WO2017220041A2 - 苯扎贝特的药物组合物及其在类风湿性关节炎中的应用 - Google Patents

苯扎贝特的药物组合物及其在类风湿性关节炎中的应用 Download PDF

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WO2017220041A2
WO2017220041A2 PCT/CN2017/097766 CN2017097766W WO2017220041A2 WO 2017220041 A2 WO2017220041 A2 WO 2017220041A2 CN 2017097766 W CN2017097766 W CN 2017097766W WO 2017220041 A2 WO2017220041 A2 WO 2017220041A2
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compound
bezafibrate
ethanol
rheumatoid arthritis
eluted
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WO2017220041A3 (zh
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崔坤峰
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赵吉永
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus

Definitions

  • the invention belongs to the field of biomedicine and relates to a new use of bezafibrate, in particular to a pharmaceutical composition of bezafibrate and its use in rheumatoid arthritis.
  • Bezafibrate belongs to the fibrate class and was first introduced in the 1970s. It has undergone numerous clinical trials and large-scale systematic clinical trials worldwide, and has been shown to significantly reduce triglycerides and increase High-density lipoprotein levels delay the progression of coronary atherosclerotic lesions and reduce the risk of coronary heart disease events. There are two mechanisms for lowering blood fat. First, the product can increase lipoprotein lipase and hepatic lipase activity, promote the catabolism of very low density lipoprotein, and lower the level of triglyceride in plasma. Secondly, this product makes The secretion of very low density lipoprotein is reduced.
  • This product lowers the role of triglyceride in plasma than cholesterol lowering, and also raises high-density lipoprotein. In addition, this product can also reduce fibrinogen. Previous studies have not found that this product has carcinogenic and mutagenic effects.
  • bezafibrate has a unique role in not only improving lipid metabolism, but also improving sugar metabolism.
  • Foreign studies have confirmed that long-term use of bezafibrate has a role in preventing diabetes and improving insulin sensitivity, and can be used for lipid metabolism disorders caused by diabetic patients, which is obviously superior to other fibrates.
  • synovial cells are both target cells and participants through a variety of pathways, and their functional changes play a vital role in the progression of the disease, fibroblast-like Membrane cells are the main component of synovial tissue, which is involved in the destruction of articular cartilage and the destruction of bone tissue around the joint. Therefore, inhibiting the proliferation of fibroblast-like synoviocytes is one of the means to treat rheumatoid arthritis.
  • the object of the present invention is to provide a pharmaceutical composition of bezafibrate, which comprises bezafibrate and a novel natural product, and bezafibrate and the natural product can synergistically treat rheumatoid arthritis.
  • a pharmaceutical composition of bezafibrate comprising bezafibrate, compound (I) as described above, and a pharmaceutically acceptable carrier.
  • the preparation method of the compound (I) as described above comprises the following steps: (a) pulverizing the dried flower buds of cloves, extracting them by hot reflux with 70-90% ethanol, combining the extracts, concentrating to an alcohol-free taste, and sequentially using petroleum.
  • step (a) n-butanol extract in step (a) is removed by macroporous resin First, elute 12 column volumes with 20% ethanol, then elute 15 column volumes with 80% ethanol, collect 80% eluent, concentrate under reduced pressure to obtain 80% ethanol eluting concentrate; (c) step (b) The 80% ethanol eluting concentrate was separated by normal phase silica gel, and sequentially eluted with a dichloromethane-methanol gradient of 80:1, 40:1, 20:1 and 10:1 by volume to obtain 4 components; (d) In step (c), component 3 is further separated by normal phase silica gel, and sequentially eluted with a dichloromethane-methanol gradient of 25:1, 20:1 and 15:1 by volume to obtain three components; e
  • step (a) is carried out by hot reflux extraction with 80% ethanol, and the extracts are combined.
  • the macroporous resin is a D101 type macroporous adsorption resin.
  • the pharmaceutical composition of bezafibrate provided by the invention comprises bezafibrate and a novel natural product isolated from the dried flower bud of clove, and the bezafibrate and the natural product have therapeutic effects when used alone.
  • Rheumatoid arthritis when the combination of the two, the effect of treating rheumatoid arthritis is further improved, and it can be developed into a drug for treating rheumatoid arthritis.
  • the present invention has outstanding substantial features and significant advances over the prior art.
  • step (c) The 80% ethanol eluting concentrate in step (b) is separated by normal phase silica gel in a volume ratio of 80:1 (10 column volumes), 40:1 (8 column volumes), 20:1 ( 4 column volumes) and 10:1 (9 column volumes) of dichloromethane-methanol gradient elution to obtain 4 components;
  • step (c) component 3 is further separated by normal phase silica gel, followed by A gradient of 25:1 (7 column volumes), 20:1 (8 column volumes) and 15:1 (7 column volumes) of dichloromethane-methanol gradient eluted to give 3 components;
  • step (d) component 2 is separated by octadecylsilane-bonded reversed phase silica gel, eluted isocratically with 65% by volume aqueous methanol solution, and 13 to 17 column volumes of eluent are collected and washed. The mixture was concentrated under reduced pressure to give Compound (I) (6
  • the 1756 cm -1 absorption band in the infrared spectrum and the 236 nm absorption band in the UV spectrum indicate that the compound contains an ⁇ , ⁇ -unsaturated ⁇ -lactone structure and passes ⁇ C 163.4, 124.6 and 174.3 in the 13 C-NMR spectrum.
  • the carbon signal is verified.
  • 13 C-NMR, DEPT and HSQC spectra show 16 carbon signals, including four methyl groups (one methoxy group), two methylene groups, four methine groups (one oxygenated carbon), and six
  • the above functional structure combined with the number of unsaturation indicates that the compound has a tricyclic structure.
  • H-12/H-3/H 2 -4, H-6/H-7 and H-6/H 3 -13 related signals are present in the 1 H- 1 H COSY spectrum, and H 2 - is shown in the HMBC spectrum.
  • 4 with C-2 and C-5, H-6 with C-1 and C-5, H-7 with C-10, H 2 -10 with C-1 and C-14, H-12 and C-2 And C-11, H 3 -13 and C-5 and C-7, H 3 -14 and C-8, C-9 and C-10, H 3 -15 and C-9 and C-10,12- OMe and C-12 related signals can be constructed by the related information in the above NMR spectrum, and the above spectral data indicates that the compound is a tremulane type sesquiterpene.
  • the correlation between the proton signal ⁇ H 3.48 and C-12 indicates that a methoxy group is attached to the C-12 position; H 2 -4, H-6 and H 3 -13 and C-5 ( ⁇ C 212.4) The correlation indicates that C-5 is a carbonyl group; the correlation between H 3 -14 and C-8 ( ⁇ C 215.6) indicates that C-8 is a ketone carbonyl group.
  • the H-6 and H-7 positions are generally in the ⁇ configuration
  • the H 3 -14 configuration is usually in the ⁇ position
  • the H 3 -15 configuration is in the ⁇ position, in the NOE test of the compound.
  • H-7 and H 3 -14 related signals reflected in the tremulane-type sesquiterpene configuration In the NOESY spectrum, the correlation signals of H-3 and H 3 -13 suggest that H-3 is the ⁇ configuration, the NOE correlation between H-3 and H-12, and the coupling constant of the two indicate that H-12 is the ⁇ configuration. Then 12-OMe is in the beta configuration.
  • Comprehensive hydrogen, carbon, HMBC and NOESY spectra, as well as the literature on the relevant types of nuclear magnetic data, can be basically determined that the compound is shown below, the stereo configuration is further determined by ECD test, the theoretical values are basically consistent with the experimental values.
  • Bezabet was purchased from the China National Institute for the Control of Pharmaceutical and Biological Products.
  • the compound (I) was prepared by itself, and the preparation method is shown in Example 1.
  • Type II collagen American Sigma
  • incomplete Freund's adjuvant Sigma
  • RPMI-1640 GIBCO
  • Type II collagen was dissolved in 0.1 mmol/L glacial acetic acid (final concentration of type II collagen was 2 g/L), and overnight at 4 °C. It is then added dropwise to a cold, incompletely incomplete Freund's adjuvant and fully emulsified. The emulsion was intradermally injected into each rat with 0.5 ml, 4 points on the back and 1 point on the heel. The injection was boosted once in the same manner 7 days later.
  • each rat joint was scored daily, divided into 0 to 4 points: 0 points, no redness; 1 point, small toe joint swelling; 2 points toe joint and ankle swelling; 3 points, ⁇ Swelling of the feet below the joints; 4 points, including all paws within the ankle joint Bulging.
  • the cumulative score of each joint is scored, which is the arthritis index of each rat.
  • each group contained 10% fetal bovine serum; the positive control group also contained 1.0 ⁇ 10 -6 mol/L methotrexate, and the bezafibrate group also contained 2.0 ⁇ 10 -6 mol/L benzene.
  • Zabeite the compound (I) group further contains 2.0 ⁇ 10 -6 mol/L of the compound (I), and the bezafibrate and the compound (I) composition group further contain 1.0 ⁇ 10 -6 mol/L of benzal. Beth and 1.0 ⁇ 10 -6 mol/L of compound (I) were cultured in a 37 ° C 5% CO 2 incubator, digested and passaged, and passaged cells 3 to 5 were used.
  • the cells were collected, resuspended in PBS and boiled into a single cell suspension, which was slowly added to pre-cooled 5 ml of 75% ethanol, fixed overnight at 4 ° C; the concentration was adjusted to 5 ⁇ 10 6 /ml with PBS, then Take 400 ⁇ l, add RAase 20 ml, 37 ° C water bath for 30 min; add PI 100 ⁇ l, stain for 20 min in the dark, filter with 300 mesh nylon mesh, upflow cytometer.
  • the experimental data were expressed as mean ⁇ standard deviation (x ⁇ s).
  • One-way ANOVA and t-test were performed using SPSS18.0 statistical software. The difference was statistically significant at P ⁇ 0.05.

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Abstract

本发明公开了苯扎贝特的药物组合物及其在类风湿性关节炎中的应用，本发明提供的苯扎贝特的药物组合物中含有苯扎贝特和一种从丁香的干燥花蕾中分离得到的结构新颖的天然产物化合物(Ⅰ)，苯扎贝特和本发明提供的化合物(Ⅰ)联合作用时，可使滑膜细胞停滞在细胞周期G1期，从而抑制滑膜细胞增殖，治疗类风湿性关节炎；治疗效果优于苯扎贝特或化合物(Ⅰ)单独作用效果。苯扎贝特与化合物(Ⅰ)组合物可以开发成治疗类风湿性关节炎的药物，与现有技术相比具有突出的实质性特点和显著的进步。

Description

苯扎贝特的药物组合物及其在类风湿性关节炎中的应用 技术领域
本发明属于生物医药领域，涉及苯扎贝特的新用途，具体涉及苯扎贝特的药物组合物及其在类风湿性关节炎中的应用。
背景技术
苯扎贝特属于贝特类药物，在上个世纪70年代首次面市，在世界范围内经过了大量临床应用考验，以及大规模系统临床试验，均被证实能明显地降低甘油三酯、升高高密度脂蛋白水平，延缓冠状动脉粥样硬化病变的进展，降低冠心病事件发生的危险性。其降血脂作用有两种机制，一是本品能提高脂蛋白脂酶和肝脂酶活性，促进极低密度脂蛋白的分解代谢，使血浆中甘油三酯的水平降低；其次是本品使极低密度脂蛋白的分泌减少。本品降低血浆中甘油三酯的作用比降低胆固醇的作用要强，也使高密度脂蛋白升高，此外本品也可降低纤维蛋白原。已有的研究未发现本品有致癌、致突变作用。
近期对苯扎贝特的深入研究显示，苯扎贝特具有很独特的作用，不仅可以改善脂代谢，更重要的对糖代谢也有改善。国外研究证实，长期服用苯扎贝特对预防糖尿病，改善胰岛素敏感性有作用，可用于糖尿病患者引起的脂代谢紊乱，这一点明显优于其他的贝特类药品。
在类风湿关节炎关节损伤和组织重构中，滑膜细胞既是靶细胞，也通过多种途径成为参与者，其功能的改变在疾病的进程中起着至关重要的作用，成纤维样滑膜细胞是滑膜组织的主要成分，参与了关节软骨破坏及关节周围的骨组织破坏，所以抑制成纤维样滑膜细胞增殖是治疗类风湿关节炎的手段之一。
目前尚未见苯扎贝特与治疗类风湿性关节炎的相关性报道。
发明内容
本发明的目的在于提供一种苯扎贝特的药物组合物，该药物组合物中含有苯扎贝特和一种结构新颖的天然产物，苯扎贝特和该天然产物可以协同治疗类风湿性关节炎。
本发明的上述目的是通过下面的技术方案得以实现的：
一种具有下述结构式的化合物(Ⅰ)，
Figure PCTCN2017097766-appb-000001
一种苯扎贝特的药物组合物，包括苯扎贝特、如上所述的化合物(Ⅰ)和药学上可以接受的载体。
如上所述的化合物(Ⅰ)的制备方法，包含以下操作步骤：(a)将丁香的干燥花蕾粉碎，用70～90％乙醇热回流提取，合并提取液，浓缩至无醇味，依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取，分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物；(b)步骤(a)中正丁醇取物用大孔树脂除杂，先用20％乙醇洗脱12个柱体积，再用80％乙醇洗脱15个柱体积，收集80％洗脱液，减压浓缩得80％乙醇洗脱浓缩物；(c)步骤(b)中80％乙醇洗脱浓缩物用正相硅胶分离，依次用体积比为80:1、40:1、20:1和10:1的二氯甲烷-甲醇梯度洗脱得到4个组分；(d)步骤(c)中组分3用正相硅胶进一步分离，依次用体积比为25:1、20:1和15:1的二氯甲烷-甲醇梯度洗脱得到3个组分；(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离，用体积百分浓度为65％的甲醇水溶液等度洗脱，收集13～17个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)。
进一步地，步骤(a)用80％乙醇热回流提取，合并提取液。
进一步地，所述大孔树脂为D101型大孔吸附树脂。
如上所述的化合物(Ⅰ)在制备治疗类风湿性关节炎的药物中的应用。
如上所述的苯扎贝特的药物组合物在制备治疗类风湿性关节炎的药物中的应用。
本发明的优点：
本发明提供的苯扎贝特的药物组合物中含有苯扎贝特和一种从丁香的干燥花蕾中分离得到的结构新颖的天然产物，苯扎贝特和该天然产物单独作用时，具有治疗类风湿性关节炎作用；二者联合作用时，治疗类风湿性关节炎效果进一步提高，可以开发成治疗类风湿性关节炎的药物。本发明与现有技术相比具有突出的实质性特点和显著的进步。
具体实施方式
下面结合实施例进一步说明本发明的实质性内容，但并不以此限定本发明保护范围。尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
实施例1：化合物(Ⅰ)分离制备及结构确证
分离方法：(a)将丁香的干燥花蕾(2kg)粉碎，用80％乙醇热回流提取(15L×3次)，合并提取液，浓缩至无醇味(3L)，依次用石油醚(3L×3次)、乙酸乙酯(3L×3次)和水饱和的正丁醇(3L×3次)萃取，分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物；(b)步骤(a)中乙酸乙酯萃取物用D101型大孔树脂除杂，先用20％乙醇洗脱12个柱体积，再用80％乙醇洗脱15个柱体积，收集80％洗脱液，减压浓缩得80％乙醇洗脱浓缩物； (c)步骤(b)中80％乙醇洗脱浓缩物用正相硅胶分离，依次用体积比为80:1(10个柱体积)、40:1(8个柱体积)、20:1(8个柱体积)和10:1(9个柱体积)的二氯甲烷-甲醇梯度洗脱得到4个组分；(d)步骤(c)中组分3用正相硅胶进一步分离，依次用体积比为25:1(7个柱体积)、20:1(8个柱体积)和15:1(7个柱体积)的二氯甲烷-甲醇梯度洗脱得到3个组分；(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离，用体积百分浓度为65％的甲醇水溶液等度洗脱，收集13～17个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)(644mg，HPLC归一化纯度大于98％)。
结构确证：HR-ESI-MS显示[M+H]+为m/z 293.1346，结合核磁特征可得分子式为C16H20O4，不饱和度为7。核磁共振氢谱数据δH(ppm，CDCl3，500MHz)：H-3(3.24，m)，H-4a(3.12，dd，J＝14.8，13.3Hz)，H-4b(2.50，d，J＝14.3Hz)，H-6(2.75，qd，J＝7.5，2.3Hz)，H-7(2.82，d，J＝2.3Hz)，H-10a(2.92，d，J＝15.7Hz)，H-10b(2.38，d，J＝15.7Hz)，H-12(5.25，d，J＝5.3Hz)，H-13(1.18，d，J＝7.2Hz)，H-14(0.97，s)，H-15(1.15，s)，12-OMe(3.48，s)；核磁共振碳谱数据δC(ppm，CDCl3，125MHz)：166.4(C，1-C)，128.3(C，2-C)42.1(CH，3-C)，41.5(CH2，4-C)，212.4(C，5-C)48.7(CH，6-C)，47.8(CH，7-C)，215.6(C，8-C)，48.6(C，9-C)，29.5(CH2，10-C)，169.7(C，11-C)，103.5(CH，12-C)，13.2(CH3，13-C)，20.4(CH3，14-C)，26.8(CH3，15-C)，56.7(CH3，12-OMe)。红外波谱中的1756cm-1吸收带与UV谱中的236nm吸收带表明该化合物含有α,β-不饱和γ-内酯结构，并通过13C-NMR谱中的δC163.4，124.6和174.3的碳信号得到验证。13C-NMR、DEPT和HSQC谱中显示有16个碳信号，包括四个甲基(一个甲氧基)，两个亚甲基，四个次甲基(一个连氧碳)，以及六个季碳(两个烯烃碳和三个羰基碳)，以上功能结构再结合不饱和数表明该化合物为三环结构。1H-NMR谱结合HSQC谱显示三个甲基质子信号δH 1.18(3H，d，J＝7.2Hz)、0.97(3H，s)、1.15(3H，s)，一个甲氧基质子信号δH 3.48(3H，s)，两组亚甲基质子信号δH 3.12(2H，dd，J＝14.8，13.3Hz)与2.50(1H，d，J＝14.3Hz)、2.92(1H，d，J＝15.7Hz)与2.38(1H，d，J＝15.7Hz)，三个次甲基质子信号δH 3.24(1H，m)、2.75(1H，qd，J＝7.5，2.3Hz)、2.82(1H，d，J＝2.3Hz)，一个连氧次甲基质子信号δH 5.25(1H，d，J＝5.3Hz)。1H-1H COSY谱中存在H-12/H-3/H2-4、H-6/H-7和H-6/H3-13相关信号，同时HMBC谱中显示有H2-4与C-2和C-5，H-6与C-1和C-5，H-7与C-10，H2-10与C-1和C-14，H-12与C-2和C-11，H3-13与C-5和C-7，H3-14与C-8、C-9和C-10，H3-15与C-9和C-10，12-OMe与C-12相关信号，通过上述NMR谱中的相关信息可以构建该化合物的连接方式，并且上述波谱数据表明该化合物为tremulane型倍半萜。HMBC谱中，质子信号δH3.48与C-12的相关性表明C-12位连有一个甲氧基；H2-4、H-6和 H3-13与C-5(δC212.4)的相关性表明C-5为羰基；H3-14与C-8(δC215.6)的相关性表明C-8为酮羰基。在tremulane型倍半萜中，H-6与H-7位一般为β构型，H3-14构型通常处是β位，H3-15构型为α位，在该化合物的NOE试验中体现的H-7与H3-14相关信号符合tremulane型倍半萜的构型关系。NOESY谱中，H-3与H3-13的相关信号暗示H-3为α构型，H-3与H-12的NOE相关性以及两者的耦合常数表明H-12为α构型，则12-OMe为β构型。综合氢谱、碳谱、HMBC谱和NOESY谱，以及文献关于相关类型核磁数据，可基本确定该化合物如下所示，立体构型进一步通过ECD试验确定，理论值与实验值基本一致。
该化合物化学式及碳原子编号如下：
Figure PCTCN2017097766-appb-000002
实施例2：药理作用
1.材料与方法
1.1动物
选用雄性Wistar大鼠(华中科技大学同济医学院动物实验中心提供)，体重120g左右。
1.2试剂与样品
苯扎贝特购自中国药品生物制品检定所。化合物(Ⅰ)自制，制备方法见实施例1。Ⅱ型胶原(美国Sigma公司)、不完全弗氏佐剂(Sigma公司)、RPMI-1640(GIBCO公司)。
1.3仪器
美国nurie CO2培养箱(NU4750型)、日本奥林巴氏倒置荧光显微镜(CKX41型)、美国Beckman Coulter流式细胞仪(EPICSXL型)、日本透射电镜(日立H-7500型)。
1.4大鼠模型制备及细胞分组
CIA大鼠模型的制备将Ⅱ型胶原溶解于0.1mmol/L的冰乙酸中(Ⅱ型胶原的终浓度为2g/L)，4℃过夜。然后将其滴加至冷的等积不完全弗氏佐剂中充分乳化。将该乳剂每只大鼠皮内注射0.5ml，分背部4点和尾跟部1点。7天后同样方法加强注射1次。根据关节肿胀指数评分系统每天对每只大鼠关节进行评分，分为0～4分：0分，无红肿；1分，小趾关节红肿；2分趾关节和足跖肿胀；3分，踝关节以下的足爪肿胀；4分，包括踝关节以内的全部足爪肿 胀。把各个关节的指数累积计分，即为每只大鼠的关节炎指数。
滑膜细胞的体外培养致炎后第25天，脱臼处死发病的大鼠，无菌条件下取得滑膜组织，剪切成约1mm3的碎块，0.5mg/ml的Ⅱ型胶原酶37℃消化6～7h，1200r/m离心8min，加入1640培养液【分为5组：模型对照组、阳性对照组、苯扎贝特组、化合物(Ⅰ)组、苯扎贝特与化合物(Ⅰ)组合物组，每组均含10％胎牛血清；阳性对照组还含有1.0×10-6mol/L的甲氨蝶呤，苯扎贝特组还含有2.0×10-6mol/L的苯扎贝特，化合物(Ⅰ)组还含有2.0×10-6mol/L的化合物(Ⅰ)，苯扎贝特与化合物(Ⅰ)组合物组还含有1.0×10-6mol/L的苯扎贝特和1.0×10-6mol/L的化合物(Ⅰ)】，置37℃5％CO2培养箱内培养，消化传代，用第3～5代细胞。
1.5流式细胞仪检测滑膜细胞周期
收集细胞，用PBS重悬吹打成单细胞悬液，将其缓慢加到预冷的5ml 75％乙醇中，4℃固定过夜；用PBS将其浓度调整到5×106个/ml，然后取400μl，加RAase 20ml，37℃水浴30min；加PI 100μl，避光染色20min，300目尼龙网过滤，上流式细胞仪。
1.6统计学方法
实验数据用均数±标准差(x±s)表示，应用SPSS18.0版统计软件进行单因素方差分析和t检验，以P＜0.05为差异有统计学意义。
2.实验结果
流式细胞仪周期分析结果显示，与模型组比较，阳性对照组G1期增加，S期减少，差异具有统计学意义(P<0.05)；苯扎贝特与化合物(Ⅰ)组合物组G1期明显增加(P<0.01)，S期明显减少(P<0.01)；苯扎贝特组和化合物(Ⅰ)组G1期增加(P<0.05)，S期减少(P<0.05)。
表1对CIA大鼠滑膜细胞周期的影响
组别 G1期(％) S期(％)
模型对照组 64.50±2.26 13.82±2.81
阳性对照组 79.24±2.33 4.52±0.73
苯扎贝特组 77.15±3.05 4.88±0.74
化合物(Ⅰ)组 78.24±2.49 4.60±0.78
苯扎贝特与化合物(Ⅰ)组合物组 84.95±5.02 1.34±0.58
上述结果表明，苯扎贝特和本发明提供的化合物(Ⅰ)联合作用时，可使滑膜细胞停滞在细胞周期G1期，从而抑制滑膜细胞增殖，治疗类风湿性关节炎；治疗效果优于苯扎贝特或化合物(Ⅰ)单独作用效果。苯扎贝特与化合物(Ⅰ)组合物可以开发成治疗类风湿性关节炎的药物。
上述实施例的作用在于说明本发明的实质性内容，但并不以此限定本发明的保护范围。 本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和保护范围。

Claims (7)

  1. 一种具有下述结构式的化合物(Ⅰ)，
    Figure PCTCN2017097766-appb-100001
  2. 一种苯扎贝特的药物组合物，其特征在于：包括苯扎贝特、如权利要求1所述的化合物(Ⅰ)和药学上可以接受的载体。
  3. 权利要求1所述的化合物(Ⅰ)的制备方法，其特征在于，包含以下操作步骤：(a)将丁香的干燥花蕾粉碎，用70～90％乙醇热回流提取，合并提取液，浓缩至无醇味，依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取，分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物；(b)步骤(a)中正丁醇取物用大孔树脂除杂，先用20％乙醇洗脱12个柱体积，再用80％乙醇洗脱15个柱体积，收集80％洗脱液，减压浓缩得80％乙醇洗脱浓缩物；(c)步骤(b)中80％乙醇洗脱浓缩物用正相硅胶分离，依次用体积比为80:1、40:1、20:1和10:1的二氯甲烷-甲醇梯度洗脱得到4个组分；(d)步骤(c)中组分3用正相硅胶进一步分离，依次用体积比为25:1、20:1和15:1的二氯甲烷-甲醇梯度洗脱得到3个组分；(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离，用体积百分浓度为65％的甲醇水溶液等度洗脱，收集13～17个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)。
  4. 根据权利要求3所述的化合物(Ⅰ)的制备方法，其特征在于：步骤(a)用80％乙醇热回流提取，合并提取液。
  5. 根据权利要求3所述的化合物(Ⅰ)的制备方法，其特征在于：所述大孔树脂为D101型大孔吸附树脂。
  6. 权利要求1所述的化合物(Ⅰ)在制备治疗类风湿性关节炎的药物中的应用。
  7. 权利要求2所述的苯扎贝特的药物组合物在制备治疗类风湿性关节炎的药物中的应用。
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