TW201823222A - Compound, pharmaceutical composition and use thereof - Google Patents

Compound, pharmaceutical composition and use thereof Download PDF

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TW201823222A
TW201823222A TW106144843A TW106144843A TW201823222A TW 201823222 A TW201823222 A TW 201823222A TW 106144843 A TW106144843 A TW 106144843A TW 106144843 A TW106144843 A TW 106144843A TW 201823222 A TW201823222 A TW 201823222A
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世安 陳
賴李應宣
楊凭勳
黃昭陵
廖柏翔
劉家瑋
劉妍希
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財團法人生物技術開發中心
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/52—Radicals substituted by nitrogen atoms not forming part of a nitro radical
    • 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
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/82—Benzo [b] furans; Hydrogenated benzo [b] furans with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/84—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C07D307/85—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
    • C07D333/14—Radicals substituted by singly bound hetero atoms other than halogen
    • C07D333/20—Radicals substituted by singly bound hetero atoms other than halogen by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The prevent invention provides a compound of Formula (I):, a pharmaceutical composition containing the compound, and the use thereof. The pharmaceutical composition containing the compound and a pharmaceutically acceptable carrier thereof, and further use in making a treatment for treating disease associated with proprotein convertase subtilisin/kexin type 9.

Description

化合物、醫藥組成物及其用途    Compound, medicinal composition and use thereof    【相關申請案】[Related applications]

本發明係主張美國專利申請案第62/438,707號(申請日：2016年12月23日)之國際優先權，該申請案之完整內容納入為本發明專利說明書的一部分以供參照。 The present invention claims the international priority of US Patent Application No. 62 / 438,707 (application date: December 23, 2016), the entire content of which is incorporated as a part of the patent specification of the present invention for reference.

本發明涉及一種化合物及醫藥組成物及其用途，特別是涉及一種用於調控PCSK9的化合物及醫藥組成物。 The invention relates to a compound and a pharmaceutical composition and uses thereof, in particular to a compound and a pharmaceutical composition for regulating PCSK9.

現有技術中已知低密度脂蛋白膽固醇(LDL-C)血漿水平的升高是造成膽固醇和脂蛋白代謝紊亂(例如冠心病)的危險因素之一，而藉由LDL受體(LDL-R)的作用，從血漿中清除LDL-C主要是在肝臟進行的。 Elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) are known in the prior art to be one of the risk factors for cholesterol and lipoprotein metabolism disorders (such as coronary heart disease). The role of LDL-C in plasma is mainly carried out in the liver.

前蛋白轉化酶枯草桿菌蛋白酶/kexin 9型(PCSK9)是一種絲氨酸蛋白酶，其與LDL-R結合以調節其在細胞表面的表達。細胞分泌的PCSK9與肝細胞上的LDL-R結合形成複合物，再於溶酶體中內化並降解。此等步驟卻抑止LDL-R再循環至細胞表面，因此抑制LDL-R在其上的表達，從而提高了LDL-C血漿水平。控制LDL-C血漿水平的方法可藉由調節降低細胞的PCSK9分泌，以降低血漿PCSK9水平，提高肝細胞上的LDL-R，或是改變PCSK9與LDL-R之間相互作用的動力學來達成。 The proprotein converting enzyme subtilisin / kexin type 9 (PCSK9) is a serine protease that binds to LDL-R to regulate its expression on the cell surface. PCSK9 secreted by cells combines with LDL-R on liver cells to form a complex, which is then internalized and degraded in the lysosome. These steps, however, prevent LDL-R from being recycled to the cell surface, thereby inhibiting LDL-R expression thereon, thereby increasing LDL-C plasma levels. The method of controlling the plasma level of LDL-C can be achieved by regulating the reduction of PCSK9 secretion of cells, reducing the plasma PCSK9 level, increasing LDL-R on liver cells, or changing the kinetics of the interaction between PCSK9 and LDL-R .

2015年7月24日，FDA批准Alirocumab(Praluent®)，2015年8月27日批准Evolocumab(RepathaTM)。而第三種藥物 Bococizumab目前正在進行第3期臨床試驗。該等藥物都是藉由每月或雙月注射針對PCSK9的單株抗體，改變PCSK9與LDL-R之間相互作用，以促進肝細胞上的LDL-R並增加肝臟LDL吸收以降低LDL-C血漿水平。目前並無降低細胞分泌PCSK9之藥物，因此，設計用於靶向PCSK9蛋白的小分子口服藥物將為新穎並具有優於抗體藥物優勢。 On July 24, 2015, the FDA approved Alirocumab (Praluent®) and on August 27, 2015 approved Evolocumab (Repatha TM ). The third drug, Bococizumab, is currently undergoing a phase 3 clinical trial. These drugs use monthly or bi-monthly injections of monoclonal antibodies against PCSK9 to alter the interaction between PCSK9 and LDL-R to promote LDL-R on liver cells and increase liver LDL absorption to reduce LDL-C. Plasma levels. There are currently no drugs that reduce PCSK9 secretion by cells. Therefore, small-molecule oral drugs designed to target PCSK9 protein will be novel and have advantages over antibody drugs.

因此，提供能有效降低細胞分泌PCSK9的新化合物已成為本領域所欲解決的重要課題。 Therefore, providing new compounds that can effectively reduce the secretion of PCSK9 by cells has become an important subject to be solved in the art.

本發明所要解決的技術問題在於，針對現有技術的不足提供一種化合物、醫藥組成物及其用途。 The technical problem to be solved by the present invention is to provide a compound, a medicinal composition and its use in response to the shortcomings of the prior art.

為了解決上述的技術問題，本發明所採用的其中一技術方案是，提供一種化合物，其具有式(I)結構： 其中，R1為雜芳基；R2、R3、R5和R6各自獨立地為H、鹵素、OH、CN、NH2、C1-6烷基、C1-6烷氧基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-6烷基氨基或C3-10環烷基氨基；R4為C1-6烷基、C1-6烷氧基、C3-10環烷基、C1-6烷基氨基、C3-10環烷基氨基或芳基；R7為C1-6烷基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-10雜環烷基、芳基或雜芳基；每個L1和L2獨立地為直接鍵、O或NH；m是1、2或3；以及n是0、1、2或3；其中，C1-6烷基、C1-6烷氧基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-10雜環烷基、C1-6烷基氨基、C3-10環烷基氨基、芳基和雜芳基中的每一個是OH、CN、NH2、C1-6烷基、C1-6烷氧基、C2-10烯基、C2-10炔基、C3-10 環烷基、C1-10雜環烷基、芳基或雜芳基。 In order to solve the above technical problem, one of the technical solutions adopted by the present invention is to provide a compound having a structure of formula (I): Wherein R 1 is a heteroaryl group; R 2 , R 3 , R 5 and R 6 are each independently H, halogen, OH, CN, NH 2 , C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-6 alkylamino, or C 3-10 cycloalkylamino; R 4 is C 1-6 alkyl, C 1-6 alkoxy, C 3-10 cycloalkyl, C 1-6 alkylamino, C 3-10 cycloalkylamino or aryl; R 7 is C 1-6 alkyl, C 2-10 ene Group, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, aryl or heteroaryl; each L 1 and L 2 is independently a direct bond, O or NH; m is 1, 2 or 3; and n is 0, 1, 2 or 3; wherein C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, Each of C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, C 1-6 alkylamino, C 3-10 cycloalkylamino, aryl, and heteroaryl is OH, CN, NH 2 , C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, aryl Or heteroaryl.

本文中的術語「烷基」意指飽和的直鏈或支鏈烴部分，如-CH3或支鏈的-C3H7；術語“烷氧基”是指-O-烷基，烷氧基的實例包括但不限於甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、異丁氧基、仲丁氧基以及第四氧基；術語“鏈烯基”是指含有一個或多個雙鍵的直鏈或支鏈烴部分，如乙烯基或丙烯基；術語“炔基”是指含有一個或多個三鍵的直鏈或支鏈烴，如丙炔基或丁炔基；術語“環烷基”是指非芳族、單環、雙環、三環或四環烴，如環己基、環己烯-3-基或金剛烷基；術語“雜環烷基”意指具有一個或多個雜原子(例如O、N、P及S)的非芳族5至8員單環、8至12員雙環或11至14員三環環，雜環烷基的實例包括但不限於哌嗪基、咪唑烷基、氮雜環庚烷基、吡咯烷基、二氫噻二唑基、二噁烷基、嗎啉基、四氫呋喃基以及四氫呋喃基；術語“烷基氨基”是指被氨基取代的烷基，例如甲氨基或乙氨基；術語“環烷基氨基”是指被氨基取代的環烷基，例如環丙基氨基或環戊基氨基；術語“芳基”是指具有一個或多個芳環的烴基，芳基的實例包括但不限於苯基、亞苯基、萘基、亞萘基、芘基、蒽基及菲基。術語“雜芳基”是指具有一個或多個含有至少一個雜原子(例如N、O或S)的芳環基，雜芳基的實例包括但不限於呋喃基、呋喃基、芴基、吡咯基、噻吩基、噁唑基、咪唑基、噻唑基、吡啶基、嘧啶基、喹唑啉基、喹啉基、異喹啉基以及吲哚基。 The term "alkyl" herein means a saturated straight or branched chain hydrocarbon moiety, such as -CH 3 or branched -C 3 H 7; term "alkoxy" refers to -O- alkyl, alkoxy Examples of radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and fourth oxy; the term "alkenyl "Means a straight or branched chain hydrocarbon moiety containing one or more double bonds, such as vinyl or propenyl; the term" alkynyl "means a straight or branched chain hydrocarbon containing one or more triple bonds, such as propylene Alkynyl or butynyl; the term "cycloalkyl" refers to a non-aromatic, monocyclic, bicyclic, tricyclic or tetracyclic hydrocarbon, such as cyclohexyl, cyclohexen-3-yl or adamantyl; the term "hetero "Cycloalkyl" means a non-aromatic 5- to 8-membered monocyclic, 8 to 12-membered bicyclic, or 11 to 14-membered tricyclic, heterocyclic ring having one or more heteroatoms (such as O, N, P, and S). Examples of alkyl include, but are not limited to, piperazinyl, imidazolidinyl, azacycloheptyl, pyrrolidinyl, dihydrothiadiazyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and tetrahydrofuranyl; terms "Alkylamino" Refers to an alkyl group substituted with an amino group, such as methylamino or ethylamino; the term "cycloalkylamino" refers to a cycloalkyl group substituted with an amino group, such as cyclopropylamino or cyclopentylamino; the term "aryl" refers to A hydrocarbon group having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, phenylene, naphthyl, naphthylene, fluorenyl, anthracenyl, and phenanthryl. The term "heteroaryl" refers to one or more aromatic ring groups containing at least one heteroatom (such as N, O, or S). Examples of heteroaryl groups include, but are not limited to, furyl, furyl, fluorenyl, pyrrolyl , Thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolinyl, isoquinolinyl, and indolyl.

除非另有說明，本文提到的烷基，烯基、炔基，環烷基、雜環烷基、烷基氨基、環烷基氨基、芳基以及雜芳基包括經取代及未經取代的基團，環烷基、雜環烷基、芳基以及雜芳基上可能的取代基包括C1-10烷基、C2-10烯基、C2-10炔基、C3-20環烷基、C3-20環烯基、C1-20雜環烷基、C1-20雜環烯基、C1-10烷基氨基、芳基氨基、二芳基氨基、羥基、氨基硫代醯基、脒基、胍、脲基、氰基、硝基、醯基、硫代醯基、醯氧基、羧基和羧酸酯。另一方面、烷 基上可能的取代基包括所有上述取代基(除C1-10烷基、C2-10烯基以及C2-10炔基之外)；環烷基、雜環烷基、芳基以及雜芳基也可以彼此稠合。 Unless otherwise stated, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylamino, cycloalkylamino, aryl, and heteroaryl groups mentioned herein include substituted and unsubstituted Groups, possible substituents on cycloalkyl, heterocycloalkyl, aryl and heteroaryl include C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-20 ring Alkyl, C 3-20 cycloalkenyl, C 1-20 heterocycloalkyl, C 1-20 heterocycloalkenyl, C 1-10 alkylamino, arylamino, diarylamino, hydroxyl, aminosulfide Substituted fluorenyl, fluorenyl, guanidine, ureido, cyano, nitro, fluorenyl, thiofluorenyl, fluorenyloxy, carboxyl, and carboxylic acid esters. On the other hand, possible substituents on alkyl include all the above substituents (except C 1-10 alkyl, C 2-10 alkenyl, and C 2-10 alkynyl); cycloalkyl, heterocycloalkyl , Aryl, and heteroaryl may be fused to each other.

上述化合物包括化合物本身及適用的其鹽類、前驅藥物及溶劑合物。舉例而言，可以在雜環化合物上的陰離子和帶正電荷的基團(例如氨基)之間形成鹽，合適的陰離子包括氯化物、溴化物、碘化物，硫酸鹽、硝酸鹽、磷酸鹽、檸檬酸鹽、甲磺酸鹽、三氟乙酸鹽、乙酸鹽、蘋果酸鹽、甲苯磺酸鹽、酒石酸鹽。同樣地，亦可在雜環化合物上的陽離子和帶負電的基團(例如羧酸酯)之間形成鹽，合適的陽離子包括鈉離子、鉀離子、鎂離子、鈣離子和銨陽離子(如四甲基銨離子)，這些化合物更包括含有季氮原子的鹽。前驅藥物的實例包括酯和其藥學上可接受的衍生物，其在給予受試者時能夠提供活性雜環化合物。溶劑合物是指在活性雜環化合物和藥學上可接受的溶劑之間形成的絡合物，藥學上可接受的溶劑的實例包括水、乙醇、異丙醇，乙酸乙酯、乙酸和乙醇胺。 The aforementioned compounds include the compounds themselves and their salts, prodrugs and solvates where applicable. For example, a salt can be formed between an anion on a heterocyclic compound and a positively charged group (such as an amino group). Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, Citrate, mesylate, trifluoroacetate, acetate, malate, tosylate, tartrate. Similarly, a salt can be formed between a cation on a heterocyclic compound and a negatively charged group (such as a carboxylic acid ester). Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations (such as Methyl ammonium ion), these compounds further include salts containing quaternary nitrogen atoms. Examples of prodrugs include esters and pharmaceutically acceptable derivatives thereof, which are capable of providing an active heterocyclic compound when administered to a subject. A solvate refers to a complex formed between an active heterocyclic compound and a pharmaceutically acceptable solvent. Examples of the pharmaceutically acceptable solvent include water, ethanol, isopropanol, ethyl acetate, acetic acid, and ethanolamine.

本發明的化合物可含有非芳族雙鍵，其可以順式或反式異構形式存在，此異構式是可預期的。 The compounds of the present invention may contain non-aromatic double bonds, which may exist in cis or trans isomeric forms, such isomeric forms are contemplated.

為了解決上述的技術問題，本發明所採用的另外一技術方案是，提供一種醫藥組成物，其包含治療有效量之如本發明上述之式(I)化合物及醫藥學上可接受的載劑。 In order to solve the above technical problems, another technical solution adopted by the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of the compound of the formula (I) as described above and a pharmaceutically acceptable carrier.

為了解決上述的技術問題，本發明所採用的另外再一技術方案是，提供一種醫藥組成物的用途，其是用於製造用以治療個體的前蛋白轉化酶枯草桿菌蛋白酶kexin 9型(PCSK9)相關疾病的藥物。 In order to solve the above-mentioned technical problem, another technical solution adopted by the present invention is to provide a use of a medicinal composition for manufacturing a preprotein-converting enzyme subtilisin kexin type 9 (PCSK9) for treating an individual. Drugs for related diseases.

本發明的一具體實施例中，所述前蛋白轉化酶枯草桿菌蛋白酶kexin 9型(PCSK9)相關疾病是膽固醇和脂蛋白代謝紊亂。 In a specific embodiment of the present invention, the pre-protein converting enzyme subtilisin kexin type 9 (PCSK9) -related disease is a disorder of cholesterol and lipoprotein metabolism.

本發明的一具體實施例中，所述膽固醇和脂蛋白代謝紊亂包 括但不限於冠心病、心肌梗塞、動脈粥樣硬化以及高膽固醇血症。 In a specific embodiment of the present invention, the disorders of cholesterol and lipoprotein metabolism include, but are not limited to, coronary heart disease, myocardial infarction, atherosclerosis, and hypercholesterolemia.

本發明中術語“治療”或“治療”是指將一種或多種上述化合物給予具有上述疾病、該疾病的症狀或對該疾病的傾向的受試者，其目的為賦予治療效果，例如治癒、緩解、改變、影響、改善或預防上述疾病、其症狀或其傾向。而術語“有效劑量”是指達到預期治療效果所需的活性劑劑量，如所屬技術領域具有通常知識者所習知的，有效劑量將根據所治療的疾病的類型、給藥途徑、賦形劑的使用以及可能同時使用其它藥物而有所差異。 The term "treating" or "treating" in the present invention refers to administering one or more of the above-mentioned compounds to a subject having the above-mentioned disease, the symptoms of the disease, or the tendency to the disease, with the purpose of conferring a therapeutic effect, such as healing, remission , Alter, affect, ameliorate or prevent the above diseases, their symptoms or their tendency. The term "effective dose" refers to the dose of the active agent required to achieve the desired therapeutic effect. As known to those skilled in the art, the effective dose will depend on the type of disease to be treated, the route of administration, and excipients. The use of and other drugs may vary.

有效劑量的測定，對於所屬技術領域具有通常知識者而言，無需過度實驗僅需要常規技能，即可確定預定用途的有效劑量。需要治療的個體可為哺乳動物。術語“哺乳動物”是指人類或非人類之哺乳動物，例如：狗、貓、豬、牛、綿羊、山羊、馬、大鼠、或小鼠。 The determination of the effective dose, for those with ordinary knowledge in the technical field, can determine the effective dose for the intended use without undue experimentation and only requiring conventional skills. The individual in need of treatment may be a mammal. The term "mammal" refers to a human or non-human mammal, such as: dog, cat, pig, cow, sheep, goat, horse, rat, or mouse.

為了實施本發明的方法，具有一種或多種上述化合物的組成物可以腸胃外、口服、經鼻、經直腸、局部或口腔給藥。本文使用的術語“腸胃外”是指皮下、皮內、靜脈內、腹膜內、肌內、關節內、動脈內、滑膜內、胸骨內、蛛網膜下腔、損傷內或顱注注射以及任何合適的注入技術。 For carrying out the method of the present invention, a composition having one or more of the aforementioned compounds may be administered parenterally, orally, nasally, rectally, topically, or orally. The term "parenteral" as used herein refers to subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, subarachnoid, intralesional or intracranial injection and any Appropriate injection technology.

無菌可注射組成物可以是在無毒腸胃外可接受的稀釋劑或溶劑中的溶液或懸浮液，例如1,3-丁二醇中的溶液。在可接受的載體和溶劑中可以使用的是甘露醇、水、林格氏溶液(Ringer’s solution)以及等滲氯化鈉溶液。此外，通常使用無揮發性油常用作為溶劑或懸浮介質(例如合成的單或雙甘油酯)。脂肪酸可用於製備注射劑，如油酸及其甘油酯衍生物，天然物藥理上可接受的油如橄欖油和蓖麻油，尤其是其聚氧乙烯化形式。這些油溶液或懸浮液還可以含有長鏈醇稀釋劑或分散劑、羧甲基纖維素或類似的分散劑。其他常用的界面活性劑通常用於製備藥學上可接受的固體、液體或其他劑型也可用於配製目的，如Tweens、Spans或其 他類似的乳化劑或生物利用度增強劑。 The sterile injectable composition may be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, non-volatile oils are commonly used as solvents or suspension media (such as synthetic mono- or diglycerides). Fatty acids are useful in the preparation of injectables such as oleic acid and its glyceride derivatives, natural pharmacologically acceptable oils such as olive oil and castor oil, and especially their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, carboxymethyl cellulose or similar dispersants. Other commonly used surfactants are commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms and can also be used for formulation purposes, such as Tweens, Spans or other similar emulsifiers or bioavailability enhancers.

用於口服給藥的組成物可以是任何口服可接受的劑型，包括但不限於膠囊、錠劑、乳液和水性懸浮液、分散體以及溶液。在錠劑的情況下，常用的載體包括乳糖和玉米澱粉，通常還可以加入潤滑劑如硬脂酸鎂；對於膠囊形式的口服給藥，適用的稀釋劑包括乳糖和乾玉米澱粉；當口服施予水性懸浮液或乳劑時，活性成分可以懸浮或溶解在與乳化劑或懸浮劑結合的油相中。視需求可加入某些甜味劑、調味劑或著色劑。鼻用氣溶膠或吸入組成物可根據藥物製劑領域熟知的技術製備。例如，可以使用任何合適的防腐劑或吸收促進劑(例如苯甲醇)或任何增溶劑或分散劑(例如氟碳化合物)將該等組成物製備成鹽水溶液。 Compositions for oral administration can be in any orally acceptable dosage form, including but not limited to capsules, lozenges, emulsions and aqueous suspensions, dispersions, and solutions. In the case of lozenges, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate can usually be added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. In the case of pre-aqueous suspensions or emulsions, the active ingredient may be suspended or dissolved in an oily phase combined with an emulsifier or suspending agent. If desired, certain sweeteners, flavoring agents or coloring agents can be added. Nasal aerosol or inhalation compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. For example, the compositions can be prepared as a saline solution using any suitable preservative or absorption enhancer (such as benzyl alcohol) or any solubilizing or dispersing agent (such as a fluorocarbon).

具有一種或多種上述化合物的藥物組成物亦適用於直腸給藥的栓劑形式給藥。 Pharmaceutical compositions having one or more of the above compounds are also suitable for rectal administration in the form of suppositories.

醫藥組成物中的載體必須是“可接受的”，即能夠與組成物中的活性成分相容(若能夠使其穩定化更加)且對待治療之個體無害。一種或多種增溶劑可以用作藥物賦形劑用於遞送活性化合物。其它載體的例子包括膠體二氧化矽、硬脂酸鎂、纖維素、十二烷基硫酸鈉。 The carrier in the pharmaceutical composition must be "acceptable", that is, compatible with the active ingredients in the composition (if it can be stabilized more) and harmless to the individual to be treated. One or more solubilizers can be used as pharmaceutical excipients for delivering the active compound. Examples of other carriers include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate.

本發明的其中一有益效果在於，本發明所提供的化合物、醫藥組成物及其用途，其能通過“式(I)的化合物”以及“施用有效量的式(I)的化合物”的技術方案，下調PCSK9表達的水平，以治療個體的前蛋白轉化酶枯草桿菌蛋白酶kexin 9型相關疾病。 One of the beneficial effects of the present invention is that the compound, the pharmaceutical composition and the application provided by the present invention can pass the technical scheme of "the compound of formula (I)" and "administer an effective amount of the compound of formula (I)" , Down-regulate the expression level of PCSK9 to treat individuals' preprotein-converting enzyme subtilisin kexin type 9-related diseases.

為使能更進一步瞭解本發明的特徵及技術內容，請參閱以下有關本發明的詳細說明與圖式，然而所提供的圖式僅用於提供參考與說明，並非用來對本發明加以限制。 In order to further understand the features and technical contents of the present invention, please refer to the following detailed description and drawings of the present invention. However, the drawings provided are only for reference and description, and are not intended to limit the present invention.

圖1為經由本發明實施例所提供的化合物處理的HepG2細胞的西方墨點法分析結果。 FIG. 1 is a result of Western blot analysis of HepG2 cells treated with a compound provided by an embodiment of the present invention.

以下是通過特定的具體實施例來說明本發明所公開有關“化合物、醫藥組成物及其用途”的實施方式，本領域技術人員可由本說明書所公開的內容瞭解本發明的優點與效果。本發明可通過其他不同的具體實施例加以施行或應用，本說明書中的各項細節也可基於不同觀點與應用，在不悖離本發明的構思下進行各種修改與變更。以下的實施方式將進一步詳細說明本發明的相關技術內容，但所公開的內容並非用以限制本發明的保護範圍。 The following is a description of specific embodiments of the "compounds, pharmaceutical compositions, and uses" disclosed by the present invention. Those skilled in the art can understand the advantages and effects of the present invention from the contents disclosed in this specification. The present invention can be implemented or applied through other different specific embodiments, and various details in this specification can also be based on different viewpoints and applications, and various modifications and changes can be made without departing from the concept of the present invention. The following embodiments will further describe the related technical content of the present invention in detail, but the disclosed content is not intended to limit the protection scope of the present invention.

為了解決上述的技術問題，本發明所採用的其中一技術方案是，提供一種化合物，其具有式(I)結構： 其中，R1為雜芳基；R2、R3、R5和R6各自獨立地為H、鹵素、OH、CN、NH2、C1-6烷基、C1-6烷氧基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-6烷基氨基或C3-10環烷基氨基；R4為C1-6烷基、C1-6烷氧基、C3-10環烷基、C1-6烷基氨基、C3-10環烷基氨基或芳基；R7為C1-6烷基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-10雜環烷基、芳基或雜芳基；每個L1和L2獨立地為直接鍵、O或NH；m是1、2或3；以及n是0、1、2或3；其中，C1-6烷基、C1-6烷氧基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-10雜環烷基、C1-6烷基氨基、C3-10環烷基氨基、芳基和雜芳基中的每一個是OH、CN、NH2、C1-6烷基、C1-6烷氧基、C2-10烯基、C2-10炔基、C3-10環烷基、C1-10雜環烷基、芳基或雜芳基。 In order to solve the above technical problem, one of the technical solutions adopted by the present invention is to provide a compound having a structure of formula (I): Wherein R 1 is a heteroaryl group; R 2 , R 3 , R 5 and R 6 are each independently H, halogen, OH, CN, NH 2 , C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-6 alkylamino, or C 3-10 cycloalkylamino; R 4 is C 1-6 alkyl, C 1-6 alkoxy, C 3-10 cycloalkyl, C 1-6 alkylamino, C 3-10 cycloalkylamino or aryl; R 7 is C 1-6 alkyl, C 2-10 ene Group, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, aryl or heteroaryl; each L 1 and L 2 is independently a direct bond, O or NH; m is 1, 2 or 3; and n is 0, 1, 2 or 3; wherein C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, Each of C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, C 1-6 alkylamino, C 3-10 cycloalkylamino, aryl, and heteroaryl is OH, CN, NH 2 , C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, aryl Or heteroaryl.

具體而言，式(I)化合物中R1是五員雜芳基(例如呋喃基或噻吩基)；R2、R3、R5及R6各自獨立地為H或C1-6烷氧基；R4是C1-6烷基或芳基；且R7為C1-6烷基、C1-10雜環烷基或雜芳基； 其中，L1是O，且L2為直接鍵或NH；m是1或2，且n是0、1或2。 Specifically, in the compound of formula (I), R 1 is a five-membered heteroaryl group (such as furyl or thienyl); R 2 , R 3 , R 5, and R 6 are each independently H or C 1-6 alkoxy R 4 is C 1-6 alkyl or aryl; and R 7 is C 1-6 alkyl, C 1-10 heterocycloalkyl or heteroaryl; wherein L 1 is O and L 2 is Direct bond or NH; m is 1 or 2 and n is 0, 1 or 2.

本發明的一具體實施例提供式(I)化合物包含：R1是呋喃基或噻吩基；R2、R3、R5及R6各自獨立為H或C1-6烷氧基；R4是C1-6烷基或芳基；且R7為C1-6烷基，C1-10雜環烷基或雜芳基；L1是O；L2是直接鍵；m是1或2，且n是0、1或2。 A specific embodiment of the present invention provides that the compound of formula (I) comprises: R 1 is furyl or thienyl; R 2 , R 3 , R 5 and R 6 are each independently H or C 1-6 alkoxy; R 4 Is C 1-6 alkyl or aryl; and R 7 is C 1-6 alkyl, C 1-10 heterocycloalkyl or heteroaryl; L1 is O; L2 is a direct bond; m is 1 or 2, And n is 0, 1, or 2.

本發明的另一具體實施例提供式(I)化合物包含：R1是呋喃基或噻吩基；R2、R3、R5及R6各自獨立為H或C1-6烷氧基；R4是C1-6烷基或芳基；且R7為C1-6烷基，C1-10雜環烷基或雜芳基；L1是O；L2是NH；m是1或2，且n是0、1或2。 Another specific embodiment of the present invention provides a compound of formula (I) comprising: R 1 is furyl or thienyl; R 2 , R 3 , R 5 and R 6 are each independently H or C 1-6 alkoxy; R 4 is C 1-6 alkyl or aryl; and R 7 is C 1-6 alkyl, C 1-10 heterocycloalkyl or heteroaryl; L 1 is O; L 2 is NH; m is 1 or 2 and n is 0, 1, or 2.

本發明式(I)化合物具有調節PCSK9的生理作用，包括其與低密度脂蛋白受體(LDL-R)的相互作用。這些小分子的PCSK9調節劑可用於降低血液中的低密度脂蛋白膽固醇(LDL-C)水平，並可用於預防及/或治療膽固醇與脂蛋白代謝紊亂，如冠心病、心肌梗塞、動脈粥樣硬化以及高膽固醇血症。 The compounds of formula (I) of the present invention have physiological effects in regulating PCSK9, including their interaction with low-density lipoprotein receptor (LDL-R). These small-molecule PCSK9 modulators can be used to lower low-density lipoprotein cholesterol (LDL-C) levels in the blood, and can be used to prevent and / or treat disorders of cholesterol and lipoprotein metabolism, such as coronary heart disease, myocardial infarction, atherosclerosis Sclerosis and hypercholesterolemia.

本發明進一步包括含有一種或多種上述式(I)化合物的醫藥組成物，以及醫藥組成物用於製造用以治療個體的前蛋白轉化酶枯草桿菌蛋白酶kexin 9型(PCSK9)相關疾病的藥物。更進一步而言，前蛋白轉化酶枯草桿菌蛋白酶kexin 9型(PCSK9)相關疾病如冠心病、心肌梗塞、動脈粥樣硬化以及高膽固醇血症。 The present invention further includes a pharmaceutical composition containing one or more compounds of the above formula (I), and a pharmaceutical composition for manufacturing a proprotein converting enzyme subtilisin kexin type 9 (PCSK9) -related disease for treating an individual. Furthermore, preprotein-converting enzyme subtilisin kexin type 9 (PCSK9) -related diseases such as coronary heart disease, myocardial infarction, atherosclerosis, and hypercholesterolemia.

本發明的式(I)化合物可藉由所屬技術領域現有的方法製備。製備方法可參閱R.Larock發表的Comprehensive Organic Transformations(第二版，VCH Publishers 1999)；P.G.M.Wuts及T.W.Greene等人發表於Organic Synthesis(第四版，John Wiley and Sons 2007)的Greene’s Protective Groups；L.Fieser以及M.Fieser的著作Fieser and Fieser’s Reagents for Organic Synthesis(John Wiley and Sons 1994)；L.Paquette著作Encyclopedia of Reagents for Organic Synthesis(第二版，John Wiley and Sons 2009)；以及G.J.Yu等人2008年發表於J.Med.Chem.第51卷，第6044 至6054頁的著作。 The compound of formula (I) of the present invention can be prepared by methods existing in the art. For the preparation method, please refer to Comprehensive Organic Transformations (Second Edition, VCH Publishers 1999) published by R. Larock; Greene's Protective Groups published by Organic Synthesis (Fourth Edition, John Wiley and Sons 2007) by PGMWuts and TW Greene et al .; L. Fieser And M. Fieser's work Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons 1994); L. Paquette's work Encyclopedia of Reagents for Organic Synthesis (Second Edition, John Wiley and Sons 2009); and GJYu et al. 2008 In J. Med. Chem. Vol. 51, pp. 6044 to 6054.

本文所提及的化合物可以含有非芳香族雙鍵和一個或多個不對稱中心，例如：位在連接於核心芳香環的取代基。因此，此等化合物可作為消旋混和物和外消旋混合物、單一對掌異構物、單個非對映立體異構物、非對映體混合物，以及順式或反式異構體形式。所有這些異構體形式都含括在本發明範圍之內。 The compounds mentioned herein may contain non-aromatic double bonds and one or more asymmetric centers, such as substituents attached to the core aromatic ring. Thus, these compounds can be used as racemic and racemic mixtures, single paraisomers, single diastereoisomers, diastereomeric mixtures, and cis or trans isomers. All these isomeric forms are included within the scope of the invention.

如此製備的式(I)化合物可以使用體外測定，例如本發明實施例2中描述的PCSK9分泌測定。隨後可以使用本領域現有的體內測定評估化合物在降低哺乳動物中的LDL-C血漿水平方面的功效。所選擇的化合物可進一步測試以驗證其在治療膽固醇和脂蛋白代謝紊亂或治療與PCSK9相關的疾病(例如冠心病、心肌梗塞、動脈粥樣硬化或高膽固醇血症)中的功效，舉例而言，可將化合物施予患有冠心病的動物(例如小鼠)，然後評估其治療效果。根據結果，可以確定合適的劑量範圍以及給藥途徑。 The compounds of formula (I) thus prepared can be used in vitro assays, such as the PCSK9 secretion assay described in Example 2 of the invention. The efficacy of a compound in reducing LDL-C plasma levels in mammals can then be assessed using in vivo assays available in the art. The selected compound can be further tested to verify its efficacy in treating disorders of cholesterol and lipoprotein metabolism or in diseases associated with PCSK9, such as coronary heart disease, myocardial infarction, atherosclerosis, or hypercholesterolemia, for example Compounds can be administered to animals (eg, mice) with coronary heart disease and then evaluated for their therapeutic effect. Based on the results, an appropriate dosage range and route of administration can be determined.

在沒有更進一步的闡述下，相信由以上之敘述足以呈現本發明。因此，接下來的實施例是純粹做為解說性之實例，無論如何不受限於其餘以任何形式公開的事情。所有在此文中被引用之刊物皆已全部整合於參考文獻中。 Without further elaboration, it is believed that the above description is sufficient to present the present invention. Therefore, the following embodiments are purely illustrative examples and are not limited in any way to the rest of what is disclosed in any form. All publications cited in this article have been incorporated into the reference.

本發明之實施例將進一步列舉以下的實例說明，本發明所屬技術領域中具通常知識者可以藉由本案說明書了解該等實例例僅用於說明本發明，各種修改和變化在不偏離本發明的範圍下為可行的，因此，下述的實施例僅作為本發明代表性的不同面向及特徵，而非用來限制本發明。 The embodiments of the present invention will further enumerate the following example descriptions. Those with ordinary knowledge in the technical field to which the present invention pertains can understand from the description of this case that these examples are only used to illustrate the present invention, and various modifications and changes do not depart from the present invention The scope is feasible. Therefore, the following embodiments are only representative of different aspects and features of the present invention, and are not intended to limit the present invention.

下列顯示了12種式(I)例示性化合物的結構。製備該等化合物的方法以及化合物的結構鑑定數據均列於實施例1中。在實施例2至6中則進一步描述這些化合物的分析測試程序。 The structures of 12 exemplary compounds of formula (I) are shown below. The methods for preparing these compounds and the structural identification data of the compounds are listed in Example 1. The analytical test procedures for these compounds are further described in Examples 2 to 6.

接者，合成式(I)化合物反應步驟流程，即反應流程1及2：反應流程1 Next, the reaction process of the compound of formula (I), namely reaction schemes 1 and 2: reaction scheme 1

本發明所有的化學品和溶劑皆可由商業供應商購得，並按原樣使用。上述合成反應均在乾燥的氮氣氣氛下進行，使用Merck 60 F 254矽膠玻璃平板(5×10cm)藉由TLC監測反應；並且在紫外線(254nm)照射下或者通過噴灑磷鉬酸試劑(Aldrich)，在80℃下加熱來目視檢測區域。所有管柱層析色譜法是使用Merck Kiesel gel 60,9385的ASTM矽膠(230至400目)作為固定相進行。 All chemicals and solvents of the present invention are available from commercial suppliers and used as is. The above synthesis reactions were all performed under a dry nitrogen atmosphere. The reaction was monitored by TLC using a Merck 60 F 254 silicone glass plate (5 × 10cm); and under ultraviolet (254nm) irradiation or by spraying a phosphomolybdic acid reagent (Aldrich), The test area was visually inspected by heating at 80 ° C. All column chromatography was performed using Merck Kiesel gel 60, 9385, ASTM silicone (230 to 400 mesh) as a stationary phase.

質子核磁共振譜(1H)以Varian Mercury-300或Varian Mercury-400核磁共振儀測量，化學位移(Chemical shifts)以相對於溶劑峰的共振的標度(δ)記錄為百萬分率(ppm)。以下縮寫用於描述耦合：s=單峰；d=雙峰；t=三重峰；q=四重峰；quin=五重峰；br=寬峰；以及m=多重峰。液相層析質譜(LCMS)數據由Agilent MSD-1100ESI-MS/MS、Agilent 1200系列LC/MSD VL以及Waters Acquity UPLC-ESI-MS/MS系統進行測量。 Proton nuclear magnetic resonance spectra (1 H) in a Varian Mercury-300 or Varian Mercury-400 NMR measuring apparatus, chemical shifts (Chemical shifts) scale with respect to the solvent peak of the resonance ([delta]) reported as parts per million (ppm ). The following abbreviations are used to describe the coupling: s = singlet; d = doublet; t = triplet; q = quartet; quin = quintet; br = broad peak; and m = multiplet. Liquid chromatography mass spectrometry (LCMS) data was measured by an Agilent MSD-1100ESI-MS / MS, an Agilent 1200 series LC / MSD VL, and a Waters Acquity UPLC-ESI-MS / MS system.

[實施例1]： 式(I)化合物的合成 [Example 1]: Synthesis of a compound of formula (I)

(a)合成第一支鏈的步驟： (a) Steps to synthesize the first branch:

在25℃下，N-((第三丁基氧基)羰基)亞氨基二乙酸(2.33g，10mmol)在二氯甲烷(DCM)(30ml)溶液中以1-乙基-3-(3-二甲基氨基丙基)碳醯二亞胺(EDCI)(1.98g，10.3mmol)處理。將反應混合物在25℃下攪拌1小時，然後加入胺類(1.26g，12mmol)，並將溶液在25℃下攪拌20小時。將反應混合物倒入10%HCl(aq)(100ml)中，並用DCM(100ml×2)萃取，混和物的有機相用10%HCl(aq)(80ml×2)及飽和食鹽水NaCl(aq)(100ml×2)洗滌、乾燥(MgSO4)、過濾並真空濃縮，得到純化的N-((第三丁基氧基)-羰基)亞氨基二乙酸單醯胺。 At 25 ° C, N-((third butyloxy) carbonyl) iminodiacetic acid (2.33 g, 10 mmol) in a solution of dichloromethane (DCM) (30 ml) with 1-ethyl-3- (3 -Treatment with dimethylaminopropyl) carbodiimide (EDCI) (1.98 g, 10.3 mmol). The reaction mixture was stirred at 25 ° C for 1 hour, then amines (1.26 g, 12 mmol) were added, and the solution was stirred at 25 ° C for 20 hours. The reaction mixture was poured into 10% HCl (aq) (100ml) and extracted with DCM (100ml × 2). The organic phase of the mixture was 10% HCl (aq) (80ml × 2) and saturated brine NaCl (aq) (100ml × 2), dried (MgSO 4), filtered and concentrated in vacuo to give the purified N - ((tert-butyl oxy) - carbonyl) iminodiacetic acid mono acyl amine.

在25℃下，N-((第三丁基氧基)-羰基)亞氨基二乙酸(2.33g，10mmol)在二氯甲烷(DCM)(30ml)溶液中用碳醯二亞胺(EDCI)(1.98g，10.3mmol)處理。將混合物在25℃下攪拌1小時，然後加入胺類化合物(1.26g，12mmol)，並將溶液在25℃下攪拌20小時。將反應混合物倒入10%HCl(aq)(100ml)中並用DCM(100ml×2)萃取。有機相用10%HCl(aq)(80ml×2)和飽和食鹽水NaCl(aq)(100ml×2)洗滌、乾燥(MgSO4)、過濾並真空濃縮，得到純化的N-((第三丁基氧基)羰基)亞氨基二乙酸單醯胺(流程圖產物2，產率82%，2.573g)。 Carbodiimide (EDCI) in a solution of N-((third butyloxy) -carbonyl) iminodiacetic acid (2.33 g, 10 mmol) in dichloromethane (DCM) (30 ml) at 25 ° C (1.98 g, 10.3 mmol). The mixture was stirred at 25 ° C for 1 hour, then an amine compound (1.26 g, 12 mmol) was added, and the solution was stirred at 25 ° C for 20 hours. The reaction mixture was poured into 10% HCl (aq) (100 ml) and extracted with DCM (100 ml x 2). The organic phase was washed with 10% HCl (aq) (80 ml × 2) and saturated brine NaCl (aq) (100 ml × 2), dried (MgSO 4 ), filtered, and concentrated in vacuo to obtain purified N-(( Methyloxy) carbonyl) iminodiacetic acid monoamidine (Scheme Product 2, yield 82%, 2.573 g).

1H NMR(500MHz，CDCl3)：δ 1.38(d,J=41.9Hz,9H),3.92(d,J=22.0Hz,2H),4.00(s,2H),4.44(s,2H),6.24(s,1H),6.28(s,1H),7.32(s,1H),7.94(d,J=326.5Hz,1H)。 1 H NMR (500 MHz, CDCl 3 ): δ 1.38 (d, J = 41.9 Hz, 9H), 3.92 (d, J = 22.0 Hz, 2H), 4.00 (s, 2H), 4.44 (s, 2H), 6.24 (s, 1H), 6.28 (s, 1H), 7.32 (s, 1H), 7.94 (d, J = 326.5Hz, 1H).

LCMS(ESI)：m/z理論值[C14H20N2O6-H]- 311.12，實測值311.30[M+H]+。 LCMS (ESI): m / z theoretical values [C 14 H 20 N 2 O 6 -H] - 311.12, found 311.30 [M + H] +.

(b)合成第二支鏈的步驟： (b) Steps to synthesize the second branch:

將N-((第三丁基氧基)-羰基)亞氨基二乙酸單醯胺(4.8mmol)溶於DCM(15ml)中。該溶液用胺類化合物(1eq)、EDCI(1.2eq)、HOBt(1.2eq)以及Et3N(1.5eq)處理，將該溶液在25℃下攪拌20小時候，將混合物倒入水中並以DCM(40ml×2)萃取。有機相用飽和食鹽水NaCl(aq)(50ml×2)洗滌，乾燥(MgSO4)，過濾並真空濃縮。通過MPLC(MeOH/DCM=1/19)純化粗產物以得到純化的二醯胺。 N-((Third-butyloxy) -carbonyl) iminodiacetic acid monofluorenylamine (4.8 mmol) was dissolved in DCM (15 ml). This solution was treated with amines (1eq), EDCI (1.2eq), HOBt (1.2eq) and Et 3 N (1.5eq). The solution was stirred at 25 ° C for 20 hours. The mixture was poured into water and DCM (40 ml x 2) extraction. The organic phase was washed with saturated brine NaCl (aq) (50 ml × 2), dried (MgSO 4 ), filtered and concentrated in vacuo. The crude product was purified by MPLC (MeOH / DCM = 1/19) to obtain purified diamidine.

以EDCI(439.3mg，2.30mmol)、HOBt(294.4mg，2.30mmol)以及Et3N(0.32ml，2.30mmol)處理前述步驟的產物2(478.8mg，1.53mmol)、4-苯氧基苯胺(340.8mg，1.84mmol)的無水DCM(15ml)混合溶液。在室溫下攪拌混合物隔夜。將混合物倒入水中並用DCM萃取。有機相用飽和食鹽水溶液洗滌、乾燥、過濾並真空濃縮。使用MPLC(DCM：MeOH=95：5)純化粗產物，得到預期 產物3(流程圖產物3，581.3mg，產率80%)。 The product 2 (478.8 mg, 1.53 mmol), 4-phenoxyaniline (4-phenoxyaniline) from the previous step was treated with EDCI (439.3 mg, 2.30 mmol), HOBt (294.4 mg, 2.30 mmol) and Et 3 N (0.32 ml, 2.30 mmol). A mixed solution of 340.8 mg, 1.84 mmol) in anhydrous DCM (15 ml). The mixture was stirred at room temperature overnight. The mixture was poured into water and extracted with DCM. The organic phase was washed with saturated saline solution, dried, filtered and concentrated in vacuo. The crude product was purified using MPLC (DCM: MeOH = 95: 5) to give the expected product 3 (Scheme Product 3, 581.3 mg, 80% yield).

1H NMR(500MHz,CDCl3)：δ 10.90以及9.86(two s,total 1H),7.70以及7.63(two m,total 2H),7.31(m,3H),7.06(t,1H,J=7.35Hz),6.98(m,4H),6.31(m,2H),4.52(d,2H,J=5Hz),4.04-3.89(three s,total 4H),1.40以及1.37(two s,total 9H)。 1 H NMR (500MHz, CDCl 3 ): δ 10.90 and 9.86 (two s, total 1H), 7.70 and 7.63 (two m, total 2H), 7.31 (m, 3H), 7.06 (t, 1H, J = 7.35Hz ), 6.98 (m, 4H), 6.31 (m, 2H), 4.52 (d, 2H, J = 5Hz), 4.04-3.89 (three s, total 4H), 1.40 and 1.37 (two s, total 9H).

LCMS(ESI)：m/z理論值[C26H29N3O6+H]+480.52，實測值480.42[M+H]+。 LCMS (ESI): m / z theoretical value [C 26 H 29 N 3 O 6 + H] + 480.52, found 480.42 [M + H] + .

將N-((第三丁基氧基)-羰基)亞氨基二乙酸單醯胺(流程圖化合物2，1.5g，4.8mmol)溶於DCM(15ml)中。該溶液用胺類化合物(1eq)、EDCI(1.2eq)、HOBt(1.2eq)以及Et3N(1.5eq)處理。將該溶液在25℃下攪拌20小時。將混合物倒入水中並用DCM(40ml×2)萃取。有機相用飽和食鹽水NaCl(aq)(50ml×2)洗滌、乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(MeOH/DCM=1/19)純化粗產物，得到純化的二醯胺(流程圖產物4，產率60%，1.432g)。 N-((Third-butyloxy) -carbonyl) iminodiacetic acid monoamidine (Scheme Compound 2, 1.5 g, 4.8 mmol) was dissolved in DCM (15 ml). The solution, EDCI (1.2eq), HOBt ( 1.2eq) and Et 3 N (1.5eq) was treated with an amine compound (1eq). The solution was stirred at 25 ° C for 20 hours. The mixture was poured into water and extracted with DCM (40 ml x 2). The organic phase was washed with saturated brine NaCl (aq) (50 ml x 2), dried (MgSO4), filtered and concentrated in vacuo. The crude product was purified by MPLC (MeOH / DCM = 1/19) to obtain purified diamidine (Scheme Product 4, yield 60%, 1.432 g).

1H NMR(500MHz,CDCl3)：δ 1.40(s,9H),3.96(t,J=34.6Hz,4H),4.52(t,J=5.1Hz,2H),6.24-6.35(m,2H),6.90-7.04(m,6H),7.35(s,1H),7.63(d,J=8.8Hz,1H),7.70(d,J=8.8Hz,1H),9.90(s,1H),10.92(s,1H)。 1 H NMR (500MHz, CDCl 3 ): δ 1.40 (s, 9H), 3.96 (t, J = 34.6Hz, 4H), 4.52 (t, J = 5.1Hz, 2H), 6.24-6.35 (m, 2H) , 6.90-7.04 (m, 6H), 7.35 (s, 1H), 7.63 (d, J = 8.8Hz, 1H), 7.70 (d, J = 8.8Hz, 1H), 9.90 (s, 1H), 10.92 ( s, 1H).

LCMS(ESI)：m/z理論值[C26H28FN3O6+H]+ 498.20，實測值498.25[M+H]+。 LCMS (ESI): m / z theoretical [C 26 H 28 FN 3 O 6 + H] + 498.20, found 498.25 [M + H] + .

合成PPC-014以及PPC-019Synthesis of PPC-014 and PPC-019

反應流程5Reaction Scheme 5

製備Boc去保護基化合物的一般反應流程：N'-((第三丁基氧基)羰基)-N,N-二取代的亞氨基二乙酸二醯胺(2.88mmol)溶於TFA：DCM(v/v)=1：1中，混合物在25℃下持續攪拌1小時。真空除去溶劑。通過MPLC(DCM/MeOH=19/1)純化殘餘物以得到預期的產物。 General reaction scheme for the preparation of Boc deprotecting compounds: N '-((third butyloxy) carbonyl) -N, N-disubstituted iminodiacetic acid dihydrazide (2.88 mmol) is dissolved in TFA: DCM ( v / v) = 1: 1, the mixture was continuously stirred at 25 ° C for 1 hour. The solvent was removed in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to give the expected product.

PPC-014     PPC-014    

無色油狀。產率：92%。 Colorless oily. Yield: 92%.

1H NMR(500MHz,MeOD)：δ 2.76(t,J=7.2Hz,2H),3.06(t,J=7.0Hz,2H),3.49(m,4H),3.73(m,4H),3.80(s,3H),3.83(s,3H),6.77(m,1H),6.85(m,3H),6.93(m,1H),7.22(d,J=5.1Hz,1H)。 1 H NMR (500MHz, MeOD): δ 2.76 (t, J = 7.2Hz, 2H), 3.06 (t, J = 7.0Hz, 2H), 3.49 (m, 4H), 3.73 (m, 4H), 3.80 ( s, 3H), 3.83 (s, 3H), 6.77 (m, 1H), 6.85 (m, 3H), 6.93 (m, 1H), 7.22 (d, J = 5.1Hz, 1H).

LCMS(ESI)：m/z理論值[C20H27N3O4S+H]+406.1722，HRMS(ESI)：m/z實測值406.1809[M+H]+。 LCMS (ESI): m / z theoretical value [C 20 H 27 N 3 O 4 S + H] + 406.1722, HRMS (ESI): m / z observed value 406.1809 [M + H] + .

PPC-019     PPC-019    

無色油狀。產率：95%。 Colorless oily. Yield: 95%.

1H NMR(500MHz,MeOD)：δ 3.05(t,J=7.0Hz,2H),3.51(t,J=7.0Hz,2H),3.88(s,2H),4.00(s,2H),6.87(s,1H),6.9(m,1H),6.9(m,1H),6.93(m,4H),7.08(t,J=7.3,1H),7.19(d,J=5.0Hz,1H),7.32(t,J=7.8,2H),7.56(d,J=8.8,2H)。 1 H NMR (500 MHz, MeOD): δ 3.05 (t, J = 7.0 Hz, 2H), 3.51 (t, J = 7.0 Hz, 2H), 3.88 (s, 2H), 4.00 (s, 2H), 6.87 ( s, 1H), 6.9 (m, 1H), 6.9 (m, 1H), 6.93 (m, 4H), 7.08 (t, J = 7.3, 1H), 7.19 (d, J = 5.0 Hz, 1H), 7.32 (t, J = 7.8,2H), 7.56 (d, J = 8.8,2H).

LCMS(ESI)：m/z理論值[C22H23N3O3S+H]+410.1460，HRMS(ESI)：m/z實測值410.1546[M+H]+。 LCMS (ESI): theoretical m / z [C 22 H 23 N 3 O 3 S + H] + 410.1460, HRMS (ESI): m / z found 410.1546 [M + H] + .

合成PPC-020以及PPC-036Synthesis of PPC-020 and PPC-036

將PPC-014(0.25mmol)、羧酸(0.25mmol)、EDCI(0.30mol)，HOBt(0.3mol)、Et3N(0.05ml)、DMF(2ml)的混合溶液在25℃下攪拌20小時。將該混合物倒入10%HCl(aq)中並用EtOAc萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的加成產物。 A mixed solution of PPC-014 (0.25 mmol), carboxylic acid (0.25 mmol), EDCI (0.30 mol), HOBt (0.3 mol), Et 3 N (0.05 ml), and DMF (2 ml) was stirred at 25 ° C. for 20 hours . The mixture was poured into 10% HCl (aq) and extracted with EtOAc. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected addition product.

PPC-020     PPC-020    

無色油狀。產率：62%。 Colorless oily. Yield: 62%.

1H NMR(500MHz,MeOD)：δ 1.99(s,9H),2.05(d,J=42.15Hz,2H), 2.79(m,2H),3.03(m,2H),3.43(m,2H),3.51(m,2H),3.76(s,3H),3.77(s,3H),3.93(s,2H),4.09(d,J=9.95Hz,2H),6.76(m,1H),6.87(m,4H),7.19(d,J=5.04,1H)。 1 H NMR (500MHz, MeOD): δ 1.99 (s, 9H), 2.05 (d, J = 42.15Hz, 2H), 2.79 (m, 2H), 3.03 (m, 2H), 3.43 (m, 2H), 3.51 (m, 2H), 3.76 (s, 3H), 3.77 (s, 3H), 3.93 (s, 2H), 4.09 (d, J = 9.95Hz, 2H), 6.76 (m, 1H), 6.87 (m , 4H), 7.19 (d, J = 5.04, 1H).

LCMS(ESI)：m/z理論值[C26H37N3O5S+H]+ 504.2454，HRMS(ESI)：m/z實測值504.2545[M+H]+。 LCMS (ESI): m / z theoretical value [C 26 H 37 N 3 O 5 S + H] + 504.2454, HRMS (ESI): m / z observed value 504.2545 [M + H] + .

PPC-036     PPC-036    

無色油狀。產率：32%。 Colorless oily. Yield: 32%.

1H NMR(500MHz,MeOD)：δ 2.77(d,J=6.4Hz,2H),3.04(m,2H),3.51(m,4H),3.66(s,3H),3.73(s,3H),4.14(m,2H),4.42(m,2H),6.66(m,3H),6.86(m,3H),7.06(t,J=7.5Hz,1H),7.16(s,1H),7.23(t,J=7.5Hz,1H),7.43(d,J=8.1Hz,1H),7.54(s,1H)。 1 H NMR (500MHz, MeOD): δ 2.77 (d, J = 6.4Hz, 2H), 3.04 (m, 2H), 3.51 (m, 4H), 3.66 (s, 3H), 3.73 (s, 3H), 4.14 (m, 2H), 4.42 (m, 2H), 6.66 (m, 3H), 6.86 (m, 3H), 7.06 (t, J = 7.5Hz, 1H), 7.16 (s, 1H), 7.23 (t , J = 7.5Hz, 1H), 7.43 (d, J = 8.1Hz, 1H), 7.54 (s, 1H).

LCMS(ESI)：m/z理論值[C29H32N4O5S+H]+ 549.2093，HRMS(ESI)：m/z實測值549.2179[M+H]+。 LCMS (ESI): m / z theoretical value [C 29 H 32 N 4 O 5 S + H] + 549.2093, HRMS (ESI): m / z observed value 549.2179 [M + H] + .

合成PPC-038、PPC-039、PPC-045、PPC069以及PPC070Synthesis of PPC-038, PPC-039, PPC-045, PPC069, and PPC070

將PPC-019(0.25mmol)、羧酸(0.25mmol)、EDCI(0.30mol)、HOBt(0.3mol)、Et3N(0.05ml)以及DMF(2ml)的混合溶液在25℃攪拌20小時。再將該混合物倒入10%HCl(aq)中並用EtOAc 萃取。飽和食鹽水NaCl(aq)洗滌，並將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以得到預期的產物。 A mixed solution of PPC-019 (0.25 mmol), carboxylic acid (0.25 mmol), EDCI (0.30 mol), HOBt (0.3 mol), Et 3 N (0.05 ml) and DMF (2 ml) was stirred at 25 ° C. for 20 hours. The mixture was poured into 10% HCl (aq) and extracted with EtOAc. It was washed with saturated brine NaCl (aq) , and the organic phase was dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to give the expected product.

PPC-038     PPC-038    

白色固體。產率：78%。 White solid. Yield: 78%.

1H NMR(500MHz,MeOD)：δ 2.05(m,2H),3.05(m,2H),3.34(m,2H),3.48(m,2H),3.55(m,1H),3.81(m,2H),4.10(d,J=17.5,2H),4.33(m,2H),6.87(m,2H),6.93(m,4H),7.10(m,1H),7.15(d,J=5.3,1H),7.29(d,J=7.6,2H),7.56(d,J=8.9,1H),7.61(d,J=8.9,1H)。 1 H NMR (500MHz, MeOD): δ 2.05 (m, 2H), 3.05 (m, 2H), 3.34 (m, 2H), 3.48 (m, 2H), 3.55 (m, 1H), 3.81 (m, 2H ), 4.10 (d, J = 17.5, 2H), 4.33 (m, 2H), 6.87 (m, 2H), 6.93 (m, 4H), 7.10 (m, 1H), 7.15 (d, J = 5.3, 1H ), 7.29 (d, J = 7.6,2H), 7.56 (d, J = 8.9,1H), 7.61 (d, J = 8.9,1H).

LCMS(ESI)：m/z理論值[C27H29N3O5S+H]+ 508.1828，HRMS(ESI)：m/z實測值508.1896[M+H]+。 LCMS (ESI): m / z theoretical value [C 27 H 29 N 3 O 5 S + H] + 508.1828, HRMS (ESI): m / z observed value 508.1896 [M + H] + .

PPC-039     PPC-039    

黃色固體。產率：25%。 Yellow solid. Yield: 25%.

1H NMR(500MHz,MeOD)：δ3.03(m,2H),3.52(m,2H),4.30(m,2H),4.56(m,2H),6.79(m,3H),6.93(m,4H),7.06(t,J=7.3,3H),7.21(t,J=7.3,3H),7.30(t,J=8.3,2H),7.42(t,J=8.3,1H),7.57(d,J=8.0,3H)。 1 H NMR (500MHz, MeOD): δ 3.03 (m, 2H), 3.52 (m, 2H), 4.30 (m, 2H), 4.56 (m, 2H), 6.79 (m, 3H), 6.93 (m, 4H), 7.06 (t, J = 7.3,3H), 7.21 (t, J = 7.3,3H), 7.30 (t, J = 8.3,2H), 7.42 (t, J = 8.3,1H), 7.57 (d , J = 8.0,3H).

LCMS(ESI)：m/z理論值[C31H28N4O4S+H]+ 553.1831，HRMS(ESI)：m/z實測值553.1904[M+H]+。 LCMS (ESI): m / z theoretical value [C 31 H 28 N 4 O 4 S + H] + 553.1831, HRMS (ESI): m / z observed value 553.1904 [M + H] + .

PPC-045     PPC-045    

白色固體。產率：78%。 White solid. Yield: 78%.

1H NMR(500MHz,MeOD)：δ 0.92(dd,J=2.0,6.6,6H),2.12(m,2H),2.21(m,1H),3.05(dt,J=6.8,21.4,2H),3.51(dt,J=6.8,29.6,2H),4.09(d,J=16.4,2H),4.24(d,J=43.7,2H),6.87(d,J=5.0,2H),9.41(m,4H),7.06(m,1H),7.15(m,1H),7.30(t,J=5.7,2H),7.59(dd,J=8.9,24.8,2H)。 1 H NMR (500MHz, MeOD): δ 0.92 (dd, J = 2.0,6.6,6H), 2.12 (m, 2H), 2.21 (m, 1H), 3.05 (dt, J = 6.8,21.4,2H), 3.51 (dt, J = 6.8, 29.6, 2H), 4.09 (d, J = 16.4, 2H), 4.24 (d, J = 43.7, 2H), 6.87 (d, J = 5.0, 2H), 9.41 (m, 4H), 7.06 (m, 1H), 7.15 (m, 1H), 7.30 (t, J = 5.7, 2H), 7.59 (dd, J = 8.9, 24.8, 2H).

LCMS(ESI)：m/z理論值[C27H31N3O4S+H]+ 494.2035，HRMS(ESI)：m/z實測值494.2109[M+H]+。 LCMS (ESI): m / z theoretical value [C 27 H 31 N 3 O 4 S + H] + 494.2035, HRMS (ESI): m / z observed value 494.2109 [M + H] + .

PPC-069     PPC-069    

白色固體。產率：78%。 White solid. Yield: 78%.

1H NMR(500MHz,MeOD)：δ 3.05(m,2H),3.51(m,2H),4.30(d,J=24.9,2H),4.49(d,J=45.7,2H),6.85(m,2H),6.87(m,4H),6.95(m,1H),7.31(d,J=7.6,1H),7.48(m,1H),7.55(m,4H),8.50(d,J=12.3,1H)。 1 H NMR (500MHz, MeOD): δ 3.05 (m, 2H), 3.51 (m, 2H), 4.30 (d, J = 24.9, 2H), 4.49 (d, J = 45.7, 2H), 6.85 (m, 2H), 6.87 (m, 4H), 6.95 (m, 1H), 7.31 (d, J = 7.6, 1H), 7.48 (m, 1H), 7.55 (m, 4H), 8.50 (d, J = 12.3, 1H).

LCMS(ESI)：m/z理論值[C26H24N4O5S+H]+ 505.1467，HRMS(ESI)：m/z實測值505.1533[M+H]+。 LCMS (ESI): m / z theoretical value [C 26 H 24 N 4 O 5 S + H] + 505.1467, HRMS (ESI): m / z observed value 505.1533 [M + H] + .

PPC-070     PPC-070    

白色固體。產率：81%。 White solid. Yield: 81%.

1H NMR(500MHz,MeOD)：δ 0.13(m,2H),0.52(t,J=6.6,2H),1.30(m,1H),2.19(m,2H),3.06(m,2H),3.52(m,2H),4.11(m,2H),4.22(m,2H),6.88(m,2H),6.95(m,4H),7.07(m,2H),7.31(m,2H),7.80(m,2H)。 1 H NMR (500MHz, MeOD): δ 0.13 (m, 2H), 0.52 (t, J = 6.6, 2H), 1.30 (m, 1H), 2.19 (m, 2H), 3.06 (m, 2H), 3.52 (m, 2H), 4.11 (m, 2H), 4.22 (m, 2H), 6.88 (m, 2H), 6.95 (m, 4H), 7.07 (m, 2H), 7.31 (m, 2H), 7.80 ( m, 2H).

LCMS(ESI)：m/z理論值[C27H29N3O5S+H]+ 492.1879，HRMS(ESI)：m/z實測值492.1944[M+H]+。 LCMS (ESI): m / z theoretical value [C 27 H 29 N 3 O 5 S + H] + 492.1879, HRMS (ESI): m / z observed value 492.1944 [M + H] + .

合成PPC-021以及PPC-027Synthetic PPC-021 and PPC-027

將PPC013(0.25mmol)、羧酸(0.25mmol)、EDCI(0.30mol)、HOBt(0.3mol)、Et3N(0.05ml)以及DMF(2ml)的溶液在25℃下攪拌20小時。將該混合物倒入10%HCl(aq)中並用EtOAc萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的加成產物。 A solution of PPC013 (0.25 mmol), carboxylic acid (0.25 mmol), EDCI (0.30 mol), HOBt (0.3 mol), Et 3 N (0.05 ml), and DMF (2 ml) was stirred at 25 ° C. for 20 hours. The mixture was poured into 10% HCl (aq) and extracted with EtOAc. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected addition product.

PPC-021     PPC-021    

無色油狀。產率：80%。 Colorless oily. Yield: 80%.

1H NMR(500MHz,DMSO)：δ 0.96(m,9H),2.09(s,1H),2.16(s,1H),4.02-4.37(m,6H),6.29(s,1H),6.39(s,1H),6.94-7.10(m,5H),7.35(m,2H),7.57-7.60(m,3H)。 1 H NMR (500MHz, DMSO): δ 0.96 (m, 9H), 2.09 (s, 1H), 2.16 (s, 1H), 4.02-4.37 (m, 6H), 6.29 (s, 1H), 6.39 (s , 1H), 6.94-7.10 (m, 5H), 7.35 (m, 2H), 7.57-7.60 (m, 3H).

LCMS(ESI)：m/z理論值[C27H31N3O5+H]+ 478.2336，HRMS(ESI)：m/z實測值478.2354[M+H]+。 LCMS (ESI): theoretical m / z [C 27 H 31 N 3 O 5 + H] + 478.2336, HRMS (ESI): m / z found 478.2354 [M + H] + .

PPC-027     PPC-027    

白色固體。產率：40%。 White solid. Yield: 40%.

1H NMR(500MHz,DMSO)：δ 4.27-4.41(m,4H),4.51(s,1H),4.60(s,1H),6.29(d,J=28.02Hz,1H),6.90(s,1H),6.69(d,J=39.94Hz,1H),6.95(d,J=8Hz,2H),7.01(m,3H),7.09(t,J=7.35Hz,1H),7.20(t,J=7.43Hz,1H),7.35(t,J=7.82Hz,2H),7.42(m,1H),7.54-7.67(m,4H),11.66(d,J=9.4Hz,1H)。 1 H NMR (500MHz, DMSO): δ 4.27-4.41 (m, 4H), 4.51 (s, 1H), 4.60 (s, 1H), 6.29 (d, J = 28.02Hz, 1H), 6.90 (s, 1H ), 6.69 (d, J = 39.94Hz, 1H), 6.95 (d, J = 8Hz, 2H), 7.01 (m, 3H), 7.09 (t, J = 7.35Hz, 1H), 7.20 (t, J = 7.43Hz, 1H), 7.35 (t, J = 7.82Hz, 2H), 7.42 (m, 1H), 7.54-7.67 (m, 4H), 11.66 (d, J = 9.4Hz, 1H).

LCMS(ESI)：m/z理論值[C30H26N4O5-H]- 523.1975，HRMS(ESI)：m/z實測值523.1984[M+H]+。 LCMS (ESI): m / z theoretical value [C 30 H 26 N 4 O 5 -H]-523.1975, HRMS (ESI): m / z observed value 523.1984 [M + H] + .

合成PPC-017以及PPC-018Synthetic PPC-017 and PPC-018

將PPC004(0.25mmol)、羧酸(0.25mmol)、EDCI(0.30mol)、HOBt(0.3mol)、Et3N(0.05ml)以及DMF(2ml)的混合溶液在25℃下攪拌20小時。將該混合物倒入10%HCl(aq)中並用EtOAc萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的產物。 A mixed solution of PPC004 (0.25 mmol), carboxylic acid (0.25 mmol), EDCI (0.30 mol), HOBt (0.3 mol), Et 3 N (0.05 ml), and DMF (2 ml) was stirred at 25 ° C. for 20 hours. The mixture was poured into 10% HCl (aq) and extracted with EtOAc. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected product.

PPC-017     PPC-017    

無色油狀。產率：40%。 Colorless oily. Yield: 40%.

1H NMR(500MHz,CDCl 3)：δ 2.80(m,2H),3.58(m,2H),3.70-3.86(m,6H),4.05(m,2H),4.36(m,2H),4.52(m,2H),6.29(m,2H),6.74(m,4H),7.12(m,1H),7.26-7.31(m,2H),7.39(d,J=8.28Hz,1H)7.63(d, J=8.04,1H)。 1 H NMR (500MHz, CDCl 3 ): δ 2.80 (m, 2H), 3.58 (m, 2H), 3.70-3.86 (m, 6H), 4.05 (m, 2H), 4.36 (m, 2H), 4.52 ( m, 2H), 6.29 (m, 2H), 6.74 (m, 4H), 7.12 (m, 1H), 7.26-7.31 (m, 2H), 7.39 (d, J = 8.28Hz, 1H) 7.63 (d, J = 8.04, 1H).

LCMS(ESI)：m/z理論值[C28H30N4O5+H]- 519.2238，HRMS(ESI)：m/z實測值519.2256[M+H]+。 LCMS (ESI): m / z theoretical values [C 28 H 30 N 4 O 5 + H] - 519.2238, HRMS (ESI): m / z found 519.2256 [M + H] +.

PPC-018     PPC-018    

無色油狀。產率：62%。 Colorless oily. Yield: 62%.

1H NMR(500MHz,CDCl3)：δ 2.00(m,2H),2.75-2.85(m,2H),3.04-3.09(m,1H),3.52-3.54(m,2H),3.76-3.84(m,2H),3.84-3.88(m,10H),4.03-4.09(m,2H),4.48(m,2H),6.24-6.33(m,2H),6.73-6.82(m,3H),7.35(d,J=14.5,1H)。 1 H NMR (500MHz, CDCl 3 ): δ 2.00 (m, 2H), 2.75-2.85 (m, 2H), 3.04-3.09 (m, 1H), 3.52-3.54 (m, 2H), 3.76-3.84 (m , 2H), 3.84-3.88 (m, 10H), 4.03-4.09 (m, 2H), 4.48 (m, 2H), 6.24-6.33 (m, 2H), 6.73-6.82 (m, 3H), 7.35 (d , J = 14.5,1H).

LCMS(ESI)：m/z理論值[C24H31N3O7+H]+ 474.2234，HRMS(ESI)：m/z實測值474.2251[M+H]+。 LCMS (ESI): m / z theoretical value [C 24 H 31 N 3 O 7 + H] + 474.2234, HRMS (ESI): m / z observed value 474.2251 [M + H] + .

合成PPC111Synthesis PPC111

將胺類產物(PPC-091，199mg，0.5mmol)、羧酸(0.5mmol)、EDCI(0.6mmol)、HOBt(0.6mmol)、Et3N(0.5ml)以及DMF(3ml)的混合溶液在25℃攪拌20小時。將該混合物倒入水中並用EtOAc萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的加成產物(PPC-111，產率43%，115mg)。 A mixed solution of amine products (PPC-091, 199 mg, 0.5 mmol), carboxylic acid (0.5 mmol), EDCI (0.6 mmol), HOBt (0.6 mmol), Et 3 N (0.5 ml) and DMF (3 ml) Stir at 25 ° C for 20 hours. The mixture was poured into water and extracted with EtOAc. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected addition product (PPC-111, 43% yield, 115 mg).

1H NMR(500MHz,MeOD)：δ 4.34-4.42(m,2H),4.43-4.47(m,2H),4.58-4.67(m,2H),6.20-6.33(m,2H),6.90-6.98(m,4H),7.03-7.09(m,2H),7.28-7.31(m,2H),7.38-7.52(m,3H),7.67-7.70(m,1H)。 1 H NMR (500MHz, MeOD): δ 4.34-4.42 (m, 2H), 4.43-4.47 (m, 2H), 4.58-4.67 (m, 2H), 6.20-6.33 (m, 2H), 6.90-6.98 ( m, 4H), 7.03-7.09 (m, 2H), 7.28-7.31 (m, 2H), 7.38-7.52 (m, 3H), 7.67-7.70 (m, 1H).

LCMS(ESI)：m/z理論值[C30H24FN3O6+H]+ 542.17，實測值542.10[M+H]+。 LCMS (ESI): m / z theoretical [C 30 H 24 FN 3 O 6 + H] + 542.17, found 542.10 [M + H] + .

合成PPC-128Synthetic PPC-128

將胺類產物(PPC-127,190mg，0.5mmol)、醯氯(0.5mmol)、Et 3 N(2.5ml)、THF(4ml)的混合溶液在25℃下攪拌16小時。將混合物倒入水中，並用EtOAc萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供期望的加成產物(PPC-128，產率7%，17.7mg)。 A mixed solution of the amine product (PPC-127, 190 mg, 0.5 mmol), amidine chloride (0.5 mmol), Et 3 N (2.5 ml), and THF (4 ml) was stirred at 25 ° C. for 16 hours. The mixture was poured into water and extracted with EtOAc. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the desired addition product (PPC-128, yield 7%, 17.7 mg).

1H NMR(500MHz,DMSO)：δ 2.26(s,3H),4.26-4.59(m,6H),4.41(s,2H),6.26以及6.32(two s,total 1H),6.39(s,1H),6.64以及6.72(two s,total 1H),6.87(d,J=8.0Hz,2H),6.96(d,J=8.0Hz,2H),7.04(m,1H),7.15-7.19(m,3H),7.41(m,1H),7.61(m,4H),8.86以及9.13(two s,total 1H)。 1 H NMR (500MHz, DMSO): δ 2.26 (s, 3H), 4.26-4.59 (m, 6H), 4.41 (s, 2H), 6.26 and 6.32 (two s, total 1H), 6.39 (s, 1H) , 6.64 and 6.72 (two s, total 1H), 6.87 (d, J = 8.0Hz, 2H), 6.96 (d, J = 8.0Hz, 2H), 7.04 (m, 1H), 7.15-7.19 (m, 3H ), 7.41 (m, 1H), 7.61 (m, 4H), 8.86 and 9.13 (two s, total 1H).

LCMS(ESI)：m/z理論值[C31H28N4O5+H]+ 536.21，實測值 536.84[M+H]+。 LCMS (ESI): m / z theoretical value [C 31 H 28 N 4 O 5 + H] + 536.21, found 536.84 [M + H] + .

合成PPC-146Synthetic PPC-146

將胺類產物(PPC-131，199mg，0.5mmol)、醯氯(0.5mmol)、Et 3 N(0.5ml)、DCM(3ml)的混合溶液在25℃下攪拌20小時。將混合物倒入水中並用DCM萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供期望的加成產物PPC-146。 A mixed solution of the amine product (PPC-131, 199 mg, 0.5 mmol), trichloromethane (0.5 mmol), Et 3 N (0.5 ml), and DCM (3 ml) was stirred at 25 ° C. for 20 hours. The mixture was poured into water and extracted with DCM. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the desired addition product PPC-146.

1H NMR(500MHz,DMSO)：δ 3.74(s,3H),4.23-4.42(m,6H),6.13-6.41(m,2H),6.63(d,J=34.3Hz,1H),7.08-7.16(m,5H),7.23-7.27(m,1H),7.48-7.66(m,4H),7.69-7.73(m,3H),8.78-8.83(m,1H),10.62(d,J=25.5Hz,1H)。 1 H NMR (500MHz, DMSO): δ 3.74 (s, 3H), 4.23-4.42 (m, 6H), 6.13-6.41 (m, 2H), 6.63 (d, J = 34.3Hz, 1H), 7.08-7.16 (m, 5H), 7.23-7.27 (m, 1H), 7.48-7.66 (m, 4H), 7.69-7.73 (m, 3H), 8.78-8.83 (m, 1H), 10.62 (d, J = 25.5Hz , 1H).

LCMS(ESI)：m/z理論值[C32H27F3N4O5+H]+ 605.19，實測值605.23[M+H]+。 LCMS (ESI): m / z theoretical value [C 32 H 27 F 3 N 4 O 5 + H] + 605.19, found 605.23 [M + H] + .

合成PPC157Synthetic PPC157

將N'-((第三丁基氧基)羰基)-N,N-二取代亞氨基二乙酸二醯胺(1.18mmol)溶解於TFA：DCM(v/v)=1：1中，將該混合物在25℃下攪拌1小時。真空除去溶劑。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的產物(PPC-157，產率99%，529mg)。 Dissolve N '-((third butyloxy) carbonyl) -N, N-disubstituted iminodiacetic acid diamidine (1.18 mmol) in TFA: DCM (v / v) = 1: 1, and The mixture was stirred at 25 ° C for 1 hour. The solvent was removed in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected product (PPC-157, 99% yield, 529 mg).

1H NMR(500MHz,DMSO)：δ 3.27-3.48(m,4H),3.50(s,2H), 3.65(s,2H),3.72(s,2H),4.12(s,2H),4.35(d,J=5.30Hz,2H),6.32(s,1H),6.42(s,1H),7.09(d,J=8.43Hz,2H),7.54(d,J=8.43Hz,2H),7.61(s,1H),8.87(s,1H),8.97(s,1H)。 1 H NMR (500MHz, DMSO): δ 3.27-3.48 (m, 4H), 3.50 (s, 2H), 3.65 (s, 2H), 3.72 (s, 2H), 4.12 (s, 2H), 4.35 (d , J = 5.30Hz, 2H), 6.32 (s, 1H), 6.42 (s, 1H), 7.09 (d, J = 8.43Hz, 2H), 7.54 (d, J = 8.43Hz, 2H), 7.61 (s , 1H), 8.87 (s, 1H), 8.97 (s, 1H).

LCMS(ESI)：m/z理論值[C20H23F3N4O3+H]+ 425.17，實測值425.25[M+H]+。 LCMS (ESI): m / z theoretical value [C 20 H 23 F 3 N 4 O 3 + H] + 425.17, found 425.25 [M + H] + .

合成PPC-163Synthetic PPC-163

將胺類產物(PPC-162，199mg，0.5mmol)、醯氯(0.5mmol)、Et3N(0.5ml)以及DCM(3ml)的混合溶液在25℃下攪拌20小時。將混合物倒入水中並用DCM萃取。有機相用飽和食鹽水NaCl(aq)洗滌。將有機相乾燥(MgSO4)、過濾並真空濃縮。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的加成產物(PPC-163，%，mg)。 A mixed solution of the amine product (PPC-162, 199 mg, 0.5 mmol), trichloromethane (0.5 mmol), Et 3 N (0.5 ml), and DCM (3 ml) was stirred at 25 ° C. for 20 hours. The mixture was poured into water and extracted with DCM. The organic phase was washed with saturated brine NaCl (aq) . The organic phase was dried (MgSO 4), filtered and concentrated in vacuo. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected addition product (PPC-163,%, mg).

1H NMR(500MHz,DMSO)：δ 4.34(d,J=9.75Hz,2H),4.52-4.64(m,4H),6.73(s,1H),6.93-7.11(m,6H),7.20(s,1H),7.34-7.38(m,2H),7.42-7.44(m,1H),7.58-7.66(m,3H),8.54-8.60(m,2H),8.69(d,J=11.65Hz,1H),9.22(d,J=110.70Hz,1H),10.72(d,J=239.20Hz,1H),11.68(d,J=21.95Hz,1H)。 1 H NMR (500MHz, DMSO): δ 4.34 (d, J = 9.75Hz, 2H), 4.52-4.64 (m, 4H), 6.73 (s, 1H), 6.93-7.11 (m, 6H), 7.20 (s , 1H), 7.34-7.38 (m, 2H), 7.42-7.44 (m, 1H), 7.58-7.66 (m, 3H), 8.54-8.60 (m, 2H), 8.69 (d, J = 11.65Hz, 1H ), 9.22 (d, J = 110.70 Hz, 1H), 10.72 (d, J = 239.20 Hz, 1H), 11.68 (d, J = 21.95 Hz, 1H).

LCMS(ESI)：m/z理論值[C30H26N6O4+H]+ 535.20，實測值535.12[M+H]+。 LCMS (ESI): m / z theoretical value [C 30 H 26 N 6 O 4 + H] + 535.20, found 535.12 [M + H] + .

合成PPC-168Synthetic PPC-168

將胺(PPC-162，199mg，0.5mmol)、異氰醯基環丙烷(0.5mmol)以及DCM(3ml)的溶液在25℃下攪拌20小時。將混合物真空濃縮去除溶劑。通過MPLC(DCM/MeOH=19/1)純化殘餘物以提供預期的加成產物PPC-168。 A solution of the amine (PPC-162, 199 mg, 0.5 mmol), isocyanatocyclopropane (0.5 mmol), and DCM (3 ml) was stirred at 25 ° C for 20 hours. The mixture was concentrated in vacuo to remove the solvent. The residue was purified by MPLC (DCM / MeOH = 19/1) to provide the expected addition product PPC-168.

1H NMR(500MHz,DMSO)：δ 0.33-0.37(m,2H),0.50-0.54(m,2H),4.03(d,J=6.65Hz,4H),4.45-4.50(m,2H),6.70(d,J=2.60Hz,1H),6.92-7.01(m,4H),7.09(t,J=7.35Hz,1H),7.36(t,J=8.35Hz,2H),7.57-7.65(m,2H),8.51-8.64(m,3H),9.02(s,1H),10.58(s,1H)。 1 H NMR (500MHz, DMSO): δ 0.33-0.37 (m, 2H), 0.50-0.54 (m, 2H), 4.03 (d, J = 6.65Hz, 4H), 4.45-4.50 (m, 2H), 6.70 (d, J = 2.60 Hz, 1H), 6.92-7.01 (m, 4H), 7.09 (t, J = 7.35Hz, 1H), 7.36 (t, J = 8.35Hz, 2H), 7.57-7.65 (m, 2H), 8.51-8.64 (m, 3H), 9.02 (s, 1H), 10.58 (s, 1H).

LCMS(ESI)：m/z理論值[C25H26N6O4+H]+ 475.20，實測值475.21[M+H]+。 LCMS (ESI): m / z theoretical value [C 25 H 26 N 6 O 4 + H] + 475.20, found 475.21 [M + H] + .

[實施例2]：使用式(I)化合物進行抑制PCSK9分泌試驗。     [Example 2]: A PCSK9 secretion test was performed using a compound of formula (I).    

建立抑制PCSK9分泌的細胞篩選試驗，藉由暫時性轉染系統於293細胞中含有人類基因完整長度PCSK9序列(NCBI參考識別，NM_1749 36.2)的pCMV-PCSK9-Myc-DDK載體(OriGene Technologies)來進行。 Establish a cell screening test for inhibiting PCSK9 secretion, by using a transient transfection system in 293 cells containing the full-length PCSK9 sequence of human genes (NCBI reference recognition, NM_1749 36.2) pCMV-PCSK9-Myc-DDK vector (OriGene Technologies) .

將293細胞培養於含有10%胎牛血清的DMEM中的6孔盤中。第二天早上用GeneJuice轉染試劑(Novagene)PCSK9重組構建體轉染細胞。24小時後，通過胰蛋白酶處理收集轉染的細胞，並重新培養於含有10%FBS的DMEM培養基的96孔盤中，以每孔2×104個細胞的密度培養。將化合物以連續稀釋液溶於DMSO中，然後加入到測定盤中，使終濃度為30或10μM，然後再培養43小時。在PCSK9測定前5小時，將細胞培養基更換為含有相同濃度的化合物或載體的無血清DMEM培養基。收集組織培養基，並根據製造商的實驗步驟，以AlphaLISA kit(Alpha Kits # AL270C，PerkinElmer)測定分泌到細胞培養基中的PCSK9的量。使用EnSpire Multilabel儀器讀取AlphaLISA®數值(每秒計數)。 同時建立含有0.5%DMSO的培養基作為對照，實驗二重複進行。 293 cells were cultured in 6-well dishes in DMEM containing 10% fetal bovine serum. Cells were transfected the next morning with GeneJuice transfection reagent (Novagene) PCSK9 recombinant construct. After 24 hours, the transfected cells were collected by trypsin treatment, and re-cultured in 96-well dishes containing 10% FBS DMEM medium, and cultured at a density of 2 × 10 4 cells per well. The compounds were dissolved in DMSO and serially diluted, and then added to the assay plate to give a final concentration of 30 or 10 μ M, and then cultured for 43 hours. Five hours before the PCSK9 assay, the cell culture medium was changed to a serum-free DMEM medium containing the same concentration of compound or carrier. Tissue culture medium was collected and the amount of PCSK9 secreted into the cell culture medium was determined with an AlphaLISA kit (Alpha Kits # AL270C, PerkinElmer) according to the manufacturer's experimental procedure. AlphaLISA® values (counts per second) were read using an EnSpire Multilabel instrument. At the same time, a medium containing 0.5% DMSO was established as a control, and experiment two was repeated.

實驗結果如下表1，是藉由DMSO對照組的百分比表示，其使用下式計算：PCSK9分泌量(%)=Stc/Sc×100(%) The experimental results are shown in Table 1 below, which is expressed by the percentage of the DMSO control group, which is calculated using the following formula: PCSK9 secretion (%) = S tc / S c × 100 (%)

Stc是待測化合物存在下的AlphaLISA®數值 S tc is the AlphaLISA® value in the presence of the test compound

Sc是DMSO對照組的AlphaLISA®數值 S c is the AlphaLISA® value of the DMSO control group

[實施例3]：式(I)化合物的細胞毒性試驗     [Example 3]: Cytotoxicity test of compound of formula (I)    

為了排除由於細胞毒性的原因造成PCSK9分泌減少而進行細胞毒性試驗，將293細胞在測定培養基中以2×104個細胞/孔的密度培養於96孔盤中。每孔加入30或10μM的化合物。培養48小時後，使用MTS試劑(3-(4,5-二甲基噻唑-2-基)-5-(3-羥基甲氧基苯基)-2-(4-磺酸基苯基)-2H-四唑，Promega)評估細胞活性並記錄於表2。 In order to exclude the cytotoxicity caused by the reduction of PCSK9 secretion due to cytotoxicity, a cytotoxicity test was performed, and 293 cells were cultured in a measurement medium at a density of 2 × 10 4 cells / well in a 96-well plate. Add 30 or 10 μM of compound to each well. After 48 hours of incubation, use MTS reagent (3- (4,5-dimethylthiazol-2-yl) -5- (3-hydroxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, Promega). Cell viability was evaluated and reported in Table 2.

[實施例4]：在HepG2細胞中檢測LDL的西方墨點法(Westem Blot)     [Example 4]: Westem Blot for detecting LDL in HepG2 cells    

HepG2細胞裂解物藉由SDS-PAGE電泳而分離，並轉移至聚偏二氟乙烯(PVDF)膜上。將膜與人類抗體LDLR培養，然後再與1：2000稀釋度的HRP綴合的第二抗體與初級Igs進行培養，西方墨點法的分析結果請參閱圖1。 HepG2 cell lysates were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was cultured with the human antibody LDLR, and then cultured with the primary antibody Igs conjugated with the HRP-conjugated secondary antibody at a 1: 2000 dilution. The results of the Western blot method are shown in Figure 1.

[實施例5]：藉由ELISA對Herceptin Ab分泌量進行定量     [Example 5]: Quantification of Herceptin Ab secretion by ELISA    

本發明實施例所提供的化合物對PCSK9分泌抑制的選擇性抑制作用藉由計數篩選進行測試，用以測試其對另一種穩定細胞系表達的活性和分泌赫賽汀(Herceptin)抗體。該測定是使用識別不同赫賽汀抗原表位的兩種抗體(A和B)的三明治免疫分析法。簡而言之，將等份的稀釋的培養基覆蓋在用抗-赫賽汀抗體A塗覆的孔上。培養1小時後，洗滌各孔，用HRP綴合的抗-Herceptin抗體B溶液覆蓋，並培養1小時。將它們再次洗滌，並用四甲基聯苯胺溶液覆蓋作為HRP的呈色受質。15分鐘後，用硫酸銨終止反應，用分光光度法在450nm測定反應混合物的吸光度。上述所有的步驟都是在室溫下進行的。 The compounds of the present invention provide selective inhibition of PCSK9 secretion inhibition by counting screening tests to test its activity on the expression of another stable cell line and secretion of Herceptin antibody. The assay is a sandwich immunoassay using two antibodies (A and B) that recognize different Herceptin epitopes. Briefly, aliquots of the diluted medium were covered on wells coated with anti-Herceptin antibody A. After 1 hour incubation, each well was washed, covered with a HRP-conjugated anti-Herceptin antibody B solution, and incubated for 1 hour. They were washed again and covered with tetramethylbenzidine solution as a color receptor for HRP. After 15 minutes, the reaction was terminated with ammonium sulfate, and the absorbance of the reaction mixture was measured spectrophotometrically at 450 nm. All the above steps are performed at room temperature.

[實施例6]：本發明實施例的化合物對LDL的攝取實驗     [Example 6]: LDL uptake experiment of the compound of the example of the present invention    

將Hep G2細胞以2×104個細胞/孔的細胞密度及0.1ml 10%FBS DMEM培養基條件下培養於96黑色底孔盤中，並在37℃培養72小時。用PBS沖洗，用0.1ml含有DMSO或10μM、3μM以及1μM濃度的化合物的10% Charcoal free FBS DMEM培養基覆蓋，再於37℃培養24小時。 Hep G2 cells were cultured in a 96-well black plate at a cell density of 2 × 10 4 cells / well and 0.1 ml of 10% FBS DMEM medium, and cultured at 37 ° C. for 72 hours. Washed with PBS containing 0.1ml DMSO or with 10 μ M, 10% Charcoal free FBS DMEM medium 3 μ M and 1 μ M concentration of the compound covered, incubated at 37 ℃ 24 hours.

為了測定LDL攝取能力，用預熱37℃的無血清DMEM沖洗細胞，並覆蓋0.1ml無血清DMEM，在37℃培養1小時。將它們用含有10μg/ml硼-二毗咯亞甲基類化合物-LDL(bodipy-LDL)的0.1ml無血清DMEM覆蓋，然後在於37℃孵育2.5小時使LDLR-介導的螢光脂蛋白產生內吞作用。使用冰冷的PBS/0.4%FBS終止該過程。使用0.2ml冰冷的PBS/0.4%FBS沖洗4次後，將細胞用0.15ml 0.1%SDS以及0.1N NaH溶液在避光條件振盪10分鐘。在SpectraMax i3X熒光板閱讀器(Molecular Devices)中分別在485nm及525nm的激發和發射波長下測量細胞內瑩光。藉由將細胞在含有bodipy-LDL(10μg/ml)以及50倍過量的非螢光LDL(500μg/ml)的培養基中培養來測量非特異性螢光，LDL攝取結果如表 3所示。 To determine the LDL uptake capacity, cells were washed with serum-free DMEM preheated at 37 ° C, covered with 0.1 ml of serum-free DMEM, and cultured at 37 ° C for 1 hour. They containing 10 μ g / ml of boron - 0.1ml serum free DMEM two adjoining covering slightly Methylene Compounds -LDL (bodipy-LDL), and that the 2.5 hours at 37 ℃ so LDLR- lipid-mediated fluorescence The protein produces endocytosis. The process was terminated using ice-cold PBS / 0.4% FBS. After washing 4 times with 0.2ml ice-cold PBS / 0.4% FBS, the cells were shaken with 0.15ml 0.1% SDS and 0.1N NaH solution for 10 minutes in the dark. Intracellular fluorescence was measured in a SpectraMax i3X fluorescent plate reader (Molecular Devices) at excitation and emission wavelengths of 485 nm and 525 nm, respectively. The cells were cultured by well in a medium containing 50-fold bodipy-LDL (10 μ g / ml) of excess non-fluorescent LDL (500 μ g / ml) to measure the non-specific fluorescence, LDL uptake results shown in Table 3 As shown.

[實施例的有益效果]     [Advantageous Effects of the Embodiment]    

本發明的其中一有益效果在於，本發明所提供的化合物、醫藥組成物及其用途，其能通過“式(I)的化合物”以及“施用有效量的式(I)的化合物”的技術方案，下調PCSK9的表達水平，以治療個體的前蛋白轉化酶枯草桿菌蛋白酶kexin 9型相關疾病。 One of the beneficial effects of the present invention is that the compound, the pharmaceutical composition and the application provided by the present invention can pass the technical scheme of "the compound of formula (I)" and "administer an effective amount of the compound of formula (I)" , Down-regulate the expression level of PCSK9 to treat individuals' preprotein-converting enzyme subtilisin kexin type 9-related diseases.

進一步而言，本發明式(I)化合物具有調節PCSK9的生理作用，包括其與低密度脂蛋白受體(LDL-R)的相互作用。這些小分子的PCSK9調節劑可用於降低血液中的低密度脂蛋白膽固醇(LDL-C)水平，並可用於預防及/或治療膽固醇與脂蛋白代謝紊亂疾病，如冠心病、心肌梗塞、動脈粥樣硬化以及高膽固醇血症。 Furthermore, the compound of formula (I) of the present invention has a physiological effect on PCSK9, including its interaction with the low-density lipoprotein receptor (LDL-R). These small-molecule PCSK9 modulators can be used to reduce low-density lipoprotein cholesterol (LDL-C) levels in the blood and can be used to prevent and / or treat disorders of cholesterol and lipoprotein metabolism disorders such as coronary heart disease, myocardial infarction, atherosclerosis Sclerosis and hypercholesterolemia.

以上所公開的內容僅為本發明的優選可行實施例，並非因此侷限本發明的申請專利範圍，所以凡是運用本發明說明書及圖式內容所做的等效技術變化，均包含於本發明的申請專利範圍內。 The contents disclosed above are only the preferred and feasible embodiments of the present invention, and therefore do not limit the scope of patent application of the present invention. Therefore, any equivalent technical changes made by using the description and drawings of the present invention are included in the application of the present invention. Within the scope of the patent.

Claims (22)

一種化合物，其具有式(I)結構： 其中，R 1為雜芳基；R 2、R 3、R 5及R 6各自獨立地為H、鹵素、OH、CN、NH 2、C 1-6烷基、C 1-6烷氧基、C 2-10烯基、C 2-10炔基、C 3-10環烷基、C 1-6烷基氨基或C 3-10環烷基氨基；R 4為C 1-6烷基、C 1-6烷氧基、C 3-10環烷基、C 1-6烷基氨基、C 3-10環烷基氨基或芳基；R 7為C 1-6烷基、C 2-10烯基、C 2-10炔基、C 3-10環烷基、C 1-10雜環烷基、芳基或雜芳基；每個L 1和L 2獨立地為直接鍵、O或NH；m是1、2或3；以及n是0、1、2或3；其中，C 1-6烷基、C 1-6烷氧基、C 2-10烯基、C 2-10炔基、C 3-10環烷基、C 1-10雜環烷基、C 1-6烷基氨基、C 3-10環烷基氨基、芳基和雜芳基中的每一個是OH、CN、NH 2、C 1-6烷基、C 1-6烷氧基、C 2-10烯基、C 2-10炔基、C 3-10環烷基、C 1-10雜環烷基、芳基或雜芳基。 A compound having the structure of formula (I): Wherein R 1 is heteroaryl; R 2 , R 3 , R 5 and R 6 are each independently H, halogen, OH, CN, NH 2 , C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-6 alkylamino, or C 3-10 cycloalkylamino; R 4 is C 1-6 alkyl, C 1-6 alkoxy, C 3-10 cycloalkyl, C 1-6 alkylamino, C 3-10 cycloalkylamino or aryl; R 7 is C 1-6 alkyl, C 2-10 ene Group, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, aryl or heteroaryl; each L 1 and L 2 is independently a direct bond, O or NH; m is 1, 2 or 3; and n is 0, 1, 2 or 3; wherein C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, Each of C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, C 1-6 alkylamino, C 3-10 cycloalkylamino, aryl, and heteroaryl is OH, CN, NH 2 , C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, C 1-10 heterocycloalkyl, aryl Or heteroaryl. 如請求項1所述的化合物，其中，R 1是五員雜芳基；R 2、R 3、R 5及R 6各自獨立地為H或C 1-6烷氧基；R 4是C 1-6烷基或芳基；且R 7為C 1-6烷基、C 1-10雜環烷基或雜芳基。 The compound according to claim 1, wherein R 1 is a five-membered heteroaryl group; R 2 , R 3 , R 5, and R 6 are each independently H or C 1-6 alkoxy; R 4 is C 1 -6 alkyl or aryl; and R 7 is C 1-6 alkyl, C 1-10 heterocycloalkyl or heteroaryl. 如請求項2所述的化合物，其中，L 1是O，且L 2為直接鍵或NH。 The compound according to claim 2, wherein L 1 is O, and L 2 is a direct bond or NH. 如請求項3所述的化合物，其中，m是1或2，且n是0、1或2。     The compound according to claim 3, wherein m is 1 or 2 and n is 0, 1 or 2.     如請求項2所述的化合物，其中，R 1是呋喃基或噻吩基。 The compound according to claim 2, wherein R 1 is furyl or thienyl. 如請求項5所述的化合物，其中，R 2、R 3、R 5及R 6各自為H， R 4是芳基，以及R 7是雜芳基。 The compound according to claim 5, wherein R 2 , R 3 , R 5, and R 6 are each H, R 4 is an aryl group, and R 7 is a heteroaryl group. 如請求項5所述的化合物，其中，L 1是O，且L 2為直接鍵或NH。 The compound according to claim 5, wherein L 1 is O, and L 2 is a direct bond or NH. 如請求項7所述的化合物，其中，m是1或2，且n是0、1或2。     The compound according to claim 7, wherein m is 1 or 2 and n is 0, 1 or 2.     如請求項8所述的化合物，其中，R 2、R 3、R 5及R 6各自為H，R 4是芳基，以及R 7是雜芳基。 The compound according to claim 8, wherein R 2 , R 3 , R 5, and R 6 are each H, R 4 is an aryl group, and R 7 is a heteroaryl group. 如請求項1所述的化合物，其中，R 1是呋喃基或噻吩基。 The compound according to claim 1, wherein R 1 is furyl or thienyl. 如請求項10所述的化合物，其中，R 2、R 3、R 5及R 6各自為H，R 4是芳基，以及R 7是雜芳基。 The compound according to claim 10, wherein R 2 , R 3 , R 5 and R 6 are each H, R 4 is an aryl group, and R 7 is a heteroaryl group. 如請求項10所述的化合物，其中，L 1是O，且L 2為直接鍵或NH。 The compound according to claim 10, wherein L 1 is O, and L 2 is a direct bond or NH. 如請求項12所述的化合物，其中，m是1或2，且n是0、1或2。     The compound according to claim 12, wherein m is 1 or 2 and n is 0, 1 or 2.     如請求項13所述的化合物，其中，R 2、R 3、R 5及R 6各自為H，R 4是芳基，以及R 7是雜芳基。 The compound according to claim 13, wherein R 2 , R 3 , R 5 and R 6 are each H, R 4 is an aryl group, and R 7 is a heteroaryl group. 如請求項1所述的化合物，其中，所述化合物是選自下列化合物結構所組成的群組： 以及 ； 其中，X 1獨立地選自取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基、取代或未取代的芳基、雜環和雜芳基；Y獨立地選自氫、鹵素、取代或未取代的烷基、 取代或未取代的烯基、取代或未取代的環烷基；Z獨立地選自氫、鹵素、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；G a獨立地選自氫、鹵素、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；G b獨立地選自氫、鹵素、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；G c獨立地選自1至2級烷氧基、1至2級烷基氨基和鹵素基團。 The compound according to claim 1, wherein the compound is selected from the group consisting of the following compound structures: as well as ; Wherein X 1 is independently selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a heterocyclic ring, and a heteroaryl group; Y Independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl; Z is independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, Substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl; G a is independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkane G b is independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl; G c is independently selected from 1 to 2 alkoxy , 1 to 2 alkylamino and halogen groups. 如請求項1所述的化合物，其中，所述化合物是選自下列化合物結構所組成的群組： 以及 ； 其中，X 2獨立地選自取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基、取代或未取代的芳基、雜環和雜芳基；Y獨立地選自氫、鹵素、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；Z獨立地選自氫、鹵素、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；G a獨立地選自氫、鹵素，取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；G b獨立地選自氫、鹵素、取代或未取代的烷基、取代或未取代的烯基、取代或未取代的環烷基；G c獨立地選自1至2級烷氧基、1至2級烷基氨基和鹵素基團。 The compound according to claim 1, wherein the compound is selected from the group consisting of the following compound structures: as well as ; Wherein X 2 is independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, heterocycle, and heteroaryl; Y Independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl; Z is independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, Substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl; G a is independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkane G b is independently selected from hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl; G c is independently selected from 1 to 2 alkoxy , 1 to 2 alkylamino and halogen groups. 如請求項1所述的化合物，其中，所述化合物是選自下列化合物： 以及 所組成 的群組。 The compound according to claim 1, wherein the compound is selected from the following compounds: as well as A group of people. 如請求項1所述的化合物，其中，所述化合物是選自下列化合物： 、 以及 所組成的群組。 The compound according to claim 1, wherein the compound is selected from the following compounds: , as well as A group of people. 一種醫藥組成物，其包含治療有效量的如請求項1之式(I)化合物及醫藥學上可接受的載劑。     A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as claimed in claim 1 and a pharmaceutically acceptable carrier.     一種如請求項19所述的醫藥組成物的用途，其是用於製造用以治療個體的前蛋白轉化酶枯草桿菌蛋白酶kexin 9型相關疾病的藥物。     The use of the pharmaceutical composition according to claim 19 is for the manufacture of a medicament for treating a proprotein-converting enzyme subtilisin kexin type 9-related disease in an individual.     如請求項20所述的醫藥組成物的用途，其中所述前蛋白轉化酶枯草桿菌蛋白酶kexin 9型相關疾病是膽固醇及脂蛋白代謝疾病。     The use of the pharmaceutical composition according to claim 20, wherein the proprotein-converting enzyme subtilisin kexin type 9-related disease is a cholesterol and lipoprotein metabolism disease.     如請求項21所述的醫藥組成物的用途，其中所述膽固醇及脂蛋白代謝疾病是選自冠心病、心肌梗塞、動脈粥樣硬化以及高膽固醇血症所組成的群組。     The use of the pharmaceutical composition according to claim 21, wherein the cholesterol and lipoprotein metabolism disease is selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, and hypercholesterolemia.    
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