WO2013029555A1 - Lavendustin a derivatives, preparation method and use thereof - Google Patents

Lavendustin a derivatives, preparation method and use thereof Download PDF

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WO2013029555A1
WO2013029555A1 PCT/CN2012/080809 CN2012080809W WO2013029555A1 WO 2013029555 A1 WO2013029555 A1 WO 2013029555A1 CN 2012080809 W CN2012080809 W CN 2012080809W WO 2013029555 A1 WO2013029555 A1 WO 2013029555A1
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methanol
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王华庆
王先火
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天津医科大学附属肿瘤医院
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/38—2-Pyrrolones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/52—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
    • C07C229/54—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C229/64—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring the carbon skeleton being further substituted by singly-bound oxygen atoms
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/65—One oxygen atom attached in position 3 or 5
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/28—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/52—Radicals substituted by nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • This invention relates to the field of medicinal chemistry, particularly to the xanthin A derivative, to its preparation and use.
  • Lavendustin A is a compound isolated from the metabolite of the genus gr i seolavendus of the genus Streptomyces and has a strong inhibitory activity against protein tyrosine kinase. Protein tyrosine kinase plays an important role in controlling tumor cell proliferation, differentiation and death. In recent years, it has been widely used as a target for anti-tumor drugs. Small molecule protein tyrosine alcohol inhibitors have become anti-tumor research. One of the important topics in the field. Inhibitory activity of spirulina A as a protein tyrosine kinase inhibitor
  • the difference between the two is the difference in donor capacity of phenolic hydroxyl hydrogen on the A ring, and the stronger the donor ability, the stronger the activity.
  • Xanthophyllin A has been reported to interact with the epidermal growth factor receptor (EGFR) in the receptor-type protein tyrosine kinase type, thereby inhibiting autophosphorylation of EGFR and exerting an anti-tumor effect.
  • EGFR epidermal growth factor receptor
  • researchers have done some modification and derivatization work on oxacin A, mainly for its A-ring structure and C-ring structure. Modifications and obtaining partially active compounds, including patents including Di subs ti tuted lavendustin A analogs and pharmaceutical composition including the analogs (Patent No.
  • the VEGF family includes VEGF (VEGFA), VEGFB, VEGFC, VEGFD, and placental growth factor (P IGF ).
  • VEGFA VEGF
  • VEGFB vascular growth factor
  • VEGFD VEGFD
  • P IGF placental growth factor
  • VEGFR3/FLT-4 was found to be involved in lymphangiogenesis.
  • the experimental results obtained by animal models found that tumor-induced lymphangiogenesis is achieved by secreting VEGFC or VEGFD, specifically binding and activating VEGFR3. Lymphangiogenesis is closely related to tumor lymphatic metastasis.
  • the technical problem to be solved by the present invention is to provide a scavenger A derivative.
  • Another technical problem to be solved by the present invention is to provide a process for preparing the ascorbate A derivative.
  • Another technical problem to be solved by the present invention is to provide an application of the ascorbate A derivative.
  • the technical solution of the present invention is:
  • the sphingosine A derivative is a compound of the formula I, a pharmaceutically acceptable salt of a compound of the formula I, a compound of the formula I is added to a cosolvent or a compound precursor, and the chemical structure of the formula I is as follows:
  • R in the formula I is an alkyl group, an ether group, a carbonyl group, a p-tolyl group, a five-membered heterocyclic ring or a five-membered substitution a heterocyclic ring, a six-membered heterocyclic ring or a six-membered substituted heterocyclic ring, wherein the compound precursor is a compound of the formula I, a formula I, which is metabolized or chemically reacted in a patient after administration by a suitable method.
  • a pharmaceutically acceptable salt or a solution of a compound of formula I added to a cosolvent.
  • the above-mentioned Kaempferol A derivative wherein R in the formula I is an alkyl group, an acid group, a carbonyl group, a p-nonylphenyl group, a five-membered heterocyclic ring or a five-membered heterocyclic ring, a six-membered heterocyclic ring or a six-membered compound Substituted heterocycle.
  • the above-mentioned oxacin A derivative wherein R in the formula I is a cyclohexyloxy group, a 4-aminopyrimidinyl group, a 2-oxo-3-pyridinyl group, a 3-pyridinyl group, a 5-pyrimidine group , 5-thiazolyl, 2-indolyl-3-hydroxy-4-pyridyl, 3-pyridyl, 3-[(1-aminodecanoyl-2-oxo)pyrrolidinyl, 2-(5-methyl Furan), 4-nonylphenyl, 2-furyl, 2-indolyl-5-pyridyl and 3-hydroxy-4-pyridyl.
  • the above-mentioned Kaempferol A derivative is obtained by the following method: using the computer drug virtual screening technology as a target for VEGFR3 to design a Kaempferol A derivative, specifically using the electronic isosterism principle through a huge fragment library A large number of xanthophyllin A derivatives were designed, sorted according to the degree of similarity of the substituted groups, and the first 99 modified xanthophyllin A derivatives were retained, and then used with the reference compound, oxacillin A.
  • the Fast Rigid Exhaustive Docking program docks to the active site of the target protein VEGFR3 tyrosine kinase domain, and evaluates the degree of interaction between the compound and VEGFR3.
  • the above-mentioned oxacin A derivative is obtained by the following method:
  • More than 1,000 small molecule drugs approved by the FDA were cut into about 300,000 pieces with different volume shapes, different sizes, different ester/water partition coefficients, and different charge properties.
  • ascorbate A was used as a reference compound, and the B-ring portion was preliminarily selected.
  • the bioisosteric search was used to replace the fragments in the fragment library to design a new xanthophyll A analog.
  • the first 99 modified new compounds were retained, together with the reference compound, oxacillin A, using the fast Rigid Exhaustive Docking program.
  • the use of the above-mentioned scavenger A derivative is lymphoma, lung cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, colon cancer, melanoma or head and neck cancer.
  • the tumor is lymphoma or lung cancer.
  • the tumor is a lymphoma.
  • Kaempferol A derivative was found to have significant inhibition of human lymphatic endothelial cells (HLEC) and LL/2 Lewis lung cancer cell line stably expressing VEGFD (VEGF-D-LL/2) by in vitro cell level experiments.
  • HLEC human lymphatic endothelial cells
  • VEGF-D-LL/2 Lewis lung cancer cell line stably expressing VEGFD
  • FIG. 15 is a diagram showing the mode of action of HQ-001 and VEGFR3.
  • VEGFR3 is represented by so l id r ibbon and is colored according to its Secondary Type
  • HQ-001 is represented by CPK model, where green is carbon atom and red is oxygen atom
  • HQ_ 001 is represented by a ball stick model , wherein green is a carbon atom and red is an oxygen atom; except for polar hydrogen (white), the remaining hydrogen atoms are not shown
  • the amino acid residue of the active site of the VEGFR3 tyrosine kinase domain is represented by a rod and is colored according to the residue.
  • the hydrogen bond formed between the compound HQ _ 001 and the active site residue is indicated by a dotted line;
  • Figure I 6 is HQ-001 induced apoptosis of human lymphatic endothelial cells (HLEC) (PI staining): a. Normal HLEC cells; b. 0. 5uM HQ_ 001 induces apoptosis in HLEC cells;
  • Figure 17 is a time-dependent relationship between HQ_ 001-induced apoptosis in human lymphatic endothelial cells (HLEC) (Hoechst 33258 staining): A. Action Oh; B. Action 2h; C. Action 4h; D. Action 8h; E. Action 12h; F. Effect 24h;
  • HLEC human lymphatic endothelial cells
  • Figure 18 shows the HX-001 inhibition of human lymphatic endothelial cell (HLEC) tube formation: a. The results of tube formation of untreated HLEC cells at matrigel surface for 6 h; b. HLEC and 0.5 uM HQ-001 co-culture for 6 h after Results of tube formation on matrigel surface; c. morphology of untreated HLEC cells cultured on gelatin surface for 6 h; d. morphology of gelatin surface after co-culture of HLEC with 0.5 uM HQ_001 for 6 h; Figure 19 shows that HQ_ 001 inhibits secretion of VEGFD by tumor cells And its mechanism of action;
  • HLEC human lymphatic endothelial cell
  • Figure 20 shows HQ_001 inhibiting the expression of VEGFR3 in HLEC cells and its mechanism of action
  • Figure 21 shows that HQ_001 inhibits the formation of zebrafish internode blood vessels.
  • test materials used in the following examples are purchased from conventional biochemical reagent stores;
  • test materials used in the following examples are purchased from conventional biochemical reagent stores;
  • % in the following examples are mass percentages unless otherwise specified.
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • Example 4 gave the white solid product (Compound 15) 5-[(2,5-dihydroxybenzyl)-(5-thiazolylmethyl)]amino-2-hydroxybenzoic acid.
  • 'H-NMR 300 Hz, DMS0-d 6 ) ⁇ : 12.19 (br, 1H), 9.87 (br, 2H), 8.94 (br, 1H), 8.67 (s, 2H), 6.88-7.33 (m, 7H) , 5.23 (s, 2H), 4.61 (s, 2H).
  • LC-ESI-MS M+H: 373.38.
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • the preparation method is as follows:
  • Novel xanthophyllin A analogue inhibits proliferation of human lymphatic endothelial cells (HLEC) and VEGFD-expressing LL/2 cells (VEGFD-LL/2)
  • Cell Culture HLEC cells were cultured in endothelial cell culture medium (ECM) containing 5% fetal bovine serum, 1% endothelial cell growth additive (ECGS), 100 IU/mL penicillin and 100 ⁇ g/mL streptomycin.
  • ECM endothelial cell culture medium
  • ECGS endothelial cell growth additive
  • the culture environment is also a saturated humidity, 5% C02 incubator at 37 °C.
  • VEGFD-LL/2 cells were routinely cultured in DMEM containing 10% fetal calf serum, 100 U/ml penicillin and 100 mg/L streptomycin in a saturated humidity, 53 ° C C52 incubator.
  • the MTT test takes the logarithmic growth phase cells, digested or digested and directly mixed and counted.
  • the cell suspension concentration was adjusted to 2 lOVmL, and 100 ul of cell suspension was added to each well of a 96-well plate; after 24 h, the original medium was aspirated. , add 200 ul of complete culture solution containing 15 compounds including oxacillin A (negative control group plus equal amount of DMS0), each group of 3 duplicate wells, continue to culture in C02 incubator; respectively after dosing After 48 h, remove the 96-well plate, add 50 ⁇ l of 5 mg/1 MTT reagent to each well, and continue to culture for 4 hours.
  • Relative inhibition rate (control hole average 0D value - dosing hole average 0D value) / control hole average 0D value X 1003 ⁇ 4.
  • HQ_ 001 induces apoptosis in human lymphatic endothelial cells (HLEC)
  • 0.5uM HQ_001 was selected to treat HLEC fine J package, DMS0 control group was added with equal volume of DMS0, blank control group was added with equal volume of complete medium, and treated with PI staining for 48h after treatment: 1) original culture Base aspirate, suck clean, pay attention to gentle operation, so as not to cause cell loss;
  • HQ _ 001 induces time-dependent apoptosis of human lymphatic endothelial cells (HLEC)
  • HQ-001 inhibits the ability of human lymphatic endothelial cells (HLEC) to establish tube
  • HQ_001 The effect of HQ_001 on the migration and differentiation of human lymphatic endothelial cells HLEC and the formation of tubular structures were studied by in vitro matrigel tube experiments.
  • the results of light microscopy (Fig. 18) showed that the HLEC cells in the control group could migrate and differentiate on Matr igel within 6 hours, stretching and interconnecting to form a better network tubular structure.
  • This structure is capillary-like and forms a multi-center node (Fig. 18a); whereas in the case of co-culture with 0.5 uM HQ_001, the formation of the tubular structure is significantly inhibited compared to the control group, resulting in the formation of HLEC cells.
  • the mesh was significantly reduced, and the HLEC cell morphology gradually became rounded by the stretched long fusiform (Fig. 18b).
  • HLEC cells grow normally adherently on the gelatin surface without migration and differentiation, nor form a tubular structure (Fig. 18c).
  • Co-culture with 0.5 uM HQ_ 001 did not significantly affect normal adherence and growth of HLEC cells (Fig. 18d). It can be seen that HQ-001 can significantly inhibit the HLEC's ability to form tubes in a short period of time. This inhibition is not due to endothelial cell damage caused by its cytotoxic effects. Therefore, it was confirmed that HQ-001 has an effect of inhibiting lymphangiogenesis.
  • HQ_001 inhibits the secretion of VEGFD from tumor cells
  • VEGFD-LL/2 the expression of VEGFD in tumor cells was first detected by Western blot.
  • the secretion of VEGFD in tumor cells is associated with the transcription factor AP-1, so the phosphorylation of the upstream kinase ERK1/2 of c_fos was tested.
  • the PI3K/AKT pathway which regulates cell survival, migration, and proliferation, was also examined.
  • the results of Western blot (Fig. 19) showed that the expression level of VEGFD was significantly increased in VEGFD-LL/2 cells compared with unloaded pcDNA-LL/2 cells. And after 12 h of action at 0.5 uM HQ_001, the VEGF-D expression level of VEGFD-LL/2 was significantly inhibited.
  • HQ_001 blocking the MEK/ERK1/2 pathway in tumor fines.
  • BRK showed constitutive activation in tumor cells
  • ERK of the control group showed significant phosphorylation of ⁇ , and the activation of this phosphorylation was blocked by HQ_001.
  • phosphorylation of AKT was also significantly inhibited. Therefore, HQ-001 has a direct inhibitory effect on the signaling pathway of VEGFD-LL/2 cells.
  • the inhibition of lymphangiogenesis and lymphatic metastasis by HQ_001 is associated with HQ-001 inhibiting the activation of ERK and AKT in tumor cells, thereby downregulating the expression of VEGFD.
  • HQ-001 inhibits the expression of VEGFR3 in human lymphatic endothelial cells (HLBC)
  • VEGFR3 in HLEC cells was detected by Western blot.
  • the phospho-4-is and PI3K/AKT pathways of the kinase BRK1/2 were also examined.
  • Western blot results (Fig. 20) showed that ERK1/2 phosphorylation and VEGFR3 expression levels were significantly inhibited in HLEC cells.
  • the VEGFR3 pathway is the main pathway regulating lymphangiogenesis. Tumor-induced lymphangiogenesis plays a role in the activation of the VEGFR3 pathway by secreting VEGFD. Therefore, HQ-001 can inhibit lymphocyte generation by inhibiting BRK1/2 phosphorylation and VEGFR3 expression, thereby down-regulating cell division, migration, and tube formation.
  • HQ_001 inhibits angiogenesis in zebrafish
  • the zebrafish model has become a new type of in vivo model biology because it can clearly observe the development of blood vessels, which is very suitable for the study of vascular biology in vivo.
  • the zebrafish eggs were collected first, and the dead eggs and unhealthy fish eggs were picked and cultured in 28.5 °C fish water. Dilute HQ_001 to the desired concentration with fish water. After 6 hours, the eggs were distributed into a six-well plate and treated with HQ_001 solution. After incubation for 24 hours, anesthetize with 0.05% hydrated chloric acid, peel the eggs under a fluorescent microscope and observe angiogenesis.
  • Fig. 21 The results are shown in Fig. 21.
  • the internode tube is very complete and is in a beautiful trapezoidal arrangement.
  • the concentration of HQ_001 is increased, it is 0.2 ⁇ , 0.4 ⁇ .
  • the germination of the zebrafish internode tube was inhibited and the formation was incomplete.
  • HQ_001 treated with the same concentration (0.4 ⁇ ) of HQ_001 and sphingosine ⁇ showed a stronger inhibition of zebrafish internode formation. This shows that HQ_001 can strongly inhibit the formation of new blood vessels.
  • the sphingosine A derivative of the present invention has a significant inhibition of human lymphatic endothelial cells (HLEC) and a stable and highly expressed VEGFD-expressing LL/2 Lewis lung cancer cell line (VEGF).
  • HLEC human lymphatic endothelial cells
  • VEGF Lewis lung cancer cell line
  • Kaempferol A derivative its preparation method and application, with reference to the specific embodiments, are illustrative and not limiting, and several embodiments may be enumerated according to the scope of the limitation, and therefore Variations and modifications that are within the spirit of the invention are intended to be within the scope of the invention.

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Abstract

Provided are lavendustin A derivatives, a preparation method and use thereof. The compounds are shown by the structure of formula (I), wherein R is alkyl, ether group, carbonyl, p-tolyl, five-membered heterocyclic group, five-membered substituted heterocyclic group, six-membered heterocyclic group, or six-membered substituted heterocyclic group. The compounds inhibit the proliferation of human lymphatic endothelial cells and LL/2 Lewis lung cancer cell strain (VEGF-D-LL/2) which stably expresses VEGFD, and can be used for the treatment of tumor lymphatic metastasis.

Description

说 明 书 薰草菌素 A衍生物及其制备方法与应用 技术领域  Description: Kaempferol A derivative, preparation method and application thereof
本发明涉及药物化学领域， 尤其是薰草菌素 A衍生物及其制备方法与应 用。  Field of the Invention This invention relates to the field of medicinal chemistry, particularly to the xanthin A derivative, to its preparation and use.
背景技术 Background technique
薫草菌素 A ( Lavendustin A)是从链霉属的 gr i seolavendus菌的代谢 产物中分离得到的化合物， 对蛋白酪氨酸激酶具有较强的抑制活性。 而蛋白 酪氨酸激酶在控制肿瘤细胞增殖、 分化及死亡中扮演着重要角色， 近年来广 泛地被作为抗肿瘤药物的靶点， 小分子的蛋白酪氨酸激醇抑制剂已成为抗肿 瘤研究领域的重要课题之一。 薰草菌素 A作为蛋白酪氨酸激酶抑制剂的抑制 活性  Lavendustin A is a compound isolated from the metabolite of the genus gr i seolavendus of the genus Streptomyces and has a strong inhibitory activity against protein tyrosine kinase. Protein tyrosine kinase plays an important role in controlling tumor cell proliferation, differentiation and death. In recent years, it has been widely used as a target for anti-tumor drugs. Small molecule protein tyrosine alcohol inhibitors have become anti-tumor research. One of the important topics in the field. Inhibitory activity of spirulina A as a protein tyrosine kinase inhibitor
Figure imgf000003_0001
Figure imgf000003_0001
薰草菌素 A 薰草菌素 B  Kaempferin A Kaempferol B
可见二者之间不同之处在于 A环上酚羟基氢的给体能力差异， 给体能力 越强， 活性越强。  It can be seen that the difference between the two is the difference in donor capacity of phenolic hydroxyl hydrogen on the A ring, and the stronger the donor ability, the stronger the activity.
薰草菌素 A被报道是与受体型蛋白酪氨酸激酶类型中表皮生长因子受体 ( EGFR )相互作用， 进而抑制 EGFR的自身磷酸化， 发挥抗肿瘤作用。 为了寻 找活性比 f草菌素 A更强的蛋白酪氨酸激酶抑制剂， 学者们已围绕薰草菌素 A作了一些修饰和衍生化工作， 主要是对其 A环结构以及 C环结构进行改造， 并获得部分活性较好的化合物， 所涉及到得专利包括 Di subs t i tuted lavendustin A analogs and pharmaceutical composition comprising the analogs (专利号为 US 6943191 Bl) ^ Pharmaceutical compositions of Lavendustin (专利号为 US 2008/0242686 Al) , 而到目前为止， 未见对薰草 菌素 A中 B环进行改造的相关报道。 恶性肿瘤细胞从原发部位直接侵入周围组织或者进入循环系统进行转 移。 肿瘤的转移是决定肿瘤患者存活的关键因素。 这种转移主要通过人体自 身的脉管系统： 血管 （血道转移）和淋巴管 （淋巴道转移） 来实现的。 肿瘤 细胞具有诱导血管和淋巴管新生的能力，这种能力是通过 VEGF家族成员来起 作用的。 VEGF家族包括 VEGF ( VEGFA )、 VEGFB, VEGFC、 VEGFD及胎盘生长因 子（P IGF )。 这些内皮细胞生长因子能够激活内皮细胞上的 VEGFR家族， 并且 启动与内皮细胞增殖、 迁移相关的细胞信号转导通路。 目前对于肿瘤血管新 生的研究已经有了极大的进展。 肿瘤在生长到一定大小的时候， 缺氧的环境 和对营养的需求刺激肺瘤细胞分泌血管生长因子 VEGF和 VEGFB, 从而诱导血 管往肿瘤方向发生， 为肿瘤提供氧气和营养， 同时也为肿瘤细胞的转移提供 重要的途径。 对于淋巴管新生的研究在近年来也取得很大的突破。 VEGFR3/FLT-4被发现与淋巴管的发生有关。 通过动物模型获得到的实验结果 发现， 肿瘤诱导的淋巴管新生是通过分泌 VEGFC或者 VEGFD , 特异性地结合 并激活 VEGFR3来实现的。 淋巴管新生与肿瘤淋巴转移息息相关。 Xanthophyllin A has been reported to interact with the epidermal growth factor receptor (EGFR) in the receptor-type protein tyrosine kinase type, thereby inhibiting autophosphorylation of EGFR and exerting an anti-tumor effect. In order to find protein tyrosine kinase inhibitors that are more active than f-organin A, scholars have done some modification and derivatization work on oxacin A, mainly for its A-ring structure and C-ring structure. Modifications and obtaining partially active compounds, including patents including Di subs ti tuted lavendustin A analogs and pharmaceutical composition including the analogs (Patent No. US 6943191 Bl) ^ Pharmaceutical compositions of Lavendustin (Patent No. US 2008/ 0242686 Al) , and so far, no reports have been made on the transformation of the B ring in the spirulina A. Malignant tumor cells directly invade surrounding tissues from the primary site or enter the circulatory system for metastasis. Tumor metastasis is a key factor in determining the survival of tumor patients. This transfer is mainly achieved by the body's own vasculature: blood vessels (hematopoiesis) and lymphatic vessels (lymphatic metastasis). Tumor cells have the ability to induce vascular and lymphangiogenesis, a function that is through the members of the VEGF family. The VEGF family includes VEGF (VEGFA), VEGFB, VEGFC, VEGFD, and placental growth factor (P IGF ). These endothelial cell growth factors activate the VEGFR family on endothelial cells and initiate cellular signaling pathways involved in endothelial cell proliferation and migration. At present, great progress has been made in the study of tumor angiogenesis. When the tumor grows to a certain size, the hypoxic environment and the need for nutrition stimulate the tumor cells to secrete vascular growth factors VEGF and VEGFB, thereby inducing blood vessels to the tumor, providing oxygen and nutrients to the tumor, and also tumor cells. The transfer provides an important avenue. Research on lymphangiogenesis has also made great breakthroughs in recent years. VEGFR3/FLT-4 was found to be involved in lymphangiogenesis. The experimental results obtained by animal models found that tumor-induced lymphangiogenesis is achieved by secreting VEGFC or VEGFD, specifically binding and activating VEGFR3. Lymphangiogenesis is closely related to tumor lymphatic metastasis.
发明内容 Summary of the invention
本发明所要解决的技术问题在于提供薰草菌素 A衍生物。  The technical problem to be solved by the present invention is to provide a scavenger A derivative.
本发明所要解决的另一技术问题在于提供所述薰草菌素 A衍生物的制备 方法。  Another technical problem to be solved by the present invention is to provide a process for preparing the ascorbate A derivative.
本发明所要解决的另一技术问题在于提供所述薰草菌素 A衍生物的应 用。  Another technical problem to be solved by the present invention is to provide an application of the ascorbate A derivative.
为解决上述技术问题， 本发明的技术方案是：  In order to solve the above technical problem, the technical solution of the present invention is:
薰草菌素 A衍生物， 为通式 I化合物、通式 I化合物药学上可接受的盐， 通式 I化合物加入助 液或化合物前体， 通式 I的化学结构如下：  The sphingosine A derivative is a compound of the formula I, a pharmaceutically acceptable salt of a compound of the formula I, a compound of the formula I is added to a cosolvent or a compound precursor, and the chemical structure of the formula I is as follows:
Figure imgf000004_0001
Figure imgf000004_0001
其中， 式 I中 R为烷基、 醚基、 羰基、 对甲苯基、 五元杂环或五元取代 杂环、 六元杂环或六元取代杂环， 所述化合物前体是指当病人用适当的方法 服用后， 化合物在病人体内进行代谢或化学反应而转变成通式 I化合物、 通 式 I化合物药学上可接受的盐或通式 I化合物加入助溶剂的溶液。 Wherein R in the formula I is an alkyl group, an ether group, a carbonyl group, a p-tolyl group, a five-membered heterocyclic ring or a five-membered substitution a heterocyclic ring, a six-membered heterocyclic ring or a six-membered substituted heterocyclic ring, wherein the compound precursor is a compound of the formula I, a formula I, which is metabolized or chemically reacted in a patient after administration by a suitable method. A pharmaceutically acceptable salt or a solution of a compound of formula I added to a cosolvent.
优选的， 上述薰草菌素 A衍生物， 所述式 I中 R为烷基、 酸基、 羰基、 对曱苯基、 五元杂环或五元取代杂环、 六元杂环或六元取代杂环。  Preferably, the above-mentioned Kaempferol A derivative, wherein R in the formula I is an alkyl group, an acid group, a carbonyl group, a p-nonylphenyl group, a five-membered heterocyclic ring or a five-membered heterocyclic ring, a six-membered heterocyclic ring or a six-membered compound Substituted heterocycle.
优选的， 上述薰草菌素 A衍生物， 所述式 I中 R为环己基氧基、 4-氨基 嘧啶基、 2 -氧代 -3-吡啶曱基、 3-吡啶曱基、 5 -嘧啶基、 5-噻唑基、 2-曱基 -3- 羟基- 4-吡啶基、 3 -吡啶基、 3_[(1-氨基曱酰基- 2-氧代）吡咯基、 2- (5-甲基 呋喃）基、 4 -曱基苯基、 2-呋喃基、 2-曱基 -5-吡啶基和 3_羟基- 4-吡啶基。  Preferably, the above-mentioned oxacin A derivative, wherein R in the formula I is a cyclohexyloxy group, a 4-aminopyrimidinyl group, a 2-oxo-3-pyridinyl group, a 3-pyridinyl group, a 5-pyrimidine group , 5-thiazolyl, 2-indolyl-3-hydroxy-4-pyridyl, 3-pyridyl, 3-[(1-aminodecanoyl-2-oxo)pyrrolidinyl, 2-(5-methyl Furan), 4-nonylphenyl, 2-furyl, 2-indolyl-5-pyridyl and 3-hydroxy-4-pyridyl.
上述薰草菌素 A衍生物，是由以下方法得到的：针对 VEGFR3为靶点采用 计算机药物虚拟筛选技术而设计出薰草菌素 A衍生物， 具体为通过巨大的碎 片库运用电子等排原理设计出大量薰草菌素 A衍生物， 依据被置换基团的相 似程度进行排序， 留取前 99个被改造的薰草菌素 A衍生物， 随后与参考化合 物薰草菌素 A—道用怏速刚性构象穷举对接程序 （Fast Rigid Exhaustive Docking)对接到靶标蛋白 VEGFR3酪氨酸激酶域的活性位点， 并对化合物与 VEGFR3间的相互作用程度进行评价， 作用越强， Total Score绝对值越大， 最终筛选出 14个化合物， 其活性优于薰草菌素 A， 依次被命名为 HQ_001、 HQ— 002、 HQ— 003、 HQ— 004、 HQ— 005、 HQ— 006、 HQ— 007、 HQ— 008、 HQ— 009、 HQ— 010, HQ 011、 HQ— 012、 HQ_013、 HQ— 014, 化学结构见表 1。  The above-mentioned Kaempferol A derivative is obtained by the following method: using the computer drug virtual screening technology as a target for VEGFR3 to design a Kaempferol A derivative, specifically using the electronic isosterism principle through a huge fragment library A large number of xanthophyllin A derivatives were designed, sorted according to the degree of similarity of the substituted groups, and the first 99 modified xanthophyllin A derivatives were retained, and then used with the reference compound, oxacillin A. The Fast Rigid Exhaustive Docking program docks to the active site of the target protein VEGFR3 tyrosine kinase domain, and evaluates the degree of interaction between the compound and VEGFR3. The stronger the effect, the absolute value of Total Score. The larger, the final screening of 14 compounds, its activity is better than the oxacin A, named HQ_001, HQ-002, HQ-003, HQ-004, HQ-005, HQ-006, HQ-007, HQ—008, HQ—009, HQ—010, HQ 011, HQ—012, HQ_013, HQ—014, chemical structure are shown in Table 1.
优选的， 上述薰草菌素 A衍生物， 是由以下方法得到的：  Preferably, the above-mentioned oxacin A derivative is obtained by the following method:
(1 )碎片库的准备  (1) Preparation of the debris library
将 FDA批准上市的一千多个小分子药物， 裁成具有不同体积形状、 不同 大小、 具有不同酯 /水分配系数、 不同荷电性质的碎片约 300000个。  More than 1,000 small molecule drugs approved by the FDA were cut into about 300,000 pieces with different volume shapes, different sizes, different ester/water partition coefficients, and different charge properties.
(2)薰草菌素 A类似物的设计、 虛拟 选与评价  (2) Design, Virtual Selection and Evaluation of Kaempferol A Analogs
以背景技术中薰草菌素 A为参考化合物， 初步选取 B环部分， 运用电子 等排原理（bioisosteric search)， 用碎片库中的碎片进行置换改造， 设计出 新的薰草菌素 A类似物； 再依据与被置换基团的相似程度排序， 共留取前 99 个被改造的新化合物， 与参考化合物薰草菌素 A—起， 采用快速刚性构象穷 举对接程序 ( Fast Rigid Exhaustive Docking )对接 ( docking )到革巴标蛋 白 VEGFR3的酸氨酸激醃域 ( tyrosine kinase domain) 的活性位点 ( active site), 并对化合物与 VEGFR3间的相互作用程度进行评价， 作用越强， Total Score绝对值越大； 根据排列顺序利用电子等排原理改造设计所获得的 99个 新化合物中， 筛选出 14个活性优于薰草菌素 A的化合物， 该新化合物依次被 命名为 HQ—001 , HQ— 002 HQ— 014， 化学结构见表 1。 In the background art, ascorbate A was used as a reference compound, and the B-ring portion was preliminarily selected. The bioisosteric search was used to replace the fragments in the fragment library to design a new xanthophyll A analog. And according to the degree of similarity with the displaced groups, the first 99 modified new compounds were retained, together with the reference compound, oxacillin A, using the fast Rigid Exhaustive Docking program. Docking to the active site of the tyrosine kinase domain of the viral protein VEGFR3, and evaluating the degree of interaction between the compound and VEGFR3, the stronger the effect, Total Score The greater the absolute value; the 99 obtained by the design of the electronic isosteric principle according to the arrangement order Among the new compounds, 14 compounds with better activity than xanthophyllin A were selected. The new compounds were named HQ-001, HQ-002 HQ-014, and the chemical structure is shown in Table 1.
表 1 Table 1
Figure imgf000006_0001
5- [ (2， 5-二羟基苯甲基 )- (3-吡 啶乙基)] 氨基 -2-羟基苯甲酸
Figure imgf000006_0001
5-[(2,5-Dihydroxybenzyl)-(3-pyridylethyl)]amino-2-hydroxybenzoic acid
Figure imgf000007_0001
Figure imgf000007_0001
5- [(2, 5-二羟基苯甲基）-(5-嘧 5- [(2, 5-dihydroxybenzyl)-(5-pyrimidine)
HQ— 005 -92.74 -93.35 18.50 -17.41 -0.48 啶甲基)] 氨基 -2-羟基苯甲酸 HQ— 005 -92.74 -93.35 18.50 -17.41 -0.48 pyridinemethyl)]amino-2-hydroxybenzoic acid
5- [(2, 5-二羟基苯甲基 )-(5-噻5- [(2, 5-dihydroxybenzyl)-(5-thia
HQ— 006 -92.09 -93.70 11.90 -9.73 -0.57 唑甲基)] 氨基 -2-羟基苯甲酸 HQ— 006 -92.09 -93.70 11.90 -9.73 -0.57 oxazomethyl)]amino-2-hydroxybenzoic acid
Figure imgf000007_0002
Figure imgf000007_0002
5- [(2， 5-二羟基苯甲基 )-(2-甲 基 -3-羟基 -4-吡啶甲基)氨 5-[(2,5-Dihydroxybenzyl)-(2-methyl-3-hydroxy-4-pyridylmethyl)ammonia
HQ— 007 -91.99 -15.04 -0.19 基] -2-羟基苯甲酸 HQ—007 -91.99 -15.04 -0.19 base]-2-hydroxybenzoic acid
■。入。 ■. In.
5- [(2， 5-二羟基苯甲基 )-(3-吡 5- [(2, 5-dihydroxybenzyl)-(3-pyridyl)
HQ— 008 -91.69 -92.62 17.31 -15.83 -0.56 啶甲基） 氨基] -2-羟基苯甲酸 HQ—008-91.69 -92.62 17.31 -15.83 -0.56 pyridinemethyl)amino]-2-hydroxybenzoic acid
Figure imgf000008_0001
5-[(2, 5-二羟基苯甲基）-(2-甲 基 -5-吡啶甲基） 氨基] -2-羟基
Figure imgf000008_0001
5-[(2,5-dihydroxybenzyl)-(2-methyl-5-pyridylmethyl)amino]-2-hydroxyl
HQ— 013 -90.32 -93.64 13.31 -9.56 -0.43 苯甲酸 HQ— 013 -90.32 -93.64 13.31 -9.56 -0.43 Benzoic acid
Figure imgf000009_0001
Figure imgf000009_0001
5- [(2, 5-二羟基苯甲基 )-(3-羟 基 -4-吡啶曱基） 氨基] -2-羟基 5-[(2,5-Dihydroxybenzyl)-(3-hydroxy-4-pyridinyl)amino]-2-hydroxyl
HQ— 014 -90.28 -86.64 20.05 -21.80 -1.88 HQ— 014 -90.28 -86.64 20.05 -21.80 -1.88
Figure imgf000009_0002
Figure imgf000009_0002
5- [[(2, 5-二羟苯基）甲基] [(2 - 羟基苯基）甲基]氨基] -2-羟基苯 薰草菌素 A -90.21 -87.80 14.66 -15.44 -1.63 甲酸 上述表 1中筛选出的 14个活性优于薰草菌素 A的化合物 （薰草菌素 A 衍生物） 的制备方法， 具体步骤为：  5-[[(2, 5-Dihydroxyphenyl)methyl][(2-hydroxyphenyl)methyl]amino]-2-hydroxyphenylsporin A -90.21 -87.80 14.66 -15.44 -1.63 Formic acid The preparation method of the 14 compounds (the sylvestin A derivative) whose activity is better than that of the spirulina A in the above Table 1 is as follows:
(A1 )每 5克 2 -羟基 _5 -硝基苯甲酸（
Figure imgf000009_0003
）， 40毫升丙酮， 1亳升(A1) every 5 grams of 2-hydroxy-5-nitrobenzoic acid (
Figure imgf000009_0003
), 40 ml of acetone, 1 liter
.氟乙酸， 回流 2小时， TLC检测反应完全， 减压除去丙酮， 柱层析， 制得
Figure imgf000010_0001
ο ο Fluoroacetic acid, reflux for 2 hours, complete reaction by TLC, removal of acetone under reduced pressure, column chromatography,
Figure imgf000010_0001
ο ο
、、 ,
( Α2 )每 2克 ¾， 50毫升乙醇， 500毫克 10%的 Pd/C, latra氢化  ( Α 2 ) every 2 g 3⁄4, 50 ml ethanol, 500 mg 10% Pd/C, latra hydrogenation
室温搅拌 2小时， TLC检测反应完全， 原料消失， 过滤， 浓缩制得
Figure imgf000010_0002
Stir at room temperature for 2 hours, complete the reaction by TLC, disappear the raw materials, filter, concentrate and obtain
Figure imgf000010_0002
( A3 )每 2克
Figure imgf000010_0003
， 0.8克二环己基碳二亚胺， 1.1克 1-羟基苯并三 氮唑， 50亳升二氯甲烷， 室温搅拌 12小时， TLC检测反应完全， 减压除去溶 (A3) every 2 grams
Figure imgf000010_0003
, 0.8 g of dicyclohexylcarbodiimide, 1.1 g of 1-hydroxybenzotriazole, 50 ml of dichloromethane, stirred at room temperature for 12 hours, completely detected by TLC, and dissolved under reduced pressure
剂， 柱层析， 制得 Preparation, column chromatography,
4 )每 1克
Figure imgf000010_0004
1.2克 NaBH4- A1C13, ：30毫升无水四氢呋 喃， 回流搅拌 2小时， TLC检测反应完全， 减压除去四氢呋喃， 残余物水洗， 二氯甲烷萃取， 浓缩， 4) Every 1 gram
Figure imgf000010_0004
1. 2 g of NaBH 4 - A1C1 3 , : 30 ml of anhydrous tetrahydrofuran, stirred under reflux for 2 hours, the reaction was completed by TLC, the tetrahydrofuran was removed under reduced pressure, and the residue was washed with water. Extracted with dichloromethane, concentrated,
5)每 500毫克
Figure imgf000011_0001
, 470毫克 3, 5 二羟基苯甲醛， 380毫 克氰基硼氢化钠， 30毫升甲醇， 回流 2小时， TLC检测反应完全， 加水淬灭 5) Every 500 mg
Figure imgf000011_0001
, 470 mg of 3,5 dihydroxybenzaldehyde, 380 mg of sodium cyanoborohydride, 30 ml of methanol, refluxed for 2 hours, complete reaction by TLC, quenched with water
反应， 减压除去甲醇， 二 Reaction, methanol removal under reduced pressure, two
fi Fi
(Α6)每 200毫克
Figure imgf000011_0002
, 含 100毫克氢氧化钠的 30毫升甲 醇溶液， 回流搅拌 2小时， TLC检测原料小时，反应完全， 2N盐酸调 PH为 5, 加压除去甲醇， 加入水， 二氯曱烷萃取， 浓缩得到 5- [[(2， 5-二羟基苯基）曱 (Α6) every 200 mg
Figure imgf000011_0002
The solution containing 100 mg of sodium hydroxide in 30 ml of methanol was stirred under reflux for 2 hours. The reaction time was determined by TLC. The reaction was completed. The pH was adjusted to 5 with 2N hydrochloric acid. The methanol was removed by pressure, water was added, and the mixture was concentrated. - [[(2, 5-Dihydroxyphenyl)anthracene
基] [(环己基氧）曱基]氛基 ] -2-羟基苯曱酸， 即
Figure imgf000011_0003
( Bl )每 2克 5-氨基水杨酸（
Figure imgf000012_0001
), 1. 8克 2， 5 -二羟基苯甲醛，]][(cyclohexyloxy)indolyl]-2-hydroxybenzoic acid, ie
Figure imgf000011_0003
(B) every 2 grams of 5-aminosalicylic acid (
Figure imgf000012_0001
), 1. 8 g of 2,5-dihydroxybenzaldehyde,
2. 1克！^硼氢化钠， 80毫升甲醇， 回流 2小时， TLC检测反应完全， 加入2. 1 gram! ^Sodium borohydride, 80 ml of methanol, reflux for 2 hours, complete reaction by TLC, add
20毫升饱和氯化铵溶液， 减压除去曱醇， 残余物水洗， 二氯曱烷萃取， 浓缩, 20 ml of saturated ammonium chloride solution, decyl alcohol was removed under reduced pressure, the residue was washed with water, extracted with dichloromethane and concentrated.
柱层析， Column chromatography,
( B2
Figure imgf000012_0002
)每 1克 , 1. 6克 4- Boc氨基- 5-嘧啶甲醛， 1. 2克氰基 硼氢化钠， 50毫升曱醇， 回流 2小时加入 50毫升饱和氯化铵溶液， 减压除 (B2
Figure imgf000012_0002
) 1 g, 1. 6 g of 4- Boc amino 5-pyrimidinecarboxaldehyde, 1. 2 g of sodium cyanoborohydride, 50 ml of decyl alcohol, refluxed for 2 hours, 50 ml of saturated ammonium chloride solution,
去曱醇， 二氯甲烷萃 Demethyl alcohol, dichloromethane
( B3 )每 500毫克
Figure imgf000012_0003
, 30毫升二氯甲烷， 1毫升三氟乙酸, 室温搅拌 10小时， TLC检测反应完全， 減压除去二氯甲烷， 饱和碳酸氢钠调 PH为 7 , 柱层析， 制得 5_ [ [ (2, 5-二羟基苯基）曱基] [5- (4_ 嘧啶）曱基 (B3) every 500 mg
Figure imgf000012_0003
, 30 ml of dichloromethane, 1 ml of trifluoroacetic acid, stirred at room temperature for 10 hours, completely detected by TLC, dichloromethane was removed under reduced pressure, saturated sodium hydrogen carbonate PH 7 , column chromatography, to obtain 5_ [ [ (2, 5-dihydroxyphenyl) fluorenyl] [5- ( 4 _ pyrimidine) fluorenyl
氨基] -2-羟基苯 或 Amino]-2-hydroxybenzene or
( C )每 1克
Figure imgf000013_0001
, 2克 3-吡啶基 _2-氧代-丙醛， 600毫克氰基硼 氢化钠， 50毫升曱醇， 回流 2小时， 加入 50毫升饱和氯化铵溶液， 减压除 去曱醇， 萃取， 浓缩， 柱层析， 制得 5- [ (2, 5 -二羟基苯甲基） （2 -氧代 _3 -吡 (C) every 1 gram
Figure imgf000013_0001
2 g of 3-pyridyl-2-oxo-propanal, 600 mg of sodium cyanoborohydride, 50 ml of decyl alcohol, refluxed for 2 hours, 50 ml of saturated ammonium chloride solution was added, decyl alcohol was removed under reduced pressure, and extracted. Concentration, column chromatography, to give 5-[(2,5-dihydroxybenzyl) (2-oxo-3-3-pyridyl)
啶乙基） ] 氨基 - 2_羟基苯甲酸， 即
Figure imgf000013_0002
Acridine ethyl)]amino- 2 hydroxybenzoic acid, ie
Figure imgf000013_0002
( D )每 1克
Figure imgf000013_0003
, 600毫克 3 -吡啶乙醛， 480毫克氰基硼氢化钠,(D) every 1 gram
Figure imgf000013_0003
, 600 mg of 3-pyridine acetaldehyde, 480 mg of sodium cyanoborohydride,
40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [ (2, 5-二羟基苯甲基 ) - (3-吡啶 乙基)] 氨基- 2-羟基苯曱酸， 即
Figure imgf000014_0001
40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, methanol was removed under reduced pressure, dichloromethane was extracted and concentrated to give 5-[(2,5-dihydroxybenzyl)- (3-pyridine Ethyl)]amino-2-hydroxybenzoic acid, ie
Figure imgf000014_0001
( E )每 1
Figure imgf000014_0002
, 600毫克 5 -嘧啶甲醛， 480毫克氰基硼氢化钠, 40毫升甲醇，回流 I小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯曱烷萃取， 浓缩制得 5- [(2， 5-二羟基苯曱基 )-(5-嘧啶 (E) every 1
Figure imgf000014_0002
, 600 mg of 5-pyrimidinecarboxaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, refluxed for 1 hour, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, methanol was removed under reduced pressure, dichloromethane was extracted and concentrated. Preparation of 5-[(2,5-dihydroxyphenyl)-(5-pyrimidine)
甲基）] 羟基苯曱酸， 即
Figure imgf000014_0003
Methyl)]hydroxybenzoic acid, ie
Figure imgf000014_0003
Figure imgf000014_0004
， 600毫克 5 塞唑甲醛， 480毫克氰基硼氢化钠, 40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 減压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [(2, 5-二羟基苯曱基 )-(5-噻唑 曱基）] 氨基 -2-羟基苯曱酸， 即
Figure imgf000015_0001
或
Figure imgf000014_0004
, 600 mg of 5-pyrazolecarboxaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, methanol was removed under reduced pressure, dichloromethane was extracted, and concentrated. 5-[(2,5-dihydroxyphenyl)-(5-thiazole) Amino-2-hydroxybenzoic acid, ie
Figure imgf000015_0001
or
( G )每 1
Figure imgf000015_0002
, 600毫克 2_甲基- 3-羟基- 4 -吡啶曱醛， 480 毫克氰基硼氢化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50 毫升饱和氯化铵溶液， 减压除去甲醇， 二氯曱烷萃取， 浓缩制得 5- [ (2, 5-二 羟基苯甲基) - (2-甲基- 3-羟基- 4-吡啶曱基)氨基] -2-羟基苯甲酸， 即 (G) every 1
Figure imgf000015_0002
, 600 mg of 2-methyl-3-hydroxy-4-pyridylfurfural, 480 mg of sodium cyanoborohydride, 40 ml of methanol, refluxed for 2 hours, complete reaction by TLC, add 50 ml of saturated ammonium chloride solution, decompress Removal of methanol, extraction with dichloromethane, concentration to give 5-[(2,5-dihydroxybenzyl)-(2-methyl-3-hydroxy-4-pyridinyl)amino]-2-hydroxybenzene Formic acid, ie
Figure imgf000015_0003
, 600毫克 3 -吡啶曱醛， 480毫克氰基硼氢化钠,
Figure imgf000015_0003
, 600 mg of 3-pyridine furfural, 480 mg of sodium cyanoborohydride,
40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [ (2, 5-二羟基苯甲基 ) - (3-吡啶 曱基） 氨基] -2-羟基苯曱酸， 即
Figure imgf000016_0001
40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, methanol was removed under reduced pressure, dichloromethane was extracted and concentrated to give 5-[(2,5-dihydroxybenzyl)- (3-pyridine Amino]-2-hydroxybenzoic acid, ie
Figure imgf000016_0001
( I )每 1克
Figure imgf000016_0002
， 600毫克 1-氨基曱酰基- 2-氧代- 3 -吡咯甲醛， 480毫克氰基硼氢化钠， 40毫升曱醇， 回流 2小时， TLC监测反应完全， 加 入 50毫升饱和氯化铵溶液，减压除去甲醇，二氯甲烷萃取，浓缩制得 5- [ (2, 5- 二羟基苯甲基) - [ 3- [ a- 甲酰基 -2-氧代)吡咯甲基）] 氨基] -2-羟基苯甲 (I) every 1 gram
Figure imgf000016_0002
, 600 mg of 1-aminodecanoyl-2-oxo-3-pyrrolidine, 480 mg of sodium cyanoborohydride, 40 ml of decyl alcohol, refluxed for 2 hours, the reaction was completely monitored by TLC, and 50 ml of saturated ammonium chloride solution was added. Methanol was removed under reduced pressure, extracted with dichloromethane and concentrated to give 5-[(2,5-dihydroxybenzyl)-[3-[a-formyl-2-oxo)pyrrolemethyl)]amino] 2-hydroxybenzol
酸， 或 Acid, or
Figure imgf000016_0003
( J )每 1克 , 600毫克 5-甲基呋喃甲醛， 480毫克氰基硼氢 化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和氯化 铵溶液， 减压除去甲醇， 二氯曱烷萃取， 浓缩制得 5- [ (2, 5-二羟基苯曱 (5-曱基呋喃）甲基]] 氨基 -2-羟基苯甲酸
Figure imgf000017_0001
Figure imgf000016_0003
(J), per gram, 600 mg of 5-methylfurfural, 480 mg of sodium cyanoborohydride, 40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, and methanol was removed under reduced pressure. , dichlorodecane extraction, concentration to give 5-[(2, 5-dihydroxyphenylhydrazine) (5-decylfuran)methyl]]amino-2-hydroxybenzoic acid
Figure imgf000017_0001
U)每 1克
Figure imgf000017_0002
, 600毫克对曱基苯甲醛， 480毫克氰基硼氢化 钠， 40毫升曱醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和氯化铵 溶液，减压除去曱醇，二氯甲烷萃取， 浓 - [(2, 5-二羟基苯甲基 )-(4- U) every 1 gram
Figure imgf000017_0002
, 600 mg of p-nonylbenzaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of decyl alcohol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, decyl alcohol was removed under reduced pressure, and dichloromethane was extracted. , concentrated - [(2, 5-dihydroxybenzyl)-(4-
曱基苯甲基） 氨基] -2-羟基苯曱酸， 即
Figure imgf000017_0003
或 Mercaptobenzyl)amino]-2-hydroxybenzoic acid, ie
Figure imgf000017_0003
or
( L )每 1克
Figure imgf000017_0004
, 600毫克 2 -呋喃曱醛， 480毫克氰基硼氢化钠,(L) every 1 gram
Figure imgf000017_0004
, 600 mg of 2-furanfurfural, 480 mg of sodium cyanoborohydride,
40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [(2, 5-二羟基苯甲基 )-(2-呋喃 曱基） tt] -2-羟基苯曱酸， 即
Figure imgf000018_0001
40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, methanol was removed under reduced pressure, dichloromethane was extracted and concentrated to give 5-[(2,5-dihydroxybenzyl)- (2-furan Thio) tt]-2-hydroxybenzoic acid, ie
Figure imgf000018_0001
(M)每 1
Figure imgf000018_0002
, 600毫克 2_甲基- 5-吡啶甲醛， 480毫克氰基 硼氢化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和 氯化铵溶液， 减压除去曱醇， 二氯甲烷萃取， 浓缩制得 5- [(2， 5-二羟基苯曱 (M) every 1
Figure imgf000018_0002
, 600 mg of 2-methyl- 5-pyridinecarboxaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, and decyl alcohol was removed under reduced pressure. Extraction with methyl chloride and concentration to give 5-[(2,5-dihydroxyphenylhydrazine)
基） -(2-曱基- 5-吡啶甲基） 氨基] -2-羟基苯甲酸， 即
Figure imgf000018_0003
-(2-mercapto-5-pyridylmethyl)amino]-2-hydroxybenzoic acid, ie
Figure imgf000018_0003
或 Or
(N)每 1克
Figure imgf000018_0004
, 600毫克 2_甲基- 5-吡啶甲醛， 480毫克氰基 硼氢化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和 氯化铵溶液， 减压除去曱醇， 二氯甲烷萃取， 浓缩制得 5- [(2， 5-二羟基苯甲 基) -（3-羟基- 4 -吡啶曱基） 氨基 ] _2-羟基苯甲酸， 即
Figure imgf000019_0001
上述薰草菌素 A衍生物在制备用于治疗肿瘤的药物方面的应用。 (N) every 1 gram
Figure imgf000018_0004
, 600 mg of 2-methyl- 5-pyridinecarboxaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, refluxed for 2 hours, the reaction was completely monitored by TLC, 50 ml of saturated ammonium chloride solution was added, and decyl alcohol was removed under reduced pressure. Extraction of methyl chloride, concentration to give 5-[(2,5-dihydroxybenzophenone) -(3-hydroxy-4-pyridinyl)amino]_2-hydroxybenzoic acid, ie
Figure imgf000019_0001
The use of the above-mentioned oxacin A derivative in the preparation of a medicament for treating a tumor.
优选的， 上述薰草菌素 A衍生物的应用， 所述肿瘤为淋巴瘤、 肺癌、 肝 癌、 乳腺癌、 卵巢癌、 胃癌、 大肠癌、 结肠癌、 黑色素瘤或头颈部癌。  Preferably, the use of the above-mentioned scavenger A derivative is lymphoma, lung cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, colon cancer, melanoma or head and neck cancer.
优选的， 上述薰草菌素 A衍生物的应用， 所述肿瘤为淋巴瘤或肺癌。 优选的， 上述薰草菌素 A衍生物的应用， 所述肿瘤为淋巴瘤。  Preferably, the use of the above-mentioned scavenger A derivative, the tumor is lymphoma or lung cancer. Preferably, in the application of the above-mentioned scavenger A derivative, the tumor is a lymphoma.
本发明的有益效果是：  The beneficial effects of the invention are:
上述薰草菌素 A衍生物， 通过体外细胞水平实验发现， 具有显著性抑制 人淋巴内皮细胞 ( HLEC ) 和稳定高表达 VEGFD的 LL/2 Lewi s肺癌细胞株 ( VEGF-D-LL/2 )增殖的作用， 并诱导 HLEC细胞凋亡， 抑制 HLEC细胞成管能 力， 作用机制研究发现该薰草菌素 A衍生物具有更强地抑制肿瘤细胞 VEGFD 的表达以及 A T和 ERK磷酸化水平， 同时更强地抑制 HLEC细胞 VEGF 3的表 达， 斑马直模型也显示新型薰草菌素 A类似物具有更强地抑制血管生成的作 用； 其制备方法筒单， 工艺条件温和， 适合规模化工业生产的应用； 可广泛 用于肿瘤淋巴转移的治疗， 为抗肿瘤药物的开发提供了一种新的选择。  The above-mentioned Kaempferol A derivative was found to have significant inhibition of human lymphatic endothelial cells (HLEC) and LL/2 Lewis lung cancer cell line stably expressing VEGFD (VEGF-D-LL/2) by in vitro cell level experiments. The role of proliferation, and induces apoptosis of HLEC cells, inhibits the ability of HLEC cells to form into a tube, and the mechanism of action has found that the scavenger A derivative has stronger inhibition of VEGFD expression and AT and ERK phosphorylation levels in tumor cells, and Strongly inhibiting the expression of VEGF 3 in HLEC cells, the zebra straight model also shows that the new phytohormone A analogue has a stronger inhibitory effect on angiogenesis; its preparation method is simple, the process conditions are mild, suitable for large-scale industrial production applications. It can be widely used in the treatment of tumor lymphatic metastasis, providing a new choice for the development of anti-tumor drugs.
附图说明 DRAWINGS
图 1-14是实施例 1-14分别对应的反应方程式；  1-14 are reaction equations corresponding to Examples 1-14, respectively;
图 15是 HQ— 001与 VEGFR3的作用模式图。 （a) : VEGFR3用 so l id r ibbon 表示， 并根据其 Secondary Type着色； HQ—001用 CPK模型表示， 其中绿色 为碳原子， 红色为氧原子； （b) : HQ_ 001用球棍模型表示， 其中绿色为碳原 子， 红色为氧原子； 除极性氢（白色）外， 其余氢原子没有显示； VEGFR3酪 氨酸激酶域活性位点的氨基酸残基， 用棒状表示， 并根据残基着色。 化合物 HQ _ 001与活性位点残基间所形成的氢键用虛线标示；  Figure 15 is a diagram showing the mode of action of HQ-001 and VEGFR3. (a): VEGFR3 is represented by so l id r ibbon and is colored according to its Secondary Type; HQ-001 is represented by CPK model, where green is carbon atom and red is oxygen atom; (b) : HQ_ 001 is represented by a ball stick model , wherein green is a carbon atom and red is an oxygen atom; except for polar hydrogen (white), the remaining hydrogen atoms are not shown; the amino acid residue of the active site of the VEGFR3 tyrosine kinase domain is represented by a rod and is colored according to the residue. . The hydrogen bond formed between the compound HQ _ 001 and the active site residue is indicated by a dotted line;
图 I 6是 HQ— 001诱导人淋巴内皮细胞（HLEC )凋亡（P I染色） ： a.正常 HLEC细胞； b. 0. 5uM HQ_ 001诱导 HLEC细胞发生凋亡现象； Figure I 6 is HQ-001 induced apoptosis of human lymphatic endothelial cells (HLEC) (PI staining): a. Normal HLEC cells; b. 0. 5uM HQ_ 001 induces apoptosis in HLEC cells;
图 17是 HQ_ 001诱导人淋巴内皮细胞（ HLEC ) 凋亡作用的时间依赖关系 ( Hoechst 33258染色）： A.作用 Oh; B.作用 2h; C. 作用 4h; D. 作用 8h; E. 作用 12h; F. 作用 24h; Figure 17 is a time-dependent relationship between HQ_ 001-induced apoptosis in human lymphatic endothelial cells (HLEC) (Hoechst 33258 staining): A. Action Oh; B. Action 2h; C. Action 4h; D. Action 8h; E. Action 12h; F. Effect 24h;
图 18是 HQ-001抑制人淋巴内皮细胞（HLEC) 的成管能力： a.未经处理 的 HLEC细胞在 matrigel表面 6h的成管结果； b. HLEC与 0.5uM HQ— 001共培 养 6h后在 matrigel表面的成管结果； c. 未经处理的 HLEC细胞在明胶表面 培养 6h的形态； d. HLEC与 0.5uM HQ_001共培养 6h后在明胶表面的形态； 图 19是 HQ_ 001抑制肿瘤细胞分泌 VEGFD及其作用机制；  Figure 18 shows the HX-001 inhibition of human lymphatic endothelial cell (HLEC) tube formation: a. The results of tube formation of untreated HLEC cells at matrigel surface for 6 h; b. HLEC and 0.5 uM HQ-001 co-culture for 6 h after Results of tube formation on matrigel surface; c. morphology of untreated HLEC cells cultured on gelatin surface for 6 h; d. morphology of gelatin surface after co-culture of HLEC with 0.5 uM HQ_001 for 6 h; Figure 19 shows that HQ_ 001 inhibits secretion of VEGFD by tumor cells And its mechanism of action;
图 20是 HQ_001抑制 HLEC细胞 VEGFR3的表达及其作用机制；  Figure 20 shows HQ_001 inhibiting the expression of VEGFR3 in HLEC cells and its mechanism of action;
图 21是 HQ_001抑制斑马鱼节间血管的生成。  Figure 21 shows that HQ_001 inhibits the formation of zebrafish internode blood vessels.
具体实施方式 detailed description
下面结合具体实施例对本发明所述技术方案作进一步的说明。  The technical solution of the present invention will be further described below in conjunction with specific embodiments.
下述实施例中的实验方法， 如无特殊说明， 均为常规方法； 下述实施例 中所用的试验材料， 如无特珠说明， 均为自常规生化试剂商店购买得到的； 下实施例中的定量试验， 均设置三次重复实验， 结果取平均值； 以下实施例 中的％, 如无特别说明， 均为质量百分含量。  The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples, if no special beads are described, are purchased from conventional biochemical reagent stores; For the quantitative test, three replicate experiments were set, and the results were averaged; the % in the following examples are mass percentages unless otherwise specified.
实施例 1  Example 1
如图 1所示， 制备方法如下：  As shown in Figure 1, the preparation method is as follows:
( 1 ) 100毫升反应瓶中加入 5克 2-羟基- 5-硝基苯曱酸 (化合物 1) , 40 毫升丙酮， 1毫升三氟乙酸， 回流 2小时， TLC检测反应完全，减压除去丙酮， 柱层析，制得无色油状液体化合物 2即 6-硝基苯并 [d] [1, 3] -2, 2-二甲基 -4- 氧代- 1， 3-二氧六环， 重 3.7克。 LC-ESI-MS (M+H+)： 224.12;  (1) Add 5 g of 2-hydroxy-5-nitrobenzoic acid (Compound 1) to a 100 ml reaction flask, 40 ml of acetone, 1 ml of trifluoroacetic acid, reflux for 2 hours, complete the reaction by TLC, and remove the acetone under reduced pressure. , column chromatography, to obtain a colorless oily liquid compound 2, 6-nitrobenzo[d] [1, 3] -2, 2-dimethyl-4-oxo-1, 3-dioxane , weighs 3.7 grams. LC-ESI-MS (M+H+): 224.12;
(2 ) 100毫升反应瓶中加入 2克化合物 2, 50毫升乙醇， 500毫克 10% 的 Pd/C, latm氢化室温搅拌 2小时， TLC检测反应完全， 原料消失。 过滤， 浓缩制得黄色固体产物化合物 3即 6-氨基苯并 [d] [1, 3] -2， 2-二甲基 -4 -氧 代- 1， 3-二氧六环。 LC-ESI-MS (Μ+ίΤ): 194.22;  (2) 2 g of compound 2, 50 ml of ethanol, 500 mg of 10% Pd/C, and latm were stirred at room temperature for 2 hours in a 100 ml reaction flask, and the reaction was completed by TLC, and the starting material disappeared. Filtration and concentration gave the yellow solid product compound 3, 6-aminobenzo[d][1,3]-2,2-dimethyl-4-oxo-1, 3-dioxane. LC-ESI-MS (Μ+ίΤ): 194.22;
( 3) 100毫升反应瓶中加入 1克化合物 3， 0.8克二环己基碳二亚胺， 1.1 克 1 -羟基苯并三氮唑， 50毫升二氯甲烷， 室温搅拌 12小时， TLC检测反应 完全， 减压除去溶剂， 柱层析， 制得白色固体化合物 4即 6- (环己基氧甲酰 胺基）苯并 [d] [1, 3] -2, 2-二甲基 -4-氧代- 1, 3-二氧六环。 LC-ESI-MS (M+H+)： 320.38;  (3) Add 1 g of compound 3, 0.8 g of dicyclohexylcarbodiimide, 1.1 g of 1-hydroxybenzotriazole, 50 ml of dichloromethane to a 100 ml reaction flask, stir at room temperature for 12 hours, and completely detect by TLC. The solvent was removed under reduced pressure and purified by column chromatography to yield white compound 4, 6-(cyclohexyloxycarbamoyl)benzo[d][1,3]-2,2-dimethyl-4-oxo - 1, 3-dioxane. LC-ESI-MS (M+H+): 320.38;
(4 ) 50毫升反应瓶中加入 1克化合物 4， 1.2克 NaBH4- A1C13, 30毫升 无水四氢呋喃， 回流搅拌 2小时， TLC检测反应完全， 减压除去四氢呋喃， 残余物水洗,二氯曱烷萃取，浓缩，柱层析得淡黄色油状液体化合物 5即 6- (环 己基氧甲基氨基)苯并 [d] [1， 3]- 2,2-二曱基 -4-氧代- 1,3-二氧六环， 重 720mgo LC-ESI-MS (M+H+)： 306.89; (4) Add 1 g of compound 4, 1.2 g of NaBH4-A1C13, 30 ml to a 50 ml reaction flask. Anhydrous tetrahydrofuran was stirred under reflux for 2 hours, and the reaction was completed by TLC. THF was evaporated, and the residue was washed with water, and the residue was washed with water, and then evaporated and evaporated to give a pale yellow oily liquid compound 5, 6-(cyclohexyloxymethyl) Amino)benzo[d][1,3]- 2,2-didecyl-4-oxo-1,3-dioxane, weight 720 mg o LC-ESI-MS (M+H + ): 306.89;
(5) 50毫升反应瓶中加入 500毫克化合物 5, 470毫克 3， 5 二羟基苯甲 醛， 380毫克氰基硼氢化钠， 30毫升甲醇， 回流 2小时， TLC检测反应完全， 加水淬灭反应， 减压除去甲醇， 二氯曱烷萃取， 柱层析，制得白色固体化合物 6即 6- [(环己基氧甲基）（2,5-二羟基苯基)氨基]苯并 [d] [1， 3] -2, 2-二甲基 -4-氧代- 1, 3-二氧六环， 重 0.67克。 LC- ESI- MS (M+H+)： 428.33;  (5) Add 500 mg of compound 5, 470 mg of 3,5 dihydroxybenzaldehyde, 380 mg of sodium cyanoborohydride, 30 ml of methanol to a 50 ml reaction flask, reflux for 2 hours, complete the reaction by TLC, and quench the reaction with water. Methanol was removed under reduced pressure, extracted with dichloromethane, and purified by column chromatography to afford compound 6 as a white solid, 6-[(cyclohexyloxymethyl)(2,5-dihydroxyphenyl)amino]benzo[d][ 1, 3] -2,2-Dimethyl-4-oxo-1, 3-dioxane, weighing 0.67 g. LC-ESI-MS (M+H+): 428.33;
(6) 50毫升反应瓶中加入 200毫克化合物 6, 100毫克氢氧化钠的 30 毫升甲醇溶液， 回流搅袢 2小时， TLC检测原料小时， 反应完全。 2N盐酸调 PH为 5, 加压除去甲醇， 加入水， 二氯甲烷萃取， 浓缩得白色固体产品（化 合物 7 )即 5- [ [ (2, 5-二羟基苯基）甲基] [ (环己基氧）曱基] -2-羟基苯甲 酸， 重 121亳克。 ^-NMR (300Hz, DMSO- d6) δ： 12.12 (s， 1H), 10.34 (br， 1H), 9.41 (br, 1H), 6.71-7.23 (m， 6H) , 5.43 (s, 2H) , 5.02 (s， 2H) , 2.79 (s, 1H), 1..43-1.71 (m, 10H) . LC-ESI-MS (M+『） : 388.17。 (6) A 50 ml reaction flask was charged with 200 mg of compound 6, 100 mg of sodium hydroxide in 30 ml of methanol, and the mixture was refluxed for 2 hours, and the reaction was completed by TLC. 2N hydrochloric acid was adjusted to pH 5, methanol was removed under pressure, water was added, and dichloromethane was extracted and concentrated to give a white solid product (Compound 7), 5-[[(2,5-dihydroxyphenyl)methyl][ Hexyloxy) indenyl]-2-hydroxybenzoic acid, weighing 121 g. ^-NMR (300 Hz, DMSO-d 6 ) δ: 12.12 (s, 1H), 10.34 (br, 1H), 9.41 (br, 1H), 6.71-7.23 (m, 6H), 5.43 (s, 2H) , 5.02 (s, 2H), 2.79 (s, 1H), 1..43-1.71 (m, 10H). LC-ESI-MS (M+): 388.17.
实施例 2  Example 2
如图 2所示， 制备方法如下：  As shown in Figure 2, the preparation method is as follows:
(1 )100毫升反应瓶中加入 2克 5-氨基水杨酸（化合物 8 ), 1.8克 2, 5- 二羟基苯甲醛， 2.1克氰基硼氢化钠， 80亳升曱醇， 回流 2小时， TLC检测 反应完全， 加入 20毫升饱和氯化铵溶液， 減压除去曱醇， 残余物水洗， 二氯 曱烷萃取， 浓缩， 柱层析， 制得白色固体化合物 9即 5-(2, 5-二羟基苯曱基） 氨基 -2-羟基苯曱酸, 重 1.9克。 LC-ESI-MS (M+H十）: 276.76;  (1) Add 100 g of 5-aminosalicylic acid (Compound 8), 1.8 g of 2, 5-dihydroxybenzaldehyde, 2.1 g of sodium cyanoborohydride, 80 liters of decyl alcohol, and reflux for 2 hours in a 100 ml reaction flask. The reaction was completely detected by TLC. 20 ml of a saturated ammonium chloride solution was added, the decyl alcohol was removed under reduced pressure, the residue was washed with water, extracted with dichloromethane, concentrated, and purified by column chromatography to give a white solid compound 9 (5-(2, 5) -Dihydroxyphenylhydrazino)amino-2-hydroxybenzoic acid, weighing 1.9 g. LC-ESI-MS (M+H): 276.76;
(2) 100毫升反应瓶中加入 1克化合物 9, 1.6克 4- Boc氨基 -5-嘧啶甲 醛， 1.2克氰基硼氢化钠， 50毫升甲醇， 回流 1小时加入 50毫升饱和氯化铵 溶液， 减压除去甲醇， 二氯甲烷萃取， 浓缩， 制得淡黄色固体化合物 10即 5- [(2, 5-二羟基苯甲基） [5_(4-Boc氨基嘧啶甲基)氨基] -2-羟基苯曱酸， 重 1.15克。 LC-ESI-MS (Μ+Η+)： 483.74; (2) Add 1 gram of compound 9, 1.6 g of 4-Boc amino-5-pyrimidinecarboxaldehyde, 1.2 g of sodium cyanoborohydride, 50 ml of methanol to a 100 ml reaction flask, and add 50 ml of saturated ammonium chloride solution under reflux for 1 hour. Methanol was removed under reduced pressure, extracted with dichloromethane, and concentrated to give pale yellow solid compound 10, 5-[(2,5-dihydroxybenzyl)[5-(4-Bocaminopyrimidinylmethyl)amino]-2- Hydroxybenzoic acid, weighing 1.15 grams. LC-ESI-MS (Μ+Η + ): 483.74;
( 3) 50毫升反应瓶中加入 500毫克化合物 10, 30毫升二氯甲烷， 1毫 升三氟乙酸， 室温搅拌 10小时， TLC检测反应完全， 减压除去二氯甲烷， 饱 和碳酸氢钠调 ΡΗ为 7， 柱层析， 制得白色固体产品（化合物 11 )即 5- [ (2, 5- 二羟基苯甲基） [5-(4-氨基嘧啶）曱基]氨基] -2-羟基苯甲酸， 重 210毫克。 'H-NMR (300HZ, DMSO— d6) δ： 12.68 (br, 1H) , 10.21 (br, 1H), 9.96 (br, 1H), 8.87 (br, 1H), 8.35 (s, 1H), 7.93 (s, 1H), 7.23 (s, 1H), 6.66—6.93 (m， 5H), 6.32 (br, 2H) , 5.32 (s, 2H) , 5.18 (s, 2H) . LC- ESI- MS (M+H ： 383. 38。 (3) Add 500 mg of compound 10, 30 ml of dichloromethane, 1 ml of trifluoroacetic acid in a 50 ml reaction flask, stir at room temperature for 10 hours, complete the reaction by TLC, remove the dichloromethane under reduced pressure, and dilute with sodium hydrogen carbonate. 7. Column chromatography to obtain a white solid product (Compound 11), ie 5-[(2,5-dihydroxybenzyl)[5-(4-aminopyrimidinyl)indolyl]amino]-2-hydroxybenzoic acid It weighs 210 mg. 'H-NMR (300HZ, DMSO-d 6 ) δ: 12.68 (br, 1H), 10.21 (br, 1H), 9.96 (br, 1H), 8.87 (br, 1H), 8.35 (s, 1H), 7.93 (s, 1H), 7.23 (s, 1H), 6.66-6.93 (m, 5H), 6.32 (br, 2H), 5.32 (s, 2H), 5.18 (s, 2H) . LC- ESI- MS (M +H : 383. 38.
实施例 3  Example 3
如图 3所示， 制备方法如下：  As shown in Figure 3, the preparation method is as follows:
100毫升反应瓶中加入 1克化合物 9， 2克 3_吡啶基 -2-氧代-丙醛， 600 毫克氰基硼氢化钠， 50毫升甲醇， 回流 2小时， 加入 50毫升饱和氯化铵溶 液， 减压除去曱醇， 萃取， 浓缩， 柱层析， 制得白色固体产品 （化合物 12 ) 即 5_[(2，5-二羟基苯曱基） U-氧代- 3-吡啶乙基） ] 氣基 _2-羟基苯甲酸， 重 225毫克。 - NMR (300Hz, DMSO- d6) δ： 12.89 (br, 1H) , 9.97 (br, 2H) , 8.97 (br, 1H), 9.15 (s， 1H), 8.82 (s， 1H), 6.66-7.23 (m， 8H) , 5.08 (s, 2H), 4.46 (s, 2H). LC- ESI- MS (M+『）： 395.74。 1 gram of compound 9, 2 g of 3-pyridyl-2-oxo-propanal, 600 mg of sodium cyanoborohydride, 50 ml of methanol, refluxed for 2 hours, and 50 ml of saturated ammonium chloride solution were added to a 100 ml reaction flask. , Yue alcohol extracted, concentrated under removed under reduced pressure, column chromatography, the product as a white solid (compound 12) i.e. 5 _ [(2,5-dihydroxyphenyl Yue-yl) oxo-U- --3- pyridin-ethyl)] Gas-based -2 -hydroxybenzoic acid, weighing 225 mg. - NMR (300Hz, DMSO-d 6 ) δ: 12.89 (br, 1H) , 9.97 (br, 2H) , 8.97 (br, 1H), 9.15 (s, 1H), 8.82 (s, 1H), 6.66-7.23 (m, 8H), 5.08 (s, 2H), 4.46 (s, 2H). LC-ESI- MS (M+ "): 395.74.
实施例 4  Example 4
如图 4所示， 制备方法如下：  As shown in Figure 4, the preparation method is as follows:
100毫升反应瓶中加入 1克化合物 9, 600毫克 3-吡啶乙醛， 480毫克氰 基硼氢化钠， 40毫升甲醇， 回流 1小时， TLC监测反应完全， 加入 50亳升饱 和氯化铵溶液， 减压除去曱醇， 二氯甲烷萃取， 浓缩制得白色固体产品（化 合物 13 ) 即 5- [ (2, 5-二羟基苯曱基 )-（3-吡啶乙基）] 氨基 -2-羟基苯甲酸， 重 290毫克。 !H-NMR (300Hz, DMSO- d6) 5： 11.87 (br, 1H) , 9.67 (br, 2H) ,1 gram of compound 9, 600 mg of 3-pyridineacetaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol were added to a 100 ml reaction flask, and refluxed for 1 hour. The reaction was completely monitored by TLC, and 50 liters of saturated ammonium chloride solution was added. The decyl alcohol is removed under reduced pressure, extracted with dichloromethane, and concentrated to give a white solid product (Compound 13), 5-[(2,5-dihydroxyphenyl)-(3-pyridylethyl)]amino-2-hydroxy Benzoic acid, weighing 290 mg. ! H-NMR (300Hz, DMSO- d 6) 5: 11.87 (br, 1H), 9.67 (br, 2H),
8.84 (br, 1H)， 8.34 (s, 1H), 8.22 (s, 1H), 6.61-7.29 (m, 8H)， 5.08 (d, J = 4. 2 Hz, 2H) , 3.68 (d, J = 4. 2 Hz, 2H) . LC- ESI- MS (M+H+) : 381.66。 8.84 (br, 1H), 8.34 (s, 1H), 8.22 (s, 1H), 6.61-7.29 (m, 8H), 5.08 (d, J = 4. 2 Hz, 2H), 3.68 (d, J = 4. 2 Hz, 2H). LC-ESI- MS (M+H+): 381.66.
实施例 5  Example 5
如图 5所示， 制备方法如下：  As shown in Figure 5, the preparation method is as follows:
将 3-吡啶乙醛换成 5-嘧啶甲醛，其余所需原料、试剂和制备方法同实施 例 4, 得白色固体产品（化合物 14 ) 即 5- [ (2, 5-二羟基苯甲基) - (5-嘧啶甲 基）] 氨基- 2-羟基苯甲酸。 NMR (300Hz, DMSO- d6) 8： 12.67 (br, 1H),3-pyridineacetaldehyde was replaced with 5-pyrimidinecarboxaldehyde, and the remaining starting materials, reagents and preparation methods were the same as in Example 4 to obtain a white solid product (Compound 14), ie 5-[(2,5-dihydroxybenzyl) - (5-pyrimidinylmethyl)]amino-2-hydroxybenzoic acid. NMR (300 Hz, DMSO-d 6 ) 8: 12.67 (br, 1H),
9.57 (br, 2H)， 9.06 (s, 1H) , 8.94 (br, 1H), 8.57 (s， 2H) , 6.51-7.23 (m, 6H), 5.23 (s, 2H) , 4.88 (s， 2H) . LC— ESI— MS (M+H+) : 368.34。 9.57 (br, 2H), 9.06 (s, 1H), 8.94 (br, 1H), 8.57 (s, 2H), 6.51-7.23 (m, 6H), 5.23 (s, 2H), 4.88 (s, 2H) LC-ESI-MS (M+H+): 368.34.
实施例 6  Example 6
如图 6所示， 制备方法如下：  As shown in Figure 6, the preparation method is as follows:
将 3-吡啶乙醛换成 5-噻，，坐甲醛，其余所需原料、试剂和制备方法同实施 例 4, 得白色固体产品（化合物 15 ) 即 5- [ (2, 5-二羟基苯甲基) - (5-噻唑甲 基）] 氨基 -2-羟基苯曱酸。 'H-NMR (300Hz, DMS0-d6) δ： 12.19 (br, 1H), 9.87 (br, 2H)， 8.94 (br, 1H) , 8.67 (s, 2H) , 6.88-7.33 (m, 7H) , 5.23 (s, 2H), 4.61 (s, 2H). LC-ESI-MS (M+H ： 373.38。 Replace 3-pyridine acetaldehyde with 5-thiene, take formaldehyde, and the other required raw materials, reagents and preparation methods are the same. Example 4 gave the white solid product (Compound 15) 5-[(2,5-dihydroxybenzyl)-(5-thiazolylmethyl)]amino-2-hydroxybenzoic acid. 'H-NMR (300 Hz, DMS0-d 6 ) δ: 12.19 (br, 1H), 9.87 (br, 2H), 8.94 (br, 1H), 8.67 (s, 2H), 6.88-7.33 (m, 7H) , 5.23 (s, 2H), 4.61 (s, 2H). LC-ESI-MS (M+H: 373.38.
实施例 Ί  Example Ί
如图 7所示， 制备方法如下：  As shown in Figure 7, the preparation method is as follows:
将 3-吡啶乙醛换成 2-曱基 -3-羟基 -4-吡啶曱醛，其余所需原料、试剂和 制备方法同实施例 4, 得淡黄色固体产品（化合物 16 )即 5- [ (2, 5-二羟基苯 曱基） -(2-曱基- 3-羟基 -4-吡啶甲基)氛基 ] -2-羟基苯曱酸。 'H-NMR (300Hz, DMS0-d6) δ： 12.38 (br, 1H), 9.68 (br, 2H)， 8.74 (br, 2H) , 8.26 (s， 1H), 6.88-7.33 (m, 7H) , 5.23 (s， 2H) , 4.61 (s， 2H) , 2.53 (s, 3H) . LC-ESI-MS (M+H+): 397.89。 3-pyridineacetaldehyde was replaced with 2-mercapto-3-hydroxy-4-pyridylfurfural. The remaining starting materials, reagents and preparation methods were the same as in Example 4 to obtain a pale yellow solid product (Compound 16), ie 5-[ (2,5-Dihydroxyphenylindenyl)-(2-indolyl-3-hydroxy-4-pyridylmethyl)aryl]-2-hydroxybenzoic acid. 'H-NMR (300 Hz, DMS0-d 6 ) δ: 12.38 (br, 1H), 9.68 (br, 2H), 8.74 (br, 2H), 8.26 (s, 1H), 6.88-7.33 (m, 7H) , 5.23 (s, 2H), 4.61 (s, 2H), 2.53 (s, 3H). LC-ESI-MS (M+H+): 397.89.
实施例 8  Example 8
如图 8所示， 制备方法如下：  As shown in Figure 8, the preparation method is as follows:
将 3-吡啶乙醛换成 3-吡啶曱醛，其余所需原料、试剂和制备方法同实施 例 4, 得淡黄色固体产品（化合物 17) 即 5_[ (2, 5-二羟基苯甲基 )-（3-吡啶 曱基）氨基] -2-羟基苯甲酸。 ^-NMR (300Hz, DMSO- d6) δ： 12.58 (br, 1H), 9.78 (br, 2H), 8.78 (br, 2H) , 8.59 (s, 1H), 8.26 (d, / = 7.8 Hz, 1H), 7.86 (s, 1H), 6.98-7.33 (m, 7H) , 5.23 (s, 2H) , 4.61 (s, 2H) . LC-ESI-MS (M+H+): 367.45。 3-pyridineacetaldehyde was replaced with 3-pyridylfurfural, and the remaining starting materials, reagents and preparation methods were the same as those in Example 4 to obtain a pale yellow solid product (Compound 17), ie 5-[(2,5-dihydroxybenzyl) )-(3-pyridinyl)amino]-2-hydroxybenzoic acid. ^-NMR (300 Hz, DMSO-d 6 ) δ: 12.58 (br, 1H), 9.78 (br, 2H), 8.78 (br, 2H), 8.59 (s, 1H), 8.26 (d, / = 7.8 Hz, 1H), 7.86 (s, 1H), 6.98-7.33 (m, 7H), 5.23 (s, 2H), 4.61 (s, 2H). LC-ESI-MS (M+H+): 367.45.
实施例 9  Example 9
如图 9所示， 制备方法如下：  As shown in Figure 9, the preparation method is as follows:
将 3-吡啶乙醛换成 1-氨基甲醜基 -2-氧代 -3-吡咯甲醛， 其余所需原料、 试剂和制备方法同实施例 4, 得白色固体产品（化合物 18 ) 即 5- [ (2, 5-二羟 基苯甲基) -[3- [(1-氨基甲酰基 -2-氧代)吡咯甲基） ] 氨基] -2-羟基苯曱酸。 'H-NMR (300Hz, DMSO— d6) δ： 12.88 (br, 1H), 9.98 (br, 1H), 8.74 (br, 1H), 6.75-7.23 (m, 7H) , 6.25 (br, 2H) , 5.23 (s, 2H) , 4.21 (s, 2H) . LC-ESI-MS (M+H ： 414.45。 The 3-pyridine acetaldehyde is replaced by 1-aminomethyl idyl-2-oxo-3-pyrrolecarboxaldehyde. The other desired starting materials, reagents and preparation methods are the same as in Example 4, to obtain a white solid product (compound 18), ie 5- [(2,5-Dihydroxybenzyl)-[3-[(1-carbamoyl-2-oxo)pyrrolemethyl)]amino]-2-hydroxybenzoic acid. 'H-NMR (300 Hz, DMSO-d 6 ) δ: 12.88 (br, 1H), 9.98 (br, 1H), 8.74 (br, 1H), 6.75-7.23 (m, 7H), 6.25 (br, 2H) , 5.23 (s, 2H), 4.21 (s, 2H). LC-ESI-MS (M+H: 414.45.
实施例 10  Example 10
如图 10所示， 制备方法如下：  As shown in Figure 10, the preparation method is as follows:
将 3-吡啶乙醛换成 5-曱基呋喃曱醛， 其余所需原料、试剂和制备方法同 实施例 4, 得白色固体产品 （化合物 19) 即 5- [(2, 5-二羟基苯甲基） [2- (5- 曱基呋喃）曱基]] 氨基 _2-羟基苯甲酸。 -丽 R (300Hz, DMS0-d6) δ: 12.48 (br, 1H), 9.78 (br, IH), 8.75 (br, 1H) , 6.75-7.23 (m, 6H) , 6.25 (d, /= 5.1 Hz, IH), 6.02 (d, /= 5.1 Hz, IH) , 2.3 (s， 3H) . LC-ESI-MS (M+H十）： 370.49。 3-pyridineacetaldehyde was replaced with 5-mercaptofuranfurfural, and the remaining desired starting materials, reagents and preparation methods were the same as in Example 4 to obtain a white solid product (Compound 19), ie 5-[(2,5-dihydroxybenzene). Methyl) [2- (5- Mercaptofuran)indolyl]]amino- 2 -hydroxybenzoic acid. -R (300Hz, DMS0-d 6 ) δ: 12.48 (br, 1H), 9.78 (br, IH), 8.75 (br, 1H), 6.75-7.23 (m, 6H), 6.25 (d, /= 5.1 Hz, IH), 6.02 (d, /= 5.1 Hz, IH), 2.3 (s, 3H). LC-ESI-MS (M+H X): 370.49.
实施例 11  Example 11
如图 11所示， 制备方法如下：  As shown in Figure 11, the preparation method is as follows:
将 3-吡啶乙醛换成对甲基苯甲醛， 其余所需原料、 试剂和制备方法同实 施例 4, 得白色固体产品（化合物 20) 即 5- [(2， 5-二羟基苯曱基 )-(4-甲基 苯曱基） 氨基] -2-羟基苯曱酸。 -匪 R (300Hz, DMS0-d6) δ： 12.78 (br, IH), 9.88 (br, IH), 8.72 (br, IH), 7.23 (d， / = 7.8 Hz， 2H)， 7.02 (d， J = 7.8 Hz, 2H), 6.75-7.23 (m, 6H) , 5.21 (s, 2H) , 2.3 (s， 3H) . LC-ESI-MS (M+H+): 380.30。 The 3-pyridineacetaldehyde was replaced with p-methylbenzaldehyde, and the remaining starting materials, reagents and preparation methods were the same as those in Example 4 to obtain a white solid product (Compound 20), ie 5-[(2,5-dihydroxyphenylhydrazino). )-(4-Methylphenylhydrazino)amino]-2-hydroxybenzoic acid. -匪R (300Hz, DMS0-d 6 ) δ: 12.78 (br, IH), 9.88 (br, IH), 8.72 (br, IH), 7.23 (d, / = 7.8 Hz, 2H), 7.02 (d, J = 7.8 Hz, 2H), 6.75-7.23 (m, 6H), 5.21 (s, 2H), 2.3 (s, 3H). LC-ESI-MS (M+H+): 380.30.
实施例 12  Example 12
如图 12所示， 制备方法如下：  As shown in Figure 12, the preparation method is as follows:
将 3-吡啶乙醛换成 2-呋喃曱醛，其余所需原料、试剂和制备方法同实施 例 4, 得白色固体产品（化合物 21 ) 即 5- [ (2, 5-二羟基苯甲基) - (2-呋喃甲 基） 氨基]—2—羟基苯曱酸。 - NMR (300Hz, DMSO- d6) 5： 12.98 (br, IH), 9.81 (br, IH), 8.70 (br, IH), 6.75-7.23 (m, 9H) , 5.21 (s, 2H) , 5.09 (s, 2H) . LC-ESI-MS (M+H+): 356.69。 3-pyridineacetaldehyde was replaced with 2-furanfurfural, and the remaining starting materials, reagents and preparation methods were the same as those in Example 4 to obtain a white solid product (Compound 21), ie 5-[(2,5-dihydroxybenzyl) ) - (2-furylmethyl)amino]-2-hydroxybenzoic acid. - NMR (300Hz, DMSO-d 6 ) 5: 12.98 (br, IH), 9.81 (br, IH), 8.70 (br, IH), 6.75-7.23 (m, 9H) , 5.21 (s, 2H) , 5.09 (s, 2H). LC-ESI-MS (M+H+): 356.
实施例 13  Example 13
如图 13所示， 制备方法如下：  As shown in Figure 13, the preparation method is as follows:
将 3-吡啶乙醛换成 2-甲基- 5-吡啶甲醛， 其余所需原料、 试剂和制备方 法同实施例 4,得白色固体产品（化合物 11 )即 5- [ (2, 5-二羟基苯甲基) - (2- 甲基- 5-吡啶曱基） 氨基] -2-羟基苯甲酸。 -匪 R (300Hz, DMS0-d6) δ: 12.77 (br, IH), 9.98 (br, IH), 8.95 (br, IH) , 8.60 (s， IH) , 6.75-7.73 (m， 8H), 5.23 (s, 2H), 5.08 (s, 2H) , 2.3 (s, 3H) . LC-ESI-MS (M+Hl： 381.90。 The 3-pyridineacetaldehyde was replaced with 2-methyl-5-pyridinecarboxaldehyde, and the remaining materials, reagents and preparation methods were the same as those in Example 4. The white solid product (Compound 11) was obtained as 5-[(2, 5- Hydroxybenzyl)-(2-methyl-5-pyridinyl)amino]-2-hydroxybenzoic acid. -匪R (300Hz, DMS0-d 6 ) δ: 12.77 (br, IH), 9.98 (br, IH), 8.95 (br, IH), 8.60 (s, IH), 6.75-7.73 (m, 8H), 5.23 (s, 2H), 5.08 (s, 2H), 2.3 (s, 3H). LC-ESI-MS (M+Hl: 381.90.
实施例 14  Example 14
如图 14所示， 制备方法如下：  As shown in Figure 14, the preparation method is as follows:
将 3-吡啶乙醛换成 2-甲基- 5-吡啶甲醛， 其余所需原料、 试剂和制备方 法同实施例 4,得白色固体产品（化合物 23 )即 5- [ (2, 5-二羟基苯甲基) - (3- 羟基- 4-吡啶曱基） 基]- 2-羟基苯曱酸。 -丽 R (300Hz, DMSO- d6) δ: 12.97 (br, 1H)， 9.68 (br, 1H) , 8.75 (br, 2H) , 8.30 (s, 1H), 6.75—7.83 (m, 8H) , 5.23 (s, 2H), 5.08 (s, 2H) . LC-ESI-MS (M+H+) : 383.39。 3-pyridineacetaldehyde was replaced by 2-methyl-5-pyridinecarboxaldehyde. The remaining starting materials, reagents and preparation methods were the same as those in Example 4. The white solid product (Compound 23) was obtained as 5-[(2, 5- Hydroxybenzyl)-(3-hydroxy-4-pyridinyl)yl]-2-hydroxybenzoic acid. - Li R (300Hz, DMSO- d 6 ) δ: 12.97 (br, 1H), 9.68 (br, 1H), 8.75 (br, 2H), 8.30 (s, 1H), 6.75-7.83 (m, 8H), 5.23 (s, 2H), 5.08 (s, 2H). LC-ESI-MS (M+H+): 353.39.
实施例 15  Example 15
新型薰草菌素 A类似物抑制人淋巴内皮细胞（ HLEC )和 VEGFD高表达的 LL/2细胞 ( VEGFD-LL/2 )增殖  Novel xanthophyllin A analogue inhibits proliferation of human lymphatic endothelial cells (HLEC) and VEGFD-expressing LL/2 cells (VEGFD-LL/2)
试验方法： 细胞培养 HLEC细胞培养在含 5%胎牛血清， 1%内皮细胞生长 添加剂（ ECGS )， 100IU/mL青霉素和 100 μ g/mL链霉素的内皮细胞培养基（ ECM) 中。 培养环境也是饱和湿度、 37°C的 5% C02孵箱。 VEGFD- LL/2细胞常规培 养于含 10%胎牛血清、 100U/ml青霉素和 100 mg/L链霉素的 DMEM培养液中， 置饱和湿度、 37 °C的 5¾ C02孵箱中培养。  Test Methods: Cell Culture HLEC cells were cultured in endothelial cell culture medium (ECM) containing 5% fetal bovine serum, 1% endothelial cell growth additive (ECGS), 100 IU/mL penicillin and 100 μg/mL streptomycin. The culture environment is also a saturated humidity, 5% C02 incubator at 37 °C. VEGFD-LL/2 cells were routinely cultured in DMEM containing 10% fetal calf serum, 100 U/ml penicillin and 100 mg/L streptomycin in a saturated humidity, 53 ° C C52 incubator.
MTT试验 取对数生长期细胞， 消化液消化或直接吹打混匀后计数； 调整细胞悬液浓度 2 lOVmL, 于 96孔板中每孔加入 lOOul细胞悬液； 24 h 后吸掉原有培养基， 分别加入 200 ul含有包括薰草菌素 A在内的 15个化合 物的完全培养液（阴性对照组加等量 DMS0 ), 每组 3复孔， 继续培养于 C02 孵箱； 分別于加药后 48 h后取出 96孔板, 每孔加入 5 mg/1 MTT试剂 50ul, 继续培养 4小时； 吸出孔中的液体， 每孔加入 15Q ul DMS0, 室温震荡 15分 钟充分溶解沉淀， 用酶标仪在 570 nm波长检测每孔细胞 0D值。 根据相对抑 制率判断薰草菌素 A类似物对 HLEC和 VEGFD-LL/2细胞的抑制作用：  The MTT test takes the logarithmic growth phase cells, digested or digested and directly mixed and counted. The cell suspension concentration was adjusted to 2 lOVmL, and 100 ul of cell suspension was added to each well of a 96-well plate; after 24 h, the original medium was aspirated. , add 200 ul of complete culture solution containing 15 compounds including oxacillin A (negative control group plus equal amount of DMS0), each group of 3 duplicate wells, continue to culture in C02 incubator; respectively after dosing After 48 h, remove the 96-well plate, add 50 μl of 5 mg/1 MTT reagent to each well, and continue to culture for 4 hours. Aspirate the liquid in the well, add 15Q ul DMS0 to each well, and lyse for 15 minutes at room temperature to fully dissolve the precipitate, using a microplate reader. The 570 nm wavelength was used to detect the 0D value of each well. The inhibitory effect of the oxacin A analogue on HLEC and VEGFD-LL/2 cells was determined based on the relative inhibition rate:
相对抑制率 = (对照孔平均 0D值 -加药孔平均 0D值） /对照孔平均 0D值 X 100¾。  Relative inhibition rate = (control hole average 0D value - dosing hole average 0D value) / control hole average 0D value X 1003⁄4.
并计算 IC5。值。 结果见表 2。 And calculate IC 5 . value. The results are shown in Table 2.
表 2 薰草菌素 A及 14个新型类似物对 HLEC和 YEGFD-LL/2增殖的抑制 作用  Table 2 Inhibition of HLEC and YEGFD-LL/2 proliferation by xanthosin A and 14 novel analogues
化合物 IC5。 (uM) for IC5。 (uM) for Compound IC 5 . (uM) for IC 5 . (uM) for
HLEC VEGFD-LL/2  HLEC VEGFD-LL/2
HQ— 001 0.38 0.42  HQ— 001 0.38 0.42
HQ— 002 1.75 2.10  HQ—002 1.75 2.10
HQ— 003 0.62 1.95  HQ— 003 0.62 1.95
HQ— 004 1.36 12.65  HQ— 004 1.36 12.65
HQ— 005 7.31 6.22  HQ— 005 7.31 6.22
HQ— 006 2.17 9.40  HQ— 006 2.17 9.40
HQ— 007 13.62 >50 HQ— 008 8.16 26.51 HQ—007 13.62 >50 HQ—008 8.16 26.51
HQ— 009 >50  HQ—009 >50
HQ— 010 3.06 8.52  HQ— 010 3.06 8.52
HQ— Oil 19.51 31.90  HQ- Oil 19.51 31.90
HQ— 012 8.07 12.63  HQ— 012 8.07 12.63
HQ— 013 5.84 19.78  HQ— 013 5.84 19.78
HQ— 014 22.08 40.12  HQ— 014 22.08 40.12
Lavendustin A 5.18 9.06 实施例 16  Lavendustin A 5.18 9.06 Example 16
HQ_001与 VEGFR3间的相互作用模式  Interaction mode between HQ_001 and VEGFR3
采用计算机药物虚拟筛选技术和细胞水平技术获得 HQ_001 HQ_002, HQ_003, HQ_005和 HQ_010抗肿瘤效果皆比母体薰草菌素 A更佳， 于是 选择活性相对最优的 HQ_001作为以下研究的新型化合物。 图 15显示了 HQ— 001与 VEGFR3间的相互作用模式。 从图 15 (b)中观察到， HQ_001的邻羟 基苯甲酸部分的羧基氧和羟基氧， 与 CyslOO的 alpha-氨基氬形成氢键； HQ-001的对二羟基苯酚部分其中一个羟基氧，为 Lys49的 alpha-氨基氢提供 氢键受体（acceptor) , 而该羟基上的氢， 为 Ala47的羰基氧， 提供氢键给体 (donor)  Using the computer drug virtual screening technology and cell level technology to obtain HQ_001 HQ_002, HQ_003, HQ_005 and HQ_010 are better than the parental oxacillin A, so the HQ_001 with relatively optimal activity was selected as the new compound for the following study. Figure 15 shows the interaction pattern between HQ-001 and VEGFR3. It is observed from Fig. 15 (b) that the carboxyl oxygen and hydroxyl oxygen of the o-hydroxybenzoic acid moiety of HQ_001 form a hydrogen bond with the alpha-amino argon of CyslOO; one of the hydroxyl groups of the p-dihydroxyphenol of HQ-001 is The alpha-amino hydrogen of Lys49 provides a hydrogen bond acceptor, and the hydrogen on the hydroxyl group, which is the carbonyl oxygen of Ala47, provides a hydrogen bond donor.
实施例 17  Example 17
HQ_ 001诱导人淋巴内皮细胞（HLEC) 凋亡  HQ_ 001 induces apoptosis in human lymphatic endothelial cells (HLEC)
按照 MTT结果， 选择 0.5uM的 HQ_001处理 HLEC细 J包， DMS0对照组加入 等体积的 DMS0, 空白对照组加入等体积的完全培养基， 处理 48h后进行 PI 染色观察： 1)将原有的培养基吸出， 吸干净， 注意轻柔操作， 以免导致细胞 丢失；  According to the MTT results, 0.5uM HQ_001 was selected to treat HLEC fine J package, DMS0 control group was added with equal volume of DMS0, blank control group was added with equal volume of complete medium, and treated with PI staining for 48h after treatment: 1) original culture Base aspirate, suck clean, pay attention to gentle operation, so as not to cause cell loss;
2)加入碘化丙锭 (PI)染色液， 避光染色 5分钟左右； 3) 于导致显微镜 下观察并保存图像； 结果见图 16。 由图 16 b可见 HLEC细胞容积变小， 染色 质固缩， 并产生核碎片和凋亡小体。  2) Add propidium iodide (PI) staining solution and stain it in the dark for about 5 minutes; 3) Observe and save the image under the microscope; the results are shown in Figure 16. It can be seen from Fig. 16b that the HLEC cell volume becomes smaller, the chromatin shrinks, and nuclear debris and apoptotic bodies are produced.
实施例 18  Example 18
HQ _ 001诱导人淋巴内皮细胞（ HLEC ) 凋亡的时间依赖性  HQ _ 001 induces time-dependent apoptosis of human lymphatic endothelial cells (HLEC)
为了探索 HQ_001诱导 HLEC细胞凋亡在时间上的规律， 实验中还对 HQ-001作用下的 HLEC细胞核形态进行了动态观察（图 17 )。 在 HQ-001分别 作用 Oh, 2h, 4h, 8h， 12h和 24h之后， 对细胞进行 Hoechst33258染色。在 荧光显 镜下观察发现， HQ_001作用 8h后， HLEC细胞开始产生凋亡的迹象， 这种迹象随着时间的延长逐渐增多。 在作用 24h之后， 能明显看到大量的细 胞产生核固缩、 染色体凝集等现象， 并能观察到凋亡小体的存在。 In order to explore the temporal regularity of HQ_001-induced apoptosis of HLEC cells, the morphological morphology of HLEC cells under HQ-001 was also observed dynamically (Fig. 17). In HQ-001 respectively After Oh, 2h, 4h, 8h, 12h and 24h, the cells were stained with Hoechst 33258. Under the fluorescence microscope, it was found that HLEC cells began to produce signs of apoptosis after 8 hours of HQ_001 treatment, and this sign gradually increased with time. After 24 hours of action, it was obvious that a large number of cells produced nuclear pyknosis, chromosome agglutination, etc., and the presence of apoptotic bodies was observed.
实施例 19  Example 19
HQ— 001抑制人淋巴内皮细胞（HLEC) 的成管能力  HQ-001 inhibits the ability of human lymphatic endothelial cells (HLEC) to establish tube
通过体外 matrigel成管实验， 研究了 HQ_001对人淋巴管内皮细胞 HLEC迁移分化并形成管状结构的影响。 光镜下观察结果（图 18 )显示， 对照 组 HLEC细胞在 6小时内能够在 Matr igel上迁移分化， 伸展并相互连接， 形 成较好的网络管状结构。 这种结构呈毛细血管状， 并形成多中心的结点（图 18 a); 而与 0.5uM HQ_001共培养的情况下， 与对照组相比， 管状结构的形 成明显受到抑制， 使 HLEC细胞形成的网格明显减少， HLEC细胞形态由拉伸 的长梭形逐渐变圓 （图 18 b)。  The effect of HQ_001 on the migration and differentiation of human lymphatic endothelial cells HLEC and the formation of tubular structures were studied by in vitro matrigel tube experiments. The results of light microscopy (Fig. 18) showed that the HLEC cells in the control group could migrate and differentiate on Matr igel within 6 hours, stretching and interconnecting to form a better network tubular structure. This structure is capillary-like and forms a multi-center node (Fig. 18a); whereas in the case of co-culture with 0.5 uM HQ_001, the formation of the tubular structure is significantly inhibited compared to the control group, resulting in the formation of HLEC cells. The mesh was significantly reduced, and the HLEC cell morphology gradually became rounded by the stretched long fusiform (Fig. 18b).
为了验证 matrigel組分的诱导作用和排除药物毒性的影响，实验中设置 了在明胶表面的对照实验。在没有药物作用和没有 matrigel的情况下， HLEC 细胞在明胶表面正常贴壁生长， 不发生迁移和分化， 也不形成管状结构（图 18 c )。 与 0.5uM HQ_ 001共培养并没有明显影响 HLEC细胞的正常贴壁和生长 (图 18 d)。由此可见， HQ— 001能够在较短时间内明显抑制 HLEC的成管能力。 这种抑制作用并不是由于其细胞毒作用导致的内皮细胞损伤。 因此证明， HQ-001具有抑制淋巴管生成的作用。  In order to verify the induction of the matrigel component and to rule out the effects of drug toxicity, a control experiment on the gelatin surface was set up in the experiment. In the absence of drug action and without matrigel, HLEC cells grow normally adherently on the gelatin surface without migration and differentiation, nor form a tubular structure (Fig. 18c). Co-culture with 0.5 uM HQ_ 001 did not significantly affect normal adherence and growth of HLEC cells (Fig. 18d). It can be seen that HQ-001 can significantly inhibit the HLEC's ability to form tubes in a short period of time. This inhibition is not due to endothelial cell damage caused by its cytotoxic effects. Therefore, it was confirmed that HQ-001 has an effect of inhibiting lymphangiogenesis.
实施例 20  Example 20
HQ_001抑制肿瘤细胞 VEGFD的分泌  HQ_001 inhibits the secretion of VEGFD from tumor cells
本实验通过 Western blot方法首先检测了肿瘤细胞中 VEGFD的表达。肿 瘤细胞中 VEGFD的分泌与转录因子 AP-1有关，因此实验对 c_fos的上游激酶 ERK1/2的磷酸化进行了检测。 同时， 也对调控细胞存活、 迁移、 增殖的 PI3K/AKT通路进行了检测。 Western blot实验结果（图 19)显示， VEGFD- LL/2 细胞较空载的 pcDNA- LL/2细胞， VEGFD的表达水平显著增高。 并且在 0.5uM HQ_001作用 12h后， VEGFD-LL/2的 VEGF-D表达水平受到明显的抑制。 这种 抑制作用与 HQ_001阻断了肿瘤细包中 MEK/ERK1/2通路有关。 从结果可以看 到， 肿瘤细胞中 BRK呈现组成性激活， 对照组的细胞中 ERK发生了明显的磷 δ交化， 这种磷酸化的激活被 HQ_001阻断。 同样， AKT的磷酸化也受到了明显 的抑制。 因此， HQ-001对于 VEGFD-LL/2细胞的信号通路有直接的抑制作用。 HQ_001抑制淋巴管新生和淋巴转移的结果与 HQ-001抑制肿瘤细胞中 ERK和 AKT的活化， 从而下调 VEGFD的表达有关。 In this experiment, the expression of VEGFD in tumor cells was first detected by Western blot. The secretion of VEGFD in tumor cells is associated with the transcription factor AP-1, so the phosphorylation of the upstream kinase ERK1/2 of c_fos was tested. At the same time, the PI3K/AKT pathway, which regulates cell survival, migration, and proliferation, was also examined. The results of Western blot (Fig. 19) showed that the expression level of VEGFD was significantly increased in VEGFD-LL/2 cells compared with unloaded pcDNA-LL/2 cells. And after 12 h of action at 0.5 uM HQ_001, the VEGF-D expression level of VEGFD-LL/2 was significantly inhibited. This inhibition is related to HQ_001 blocking the MEK/ERK1/2 pathway in tumor fines. As can be seen from the results, BRK showed constitutive activation in tumor cells, and ERK of the control group showed significant phosphorylation of δ, and the activation of this phosphorylation was blocked by HQ_001. Similarly, phosphorylation of AKT was also significantly inhibited. Therefore, HQ-001 has a direct inhibitory effect on the signaling pathway of VEGFD-LL/2 cells. The inhibition of lymphangiogenesis and lymphatic metastasis by HQ_001 is associated with HQ-001 inhibiting the activation of ERK and AKT in tumor cells, thereby downregulating the expression of VEGFD.
实施例 21  Example 21
HQ— 001抑制人淋巴内皮细胞 ( HLBC ) VEGFR3的表达  HQ-001 inhibits the expression of VEGFR3 in human lymphatic endothelial cells (HLBC)
本实验通过 Western blot方法检测了 HLEC细胞中 VEGFR3的表达。 同时 也对激酶 BRK1/2的磷酸 4匕和 PI3K/AKT通路进行了检测。 Western blot 实验 结果（图 20 )显示， 在 HLEC细胞中 ERK1/2的磷酸化和 VEGFR3表达水平受 到了明显抑制。 VEGFR3通路是调控淋巴管新生的主要途径， 肿瘤诱导的淋巴 管生成作用就是通过分泌 VEGFD来激活 VEGFR3通路起作用的。 故 HQ-001可 通过抑制 BRK1/2磷酸化、 VEGFR3表达， 从而下调细胞分裂、 迁移、 成管， 进而达到抑制淋巴管生成的作用。  In this experiment, the expression of VEGFR3 in HLEC cells was detected by Western blot. The phospho-4-is and PI3K/AKT pathways of the kinase BRK1/2 were also examined. Western blot results (Fig. 20) showed that ERK1/2 phosphorylation and VEGFR3 expression levels were significantly inhibited in HLEC cells. The VEGFR3 pathway is the main pathway regulating lymphangiogenesis. Tumor-induced lymphangiogenesis plays a role in the activation of the VEGFR3 pathway by secreting VEGFD. Therefore, HQ-001 can inhibit lymphocyte generation by inhibiting BRK1/2 phosphorylation and VEGFR3 expression, thereby down-regulating cell division, migration, and tube formation.
实施例 22  Example 22
HQ_001抑制斑马鱼节间血管的生成  HQ_001 inhibits angiogenesis in zebrafish
肿瘤的生长、 侵袭和转移的过程都依赖新生血管的生成， 而斑马鱼模型 由于能清晰地观察到血管的发生发展， 已成为一种新型体内模式生物学， 非 常适合体内血管生物学的研究。 本实验采取先收集斑马鱼鱼卵， 挑除死卵和 不健康的鱼卵， 置 28.5°C鱼水中培养。 用鱼水稀释 HQ_001到所需浓度， 6 小时后，将鱼卵分配到六孔板中，加入 HQ_001溶液处理。孵育 24h后，用 0.05% 水合氯酸麻醉， 荧光显微镜下剥卵并观察血管新生情况。  Tumor growth, invasion and metastasis depend on the formation of new blood vessels. The zebrafish model has become a new type of in vivo model biology because it can clearly observe the development of blood vessels, which is very suitable for the study of vascular biology in vivo. In this experiment, the zebrafish eggs were collected first, and the dead eggs and unhealthy fish eggs were picked and cultured in 28.5 °C fish water. Dilute HQ_001 to the desired concentration with fish water. After 6 hours, the eggs were distributed into a six-well plate and treated with HQ_001 solution. After incubation for 24 hours, anesthetize with 0.05% hydrated chloric acid, peel the eggs under a fluorescent microscope and observe angiogenesis.
结果见图 21, 在 Control组中， 节间管非常完整， 成漂亮的梯形排布， 0.1 μΜ HQ_001处理时， 药效作用并不明显， 但当 HQ_001作用浓度提高后，在 0.2 μΜ、 0.4 μΜ时， 斑马鱼节间管的萌芽被抑制， 形成并不完整。 相同浓度 ( 0.4 μΜ)的 HQ_001与薰草菌素 Α处理后， HQ_001体现了更强地抑制斑马鱼节 间管形成的作用。 这说明 HQ_001能很强抑制新血管的形成。  The results are shown in Fig. 21. In the Control group, the internode tube is very complete and is in a beautiful trapezoidal arrangement. When 0.1 μΜ HQ_001 is treated, the pharmacodynamic effect is not obvious, but when the concentration of HQ_001 is increased, it is 0.2 μΜ, 0.4 μΜ. At the time, the germination of the zebrafish internode tube was inhibited and the formation was incomplete. HQ_001 treated with the same concentration (0.4 μΜ) of HQ_001 and sphingosine 体现 showed a stronger inhibition of zebrafish internode formation. This shows that HQ_001 can strongly inhibit the formation of new blood vessels.
综上所述， 通过体外细胞水平实验可以发现， 本发明所述薰草菌素 A衍 生物具有显著性抑制人淋巴内皮细胞（HLEC )和稳定高表达 VEGFD的 LL/2 Lewis肺癌细胞株 ( VEGF-D-LL/2 )增殖的作用， 并诱导 HLBC细胞凋亡， 抑 制 HLEC细胞成管能力，作用机制研究发现该薰草菌素 A衍生物具有更强地抑 制肿瘤细胞 VEGFD的表达以及 AKT和 ERK磚酸化水平， 同时更强地抑制 HLEC 细胞 VEGFR3的表达；斑马鱼模型也显示了该薰草菌素 A衍生物具有更强地抑 制血管生成的作用。 上述参照具体实施方式对该薰草菌素 A衍生物及其制备方法与应用进行 的详细描述， 是说明性的而不是限定性的， 可按照所限定范围列举出若干个 实施例， 因此在不脱离本发明总体构思下的变化和修改， 应属本发明的保护 范围之内。 In summary, it can be found by in vitro cell level experiments that the sphingosine A derivative of the present invention has a significant inhibition of human lymphatic endothelial cells (HLEC) and a stable and highly expressed VEGFD-expressing LL/2 Lewis lung cancer cell line (VEGF). -D-LL/2) Proliferation, induces apoptosis of HLBC cells, inhibits the ability of HLEC cells to form tubes, and studies the mechanism to find that the scavenger A derivative has stronger inhibition of VEGFD expression in tumor cells and AKT and ERK brick acidification level, while more strongly inhibiting the expression of VEGFR3 in HLEC cells; the zebrafish model also showed that the Kaempferol A derivative has a stronger inhibitory effect on angiogenesis. The above detailed description of the Kaempferol A derivative, its preparation method and application, with reference to the specific embodiments, are illustrative and not limiting, and several embodiments may be enumerated according to the scope of the limitation, and therefore Variations and modifications that are within the spirit of the invention are intended to be within the scope of the invention.

Claims

权利 要 求 书 claims
1、 薰草菌素 A衍生物， 其特征在于： 为通式 I化合物、 通式 I化合物药 学上可接受的盐、 通式 I化合物加入助溶剂的溶液或化合物前体， 通式 I的 化学结构如下： 1. A derivative of lavavidin A, which is characterized in that: it is a compound of general formula I, a pharmaceutically acceptable salt of a compound of general formula I, a solution of a compound of general formula I adding a cosolvent, or a compound precursor, the chemical formula of general formula I The structure is as follows:
Figure imgf000030_0001
Figure imgf000030_0001
其中， 式 I中 R为烷基、 酸基、 羰基、 对甲苯基、 五元杂环或五元取代 杂环、 六元杂环或六元取代杂环。 Among them, R in formula I is an alkyl group, an acid group, a carbonyl group, a p-tolyl group, a five-membered heterocycle or a five-membered substituted heterocycle, a six-membered heterocycle or a six-membered substituted heterocycle.
2、 根据权利要求 1所述的薰草菌素 A衍生物， 其特征在于： 式 I中 R 为烷基、 醚基、 羰基、 对曱苯基、 五元杂环或五元取代杂环、 六元杂环或六 元取代杂环。 2. The lavonidin A derivative according to claim 1, characterized in that: in formula I, R is an alkyl group, an ether group, a carbonyl group, a p-tolyl group, a five-membered heterocyclic ring or a five-membered substituted heterocyclic ring, Six-membered heterocycle or six-membered substituted heterocycle.
3、 根据权利要求 1所述的薰草菌素 A衍生物， 其特征在于： 式 I中 R 为环己基氧基、 4_氨基嘧 、 2 -氧代 -3-吡啶甲基、 3-吡啶曱基、 5-嘧啶基， 5-噻唑基、 2-曱基- 3-羟基- 4-吡啶基、 3-吡啶基、 3- [ α-氨基甲酰基- 2-氧代） 吡咯基、 2- (5-曱基呋喃)基、 4 -曱基苯基、 2-呋喃基、 2 -曱基 -5-吡^^和 3-羟基- 4-吡啶基。 3. The lavonidin A derivative according to claim 1, characterized in that: in formula I, R is cyclohexyloxy, 4-aminopyrimidine, 2-oxo-3-pyridylmethyl, 3-pyridine Methyl, 5-pyrimidinyl, 5-thiazolyl, 2-methyl-3-hydroxy-4-pyridyl, 3-pyridyl, 3-[α-carbamoyl-2-oxo)pyrrolyl, 2 - (5-methylfuran)yl, 4-methylphenyl, 2-furyl, 2-methyl-5-pyridyl and 3-hydroxy-4-pyridyl.
4、权利要求 1-3之一所述的薰草菌素 Α衍生物的制备方法，其特征在于： 具体步骤为：
Figure imgf000030_0002
 4. The preparation method of lavonidin A derivatives according to one of claims 1 to 3, characterized in that: the specific steps are:
Figure imgf000030_0002
( A1 )每 5克 2-羟基 -5-硝基苯甲酸（ ) , 40毫升丙酮， 1毫升 三氟乙酸， 回流 2小时， TLC检测反应完全， 减压除去丙酮， 柱层析， 制得
Figure imgf000031_0001
ο ο (A1) For every 5 grams of 2-hydroxy-5-nitrobenzoic acid (), 40 ml of acetone, 1 ml of trifluoroacetic acid, reflux for 2 hours, TLC detects that the reaction is complete, remove the acetone under reduced pressure, and perform column chromatography to prepare
Figure imgf000031_0001
ο ο
、、 ,,
( Α2 )每 2克 ¾， 50毫升乙醇， 500毫克 10%的 Pd/C, latra氢化 (Α2) per 2 g ¾, 50 ml ethanol, 500 mg 10% Pd/C, latra hydrogenation
室温搅拌 2小时， TLC检测反应完全， 原料消失， 过滤， 浓缩制得
Figure imgf000031_0002
Stir at room temperature for 2 hours. TLC detects that the reaction is complete and the raw materials disappear. Filter and concentrate to obtain
Figure imgf000031_0002
( A3 )每 2克
Figure imgf000031_0003
， 0.8克二环己基碳二亚胺， 1.1克 1-羟基苯并三 氮唑， 50亳升二氯甲烷， 室温搅拌 12小时， TLC检测反应完全， 减压除去溶 (A3) per 2 grams
Figure imgf000031_0003
, 0.8 g dicyclohexylcarbodiimide, 1.1 g 1-hydroxybenzotriazole, 50 ml methylene chloride, stir at room temperature for 12 hours, TLC detects that the reaction is complete, remove the solvent under reduced pressure
剂， 柱层析， 制得 agent, column chromatography, prepared
4 )每 1克
Figure imgf000031_0004
1.2克 NaBH4- A1C13, ：30毫升无水四氢呋 喃， 回流搅拌 2小时， TLC检测反应完全， 减压除去四氢呋喃， 残余物水洗， 二氯甲烷萃取， 浓缩， 4) per 1 gram
Figure imgf000031_0004
1. 2 grams of NaBH 4 - A1C1 3 , : 30 ml of anhydrous tetrahydrofuran, stir under reflux for 2 hours, TLC detects that the reaction is complete, remove tetrahydrofuran under reduced pressure, and wash the residue with water. Dichloromethane extraction, concentration,
5)每 500毫克
Figure imgf000032_0001
, 470毫克 3, 5 二羟基苯甲醛， 380毫 克氰基硼氢化钠， 30毫升甲醇， 回流 2小时， TLC检测反应完全， 加水淬灭 5) per 500 mg
Figure imgf000032_0001
, 470 mg 3,5 dihydroxybenzaldehyde, 380 mg sodium cyanoborohydride, 30 ml methanol, reflux for 2 hours, TLC detects that the reaction is complete, add water to quench
反应， 减压除去甲醇， 二 Reaction, remove methanol under reduced pressure, 2
fi fi
(Α6)每 200毫克
Figure imgf000032_0002
, 含 100毫克氢氧化钠的 30毫升甲 醇溶液， 回流搅拌 2小时， TLC检测原料小时，反应完全， 2N盐酸调 PH为 5, 加压除去甲醇， 加入水， 二氯曱烷萃取， 浓缩得到 5- [[(2， 5-二羟基苯基）曱 (Α6) per 200 mg
Figure imgf000032_0002
, 30 ml of methanol solution containing 100 mg of sodium hydroxide, reflux and stir for 2 hours, TLC detects the raw material hours, the reaction is complete, adjust the pH to 5 with 2N hydrochloric acid, remove the methanol under pressure, add water, extract with dichloromethane, and concentrate to obtain 5 - [[(2, 5-dihydroxyphenyl)meth
基] [(环己基氧）曱基]氛基 ] -2-羟基苯曱酸， 即
Figure imgf000032_0003
( Bl )每 2克 5-氨基水杨酸（
Figure imgf000033_0001
), 1. 8克 2， 5 -二羟基苯甲醛，base][(cyclohexyloxy)methyl]cyano]-2-hydroxybenzoic acid, i.e.
Figure imgf000032_0003
(Bl) per 2 grams of 5-aminosalicylic acid (
Figure imgf000033_0001
), 1.8 grams of 2,5-dihydroxybenzaldehyde,
2. 1克！^硼氢化钠， 80毫升甲醇， 回流 2小时， TLC检测反应完全， 加入2. 1 gram! ^Sodium borohydride, 80 ml of methanol, reflux for 2 hours, TLC detects that the reaction is complete, add
20毫升饱和氯化铵溶液， 减压除去曱醇， 残余物水洗， 二氯曱烷萃取， 浓缩, 20 ml of saturated ammonium chloride solution, remove methanol under reduced pressure, wash the residue with water, extract with dichloromethane, and concentrate.
柱层析， column chromatography,
( B2
Figure imgf000033_0002
)每 1克 , 1. 6克 4- Boc氨基- 5-嘧啶甲醛， 1. 2克氰基 硼氢化钠， 50毫升曱醇， 回流 2小时加入 50毫升饱和氯化铵溶液， 减压除 (B2
Figure imgf000033_0002
) per 1 gram, 1.6 grams of 4-Boc amino-5-pyrimidine carboxaldehyde, 1.2 grams of sodium cyanoborohydride, 50 ml of methanol, reflux for 2 hours, add 50 ml of saturated ammonium chloride solution, and remove under reduced pressure.
去曱醇， 二氯甲烷萃 Remove methanol, extract with methylene chloride
( B3 )每 500毫克
Figure imgf000033_0003
, 30毫升二氯甲烷， 1毫升三氟乙酸, 室温搅拌 10小时， TLC检测反应完全， 減压除去二氯甲烷， 饱和碳酸氢钠调 PH为 7 , 柱层析， 制得 5_ [ [ (2, 5-二羟基苯基）曱基] [5- (4_ 嘧啶）曱基 (B3) per 500 mg
Figure imgf000033_0003
, 30 ml of methylene chloride, 1 ml of trifluoroacetic acid, stir at room temperature for 10 hours, TLC detects that the reaction is complete, remove the methylene chloride under reduced pressure, and adjust with saturated sodium bicarbonate. The pH is 7. Column chromatography produces 5_[[(2,5-dihydroxyphenyl)methyl][5-( 4_pyrimidine )methyl]
氨基] -2-羟基苯 或 Amino]-2-hydroxybenzene or
( C )每 1克
Figure imgf000034_0001
, 2克 3-吡啶基 _2-氧代-丙醛， 600毫克氰基硼 氢化钠， 50毫升曱醇， 回流 2小时， 加入 50毫升饱和氯化铵溶液， 减压除 去曱醇， 萃取， 浓缩， 柱层析， 制得 5- [ (2, 5 -二羟基苯甲基） （2 -氧代 _3 -吡 (C) per 1 gram
Figure imgf000034_0001
, 2 grams of 3-pyridyl-2-oxo-propionaldehyde, 600 mg of sodium cyanoborohydride, 50 ml of methanol, reflux for 2 hours, add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, and extract. Concentrate and column chromatography to prepare 5-[(2, 5-dihydroxybenzyl) (2-oxo_3-pyridine
啶乙基） ] 氨基 - 2_羟基苯甲酸， 即
Figure imgf000034_0002
ethyl] amino- 2 -hydroxybenzoic acid, i.e.
Figure imgf000034_0002
( D )每 1克
Figure imgf000034_0003
, 600毫克 3 -吡啶乙醛， 480毫克氰基硼氢化钠,(D) per 1 gram
Figure imgf000034_0003
, 600 mg 3-pyridineacetaldehyde, 480 mg sodium cyanoborohydride,
40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [ (2, 5-二羟基苯甲基 ) - (3-吡啶 乙基)] 氨基- 2-羟基苯曱酸， 即
Figure imgf000035_0001
40 ml of methanol was refluxed for 2 hours. TLC monitored the reaction to be complete. Add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, extract with dichloromethane, and concentrate to obtain 5-[(2, 5-dihydroxybenzyl)- (3-pyridine Ethyl)] amino-2-hydroxybenzoic acid, i.e.
Figure imgf000035_0001
( E )每 1
Figure imgf000035_0002
, 600毫克 5 -嘧啶甲醛， 480毫克氰基硼氢化钠, 40毫升甲醇，回流 I小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯曱烷萃取， 浓缩制得 5- [(2， 5-二羟基苯曱基 )-(5-嘧啶 (E) per 1
Figure imgf000035_0002
, 600 mg of 5-pyrimidinecarbaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, reflux for 1 hour, TLC monitors that the reaction is complete, add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, extract with dichloromethane, and concentrate. Preparation of 5-[(2, 5-dihydroxyphenylmethyl)-(5-pyrimidine
甲基）] 羟基苯曱酸， 即
Figure imgf000035_0003
Methyl)] hydroxybenzoic acid, i.e.
Figure imgf000035_0003
Figure imgf000035_0004
， 600毫克 5 塞唑甲醛， 480毫克氰基硼氢化钠, 40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 減压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [(2, 5-二羟基苯曱基 )-(5-噻唑 曱基）] 氨基 -2-羟基苯曱酸，
Figure imgf000036_0001
或
Figure imgf000035_0004
, 600 mg of 5-thazole formaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, reflux for 2 hours, TLC monitors that the reaction is complete, add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, extract with dichloromethane, and concentrate. Obtain 5-[(2, 5-dihydroxyphenylmethyl)-(5-thiazole) Methyl)] amino-2-hydroxybenzoic acid,
Figure imgf000036_0001
or
( G )每 1
Figure imgf000036_0002
, 600毫克 2_甲基- 3-羟基- 4 -吡啶曱醛， 480 毫克氰基硼氢化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50 毫升饱和氯化铵溶液， 减压除去甲醇， 二氯曱烷萃取， 浓缩制得 5- [ (2, 5-二 羟基苯甲基) - (2-甲基- 3-羟基- 4-吡啶曱基)氨基] -2-羟基苯甲酸， 即 (G) per 1
Figure imgf000036_0002
, 600 mg of 2_methyl-3-hydroxy-4-pyridine formaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, reflux for 2 hours, TLC monitors that the reaction is complete, add 50 ml of saturated ammonium chloride solution, and reduce the pressure Remove methanol, extract with dichloromethane, and concentrate to obtain 5-[(2, 5-dihydroxybenzyl)-(2-methyl-3-hydroxy-4-pyridylmethyl)amino]-2-hydroxybenzene Formic acid, i.e.
Figure imgf000036_0003
, 600毫克 3 -吡啶曱醛， 480毫克氰基硼氢化钠,
Figure imgf000036_0003
, 600 mg of 3-pyridine formaldehyde, 480 mg of sodium cyanoborohydride,
40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [ (2, 5-二羟基苯甲基 ) - (3-吡啶 曱基） 氨基] -2-羟基苯曱酸， 即
Figure imgf000037_0001
40 ml of methanol was refluxed for 2 hours. TLC monitored the reaction to be complete. Add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, extract with dichloromethane, and concentrate to obtain 5-[(2, 5-dihydroxybenzyl)- (3-pyridine Methyl) amino]-2-hydroxybenzoic acid, i.e.
Figure imgf000037_0001
( I )每 1克
Figure imgf000037_0002
， 600毫克 1-氨基曱酰基- 2-氧代- 3 -吡咯甲醛， 480毫克氰基硼氢化钠， 40毫升曱醇， 回流 2小时， TLC监测反应完全， 加 入 50毫升饱和氯化铵溶液，减压除去甲醇，二氯甲烷萃取，浓缩制得 5- [ (2, 5- 二羟基苯甲基) - [ 3- [ a- 甲酰基 -2-氧代)吡咯甲基）] 氨基] -2-羟基苯甲 (I) per 1 gram
Figure imgf000037_0002
, 600 mg of 1-aminoformyl-2-oxo-3-pyrrolecarbaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, reflux for 2 hours, TLC monitors that the reaction is complete, add 50 ml of saturated ammonium chloride solution, Methanol was removed under reduced pressure, extracted with dichloromethane, and concentrated to obtain 5-[(2, 5-dihydroxybenzyl)-[3-[a-formyl-2-oxo)pyrrolemethyl)]amino]- 2-Hydroxybenzyl
酸， 或 acid, or
Figure imgf000037_0003
( J )每 1克 , 600毫克 5-甲基呋喃甲醛， 480毫克氰基硼氢 化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和氯化 铵溶液， 减压除去甲醇， 二氯曱烷萃取， 浓缩制得 5- [ (2, 5-二羟基苯曱 (5-曱基呋喃）甲基]] 氨基 -2-羟基苯甲酸
Figure imgf000038_0001
Figure imgf000037_0003
(J) For every 1 gram, 600 mg of 5-methylfurancarbaldehyde, 480 mg of sodium cyanoborohydride, and 40 ml of methanol were refluxed for 2 hours. TLC monitored the reaction to be complete. Add 50 ml of saturated ammonium chloride solution and remove the methanol under reduced pressure. , extracted with dichloromethane and concentrated to obtain 5-[(2, 5-dihydroxybenzene (5-Methylfuran)methyl]] amino-2-hydroxybenzoic acid
Figure imgf000038_0001
U)每 1克
Figure imgf000038_0002
, 600毫克对曱基苯甲醛， 480毫克氰基硼氢化 钠， 40毫升曱醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和氯化铵 溶液，减压除去曱醇，二氯甲烷萃取， 浓 - [(2, 5-二羟基苯甲基 )-(4- U) per 1 gram
Figure imgf000038_0002
, 600 mg p-methylbenzaldehyde, 480 mg sodium cyanoborohydride, 40 ml methanol, reflux for 2 hours, TLC monitors the reaction is complete, add 50 ml saturated ammonium chloride solution, remove the methanol under reduced pressure, and extract with dichloromethane , concentrated-[(2, 5-dihydroxybenzyl)-(4-
曱基苯甲基） 氨基] -2-羟基苯曱酸， 即
Figure imgf000038_0003
或 Methylbenzyl) amino]-2-hydroxybenzoic acid, i.e.
Figure imgf000038_0003
or
( L )每 1克
Figure imgf000038_0004
, 600毫克 2 -呋喃曱醛， 480毫克氰基硼氢化钠,(L) per 1 gram
Figure imgf000038_0004
, 600 mg of 2-furandehyde, 480 mg of sodium cyanoborohydride,
40毫升甲醇，回流 2小时， TLC监测反应完全，加入 50毫升饱和氯化铵溶液, 减压除去甲醇， 二氯甲烷萃取， 浓缩制得 5- [(2, 5-二羟基苯甲基 )-(2-呋喃 曱基） tt] -2-羟基苯曱酸， 即
Figure imgf000039_0001
40 ml of methanol was refluxed for 2 hours. TLC monitored that the reaction was complete. Add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, extract with dichloromethane, and concentrate to obtain 5-[(2, 5-dihydroxybenzyl)- (2-furan Methyl) tt] -2-Hydroxybenzoic acid, i.e.
Figure imgf000039_0001
(M)每 1
Figure imgf000039_0002
, 600毫克 2_甲基- 5-吡啶甲醛， 480毫克氰基 硼氢化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和 氯化铵溶液， 减压除去曱醇， 二氯甲烷萃取， 浓缩制得 5- [(2， 5-二羟基苯曱 (M)Every 1
Figure imgf000039_0002
, 600 mg of 2-methyl-5-pyridinecarboxaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, reflux for 2 hours, TLC monitors that the reaction is complete, add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, 2 Extracted with methyl chloride and concentrated to obtain 5-[(2, 5-dihydroxyphenyl
基） -(2-曱基- 5-吡啶甲基） 氨基] -2-羟基苯甲酸， 即
Figure imgf000039_0003
base) -(2-methyl-5-pyridylmethyl) amino]-2-hydroxybenzoic acid, i.e.
Figure imgf000039_0003
或 or
(N)每 1克
Figure imgf000039_0004
, 600毫克 2_甲基- 5-吡啶甲醛， 480毫克氰基 硼氢化钠， 40毫升甲醇， 回流 2小时， TLC监测反应完全， 加入 50毫升饱和 氯化铵溶液， 减压除去曱醇， 二氯甲烷萃取， 浓缩制得 5- [(2， 5-二羟基苯甲 基) -（3-羟基- 4 -吡啶曱基） 氨基 ] _2-羟基苯甲酸， 即
Figure imgf000040_0001
(N) per 1 gram
Figure imgf000039_0004
, 600 mg of 2-methyl-5-pyridinecarboxaldehyde, 480 mg of sodium cyanoborohydride, 40 ml of methanol, reflux for 2 hours, TLC monitors that the reaction is complete, add 50 ml of saturated ammonium chloride solution, remove the methanol under reduced pressure, 2 Extract with methyl chloride and concentrate to obtain 5-[(2, 5-dihydroxybenzyl base)-(3-hydroxy-4-pyridylmethyl)amino]-2-hydroxybenzoic acid, i.e.
Figure imgf000040_0001
5、权利要求 1-3之一所述的薰草菌素 A衍生物在制备用于治疗肿瘤淋巴 转移的药物方面的应用。 5. Application of the lavonidin A derivative according to any one of claims 1 to 3 in the preparation of drugs for treating lymphatic metastasis of tumors.
6、根据权利要求 5所述的薰草菌素 A衍生物的应用， 其特征在于： 所述 肿瘤为淋巴瘤、 肺癌、 肝癌、 乳腺癌、 卵巢癌、 胃癌、 大肠癌、 结肠癌、 黑 色素瘤或头颈部癌。 6. The application of lavandin A derivatives according to claim 5, characterized in that: the tumor is lymphoma, lung cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, large intestine cancer, colon cancer, melanoma or head and neck cancer.
7、根据权利要求 6所述的薰草菌素 A衍生物的应用， 其特征在于： 所述 肿瘤为淋巴瘤或肺癌。 7. The application of the lavonidin A derivative according to claim 6, characterized in that: the tumor is lymphoma or lung cancer.
8、 根据权利要求 6或 7所述的薰草菌素 A衍生物的应用， 其特征在于： 所述肿瘤为淋巴瘤。 8. Application of the lavonidin A derivative according to claim 6 or 7, characterized in that: the tumor is lymphoma.
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