WO2022063071A1 - 一种化合物及在制备靶向抑制ptl和/或npc1l1药物中的应用 - Google Patents

一种化合物及在制备靶向抑制ptl和/或npc1l1药物中的应用 Download PDF

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WO2022063071A1
WO2022063071A1 PCT/CN2021/119276 CN2021119276W WO2022063071A1 WO 2022063071 A1 WO2022063071 A1 WO 2022063071A1 CN 2021119276 W CN2021119276 W CN 2021119276W WO 2022063071 A1 WO2022063071 A1 WO 2022063071A1
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compound
npc1l1
ptl
inhibiting
formula
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邢东明
张仁帅
宋正明
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青岛大学
青岛大学附属医院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
    • C07D305/02—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D305/10—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms not condensed with other rings having one or more double bonds between ring members or between ring members and non-ring members
    • C07D305/12—Beta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

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  • the present invention belongs to the field of targeted compounds, and particularly relates to a compound for inhibiting PTL/NPC1L1 and uses thereof.
  • Pancreatic triglyceride lipase is the main component of pancreatic lipase, which is secreted by pancreatic acinar cells and plays a role in digesting fat in the duodenum.
  • PTL has the effect of high-efficiency hydrolysis of fat.
  • triglycerides are hydrolyzed into diglycerides, which are further hydrolyzed into monoglycerides and fatty acids, which are absorbed by small intestinal epithelial cells. Both have high hydrolysis rates, which, combined with their high secretion levels, allow them to efficiently hydrolyze almost all long-chain fatty acids.
  • Changes in PTL expression are closely related to obesity and other metabolic diseases, and pancreatic lipase has become a target for the treatment of obesity and other metabolic diseases.
  • NPC1L1 (Niemann-Pick-C1-Like 1, Niemann-Pick C1-like protein 1) is a key protein in cholesterol transmembrane transport. one of the key nodes. NPC1L1 interacts with the related cholesterol transporters Flotillin-1 and Flotillin-2 to transport cholesterol and other sterols across the brush border of the intestinal epithelium and into the intestinal epithelium. A large number of cell and animal experiments have shown that NPC1L1 can play an important role in intestinal cholesterol absorption and metabolism, and has become one of the important new targets found in recent years to regulate cholesterol absorption.
  • the cholesterol-lowering drug Ezetimibe blocks the interaction of NPC1L1 with Flotillin-1 and Flotillin-2, thereby inhibiting cholesterol absorption.
  • NPC1L1 can further maintain the dynamic balance of cholesterol in the body by regulating the level of cellular cholesterol, and then regulating genes such as SREBP1c, FAS and ABCT1 related to cholesterol metabolism.
  • the present invention provides a compound for inhibiting PTL/NPC1L1 and its use.
  • a compound, the compound is a compound represented by the following formula (I),
  • X is -O-, -S- or -NH-;
  • R can be a benzene ring, a benzene ring containing a substituted group, a five-membered heterocycle, or a six-membered heterocycle;
  • R is a benzene ring containing a substituent group
  • the substituent group is selected from the substituent groups of the following group, including: OH, CN, NO2, C1-C6 alkoxy, halogen (F, Cl, Br, I in one or two or more), 1-5 of C1-C6 alkoxy groups with one or more halogen (one or two or more of F, Cl, Br, I) substituents, Preferably 1-3; the number of substituent groups on the benzene ring is 1-5, preferably 1-3;
  • R is a five-membered heterocycle or a six-membered heterocycle
  • the heteroatoms other than C contained in the heterocycle are any one or two of O, S, and N, and the number of heteroatoms on the heterocycle is 1 or 1. one or two.
  • the compound represented by the formula (I) reduces the absorption of cholesterol by inhibiting the activity of NPC1L1.
  • the compound represented by the formula (I) reduces the hydrolysis rate of triglyceride by inhibiting the activity of PTL.
  • the compound represented by the formula (I) inhibits intestinal triglyceride and cholesterol absorption by simultaneously inhibiting PTL and NPC1L1.
  • the compound represented by the formula (I) inhibits intestinal triglyceride and cholesterol absorption by simultaneously inhibiting PTL and NPC1L1.
  • the medicines include powders, pills and capsules prepared by mixing one or more of the compounds represented by the formula (I) and their pharmaceutically acceptable salts with a medically or pharmaceutically acceptable drug carrier.
  • the compound of the invention can simultaneously act on pancreatic triglyceride lipase (PTL) and NPC1L1, a key protein of cholesterol transmembrane transport, with obvious effect, and can better solve the absorption of triglyceride and cholesterol.
  • the compound can act as a dual-targeted inhibitor of PTL/NPC1L1, and simultaneously act on PTL and NPC1L1, which are of great value in the absorption of triglycerides and cholesterol in the intestine. Maibu comparison shows its advantages of double-effect characteristics.
  • novel compound prepared by the present invention exhibits good activity of simultaneously inhibiting PTL and NPC1L1, and has application value as a preparation for treating and/or preventing obesity, hypercholesterolemia and related cardiovascular and cerebrovascular diseases.
  • Figure 1 is the surface plasmon resonance method to evaluate the inhibitory effect of compound 1-compound 5 on NPC1L1, with Orlistat and Ezetimibe as controls.
  • FIG. 2 is a graph showing the effects of compound 2 and compound 3 on cholesterol absorption by using Caco2 cells as a model and Orlistat and Ezetimibe as controls in Example 8.
  • FIG. 1 is the surface plasmon resonance method to evaluate the inhibitory effect of compound 1-compound 5 on NPC1L1, with Orlistat and Ezetimibe as controls.
  • FIG. 2 is a graph showing the effects of compound 2 and compound 3 on cholesterol absorption by using Caco2 cells as a model and Orlistat and Ezetimibe as controls in Example 8.
  • a substance that inhibits PTL/NPC1L1 is a compound having the structure shown in the formula (I) or a pharmaceutically acceptable salt thereof:
  • X is -O-, -S- or -NH-;
  • R can be a benzene ring or a benzene ring containing a substituted group, a five-membered heterocycle, or a six-membered heterocycle;
  • R is a benzene ring containing a substituent group
  • the substituent group is selected from the substituent groups of the following group, including: OH, CN, NO2, C1-C6 alkoxy, halogen (F, Cl, Br, I in one or two or more), 1-5 of C1-C6 alkoxy groups with one or more halogen (one or two or more of F, Cl, Br, I) substituents, Preferably 1-3; the number of substituent groups on the benzene ring is 1-5, preferably 1-3;
  • R is a five-membered heterocycle or a six-membered heterocycle
  • the heteroatoms other than C contained in the heterocycle are any one or two of O, S, and N, and the number of heteroatoms on the heterocycle is 1 or 1. one or two.
  • the preparation method of the compound having the structure represented by the formula (I) includes: synthesizing the compound by chemical synthesis method and synthesizing the compound by biosynthesis method.
  • the use of the compound with the structure shown in formula (I) includes: acting on NPC1L1 alone, inhibiting intestinal cholesterol absorption, and being used for the prevention and treatment of hypercholesterolemia and related cardiovascular and cerebrovascular diseases;
  • PTL/NPC1L1 Simultaneously acts on PTL/NPC1L1, inhibits intestinal triglyceride and cholesterol absorption, and is used for the prevention and treatment of obesity, hypercholesterolemia and related cardiovascular and cerebrovascular diseases;
  • the prepared medical drug for treating and/or preventing obesity and diseases related to hypersteroidemia can act on NPC1L1 alone or on PTL/NPC1L1 simultaneously.
  • the synthesis process of compound (3) is the same as that in Example 2, except that the reaction raw material 3-tert-butyldimethylsilyloxybenzoic acid is replaced with 4-tert-butyldimethylsilyloxybenzoic acid, and the rate 67%.
  • Different concentrations final concentrations in the mixed solution were 100 ⁇ M, 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M, respectively) of the test compounds or positive controls of different concentrations orlistat (Orlistat) or ezetimibe (Ezetimibe) (in
  • the final concentrations in the mixed solution were 100 ⁇ M, 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M, respectively, and PTL (the final concentration of PTL was 1 ⁇ g/mL) was mixed, and kept at 37 degrees for 30 minutes to obtain a mixed solution.
  • the blank control group was an equal volume and equal concentration of PTL solution without adding the test substance and positive control.
  • p-Nitrophenyl acetate at a final concentration of 0.5 mM in the mixed solution was added to the mixed solution, and kept at 37 degrees for 60 minutes. Measure the absorbance A at a wavelength of 405 nm.
  • the IC50 was calculated by using the logarithmic value of the compound concentration to make a regression equation on the inhibition rate (ie, the logarithmic value of the compound concentration was on the abscissa, and the inhibition rate was on the ordinate).
  • the results of the compound's inhibitory activity on PTL are shown in Table 1.
  • inhibition rate (A value of the experimental group - A value of the blank control group) / (A value of the control group - A value of the blank control group) ⁇ 100%
  • Table 1 shows the inhibitory effect of compounds 1-5 on PTL according to an embodiment of the present invention. Wherein the structure of compound 1-compound 5 is
  • the binding activity of the compounds to NPC1L1 was determined by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • CM5 series sensor chips, coupling reagents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from GE.
  • Control group activated and blocked as described above, but without the addition of NPC1L1 solution (unconjugated).
  • test compound 1-compound 5 final concentration of 100 ⁇ M
  • positive control Orlistat final concentration of 100 ⁇ M
  • Ezetimibe final concentration of 100 ⁇ M
  • Binding response values are reported in response units (RU), which are defined as the response of the flow chamber (experimental chip) containing the immobilized receptor minus the response of the control flow chamber (control chip).
  • Inhibitors Compound 2, Compound 3, Orlistat, Ezetimibe
  • Cholesterol-d6 was added at a final concentration of 10 ⁇ M and the incubation was continued for 1 hour at 37°C.
  • the medium was removed, washed three times with PBS buffer (3 mL each time), 1 mL of methanol was added, sonicated, and quantitatively analyzed by high performance liquid chromatography-mass spectrometry (Agilent Technologies, Santa Clara, CA, USA).
  • Example 6 in the test of inhibitory activity against PTL, all tested compounds showed good PTL inhibitory activity at a concentration of 100 ⁇ M, and compounds 1, 3, and 4 inhibited PTL.
  • the active IC 50s were 13.5 ⁇ M, 5.3 ⁇ M, and 15.8 ⁇ M, respectively; in the inhibitory activity test against NPC1L1, compounds 1, 2, 3, and 4 all showed a certain binding activity to NPC1L1, and compound 2 and compound 3 bound to NPC1L1. The best; in the cholesterol absorption inhibition test, Compound 2 and Compound 3 can significantly inhibit the absorption of cholesterol.

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Abstract

本发明提供了一种新型的同时靶向PTL和NPC1L1的抑制剂及其应用。即具有(Ⅰ)所示结构的化合物及其在抑制PTL和/或NPC1L1的药物中的用途，所述药物通过同时抑制PTL和NPC1L1来抑制肠道甘油三酯及胆固醇吸收，所述药物用于治疗和/或预防肥胖、高胆固醇血症及相关心脑血管疾病。本发明所制备的新型化合物表现出良好的同时抑制PTL和NPC1L1的活性作用，有作为制备治疗和/或预防肥胖、高胆固醇血症及相关心脑血管疾病的应用价值。

Description

一种化合物及在制备靶向抑制PTL和/或NPC1L1药物中的应用 技术领域
本发明属于靶向化合物领域，特别涉及一种抑制PTL/NPC1L1的化合物及其用途。
背景技术
胰腺甘油三酯脂酶(pancreatic triglyceride lipase,PTL)是胰脂肪酶主要成份，由胰腺腺泡细胞分泌，并在十二指肠中起到消化脂肪的作用。PTL具有高效水解脂肪的作用，在辅酶和胆盐的存在下将甘油三酯水解成甘油二酯，进一步水解成甘油单酯和脂肪酸，从而被小肠上皮细胞吸收，其对不同长度的甘油三酯均具有较高的水解速率，结合其高分泌水平，使其可以高效水解几乎所有的长链脂肪酸。PTL表达的改变与肥胖等代谢性疾病有着密切的关系，胰腺脂肪酶已成为治疗肥胖等代谢性疾病的靶点。
NPC1L1(Niemann-Pick-C1-Like 1，尼曼-匹克C1型类似蛋白1)是胆固醇跨膜转运的关键蛋白，在人肠表皮细胞膜和肝实质细胞膜上大量表达，是人体胆固醇代谢调节网络的关键节点之一。NPC1L1与相关胆固醇转运蛋白Flotillin-1和Flotillin-2相互作用，将胆固醇和其他甾醇转运通过肠上皮细胞刷状缘、运送到肠上皮细胞内。大量的细胞及动物实验表明，NPC1L1能在肠道胆固醇吸收及代谢过程中发挥重要作用，已经成为近年发现的对胆固醇的吸收有调控作用的重要新靶点之一。降胆固醇药物Ezetimibe(依折麦布)正是阻断了NPC1L1和Flotillin-1和Flotillin-2的相互作用，从而抑制胆固醇吸收。NPC1L1能够通过调节细胞胆固醇水平，进而调控胆固醇代谢相关的SREBP1c、FAS和ABCT1等基因，进一步维持体内胆固醇的动态平衡。
由于高脂血症、肥胖经常伴随发生，且患者群体庞大。需要研制新的有效药物。
发明内容
针对上述问题，本发明提供了一种抑制PTL/NPC1L1的化合物及其用途。
一种化合物，所述化合物为下述式(Ⅰ)所示的化合物，
Figure PCTCN2021119276-appb-000001
其中，X是-O-、-S-或-NH-；
R可以是苯环、含有取代基团的苯环、五元杂环、或六元杂环；
当R是含有取代基团的苯环时，取代基团选自下组的取代基团，包括：OH、CN、NO2、C1-C6烷氧基、卤素(F、Cl、Br、I中的一种或二种以上)、带有1个或2个以上卤素(F、Cl、Br、I中的一种或二种以上)取代基的C1-C6烷氧基中的1-5个，优选1-3个；苯环上取代基团的个数为1-5个，优选1-3个；
当R是五元杂环或者六元杂环时，其杂环上含有的除C之外的杂原子为O、S、N中的任何一个或两个，杂环上杂原子个数或1个或2个。
所述的化合物或其药学上可接受的盐中的一种或二种以上在制备用于抑制PTL和/或NPC1L1活性的药物中的应用。
所述的化合物或其药学上可接受的盐中的一种或二种以上在制备用于抑制肠道甘油三酯和/或胆固醇吸收的药物中的应用。
所述式(Ⅰ)所示的化合物通过抑制NPC1L1的活性降低胆固醇的吸收。
所述式(Ⅰ)所示的化合物通过抑制PTL的活性降低甘油三酯水解率。
所述式(Ⅰ)所示的化合物通过同时抑制PTL和NPC1L1来抑制肠道甘油三酯及胆固醇吸收。
所述的化合物或其药学上可接受的盐中的一种或二种以上在制备用于治疗和/或预防肥胖、高胆固醇血症或相关心脑血管疾病的药物中的应用。
所述式(Ⅰ)所示的化合物通过同时抑制PTL和NPC1L1来抑制肠道甘油三酯及胆固醇吸收。
所述药物包括具有(Ⅰ)式所示的化合物及其药学上可接受的盐中的一种或二种以上与医学上或药物学上可接受的药物载体混合制成的散剂、丸剂、胶囊剂、片剂、微囊剂、软胶囊剂、膜剂、栓剂、注射剂、膏剂、酊剂、散剂或冲剂。
本发明的化合物能够同时作用于胰腺甘油三酯脂酶(PTL)和胆固醇跨膜转运的关键蛋白NPC1L1，作用效果明显，能够较好的解决甘油三酯和胆固醇的吸收。化合物能够作为PTL/NPC1L1双靶向抑制剂，同时作用于肠道对甘油三酯及胆固醇吸收具有重要价值的PTL和NPC1L1，与现有减肥、调脂领域的经典药物奥利司他、依泽麦布比较显现其双效特征优势。
本发明所制备的新型化合物表现出良好的同时抑制PTL和NPC1L1的活性作用，有作为制备治疗和/或预防肥胖、高胆固醇血症及相关心脑血管疾病的应用价值。
本发明的其它特征和优点将在随后的说明书中阐述，并且，部分地从说明书中变得显而易见，或者通过实施本发明而了解。本发明的目的和其他优点可通过在说明书、权利要求书以及附图中所指出的结构来实现和获得。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
[根据细则91更正 15.12.2021]　
图1为表面等离子共振法评价化合物1-化合物5对NPC1L1的抑制作用，以Orlistat和Ezetimibe作为对照。
图2为实施例8中以Caco2细胞为模型，以Orlistat和Ezetimibe为对照，测试化合物2、化合物3对胆固醇吸收的影响图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地说明，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一种抑制PTL/NPC1L1的物质，该物质为具有如(Ⅰ)式所示结构的化合物或其在药学上可接受的盐：
Figure PCTCN2021119276-appb-000002
其中，X是-O-、-S-或-NH-；
R可以是苯环或含有取代基团的苯环、五元杂环、或六元杂环；
当R是含有取代基团的苯环时，取代基团选自下组的取代基团，包括：OH、CN、NO2、C1-C6烷氧基、卤素(F、Cl、Br、I中的一种或二种以上)、带有1个或2个 以上卤素(F、Cl、Br、I中的一种或二种以上)取代基的C1-C6烷氧基中的1-5个，优选1-3个；苯环上取代基团的个数为1-5个，优选1-3个；
当R是五元杂环或者六元杂环时，其杂环上含有的除C之外的杂原子为O、S、N中的任何一个或两个，杂环上杂原子个数或1个或2个。
具有如(Ⅰ)式所示结构的化合物制备方法包括：采用化学合成方法合成所述化合物和采用生物合成方法合成所述化合物。
具有如(Ⅰ)式所示结构的化合物的用途包括：单独作用于NPC1L1，抑制肠道胆固醇吸收，用于高胆固醇血症及相关心脑血管疾病的预防和治疗；
同时作用于PTL/NPC1L1，抑制肠道甘油三酯及胆固醇吸收，用于肥胖、高胆固醇血症及相关心脑血管疾病的预防和治疗；
具有如(Ⅰ)式所示结构的化合物及其在药学上可接受的盐、酯、醚和其他明显的化学等价物的任意单一体或混合物可用于制备治疗和/或预防肥胖及高单股固醇血症有关疾病的医学药物中。
所制备的治疗和/或预防肥胖及高单股固醇血症有关疾病的医学药物可单独作用于NPC1L1或同时作用于PTL/NPC1L1。
单独作用于NPC1L1或者同时作用于PTL/NPC1L1的化合物及其药物学上可接受的盐、酯、醚和其它明显的化学等价物，可与医学上可接受的药物载体混合制成的散剂、丸剂、胶囊剂、片剂、微囊剂、软胶囊剂、膜剂、栓剂、注射剂、膏剂、酊剂、散剂或冲剂。
实施例1
(Ⅰ)式所示结构中，若R为
Figure PCTCN2021119276-appb-000003
则化合物(1)的结构式如下：
Figure PCTCN2021119276-appb-000004
化合物(1)的合成路线如下所示：
Figure PCTCN2021119276-appb-000005
取1摩尔化合物A-1加入1.5摩尔间甲氧基苯甲酸，加入20毫升干燥的四氢呋喃(或二氯甲烷)作为溶剂，加入1.2摩尔1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐作为缩合剂，室温下反应12小时。减压浓缩后硅胶色谱柱纯化(体积比，石油醚：乙酸乙酯：二氯甲烷＝3:1:1)得到化合物1(366毫克白色固体，收率75％)。1H NMR(500MHz,Chloroform-d)δ7.62(d,J＝7.6Hz,1H),7.55(s,1H),7.36(t,J＝7.9Hz,1H),7.12(d,J＝8.2Hz,1H),5.25–5.19(m,1H),4.39(q,J＝5.9Hz,1H),3.86(s,3H),3.24 (td,J＝7.5,3.8Hz,1H),2.33(dt,J＝14.7,7.3Hz,1H),2.11(dt,J＝14.8,5.0Hz,1H),1.78(p,J＝8.6,7.9Hz,2H),1.71(ddd,J＝15.5,9.0,5.0Hz,2H),1.24(s,26H),0.86(dt,J＝11.2,6.8Hz,6H).13C NMR(125MHz,cdcl3)δ171.06,165.93,159.60,131.35,129.46,121.89,119.41,114.26,74.68,71.83,56.90,55.44,38.68,34.20,31.89,31.41,29.59,29.51,29.43,29.34,29.31,28.93,27.66,26.66,25.28,22.67,22.45,14.10,13.99.HR-ESI-MS,calcd for C30H48O5[M+H]+:m/z 489.3575；found:m/z 489.3574.
实施例2
(Ⅰ)式所示结构中，若R为
Figure PCTCN2021119276-appb-000006
则化合物(2)的结构式如下：
Figure PCTCN2021119276-appb-000007
化合物(2)的合成路线如下所示：
Figure PCTCN2021119276-appb-000008
取1摩尔化合物A-1加入1.5摩尔3-叔丁基二甲基硅烷基氧基苯甲酸，加入20毫升干燥的四氢呋喃(或二氯甲烷)作为溶剂，加入1.2摩尔1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐作为缩合剂，室温下反应12小时。减压浓缩后溶解到10毫升四氢呋喃中，加入2摩尔4-丁基氟化铵，室温反应12小时。减压浓缩后硅胶色谱柱纯化(体积比，乙酸乙酯：二氯甲烷＝1:3)得到化合物2(289毫克白色固体，收率61％)。1H NMR(500MHz,Chloroform-d)δ7.58(d,J＝7.6Hz,1H),7.54(s,1H),7.31(t,J＝7.8Hz,1H),7.07(d,J＝7.9Hz,1H),6.08(s,1H),5.21(s,1H),4.40(q,J＝5.8Hz,1H),3.25(q,J＝7.1Hz,1H),2.32(dt,J＝15.0,7.5Hz,1H),2.11(dt,J＝14.9,4.9Hz,1H),1.81–1.64(m,4H),1.24(s,26H),0.86(dt,J＝13.6,6.7Hz,6H).13C NMR(125MHz,cdcl3)δ171.58,166.10,155.99,131.34,129.73,121.78,120.44,116.32,75.00,72.09,56.84,38.62,34.20,31.89,31.39,29.59,29.52,29.44,29.36,29.32,28.91,27.62,26.66,25.25,22.67,22.45,14.11,13.99.HR-ESI-MS,calcd for C29H46O5[M+H]+:m/z 475.3418；found:m/z 475.3416.
实施例3
(Ⅰ)式所示结构中，若R为
Figure PCTCN2021119276-appb-000009
则化合物(3)的结构式如下：
Figure PCTCN2021119276-appb-000010
化合物(3)的合成过程同实施例2，区别在于：将反应原料3-叔丁基二甲基硅烷基氧基苯甲酸替换为4-叔丁基二甲基硅烷基氧基苯甲酸，收率67％。1H NMR(500MHz,Chloroform-d)δ7.94(d,J＝8.1Hz,2H),6.87(d,J＝8.2Hz,2H),5.19(s,1H),4.39(q,J＝5.8Hz,1H),3.24(q,J＝6.5,6.1Hz,1H),2.31(dt,J＝14.8,7.4Hz,1H),2.10(dt,J＝15.0,4.9Hz,1H),1.77(q,J＝7.3Hz,2H),1.67(d,J＝8.3Hz,2H),1.24(s,26H),0.86(dt,J＝12.1,6.9Hz,6H).13C NMR(125MHz,cdcl3)δ171.38,165.83,160.12,131.91,122.44,115.27,74.88,71.37,56.85,38.73,34.28,31.89,31.40,29.69,29.59,29.51,29.44,29.36,29.32,28.92,27.64,26.64,25.29,22.67,22.45,14.11,14.00.HR-ESI-MS,calcd for C29H46O5[M+H]+:m/z 475.3418；found:m/z 475.3412.
实施例4
(Ⅰ)式所示结构中，若R为
Figure PCTCN2021119276-appb-000011
则化合物(4)的结构式如下：
Figure PCTCN2021119276-appb-000012
化合物(4)的合成过程同实施例1，区别在于：将反应原料间甲氧基苯甲酸换为对甲氧基苯甲酸，收率83％。1H NMR(500MHz,Chloroform-d)δ7.98(d,J＝8.2Hz,2H),6.93(d,J＝8.4Hz,2H),5.19(dp,J＝7.9,4.5Hz,1H),4.39(q,J＝6.0Hz,1H),3.86(s,3H),3.24(td,J＝7.5,3.8Hz,1H),2.31(dt,J＝14.6,7.3Hz,1H),2.09(dt,J＝14.7,5.1Hz,1H),1.76(tt,J＝13.5,6.8Hz,2H),1.72–1.61(m,2H),1.24(d,J＝8.3Hz,26H),0.86(dt,J＝11.6,6.9Hz,6H).13C NMR(125MHz,cdcl3)δ171.18,165.77,163.50,131.61,122.40,113.67,74.73,71.26,56.85,55.45,38.70,34.26,31.89,31.41,29.60,29.51,29.50,29.44,29.36,29.32,28.94,27.65,26.64,25.30,22.67,22.46,14.12,14.01.HR-ESI-MS,calcd for C30H48O5[M+H]+:m/z 489.3575；found:m/z 489.3562.
实施例5
(Ⅰ)式所示结构中，若R为
Figure PCTCN2021119276-appb-000013
则化合物(5)的结构式如下：
Figure PCTCN2021119276-appb-000014
化合物(5)的合成过程同实施例1，区别在于：将反应原料间甲氧基苯甲酸换为3,4-二甲氧基苯甲酸，收率76％。1H NMR(500MHz,Chloroform-d)δ7.65(t,J＝9.3Hz,1H),7.52(d,J＝13.1Hz,1H),6.89(d,J＝8.4Hz,1H),5.19(tt,J＝7.9,4.6Hz,1H),4.39(q,J＝6.0Hz,1H),3.96–3.90(m,6H),3.24(td,J＝7.5,3.9Hz,1H),2.32(dt,J＝14.8,7.5Hz,1H),2.10(dt,J＝14.8,5.0Hz,1H),1.78(dt,J＝14.3,7.0Hz,2H),1.70(ddd,J＝14.0,9.7,4.8Hz,2H),1.24(d,J＝9.0Hz,26H),0.86(dd,J＝12.0,4.8Hz,6H).13C NMR(125MHz,cdcl3)δ171.13,165.87,153.14,148.69,123.52,122.51,111.98,110.22,74.80,71.55,56.87,56.04,56.01,38.75,34.30,31.89,31.48,31.42,29.69,29.60,29.55,29.52,29.50,29.44,29.36,29.32,28.94,27.65,26.65,25.29,22.67,22.46,14.11,14.01.HR-ESI-MS,calcd for C30H50O6[M+H]+:m/z 519.3680；found:m/z 519.3668.
实施例6
检测化合物1、化合物2、化合物3、化合物4、化合物5对PTL的抑制活性。
实验过程：
将PTL溶解到磷酸盐缓冲溶液中(PH＝7.4)。不同浓度(于混合溶液中的终浓度分别为100μM,10μM,1μM,0.1μM,0.01μM)的待测试化合物或者不同浓度阳性对照奥利司他(Orlistat)或依折麦布(Ezetimibe)(于混合溶液中的终浓度分别为100μM,10μM,1μM,0.1μM,0.01μM)分别与PTL(PTL的终浓度为1μg/mL)混合，37度条件下保持30分钟，得混合溶液。空白对照组为不添加待测物及阳性对照的等体积、等浓度的PTL溶液。于混合溶液中的终浓度为0.5mM的对硝基苯基乙酸酯添加到混合溶液中，在37度条件下保持60分钟。测定405纳米波长的吸光度A。采用化合物浓度的对数值对抑制率作回归方程(即化合物浓度的对数值为横坐标，抑制率为纵坐标)，计算IC 50。化合物对PTL抑制活性结果见表1。
其中抑制率的计算方式为：抑制率＝(实验组A值-空白对照组A值)/(对照组A值-空白对照组A值)×100％
实验结果：如表1所示，化合物1-化合物5均表现出较好的PTL抑制活性，在100μM浓度下的抑制率均大于50％。其中化合物1、化合物3、化合物4对PTL的抑制活性尤为突出，IC 50值分别为13.5μM，5.3μM，15.8μM。
表1示出了根据本发明实施例的化合物1-5对PTL的抑制作用。其中化合物1-化合物5的结构为
Figure PCTCN2021119276-appb-000015
表1.化合物1-化合物5对PTL的抑制活性评价(以Orlistat和Ezetimibe作为对 照)。
Figure PCTCN2021119276-appb-000016
实施例7
检测化合物1、化合物2、化合物3、化合物4、化合物5与NPC1L1的结合活性。
实验过程：
利用表面等离子共振技术(SPR)测定化合物与NPC1L1的结合活性。CM5系列传感器芯片、偶联试剂1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)均购自GE。
实验组：将NPC1L1(终浓度50μg/ml)溶解于10mM醋酸钠缓冲液(pH＝4.56)中。使用100mM的EDC和100mM的NHS的体积比1:1的混合物，以10μL/分钟的流量活化7分钟，随后注入NPC1L1溶液以10μL/分钟的流量偶联7分钟。将传感器芯片表面多余的反应酯用1M乙醇胺(pH 8.5)以10μL/分钟的流量阻断7min。
对照组：按上述方法活化和阻断，但不加入NPC1L1溶液(不偶联)。
分别于实验组和对照组的芯片上，分别以10μL/分钟的流量注入待测化合物1-化合物5(终浓度分别为100μM)、阳性对照Orlistat(终浓度为100μM)和Ezetimibe(终浓度为100μM)4分钟。结合响应值以响应单位(RU)来报道，其定义为包含固定化受体的流室(实验组芯片)的响应减去对照流室(对照组芯片)的响应。
实验结果(图1)：化合物1、2、3、4均展现出一定的NPC1L1结合活性，其中化合物2和化合物3对NPC1L1的结合最好。
实施例8
以Caco2细胞为模型，以Orlistat和Ezetimibe为对照，测试化合物2、化合物3对胆固醇吸收的影响。
实验过程：Caco2细胞使用DMEM培养基培养在体积浓度为5％二氧化碳的空气37摄氏度培养箱中。
分别使用终浓度为50μM的抑制剂(化合物2、化合物3、Orlistat、Ezetimibe)在37度共孵育1小时。添加终浓度为10μM的胆固醇-d6，在37度继续孵育1小时。分别移除培养基，PBS缓冲液洗三遍(每次3mL)，加入甲醇1mL，超声破碎，通过高效液相色谱-质谱联用(Agilent Technologies,Santa Clara,CA,USA)进行定量分析。
对照组(control)不添加抑制剂，仅添加终浓度为10μM的胆固醇-d6，在37度 继续孵育1小时。移除培养基，PBS缓冲液洗三遍(每次3mL)，加入甲醇1mL，超声破碎，通过高效液相色谱-质谱联用(Agilent Technologies,Santa Clara,CA,USA)进行定量分析。对照组胆固醇-d6的细胞摄取量设为100％。
实验结果(图2)：化合物2和化合物3可以显著抑制胆固醇的吸收，其抑制效果优于Orlistat，与Ezetimibe相当。
由实例6，实例7和实例8的结果可知：在对PTL的抑制活性测试中，所有测试化合物在100μM浓度下均展现出较好的PTL抑制活性，其中化合物1、3、4对PTL的抑制活性IC 50分别为13.5μM，5.3μM，15.8μM；在对NPC1L1的抑制活性测试中，化合物1、2、3、4均展现出一定的NPC1L1结合活性，其中化合物2和化合物3对NPC1L1的结合最好；在对胆固醇吸收抑制测试中，化合物2和化合物3能显著抑制胆固醇的吸收。
通过上述实施例可知，具有通式(Ⅰ)所示结构的化合物其在药学上可接受的盐、酯、醚和其他明显的化学等价物能够作用于胰腺甘油三酯脂酶(PTL)和/或胆固醇跨膜转运的关键蛋白NPC1L1，作用效果明显。
尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (9)

  1. 一种化合物，所述化合物为下述式(Ⅰ)所示的化合物，
    Figure PCTCN2021119276-appb-100001
    其中，X是-O-、-S-或-NH-；
    R可以是苯环、含有取代基团的苯环、五元杂环、或六元杂环；
    当R是含有取代基团的苯环时，取代基团选自下组的取代基团，包括：OH、CN、NO 2、C 1-C 6烷氧基、卤素(F、Cl、Br、I中的一种或二种以上)、带有1个或2个以上卤素(F、Cl、Br、I中的一种或二种以上)取代基的C 1-C 6烷氧基中的1-5个，优选1-3个；苯环上取代基团的个数为1-5个，优选1-3个；
    当R是五元杂环或者六元杂环时，其杂环上含有的除C之外的杂原子为O、S、N中的任何一个或两个，杂环上杂原子个数或1个或2个。
  2. 一种权利要求1所述的化合物或其药学上可接受的盐中的一种或二种以上在制备用于抑制PTL和/或NPC1L1活性的药物中的应用。
  3. 一种权利要求1所述的化合物或其药学上可接受的盐中的一种或二种以上在制备用于抑制肠道甘油三酯和/或胆固醇吸收的药物中的应用。
  4. 如权利要求3所述的应用，其特征在于，所述式(Ⅰ)所示的化合物通过抑制NPC1L1的活性降低胆固醇的吸收。
  5. 如权利要求3所述的应用，其特征在于，所述式(Ⅰ)所示的化合物通过抑制PTL的活性降低甘油三酯水解率。
  6. 如权利要求4或5所述的应用，其特征在于，所述式(Ⅰ)所示的化合物通过同时抑制PTL和NPC1L1来抑制肠道甘油三酯及胆固醇吸收。
  7. 一种权利要求1所述的化合物或其药学上可接受的盐中的一种或二种以上在制备用于治疗和/或预防肥胖、高胆固醇血症或相关心脑血管疾病的药物中的应用。
  8. 如权利要求7所述的应用，其特征在于，所述式(Ⅰ)所示的化合物通过同时抑制PTL和NPC1L1来抑制肠道甘油三酯及胆固醇吸收。
  9. 如权利要求2-8任一项所述应用，其特征在于，所述药物包括具有(Ⅰ)式所示的化合物及其药学上可接受的盐中的一种或二种以上与医学上或药物学上可接受的药物载体混合制成的散剂、丸剂、胶囊剂、片剂、微囊剂、软胶囊剂、膜剂、栓剂、注射剂、膏剂、酊剂、散剂或冲剂。
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