CN114075213B - Cephalotaxine N-oxide derivative and its preparation method and use - Google Patents
Cephalotaxine N-oxide derivative and its preparation method and use Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
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- -1 Cephalotaxine N-oxide Chemical class 0.000 title abstract description 13
- 229930013930 alkaloid Natural products 0.000 claims abstract description 13
- 150000003797 alkaloid derivatives Chemical class 0.000 claims abstract description 11
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- 150000001875 compounds Chemical class 0.000 claims description 12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/16—Peri-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a cephalotaxine N-oxide derivative, a preparation method thereof and application thereof in preparing antitumor medicines. The N-oxide cephalotaxine component (cephalotaxine A-D) is separated from cephalotaxine branches and leaves, and proved by in vitro activity tests, the alkaloid component (cephalotaxine A-D) has remarkable inhibitory activity on proliferation of cell strains such as human non-small cell lung cancer (A549), human large cell lung cancer (NCI-H460), human promyelocytic leukemia (HL 60), human plasma cell leukemia (NCI-H929) and human myeloma cells (RPMI-8226), so that the cephalotaxine component can be used for preparing medicines for treating tumor diseases such as lung cancer, leukemia and myeloma.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a cephalotaxine N-oxide derivative in cephalotaxine, a preparation method thereof and application thereof in preparing antitumor medicines, in particular to application in preparing medicines for treating tumor diseases such as lung cancer, leukemia or myeloma.
Background
It is known that malignant tumors are seriously threatening the health and life of human beings, and have become one of the most important causes of death worldwide. The current situation of malignant tumor disease in China is particularly severe, and the trend is rising in recent years. According to the latest data published by the national cancer center, the new incidence of cancer in China is about 380.4 tens of thousands of cases each year, wherein the incidence rate and the mortality rate of lung cancer are all the first (Siegel et al, CACancer J Clin 2017,67,7-30). The treatment scheme for malignant tumor diseases at the present stage still takes chemotherapy and radiotherapy as main treatment, and medical practice shows that the treatment measures have large side effects and high recurrence rate, are easy to generate drug resistance and have unsatisfactory clinical curative effects. Therefore, the development of antitumor drugs with high efficiency and low toxicity is an important subject that the pharmaceutical community is actively striving for and is urgently expected to break through. Traditional Chinese medicines are increasingly valued by medical workers according to the unique theory of medicine properties and exact clinical curative effects. Meanwhile, natural products derived from traditional Chinese medicines often have the characteristics of structural complexity and diversity, and are an important source for developing innovative medicines. Therefore, the development of the novel anti-tumor drug from the active ingredients of the traditional Chinese medicine has wide research prospect (Newman and Cragg, JNAT Prod 2020,83,770-803).
Cephalotaxus hainanensis (Cephalotaxus hainanensis Mast.) is a rare endangered plant of Cephalotaxus genus of Cephalotaxus family, and sporadically grows in primary and secondary evergreen broad-leaved forest with altitude of 300-1800 m, and is distributed in Sichuan, yue, guangdong, gui, gangxi, hunan, hubei province, qian, yunnan and other provinces in China. Due to the low level of genetic diversity, limited ability to proliferate itself, and the addition of manual harvesting and ecological environmental disruption, the natural number of Cephalotaxus cinerea has been drastically reduced, and has been defined as a progressive species by the Chinese redbook and is listed in 1999 as a national secondary protective plant (Fu et al China Plant Red Data Book: rare and Endangered Plants, science Press: beijin, 1992, pp 26). Cephalotaxus has been belonging to the family Ponciraceae, but later on, based on its special plant morphology, tissue anatomy, geographical distribution etc., especially the uniqueness of the phytochemicals of this genus, this genus was subsequently raised to a separate family (Cephalotaxus, cephalotaxus only 1) (Aldelkafi et al, nat Prod Rep 2012,29,845-869). The seeds of Cephalotaxus plants have effects of resolving food stagnation, moistening lung, expelling parasites, and can be used for treating ascariasis, ancylostomiasis, dyspepsia, etc. in traditional application in China. Meanwhile, researches show that cephalotaxus plants are rich in alkaloids and biflavone components, wherein alkaloids are main active components. At the beginning of the 60 s of the 20 th century, pauder et al first isolated alkaloid components from cephalotaxus fortunei and cephalotaxus japonica plants (Pauder et al, J Org Chem 1963,28,2194-2197), but until 1969 Powell et al determined their structure by X-ray single crystal diffraction and found that cephalotaxine compounds therein had significant inhibitory activity on mouse P388, L-1210 leukemia cells (Powell et al, tetrahedron letters 1969,46,4081-4084;Mikolajczak et al, tetrahedron 1972,28,1995-2001). The unique five-membered condensed ring structure and remarkable pharmacological activity of cephalotaxine have attracted attention of researchers in various countries of the world, and research results are endlessly and continuously deep. China established a cephalotaxus study collaboration group nationwide in the beginning of the 70 s of the 20 th century, conducted resource screening and chemical composition study on cephalotaxus plants distributed in China, and developed cephalotaxine (harringtonine) and homoharringtonine into anticancer drugs for the first time, which are now listed as 36 common anticancer drugs in the world, clinically used for treating Chronic Myelogenous Leukemia (CML) and acute non-lymphoblastic leukemia (ANLL) (Mei et al, chinese Herb Med 2006, 452-458). The us FDA approved homoharringtonine (HHT, synribo) for the treatment of Chronic Myelogenous Leukemia (CML) at 10 months 2012, and was applicable to chronic or accelerated phase CML adult patients (Kantarjian et al Lymph Myelom Leuk 2013,13,530-533) who are resistant and/or tolerant to two or more Tyrosine Kinase Inhibitors (TKIs).
However, researches on chemical components of special plant Cephalotaxus fortunei in China are recently reported. Based on the current state of the art, the inventor of the application is to search a novel antitumor drug lead compound and further explore and expand the clinical application range of cephalotaxine components (not limited to leukemia), and is to study the novel alkaloid components with N-oxide functional groups in cephalotaxine and the antitumor activity of the alkaloid components.
Disclosure of Invention
The invention aims to provide a cephalotaxine N-oxide derivative in Cephalotaxus fortunei, a preparation method thereof and application thereof in preparing anti-tumor drugs, in particular to application in preparing drugs for treating tumor diseases such as lung cancer, leukemia, myeloma and the like.
The invention extracts and separates cephalotaxine N-oxide derivatives from special rare or endangered medicinal plant Cephalotaxine in China, and in vitro anti-tumor activity tests show that the alkaloid components have remarkable cytotoxicity on various tested human tumor cell strains, and can be used for preparing anti-tumor drugs or lead compounds of the drugs.
The cephalotaxine N-oxide derivative (cephalotaxine A-D, 1-4) takes nitrogen atoms in cephalotaxine mother nucleus to form N-oxide form (nitrogen oxide, N-O) as main structural characteristics, has the following structural formula, and cephalotaxine A-D is a natural cephalotaxine (cephalotaxine ester-type) N-oxide derivative reported for the first time.
The structural formula of the cephalotaxine N-oxide derivative is any one of the following (cephalotaxine A-D, (1) - (4):
the preparation method of the cephalotaxine N-oxide derivative provided by the invention comprises the following specific steps:
pulverizing branches and leaves of Cephalotaxus fortunei, soaking in 95% ethanol at room temperature, concentrating the extractive solution under reduced pressure, suspending in 3% tartaric acid aqueous solution, extracting with equal volume of ethyl acetate for 3-5 times, and concentrating the extractive solution with Na 2 CO 3 Regulating pH to 10, and extracting with chloroform to obtain chloroform extract (i.e. total alkaloid fraction); separating chloroform extract by MCI resin, polyamide resin, sephadex LH-20 gel column chromatography and reversed-phase semi-preparative HPLC chromatography to obtain compounds (cephalofeines A-D).
The invention provides an application of cephalotaxine N-oxide derivatives in preparing medicines for treating tumor.
In the present invention, the tumor disease is any one of lung cancer, leukemia or myeloma.
In the invention, the cephalotaxine N-oxide derivative is singly or jointly used for preparing medicines for treating tumor diseases.
In the invention, the cephalotaxine N-oxide derivative is combined with a pharmaceutically acceptable carrier or excipient to prepare an oral or non-oral dosage form.
The compound can be obtained by separating and purifying branches and leaves of cephalotaxus hainanensis; or can be obtained synthetically by chemical methods well known to those skilled in the art.
The invention tests the in vitro anti-cytotoxicity activity of the cephalotaxine derivatives (cephalotaxine A-D), and the results show that the compounds have remarkable inhibitory activity on the proliferation of various tested human tumor cell strains (human non-small cell lung cancer A549, human large cell lung cancer NCI-H460, human promyelocytic leukemia HL60, human plasma cell leukemia NCI-H929 and human myeloma cell RPMI-8226), so that the compounds can be used for preparing anti-tumor drugs, especially for preparing drugs for treating tumor diseases such as lung cancer, leukemia and myeloma, or as lead compounds of the drugs.
The invention has the beneficial effects that:
the target compound (cephalotaxine A-D, 1-4) is a first discovered natural cephalotaxine (cephalotaxine ester-type) component with an N-oxide form, has a novel chemical structure, and has not been reported in any literature so far; and cephalovertesea-D is the first reported N-oxide derivative with significant antitumor activity in cephalotaxus alkaloid (Cephalotaxus alkaloids). In addition, the invention evaluates the antitumor activity of cephalotaxine components on human plasma cell leukemia (NCI-H929) cell line for the first time. The compounds (cephalverines A-D) show remarkable cytotoxic activity on 5 tested human tumor cells, and have considerable application prospects in the treatment of increasingly severe tumor diseases (such as lung cancer).
Detailed Description
The invention is further illustrated below with reference to examples, which are in no way limiting of the invention. Any alterations in the practice of this invention that come within the scope of the claims will be within the purview of those skilled in the art in light of the present description.
In the following examples, specific rotation testing was performed by JASCO P-1020 polarimeter; the ultraviolet and infrared spectrum data are respectively obtained by a Shimadzu UV-2550 ultraviolet spectrometer and a Nicolet AVATAR 360 infrared spectrometer; NMR was measured using a bruker avance II type 400 and type 600 nuclear magnetic instruments; MS was determined by an Agilent 1100Series LC/MSD G1946D type instrument and HR-MS was determined by a AB Sciex TripleTOF 5600 type instrument; sephadex LH-20 Gel is manufactured by Switzerland GE Healthcare Bio-Sciences, MCI Gel CHP 20P (75-150 μm) is manufactured by Mitsubishi corporation of Japan, and polyamide resin (30-60 mesh) is manufactured by Taizhou road bridge tetramethyo chemical corporation; semi-preparative HPLC was a Waters e2695 equipped with 2998PDA and 2424 evaporative light scattering double detectors and SunFire ODS (5 μm, 250X 10 mm) semi-preparative column; all reagents were manufactured by Shanghai national pharmaceutical Congress chemical reagent Co.
Example 1: preparation of Natural cephalotaxine N-oxide derivatives (cephalofinase A-D, 1-4)
Dried branches and leaves (9.0 kg) of cephalotaxus hainanensis are fully crushed, extracted for 4 times by 95% ethanol at room temperature, and the extracting solutions are combined and concentrated under reduced pressure to obtain 700g of total extract. Dispersing the total extract with 3% tartaric acid aqueous solution, extracting with ethyl acetate (4 times), collecting ethyl acetate layer as non-alkaloid part, and collecting the rest water layer with Na 2 CO 3 The pH was adjusted to 10 and extracted with chloroform, and the concentrated chloroform layer was the total alkaloid fraction (14.0 g). First, MCI column chromatography (MeOH-H) 2 O,1:1-1:0, v/v) the total alkaloid fraction was coarsely separated to give 6 fractions (Fr. A-Fr. F). Fr.A (800 mg) was crude separated by polyamide column chromatography, followed by MeOH-H 2 The O system is eluted in a gradient way (1:2-1:0, v/v) to obtain the subfractions A1-A3. Compound 3 (4.0 mg, t) R =22.0 min) and 4 (3.1 mg, t R =14.6 min) from fr.a3 via semi-preparative HPLC [ MeOH-H 2 O (containing 0.05% Et) 2 NH,v/v)11:89,v/v；flow rate,3.0mL/min]Is prepared. Fr.B (600 mg) was crude separated by Sephadex LH-20 (MeOH) to give Fr.B1-B3. Compound 1 (6.5 mg, t) R =13.9 min) from fr.b3 via semi-preparative HPLC [ MeOH-H 2 O (containing 0.05% Et) 2 NH,v/v)40:60,v/v；flow rate,3.0mL/min]Purifying to obtain the final product. Fr.D (1.0 g) was subjected to Sephadex LH-20 (MeOH) column chromatography to give four sub-components D1-D4, fr.D2 was then purified by semi-preparative HPLC to give compound 2 (9.4 mg, t) R ＝14.7min；MeOH/H 2 O=48/52, v/v) the cephalofeinesa-D (1-4) obtained by separation is all new compounds, the spectrum and physicochemical data of which are as follows:
cephalovertine A (1) is colorless jelly; [ alpha ]] D 20 –57(c 0.10,MeOH)；UV(MeOH)λ max (logε)291(3.47)nm；IR(film)ν max :3386,2970,2921,2853,1747,1656,1591,1500and 1488cm -1 ； 1 H NMR(400MHz,CDCl 3 ):δ6.67(1H,s,H-17),6.47(1H,s,H-14),5.99(1H,d,J＝9.6Hz,H-3),5.92(1H,d,J＝1.2Hz,H-18),5.86(1H,d,J＝1.2Hz,H-18),5.13(1H,s,H-1),3.87(1H,d,J＝9.6Hz,H-4),3.80(3H,s,OMe-19),3.78(1H,m,H-8b),3.75(1H,m,H-10a),3.73(1H,m,H-10b),3.60(3H,s,OMe-5'),3.58(1H,m,H-8a),3.44(1H,ddd,J＝14.0,13.5,6.0Hz,H-11b),2.69(1H,m,H-6a),2.54(1H,ddd,J＝14.0,6.0,2.0Hz,H-11a),2.29(1H,d,J＝16.5Hz,H-3'),1.97(1H,m,H-6b),1.97(1H,m,H-7a),1.93(1H,m,H-7b),1.81(1H,d,J＝16.5Hz,H-3'),1.57(2H,m,H 2 -1"),1.53(1H,dt,J＝4.6,13.5Hz,H-2"),1.25(1H,m,H-2"),1.16(3H,s,OMe-5"),1.14(3H,s,OMe-4")； 13 C NMR(100MHz,CDCl 3 ):δ173.7(C-1'),170.7(C-4'),162.5(C-2),146.4(C-16),146.2(C-15),130.6(C-12),128.4(C-13),111.8(C-14),109.6(C-17),101.2(C-18),99.5(C-1),86.3(C-5),74.9(C-2'),72.9(C-3),70.1(C-3"),69.3(C-8),62.2(C-10),58.6(C-19),52.3(C-4),51.9(C-5'),42.8(C-3'),39.2(C-6),36.8(C-2"),33.3(C-1"),29.9(C-5"),29.0(C-4"),28.4(C-11),18.2(C-7)；ESIMS m/z 548[M+H] + ；HRESIMS m/z 548.2486[M+H] + (calcd for C 28 H 38 NO 11 ,548.2490)；
Cephalovertine B (2) is colorless jelly; [ alpha ]] D 20 –77(c 0.10,MeOH)；UV(MeOH)λ max (logε)292(3.55)nm；IR(film)ν max :3385,2967,2920,2850,1747,1650,1589,1500and 1489cm -1 ； 1 H NMR(400MHz,CDCl 3 ):δ6.61(1H,s,H-17),6.60(1H,s,H-14),5.94(1H,br s,H-18),5.93(1H,br s,H-18),5.88(1H,d,J＝8.5Hz,H-3),5.40(1H,s,H-1),4.04(1H,ddd,J＝13.0,11.6,6.0Hz,H-10a),3.82(3H,s,OMe-19),3.73(1H,m,H-10b),3.70(1H,d,J＝8.5Hz,H-4),3.58(1H,m,H-11b),3.53(3H,s,OMe-5'),3.50(1H,m,H-8b),3.07(1H,dd,J＝16.0,8.6Hz,H-8a),2.94(1H,ddd,J＝14.5,10.3,7.0Hz,H-6a),2.83(1H,ddd,J＝15.0,8.5,6.0Hz,H-11a),2.45(1H,d,J＝16.2Hz,H-3'),2.28(1H,d,J＝16.2Hz,H-3'),1.87(1H,ddd,J＝14.5,9.3,3.2Hz,H-6b),2.27(1H,m,H-7a),1.72(1H,m,H-7b),1.68(2H,m,H 2 -1"),1.51(1H,dt,J＝5.1,13.0Hz,H-2"),1.22(1H,m,H-2"),1.17(3H,s,OMe-5"),1.13(3H,s,OMe-4")； 13 C NMR(100MHz,CDCl 3 ):δ173.9(C-1'),170.3(C-4'),161.1(C-2),148.2(C-16),146.9(C-15),130.4(C-12),125.4(C-13),112.3(C-14),109.6(C-17),101.6(C-18),101.8(C-1),90.3(C-5),75.0(C-2'),75.8(C-3),69.6(C-3"),65.7(C-8),61.6(C-10),58.0(C-19),56.2(C-4),51.7(C-5'),43.4(C-3'),38.1(C-6),36.7(C-2"),33.5(C-1"),30.2(C-5"),28.8(C-4"),30.4(C-11),19.7(C-7)；ESIMS m/z 548[M+H] + ,1095[2M+H] + ；HRESIMS m/z 548.2486[M+H] + (calcd for C 28 H 38 NO 11 ,548.2490)；
Cephalovertine C (3) is colorless jelly; [ alpha ]] D 20 –68(c 0.10,MeOH)；UV(MeOH)λ max (logε)291(3.50)nm；IR(film)ν max :3384,2970,2922,2850,1753,1649,1594and 1488cm -1 ； 1 H NMR(400MHz,DMSO-d 6 ):δ6.74(1H,s,H-17),6.51(1H,s,H-14),5.90(1H,br s,H-18),5.82(1H,br s,H-18),5.78(1H,d,J＝9.6Hz,H-3),5.31(1H,s,H-1),4.03(1H,d,J＝9.6Hz,H-4),3.79(1H,m,H-8b),3.76(1H,m,H-8a),3.70(1H,m,H-10a),3.67(3H,s,OMe-19),3.66(1H,m,H-10b),3.31(1H,m,H-11b),2.56(1H,dd,J＝14.0,6.5Hz,H-11a),2.45(1H,m,H-6a),1.98(1H,m,H-6b),1.91(1H,m,H-7a),1.74(1H,d,J＝16.2Hz,H-3'),1.72(1H,m,H-7b),1.45(1H,d,J＝16.2Hz,H-3'),1.35(1H,m,H-1"),1.34(1H,m,H-2"),1.30(1H,m,H-1"),1.08(1H,m,H-2"),0.99(3H,s,OMe-5"),0.95(3H,s,OMe-4")； 13 C NMR(100MHz,DMSO-d 6 ):δ172.8(C-1'),170.9(C-4'),162.0(C-2),145.5(C-16),145.3(C-15),130.6(C-12),128.6(C-13),111.3(C-14),109.1(C-17),100.5(C-18),99.4(C-1),86.5(C-5),74.4(C-2'),71.6(C-3),68.3(C-3"),67.5(C-8),60.7(C-10),57.8(C-19),50.7(C-4),43.0(C-3'),38.0(C-6),36.7(C-2"),33.5(C-1"),29.9(C-5"),28.5(C-4"),27.2(C-11),17.3(C-7)；ESIMS m/z 534[M+H] + ；HRESIMS m/z534.2329[M+H] + (calcdfor C 27 H 36 NO 10 ,534.2334)；
Cephalovertine D (4) is colorless jelly; [ alpha ]] D 20 –45(c 0.10,MeOH)；UV(MeOH)λ max (logε)291(3.48)nm；IR(film)ν max :3386,2970,2924,2853,1731,1649,1500and 1488cm -1 ； 1 H NMR(600MHz,DMSO-d 6 ):δ6.74(1H,s,H-17),6.57(1H,s,H-14),5.89(1H,br s,H-18),5.84(1H,d,J＝9.6Hz,H-3),5.80(1H,br s,H-18),5.34(1H,s,H-1),4.10(1H,d,J＝9.6Hz,H-4),3.95(1H,m,H-8b),3.89(1H,m,H-8a),3.84(1H,m,H-10a),3.80(1H,m,H-10b),3.70(3H,s,OMe-19),3.38(1H,m,H-11b),2.85(1H,s,H-3'),2.62(1H,dd,J＝14.0,6.5Hz,H-11a),2.51(1H,m,H-6a),2.07(1H,m,H-6b),2.04(1H,m,H-7a),2.01(1H,m,H-7b),1.45(1H,ddd,J＝12.4,12.0,6.0Hz,H-1"),1.33(1H,m,H-1"),1.32(1H,m,H-2"),1.11(1H,m,H-2"),1.01(3H,s,OMe-5"),0.97(3H,s,OMe-4")； 13 C NMR(150MHz,DMSO-d 6 ):δ173.3(C-4'),171.9(C-1'),162.6(C-2),145.7(C-16),145.4(C-15),130.4(C-12),128.1(C-13),111.6(C-14),109.4(C-17),100.5(C-18),98.9(C-1),87.2(C-5),79.2(C-2'),73.6(C-3'),71.7(C-3),68.4(C-3"),67.6(C-8),60.6(C-10),57.9(C-19),50.6(C-4),37.7(C-6),36.7(C-2"),29.2(C-1"),29.8(C-5"),28.7(C-4"),27.2(C-11),17.4(C-7)；ESIMS m/z 550[M+H] + ；HRESIMS m/z 550.2253[M+H] + (calcd for C 27 H 36 NO 11 ,550.2283)。
Example 2: determination of inhibition of tumor cell proliferation by cephalotaxine N-oxide derivatives
Experimental materials and methods: human non-small cell lung cancer A549, human large cell lung cancer NCI-H460, human promyelocytic leukemia HL60, human plasma cell leukemia NCI-H929 and human myeloma cell RPMI-8226 were all purchased from ATCC. All cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (FBS), 100units/mL penicillin and 100units/mL streptomycin (Invitrogen). All cells were kept at 37℃in 5% CO 2 Culturing under the condition. Cell viability was tested using the standard MTT method: cells were grown in 96 wells at a final density of 5000cells/wellAfter 12h of incubation on the plate, the concentration gradient (10 -2 -10 2 Mu M) is added, and in order to eliminate the influence of phototoxic effect, strong illumination is avoided and the whole operation process is cultivated in a dark incubator. Harringtonine was used as positive control. After 48 hours, the cells were washed with fresh medium (without FBS), then incubated with MTT solution (5 mg/mL) for 4 hours, the suspension was discarded, 100. Mu.L of DMSO (Sigma-Aldrich) was added, and the absorbance at 570nm was measured with a microplate reader (FLX 800, bio-TEK instruments, INC. USA), IC 50 Values were calculated by SigmaPlot10.0 software for the ratio of cell viability to log concentration in the curve and the test results are shown in Table 1.
Table 1 cytotoxic Activity of CephaloverteseA-D (1-4)
a IC 50 value of each tested compound was defined as the concentration that caused 50％inhibition of cell growth.The results are averages of six independentexperiments. b Positive control.。
The test result of the anti-tumor activity shows that the compound cephalverinesA-D (1-4) has obvious inhibition activity on the growth of 5 tested human tumor cells (human non-small cell lung cancer A549, human large cell lung cancer NCI-H460, human promyelocytic leukemia HL60, human plasma cell leukemia NCI-H929 and human myeloma cell RPMI-8226) (table 1), IC 50 The main range of the values is 0.45-9.34 mu M, which shows that the compound can be used for preparing medicaments for treating lung cancer, leukemia or myeloma and other tumor diseases or can be used as lead compounds of the medicaments.
Claims (1)
1. CephalotaxineN-a process for the preparation of an oxide derivative, characterized by the following specific steps:
taking branches and leaves of Cephalotaxus fortunei as raw materials, pulverizing, soaking and extracting with 95% ethanol at room temperature, concentrating the extractive solution under reduced pressure, and suspending in appropriate amountExtracting with 3% tartaric acid aqueous solution with equal volume of ethyl acetate for 3-5 times, and treating the extracted tartaric acid aqueous solution with Na 2 CO 3 Regulating pH to 10, and extracting with chloroform to obtain chloroform extract as total alkaloid fraction; separating the chloroform extract by MCI resin, polyamide resin, sephadex LH-20 gel column chromatography and reversed-phase semi-preparative HPLC chromatography to obtain compound cephalverines A ‒ D, wherein the structural formula of the compound cephalverines A ‒ D is shown as follows:
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