CN101113148A

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Publication number: CN101113148A
Application number: CNA2006101036557A
Authority: CN
Inventors: 顾谦群; 刘为忠; 朱伟明; 朱天骄; 方玉春
Original assignee: CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION; Ocean University of China
Current assignee: CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION; Ocean University of China
Priority date: 2006-07-26
Filing date: 2006-07-26
Publication date: 2008-01-30

Abstract

本发明涉及两种二氧哌嗪类化合物的制备方法和用途。本发明用采自福建红树林根际海泥中的疏展曲霉菌H1－1(Aspergillus effuses H1－1)生产出结构新颖的二氧哌嗪类化合物。经实验证实，该类化合物可作为细胞增殖抑制剂或抗肿瘤剂。The present invention relates to the preparation method and application of two kinds of dioxypiperazine compounds. The invention uses Aspergillus effuses H1-1 collected from the rhizosphere sea mud of Fujian mangroves to produce dioxypiperazine compounds with novel structure. Experiments have proved that the compound can be used as a cell proliferation inhibitor or an antitumor agent.

Description

Translated fromChinese

二氧哌嗪类化合物及其制备方法和用途Dioxypiperazine compounds and their preparation method and use

技术领域：Technical field:

本发明涉及用疏展曲霉菌H1-1(Aspergillus effuses H1-1)(该菌种已于2006年7月13日在中国典型培养物保藏中心保藏，保藏编号是：CCTCC M 206066)生产二氧哌嗪类化合物的方法；本发明还涉及该类化合物在制备细胞增殖抑制剂或抗肿瘤剂中的用途。The present invention relates to the use of Aspergillus effuses H1-1 (Aspergillus effuses H1-1) (this strain has been preserved in the China Typical Culture Collection Center on July 13, 2006, and the preservation number is: CCTCC M 206066) to produce dioxygen A method for piperazine compounds; the present invention also relates to the use of such compounds in the preparation of cell proliferation inhibitors or antitumor agents.

背景技术：Background technique:

有关二氧哌嗪类化合物的文献报道较多，但是这两种新结构的二氧哌嗪类化合物未见报道。本发明人研究得知，疏展曲霉菌H1-1(Aspergillus effuses H1-1)液体发酵产物经超声破碎后的粗提物有很好的海虾致死活性，遂对其活性成分进行了研究。研究发现，所示二氧哌嗪类化合物具有抗肿瘤活性，目前尚未见对该菌株的抗肿瘤活性成分研究、该化合物的化学结构及细胞增殖抑制活性的报道，因此市场上也尚未见有与此有关的药物。There are many reports in the literature on dioxopiperazine compounds, but there are no reports on these two new structures of dioxypiperazine compounds. The inventors have learned from studies that the crude extract of Aspergillus effuses H1-1 (Aspergillus effuses H1-1) liquid fermentation product after ultrasonic crushing has good sea shrimp lethal activity, so its active components have been studied. Studies have found that the shown dioxopiperazine compounds have antitumor activity, and there is no report on the antitumor active ingredients of the strain, the chemical structure of the compound and the inhibitory activity of cell proliferation, so there is no report on the market. related drugs.

发明内容：Invention content:

本发明旨在提供两种具有细胞增殖抑制以及直接杀伤癌细胞等抗肿瘤活性的新化合物。其结构式分别是：The present invention aims to provide two novel compounds with anti-tumor activities such as inhibiting cell proliferation and directly killing cancer cells. Its structural formula is respectively:

式IFormula I

式IIFormula II

其结构特征是：式I可以看作是由二氧哌嗪和苯甲醛衍生物两部分构成。二氧哌嗪部分是由色氨酸与丙氨酸构成；苯甲醛衍生物的苯环上连有一个异戊烯基、一个7个碳的烃基和两个R基，R₁和R₂是氨基、羟基、烷氧基、酰氧基或酰氨基等基团。式II可看作是由色氨酸和丙氨酸构成的二氧哌嗪，其中色氨酸部分上连有两个异戊烯基和一个反异戊烯基；丙氨酸部分为丙氨酸的脱氢产物。Its structural features are: formula I can be regarded as composed of two parts: dioxypiperazine and benzaldehyde derivatives. The dioxypiperazine part is composed of tryptophan and alanine; the benzene ring of the benzaldehyde derivative is connected with an isopentenyl group, a 7-carbon hydrocarbon group and two R groups, R₁ and R₂ are Amino, hydroxyl, alkoxy, acyloxy or amido groups. Formula II can be regarded as a dioxypiperazine composed of tryptophan and alanine, wherein the tryptophan part is connected with two prenyl groups and a trans-prenyl group; the part of alanine is alanine acid dehydrogenation products.

本发明采用MTT法测试了式I和式II化合物对P388和HL60细胞株的抗肿瘤活性；采用SRB法测试了式I和式II化合物对A549和BEL7402细胞株的抗肿瘤活性。实验证实，式I化合物对P388、HL60和A549这三种肿瘤细胞有抗肿瘤作用；式II化合物对A549、HL60和BEL7402这三种肿瘤细胞有抗肿瘤作用。The present invention adopts MTT method to test the antitumor activity of formula I and formula II compounds on P388 and HL60 cell lines; adopts SRB method to test the antitumor activity of formula I and formula II compounds on A549 and BEL7402 cell lines. Experiments have proved that the compound of formula I has anti-tumor effect on three tumor cells of P388, HL60 and A549; the compound of formula II has anti-tumor effect on three tumor cells of A549, HL60 and BEL7402.

因此本发明的式I或II化合物可用作细胞增殖抑制剂或肿瘤细胞杀伤剂。The compounds of formula I or II according to the invention are therefore useful as cell proliferation inhibitors or tumor cell killers.

式I或式II化合物与各种药物可接受的载体、赋形剂或辅料配伍，可制成抗肿瘤药物，用于肿瘤的治疗。Compounds of formula I or formula II are compatible with various pharmaceutically acceptable carriers, excipients or auxiliary materials, and can be made into antitumor drugs for the treatment of tumors.

式I或II化合物还可作为抑制细胞增殖的低分子生物探针用于生命科学研究，作为探针应用时，式I或II化合物可溶于甲醇、水或含水甲醇中，也可溶于二甲基亚砜的含水溶液中加以应用。The compound of formula I or II can also be used as a low-molecular biological probe for inhibiting cell proliferation for life science research. When used as a probe, the compound of formula I or II can be dissolved in methanol, water or aqueous methanol, and can also be dissolved in two It is used in aqueous solution of methyl sulfoxide.

本发明的式I或II化合物可通过微生物发酵培养来获取含有该二氧哌嗪类化合物的发酵物，然后从发酵物中采用硅胶柱层析、制备薄层层析、HPLC和重结晶等方法分离纯化得到。The compound of formula I or II of the present invention can be fermented to obtain the fermented product containing the dioxypiperazine compound through microbial fermentation, and then adopt methods such as silica gel column chromatography, preparative thin-layer chromatography, HPLC and recrystallization from the fermented product obtained by separation and purification.

本发明的下述实施例中列举了利用疏展曲霉菌H1-1(Aspergillus effuses H1-1)制备优选的本发明式I或式II化合物的实例。The following examples of the present invention enumerate the example of utilizing Aspergillus effuses H1-1 (Aspergillus effuses H1-1) to prepare preferred compounds of formula I or formula II of the present invention.

因此，本发明的再一方面提供了一种疏展曲霉菌H1-1(Aspergillus effuses H1-1)，保藏编号是：CCTCC M206066。Therefore, another aspect of the present invention provides a kind of Aspergillus effuses H1-1 (Aspergillus effuses H1-1), the preservation number is: CCTCC M206066.

需要特别说明的是，凡是能产生本发明式I或II化合物的任何微生物，都能用于生产该二氧哌嗪类化合物；也可经过化学方法合成该类化合物。It should be noted that any microorganism capable of producing the compound of formula I or II of the present invention can be used to produce the dioxopiperazine compound; the compound can also be synthesized by chemical methods.

具体实施方式：Detailed ways:

在如下的实施例中所指的化合物I和II的化学结构(结构式中的阿拉伯数字是化学结构中碳原子的标位)分别是：The chemical structures of compounds I and II indicated in the following examples (the Arabic numerals in the structural formula are the positions of the carbon atoms in the chemical structure) are respectively:

化合物ICompound I

化合物IICompound II

实施例1化合物I和II的发酵生产及分离精制Fermentative production and separation and purification of compound I and II of embodiment 1

1发酵生产1 Fermentation production

生产菌的发酵培养：按培养微生物的常规方法，取疏展曲霉菌H1-1(Aspergillus effusesH1-1)(保藏编号是：CCTCC M 206066)适量，接种到PDA固体斜面培养基上，在28摄氏度培养箱中培养4天。Fermentation culture of production bacteria: according to the conventional method of cultivating microorganisms, get Aspergillus effuses H1-1 (Aspergillus effuses H1-1) (preservation number is: CCTCC M 206066) in an appropriate amount, inoculate on the PDA solid slant medium, at 28 degrees Celsius Cultured in the incubator for 4 days.

取斜面培养4天的疏展曲霉菌H1-1(Aspergillus effuses H1-1)适量，接种到装有120mL培养液[培养基组成(克/升)：麦芽糖20.0，甘露醇20.0，味精10.0，KH₂PO₄0.5，MgSO₄·H₂O0.3，酵母膏3.0，玉米浆1，pH自然]的500mL锥型瓶中，在28℃、120转/分钟条件下摇床培养4天，获得疏展曲霉菌的种子培养液。按10％接种量将该种子培养液接种于装有150毫升生产培养液[培养基组成(克/升)：麦芽糖20.0，甘露醇20.0，味精10.0，KH₂PO₄0.5，MgSO₄·H₂O0.3，酵母膏3.0，玉米浆1，pH自然]的500mL三角烧瓶中，装载于28℃、120转/分钟的摇床上，进行为期11天的生产发酵，获得菌丝体和发酵液。Take an appropriate amount of Aspergillus effuses H1-1 (Aspergillus effuses H1-1) that has been cultured on a slant for 4 days, and inoculate it into 120 mL of culture solution [medium composition (g/L): maltose 20.0, mannitol 20.0, monosodium glutamate 10.0, KH₂ PO₄ 0.5, MgSO₄ ·H₂ O0.3, yeast extract 3.0, corn steep liquor 1, pH natural] in a 500mL Erlenmeyer flask, cultured on a shaker at 28°C and 120 rpm for 4 days to obtain sparse Seed culture solution of Aspergillus sp. This seed culture solution is inoculated into 150 milliliters of production culture solution [substrate composition (gram/liter): maltose 20.0, mannitol 20.0, monosodium glutamate 10.0, KH₂ PO₄ 0.5, MgSO₄ H₂ 0.3, yeast extract 3.0, corn steep liquor 1, pH natural] in a 500mL Erlenmeyer flask loaded on a shaker at 28°C and 120 rpm for 11 days of production fermentation to obtain mycelium and fermentation broth.

2浸膏的获得2 Obtaining the extract

用棉布将菌丝体和发酵液分离。将菌丝体用甲醇浸提4次，减压浓缩至不含甲醇，所得水层用等体积乙酸乙酯萃取三次，合并乙酸乙酯萃取液减压浓缩，得粗浸膏。发酵液减压浓缩为四分之一体积后，用乙酸乙酯萃取三次，合并菌丝体和发酵液的浸膏，共75克。Separate the mycelium and fermentation broth with a cotton cloth. The mycelium was leached 4 times with methanol, concentrated under reduced pressure to no methanol, the obtained aqueous layer was extracted three times with an equal volume of ethyl acetate, and the combined ethyl acetate extracts were concentrated under reduced pressure to obtain a crude extract. After the fermented liquid was concentrated under reduced pressure to a quarter volume, it was extracted three times with ethyl acetate, and the extracts of mycelia and fermented liquid were combined to a total of 75 grams.

3化合物的分离精制3 Separation and purification of compounds

浸膏(75克)用氯仿-甲醇(9∶1)混合溶剂溶解后，加100克200-300目硅胶H(青岛海洋化工集团公司产品)拌样，减压除去溶剂后，用硅胶柱层析，以石油醚、石油醚-氯仿，氯仿-甲醇为溶剂进行梯度洗脱，分为8个流份。Fr-4以氯仿-甲醇(100∶1～10∶1)为溶剂，再进行硅胶柱层析，在氯仿-甲醇(100∶1)的洗脱溶剂中得化合物I(28mg)；由氯仿-甲醇(90∶1)的洗脱溶剂所得的流份进一步以硅胶柱层析，以氯仿-甲醇(95∶1)为洗脱溶剂，得化合物II(22mg)。After the extract (75 grams) was dissolved in a mixed solvent of chloroform-methanol (9:1), add 100 grams of 200-300 mesh silica gel H (product of Qingdao Ocean Chemical Group Co., Ltd.) to mix the sample, remove the solvent under reduced pressure, and use a silica gel column layer Analysis, using petroleum ether, petroleum ether-chloroform, chloroform-methanol as solvent for gradient elution, divided into 8 fractions. Fr-4 takes chloroform-methanol (100:1～10:1) as solvent, and then carries out silica gel column chromatography, obtains compound I (28mg) in the elution solvent of chloroform-methanol (100:1); The obtained fraction was further subjected to silica gel column chromatography using chloroform-methanol (95:1) as the elution solvent to obtain compound II (22 mg).

化合物I：分子式C₃₈H₄₃N₃O₅，黄色固体，mp148-150℃，HR-ESI-MS m/z：622.3256[M+H]⁺(calc.622.3281)；UVλ_max^MeOH nm(lgε)：348(3.76)，275(3.86)，224(4.21)；IR(KBr)cm^-1：3350，3240，3044，2967，2928，1670，1637，1428，1380，1271，1253，747。¹H及¹³C NMR数据见表1。Compound I: molecular formula C₃₈ H₄₃ N₃ O₅ , yellow solid, mp148-150℃, HR-ESI-MS m/z: 622.3256[M+H]⁺ (calc.622.3281); UVλ_max^MeOH nm(lgε) : 348 (3.76), 275 (3.86), 224 (4.21); IR (KBr) cm^-1 : 3350, 3240, 3044, 2967, 2928, 1670, 1637, 1428, 1380, 1271, 1253, 747. See Table 1 for¹ H and¹³ C NMR data.

表1.化合物I的¹H和¹³CNMR数据(600，150MHz，DMSO-d₆，TMS，δppm)Table 1.¹ H and¹³ CNMR data of compound I (600, 150 MHz, DMSO-d₆ , TMS, δppm)

No.No. δ_H(Jin Hz)δ_H (Jin Hz) δ_C_δC No.No. δ_H(Jin Hz)δ_H (Jin Hz)δ_C_δC1233a45677a8910111233a45677a891011 11.05(1H，s)7.07(1H，m)7.00(1H，dd，7.8，6.9)7.07(1H，m)7.41(1H，d，8.2)7.71(1H，s)11.05(1H,s)7.07(1H,m)7.00(1H,dd,7.8,6.9)7.07(1H,m)7.41(1H,d,8.2)7.71(1H,s)144.2103.6127.1118.8119.4120.7111.6135.1110.7124.4168.3144.2103.6127.1118.8119.4120.7111.6135.1110.7124.4168.3 21222324252627282930313233212223242526272829303132336.92(1H，s)2.88(1H，m)；3.07(1H，m)1.54(2H，m)2.74(1H，m)5.78(1H，bd，10.3)5.71(1H，bd，10.3)2.58(1H，m)1.74(1H，dd，13.7，2.8，H_a)2.08(1H，dd，13.7，7.3，H_b)1.10(3H，d，7.4)6.92 (1H, s) 2.88 (1H, m); 3.07 (1H, m) 1.54 (2H, m) 2.74 (1H, m) 5.78 (1H, bd, 10.3) 5.71 (1H, bd, 10.3) 2.58 (1H , m) 1.74 (1H, dd, 13.7, 2.8, H_a ) 2.08 (1H, dd, 13.7, 7.3, H_b ) 1.10 (3H, d, 7.4)153.4127.1125.2146.8128.020.932.739.7126.1131.828.038.621.6153.4127.1125.2146.8128.020.932.739.7126.1131.828.038.621.6

1213141516171819201213141516171819208.86(1H，s)6.05(1H，dd，17.2，10.3)5.01(1H，d，17.2)5.03(1H，d，10.3)1.45(1H，s)1.47(1H，s)8.86 (1H, s) 6.05 (1H, dd, 17.2, 10.3) 5.01 (1H, d, 17.2) 5.03 (1H, d, 10.3) 1.45 (1H, s) 1.47 (1H, s) 59.7161.339.2145.1111.727.527.6117.259.7161.339.2145.1111.727.527.6117.2 34353637383921-OH24-OH34353637383921-OH24-OH10.18(1H，s)3.12(2H，m)5.16(1H，bt，7.3)1.62(3H，s)1.67(3H，s)11.76(1H，s)9.05(1H，s)10.18 (1H, s) 3.12 (2H, m) 5.16 (1H, bt, 7.3) 1.62 (3H, s) 1.67 (3H, s) 11.76 (1H, s) 9.05 (1H, s) 196.926.6121.7132.517.625.5196.926.6121.7132.517.625.5

化合物II：分子式C₂₉H₃₇N₃O₂，无色固体；mp 168～169℃(丙酮)；[α]_D²⁰-75.9(c0.08，MeOH)；HRESI-MSm/z：460.2981[M+H]⁺(calc.460.2964)；UVλ_max^MeOH nm(lgε)：296(3.37)，287(3.47)，228(4.15)；IR(KBr)cm^-1：3468，3184，2968，2912，1682，1625，1438，1324，1096，923，873，839。¹H及¹³C NMR数据见表2。Compound II: molecular formula C₂₉ H₃₇ N₃ O₂ , colorless solid; mp 168～169°C (acetone); [α]_D²⁰ -75.9 (c0.08, MeOH); HRESI-MSm/z: 460.2981[M +H]⁺ (calc.460.2964); UVλ_max^MeOH nm(lgε): 296(3.37), 287(3.47), 228(4.15); IR(KBr)cm^-1 : 3468, 3184, 2968, 2912, 1682 , 1625, 1438, 1324, 1096, 923, 873, 839. See Table 2 for¹ H and¹³ C NMR data.

表2化合物II的¹H和¹³C NMR数据(600，150MHz，DMSO-d₆，TMS，δppm)Table 2¹ H and¹³ C NMR data of Compound II (600, 150 MHz, DMSO-d₆ , TMS, δppm)

No.No. δ_H(Jin Hz)δ_H (Jin Hz) δ_C_δC 1233a45677a89101112131233a45677a8910111213 8.07(1H，s)7.14(1H，bs)6.81(1H，bs)3.62(1H，dd，14.4，3.9Hz)；3.26(1H，dd，14.4，11.7Hz)4.54(1H，ddd，11.7，3.9，1.8Hz)5.80(1H，bs)8.07(1H,s)7.14(1H,bs)6.81(1H,bs)3.62(1H,dd,14.4,3.9Hz); 3.26(1H,dd,14.4,11.7Hz)4.54(1H,ddd,11.7,3.9 , 1.8Hz) 5.80 (1H, bs)141.5103.6128.8115.0133.9122.8^d123.4132.230.855.6166.7132.9^a158.0141.5103.6128.8115.0133.9122.8^d 123.4132.230.855.6166.7132.9^a 158.0

14151617181920212223242526272829301415161718192021222324252627282930 8.99(1H，bs)6.08(1H，dd，17.4，10.5Hz)5.15(1H，dd，17.4，0.9Hz)；5.14(1H，dd，10.5，0.9Hz)1.49(3H，s)1.50(3H，s)5.61(1H，bs)；4.94(1H，bs)3.40(2H，bd，7.3Hz)5.35(1H，tm，7.3Hz)1.74(3H，bs)1.75(3H，bs)3.54(2H，m)5.43(1H，tm，7.3Hz)1.81(3H，bs)1.87(3H，bs)8.99 (1H, bs) 6.08 (1H, dd, 17.4, 10.5Hz) 5.15 (1H, dd, 17.4, 0.9Hz); 5.14 (1H, dd, 10.5, 0.9Hz) 1.49 (3H, s) 1.50 (3H, s) 5.61 (1H, bs); 4.94 (1H, bs) 3.40 (2H, bd, 7.3Hz) 5.35 (1H, tm, 7.3Hz) 1.74 (3H, bs) 1.75 (3H, bs) 3.54 (2H, m )5.43 (1H, tm, 7.3Hz) 1.81 (3H, bs) 1.87 (3H, bs)38.9145.6112.127.7^b27.8^b102.334.6124.5131.625.7^c17.931.4122.9^d133.0^a25.8^c17.938.9145.6112.127.7^b 27.8^b 102.334.6124.5131.625.7^c 17.931.4122.9^d 133.0^a 25.8^c 17.9

注：具有相同上标的数据可以互换。Note: Data with the same superscript are interchangeable.

实施例2抗肿瘤活性的测试The test of embodiment 2 antitumor activity

1实验样品及实验方法1 Experimental samples and experimental methods

被测样品溶液的配制：测试样品为上述实施例1中分离精制的纯品化合物I-II。准确称取适量样品，用DMSO配制成所需浓度的溶液，供活性测试。Preparation of the test sample solution: the test sample is the pure compound I-II isolated and refined in the above-mentioned Example 1. Accurately weigh an appropriate amount of sample and prepare a solution with the required concentration with DMSO for activity testing.

细胞系及细胞的继代培养：活性测试采用P388、A-549、HL60和BEL7402细胞系。各种细胞均用含10％FBS的RPMI-1640培养基，在37℃通入5％二氧化碳的培养箱中继代培养。Cell lines and subculture of cells: P388, A-549, HL60 and BEL7402 cell lines were used for activity testing. All kinds of cells were subcultured in RPMI-1640 medium containing 10% FBS at 37°C in an incubator filled with 5% carbon dioxide.

1-1MTT法活性测试方法1-1 MTT method activity test method

本发明采用MTT法，测试评价了被测试样品对P388和HL60癌细胞增殖的抑制活性。活细胞线粒体中脱氢酶能够代谢还原黄色的溴化3-(4，5-二甲基噻唑)-2，5-二苯基四氮唑为蓝紫色的不溶于水的formazan，formazan的多少可通过酶标仪测定其吸收度求得。由于formazan的量与活细胞数成正比，所以可根据吸收度求出活细胞的数目，从而了解药物抑制或杀伤肿瘤细胞的能力。The invention adopts the MTT method to test and evaluate the inhibitory activity of the tested samples on the proliferation of P388 and HL60 cancer cells. The dehydrogenase in the mitochondria of living cells can metabolize and reduce the yellow 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide to blue-purple water-insoluble formazan, the amount of formazan It can be obtained by measuring its absorbance with a microplate reader. Since the amount of formazan is directly proportional to the number of living cells, the number of living cells can be calculated according to the absorbance, so as to understand the ability of the drug to inhibit or kill tumor cells.

活性测试时，取对数生长期的P388和HL60细胞，用新鲜的RPMI-1640培养基配制成密度为每毫升5×10⁴个细胞的细胞悬液，按每孔200微升接种于96孔板中，在37℃下培养24小时后，每孔加入2微升不同浓度的样品溶液，继续培养72小时。然后加入20μL含MTT的IPMI-1640溶液(5mg/L)，再培养4小时，移出150μL培养液后加入150μLDMSO溶解formazan，在540nm处测定其吸收度。按照IR％＝(OD_空白对照-OD_样品)/OD_空白对照X100％式计算每个浓度下的细胞增殖抑制率(IR％)，并采用bliss法计算出半数抑制浓度IC₅₀。For the activity test, take the P388 and HL60 cells in the logarithmic growth phase, use fresh RPMI-1640 medium to prepare a cell suspension with a density of 5×¹⁰⁴ cells per milliliter, and inoculate 200 microliters per well in 96 wells In the plate, after culturing at 37° C. for 24 hours, 2 microliters of sample solutions of different concentrations were added to each well, and the culture was continued for 72 hours. Then add 20 μL of MTT-containing IPMI-1640 solution (5 mg/L), and culture for another 4 hours, remove 150 μL of culture solution, add 150 μL of DDMSO to dissolve formazan, and measure its absorbance at 540 nm. The cell proliferation inhibition rate (IR%) at each concentration was calculated according to the formula IR%=(OD_{blank control} -OD_sample )/OD_{blank control} X100%, and the half inhibitory concentration IC₅₀ was calculated by bliss method.

1-2SRB活性测试方法1-2 SRB activity test method

本发明采用SRB法，测试评价了被测试样品对A-549和BEL7402癌细胞增殖的抑制活性。SRB是一种粉红色的，含有两个硫酸基的氨基甲氧杂蒽类碱性蛋白结合染料，弱酸性条件下，SRB能与被三氯乙酸(TCA)固定的细胞表面生物大分子的碱性氨基酸残基(Lys、Arg或His)通过静电引力结合，被结合染料的量在细胞密度为2.5×10⁴～6×10⁵/cm³范围内，与蛋白含量呈正比，而蛋白含量与细胞数量呈正比。而在弱碱性条件下，经Tris非缓冲液洗涤后，SRB与碱性氨基酸残基分离。故可根据细胞染色程度推测细胞数。The present invention uses the SRB method to test and evaluate the inhibitory activity of the tested samples on the proliferation of A-549 and BEL7402 cancer cells. SRB is a pink aminomethoxanthene basic protein-binding dye containing two sulfate groups. Under weakly acidic conditions, SRB can bind to the base of biomacromolecules on the cell surface immobilized by trichloroacetic acid (TCA). Active amino acid residues (Lys, Arg or His) are combined by electrostatic attraction, and the amount of the bound dye is in direct proportion to the protein content in the range of cell density 2.5×10⁴ ～6×10⁵ /cm³ , while the protein content is proportional to the The number of cells is proportional. While under weakly alkaline conditions, SRB was separated from basic amino acid residues after washing with Tris non-buffer. Therefore, the number of cells can be estimated according to the degree of cell staining.

取对数生长期的A-549和BEL7402肿瘤细胞，用新鲜RPMI-1640培养基配成浓度为2×10⁵个/mL的细胞悬液，按每孔200μL接种于96孔板中，每孔加入2μL不同浓度样品溶液，37℃培养72小时。然后在4℃、3000prm条件下离心3min，吸去上清。每孔细胞中加入预冷的80％三氯乙酸(TCA)50μL，置于4℃冰箱固定1.5小时，再用水冲洗5次并空气干燥，每孔加入0.5％SRB的1％TCA溶液50μL并于室温静置30min，用预冷的1％TCA水溶液清洗4次，除去未结合的SRB染料。每孔加入150μLTris溶液(10mmol/L，pH10.5)溶解蛋白结合染料，用酶标仪于520nm下测定每孔吸光度OD值。按细胞增殖抑制率IR(％)＝(OD_空白-OD_样品)/OD_空白×100％计算，再利用Bliss方法由求出半数抑制浓度(IC₅₀)。Take the A-549 and BEL7402 tumor cells in the logarithmic growth phase, use fresh RPMI-1640 medium to prepare a cell suspension with a concentration of 2×10⁵ cells/mL, and inoculate 200 μL per well in a 96-well plate. Add 2 μL of sample solutions of different concentrations and incubate at 37°C for 72 hours. Then centrifuge at 4°C and 3000prm for 3min, and suck off the supernatant. Add 50 μL of pre-cooled 80% trichloroacetic acid (TCA) to each well of the cells, place them in a refrigerator at 4°C for 1.5 hours, wash them with water for 5 times and air-dry them, add 50 μL of 0.5% SRB in 1% TCA solution to each well and place in the Let it stand at room temperature for 30 min, wash with pre-cooled 1% TCA aqueous solution 4 times to remove unbound SRB dye. Add 150 μL Tris solution (10 mmol/L, pH 10.5) to each well to dissolve the protein-binding dye, and measure the absorbance OD value of each well at 520 nm with a microplate reader. The cell proliferation inhibition rate IR (%)=(OD_blank -OD_sample )/OD_blank ×100% was calculated, and then the half inhibitory concentration (IC₅₀ ) was calculated by Bliss method.

2实验结果2 Experimental results

表3.化合物I对P388、A-549、HL60和BEL7402细胞的抑制活性Table 3. The inhibitory activity of compound I on P388, A-549, HL60 and BEL7402 cells

cellcell Inhibition Rates(％)Inhibition Rates(%)IC₅₀(μM)_IC50 (μM) 10^-4M10^-4 M10^-5M10^-5 M10^-6M10^-6 M10^-7M10^-7 M10^-8M10^-8 M P388A549HL60BEL7402P388A549HL60BEL7402 86.563.593.036.386.563.593.036.3 79.962.086.922.079.962.086.922.0012.76.014.5012.76.014.508.306.808.306.8 2.03.508.02.03.508.0 1.814.54.8＞1001.814.54.8＞100

表4.化合物II对P388、A-549、HL60和BEL7402细胞的抑制活性Table 4. Inhibitory activity of compound II on P388, A-549, HL60 and BEL7402 cells

cellcell Inhibition Rates(％)Inhibition Rates(%) IC₅₀(μM)_IC50 (μM) 10^-4M10^-4 M 10^-5M10^-5 M 10^-6M10^-6 M 10^-7M10^-7 M 10^-8M10^-8 M P388A549HL60BEL7402P388A549HL60BEL7402 31.498.010094.431.498.010094.4 6.290.048.056.26.290.048.056.2 030.636.415.1030.636.415.1 06.713.211.806.713.211.8 04.515.812.204.515.812.2 ＞1001.415.64.2＞1001.415.64.2

3结论3 Conclusion

化合物I对P388、A-549和HL60癌细胞具有抗肿瘤作用；化合物II对A-549、HL60和BEL7402细胞具有增殖抑制作用。化合物I和化合物II可作为抗肿瘤剂(即抗肿瘤药物)用于抗肿瘤的研究中，具有开发为抗肿瘤药物的潜力；也可作为抑制细胞增殖抑制的低分子生物探针用于生命科学研究。Compound I has anti-tumor effect on P388, A-549 and HL60 cancer cells; compound II has proliferation inhibitory effect on A-549, HL60 and BEL7402 cells. Compound I and Compound II can be used as anti-tumor agents (i.e. anti-tumor drugs) in anti-tumor research, and have the potential to be developed as anti-tumor drugs; they can also be used in life sciences as low-molecular-weight biological probes that inhibit cell proliferation Research.

Claims

1. formula I compound

Formula I

The described formula I compound of claim 1, wherein R₁And R₂Be hydroxyl.

2. formula II compound

Formula II

3. the preparation method of described formula I compound of claim 1 or the described formula II compound of claim 3, it is characterized in that fermentation culture aspergillus effusus bacterium H1-1, obtain the fermented product that contains above-mentioned formula I and formula II compound, separation and purification goes out formula I and formula II compound from fermented product then.

4. the described preparation method of claim 3, wherein with described fermented product through silica gel column chromatography, with sherwood oil, sherwood oil-chloroform, chloroform-methanol is that solvent carries out gradient elution, wherein Fr-4 is a solvent with chloroform-methanol (100: 1～10: 1), carry out silica gel column chromatography again, in the eluting solvent of chloroform-methanol (100: 1), get Compound I; Further with silica gel column chromatography, is eluting solvent with chloroform-methanol (95: 1) by stream part of the eluting solvent gained of chloroform-methanol (90: 1), Compound I I.

5. claim 1 or 2 described formula I or formula II compound are in the purposes of preparation in the antitumor drug.

6. purposes according to claim 5, wherein said antitumor drug are meant the purposes of described formula I formula II compound in preparation inhibition of cell proliferation or tumor cytotoxicity agent.

7. purposes according to claim 5, wherein said antitumor drug are meant described formula I or the purposes of formula II compound in preparation inhibition tumor cell proliferation medicine.