Evodiamide B, a preparation method and use thereof

Abstract

The application relates to the technical field of medicines, and provides a preparation method of a new amide alkaloid compound, i.e., wuzhuyu amide B (1-methyl-3-(2-(quinazolin-4-yl)ethyl)quinazoline-2,4(1 H ,3 H )‑dione) with tumor disease treatment and the use in the medical field for treating cancer, which can be used in the medical field.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel amide alkaloid compound extracted and isolated from Evodia rutaecarpa that has therapeutic effects on tumors—Evodia rutaecarpa amide B (1-methyl-3-(2-(quinazolin-4-yl)ethyl)quinazoline-2,4 (1... H , 3 H Preparation method of )-dione and its application in cancer treatment. Background Technology

[0002] Natural indoloquinazoline alkaloids are extremely rare. Currently, the quinazoline alkaloids reported in the literature are mainly distributed in the genus *Evodia* of the family Rutaceae. Evodia In plants.

[0003] Evodia rutaecarpa, an ancient traditional Chinese medicinal plant, has its fruit used medicinally as early as the Western Han Dynasty, as recorded in the "Fifty-Two Prescriptions" unearthed from the Mawangdui Ruanhou Tomb in Changsha. The classic Chinese medicine Evodia rutaecarpa has a long history of use. Its nearly mature fruits are typically harvested, washed with salt water or stir-fried with vinegar, and then dried for medicinal use. It is also sometimes processed using honey-processed licorice root. Records of Evodia rutaecarpa can be found in major herbal classics from the Jin and Tang dynasties and later, such as the *Shennong Bencao Jing*, *Xinxiu Bencao*, *Daguan Bencao*, *Zhenglei Bencao*, *Zhenghe Bencao*, and *Qimin Yaoshu*. Li Shizhen, in his *Compendium of Materia Medica*, provided a detailed description of the medicinal effects of Evodia rutaecarpa, inheriting and developing the records of Tao Hongjing in *Mingyi Bielu*.

[0004] Evodia rutaecarpa, a plant in the genus Evodia ( E . rutaecarpa (Juss.) Benth.), Leopard ( E . rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang) and sparse hairs of Evodia rutaecarpa ( E . rutaecarpa (Juss.) Benth. var. bodinieri(Dode) Huang is listed in the Chinese Pharmacopoeia and is the source plant of the legally recognized traditional Chinese medicine Evodia rutaecarpa. Evodia rutaecarpa has attracted considerable attention due to its effects of dispelling cold and relieving pain, suppressing nausea and vomiting, tonifying yang and stopping diarrhea, soothing the liver and relieving depression, and promoting qi circulation and relieving pain. Since Asahina et al. first isolated the main components—evodiamine and evodiamine with an indolequinazoline skeleton—from Evodia rutaecarpa in 1929 and determined their structures, modern research on plants of the Evodia genus has made significant progress. Modern pharmacological studies have found that Evodia rutaecarpa has significant effects on the cardiovascular system, central nervous system, and digestive system, and can be used to treat symptoms such as angina pectoris, headache, abdominal pain, and postpartum hemorrhage. In addition, it also has antibacterial and antitumor activities. Summary of the Invention

[0005] This invention provides a novel amide alkaloid compound with significant tumor-inhibiting activity—Evodia amide B (1-methyl-3-(2-(quinazolin-4-yl)ethyl)quinazoline-2,4 (1 H , 3 H Preparation methods of α-dione, euodiamine B) and its use in the medical field for treating cancer.

[0006] The present invention discloses the structural formula of the compound as follows:

[0007]

[0008] The molecular formula is C19H16N4O2, and the degree of unsaturation is 14.

[0009] Molecular weight: 332.

[0010] It is named: Evodiaein B.

[0011] The preparation method of the above-mentioned compound in this invention is as follows:

[0012] The compound is characterized by using *Evodia rutaecarpa*, *Evodia rutaecarpa*, and *Evodia pubescens* (all belonging to the Rutaceae family) as raw materials. First, HPLC was used to determine the content and composition of the main components. Then, the compound was repeatedly purified by silica gel or spherical silica gel, Sephadex LH-20 gel column chromatography, one or more of the reversed-phase material ODS, and recrystallization. MTT assay showed a certain anticancer effect.

[0013] The novel compounds and their derivatives of this invention, together with medically acceptable pharmaceutical excipients, form pharmaceutical formulations for the treatment of tumors. Attached Figure Description

[0014] Figure 1 Chemical structure of compounds Figure 2 1H-NMR spectra of the compounds

[0015] Figure 3 13C-NMR spectrum of the compound Figure 4 1H-1H COSY spectra of the compounds

[0016] Figure 5 HSQC spectra of compounds Figure 6 HMBC spectra of compounds

[0017] Figure 7 ESI-MS spectra of compounds Detailed Implementation

[0018] The present invention will be further described in conjunction with specific embodiments, but the content of the present invention is not limited to the listed embodiments.

[0019] Example 1. Isolation and identification of the new compound from Evodia rutaecarpa

[0020] With Evodia rutaecarpa ( E.rutaecarpa Using quinazoline alkaloids from (Juss.) Benth as the research object, we employed liquid chromatography-mass spectrometry (LC-MS) and preparative liquid chromatography (PCLC) techniques to detect and purify quinazoline alkaloids. 4 kg of Evodia rutaecarpa was used. 200 g of the extracted oil was extracted using a volatile oil extractor to obtain 2 ml of volatile oil, which was then used for GC-MS. The residue was combined with the remaining raw Evodia rutaecarpa and extracted three times by reflux with 80% ethanol, each time for 3 hours. The ethanol extracts were combined and concentrated under reduced pressure to obtain 800 g of a thick extract. The extract was dissolved in water to form a suspension and then extracted sequentially with petroleum ether, chloroform, ethyl acetate, and water-saturated n-butanol. The extracts were concentrated to obtain 14 g of the petroleum ether fraction, 209 g of the chloroform fraction, 26 g of the ethyl acetate fraction, 210 g of the n-butanol fraction, and 316 g of the water fraction. The chloroform fraction was repeatedly purified by silica gel column chromatography at normal, low, and medium pressures using a petroleum ether-ethyl acetate-acetone solvent system or a chloroform-methanol solvent system, Sephadex LH-20 gel column chromatography, or reversed-phase ODS column chromatography using methanol, ethanol, or methanol-water or ethanol-water solutions in different proportions. Recrystallization was then used to separate and purify the monomer compound to 13.0 mg.

[0021] The structure of this compound was identified by nuclear magnetic resonance spectroscopy as follows:

[0022] Evodiamide B is a white powder, soluble in chloroform, methanol, and DMSO. It shows a dark spot under UV254 nm light and no fluorescence under UV365 nm light. It does not show color with modified bismuth potassium iodide reagent, nor does it show color after being sprayed with 5% vanillin-sulfuric acid solution and heated.

[0023] ESI-MS showed that the quasi-molecular ion peak of this compound was m / z 333.2282 [M+H] + (calcd forC) 19 H 17 N4O2 (333.1307). The molecular formula of the compound is deduced to be C. 19 H 16 N4O2, with an unsaturation degree of 14.

[0024] Evodiamide B 1 The 1H NMR spectrum showed 16 hydrogen signals: 8 aromatic hydrogen signals, 1 nitrogen-hydrogen proton signal, 1 nitrogen-methyl proton signal, and a -CH2-CH2- fragment. 1 H- 1 H COSY discovered that these 8 aromatic hydrogens are hydrogens from two ortho-disubstituted benzene rings. 13 C NMR (125 MHz, DMSO- d 6) and 1 H NMR (500 MHz, DMSO- d 6) Data is shown in Table 1. H-23 ( δ H 3.52) with C-2 (150.2) and C-8 (114.5), H-5 ( δ H The HMBC long-range correlation between C-4 (161.0) and C-4 (8.03) also confirms that evodiamide B has a methylquinazoline dione structure, which is corroborated by its secondary mass spectrometry data. Based on the previously deduced molecular formula, the methylquinazoline dione fragment, and the -CH2-CH2- fragment, it can be inferred that evodiamide B also has a quinazoline fragment. Through H-11 ( δ H 7.79) and C-4 (161.0), C-2 (150.2) and C-13 (168.3), H-12 ( δ H The correlation signals of HMBCs at C-13 (168.3) and C-21 (123.5) revealed that the two quinazoline fragments of euodiamide B are connected at the N-3 and C-13 positions by -CH2-CH2-. Thus, the structure of euodiamide B has been determined, and it is a novel bisquinazoline alkaloid, named euodiamide B.

[0025] Table 1. Evodiamide B 1 H NMR, 13 C NMR data

[0026] Position <![CDATA[ δ C ]]> <![CDATA[ δ H ( J in Hz)]]> <![CDATA[ 1 H- 1 H COSY]]> HMBC (H to C) 2 150.2 4 161.0 5 127.7 8.03 (1H, dd, 7.7, 1.5) H-6, H-7 C-4, 7, 9 6 122.7 7.31 (1H, dd, 7.7, 7.3) H-5, H-7 C-5, 7, 9, 10 7 135.2 7.79 (1H, ddd, 7.3, 8.4, 1.5) H-5, H-6, H-8 C-5, 8, 9 8 114.5 7.46 (1H, d, 8.4) H-7 C-4, 5, 6, 9, 10 9 140.2 10 114.6 11 40.0 4.45 (2H, dd, 7.7, 7.5) H-12 C-2, 4, 12, 13 12 31.9 3.60 (2H, dd, 7.7, 7.5) H-11 C-11, 13, 21 13 168.3 15 154.0 9.13 (1H, s) C-13, 21, 22 17 128.3 8.00 (1H, dd, 6.3,) H-18 C-19, 20, 21, 22 18 127.8 7.75 (1H, dd, 6.3, 7.2) H-17, H-19 C-19, 20, 21, 22 19 133.9 7.99 (1H, dd, 7.2, 8.3) H-18, H-20 C-17, 21, 22 20 124.8 8.39 (1H, d, 8.3) H-19 C-13, 17, 18, 21, 22 21 123.5 22 149.1 23 30.5 3.52 (3H, s) C-2, 8, 9

[0027] Example 2. Isolation and identification of the new compound from leopard cat

[0028] Leopard fruit (5 kg) was extracted three times by reflux in a round-bottom flask (5 L). For the first extraction, 2.5 L of 50% ethanol was added and refluxed for 3 h; for the second extraction, 2 L of 50% ethanol was added and refluxed for 2 h; and for the third extraction, 2 L of 50% ethanol was added and refluxed for 1 h. The extracts were combined and concentrated under reduced pressure to obtain a thick extract (1 kg). This extract was mixed with silica gel (100-200 mesh) at a 1:1 ratio, and then coarsely separated by silica gel column chromatography. It was divided into seven fractions (A→G): petroleum ether fraction 52 g (A), petroleum ether-dichloromethane (1:1 v / v) fraction 86 g (B), dichloromethane fraction 93 g (C), dichloromethane-ethyl acetate (1:1 v / v) fraction 74 g (D), ethyl acetate fraction 75 g (E), ethyl acetate-methanol (1:1 v / v) fraction 83 g (F), and methanol fraction 176 g (G). The dichloromethane fraction was repeatedly separated and purified by silica gel column chromatography at normal pressure, low pressure and medium pressure, Sephadex LH-20 gel column chromatography, reversed-phase material ODS column chromatography and recrystallization to obtain 13.7 mg of the monomer compound.

[0029] Example 3. Isolation and identification of the new compound from Evodia rutaecarpa.

[0030] The nearly mature dried fruit of *Evodia rutaecarpa* was pulverized into coarse powder (1.5 kg) and extracted three times by refluxing with 70% ethanol for 2 h each time. The extracts were combined and concentrated under reduced pressure to obtain a thick extract (308 g). The extract was subjected to silica gel column chromatography (100-200 mesh). The petroleum ether fraction (Fr. A, 83 g) was eluted successively with petroleum ether to obtain the petroleum ether fraction (Fr. A, 34 g), with dichloromethane to obtain the dichloromethane fraction (Fr. B, 53 g), with ethyl acetate to obtain the ethyl acetate fraction (Fr. C, 53 g), and with methanol to obtain the methanol fraction (Fr. D, 60 g). During the separation process, guided by TLC and HPLC-Q-TOF-MS detection, the dichloromethane fraction was repeatedly separated and purified by silica gel column chromatography at normal pressure, low pressure and medium pressure, eluted with petroleum ether-ethyl acetate-acetone solvent system or chloroform-methanol solvent system, Sephadex LH-20 gel column chromatography or reversed-phase material ODS column chromatography, eluted with methanol, ethanol or methanol-water, ethanol-water solutions in different proportions, and recrystallization to obtain 2.3 mg of the monomer compound.

[0031] Example 4. Screening of in vitro antitumor activity of evodiamide B

[0032] 1. Materials and Methods

[0033] Breast cancer cell line (MDA-MB-231), cervical cancer cell line (HeLa), and colon cancer cell line (LoVo) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. High-glucose DMEM, fetal bovine serum (FBS), penicillin, and streptomycin were Gibco / Invitrogen products.

[0034] Thermo Nap-flow clean bench (Thermo Scientific, USA), Thermo-6500 CO2 incubator, IX5 inverted microscope (Olympus, Japan), Cerifuge 5804 R high-speed centrifuge (Eppendorf, Germany), MS3digital timer microoscillator (IKA, Germany), Infinite M200 microplate reader (TECAN, USA).

[0035] MTT assay (96-well plate): used for initial screening of the antitumor activity of the test compounds.

[0036] (1) Preparation of test samples: The monomer compound to be tested is prepared into a stock solution with a concentration of 10 mM using dimethyl sulfoxide (DMSO) and stored in a refrigerator at 40 ℃. The working solution is prepared fresh for use, and the concentration of DMSO does not exceed 0.2% (v / v).

[0037] (2) Cell culture: Breast cancer cells MDA-MB-231 and cervical cancer cells HeLa were cultured in a solution containing 10% fetal bovine serum and 100 U / mL. -1 Penicillin, 100 U·mL -1 Colon cancer cells (LoVo) were cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL streptomycin. -1 Penicillin, 100 U·mL -1 Cells were incubated in streptomycin 1640 medium at 37 °C in a humidified incubator containing 5% CO2. The culture medium was changed every other day, and cells in the logarithmic growth phase were used for experiments.

[0038] (3) Plating: MDA-MB-231 cells, HeLa cells, and LoVo cells in the logarithmic growth phase were taken from culture flasks, observed under a microscope, and counted. Then, they were plated into 96-well plates, with 100 μL of MDA-MB-231 cells per well containing 10,000 cells, and 100 μL of HeLa cells and LoVo cells per well containing 5,000 cells. The plates were incubated at 37 ℃ in a saturated humidity incubator containing 5% CO2 for 24 h.

[0039] (4) Adding reagents: In a clean bench, add different test samples to a 96-well plate, repeating each concentration 3 times. Incubate at 37 ℃ in a saturated humidity incubator containing 5% CO2 for 72 h.

[0040] (5) Add MTT: Add 10 μL of MTT (5 mg / mL) to each well. -1 , PBS), and incubate in an incubator for 4 h.

[0041] (6) Measurement and Calculation: Aspirate the culture medium, add 100 μL of DMSO to each well, shake for 5 min, and measure the absorbance of each well using a microplate reader at a wavelength of 570 nm. The absorbance of the test compound is (A). i The absorbance of the blank control was (A0). The cell growth inhibition rate was calculated using the formula below, and the final inhibition rate was expressed as the average value. The concentration was 10 μg / mL. -1 Taxol was used as a positive control.

[0042] Inhibition rate (%) =

[0043] 2. Results and Analysis

[0044] Evodiamide B exhibited moderate to low levels of antitumor activity against breast cancer cell line (MDA-MB-231), cervical cancer cell line (HeLa), and colon cancer cell line (LoVo).

Claims

1. An amide alkaloid compound, characterized in that... The chemical name of this compound is Evodia amide B, and its chemical structural formula is: 。 2. A method for preparing the compound of claim 1, characterized in that... Using Evodia rutaecarpa, Evodia rutaecarpa var. sparse-haired as raw materials, the compound was first extracted by reflux with alcohol, the alcohol extracts were combined, concentrated under reduced pressure, and then separated and purified by multiple silica gel column chromatography with petroleum ether-ethyl acetate-acetone solvent system or dichloromethane-methanol solvent system, Sephadex LH-20 gel column chromatography or reversed-phase column chromatography with methanol, ethanol or methanol-water or ethanol-water solutions in different proportions, and recrystallization to obtain the compound of claim 1.

3. The preparation method according to claim 2, characterized in that... The support used in column chromatography is selected from one or more of silica gel, gel, and reversed-phase ODS.

4. The compound of claim 1, together with a medically acceptable pharmaceutical excipient, constitutes a pharmaceutical preparation.

5. Use of the compound of claim 1 in the preparation of a medicament for treating tumor diseases.

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