CN114805382B - A sesquiterpene ketone compound and its separation and application in the preparation of anti-pancreatic cancer drugs - Google Patents
A sesquiterpene ketone compound and its separation and application in the preparation of anti-pancreatic cancer drugs Download PDFInfo
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
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Abstract
Description
技术领域technical field
本发明涉及一种天然倍半萜色酮类化合物及其分离方法,同时还涉及该天然倍半萜色酮类化合物的抗胰腺癌生物活性及作为制备抗胰腺癌药物活性物质的应用,属于天然药物化学领域。The present invention relates to a natural sesquiterpene ketone compound and its separation method, and also relates to the anti-pancreatic cancer biological activity of the natural sesquiterpene ketone compound and its application as an active substance for preparing anti-pancreatic cancer drugs, belonging to natural field of medicinal chemistry.
背景技术Background technique
胰腺癌(Pancreatic cancer,PC)具有很高的侵袭性和致命性,是目前已知预后最差的恶性肿瘤之一。近年来,我国胰腺癌的发病率呈上升趋势,其死亡率居恶性肿瘤的前10位,5年生存率只有10%。胰腺癌患者中大约有80%-85%的患者无法手术切除或者已经发生转移,即使可以局部手术切除的患者,预后仍然很差,通过手术得到控制的患者只有10%~15%。药物治疗是胰腺癌治疗的主要手段之一,但由于耐药性和缺乏药物特异性,药物总体有效率不到20%。为了延长胰腺癌患者的生存期,迫切需要开发寻找更加安全有效的胰腺癌化疗药物。Pancreatic cancer (PC) is highly invasive and fatal, and is one of the malignant tumors with the worst prognosis currently known. In recent years, the incidence of pancreatic cancer in my country has been on the rise, and its mortality rate ranks among the top 10 malignant tumors, with a 5-year survival rate of only 10%. About 80%-85% of pancreatic cancer patients are unresectable or have metastasized. Even for patients who can be locally surgically resected, the prognosis is still poor, and only 10%-15% of patients can be controlled by surgery. Drug therapy is one of the main means of pancreatic cancer treatment, but due to drug resistance and lack of drug specificity, the overall effective rate of drugs is less than 20%. In order to prolong the survival period of patients with pancreatic cancer, it is urgent to develop safer and more effective chemotherapy drugs for pancreatic cancer.
天然产物作为药物的重要来源,在新药的发现和开发中发挥着重要的作用。60%以上的抗肿瘤药物与天然产物密切相关。虽然小分子靶向药物在癌症治疗中占主导地位。因此,寻找天然活性化合物,发现新型抗胰腺癌药物,有助于推动寻找高效抗胰腺癌药物。新疆阿魏(Ferula sinkiangensis K. M. Shen)是伞形科阿魏属植物,多年生一次结果的草本,高0.5~1.5米,全株有强烈的葱蒜样臭味。根纺锤形或圆锥形,粗壮,根颈上残存有枯萎叶鞘纤维。新疆阿魏所分泌的具有特殊葱蒜样臭味的油胶树脂,被历代本草及《中华人民共和国药典》收载,具有消积、散痞、杀虫的功效,可用于治疗胃病、消化不良、虫积腹痛等疾病。As an important source of drugs, natural products play an important role in the discovery and development of new drugs. More than 60% of antineoplastic drugs are closely related to natural products. Although small-molecule targeted drugs dominate cancer therapy. Therefore, searching for natural active compounds and discovering new anti-pancreatic cancer drugs will help promote the search for highly effective anti-pancreatic cancer drugs. Xinjiang Ferula ( Ferula sinkiangensis KM Shen) is a plant of the genus Ferula in the family Umbelliferae. It is a perennial herb that bears fruit once. It is 0.5-1.5 meters high. The whole plant has a strong onion and garlic-like odor. Root spindle-shaped or conical, stout, with withered leaf sheath fibers remaining on the root neck. The oil gum resin secreted by Ferulicum ferulae in Xinjiang has a special onion and garlic-like odor, which has been recorded in successive dynasties of herbal medicines and "Pharmacopia of the People's Republic of China". , Insect accumulation abdominal pain and other diseases.
发明内容Contents of the invention
本发明的目的是提供一种倍半萜色酮类化合物及其分离方法;The object of the present invention is to provide a kind of sesquiterpene ketone compound and separation method thereof;
本发明的另一目的是对上述分离的倍半萜色酮类化合物的抗胰腺癌活性进行研究,以期用于制备治疗抗胰腺癌的药物。Another object of the present invention is to study the anti-pancreatic cancer activity of the above-mentioned isolated sesquiterpene ketones, in order to prepare anti-pancreatic cancer drugs.
一、倍半萜色酮类化合物的分离1. Separation of sesquiterpene ketones
本发明倍半萜色酮类化合物的分离,包括以下步骤:The separation of sesquiterpene ketones of the present invention comprises the following steps:
(1)将干燥的新疆阿魏根用甲醇室温提取3次,每次7天,合并提取液,蒸发浓缩至无醇味,得到总提取液;(1) Extract the dried Xinjiang ferulae root with methanol at room temperature for 3 times, each time for 7 days, combine the extracts, evaporate and concentrate until there is no alcohol smell, and obtain the total extract;
(2)将总提取物用水分散后,分别用石油醚(60~90℃)、二氯甲烷、乙酸乙酯、正丁醇萃取,得到石油醚相、二氯甲烷相、乙酸乙酯相、正丁醇相和水相;(2) After dispersing the total extract with water, extract with petroleum ether (60~90℃), dichloromethane, ethyl acetate and n-butanol respectively to obtain petroleum ether phase, dichloromethane phase, ethyl acetate phase, n-butanol phase and water phase;
(3)乙酸乙酯相进行硅胶色谱分离,并用石油醚-乙酸乙酯 (40:1~1:1)洗脱,依次得到12个组分FA-FL。(3) The ethyl acetate phase was separated by silica gel chromatography and eluted with petroleum ether-ethyl acetate (40:1~1:1) to obtain 12 components FA-FL in sequence.
对组分FH进行硅胶柱色谱,并用二氯甲烷-乙酸乙酯(50:1)洗脱,得到五个组分FH-1 至 FH-5;Silica gel column chromatography was performed on component FH, and eluted with dichloromethane-ethyl acetate (50:1), to obtain five components FH-1 to FH-5;
对组分 FH-1进行硅胶柱色谱,并用石油醚-乙酸乙酯(10:1)洗脱,得到 12 个组分 FH-1-1 至 FH-1-12。Fraction FH-1 was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate (10:1) to obtain 12 fractions FH-1-1 to FH-1-12.
对组分FH-1-7用二氯甲烷-乙酸乙酯(60:1)洗脱,得到八个组分FH-1-7-1至FH-1-7-8;Fraction FH-1-7 was eluted with dichloromethane-ethyl acetate (60:1) to obtain eight fractions FH-1-7-1 to FH-1-7-8;
对组分FH-1-7-5用二氯甲烷-乙酸乙酯(40:1)得到四个组分FH-1-7-5-1到 FH-1-7-5-4;Use dichloromethane-ethyl acetate (40:1) for component FH-1-7-5 to obtain four components FH-1-7-5-1 to FH-1-7-5-4;
(4)对组分FH-1-7-5-1使用HPLC(C18,MeOH/H2O= 76/24,流速=4 ml/min)纯化,得到化合物1;(4) Purify fraction FH-1-7-5-1 by HPLC (C 18 , MeOH/H 2 O=76/24, flow rate=4 ml/min) to obtain
(5)对化合物1使用手性HPLC(Chiral CD-Ph,石油醚:乙酸乙酯=84/16,流速= 3ml/min)纯化,依次得到化合物 (+)-1、化合物 (- )-1。(5)
通过旋光值、紫外光谱UV、圆二色光谱C、光谱IR、红外(KBr)、氢谱1H 和碳谱13C、NMR高分辨质谱HRESIMS对上述分离得到的化合物(+)-1、化合物 (- )-1进行分析,其1H(400 MHz)和13C (100MHz) NMR 数据见表1,并确定其结构式如下:Compound ( + )-1 and compound (-)-1 is analyzed, and its 1H (400 MHz) and 13C (100MHz) NMR data are shown in Table 1, and its structural formula is determined as follows:
化合物(+)-1和化合物 (- )-1是无色油状,分子式是C24H30O5,质谱数据(m/z399.2158, [M + H]+ 399.2158) 可以推算出有5个不饱和度。3424-1和1723.9 cm-1处的强红外吸收表明存在羟基和羰基。1D NMR数据表明5,6,8-三取代苯环[δH7.77(d,J=8.5Hz,H-5),6.50(dd,J=8.5,2.16Hz,H-6),6.37(d,J=2.04Hz,H-8)和δC129.5(C-5),110.3(C-6),162.4(C-7),103.6(C-8),114.39C-9),160.69C-10)],以及两个三取代烯烃键[5.11(d,J=9.76Hz,H-2,H-6')和 δC126.1(C-2'), 129.3(C-6')]。两个H质子[δH3.14(d,J=10.5Hz,H-3),3.14(d,J=10.52Hz,H-10') 和 δC55.0(C-3),83.7(C-10')]。1D NMR数据也显示了24个碳信号,包括一个羰基δC 191.34(C-4)、四个甲基[δH 1.19(s,H3-13')、1.29(s,H3-12')、1.51(s,H3-14')和1.53(s,H3-15')]。Compound (+ ) -1 and compound (- ) -1 are colorless oils, and the molecular formula is C 24 H 30 O 5 saturation. Strong infrared absorption at 3424 -1 and 1723.9 cm -1 indicated the presence of hydroxyl and carbonyl groups. 1D NMR data indicated 5,6,8-trisubstituted phenyl ring [δH7.77(d, J=8.5Hz, H-5), 6.50(dd, J=8.5,2.16Hz, H-6), 6.37(d , J=2.04Hz, H-8) and δC129.5(C-5), 110.3(C-6), 162.4(C-7), 103.6(C-8), 114.39C-9), 160.69C- 10)], and two trisubstituted olefin bonds [5.11 (d, J=9.76Hz, H-2, H-6') and δ C 126.1(C-2'), 129.3(C-6')]. Two H protons [δH3.14(d, J=10.5Hz, H-3), 3.14(d, J=10.52Hz, H-10') and δC55.0(C-3), 83.7(C-10 ')]. 1D NMR data also showed 24 carbon signals, including one carbonyl δC 191.34 (C-4), four methyl groups [δH 1.19 (s, H3-13'), 1.29 (s, H3-12'), 1.51 ( s, H3-14') and 1.53 (s, H3-15')].
通过对2D NMR谱的综合解释,进一步阐明了化合物(+)-1和化合物 (- )-1的平面结构。对1H-1H COSY谱的分析表明存在四个相关体系,即CH(10’)-CH(8’)、CH(5)-CH(6)、CH(9’)-CH(1’)、CH(6’)-CH(5’)。HMBC数据在H-5/C-4/ C-7/C-9和 H-6/C-8/C-10, 和 H-8/C-6/C-10 的相关性表明A环的存在(如图1所示),在H-3/C-/C-2'/C-2/C-1' 、H-2'/ C-4'/C-15'、H3-15'/C-3'/C-2'/C-4'、H3-14'/C-7'/C-8'、H-6'/C-14'/C-8'、H-4'/C-6'、H-10'/C-11'/C-8'、H3-13'/C-10'/C-11'/C-12'和H-12'/C-13'/C-11'的相关性表明,B、C、D环的存在(如图1所示)。绝对构型的测定通过ECD计算(图2-图7)。The planar structures of compound (+)-1 and compound (- )-1 were further elucidated by comprehensive interpretation of the 2D NMR spectra. The analysis of 1 H- 1 H COZY spectrum shows that there are four related systems, namely CH(10')-CH(8'), CH(5)-CH(6), CH(9')-CH(1' ), CH(6')-CH(5'). The correlation of HMBC data at H-5/C-4/C-7/C-9 and H-6/C-8/C-10, and H-8/C-6/C-10 indicates that the A ring Exist (as shown in Figure 1), in H-3/C-/C-2'/C-2/C-1', H-2'/C-4'/C-15', H 3 -15 '/C-3'/C-2'/C-4', H 3 -14'/C-7'/C-8', H-6'/C-14'/C-8', H- 4'/C-6', H-10'/C-11'/C-8', H 3 -13'/C-10'/C-11'/C-12' and H-12'/C The -13'/C-11' correlation indicated the presence of B, C, and D loops (as shown in Figure 1). The absolute configuration was determined by ECD calculations (Fig. 2-Fig. 7).
二、倍半萜色酮类化合物的抗胰腺癌活性2. Anti-pancreatic cancer activity of sesquiterpene ketones
本实验用人源胰腺癌细胞SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞,取对数生长期的SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞,接种于96孔板(每孔1×104个细胞),待细胞贴壁后,分别加入浓度范围为0.1-100 µM梯度的待测化合物,培养72 h,然后每孔加入10 µL CCK-8溶液继续培养4小时,振荡10 min,酶标仪450 nm测定吸光度值,采用MTT法检测新疆阿魏倍半萜色酮类化合物 (+)-1和 (-)-1对SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞的增殖抑制作用(图6),根据吸光值计算细胞存活率,并计算测试化合物IC50值。其中,紫杉醇(Taxol)作为阳性对照药物。In this experiment, human pancreatic cancer cells SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells were used, and SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells in logarithmic growth phase were used to inoculate In a 96-well plate (1×10 4 cells per well), after the cells adhere to the wall, add the compound to be tested in a gradient concentration range of 0.1-100 µM, incubate for 72 h, and then add 10 µL CCK-8 to each well The solution was incubated for 4 hours, shaken for 10 min, and the absorbance value was measured at 450 nm by a microplate reader. The MTT method was used to detect the effects of Xinjiang ferula sesquiterpene ketones (+)-1 and (-)-1 on SW1990 cells, CFPAC- 1 cell, Capan-2 cell and PANC-1 cell proliferation inhibitory effect (Figure 6), the cell survival rate was calculated according to the absorbance value, and the IC 50 value of the test compound was calculated. Among them, paclitaxel (Taxol) was used as a positive control drug.
表2为化合物 (+)-1和(-)-1对胰腺癌SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞的抑制作用。其中,化合物 (+)-1抑制SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞的IC50值为11.77±1.83 µM、6.12±0.52 µM、8.57±0.59 µM、2.24±0.83 µM,化合物(-)-1抑制SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞的IC50值为15.67±1.53 µM、19.13±2.99 µM、14.57±0.65 µM、0.70±0.47 µM;结果显示,化合物 (+)-1和(-)-1对胰腺癌SW1990细胞、CFPAC-1细胞、Capan-2细胞和PANC-1细胞都有显著的增殖抑制作用,表明化合物 (+)-1和(-)-1能够明显抑制胰腺癌细胞的增殖,具有抗胰腺癌潜在作用,具有开发胰腺癌治疗药物的潜能,可作为活性成分用于制备抗胰腺癌药物。即以新疆阿魏倍半萜色酮类化合物为活性组分,以药学或生理学可接受的辅料及常规药物制剂的制备工艺制成内服制剂或注射剂。内服制剂为散剂、颗粒剂、胶囊剂、软胶囊、粉剂、丸剂、片剂、口服液。Table 2 shows the inhibitory effects of compounds (+)-1 and (-)-1 on pancreatic cancer SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells. Among them, compound (+)-1 inhibited SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells with IC 50 values of 11.77±1.83 µM , 6.12±0.52 µM , 8.57±0.59 µM , 2.24 ±0.83 µM , compound (-)-1 inhibited SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells with IC 50 values of 15.67±1.53 µM , 19.13±2.99 µM , 14.57±0.65 µM M, 0.70±0.47 µM ; The results showed that compounds (+)-1 and (-)-1 had significant inhibitory effects on pancreatic cancer SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells , indicating that compounds (+)-1 and (-)-1 can significantly inhibit the proliferation of pancreatic cancer cells, have potential anti-pancreatic cancer effects, and have the potential to develop therapeutic drugs for pancreatic cancer, and can be used as active ingredients for the preparation of anti-pancreatic cancer drugs . That is to use Xinjiang ferulicum sesquiterpene ketones as active components, and make internal preparations or injections with pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparations. The internal preparations are powders, granules, capsules, soft capsules, powders, pills, tablets, and oral liquids.
附图说明Description of drawings
图1为化合物(+)-1、化合物(-)-1的1H-1H COSY(黑色加粗) 和HMBC (黑色箭头)。Figure 1 shows the 1 H -1 H COZY (black bold) and HMBC (black arrow) of compound (+)-1 and compound (-)-1.
图2为化合物(+)-1的绝对构型。Figure 2 is the absolute configuration of compound (+)-1.
图 3为化合物(-)-1的ECD谱。Figure 3 is the ECD spectrum of compound (-)-1.
图 4为化合物(-)-1的CD谱。Figure 4 is the CD spectrum of compound (-)-1.
图 5为化合物(+)-1的绝对构型。Figure 5 shows the absolute configuration of compound (+)-1.
图 6为化合物(+)-1的ECD谱。Figure 6 is the ECD spectrum of compound (+)-1.
图 7为化合物(+)-1的CD谱。Figure 7 is the CD spectrum of compound (+)-1.
图 8为化合物 (+)-1和 (-)-1抑制胰腺癌细胞存活率。Figure 8 shows the inhibition of the survival rate of pancreatic cancer cells by compounds (+)-1 and (-)-1.
具体实施方式Detailed ways
(1)将干燥的新疆阿魏根用甲醇室温提取3次,每次7天,提取液合并,蒸发浓缩至无醇味,得到总提取物;(1) Extract the dried Xinjiang ferulae root with methanol at room temperature for 3 times, each time for 7 days, combine the extracts, evaporate and concentrate until there is no alcohol smell, and obtain the total extract;
(2)将总提取物分别用水分散溶解后分别用石油醚、二甲烷氯、乙酸乙酯和正丁醇进行萃取,得到石油醚相、二氯甲烷相、乙酸乙酯相、正丁醇相和水相;(2) Disperse and dissolve the total extracts in water and then extract with petroleum ether, dimethyl chloride, ethyl acetate and n-butanol to obtain petroleum ether phase, dichloromethane phase, ethyl acetate phase, n-butanol phase and water box;
(3)乙酸乙酯相(260 g)在硅胶柱上进行色谱分离,用石油醚:乙酸乙酯(石油醚:乙酸乙酯体积比依次为40:1、30:1、20:1、10:1、8:1、6:1、5:1、4:1、3:1、2:1、1:1)进行洗脱,依次得到12个组分FA-FL;(3) The ethyl acetate phase (260 g) was chromatographically separated on a silica gel column, using petroleum ether: ethyl acetate (the volume ratio of petroleum ether: ethyl acetate was 40:1, 30:1, 20:1, 10 :1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1) for elution to obtain 12 components FA-FL in sequence;
对组分FH(61.3g) 进行硅胶柱色谱,用二氯甲烷:乙酸乙酯(50:1)洗脱,依次得到五个组分 FH-1至FH-5;Component FH (61.3g) was subjected to silica gel column chromatography, eluted with dichloromethane:ethyl acetate (50:1), to obtain five components FH-1 to FH-5 in sequence;
对组分 FH-1(14.05 g)进行硅胶柱色谱,用石油醚:乙酸乙酯(10:1)洗脱,依次得到 12 个组分 FH-1-1 至 FH-1-12;Component FH-1 (14.05 g) was subjected to silica gel column chromatography, eluted with petroleum ether: ethyl acetate (10:1), and 12 components FH-1-1 to FH-1-12 were obtained in sequence;
对组分FH-1-7(393.6 mg)用二氯甲烷:乙酸乙酯(60:1)洗脱,得到八个组分FH-1-7-1至FH-1-7-8;Fraction FH-1-7 (393.6 mg) was eluted with dichloromethane:ethyl acetate (60:1) to obtain eight fractions FH-1-7-1 to FH-1-7-8;
对组分FH-1-7-5(67mg)用二氯甲烷:乙酸乙酯(40:1)得到四个组分FH-1-7-5-1到FH-1-7-5-4;Fraction FH-1-7-5 (67mg) was used dichloromethane:ethyl acetate (40:1) to obtain four fractions FH-1-7-5-1 to FH-1-7-5-4 ;
(4)对组分FH-1-7-5-1(24 mg)使用HPLC(C18,MeOH/H2O= 76/24,流速=4 ml/min)纯化,得到化合物1(4 mg,tR = 65.052 min);(4) Fraction FH-1-7-5-1 (24 mg) was purified by HPLC (C 18 , MeOH/H 2 O = 76/24, flow rate = 4 ml/min) to obtain compound 1 (4 mg , t R = 65.052 min);
(5)对化合物1使用手性HPLC(Chiral CD-Ph,石油醚:乙酸乙酯= 84/16,流速=3ml/min)纯化,依次得到化合物 (+)-1(1mg,tR=26.180min)和化合物(-)-1(0.9mg,tR=31.917 min)。(5)
化合物[(±)-1]:白色油状;mp 218-221 °C; UV (CH3CN) λmax (log ε) 204(3.131)nm, 274(2.356) nm; IR (KBr) νmax = 3424, 2926.9, 1723.9, 1612.2,1463.6, 1272.3, 1272.3, 1111.5, 1022.5, 1000.8, 972.0, 973.7 cm−1; 1H NMR 和13CNMR 数据如表1; 化合物 [(±)-1]的质谱 m/z 399.2158 [M + H]+ (C24H30O5,399.2158)。Compound [(±)-1]: white oil; mp 218-221 °C; UV (CH 3 CN) λmax (log ε) 204(3.131)nm, 274(2.356) nm; IR (KBr) ν max = 3424 , 2926.9, 1723.9, 1612.2,1463.6, 1272.3, 1272.3, 1111.5, 1022.5, 1000.8, 972.0, 973.7 cm −1 ; 1 H NMR and 13 CNMR data are shown in Table 1; spectrum m/z 399.2158 [M + H] + (C 24 H 30 O 5 , 399.2158).
(+)-1:白色油状;+52.381° (c 0.1, CH3OH); ECD (CH3OH) λmax (Δε):207(Δε -38.385), 257 (Δε -2.98), 268 (Δε -4.96), 301 (Δε 6.35), 328 (Δε-1.44) nm。(+)-1: white oil; +52.381° (c 0.1, CH 3 OH); ECD (CH 3 OH) λ max (Δε):207(Δε -38.385), 257 (Δε -2.98), 268 (Δε -4.96), 301 (Δε 6.35) , 328 (Δε-1.44) nm.
(-)-1:白色油状;-48.531° (c 0.1, CH3OH); ECD (CH3OH)λmax (Δε):208 (Δε 33.67), 249 (Δε 0.77), 271 (Δε 3.67), 300 (Δε -5.54), 327(Δε0.96) nm。(-)-1: white oil; -48.531° (c 0.1, CH 3 OH); ECD (CH 3 OH) λ max (Δε):208 (Δε 33.67), 249 (Δε 0.77), 271 (Δε 3.67), 300 (Δε -5.54), 327 (Δε0.96) nm.
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