CN103288614A - Monocyclic phloroglucinol compounds with antineoplastic activity and pharmaceutical composition thereof - Google Patents

Abstract

The invention relates to monocyclic phloroglucinol compounds Rxd-41 and Rxd-43 or officinal salt and a pharmaceutical composition consisting of an effective component and at least one pharmaceutically acceptable carrier, a preparation method thereof and an application of the monocyclic phloroglucinol compounds in preparation of antineoplastic drugs. Rxd-41 and Rxd-43 are monocyclic phloroglucinol compounds with five-membered ring side chains and are remarkable in vitro cytotoxic activity, novel in chemical structure and strong in activity. The compounds used as antineoplastic drugs have many advantages.

Description

Monocyclic phloroglucinol compound with antitumor activity and pharmaceutical composition thereof

Technical field:

The present invention belongs to the technical field of medicines, and in particular relates to active compounds Rxd41 and Rxd43 with anti-tumor activity and their preparation methods, pharmaceutical compositions with the compounds as active ingredients, and their application in treating tumor diseases.

Background technique:

Hypericum (Hypericum) is a large genus of Garcinia (Guttiferae), with about 400 species in the world, and it is widely distributed in the world except the north and south poles or deserts and most tropical lowlands. There are about 55 species and 8 subspecies in my country, which are produced in various parts of the country, but mainly concentrated in the southwest. This genus is an important group of medicinal and ornamental plants. Many species of Hypericum have medicinal value, among which there are about 18 species of folk medicinal plants. It is mainly used for antibacterial and anti-inflammatory, astringent and hemostasis, detoxification and swelling, dampness and pain relief. In foreign folk or clinical medicine, plants of this genus are often used to treat nervous system diseases, trauma, and antibacterial. It is precisely because many species of plants in this genus have a long-term basis for folk medicine that many countries have carried out a lot of research work on plants in this genus, especially in pharmaceutical research. Successively discovered from different Hypericum plants such as hypericin, quercetin, hyperin, rutin, flavanone alcohols, flavonoids, ketones, coumarins and m-benzene Active ingredients such as triphenol derivatives (hyperforin). Among them, the structural types of phloroglucinol compounds are very rich and varied, and many compounds with new skeleton types are reported. Among them, monocyclic phloroglucinol has various structural types and good biological activity, and has always been a research hotspot in related fields in the world. However, so far, there are no reports on monocyclic phloroglucinol compounds Rxd41 and Rxd43 and their activities in the prior art.

Invention content:

The object of the present invention is to provide a class of monocyclic phloroglucinol compounds with a six-membered ring side chain isolated from Hypericum henryi levl., and its preparation method, especially in antitumor drugs Applications.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

Monocyclic phloroglucinol compounds Rxd-41 and Rxd-43 represented by the following structural formula or pharmaceutically acceptable salts thereof,

The pharmaceutically acceptable salt refers to a pharmaceutically acceptable salt formed with an organic acid such as tartaric acid or citric acid or formic acid or oxalic acid, or with an inorganic acid such as hydrochloric acid or sulfuric acid or phosphoric acid.

A pharmaceutical composition for preventing or treating tumor diseases, comprising the compound Rxd-41, Rxd-43 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

The preparation method of the shown compound Rxd-41, Rxd-43 comprises using 100% acetone or ethanol or methanol solvent, cold soaking or hot reflux extracting the coarse powder of the aerial part of Miscanthus awnicae to obtain the total extract, and the total extract is subjected to various The compound was obtained after forward and reverse phase column chromatography and preparative and semi-preparative HPLC separation.

Application of the compounds Rxd-41, Rxd-43 or pharmaceutically acceptable salts thereof in the preparation of drugs for preventing or treating tumors.

Application of the compounds Rxd-41, Rxd-43 or pharmaceutically acceptable salts thereof in the preparation of drugs for preventing or treating leukemia, lung cancer and colon cancer.

The compounds Rxd-41 and Rxd-43 of the present invention can be directly used as medicine, and the other auxiliary materials are pharmaceutically acceptable, non-toxic and inert pharmaceutical carriers or excipients to humans and animals.

The pharmaceutical carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, superfillers and pharmaceutical product adjuvants. The effective extract or effective part is used in the form of dosage per unit body weight. The medicine of the present invention can be administered in two forms of oral administration and oral spraying.

Its solid or liquid preparations can be used orally, such as powders, tablets, sugar-coated tablets, capsules, tinctures, syrups, dropping pills, etc.

Oral spray can be used in its solid or liquid formulations.

The medicine of the invention can be used for treating tumor diseases.

Detailed ways:

The following examples of the present invention are used to further illustrate the substantive content of the present invention, but the present invention is not limited thereto.

Example 1:

Preparation of compounds Rxd-41, Rxd-43:

Separation process: dry 18kg of the aboveground part of Miscanthus radix, crush it, soak it three times at room temperature with 100% methanol (50 liters each time), and soak it for 2 days each time. After the extract is concentrated, mix the sample with silica gel and put it on a silica gel column. Ethyl acetate flush. 125 g of the chloroform part was dissolved in acetone, and the polyamide mixed sample was loaded on the MCI column, and methanol/water was used as the gradient elution of the mobile phase, detected by TLC, and the same fractions were combined to obtain seven parts. The third part of the silica gel mixed sample was divided into three parts through a silica gel column. After the component 2 was passed through a Sephadex LH-20 column, it was separated by MPLC to obtain Rxd-41 (18 mg) and Rxd-43 (10 mg).

Compound structure analysis:

ESI-MS谱给出准分子离子峰为m/z507[M+Na]⁺，结合高分辨正离子HR-ESIMS(m/z[M+Na]⁺507.3093,calcd for507.3086)和NMR谱提供的信息，确定其分子式为C₃₀H₄₄O₅。UV光谱在206(3.3),224(3.3),356（3.2）nm处有吸收，说明分子中存在多个共轭基团。IR光谱显示存在羟基（3419cm^-1，br）和羰基（1727and1637cm^-1）等基团。分析¹H和¹³C NMR谱，再结合HSQC谱表明化合物43中含有30个碳信号，其中包括8个甲基，6个亚甲基，5个次甲基;11个季碳(包括9个不饱和季碳)。Compound Rxd-43, colorless oil,

The ESI-MS spectrum gives the quasi-molecular ion peak as m/z507[M+Na] ⁺ , combined with high-resolution positive ion HR-ESIMS (m/z[M+Na] ⁺ 507.3093, calcd for507.3086) and NMR spectrum to provide According to the information, its molecular formula is determined to be C ₃₀ H ₄₄ O ₅ . The UV spectrum has absorption at 206 (3.3), 224 (3.3), 356 (3.2) nm, indicating that there are multiple conjugated groups in the molecule. The IR spectrum showed the presence of groups such as hydroxyl (3419cm ^-1 , br) and carbonyl (1727and1637cm ^-1 ). Analysis of ¹ H and ¹³ C NMR spectra combined with HSQC spectra showed that compound 43 contained 30 carbon signals, including 8 methyl groups, 6 methylene groups, 5 methine groups; 11 quaternary carbons (including 9 unsaturated quaternary carbon).

Table-1. NMR spectral data of Rxd-43 (Me ₂ CO)

ESI-MS谱给出准分子离子峰为m/z429[M-H]^-，结合高分辨正离子HR-EI(m/z[M]⁺430.2726,calcd for430.2719)和NMR谱提供的信息，确定其分子式为C₂₆H₃₈O₅。UV光谱在202(3.0),223(2.9),290（2.7），356（2.8）nm处有吸收，说明分子中存在多个共轭基团。IR光谱显示存在羟基（3426cm^-1，br）和羰基（1712and1632cm^-1）等基团。分析¹H和¹³C NMR谱，再结合HSQC谱表明化合物41中含有26个碳信号，其中包括7个甲基，5个亚甲基，4个次甲基;10个季碳(包括8个不饱和季碳)。Compound Rxd-41, colorless oil,

The ESI-MS spectrum gives the quasi-molecular ion peak as m/z429[MH] ^- , combined with the information provided by the high-resolution positive ion HR-EI (m/z[M] ⁺ 430.2726, calcd for430.2719) and the information provided by the NMR spectrum, it is determined that Its molecular formula is C ₂₆ H ₃₈ O ₅ . The UV spectrum has absorption at 202 (3.0), 223 (2.9), 290 (2.7), 356 (2.8) nm, indicating that there are multiple conjugated groups in the molecule. The IR spectrum showed the presence of groups such as hydroxyl (3426cm ^-1 , br) and carbonyl (1712and1632cm ^-1 ). Analysis of ¹ H and ¹³ C NMR spectrum combined with HSQC spectrum shows that compound 41 contains 26 carbon signals, including 7 methyl groups, 5 methylene groups, 4 methine groups; 10 quaternary carbons (including 8 unsaturated quaternary carbon).

Table-2. NMR spectral data (MeOD) of Rxd-41

Example 2:

In vitro anti-tumor activity evaluation of Rxd41 and Rxd43:

Drugs and reagents: RPMI1640 medium, DMEM medium, fetal bovine serum (FBS), and trypsin for cell experiments are GibcoBRL products (Invitrogen Corporation, USA); cell counting kit CCK-8 is a product of Japan Tongjin Co., Ltd. Other chemical reagents were purchased from Sigma/Aldrich Company.

Cell lines and cell culture: the culture medium of human non-small cell lung cancer cell line NCI-H460 and human prostate cancer cell line PC3 was RPMI1640 medium supplemented with 10% FBS, and the culture medium of human breast cancer cell line MCF-7 was Added 10% FBS high sugar DMEM medium, human colorectal adenocarcinoma cell line HCT-15 culture medium was added 10% FBS, 2mM L-glutamine, 1.5g/L sodium bicarbonate, 4.5g/L Glucose, 10mM HEPES and 1.0mM sodium pyruvate were cultured in RPMI1640 medium. Cell culture conditions: 37°C, saturated humidity, 5% carbon dioxide incubator.

Drug action: Collect tumor cells in the logarithmic growth phase, use culture medium to prepare a single cell suspension containing 5×10 ⁴ cells per ml, inoculate 100 μL/well in a 96-well plate, and then place it in a 37°C incubator for incubation Allow cells to attach for 24 hours. Then replace the culture solution in each well with fresh culture solution containing different concentrations of the drug to be tested, and every 3 wells is a detection concentration. The experiment also set up a blank control group of culture medium and a control group of cells without drug treatment. After the drug interacted with the cells for 72 hours, the culture plate was taken out, 10 μL/well of CCK-8 was added, and then placed in the cell culture incubator for 1-2 hours. Take it out, and measure the optical density value OD of each well at 450 nm on a microplate reader. The optical density value is positively correlated with the number of living cells. According to the OD value, the viability (%) of the cells under the action of different concentrations of drugs relative to the cells in the control wells can be obtained, and the drug concentration when the cell viability is 50% is the half inhibitory concentration IC ₅₀ of the drug on cell proliferation. The smaller the IC ₅₀ value, the stronger the ability of the drug to inhibit the proliferation of tumor cells.

Results: Rxd41 and Rxd43 showed inhibitory activity on the proliferation of human non-small cell lung cancer cell line NCI-H460, human colorectal adenocarcinoma cell line HCT-15, human breast cancer cell line MCF7 and human prostate cancer cell line PC3. Among them, Rxd43 had the strongest activity, and showed significant proliferation inhibitory ability on all four cell lines (IC ₅₀ 1.3-7.9μM), and had the strongest inhibitory ability on HCT-15 cells. Rxd41 also showed significant inhibitory activity on the proliferation of NCI-H460, HCT-15 and MCF-7 cells (IC ₅₀ 2.4～7.0μM), and also showed stronger inhibitory activity on HCT-15 cells, but its The ability to inhibit the proliferation of PC3 cells is slightly weaker. See Table 3 for specific data.

Table 3. Inhibitory effect of Rxd41 and Rxd43 on tumor cell proliferation (IC ₅₀ , μM)

Note: The table shows the mean ± standard deviation of three independent test results

Conclusion: Rxd41 and Rxd43 have significant anti-tumor effects, and have certain anti-tumor effects.

Example 3:

Rxd41 and Rxd43 were prepared according to Example 1, and excipients were added at a weight ratio of 1:1 to the excipients, granulated and compressed into tablets.

Example 4:

Rxd41 and Rxd43 were prepared according to Example 1, and excipients were added at a weight ratio of 1:2 to the excipients, granulated and compressed into tablets.

Example 5:

Prepare Rxd41 and Rxd43 according to Example 1, and make capsules according to the conventional capsule preparation method.

Embodiment 6:

Obtain Rxd41, Rxd43 according to embodiment 1, then make tablet according to the following method:

tablet:

Embodiment 7:

Capsules: Rxd41, Rxd43 100mg

Appropriate amount of starch

Magnesium Stearate Appropriate amount

Preparation method: mix Rxd41 and Rxd43 with additives, sieve, mix evenly in a suitable container, and put the obtained mixture into hard gelatin capsules.

Embodiment 8:

Nasal spray:

Preparation method: Add one ingredient at a time to an appropriate volume of double-distilled water with stirring until completely decomposed, and then add another ingredient. After making up to 2 ml with water, the solution is filtered on a sterile filter, filled into bottles and divided according to the appropriate doses.

Embodiment 9:

Dropping pills: Rxd41, Rxd43 1g

Macrogol 6000 9g

Preparation method: Preparation of Rxd41, Rxd43 and Polyethylene Glycol 6000 melt solution: Weigh Rxd41 and Rxd43 according to the above prescription amount, add appropriate amount of absolute ethanol, after slightly heating to dissolve, add the prescription amount into the polyethylene glycol melt solution ( 60°C water bath for heat preservation), stir and mix evenly until the ethanol evaporates completely, then place it in a 60°C water bath for 30 minutes, wait for the air bubbles to be removed, and then transfer the above-mentioned mixed molten liquid that has been removed from the air bubbles into the liquid storage cylinder. Under the condition of heat preservation of 80-85 ℃, control the dripping speed, drip into the condensate drop by drop, wait until the condensation is complete, pour off the condensate, collect the dropping pills, drain and remove the condensate on the pills with filter paper, and place silica gel Dry in a desiccator or naturally.

Claims (6)

1. Monocyclic phloroglucinol compounds Rxd-41 and Rxd-43 shown in the following structural formula or their pharmaceutically acceptable salts,

2. The pharmaceutically acceptable salt as claimed in claim 1, refers to a pharmaceutically acceptable salt formed with an organic acid such as tartaric acid or citric acid or formic acid or oxalic acid, or with an inorganic acid such as hydrochloric acid or sulfuric acid or phosphoric acid Salt. 3. A pharmaceutical composition for preventing or treating tumor diseases, which comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier. 4. The preparation method of the compound shown in claim 1 comprises using 100% acetone or ethanol or methanol solvent, cold soaking or hot reflux leaching aerial part coarse powder of Miscanthus awnicae to obtain total extract, total extract after various positive and negative The compound was obtained after column chromatography and preparative and semi-preparative HPLC separation. 5. The compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating tumors. 6. The application of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention or treatment of leukemia, lung cancer and colon cancer.