A class of 2-(2-phenylethyl)chromone compounds, their pharmaceutical compositions, and their applications in pharmaceutical manufacturing.

Abstract

This invention provides a class of 2-(2-phenylethyl)chromone compounds and their application in the preparation of anti-inflammatory or antitumor drugs. It belongs to the field of pharmaceutical technology. This invention employs column chromatography and semi-preparative high-performance liquid chromatography for separation, and mass spectrometry and nuclear magnetic resonance spectroscopy for identification. For the first time, 12 2-(2-phenylethyl)chromone compounds were isolated and identified from the ethanol extract of Aquilaria sinensis heartwood. These include five new compounds, PGE63, PGE76, PGE66b, PGE75, and PGE67a, and seven known compounds, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60, and PGE62. Furthermore, it demonstrates for the first time that these compounds possess antitumor and anti-inflammatory activities and can be used to prepare drugs for the prevention or treatment of tumors and inflammation-related diseases. The preparation method of this invention allows for the simple and rapid extraction of the above chemical components from Aquilaria sinensis heartwood.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry and pharmaceutical technology, specifically involving five new 2-(2-phenylethyl)chromone compounds isolated from the heartwood of Aquilaria sinensis and their pharmaceutical uses, as well as seven known 2-(2-phenylethyl)chromone compounds and their new applications in the preparation of anti-inflammatory and anti-tumor drugs. Background Technology

[0002] Cancer is a major public health problem worldwide, and its treatment remains a serious challenge. Chemotherapy, as one of the main methods of cancer treatment, relies heavily on the chemical synthesis of most anticancer drugs. Currently, commonly used cancer drugs suffer from unsatisfactory efficacy, significant side effects, and high costs. Long-term drug use can lead to drug resistance, severely restricting cancer treatment and impacting patients' quality of life. Natural products and their metabolites, with their unique activity, multi-target effects, and synergistic and toxicity-reducing properties, have long been considered the first choice for screening anticancer drugs. Many marketed antitumor drugs, such as paclitaxel and vincristine, are derived from natural products.

[0003] Aquilaria sinensis (Lour.) Spreng., also known as white agarwood, is a plant belonging to the genus Aquilaria in the family Thymelaeaceae. Its resinous heartwood is the traditional Chinese medicine agarwood. Existing research on natural products of agarwood shows that its sesquiterpenes and phenylethyl chromone derivatives are the main compounds related to its medicinal properties. More than 180 sesquiterpenes and more than 240 2-(2-phenylethyl) chromone compounds have been isolated from plants of the genus Aquilaria [W. Li, HQ Chen, H. Wang, et al., Natural products in agarwood and Aquilaria plants: chemistry, biological activities and biosynthesis, Natural Product Reports. 2021, 38(3): 528–565]. However, the anti-inflammatory and antitumor activities of most of these compounds have not yet been systematically explored, and a large number of such compounds remain undiscovered.

[0004] Currently, no (5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome (PGE63) or (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochrome (PGE76) or (5R,6S,7S,8S)- 7,8-Epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome (PGE66b), (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome (PGE75 and (5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy) The five 2-(2-phenylethyl)chromone compounds, namely 2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone (PGE67a), have not been reported to have anti-inflammatory or antitumor activities. Furthermore, seven known compounds, (5R,6R,7R,8R)-agaric chromone A (PGE22), (5S,6S,7S,8S)-agaric chromone B (PGE26), (5S,6S,7S,8S)-agaric chromone B (PGE26), and (5S,6S,7S,8S)-agaric chromone A (PGE22), (5S,6S,7S,8S)-agaric chromone B (PGE26), ...3), (5S,6S,7S,8S)-agaric chromone A (PGE24), (5S, The anti-inflammatory and antitumor activities and other medicinal values ​​of 8-chloro-2-(2-phenylethyl)-5,6,7'-trihydroxy-5,6,7,8-tetrahydrochromene (PGE62) have not been publicly reported, and there is a gap in the research on related isolation and preparation methods and medicinal applications. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention aims to provide a dichloromethane extract of 2-(2-phenylethyl) chromone compounds, specifically (5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone (PGE63) and (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl]-5,6,7,8-tetrahydrochromone (PGE63), from the PXS272 fraction of the heartwood extract of *Aquilaria sinensis* (Lour.) Spreng. Hydroxychrome (PGE76), (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome (PGE66b), (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome (PGE75 and (5R,6S,7R,8R)- 7,8-Epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene (PGE67a) (the above 5 compounds are the first discovery from Aquilaria species and belong to the 2-(2-phenylethyl)chromene class), their drug combinations and preparation methods, and their applications in the preparation of anti-inflammatory and antitumor drugs. Also provided are 7 known compounds (5R, 6R, 7R, 8R)-oxidized aquilaria chromene A (PGE2) 2) Application of (5S,6S,7S,8S)-oxidized agarwood chromone B (PGE26), (5S,6S,7S,8S)-oxidized agarwood chromone C (PGE21), tetrahydroagarwood chromone M (PGE34), tetrahydroagarwood chromone K (PGE57), tetrahydroagarwood chromone L (PGE60), and 8-chloro-2-(2-phenylethyl)-5,6,7,-trihydroxy-5,6,7,8-tetrahydrochromone (PGE62) in the preparation of anti-inflammatory and antitumor drugs.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] The following structural formulas represent 2-(2-phenylethyl)chromone compounds PGE63, PGE76, PGE66b, PGE75, and PGE67a, wherein:

[0008] PGE63 is (5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone {(5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone}.

[0009] PGE76 is (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone.

[0010] PGE66b is (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone {(5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone}.

[0011] PGE75 is (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone.

[0012] PGE67a is (5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone {(5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone}. .

[0013] The preparation methods of the 2-(2-phenylethyl)chromone compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a include the following steps:

[0014] I. Extraction of dichloromethane fraction PXS272 from the heartwood of *Aquilaria sinensis*: The heartwood of *Aquilaria sinensis* was taken, pulverized into powder, and then 90% ethanol was added. The mixture was ultrasonically extracted in a 60 °C water bath for 1 hour, repeated 8 times. The extract was recovered under reduced pressure and concentrated to obtain the ethanol extract (number PXS264). The crude extract was dissolved in an appropriate amount of water and extracted 3-5 times in sequence with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction (number: PXS272), ethyl acetate fraction, n-butanol fraction, and water fraction, respectively.

[0015] II. PGE63, PGE76, PGE66b, and PGE75 were isolated and extracted from the dichloromethane extract PXS272.

[0016] (1) PXS272 was separated by silica gel column chromatography, with petroleum ether-ethyl acetate ratio of 20:1-0:1, to obtain 12 fractions including Fr.B1-Fr.B12;

[0017] (2) The Fr. B10 fraction was subjected to RP-18 reversed-phase silica gel column chromatography with a gradient elution of methanol-water 30%-100%. The fractions were analyzed by TLC plate, and the same fractions were combined to obtain 14 fractions from Fr. B10-1 to Fr. B10-14.

[0018] (3) Fr. B10-2 was recrystallized from methanol to obtain two parts, Fr. B10-2-1 and Fr. B10-2-2.

[0019] (4) Fr. B10-2-2 was separated by elution with a normal-phase silica gel column (200-300 mesh) and a petroleum ether-acetone system at concentration gradients of 9:1, 3:1, 2:1, 1:1, and 0:1 to obtain a total of 12 components, Fr. B10-2-2-1 to Fr. B10-2-2-12.

[0020] (5) Fr. B10-2-2-4 was separated into four fractions, Fr. B10-2-2-4-1 ~ Fr. B10-2-2-4-4, by Sephadex LH-20 gel chromatography;

[0021] (6) Fr. B10-2-2-4-1 was separated by normal phase silica gel column chromatography. After elution in dichloromethane-acetone systems of 20:1, 15:1, 10:1, 5:1, and 3:1, compound PGE57 and the remaining 6 fractions Fr. B10-2-2-4-1-1 ~ Fr. B10-2-2-4-6 were obtained.

[0022] (7) Fr. B10-2-2-4-1-1 was further purified by reverse-phase semi-preparative HPLC to obtain compounds PGE76, PGE75 and PGE66b;

[0023] (8) Fr. B10-2-2-4-1-2 was further purified by semi-preparative HPLC to obtain compounds PGE76, PGE66b, PGE57 and PGE67a;

[0024] (9) Fr. B10-2-2-6 was separated into six fractions, Fr. B10-2-2-6-1 ~ Fr. B10-2-2-6-6, by Sephadex LH-20 gel chromatography. Fr. B10-2-2-6-5 was separated by normal phase silica gel column chromatography. After elution in dichloromethane-acetone system at ratios of 20:1, 15:1, 10:1, 5:1, and 3:1, 2 fractions, Fr. B10-2-2-6-5-1 ~ B10-2-2-6-5-12 fractions were obtained. Fr. B10-2-2-6-5-1-2 was further purified by reversed-phase semi-preparative HPLC to obtain compound PGE63.

[0025] Among them, (7) the semi-preparative high performance liquid chromatography is performed using an Agilent 1200 liquid chromatograph with a Chiral CD-PH column, the column size is 4.6 × 250 mm, the eluent is methanol-water in a ratio of 60:40, and the flow rate is 1 mL / min; (8) the semi-preparative high performance liquid chromatography is performed using an Agilent 1200 liquid chromatograph with a Chiral CD-PH column, the column size is 4.6 × 250 mm, the eluent is n-hexane-isopropanol in a ratio of 80:20, and the flow rate is 1 mL / min; (9) the semi-preparative high performance liquid chromatography is performed using an Agilent 1200 liquid chromatograph with a Chiral CD-PH column, the column size is 4.6 × 250 mm, the eluent is methanol-water in a ratio of 90:10, and the flow rate is 1 mL / min.

[0026] This invention involves a total of 12 novel and known compounds, the structural formulas of which are shown below:

[0027]

[0028] The preparation methods of these 12 compounds are as follows: Dichloromethane extract PXS272 was extracted from the heartwood of *Aquilaria sinensis*: 1.59 kg of *Aquilaria sinensis* heartwood was pulverized into powder. After pulverization, 90% ethanol was added, and the mixture was ultrasonically extracted in a 60 °C water bath for 1 hour. This process was repeated 8 times. The extract was recovered under reduced pressure and concentrated to obtain the ethanol extract (PXS264). The crude extract was dissolved in an appropriate amount of water and extracted 3-5 times sequentially with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction (PXS272), ethyl acetate fraction, n-butanol fraction, and water fraction, respectively.

[0029] (1) PXS272 was separated by silica gel column chromatography, petroleum ether-ethyl acetate (20:1-0:1), to obtain 12 fractions including Fr.B1-Fr.B12;

[0030] (2) The Fr. B10 fraction was subjected to RP-18 reversed-phase silica gel column chromatography with methanol-water (30%-100%) gradient elution. The fractions were analyzed by TLC plate, and the same fractions were combined to obtain 14 fractions from Fr. B10-1 to Fr. B10-14.

[0031] (3) Fr. B10-2 was recrystallized from methanol to obtain two parts: Fr. B10-2-1 and Fr. B10-2-2;

[0032] (4) Fr. B10-2-1 was subjected to Sephadex LH-20 gel chromatography (MeOH) to obtain compounds PGE21 and Fr. B10-2-1-2;

[0033] (5) Fr. B10-2-1-2 was separated by normal phase silica gel column chromatography (H silica gel) and eluted with a gradient of dichloromethane-ethyl acetate system (15:1) to obtain compound PGE34;

[0034] (6) Fr. B10-2-2 was separated by elution with a normal-phase silica gel column (200 ~ 300 mesh) and a petroleum ether-acetone system (9:1, 3:1, 2:1, 1:1, 0:1) concentration gradient to obtain a total of 12 components, Fr. B10-2-2-1 ~ Fr. B10-2-2-12;

[0035] (7) Fr. B10-2-2-4 was separated into four fractions, Fr. B10-2-2-4-1 ~ Fr. B10-2-2-4-4, by Sephadex LH-20 gel chromatography (MeOH);

[0036] (8) Fr. B10-2-2-4-1 was separated by normal phase silica gel column chromatography (H silica gel). After elution with dichloromethane-acetone system (20:1, 15:1, 10:1, 5:1, 3:1), compound PGE57 and the remaining 6 fractions Fr. B10-2-2-4-1-1 ~ Fr. B10-2-2-4-6 were obtained.

[0037] (9) Fr. B10-2-2-4-1-1 was further purified by reverse-phase semi-preparative HPLC to obtain compounds PGE76, PGE75 and PGE66b;

[0038] (10) Fr. B10-2-2-4-1-2 was further purified by semi-preparative HPLC to obtain compounds PGE76, PGE66b, PGE57 and PGE67a;

[0039] (11) Fr. B10-2-2-5-4 was recrystallized from methanol to obtain compound PGE60;

[0040] (12) Fr. B10-2-2-6 was separated into six fractions, Fr. B10-2-2-6-1 ~ Fr. B10-2-2-6-6, by Sephadex LH-20 gel chromatography (MeOH). Fr. B10-2-2-6-5 was separated by normal phase silica gel column chromatography (H silica gel), and after elution with dichloromethane-acetone system (20:1, 15:1, 10:1, 5:1, 3:1), 2 fractions, Fr. B10-2-2-6-5-1 ~ B10-2-2-6-5-1, were obtained. Fr. B10-2-2-6-5-1-2 was further purified by reversed-phase semi-preparative HPLC to obtain compounds PGE63 and PGE22. Fr. E10 was further purified by reversed-phase semi-preparative HPLC to obtain compound PGE62.

[0041] (13) Fr. B10-3 was separated into six fractions, Fr. B10-3-1 to Fr. B10-3-6, by Sephadex LH-20 gel chromatography (MeOH). Fr. B10-3-3 was recrystallized from methanol to give compound PGE26;

[0042] Among them, (9) the semi-preparative high-performance liquid chromatography (HPLC) was performed using an Agilent 1200 HPLC system with a Chiral CD-PH column, a column size of 4.6 × 250 mm, and methanol-water as the eluent in a ratio of 60:40, with a flow rate of 1 mL / min; (10) the semi-preparative HPLC was performed using an Agilent 1200 HPLC system with a Chiral CD-PH column, a column size of 4.6 × 250 mm, and n-hexane-isopropanol as the eluent in a ratio of 80:20, with a flow rate of 1 mL / min; (12) the semi-preparative HPLC was performed using an Agilent 1200 HPLC system with a Chiral CD-PH column, a column size of 4.6 × 250 mm, and methanol-water as the eluent in a ratio of 90:10, with a flow rate of 1 mL / min. mm, the eluent is methanol-water in a ratio of 60:40, and the flow rate is 1 mL / min.

[0043] In this invention, 12 compounds were isolated from PXS272, and their chemical structures were determined by mass spectrometry (MS) and nuclear magnetic resonance (NMR).

[0044] The present invention also provides pharmaceutical compositions comprising therapeutically effective amounts of the compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a, and pharmaceutically acceptable carriers.

[0045] The preparation method of the pharmaceutical composition involves first obtaining compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a using the aforementioned preparation method, and then taking one or any of them and adding a pharmaceutically acceptable carrier.

[0046] Further, the use of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, or the pharmaceutical compositions thereof in the preparation of medicaments for the prevention and / or treatment of leukemia, lung cancer, breast cancer, and colon cancer is provided.

[0047] And the use of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a or the pharmaceutical composition thereof in the preparation of anti-inflammatory drugs.

[0048] The present invention also provides the use of compounds PGE22 [(5R,6R,7R,8R)-agaricone A], PGE26 [(5S,6S,7S,8S)-agaricone B], PGE21 [(5S,6S,7S,8S)-agaricone C], PGE34 (tetrahydroagaricone M), PGE57 (tetrahydroagaricone K), PGE60 (tetrahydroagaricone L), PGE62 [8-chloro-2-(2-phenylethyl)-5,6,7'-trihydroxy-5,6,7,8-tetrahydroagaricone] or pharmaceutical compositions thereof, as shown in the following structural formulas, in the preparation of antitumor drugs and in the preparation of anti-inflammatory drugs.

[0049] .

[0050] And their application in the preparation of drugs for the prevention and / or treatment of leukemia, lung cancer, breast cancer, and colon cancer.

[0051] The dichloromethane extract (PXS272) of the heartwood of *Aquilaria sinensis* and the 2-(2-phenylethyl)chromone compound isolated and identified therefrom exhibit significant antitumor and anti-inflammatory activities. Specific technical effects are as follows:

[0052] 1. Antitumor activity: In tests on five tumor cell lines—HL-60 leukemia, K562 leukemia, A549 lung cancer, MDA-MB-231 triple-negative breast cancer, and SW480 colon cancer—PGE21, PGE22, and PGE26 exhibited potent and broad-spectrum cytotoxic activity, with IC50 values ​​of [missing data]. 50 All values ​​were less than 7 μM, indicating activity close to or superior to the positive control doxorubicin; among them, PGE22 showed the best activity, with an IC50 value of less than 7 μM against SW480 colon cancer cells. 50 The value was only 0.18 μM, with an IC50 value of only 0.18 μM for leukemia K562, HL-60 cells and MDA-MB-231 cells. 50 All values ​​were less than 1 μM. PGE60 showed inhibitory activity against all five tumor cell types, and its IC50 values ​​against HL-60 and SW480 cells were also less than 1 μM. 50 The value is less than 10 μM, and it is the only compound tested that is active against triple-negative breast cancer MDA-MB-231 cells (IC50). 50 = 20.63μM); PGE57 showed significant inhibitory activity against HL-60, A549, and SW480 cells, IC50 = 20.63μM. 50 The values ​​were all less than 20 μM; PGE67a and PGE62 showed moderate and weak inhibitory activity against SW480 cells, respectively, while PGE63, PGE76, PGE66b and PGE75 had no obvious cytotoxic effects.

[0053] 2. Validation of antitumor universality: In tests on six newly added tumor cell types—HepG2 liver cancer, HCT116 colon cancer, Caco2 colon cancer, SK-OV-3 ovarian cancer, MG-63 osteosarcoma, and U251 glioma—PGE21, PGE22, and PGE26 maintained potent and broad-spectrum inhibitory activity, further confirming their universal antitumor efficacy. Among them, PGE21 showed the best activity, with an IC50 concentration of 12.5% ​​for MG-63 and HCT116 cells. 50 The values ​​were 0.56 μM and 1.02 μM, respectively, and the IC50 values ​​for the other four cell types were [not specified]. 50 The values ​​were all less than 4 μM; PGE22 and PGE26 activities were stable, and the IC50 values ​​for 6 cell types were all less than 4 μM. 50 The values ​​were all less than 6 μM, and PGE26 showed outstanding inhibitory effects on Caco2, MG-63, and U251 cells, while PGE22 exhibited excellent activity against HCT116 and MG-63 cells.

[0054] 3. Anti-inflammatory activity: Among the tested compounds, all except PGE63 exhibited certain anti-inflammatory effects. PGE34, PGE62, PGE67a, and PGE57 showed higher anti-inflammatory activity than the positive control drug L-NMMA. PGE67a, PGE57, and PGE60 demonstrated strong NO production inhibition activity, with IC50 values ​​of [missing information]. 50 The values ​​were 12.06 μM, 10.41 μM, and 3.55 μM, respectively. Particularly noteworthy were the significant anti-inflammatory effects of PGE21, PGE22, and PGE26, with PGE22 and PGE26 exhibiting significantly higher inhibitory activity against NO production than the positive control, IC50... 50 The values ​​range from 0.60 to 1.01 μM.

[0055] Compared with the prior art, the present invention has the following advantages:

[0056] 1. This invention elucidates the structures of five novel compounds for the first time. Specifically, it isolates and identifies five novel sesquiterpene compounds from the heartwood of *Aquilaria sinensis*, namely PGE63{(5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene}, PGE76{(5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5- Methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochrome]}, PGE66b{(5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome}, PGE75{(5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl] [Ethyl]-5,6,7,8-tetrahydrochrome ketone} and PGE67a{(5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome ketone}, as well as 7 known compounds with the best activity, PGE22[(5R,6R,7R,8R)-oxidized agaric chrome ketone A], PGE26[(5S,6S,7S,8S)- The compounds PGE21 ((5S,6S,7S,8S)-oxidized agaric chromone B), PGE34 (tetrahydroagaric chromone M), PGE57 (tetrahydroagaric chromone K), PGE60 (tetrahydroagaric chromone L), and PGE62 (8-chloro-2-(2-phenylethyl)-5,6,7'-trihydroxy-5,6,7,8-tetrahydrochromone) are original and novel, enriching the chemical composition library of Aquilaria species.

[0057] 2. Novel antitumor and anti-inflammatory applications of the known compounds PGE22 [(5R,6R,7R,8R)-agaric chromone A], PGE26 [(5S,6S,7S,8S)-agaric chromone B], PGE21 [(5S,6S,7S,8S)-agaric chromone C], PGE34 (tetrahydroagaric chromone M), PGE57 (tetrahydroagaric chromone K), PGE60 (tetrahydroagaric chromone L), and PGE62 [8-chloro-2-(2-phenylethyl)-5,6,7'-trihydroxy-5,6,7,8-tetrahydrochromone] were discovered for the first time, filling the research gaps related to these seven compounds.

[0058] 3. For the first time, compounds (5R,6R,7R,8R)-agaric chromone A (PGE22), (5S,6S,7S,8S)-agaric chromone B (PGE26), and (5S,6S,7S,8S)-agaric chromone C (PGE21) were discovered to possess broad-spectrum cytotoxic activity against 11 cancer cell lines (HL-60, K562, A549, MDA-MB-231, SW480, HepG2, HCT116, Caco2, SK-OV-3, MG-63, and U251), with significant IC50 values. 50 With a value between 0.18 and 6.35 μM, its potential for medicinal development far exceeds that of existing compounds of the same type.

[0059] 4. Significant differences in compound activity, offering advantages for diversified development: Compounds PGE67a and PGE62 are selective for colorectal cancer SW480 and can serve as lead compounds for colorectal cancer-specific targeted drugs, avoiding the non-specific toxic side effects of broad-spectrum drugs. Broad-spectrum active compounds PGE21, PGE22, and PGE26 can be developed into universal anti-tumor drugs, adaptable to the treatment needs of multiple tumor types.

[0060] 5. This study marks the first discovery of novel anti-inflammatory and antitumor effects in the known compounds PGE22, PGE26, and PGE60. The anti-inflammatory activity of these three compounds is significantly superior to that of the positive control L-NMMA, and they also exhibit good antitumor activity, expanding the application scope of this type of compound. Attached Figure Description

[0061] Figure 1 Schematic diagram of the chemical structure of PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE22, PGE26, PGE21, PGE34, PGE57, PGE60 and PGE62.

[0062] Figure 2 Key two-dimensional NMR correlation diagrams of compounds PGE63, PGE76, PGE66b, PGE75, and PGE67a;

[0063] Figure 3 Crystal diffraction structures of compounds PGE21, PGE22, PGE26, PGE34, and PGE57;

[0064] Figure 4 Calculated and experimental electron circular dichroism (ECD) spectra of compounds PGE63, PGE76, PGE66b, PGE75, and PGE67a.

[0065] Figure 5Schematic diagrams of the chemical structures of compounds PGE63, PGE76, PGE66b, PGE75, and PGE67a. Detailed Implementation

[0066] The following description, in conjunction with the accompanying drawings, uses embodiments of the present invention to further illustrate the substantive content of the invention, but does not limit the invention thereto.

[0067] Example 1

[0068] Preparation of PXS272:

[0069] 1.59 kg of agarwood heartwood was collected, pulverized into powder, and then extracted with 90% ethanol. The mixture was ultrasonically extracted in a 60 °C water bath for 1 hour, repeated 8 times. The extract was recovered under reduced pressure and concentrated to obtain the ethanol extract (PXS264). The crude extract was dissolved in an appropriate amount of water and extracted 3-5 times sequentially with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction (PXS272), ethyl acetate fraction, n-butanol fraction, and water fraction, respectively.

[0070] Example 2

[0071] PXS272 was prepared in fractions using silica gel column chromatography, and the chemical composition of 2-(2-phenylethyl)chromone compounds was separated:

[0072] (1) PXS272 was separated by silica gel column chromatography, petroleum ether-ethyl acetate (20:1-0:1), to obtain 12 fractions including Fr.B1-Fr.B12;

[0073] (2) The Fr. B10 fraction was subjected to RP-18 reversed-phase silica gel column chromatography with methanol-water (30%-100%) gradient elution. The fractions were analyzed by TLC plate, and the same fractions were combined to obtain 14 fractions from Fr. B10-1 to Fr. B10-14.

[0074] (3) Fr. B10-2 was recrystallized from methanol to obtain two parts: Fr. B10-2-1 and Fr. B10-2-2;

[0075] (4) Fr. B10-2-1 was subjected to Sephadex LH-20 gel chromatography (MeOH) to obtain compounds PGE21 and Fr. B10-2-1-2;

[0076] (5) Fr. B10-2-1-2 was separated by normal phase silica gel column chromatography (H silica gel) and eluted with a gradient of dichloromethane-ethyl acetate system (15:1) to obtain compound PGE34;

[0077] (6) Fr. B10-2-2 was separated by elution with a normal-phase silica gel column (200 ~ 300 mesh) and a petroleum ether-acetone system (9:1, 3:1, 2:1, 1:1, 0:1) concentration gradient to obtain a total of 12 components, Fr. B10-2-2-1 ~ Fr. B10-2-2-12;

[0078] (7) Fr. B10-2-2-4 was separated into four fractions, Fr. B10-2-2-4-1 ~ Fr. B10-2-2-4-4, by Sephadex LH-20 gel chromatography (MeOH);

[0079] (8) Fr. B10-2-2-4-1 was separated by normal phase silica gel column chromatography (H silica gel). After elution with dichloromethane-acetone system (20:1, 15:1, 10:1, 5:1, 3:1), compound PGE57 and the remaining 6 fractions Fr. B10-2-2-4-1-1 ~ Fr. B10-2-2-4-6 were obtained.

[0080] (9) Fr. B10-2-2-4-1-1 was further purified by reverse-phase semi-preparative HPLC to obtain compounds PGE76, PGE75 and PGE66b;

[0081] (10) Fr. B10-2-2-4-1-2 was further purified by semi-preparative HPLC to obtain compounds PGE76, PGE66b, PGE57 and PGE67a;

[0082] (11) Fr. B10-2-2-5-4 was recrystallized from methanol to obtain compound PGE60;

[0083] (12) Fr. B10-2-2-6 was separated into six fractions, Fr. B10-2-2-6-1 ~ Fr. B10-2-2-6-6, by Sephadex LH-20 gel chromatography (MeOH). Fr. B10-2-2-6-5 was separated by normal phase silica gel column chromatography (H silica gel), and after elution with dichloromethane-acetone system (20:1, 15:1, 10:1, 5:1, 3:1), 2 fractions, Fr. B10-2-2-6-5-1 ~ B10-2-2-6-5-1, were obtained. Fr. B10-2-2-6-5-1-2 was further purified by reversed-phase semi-preparative HPLC to obtain compounds PGE63 and PGE22. Fr. E10 was further purified by reversed-phase semi-preparative HPLC to obtain compound PGE62.

[0084] (13) Fr. B10-3 was separated into six fractions, Fr. B10-3-1 to Fr. B10-3-6, by Sephadex LH-20 gel chromatography (MeOH). Fr. B10-3-3 was recrystallized from methanol to give compound PGE26;

[0085] Among them, (9) the semi-preparative high-performance liquid chromatography (HPLC) was performed using an Agilent 1200 HPLC system with a Chiral CD-PH column, a column size of 4.6 × 250 mm, and methanol-water as the eluent in a ratio of 60:40, with a flow rate of 1 mL / min; (10) the semi-preparative HPLC was performed using an Agilent 1200 HPLC system with a Chiral CD-PH column, a column size of 4.6 × 250 mm, and n-hexane-isopropanol as the eluent in a ratio of 80:20, with a flow rate of 1 mL / min; (12) the semi-preparative HPLC was performed using an Agilent 1200 HPLC system with a Chiral CD-PH column, a column size of 4.6 × 250 mm, and methanol-water as the eluent in a ratio of 90:10, with a flow rate of 1 mL / min. mm, the eluent is methanol-water in a ratio of 60:40, and the flow rate is 1 mL / min.

[0086] Example 3

[0087] Compounds PGE63{(5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromen}, PGE76{(5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl]-5,6,7,8-tetrahydrochromen}, PGE66b{(5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromen}, and PGE75{(5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7 Spectroscopic data and structural analysis of the following compounds: PGE67a[(5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene], PGE22[(5R,6R,7R,8R)-oxidized agarwood chromene A], PGE26[(5S,6S,7S,8S)-oxidized agarwood chromene B], PGE21[(5S,6S,7S,8S)-oxidized agarwood chromene C], PGE34(tetrahydroagarwood chromene M), PGE57(tetrahydroagarwood chromene K), PGE60(tetrahydroagarwood chromene L), and PGE62[8-chloro-2-(2-phenylethyl)-5,6,7,-trihydroxy-5,6,7,8-tetrahydrochromene].

[0088] Spectral analysis of compound PGE63:

[0089] Compound PGE63 is a pale yellow amorphous solid. Its molecular formula, determined by high-resolution mass spectrometry, is C63. 18 H 16 O6, quasi-molecular ion peak m / z 351.084 0 [M + Na] + (Calculated value C) 18 H 16 NaO6, 351.080 5), unsaturation 10. ¹H NMR data (Table 1) show: a hydroxyl proton signal δ H 5.52 (1H, s), a methoxyl signal δ H 3.87 (3H,s), and an ABX spin system [δ H 6.85 (1H, d, J = 8.0 Hz), 6.68 (1H, dd, J = 8.0, 1.7 Hz), 6.65 (1H, d, J = 1.7 Hz)], this is the characteristic signal of the 1,2,4-trisubstituted benzene ring. ¹³C NMR data (Table 1) show: one methoxy group (δ...C 56.1), a 3,4-disubstituted phenethyl (δ C 146.9, 144.7, 131.2, 114.7, 110.9, 121, 35.8, 32.8) and a diepoxytetrahydrochromene unit (δ C 177.8, 168.4, 161.4, 121.0, 114.3, 48.8, 47.9, 47.0, 46.6) [16,17]. Comparing its ¹H and ¹³C NMR data with literature reports, it was found that it was similar to the chromone derivatives (5R,6R,7R,8R)-agarwood chromone A (PGE22), (5S,6S,7S,8S)-agarwood chromone B (PGE26) [T. Yagura, N. Shibayama, M. Ito, F. Kiuchi, G. Honda, Three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding, Tetrahedron Letters. 2005, 46(25): 4395–4398.] and (5S,6S,7S,8S)-agarwood chromone C (PGE21) [Y. Yang, WL Mei, WH Dong, W. Li, J.Wang, HN Wang, ZB Cheng, HF Dai, Bioactive components from agarwood originating from Aquilaria crassna, Journal of Tropical and SubtropicalBotany. 2016, 24(5), 577–583 and Q. He, DB Hu, L. Zhang, MY Xia, H. Yan, XN Li, JF Luo, YS Wang, JH Yang, YH Wang, Neuroprotective compounds from the resinous heartwood of Aquilaria sinensis, Phytochemistry. 2021, 181,112554.] are highly similar, indicating that compound PGE63 has the same 5,6:7,8-bisepoxytetrahydrochromone core.

[0090] Through COSY and HMBC related ( Figure 2 The planar structure was further clarified. The H-5 / H-6 / H-7 / H-8, H-5' / H-6', and H2-7' / H2-8' structural fragments were constructed by the H-1H-1H COSY correlation. HMBC related: H-3 with C-4, C-4a and C-8'; H-5 with C-4, C-7 and C-8a; H-8 with C-4a; H-2' with C-4'; H-5' with C-1' and C-3'; H-6' with C-2' and C-4'; H2-7' with C-2, C-2' and C-6'; H2-8' with C-3 and C-1'; 3'-OMe with C-3'; 4'-OH with C-5', thus determining the planar structure as 5,6:7,8-bisepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene. The relative configuration of the 5,6:7,8-bisepoxytetrahydrochromene unit was determined by analyzing the proton coupling constant. The observed ortho-coupling constant J 5，6 =3.5 Hz, J 6，7 = 3.3 Hz and J 7，8 = 3.3 Hz, consistent with typical reported values ​​for cis-epoxide rings in similar systems [e.g., cis-naphthalene-1,2:3,4-dioxide (J 1，2 = J 3，4 = 3.6 Hz, J 2，3 = 3.0 Hz)], and with trans analogs [e.g., trans-naphthalene dioxide (J 1，2 = J 3，4 = 4.2 Hz, J 2，3= 1.8 Hz)][ Q. He, DB Hu, L. Zhang, MY Xia, H. Yan, XN Li, JF Luo, YS Wang, JH Yang, YH Wang, Neuroprotective compounds from the resinous heartwood of Aquilaria sinensis, Phytochemistry. 2021, 181, 112554 and E. Vogel, HH Klug, M. Schäfer‐Ridder, Syn‐und anti‐Naphtahalin‐1,2;3,4‐dioxid, Angewandte Chemie. 1976, 88(8), 268–269.] are significantly different. This inference was further confirmed by comparison with the ¹H NMR data of compounds PGE22, PGE26 and PGE21, whose absolute configurations have been clearly determined by single-crystal X-ray diffraction ( Figure 3 ).

[0091] The absolute configuration of compound PGE63 was derived by electronic circular dichroism (ECD) calculations. Its experimental ECD spectrum and the calculated ECD spectrum of the (5S,6S,7S,8S)-enantiomers are compared. Figure 4 The configuration of compound PGE63 is thus determined to be (5S,6S,7S,8S)-5,6:7,8-bisepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome.

[0092] Spectral data for {(5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene} (PGE63):

[0093] {(5S,6S,7S,8S)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene} (PGE63), pale yellow amorphous solid; [α]20 D +5 (c = 0.1, MeOH); UV (MeOH)λ max (log ε) 306 (2.06), 279 (2.77), 248 (3.04), 218 (3.44), 202 (3.77); ECD (1.43 mM, MeOH) λ max(Δε) 291 (+0.18), 233 (+0.12), 204 (–0.79); 1 H and 13 C NMR data, see Table 1; ESIMS (positive) m / z 351 [M + Na] + ;HRESIMS (positive) m / z 351.084 0 [M + Na] + (calcd for C 18 H 16 O6Na, m / z 351.084 5).

[0094] Spectral analysis of compound PGE76:

[0095] Compound PGE76 is a pale yellow amorphous solid. It is bound to HRESIMS (m / z 337.1044 [M+Na)). + Calculated value C 18 H 18 NaO (337.1046) and its ¹H and ¹³C NMR data (Table 1) were used to determine its molecular formula as C. 18 H 18 O5 (unsaturation 10). Detailed NMR analysis showed a methoxy group signal [δ]. H 3.55 (3H, s); δ C 61.0], a phenylethyl structural unit [δ H 7.31 (2H, m), 7.24 (1H, m), 7.19 (2H, m), 2.99 (2H, m), 2.86 (2H, m); δ C [128.8×2, 128.2×2, 126.7, 35.1, 32.9] and a 5,6,7,8-tetrahydrochromene unit containing an epoxy group (δ C57.2 and 50.1). The NMR data of compound PGE76 are highly similar to those of tetrahydrochromone M (PGE34) and tetrahydrochromone K (PGE57) [G. Liao, W.-L. Mei, F.-D. Kong, W. Li, J.-Z.Yuan, H.-F. Dai, 5,6,7,8-Tetrahydro-2-(2-phenylethyl)chromones from artificial agarwood of Aquilaria sinensis and their inhibitory activity against acetylcholinesterase, Phytochemistry. 2017, 139, 98–108.], confirming that it has the same 7,8-epoxytetrahydrochromone skeleton.

[0096] Key HMBC related ( Figure 2 Display: δ H 3.55 (5-OMe) is associated with C-5 (δC 69.6) and H-5 (δ H The correlation between 4.70 and the methoxy carbon (δC 61.0) indicates the presence of a methoxy group at the C-5 position. This is based on the COSY correlation and other HMBC correlations ( Figure 2 The planar structure of compound PGE76 was determined to be 7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone. The coupling constants of H-5 to H-8 in compound PGE76 are consistent with those of compounds PGE34 and PGE57 [G. Liao, W.-L. Mei, F.-D. Kong, W. Li, J.-Z. Yuan, H.-F. Dai, 5,6,7,8-Tetrahydro-2-(2-phenylethyl)chromones from artificial agarwood of Aquilaria sinensis and their inhibitory activity against acetylcholinesterase, Phytochemistry. 2017, 139, 98–108.], indicating that these compounds have the same relative configuration. The experimental ECD spectrum is consistent with the calculated curve of the (5R,6S,7S,8S)-isomer ( Figure 4The agreement is good, and based on this, the absolute configuration of compound PGE76 is determined to be (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone.

[0097] Spectral data of compound (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl]-5,6,7,8-tetrahydrochromene (PGE76):

[0098] (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochrome (PGE76), pale yellow amorphous solid; [α]25D–7 (c = 0.1, MeOH); UV (MeOH)λ max (log ε) 252(3.52), 204 (3.80); ECD (0.83 mM, MeOH) λ max (Δε) 305 (+0.27), 238 (–1.11), 195 (+1.93); 1 H and 13 C NMR data, see Table 4; ESIMS (positive) m / z 337 [M + Na] + ;HRESIMS (positive) m / z 337.104 4 [M + Na] + (calcd for C 18 H 18 O5Na, m / z 337.1046).

[0099] Spectral analysis of compound PGE66b:

[0100] Compound PGE66b is a pale yellow amorphous solid. According to HRESIMS (m / z 367.1157 [M+Na]), its molecular structure is... + Calculated value C 19 H 20 Based on the ¹H and ¹³C NMR data (Table 1), its molecular formula was determined to be C6367.1158. 19 H20 O6 (unsaturation 10). ¹H NMR spectrum shows characteristic signals: two methoxy groups (δ¹⁰). H 3.79 and 3.55), an olefin proton (δH 6.16), and a typical AA′BB′ coupling system [δ H The 6.84 (2H, br d, J = 8.7 Hz) and 7.09 (2H, br d, J = 8.7 Hz) values ​​indicate the presence of a 1,4-disubstituted benzene ring. The ¹³C NMR spectrum shows 19 carbon signals, including two methoxy groups (δ¹³C). C 61.0 and 55.3), a 4-substituted phenethyl fragment, and a dihydroxy-5,6,7,8-tetrahydrochromene unit containing an epoxy group (δ C 57.2 and 50.1). Its NMR data are similar to those of compound PGE76, the difference being that compound PGE66b has an additional methoxy group signal (δ). C 55.3).

[0101] Two-dimensional NMR analysis (including HMBC and COSY) was performed. Figure 2 ), and all ¹H and ¹³C NMR signals were fully assigned. Key HMBC correlation: δ H 3.79 (4′-OMe) and C-4′ (δ C 158.4) related, δ H 3.55 (5-OMe) is associated with C-5 (δC69.5) and H-5 (δ H 4.19) and 5-OMe carbon (δ C Related to 61.0), the methoxy substitution at C-4′ and C-5 positions was confirmed. Based on this, the planar structure of compound 6 was determined to be 7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome.

[0102] The coupling constants of H-5 to H-8 in compound PGE66b are consistent with those in compounds PGE76, PGE34, and PGE57, indicating that these compounds have the same relative configuration. The absolute configuration was determined by ECD calculations at the B3LYP / 6-311G(d,p) level. (Calculated ECD spectra of the (5R,6S,7S,8S)-isomers are compared with experimental data. Figure 4The structure matches perfectly. Therefore, the chemical structure of compound PGE66b is determined to be (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone {(5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone, PGE66b}.

[0103] Spectral data of compound (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene (PGE66b):

[0104] The compound (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene (PGE66b) is a pale yellow amorphous solid; [α]25D+15 (c = 0.2, MeOH); UV(MeOH)λ max (logε) 252 (3.92), 222 (4.17), 202 (4.26); ECD (0.33 mM, MeOH) λ max (Δε) 347 (–0.001), 298 (+1.87), 240 (–2.95), 196 (+3.04); 1 H and 13 C NMR data, seeTable 4; ESIMS (positive) m / z 367 [M + Na] + ;HRESIMS (positive) m / z 367.115 7[M + Na] + (calcd for C 19 H 20 O6Na, m / z 367.115 8).

[0105] Spectral analysis of compound PGE75:

[0106] Compound PGE75 is a pale yellow amorphous solid. According to HRESIMS (m / z 367.1159 [M+Na]), it was... + Calculated value C 19 H 20 Based on the ¹H and ¹³C NMR data (Table 2), its molecular formula was determined to be C6367.1158.19 H 20 O6 (unsaturation 10). Detailed NMR analysis revealed characteristic signals from two methoxy groups, one 4-phenylethyl structural unit, and one dihydroxy-5,6,7,8-tetrahydrochromene unit containing an epoxy group. The NMR data for PGE75 were identical to those for PGE66b, indicating that PGE66b, with positive optical rotation, and PGE75, with negative optical rotation, are optical enantiomers. Based on their two-dimensional NMR correlation ( Figure 2 Based on this, it is inferred that the planar structure of PGE75 is 7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome.

[0107] Furthermore, compared to compound PGE66b, the experimental and calculated ECD spectra of compound PGE75 exhibit a mirror Cotton effect (…). Figure 4 This indicates that the two compounds are enantiomer pairs ( Figure 4 Therefore, the chemical structure of compound PGE75 is determined to be (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone {(5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone, PGE75}.

[0108] Spectral data of compound (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene (PGE75):

[0109] (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrome (PGE75), a pale yellow amorphous solid; [α]25D–4 (c = 0.2, MeOH); UV (MeOH)λ max (logε) 252 (3.48), 222 (3.72), 202 (3.80); ECD (1.31 mM, MeOH) λ max (Δε) 347 (+0.06), 298 (–0.29), 240 (+0.92), 196 (–0.78); 1 H and 13C NMR data, see Table 5; ESIMS (positive) m / z 367 [M + Na] + ;HRESIMS (positive) m / z 367.115 9 [M + Na] + (calcd for C 19 H 20 O6Na, m / z 367.115 8).

[0110] Spectral analysis of compound PGE67a:

[0111] Compound PGE67a is a pale yellow amorphous solid. According to HRESIMS (m / z 367.1150 [M+Na]), it was... + Calculated value C 19 H 20 Based on the ¹H and ¹³C NMR data of NaO6 (367.1152) and combined with the ¹H and ¹³C NMR data of CDCl3 and DMSO-d6 (Table 3), its molecular formula was determined to be C. 19 H 20 O6 (unsaturation 10). In CDCl3, J cannot be calculated because both H-6 and H-7 are multiplets. 6，7 Value; while J can be measured in DMSO-d6. 6，7 = 4.1 Hz. Detailed NMR analysis (DMSO-d6) showed: two methoxy groups [δ] H 3.70 (3H, s) and 3.34 (3H, s); δ C 58.3 and 55.0], a 4-substituted phenethyl [δ H 7.10 (1H, br d, J = 8.6 Hz) and 6.84 (2H, br d, J = 8.6 Hz)] and a dihydroxy-5,6,7,8-tetrahydrochromene unit containing an epoxy group (δ C 53.6 and 48.3). The NMR data of compound PGE67a are highly similar to those of compounds PGE66b and PGE75, indicating that compound PGE67a has the same planar structure as 7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene, which is correlated by COSY and HMBC ( Figure 2 This has been further confirmed.

[0112] The ROESY spectrum of compound PGE67a in CDCl3 shows correlation signals for 6-OH / H-7 and 6-OH / H-5. Figure 2This indicates that H-5, 6-OH, and H-7 should be α-oriented, while 5-OMe, H-6, and the epoxy group should be β-oriented. The experimental ECD spectrum of compound PGE67a and the calculated curve of the (5R,6S,7R,8R)-isomer (…) Figure 4 Therefore, compound PGE67a was identified as (5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone.

[0113] Spectral data of compound (5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene (PGE67a):

[0114] The compound (5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromene (PGE67a) is a pale yellow amorphous solid; [α]25D+5 (c = 0.1, MeOH); UV(MeOH)λ max (logε) 252 (3.48), 222 (3.72), 202 (3.78); ECD (0.60 mM, MeOH) λ max (Δε) 308 (–0.18), 235 (+2.06); 1 H and 13 C NMR data, see Table 5; ESIMS (positive) m / z 367 [M + Na] + ;HRESIMS (positive) m / z 367.115 0 [M + Na] + (calcd forC 19 H 20 O6Na, m / z 367.115 2).

[0115] Table 1. Compounds PGE63, PGE76, and PGE66b 1 H NMR and 13 C NMR (500, 126 MHz, CDCl3) data (δ in ppm)

[0116]

[0117] Table 2. Compounds PGE75 and PGE67a 1 H NMR (500M) and 13 C NMR (126 MHz) data (δ inppm)

[0118]

[0119] a CDCl3.

[0120] b DMSO-d6.

[0121] Spectroscopic data of compound PGE22:

[0122] (5R,6R,7R,8R)-oxidoagarochromone A [(5R,6R,7R,8R)-oxidoagarochromone A, PGE22], White needles (MeOH), C 17 H 14 O4; [α]25 D+19 (c 0.3, MeOH); UV (MeOH) λ max (logε) 254 (1.93), 195 (2.45) nm; ECD (3.53 mM, MeOH) λ max (Δε) 297 (–0.18), 258 (+0.24), 197 (+0.61) nm; 1 H NMR (CDCl3, 500 MHz) δ H 7.31 (2H, m, H-3′,5′), 7.23 (1H, m, H-4′), 7.18 (2H, m, H-2′,6′), 6.17 (1H, s, H-3), 4.34 (1H, br d, J = 3.6 Hz, H-5), 3.97 (1H, m, H-7), 3.82 (1H, dd, J = 3.6, 3.0 Hz, H-6), 3.81 (1H, d, J = 3.5 Hz, H-8), 2.98 (2H, m, H2-7′, 2.86 (2H, m, H2-8′); 13 C NMR (CDCl3, 125 MHz) δ C177.6 (C-4), 168.1 (C-2), 161.2 (C-8a), 139.2 (C-1′), 128.8 (C-3′,5′), 128.3 (C-2′,6′), 126.8 (C-4′), 120.8 (C-4a), 114.2 (C-3), 48.7 (C-7), 47.7 (C-8), 46.9 (C-5), 46.5 (C-6), 35.3 (C-8′), 32.9 (C-7′); ESIMS m / z 283 [M + H] + The NMR data is basically consistent with the data in the root literature [T. Yagura, N. Shibayama, M. Ito, F. Kiuchi, G. Honda, Three novel diepoxytetrahydrochromones from agarwood artificially produced by intentionalwounding, Tetrahedron Letters. 2005, 46(25): 4395–4398.]. This study supplements the crystal diffraction data of this compound and determines its absolute configuration as (5R,6R,7R,8R)-oxidoagarochromone A [(5R,6R,7R,8R)-oxidoagarochromone A] ( Figure 3 ).

[0123] PGE22 crystal data: C 17 H 14 O4, M = 282.28, a = 8.9880(5) Å, b = 13.5271(9)Å, c = 22.2582(14) Å, α = 90°, β = 90°, γ = 90°, V = 2706.2(3) Å 3 , T = 150.(2) K, space group P212121, Z = 8, μ (Cu Kα) = 0.814 mm –1 , 26812 reflectionsmeasured, 4963 independent reflections (R int = 0.0584). The final R1 values ​​were 0.0292 (I > 2σ(I)). The final wR (F 2) values ​​were 0.0762 (I > 2σ(I)).The final R1 values ​​were 0.0302 (all data). The final wR (F 2 ) values ​​were0.0769 (all data). The goodness of fit on F 2 was 1.032. Flack parameter =0.04(5). The supplementary crystallographic data can be obtained free ofcharge from the Cambridge Crystallographic Data Center (CCDC) (depositionnumber CCDC 2501088) via http: / / www.ccdc.cam.ac.uk.

[0124] Spectroscopic data of compound PGE26:

[0125] (5S,6S,7S,8S)-oxidoagarochromone B, white flaky crystals (MeOH), C 18 H 16 O5; [α]25 D+1 (c 0.2, MeOH); UV (MeOH) λ max (log ε) 254(2.94), 223 (3.23), 195 (3.63) nm; ECD (0.25 mM, MeOH) λ max (Δε) 294 (+1.94), 256 (–2.01), 201 (–5.54) nm; 1 H NMR (CDCl3, 500 MHz) δ H 7.07 (2H, m, H-2′,6′), 6.82 (2H, m, H-3′, 5′), 6.14 (1H, s, H-3), 4.31 (1H, br d, J = 3.5 Hz, H-5), 3.95 (1H, m, H-7), 3.81 (2H, m, H-6,8), 3.77 (3H, s, 4′-OMe), 2.90 (2H, m, H2-7′), 2.81 (2H, m, H2-8′); 13 C NMR (CDCl3, 125 MHz) δC 177.5 (C-4), 168.2 (C-2), 161.1 (C-4a), 158.2 (C-4′), 131.1 (C-1′), 129.1 (C-2′,6′), 120.6 (C-8a), 114.0 (C-3), 114.0 (C-3′,5′), 55.2 (4′

[0126] -OMe), 48.6 (C-7), 47.6 (C-8), 46.8 (C-5), 46.4 (C-6), 35.6 (C-8′), 32.2(C-7′); ESIMS m / z 313 [M + H] + The NMR data is basically consistent with the data in the literature [T. Yagura, N. Shibayama, M. Ito, F. Kiuchi, G. Honda, Three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding, Tetrahedron Letters. 2005, 46(25): 4395–4398.]. This study supplements the crystal diffraction data of this compound and determines its absolute configuration as (5S,6S,7S,8S)-oxidoagarochromone B [(5S,6S,7S,8S)-oxidoagarochromone B] ( Figure 3 ).

[0127] PGE26 crystal data: C 18 H 16 O5, M = 312.31, a = 8.2849(3) Å, b = 10.5491(4) Å, c = 8.8684(3) Å, α = 90°, β = 109.0820(10)°, γ = 90°, V = 732.49(5) Å 3 , T =150.(2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.860 mm –1 , 16608 reflectionsmeasured, 2659 independent reflections (R int= 0.0671). The final R1 values were 0.0412 (I > 2σ(I)). The final wR(F 2 ) values were 0.1147 (I > 2σ(I)). The final R1 values were 0.0424 (all data). The final wR(F 2 ) values were 0.1163 (all data). The goodness of fit on F 2 was 0.998. Flack parameter = 0.14(11). The supplementary crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre (CCDC) (deposition number CCDC 2501089) via http: / / www.ccdc.cam.ac.uk.

[0128] Spectral data of compound PGE21:

[0129] (5S,6S,7S,8S)-Oxidoagarochromone C [(5S,6S,7S,8S)-oxidoagarochromone C, PGE21], white needle crystals (MeOH), C 18 H 16 O6; [α]25D –9 (c 0.4, MeOH); UV (MeOH) λ max (logε) 252 (1.62), 210 (1.98) 199 (2.32) nm; ECD (4.73 mM, MeOH) λ max (Δε) 294 (+1.94), 256 (–2.01), 201 (–5.54) nm; 1 1H NMR (CDCl3, 500 MHz) δ H6.76 (1H,d,J = 8.3Hz,H-5′),6.75 (1H,d,J = 2.3 Hz,H-2′),6.62 (1H,dd,J = 8.3,2.3 Hz,H-6′),6.14(1H,s,H-3),5.70 (1H,s,3′-OH),4.33 (1H,br d,J = 3.6 Hz,H-5),3.97 (1H,m,H-7),3.86 (3H,s,4′-OMe),3.83 (2H,m,H-6,8),2.88 (2H,m,H2-7′),2.82 (2H,m,H2-8′); 13 CNMR (CDCl3,125 MHz) δ C 177.7 (C-4),168.3 (C-2),161.2 (C-8a),145.8 (C-3′),145.4(C-4′),132.4 (C-1′),120.7 (C-4a),119.7 (C-6′),114.4 (C-2),114.2 (C-3),110.9(C-5′),56.0 (4′-OMe),48.7 (C-7),47.8 (C-8),46.9 (C-5),46.5 (C-6),35.4 (C-8′),32.2 (C-7′);ESIMS m / z 351 [M + Na] +The NMR data are largely consistent with those in the root literature [T. Yagura, N. Shibayama, M. Ito, F. Kiuchi, G. Honda, Three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding, Tetrahedron Letters.2005, 46(25): 4395–4398 and Y. Yang, WL Mei, WH Dong, W. Li, J. Wang, HNWang, ZB Cheng, HF Dai, Bioactive components from agarwood originating from Aquilaria crassna, Journal of Tropical and Subtropical Botany. 2016, 24(5): 577-583.]. This study supplemented the crystal diffraction data of this compound, determining its absolute configuration to be (5S,6S,7S,8S)-agaric chromone C{(5S,6S,7S,8S)-diepoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochrmone}. Figure 3 ).

[0130] PGE21 crystal data: C 18 H 16 O6•CH4O, M = 360.35, a = 9.7750(3) Å, b = 6.1835(2)Å, c = 13.9591(4) Å, α = 90°, β = 91.4200(10)°, γ = 90°, V = 843.48(4) Å 3 , T =150.(2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.913 mm –1 , 13183 reflectionsmeasured, 3066 independent reflections (R int = 0.0484). The final R1 values ​​were 0.0314 (I > 2σ(I)). The final wR(F 2) values ​​were 0.0829 (I > 2σ(I)). Thefinal R1 values ​​were 0.0319 (all data). The final wR(F 2 ) values ​​were 0.0833(all data). The goodness of fit on F 2 was 1.028. Flack parameter = 0.07(7). The supplementary crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Center (CCDC) (deposition number CCDC2498987) via http: / / www.ccdc.cam.ac.uk.

[0131] Spectroscopic data of compound PGE34:

[0132] Tetrahydrochromone M [PGE34], white needle-like crystals (MeOH), C 18 H 18 O7; [α]20 D+81 (c 0.1, MeOH); UV (MeOH) λ max (log ε) 252 (3.23), 199 (3.84) nm; ECD (0.13 mM, MeOH) λ max (Δε) 284 (+1.52), 248 (+2.58), 214 (+1.50) nm; 1 H NMR (methanol-d4, 500 MHz) δ H6.80 (1H, d, J = 8.2 Hz, H-5′), 6.67 (1H, d, J = 2.1 Hz, H-2′), 6.62 (1H, d, J = 8.2, 2.1 Hz, H-6′), 6.15 (1H, s, H-3), 4.56 (1H, d, J = 7.4 Hz, H-5), 3.99 (1H, dd, J = 7.4, 1.3 Hz, H-6), 3.89 (1H, d, J = 4.3 Hz, H-8), 3.76 (1H, dd, J =4.3, 1.3 Hz, H-7), 3.79 (3H, s, 4′-OMe), 2.88 (4H, br s, H2-7′,8′); 13 C NMR (methanol-d4, 125 MHz) δ C 182.7 (C-4), 171.8 (C-2), 161.4 (C-8a), 147.7 (C-3′), 147.7 (C-4′), 133.9 (C-1′), 121.5 (C-4a), 120.5 (C-6′), 116.4 (C-2′), 114.9 (C-3), 112.9 (C-5′), 73.1 (C-6), 70.0 (C-5), 56.5 (C-7), 56.4 (4′-OMe), 50.8 (C-8), 36.2 (C-8′), 33.2(C-7′); ESIMS m / z 369 [M + Na] + The NMR data are consistent with those in the original literature [G. Liao, W.-L. Mei, F.-D. Kong, W. Li, J.-Z. Yuan, H.-F. Dai, 5, 6, 7, 8-Tetrahydro-2-(2-phenylethyl)chromones from artificial agarwood of Aquilaria sinensis and their inhibitory activity against acetylcholinesterase, Phytochemistry. 2017, 139: 98–108.].

[0133] PGE34 crystal data: C 18 H 18O7, M = 346.32, a = 7.4229(12) Å, b = 7.5970(13) Å, c = 28.084(5) Å, α = 90°, β = 90°, γ = 90°, V = 1583.7(5) Å 3 , T = 150.(2) K, spacegroup P212121, Z = 4, μ(Cu Kα) = 0.949 mm –1 , 6564 reflections measured, 2707 independent reflections (R int = 0.1252). The final R1 values were 0.1149 (I > 2σ(I)). The final wR(F 2 ) values were 0.2875 (I > 2σ(I)). The final R1 values were 0.1689 (all data). The final wR(F 2 ) values were 0.3157 (all data). The goodness of fit on F 2 was 1.101. Flack parameter = 0.1(3). The supplementary crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre (CCDC) (deposition number CCDC 2501152) via http: / / www.ccdc.cam.ac.uk.

[0134] Spectral data of compound PGE57:

[0135] Tetrahydrochromone K [Tetrahydrochromone K, PGE57, colorless square crystals (MeOH), C 19 H 16 O5; [α]20 D –1 (c 0.1, MeOH); UV (MeOH) λ max (log ε) 253 (3.96), 204 (4.27) nm; ECD(0.59 mM, MeOH) λ max(Δε) 294 (–2.30),247 (–4.68),196 (8.05) nm; 1 H NMR(pyridine-d5,500 MHz) δ H 8.15 (1H,br s,5-OH),7.31 (2H,m,H-3′,5′),7.24 (2H,m,H-2′,6′),7.23 (1H,m,H-4′),7.03 (1H,br s,6-OH),6.32 (1H,s,H-3),5.24 (1H,dd,J =7.0,1.7 Hz,H-5),4.50 (1H,br d,J = 7.0 Hz,H-6),4.10 (1H,m,H-7),3.97 (1H,dd,J =4.2,1.7 Hz,H-8),2.89 (2H,m,H2-7′),2.78 (2H,m,H2-8′); 13 C NMR (pyridine-d5,125MHz) δ C 181.3 (C-4),169.2 (C-2),159.5 (C-8a),140.4 (C-1′),129.1 (C-3′,5′),128.8 (C-2′,6′),126.9 (C-4′),121.6 (C-4a),114.3 (C-3),72.4 (C-6),70.4 (C-5),56.8 (C-7),50.2 (C-8),34.9 (C-8′),32.7 (C-7′); 1 H NMR (CDCl3,500 MHz) δ H 7.31(2H,m,H-3′,5′),7.24 (1H,m,H-4′),7.17 (2H,m,H-2′,6′),6.21 (1H,s,5-OH),6.13(1H,s,H-3),4.71 (1H,d,J = 7.7,H-5),4.12 (1H,br d,J = 7.7,H-6),3.85 (1H,br d,J= 4.3 Hz,H-7),3.78 (1H,d,J = 4.3 Hz,H-8),2.99 (2H,m,H2-7′),2.88 (2H,m,H2-8′); 13 C NMR (CDCl3,125 MHz) δ C180.8 (C-4), 169.1 (C-2), 158.5 (C-8a), 139.1 (C-1′), 128.8 (C-3′,5′), 128.2 (C-2′,6′), 126.8 (C-4′), 120.4 (C-4a), 114.3 (C-3), 72.9(C-6), 69.0 (C-5), 54.8 (C-7), 49.5 (C-8), 35.2 (C-8′), 32.9 (C-7′); ESIMS m / z 301[M + H] + The NMR data are consistent with those in the literature [G. Liao, W.-L. Mei, F.-D. Kong, W. Li, J.-Z. Yuan, H.-F. Dai, 5, 6, 7, 8-Tetrahydro-2-(2-phenylethyl)chromones from artificialagarwood of Aquilaria sinensis and their inhibitory activity against acetylcholinesterase, Phytochemistry. 2017, 139: 98–108.].

[0136] PGE57 crystal data: C 17 H 16 O5, M = 300.30, a = 9.4609(5) Å, b = 5.9668(3)Å, c = 13.2124(7) Å, α = 90°, β = 110.288(2)°, γ = 90°, V = 699.59(6) Å 3 , T= 150.(2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.874 mm –1 , 12065 reflections measured, 2527 independent reflections (R int = 0.0646). The finalR1 values ​​were 0.0370 (I > 2σ(I)). The final wR(F 2) values ​​were 0.0888 (I > 2σ(I)). The final R1 values ​​were 0.0406 (all data). The final wR(F 2 ) values ​​were0.0910 (all data). The goodness of fit on F 2 was 1.099. Flack parameter =0.04(11). The supplementary crystallographic data can be obtained free ofcharge from the Cambridge Crystallographic Data Center (CCDC) (depositionnumber CCDC 2501090) via http: / / www.ccdc.cam.ac.uk.

[0137] Spectroscopic data of compound PGE60:

[0138] The compound tetrahydrochromone L [PGE60] is a pale yellow needle-like crystal (MeOH), C 18 H 18 O6; [α]25 D+55 (0.1, MeOH); UV (MeOH) λ max (logε) 241 (1.05), 220 (1.38), 195 (1.89) nm; ECD (8.18 mM, MeOH) λ max (Δε) 294 (+0.02), 245 (+0.03), 199 (+0.04) nm; 1 H NMR (CDCl3, 500 MHz) δ H7.09 (2H, br d, J = 8.7 Hz, H-2′, 6′), 6.84 (2H, br d, J = 8.7 Hz, H-3′, 5′), 6.20 (1H, s, 5-OH), 6.12 (1H, s, H-3), 4.71 (1H, d, J =7.7 Hz, H-5), 4.12 (1H, br d, J = 7.7 Hz, H-6), 3.86 (1H, br d, J = 4.4 Hz, H-7), 3.79 (3H, s, 4′-OMe), 3.78 (1H, d, J = 4.4 Hz, H-8), 2.93 (2H, m, H2-7′), 2.85 (2H, m, H2-8′); 13 C NMR (CDCl3, 125 MHz) δ C 180.9 (C-4), 169.3 (C-2), 158.6 (C-8a), 158.4 (C-4′), 131.1 (C-1′), 129.2 (C-2′, 6′), 120.3 (C-4a), 114.3 (C-3), 114.2 (C-3′, 5′), 72.8 (C-6), 69.1 (C-5), 55.3 (4′- OMe), 54.8 (C-7), 49.6 (C-8), 35.5 (C-8′), 32.1 (C-7′); ESIMS m / z 331 [M + H] + The NMR data are consistent with those in the literature [G. Liao, W.-L. Mei, F.-D. Kong, W. Li, J.-Z. Yuan, H.-F. Dai, 5,6,7,8-Tetrahydro-2-(2-phenylethyl)chromones from artificial agarwood of Aquilaria sinensis and their inhibitory activity against acetylcholinesterase, Phytochemistry. 2017, 139: 98–108.].

[0139] Spectroscopic data of compound PGE62:

[0140] Compound 8-chloro-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone [PGE62], white amorphous powder, C 17 H 17 ClO5; [α]20 D +1 (c 0.3, MeOH); UV (MeOH) λ max (logε) 253 (2.94), 195 (3.38) nm; ECD (0.41 mM, MeOH) λ max (Δε) 309 (+0.49), 234 (+1.72), 213 (+0.40)nm; 1 H NMR (methanol-d4, 500 MHz) δ H 7.25 (2H, m, H-3′, 5′), 7.20 (2H, m, H-2′, 6′), 7.17 (1H, m, H-4′), 6.12 (1H, s, H-3), 4.84 (1H, d, J = 7.4 Hz, H-8), 4.75 (1H, d, J = 4.4 Hz, H-5), 4.20 (1H, dd, J = 7.4, 2.2 Hz, H-7), 4.02 (1H, dd, J = 4.4, 2.2 Hz, H-6), 3.01 (2H, m, H2-7′), 2.92 (2H, m, H2-8′); 13 C NMR (methanol-d4, 125 MHz) δ C 181.5 (C-4), 171.2 (C-2), 161.1 (C-8a), 141.0 (C-1′), 129.6 (C-3′, 5′), 129.5 (C-2′, 6′), 127.5 (C-4′), 122.9 (C-4a), 114.3 (C-3), 73.6 (C-6), 73.6 (C-7), 66.4 (C-5), 58.1 (C-8), 36.2 (C-8′), 33.7 (C-7′); ESIMS m / z 359 [M + Na] +. Its NMR data root literature [J. Ma, H. Huo, H. Zhang, L. Wang, Y. Meng, F. Jin, X. Wang, Y. Zhao, Y. Zhao, P. Tu, Y. Song, J. Zheng, J. Li, 2-(2-phenylethyl)chromone-enriched extract of the resinousheartwood of Chinese agarwood (Aquilaria sinensis) protects against taurocholic acid-induced gastric epithelial Cell apoptosis through Perk / eIF2alpha / CHOP pathway, Phytomedicine. 2022, 98: 153935 and T. Yagura, M. Ito, F. Kiuchi, G. Honda, Y. Shimada, Four new 2-(2-phenylethyl)chromone derivatives from withered wood of Aquilaria sinensis, Chemical & Pharmaceutical The data in Bulletin.2003, 51(5): 560–564.] are consistent.

[0141] Example 4:

[0142] The inhibitory effects of compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 on cancer cell growth.

[0143] Cancer cytotoxicity activity was assessed using the MTS method [Yang J, Hu DB, Xia MY, Luo JF, Li XY, Wang Y-H. Bioassay-guided isolation of cytotoxic constituents from the flowers of Aquilaria sinensis. Natural Products and Bioprospecting, 2022, 12, 1-11], with doxorubicin and paclitaxel as positive controls. The IC50 of the dichloromethane extract (PXS272) of Aquilaria sinensis heartwood extract and the 2-(2-phenylethyl)chromone compound isolated and identified therefrom on the growth inhibition of five tumor cell lines (leukemia HL-60, leukemia K562, lung cancer A549, triple-negative breast cancer MDA-MB-231, and colon cancer SW480) was determined. 50 The values ​​are shown in Tables 3 and 4. Table 3 shows that PGE63, PGE76, PGE66b, and PGE75 had no significant cytotoxic effect on the five tumor cell types. PGE67a (IC50) 50 = 25.75 μM) and PGE62 (IC 50 = 31.35 μM) showed moderate to weak inhibitory activity against SW480 colorectal cancer. PGE57 and PGE60 had inhibitory effects on various tumor cell types, with PGE60 showing activity against all five cell types. The best activity was observed against HL-60 and SW480. 50 The value was less than 10 μM, and it was the only compound among the nine compounds tested in this batch that showed activity against triple-negative breast cancer MDA-MB-232 (IC50). 50 = 20.63 μM). Compound PGE57 showed significant activity against leukemia HL-60, lung cancer A549, and colon cancer SW480 cell lines, with an IC50 concentration of 20.63 μM. 50 The value is less than 20 μM.

[0144] Table 4 shows that PGE21, PGE22, and PGE26 all exhibited potent and broad-spectrum cytotoxic activity, with IC50 values ​​of [missing information]. 50 The concentration was less than 7 μM, and close to or better than some positive control doxorubicin. PGE22 activity was particularly optimal, exhibiting extremely strong inhibitory effect on SW480 colon cancer cells (IC50). 50 = 0.18 μM), also showed highly efficient inhibitory effects on leukemia K562 and HL-60 cells as well as MDA-MB-231, IC50 50 The value is less than 1 μM.

[0145] To further verify the broad-spectrum inhibitory effects and selectivity of PGE21, PGE22, and PGE26 on cancer cells, six additional tumor cell lines were tested: HepG2 liver cancer, HCT116 colon cancer, Caco2 colon cancer, SK-OV-3 ovarian cancer, MG-63 osteosarcoma, and U251 glioma. The results are shown in Table 5. The results indicate that these three compounds maintained potent and broad-spectrum inhibitory activity against the six newly added tumor cell lines, further validating their antitumor universality. Specifically, PGE21 showed the best activity against osteosarcoma MG-63 (IC50). 50 = 0.56 μM) and HCT116 (IC) for colorectal cancer 50 = 1.02 μM) showed extremely strong inhibitory activity, and also exhibited highly efficient inhibition against the other four cell types, with an IC50 of 1.02 μM. 50 The value was less than 4 μM. PGE22 and PGE26 activities were stable, with IC50 values ​​for 6 cell lines. 50 The values ​​were all less than 6 μM. Among them, PGE26 showed outstanding inhibitory effects on colorectal cancer Caco2, osteosarcoma MG-63 and glioma U251, while PGE22 showed excellent activity against colorectal cancer HCT116 and osteosarcoma MG-63.

[0146] Table 3. Cytotoxic activities of PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE34, PGE57, PGE60 and PGE62

[0147]

[0148] Table 4. Cytotoxic activities of PGE21, PGE22, and PGE26

[0149]

[0150] Table 5. Cytotoxic activities of PGE21, PGE22, and PGE26

[0151]

[0152] Example 5:

[0153] The inhibitory effects of compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 on the formation of nitric oxide (NO).

[0154] The anti-inflammatory test method is as follows:

[0155] 1. Experimental Principle

[0156] Nitric oxide (NO) has a wide range of important biological regulatory functions, playing a crucial role in inflammation, tumors, and the cardiovascular system. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., they generate large amounts of induced NO synthase (iNOS), producing NO for the immune response. Therefore, inhibiting NO production is a direct indicator of the anti-inflammatory activity of compounds. Mouse RAW264.7 mononuclear macrophages were induced to produce nitric oxide synthase using LPS lipopolysaccharide, and the test compound was added simultaneously. The absorbance of the culture medium was measured at 570 nm using the Griess method to detect nitrite (NO). 2- ).

[0157] 2. Reagents

[0158] Mouse monocytes (RAW264.7) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. DMEM culture medium and fetal bovine serum were purchased from BI. Griess Reagent, lipopolysaccharide (LPS), and the positive control drug NG-monomethyl-L-arginine (L-NMMA) were purchased from Sigma.

[0159] 3. Experimental Methods

[0160] RAW264.7 cells were seeded into 96-well plates and induced with 1 μg / mL LPS. Simultaneously, the test substances (PGE90, PGE93a, PGE81, PGE82, and PGE83) were added (final concentrations were 2-fold diluted from 50 μM). A drug-free group and an L-NMMA-positive drug group were set up as controls. After overnight culture, the culture medium was used to detect NO production, and the absorbance was measured at 570 nm. MTS was added to the remaining culture medium to detect cell viability and rule out the toxic effects of the compounds on the cells.

[0161] NO generation inhibition rate (%) = (OD in non-drug treatment group) 570 nm – Sample group OD 570 nm ) / Non-drug treatment group OD 570 nm × 100%

[0162] IC 50 (50% concentration of inhibition) calculated according to the Reed & Muench method.

[0163] The anti-inflammatory results of PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE34, PGE57, PGE60, and PGE62 are shown in Table 6. As can be seen from the table, all compounds except PGE63 exhibited certain anti-inflammatory effects, especially PGE34, PGE62, PGE67a, PGE57, and PGE62, whose activities were higher than those of the positive control drug L-NMMA. Among them, compound PGE67a (IC50) showed the best anti-inflammatory effect. 50 = 12.06 μM), PGE57 (IC 50 = 10.41 μM) and PGE60 (IC 50 = 3.55 μM) exhibited strong NO generation inhibition activity.

[0164] The anti-inflammatory results of PGE22, PGE26, and PGE21 are shown in Table 7. The results indicate that all three compounds have certain anti-inflammatory effects, especially compounds PGE22 and PGE26, which show significantly higher inhibitory activity against NO production than the positive control (IC50, IC50, and C60). 50 The values ​​range from 0.60 to 1.01 μM.

[0165] Table 6 IC50 values ​​for inhibiting NO generation in samples PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE34, PGE57, PGE60, and PGE62. 50 value

[0166]

[0167] “ 1) "This indicates that the compound is cytotoxic at this concentration."

[0168] "–" indicates that the inhibition rate did not exceed 50%, and IC testing was not continued. 50 value.

[0169] Table 7 IC50 values ​​for inhibiting NO generation by samples PGE22, PGE26, and PGE21 50 value

[0170]

[0171] “ 1) "This indicates that the compound is cytotoxic at this concentration."

[0172] "–" indicates that the inhibition rate did not exceed 50%, and IC testing was not continued. 50 value.

[0173] Formulation Examples 1–7:

[0174] In the following formulation examples, conventional reagents were selected and formulations were prepared according to existing conventional methods. These formulation examples only demonstrate that at least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60, and PGE62 described in this invention can be prepared into different formulations. Specific reagents and operations are not specifically limited.

[0175] 1. Dissolve at least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 in DMSO, add water for injection according to conventional methods, filter, fill and sterilize to prepare an injection solution with a concentration of 0.5–5 mg / mL.

[0176] 2. Dissolve at least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 in DMSO, then dissolve them in sterile water for injection, stir until dissolved, filter using a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then aseptically seal to obtain a powder for injection.

[0177] 3. At least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 are added to the excipient at a mass ratio of 9:1 to prepare a powder.

[0178] 4. Add at least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 to the excipient at a mass ratio of 5:1, and granulate and compress the mixture into tablets.

[0179] 5. Prepare an oral liquid by taking at least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 according to conventional oral liquid preparation methods.

[0180] 6. At least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 are added to the excipient at a mass ratio of 5:1 to prepare capsules.

[0181] 7. Add at least one or any of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a, PGE21, PGE22, PGE26, PGE34, PGE57, PGE60 and PGE62 to the excipient at a mass ratio of 5:1 to prepare granules.

[0182] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The 2-(2-phenylethyl)chromone compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a shown in the following structural formulas, wherein: PGE63 is (5R,6R,7R,8R)-5,6:7,8-diepoxy-2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone. PGE76 is (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone. PGE66b is (5R,6S,7S,8S)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone. PGE75 is (5S,6R,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone. PGE67a is (5R,6S,7R,8R)-7,8-epoxy-6-hydroxy-5-methoxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone. 。 2. The method for preparing the 2-(2-phenylethyl)chromone compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a according to claim 1, characterized in that, The method includes the following steps: I. Extraction of dichloromethane fraction PXS272 from the heartwood of *Aquilaria sinensis*: The heartwood of *Aquilaria sinensis* was taken, pulverized into powder, and then 90% ethanol was added. The mixture was ultrasonically extracted in a 60 °C water bath for 1 hour, repeated 8 times. The extract was recovered under reduced pressure and concentrated to obtain the ethanol extract (PXS264). The crude extract was dissolved in an appropriate amount of water and extracted 3-5 times in sequence with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction (PXS272), ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. II. PGE63, PGE76, PGE66b, and PGE75 were isolated and extracted from the dichloromethane extract PXS272. (1) PXS272 was separated by silica gel column chromatography, with petroleum ether-ethyl acetate ratio of 20:1-0:1, to obtain 12 fractions including Fr.B1-Fr.B12; (2) The Fr. B10 fraction was subjected to RP-18 reversed-phase silica gel column chromatography with a gradient elution of methanol-water (30%-100%). The fractions were analyzed by TLC plate, and fractions of the same type were combined to obtain 14 fractions, from Fr. B10-1 to Fr. B10-14. (3) Fr. B10-2 was recrystallized from methanol to obtain two parts: Fr. B10-2-1 and Fr. B10-2-2; (4) Fr. B10-2-2 was separated by elution with a normal-phase silica gel column (200-300 mesh) and a petroleum ether-acetone system with concentration gradients of 9:1, 3:1, 2:1, 1:1, and 0:1 to obtain a total of 12 components, Fr. B10-2-2-1 to Fr. B10-2-2-12. (5) Fr. B10-2-2-4 was separated into four fractions, Fr. B10-2-2-4-1 to Fr. B10-2-2-4-4, by Sephadex LH-20 gel chromatography; (6) Fr. B10-2-2-4-1 was separated by normal phase silica gel column chromatography. After elution in dichloromethane-acetone systems of 20:1, 15:1, 10:1, 5:1, and 3:1, compound PGE57 and the remaining 6 fractions Fr. B10-2-2-4-1-1 ~ Fr. B10-2-2-4-6 were obtained. (7) Fr. B10-2-2-4-1-1 was further purified by reverse-phase semi-preparative HPLC to obtain compounds PGE76, PGE75 and PGE66b; (8) Fr. B10-2-2-4-1-2 was further purified by semi-preparative HPLC to obtain compounds PGE76, PGE66b, PGE57 and PGE67a; (9) Fr. B10-2-2-6 was separated into six fractions, Fr. B10-2-2-6-1 ~ Fr. B10-2-2-6-6, by Sephadex LH-20 gel chromatography. Fr. B10-2-2-6-5 was separated by normal phase silica gel column chromatography. After elution in dichloromethane-acetone system at ratios of 20:1, 15:1, 10:1, 5:1, and 3:1, 2 fractions, Fr. B10-2-2-6-5-1 ~ B10-2-2-6-5-12 fractions were obtained. Fr. B10-2-2-6-5-1-2 was further purified by reversed-phase semi-preparative HPLC to obtain compound PGE63. Among them, (7) the semi-preparative high performance liquid chromatography is performed using an Agilent 1200 liquid chromatograph with a Chiral CD-PH column, the column size is 4.6 × 250 mm, the eluent is methanol-water in a ratio of 60:40, and the flow rate is 1 mL / min; (8) the semi-preparative high performance liquid chromatography is performed using an Agilent 1200 liquid chromatograph with a Chiral CD-PH column, the column size is 4.6 × 250 mm, the eluent is n-hexane-isopropanol in a ratio of 80:20, and the flow rate is 1 mL / min; (9) the semi-preparative high performance liquid chromatography is performed using an Agilent 1200 liquid chromatograph with a Chiral CD-PH column, the column size is 4.6 × 250 mm, the eluent is methanol-water in a ratio of 90:10, and the flow rate is 1 mL / min.

3. A pharmaceutical composition comprising a therapeutically effective amount of the compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a of claim 1, and a pharmaceutically acceptable carrier.

4. The method for preparing the pharmaceutical composition according to claim 3, characterized in that, First, compounds PGE63, PGE76, PGE66b, PGE75 and PGE67a are obtained using the preparation method described in claim 2. Then, one or any combination of these compounds is added to a pharmaceutically acceptable carrier.

5. The use of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a of claim 1 or the pharmaceutical composition of claim 3 in the preparation of a medicament for the prevention and / or treatment of leukemia, lung cancer, breast cancer, and colon cancer.

6. The use of the compounds PGE63, PGE76, PGE66b, PGE75, PGE67a of claim 1 or the pharmaceutical composition of claim 3 in the preparation of an anti-inflammatory drug.

7. The use of compounds PGE22 [(5R,6R,7R,8R)-agaricone A], PGE26 [(5S,6S,7S,8S)-agaricone B], PGE21 [(5S,6S,7S,8S)-agaricone C], PGE34 (tetrahydroagaricone M), PGE57 (tetrahydroagaricone K), PGE60 (tetrahydroagaricone L), PGE62 (8-chloro-2-(2-phenylethyl)-5,6,7'-trihydroxy-5,6,7,8-tetrahydroagaricone), or pharmaceutical combinations thereof, as shown in the following structural formulas, in the preparation of antitumor drugs and in the preparation of antiinflammatory drugs. 。 8. The use of compounds PGE22 [(5R,6R,7R,8R)-agaricone A], PGE26 [(5S,6S,7S,8S)-agaricone B], PGE21 [(5S,6S,7S,8S)-agaricone C], PGE34 (tetrahydroagaricone M), PGE57 (tetrahydroagaricone K), PGE60 (tetrahydroagaricone L), PGE62 (8-chloro-2-(2-phenylethyl)-5,6,7'-trihydroxy-5,6,7,8-tetrahydroagaricone), or pharmaceutical compositions thereof, as shown in the following structural formulas, in the preparation of medicaments for the prevention and / or treatment of leukemia, lung cancer, breast cancer, and colon cancer. 。

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