Polyisopentenyl acyl phloroglucinol compound and preparation method thereof

Abstract

The invention discloses a preparation method of a polyisopentenyl acyl phloroglucinol compound, the compound is subjected to a tumor multidrug resistance reversing activity test, under the same concentration, hyperiumono C shows a reversing effect superior to that of a positive drug verapamil, the reversing multiple reaches 56, and the compound has a good anti-tumor effect. A lead compound with development potential is provided for overcoming breast cancer chemotherapy drug resistance.

Description

Technical Field

[0001] This invention relates to a method for preparing polyisoprenyl acyl phloroglucinol compounds, belonging to the technical field of multidrug resistance reversal agents. Background Technology

[0002] According to data from the International Agency for Research on Cancer (IARC) of the World Health Organization, breast cancer saw 2.31 million new cases and 670,000 deaths globally in 2022, surpassing other malignant tumors to become the leading threat to women's health. Despite continuous advancements in treatment strategies, chemotherapy remains the cornerstone of first-line treatment for breast cancer and the prevention of postoperative recurrence, with doxorubicin being widely used as the preferred chemotherapy drug. However, the emergence of multidrug resistance severely limits the clinical efficacy of doxorubicin. While preclinical research on multidrug resistance chemosensitizers continues to progress, their clinical translation is often limited by dose toxicity and off-target effects. Therefore, the development of highly effective and low-toxicity multidrug resistance reversal agents is urgently needed. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for preparing polyisoprenyl acyl phloroglucinol compounds (PPAPs) that has the activity of reversing multidrug resistance in breast cancer cells, enriching the structural diversity of active PPAPs compounds; and overcoming the shortcomings of the prior art.

[0004] The objective of this invention is achieved through the following technical solution: This invention discloses a polyisoprene acyl phloroglucinol compound, which is represented by the following formula (1): (1) In the formula, the numbers represent carbon atom labels.

[0005] The above-mentioned compound has the molecular formula C. 26 H 37 O6.

[0006] The preparation method of the polyisoprenyl acyl phloroglucinol compound includes the following steps: Step 1, take dried Hypericum perforatum flowers, extract with methanol at room temperature, and concentrate under reduced pressure to obtain methanol extract; Step 2: The extract was separated by silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient, to obtain 7 major fractions A-G; Step 3: Separate fraction D from fractions A–G by reversed-phase C18 column chromatography with methanol–water gradient elution to obtain 5 subfractions Fr.D1–Fr.D5; Step 4: Separate Fr.D3 in subfractions Fr.D1–Fr.D5 by Sephadex LH-20 gel column chromatography to obtain 8 secondary fractions Fr.D3a–Fr.D3h; Step 5: Further separate Fr.D3b in the secondary components Fr.D3a–Fr.D3h by repeated silica gel column chromatography to obtain 9 sub-fractions Fr.D3b1–Fr.D3b9; Step 6: Fr.D3b6 from the nine subdivisions Fr.D3b1–Fr.D3b9 was purified by preparative high performance liquid chromatography to finally obtain the polyisoprenyl acyl phloroglucinol compound hypericumono C.

[0007] In step 2 above, the petroleum ether–ethyl acetate gradient elution is performed, with the petroleum ether–ethyl acetate gradient changing from 100:0 to 0:100, v / v.

[0008] In step 3 above, methanol-water gradient elution is performed, with the methanol-water gradient changing from 40:60 to 100:0, v / v.

[0009] In step 6 above, the volume ratio of methanol to water used for purification is 75:25.

[0010] Compared with the prior art, the beneficial effects of the present invention are: 1. The compound of this invention was tested for its activity in reversing multidrug resistance in tumors. At the same concentration, the compound hypericumono C showed a superior reversal effect compared to the positive control drug verapamil, with a reversal fold of 56 times.

[0011] 2. This work not only expands the chemical diversity of polycyclic polyisoprenyl acyl phloroglucinol compounds (PPAPs), but also provides promising lead compounds for overcoming chemotherapy resistance in breast cancer, and offers valuable information for finding active ingredients with multidrug resistance reversal effects from Hypericum plants.

[0012] 3. This compound has had a profound and positive impact on multiple dimensions, including expanding chemical diversity, discovering lead compounds, and conducting natural resource-oriented research, and has significant scientific value and potential application value.

[0013] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will now be described in further detail with reference to the accompanying drawings, wherein: Figure 1These are the hydrogen-hydrogen chemical shift correlation spectra and heteronuclear multibond correlation spectra of compound 1 of the present invention.

[0015] Figure 2 This is the Fröhlberg effect spectrum of compound 1 of the present invention.

[0016] Figure 3 This is the electronic circular dichroism spectroscopy of compound 1 of the present invention. Detailed Implementation

[0017] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be understood that the preferred embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0018] The present invention discloses a polyisopentenyl acyl phloroglucinol compound, which is represented by the following formula (1): (1) In the formula, the carbon atom is labeled.

[0019] Its compound has the molecular formula C 26 H 37 O6.

[0020] The preparation method of the polyisoprenyl acyl phloroglucinol compound includes the following steps: Nine kg of dried Hypericum flowers were extracted with methanol (3 × 100.0 L) at room temperature, and the extract was concentrated under reduced pressure to obtain a methanol extract (3 kg). The extract was separated by silica gel column chromatography (100–200 mesh, 4.5 kg) with a petroleum ether–ethyl acetate gradient elution (100:0→0:100, v / v) to obtain seven major fractions (A–G).

[0021] Fraction D (220.0 g) was separated by reversed-phase C18 column chromatography with a methanol-water gradient elution (40:60 → 100:0, v / v) to obtain 5 subfractions (Fr.D1–Fr.D5). Fr.D3 (42 g) was separated by Sephadex LH-20 gel column chromatography to obtain 8 secondary fractions (Fr.D3a–Fr.D3h). Fr.D3b (12 g) was further separated by repeated silica gel column chromatography (300–400 mesh) to obtain 9 fine fractions (Fr.D3b1–Fr.D3b9). Fr.D3b6 (60 mg) was purified by preparative high-performance liquid chromatography (methanol-water, 75:25, v / v) to finally obtain hypericumono C ( ). Process and related experimental data Materials and methods

[0022] The Hypericum flowers were collected in June 2018 from Guiyang, Guizhou Province, China (26°11′–27°22′ N, 106°07′–107◦17′ E). The voucher specimen (No. H20180601) is deposited in the specimen bank of the Guizhou Provincial Key Laboratory of Natural Product Chemistry.

[0023] Spectral data

[0024] ECD calculations for compound hypericumono C The theoretical electron circular dichroism calculations for Hypericumono C were performed using the Gaussian16 package. First, an initial conformational distribution search was performed using HyperChemRelease 8.0, selecting conformations with a population greater than 1% for subsequent optimization. Geometric optimization of all conformations was performed in Gaussian16 using density functional theory. The process was conducted at a horizontal level. Subsequently, based on time-dependent density functional theory, in... The ECD transitions of the optimized conformation were calculated at the horizontal level, and a polarization continuum model was used to simulate the methanol solvent environment. Finally, the theoretical ECD spectrum of the compound was obtained by averaging the Boltzmann weights of the spectra of each conformation and processing it with SpecDis 1.64 software.

[0025] Cell culture MCF7 / ADR cells (doxorubicin-resistant subline) were purchased from Sun Yat-sen University. The cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum and incubated at 37°C in a humidifier containing 5% CO2.

[0026] Cytotoxicity assay Logarithmically growing cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured for 24 h. Then, graded concentrations of the test compound were added, and the cells were cultured for another 48 h. 20 μL of LMTT reagent solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 h. The supernatant was discarded, and 160 μL of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Wells containing an equal volume of DMSO (<0.1%) served as blank controls. All experiments were independently repeated three times.

[0027] Research on reversing multidrug resistance in tumors The multidrug resistance reversal activity assay was performed according to previously reported methods. The simplified procedure is as follows: MCF-7 / ADR cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured for 24 h. Then, pre-set concentrations of reversal agent and six gradient concentrations of doxorubicin were added, and the cells were cultured for another 48 h. Cell viability was assessed using the MTT assay to evaluate the cytotoxicity of doxorubicin in the presence or absence of the reversal agent. Verapamil was used as a positive control, and DMSO (final concentration <0.1%) was used as a negative control. The reversal fold was calculated using the formula... The IC50 ratio (doxorubicin alone) / (doxorubicin + reversal agent) was calculated. All experiments were independently repeated three times. result

[0028] Compound 1 (hypericumono C) is a white amorphous powder. High-resolution electrospray ionization mass spectrometry (HRESIMS) shows its molecular formula as follows: Measured value (Calculated value) (445.2585). Its one-dimensional NMR data are basically similar to those of the known compound hyperpatulol C (from the microspectral database, and this reference Liu YY, Ao Z, Xu QQ, Zhu DR, Chen C, Wang XB, Luo JG, Kong LY (2019) Hyperpatulols A–I, spirocyclic acylphloroglucinolderivatives with anti-migration activities from the flowers of Hypericum patulum. Bioorg Chem 87:409–416. (This known compound can be found at https: / / doi.org / 10.1016 / j.bioorg.2019.03.025.) The main difference lies in the carbonyl group in hyperpatulol C. Compound 1 is enol group Substitution, and the enol group in hyperpatulol C In compound 1, it is carbonyl group replace( Figure 1 The above structural hypothesis was confirmed by HMBC-related signals: and and Related; and , , and Related. Among them, the heteronuclear multi-bond correlation spectrum of compound 1, the hydrogen-hydrogen chemical shift correlation spectrum of compound 1, and the nuclear Overhauser effect spectrum are as follows: Figure 1 , Figure 2 As shown.

[0029] Table 1 shows compound 1 in In and data

[0030] Based on two-dimensional nuclear magnetic resonance spectroscopy data analysis, the remaining structural features of compound 1 are consistent with those of hyperpatulol C. Figure 2 Its relative configuration was determined by the NOESY experiment: The presence of key correlation peaks between them indicates that these groups are all β - orientation; and The NOESY correlation between them indicates that these groups are α - Orientation, thus inferring the relative configuration of compound 1. Furthermore, the calculated ECD spectrum agrees well with the experimentally determined ECD spectrum, further confirming its absolute configuration. In summary, the structure of compound 1 is thus clarified, and its structural formula is as follows: Figure 3 As shown.

[0031] Given the reported moderate to significant multidrug resistance reversal activity of polycyclic isopentenyl acyl phloroglucinol derivatives, this study used the MTT assay to evaluate the multidrug resistance reversal ability of all compounds against MCF-7 / ADR cells. First, the cytotoxicity of all compounds against MCF-7 / ADR cells was tested using the MTT assay, and the results showed no significant toxicity at the tested concentrations. Subsequently, MDR reversal activity was evaluated by combining different concentrations of doxorubicin with either 20 μM or 10 μM of compound 1. The results showed that the half-maximal inhibitory concentration (IC50) of doxorubicin against MCF-7 / ADR cells was 283.54 μM. However, when combined with compound 1, its cytotoxicity was significantly enhanced, with an IC50 of 5.04 μM and a reversal fold of 56-fold, slightly higher than the positive control verapamil.

[0032] Table 2. Cytotoxicity and anti-tumor multidrug resistance activity of compound 1 against MCF-7 / ADR.

[0033] a The concentration for testing the reversal activity of the compound or verapamil is 20 or 10 μM. b IC 50 Values ​​are the results of three independent experiments, expressed as mean ± standard deviation; c The compound's autocytotoxic effects on drug-resistant cancer cell lines.d Doxorubicin was used as a single-drug control group; e Verapamil was used as a positive control.

[0034] This work reveals a novel PPAP with the activity of reversing multidrug resistance in breast cancer cells, enriching the structural diversity of active PPAPs and providing valuable information for finding active ingredients with multidrug resistance reversal effects from Hypericum plants.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A polyisopentenyl acyl phloroglucinol compound, characterized in that, It is represented by the following formula (1): （1） In the formula, the numbers represent carbon atom labels.

2. The polyisopentenyl acyl phloroglucinol compound according to claim 1, characterized in that, Its compound molecular formula is .

3. A method for preparing a polyisoprenyl acyl phloroglucinol compound, characterized in that, Includes the following steps: Step 1: Take dried Hypericum flowers, extract with methanol at room temperature, and concentrate under reduced pressure to obtain methanol extract; Step 2: The extract was separated by silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient, to obtain 7 major fractions A-G; Step 3: Separate fraction D from fractions A–G by reversed-phase C18 column chromatography with methanol–water gradient elution to obtain 5 subfractions Fr.D1–Fr.D5; Step 4: Separate Fr.D3 in subfractions Fr.D1–Fr.D5 by Sephadex LH-20 gel column chromatography to obtain 8 secondary fractions Fr.D3a–Fr.D3h; Step 5: Further separate Fr.D3b in the secondary components Fr.D3a–Fr.D3h by repeated silica gel column chromatography to obtain 9 sub-fractions Fr.D3b1–Fr.D3b9; Step 6: Fr.D3b6 from the nine subdivisions Fr.D3b1–Fr.D3b9 was purified by preparative high performance liquid chromatography to finally obtain the polyisoprenyl acyl phloroglucinol compound hypericumono C.

4. The method for preparing polyisoprenyl acyl phloroglucinol compounds according to claim 3, characterized in that, In step 2, the petroleum ether–ethyl acetate gradient elution is performed, with the petroleum ether–ethyl acetate gradient changing from 100:0 to 0:100, v / v.

5. The method for preparing polyisoprenyl acyl phloroglucinol compounds according to claim 3, characterized in that, In step 3, methanol-water gradient elution is performed, with the methanol-water gradient changing from 40:60 to 100:0, v / v.

6. The method for preparing polyisoprenyl acyl phloroglucinol compounds according to claim 3, characterized in that, In step 6, the purification process uses a methanol to water volume ratio of 75:25.

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