WO2023241290A1 - 基于喹诺里西啶类衍生物的药物化合物及其制备方法和应用 - Google Patents

基于喹诺里西啶类衍生物的药物化合物及其制备方法和应用 Download PDF

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WO2023241290A1
WO2023241290A1 PCT/CN2023/094631 CN2023094631W WO2023241290A1 WO 2023241290 A1 WO2023241290 A1 WO 2023241290A1 CN 2023094631 W CN2023094631 W CN 2023094631W WO 2023241290 A1 WO2023241290 A1 WO 2023241290A1
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acid
alkyl
substituted
group
alkenyl
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French (fr)
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尹小英
王延青
刘庆山
单宏丽
唐莉
陈玲艳
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上海工程技术大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/18—Bridged systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of anti-tumor and nerve damage drug synthesis, and in particular to a quinolizidine derivative with anti-tumor or anti-nerve damage effects.
  • Kudouzi was first recorded in "Shen Nong's Materia Medica” and is now included in "Chinese Pharmacopoeia", “National Collection of Chinese Herbal Medicine”, “Chinese Materia Medica”, etc.
  • the whole plant is bitter in taste and cold in nature, and acts on the heart and lung meridians.
  • Kudouzi is usually used as medicine in the form of dried whole plant or mature fruit. It can soothe the five internal organs, stabilize the mind and improve essence. It has the functions of clearing away heat and detoxification, dispelling wind and dampness, relieving pain and killing insects. It is often used for sore throat, acute dysentery, itchy skin and stomachache. treatment of many diseases.
  • Sophora sophora seeds have anti-tumor effects and effects on the central nervous system. They have obvious inhibitory effects on liver cancer cells, and also have inhibitory effects on cervical cancer, lymphosarcoma, and lung cancer.
  • the inhibitory effects on the central nervous system are mainly related to pharmacological effects such as sedation, hypnosis, and Mainly analgesic and cooling, its analgesic characteristics are mainly central, without addiction and drug resistance.
  • These medicinal active substances are alkaloids and flavonoids, including cytisine, matrine, sophoridine and sophorocarpine. These alkaloids have a quinolizidine structure, but these compounds have low activity.
  • CN109970738A and CN110804056A disclose several cytisine derivatives. Among them, the structural modification of the isoflavone part of the compound mentioned in CN109970738A is insufficient, resulting in its limited binding affinity to anti-cancer targets, and its activity performance in anti-tumor activity experiments is not excellent.
  • the compound mentioned in CN110804056A improves its biological activity by modifying the 2-pyridone part of cytisine, but its isoflavone part is not further modified, and its activity improvement is limited.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings in the prior art and provide a pharmaceutical compound based on quinolizidine derivatives that is highly active and can be used for anti-tumor or anti-nerve damage effects, as well as its preparation method and application.
  • a pharmaceutical compound based on quinolizidine derivatives the structural formula of the pharmaceutical compound is:
  • R 2 and R 3 are functional groups that are easy to form hydrogen bonds, including hydroxyl and its substituents, amino and its substituents, azide, nitro, cyano, mercapto and its substituents or halogen, etc.
  • the substituents have been modified Finally, the stability of the compound targeting anti-cancer targets can be improved;
  • R 4 , R 5 , R 6 , R 8 , and R 9 are smaller functional groups, including hydrogen, hydroxyl and its substituents, amino and its substituents, azide group, nitro, cyano group, mercapto group and their substitutions group or halogen, etc. After modification of this substituent, the compound will have more interactions with the amino acid residues in the active pocket of the anti-cancer target;
  • R 1 and R 7 are functional groups with diverse structures, including hydrogen, hydroxyl group and its derivatives, amino group and its derivatives, azide group, nitro group, cyano group, mercapto group and its derivatives, halogen, alkyl group, carbocyclic ring Alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl or arylalkyl, etc. After modification of the substituent, the compound can interact with both the front pocket and the back pocket of the anti-cancer target. This key region produces a stronger interaction, improving its selectivity and biological activity with the target.
  • R 2 and R 3 are respectively OR a , N(R a ) 2 , N 3 , CN, NO 2 , S(O) n R a or halogen;
  • R 4 , R 5 , R 6 , R 8 and R 9 are respectively H, OR a , N(R a ) 2 , N 3 , CN, NO 2 , S(O) n R a or halogen;
  • n is independently 0, 1 or 2;
  • Each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkyne base, (C2-C8) substituted alkynyl, (C6-C20) aryl, (C6-C20) substituted aryl, (C2-C20) heterocyclyl, (C2-C20) substituted heterocyclyl, (C6-C20)aryl(C1-C8)alkyl or substituted (C6-C20)aryl(C1-C8)alkyl;
  • Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or (C6-C20)aryl (C1-C8)alkyl group of R 10 or R 11 is independently optional Substituted by one or more halogen, hydroxyl, CN, N 3 , N(R a ) 2 or OR a ; wherein one or more non-terminal carbon atoms of each said (C1-C8) alkyl group may optionally Replaced by -O-, -S- or -NR a -; and R 10 and R 11 together with the nitrogen to which they are both connected form a 3-7 membered heterocyclic ring, in which any one of the carbon atoms of the heterocyclic ring may optionally be -O, -S- or -NR a - instead.
  • the quinolizidine derivatives include one of the compounds corresponding to the formulas CNI3-1 to CNI3-4, and also include salts corresponding to the structural formulas CNI3-1 to CNI3-4,
  • the salts corresponding to the structural formulas CNI3-1 to CNI3-4 are all corresponding compounds with no Prepared by the reaction of organic acids or organic acids;
  • the inorganic acid is one of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid or phosphoric acid;
  • the organic acids are citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, Malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, parapic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-amine One of benzene sulfonic acid, 2-acetamidobenzoic acid or isethionic acid.
  • Another object of the present invention is to provide a method for preparing the above-mentioned pharmaceutical compounds based on quinolizidine derivatives, which includes the following steps:
  • step S01 the 2,4,6-trihydroxyacetophenone derivative is protected by hydroxyl group using chloromethyl methyl ether, and the molar ratio of 2,4,6-trihydroxyacetophenone to chloromethyl methyl ether is 1: (2 ⁇ 3), the reaction temperature is 0°C, and the reaction temperature is 0°C.
  • the response time is 3 to 4 hours;
  • the volume ratio of the molar mass of 2,4,6-trihydroxyacetophenone to N,N-dimethylformamide dimethyl acetal is 1 mmol: (5-7) ml, and the condensation reaction temperature is 70 ⁇ 80°C, reaction time is 4 ⁇ 8h;
  • step S02 the molar ratio of aminoketene to elemental iodine is 1: (1 ⁇ 2), the reaction temperature is 20 ⁇ 35°C, and the reaction time is 12 ⁇ 48h; the molar ratio of 3-iodochromone and the phenylboronic acid derivative is 1: (1 ⁇ 2), the reaction temperature is 40 ⁇ 60°C respectively, the reaction time is 4 ⁇ 7h respectively;
  • HCl is used for isoflavones and the methoxymethyl ether protecting groups at positions 5 and 7 are removed.
  • the volume ratio of the mole of isoflavones to 6M HCl is 1 mmol: (7-10) ml.
  • the reaction temperature is 80-110°C. Time is 1 ⁇ 3h;
  • step S03 the isoflavones with the methoxymethyl ether protection group are subjected to Mannich reaction with cytisine and formaldehyde, and the molar ratio of the isoflavones with the methoxymethyl ether protection group to cytisine and formaldehyde is It is 1: (1 ⁇ 2): (0.5 ⁇ 0.8), the reaction temperature is 50 ⁇ 80°C, and the reaction time is 5 ⁇ 8h.
  • the present invention also provides the application of the above-mentioned pharmaceutical compounds based on quinolizidine derivatives.
  • the quinolizidine derivatives are used to prepare anti-tumor drugs, including in the preparation of anti-epidermal squamous cell carcinoma, breast cancer, and lung cancer drugs. , gastric cancer, ovarian cancer or oral cancer drugs.
  • Another object of the present invention is to provide a pharmaceutical compound based on quinolizidine derivatives.
  • the structural formula of the pharmaceutical compound is:
  • R 2 and R 3 are functional groups that are difficult to form hydrogen bonds, including hydrogen, alkyl, carbocyclylalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl or arylalkyl etc., after modification of this substituent, the toxic effect of the compound on cells is reduced;
  • R 4 is a smaller functional group, including hydrogen, hydroxyl and its substituents, amino and its substituents, azide, nitro, cyano, mercapto and its substituents, halogen, etc. After modification, the substituent Balance the lipid-water partition coefficient of the compound, thereby enhancing the oral absorption and utilization of the drug;
  • R 1 , R 5 , R 6 , R 7 , R 8 , and R 9 are functional groups with diverse structures, including hydrogen, hydroxyl and its substituents, amino and its substituents, azide group, nitro group, cyano group, and mercapto group and its derivatives, halogen, alkyl, carbocyclylalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl or arylalkyl, etc., after the substituent is modified, The compound's fat solubility is improved, making it easier to penetrate the blood-brain barrier and act on the central nervous system, thereby exerting anti-nerve damage effects.
  • R 4 is H, OR a , N(R a ) 2 , N 3 , CN, NO 2 , S(O) n R a or halogen;
  • n is independently 0, 1 or 2;
  • Each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkyne base, (C2-C8) substituted alkynyl, (C6-C20) aryl, (C6-C20) substituted aryl, (C2-C20) heterocyclyl, (C2-C20) substituted heterocyclyl, (C6-C20)aryl(C1-C8)alkyl or substituted (C6-C20)aryl(C1-C8)alkyl;
  • Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or (C6-C20)aryl (C1-C8)alkyl group of R 10 or R 11 is independently optional Substituted by one or more halogen, hydroxyl, CN, N 3 , N(R a ) 2 or OR a ; wherein one or more non-terminal carbon atoms of each said (C1-C8) alkyl group may optionally Replaced by -O-, -S- or -NR a -; and R 10 and R 11 together with the nitrogen to which they are both connected form a 3-7 membered heterocyclic ring, in which any one of the carbon atoms of the heterocyclic ring may optionally be -O-, -S- or -NR a - instead.
  • Another object of the present invention is to provide a method for preparing the above-mentioned pharmaceutical compounds based on quinolizidine derivatives.
  • 4-benzyloxy-2-hydroxyacetophenone and N,N-dimethylformamide Aminoketene is obtained from methylal through condensation reaction.
  • aminoketene is iodized and cyclized under the action of elemental iodine to form 3-iodochromone.
  • Mannich reaction was used to obtain quinolizidine derivatives. Specifically, it includes the following steps:
  • Aminoketene is iodinated and cyclized under the action of elemental iodine.
  • the compound is further catalyzed by sodium carbonate/potassium carbonate and palladium acetate. Obtained by Suzuki coupling reaction And use hydrogen bromide-acetic acid solution and water to remove the protecting group;
  • step S11 The molar mass and the volume of N,N-dimethylformamide dimethyl acetal are 1mmol and (6 ⁇ 8)mL respectively, the reaction temperature is 74°C, and the reaction time is 3-5h;
  • step S12 The molar ratio with elemental iodine is 1:1.5, the reaction temperature is room temperature, and the reaction time is 12 to 24 hours;
  • the molar ratio is 1:1.5, the reaction temperature is 50°C, and the reaction time is 5 to 12h.
  • a mixed solution of tetrahydrofuran and methanol is used as the solvent, alkali and palladium acetate are used as the catalyst, and the alkali is anhydrous sodium carbonate/ carbonic acid base;
  • the molar volume ratio of hydrogen bromide in acetic acid solution and water is 1mmol:3.5mL:0.88mL, where the mass fraction of hydrogen bromide in hydrogen bromide in acetic acid solution is 33%, the reaction temperature is 50°C, and the reaction time is 3 ⁇ 12h;
  • step S13 The molar ratios with cytisine, DMAP and formalin are 1mmol, 1.25mmol, 2.4mg and 0.5mL respectively.
  • the reaction temperature is 70°C and the reaction time is 6-10h.
  • Another object of the present invention is to provide the application of the above-mentioned pharmaceutical compounds based on quinolizidine derivatives.
  • the quinolizidine derivatives are used to prepare anti-nerve injury drugs, including in the preparation of anti-brain trauma, Application of nerve injury drugs in spinal cord injury, postoperative nerve injury, stroke, hypoxic nerve injury, neurodegenerative diseases.
  • the present invention has the following beneficial effects:
  • the present invention found that the structural modification of the isoflavone part can greatly enhance the selectivity and affinity of the compound for anti-cancer targets, and greatly improve the inhibitory activity against various solid tumor cells. Therefore, the present invention greatly improves its selectivity and affinity with anti-cancer targets by structurally modifying the N-isoflavone isoflavone part of cytisine, and can effectively treat various solid tumor cells in anti-tumor activity experiments. It shows extremely strong inhibitory activity and anti-migration effect. Compared with existing technologies such as CN109970738A and CN110804056A, the structure modification method of the compound of the present invention is more advanced and comprehensive, and the new compound obtained has stronger anti-tumor activity and wider application range.
  • the present invention enables the compound to produce extremely potent inhibitory activity and anti-migration effects against a variety of cancer cells, and is a very potential lead compound against a variety of solid tumor cells.
  • the present invention obtains a series of compounds with neuroprotective effects through ingenious structural design, which is another major innovation in the pharmacological activity application of such compounds. specifically:
  • the compounds provided by the present invention are based on natural cytisine-isoflavones and structurally modify the isoflavones to obtain a series of derivatives.
  • compound 1 CNI1-1
  • compound 3 CNI1- 3
  • Compounds 9-13 CNI1-9-13
  • CNI1-1, CNI1-3, CNI1-9, CNI1-10, CNI1-11, CNI1-12, and CNI1-13 synthesized in the compounds provided by the invention cause OGD damage to primary cultured neuronal cells and BV2 cells. It has strong protective activity and has statistical difference.
  • the compound provided by the present invention utilizes the rational design strategy of pharmacophore combination to improve the effectiveness of quinolizidines.
  • Derivatives undergo innovative structural modifications and transformations to obtain new compounds with higher anti-tumor activity.
  • the preparation method is efficient, easy to operate, and the product has high purity.
  • the compounds of the present invention can effectively inhibit the growth and migration of tumor cells and have good anti-tumor therapeutic effects.
  • Two of the compounds showed significant antiproliferative activity and cytotoxicity against both cancer cells.
  • the IC50 values of the two compounds CNI3-2 and CNI3-4 against A431 cells were 53.39 ⁇ M and 26.66 ⁇ M respectively, and the IC50 values against SK-BR-3 cells were 47.91 ⁇ M and 29.57 ⁇ M respectively, which were comparable to the positive control.
  • the results further demonstrated that compared with positive drugs, these two compounds showed strong anti-proliferative activity and cytotoxicity on A431 cells and SK-BR-3 cells.
  • the present invention also has the following outstanding substantive features and significant progress.
  • the specific analysis is as follows:
  • the present invention greatly improves the selectivity and affinity with anti-cancer targets through the structural modification of the isoflavone part of the compound, and shows extremely strong inhibitory activity and activity against a variety of solid tumor cells in anti-tumor activity experiments. Anti-migration effect.
  • the structural modification of the isoflavone part of the above compound is insufficient, resulting in limited binding affinity to anti-cancer targets and poor performance in anti-tumor activity experiments.
  • the present invention greatly improves selectivity and affinity with anti-cancer targets, and exhibits extremely strong inhibitory activity and anti-migration against a variety of solid tumor cells in anti-tumor activity experiments. Effect.
  • the invention fills the research gap at home and abroad by applying quinolizidine derivatives to anti-nerve injury drugs, and is original. There is a lack of effective drugs for clinical diseases such as stroke, hypoxic nerve damage, neurodegenerative diseases, and nerve damage at home and abroad.
  • the technology developed by the present invention has good application prospects.
  • Figure 1 is a flow chart 1 for the preparation of cytisine-isoflavone derivatives among the compounds provided by the embodiments of the present invention
  • Figure 2 is a 1 H NMR chart corresponding to CNI1-1 synthesized in the embodiment
  • Figure 3 is a 13 C NMR chart corresponding to CNI1-1 synthesized in the example.
  • Figure 4 is a 1 H NMR chart corresponding to CNI1-3 synthesized in the embodiment.
  • Figure 5 is a 13 C NMR chart corresponding to CNI1-3 synthesized in the example.
  • Figure 6 is a 1 H NMR chart corresponding to CNI1-9 synthesized in the embodiment.
  • Figure 7 is a 13 C NMR chart corresponding to CNI1-9 synthesized in the example.
  • Figure 8 is a 1 H NMR chart corresponding to CNI1-10 synthesized in the embodiment.
  • Figure 9 is a 13 C NMR chart corresponding to CNI1-10 synthesized in the example.
  • Figure 10 is the 1 H NMR chart corresponding to CNI1-11 synthesized in the embodiment.
  • Figure 11 is a 13 C NMR chart corresponding to CNI1-11 synthesized in the example.
  • Figure 12 is the 1 H NMR chart corresponding to CNI1-12 synthesized in the embodiment.
  • Figure 13 is a 13 C NMR chart corresponding to CNI1-12 synthesized in the example.
  • Figure 14 is the 1 H NMR chart corresponding to CNI1-13 synthesized in the embodiment.
  • Figure 15 is a 13 C NMR chart corresponding to CNI1-13 synthesized in the example.
  • Figure 16 is a 9 F NMR chart corresponding to CNI1-13 synthesized in the example.
  • Figure 17 is a comparison of cell morphology (a-c), which is a comparison of cell morphology in the control group of the first experiment, a comparison of cell morphology in the model group and the treatment group 24 hours after drug administration, and (e-f) is a comparison of cell morphology in the second experiment. Comparison of cell morphology in the control group of the second experiment, comparison of cell morphology in the model group and the treatment group 24 hours after administration;
  • Figure 18 is a graph showing the cell viability detection results of compound 1 (CNI1-1), compound 3 (CNI1-3), and compounds 9 to 13 (CNI1-9 to 13);
  • Figure 19 is a flow chart 2 for the preparation of cytisine-isoflavone derivatives among the compounds provided by the embodiments of the present invention.
  • Figure 20 is a 1 H NMR chart corresponding to CNI3-1 synthesized in the embodiment.
  • Figure 21 is a 13 C NMR chart corresponding to CNI3-1 synthesized in the example.
  • Figure 22 is a 1 H NMR chart corresponding to CNI3-2 synthesized in the embodiment.
  • Figure 23 is a 13 C NMR chart corresponding to CNI3-2 synthesized in the example.
  • Figure 24 is a 1 H NMR chart corresponding to CNI3-3 synthesized in the embodiment.
  • Figure 25 is the 13 C NMR chart corresponding to CNI3-3 synthesized in the embodiment.
  • Figure 26 is the 1 H NMR chart corresponding to CNI3-4 synthesized in the embodiment.
  • Figure 27 is a 13 C NMR chart corresponding to CNI3-4 synthesized in the example.
  • Figure 28 is a schematic diagram of the anti-migration effects of compounds CNI3-2 and CNI3-4 on 4T1 breast cancer cells.
  • Figure 29 shows the results of BV2 hypoxic and glucose deprivation injury time screening.
  • Figure 30 Comparison of cell morphology (a-c) shows the control group, model group and administration group 24 hours after administration in the first experiment respectively.
  • Figures (e-f) shows the control group, model group and administration group in the second experiment respectively. group administered 24 hours after the drug;
  • Figure 31 is a graph showing the cell viability detection results of compounds 1 to 13;
  • FIG. 32 Discovery of quinolizidine derivatives (CNIs) from sophora sophora seeds
  • A Schematic diagram describing the source of CNI1 and its derivatives
  • B EGFR and HER2 bound by TAK-285
  • PDB codes: 3POZ and 3RCD Structural overlay and comparison of the binding mode of TAK-285 in the binding pocket of EGFR and HER2
  • C Structural overlap of CNI3 and TAK-285 bound in the EGFR/HER2 kinase domain;
  • Figure 33 shows the dose-response curves of CNI and TAK-285 on EGFR (left picture) and HER2 (right picture) measured by HTRF;
  • Figure 34 shows that CNI3 and CNI4 inhibit the proliferation and migration of different cancer cells.
  • A-B Growth inhibition assay of A431 (A) and BT-474 (B) cancer cell lines by CNI3 (left), CNI4 (middle) and TAK-285 (right), as assessed by CCK-8.
  • C Live/dead cell double staining method to measure the effects of CNI3, CNI4 and TAK-285 on the viability of BT-474 cancer cells at their respective IC50 concentrations;
  • Figure 35 shows the linear correlation between the experimentally determined pKi values and the calculated binding free energies ( ⁇ Gcalc b) of CNIs and TAK-285 for EGFR and HER2 kinase.
  • the graph shows the correlation coefficient (R2) for each series;
  • Figure 36 shows the experimental results; breast cancer cells in the blank group migrate relatively quickly to the edge of the scratch, and the width of the scratch narrows more obviously, while the change in scratch width in the CNI3-2 and CNI3-4 treatment groups is not as good as in the blank group The group is obvious;
  • FIG. 37 shows the experimental results; CNI3-2 and CNI3-4 can induce higher death rates in BT-474, SKBR-3 and A431 cells than other compounds;
  • Figure 38 is a flow chart of neuroprotection experiment.
  • Sophora sophora seeds have anti-tumor effects and effects on the central nervous system. They have obvious inhibitory effects on liver cancer cells, and also have inhibitory effects on cervical cancer, lymphosarcoma, and lung cancer.
  • the inhibitory effects on the central nervous system are mainly related to pharmacological effects such as sedation, hypnosis, and analgesia. Its analgesic characteristics are mainly central and have no addiction or drug resistance.
  • These medicinal active substances are alkaloids and flavonoids, including cytisine, matrine, sophoridine and sophorocarpine. These alkaloids have a quinolizidine structure.
  • Flavones and isoflavones mainly include genistein, 5,7-dihydroxyflavone, formononetin, etc.
  • the present invention isolated cytisine N-methylene-(4,7-dihydroxy-3-methoxy)isoflavone from the traditional Chinese medicine Sophora sophora for the first time, which belongs to the quinol Risidine compounds have good anti-tumor activity and anti-nerve damage effects.
  • the research object in the present invention is the natural product quinolizidine derivatives and their derivatives.
  • the natural product quinolizidine derivative CNI1 is a new isoflavone alkaloid extracted from Sophora sophora fruit. It mainly consists of two main structures, cytisine and isoflavone, connected through methylene groups. In the synthesis, the isoflavone structure is mainly synthesized. It is mainly synthesized from commercially available 4-benzyloxy-2-hydroxyacetophenone derivatives through aldol condensation reaction, cyclization reaction, Suzuki coupling reaction and Mannich reaction.
  • the preparation method of cytisine-isoflavone derivatives includes the following steps:
  • the molar mass and volume of N,N-dimethylformamide dimethyl acetal are 1 respectively. mmol, 7mL, the reaction temperature is 74°C, and the reaction time is 3-5h (after comparing multiple sets of experiments by the applicant, any time in the range has little impact on the synthesis);
  • the molar amounts of elemental iodine are 1 mmol and 1.5 mmol respectively, the reaction temperature is room temperature, and the reaction time is 12 to 24 hours (after comparing multiple sets of experiments by the applicant, any time in the range has little impact on the synthesis).
  • step (1) Prepare step (1) to obtain and (In this example, R1-R4 are selected from the corresponding commercially available phenylboronic acid derivatives according to the different products of CNI1-1, CNI1-3 and CNI1-9 ⁇ 13 in the above structural formula) and mixed, and the reaction is prepared.
  • the molar amounts are 1 mmol and 1.5 mmol respectively, the reaction temperature is 50°C, and the reaction time is 5 to 12 hours (after comparison by the applicant in multiple sets of experiments, any time in the range has little impact on the synthesis).
  • the reaction uses a mixed solution of tetrahydrofuran and methanol as the solvent, alkali and palladium acetate as the catalyst, and the alkali is anhydrous sodium carbonate.
  • the volumes of acetic acid solution and water with hydrogen bromide are 1mmol, 3.5mL, and 0.88mL respectively.
  • the mass fraction of hydrogen bromide in the acetic acid solution of hydrogen bromide is 33%, the reaction temperature is 50°C, and the reaction time is 3 to 12 hours (after comparison by the applicant in multiple groups of experiments, any time in the range has little impact on the synthesis).
  • the molar ratios with cytisine, DMAP and formalin are 1mmol, 1.25mmol, 2.4mg and 0.5mL respectively.
  • the reaction temperature is 70°C and the reaction time is 6-10h (after The applicant has compared multiple sets of experiments and found that any time within the range has little impact on the synthesis).
  • BV2 cells were seeded into a 96-well plate at a density of 1 ⁇ 10 5 cells/mL. After 24 hours of adhesion, OGD modeling was performed. After treatment, the cells were administered and cultured for 24 hours. Then, 10% of the medium volume of CCK-8 was added to each well. After continuing to incubate for 2 hours in the cell culture incubator, use a microplate reader to measure the optical density (OD) value at 450 nm, and calculate the cell viability (%).
  • OD optical density
  • BV2 cells The morphological changes of BV2 cells in different groups were observed under an inverted microscope. Microglia without OGD/R treatment grew well. The cells were oval, the cell membrane was intact, the shape was clear, and the cells were normal. After OGD, microglia were significantly damaged, their number decreased, some cells were activated, antennae and synapses appeared, cell morphology changed significantly, and more cells died. After administration, the cell morphology became clear and the cell membrane began to become complete. Morphologically, cell damage has been rescued to a certain extent (see Figure 17). The comparison of cell morphology between the control group, model group and drug treatment group (* indicates P ⁇ 0.05, **P ⁇ 0.05) is statistically significant.
  • the BV2 cell viability test results of different concentrations of compounds 24 hours after administration are shown in Figure 3.
  • the cell viability of the model group decreased significantly.
  • compound 1, compound 3, compound 9, and compound 10 , Compound 11, Compound 12 and Compound 13 significantly increased cell viability.
  • These compounds have a strong rescue effect on BV2 cell damage and have good activity.
  • Compound 2 and Compound 5 had cell viability between 50 ⁇ mol and 150 ⁇ mol.
  • the cell viability of compound 6 also increased significantly at 6.25 ⁇ mol.
  • the cell viability of compound 2, compound 4, compound 6, compound 7 and compound 8 did not increase significantly at other concentrations.
  • Compound 1, Compound 3, Compound 9, Compound 10, Compound 11, Compound 12 and Compound 13 have better ODG damage activity on BV2 cells. See Figure 18 for specific results.
  • BV2 cells retain various morphologies and functions of microglia and are a glial cell line derived from mice.
  • the present invention found that BV2 cell morphology shrank and cell viability significantly decreased after 2-5 hours of OGD, and some BV2 cells changed from resting state to activated state after OGD stimulation.
  • Compound 1, Compound 3, Compound 9, Compound 10, Compound 11, Compound 12, and Compound 13 effectively protect BV2 cells from OGD damage, normalize the morphology, increase cell viability, and reduce the number of activated cells. This compound is effective in the BV2 cell OGD model. Has better activity.
  • HTRF time-resolved fluorescence technology
  • the preparation method of cytisine-isoflavone derivatives includes the following steps:
  • step (1) The molar ratio with chloromethyl methyl ether is 1:2.5, the reaction The temperature is 0°C, and the reaction time is 3 to 4 hours; The volume ratio of molar mass to N,N-dimethylformamide dimethyl acetal is 1mmol:6ml, the reaction temperature is 70 ⁇ 80°C, and the reaction time is 4 ⁇ 8h; The molar ratio with elemental iodine is 1:1.5, the reaction temperature is 20 ⁇ 35°C, and the reaction time is 12 ⁇ 48h.
  • Human epidermal cancer cell line (A431), human breast cancer cell line (SK-BR-3) and mouse breast cancer cell line (4T1) were provided by Shanghai Sabikon Biotechnology.
  • Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Cells were seeded into a 96-well plate at 8 ⁇ 10 3 /well and cultured in a 37°C constant-temperature incubator with 5% CO 2 until cells adhered.
  • the CCK-8 method was used to detect the anti-proliferative effects and cytotoxic effects of compounds on cell lines (human epidermal squamous cell carcinoma A431 cell line and human breast cancer SK-BR-3 cell line).
  • Test compounds were dissolved in DMSO and diluted to different concentrations with culture medium (final concentration of DMSO was 0.5%). Different concentrations of the compounds to be tested were added to the reaction wells, and the cells were cultured for 72 hours.
  • CNI3-2 and CNI3-4 were 53.39 ⁇ M and 26.66 ⁇ M respectively, and the IC 50 values against SK-BR-3 cells were 47.91 ⁇ M and 29.57 ⁇ M respectively, which were comparable to the positive control.
  • Mouse breast cancer 4T1 cells in the logarithmic growth phase were inoculated into a 6-well plate at 1.5 ⁇ 10 5 cells/well and cultured until the cells were confluent. Draw a longitudinal straight line (wound area) to simulate a wound on the cells in the center of the bottom of each well, and use a sterile inoculation loop. Wound zone width was measured using a 37XC inverted biomicroscope. Cells were washed with PBS, and one group of cells was cultured in complete medium as a control group, while another group of cells was treated with samples (10, 20, and 40 ⁇ M) dissolved in complete medium.
  • Normal control group (Nor, cultured with DMEM solution, without sorbitol injury, the other groups were injured with sorbitol);
  • Model control group (Model, no treatment);
  • Positive control group (Con, using the positive control drug ganglioside 1 ⁇ M);
  • the cells were washed twice with serum-free DMEM culture medium, and then serum-free medium, positive drugs and drugs to be tested were added.
  • sorbitol solution was added to each group to a final concentration of 25mM.
  • Neurons were cultured for 24 hours in a constant temperature incubator at 37°C and 5% carbon dioxide.
  • the medium was aspirated, and 0.04% MTT (prepared in serum-free medium) was added to each well and incubated for 4 hours.
  • discard the medium containing MTT add 150 ⁇ l/well of DMSO, shake thoroughly to dissolve the formazan particles, and measure the absorbance value at 540 nm.
  • the viability of cells in each group was expressed as the ratio of the absorbance value of each group to the absorbance value of the fixed well of the normal control group multiplied by 100%.
  • Nor (normal control group), Modle (model control group), Con (positive control group), and CNI1-1 to CNI1-13 are compounds 1-13.
  • the results show that 7 compounds including compound 1, compound 3, compound 9, compound 10, compound 11, compound 12 and compound 13 have obvious neuron protective activity, and at the same molar concentration, the efficacy is higher than that of the positive control drug. Converted into mass concentration (ng/L), the medicinal effect is more prominent.
  • BV2 cells were seeded into a 96-well plate at a density of 1 ⁇ 10 5 cells/mL. After 24 hours of adhesion, OGD modeling was performed. After treatment, the cells were administered and cultured for 24 hours. Then, 10% of the medium volume of CCK-8 was added to each well. After continuing to incubate for 2 hours in the cell culture incubator, use a microplate reader to measure the optical density (OD) value at 450 nm, and calculate the cell viability (%).
  • OD optical density
  • Cell viability (%) [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] ⁇ 100 (A (drug added): with cells, CCK-8 Wells for solutions and drug solutions OD value, A (0 plus drug): OD value of the well with cells, CCK-8 solution but no drug solution, A (blank): OD value of the well without cells, cell viability: cell proliferation activity or cytotoxicity vitality).
  • the drug was dissolved by gradually increasing the amount of DMSO until dissolution was the end point. Except for compound 9, compound 11, and compound 12 which are insoluble at 10 ⁇ l, other compounds are dissolved at 10 ⁇ l. Compound 9, compound 11, and compound 12 are dissolved at 30 ⁇ l. At the same time, the maximum concentration is determined based on the IC 50 values of compounds 4 and 6. is 200 ⁇ mol.
  • the drug group was divided into the following 6 concentration gradients: "6.25 ⁇ mol ⁇ L -1 , 12.5 ⁇ mol ⁇ L -1 , 50 ⁇ mol ⁇ L - 1 , 100 ⁇ mol ⁇ L -1 , 150 ⁇ mol ⁇ L -1 , 200 ⁇ mol ⁇ L -1 ”, set 1 to 6 duplicate holes in each group, and repeat 3 to 4 times.
  • BV2 cells were cultured in high-glucose DMEM + 10% inactivated fetal bovine serum in a 37°C, 5% CO 2 incubator, and the culture medium was replaced every other day. When the cell confluence reaches 80% to 85%, digest and passage with 0.25% trypsin. Cells in the logarithmic growth phase were seeded into a 96-well plate at an appropriate density.
  • Figure 29 shows the results of BV2 hypoxic and glucose deprivation injury time screening.
  • Figure 30 Comparison of cell morphology (a-c) shows the control group, model group and administration group 24 hours after administration in the first experiment respectively.
  • Figures (e-f) shows the control group, model group and administration group in the second experiment respectively. The group was administered 24 hours after the drug.
  • BV2 cells The morphological changes of BV2 cells in different groups were observed under an inverted microscope without OGD/R treatment.
  • the microglia grow well, the cells are oval, the cell membrane is intact, the shape is clear, and the cells are normal.
  • OGD microglia were significantly damaged, their number decreased, some cells were activated, antennae and synapses appeared, cell morphology changed significantly, and more cells died.
  • the cell morphology became clear and the cell membrane began to become complete. Morphologically, cell damage was somewhat rescued (see Figure 30).
  • the BV2 cell viability test results of different concentrations of compounds 24 hours after administration are shown in Figure 31.
  • the cell viability of the model group decreased significantly.
  • the cell viability of Compound 1, Compound 3, Compound 9, and Compound 10 , Compound 11, Compound 12 and Compound 13 significantly increased cell viability.
  • These compounds have a strong rescue effect on BV2 cell damage and have good activity.
  • Compound 2 and Compound 5 had cell viability between 50 ⁇ mol and 150 ⁇ mol.
  • the cell viability of compound 6 also increased significantly at 6.25 ⁇ mol.
  • the cell viability of compound 2, compound 4, compound 6, compound 7 and compound 8 did not increase significantly at other concentrations.
  • Compound 1, Compound 3, Compound 9, Compound 10, Compound 11, Compound 12 and Compound 13 have good ODG damage activity on BV2 cells.
  • Figure 31 cell viability test results of Compounds 1-13. 4.Experimental discussion
  • BV2 cells retain various morphologies and functions of microglia and are a glial cell line derived from mice. This study found that BV2 cell morphology shrunk and cell viability significantly decreased after 2-5 hours of OGD, and some BV2 cells changed from resting state to activated state after OGD stimulation. Compound 1, Compound 3, Compound 9, Compound 10, Compound 11, Compound 12, and Compound 13 effectively protect BV2 cells from OGD damage, normalize the morphology, increase cell viability, and reduce the number of activated cells. This compound is effective in the BV2 cell OGD model. Has better activity. Of course, we are only exploring the activity of this series of compounds against OGD damage to BV2 cells from the dimensions of morphology and cell viability. We may still need to study other organelles or the PCR or western blot expression levels of different factors after OGD damage to the organelle to verify.
  • CNIs quinolizidine derivatives
  • A describes the source of CNI1 and its derivatives. The cytisine and isoflavone fractions appear red and blue respectively.
  • B Structural superposition of TAK-285 bound EGFR and HER2 (PDB codes: 3POZ and 3RCD) and comparison of the binding modes of TAK-285 within the binding pockets of EGFR and HER2.
  • the EGFR and HER2 kinase domains are represented as cyan and yellow cartoons, respectively, with the regulatory secondary structures ⁇ -helix C and A-loop in red and green respectively.
  • the inhibitor TAK-285 and its interacting residues are depicted as purple and gray sticks, respectively, with the DFG motif highlighted in orange. Black dashed lines represent hydrogen bonds.
  • C Structural overlap of CNI3 and TAK-285 bound within the EGFR/HER2 kinase domain. Black dashed circles outline the binder pockets occupied by different moieties of the kinase inhibitor.
  • Figure 33 shows the dose-response curves of CNI and TAK-285 for EGFR (left panel) and HER2 (right panel) as measured by HTRF.
  • compounds CNI3 and CNI4 may serve as promising EGFR/HER2 dual-target inhibitors
  • MDA-MB-231 and mouse breast cancer cell line 4T1 are both triple-negative breast cancers (TNBC) that do not produce excess HER2 receptors, and the former cell line is known to normally express EGFR.
  • TNBC triple-negative breast cancers
  • the migration rate of both types of cancer cells at the wound edge was significantly attenuated when treated with CNI3 and CNI4 (*P ⁇ 0.05 and **P ⁇ 0.01 compared to the control group, respectively). And showed strong dose dependence (Figure 34). Therefore, the results indicate that CNI3 and CNI4 have encouraging in vitro anti-proliferative and anti-migration activities against different breast cancer cells and have the potential for further development as lead drugs.
  • CNI3 and CNI4 inhibit the proliferation and migration of different cancer cells.
  • A-B Inhibition of A431 (A) by CNI3 (left), CNI4 (middle) and TAK-285 (right) as assessed by CCK-8 and growth assay of BT-474(B) cancer cell line.
  • C Live/dead cell double staining method measures the effects of CNI3, CNI4 and TAK-285 on the viability of BT-474 cancer cells at their respective IC50 concentrations. It is worth noting that most of the dead cells (stained red) have been washed away with buffer solution.
  • D Dose-dependent inhibition of migration of breast cancer cell lines MDA-MB-231 (left) and 4T1 (right) by CNI3 and CNI4. Compared with the control group, *P ⁇ 0.05 and **P ⁇ 0.01.
  • the anti-cancer cell proliferation activity of the compound was used as the screening condition. According to the analysis of its IC 50 value, it can be seen that compound CNI3-4 has good anti-cell proliferation on BT-474, SK-BR-3, MDA-MB-231, and A431 cell lines. active.
  • CNI3-1, CNI3-2, CNI3-3, and CNI3-4 are as follows:
  • Scratch mobility (%) 1 - (scratch area value at corresponding time/scratch area value at 0) x 100%.
  • the experimental results Breast cancer cells in the blank group migrated to the scratch edge relatively quickly, and the width of the scratch narrowed significantly.
  • the change in scratch width of the CNI3-4 treatment group was not as obvious as that of the blank group.
  • the scratch area value of the CNI3-4 group was significantly larger than that of the control group and CNI3-2 group. Therefore, it can be inferred that the CNI3-4 compound has the physiological activity to inhibit the migration of breast cancer cells.
  • the dual dyes of Calcein-AM and PI are used for dual staining and labeling of living cells and dead cells, so as to analyze the levels of living cells and dead cells.
  • Compounds CNI3-1, CNI3-2, CNI3-3 and CNI3-4 treated cells at their corresponding half inhibitory concentrations to treat cells BT-474, SK-BR-3 and A431 respectively. After the sample is stained, take pictures with a fluorescence inverted microscope: green fluorescence - living cells, red fluorescence - dead cells, as shown in Figure 37. Experimental results: Compared with other compounds, CNI3-4 can induce BT-474, SK-BR-3, A431 cell death rate was higher.

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Abstract

本发明涉及属于抗肿瘤和神经损伤药物合成技术领域，公开了一种基于喹诺里西啶类衍生物的抗肿瘤和抗神经损伤药物化合物、应用及制备方法，药物化合物的结构式与现有技术相比，本发明提供的化合物在天然喹诺里西啶生物碱－异黄酮骨架基础上，对异黄酮类部位进行结构修饰得到一系列衍生物，实验证明R2＝R3＝H系列化合物能够抗神经损伤，在防治神经损伤相关疾病方面有很好的应用前景；当R2、R3为易形成氢键的官能团系列化合物具有抗肿瘤活性，有应用前景。具有制备方法高效、易于操作，且产物纯度高、活性高等优点。

Description

基于喹诺里西啶类衍生物的药物化合物及其制备方法和应用 技术领域
本发明涉及抗肿瘤和神经损伤药物合成技术领域，尤其是涉及一种具备抗肿瘤或抗神经损伤作用的喹诺里西啶类衍生物。
背景技术
苦豆子最早记载于《神农本草经》，现收载于《中国药典》、《全国中草药汇编》、《中华本草》等。全株味苦、性寒，归心、肺经。苦豆子通常以干燥全草或成熟的果实入药，能安五脏、定志益精，具有清热解毒、祛风燥湿、止痛杀虫等作用，常用于咽喉肿痛、急性痢疾、皮肤瘙痒、胃痛等多种疾病的治疗。文献报道苦豆子具有抗肿瘤作用和对中枢神经系统的作用，对肝癌细胞抑制作用明显，对宫颈癌、淋巴肉瘤、肺癌也有抑制作用；对中枢神经系统的抑制作用主要与药理作用以镇静催眠、镇痛及降温为主，其镇痛特征是以中枢性为主、没有成瘾性和耐药性。这些药效作用物质成分是生物碱和黄酮类成分，包括金雀花碱、苦参碱、槐定碱和槐果碱，这些生物碱具有喹诺里西啶结构，但是这些化合物活性较低。例如，CN109970738A与CN110804056A公开了几种金雀花碱衍生物。其中CN109970738A中所提到的化合物在异黄酮部分的结构修饰并不充分，导致其与抗癌靶标的结合亲和力有限，且在抗肿瘤活性实验中活性表现并不出色。CN110804056A中所提的化合物是通过修饰金雀花碱的2－吡啶酮部分来提高其生物活性，但对于其异黄酮部分并没有进一步修饰，其活性提高有限。
目前，在临床上国内外在脑卒中、缺氧性神经损伤神经退行性疾病的神经损伤等方面疾病缺乏有效的药物。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种活性高、可用于抗肿瘤或抗神经损伤作用的基于喹诺里西啶类衍生物的药物化合物及其制备方法和应用。
本发明的目的可以通过以下技术方案来实现：
一种基于喹诺里西啶类衍生物的药物化合物，该药物化合物的结构式为：
其中：
R2、R3为易形成氢键的官能团，包括羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其取代基或卤素等，该取代基经过修饰后，可以提高化合物靶向抗癌靶标的稳定性；
R4、R5、R6、R8、R9为体积较小的官能团，包括氢、羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其取代基或卤素等，该取代基经过修饰后，化合物与抗癌靶标的活性口袋中氨基酸残基产生更多的相互作用；
R1、R7为结构较为多样的官能团，包括氢、羟基及其衍生物、氨基及其衍生物、叠氮基、硝基、氰基、巯基及其衍生物、卤素、烷基、碳环基烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基或芳基烷基等，该取代基经过修饰后，化合物与抗癌靶标的前口袋和后口袋两个关键区域产生更强的相互作用，提高其与靶标的选择性和生物活性。
进一步地，所述喹诺里西啶类衍生物中：
R1、R7分别为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa、－C(＝O)R10、－C(＝O)OR10、－C(＝O)NR10R11、－C(＝O)SR10、－S(O)R10、－S(O)2R10、－S(O)(OR10)、－S(O)2(OR10)、－SO2NR10R11、卤素、(C1－C8)烷基、(C4－C8)碳环基烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基或(C6－C20)芳基(C1－C8)烷基；
R2、R3分别为ORa、N(Ra)2、N3、CN、NO2、S(O)nRa或卤素；
R4、R5、R6、R8、R9分别为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa或卤素；
每个n独立地为0、1或2；
每个R独立地为H、(C1－C8)烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基、(C6－C20)芳基、(C6－C20)取代的芳基、(C2－C20)杂环基、(C2－C20)取代的杂环基、(C6－C20)芳基(C1－C8)烷基或取代的(C6－C20)芳基(C1－C8)烷基；
每个Ra独立地为H、(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基、(C6－C20)芳基(C1－C8)烷基、(C4－C8)碳环基烷基、－C(＝O)R、－C(＝O)OR、－C(＝O)NR2、－C(＝O)SR、－S(O)R、－S(O)2R、－S(O)(OR)、－S(O)2(OR)或－SO2NR2；
每个R10或R11的(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基或(C6－C20)芳基(C1－C8)烷基独立的任选被一个或多个卤素、羟基、CN、N3、N(Ra)2或ORa取代；其中每个所述(C1－C8)烷基的一个或多个非末端碳原子可以任选地被－O－、－S－或－NRa－代替；并且R10和R11与它们都连接的氮一起形成3－7元杂环，其中所述杂环的任何一个碳原子可以任选被－O、－S－或－NRa－代替。
进一步优选地，所述喹诺里西啶类衍生物包括式CNI3-1～CNI3-4对应的化合物中的一种，还包括结构式CNI3-1～CNI3-4对应的盐，
更进一步优选地，结构式CNI3-1～CNI3-4所对应的盐均为对应化合物与无 机酸或有机酸反应制得；
所述无机酸为盐酸、氢溴酸、硫酸、硝酸、氨基磺酸或磷酸中的一种；
所述有机酸为柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2－乙酰氨基苯甲酸或羟乙磺酸中的一种。
本发明的另一目的在于提供上述基于喹诺里西啶类衍生物的药物化合物的制备方法，包括以下步骤：
S01、将2,4,6－三羟基苯乙酮的4和6位羟基进行保护，加入N,N－二甲基甲酰胺二甲缩醛通过缩合反应得到氨烯酮；
首先，使用氯甲基甲醚将(即2,4,6－三羟基苯乙酮衍生物)进行保护，并N,N－二甲基甲酰胺二甲缩醛通过缩合反应得到即(氨烯酮)；
S02、在单质碘的作用下氨烯酮碘化、环合形成3－碘色酮进一步与多种苯硼酸衍生物通过Suzuki偶联反应得到异黄酮并除去5和7位的甲氧基甲醚保护基；
S03、利用曼尼希反应得到喹诺里西啶类衍生物。
进一步地，
步骤S01中，2,4,6－三羟基苯乙酮衍生物采用氯甲基甲醚进行羟基保护，2,4,6－三羟基苯乙酮与氯甲基甲醚的摩尔比为1：(2～3)，反应温度为0℃，反 应时间3～4h；
所述的2,4,6－三羟基苯乙酮的摩尔质量与N,N－二甲基甲酰胺二甲缩醛的体积比为1mmol：(5～7)ml，缩合反应反应温度为70～80℃，反应时间为4～8h；
步骤S02中，氨烯酮与单质碘的摩尔比为1：(1～2)，反应温度为20～35℃，反应时间12～48h；3－碘色酮与苯硼酸衍生物的摩尔比为1：(1～2)，反应温度分别为40～60℃，反应时间分别为4～7h；
异黄酮采用HCl并除去5和7位的甲氧基甲醚保护基，其中，异黄酮的摩尔与6M HCl的体积比为1mmol：(7～10)ml，反应温度为80～110℃，反应时间为1～3h；
步骤S03中，去甲氧基甲醚保护基的异黄酮与与金雀花碱及甲醛进行曼尼希反应，去甲氧基甲醚保护基的异黄酮与金雀花碱及甲醛的摩尔比为1：(1～2)：(0.5～0.8)，反应温度为50～80℃，反应时间为5～8h。
本发明还提供上述基于喹诺里西啶类衍生物的药物化合物的应用，将所述喹诺里西啶类衍生物用于制备抗肿瘤药物，包括在制备抗表皮鳞癌、乳腺癌、肺癌、胃癌、卵巢癌或口腔癌药物中的应用。
综述所述，针对该母核骨架中异黄酮结构，基于抗肿瘤靶标口袋的整体空间进行了全方位的设计，提高化合物与靶标的可逆结合能力，提高化合物的抗肿瘤活性。同时通过化合物的活性官能团修饰，大大提高了化合物的极性和水溶性等理化性质，提升化合物的成药性。
本发明的目的还在于提供一种基于喹诺里西啶类衍生物的药物化合物，该药物化合物的结构式为：
其中：
R2、R3为不易形成氢键的官能团，包括氢、烷基、碳环基烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基或芳基烷基等，该取代基经过修饰后，降低了化合物对细胞的毒害作用；
R4为体积较小的官能团，包括氢、羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其取代基、卤素等，该取代基经过修饰后，平衡化合物的脂水分配系数，进而增强药物的口服吸收利用度；
R1、R5、R6、R7、R8、R9为结构较为多样的官能团，包括氢、羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其衍生物、卤素、烷基、碳环基烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基或芳基烷基等，该取代基经过修饰后，提高了化合物的脂溶性，使其更易透过血脑屏障作用于中枢神经系统，进而发挥抗神经损伤作用。
进一步地，所述喹诺里西啶类衍生物中：
R2、R3分别为H、－C(＝O)R10、－C(＝O)OR10、－C(＝O)NR10R11、－C(＝O)SR10、－S(O)R10、－S(O)2R10、－S(O)(OR10)、－S(O)2(OR10)、－SO2NR10R11、(C1－C8)烷基、(C4－C8)碳环基烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基或(C6－C20)芳基(C1－C8)烷基；
R4为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa或卤素；
R1、R5、R6、R7、R8、R9分别为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa、卤素、－C(＝O)R10、－C(＝O)OR10、－C(＝O)NR10R11、－C(＝O)SR10、－ S(O)R10、－S(O)2R10、－S(O)(OR10)、－S(O)2(OR10)、－SO2NR10R11、卤素、(C1－C8)烷基、(C4－C8)碳环基烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基或(C6－C20)芳基(C1－C8)烷基；
每个n独立地为0、1或2；
每个R独立地为H、(C1－C8)烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基、(C6－C20)芳基、(C6－C20)取代的芳基、(C2－C20)杂环基、(C2－C20)取代的杂环基、(C6－C20)芳基(C1－C8)烷基或取代的(C6－C20)芳基(C1－C8)烷基；
每个Ra独立地为H、(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基、(C6－C20)芳基(C1－C8)烷基、(C4－C8)碳环基烷基、－C(＝O)R、－C(＝O)OR、－C(＝O)NR2、－C(＝O)SR、－S(O)R、－S(O)2R、－S(O)(OR)、－S(O)2(OR)或－SO2NR2；
每个R10或R11的(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基或(C6－C20)芳基(C1－C8)烷基独立的任选被一个或多个卤素、羟基、CN、N3、N(Ra)2或ORa取代；其中每个所述(C1－C8)烷基的一个或多个非末端碳原子可以任选地被－O－、－S－或－NRa－代替；并且R10和R11与它们都连接的氮一起形成3－7元杂环，其中所述杂环的任何一个碳原子可以任选被－O－、－S－或－NRa－代替。
进一步地，所述喹诺里西啶类衍生物的化学结构式包括CNI1-1、CNI1-3、CNI1-9～13：
本发明的另一目的在于提供上述基于喹诺里西啶类衍生物的药物化合物的制备方法，首先，4－苄氧基－2－羟基苯乙酮与N,N－二甲基甲酰胺二甲缩醛通过缩合反应得到氨烯酮。随后，在单质碘的作用下氨烯酮碘化、环合形成3－碘色酮。进一步与多种苯硼酸衍生物通过Suzuki偶联反应得到异黄酮，并除去7位的苄基保护基。最终，利用曼尼希反应得到喹诺里西啶类衍生物。具体包括以下步骤：
S11、与N,N－二甲基甲酰胺二甲缩醛通过缩合反应得到
S12、在单质碘的作用下氨烯酮碘化、环合形成化合物在碳酸钠/碳酸钾和醋酸钯的催化下进一步与通过Suzuki偶联反应得到 并使用溴化氢－醋酸溶液和水除去保护基；
S13、化合物与金雀花碱、甲醛在DMAP的催化下通过曼尼希反应得到
进一步地，
步骤S11中，的摩尔质量和N,N－二甲基甲酰胺二甲基缩醛的体积分别为1mmol、(6～8)mL，反应温度为74℃，反应时间为3－5h；
步骤S12中，与单质碘的摩尔比为1：1.5，反应温度为室温，反应时间为12～24h；
的摩尔比为1：1.5，反应温度为50℃，反应时间为5～12h，其中反应中以四氢呋喃和甲醇的混合溶液为溶剂，碱和醋酸钯为催化剂，所述碱为无水碳酸钠/碳酸碱；与溴化氢的醋酸溶液和水的摩尔体积比为1mmol：3.5mL：0.88mL，其中溴化氢的醋酸溶液中溴化氢的质量分数为33％，反应温度为50℃，反应时间为3～12h；
步骤S13中，与金雀花碱、DMAP及福尔马林的摩尔比分别为1mmol、1.25mmol、2.4mg、0.5mL，反应温度为70℃，反应时间为6－10h。
本发明的另一目的在于提供上述基于喹诺里西啶类衍生物的药物化合物的应用，将所述喹诺里西啶类衍生物用于制备抗神经损伤药物，包括在制备抗脑外伤、脊髓伤、术后神经损伤、脑卒中、缺氧性神经损伤神经退行性疾病的神经损伤药物中的应用。
与现有技术相比，本发明具有以下有益效果：
本发明在抗癌靶标的研究中发现，对异黄酮部分的结构修饰可以大大增强化合物对抗癌靶标的选择性和亲和性，并大幅度提高对多种实体肿瘤细胞的抑制活性。所以，本发明通过对金雀花碱N-异黄酮异黄酮部分的结构修饰，极大地提高了其与抗癌靶标的选择性和亲和性，并在抗肿瘤活性实验中对多种实体肿瘤细胞表现出极强的抑制活性和抗迁移效果，与如CN109970738A、CN110804056A等现有技术相比，本发明化合物结构修饰手段更加先进和全面，获得的新化合物抗肿瘤活性更强、应用范围更广谱，具有有较大的开发潜力。本发明通过适当合理的结构修饰，使得化合物针对多种癌细胞产生极强效地抑制活性和抗迁移效果，是一类针对多种实体瘤细胞非常有潜力的先导化合物。本发明通过巧妙的结构设计得到系列具有神经保护效果的化合物，是此类化合物在药理活性应用方面的又一大创新。具体地：
1)本发明提供的化合物在天然金雀花碱－异黄酮类化合物基础上，对异黄酮类部位进行结构修饰得到一系列衍生物，实验证明化合物1(CNI1-1)，化合物3(CNI1-3)，化合物9～13(CNI1-9～13)可抗神经损伤，在防治神经损伤相关疾病方面有很好的应用前景。
2)本发明提供的化合物中合成的CNI1-1、CNI1-3、CNI1-9、CNI1-10、CNI1-11、CNI1-12、CNI1-13对原代培养的神经元细胞、BV2细胞OGD损伤具有较强的保护活性，有统计学差异。
3)本发明提供的化合物利用药效团组合的理性设计策略，对喹诺里西啶类 衍生物进行创新性结构修饰、改造得到抗肿瘤活性更高的新化合物，制备方法高效、易于操作，且产物纯度高。
4)本发明化合物可有效抑制肿瘤细胞的生长和迁移，具有良好的抗肿瘤治疗效果。其中两种化合物对于两种癌细胞都表现出明显的抗增殖活性以及细胞毒性。两个化合物CNI3-2和CNI3-4对A431细胞的IC50值分别为53.39μM和26.66μM，对SK-BR-3细胞的IC50值分别为47.91μM和29.57μM，与阳性对照相当。结果进一步说明了相较于阳性药物，这两个化合物对于A431细胞和SK-BR-3细胞都表现出极强的抗增殖活性以及细胞毒性。
此外，本发明与现有技术相比，还具备以下突出的实质性特点和显著的进步，具体分析如下：
与CN201610390193涉及化合物区别：相比于上述化合物，本发明在化合物的基础上进行了更加全面、细致的结构修饰。经过一系列结构优化后，化合物整体的抗神经损伤效果得到了极大地提升，经大鼠脑皮层神经元细胞活力测试实验比较，本发明优化的化合物CNI1-3，CNI1-12等化合物神经元保护活性远高于上述化合物30％以上。进一步开辟了喹诺里西啶类衍生物化合物应用于抗神经损伤药物的新路线。
与CN201911076190涉及化合物区别：上述的化合物是通过修饰金雀花碱的2－吡啶酮部分来提高其生物活性，但对于此类化合物的异黄酮部分并没有进一步修饰。然而，本发明对该类化合物与抗癌靶标的研究中发现，对此类化合物异黄酮部分的结构修饰可以大大增强化合物对抗癌靶标的选择性和亲和性，并大幅度提高对多种实体肿瘤细胞的抑制活性。所以，本发明通过对化合物异黄酮部分的结构修饰，极大地提高了与抗癌靶标的选择性和亲和性，并在抗肿瘤活性实验中对多种实体肿瘤细胞表现出极强的抑制活性和抗迁移效果。
与相关文献(Doi：https://doi.org/10.1007/s10600-011-0006-2)区别：上述的化合物是金雀花碱与异黄酮通过氨甲基化反应得到的。文中只提到了通过该步骤的合成方法得到了一些化合物，其中并没有进行生物活性实验和构效关系分析，没有发现该类化合物的抗肿瘤活性。然而，本发明中提到的化合物在R2、R3修饰了一些已形成氢键的官能团，这是抗肿瘤活性的关键药效基团，经分子对接理论计算以及CCK8活性测试结果证明，每加上一个氢键，抑制肿瘤活性 提高10倍以上，该取代基经过修饰优化后，极大地提高了此类化合物靶向抗癌靶标的稳定性和选择性。
与CN201910147340涉及化合物区别：上述的化合物在异黄酮部分的结构修饰并不充分，导致其与抗癌靶标的结合亲和力有限，且在抗肿瘤活性实验中表现并不出色。本发明通过对化合物异黄酮部分的结构修饰，极大地提高了与抗癌靶标的选择性和亲和性，并在抗肿瘤活性实验中对多种实体肿瘤细胞表现出极强的抑制活性和抗迁移效果。
本发明将喹诺里西啶类衍生物应用于抗神经损伤药物填补了国内外研究空白，有独创性。国内外临床在脑卒中、缺氧性神经损伤神经退行性疾病的神经损伤等方面疾病缺乏有效的药物，本发明研发的技术具有很好的应用前景。
附图说明
图1为本发明实施例提供的化合物中金雀花碱－异黄酮类衍生物的制备流程图1；
图2为实施例中合成的CNI1-1对应的1H NMR图；
图3为实施例中合成的CNI1-1对应的13C NMR图；
图4为实施例中合成的CNI1-3对应的1H NMR图；
图5为实施例中合成的CNI1-3对应的13C NMR图；
图6为实施例中合成的CNI1-9对应的1H NMR图；
图7为实施例中合成的CNI1-9对应的13C NMR图；
图8为实施例中合成的CNI1-10对应的1H NMR图；
图9为实施例中合成的CNI1-10对应的13C NMR图；
图10为实施例中合成的CNI1-11对应的1H NMR图；
图11为实施例中合成的CNI1-11对应的13C NMR图；
图12为实施例中合成的CNI1-12对应的1H NMR图；
图13为实施例中合成的CNI1-12对应的13C NMR图；
图14为实施例中合成的CNI1-13对应的1H NMR图；
图15为实施例中合成的CNI1-13对应的13C NMR图；
图16为实施例中合成的CNI1-13对应的9F NMR图；
图17为细胞形态对比图(a－c)分别为第一次实验对照组细胞形态对比图，模型组和给药24h后给药组细胞形态对比图，图(e－f)分别为第二次实验对照组细胞形态对比图，模型组和给药24h后给药组细胞形态对比图；
图18为化合物1(CNI1-1)、化合物3(CNI1-3)、化合物9～13(CNI1-9～13)细胞活力检测结果图；
图19为本发明实施例提供的化合物中金雀花碱－异黄酮类衍生物的制备流程图2；
图20为实施例中合成的CNI3-1对应的1H NMR图；
图21为实施例中合成的CNI3-1对应的13C NMR图；
图22为实施例中合成的CNI3-2对应的1H NMR图；
图23为实施例中合成的CNI3-2对应的13C NMR图；
图24为实施例中合成的CNI3-3对应的1H NMR图；
图25为实施例中合成的CNI3-3对应的13C NMR图；
图26为实施例中合成的CNI3-4对应的1H NMR图；
图27为实施例中合成的CNI3-4对应的13C NMR图；
图28为化合物CNI3-2、CNI3-4对4T1乳腺癌细胞的抗迁移效果示意图。
图29示出了BV2缺氧缺糖损伤时间筛选结果。
图30细胞形态对比图(a－c)分别为第一次实验对照组，模型组和给药24h后给药组，图(e－f)分别为第二次实验对照组，模型组和给药24h后给药组；
图31为化合物1～13细胞活力检测结果图；
图32从苦豆子中发现喹诺里西啶类衍生物(CNIs)(A)描述CNI1及其衍生物来源的示意图；(B)TAK-285结合的EGFR和HER2(PDB代码：3POZ和3RCD)的结构叠加以及TAK-285的结合模式在EGFR和HER2的结合口袋内的比较；(C)结合在EGFR/HER2激酶结构域内的CNI3和TAK-285的结构重叠；
图33所示通过HTRF测定，CNI和TAK-285对EGFR(左图)和HER2(右图)的剂量－反应曲线；
图34所示CNI3和CNI4抑制不同癌细胞的增殖和迁移。(A－B)通过CCK-8评估，通过CNI3(左)，CNI4(中)和TAK-285(右)抑制A431(A)和BT-474(B)癌细胞系的生长测定。(C)活/死细胞双染色法测量CNI3，CNI4和TAK-285在其各自IC50浓度下对BT-474癌细胞活力的影响；
图35所示实验测定的pKi值与计算所得的CNIs和TAK-285对于EGFR和HER2激酶的结合自由能(ΔGcalc b)之间的线性相关性。图中显示了每个系列的相关系数(R2)；
图36为实验结果图；空白组的乳腺癌细胞向划痕边缘迁移的速度相对较迅速，划痕的宽度变窄较为明显，而CNI3-2、CNI3-4处理组的划痕宽度变化不如空白组明显；
图37为实验结果图；CNI3-2、CNI3-4相比其他化合物可诱导BT-474、SKBR-3、A431细胞死亡率更高；
图38为神经保护实验流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
苦豆子具有抗肿瘤作用和对中枢神经系统的作用，对肝癌细胞抑制作用明显，对宫颈癌、淋巴肉瘤、肺癌也有抑制作用；对中枢神经系统的抑制作用主要与药理作用以镇静催眠、镇痛及降温为主，其镇痛特征是以中枢性为主、没有成瘾性和耐药性。这些药效作用物质成分是生物碱和黄酮类成分，包括金雀花碱、苦参碱、槐定碱和槐果碱，这些生物碱具有喹诺里西啶结构。黄酮及异黄酮类化合物主要有金雀异黄素，5，7－二羟基黄酮，芒柄花黄素等。在前期对苦豆子进行系统分离研究中，本发明首次从中药苦豆子分离出金雀花碱N－亚甲基－(4,7－二羟基－3－甲氧基)异黄酮，属喹诺里西啶类化合物，该化合物具有较好地抗肿瘤活性和抗神经损伤作用。为了进一步提高该化合物的活性，分别以抗肿瘤活性和抗神经损伤活性为导向，利用计算机辅助设计结合药效实验，对该化合物进行了系统的结构修饰和优化，获得了一系列新型喹诺里 西啶类衍生物。
实施例1～7
本发明中的研究对象是天然产物喹诺里西啶类衍生物及其衍生物。天然产物喹诺里西啶类衍生物CNI1是从苦豆子果实中提取出的新型异黄酮生物碱，它主要是由金雀花碱和异黄酮两个主体结构通过亚甲基相连接。在合成中，主要对异黄酮结构进行合成。主要以市售的4－苄氧基－2－羟基苯乙酮衍生物为原料，通过羟醛缩合反应、环化反应、Suzuki偶联反应以及Mannich反应来合成。通过结构修饰，改变Suzuki偶联反应中苯硼酸的种类，合成了一系列喹诺里西啶类衍生物衍生物(CNI1-1、CNI1-3和CNI1-9～13)，(得到七个具有活性的化合物，图1)。
金雀花碱－异黄酮类衍生物的制备方法，包括以下步骤：
(1)在中加入NN－二甲基甲酰胺二甲基缩醛，反应得到然后在单质碘的作用下反应生成
的摩尔质量和N,N－二甲基甲酰胺二甲基缩醛的体积分别为1 mmol、7mL，反应温度为74℃，反应时间为3－5h(经过申请人多组实验对比，范围中任一时间对合成影响不大)；
与单质碘的摩尔量分别1mmol、1.5mmol，反应温度为室温，反应时间为12～24h(经过申请人多组实验对比，范围中任一时间对合成影响不大)。
(2)将步骤(1)制备得到与(本实施例中R1-R4按上述结构式中CNI1-1、CNI1-3和CNI1-9～13不同产物选择对应的市售苯硼酸衍生物)混合，反应制备得到
的摩尔量分别为1mmol、1.5mmol，反应温度为50℃，反应时间为5～12h(经过申请人多组实验对比，范围中任一时间对合成影响不大)。
的反应以四氢呋喃和甲醇的混合溶液为溶剂，碱和醋酸钯为催化剂，所述碱为无水碳酸钠。
(3)将步骤(2)制备得到的在溴化氢－醋酸溶液和水的作用下反应生成
与溴化氢的醋酸溶液和水的体积分别为1mmol、3.5mL、0.88mL。
溴化氢的醋酸溶液中溴化氢的质量分数为33％，反应温度为50℃，反应时间为3～12h(经过申请人多组实验对比，范围中任一时间对合成影响不大)。
(4)将步骤(3)制备得到的与金雀花碱、福尔马林在DMAP的催化下，反应得到
与金雀花碱、DMAP及福尔马林的摩尔比分别为1mmol、1.25mmol、2.4mg、0.5mL，反应温度为70℃，反应时间为6－10h(经 过申请人多组实验对比，范围中任一时间对合成影响不大)。
得到七个具有活性的化合物的1H NMR、13C NMR和19F NMR结果如下，分别对应图2～16。
化合物1(CNI1-1)：
1H NMR(400MHz,Chloroform-d)δ8.02(d,J＝8.8Hz,1H),7.80(s,1H),7.32–7.26(m,3H),6.83(d,J＝9.1Hz,1H),6.76(d,J＝8.8Hz,1H),6.51(d,J＝8.9Hz,1H),5.99(d,J＝6.5Hz,1H),4.15(d,J＝15.6Hz,1H),3.97–3.82(m,3H),3.82(s,3H),3.07(tt,J＝14.0,6.6Hz,3H),2.67–2.41(m,3H),2.22(s,3H),2.08–1.78(m,2H).13C NMR(101MHz,CDCl3)δ176.02,163.63,163.08,158.01,155.19,151.31,149.11,138.93,131.38,127.61,127.17,126.85,124.96,123.93,118.07,117.53,115.72,110.12,106.77,105.28,60.99,59.75,55.57,53.56,49.60,35.18,27.86,25.89,16.39.
化合物3(CNI1-3)：
1H NMR(400MHz,Chloroform-d)δ8.10(d,J＝8.9Hz,1H),8.00(s,1H),7.80(t,J＝1.7Hz,1H),7.74(d,J＝1.7Hz,2H),7.70–7.65(m,4H),7.49–7.42(m,4H),7.40–7.29(m,3H),6.82(d,J＝8.9Hz,1H),6.56(m,1H),6.01(m,1H),4.20(d,J ＝15.7Hz,1H),4.06–3.80(m,3H),3.22–3.03(m,3H),2.69–2.42(m,3H),2.11–1.84(m,2H).13C NMR(101MHz,Chloroform-d)δ175.54,163.46,163.19,155.09,151.98,148.96,142.04,140.94,138.77,132.89,128.80,127.52,127.37,127.01,126.84,125.95,124.93,117.90,117.38,115.81,106.79,105.11,60.85,59.59,53.41,49.43,35.01,27.70,25.70.
化合物9(CNI1-9)：
1H NMR(400MHz,DMSO-d6)δ8.10(s,1H),7.84(d,J＝8.8Hz,1H),7.29(m,1H),7.11(s,2H),6.81(d,J＝8.8Hz,1H),6.19(m,1H),6.04(m,1H),3.83(d,J＝15.6Hz,3H),3.75–3.65(m,1H),3.08–2.96(m,2H),2.93–2.84(m,1H),2.45(m,3H),2.19(s,6H),1.90–1.64(m,2H).13C NMR(101MHz,DMSO-d6)δ175.34,162.69,162.30,155.77,153.52,152.79,151.65,139.18,129.27,126.10,124.28,123.83,123.14,116.99,116.13,114.97,109.36,104.49,60.15,59.44,51.27,49.85,34.82,27.65,25.24,17.10.
化合物10(CNI1-10)：
1H NMR(400MHz,DMSO-d6)δ8.26(d,J＝23.4Hz,1H),7.85(d,J＝8.8Hz,1H),7.68(d,J＝2.2Hz,1H),7.53(m,1H),7.27(m,1H),7.19(d,J＝8.6Hz,1H),6.83(d,J＝8.8Hz,1H),6.17(d,J＝9.0Hz,1H),6.02(d,J＝7.0Hz,1H),3.88(s, 3H),3.86–3.77(m,3H),3.69(dd,J＝15.4,6.6Hz,1H),3.09–2.81(m,3H),2.49–2.45(m,1H),2.45–2.40(m,2H),1.89–1.66(m,2H).13C NMR(101MHz,DMSO-d6)δ174.95,162.67,162.38,155.83,154.62,153.72,151.69,139.15,130.52,129.08,126.11,125.77,121.98,121.15,116.89,116.05,115.12,113.00,109.67,104.44,60.10,59.49,56.65,51.09,49.87,34.85,27.70,25.25.
化合物11(CNI1-11)：
1H NMR(400MHz,Chloroform-d)δ8.02(d,J＝8.8Hz,1H),7.82(s,1H),7.35–7.26(m,3H),7.05–6.91(m,2H),6.75(d,J＝8.5Hz,1H),6.51(d,J＝9.2Hz,1H),5.98(d,J＝6.8Hz,1H),4.15(d,J＝15.6Hz,1H),3.98–3.79(m,3H),3.76(s,3H),3.20–2.96(m,3H),2.60–2.39(m,3H),2.02–1.79(m,2H).13C NMR(101MHz,Chloroform-d)δ175.36,163.44,162.86,157.51,155.11,153.13,149.28,138.86,131.71,129.64,126.73,122.09,120.96,120.46,117.52,117.30,115.30,111.28,106.97,105.21,60.73,59.44,55.76,53.27,49.46,34.94,27.62,25.54.
化合物12(CNI1-12)：
1H NMR(400MHz,Chloroform-d)δ8.02(d,J＝8.8Hz,1H),7.85(s,1H),7.45(d,J＝8.2Hz,2H),7.38–7.27(m,3H),6.78(d,J＝8.8Hz,1H),6.52(d,J＝9.2Hz,1H),5.99(d,J＝6.8Hz,1H),4.16(d,J＝15.6Hz,1H),3.98–3.77(m,3H),3.16–3.03(m,3H),2.61–2.45(m,3H),2.04–1.84(m,2H).13C NMR(101MHz,CDCl3) δ175.25,163.44,163.22,155.03,151.77,149.01,138.79,134.06,130.42,130.20,128.60,126.88,123.85,117.80,117.17,115.79,106.83,105.13,60.83,59.53,53.33,49.42,34.98,27.67,25.65.
化合物13(CNI1-13)：
1H NMR(400MHz,Chloroform-d)δ8.02(d,J＝8.9Hz,1H),7.90(s,1H),7.30(m,1H),7.17–7.01(m,2H),6.79(d,J＝8.8Hz,2H),6.52(d,J＝9.1Hz,1H),6.00(d,J＝6.8Hz,1H),4.17(d,J＝15.6Hz,1H),4.04–3.74(m,3H),3.25–2.92(m,3H),2.69–2.40(m,3H),2.18–1.78(m,2H).19F NMR(376MHz,Chloroform-d)δ-109.78.13C NMR(101MHz,Chloroform-d)δ174.75,164.12(d,J＝13.1Hz),163.42,161.65(d,J＝13.0Hz),154.93,152.27,148.95,138.78,126.95,123.02,117.85,117.09,116.00,111.87(d,J＝7.1Hz),111.69(d,J＝7.1Hz),106.87,105.12,103.30(d,J＝25.0Hz),60.84,59.56,53.32,49.42,34.99,27.68,25.66.
从中上述表征数据可以分析出：从上述氢谱和碳谱可以确证其结构为化合物CNI1-1、CNI1-3和CNI1-9～13。
药理活性实验及结果
利用CCK8法检测细胞活性
将BV2细胞按1×105个/mL密度接种于96孔板，贴壁24h后，进行OGD造模，处理后给药培养24小时，然后每孔加入10％培养基体积的CCK-8，在细胞培养箱中继续孵育2h后用酶标仪测定在450nm处的光密度(OD)值，计算细胞活性(％)。
活力计算：细胞活力(％)＝[A(加药)－A(空白)]/[A(0加药)－A(空白)]×100(A(加药)：具有细胞、CCK-8溶液和药物溶液的孔的OD值、A(0加药)：具有细胞、CCK-8溶液而没有药物溶液的孔的OD值、A(空白)：没有细胞的孔的OD值、细胞活力：细胞增殖活力或细胞毒性活力)
不同组别BV2细胞形态学改变于倒置显微镜下观察，未进行OGD/R处理的小胶质细胞生长良好，细胞呈椭圆，胞膜完整，形态清晰，细胞正常。OGD后小胶质细胞损伤明显，数目减少，部分细胞激活，出现触角和突触，细胞形态出现明显变化，且出现较多细胞死亡，给药后，细胞形态变清晰，胞膜开始趋于完整从形态上细胞损伤得到一定挽救(见图17)，对照组、模型组和给药组细胞形态对比(*表示P<0.05,**P<0.05)有统计学显著性。
不同浓度化合物在给药24h后BV2细胞活力检测结果如图3所示，与正常组相比，模型组细胞活力极显著下降，与模型组相比，化合物1、化合物3、化合物9、化合物10、化合物11、化合物12及化合物13细胞活力显著上升，这几个化合物对BV2细胞损伤有较强的挽救作用，活性较好；与模型组相比，化合物2和化合物5在50μmol－150μmol细胞活力显著升高，化合物6在6.25μmol时细胞活力也显著升高，除此以外化合物2、化合物4、化合物6、化合物7和化合物8在其他浓度细胞活力上升不是很明显。综上所述，化合物1、化合物3、化合物9、化合物10、化合物11、化合物12及化合物13对BV2细胞的ODG损伤活性较好，具体结果参见图18。
BV2细胞保留了小胶质细胞的各种形态和功能，是来源于小鼠的神经胶质细胞系。本发明发现OGD 2－5小时后BV2细胞形态固缩且细胞活力显著下降，且OGD刺激后部分BV2细胞由静息态均变为激活态。化合物1、化合物3、化合物9、化合物10、化合物11、化合物12、化合物13有效保护BV2细胞OGD损伤，使形态趋于正常，细胞活力上升，激活态细胞数目减少，该化合物在BV2细胞OGD模型有较好的活性。
以下是对本发明实施例提供的化合物抗肿瘤作用的实例及其良好的效果实验过程。
本发明实施例提供的化合物中利用均相时间分辨荧光技术(HTRF)发现这些新化合物对肿瘤细胞的关键靶点EGFR和HER2均具有抑制活性，且为一种 新作用机制。细胞增殖与细胞毒性实验表明该化合物对人乳腺癌细胞和人皮肤鳞癌细胞生长有较强的抑制作用，细胞划痕实验显示该化合物对高转移乳腺癌细胞有明显的抗转移效果。因此，本发明提供的喹诺里西啶类衍生物是一个新型的、极具前景的EGFR/HER2双靶抑制剂。
实施例8
本发明中的研究对象是天然产物喹诺里西啶类衍生物及其衍生物。天然产物喹诺里西啶类衍生物CNI1是从苦豆子果实中提取出的新型异黄酮生物碱，它主要是由金雀花碱和异黄酮两个主体结构通过亚甲基相连接。在合成中，主要对异黄酮结构进行合成。在本实施例中主要以2,4,6－三羟基苯乙酮衍生物为原料，通过羟醛缩合反应、环化反应、Suzuki偶联反应以及Mannich反应来合成。通过结构修饰，改变Suzuki偶联反应中苯硼酸的种类，合成了一系列喹诺里西啶类衍生物衍生物(CNI3-1～CNI3-4)，(得到四个具有活性的化合物，图20)。
金雀花碱－异黄酮类衍生物的制备方法，包括以下步骤：
(1)使用氯甲基甲醚将进行保护，得到加入N,N－二甲基甲酰胺二甲缩醛，反应得到在单质碘的作用下获得
(2)将与步骤(1)获得的在碳酸钠/碳酸钾和醋酸钯的催化下，分别 制备得到 并利用盐酸进行脱保护分别得到
(3)将步骤(2)获得的 与金雀花碱和甲醛在DMAP的催化下最终得到
其中，步骤(1)中，与氯甲基甲醚的摩尔比为1：2.5，反应 温度为0℃，反应时间3～4h；的摩尔质量与N,N－二甲基甲酰胺二甲缩醛的体积比为1mmol：6ml，反应温度为70～80℃，反应时间为4～8h；与单质碘的摩尔比为1：1.5，反应温度为20～35℃，反应时间12～48h。
步骤(2)中，与 的摩尔比为1：1.5，反应温度分别为40～60℃，反应时间分别为4～7h； 的摩尔与6M HCl的体积比为1mmol：8ml，反应温度为80～110℃，反应时间为1～3h。
优选的，步骤(3)中， 与金雀花碱及甲醛的摩尔比为1：1.5：0.6，反应温度为50～80℃，反应时间为5～8h。
得到四个具有活性的化合物的1H NMR和13C NMR结果如下，分别对应图 21～27。
化合物14(CNI3-1)：
1H NMR(400MHz,CDCl3)δ7.79(s,1H),7.41(d,J＝8.7Hz,2H),7.32–7.27(m,1H),6.95(d,J＝8.7Hz,2H),6.52(dd,J＝9.1,1.4Hz,1H),6.18(s,1H),5.98(dd,J＝6.9,1.5Hz,1H),4.16(d,J＝15.7Hz,1H),3.90(dd,J＝15.7,6.5Hz,1H),3.82(s,3H),3.82(d,J＝14.1Hz,1H),3.74(d,J＝14.2Hz,1H),3.18–3.02(m,3H),2.59–2.43(m,3H),1.99(d,J＝9.1Hz,2H),1.88(d,J＝12.9Hz,1H).13C NMR(101MHz,CDCl3)δ180.72,165.02,163.45,162.14,159.79,154.84,151.81,148.99,138.82,130.10,123.43,122.90,117.80,114.09,105.30,105.17,100.30,97.89,60.57,59.44,55.37,53.02,49.43,34.94,27.66,25.71.
化合物15(CNI3-2)：
1H NMR(400MHz,CDCl3)δ7.83(s,1H),7.53(d,J＝8.7Hz,2H),7.30(dd,J＝9.1,6.8Hz,1H),7.28(d,J＝8.7Hz,2H),6.53(dd,J＝9.1,1.4Hz,1H),6.19(s,1H),5.99(dd,J＝6.8,1.4Hz,1H),4.17(d,J＝15.5Hz,1H),3.90(dd,J＝15.8,6.7Hz,1H),3.85–3.74(m,2H),3.17–3.05(m,3H),2.58–2.46(m,3H),2.01(d,J＝13.1Hz,0H),1.89(d,J＝13.0Hz,1H).13C NMR(100MHz,CDCl3)δ180.16,165.34,163.45,162.12,154.79,152.42,149.27,148.94,138.82,130.40,122.67,121.09,120.47(q,J＝257.4Hz),117.86,105.21,105.16,100.59,98.09,60.61,59.46,53.03,49.43,34.93,27.66,25.70.
化合物16(CNI3-3)：
1H NMR(400MHz,CDCl3)δ7.78(s,1H),7.33–7.27(m,2H),7.25(d,J＝2.1Hz,1H),6.87(d,J＝8.4Hz,1H),6.52(dd,J＝9.1,1.4Hz,1H),6.18(s,1H),5.98(dd,J＝6.8,1.4Hz,1H),4.16(d,J＝15.6Hz,1H),3.90(dd,J＝15.6,6.5Hz,1H),3.85(s,3H),3.82(d,J＝13.9Hz,1H),3.74(d,J＝14.2Hz,1H),3.22–2.99(m,3H),2.61–2.41(m,3H),2.24(s,3H),2.00(d,J＝12.9Hz,1H),1.88(d,J＝12.9Hz,1H).13C NMR(100MHz,CDCl3)δ180.83,164.97,163.45,162.16,158.04,154.84,151.76,148.98,138.80,131.12,127.51,126.91,123.67,122.43,117.83,109.98,105.33,105.14,100.28,97.84,60.59,59.47,55.43,53.04,49.43,34.95,27.67,25.73,16.34.
化合物17(CNI3-4)：
1H NMR(400MHz,CDCl3)δ7.81(s,1H),7.39(d,J＝2.2Hz,1H),7.33–7.27(m,2H),7.24(d,J＝1.7Hz,1H),6.52(dd,J＝9.2,1.4Hz,1H),6.19(s,1H),5.99(dd,J＝6.9,1.3Hz,1H),4.16(d,J＝15.6Hz,1H),3.90(dd,J＝15.7,6.5Hz,1H),3.79(q,J＝14.1Hz,2H),3.20–3.02(m,3H),2.59–2.45(m,3H),2.34(s,3H),2.01(d,J＝13.0Hz,1H),1.88(d,J＝12.9Hz,1H).13C NMR(101MHz,CDCl3)δ180.25,165.28,163.45,162.12,154.79,152.40,148.95,147.91,138.82,132.15,131.30,129.34,127.63,122.82,121.06,120.68(q,J＝257.2Hz)117.83,105.22,105.16,100.53,98.06,60.59,59.45,53.01,49.42,34.93,27.66,25.70,16.26.
从中上述表征数据可以分析出：从上述氢谱和碳谱可以确证其结构为化合物CNI3-1～CNI3-4。
药理活性检测：
以下是化合物CNI3-1～CNI3-4两个化合物的药理活性实验结果：
利用多种肿瘤细胞系的细胞增殖/细胞毒性模型和细胞划痕模型研究化合物对肿瘤细胞的生长迁移能力的影响。
人表皮癌细胞(A431)、人乳腺癌细胞系(SK-BR-3)和小鼠乳腺癌细胞系(4T1)由上海赛百慷生物提供。在补充由10％的胎牛血清(FBS)的RPMI 1640培养基中培养细胞。细胞以8×103/孔被接种到96孔板中，在5％CO2的37℃恒温培养箱中培养至细胞贴壁。
使用CCK-8法来检测化合物对细胞系(过人表皮鳞癌A431细胞系和人乳腺癌SK-BR-3细胞系)的抗增殖作用以及细胞毒性影响。
将测试化合物溶解在DMSO中，并用培养基稀释至不同浓度(DMSO的终浓度为0.5％)。将不同浓度的待测化合物加入反应孔，并培养细胞72小时。
移除培养基，向每孔加入100μL含0.5mg/mL CCK-8的培养基，并在5％CO2的37℃恒温培养箱中培养2小时。使用酶标仪于450nm处检测吸光度，并计算相对活力。使用Grap Pad Prism 6.01计算化合物的IC50值。数据表示为来自三个独立实验的平均值。CNI3-2～CNI3-4对于两种癌细胞都表现出明显的抗增殖活性以及细胞毒性。其中CNI3-2和CNI3-4对A431细胞的IC50值分别为53.39μM和26.66μM，对SK-BR-3细胞的IC50值分别为47.91μM和29.57μM，与阳性对照相当。结果进一步说明了相较于阳性药物，CNI3-2和CNI3-4对于A431细胞和SK-BR-3细胞都表现出极强的抗增殖活性以及细胞毒性。
取对数生长期的小鼠乳腺癌4T1细胞以1.5×105个/孔接种到6孔板中，培养至细胞铺满。在每孔底部中央的细胞上划上一条纵向模拟创伤的直线(创伤区)，使用无菌接种环。创伤区宽度被测量使用37XC倒置生物显微镜。使用PBS清洗细胞，一组细胞被培养在完全培养基中作为对照组，另外一组细胞被处理在溶于完全培养基的样品(10、20和40μM)中。当观察到对照组的创伤区即将消失时，各组创伤区的宽度被测量，使用37XC倒置生物显微镜以评估相对迁移比率。数据表示为来自三个独立实验的平均值。如图7所示，与空白组相比，被加入两个化合物的创伤区癌细胞迁移距离缩短，迁移比率明显下降(****P<0.0001)，并且随着化合物剂量的增大，迁移比率下降更大，表明两种化合物的抑制作用具有剂量依赖性。这些结果表明了CNI3-2和CNI3-4都具有 极佳的体外抗乳腺癌肿瘤细胞转移活性。
为了进一步验证本发明实施例提供的化合物的在抗神经损伤方面的作用，在上述实验的基础上，又进行了以下试验，充分证明了本发明权利要求所保护的技术方案的良好的技术效果。
以下是CNI1-1～CNI1-13共13个新化合物药效评价报告。其中，CNI1-2、CNI1-4～CNI1-8通过与实施例1-7相同的方法制备得到。以下为相关的结构式。
一、神经保护实验
1大鼠脑皮层神经元的原代培养
按常规方案培养原代神经元，每次取一日龄大鼠3～4只，无菌法取大脑，剔除脑膜和小血管。剥离皮层，用D-Hanks液洗3次。
将纯净的脑皮层剪成1mm3的小块，加入0.125％胰蛋白酶D-Hanks消化，37℃震荡孵育10min，加入DMEM完全培养液终止消化。600g离心5min，弃上清。然后加入DMEM完全培养液，轻轻吹打分散细胞，过200目筛，取细胞悬液接种于多聚赖氨酸包被的96孔板中，细胞密度1×106个/ml，每孔100μl。次日换半量DMEM培养基(其中含马血清和FCS各10％，青霉素100U/ml)。3天后加阿糖胞苷至10μM抑制杂细胞，24h后换全液，去除阿糖胞苷。以后每3天换1/2液，培养成熟后用于抗损伤试验。
2细胞分组和给药方法
原代神经元细胞分组和处理如下：
正常对照组(Nor，用DMEM液培养，不用山梨醇损伤，其余各组均用山梨醇损伤)；
模型对照组(Model，不治疗)；
阳性对照组(Con，用阳性对照药物神经节苷脂1μM)；
药物高剂量组(CNI1-1～13，新化合物终浓度为1μM)。
每组3个平行孔(n＝1)，试验重复5次(n＝5)
3 MTT法测定细胞活力和结果
细胞接种后第4天，用无血清DMEM培养液洗2遍，然后加入无血清培养基、阳性药和待测药物，同时除正常对照组外，各组加入山梨醇溶液至终浓度为25mM损伤神经元，于37℃、5％二氧化碳恒温培养箱继续培养24h，吸弃培养基，每孔加入0.04％MTT(以无血清培养基配制)，孵育4h。测定时弃去含MTT的培养基，加入DMSO 150μl/孔，震荡，充分震荡溶解甲簪颗粒，于540nm处测定吸光度值。以各组吸光度值与正常对照组固定孔的吸光度值之比乘以100％表示各组细胞的活力。

注：上表中Nor(正常对照组)，Modle(模型对照组)，Con(阳性对照组)，CNI1-1～CNI1-13为化合物1-13。结果可见化合物1、化合物3、化合物9、化合物10、化合物11、化合物12及化合物13等7化合物具有明显的神经元保护活性，在同等摩尔浓度下，药效高于阳性对照药。换算成质量浓度(ng/L)则药效更突出。
二、神经保护实验
1.实验目的
观察新结构化合物1-13对氧糖剥夺细胞活力的影响，探究新结构化合物1-13的活性。
2.实验材料
2.2仪器

3.方法与过程
3.1实验流程
如图38所示，为了考察新结构化合物1-13对氧糖剥夺细胞活力的影响，探究新结构化合物1-13的活性，设计了实验流程，包括细胞复苏、细胞传代、造模时间筛选和药物筛选四个环节，具体实验如下。
3.2细胞培养
(1)细胞复苏
将取出的冻存管立即放入水浴锅(37℃)内并迅速摇晃解冻。75％酒精消毒后，将冻存管放在超净工作台上，将BV2细胞转移至15mL的无菌离心管中，立即加入5mLDMEM培养基(89％的DMEM高糖培养基，10％的胎牛血清，1％青霉素－链霉素)，混合均匀后，离心(4min，800rpm)，弃去上清液，随后加入2mL的含血清培养基，捶打至混悬。接种于培养皿上加入5mL的含血清培养基，然后将5mL细胞混悬液转入培养皿上，前后左右轻轻摇晃，使细胞分散均匀，放入37℃、5％CO2培养箱中培养。次日在显微镜下观察细胞生长状态，若培养皿中漂浮的细胞过多，进行换液操作，即将培养皿中旧的培养 基吸弃，用1.5mL的PBS洗2次，然后加10mL的含血清培养基继续培养。
(2)细胞传代
显微镜观察细胞状态，细胞状态稳定，并长至80％，即可传代。倒弃旧培养液。用PBS溶液洗涤细胞2次。加入0.8mL0.25％胰蛋白酶消化细胞(37℃，消化1－3min)，当细胞开始脱落时，加入2mLH-DMEM终止消化。用移液枪吹打细胞成细胞悬液，并转移至离心管中。1000rpm离心5min，弃上清。1：3传代，将细胞用3mL完全培养液轻轻重悬，均匀接种在含有9mL完全培养液的3个10mL培养皿中，在37℃、5％CO2的培养箱中过夜培养。(或者细胞加入培养皿中，观察细胞生长状态，根据不同实验要求培养适当时间后进行后续实验。)
(3)细胞计数
将BV2细胞按细胞传代的操作方法获得细胞沉淀。用适量的培养基稀释到合适的细胞密度，混匀，吸取细胞悬液(10μL细胞悬液与10μL台盼蓝混合)从计数板和盖玻片的下边缘打入细胞悬液，使液体充满在整个盖玻片上，防止出现气泡，用计数仪计数。
(4)细胞冻存
在培养箱中取出细胞，当培养皿中的细胞密度达到80－90％时，用2mL的PBS清洗两遍，加入1mL的胰蛋白酶进行消化，显微镜下观察细胞变圆并脱落时，加入4mL的含血清的培养基终止消化，离心(3min，1000rpm)，弃去上清液，加入冻存液(胎牛血清：DMSO＝9：1)重悬细胞。充分混匀后将细胞悬液转移至冻存管中，每支冻存管冻存1mL的细胞悬液(细胞密度≥1×106个/mL)。冻存管做好标识后，依次降温(4℃，30min；-20℃，1h；-80℃，过夜；液氮保存)。
3.3利用CCK8法检测细胞活性
将BV2细胞按1×105个/mL密度接种于96孔板，贴壁24h后，进行OGD造模，处理后给药培养24小时，然后每孔加入10％培养基体积的CCK-8，在细胞培养箱中继续孵育2h后用酶标仪测定在450nm处的光密度(OD)值，计算细胞活性(％)。活力计算：细胞活力(％)＝[A(加药)－A(空白)]/[A(0加药)－A(空白)]×100(A(加药)：具有细胞、CCK-8溶液和药物溶液的孔 的OD值、A(0加药)：具有细胞、CCK-8溶液而没有药物溶液的孔的OD值、A(空白)：没有细胞的孔的OD值、细胞活力：细胞增殖活力或细胞毒性活力)。
3.4化合物－配制
根据化合物结构和分子量及预实验后发现化合物较难溶于水，因此通过逐量增加DMSO的方法来溶解药物以溶清为终点。除化合物9、化合物11、化合物12在10μl时不溶外，其他化合物均在10μl时溶解，化合物9、化合物11、化合物12在30μl时溶解，同时根据化合物4和6的IC50值，确定最大浓度为200μmol。
3.5实验分组及过程
首先取一瓶状态良好的BV2细胞，制成细胞悬液，并以每孔约1x105个细胞接种于96孔板内(96孔板外周用1×PBS填补，以防边缘效应)，每孔100μL，轻敲孔板使悬液混合均匀，过夜培养约24h。镜下观察细胞贴壁良好生长，分为对照组、模型组和给药组，给药组分为以下6个浓度梯度“6.25μmol·L-1，12.5μmol·L-1，50μmol·L-1，100μmol·L-1，150μmol·L-1，200μmol·L-1”，每组设置1～6个复孔，重复3－4次。BV2细胞以高糖DMEM+10％灭活的胎牛血清为完全培养基，置于37℃，5％CO2的孵箱中进行培养，隔日更换培养液。当细胞融合度达到80％～85％时，用0.25％胰酶消化传代。取对数生长期细胞以适当密度接种于96孔板中，
3.6缺氧缺糖造模时间筛选
根据文献调，详见下表，模型组及各剂量实验组均使用无糖Earle＇s平衡盐溶液清洗细胞3遍后，每个孔加入Earle＇s溶液100μL，设置3－6个复孔，置于37℃恒温箱的密封缺氧装置中，快速通入N2与CO2的混合气体30min，赶尽装置内空气，然后调小气体流量，保持培养箱为(94％N2、5％CO2和1％O2)开始计时缺糖缺氧时间(2－5h)，OGD处理后，小心吸弃无糖缓冲液，置于37℃细胞培养箱中继续培养24h后CCK8测定细胞活力。对照组以有糖DMEM清 洗细胞3遍并孵育，不进行缺氧处理，其他操作同模型组。

3.7统计学分析
应用SSSS22.0软件进行统计学分析，数据以表示，组内两两比较采用独立样本t检验，组间比较采用单因素方差分析，P＜0.05为差异有统计学意义。
4.实验结果
4.1缺氧缺糖造模时间筛选结果
通过文献调研，发现一个问题，对于BV2细胞OGD模型造模时间并不完全一样，基于此我们研究了缺氧缺糖不同时间对细胞活力的影响。研究发现缺氧缺糖2－5h后与正常组相比，各组均有显著性差异，其中缺氧缺糖5h均值差异最大，且模型组细胞活力依旧较好。因此后续实验中选取5小时为造模终点。
图29显示出了BV2缺氧缺糖损伤时间筛选结果。
4.2药物筛选实验结果
4.2.1对照组、模型组和给药组细胞形态对比
图30细胞形态对比图(a－c)分别为第一次实验对照组，模型组和给药24h后给药组，图(e－f)分别为第二次实验对照组，模型组和给药24h后给药组。
不同组别BV2细胞形态学改变于倒置显微镜下观察，未进行OGD/R处理 的小胶质细胞生长良好，细胞呈椭圆，胞膜完整，形态清晰，细胞正常。OGD后小胶质细胞损伤明显，数目减少，部分细胞激活，出现触角和突触，细胞形态出现明显变化，且出现较多细胞死亡，给药后，细胞形态变清晰，胞膜开始趋于完整从形态上细胞损伤得到一定挽救(见图30)。
4.2.2对照组、模型组和给药组细胞形态对比(*表示P<0.05,**P<0.05有统计学显著性)
不同浓度化合物在给药24h后BV2细胞活力检测结果如图31所示，与正常组相比，模型组细胞活力极显著下降，与模型组相比，化合物1、化合物3、化合物9、化合物10、化合物11、化合物12及化合物13细胞活力显著上升，这几个化合物对BV2细胞损伤有较强的挽救作用，活性较好；与模型组相比，化合物2和化合物5在50μmol－150μmol细胞活力显著升高，化合物6在6.25μmol时细胞活力也显著升高，除此以外化合物2、化合物4、化合物6、化合物7和化合物8在其他浓度细胞活力上升不是很明显。综上所述，化合物1、化合物3、化合物9、化合物10、化合物11、化合物12及化合物13对BV2细胞的ODG损伤活性较好，具体结果参见图31，化合物1-13细胞活力检测结果。4.实验讨论
BV2细胞保留了小胶质细胞的各种形态和功能，是来源于小鼠的神经胶质细胞系。本研究发现OGD 2－5小时后BV2细胞形态固缩且细胞活力显著下降，且OGD刺激后部分BV2细胞由静息态均变为激活态。化合物1、化合物3、化合物9、化合物10、化合物11、化合物12、化合物13有效保护BV2细胞OGD损伤，使形态趋于正常，细胞活力上升，激活态细胞数目减少，该化合物在BV2细胞OGD模型有较好的活性。当然，我们只是从形态学和细胞活力维度探讨该系列化合物对BV2细胞OGD损伤的活性，后续可能仍需要研究其他细胞器或者该细胞器OGD损伤后不同因子的PCR或western blot表达水平来验证。
为了进一步验证本发明实施例提供的化合物的在抗肿瘤方面的作用，在上述实验的基础上，又进行了以下实验，充分证明了本发明权利要求所保护的技术方案的良好的技术效果。
一、CNIs作为潜在EGFR/HER2抑制剂的分子对接研究
如图32所示，从苦豆子中发现喹诺里西啶类衍生物(CNIs)(A)描述CNI1及其衍生物来源。金雀花碱和异黄酮的部分分别呈现红色和蓝色。(B)TAK-285结合的EGFR和HER2(PDB代码：3POZ和3RCD)的结构叠加以及TAK-285的结合模式在EGFR和HER2的结合口袋内的比较。EGFR和HER2激酶结构域分别表示为青色和黄色卡通，其中调节的二级结构α－螺旋C和A－环分别为红色和绿色。抑制剂TAK-285及其相互作用的残基分别描绘为紫色和灰色棒，DFG基序以橙色突出显示。黑色虚线表示氢键。(C)结合在EGFR/HER2激酶结构域内的CNI3和TAK-285的结构重叠。黑色虚线圆圈勾勒出由激酶抑制剂的不同部分占据的结合子口袋。
我们首先利用分子对接软件AutoDock Vina来预测一系列CNIs化合物与激酶EGFR和HER2的结合相互作用。根据内置评分功能，每种CNIs化合物与EGFR和HER2的最佳结合姿势分别获得了约－10和－9kcal/mol的结合能，而TAK-285(先前报道的强效EGFR/HER2抑制剂，且TAK-285与EGFR/HER2的共晶体结构已被解析)约为－11和－10kcal/mol(图2－1B)。因此，我们计算预测这些CNIs分子对EGFR和HER2都具有较高结合亲和力，尽管相对低于TAK-285。与TAK-285的进一步比较使我们假设这些CNIs化合物可能是潜在的EGFR/HER2双靶抑制剂，因为它们与TAK-285的拓扑学和药效学相似(图2－1C)。
如图33，所示通过HTRF测定，CNI和TAK-285对EGFR(左图)和HER2(右图)的剂量－反应曲线。
表2－3.CNIs和TAK-285的激酶、细胞和计算数据。


aBiochemical IC50and Ki determined through the HTRF assay(N＝3).
b Cellular IC50measured by the CCK-8 analysis(N＝3).A431,HER2－
overexpressing human epidermoid carcinoma cell line；BT-474,EGFR－overexpressing human breast cancer cell line.
cFree energy of binding(ΔGcalc b)estimated by the MM－GBSA method.
在CNIs系列中，CNI3在抑制EGFR(IC50＝1.12μM；Ki＝0.59μM)和HER2(IC50＝3.51μM；Ki＝1.77μM)的激酶活性方面最为有效。CNI4对HER2也表现出高效力(IC50＝6.78μM；Ki＝3.41μM)，而其对EGFR的活性(IC50＝32.86μM；Ki＝17.30μM)相对较低但仍然相当可观。总之，化合物CNI3和CNI4可能作为有希望的EGFR/HER2双靶抑制剂，
测定待测化合物是否可以对人乳腺癌细胞系MDA-MB-231和小鼠乳腺癌细胞系4T1的迁移产生影响。MDA-MB-231和4T1均属于三阴性乳腺癌(TNBC)，其不会产生过多的HER2受体，而已知前一种细胞系正常表达EGFR。从我们的伤口愈合测定中，当用CNI3和CNI4处理时，两种类型的癌细胞在伤口边缘的迁移率显著减弱(分别与对照组相比，*P<0.05和**P<0.01)。并表现出较强的剂量依赖性(图34)。因此，结果表明CNI3和CNI4对不同的乳腺癌细胞具有令人鼓舞的体外抗增殖和抗迁移活性，并且具有作为先导药物进一步发展的潜力。
如图34所示，CNI3和CNI4抑制不同癌细胞的增殖和迁移。(A－B)通过CCK-8评估，通过CNI3(左)，CNI4(中)和TAK-285(右)抑制A431(A) 和BT-474(B)癌细胞系的生长测定。(C)活/死细胞双染色法测量CNI3，CNI4和TAK-285在其各自IC50浓度下对BT-474癌细胞活力的影响。值得注意的是，大多数死细胞(染成红色)已用缓冲溶液洗掉。(D)通过CNI3和CNI4剂量依赖性抑制乳腺癌细胞系MDA-MB-231(左)和4T1(右)的迁移。与对照组相比，*P<0.05和**P<0.01。
接下来开始使用分子动力学(MD)模拟方法阐明CNI对EGFR和HER2的作用机制。通过MM-GBSA方法估计每种激酶化合物复合体系的结合自由能。总体而言，计算表明亲和力的强弱顺序与所做的激酶抑制数据一致(表2－3)，计算结果表明TAK-285是最强的EGFR/HER2的抑制剂，其次是CNI3。计算的结合能和实验抑制常数之间的高度相关性(图35)表明我们模拟的有效性。
如图35所示，实验测定的pKi值与计算所得的CNIs和TAK-285对于EGFR和HER2激酶的结合自由能(ΔGcalc b)之间的线性相关性。图中显示了每个系列的相关系数(R2)。
二、细胞水平抗肿瘤活性实验
1、以化合物CNI3-1～CNI3-4抗癌细胞增殖活性作为筛选条件CCK-8法评估BT-474、SK-BR-3、MDA-MB-231、A431的抗增殖活性。
实验方法：
1)细胞处理：待细胞贴壁密度达到80％时，胰酶消化传代。传至第3代的细胞，胰酶消化获得单细胞悬液，调整细胞浓度，按照5×103/孔接种到96孔板中，5％CO2，37℃恒温培养箱中培养至细胞贴壁。
2)样本处理：称取化合物各45mg，溶解至DMSO中得到C原，用完全培养基稀释C原使得溶液中DMSO浓度为0.5％，稀释后浓度作为最大浓度。按照下表进行样品稀释配置。每个处理组均做3个复孔。
3)CCK-8实验：96孔板培养细胞至贴壁后移除旧培养基，将培养基与样品溶 液按照一定比例加入孔板使得孔中总体积为100ul，使得孔板中样品浓度为下表梯度，培养48h后加入10ul的CCK-8试剂，5％CO2，37℃恒温培养箱中培养2小时。于450nm处检测吸光度计算细胞的增殖抑癌率。公式如下：细胞增殖抑制率(％)＝[(空白组A值－牛蒡子苷处理组A值)/空白组A值]×100％
化合物抗癌细胞增殖活性-CCK8
以化合物抗癌细胞增殖活性为筛选条件，根据其IC50值分析可知，化合物CNI3-4对BT-474、SK-BR-3、MDA-MB-231、A431细胞株均有良好的抗细胞增殖活性。
其中CNI3-1,CNI3-2,CNI3-3,CNI3-4的结构式如下：
2.细胞划痕实验：
1)取一个6孔板，用直尺及马克笔在板背后画出匀称的直线。
2)取一瓶细胞，胰酶消化，经计数后，在每个孔中加入约5x105个4T1细胞。
3)待细胞铺满板后，用枪头比着尺子，垂直于之前画的横线划横。
4)用无菌PBS洗细胞3次，去除划下的细胞。0uM、10uM、20uM、40uM 处理4T1细胞，加入无血清的专用培养基。拍下0h的划痕，每个时间点取1视野，
5)在培养温度为37℃，CO2浓度为5％的条件下培养48小时后再次拍照记录。最后以及得出的两组不同的划痕面积算出细胞划痕迁移率。
划痕迁移率(％)＝1－(相应时间划痕面积值/0时划痕面积值)x100％。
如图36所示，实验结果：空白组的乳腺癌细胞向划痕边缘迁移的速度相对较迅速，划痕的宽度变窄较为明显。而CNI3-4处理组的划痕宽度变化不如空白组明显。如图所示在处理48小时后，CNI3-4作用组的划痕面积值明显大于control组及CNI3-2组，因此可推断CNI3-4化合物具有抑制乳腺癌细胞迁移的生理活性。
3.Calcein-AM/PI活死细胞染色实验
选用Calcein-AM和PI的双重染料，来进行活细胞和死细胞的双重染色标记，从而进行活细胞和死细胞水平的分析。
化合物CNI3-1、CNI3-2、CNI3-3和CNI3-4以其相对应的半抑制浓度处理细胞分别处理细胞BT-474、SK-BR-3、A431。样本染色后，荧光倒置显微镜进行拍照：绿色荧光－活细胞，红色荧光－死细胞，如图37所示，实验结果：CNI3-4相比其他化合物可诱导BT-474、SK-BR-3、A431细胞死亡率更高。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，凡在本发明的精神和原则之内所做的任何修改、等同替换和改进等，都应涵盖在本发明的保护范围之内。

Claims (13)

  1. 一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，该药物化合物的结构式为：
    其中：
    R2、R3为易形成氢键的官能团，包括羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其取代基或卤素；
    R4、R5、R6、R8、R9为体积较小的官能团，包括氢、羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其取代基或卤素；
    R1、R7为结构较为多样的官能团，包括氢、羟基及其衍生物、氨基及其衍生物、叠氮基、硝基、氰基、巯基及其衍生物、卤素、烷基、碳环基烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基或芳基烷基。
  2. 根据权利要求1所述的一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，所述喹诺里西啶类衍生物中：
    R1、R7分别为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa、－C(＝O)R10、－C(＝O)OR10、－C(＝O)NR10R11、－C(＝O)SR10、－S(O)R10、－S(O)2R10、－S(O)(OR10)、－S(O)2(OR10)、－SO2NR10R11、卤素、(C1－C8)烷基、(C4－C8)碳环基烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基或(C6－C20)芳基(C1－C8)烷基；
    R2、R3分别为ORa、N(Ra)2、N3、CN、NO2、S(O)nRa或卤素；
    R4、R5、R6、R8、R9分别为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa或卤素；
    每个n独立地为0、1或2；
    每个R独立地为H、(C1－C8)烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基、(C6－C20)芳基、(C6－C20)取代的芳基、(C2－C20)杂环基、(C2－C20)取代的杂环基、(C6－C20)芳基(C1－C8)烷基或取代的(C6－C20)芳基(C1－C8)烷基；
    每个Ra独立地为H、(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基、(C6－C20)芳基(C1－C8)烷基、(C4－C8)碳环基烷基、－C(＝O)R、－C(＝O)OR、－C(＝O)NR2、－C(＝O)SR、－S(O)R、－S(O)2R、－S(O)(OR)、－S(O)2(OR)或－SO2NR2；
    每个R10或R11的(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基或(C6－C20)芳基(C1－C8)烷基独立的任选被一个或多个卤素、羟基、CN、N3、N(Ra)2或ORa取代；其中每个所述(C1－C8)烷基的一个或多个非末端碳原子任选地被－O－、－S－或－NRa－代替；并且R10和R11与它们都连接的氮一起形成3－7元杂环，其中所述杂环的任何一个碳原子任选被－O、－S－或－NRa－代替。
  3. 根据权利要求1所述的一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，所述喹诺里西啶类衍生物包括式CNI3-1～CNI3-4对应的化合物中的一种，还包括结构式CNI3-1～CNI3-4对应的盐，
  4. 根据权利要求3所述的一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，结构式CNI3-1～CNI3-4所对应的盐均为对应化合物与无机酸或有机酸反应制得；
    所述无机酸为盐酸、氢溴酸、硫酸、硝酸、氨基磺酸或磷酸中的一种；
    所述有机酸为柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2－乙酰氨基苯甲酸或羟乙磺酸中的一种。
  5. 一种如权利要求1-4中任一所述的基于喹诺里西啶类衍生物的药物化合物的制备方法，其特征在于，包括以下步骤：
    S01、将2,4,6－三羟基苯乙酮的4和6位羟基进行保护，加入N,N－二甲基甲酰胺二甲缩醛通过缩合反应得到氨烯酮；
    S02、在单质碘的作用下氨烯酮碘化、环合形成3－碘色酮，进一步与多种苯硼酸衍生物通过Suzuki偶联反应得到异黄酮，并除去5和7位的甲氧基甲醚保护基；
    S03、利用曼尼希反应得到喹诺里西啶类衍生物。
  6. 根据权利要求5所述的基于喹诺里西啶类衍生物的药物化合物的制备方法，其特征在于，
    步骤S01中，2,4,6－三羟基苯乙酮衍生物采用氯甲基甲醚进行羟基保护，2,4,6－三羟基苯乙酮与氯甲基甲醚的摩尔比为1：(2～3)，反应温度为0℃，反应时间3～4h；
    所述的2,4,6－三羟基苯乙酮衍生物的摩尔质量与N,N－二甲基甲酰胺二甲缩醛的体积比为1mmol：(5－7)ml，缩合反应反应温度为70～80℃，反应时间为4～8h；
    步骤S02中，氨烯酮与单质碘的摩尔比为1：(1～2)，反应温度为20～35℃，反应时间12～48h；3－碘色酮与苯硼酸衍生物的摩尔比为1：(1～2)，反应温度分别为40～60℃，反应时间分别为4～7h；
    异黄酮采用HCl并除去5和7位的甲氧基甲醚保护基，其中，异黄酮的摩 尔与6M HCl的体积比为1mmol：(7～10)ml，反应温度为80～110℃，反应时间为1～3h；
    步骤S03中，去甲氧基甲醚保护基的异黄酮与与金雀花碱及甲醛进行曼尼希反应，去甲氧基甲醚保护基的异黄酮与金雀花碱及甲醛的摩尔比为1：(1～2)：(0.5～0.8)，反应温度为50～80℃，反应时间为5～8h。
  7. 一种如权利要求1-4中任一所述的基于喹诺里西啶类衍生物的药物化合物的应用，其特征在于，将所述喹诺里西啶类衍生物用于制备抗肿瘤药物，包括在制备抗表皮鳞癌、乳腺癌、肺癌、胃癌、卵巢癌或口腔癌药物中的应用。
  8. 一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，该药物化合物的结构式为：
    其中：
    R2、R3为不易形成氢键的官能团，包括氢、烷基、碳环基烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基或芳基烷基；
    R4为体积较小的官能团，包括氢、羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其取代基、卤素；
    R1、R5、R6、R7、R8、R9为结构较为多样的官能团，包括氢、羟基及其取代基、氨基及其取代基、叠氮基、硝基、氰基、巯基及其衍生物、卤素、烷基、碳环基烷基、取代的烷基、烯基、取代的烯基、炔基、取代的炔基或芳基烷基。
  9. 根据权利要求8所述的一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，所述喹诺里西啶类衍生物中：
    R2、R3分别为H、－C(＝O)R10、－C(＝O)OR10、－C(＝O)NR10R11、－C(＝O)SR10、－S(O)R10、－S(O)2R10、－S(O)(OR10)、－S(O)2(OR10)、－SO2NR10R11、(C1－C8)烷基、(C4－C8)碳环基烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基或(C6－C20)芳基(C1－C8)烷基；
    R4为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa或卤素；
    R1、R5、R6、R7、R8、R9分别为H、ORa、N(Ra)2、N3、CN、NO2、S(O)nRa、卤素、－C(＝O)R10、－C(＝O)OR10、－C(＝O)NR10R11、－C(＝O)SR10、－S(O)R10、－S(O)2R10、－S(O)(OR10)、－S(O)2(OR10)、－SO2NR10R11、卤素、(C1－C8)烷基、(C4－C8)碳环基烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基或(C6－C20)芳基(C1－C8)烷基；
    每个n独立地为0、1或2；
    每个R独立地为H、(C1－C8)烷基、(C1－C8)取代的烷基、(C2－C8)烯基、(C2－C8)取代的烯基、(C2－C8)炔基、(C2－C8)取代的炔基、(C6－C20)芳基、(C6－C20)取代的芳基、(C2－C20)杂环基、(C2－C20)取代的杂环基、(C6－C20)芳基(C1－C8)烷基或取代的(C6－C20)芳基(C1－C8)烷基；
    每个Ra独立地为H、(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基、(C6－C20)芳基(C1－C8)烷基、(C4－C8)碳环基烷基、－C(＝O)R、－C(＝O)OR、－C(＝O)NR2、－C(＝O)SR、－S(O)R、－S(O)2R、－S(O)(OR)、－S(O)2(OR)或－SO2NR2；
    每个R10或R11的(C1－C8)烷基、(C2－C8)烯基、(C2－C8)炔基或(C6－C20)芳基(C1－C8)烷基独立的任选被一个或多个卤素、羟基、CN、N3、N(Ra)2或ORa取代；其中每个所述(C1－C8)烷基的一个或多个非末端碳原子任选地被－O－、－S－或－NRa－代替；并且R10和R11与它们都连接的氮一起形成3－7元杂环，其中所述杂环的任何一个碳原子可以任选被－O－、－S－或－NRa－代替。
  10. 根据权利要求8所述的一种基于喹诺里西啶类衍生物的药物化合物，其特征在于，所述喹诺里西啶类衍生物的化学结构式包括CNI1-1、CNI1-3、 CNI1-9～13：
  11. 一种如权利要求8-10中任一所述的基于喹诺里西啶类衍生物的药物化合物的制备方法，其特征在于，包括以下步骤：
    S11、与N,N－二甲基甲酰胺二甲缩醛通过缩合反应得到
    S12、在单质碘的作用下氨烯酮碘化、环合形成化合物在碳酸钠/碳酸钾和醋酸钯的催化下进一步与通过Suzuki偶联反应得到并使用溴化氢－醋酸溶液和水除去保护基；
    S13、化合物与金雀花碱、甲醛在DMAP的催化下通过曼尼希反应得到
  12. 根据权利要求11所述的基于喹诺里西啶类衍生物的药物化合物的制备方法，其特征在于，
    步骤S11中，的摩尔质量和N,N－二甲基甲酰胺二甲基缩醛的体积分别为1mmol、(6～8)mL，反应温度为74℃，反应时间为3－5h；
    步骤S12中，与单质碘的摩尔比为1：1.5，反应温度为室温，反应时间为12～24h；
    的摩尔比为1：1.5，反应温度为50℃，反应时间为5～12h，其中反应中以四氢呋喃和甲醇的混合溶液为溶剂，碱和醋酸钯为催化剂，所述碱为无水碳酸钠/碳酸碱；与溴化氢的醋酸溶液和水的摩尔体积比为1mmol：3.5mL：0.88mL，其中溴化氢的醋酸溶液中溴化氢的质量分数为33％，反应温度为50℃，反应时间为3～12h；
    步骤S13中，与金雀花碱、DMAP及福尔马林的摩尔比分别为1mmol、1.25mmol、2.4mg、0.5mL，反应温度为70℃，反应时间为6－10h。
  13. 一种如权利要求8-10中任一所述的基于喹诺里西啶类衍生物的药物化合物的应用，其特征在于，将所述喹诺里西啶类衍生物用于制备抗神经损伤药物，包括在制备抗脑外伤、脊髓伤、术后神经损伤、脑卒中、缺氧性神经损伤神经退行性疾病的神经损伤药物中的应用。
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