Xanthone compound as well as preparation method and application thereof

Abstract

The invention relates to the field of natural medicines, and discloses a xanthone compound which is derived from leaves and branches of cratoxylum cochinchinensis and has a novel chemical structure, a preparation method of the xanthone compound and application of the xanthone compound in antitumor medicines, and various in-vitro antitumor activity evaluation results of the compound cratocochinone show that the compound has remarkable antitumor activity and can be used for preparing antitumor drugs. The compound has the protein tyrosine kinase inhibition activity equivalent to that of a positive control drug, can be further developed into an anti-tumor drug taking protein tyrosine kinase as a target, has a wide application prospect, is simple in separation and purification process, and has practical significance.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and specifically relates to a preparation method of a xanthone compound with a novel chemical structure derived from the branches and leaves of the Hypericaceae family Acanthus strychnifolia and its application in anti-tumor drugs. Background Art

[0002] Malignant tumors have become the leading cause of death in humans, and the incidence and mortality of malignant tumors are on a continuous upward trend worldwide. With the continuous elucidation of the mechanism of tumorigenesis and the continuous discovery of anti-tumor targets, targeted inhibition of tumor signal transduction has become an important direction for the development of new anti-tumor drugs. Among various molecular targets, protein tyrosine kinase (PTK) is one of the most studied and effective anti-tumor drug targets. It has become the research focus and hotspot of anti-tumor targeted therapeutic drugs and has broad application prospects. Currently, the small molecule targeted anti-tumor drugs used in clinical practice are mainly tyrosine kinase inhibitors, including single-target tyrosine kinase inhibitors and multi-target tyrosine kinase inhibitors. Tyrosine kinase controls cell proliferation, survival, apoptosis, angiogenesis, invasion and metastasis through a complex intracellular network pathway. Tumor tissues may initially respond to single-target tyrosine kinase inhibitors, but can acquire antagonistic ability through multiple mechanisms. The existence of these escape mechanisms in tumors is the basis for the necessity of multi-target therapy. Clinical practice also shows that single-target tyrosine kinase inhibitor anti-tumor drugs, such as erlotinib and gefitinib, are highly selective and have few toxic side effects, but they are prone to drug resistance during use and cannot completely kill tumor cells. Combination therapy will cause serious adverse reactions and affect their respective pharmacokinetic properties. Compared with single-target drugs and combination therapy of multiple single-target drugs, multi-target drugs have more advantages. Multi-target drugs can effectively avoid drug interactions, reduce adverse reactions, and have more comprehensive therapeutic effects. Many multi-target tyrosine kinase inhibitor anti-tumor drugs have gradually become the first-line drugs for the treatment of certain tumors due to their clinical characteristics of high efficacy and low toxicity, such as imatinib and other targeted anti-tumor drugs.

[0003] There are about 6 species of Cratoxylum in the Clusiaceae family. Modern phytochemical studies have shown that Cratoxylum plants are rich in compounds of various structural types, such as xanthones, anthraquinones, flavonoids, triterpenoids and steroids. Modern pharmacological studies have shown that the chemical components isolated from Cratoxylum plants have various biological activities, such as anti-tumor, anti-inflammatory, antibacterial, antioxidant, anti-malarial, anti-HIV, α-glucosidase inhibition, vascular protection, retinol X receptor-α transcription inhibition and protein tyrosine phosphatase 1B inhibition [Bennett, GJ; Harrison, LJ; Sia, GL; Sim, KY Triterpenoids, tocotrienols and xanthones from the bark of Cratoxylum cochinchinense. Phytochemistry 1993, 32, 1245-1251. Boonnak, N., Karalai, C., Chantrapromma, S., Ponglimanont, C., Fun, HK, Kanjana-Opas, A., Chantrapromma, K., Kato, S. Anti-pseudomonas aeruginosa xanthones from the resin and green fruits of Cratoxylum cochinchinense. 2015,17,519-531.;Kaewpiboon,C.;Boonnak,N.;Yawut,N.;Kaowinn,S.;Chung,YHCaged-xanthone from Cratoxylum formosumssp.prunifloruminhibits malignant cancer phenotypes in multidrug-resistanthuman A549 lung cancer cells through down-regulation of NF-κB.Bioorganic&Medicinal Chemistry 2019,27,2368-2375.;Kukongviriyapan,U.;Luangaram,S.;Leekhaosoong,K.;Kukongviriyapan,V.;Preeprame,S.Antioxidant and vascularprotective activities of Cratoxylum formosum,Syzygium gratum and Limnophilaaromatica.Biological&Pharmaceutical Bulletin 2007,30,661-666.;Boonnak,N.;Karalai,C.;Chantrapromma,S.;Ponglimanont,C.;Fun,H.K.;Kanjana-Opas,A.;Laphookhieo,S.Bioactive prenylated xanthones and anthraquinones fromCratoxylum formosum ssp.pruniflorum.Tetrahedron 2006,62,8850-8859.;Laphookhieo,S.;Maneerat,W.;Koysomboon,S.Antimalarial and cytotoxic phenoliccompounds from Cratoxylummaingayi and Cratoxylum cochinchinense.Molecules2009,14,1389-1395.;Li,Z.P.;Lee,H.H.;Uddin,Z.;Song,Y.H.;Park,K.H.Cagedxanthones displaying protein tyrosine phosphatase 1B(PTP1B)inhibition fromCratoxylum cochinchinense.Bioorganic Chemistry 2018,78,39-45.;Mahabusarakam,W.;Rattanaburi,S.;Phongpaichit,S.;Kanjana-Opas,A.Antibacterial and cytotoxicxanthones from Cratoxylum cochinchinense.Phytochemistry Letters 2008,1,211-214.;Ren,Y.L;Matthew,S.;Lantvit,D.D.;Ninh,T.N.;Chai,H.;Fuchs,J.R.;Soejarto,D.D.;Carcache de Blanco,E.J.;Swanson,S.M.;Kinghorn,A.D.Cytotoxic and NF-κBinhibitory constituents of the stems of Cratoxylum cochinchinense and theirsemisynthetic analogs.Journal ofNatural Products 2011,74,1117-1125.;Reutrakul,V.;Chanakul,W.;Pohmakotr,M.;Jaipetch,T.;Yoosook,C.;Kasisit,J.;Napaswat,C.;Santisuk,T.;Prabpai,S.;Kongsaeree,P.;Tuchinda,P.Anti-HIV-1constituents from leaves and twigs of Cratoxylum arborescens.Planta Medica2006,72,1433-1435.;Tantapakul,C.;Maneerat,W.;Sripisut,T.;Ritthiwigrom,T.;Andersen,Raymond J.;Cheng,P.;Cheenpracha,S.;Raksat,A.;Laphookhieo,S.Newbenzophenones and xanthones from Cratoxylum sumatranum ssp.neriifolium andtheir antibacterial and antioxidant activities.Journal of Agricultural andFood Chemistry 2016,64,8755-8762.Seo, EK; Kim, NC; Wani, MC; Wall, ME; Navarro, HA; Burgess, JP; Kawanishi, K.; Kardono, LBS; Riswan, S.; Rose, WC; Fairchild, CR; Farnsworth, NR; ofNatural Products 2002,65,299-305.]. .

[0004] C. cochinchinense is a plant of the genus C. cochinchinense of the family Lutaceae, distributed in Hainan, Guangdong, Guangxi and Yunnan provinces in my country. C. cochinchinense is a traditional folk medicinal plant, with roots, bark and young leaves used as medicine, which has the effects of clearing away heat and detoxifying, removing dampness and stagnation, and removing blood stasis and swelling. It is mainly used to treat colds, heatstroke and fever, diarrhea, jaundice, traumatic injuries, carbuncles, sores and boils; the young leaves are used as a refreshing drink to relieve summer heat and thirst. At present, there is no research on its application in anti-tumor. Summary of the invention

[0005] The purpose of the present invention is to provide a xanthone compound cratocochinone with a novel chemical structure isolated from the branches and leaves of the plant Cratocochinus of the Hypericaceae family. The compound has significant anti-tumor activity and protein tyrosine kinase inhibitory activity equivalent to that of a positive control drug, and can be further developed into an anti-tumor drug targeting protein tyrosine kinase.

[0006] In order to achieve the above object, the technical solution of the present invention is to provide a xanthone compound, the chemical name of which is cratocochinone, and the chemical structure of which is as follows:

[0007]

[0008] Another object of the present invention is to provide a method for preparing a xanthone compound, cratocochinone, comprising the following steps:

[0009] A. Cold-soak the branches and leaves of the yellow cattle tree with 75% ethanol or methanol solution for four times, filter, collect the filtrate, and then reduce the pressure and concentrate until there is no alcohol taste to obtain an alcohol extract;

[0010] B. Add distilled water to the alcohol extract to prepare a suspension, extract with petroleum ether and ethyl acetate in sequence, and concentrate the ethyl acetate extract under reduced pressure to obtain an ethyl acetate extract;

[0011] C. The ethyl acetate extract is separated and purified by column chromatography to obtain a monomer compound, cratocochinone.

[0012] Furthermore, the above step C specifically includes:

[0013] (1) The ethyl acetate extract was fractionated by silica gel column chromatography, and chloroform-acetone gradient elution was performed at volume ratios of 90:10, 80:20, 70:30 and 50:40, respectively, and the chloroform-acetone eluate at a volume ratio of 70:30 was collected;

[0014] (2) using an MCI resin column to remove the pigment from the chloroform-acetone eluate at a volume ratio of 70:30, and then using a methanol-water gradient elution at a volume ratio of 40:60, 50:50, and 65:35, and collecting the methanol-water eluate at a volume ratio of 50:50;

[0015] (3) subjecting the eluate with a volume ratio of 50:50 of methanol to water to reverse phase silica gel column chromatography, eluting with a gradient of methanol to water with a volume ratio of 45:55, 50:50 and 55:45, respectively, collecting the eluate with a volume ratio of 50:50 of methanol to water for concentration;

[0016] (4) The concentrated methanol-water eluate was separated by preparative high performance liquid chromatography using methanol-water as the mobile phase in a volume ratio of 67:33 to obtain the monomer compound cratocochinone.

[0017] Another object of the present invention is to provide the use of cratocochinone, a xanthone compound, in the preparation of anti-tumor drugs, especially to provide the use of cratocochinone, a xanthone compound, in the preparation of targeted anti-tumor drugs targeting protein tyrosine kinase.

[0018] Furthermore, the tumor cell lines include six tumor cell lines: A375 (human malignant melanoma cells), JeKo-1 (human mantle cell lymphoma cells), MCF-7 (human breast cancer cells), A549 (human lung cancer cells), SW480 (human colon cancer cells) and SMMC-7721 (human liver cancer cells).

[0019] Compared with the prior art, the present invention has the following effects:

[0020] The present invention is the first to separate and identify a xanthone compound cratocochinone with a novel chemical structure from the petroleum ether extract of the alcohol extract of the branches and leaves of the yellow cow wood. The compound has significant anti-tumor activity, especially against A375 (human malignant melanoma cells) and JeKo-1 (human mantle cell lymphoma cells). Its IC 50 They are 1.28 and 2.62 μg / mL respectively. They also have the inhibitory activity of protein tyrosine kinase comparable to that of the positive control drug imatinib. They can be further developed into targeted anti-tumor drugs targeting protein tyrosine kinase and have broad application prospects. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in further detail below in conjunction with the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually based on conventional experimental conditions.

[0022] 1. Preparation method of compound cratocochinone

[0023] Example 1

[0024] The preparation method of this embodiment comprises the following steps:

[0025] A. The powder of dried yellow cattle wood branches and leaves (8.6 kg, Hainan) was cold-extracted with 75% ethanol solution for four times, each time for four days, filtered, the filtrate was collected, and concentrated under reduced pressure until there was no alcohol, to obtain an alcohol extract;

[0026] B. The alcohol extract was added with distilled water to prepare a suspension, and extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate extract was concentrated under reduced pressure to obtain 683.6 g of petroleum ether extract and 458.7 g of ethyl acetate extract;

[0027] C. The ethyl acetate extract is subjected to column chromatography separation and purification, and the steps are as follows:

[0028] (1) The ethyl acetate extract was fractionated by silica gel column chromatography, and gradient elution was performed using chloroform-acetone (volume ratio of 90:10, 80:20, 70:30 and 50:50) as eluent, and the chloroform-acetone (volume ratio of 70:30) eluate was collected;

[0029] (2) The chloroform-acetone (volume ratio 70:30) eluate was subjected to MCI resin column chromatography to remove the pigment, and gradient elution was performed using methanol-water (volume ratio 40:60, 50:50 and 65:35) as the eluent, and the methanol-water (volume ratio 50:50) eluate was collected;

[0030] (3) subjecting the methanol-water (volume ratio of 50:50) eluate to reverse phase silica gel column chromatography, using methanol-water (volume ratios of 45:55, 50:50 and 55:45) as eluent for gradient elution, and collecting the methanol-water (volume ratio of 50:50) eluate for concentration;

[0031] (4) The concentrated methanol-water eluate was separated by preparative HPLC using acetonitrile-water (volume ratio 67:33) as the mobile phase to obtain pure cratocochinone (58.6 mg).

[0032] Structural confirmation: The chemical structure of the compound cratocochinone was determined through comprehensive analysis of a variety of modern spectral techniques such as optical rotation spectroscopy, ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

[0033] Cratocochinone: Pale yellow amorphous powder, (c 0.12,CH3OH); IR(KBr)v max 3362,3029,2928,1662,1610,1567,1479,1380,1262,1151,1070,959,865 and 731cm –1 ;UV(CH3OH)λmax(logε)233(4.58),271(4.62),319(3.98) and 381(3.79)nm; HR-ESI-MS m / z393.1698[M+H] + (Calcd for C 24 H 25 O5 + ,393.1697); 1H-NMR (400MHz, CDCl3) δ: 12.94 (1H, s, 1-OH), 7.78 (1H, d, J = 7.4Hz, H-8), 7.27 (1H, dd, J = 7.4, 7.0Hz ,H-7),7.21(1H,d,J=7.0Hz,H-6),6.94(1H,d,J=10.0Hz,H-1'),6.26(1H,s,H-2),5.56(1H,d,J=10 .0Hz,H-2'),5.10(1H,t,J=7.0Hz,H-6'),4.01(3H,s,5-OCH3),2.09-2.15(1H,m,H-5'),1.81-1.85 (1H,m,H-4'α),1.73-1.67(1H,m,H-4'β),1.65(3H,s,H-9'),1.58(3H,s,H-8'),1.46(3H,s,H-10'); 13 C-NMR (100MHz, CDCl3) δ: 180.8 (C-9), 163.0 (C-1), 161.2 (C-3), 151.6 (C-4a), 148.5 (C -5),146.1(C-10a),132.0(C-7'),125.8(C-2'),123.7(C-6'),123.6(C-7),121.3(C-8a ),116.6(C-8),115.7(C-6),115.6(C-1'),103.6(C-9a),101.1(C-4),99.1(C-2),80.8 (C-3'),56.4(C-9'),41.6(C-4'),27.1(C-10'),25.6(C-9'),22.6(C-5'),17.6(C-8').

[0034] Example 2

[0035] The preparation method of this embodiment comprises the following steps:

[0036] A. The dried powder of the branches and leaves of the yellow cattle tree dried in the shade (90.3 kg, Hainan) was extracted with methanol cold soaking six times, three days each time, filtered, the filtrate was collected, and concentrated under reduced pressure until there was no alcohol taste, to obtain a methanol extract;

[0037] B. Add distilled water to the methanol extract to prepare a suspension, extract with petroleum ether and ethyl acetate in sequence, and concentrate the petroleum ether extract under reduced pressure to obtain 6568.2 g of petroleum ether extract and 4643.6 g of ethyl acetate extract;

[0038] C. separating and purifying the ethyl acetate extract by column chromatography;

[0039] The specific steps are as follows:

[0040] (1) Separating the ethyl acetate extract by silica gel column chromatography, using chloroform-acetone (volume ratio of 90:10, 80:20, 70:30 and 50:50) as eluent for gradient elution, and collecting the chloroform-acetone (volume ratio of 70:30) eluate;

[0041] (2) The chloroform-acetone (volume ratio 70:30) eluate was subjected to MCI resin column chromatography to remove the pigment, and gradient elution was performed using methanol-water (volume ratio 40:60, 50:50 and 65:35) as the eluent, and the methanol-water (volume ratio 50:50) eluate was collected;

[0042] (3) subjecting the methanol-water (volume ratio of 50:50) eluate to reverse phase silica gel column chromatography, using methanol-water (volume ratios of 45:55, 50:50 and 55:45) as eluent for gradient elution, and collecting the methanol-water (volume ratio of 50:50) eluate for concentration;

[0043] (4) The concentrated methanol-water eluate was separated by preparative high performance liquid chromatography using acetonitrile-water (volume ratio 67:33) as the mobile phase to obtain monomer compound II (598.7 mg).

[0044] The structure of compound II was confirmed as pale yellow amorphous powder; HR-ESI-MS showed that [M+H] + m / z 393.1698; Compound II and the compound cratocochinone prepared by the preparation method in Example 1 were analyzed by TLC, and they were all uniform spots under three developing systems [petroleum ether-acetone (70:30), chloroform-acetone (90:10) and chloroform-methanol (95:5)], indicating that the monomer compound II and the compound cratocochinone prepared by the method in Example 1 are the same compound.

[0045] 2. Study on the antitumor activity of the compound cratocochinone

[0046] 1. Experimental methods

[0047] Six common tumor cell lines A375, JeKo-1, MCF-7, A549, SW480 and SMMC-7721 were cultured in RPMI-1640 medium containing 10.0% calf serum at 37.0°C and 5.0% CO2 incubator. The cell proliferation inhibition test was performed using the MTT method. The main operation was as follows: tumor cell lines in the logarithmic growth phase were digested with 0.25% trypsin, and RPMI-1640 culture medium containing 10% newborn calf serum was adjusted to 5.0×10 4Cell suspension of 100 μg / mL was inoculated in a 96-well plate, with 180.0 μL in each well. Cultured at 37.0°C, 5% CO2 saturated humidity for 8.0-10.0 h, and after the cells adhered to the wall, a sample solution prepared with PBS was added to each well, so that the final concentrations of the samples were 0.1, 1.0, and 10.0 μg / mL, respectively. Three wells were paralleled for each concentration, and cultured for 44.0 h. 50.0 μL MTT (1.0 mg / mL -1 , PBS), continue to incubate at 37.0℃, 5.0% CO2 for 4.0h, discard the culture supernatant in the well, add 150.0μL DMSO to each well, shake on a micro-oscillator for 15.0min, after the crystals are dissolved, select 570.0nm on the enzyme-linked immunosorbent assay, measure the absorbance of each well, and set up a blank group (only add culture medium containing cells) and a control group (with culture medium instead of drugs) to calculate the cell proliferation inhibition rate. Inhibition rate (%) = (1-average OD value of 3 wells in the experimental group / average OD value of 3 wells in the control group) × 100%. Use the inhibition rate as the ordinate, draw a regression curve, and calculate the sample IC 50 SPSS13.0 statistical software package was used for data processing and statistical analysis.

[0048] 2. Antitumor activity experimental results (see Table 1)

[0049] The compound cratocochinone obtained in Example 1 of the present invention showed different degrees of proliferation inhibition activity (IC 50 The smaller the value, the better the activity).

[0050] Table 1 Evaluation results of the antitumor activity of the compound cratocochinone

[0051]

[0052] 3. Inhibition of protein tyrosine kinase activity by the compound cratocochinone

[0053] Extraction of PTKs from rat brain tissue: Remove the rat brain, remove the meninges, weigh it, and add 4 times the amount of cold homogenate. Homogenize at high speed with a glass homogenizer in an ice bath, centrifuge, collect the supernatant, and centrifuge again for 10.0 minutes. Collect the supernatant, which contains cytoplasmic tyrosine kinase, and the precipitate can be used as receptor tyrosine kinase. Keep a small amount of supernatant for the determination of protein content in the extract, and divide the rest into aliquots and store at -70.0℃ for later use.

[0054] ELISA plate coating: Add the substrate dilution to a 96-well ELISA plate (125.0 μL per well) and incubate overnight at 37.0°C. Remove excess substrate solution from the plate, wash with phosphate buffered saline (PBS-Tween20), and dry at 37.0°C for 2.0 h. Store at 4.0°C for later use.

[0055] Evaluation of PTK inhibitors: first add the sample to the ELISA plate, incubate at 37.0°C, add ATP diluted with kinase buffer, incubate at 37.0°C, remove the reaction solution from the plate, wash; add the antibody complex, incubate at 37.0°C; remove the antibody complex from the plate, wash, add tetramethylbenzidine (TMB) colorimetric solution, react at room temperature in the dark, add the stop solution, and measure the absorbance (A) value at a wavelength of 450.0nm. The positive control drug is imatinib. The inhibition rate of the compound cratocochinone is calculated according to the following formula: Inhibition rate % = (A 正常 -A 样品 ) / (A 正常 -A 空白 )*100%

[0056] The results showed that the compound cratocochinone had a significant inhibitory effect on protein tyrosine kinase (inhibition rate 83.19%), and the inhibitory activity was equivalent to that of the positive control drug imatinib (inhibition rate 75.89%).

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A xanthone compound, characterized in that: The chemical name is cratocochinone, and its chemical structure is as follows:

2. A method for preparing xanthone compounds according to claim 1, characterized in that: The following steps are involved: A. Cold-soak the branches and leaves of the yellow cattle tree with 75% ethanol or methanol solution for four times, filter, collect the filtrate, and then reduce the pressure and concentrate until there is no alcohol taste to obtain an alcohol extract; B. Add distilled water to the alcohol extract to prepare a suspension, extract with petroleum ether and ethyl acetate in sequence, and concentrate the ethyl acetate extract under reduced pressure to obtain an ethyl acetate extract; C. The ethyl acetate extract is separated and purified by column chromatography to obtain a monomer compound, cratocochinone.

3. The method for preparing xanthone compounds according to claim 2, characterized in that: Step C includes: (1) separating the ethyl acetate extract by silica gel column chromatography, performing gradient elution with chloroform-acetone at volume ratios of 90:10, 80:20, 70:30 and 50:50, respectively, and collecting the chloroform-acetone eluate at a volume ratio of 70:30; (2) using an MCI resin column to remove the pigment from the chloroform-acetone eluate at a volume ratio of 70:30, and then using a methanol-water gradient elution at a volume ratio of 40:60, 50:50, and 65:35, and collecting the methanol-water eluate at a volume ratio of 50:50; (3) subjecting the eluate with a volume ratio of 50:50 of methanol to water to reverse phase silica gel column chromatography, eluting with a gradient of methanol to water with a volume ratio of 45:55, 50:50 and 55:45, respectively, collecting the eluate with a volume ratio of 50:50 of methanol to water for concentration; (4) The concentrated methanol-water eluate was separated by preparative high performance liquid chromatography using acetonitrile-water as the mobile phase in a volume ratio of 67:33 to obtain the monomer compound cratocochinone.

4. Use of the xanthone compound according to claim 1 in the preparation of anti-tumor drugs.

5. Use of the xanthone compound according to claim 4 in the preparation of targeted anti-tumor drugs targeting protein tyrosine kinase.

6. Use of the xanthone compounds according to claim 5 in the preparation of targeted anti-tumor drugs targeting protein tyrosine kinase, wherein the tumor cell lines include six tumor cell lines: A375, JeKo-1, MCF-7, A549, SW480 and SMMC-7721.

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