Butyrolactone compound with multidrug resistance reverse transcription activity, its preparation method and uses

Abstract

This invention discloses a butyrolactone compound with multidrug resistance reverse transcription activity, its preparation method, and its uses. The compound's structural formula is shown in Figure I. The preparation method includes the following steps: fermenting *Aspergillus pyrolyticus* (accession number CCTCC NO: M2014086) to obtain a butyrolactone fermentation product; extracting the fermentation product with ethyl acetate to obtain a crude extract; degreasing the crude extract with hexane and dichloromethane to obtain a final extract; and purifying the final extract using normal-phase silica gel column chromatography, reversed-phase medium-pressure column chromatography, and reversed-phase semi-preparative high-performance liquid chromatography. The advantage of this butyrolactone compound is that it possesses multidrug resistance reverse transcription activity and the ability to regulate P-gp-mediated drug efflux in multidrug-resistant cell lines, making it a potential drug for treating multidrug-resistant cancers.

Description

Technical Field

[0001] This invention relates to butyrolactone compounds, and more particularly to butyrolactone compounds with multidrug-resistant reverse transcription activity extracted from marine fungal fermentation products, their preparation methods, and uses. Background Technology

[0002] Multidrug resistance (MDR) refers to the phenomenon where some bacteria develop resistance to several different antibiotics. The high incidence of MDR is associated with the overexpression of ATP-binding cassette (ABC) efflux transporters, particularly the efflux transporter P-glycoprotein (P-gp); biomembranes are important permeability barriers and therefore play a crucial role in protecting pharmacokinetics. However, drug activity ultimately depends on the compound's ability to reach its target, which is regulated by the drug's fundamental physical properties and its interactions with membrane transporters. Overexpression of ATP-binding cassette (ABC) efflux transporters (including P-gp, MRPs, and BCRPs) leads to reduced bioavailability of chemotherapeutic drugs in cancer cells through drug efflux mechanisms at the cost of ATP hydrolysis. Therefore, the discovery of effective P-gp inhibitors from marine natural products to reverse resistance offers new insights for improving the efficacy of chemotherapy.

[0003] Marine microorganisms possess unique metabolic pathways and defense systems distinct from terrestrial microorganisms, resulting in a large number of novel and bioactive secondary metabolites. The search for novel bioactive compounds from marine microorganisms has become an important aspect of contemporary natural product research, and marine fungi, as a significant source of structurally diverse bioactive compounds, have attracted increasing attention. Butyrolactones, as an important class of natural secondary metabolites, have a unique five-membered unsaturated lactone ring as their main structural framework. Recent in-depth studies of butyrolactones have revealed that butyrolactone structures are commonly found in the metabolites of various Aspergillus fungi. These compounds possess a variety of pharmacological activities, mainly manifested in anti-inflammatory, anti-diabetic, antioxidant, and neuroprotective activities, making them important raw materials for pharmaceutical applications. In a chemical investigation of the ethyl acetate extract of the marine fungus Aspergillus sp. NBU4698 under culture medium fermentation, the inventors discovered a novel butyrolactone-based natural product. Currently, there are no reports on the chemical structure of this compound or its multidrug-resistant reverse transcription activity; therefore, no related drugs are available on the market. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a butyrolactone compound with multiple drug resistance reverse transcription activity, its preparation method and uses.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] 1. A butyrolactone compound with multiple drug resistance reverse transcription activity, the structural formula of which is shown in (I);

[0007]

[0008] 2. The preparation method of the above-mentioned butyrolactone compounds with multiple drug resistance reverse transcription activities includes the following steps:

[0009] (1) Fermentation production

[0010] Aspergillus ustus with accession number CCTCC NO: M2014086 was streaked on a PDA solid medium plate and incubated upside down in an incubator at 28℃ for 3 days. After that, a single colony was picked and inoculated into PDB liquid medium and cultured on a shaker at 28℃ and 150 rpm for 3 days to obtain the seed liquid. Rice medium was then placed in Erlenmeyer flasks and autoclaved at 121℃ for 15 minutes. 15 mL of seed liquid was then inoculated into each flask of rice medium and cultured at 28℃ under static conditions for 30 days to obtain the fermentation product.

[0011] (2) Extraction of crude extract

[0012] Add an equal volume of ethyl acetate to the fermentation product obtained in step (1), and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract.

[0013] (3) Degreasing and dehydration of crude extract

[0014] The crude extract obtained in step (2) is first dissolved in 95% methanol, and then hexane of the same volume as methanol is added for extraction to remove oil. After repeating the operation three times, the methanol solution is taken and vacuum evaporated to dryness; then it is dissolved in dichloromethane, and water of the same volume as dichloromethane is added for extraction to remove water. The dichloromethane solution is then vacuum evaporated to dryness to obtain the extract.

[0015] (4) Isolation and preparation of compounds

[0016] The extract obtained in step (3) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 100:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution was performed using a methanol-water solution with a methanol volume percentage of 35-100% as the eluent, and the eluent fractions were collected and arranged in descending order of polarity to obtain 6 components. The fourth component was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 65:35 mixture of acetonitrile and water as the mobile phase to obtain a monomeric compound, the structure of which is shown in (I).

[0017]

[0018] Furthermore, the PDA solid culture medium preparation method described in step (1) is as follows: 6g of potato starch, 20g of glucose and 20g of agar are added to 1000mL of distilled water to prepare the medium.

[0019] Furthermore, the preparation method of the PDB liquid culture medium in step (1) is as follows: dissolve 6g of potato starch and 20g of glucose in 1000mL of water.

[0020] Furthermore, the rice culture medium preparation method described in step (1) is as follows: 90g rice, 3g sea salt, 110mL water.

[0021] Furthermore, in step (4), the volume percentage of methanol in the reversed-phase medium-pressure column chromatography linear gradient elution ranges from 35% to 100%, and the elution time is 120 min.

[0022] Furthermore, the flow rate for the semi-preparative reversed-phase high-performance liquid chromatography compound separation preparation described in step (4) is 2.0 mL / min.

[0023] 3. The uses of the above-mentioned butyrolactone compounds in the preparation of multidrug-resistant reverse transcription inhibitors.

[0024] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses a butyrolactone compound, its preparation method and uses. The fermentation product is obtained by fermenting isolated microorganisms, then the fermentation product is extracted with ethyl acetate to obtain a crude extract. The crude extract is then deoiled with hexane and dehydrated with dichloromethane to obtain a final extract. This final extract is then purified by medium-pressure normal-phase column chromatography, medium-pressure reverse-phase column chromatography, and semi-preparative high-performance liquid chromatography to obtain a monomeric compound. This compound possesses multidrug-resistant reverse transcription activity and the ability to regulate P-gp-mediated drug efflux in multidrug-resistant (MDR) cell lines. It can be used as a candidate new drug for the development of potential drugs to treat multidrug-resistant cancers.

[0025] The aforementioned Aspergillus ustus strain DJ003, with accession number CCTCC NO: M2014086, was deposited on March 14, 2014, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. Attached Figure Description

[0026] Figure 1 The proton NMR spectrum of the compound of this invention;

[0027] Figure 2 The carbon NMR spectrum of the compound of this invention;

[0028] Figure 3 The DEPT-135 NMR spectrum of the compound of this invention;

[0029] Figure 4 The COSY NMR spectrum of the compound of this invention;

[0030] Figure 5 The HSQC nuclear magnetic resonance spectrum of the compound of this invention;

[0031] Figure 6 The nuclear magnetic resonance HMBC spectrum of the compound of this invention;

[0032] Figure 7 The NMR NOESY spectrum of the compound of this invention;

[0033] Figure 8 This diagram illustrates the drug resistance reversal activity of the compounds of this invention. A: Fluorescence image after Rhodamine B staining; B: Fluorescence statistics after Rhodamine B staining; C: Real-time quantitative PCR of pxr, abcd4, and cyp3a65. *Significant difference (P<0.05), **Extremely significant difference (P<0.01), ***Highly significant difference (P<0.001). Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0035] Example 1

[0036] The structural formula of a butyrolactone compound with multidrug resistance reverse transcription activity is shown in (I):

[0037]

[0038] Example 2

[0039] The preparation method of the butyrolactone compound as shown in Structural Formula (I) of Example 1 specifically includes the following steps:

[0040] (1) Fermentation production

[0041] Aspergillus ustus with accession number CCTCC NO: M2014086 was streaked onto a plate of PDA solid medium (prepared by adding 6g potato starch, 20g glucose, and 20g agar to 1000mL distilled water). After incubating upside down in a 28℃ incubator for 3 days, a single colony was picked and inoculated into PDB liquid medium (prepared by dissolving 6g potato starch and 20g glucose in 1000mL water). The culture was then incubated on a shaker at 28℃ and 150rpm / min for 3 days to obtain the seed culture. Rice culture medium (90g rice, 3g sea salt, and 110mL water) was added to a 1L Erlenmeyer flask and autoclaved at 121℃ for 15 minutes. Finally, 15mL of the seed culture was inoculated into each flask of rice culture medium and cultured at 28℃ for 30 days to obtain the fermentation product.

[0042] (2) Extraction of crude extract

[0043] Add an equal volume of ethyl acetate to the fermentation product obtained in step (1), and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract.

[0044] (3) Degreasing and dehydration of crude extract

[0045] The crude extract obtained in step (2) is first dissolved in 95% methanol, and then hexane of the same volume as methanol is added for extraction to remove oil. After repeating the operation three times, the methanol solution is taken and vacuum evaporated to dryness; then it is dissolved in dichloromethane, and water of the same volume as dichloromethane is added for extraction to remove water. The dichloromethane solution is then vacuum evaporated to dryness to obtain the extract.

[0046] (4) Isolation and preparation of compounds

[0047] The extract obtained in step (3) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 100:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution was performed using a methanol-water solution with a methanol volume percentage of 35-100% as the eluent, and the eluent fractions were collected and arranged in descending order of polarity to obtain 6 components. The fourth component was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 65:35 mixture of acetonitrile and water as the mobile phase to obtain a monomeric compound, the structure of which is shown in (I).

[0048]

[0049] Example 3

[0050] Compound I of this invention is a colorless oily substance. Cation HR-ESI-MS yielded a quasi-molecular ion peak at m / z 423.1824 [M+H]. + Its molecular formula was determined to be C 25 H 27 O6 (calculated molecular weight 423.1808) indicates an unsaturation degree of 13. This compound... 1 H and 13 C NMR spectra and two-dimensional data are shown below. Figure 1-7 And Table 1: Figure 3-7 The main two-dimensional correlation of the compounds of this invention; Figure 1 of 1 The H NMR spectrum showed a multi-peak signal (δ). H 7.52), and also showed the aromatic proton signal (δ) of 1″,3″,4″-trisubstituted benzene. H 6.57, 6.51, 6.40), 2 methoxy signals (δ H 3.76, 3.67), 1 non-equivalent methylene proton (δ H 3.30) and one alkynyl group (δ) H (5.04, 3.02, 1.62, 1.54); Yes Figure 2 and Figure 3 It is known that this compound contains 25 carbons, among which, according to 13 The C and DEPT 135 spectra indicate the presence of 10 quaternary carbons (C-1, C-2, C-3, C-4, C-6, C-1′, C-1″, C-3″, C-4″, C-9″), 2 methylene groups (C-5, C-7″), and 9 methine groups (C-2′, C-3′, C-4′, C-5′, C-6′, C-2″, C-5″, C-6″, C-8″), along with... Figure 1 of 1 Four methyl carbon signals (2-OMe, 6-OMe, C-10″, C-11″) were detected by high-field ¹H NMR signal analysis; Figure 4 The COSY spectrum shows that H-2′ and H-3′, H-3′ and H-4′, H-4′ and H-5′, H-5′ and H-6′, H-5″ and H-6″ are coupled aromatic hydrogen protons, simultaneously bound to... Figure 5 The HSQC spectrum reveals the chemical shift relationship between aromatic hydrogen protons and aromatic carbon atoms. Figure 2The given low-field chemical shifts (C-1, C-2, C-3, C-4, C-5, C-6) in the six carbon spectra indicate the presence of a butyrolactone skeleton in the compound. A comparison with the NMR spectra of the known compound butyrolactone I reveals that they share the same basic skeleton. The main difference lies in the substitution of the hydroxyl group (C-2) with a methoxy group in butyrolactone I, and the substitution of the 1,4-disubstituted benzene group (C-3) with the benzene group in butyrolactone I. Figure 6 The HMBC spectrum gives 2-OMe(δ) H 3.67) to C-2(δ C HMBC related to 141.9) and Figure 1 of 1 The δ given by the HNMR spectrum H The multiple peak signal at 7.52 (m, 5H) further confirmed the above inference, thus determining the planar structure of I and finally identifying the compound as a new compound. Figure 7 The NOESY spectrum did not show a NOESY correlation. The absolute configuration of I was determined by comparative analysis of experimental and simulated electronic circular dichroism (ECD) spectra and positive rotation values.

[0051] Table 1. Compound I 1 H and 13 C10 NMR data (DMSO-d6)

[0052]

[0053] Note 1: s - singlet, d - doublet, t - triplet, dd - quartet, m - multiplet.

[0054] Note 2: 1 H was obtained by 600MHz NMR; 13 C was obtained by 150MHz NMR.

[0055] Example 4

[0056] Butyrolactone compounds multidrug resistance reverse transcription activity

[0057] (1) Experimental Samples

[0058] Preparation of the test sample solution: The test sample was the pure compound I isolated and purified in Example 3 above. An appropriate amount of sample was accurately weighed and prepared into a solution of the required concentration using dimethyl sulfoxide (DMSO) for testing multidrug resistance reverse transcription activity. The zebrafish used in this experiment were housed in a flow-through tank, with the water temperature maintained at 28±0.5℃ and a photoperiod of 14 hours light: 10 hours darkness. The zebrafish were fed newly hatched brine shrimp.

[0059] (2) Experimental methods

[0060] Six hours after fertilization, zebrafish embryos were placed in six-well plates, with 20 embryos per well. Zebrafish larvae were randomly divided into three groups: a control group, a compound I treatment group (final concentration 5 mg / L), and a positive control group (5 μM cyclosporin A), with each experimental condition performed in triplicate. After 20 hours of incubation, the embryos were treated with 0.1 mM Rhodamine B (RhB) in the dark for 2 hours. Subsequently, the embryos were washed with fish water to remove residual dye from the chorion. Finally, the embryos were decapitated, photographed using a Zeiss stereomicroscope (AXIO Zoom.V16, Oberkochen, Germany), and quantified using the open-source software FIJI (ImageJ). RNA extraction and cDNA synthesis were performed according to the instructions of the purification kit (B518651, Sangon Biotech, Shanghai, China) and the MightyScript Plus First Strand cDNA Synthesis Master Mix (B639252, Sangon Biotech, Shanghai, China). SGExcel FastSYBR Mixture (B532955, Sangon Biotech, Shanghai, China) is used for real-time quantitative PCR analysis.

[0061] (3) Experimental Results

[0062] In the zebrafish model test, by Figure 8 The fluorescence image of compound I showed that the accumulation of rhodamine B (RhB) was comparable to that of the positive control group (5 μM cyclosporin A), indicating that compound I has strong resistance reversal activity. Figure 8 A and Figure 8 B); Simultaneously, real-time quantitative PCR data showed ( Figure 8 C) Compared with the control group, the mRNA expression levels of the pregnane X receptor (pxr) and ATP binding cassette (ABC) efflux transporter gene abcd4 were significantly decreased. This downregulation suggests that compound I may inhibit PXR-mediated downstream gene transcriptional activation, thereby reducing the ability to synthesize P-gp and related transporters.

[0063] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A butyrolactone compound with multidrug resistance reverse transcription activity, characterized in that... The structural formula of the compound is shown in (I); 2. A method for preparing a butyrolactone compound with multidrug resistance reverse transcription activity as described in claim 1, characterized in that... The steps include: (1) Fermentation production Aspergillus ustus with accession number CCTCC NO: M2014086 was streaked on a PDA solid medium plate and incubated upside down in an incubator at 28℃ for 3 days. After that, a single colony was picked and inoculated into PDB liquid medium and cultured on a shaker at 28℃ and 150 rpm for 3 days to obtain the seed liquid. Rice medium was then placed in Erlenmeyer flasks and autoclaved at 121℃ for 15 minutes. 15 mL of seed liquid was then inoculated into each flask of rice medium and cultured at 28℃ under static conditions for 30 days to obtain the fermentation product. (2) Extraction of crude extract Add an equal volume of ethyl acetate to the fermentation product obtained in step (1), and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract. (3) Degreasing and dehydration of crude extract The crude extract obtained in step (2) is first dissolved in 95% methanol, and then hexane of the same volume as methanol is added for extraction to remove oil. After repeating the operation three times, the methanol solution is taken and vacuum evaporated to dryness; then it is dissolved in dichloromethane, and water of the same volume as dichloromethane is added for extraction to remove water. The dichloromethane solution is then vacuum evaporated to dryness to obtain the extract. (4) Isolation and preparation of compounds The extract obtained in step (3) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 100:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution was performed using a methanol-water solution with a methanol volume percentage of 35-100% as the eluent, and the eluent fractions were collected and arranged in descending order of polarity to obtain 6 components. The fourth component was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 65:35 mixture of acetonitrile and water as the mobile phase to obtain a monomeric compound, the structure of which is shown in (I).

3. The method for preparing a butyrolactone compound with multiple drug resistance reverse transcription activity according to claim 2, characterized in that... The method for preparing the PDA solid culture medium in step (1) is as follows: 6g of potato starch, 20g of glucose and 20g of agar are added to 1000mL of distilled water to prepare the medium.

4. The method for preparing a butyrolactone compound with multiple drug resistance reverse transcription activity according to claim 2, characterized in that... The preparation method of PDB liquid culture medium in step (1) is as follows: dissolve 6g of potato starch and 20g of glucose in 1000mL of water.

5. The method for preparing a butyrolactone compound with multiple drug resistance reverse transcription activity according to claim 2, characterized in that... The rice culture medium preparation method described in step (1) is as follows: 90g rice, 3g sea salt, 110mL water.

6. The method for preparing a butyrolactone compound with multiple drug resistance reverse transcription activity according to claim 2, characterized in that: In step (4), the methanol volume percentage in the linear gradient elution of the reversed-phase medium-pressure column chromatography ranges from 35% to 100%, and the elution time is 120 min.

7. The method for preparing a butyrolactone compound with multiple drug resistance reverse transcription activity according to claim 2, characterized in that: The flow rate for the semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) preparation of compounds in step (4) is 2.0 mL / min.

8. Use of the butyrolactone compound of claim 1, which has multidrug resistance reverse transcription activity, in the preparation of multidrug resistance inhibitors.

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