A schizocarp-like diterpenoid compound, its preparation method and uses

Abstract

This invention discloses a secolathyrenoic acid B compound, its preparation method, and its uses, belonging to the field of natural product chemistry. The compound is characterized by being extracted, separated, and purified from *Seolathyrenoic acid* seeds using a chemical method with water or organic solvents. The invention also describes the preparation method of this compound and its uses in the pharmaceutical field, particularly in the preparation of antitumor drugs.

Description

I. Technical Field:

[0001] This invention relates to the field of natural medicines, specifically to a novel secolathyrenoic acid (2S,3R,4S,5S,9S,11S)-Secolathyrenoic acid B, extracted and isolated from the seeds of *Euphorbia hirta*, its preparation method, cytotoxic activity, and use in the preparation of antitumor drugs or health products. II. Technical Background:

[0002] Leukemia is a malignant clonal disease of hematopoietic stem cells, with complex classifications and a poor prognosis. Furthermore, existing chemotherapy drugs, in addition to killing leukemia cells, also affect normal hematopoietic cells, leading to a series of toxic side effects such as anemia, bleeding, infection, and bone marrow suppression. Therefore, finding novel drugs that selectively kill leukemia cells is an urgent problem to be solved in current leukemia treatment.

[0003] Diterpenoids, also known as sucralose diterpenoids, are a class of macrocyclic diterpenoid compounds with significant pharmacological activity. They are highly oxidized, structurally complex, and specifically distributed in plants of the genus *Euphorbia*. They often exhibit good cytotoxicity (Teng YN, Wang Y, Hsu PL, et al. Mechanism of action of cytotoxic compounds from the seeds of *Euphorbia lathyris* [J]. Phytomedicine, 2018, 41: 62-66.), reversal of multidrug resistance in tumor cells (Xiao Y, Ji WS, Jin WK, et al. Synthesis, antiproliferative and anti-MDR activities of *Lathyrane diterpene* derivatives based on configuration inversion strategy [J]. Bioorg Chem. 2023, 131: 106329.), and anti-inflammatory activity (Wang Y, Song Z, Guo Y, et al. Diterpenoids from the seeds of *Euphorbia lathyris* and their anti-inflammatory activity [J]. Bioorg Chem. 2023, 131: 106329.). (Chem. 2021, 112: 104944.) is an important source of lead compounds for antitumor drugs and has attracted widespread attention. III. Summary of the Invention:

[0004] The purpose of this invention is to provide a novel sucralose-type cleaved diterpenoid compound, its preparation method, and its use in anti-leukemia drugs.

[0005] The technical solution adopted in this invention is: a novel secoidan-type secolathyrenoic acid B, characterized by its rare secolathyrenoic structure, with the chemical name (2S,3R,4S,5S,9S,11S)-secolathyrenoic acid B. Figure 1 )

[0006] Furthermore, the Secolathyrenoic acid B is prepared as follows: using the seeds of Euphorbia alabaster L. as raw material, it is extracted 2-3 times with 3-5 times the volume of the raw material in alcohol or a mixture of alcohol and water, each time for 1 week. The extracts are combined and concentrated under reduced pressure to 0.5-1.5 times the volume of the raw material. Then, it is extracted sequentially with petroleum ether, dichloromethane, and ethyl acetate in a 1:1-3 volume ratio, each solvent being extracted 3 times. The dichloromethane extract is separated by column chromatography.

[0007] Furthermore, secolathyrenoic acid B showed an inhibitory concentration (IC50) of 50% for human acute myeloid leukemia cells U937. 50 The concentration was 22.18 ± 2.04 μM. This invention provides a new lead compound for the research of anti-leukemia drugs. IV. Description of the attached drawings:

[0008] The following image can be submitted as an attachment.

[0009] Figure 1 Here is the chemical structural formula of the secoirsiviridoditerpenoid compound of this invention;

[0010] Figure 2 This is a flowchart illustrating the preparation process of the secoired diterpenoid compounds of the present invention;

[0011] Figure 3 This is the mass spectrum of the secoirsivirid diterpenoids of the present invention;

[0012] Figure 4 The hydrogen spectrum of the cleaved diterpenoid compounds of this invention;

[0013] Figure 5 The carbon spectra of the cleaved diterpenoid compounds of this invention are shown below.

[0014] Figure 6 The secoirs of the present invention are diterpenoids. 1 H- 1 H COSY spectrum;

[0015] Figure 7 This is the HMBC spectrum of the secoirsivirid diterpenoids of this invention;

[0016] Figure 8 The HMQC spectra of the secoirsivirid diterpenoids of this invention are shown below.

[0017] Figure 9 This is the ROESY spectrum of the secoirsiate diterpenoids of this invention;

[0018] Figure 10 This is the ECD spectrum of the secoirsivirid diterpenoid compound of the present invention. V. Detailed Implementation Methods

[0019] The present invention will be further described below with reference to specific embodiments, but the content of the present invention is not limited to the listed embodiments.

[0020] Example 1

[0021] Preparation Example of Secolathyrenoic acid B

[0022] like Figure 2 As shown, 10.0 kg of dried *Eupatorium fortunei* seeds were crushed and extracted three times with three times their volume of 90% methanol (MeOH), ensuring the seeds were completely submerged in the solvent. Each extraction lasted one week. The three extracts were combined and concentrated under reduced pressure using a rotary evaporator until no methanol odor remained, yielding a total extract of 1.8 kg. An appropriate amount of ultrapure water was added for suspension, and the extract was sequentially extracted three times with equal volumes of petroleum ether (PE), dichloromethane (CH2Cl2), and ethyl acetate (EtOAc), yielding 150.3 g of the dichloromethane fraction extract. The dichloromethane fraction extract was initially separated by silica gel column chromatography. The sample was mixed with 100–200 mesh silica gel, packed into a 200–300 mesh silica gel column, and eluted using a gradient PE / EtOAc solvent system (100:0–0:100). After TLC thin-layer chromatography, fractions of the same type were combined, yielding nine fractions Fr.1–Fr.9. Fraction Fr.6 was separated by isocratic elution on a Sephadex LH-20 column (CH2Cl2:MeOH / 4:1) to obtain subfractions Fr.6.1–Fr.6.5. Fraction Fr.6.4 was purified by semi-preparative high-performance liquid chromatography (60% MeOH) to obtain compound secolathyrenoic acid B (7.1 mg), with a yield of 0.000071%. The structure of compound secolathyrenoic acid B was then identified using various spectroscopic techniques (MS, NMR, UV, IR).

[0023] The structure of the obtained compound was identified.

[0024] Colorless powder, detected by TLC with EtOAc as the developing solvent, showing absorption at 254 nm and weak fluorescence. Vanillin turns dark red upon heating after treatment with concentrated sulfuric acid. Easily soluble in chloroform and methanol, presumably a terpene compound. HR-ESI-MS data: m / z 371.1857 [M+Na] + (Calcd.for C 20 H 28 O5Na 371.1834), the molecular formula of the compound is deduced to be C 20 H 28 O5 has an unsaturation degree of 7. 1 1H NMR data (Table 1) show the presence of four methyl δ H 1.05 (3H, d, CH3-16), δ H 1.07 (3H, s, CH3-18), δ H 1.15 (3H, s, CH3-19) and δ H 1.90 (3H, s, CH3-20), two oxidized methine δ H 4.53 (1H, s, H-5) and δ H 3.70 (1H, d, H-3), three olefinic protons δ H 6.71(1H,d,J=10.6Hz,H-12), δ H 5.15 (1H, s, H-17α) and δ H 4.99 (1H, s, H-17β). 13 The 12C NMR data (Table 1) show the presence of 20 carbon signals, including one ketone carbonyl carbon δ. C 210.80 (C-15), one carboxyl carbon δ C 173.07 (C-14), four olefinic carbons δ C 147.02(C-6), δ C 143.47 (C-12), δ C 127.22(C-13) and δ C 112.21 (C-17), three oxygen-containing carbon δ C 66.56 (C-3), δ C 63.8 (C-4) and δ C 69.20(C-5). From its 1 H- 1The correlation between H COSY and HSQC proves the existence of two spin systems: H-1 / H-2 / H-3 / H3-16 and H-7 / H-8 / H-9 / H-11 / H-12. HMBC correlations at H2-1, H-3, H-5 / C-15 and H-5 / C-3, C-4 suggest the presence of a cyclopentenone and a connection between C-4 and C-5; HMBC correlations at H2-17 / C-5, C-6, C-7, H-5 / C-6 and H-7 / C-6 show a terminal olefin carbon at C-6 and a connection between C-5, C-6, and C-7; HMBC correlations at H3-18 and H3-19 with C-9, C-10, C-11 confirm the presence of dimethylcyclopropane; furthermore, HMBC correlations at H-12 with C-14 and H3-20 with C-12, C-13, C-14 indicate that the carboxyl carbon (C-14) and methyl carbon (C-20) are connected through an olefin quaternary carbon (C-13). These NMR data analyses indicate that this compound is a rare secoisic diterpene. Furthermore, based on its molecular weight and δ C 66.56(C-3) and δ C The chemical shift of 63.83 (C-4) suggests the existence of an ethylene oxide structure between C-3 and C-4.

[0025] The ROESY correlation between H-3 and H3-16β suggests that the oxygen atom of ethylene oxide is connected to C-3 and C-4 by a dashed line, located within the paper. To further confirm this inference, the absolute configuration of the compound was determined by comparing experimental measurements with the electronic circular dichroism curves predicted by TD-DFT theory. The results show that the calculated ECD curves (2S,3R,4S,5S,9S,11S) are consistent with the experimental ECD curves. In summary, the absolute configuration of this compound is 2S,3R,4S,5S,9S,11S, and it is named Secolathyrenoic acid B.

[0026] Table 1. Nuclear magnetic resonance spectral data of compound Secolathyrenoic acid B

[0027]

[0028] Example 2: Determination of the inhibitory activity of compound secolathyrenoic acid B against human acute myeloid leukemia cells U937. This invention uses the CCK-8 assay to test the in vitro activity of secolathyrenoic acid B, with cisplatin as a positive control. Tumor cells were human acute myeloid leukemia cells U937. U937 cells in logarithmic growth phase were seeded into 96-well culture plates at a density of 5000 cells per well (100 μL). Simultaneously, the cells were treated with test samples at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μmol / L, and incubated at 37°C in a 5% CO2 incubator. After 72 h, 10 μL of CCK-8 was added to each well, and incubation continued for another 4 h. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm, and the cell growth inhibition rate was calculated.

[0029] Cell growth inhibition rate (%) = (1 - OD value of experimental group / OD value of control group) × 100

[0030] Positive controls were cisplatin at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μmol / L. IC50 values ​​were calculated using DPS software. The results are shown in Table 2 below.

[0031] Table 2. Inhibitory activity of compound Secolathyrenoic acid B against human acute myeloid leukemia cells U937 (IC50, μM)

[0032]

Claims

1. A diterpenoid compound derived from *Symplocos lucida*, named (2) S , 3 R , 4 S , 5 S , 9 S , 11 S )-Secolathyrenoic acid B, chemical structural formula is: Secolathyrenoic acid B。 2. The method for preparing the diterpenoid compound according to claim 1, characterized in that, Includes the following steps: To continue following the son Euphorbia lathyris Using L. seeds as raw material, the extract was soaked 2-3 times with 3-5 times the volume of alcohol or a mixture of alcohol and water, each soaking for 1 week. The extracts were combined and concentrated under reduced pressure to 0.5-1.5 times the volume of the raw material. The extracts were then extracted sequentially with petroleum ether, dichloromethane and ethyl acetate in a 1:1-3 volume ratio, each solvent being extracted 3 times. The dichloromethane extract was separated by column chromatography.

3. Use of the compound of claim 1 in the preparation of an anti-leukemia drug.

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