A xanthone compound and its preparation method and application

Abstract

The present invention relates to the field of natural medicines, and discloses a xanthone compound with a novel chemical structure derived from the branches and leaves of the yellow cattle wood, a preparation method thereof, and an application of the compound in anti-tumor drugs. The compound, cratocochinone, has been evaluated by multiple in vitro anti-tumor activities and has significant anti-tumor activity and an activity of inhibiting protein tyrosine kinase equivalent to that of a positive control drug. The compound can be further developed into an anti-tumor drug targeting protein tyrosine kinase, has broad application prospects, and has a simple separation and purification process, thus having practical significance.

Description

Technical Field

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

[0002] Malignant tumors are currently the leading cause of death in humans, with both morbidity and mortality rates continuing to rise worldwide. With the continuous elucidation of tumorigenesis mechanisms and the discovery of anti-tumor targets, targeted inhibition of tumor signaling has become an important direction for the development of novel anti-tumor drugs. Among various molecular targets, protein tyrosine kinases (PTKs) are one of the most extensively studied and most effective anti-tumor drug targets. They have become a research focus and hotspot for targeted anti-tumor therapies and hold broad application prospects. Currently, small molecule targeted anti-tumor drugs in clinical use primarily belong to the tyrosine kinase inhibitor class, including single-target tyrosine kinase inhibitors and multi-target tyrosine kinase inhibitors. Tyrosine kinases control cell proliferation, survival, apoptosis, angiogenesis, invasion, and metastasis through a complex intracellular network. Tumors may initially respond to single-target tyrosine kinase inhibitors but can acquire resistance through multiple mechanisms. The existence of these tumor escape mechanisms underlies the necessity of multi-targeted therapies. Clinical practice has also demonstrated that single-target tyrosine kinase inhibitors, such as erlotinib and gefitinib, despite their strong selectivity and minimal toxicity, are susceptible to developing drug resistance during use, failing to completely eliminate tumor cells. Combination therapy can also lead to severe adverse reactions, impacting the pharmacokinetic properties of each drug. Multi-targeted drugs offer numerous advantages over single-target drugs and combinations of multiple single-target drugs. These drugs can effectively avoid drug-drug interactions, reduce adverse reactions, and offer a more comprehensive therapeutic effect. Many multi-targeted tyrosine kinase inhibitors, such as imatinib, have become first-line treatments for certain tumors due to their high efficacy and minimal toxicity.

[0003] There are approximately six species of Cratoxylum plants in the Clusiaceae family worldwide. Modern phytochemical studies have shown that Cratoxylum plants are rich in compounds of various structural types, including xanthones, anthraquinones, flavonoids, triterpenes, and steroids. Modern pharmacological studies have shown that chemical components isolated from Cratoxylum plants have diverse biological activities, including 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]. 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, a plant of the genus C. cochinchinense in the family Garcinia, is found in Hainan, Guangdong, Guangxi, and Yunnan provinces of my country. C. cochinchinense is a traditional folk medicinal plant, with its roots, bark, and young leaves used as medicine. It has the properties of clearing heat and detoxifying, dissipating dampness and relieving stagnation, and removing blood stasis and swelling. It is used to treat colds, heatstroke, fever, diarrhea, jaundice, injuries from falls, and carbuncles. Its young leaves can be used as a refreshing beverage to relieve summer heat and thirst. However, its anti-tumor application has not yet been studied. Summary of the Invention

[0005] The present invention aims to provide a xanthone compound, cratocochinone, with a novel chemical structure, isolated from the branches and leaves of the plant Cratocochinus genus of the Hypericaceae family. The compound has significant anti-tumor activity and protein tyrosine kinase inhibitory activity comparable 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-extract the branches and leaves of the yellow cattle tree with 75% ethanol or methanol solution four times, filter, collect the filtrate, and then concentrate under reduced pressure until there is no alcohol smell to obtain the 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 was separated and purified by column chromatography to obtain the 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) The chloroform-acetone eluate with a volume ratio of 70:30 was subjected to MCI resin column chromatography to remove the pigment, and then gradient eluted with methanol-water at a volume ratio of 40:60, 50:50, and 65:35, respectively, and the methanol-water eluate with a volume ratio of 50:50 was collected;

[0015] (3) The methanol-water eluate with a volume ratio of 50:50 was subjected to reverse phase silica gel column chromatography, and the eluate was gradient eluted with methanol-water at a volume ratio of 45:55, 50:50, and 55:45, respectively. The methanol-water eluate with a volume ratio of 50:50 was collected and concentrated;

[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 a xanthone compound, cratocochinone, in the preparation of anti-tumor drugs, and in particular to provide the use of a xanthone compound, cratocochinone, 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 isolate and identify a new xanthone compound, cratocochinone, from the petroleum ether extract of the alcohol extract of the branches and leaves of the yellow cattle 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 The concentrations of 1.28 and 2.62 μg / mL, respectively, are also comparable to those of the positive control drug imatinib in terms of protein tyrosine kinase inhibitory activity. These drugs can be further developed into targeted anti-tumor drugs targeting protein tyrosine kinase, with broad application prospects. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention but are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally 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. Shade-dried powder of yellow cattle wood branches and leaves (8.6 kg, Hainan) was extracted with 75% ethanol solution four times, each time for four days. The filtrate was collected and concentrated under reduced pressure until alcohol-free to obtain an alcohol extract.

[0026] B. The alcohol extract was added with distilled water to prepare a suspension, which was 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, the steps of which 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 ratios of 90:10, 80:20, 70:30, and 50:50) as eluents, 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 ratios 40:60, 50:50, and 65:35) as the eluent, and the methanol-water (volume ratio 50:50) eluate was collected;

[0030] (3) The methanol-water (volume ratio 50:50) eluate was subjected to reverse-phase silica gel column chromatography, and gradient elution was performed using methanol-water (volume ratios 45:55, 50:50, and 55:45) as eluents. The methanol-water (volume ratio 50:50) eluate was collected and concentrated;

[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] Structure confirmation: The chemical structure of the compound cratocochinone was determined through comprehensive analysis of multiple modern spectroscopic 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 731 cm –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. Shade-dried powder of yellow cattle wood branches and leaves (90.3 kg, Hainan) was extracted with methanol cold soak six times, three days each time, filtered, and the filtrate was collected and concentrated under reduced pressure until there was no alcohol smell to obtain a methanol extract;

[0037] B. The methanol extract was added with distilled water to prepare a suspension, which was extracted with petroleum ether and ethyl acetate in sequence. The petroleum ether extract was concentrated under reduced pressure to obtain 6568.2 g of petroleum ether extract and 4643.6 g of ethyl acetate extract;

[0038] C. subjecting the ethyl acetate extract to column chromatography for separation and purification;

[0039] The specific steps are as follows:

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

[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 ratios 40:60, 50:50, and 65:35) as the eluent, and the methanol-water (volume ratio 50:50) eluate was collected;

[0042] (3) The methanol-water (volume ratio 50:50) eluate was subjected to reverse-phase silica gel column chromatography, and gradient elution was performed using methanol-water (volume ratios 45:55, 50:50, and 55:45) as eluents. The methanol-water (volume ratio 50:50) eluate was collected and concentrated;

[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: pale yellow amorphous powder; HR-ESI-MS showed that the [M+H] + m / z 393.1698; Compound II and the compound cratocochinone prepared by the method of Example 1 were co-TLC analyzed, and the results were 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 in Example 1 are the same compound.

[0045] 2. Study on the anti-tumor 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 supplemented with 10.0% calf serum at 37.0°C in a 5.0% CO2 incubator. The MTT assay was used to investigate cell proliferation inhibition. The main steps were as follows: Tumor cell lines in the logarithmic growth phase were digested with 0.25% trypsin and then cultured in RPMI-1640 medium supplemented with 10% newborn calf serum to a volume of 5.0 × 10 4Cell suspensions of 100 μg / mL were inoculated into 96-well plates, with 180.0 μL in each well. Cultured at 37.0°C, 5% CO2, and saturated humidity for 8.0-10.0 h. After adherence, sample solutions prepared with PBS were added to each well, with final concentrations of 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 of MTT (1.0 mg / mL) was added to each well. -1 , PBS), continue incubation at 37.0℃, 5.0% CO2 for 4.0h, aspirate and discard the culture supernatant in the wells, 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 value of each well, and set up a blank group (only add culture medium containing cells) and a control group (with culture medium instead of drug) at the same time, and 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 vertical axis, draw a regression curve, and calculate the sample IC 50 SPSS 13.0 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 a 4-fold volume of cold homogenate. Homogenize the mixture at high speed using a glass homogenizer in an ice bath, centrifuge, collect the supernatant, and centrifuge again for 10 minutes. The supernatant contains cytoplasmic tyrosine kinases, while the precipitate can be used as receptor tyrosine kinases. Reserve a small amount of the supernatant for protein determination in the extract; aliquot the remainder and store at -70°C until further use.

[0054] ELISA plate coating: Add the substrate dilution to a 96-well ELISA plate (125.0 μL per well) and incubate at 37.0°C overnight. Remove excess substrate from the plate, wash with phosphate-buffered saline (PBS-Tween 20), and dry at 37.0°C for 2.0 h. Store at 4.0°C until needed.

[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, and 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 comparable 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 within the scope of protection of the present invention.

Claims

1. Use of xanthone compounds in the preparation of targeted anti-tumor drugs targeting protein tyrosine kinase. The tumor cell lines include six tumor cell lines: A375, JeKo-1, MCF-7, A549, SW480, and SMMC-7721. The chemical name of the xanthone compound is cratocochinone, and its chemical structure is as follows: 。

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