A norosequiterpenoid compound and its preparation method and application

Abstract

The present invention belongs to the field of natural medicines and relates to a norosequiterpenoid compound, mycehainanic acid, with a novel chemical structure and derived from the branches and leaves of a plant of the genus Adenocalyx in the Rubiaceae family, as well as a preparation method thereof and an application thereof in anti-tumor drugs. The results of multiple in vitro activity evaluations of the compound mycehainanic acid show that the compound has significant anti-tumor activity and protein tyrosine kinase inhibitory activity comparable to that of a positive control drug. The compound can be further developed into an anti-tumor drug targeting protein tyrosine kinase, thereby fully realizing its medicinal and economic value. The separation and purification process is simple, and the compound has practical significance.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine preparation, and relates to a norosequiterpenoid compound with a novel chemical structure derived from the Hainan adenium, and particularly relates to a norosequiterpenoid compound, a preparation method thereof, and application thereof in anti-tumor drugs. Background Art

[0002] Malignant tumors remain the second leading cause of death and serious threat to human life and health, second only to cardiovascular disease. Modern medical treatments for tumors primarily include surgery, chemotherapy, and radiotherapy, as well as the more recently developed biological therapies. However, malignant tumors are systemic diseases that often metastasize by the time of clinical diagnosis. Surgery can only address localized tumor burdens, necessitating adjuvant chemotherapy after surgery. However, currently used chemotherapy drugs are primarily anti-tumor drugs, which induce apoptosis by directly targeting components shared with normal human cells, such as DNA, RNA, or tubulin. This results in poor selectivity and significant side effects. With advances in modern life sciences, the mechanisms of tumor development and progression are increasingly understood. Leveraging these biomedical research findings, the search for anti-tumor active substances with novel mechanisms of action, strong selectivity, minimal side effects, and proprietary intellectual property rights is a crucial task in the development of new anti-tumor drugs.

[0003] Molecularly targeted anti-tumor drugs can selectively kill tumor cells by targeting specific targets during tumor cell growth and proliferation without damaging normal tissue, bringing revolutionary progress to the field of cancer treatment. Unlike traditional cytotoxic drugs, these drugs primarily target specific molecules or targets that are not expressed or only rarely expressed by normal cells but are present during tumor cell growth and proliferation. Therefore, they can act specifically on tumor cells without damaging normal tissues and cells. The emergence of molecularly targeted anti-tumor drugs has brought hope for the development of new anti-tumor drugs with excellent selectivity and minimal toxic side effects. Among various molecular targets, protein tyrosine kinase (PTK) is one of the most studied and effective anti-tumor drug targets. It has become a research focus and hot topic for targeted anti-tumor therapies and has broad application prospects. Currently, small molecule targeted anti-tumor drugs in clinical use are primarily tyrosine kinase inhibitors, 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 a variety of mechanisms, including autocrine and paracrine regulation, ligand production, receptor mutations, activation of downstream signaling pathways, and activation of alternative signaling pathways. These escape mechanisms underlie 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 side effects, are susceptible to drug resistance and inability to completely eliminate tumor cells. Combination therapy can also lead to severe adverse reactions, impacting the pharmacokinetic properties of each drug. Multi-target tyrosine kinase inhibitors offer the advantages of reducing the number of drugs used, enabling multi-faceted efficacy, combating drug resistance, avoiding drug interactions, and minimizing adverse reactions. Many multi-target tyrosine kinase inhibitors have become first-line treatments for certain cancers due to their high selectivity, efficacy, and minimal toxicity.

[0004] There are approximately 30 species of Mycetia in the Rubiaceae family worldwide, most of which are found in tropical Asia. In my country, there are 15 species, one variety, and three forms of Mycetia, distributed from southwest to southeast China [Editorial Committee of Flora of China, Chinese Academy of Sciences. Flora of China. Vol. 71(1). Science Press. Beijing: 1999, pp. 313-325.]. Due to the regional distribution and endemicity of Mycetia, research reports on the chemical components and pharmacological activities of Mycetia are extremely rare. To date, there are only two preliminary research reports on the anti-inflammatory activity and preliminary mechanism of action of crude extracts of Mycetia longifolia (collected in Bangladesh) and Mycetia cauliflora (collected in South Korea). There are no reports on the chemical components of Mycetia longifolia [Jain, P.; Hossain, MA; Fatema, K.; Mazumder, KU; Hossain, MS; Hossain, H.; Alam, MA; Reza, HB. Anti-inflammatory and anti-inflammatory activities of Mycetia longifolia (Wall.) O. Kuntze. J. Herbs Spices Med. Plants. 2015, 21, 321–331.; Jeong, SG; Kim, S.; Kim, HG; Kim, E.; Yang, WS; Oh, J.; Sung, GH; Hossain, MA; Lee, J.; Kim, JH; Cho, JY Mycetia caulifloramethanol extract exerts anti-inflammatory activity by directly targeting PDK1inthe NF-κB pathway. J. Ethnopharmacol. 2019, 231, 1–9.]. There are three species of Mycetia caulifloram distributed in Hainan, namely M. hainanensis, M. hirta and M. sinensis [Editorial Committee of Flora of China, Chinese Academy of Sciences. Flora of China. Vol. 71(1). Science Press. Beijing: 1999, pp 313-325.]. Among them, Hainan glandular calyx wood is a plant endemic to Hainan. So far, there has been no research report on the chemical components and pharmacological activities of Hainan glandular calyx wood.

[0005] M. hainanensis is a plant of the genus Cynodonium in the family Apocynaceae. It is mainly distributed in Hainan and Yunnan provinces. It is a common herbal medicine used by the Li people to treat inflammatory diseases, mainly arthritis and back pain. It has a long history of medicinal use and significant efficacy [Editorial Committee of Flora of China, Chinese Academy of Sciences. Flora of China. Vol. 71(1). Science Press. Beijing: 1999, pp 313-325.]. So far, there has been no research on its application in anti-tumor treatment. Summary of the Invention

[0006] The present invention aims to provide a mycehainanic acid, a norosequiterpenoid compound with a novel chemical structure, isolated from the branches and leaves of Adenocalyx hainanensis. 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.

[0007] In order to achieve the above object, the technical solution of the present invention is to provide a norosequiterpenoid compound, the chemical name of which is mycehainanic acid, and the chemical structure of which is as follows:

[0008]

[0009] Another object of the present invention is to provide a method for preparing a norosequiterpenoid compound, comprising the following steps:

[0010] A. Shade-dried branches and leaves of Glandularia hainanensis were crushed and extracted six times with 85% ethanol or methanol, filtered, and the filtrate was collected and concentrated under reduced pressure to dryness to obtain an alcohol extract;

[0011] B. adding water to the alcohol extract to prepare a suspension, extracting with petroleum ether and ethyl acetate in sequence, and concentrating the petroleum ether extract under reduced pressure to obtain a petroleum ether extract;

[0012] C. The petroleum ether extract was separated and purified by column chromatography to obtain the monomer compound mycehainanic acid.

[0013] Furthermore, the step C is specifically as follows: ① the petroleum ether extract is separated by silica gel column chromatography, and petroleum ether-acetone gradient elution is performed at a volume ratio of 95:5, 90:10, 80:20, 70:30 and 40:60, respectively, and the petroleum ether-acetone eluate with a volume ratio of 70:30 is collected; ② the petroleum ether-acetone (volume ratio 70:30) eluate is subjected to MCI resin column chromatography to remove the pigment, and the methanol-water gradient elution is performed at a volume ratio of 40:60, 55:45 and 70:30, and the volume ratio is collected. The methanol-water eluate was separated by preparative high performance liquid chromatography (HPLC) using a mobile phase of methanol-water in a volume ratio of 48:52 to obtain the monomer compound mycehainanic acid.

[0014] Another object of the present invention is to provide the use of a norosequiterpenoid compound in the preparation of an anti-tumor drug, in particular to provide the use of a norosequiterpenoid compound, mycehainanic acid, in the preparation of a targeted anti-tumor drug targeting protein tyrosine kinase.

[0015] Furthermore, the tumor cell lines include five tumor cell lines: JeKo-1 (human mantle cell lymphoma cells), A549 (human lung cancer cells), SMMC-7721 (human liver cancer cells), MCF-7 (human breast cancer cells) and SW480 (human colon cancer cells).

[0016] Compared with the prior art, the present invention has the following effects:

[0017] This study, based on a systematic study of the chemical constituents of the branches and leaves of the plant Mycena hainanensis, isolated and identified, for the first time, a novel sesquiterpenoid compound, Mycena hainanensis, from its petroleum ether extraction. Multiple in vitro activity evaluations demonstrated that the compound exhibited significant antitumor activity and comparable protein tyrosine kinase inhibitory activity to that of a positive control drug. This compound could be further developed into an antitumor drug targeting protein tyrosine kinase, fully realizing its medicinal and economic value. DETAILED DESCRIPTION

[0018] 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.

[0019] 1. Preparation Method of Mycehainanic Acid

[0020] Example 1

[0021] The preparation method of this embodiment comprises the following steps:

[0022] 1. Shade-dried leaves and branches of Adenophora hainanensis (10.2 kg, Hainan) were crushed and then cold-extracted with 85% ethanol solution six times, each time for three days. The filtrate was collected and concentrated under reduced pressure to obtain an ethanol extract.

[0023] 2. Add distilled water to the ethanol 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 568.9 g of petroleum ether extract;

[0024] 3. Column chromatography separation and purification of the petroleum ether extract: The petroleum ether extract was separated by silica gel column chromatography, and eluted with petroleum ether-acetone (volume ratio 70:30) as eluent (95:5, 90:10, 80:20, 70:30 and 40:60), and the petroleum ether-acetone (volume ratio 70:30) eluate was collected, and the petroleum ether-acetone (volume ratio 70:30) eluate was subjected to MCI resin column chromatography to remove the pigment, and the extract was eluted with methanol-water gradient (volume ratio 40:60, 55:45 and 7 The eluate of methanol-water (volume ratio 55:45) was collected and subjected to ODS column chromatography with (volume ratios 50:50, 55:45 and 65:35), the eluate of methanol-water (volume ratio 55:45) was collected and concentrated, and the eluate of methanol-water (volume ratio 55:45) was separated by preparative high performance liquid chromatography with methanol-water (volume ratio 48:52) as the mobile phase to obtain pure compound mycehainanic acid (48.7 mg).

[0025] Structure confirmation: The chemical structure of the compound mycehainanic acid was determined through comprehensive analysis of multiple spectral identification techniques such as UV spectroscopy, IR spectroscopy, MS and NMR spectroscopy.

[0026] Mycehainanic acid: colorless oil; UV(CH3OH)λ max (logε)206(3.56) and 230(4.08)nm; IR(KBr)v max 3428, 2948, 2872, 1718, 1456, 1379, 1269, 1227, 1153, 1120, 1091 and 1046 cm –1 ;HR-ESI-MS m / z 239.1648[M+H] + (Calcd for C14 H 23 O3,239.1642); 1 H-NMR (400MHz, CDCl3) δ: 5.62 (1H, s, H-2), 5.16 (1H, t, J = 6.8 Hz, H-6), 2.66 (2H, t, J = 7.6 Hz, H-4), 2.38 (2H, t, J = 7.4 Hz, H-10), 2.18 (2H, d t,7.6,6.8Hz,H-5),2.13(3H,s,H-12),1.96(2H,t,J=7.6Hz,H-8),1.85(3H,s,H-13),1.67(2H,tt,J=7.6,7.4Hz,H-9),1.60(3H,s,H-14); 13 C-NMR(100MHz, CDCl3)δ:209.3(C-11),168.5(C-1),156.0(C-3),134.9(C-7),124.5(C-6),117.8(C-2 ),43.0(C-10),38.8(C-8),32.8(C-4),29.9(C-12),26.5(C-5),24.8(C-13),21.8(C-9),15.7(C-14).

[0027] Example 2

[0028] The preparation method of this embodiment comprises the following steps:

[0029] 1. Shade-dried leaves and branches of Adenocalyx hainanensis (108.6 kg, Hainan) were crushed and extracted with methanol for six times, each time for five days. The filtrate was collected and concentrated under reduced pressure to obtain a methanol extract.

[0030] 2. Add 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 5963.2 g of petroleum ether extract;

[0031] 3. The petroleum ether extract was separated and purified by column chromatography: the petroleum ether extract was separated by silica gel column chromatography, and petroleum ether-acetone gradient elution (95:5, 90:10, 80:20, 70:30 and 40:60) was performed, and the petroleum ether-acetone (volume ratio 70:30) eluate was collected, and the petroleum ether-acetone (volume ratio 70:30) eluate was subjected to MCI resin column chromatography to remove the pigment, and methanol-water gradient elution (volume ratio 40:60, 55:45 and 70:30) was performed, and the The eluate of methanol-water (volume ratio of 55:45) was collected, and the eluate of methanol-water (volume ratio of 55:45) was subjected to ODS column chromatography, and gradient eluted with methanol-water (volume ratio of 50:50, 55:45 and 65:35). The eluate of methanol-water (volume ratio of 55:45) was collected and concentrated, and the eluate of methanol-water (volume ratio of 55:45) was separated by preparative high performance liquid chromatography with methanol-water (volume ratio of 48:52) as the mobile phase to obtain monomer compound II (506.2 mg).

[0032] The structure of compound II was confirmed: colorless oil; HR-ESI-MS showed that the [M+H] + m / z 239.1648; Compound II and mycehainanic acid prepared in Example 1 were analyzed by TLC, and the results were uniform spots under three developing systems [petroleum ether-ethyl acetate (5:5), petroleum ether-acetone (7:3), and chloroform-acetone (9:1)], indicating that Compound II and mycehainanic acid prepared in Example 1 were the same compound.

[0033] 2. Study on the Antitumor Activity of Mycehainanic Acid

[0034] 1. Experimental methods

[0035] Five common tumor cell lines, JeKo-1, A-549, SMMC-7721, SW480, and MCF-7, were cultured in RPMI-1640 medium supplemented with 10% calf serum at 37.0°C in a 5% 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 × 10 4Cell suspensions of 100 μg / mL were inoculated into 96-well plates, with 180.0 μL in each well. Cultured at 37°C, 5% CO2, and saturated humidity for 8.0-10.0 hours. After the cells adhered to the wall, a sample solution prepared with PBS was added to each well, with the final concentrations of the samples being 0.1, 1.0, and 10.0 μg / mL, respectively. Three wells were cultured for each concentration, and 50.0 μL of MTT (1.0 mg / mL) was added to each well after 44.0 hours of culture. -1 , prepared with PBS), incubate for 4.0 hours at 37.0°C and 5% CO2, discard the culture supernatant in the wells, add 150.0μL DMSO to each well, shake on a micro-oscillator for 15.0 minutes, and after the crystals dissolve, select 570.0nm on the enzyme-linked immunosorbent assay to measure the absorbance of each well. At the same time, set up a blank group (only culture medium containing cells) and a control group (culture medium instead of drug) 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 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.

[0036] 2. Antitumor activity experimental results (see Table 1)

[0037] The compound mycehainanic acid obtained in Example 1 showed different degrees of proliferation inhibitory activity (IC 50 The smaller the value, the better the activity).

[0038] Table 1 Evaluation results of the antitumor activity of compound mycehainanic acid

[0039]

[0040] 3. Inhibition of protein tyrosine kinase activity by the compound mycehainanic acid

[0041] 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.

[0042] 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.

[0043] Evaluation of PTK inhibitors: First, add the sample to the ELISA plate and incubate at 37.0°C. Add ATP diluted in kinase buffer and incubate at 37.0°C. Remove the reaction solution from the plate and wash. Add the antibody complex and incubate at 37.0°C. Remove the antibody complex from the plate and wash. Add tetramethylbenzidine (TMB) colorimetric solution and react at room temperature in the dark. Add the stop solution and measure the absorbance (A) at a wavelength of 450.0 nm. The positive control drug is imatinib. The inhibition rate of the compound mycehainanic acid is calculated according to the following formula: Inhibition rate % = (A 正常 -A 样品 ) / (A 正常 -A 空白 )*100%.

[0044] The results showed that the compound of the present invention, mycehainanic acid, had a significant inhibitory effect on protein tyrosine kinase (inhibition rate 82.79%), and the inhibitory activity was comparable to that of the positive control drug imatinib (inhibition rate 69.98%).

[0045] The above disclosure is merely a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope of the present invention.

Claims

1. A norsesquiterpenoid compound, characterized in that: The chemical name is mycehainanic acid, and the chemical structure is as follows:

2. A method for preparing a norosequiterpenoid compound according to claim 1, characterized in that: The following steps are involved: A. Shade-dried branches and leaves of Glandularia hainanensis were crushed and extracted six times with 85% ethanol or methanol solution, filtered, and the filtrate was collected and concentrated under reduced pressure to dryness to obtain an alcohol extract; B. adding distilled water to the alcohol extract to prepare a suspension, extracting with petroleum ether and ethyl acetate in sequence, and concentrating the petroleum ether extract under reduced pressure to obtain a petroleum ether extract; C. Purifying the petroleum ether extract by column chromatography to obtain the monomer compound mycehainanic acid; Wherein, the step C is specifically as follows: (1) The petroleum ether extract was separated by silica gel column chromatography, and petroleum ether-acetone gradient elution was performed at volume ratios of 95:5, 90:10, 80:20, 70:30, and 40:60, respectively, and the petroleum ether-acetone eluate with a volume ratio of 70:30 was collected; (2) The eluate with a volume ratio of 70:30 petroleum ether-acetone was subjected to MCI resin column chromatography to remove the pigment, and then gradient eluted with methanol-water with a volume ratio of 40:60, 55:45, and 70:30, and the methanol-water eluate with a volume ratio of 55:45 was collected; (3) The methanol-water eluate with a volume ratio of 55:45 was subjected to ODS column chromatography, and gradient elution was performed with methanol-water with a volume ratio of 50:50, 55:45, and 65:

35. The methanol-water eluate with a volume ratio of 55:45 was collected and concentrated; (4) The methanol-water eluate was separated by preparative HPLC using a mobile phase of methanol-water in a volume ratio of 48:52 to obtain the monomer compound mycehainanic acid.

3. Use of the norosequiterpenoid compound according to claim 1 in the preparation of targeted anti-tumor drugs targeting protein tyrosine kinase.

4. Use of the norosequiterpenoid compound according to claim 3 in the preparation of a targeted anti-tumor drug targeting protein tyrosine kinase, characterized in that: The tumor cell lines are five tumor cell lines: JeKo-1, A-549, SMMC-7721, SW480 and MCF-7.

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