula (A2) in the skin tissue of the transdermal administration group was 1713986.60, 2243680.95, 688818.18ng/g in 3 hours, 8 hours and 24 hours respectively; the compound of formula (A2) in the bone tissue in 3 hours, 8 hours and 24 hours
  • the average concentrations were 59818.79, 48116.10, and 15321.25ng/g, respectively.
  • NCA non-compartmental model
  • Each experimental animal was given a single administration of 2 mg/only of the test product (62.5 mg/mL aqueous solution of the compound of formula (A)).
  • the administration route is smearing on the skin, removing the hair on the back skin near the neck, and absorbing the aqueous solution of the compound of formula (A) with a 200 ⁇ L pipette.
  • the animals were reared in single cages in metabolic cages.
  • mice in the administration group were equally divided into four groups, and the four groups collected blood samples 0.5 hours and 1 hour, 2 hours and 3 hours, 5 hours and 8 hours, 12 hours and 24 hours after administration, respectively. 0.5mL.
  • the four groups were euthanized 1 hour, 3 hours, 8 hours, and 24 hours after administration, and the skin of the administration site was collected after euthanasia.
  • phase A is an aqueous solution of 0.1% formic acid and 5 mM ammonium acetate
  • phase B is acetonitrile.
  • the flow rate was 0.70 mL/min
  • the sample injection volume was 2 ⁇ L.
  • mobile phase gradient is 80% A phase+20% B phase when 0.50min, is 30%A phase+70%B phase when 1.00min, At 1.70min, it is 30% A phase + 70% B phase, at 1.80min it is 80% A phase + 20% B phase, and the operation stops at 2.50min.
  • mobile phase gradient is 80% A phase+20% B phase when 0.01min, is 30% A phase+70% B phase when 1.80min, is 30% when 1.70min Phase A + 70% Phase B, 80% Phase A + 20% Phase B at 1.80min, and stop at 2.50min.
  • Mass spectrometry was performed using an API 4000 mass spectrometer with an ESI source and positive ion MRM scanning.
  • the ion source is Turbo spray.
  • the concentration of the compound of formula (A2) was detected in vivo was low, and the plasma sample concentration of its theoretical metabolite colcemid was lower than the lower limit of quantification (1.25 ng/mL).
  • the drug residue of the compound of formula (A2) on the skin within 24 hours was 10.49%.
  • the graph of transdermal absorption rate of the compound of formula (A2) transdermal administration is shown in FIG. 19 .
  • skin standard curve and quality control samples take 100 ⁇ L sample and add 1500 ⁇ L methanol to mix well, then take 20 ⁇ L and add 1500 ⁇ L internal standard working solution (200 ng/mL terfenadine + 10 ⁇ g/mL hesperetin + 5 ⁇ g/ ml of tolbutamide in acetonitrile), vortexed for 1 min, and then centrifuged at 15400 g for 10 min at 4°C.
  • phase A is an aqueous solution of 0.1% formic acid and 5 mM ammonium acetate
  • phase B is acetonitrile.
  • the flow rate was 0.70 mL/min, and the sample injection volume was 0.5 ⁇ L.
  • the mobile phase gradient is 70% Phase A + 30% Phase B at 0.30min, 20% Phase A + 80% Phase B at 0.90min, 20% Phase A + 80% Phase B at 1.70min, and Phase A at 1.90 Min is 70% A phase + 30% B phase, and the operation stops at 2.50 min.
  • Mass spectrometry was performed using an API 4000 mass spectrometer with an ESI source and positive ion MRM scanning.
  • CAD 10 psi
  • CUR 30 psi
  • Gas1 55 psi
  • Gas2 55 psi
  • IS 5500V
  • the transdermal absorption rate diagram of the compound of formula (A1) administered through the skin is shown in FIG. 20A ; the diagram of the transdermal absorption rate of the compound of formula (A3) administered through the skin is shown in FIG. 20B .
  • skin standard curve and quality control samples take 100 ⁇ L sample and add 1500 ⁇ L methanol to mix well, then take 20 ⁇ L and add 1500 ⁇ L internal standard working solution (200 ng/mL terfenadine + 10 ⁇ g/mL hesperetin + 5 ⁇ g/ ml of tolbutamide in acetonitrile), vortexed for 1 min, and then centrifuged at 15400 g for 10 min at 4°C.
  • phase A is an aqueous solution of 0.1% formic acid and 5 mM ammonium acetate
  • phase B is acetonitrile.
  • the flow rate was 0.70 mL/min, and the sample injection volume was 0.5 ⁇ L.
  • the mobile phase gradient is 70% Phase A + 30% Phase B at 0.30min, 20% Phase A + 80% Phase B at 0.90min, 20% Phase A + 80% Phase B at 1.70min, and Phase A at 1.90 Min is 70% A phase + 30% B phase, and the operation stops at 2.50 min.
  • Mass spectrometry was performed using an API 4000 mass spectrometer with an ESI source and positive ion MRM scanning.
  • CAD 10 psi
  • CUR 30 psi
  • Gas1 55 psi
  • Gas2 55 psi
  • IS 5500V
  • the transdermal absorption rate graph of the compound of formula (A4) administered through the skin is shown in Figure 20C.
  • skin standard curve and quality control samples take 100 ⁇ L sample and add 1500 ⁇ L methanol to mix well, then take 20 ⁇ L and add 1500 ⁇ L internal standard working solution (200 ng/mL terfenadine + 10 ⁇ g/mL hesperetin + 5 ⁇ g/ ml of tolbutamide in acetonitrile), vortexed for 1 min, and then centrifuged at 15400 g for 10 min at 4°C.
  • phase A is an aqueous solution of 0.1% formic acid and 5 mM ammonium acetate
  • phase B is acetonitrile containing 0.1% formic acid.
  • the flow rate was 0.70 mL/min, and the sample injection volume was 0.5 ⁇ L.
  • the mobile phase gradient is 80% A phase + 20% B phase at 0.30min, 15% A phase + 85% B phase at 1.50min, 15% A phase + 85% B phase at 1.90min Min is 80% A phase + 20% B phase, and the operation stops at 2.50 min.
  • Mass spectrometry was performed using an API 5000 mass spectrometer with an ESI source and positive ion MRM scanning.
  • CAD 10 psi
  • CUR 35 psi
  • Gas1 55 psi
  • Gas2 55 psi
  • IS 5500V
  • the transdermal absorption rate graph of the compound of formula (A6) administered through the skin is shown in Figure 20D.
  • skin standard curve and quality control samples take 100 ⁇ L sample and add 1500 ⁇ L methanol to mix well, then take 20 ⁇ L and add 1500 ⁇ L internal standard working solution (200 ng/mL terfenadine + 10 ⁇ g/mL hesperetin + 5 ⁇ g/ ml of tolbutamide in acetonitrile), vortexed for 1 min, and then centrifuged at 15400 g for 10 min at 4°C.
  • phase A is an aqueous solution of 0.1% formic acid and 5 mM ammonium acetate
  • phase B is acetonitrile containing 0.5% formic acid.
  • the flow rate was 0.60 mL/min, and the sample injection volume was 0.5 ⁇ L.
  • the mobile phase gradient is 85% phase A + 15% phase B at 0.30min, 5% phase A + 95% phase B at 1.00min, 5% phase A + 95% phase B at 1.70min, and phase 1.85 Min is 85% A phase + 15% B phase, and the operation stops at 2.50 min.
  • Mass spectrometry was performed using an API 4000 mass spectrometer with an ESI source and positive ion MRM scanning.
  • CAD 10 psi
  • CUR 30 psi
  • Gas1 55 psi
  • Gas2 55 psi
  • IS 5500V
  • the transdermal absorption rate diagram of the compound of formula (A8) administered through the skin is shown in FIG. 20E ; the diagram of the transdermal absorption rate of the compound of formula (A9) administered through the skin is shown in FIG. 20F .
  • transdermal absorption rates of the compound of formula (A1), compound of formula (A3), compound of formula (A4), compound of formula (A6), compound of formula (A8) and compound of formula (A9) are shown in Table 6.
  • formula (A1) compound, formula (A2) compound, formula (A3) compound, formula (A4) compound, formula (A6) compound, formula (A8) compound, formula (A9) compound are all Showed good transdermal absorption rate, the transdermal absorption rate after transdermal administration 24 hours is all higher than 50%, wherein formula (A2) compound, formula (A6) compound, formula (A8) compound are in transdermal The transdermal absorption rate after 24 hours of administration is higher than 80%, and the transdermal absorption rate of the compound of formula (A2) is the most prominent.
  • Example 8 N-glycyl deacetylcolchicine, N-(N,N-diethyl) glycyl deacetylcolchicine, N-glycyl deacetyl-10-demethylcolchicine , N-(N,N-diethyl)glycyl deacetyl-10-demethylcolchicine in vitro efficacy test
  • leukocyte chemoattractant peptide (fMLP) (F3506-5MG sigma, MW 473.5) with a working concentration of 10 ⁇ M, and DMSO solution as a positive control; respectively prepare N-glycyl deacetylcolchicine (formula (A0) compound with a working concentration of 10 ⁇ M ), N-(N,N-diethyl)glycyl deacetylcolchicine (compound of formula (A2)), N-glycyl deacetyl-10-demethylcolchicine (formula (A5) compound), N-(N,N-diethyl)glycyl deacetyl-10-demethylcolchicine (compound of formula (A7)) as experimental drugs.
  • fMLP leukocyte chemoattractant peptide
  • Chemokines and serum-free medium system were added to each well, grouped as follows:
  • formula (A0) compound, formula (A2) compound, formula (A5) compound, formula (A7) compound can all inhibit the growth of leukemia cell line, and wherein formula (A0) compound and formula (A2) compound can Very significantly inhibit the growth of leukemia cell lines, p ⁇ 0.01.
  • leukocyte chemoattractant peptide (fMLP) (F3506-5MG sigma, MW 473.5) with a working concentration of 10 ⁇ M, and DMSO solution as a positive control; respectively prepare N-glycyl deacetylcolchicine (formula (A0) compound with a working concentration of 10 ⁇ M ), N-(N,N-diethyl)glycyl deacetylcolchicine (compound of formula (A2)), N-glycyl deacetyl-10-demethylcolchicine (formula (A5) compound), N-(N,N-diethyl)glycyl deacetyl-10-demethylcolchicine (compound of formula (A7)) as experimental drugs.
  • fMLP leukocyte chemoattractant peptide
  • Chemokines and serum-free medium system were added to each well, grouped as follows:
  • Colchicine solution preparation Weigh 1mg of colchicine, dissolve in 100mL of pure water, vortex and ultrasonically mix to form a uniform suspension (0.01mg/mL).
  • N-(N,N-diethyl)glycyl deacetylcolchicine solution Weigh 125mg of N-(N,N-diethyl)glycyl deacetylcolchicine (compound of formula (A2) ), dissolved in 2mL of pure water, vortexed and ultrasonically mixed into a uniform suspension solution (62.5mg/ml).
  • Rats in the compound group of formula (A2) were applied to the right ankle joint 2 hours before modeling, with a dose of 4 mg/rat, and then applied to the swollen toe once a day.
  • Rats in the other groups were orally given the corresponding drug solution 2 hours after modeling; the first dose of colchicine was 0.1 mg/kg, and then administered once every 2 hours, with a dose of 0.05 mg/kg, for a total of 5 days. After that, 0.05 mg/kg was administered once a day for 4 consecutive days.
  • the compound group of formula (A2) collected blood from the jugular vein 2 hours and 8 hours after the administration, placed it in a sodium heparin anticoagulant tube, centrifuged the plasma, and detected the concentration of the plasma test substance.
  • the paw volume was measured before inflammation and 2 hours, 5 hours, 8 hours, 11 hours, 24 hours, 48 hours, 72 hours, and 96 hours after inflammation with a toe swelling instrument.
  • a marker pen to mark the position of the ankle joint of the rat.
  • the instrument value is reset to zero and ready for measurement. Put the rat's hind legs into the water so that the marking line at the ankle joint is on the surface of the liquid.
  • step on the foot pedal and take a reading, which is the volume of the rat's foot.
  • step on the pedal again to reset and prepare for the next measurement.
  • arthritis index (AI) was scored for the swelling of the toes. Scoring criteria for arthritis index: 0 points: no swelling, normal appearance; 1 point: joint skin erythema, mild swelling, and visible bony markers; 2 points: obvious joint redness and swelling, bony markers disappear, but swelling is limited to the joint area; 3 points: Swelling of extremities outside the joints.
  • the colchicine group had significantly lower arthritis index scores (**p ⁇ 0.05 , ***p ⁇ 0.001).
  • the compound group of formula (A2) had a significant decrease in the rat arthritis index score (*p ⁇ 0.05, **p ⁇ 0.05) .
  • the rats were deeply anesthetized with CO 2 and blood was collected from the heart, and the serum IL-6 level was detected by ELISA.

Abstract

公开了摘要附图所示的化合物或其药学上可接受的盐，其中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；且R 3选自由H和甲基所组成的组。公开了摘要附图所示的化合物或其药学上可接受的盐的制备方法。公开了摘要附图所示的化合物在治疗疾病和/或症状的药物中的用途。

Description

秋水仙碱衍生物的制备方法及其用途 技术领域
本申请涉及有机化合物及其生物用途，更具体涉及秋水仙碱衍生物、其制备方法，及其在制备药物中的用途，例如制备抗炎镇痛药物的用途。
背景技术
秋水仙碱(Colchicine)是一种植物碱，最初从百合科植物秋水仙的种子和球茎所萃取。其外表一般呈现白色或淡黄色的粉末或针状晶体，有剧毒。秋水仙碱目前主要用于治疗痛风的急性发作。
秋水仙碱治疗痛风的机制为，在痛风急性发作期，关节腔内的尿酸盐结晶会引起白细胞趋化富集，但白细胞吞噬尿酸盐后却没有合适的酶来处理，反而被尿酸盐结晶损伤细胞膜，并快速释放出大量的炎性因子，这些炎性因子会使更多的白细胞富集，导致释放更多的炎性因子，从而引发严重的关节疼痛。秋水仙碱能够有效地抑制白细胞的趋化作用及吞噬活动，达到快速镇痛消炎的目的。
虽然秋水仙碱已经广泛使用，但因其具有恶心、呕吐、腹泻等副作用，而且细胞毒性大，作为临床药物使用的安全窗过窄，剂量稍大就会产生严重的副作用，故限制了其使用。因此，本领域技术人员仍然在研究寻找替代药物，甚至是效果更好的类似药物。
发明内容
在一方面，本申请公开了式(A)化合物或其药学上可接受的盐，
Figure PCTCN2022119478-appb-000001
本申请公开了式(A)化合物或其药学上可接受的盐，其中所述R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组，且所述R 3选自由H和甲基所组成的组。
在一方面，本申请公开了一种化合物或其药学上可接受的盐，例如式(A)化合物或其药学上可接受的盐的制备方法，包括将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，
Figure PCTCN2022119478-appb-000002
本申请公开了一种制备化合物或其药学上可接受的盐，例如式(A)化合物或其药学上可接受的盐，的方法，包括将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，其中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；R 3′选自由H和甲基所组成的组；且M选自由H、Li、Na、K、Rb、和Cs所组成的组。
在一方面，本申请公开了一种制备式(A)化合物的药学上可接受的盐的方法，将式(A)化合物与酸接触。
在一方面，本申请公开了式(A)化合物或其药学上可接受的盐，在制备治疗癌症、镇痛和/或抗炎的药物中的用途。
本申请的一些实施例中，提供的式(A)化合物的药学上可接受的盐，可以在一方面取得不亚于、或甚至更胜于秋水仙碱的技术效果，为这一类型的药物提供了更多样或甚至更优异的替代技术方案。
附图说明
图1示出了式(A)化合物。
图2为本申请的一些实施例中，合成N-甘氨酰去乙酰秋水仙碱的合成途径。
图3为本申请的一些实施例中所合成的N-叔丁氧羰基秋水仙碱的 1H NMR谱图。
图4为本申请的一些实施例中所合成的去乙酰秋水仙碱的 1H NMR谱图。
图5为本申请的一些实施例中所合成的N-(N-(叔丁氧羰基)甘氨酰)去乙酰秋水仙碱的 1H NMR谱图。
图6A–6C分别为本申请的一些实施例中所合成的N-甘氨酰去乙酰秋水仙碱的 1H NMR谱图、质谱检测结果、和高效液相层析测定结果。
图7为本申请的一些实施例中，合成N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱及其盐酸盐的合成途径。
图8A–8B分别为本申请的一些实施例中所合成的N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱的高效液相层析测定结果和质谱检测结果。
图8C–8E分别为本申请的一些实施例中所合成的N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱盐酸盐的 1H NMR谱图、高效液相层析测定结果、和质谱检测结果。
图9为本申请的一些实施例中所合成的N-(N,N-二甲基)甘氨酰去乙酰秋水仙碱的 1H NMR谱图。
图10为本申请的一些实施例中所合成的N-(N,N-二正丙基)甘氨酰去乙酰秋水仙碱的 1H NMR谱图。
图11A–11B分别为本申请的一些实施例中所合成的N-(N,N-二异丙基)甘氨酰去乙酰秋水仙碱的 1H NMR谱图和高效液相层析测定结果。
图12为本申请的一些实施例中，由N-甘氨酰去乙酰秋水仙碱合成N-甘氨酰去乙酰-10-去甲基秋水仙碱的合成途径。
图13A–13C分别为本申请的一些实施例中所合成的N-甘氨酰去乙酰-10-去甲基秋水仙碱的 1H NMR谱图、ESI-MS正离子模式谱图、和ESI-MS负离子模式谱图。
图14A–14F分别为本申请的一些实施例中所合成的N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱的 1H NMR谱图、 13C NMR谱图、异核单量子相关(HSQC)NMR谱图、异核多键相关(HMBC)NMR谱图、ESI-MS正离子模式谱图、和ESI-MS负离子模式谱图。
图15为本申请的一些实施例中所合成的N-(N,N-二甲基)甘氨酰去乙酰-10-去甲基秋水仙碱的 1H NMR谱图。
图16为本申请的一些实施例中所合成的N-(N,N-二正丙基)甘氨酰去乙酰-10-去甲基秋水仙碱的 1H NMR谱图。
图17为本申请的一些实施例中所合成的N-(N,N-二异丙基)甘氨酰去乙酰-10-去甲基秋水仙碱的 1H NMR谱图。
图18为本申请的一些实施例的微管蛋白聚合检测试验中，各组微管蛋白聚合反应的340nm光吸收度随时间的变化图。
图19和图20A–20F为本申请的一些实施例的透皮给药吸收效率研究中，透皮给药的透皮吸收率图。
图21为本申请的一些实施例的中性粒细胞趋化实验中，CCK8法评估细胞数目，紫外分光光度计测定OD 450 nm读数的图。
图22为本申请的一些实施例的模式细胞迁移实验中，评估迁移细胞数目的结果比较图。
图23为本申请的一些实施例的模式细胞迁移实验中，结晶紫染色后的显微镜照片。
图24A–24D为本申请的一些实施例的体内药效试验中，各组实验大鼠体重、足容积、关节炎指数、血清IL-6水平的结果比较图。
具体实施方式
为更进一步阐述本申请为了达成预定目的所采取的技术手段及功效，以下结合附图及较佳实施例，对依据本申请的具体实施方式、结构、特征及其功效，详细说明如下。
本申请中的术语“秋水仙碱衍生物”，至少包括本申请所公开的式(A)化合物(图1所示化合物)或其药学上可接受的盐，但不限于此。
在一些实施方式中，本申请公开了式(A)化合物或其药学上可接受的盐，
Figure PCTCN2022119478-appb-000003
在一些实施方式中，R 1选自由H、甲基、乙基、正丙基、和异丙基所组成的组。在一些实施方式中，R 2选自由H、甲基、乙基、正丙基、和异丙基所组成的组。在一些实施方式中，R 3选自由H和甲基所组成的组。在一些实施方式中，R 1为H。在一些实施方式中，R 1为甲基。在一些实施方式中，R 1为乙基。在一些实施方式中，R 1为正丙基。在一些实施方式中，R 1为异丙基。在一些实施方式中，R 2为H。在一些实施方式中，R 2为甲基。在一些实施方式中，R 2为乙基。在一些实施方式中，R 2为正丙基。在一些实施方式中，R 2为异丙基。在一些实施方式中，R 3为H。在一些实施方式中，R 3为甲基。在一些实施方式中，R 1选自由H以和乙基所组成的组。在一些实施方式中，R 2选自由H以和乙基所组成的组。在一些实施方式中，R 1和R 2皆为H，或R 1和R 2皆为乙基。在一些实施方式中，R 1和R 2皆为H。在一些实施方式中，R 1和R 2皆为甲基。在一些实施方式中，R 1和R 2皆为乙基。在一些实施方式中，R 1和R 2皆为正丙基。在一些实施方式中，R 1和R 2皆为异丙基。
在一些实施方式中，所述R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组，且所述R 3选自由H和甲基所组成的组。在一些实施方式中，R 1和R 2相同。在一些实施方式中，R 1和R 2相同且选自由H、甲基、乙基、正丙基、和异丙基所组成的组。在一些实施方式中，R 1和R 2相同。在一些实施方式中，R 1和R 2相同且选自由甲基、乙基、正丙基、和异丙基所组成的组。
在一些实施方式中，式(A)化合物选自由式(A0)化合物至式(A9)化合物所组成的组，
Figure PCTCN2022119478-appb-000004
Figure PCTCN2022119478-appb-000005
在一些实施方式中，式(A)化合物选自由式(A0)化合物、式(A1)化合物、式(A2)化合物、式(A3)化合物、式(A4)化合物、式(A6)化合物、式(A7)化合物、式(A8)化合物、和式(A9)化合物所组成的组，
在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，选自由甲磺酸盐、硫酸盐、对甲苯磺酸盐、盐酸盐、琥珀酸盐、马来酸盐、富马酸盐所组成的组。在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的甲磺酸盐。在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的硫酸盐。在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的对甲苯磺酸盐。在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的琥珀酸盐。在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的马来酸盐。在一些实施方式中，本申请的式(A)化合物的药学上可接受的盐，是式(A)化合物的富马酸盐。
在一些实施方式中，本申请公开了一种化合物或其药学上可接受的盐，例如式(A)化合物或其药学上可接受的盐的制备方法，包括将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，
Figure PCTCN2022119478-appb-000006
在一些实施方式中，R 3′选自由H及甲基所组成的组。在一些实施方式中，M选自由H、Li、Na、K、Rb、及Cs所组成的组。在一些实施方式中，R 3′为甲基。在一些实施方式中，R 3选自由H和甲基所组成的组，且R 3′为甲基。在一些实施方式中，R 3为H，且R 3′为甲基。在一些实施方式中，R 3与R 3′相同。在一些实施方式中，R 3和R 3′皆为甲基。在一些实施方式中，M为H。在一些实施方式中，M选自由Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，所述式(C)化合物中，M选自由Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，M为Li。在一些实施方式中，M为Na。在一些实施方式中，M为K。在一些实施方式中，M为Rb。在一些实施方式中，M为Cs。在一些实施方式中，所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物是式(C)中R 1和R 2中的H的至少一个被保护基保护。在一些实施方式中，所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物是式(C)中R 1和R 2中的一个或二个H的其中一个被保护基保护。在一些实施方式中，在所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物中，M为H。在一些实施方式中，所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组。在一些实施方式中，所述保护基为叔丁氧羰基。在一些实施方式中，所述保护基为苄氧羰基。在一些实施方式中，所述保护基为9-芴甲氧羰基。在一些实施方式中，所述保护基为苯甲基。在一些实施方式中，所述保护基为对甲氧苯基。
在一些实施方式中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组；R 3′选自由H和甲基所组成的组；且M选自由H、Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，所述式(C)化合物中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；R 3′选自由H和甲基所组成的组；且M选自由Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物中，R 1 和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组；R 3′选自由H和甲基所组成的组；且M为H。在一些实施方式中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组；R 3′选自由H和甲基所组成的组；且M选自由H、Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，式(B)化合物是与式(C)化合物接触，且所述式(C)化合物中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；R 3′选自由H和甲基所组成的组；且M选自由Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，式(B)化合物是与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物中，R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组；R 3′选自由H和甲基所组成的组；且M为H。
在一些实施方式中，将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，包括将式(B)化合物、和式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物，与选自由2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯、1-羟基苯并三唑、和4-二甲氨基吡啶所组成的组的至少一个接触。在一些实施方式中，M为H，且将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，包括将式(B)化合物、和式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物，与选自由2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯、1-羟基苯并三唑、和4-二甲氨基吡啶所组成的组的至少一个接触，特别是与2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯接触。
在一些实施方式中，式(B)化合物是与式(C)化合物接触。在一些实施方式中，式(B)化合物是与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触。在一些实施方式中，所述的式(B)化合物与式(C)化合物接触，是在碱性环境下。在一些实施方式中，所述的式(B)化合物与式(C)化合物接触，是在N,N-二异丙基乙基胺、三乙胺或2,2,6,6-四甲基哌啶存在的环境下，特别是在N,N-二异丙基乙基胺存在的环境下。在一些实施方式中，式(B)化合物与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触时，本申请的制备式(A)化合物的方法还包括将式(B)化合物与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触的产物，与选自由三氟乙酸、盐酸、和氢溴酸所组成的组中的至少一个接触，特别是与三氟乙酸接触。在一些实施方式中，式(B)化合物与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，且本申请的制备式(A)化合物的方法还包括将式(B)化合物与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触的产物，与选自由三氟乙酸、盐酸、和氢溴酸所组成的组中的至少一个接触，特别是与三氟乙酸接触。在一些实施方式中，当R 3′为甲基且R 3为H时，本申请的制备式(A)化合物的方法还包括将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触的产物，与盐酸，特别是盐酸和乙酸，接触。在一些实施方式中，R 3′为甲基，R 3为H，且本申请的制备式(A)化合物的方法还包括将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触的产物，与盐酸，特别是盐酸和乙酸，接触。
在一些实施方式中，本申请的制备式(A)化合物的方法还包括式(B1)化合物与选自由二碳酸二叔丁酯、氯甲酸苄酯、氯甲酸-9-芴甲酯所组成的组所组成的组中的至少一个接触，得到式(B2)化合物，
Figure PCTCN2022119478-appb-000007
在一些实施方式中，所述L选自由叔丁氧羰基、苄氧羰基、和9-芴甲氧羰基所组成的组，特别是叔丁氧羰基。在一些实施方式中，本申请的制备式(A)化合物的方法包括式(B1)化合物与二碳酸二叔丁酯接触，所述L为叔丁氧羰基。在一些实施方式中，本申请的制备式(A)化合物的方法包括式(B1)化合物与氯甲酸苄酯接触，所述L为苄氧羰基。在一些实施方式中，本申请的制备式(A)化合物的方法包括式(B1)化合物与氯甲酸-9-芴甲酯接触，所述 L为9-芴甲氧羰基。
在一些实施方式中，本申请的制备式(A)化合物的方法还包括式(B2)化合物与选自由R′OM′、水合肼、硼氢化钠所组成的组的至少一个接触，特别是与R′OM′接触，得到式(B3)化合物，
Figure PCTCN2022119478-appb-000008
在一些实施方式中，R′选自由甲基、乙基、正丙基、和异丙基所组成的组。在一些实施方式中，R′为乙基。在一些实施方式中，M′选自由Li、Na、K、Rb、和Cs所组成的组。在一些实施方式中，M′为钠。在一些实施方式中，R′OM′为乙醇钠。
在一些实施方式中，本申请的制备式(A)化合物的方法还包括式(B3)化合物与选自由三氟乙酸、盐酸、和氢溴酸所组成的组中的至少一个接触，特别是与三氟乙酸接触，得到式(B)化合物。在一些实施方式中，盐酸的浓度大于等于1M。在一些实施方式中，氢溴酸的浓度大于等于1M。在一些实施方式中，盐酸的浓度为1M至2M。在一些实施方式中，氢溴酸的浓度为1M至2M。
在一些实施方式中，本申请公开了一种制备式(A)化合物的药学上可接受的盐的方法，将式(A)化合物与酸接触。在一些实施方式中，本申请的制备式(A)化合物的药学上可接受的盐的方法还包括制备式(A)化合物。在一些实施方式中，本申请的制备式(A)化合物的药学上可接受的盐的方法还包括根据本申请的制备式(A)化合物的方法制备式(A)化合物。
在一些实施方式中，本申请公开了一种治疗疾病和/或症状的方法，其中包括施用式(A)化合物或其药学上可接受的盐至有需要的个体。在一些实施方式中，本申请公开了一种用于治疗疾病和/或症状的药物，其中包括式(A)化合物或其药学上可接受的盐。在一些实施方式中，本申请公开了式(A)化合物或其药学上可接受的盐，在制备治疗疾病和/或症状的药物中的用途。在一些实施方式中，本申请公开了式(A)化合物或其药学上可接受的盐，在治疗疾病和/或症状中的用途。
在一些实施方式中，所述的治疗疾病和/或症状选自治疗癌症、镇痛和抗炎中的至少一种。在一些实施方式中，所述的治疗疾病和/或症状选自抗炎和镇痛中的至少一种。在一些实施方式中，所述疾病和/或症状选自癌症和关节炎所组成的组中的至少一种。在一些实施方式中，所述疾病和/或症状为癌症。在一些实施方式中，所述疾病和/或症状为炎症。在一些实施方式中，所述疾病和/或症状为关节炎。在一些实施方式中，所述疾病和/或症状选自卵巢癌、乳癌、肺癌、卡波西氏肉瘤、子宫颈癌、胰脏癌、风湿病、急性关节炎、和急性痛风性关节炎中的至少一种。在一些实施方式中，所述疾病和/或症状选自卵巢癌、乳癌、肺癌、卡波西氏肉瘤、子宫颈癌、和胰脏癌中的至少一种。在一些实施方式中，所述疾病和/或症状选自风湿病、急性关节炎、和急性痛风性关节炎中的至少一种。
实施例1 N-甘氨酰去乙酰秋水仙碱的合成
在一些实施方式中，由秋水仙碱合成N-甘氨酰去乙酰秋水仙碱的合成途径，如图2所示。具体为：
秋水仙碱(式(I)化合物)
→N-叔丁氧羰基秋水仙碱(式(II)化合物)
→N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)
→去乙酰秋水仙碱(式(IV)化合物)
→N-(N-叔丁氧羰基)甘氨酰去乙酰秋水仙碱(式(V)化合物)
→N-甘氨酰去乙酰秋水仙碱(式(A0)化合物)
在一些实施方式中，由秋水仙碱合成N-甘氨酰去乙酰秋水仙碱的合成途径，也可以由N-叔丁氧羰基秋水仙碱(式(II)化合物)、N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)、去乙酰秋水仙碱(式(IV)化合物)、或N-(N- 叔丁氧羰基)甘氨酰去乙酰秋水仙碱(式(V)化合物)起始。
具体的合成步骤如下所述。
1、秋水仙碱引入叔丁氧羰基(Boc)保护基，得到N-叔丁氧羰基秋水仙碱
【反应步骤】
在三口烧瓶中，加入秋水仙碱(式(I)化合物)3.1g(1当量)、4-二甲氨基吡啶(DMAP)0.95g(1当量)、三乙胺2.2mL(2.0当量)、乙腈40mL，氮气置换三次，氮气保护。
接着，分三次加入二碳酸二叔丁酯(Boc 2O)7.54g(4.5当量)，每次各反应2小时，再将体系升温到90℃回流，共反应7小时。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1(v/v)作为溶剂进行薄层色谱(TLC)，可以看到产生一极性较小的点，即为产物。
【反应后处理】
回旋浓缩去除体系中的乙腈。
剩余物料加入二氯甲烷稀释，并使用10％柠檬酸水溶液洗涤一次、饱和盐水洗涤一次。
所得有机相加入无水硫酸钠干燥，旋干。
所得粗品进行柱层析，以二氯甲烷∶甲醇＝50∶1的流动相过出产品；并可以用二氯甲烷∶甲醇＝20∶1的流动相进一步冲出未反应的反应物。
最终所得产物N-叔丁氧羰基秋水仙碱(式(II)化合物)约2.2g。
【产物检测】
所得产物使用 1H NMR检测，结果如图3，确认为N-叔丁氧羰基秋水仙碱(式(II)化合物)。
2、N-叔丁氧羰基秋水仙碱去除乙酰基，得到N-叔丁氧羰基去乙酰秋水仙碱
【反应步骤】
在三口烧瓶中，加入N-叔丁氧羰基秋水仙碱(式(II)化合物)3.8g(1当量)、甲醇20mL，氮气置换三次，氮气保护。
将体系冷至0℃，滴加甲醇钠甲醇溶液(甲醇钠0.62g，1.5当量)，搅拌30分钟。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1(v/v)作为溶剂进行薄层色谱，可以看到产生一离得很近的极性大的点，即为产物。
【反应后处理】
加10％柠檬酸水溶液，将pH调至6左右。
再加二氯甲烷萃取三次，有机相加饱和盐水洗涤一次，加无水硫酸钠干燥，旋干，得N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)粗品3.9g。
3、N-叔丁氧羰基去乙酰秋水仙碱脱保护，得到去乙酰秋水仙碱
【反应步骤】
在单口烧瓶中，加入N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)粗品3.9g(1当量)、二氯甲烷20mL、三氟乙酸9mL，室温搅拌3小时。
【反应后处理】
体系加入二氯甲烷稀释，加入饱和碳酸钠水溶液将pH值调至10。
将所得体系分液，其中水相加入二氯甲烷萃取多次，将萃取所得的多次有机相与分液所得的有机相合并。
加入无水硫酸钠干燥，旋干，得去乙酰秋水仙碱(式(IV)化合物)粗品2.8g。
【产物检测】
所得产物使用 1H NMR检测，结果如图4，确认为去乙酰秋水仙碱(式(IV)化合物)。
4、去乙酰秋水仙碱与N-(叔丁氧羰基)甘氨酸反应，得到N-(N-叔丁氧羰基)甘氨酰去乙酰秋水仙碱
【反应步骤】
在三口烧瓶中，加入N-(叔丁氧羰基)甘氨酸1.51g(1.1当量)、2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯(HATU)3.28g(1.1当量)、三乙胺1.59g(2.0当量)、二氯甲烷30mL，氮气保护，室温搅拌1小时。
再加入去乙酰秋水仙碱(式(IV)化合物)2.8g(1当量)，室温搅拌过夜。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1(v/v)作为溶剂进行薄层色谱，可以看到产生一极性小的点，即为产物。反应物的点消失，反应即可结束。
【反应后处理】
体系加入二氯甲烷稀释，并使用10％柠檬酸水溶液洗涤一次、饱和盐水洗涤一次。
有机相加无水硫酸钠干燥，旋干。
所得粗品进行柱层析，以二氯甲烷∶甲醇＝40∶1的流动相过出产物N-(N-(叔丁氧羰基)甘氨酰)去乙酰秋水仙碱(式(V)化合物)2g。
【产物检测】
所得产物使用 1H NMR检测，结果如图5，确认为N-(N-(叔丁氧羰基)甘氨酰)去乙酰秋水仙碱(式(V)化合物)。
5、N-(N-(叔丁氧羰基)甘氨酰)去乙酰秋水仙碱脱保护，得到N-甘氨酰去乙酰秋水仙碱
【反应步骤】
在250mL单口烧瓶中，加入二氯甲烷10mL、N-(N-(叔丁氧羰基)甘氨酰)去乙酰秋水仙碱2.0g(1当量)、三氟乙酸4mL，氮气保护，室温搅拌3小时。
【反应检测】
使用薄层色谱可以看到产生一极性大的点，即为产物。反应物的点消失，反应即可结束。
【反应后处理】
体系加入二氯甲烷稀释，加入饱和碳酸钠水溶液将pH值调至10。
将所得体系分液，其中水相加入二氯甲烷萃取三次，将萃取所得的三次有机相与分液所得的有机相合并。
加入无水硫酸钠干燥，旋干。
所得粗品进行柱层析，以二氯甲烷∶甲醇＝8∶1，外加2滴三乙胺的流动相过出产物N-甘氨酰去乙酰秋水仙碱(式(A0)化合物)1.15g。
【产物检测】
所得产物使用 1H NMR检测，结果如图6A，确认为N-甘氨酰去乙酰秋水仙碱(式(A0)化合物)。
使用质谱检测结果如图6B，最高峰415.20所推得的分子量414.2能确认产物为N-甘氨酰去乙酰秋水仙碱。
使用高效液相层析(HPLC)测定，结果如图6C，产物纯度为97.3％。
实施例2 N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱及其盐酸盐的合成
在一些实施方式中，由秋水仙碱合成N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱及其盐酸盐的合成途径，如图7所示。此处所述“烷基”，即图7中的R 1和R 2，例如可以是H、甲基、乙基、正丙基或异丙基，但不限于此。具体为：
秋水仙碱(式(I)化合物)
→N-叔丁氧羰基秋水仙碱(式(II)化合物)
→N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)
→去乙酰秋水仙碱(式(IV)化合物)
→N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱(式(A′)化合物)
此外，还可以对N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱转盐，得到N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱盐酸盐。
本领域技术人员应当可以理解，实施例1及实施例2中由秋水仙碱制备去乙酰秋水仙碱的方法，可以相互替换、或其等效替换，而不会导致无法得到之后对应的实施例的化合物。
制备实施例2-1 N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱及其盐酸盐的合成
以下以烷基为乙基为例，即合成N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(其R 1和R 2为乙基的式(A′)化合物，式(A2)化合物)及其盐酸盐。但本实施例不限于此，应视为烷基为H、甲基、正丙基、异丙基，也可以通过对应的方法进行反应，得到对应的N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱及其盐酸盐，例如是N-(N,N-二甲基)甘氨酰去乙酰秋水仙碱及其盐酸盐、N-(N,N-二正丙基)甘氨酰去乙酰秋水仙碱及其盐酸盐、以及N-(N,N-二异丙基)甘氨酰去乙酰秋水仙碱及其盐酸盐。
本制备实施例具体的合成步骤如下所述。
1、秋水仙碱引入叔丁氧羰基(Boc)保护基，得到N-叔丁氧羰基秋水仙碱
【反应步骤】
在三口烧瓶中，加入秋水仙碱(式(I)化合物)30g(1当量)、4-二甲氨基吡啶(DMAP)9.6g(1.08当量)、三乙胺20.4mL(1.94当量)、乙腈300mL(10V)中，氮气置换三次，氮气保护。
接着，分三次加入二碳酸二叔丁酯(Boc 2O)69g(4.2当量)，每次各反应2小时，再将体系升温到90℃回流，共反应7小时。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1(v/v)作为溶剂进行薄层色谱，可以看到产生一极性较小的点，即为产物。
【反应后处理】
回旋浓缩去除体系中的乙腈。
剩余物料加入二氯甲烷稀释，并使用10％柠檬酸水溶液洗涤一次、饱和盐水洗涤一次。
所得有机相加入无水硫酸钠干燥，旋干。
所得粗品进行柱层析，以二氯甲烷∶甲醇＝40∶1的流动相过出产品；并可以用二氯甲烷∶甲醇＝10∶1的流动相进一步冲出未反应的反应物。
最终所得产物N-叔丁氧羰基秋水仙碱(式(II)化合物)约32.5g，回收原料6.2g。
2、N-叔丁氧羰基秋水仙碱去除乙酰基，得到N-叔丁氧羰基去乙酰秋水仙碱
【反应步骤】
在三口烧瓶中，加入N-叔丁氧羰基秋水仙碱(式(II)化合物)32.5g(1当量)、甲醇300mL(10V)，氮气置换三次，氮气保护。
将体系冷至0℃，滴加甲醇钠甲醇溶液(甲醇钠5.3g，1.5当量)，0℃搅拌2小时。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1(v/v)作为溶剂进行薄层色谱，可以看到原料几乎消失，而产生一离得很近的极性大的点，即为产物。
【反应后处理】
加10％柠檬酸水溶液，将pH调至6左右。
再加二氯甲烷萃取三次，有机相加饱和盐水洗涤一次，加无水硫酸钠干燥，旋干，得N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)粗品31.6g。
3、N-叔丁氧羰基去乙酰秋水仙碱脱保护，得到去乙酰秋水仙碱
【反应步骤】
在单口烧瓶中，加入N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)粗品31.6g(1当量)、二氯甲烷300mL(10V)、三氟乙酸120mL(4V)，室温搅拌3小时。
【反应后处理】
体系加入二氯甲烷稀释，加入饱和碳酸钠水溶液将pH值调至10。
将所得体系分液，其中水相加入二氯甲烷萃取三次，将萃取所得的多次有机相与分液所得的有机相合并。
加入无水硫酸钠干燥，旋干，得去乙酰秋水仙碱(式(IV)化合物)粗品24.8g。
4、去乙酰秋水仙碱与N,N-二乙基甘氨酸钠反应，得到N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱
【反应步骤】
在三口烧瓶中，加入去乙酰秋水仙碱(式(IV)化合物)24g(1当量)、N,N-二乙基甘氨酸钠12.9g(1.25当量)、2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯(HATU)38.2g(1.5当量)、三乙胺74.6g(11当量)、N,N-二甲基甲酰胺(DMF)300mL，氮气保护，升温至80℃反应过夜。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1(v/v)作为溶剂进行薄层色谱，原料和产品几乎分不开。使用HPLC作为中控手段。
【反应后处理】
体系回旋浓缩去除DMF，过柱纯化，得到产品点。
加入HCl水溶液成盐。
加入乙酸乙酯萃取杂质，取水相，加入饱和碳酸钠水溶液调整pH值至8左右。
加入二氯甲烷萃取产品，取有机相，加入无水硫酸钠干燥，回旋浓缩除去溶剂，得到褐色粘稠固体，为N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)6.17g。【产物检测】
所得产物使用液相层析－质谱(LC-MS)检测，高效液相层析结果如图8A、质谱结果如图8B。确认为N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)。
5、N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱成盐
【反应步骤】
在单口烧瓶中，加入N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱17g(1当量)、1N稀盐酸38mL(1.05当量)以调整pH值至1左右。加入乙酸乙酯萃取杂质，取水相，回旋浓缩去除溶剂，得到N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱盐酸盐。加石油醚打浆一次，油泵拉干产品，得产品重量14.7g。
【产物检测】
产物为N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱盐酸盐。
1H NMR(400MHz,D 2O)δ:7.26(s,1H),7.24(s,1H),7.04(d,J＝8Hz,1H),6.64(s,1H),4.33-4.29(t,1H),4.02-3.93(q,2H),3.81(s,3H),3.79(s,3H),3.78(s,3H),3.51(s,3H),3.13(d,J＝4Hz,4H),2.40(s,1H),2.15(s,1H),1.84(s,2H),1.15-1.12(t,6H)。
1H NMR谱图如图8C。使用液相层析－质谱(LC-MS)检测，高效液相层析结果如图8D、质谱结果如图8E。
制备实施例2-2 N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱及其盐酸盐的合成
本领域技术人员应当可以理解，制备实施例2-1及制备实施例2-2中的对应步骤可以相互替换或组合，而不会导致无法得到所期望得到的化合物。
本制备实施例具体的合成步骤如下所述。
1、秋水仙碱引入叔丁氧羰基(Boc)保护基，得到N-叔丁氧羰基秋水仙碱
【反应步骤】
秋水仙碱(式(I)化合物)1当量、4-二甲氨基吡啶2当量)溶于乙腈中，然后加入二碳酸二叔丁酯3.5当量和三乙胺1当量)，在100℃下反应1.5小时。
【反应后处理】
加入10％柠檬酸洗涤。
用二氯甲烷多次萃取，收集有机相。
有机相用饱和氯化钠洗涤，用无水硫酸钠干燥。减压浓缩得到粗品，以乙酸乙酯∶丙酮＝10∶1进行柱层析，得到棕黄色固体，为N-叔丁氧羰基秋水仙碱(式(II)化合物)。
2、N-叔丁氧羰基秋水仙碱去除乙酰基，得到N-叔丁氧羰基去乙酰秋水仙碱
【反应步骤】
将前一步骤所得的N-叔丁氧羰基秋水仙碱(式(II)化合物)1当量溶解于无水甲醇中，加入甲醇钠(4当量)，40℃搅拌40分钟。
【反应后处理】
加入适量饱和氯化铵溶液，减压以去除甲醇。
水溶液用二氯甲烷萃取，收集有机相。
有机相用饱和氯化钠洗涤，用无水硫酸钠干燥。溶剂减压蒸发得到棕黄色固体，为N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)。
3、N-叔丁氧羰基去乙酰秋水仙碱脱保护，得到去乙酰秋水仙碱
【反应步骤】
将N-叔丁氧羰基去乙酰秋水仙碱(式(III)化合物)1当量放入圆底烧瓶中，溶解于30％体积比的三氟乙酸/二氯甲烷溶液，室温搅拌1.5小时。
【反应后处理】
加入饱和碳酸钠水溶液将pH值调至8。
将所得体系分液，其中水相加入二氯甲烷萃取三次。
合并各有机相用饱和氯化钠洗涤，加入无水硫酸钠干燥。
溶剂减压蒸发，以甲醇∶二氯甲烷＝30∶1层析，得到淡黄色固体，为去乙酰秋水仙碱(式(IV)化合物)。
4、去乙酰秋水仙碱与N,N-二烷基甘氨酸反应，得到N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱
【反应步骤】
将N,N-二烷基甘氨酸1.2当量和2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯1.2当量溶于适量二氯甲烷中，将N,N-二异丙基乙基胺(DIPEA)3当量滴加入反应体系，室温搅拌10分钟。再将去乙酰秋水仙碱(式(IV)化合物)1当量溶于适量二氯甲烷中并滴加到反应体系中，室温搅拌3小时。
【反应检测】
使用薄层色谱检测反应。
【反应后处理】
加入水淬灭反应，用乙酸乙酯萃取3次，合并各有机相并用无水硫酸钠干燥，减压蒸干，得到油状粗产物。
粗产物以甲醇∶二氯甲烷＝1∶100–1∶70硅胶柱层析，得到N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱(式(A′)化合物)。
【产物检测】
N-(N,N-二甲基)甘氨酰去乙酰秋水仙碱(式(A1)化合物)的 1H NMR谱图如图9。
N-(N,N-二正丙基)甘氨酰去乙酰秋水仙碱(式(A3)化合物)的 1H NMR谱图如图10。
N-(N,N-二异丙基)甘氨酰去乙酰秋水仙碱(式(A4)化合物)的 1H NMR谱图如图11A，高效液相层析测定结果如图11B。
实施例3 N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱脱甲基，形成N-(N,N-二烷基)甘氨酰去乙酰-10-去甲基秋水仙碱
在一些实施方式中，由N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱合成N-(N,N-二烷基)甘氨酰去乙酰-10-去甲基秋水仙碱的合成途径，如图12所示。此处所述“烷基”，即图12中的R 1和R 2，例如可以是H、甲基、乙基、正丙基或异丙基，但不限于此。具体为：
N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱(式(A′)化合物)
→N-(N,N-二烷基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A″)化合物)
本领域技术人员应当可以理解，制备实施例3-1、制备实施例3-2和制备实施例3-3中的对应步骤可以相互替换或组合，而不会导致无法得到所期望得到的化合物。
制备实施例3-1 N-甘氨酰去乙酰秋水仙碱脱甲基，形成N-甘氨酰去乙酰-10-去甲基秋水仙碱
以下以烷基为H为例。具体为：
N-甘氨酰去乙酰秋水仙碱(式(A0)化合物)
→N-甘氨酰去乙酰-10-去甲基秋水仙碱(式(A5)化合物)
本制备实施例具体的合成步骤如下所述。
【反应步骤】
将N-甘氨酰去乙酰秋水仙碱300mg(0.72mmol)置于圆底烧瓶中，在常温下加入0.1M HCl 18mL，搅拌溶解至澄清，加入乙酸0.3mL(8.75mmol)，加热至100℃反应。
【反应检测】
使用二氯甲烷∶甲醇＝10∶1作为溶剂进行薄层色谱。反应24小时，发现原料消失。
【反应后处理】
降温至常温，加入饱和碳酸氢钠溶液8mL调节pH值至7。
用二氯甲烷20mL萃取3次，合并有机相。
将有机相以饱和氯化钠溶液20mL洗涤，无水硫酸镁干燥，过滤，减压蒸除二氯甲烷，得到棕色油状物粗品。
使用HPLC制备色谱纯化，得到棕色固体43mg，收率15％。
【产物检测】
熔点173.8–174.9℃。
1H NMR(400MHz,DMSO-d 6)δ:8.84(s,1H),7.11(m,2H),6.86(m,1H),6.77(d,J＝10.1Hz,1H),4.56–4.24(m,1H),3.82(s,3H),3.77(s,3H),3.76(s,3H),2.29–2.10(m,2H),2.08-2.01(m,2H)。
电喷雾电离源质谱(ESI-MS)(m/z):401[M+H] +,423[M+Na] +,399[M-H] -。
1H NMR谱图见图13A。ESI-MS正离子模式谱图见图13B，ESI-MS负离子模式谱图见图13C。
制备实施例3-2 N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱脱甲基，形成N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱
以下以烷基为乙基为例。具体为：
N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)
→N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A7)化合物)
本制备实施例具体的合成步骤如下所述。
【反应步骤】
将N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱300mg(0.72mmol)置于圆底烧瓶中，在常温下加入0.1M HCl 20mL，搅拌溶解至澄清，加入乙酸3mL，加热至100℃反应。
【反应检测】
使用二氯甲烷∶甲醇＝7∶1作为溶剂进行薄层色谱。反应48小时，发现原料消失。
【反应后处理】
降温至常温，加入饱和碳酸氢钠溶液8mL调节pH值至7。
用二氯甲烷30mL萃取3次，合并有机相。
将有机相以饱和氯化钠溶液30mL洗涤，无水硫酸镁干燥，过滤，减压蒸除二氯甲烷，得到棕色油状物粗品。
使用HPLC制备色谱纯化，得到棕色固体191mg，为N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱，收率71％。
【产物检测】
产物为N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A7)化合物)。
熔点83.7–84.8℃。
1H NMR(400MHz,DMSO-d 6)δ:8.79(s,1H),7.36(d,J＝12.7Hz,2H),7.17(d,J＝11.8Hz,1H),6.81(s,1H),4.42(dt,J＝13.9,7.1Hz,1H),3.85(s,3H),3.80(s,3H),3.54(s,3H),2.85–2.46(m,6H),2.30–1.85(m,4H),1.05(t,J＝7.0Hz,6H)。
13C NMR(151MHz,DMSO-d 6)δ:172.69,168.76,153.61,150.66,149.79,141.21,140.72,135.21,134.57,126.14,125.06,118.55,108.37,61.23,61.20,56.36,52.08,48.38,37.26,29.70,11.58。
ESI-MS(m/z):457[M+H] +,455[M-H] -。
1H NMR谱图见图14A， 13C NMR谱图见图14B。异核单量子相关(HSQC)NMR谱图见图14C，异核多键相关(HMBC)NMR谱图见图14D。ESI-MS正离子模式谱图见图14E，ESI-MS负离子模式谱图见图14F。
制备实施例3-3 N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱脱甲基，形成N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱
以下以烷基为甲基、正丙基、异丙基为例。具体为：
N-(N,N-二甲基)甘氨酰去乙酰秋水仙碱(式(A1)化合物)
→N-(N,N-二甲基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A6)化合物)
N-(N,N-二正丙基)甘氨酰去乙酰秋水仙碱(式(A3)化合物)
→N-(N,N-二正丙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A8)化合物)
N-(N,N-二异丙基)甘氨酰去乙酰秋水仙碱(式(A4)化合物)
→N-(N,N-二异丙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A9)化合物)
本制备实施例具体的合成步骤如下所述。
【反应步骤】
将N-(N,N-二烷基)甘氨酰去乙酰秋水仙碱溶于适量的0.15N稀盐酸，加热至100℃反应约4小时。
【反应检测】
使用薄层色谱检测反应，直到反应物消失。
【反应后处理】
将稀盐酸减压蒸干、柱层析纯化，得到N-(N,N-二烷基)甘氨酰去乙酰-10-去甲基秋水仙碱。
【产物检测】
N-(N,N-二甲基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A6)化合物)的 1H NMR谱图如图15。
N-(N,N-二正丙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A8)化合物)的 1H NMR谱图如图16。
N-(N,N-二异丙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A9)化合物)的 1H NMR谱图如图17。
实施例4 N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱的成盐研究
1、与不同酸的成盐能力
取适量的N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)，溶于1.5倍有机溶剂中。
滴加酸，60℃搅拌30分钟，缓慢冷却至0至10℃，观察有无固体析出。
加入的酸以及其成盐结果，如表1。
表1
Figure PCTCN2022119478-appb-000009
式(A2)化合物与常见的酸都能成盐。实施例中的盐制备量较少，因此未观察到盐直接从溶剂中析晶出来，而是通过减压浓缩后得到。
2、盐的重结晶能力
取适量的以下七种盐溶于适量溶剂中：
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱甲磺酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱硫酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱对甲苯磺酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱盐酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱琥珀酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱马来酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱富马酸盐
加热至60℃搅拌30分钟，缓慢冷却至0至10℃，同时滴加适量反相溶剂，观察有无晶体析出。实验结果显示：
1)溶剂乙酸乙酯、甲基叔丁基醚或正庚烷不能使盐(以上七种盐)溶解，因此不适合选为重结晶的良溶剂；
2)溶剂丙酮、二氯甲烷、甲醇、异丙醇或水能使盐溶解，但实施例中的盐用量较少，直接冷却或滴加反相溶剂后都不能结晶析出固体。如果反相溶剂滴加稍微过量，体系立即变成粘稠状。
3)打浆实验：盐酸盐粉末固体用乙酸乙酯打浆，能够很好分散，但抽滤时滤饼很快变软，说明晶体形态不好；甲磺酸盐粉末固体用乙醚打浆，能够很好分散，但抽滤时滤饼很快变软变粘稠。
3、盐的吸湿性
取适量的以下七种盐：
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱甲磺酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱硫酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱对甲苯磺酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱盐酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱琥珀酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱马来酸盐
-N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱富马酸盐
称重，放在烧瓶里，敞口放置一段时间，观测粉末形态是否变化(与密封保存的样品比对)及是否增重。结果如表2。
表2
Figure PCTCN2022119478-appb-000010
所有7种盐都有吸湿性，吸湿性从8.3％至20.8％不等。对甲苯磺酸盐相对最少，但也有8.29％。
4、盐酸盐的水溶性
室温下，称取100mg的N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱盐酸盐放于小的玻璃容器中，振荡下边加入适量水(根据溶解度加入0.1–10mL)，观察是否溶清。
结果加入水的量为0.1mL，样品即已溶清，说明盐的溶解度>1g/ml，为极易溶解。
实施例5 N-甘氨酰去乙酰秋水仙碱微管蛋白聚合试验
在一些实施方式中，选择了在实施例1中所制造的N-甘氨酰去乙酰秋水仙碱，以及紫杉醇(paclitaxel)、长春新碱(vincristine)以及秋水仙碱作为对照组，再加上空白对照，进行微管蛋白聚合试验。
实验步骤参照Cytoskeleton公司的微管蛋白聚合试验试剂盒BK006P，具体如下：
1、试剂准备
1)通用微管蛋白缓冲液(general tubulin buffer)：pH值6.9，终浓度为哌嗪-1,4-二乙磺酸(PIPES)80mM、氯化镁2mM、乙二醇-双-(2-氨基乙醚)四乙酸(EGTA)0.5mM。存放于4℃。
2)微管蛋白甘油缓冲液(tubulin glycerol buffer)：终浓度为甘油15％的通用微管蛋白缓冲液。存放于4℃。
3)三磷酸鸟苷母液(GTP stock)：三磷酸鸟苷的100mM溶液。10μL每管等分为10份存放于-70℃。
4)微管蛋白(tubulin protein)：加入10μL三磷酸鸟苷母液和1.1mL预冷的通用微管蛋白缓冲液混匀，置于 冰上，200μL每管等分为5份存放于-70℃。
5)紫杉醇母液：配制100μL的200μM紫杉醇DMSO溶液，作为紫杉醇母液。
6)长春新碱母液：配制100μL的200μM长春新碱DMSO溶液，作为长春新碱母液。
7)秋水仙碱母液：配制100μL的200μM秋水仙碱DMSO溶液，作为秋水仙碱母液。
8)N-甘氨酰去乙酰秋水仙碱母液：配制1mL的200mM N-甘氨酰去乙酰秋水仙碱DMSO溶液，取10μL的200mM溶液加入到990μL DMSO中配制成2mM溶液；取10μL的2mM溶液加入到90μL的DMSO中配制成200μM溶液；取10μL的200μM的母液加入到90μL的DMSO中配制成20μM溶液。
2、实验步骤
1)仪器设定：开始分析之前，将平板预热至37℃，30分钟，按说明书设定酶标仪的动力学检测。
2)制备冷(4℃)的微管蛋白聚合缓冲液(tubulin polymerization buffer)：750μL通用微管蛋白缓冲液加250μL微管蛋白甘油缓冲液和10μL三磷酸鸟苷母液混合在一起，放在冰上。
3)温浴500μL通用微管蛋白缓冲液到室温。
4)将10μL的通用微管蛋白缓冲液到空白对照孔，将平板在37℃孵育2分钟。
5)将上述试剂准备中配制的200μM紫杉醇溶液、200μM长春新碱溶液、200μM秋水仙碱溶液、浓度2mM、200μM、20μM的N-甘氨酰去乙酰秋水仙碱溶液各10μL分别与各自的190μL室温通用微管蛋白缓冲液混合，得到分别含有10μM紫杉醇、10μM长春新碱、10μM秋水仙碱、100μM N-甘氨酰去乙酰秋水仙碱、10μM N-甘氨酰去乙酰秋水仙碱、1μM N-甘氨酰去乙酰秋水仙碱的各种混合液。取10μL各种混合液加入到各自的样本孔。将平板在37℃孵育2分钟。
6)解冻一瓶或数瓶200μL微管蛋白在室温中水浴1分钟，置于冰上。
7)取420μL冷的微管蛋白聚合缓冲液加入到200μL步骤(6)的微管蛋白中混合，得到终浓度为：哌嗪-1,4-二乙磺酸(PIPES)80mM、氯化镁2mM、乙二醇-双-(2-氨基乙醚)四乙酸(EGTA)0.5mM、三磷酸鸟苷1mM、甘油10.2％、3mg/mL微管蛋白。
8)每孔加入100μL的在步骤(7)中稀释好的微管蛋白溶液。
9)立即在37℃下放入读卡器，并使用上述动力学设置开始记录，测量各组微管蛋白聚合反应的340nm光吸收度随时间的变化。
3、实验结果
各组微管蛋白聚合反应的340nm光吸收度随时间的变化图，如图18所示。
可以看到，与空白组相比，紫杉醇可以促进微管蛋白聚合，属于微管蛋白聚合剂；长春新碱和秋水仙碱可以抑制微管蛋白聚合，属于微管蛋白抑制剂。
本申请实施例的N-甘氨酰去乙酰秋水仙碱可以促进微管蛋白聚合，且在相同使用量(例如浓度同样为10μM)下的效果明显地强于紫杉醇。其中可以看到，当提高N-甘氨酰去乙酰秋水仙碱的浓度时，例如以浓度为100μM进行实验时，所得到的微管蛋白聚合效果更好。
实施例6 N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱的药代动力学及组织分布试验
本实施例公开了N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)单次静脉/透皮给予SD大鼠体内的药代动力学特征，并将透皮与静脉注射给予SD大鼠的给药方式进行比较，计算生物利用度，同时计算给药部位皮肤和关节处的药物含量。
称取适量HPLC纯度99％的式(A2)化合物，加入适量的灭菌注射用水定容至终体积，使配制后终浓度为0.5和62.5mg/mL，作为静脉及透皮给药供试品。配制后的静脉给药的供试品用0.22μm的PVDF滤膜过滤后，用于给药。
试验动物为20只SD大鼠，雌雄各半，给药时体重范围为雄性229–285g，雌性185–217g。
给药方法
雌雄大鼠各2只使用静脉注射给药，各8只使用透皮涂抹给药。静脉给药组大鼠尾静脉注射给予相应剂量的供试品(选择1mL的无菌注射器)，给药量根据最近获得的体重进行计算。透皮给药组大鼠右踝关节处皮肤均匀涂抹给予供试品，给药后技术员抓住大鼠，固定给药部位，凉置5min左右。给药部位覆盖上2至4层纱布，用无刺激性的胶布进行固定，固定时间到采集完末次时间点样本，给药后多次观察给药固定部位，避免动物磨擦。
样品采集
血样采集：静脉给药组(雌雄各2只)采血时间点为给药前、给药后5分钟、15分钟、30分钟、1小时、2小时、3小时、5小时、8小时、24小时。透皮给药组(雌雄各2只)采血时间点为给药前、给药后30分钟、1小时、2小时、3小时、5小时、8小时、12小时、24小时。采血前将EDTA-K2包被的离心管置于2–8℃冰箱中或冰浴中。采血后，将采集血液加入离心管中，手动颠倒至少3次；2–8℃ 4000rpm离心10分钟，采血后2小时内完成离心。离心后，收集血浆，超低温冰箱保存。
皮肤组织采集：给药后3小时、8小时、24小时将动物安乐死，采集给药部位皮肤组织，每个时间点4只大鼠(雌雄各2只)。所有空白EP管均提前称重，置于碎冰中。加入采集后的组织后，超低温冰箱保存。向称量的组织中加入9倍体积的生理盐水，4–6粒磁珠，于均质仪上匀浆，检查是否有未破碎均匀的样本，重复操作，直至肉眼观察样本破碎均匀。匀浆液超低温冰箱保存待分析。
骨骼组织采集：给药后3小时、8小时、24小时将动物安乐死，在采集给药部位皮肤组织后，采集右踝关节骨骼组织，每个时间点4只大鼠(雌雄各2只)。所有空白EP管均提前称重，置于碎冰中。加入采集后的组织后，超低温冰箱保存。向称量的组织中加入4倍体积的生理盐水，4–6粒磁珠，于均质仪上匀浆，检查是否有未破碎均匀的样本，重复操作，直至肉眼观察样本破碎均匀。匀浆液超低温冰箱保存待分析。
血药浓度测定
采用LC-MS/MS法检测血浆中式(A2)化合物的含量。血浆方法的定量下限为1ng/mL；皮肤组织的定量下限为1000ng/g；骨骼组织的定量下限为50ng/g。
试验结果：
式(A2)化合物以0.5mg/kg的剂量静脉注射给予SD大鼠后，平均消除半衰期t 1/2为3.26h，外推起始浓度为249.46ng/mL，AUC (0-24h)为172.28h·ng/mL。以4mg/kg的剂量透皮给予SD大鼠后，血药浓度较低，平均系统暴露水平C max和AUC (0-24h)分别为3.56ng/mL、25.50h*ng/mL，绝对生物利用度为0.44％。说明有较少量的药物透皮进入血液循环。
透皮给药组皮肤组织3小时、8小时、24小时的式(A2)化合物平均浓度分别为1713986.60、2243680.95、688818.18ng/g；骨骼组织3小时、8小时、24小时的式(A2)化合物平均浓度分别为59818.79、48116.10、15321.25ng/g。
平均系统暴露水平皮肤组织与骨骼组织(AUC (0-24h)皮肤/AUC (0-24h)血浆)的比值为35925141.82/25.50＝34002.52；平均系统暴露水平骨骼组织与骨骼组织(AUC (0-24h)骨骼/AUC (0-24h)血浆)的比值为867064.21/25.50＝1408829.09；可见药物透皮给药后，有一部分药物可以进入关节组织中。
实施例7 式(A)化合物的透皮给药吸收效率研究
本实验考察大鼠皮肤给予式(A)化合物后，评价式(A)化合物及其代谢物的皮肤、血浆暴露量和式(A)化合物皮肤剩余载药量，初步研究大鼠透皮给药的吸收效率。
实验选用32只体重相近的雄性SD大鼠，给予剂量为2mg/只，皮肤涂抹单次给药。各组动物给药后在不同 时间点采集血液及给药部位皮肤、肌肉、骨骼。采用LC-MS/MS分析方法检测血浆和皮肤组织中式(A)化合物及秋水仙胺的含量。
统计血浆浓度数据，利用非房室模型法(NCA)计算药代参数。
供试品配制
配制终浓度为62.5mg/mL式(A)化合物的水溶液。
实验动物与给药
选用20只雌雄各半SD大鼠进行实验。给药时周龄6-8周。给药前测定动物体重，选择体重相近的健康动物纳入实验，体重193–206g，其中16只为给药组，4只为空白组。
每只实验动物单次给药2mg/只的供试品(62.5mg/mL式(A)化合物水溶液)。给药途径为皮肤涂抹，近颈部的背部皮肤去除被毛，200μL移液器吸取式(A)化合物水剂，给药面积大约为1.5×1.5cm 2(剃毛面积2×2cm 2)。给药后，动物代谢笼单笼饲养。
实验动物样品采集与配制
将给药组16只小鼠均分四组，四组分别在给药后0.5小时和1小时，2小时和3小时，5小时和8小时，12小时和24小时，静脉或其他方式采集血样0.5mL。四组分别在给药后1小时、3小时、8小时、24小时安乐死，安乐死后采集给药部位皮肤。
血样采集后，放入含有肝素钠和1μL PMSF乙醇溶液(40mg/mL)的EP管中，立即置于碎冰中，之后在4500rpm低温(4℃)的条件下离心5min，取上清血浆，加入4μL PMSF乙醇溶液(40mg/mL)，超低温冰箱保存待分析。给药部位皮肤采集后，取样后皮肤分别立即放入匀浆管中，加入2mL甲醇，40μL PMSF乙醇溶液(40mg/mL)，并置于干冰中，尽快转入超低温冰箱冷冻。
标准曲线及质控样品配制
以80％甲醇水溶液为稀释溶剂，配制式(A)化合物和秋水仙胺的标准曲线工作溶液和质控工作溶液。
取工作溶液5μL与45μL空白血浆混合，配制成血浆标准曲线和质控样品。取工作溶液10μL与90μL空白皮肤匀浆液混合，配制成组织标准曲线和质控样品。
液相色谱－质谱分析
【式(A2)化合物】
前述实验动物血浆样品、血浆标准曲线和质控样品，加入200μL的内标工作液(10ng/mL的特非那定的乙腈溶液)，涡旋混匀1min后，4℃，13000g条件下离心10min。取上清液进样分析。
实验动物皮肤样品、皮肤标准曲线和质控样品，加入400μL的内标工作液(10ng/mL的特非那定的乙腈溶液)，涡旋混匀1min后，4℃，13000g条件下离心10min。取上清液用50％乙腈水溶液稀释75倍后进样分析。
高效能液相色谱LC-20A,SHIMADZU；液相泵：LC-20AD；柱温箱：CTO-20AC；自动进样器：SIL-20AC；控制器：CBM-20A；脱气机：DGU-20A 5；色谱柱：ZORBAX Eclipse Plus C18 2.1*50mm，3.5μm,Agilent；预柱：Security Guard Cartridges C18 4*2.00mm,Phenomenex；柱温：室温；自动进样器洗针液：80％乙腈水；自动进样器洗针程序：Rinse Mode:Before and after aspiration；自动进样器温度：4℃。
流动相中，A相为甲酸0.1％、乙酸铵5mM的水溶液，B相为乙腈。流速为0.70mL/min，样品进样体积为2μL。对于检测式(A2)化合物血浆与秋水仙胺血浆与皮肤样品，流动相梯度在0.50min时为80％A相+20％B相，在1.00min时为30％A相+70％B相，在1.70min时为30％A相+70％B相，在1.80min时为80％A相+20％B相，至2.50min运行停止。对于检测式(A2)化合物皮肤样品，流动相梯度在0.01min时为80％A相+20％B相， 在1.80min时为30％A相+70％B相，在1.70min时为30％A相+70％B相，在1.80min时为80％A相+20％B相，至2.50min运行停止。
质谱采用带有ESI源的API 4000质谱，采用正离子MRM扫描。离子源为Turbo spray。
源参数：CAD＝10psi；CUR＝20psi；Gas1＝60psi；Gas2＝60psi；IS＝5500V；TEM＝600℃；Resolution Q1＝Unit；Resolution Q3＝Unit/Low；Pause between mass＝5.007。
化合物参数如表3。
表3
Figure PCTCN2022119478-appb-000011
结果
药代动力学参数见表4。
表4
Figure PCTCN2022119478-appb-000012
SD大鼠皮肤给予式(A2)化合物水剂2mg后，雌性、雄性及总体血浆中秋水仙碱衍生物C max均值分别为15.9ng/mL、19.5ng/mL、12.8ng/mL；AUC 0-t均值分别为211g·h/mL、162g·h/mL、186g·h/mL；t 1/2均值分别为5.36h、59.4h、10.2h。秋水仙胺的血浆样品浓度均低于定量下限(1.25ng/mL)，无法计算其药代动力学参数。
皮肤给予SD大鼠式(A2)化合物水剂后，体内检测到式(A2)化合物浓度较低，其理论代谢产物秋水仙胺的血浆样品浓度均低于定量下限(1.25ng/mL)。24小时内皮肤式(A2)化合物药物残留为10.49％。
式(A2)化合物透皮给药的透皮吸收率图，如图19所示。
【式(A1)化合物、式(A3)化合物】
实验动物皮肤样品、皮肤标准曲线和质控样品，取100μL样本加入1500μL甲醇混匀后，取20μL加入1500μL的内标工作液(200ng/mL特非那定+10μg/mL橙皮素+5μg/ml甲苯磺丁脲的乙腈溶液)，涡旋混匀1min后，4℃，15400g条件下离心10分钟。
高效能液相色谱LC0A,SHIMADZU；色谱柱：BR-C18
Figure PCTCN2022119478-appb-000013
2.1*50mm,3μm,Sepax；柱温：室温。
流动相中，A相为甲酸0.1％、乙酸铵5mM的水溶液，B相为乙腈。流速为0.70mL/min，样品进样体积为0.5μL。流动相梯度在0.30min时为70％A相+30％B相，在0.90min时为20％A相+80％B相，在1.70min时为20％A相+80％B相，在1.90min时为70％A相+30％B相，至2.50min运行停止。
质谱采用带有ESI源的API 4000质谱，采用正离子MRM扫描。
源参数：CAD＝10psi；CUR＝30psi；Gas1＝55psi；Gas2＝55psi；IS＝5500V；TEM＝550℃。
化合物参数如表5A。
表5A
Figure PCTCN2022119478-appb-000014
式(A1)化合物透皮给药的透皮吸收率图，如图20A所示；式(A3)化合物透皮给药的透皮吸收率图，如图20B所示。
【式(A4)化合物】
实验动物皮肤样品、皮肤标准曲线和质控样品，取100μL样本加入1500μL甲醇混匀后，取20μL加入1500μL的内标工作液(200ng/mL特非那定+10μg/mL橙皮素+5μg/ml甲苯磺丁脲的乙腈溶液)，涡旋混匀1min后，4℃，15400g条件下离心10分钟。
高效能液相色谱LC0A,SHIMADZU；色谱柱：BR-C18
Figure PCTCN2022119478-appb-000015
2.1*50mm,3μm,Sepax；柱温：室温。
流动相中，A相为甲酸0.1％、乙酸铵5mM的水溶液，B相为乙腈。流速为0.70mL/min，样品进样体积为0.5μL。流动相梯度在0.30min时为70％A相+30％B相，在0.90min时为20％A相+80％B相，在1.70min时为20％A相+80％B相，在1.90min时为70％A相+30％B相，至2.50min运行停止。
质谱采用带有ESI源的API 4000质谱，采用正离子MRM扫描。
源参数：CAD＝10psi；CUR＝30psi；Gas1＝55psi；Gas2＝55psi；IS＝5500V；TEM＝550℃。
化合物参数如表5B。
表5B
Figure PCTCN2022119478-appb-000016
式(A4)化合物透皮给药的透皮吸收率图，如图20C所示。
【式(A6)化合物】
实验动物皮肤样品、皮肤标准曲线和质控样品，取100μL样本加入1500μL甲醇混匀后，取20μL加入1500μL的内标工作液(200ng/mL特非那定+10μg/mL橙皮素+5μg/ml甲苯磺丁脲的乙腈溶液)，涡旋混匀1min后，4℃，15400g条件下离心10分钟。
高效能液相色谱LC0A,SHIMADZU；色谱柱：Sapphire-C18
Figure PCTCN2022119478-appb-000017
2.1*50mm,5μm,Sepax；柱温：室温。
流动相中，A相为甲酸0.1％、乙酸铵5mM的水溶液，B相为含0.1％甲酸的乙腈。流速为0.70mL/min，样品进样体积为0.5μL。流动相梯度在0.30min时为80％A相+20％B相，在1.50min时为15％A相+85％B相，在1.70min时为15％A相+85％B相，在1.90min时为80％A相+20％B相，至2.50min运行停止。
质谱采用带有ESI源的API 5000质谱，采用正离子MRM扫描。
源参数：CAD＝10psi；CUR＝35psi；Gas1＝55psi；Gas2＝55psi；IS＝5500V；TEM＝550℃。
化合物参数如表5C。
表5C
Figure PCTCN2022119478-appb-000018
Figure PCTCN2022119478-appb-000019
式(A6)化合物透皮给药的透皮吸收率图，如图20D所示。
【式(A8)化合物、式(A9)化合物】
实验动物皮肤样品、皮肤标准曲线和质控样品，取100μL样本加入1500μL甲醇混匀后，取20μL加入1500μL的内标工作液(200ng/mL特非那定+10μg/mL橙皮素+5μg/ml甲苯磺丁脲的乙腈溶液)，涡旋混匀1min后，4℃，15400g条件下离心10分钟。
高效能液相色谱LC0A,SHIMADZU；色谱柱：ZORBAX Eclipse Plus C18,2.1*50mm,3.5μm,Agilent；柱温：室温。
流动相中，A相为甲酸0.1％、乙酸铵5mM的水溶液，B相为含0.5％甲酸的乙腈。流速为0.60mL/min，样品进样体积为0.5μL。流动相梯度在0.30min时为85％A相+15％B相，在1.00min时为5％A相+95％B相，在1.70min时为5％A相+95％B相，在1.85min时为85％A相+15％B相，至2.50min运行停止。
质谱采用带有ESI源的API 4000质谱，采用正离子MRM扫描。
源参数：CAD＝10psi；CUR＝30psi；Gas1＝55psi；Gas2＝55psi；IS＝5500V；TEM＝550℃。
化合物参数如表5D。
表5D
Figure PCTCN2022119478-appb-000020
式(A8)化合物透皮给药的透皮吸收率图，如图20E所示；式(A9)化合物透皮给药的透皮吸收率图，如图20F所示。
式(A1)化合物、式(A3)化合物、式(A4)化合物、式(A6)化合物、式(A8)化合物、式(A9)化合物的透皮吸收率比较，如表6所示。
表6
Figure PCTCN2022119478-appb-000021
请参照表6，可以看到式(A1)化合物、式(A2)化合物、式(A3)化合物、式(A4)化合物、式(A6)化合物、式(A8)化合物、式(A9)化合物都表现出了良好的透皮吸收率，在透皮给药24小时后的透皮吸收率都高于50％，其中式(A2)化合物、式(A6)化合物、式(A8)化合物在透皮给药24小时后的透皮吸收率则更高于80％，而以式(A2)化合物的透皮吸收率最为突出。
实施例8 N-甘氨酰去乙酰秋水仙碱、N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱、N-甘氨酰去乙酰-10-去甲基秋水仙碱、N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱的体外药效试验
中性粒细胞趋化实验
配制工作浓度10μM的白细胞趋化肽(fMLP)(F3506-5MG sigma，MW 473.5)，DMSO溶液作为阳性对照；分别配制工作浓度10μM的N-甘氨酰去乙酰秋水仙碱(式(A0)化合物)、N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)、N-甘氨酰去乙酰-10-去甲基秋水仙碱(式(A5)化合物)、N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A7)化合物)作为实验药物。
24孔transwell chamber各孔加入100μL无血清人急性早幼粒白血病细胞株HL-60细胞悬液(每个chamber含约5×10 4个细胞，以含0.1％FBS的RPMI-1640为基底)。
各孔加入趋化因子及无血清培养基体系(以含0.1％FBS的RPMI-1640为基底，600μL)，分组如下：
1‰DMSO(阴性对照组)
10μM fMLP处理组(阳性对照组)
10μM fMLP+10μM式(A0)化合物处理组(式(A0)化合物组)
10μM fMLP+10μM式(A2)化合物处理组(式(A2)化合物组)
10μM fMLP+10μM式(A5)化合物处理组(式(A5)化合物组)
10μM fMLP+10μM式(A7)化合物处理组(式(A7)化合物组)
37℃ CO 2细胞培养箱孵育。孵育6小时后可明显观察到阳性对照组下室中存在细胞。取出chamber，将膜上细胞擦去后，置入原孔，离心，将膜下粘附细胞离心至下室，CCK8法(细胞重新悬浮，吹匀后种96孔板，100μL/孔，3复孔，体系中加入10μL CCK8，37℃ CO 2细胞培养箱孵育2小时，紫外分光光度计测定OD 450nm读数)评估细胞数目，结果如图21。
可以看到，式(A0)化合物、式(A2)化合物、式(A5)化合物、式(A7)化合物都可以抑制白血病细胞株的生长，而其中式(A0)化合物和式(A2)化合物可以非常显著抑制白血病细胞株的生长，p<0.01。
模式细胞迁移实验
配制工作浓度10μM的白细胞趋化肽(fMLP)(F3506-5MG sigma，MW 473.5)，DMSO溶液作为阳性对照；分别配制工作浓度10μM的N-甘氨酰去乙酰秋水仙碱(式(A0)化合物)、N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)、N-甘氨酰去乙酰-10-去甲基秋水仙碱(式(A5)化合物)、N-(N,N-二乙基)甘氨酰去乙酰-10-去甲基秋水仙碱(式(A7)化合物)作为实验药物。
24孔transwell chamber各孔加入100μL无血清人胚肾细胞株HEK-293T细胞细胞悬液(每个chamber含约5×10 4个细胞，以含0.1％FBS的RPMI-1640为基底)。
各孔加入趋化因子及无血清培养基体系(以含0.1％FBS的RPMI-1640为基底，600μL)，分组如下：
1‰DMSO(阴性对照组)
10μM fMLP处理组(阳性对照组)
10μM fMLP+10μM式(A0)化合物处理组(式(A0)化合物组)
10μM fMLP+10μM式(A2)化合物处理组(式(A2)化合物组)
10μM fMLP+10μM式(A5)化合物处理组(式(A5)化合物组)
10μM fMLP+10μM式(A7)化合物处理组(式(A7)化合物组)
37℃ CO 2细胞培养箱孵育。孵育16小时后可明显观察到阳性对照组已有细胞穿过chamber并附于膜下表面。取出chamber，棉签擦去上槽面未跨膜细胞，甲醇固定10分钟，5％结晶紫染色30分钟，清洗后晾干，镜下观察是否有细胞跨膜，倒置显微镜400×拍照，评估迁移细胞数目。
评估结果比较图如图22，照片如图23。由图22可以看到，式(A0)化合物、式(A2)化合物、式(A5)化合物、 式(A7)化合物都可以非常显著抑制HEK-293T细胞株的生长，p<0.01。
实施例9 N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱的体内药效试验
秋水仙碱溶液配制：称取1mg秋水仙碱，溶解于100mL纯水中，涡旋超声混匀成均匀的混悬溶液(0.01mg/mL)。
N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱溶液配制：称取125mg N-(N,N-二乙基)甘氨酰去乙酰秋水仙碱(式(A2)化合物)，溶解于2mL纯水中，涡旋超声混匀成均匀的混悬溶液(62.5mg/ml)。
实验分组
24只大鼠随机分成4组，分别为空白组
Figure PCTCN2022119478-appb-000022
模型组(Vehicle)、秋水仙碱(阳性对照药)组，式(A2)化合物(受试物)组，每组6只。各组给药方式如表7。
表7
Figure PCTCN2022119478-appb-000023
适应期过后大鼠以异氟烷麻醉，于右踝关节外侧后方为穿刺点向关节腔内注入50μL的50mg/mL尿酸钠混合液造模。式(A2)化合物组大鼠于造模前2小时涂抹于右足踝关节，剂量4mg/只，之后每天一次涂抹于足趾肿胀部位。其余组别大鼠则分别于造模后2小时经口灌胃给予相应药液；秋水仙碱首剂量是0.1mg/kg，之后每隔2小时给药一次，剂量0.05mg/kg，共5次，之后每日一次给药0.05mg/kg，连续4天。
最后一次给药后，式(A2)化合物组分别于给药后的2h、8h颈静脉采集血液置于肝素钠抗凝管中，离心分离血浆，检测血浆受试物的浓度。
式(A2)化合物对实验大鼠体重的影响
实验时每天1次称量动物体重并记录。
如表8、图24A所示，实验动物在实验的96小时内，各组大鼠体重和模型对照组相比较均未出现统计学上的显著性差异。
表8
Figure PCTCN2022119478-appb-000024
式(A2)化合物对实验大鼠足容积的影响
采用足趾肿胀仪测量致炎前和致炎后2小时、5小时、8小时、11小时、24小时、48小时、72小时、96小时的足容积。测量前，用记号笔在大鼠踝关节处画线标记位置，仪器内加入干净清水后仪器数值清零准备测量。将大鼠后肢放入水中使踝关节处的标记线位于液体表面，此时踩下脚踏读数，为大鼠足容积。测量结束后再次踩下脚踏清零准备测量下一只。
如表9、图24B所示，秋水仙碱组在造模后的8小时、11小时、24小时、48小时和72小时与模型组相比，大鼠足容积均显著降低(*p<0.05，**p<0.05，***p<0.001)。式(A2)化合物组在造模后的8小时、11小时、24小时、48小时和72小时与模型组相比，同样大鼠足容积均显著降低(*p<0.05，**p<0.05，***p<0.001)。
表9
Figure PCTCN2022119478-appb-000025
*p<0.05、 **p<0.01、 ***p<0.001，相对于模型组
式(A2)化合物对实验大鼠关节炎指数的影响
检测足容积同时对足趾肿胀进行关节炎指数(Arthritis Index，AI)评分。关节炎指数评分标准：0分：无肿胀，外观正常；1分：关节皮肤红斑，轻度肿胀，骨性标志可见；2分：关节红肿明显，骨性标志消失，但肿胀局限于关节部位；3分：关节以外肢体肿胀。
如表10、图24C所示，秋水仙碱组在造模后的8小时、11小时、24小时和48小时与模型组相比，大鼠关节炎指数评分均显著降低(**p<0.05，***p<0.001)。式(A2)化合物组在造模后的8小时、11小时、24小时和48小时与模型组相比，大鼠关节炎指数评分同样均显著降低(*p<0.05，**p<0.05)。
表10
Figure PCTCN2022119478-appb-000026
*p<0.05、 **p<0.01、 ***p<0.001，相对于模型组
式(A2)化合物对实验大鼠血清IL-6水平的影响
实验结束后，大鼠CO 2深度麻醉后心脏采血，ELISA法检测血清IL-6水平。
如表11、图24D所示，在本次实验中各组大鼠的血清IL-6和模型对照组相比较均未出现统计学上的显著性差异。
表11
Figure PCTCN2022119478-appb-000027
上述实施方式仅为本申请的优选实施方式，不能以此来限定本申请保护的范围，本领域的技术人员在本申请的基础上所做的任何非实质性的变化及替换均属于本申请所要求保护的范围。

Claims (10)

  1. 式(A)化合物或其药学上可接受的盐，
    Figure PCTCN2022119478-appb-100001
    其中，
    所述R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；且
    所述R 3选自由H和甲基所组成的组。
  2. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于，
    所述R 1与所述R 2相同；
    优选地，所述R 1和所述R 2为H；或
    优选地，所述R 1和所述R 2为甲基；或
    优选地，所述R 1和所述R 2为乙基；或
    优选地，所述R 1和所述R 2为正丙基；或
    优选地，所述R 1和所述R 2为异丙基；或
    优选地，所述R 3为H；或
    优选地，所述R 3为甲基。
  3. 如权利要求1或2所述的化合物或其药学上可接受的盐，其特征在于，
    所述药学上可接受的盐选自由甲磺酸盐、硫酸盐、对甲苯磺酸盐、盐酸盐、琥珀酸盐、马来酸盐、富马酸盐所组成的组，
    优选地，所述药学上可接受的盐为盐酸盐。
  4. 一种制备如权利要求1至3任一所述的化合物的方法，其特征在于，包括：
    将式(B)化合物，与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，
    Figure PCTCN2022119478-appb-100002
    其中，
    所述R 1和所述R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组；
    所述R 3′选自由H和甲基所组成的组，优选地所述R 3′为甲基；且
    所述M选自由H、Li、Na、K、Rb和Cs所组成的组；
    优选地，所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组。
  5. 如权利要求4所述的方法，其特征在于，
    所述式(C)化合物中，所述R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组，所述R 3′选自由H和甲基所组成的组，且M选自由Li、Na、K、Rb、和Cs所组成的组；且/或
    所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物中，所述R 1和所述R 2各自独立地选自由H、甲基、 乙基、正丙基、和异丙基所组成的组，所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组，所述R 3′选自由H和甲基所组成的组，且所述M为H；
    优选地，所述式(B)化合物是与所述式(C)化合物接触；且所述式(C)化合物中，所述R 1和R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组，所述R 3′选自由H和甲基所组成的组，且M选自由Li、Na、K、Rb、和Cs所组成的组；
    优选地，所述式(B)化合物是与所述式(C)中R 1和R 2的至少一个被保护基保护的化合物接触；且所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物中，所述R 1和所述R 2各自独立地选自由H、甲基、乙基、正丙基、和异丙基所组成的组，所述保护基选自由叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苯甲基、和对甲氧苯基所组成的组，所述R 3′选自由H和甲基所组成的组，且所述M为H。
  6. 如权利要求4或5所述的方法，其特征在于，
    所述M为H，且所述的将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，包括将所述式(B)化合物，和所述式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物，与选自由2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯、1-羟基苯并三唑、和4-二甲氨基吡啶所组成的组的至少一个接触，优选地与2-(7-氮杂苯并三氮唑)-N,N,N′,N′-四甲基脲六氟磷酸酯接触；且/或
    所述式(B)化合物是与所述式(C)化合物接触，且是在N,N-二异丙基乙基胺、三乙胺或2,2,6,6-四甲基哌啶存在的环境下，特别是在N,N-二异丙基乙基胺存在的环境下；或所述式(B)化合物是与所述的式(C)中R 1和R 2的至少一个被保护基保护的化合物接触，且所述方法还包括将所述的将式(B)化合物与式(C)中R 1和R 2的至少一个被保护基保护的化合物接触的产物，与选自由三氟乙酸、盐酸、和氢溴酸所组成的组中的至少一个接触，优选地与三氟乙酸接触；且/或
    所述R 3′为甲基，所述R 3为H，且所述方法还包括将所述的将式(B)化合物与式(C)化合物或式(C)中R 1和R 2的至少一个被保护基保护的化合物接触的产物，与盐酸接触，优选地与盐酸和乙酸接触。
  7. 如权利要求4所述的方法，其特征在于，所述方法还包括：
    式(B1)化合物与选自由二碳酸二叔丁酯、氯甲酸苄酯、氯甲酸-9-芴甲酯所组成的组中的至少一个，优选地与二碳酸二叔丁酯接触，得到式(B2)化合物，其中所述L选自由叔丁氧羰基、苄氧羰基和9-芴甲氧羰基所组成的组，优选地所述L为叔丁氧羰基；和/或
    式(B2)化合物与选自由R′OM′、水合肼、硼氢化钠所组成的组的至少一个接触，得到式(B3)化合物，其中所述R′选自由甲基、乙基、正丙基、和异丙基所组成的组，优选地所述R′为甲基，M′选自由Li、Na、K、Rb、和Cs所组成的组，优选地所述M′为Na；和/或
    式(B3)化合物与选自由三氟乙酸、盐酸、和氢溴酸所组成的组中的至少一个接触，优选地与三氟乙酸接触，得到所述式(B)化合物，
    Figure PCTCN2022119478-appb-100003
  8. 一种制备如权利要求1至3任一所述的化合物的药学上可接受的盐的方法，其特征在于，包括：
    进行如权利要求4至7任一所述的制备如权利要求1至3任一所述的化合物的方法；以及
    将如权利要求1至3任一所述的化合物与酸接触。
  9. 权利要求1至3任一所述的化合物或其药学上可接受的盐在制备治疗疾病和/或症状的药物中的用途，其中，所述疾病和/或症状选自由治疗癌症、镇痛、和抗炎所组成的组中的至少一种。
  10. 如权利要求9所述的用途，其特征在于，所述疾病和/或症状选自由卵巢癌、乳癌、肺癌、卡波西氏肉瘤、子宫颈癌、胰脏癌、风湿病、急性关节炎、和急性痛风性关节炎所组成的组中的至少一种。
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