C containing an oxazole ring 30 Terpene polymer compounds and their preparation methods and applications

Abstract

The present invention belongs to the field of medical technology and discloses a C-containing oxazole ring. 30 Terpene polymer compounds and their preparation methods and applications. The present invention utilizes various separation techniques and methods, takes pond cypress seeds as the separation target, utilizes the molecular sieve principle of gel to achieve the enrichment of terpene polymers, and then sequentially performs silica gel column chromatography and ODS column chromatography to obtain the target fraction, and then sequentially uses reversed-phase high-performance liquid chromatography and normal-phase chiral column chromatography to obtain C shown in formula I and formula II. 30 Terpenoid polymer compounds. In vitro anti-tumor cell experiments confirmed that the C 30 Terpenoid polymer compounds have significant anti-tumor activity and can be used as active ingredients in anti-tumor drugs with a wide range of uses.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to a C-containing oxazole ring. 30 Terpene polymer compounds, preparation methods and applications thereof. Background Art

[0002] Terpenoid polymers are a relatively rare type of terpenoid polymers formed by the polymerization of monomeric terpenes. They are widely distributed in a variety of medicinal plants such as the Taxodiaceae, Leguminosae, and Lamiaceae. Common terpenoid polymers are mostly formed by the polymerization of sesquiterpenes and sesquiterpenes, sesquiterpenes and diterpenes, monoterpenes and diterpenes, diterpenes and diterpenes, diterpenes and triterpenes through different linking methods such as DA cyclization, [2+2], [5+2], CC single bonds, ether bonds, and ester bonds to form complex and variable skeleton types, thereby showing diverse and significant biological activities. The C 30 Terpenoid polymers are a relatively rare type of polymer. Modern pharmacological research shows that C 30 Terpenoid polymers exhibit a variety of activities, including anti-tumor, anti-inflammatory, anti-tumor cell migration, inhibition of non-alcoholic fatty liver disease, and hypoglycemic activity. Monoterpenes and diterpenes linked by oxazole rings are a novel class of terpenoid polymers that have not been reported before and are characteristic components of the Taxodium family. Studies have found that terpenoid polymers found in the Taxodium family have significant therapeutic potential for diseases such as cancer, diabetes, and neuroprotection.

[0003] In terms of chemical structure, C 30 Terpenoid polymers are polymers composed of diterpenes and monoterpenes connected by oxazole rings. They are a relatively rare type of terpenoid polymers. Due to the novelty of their structure and significant biological activity, they are increasingly attracting the interest of scientific researchers. Due to the special structure of this type of terpenoid, although it contains 30 carbon atoms, it is not a terpenoid composed of five isoprenes connected end to end as defined by triterpenes. Instead, it is composed of a rosinane-type diterpene unit connected to a monoterpene through an oxazole ring to form a C 30 The terpene polymer has a hydroxyethyl substitution at the 7-position. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a C containing oxazole ring 30 Terpene polymer compounds, preparation methods and applications thereof.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] In the first aspect of the present invention, the present invention provides a C containing oxazole ring 30 The terpene polymer compound is characterized in that its structural formula is as shown in Formula I or Formula II:

[0007]

[0008] In the second aspect of the present invention, the present invention provides a C containing an oxazole ring 30 The preparation method of terpenoid polymer compounds is specifically as follows: the C containing oxazole ring is isolated from the mature seeds of pool cypress 30 Terpenoid polymer compounds.

[0009] As the C containing oxazole ring of the present invention 30 A preferred embodiment of the method for preparing terpenoid polymer compounds comprises the following steps:

[0010] S1. The mature seeds of the pond cypress are extracted with a first organic solvent, and the resulting extract is concentrated in vacuo to an alcohol-free state to obtain an extract;

[0011] S2. The extract is suspended in water and extracted with a second organic solvent to obtain an extract;

[0012] S3. The extract is separated by column chromatography to obtain the C containing oxazole ring 30 Terpenoid polymer compounds.

[0013] As the C containing oxazole ring of the present invention 30 In a preferred embodiment of the method for preparing terpenoid polymer compounds, the first organic solvent is ethanol, preferably 95% ethanol; and the second organic solvent is ethyl acetate.

[0014] As the C containing oxazole ring of the present invention 30 In a preferred embodiment of the method for preparing terpenoid polymer compounds, step S3 comprises the following steps:

[0015] S31. The extract is separated by gel column chromatography to obtain an enriched portion of a terpenoid polymer;

[0016] S32. The enriched portion was sequentially subjected to silica gel column chromatography and ODS column chromatography to obtain a target fraction;

[0017] S33. The target fractions were purified by reverse phase high performance liquid chromatography and separated by normal phase chiral column chromatography to obtain C shown in formula I and formula II. 30 Terpenoid polymer compounds.

[0018] As the C containing oxazole ring of the present invention 30 In a preferred embodiment of the method for preparing terpenoid polymer compounds, the gel column chromatography uses methanol as the elution solvent, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution, and the ODS column chromatography uses a methanol / water mixed solvent for gradient elution.

[0019] As the C containing oxazole ring of the present invention 30 A preferred embodiment of the preparation method of terpene polymer compounds is that the reversed-phase high-performance liquid chromatography uses an XB-C18 chromatographic column with a specification of 5μm, 21.2×250mm, and the mobile phase of the reversed-phase high-performance liquid chromatography is a methanol-water solution, in which the volume ratio of methanol to water is 95:5.

[0020] In the third aspect of the present invention, the present invention also provides a C containing an oxazole ring as described above. 30 Application of terpenoid polymer compounds in the preparation of anti-tumor drugs.

[0021] The above-mentioned C containing oxazole ring 30 Terpene polymer compounds include C shown in formula I and / or formula II 30 Terpenoid polymer compounds.

[0022] In the fourth aspect of the present invention, the present invention also provides a drug, which comprises the above-mentioned C containing oxazole ring 30 A terpenoid polymer compound or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the above-mentioned drug further includes pharmaceutically acceptable excipients and carriers.

[0024] In some embodiments, the pharmaceutical preparation of the above-mentioned drug is granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels or injections.

[0025] Compared with the prior art, the present invention selects mature pond cypress seeds, performs reflux extraction with ethanol, extracts with ethyl acetate multiple times, and then uses various separation techniques and means to obtain the above-mentioned terpene polymers containing oxazole rings of formula I to formula II. The C 30 Terpenoid polymers are a new type of terpenoid polymers discovered for the first time. The discovery of this novel structure has enriched the skeleton and types of terpenoid polymers, and also provided ideas and sources for the discovery of natural drugs. They have significant anti-tumor activity and can be used as active ingredients in anti-tumor drugs, providing alternative compounds for the development of new anti-tumor drugs. DETAILED DESCRIPTION

[0026] The scheme of the present invention will be explained below in conjunction with embodiment. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where specific conditions are not specified in the following examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The methods used are conventional methods well known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar to or equivalent to the described content may also be applied to the present invention.

[0027] Example 1: Preparation of compounds of formula I-II

[0028] The present invention uses mature Taxodium truncatum seeds of Taxodia genus of Taxodiaceae to carry out reflux extraction with 95% ethanol, and prepares the compound of the present invention through the steps of extraction, enrichment and separation. The Taxodium truncatum seeds are collected from Nantong, Jiangsu.

[0029] Taking the seeds of pond cypress as an example, the process for preparing the compounds of formula I-II is as follows:

[0030] 1) Extraction, enrichment and initial separation

[0031] Mature, dried pond cypress seeds (10 kg) were crushed and extracted three times with 95% ethanol under reflux. The mixture was then vacuum concentrated to a concentration free of alcohol, mixed with hot water (60°C), and extracted multiple times with equal proportions of ethyl acetate. The extract (0.5 kg) was subjected to gel column chromatography using methanol as the elution solvent, yielding fractions Fr. 1-8 (200 mL each). After LC-MS identification, the Fr.1-3 fractions were combined and separated by silica gel column chromatography. The elution solvents were successively petroleum ether / ethyl acetate with a volume ratio of 100:0, petroleum ether / ethyl acetate with a volume ratio of 100:5, petroleum ether / ethyl acetate with a volume ratio of 100:10, petroleum ether / ethyl acetate with a volume ratio of 100:20, petroleum ether / ethyl acetate with a volume ratio of 100:30, petroleum ether / ethyl acetate with a volume ratio of 100:50, petroleum ether / ethyl acetate with a volume ratio of 100:100, and petroleum ether / ethyl acetate with a volume ratio of 0:100, to obtain eight fractions Fr.AH, and each fraction was detected by LC-MS, and the fraction Fr.C was locked.

[0032] 2) ODS-C18 column chromatography separation

[0033] Fraction Fr.C was separated by ODS-C18 column chromatography. The mixture was sequentially separated using methanol / water (volume ratio) of 60:40, 70:30, 80:20, 85:15, 90:10, 95:5, and 100:0. Fractions Fr.C1 to Fr.C7 were collected and analyzed by HPLC and LC-MS, identifying the target fraction, Fr.C5.

[0034] 3) Reverse-phase HPLC purification and chiral column separation

[0035] Fraction Fr.C5 was purified by reverse-phase HPLC. Preparation conditions were as follows: XB-C18 (5μm, 21.2×250mm), methanol-water (95:5, v / v), flow rate 10.0 mL / min, detection wavelength 230 nm. NMR data showed a 1:1 mixture of Formula I and Formula II with a very similar structure. Chiral separation using a Daicel AD-H column (5μm, 10.0×250mm), n-hexane-isopropanol (98:2), detection wavelength 230 nm, yielded Formula I and Formula II. Mass spectrometric and spectral data for Formulas I and II are shown below.

[0036] Compound Ⅰ: taxodascaloid A; optical rotation (c 0.18MeOH); UV absorption (MeOH)λ max (logε)215(3.58)nm; the main absorption peak of infrared spectrum (KBr)ν max 2923, 2853, and 1659cm -1 ; High-resolution mass spectrum m / z 494.3626 [M+H] + (calcd for C 32 H 48 NO3), the H NMR and C NMR data are shown in Table 1.

[0037] Compound II: taxodascaloid B; optical rotation (c 0.12MeOH); UV absorption (MeOH)λ max (logε)205(3.77)nm; the main absorption peak of infrared spectrum (KBr)ν max 2922, 2851, and 1658cm -1 ; High-resolution mass spectrum m / z 494.3640 [M+H] + (calcd for C 32 H 48 NO3), the H NMR and C NMR data are shown in Table 1.

[0038] Table 1. H NMR and C NMR data of compounds of formula I-II

[0039]

[0040]

[0041] The above results show that the structures of the obtained compounds of formula I to formula II are correct.

[0042] Example 2: Antitumor activity of compounds of formula I-II

[0043] 1) Experimental Materials

[0044] Instruments and reagents: CO2 incubator (Thermo Fisher Scientific, USA); CKX41 microscope (Olympus Optical Industry Co., Ltd., Japan); Easy pure II laboratory-grade ultrapure water instrument (Thermo Fisher Scientific, USA); DMSO (Biosharp, USA); DMEM / RPMI 1640 culture medium (Invitrogen, USA); DMSO, MTT, trypsin, and cisplatin (Sigma, USA); fetal bovine serum (Hangzhou Sijiqing Biomaterials Research Institute); other commonly used inorganic salts and other reagents were of analytical grade (Nanjing Chemical Reagent Co., Ltd.).

[0045] The tumor cell lines used in the test: HCT-116 cells (colon cancer cells), MDA-MB-231 cells (human breast cancer cells), A549 cells (non-small cell lung cancer cell line), were purchased from the Institute of Oncology, Chinese Academy of Medical Sciences.

[0046] Test sample: C obtained from pond cypress seeds 30 The terpenoid polymer compounds of formula I and formula II have a purity of more than 95%. Cisplatin is selected as a positive control drug, and each compound is dissolved in DMSO and then diluted.

[0047] 2) Experimental methods

[0048] The MTT method was used to determine the half inhibition rate IC of each test compound on tumor cell lines. 50 Value: Tumor cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 5×10 5 / mL, inoculated into 96-well culture plates, 100 μL of cell suspension was added to each well of the drug-treated group and the cell control group, and 3 replicates were set up for each group. Only RPMI 1640 complete culture medium was added to the blank control group, 100 μL per well, and 3 replicates were set up. After the 96-well culture plates were placed in a 37°C, 5% CO2 incubator for 24 hours, different concentrations of test samples were added to make the final concentrations of 0, 10, 20, and 40 μM, and the culture was continued for 24 hours. The absorbance (A) value at 570 nm was measured on a microplate reader according to the MTT method, and the inhibition rate was calculated [inhibition rate = (1-A value of the experimental group / A value of the control group) × 100%]. The experiment was repeated 3 times. SPSS11.5 software was used to make a regression equation to calculate the half-maximal inhibitory concentration (IC50) of each test sample on tumor cells for 24 hours. 50 ).

[0049] 3) Experimental results

[0050] According to the MTT test results, the IC values ​​of the test samples for the above cells were calculated. 50 The results are shown in Table 2.

[0051] Table 2. In vitro cytotoxic activity screening results of test samples

[0052]

[0053] a The above controls and samples were repeated three times.

[0054] The results show that the compounds of formula I and formula II of the present invention have good anti-tumor activity and can be used as active ingredients of anti-tumor drugs.

[0055] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A C containing an oxazole ring 30 Terpene polymer compound, characterized in that Its structural formula is shown in Formula I or Formula II:

2. A C containing an oxazole ring as claimed in claim 1 30 The preparation method of terpenoid polymer compound is characterized in that: The preparation method comprises the following steps: isolating the C-terminal compound containing an oxazole ring from mature seeds of Pool Cypress; 30 terpenoid polymers; The preparation method comprises the following steps: S1. The mature seeds of the pond cypress were extracted with ethanol, and the resulting extract was concentrated under vacuum until it was alcohol-free to obtain an extract; S2. The extract was suspended in water and extracted with ethyl acetate to obtain an extract; S3. The extract is separated by column chromatography to obtain the C containing oxazole ring 30 terpenoid polymers; Wherein, step S3 specifically includes the following steps: S31. The extract is separated by gel column chromatography to obtain an enriched portion of a terpenoid polymer; S32. The enriched portion was sequentially subjected to silica gel column chromatography and ODS column chromatography to obtain a target fraction; S33. The target fractions were purified by reverse phase high performance liquid chromatography and separated by normal phase chiral column chromatography to obtain C shown in formula I and formula II. 30 terpenoid polymers; The gel column chromatography uses methanol as an elution solvent, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution, and the ODS column chromatography uses a methanol / water mixed solvent for gradient elution.

3. The preparation method according to claim 2, characterized in that The reversed-phase high-performance liquid chromatography uses an XB-C18 chromatographic column with a specification of 5 μm and 21.2×250 mm. The mobile phase of the reversed-phase high-performance liquid chromatography is a methanol-water solution, in which the volume ratio of methanol to water is 95:

5.

4. A C containing an oxazole ring according to claim 1 30 Application of terpenoid polymer compounds in the preparation of anti-tumor drugs, wherein the tumor is colon cancer.

5. The use according to claim 4, characterized in that The C containing oxazole ring 30 Terpene polymer compounds include C shown in formula I and / or formula II 30 Terpenoid polymer compounds.

6. A drug, characterized in that The drug comprises the C containing oxazole ring according to claim 1 30 A terpenoid polymer compound or a pharmaceutically acceptable salt thereof.

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