A novel pentophoretic compound with antitumor activity, its preparation method and application

Abstract

This invention discloses a novel antitumor compound from *Pterocarya stenoptera*, its preparation method, and its applications. The invention involves in-depth research on the chemical composition of *Pterocarya stenoptera* root bark, followed by ethanol extraction, acid hydrolysis, chloroform extraction, repeated silica gel resin column chromatography, and finally, semi-preparative high-performance liquid chromatography (HPLC) for separation and purification to obtain the new compound. In vitro antitumor inhibition experiments demonstrated that the compound obtained by this invention exhibits significant antitumor activity, particularly against myeloma, human cervical cancer, and lung adenocarcinoma. This compound has a novel structure, low cross-resistance with existing antitumor drugs, and shows promise as a potential antitumor drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel compound with antitumor activity isolated from the root bark of *Pterocarya stenoptera* and its preparation method. Background Technology

[0002] *Meliosma rigida*, also known as the rigida shrub, is a small evergreen tree belonging to the genus *Meliosma* in the family Sabiaceae. It grows to a height of 10-20 meters. The trunk is straight with grayish-brown bark; young branches are stout and densely covered with rusty-brown hairs. Leaves are simple, alternate, leathery, oblanceolate or elliptic, 8-25 cm long and 3-5 cm wide, with an acute or obtuse apex, a cuneate base, and entire or sparsely serrated upper margins. The upper surface is hairy when young, and the lower surface has rusty-brown tomentum; lateral veins are prominent on the abaxial surface. The petiole is 2.5-3 cm long and has short hairs. The panicles are terminal and covered with rusty-brown hairs; flowers are small and white; sepals are 5; petals are 5; stamens are 5. The drupe is nearly globose, oblique at the base, 5-8 mm in diameter, with a persistent calyx.

[0003] The root bark and other parts of *Prunus armeniaca* are used medicinally. The root bark is harvested in autumn when mature and dried. It grows on mountain slopes, streamsides, forest edges, or in broad-leaved mixed forests and thickets below 1500m altitude. It is distributed in Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, Guangxi, Guizhou, and Yunnan provinces. Root bark: sour, neutral. Detoxifies, promotes diuresis, and reduces swelling. Mainly treats edema, abdominal distension, boils of unknown origin, and snake bites. Root bark: bitter; neutral. Relieves exterior syndromes and stops coughs. Mainly treats colds and coughs.

[0004] Modern research indicates that *Pteris vittata* possesses various physiological activities, including dispelling wind and dampness, lowering blood sugar, anticonvulsant, sedative, analgesic, and antibacterial effects. Currently, research on the chemical components responsible for the antitumor activity of *Pteris vittata* is limited.

[0005] The applicant previously conducted research on the supercritical extract of *Prunus armeniaca* fruit (application number 2023112419621) and found that it has certain anti-tumor activity. Based on the existing research, this application conducts a more in-depth study on the active components of its root bark and develops monomeric compounds with certain activities, providing a scientific basis for the resource utilization of *Prunus armeniaca*. Summary of the Invention

[0006] The purpose of this invention is to conduct in-depth research on the active ingredients of *Pteris vittata* root bark, extract and isolate its potential active ingredients, and provide a scientific basis for its clinical application.

[0007] Technical Solution: This invention discloses a novel compound with antitumor activity isolated from the root bark of the plant *Pteris vittata*. The structural formula of the purified and isolated novel compound is shown below:

[0008] .

[0009] Its molecular formula is C 17 H 24 O5.

[0010] A method for preparing a novel pentophore compound with antitumor activity, comprising the following steps:

[0011] (1) Weigh the dried root bark of *Pteris vittata*, crush it appropriately, add a certain amount of ethanol to extract it, combine the extracts, concentrate it, add a certain volume concentration of acid to the concentrate for water bath hydrolysis, after hydrolysis, filter it, wash it with water until neutral, and dry it to obtain the hydrolysis product. Take the hydrolysis product and add chloroform or ethyl acetate to extract it. Combine the filtrates and concentrate them under reduced pressure to obtain the chloroform or ethyl acetate extract.

[0012] (2) Take the chloroform or ethyl acetate extract from step (1) and use silica gel column chromatography at normal pressure to elute sequentially with different volume ratios of petroleum ether-ethyl acetate as eluent; after thin-layer chromatography spot analysis, combine the same eluent fractions, concentrate, and obtain 6 eluent fractions LF-1~LF-6.

[0013] (3) After dry processing, the fraction LF-2 obtained in step (2) is subjected to silica gel column chromatography again, and eluted sequentially with different volume ratios of chloroform-methanol as the eluent. After thin-layer chromatography spot analysis, the same eluent is combined and concentrated to obtain 4 elution fractions BLZ-1~BLZ-4.

[0014] (4) The BLZ-4 eluent obtained in step (3) is separated and purified by semi-preparative high performance liquid chromatography to obtain the monomer compound.

[0015] As a preferred embodiment, the method for preparing the above-mentioned novel pentosan compound with antitumor activity includes the following steps:

[0016] (1) Weigh the dried root bark of *Pteris vittata*, pulverize it appropriately, add 6-15 times the volume of ethanol, and extract by continuous reflux 1-3 times, 1-2 hours each time. Combine the extracts, concentrate them, add sulfuric acid of a certain concentration to the concentrate and hydrolyze it in a water bath for 1-3 hours. After hydrolysis, filter it, wash it with water until neutral, and dry it to obtain the hydrolysate. Take the hydrolysate and add 6-12 times the volume of chloroform to extract by continuous reflux 1-3 times, 0.5-2 hours each time. After filtration, collect the filtrate, combine the filtrates, concentrate them under reduced pressure, and obtain the chloroform extract.

[0017] (2) Take the chloroform extract from step (1) and perform silica gel column chromatography at normal pressure. Elute sequentially with petroleum ether-ethyl acetate eluent at volume ratios of 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4, with each gradient eluent being 4 to 6 column volumes. After thin-layer chromatography spot analysis, combine the same eluents, concentrate, and obtain 6 eluent fractions LF-1 to LF-6.

[0018] (3) After dry processing, the fraction LF-2 obtained in step (2) is subjected to silica gel column chromatography again. The fraction is eluted sequentially with trichloromethane-methanol in volume ratios of 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4:1:8, with each gradient eluting for 3 to 6 column volumes. After thin-layer chromatography spot analysis, the same eluents are combined and concentrated to obtain four eluent fractions BLZ-1 to BLZ-4.

[0019] (4) The BLZ-4 eluent obtained in step (3) was separated and purified by semi-preparative high performance liquid chromatography with acetonitrile (A)-water (B) as the mobile phase gradient elution, and the monomer compound was collected.

[0020] As a more preferred embodiment, the method for preparing the novel pentosan compound with antitumor activity described above includes the following steps:

[0021] (1) Weigh the dried root bark of *Pteris vittata*, pulverize it appropriately, add 15 times the volume of ethanol, and extract it by continuous reflux three times, the first time for 2 hours and the next two times for 1 hour. Combine the three extracts and concentrate them. Add 10% sulfuric acid at a volume ratio of 5:1 to the concentrate and hydrolyze it in a water bath for 2 hours. After hydrolysis, wash it with water until it is neutral and then dry it. Take the hydrolysis product and add 10 times the volume of chloroform to extract it by continuous reflux three times, the first two times for 1 hour and the last time for 0.5 hours. After filtration, collect the filtrate, combine the filtrates, concentrate them under reduced pressure, and obtain the chloroform extract.

[0022] (2) Take the chloroform extract from step (1) and perform silica gel column chromatography at atmospheric pressure. Elute sequentially with petroleum ether-ethyl acetate in volume ratios of 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4, with each gradient eluting for 5 column volumes. After spot analysis by thin-layer chromatography, combine the same eluents, concentrate, and obtain 6 eluent fractions LF-1 to LF-6.

[0023] (3) After dry processing, the fraction LF-2 obtained in step (2) was subjected to silica gel column chromatography again. The fraction was eluted sequentially with trichloromethane-methanol in volume ratios of 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4:1:8, with each gradient eluting for 3 column volumes. After thin-layer chromatography spot analysis, the same eluent was combined and concentrated to obtain 4 eluent fractions BLZ-1 to BLZ-4.

[0024] (4) The BLZ-4 eluent obtained in step (3) was separated and purified by semi-preparative high performance liquid chromatography with acetonitrile (A)-water (B) as the mobile phase gradient elution, and the monomer compound was collected.

[0025] As a preferred embodiment, the chromatographic conditions for the semi-preparative high-performance liquid chromatography (HPLC) in the above-described method for preparing the novel pentophore compound are: EclipseXDB Prep C. 18 A chromatographic column with dimensions of 10 nm, 5 μm, and 10 × 250 mm was prepared. The column temperature was 25–30 °C, and the injection volume was 40 μL. Acetonitrile (A)-water (B) was used as the mobile phase, with gradient elution (0–5 min, 80% A; 5–12 min, 40% A; 12–21 min, 20% A; 21–40 min, 45% A). The flow rate was 3 mL / min, and the detection wavelength was 248 nm. Monomer compounds were collected in the time interval of 21–34 min.

[0026] Beneficial effects: This invention has the following innovative aspects:

[0027] This invention conducts an in-depth study of the chemical composition of *Pteris vittata* root bark. Ethanol extraction was employed, followed by acid hydrolysis of glycosidic bonds and chloroform extraction. Repeated column chromatography with silica gel resin was then used, and finally, semi-preparative high-performance liquid chromatography (HPLC) was employed for separation and purification to obtain new compounds. In vitro antitumor inhibition experiments demonstrated that the compounds isolated and purified in this invention possess significant antitumor activity, particularly against myeloma, human cervical cancer, and lung adenocarcinoma. The compounds extracted and isolated in this invention can be formulated into various dosage forms with pharmaceutically acceptable carriers. Attached Figure Description

[0028] Figure 1 (+)-HR-ESI-MS chromatogram of the compound of the present invention.

[0029] Figure 2 The compounds of this invention 1 H-NMR spectrum.

[0030] Figure 3 The compounds of this invention 13 C-NMR spectrum.

[0031] Figure 4 The structural formula of the compound to be invented. Detailed Implementation

[0032] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims. All materials and reagents used in this invention are commercially available and conventionally obtained.

[0033] Example 1

[0034] 1. Experimental instruments and materials

[0035] The Agilent 1260 Infinity II preparative liquid chromatography system, EclipseXDB Prep C18 preparative column (10 nm, 5 μm, 10 × 250 mm); column chromatography silica gel (200–300 mesh); chromatographic grade acetonitrile purchased from Merck & Co., Ltd.; analytical grade petroleum ether, chloroform, ethyl acetate, methanol, etc. purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0036] 2. The preparation method of the monomeric compound includes the following steps:

[0037] (1) Weigh the dried root bark of *Pteris vittata*, pulverize it appropriately, add 15 times the volume of ethanol to the medicinal material, and extract it by continuous reflux three times, the first time for 2 hours and the next two times for 1 hour. Combine the three extracts, concentrate them to obtain the concentrate, add 10% sulfuric acid at a volume ratio of 5:1 and hydrolyze it in a water bath for 2 hours. After the hydrolysis is completed, filter it and wash it with water until it is nearly neutral. Dry it and take the hydrolysis product. Add 10 times the volume of chloroform to extract it by continuous reflux three times, the first two times for 1 hour and the last time for 0.5 hours. After filtration, collect the filtrate, combine the filtrates, concentrate them under reduced pressure to obtain the chloroform extract.

[0038] (2) Take the chloroform extract from step (1), mix it with silica gel dry method, and perform silica gel column chromatography at normal pressure. Elute sequentially with petroleum ether-ethyl acetate eluent at volume ratios of 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4, with each gradient eluting for 5 column volumes. After thin-layer chromatography spot analysis, combine the same eluent, concentrate, and obtain 6 eluent fractions LF-1 to LF-6.

[0039] (3) After the fraction LF-2 obtained in step (2) was dried on silica gel, it was subjected to silica gel column chromatography again. The fraction was eluted in a gradient with chloroform-methanol in volume ratios of 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4:1:8, with each gradient eluting for 3 column volumes. After thin-layer chromatography spot analysis, the same eluent was combined and concentrated to obtain 4 eluent fractions BLZ-1 to BLZ-4.

[0040] (4) The BLZ-4 eluent obtained in step (3) was separated and purified by semi-preparative high performance liquid chromatography. Acetonitrile (A)-water (B) was used as the mobile phase for gradient elution (0-5 min, 80% A, 5-12 min, 40% A, 12-21 min, 20% A, 21-40 min, 45% A). The monomer compound was collected in the time period of 21-34 min with a purity of 99%.

[0041] 3. Structural identification of compounds

[0042] The compound is a white amorphous powder. +40.0 (c 0.01, MeOH). Figure 1 According to the high-resolution mass spectrometry (Waters Synapt G2-Si QTOF) of this compound, the quasi-molecular ion peak m / z is 309.1701 ([M + H)). + C 17 H 25 O5 + The calculated value is 309.1704, from which the molecular formula of the compound is deduced to be C. 17 H 24 O5.

[0043] The following is based on... Figure 2 and Figure 3 Structural analysis was performed using hydrogen and carbon NMR data:

[0044] As shown in Table 1, the structural analysis of the proton and carbon spectra of this compound... 1 The H-NMR spectrum showed a signal δ of two methyl protons. H (1.10, CH3-15; 1.02, CH3-16), one oxygen-containing methylene proton signal δ H (4.00 / 3.81, H-17), 2 oxygen-containing methine proton signals δ H (3.68, H-1; 3.75, H-6) and an olefin proton signal (δ) H 6.24, CH-14). Proton signals δ of multiple methylene groups. H(1.50, 1.51, 1.52, 1.54, H-3 and H-4), δ H (2.09, 2.12, 2.42, 2.53, H-11 and H-12).

[0045] The compound 13 C-NMR and DEPT spectra indicate signals from 17 carbons, including two methyl carbons in the carbon spectrum. C 21.6 (CH3-15), 32.0 (CH3-16); 4 methylene carbons, each with a δ-carbon atom. C 30.1 (C-2), 32.0 (C-3), 28.0 (C-11), 39.5 (C-12) and one hydroxymethylene carbon 65.1 (C-17); three methine carbons δ C 73.3 (C-1), 61.1 (C-5), 72.6 (C-6), one olefinic methylene carbon signal 124.2 (C-14); one carbonyl carbon 202.8 (C-13).

[0046] By combining HMBC and NOE to determine the linking position and configuration of the substituents, the H-5 (δ) group can be deduced. H 1.27) / H-6(δ H 3.75) / H-9 (δ H 2.55) / CH3-15 (δ H The correlation between 1.10) and H-1(δ) H 3.68) / CH3-16(δ H 1.02) / H-17 (δ H The correlation signals between 4.00 and 3.82 indicate that H-5, H-6, H-9, and CH3-15 are all β-oriented, while H-1, CH3-16, and CH2-17 are α-oriented. The structural formula is determined as follows:

[0047]

[0048] Table 1. Compounds 1 H-NMR and 13 C-NMR data ( 1 H, 500 MHz; 13 C, 125 MHz,

[0049] .

[0050] Example 2: Experiment on in vitro antitumor activity

[0051] 1. Experimental materials

[0052] Tumor cell lines: ARP-1 cells (multiple myeloma (MM) cell line), A549 lung adenocarcinoma cell line, SW620 human colon cancer cell line, Hela human cervical cancer cell line, and SMMC-7721 human liver cancer cell line.

[0053] Test drug: The pentozo monomer compound prepared in Example 1.

[0054] 96-well plates were purchased from Costar; RMPI 1640 medium was purchased from Gibco; DMEM medium was purchased from Gibco; and thiazolyl blue MTT was purchased from Amresco. Newborn calf serum was purchased from Hangzhou Sijiqing Pharmaceutical Co., Ltd.

[0055] The experimental instruments used in the following examples are as follows:

[0056] Experimental instrument: BIO-RAD Bio-Rad 680 microplate reader.

[0057] 2. Experimental Methods

[0058] This invention employs a modified MTT assay to culture multiple myeloma (MM) cell line ARP-1, lung adenocarcinoma cell line A549, human colon cancer cell line SW620, human cervical cancer cell line HeLa, and human liver cancer cell line SMMC-7721 in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin in a 37°C, 5% CO2 incubator. Cells are then digested with 0.25% trypsin and 0.02% EDTA, passaged, and then in logarithmic growth phase. Cells are then digested with trypsin and a cell suspension is prepared using RPMI-1640 medium containing 10% fetal bovine serum to achieve a cell concentration of 1 × 10⁻⁶ cells / mL. 5 Approximately [number] cells / ml were then seeded into 96-well plates, with 100 μl of cell suspension added to each well. Three replicates were set up for each group. The test drug (the pentosan monomer compound prepared in Example 1) was diluted with incomplete culture medium to different desired concentration gradients, and 100 μl of the corresponding drug-containing medium was added to each well. A negative control group without drug was also set up. The plates were incubated at 37°C and 5% CO2 for 48 h. Freshly prepared MTT reagent (5 mg / ml) was added to each well, and the plates were incubated at 37°C and 5% CO2 for another 4 h. The plates were then centrifuged, and 150 μl of DMSO was added to each well. The OD value of each well was then measured at 570 nm using a microplate reader. After data processing, cell growth curves were plotted with the concentration of each group on the x-axis and cell viability on the y-axis. The IC50 of the test drug in each group was calculated using the Reed-Muench method. 50 value.

[0059] 3. Experimental Results

[0060] The specific experimental results of the inhibition rate of the monomeric compounds of the present invention against various tumor cells are shown in Table 1 below.

[0061] Table 1. Inhibitory effect of monomeric compounds on tumor cells (IC50) 50 value)

[0062] .

[0063] The experimental results in Table 1 above show that the novel monomeric compounds isolated in this invention have good anti-proliferative activity against multiple myeloma (MM) cell line ARP-1, human cervical cancer cell line HeLa, and lung adenocarcinoma cell line A549, and have the potential to be developed into anti-tumor agents.

Claims

1. A novel pentophore compound with antitumor activity, characterized in that, The structural formula of the compound is as follows: 。 2. The method for preparing the novel pentophore compound with antitumor activity according to claim 1, characterized in that, Includes the following steps: (1) Weigh the dried root bark of *Pteris vittata*, pulverize it appropriately, add 6-15 times the volume of ethanol, and extract by continuous reflux 1-3 times, 1-2 hours each time. Combine the extracts, concentrate them, add sulfuric acid of a certain concentration to the concentrate and hydrolyze it in a water bath for 1-3 hours. After hydrolysis, filter it, wash it with water until neutral, and dry it to obtain the hydrolysate. Take the hydrolysate and add 6-12 times the volume of chloroform to extract by continuous reflux 1-3 times, 0.5-2 hours each time. After filtration, collect the filtrate, combine the filtrates, concentrate them under reduced pressure, and obtain the chloroform extract. (2) Take the chloroform extract from step (1) and perform silica gel column chromatography at normal pressure. Elute sequentially with petroleum ether-ethyl acetate eluent at volume ratios of 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4, with each gradient eluent being 4 to 6 column volumes. After thin-layer chromatography spot analysis, combine the same eluents, concentrate, and obtain 6 eluent fractions LF-1 to LF-6. (3) After dry processing, the fraction LF-2 obtained in step (2) is subjected to silica gel column chromatography again. The fraction is eluted sequentially with trichloromethane-methanol in volume ratios of 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4:1:8, with each gradient eluting for 3 to 6 column volumes. After thin-layer chromatography spot analysis, the same eluents are combined and concentrated to obtain four eluent fractions BLZ-1 to BLZ-4. (4) The BLZ-4 eluent obtained in step (3) was separated and purified by semi-preparative high performance liquid chromatography. Acetonitrile was used as phase A and water as phase B as the mobile phase, and gradient elution was performed to collect the monomer compound.

3. The method for preparing the novel pentophore compound with antitumor activity according to claim 2, characterized in that, Includes the following steps: (1) Weigh the dried root bark of *Pteris vittata*, pulverize it appropriately, add 15 times the volume of ethanol, and extract it by continuous reflux three times, the first time for 2 hours and the next two times for 1 hour. Combine the three extracts and concentrate them. Add 10% sulfuric acid at a volume ratio of 5:1 to the concentrate and hydrolyze it in a water bath for 2 hours. After hydrolysis, wash it with water until it is neutral and then dry it. Take the hydrolysis product and add 10 times the volume of chloroform to extract it by continuous reflux three times, the first two times for 1 hour and the last time for 0.5 hours. After filtration, collect the filtrate, combine the filtrates, concentrate them under reduced pressure, and obtain the chloroform extract. (2) Take the chloroform extract from step (1) and perform silica gel column chromatography at atmospheric pressure. Elute sequentially with petroleum ether-ethyl acetate in volume ratios of 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4, with each gradient eluting for 5 column volumes. After spot analysis by thin-layer chromatography, combine the same eluents, concentrate, and obtain 6 eluent fractions LF-1 to LF-6. (3) After dry processing, the fraction LF-2 obtained in step (2) was subjected to silica gel column chromatography again. The fraction was eluted sequentially with trichloromethane-methanol in volume ratios of 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 1:4:1:8, with each gradient eluting for 3 column volumes. After thin-layer chromatography spot analysis, the same eluent was combined and concentrated to obtain 4 eluent fractions BLZ-1 to BLZ-4. (4) The BLZ-4 eluent obtained in step (3) was separated and purified by semi-preparative high performance liquid chromatography. Acetonitrile was used as phase A and water as phase B as the mobile phase, and gradient elution was performed to collect the monomer compound.

4. The method for preparing the novel penrose compound according to claim 2 or 3, characterized in that, The chromatographic conditions for semi-preparative high-performance liquid chromatography were: EclipseXDB Prep C 18 A chromatographic column with dimensions of 10 nm, 5 μm, and 10 × 250 mm was prepared. The column temperature was 25–30 °C, and the injection volume was 40 μL. Acetonitrile (phase A) and water (phase B) were used as the mobile phase. Gradient elution was performed: 0–5 min, 80% A; 5–12 min, 40% A; 12–21 min, 20% A; 21–40 min, 45% A; flow rate was 3 mL / min; and the detection wavelength was 248 nm. Monomer compounds were collected during the 21–34 min time period.

5. The use of the compound of claim 1 in the preparation of antitumor drugs.

6. The use of the compound of claim 1 in the preparation of a medicament for treating myeloma, lung adenocarcinoma, colon cancer, cervical cancer, or liver cancer.

7. The application according to claim 5 or 6, wherein the compound is prepared into a pharmaceutical formulation with a pharmaceutically acceptable carrier.

8. The application according to claim 7, wherein the pharmaceutical preparation is a tablet, capsule, granule, pill, injection, mixture, or oral liquid.

9. The use of the compound of claim 1 in combination with other antitumor drugs in the preparation of antitumor drugs.

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