TW201915000A - Fused heteroaryl derivatives, a preparation method thereof and pharmaceutical use thereof - Google Patents
Fused heteroaryl derivatives, a preparation method thereof and pharmaceutical use thereof Download PDFInfo
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
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- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
Description
本發明屬於醫藥領域，涉及一種通式(I)所示的新的稠合雜芳基衍生物、其製備方法及含有該衍生物的醫藥組成物以及其作為治療劑，特別是作為TLR7激動劑的用途。 The present invention belongs to the field of medicine, and relates to a new fused heteroaryl derivative represented by the general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative, and a therapeutic agent, particularly a TLR7 agonist. the use of.
類Toll受體(toll-like receptors；TLRs)是參與先天免疫的一類重要蛋白質分子。TLRs是單體跨膜的非催化性受體，通常在崗哨細胞如巨噬細胞和樹突狀細胞中表達，可以識別由微生物產生的結構保守的分子。一旦這些微生物突破如皮膚或腸道黏膜的物理屏障，就會被TLRs識別，繼而激活免疫細胞應答(Mahla,R S.等人，Front Immunol.4：248(2013))。免疫系統之所以具有廣泛識別病原微生物的能力，某種程度上是由於Toll樣免疫受體的廣泛存在。 Toll-like receptors (TLRs) are an important class of protein molecules involved in innate immunity. TLRs are monomeric non-catalytic receptors that are commonly expressed in sentinel cells such as macrophages and dendritic cells, and can recognize structurally conserved molecules produced by microorganisms. Once these microorganisms break through physical barriers such as the skin or intestinal mucosa, they are recognized by TLRs, which in turn activates the immune cell response (Mahla, RS, et al., Front Immunol. 4: 248 (2013)). The ability of the immune system to recognize pathogenic microorganisms is partly due to the widespread presence of Toll-like immune receptors.
在哺乳動物中至少有10種不同的TLRs。一些此類受體的配體和相應的信號級聯放大已經被鑒定出。TLR7是TLRs(TLRs 3、7、8和9)亞組的成員，侷限於專門檢測非己核酸的細胞的內涵體隔室。TLR7在藉由識別ssRNA抗 病毒防禦方面起關鍵作用(Diebold S.S.等，Science,2004：303,1529-1531；和Lund J.M.等，PNAS,2004：101,5598-5603)。TLR7在人身上具有有限的表達分佈，並主要藉由B細胞和類漿細胞樹突細胞(pDC)表達，而較低程度地藉由單核細胞表達。類漿細胞DCs是淋巴衍生的樹突細胞的唯一群體(0.2-0.8%的外周血單核細胞(PBMCs))，它是響應病毒感染而分泌高水平干擾素-α(IFNα)和干擾素-β(IFNβ)的最初的I型干擾素生成細胞(Liu Y-J,Annu.Rev.Immunol.,2005：23,275-306)。 There are at least 10 different TLRs in mammals. Ligands and corresponding signal cascades for some of these receptors have been identified. TLR7 is a member of the TLRs (TLRs 3, 7, 8, and 9) subgroup and is restricted to the endosome compartment of cells that specifically detect non-nucleic acids. TLR7 plays a key role in antiviral defense by identifying ssRNAs (Diebold S.S. et al., Science, 2004: 303, 1529-1531; and Lund J.M. et al., PNAS, 2004: 101, 5598-5603). TLR7 has a limited expression profile in humans and is mainly expressed by B cells and plasma-like dendritic cells (pDC), and to a lesser extent by monocytes. Plasmaid DCs are the only population of lymphoid-derived dendritic cells (0.2-0.8% of peripheral blood mononuclear cells (PBMCs)) that secrete high levels of interferon-α (IFNα) and interferon- β (IFNβ) was the first type I interferon-producing cell (Liu YJ, Annu. Rev. Immunol., 2005: 23, 275-306).
很多疾病、障礙與TLRs的異常有關，比如黑色素瘤、非小細胞肺癌、肝細胞癌、基底細胞癌(basalcellcarcinoma)、腎細胞癌、骨髓瘤、變應性鼻炎、哮喘、慢性阻塞性肺炎(COPD)、潰瘍性結腸炎、肝纖維化，HBV、黃病毒科(Flaviviridae)病毒、HCV、HPV、RSV、SARS、HIV或流行性感冒的病毒感染等。因此運用TLRs的激動劑治療相關疾病是很有前景的。 Many diseases and disorders are related to abnormal TLRs, such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, basalcellcarcinoma, renal cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia (COPD ), Ulcerative colitis, liver fibrosis, HBV, Flaviviridae virus, HCV, HPV, RSV, SARS, HIV or influenza virus infection, etc. Therefore, the use of agonists of TLRs to treat related diseases is very promising.
由於TLR7和TLR8高度同源，因此TLR7配體，在大多數情況下也是TLR8配體。TLR8刺激主要誘導產生細胞因子如腫瘤壞死因子α(TNF-α)和趨化因子。干擾素α是治療慢性乙型肝炎或丙型肝炎的主要藥物之一，而TNF-α是一種促炎細胞因子，過多分泌可能導致嚴重的副作用。所以對TLR7和TLR8的選擇性對於開發TLR7激動劑用於治療病毒感染性疾病至關重要。 Because TLR7 and TLR8 are highly homologous, TLR7 ligands are also TLR8 ligands in most cases. TLR8 stimulation mainly induces the production of cytokines such as tumor necrosis factor alpha (TNF-α) and chemokines. Interferon alpha is one of the main drugs for the treatment of chronic hepatitis B or C, and TNF-α is a pro-inflammatory cytokine. Excessive secretion may cause serious side effects. Therefore, selectivity for TLR7 and TLR8 is essential for developing TLR7 agonists for the treatment of viral infectious diseases.
目前已有相關的TLR7激動劑專利申請，如 WO2005025583、WO2007093901、WO2008011406、WO2009091032、WO2010077613、WO2010133882、WO2011031965、WO2012080730。但是仍有必要繼續研發安全的和治療上更有效的TLR7激動劑。 There are currently related patent applications for TLR7 agonists, such as WO2005025583, WO2007093901, WO2008011406, WO2009091032, WO2010077613, WO2010133882, WO2011031965, WO2012080730. However, there is still a need to continue to develop safer and more therapeutically effective TLR7 agonists.
本發明針對上述技術問題，提供一種起效濃度更低，選擇性更好，激活效果更明顯的藥物化合物，同時，其對CYP和hERG沒有抑制作用或抑制作用弱，是更安全和更有效的TLR7激動劑。 The invention aims at the above technical problems, and provides a drug compound with a lower effective concentration, better selectivity, and more obvious activation effect. At the same time, it has no inhibitory effect or weak inhibitory effect on CYP and hERG, and is safer and more effective. TLR7 agonist.
本發明的目的在於提供一種通式(I)所示的化合物：
在本發明一個較佳的實施方案中，所述的通式(I)所示的化合物，其為通式(II)所示的化合物：
在本發明一個較佳的實施方案中，所述的通式(I)所示 的化合物，其為通式(IIAa)所示的化合物：
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其中該R2選自芳基、雜芳基、雜環基和-NR6R7，其中該芳基、雜芳基和雜環基各自獨立地視需要被選自鹵素、烷基、烷氧基、鹵烷基、鹵烷氧基、羥基、羥烷基、氰基、胺基、硝基、環烷基、雜環基、雜環基烷基和-X2-NR6R7中的一個或多個取代基所取代；X2、R6和R7如通式(I)中所定義。 In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein the R 2 is selected from the group consisting of an aryl group, a heteroaryl group, a heterocyclic group, and -NR 6 R 7 , wherein the aryl group, Heteroaryl and heterocyclyl are each independently selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, and cycloalkane , Heterocyclyl, heterocyclylalkyl, and -X 2 -NR 6 R 7 are substituted with one or more substituents; X 2 , R 6 and R 7 are as defined in general formula (I).
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其中該R2選自苯基、吡啶基、吡咯烷基和-NR6R7，其中該苯基、吡啶基和吡咯烷基各自獨立地視需要被選自鹵素、烷基、烷氧基、鹵烷基、鹵烷氧基、羥基、羥烷基、氰基、胺基、硝基、雜環基烷基和-X2-NR6R7中的一個或多個取代基所取代，該雜環基烷基較佳吡咯烷基伸甲基；X2、R6和R7如通式(I)中所定義。 In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein R 2 is selected from phenyl, pyridyl, pyrrolidinyl, and -NR 6 R 7 , wherein the phenyl, pyridine And pyrrolidinyl are each independently selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, heterocycloalkyl And -X 2 -NR 6 R 7 are substituted by one or more substituents, and the heterocyclyl alkyl group is preferably pyrrolidinylmethyl; X 2 , R 6 and R 7 are as in the general formula (I) As defined.
在本發明一個較佳的實施方案中，該通式(I)所示的化 合物，其為通式(III)或通式(IV)所示的化合物：
在本發明一個較佳的實施方案中，所述的通式(I)所示的化合物，其中該X1為伸烷基。 In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein X 1 is an alkylene group.
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其中該G1為N。 In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein G 1 is N.
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其為通式(V)或通式(VI)所示的化合物：
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其為通式(VII)所示的化合物：
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其中該L1為-O-。 In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein the L 1 is -O-.
在本發明一個較佳的實施方案中，該通式(I)所示的化合物，其中該R1為烷基。 In a preferred embodiment of the present invention, the compound represented by the general formula (I), wherein R 1 is an alkyl group.
本發明的典型化合物包括但不限於：
本發明的另一方面涉及一種通式(IA)所示的化合物，
通式(IA)所示的化合物包括，但不限於：
本發明的另一方面涉及一種通式(IIA)所示的化合物，
本發明的另一方面涉及一種通式(IIB)所示的化合物，
通式(IIB)所示的化合物包括，但不限於：
本發明的另一方面涉及一種製備通式(I)所示的化合物的方法，該方法包括：
通式(IA)的化合物脫去保護基得到通式(I)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；G1、L1、X1、R1和R2如通式(I)中所定義。 The compound of the general formula (IA) is deprotected to obtain a compound of the general formula (I); wherein: W is an amine protecting group, and is selected from the group consisting of a third butoxycarbonyl group, an ethylfluorenyl group, a benzyl group, an allyl group and p-formyl methoxybenzyl, preferably p-methoxybenzyl; R a is a hydrogen atom or an amino protecting group, the amino protecting group is selected from tertiary-butoxycarbonyl, acetyl, benzyl, allyl, and p-toluenesulfonic Oxybenzyl, preferably p-methoxybenzyl; G 1 , L 1 , X 1 , R 1 and R 2 are as defined in general formula (I).
本發明的另一方面涉及一種製備通式(IIA)所示的化合物的方法，該方法包括：
通式(IIB)的化合物在鹼性條件下，發生消除反應得到通式(IIA)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁 氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；G1、L1、X1、R1和R2如通式(IIA)中所定義。 The compound of the general formula (IIB) undergoes an elimination reaction under basic conditions to obtain a compound of the general formula (IIA); wherein: W is an amine protecting group, and is selected from the group consisting of a third butoxycarbonyl group, an ethylfluorenyl group, a benzyl group, and an alkylene group. and propyl p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is a hydrogen atom or an amino protecting group, the amino protecting group is selected from tertiary-butoxycarbonyl, acetyl, benzyl, alkenyl and propyl p-methoxybenzyl group, preferably a p-methoxybenzyl group; G 1, L 1, X 1, R 1 and R 2 as in formula (IIA) as defined above.
本發明的另一方面涉及一種製備通式(II)所示的化合物的方法，該方法包括：
通式(IIA)的化合物在酸性條件下脫去保護基得到通式(II)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；G1、L1、X1、R1和R2如通式(II)中所定義。 A compound of the general formula (IIA) is deprotected under acidic conditions to obtain a compound of the general formula (II); wherein: W is an amine protecting group, and is selected from the group consisting of a third butoxycarbonyl group, an ethylfluorenyl group, a benzyl group, and an allyl group. group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is a hydrogen atom or an amino protecting group, the amino protecting group is selected from tertiary-butoxycarbonyl, acetyl, benzyl, allyl And p-methoxybenzyl, preferably p-methoxybenzyl; G 1 , L 1 , X 1 , R 1 and R 2 are as defined in general formula (II).
本發明的另一方面涉及一種醫藥組成物，該醫藥組成物含有治療有效量的通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式或其可藥用的鹽，以及一種或多種藥學上可接受的載體、稀釋劑或賦形劑。 Another aspect of the present invention relates to a pharmaceutical composition containing a therapeutically effective amount of a compound represented by the general formula (I) or a tautomer, a meso, a racemate, an enantiomer thereof. A conformer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
本發明進一步涉及通式(I)所示化合物或其互變異構 體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用的鹽或包含其的醫藥組成物在製備用於激動TLR7的藥物中的用途。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a Use of a pharmaceutically acceptable salt or a pharmaceutical composition containing the same in the manufacture of a medicament for stimulating TLR7.
本發明進一步涉及通式(I)所示化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用的鹽或包含其的醫藥組成物在製備用於治療由病毒引起的感染的藥物中的用途。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a Use of a pharmaceutically acceptable salt or a pharmaceutical composition comprising the same in the manufacture of a medicament for the treatment of an infection caused by a virus.
本發明進一步涉及通式(I)所示化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用的鹽或包含其的醫藥組成物在製備用於治療或預防腫瘤的藥物中的用途。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a Use of a pharmaceutically acceptable salt or a pharmaceutical composition comprising the same in the manufacture of a medicament for treating or preventing a tumor.
本發明進一步涉及一種激動TLR7的方法，其包括將通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的醫藥組成物與TLR7接觸的步驟。 The present invention further relates to a method for stimulating TLR7, which comprises converting a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer A step of contacting a body, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same with TLR7.
本發明進一步涉及一種治療由病毒引起的感染的方法，該方法包括給予所需患者治療有效量的通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的醫藥組成物。 The present invention further relates to a method for treating an infection caused by a virus, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound represented by the general formula (I) or a tautomer, a meso, a racemate thereof , Enantiomers, diastereomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.
本發明進一步涉及一種治療或預防腫瘤的方法，其包括給予所需患者治療有效量的通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的醫藥 組成物。 The invention further relates to a method for treating or preventing tumors, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer thereof Isomers, diastereomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same.
本發明進一步涉及一種通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的藥物，其用作藥物。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt thereof or a medicament containing the same, which is used as a medicament.
本發明進一步涉及一種通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的藥物，其用於激動TLR7。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a drug containing the same, which is used to agonize TLR7.
本發明進一步涉及一種通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的藥物，其用於治療或預防由病毒引起的感染。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Its pharmaceutically acceptable salt or a medicament containing the same is used for treating or preventing an infection caused by a virus.
本發明中所述的病毒選自：登革熱病毒、黃熱病毒、西尼羅病毒、日本腦炎病毒、蜱傳腦炎病毒、昆津病毒、墨累山谷腦炎病毒、聖路易腦炎病毒、鄂木斯克出血熱病毒、牛病毒性腹瀉病毒、濟卡病毒、HIV、HBV、HCV、HPV、RSV、SARS和流感病毒。 The virus in the present invention is selected from the group consisting of dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Murray valley encephalitis virus, Saint Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza viruses.
本發明進一步涉及一種通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式、或其可藥用鹽或包含其的藥物，其用於治療或預防腫瘤。 The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or A pharmaceutically acceptable salt or a medicament containing the same, which is used for treating or preventing a tumor.
本發明中所述的腫瘤選自：黑色素瘤、非小細胞肺癌、肝細胞癌、基底細胞癌、腎細胞癌和骨髓瘤。 The tumor in the present invention is selected from the group consisting of melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal cell carcinoma, renal cell carcinoma, and myeloma.
含活性成分的醫藥組成物可以是適用於口服的形式，例如片劑、糖錠劑、錠劑、水或油混懸液、可分散粉末或顆粒、乳液、硬或軟膠囊，或糖漿劑或酏劑。可按照本領域任何已知製備藥用組合物的方法製備口服組合物，此類組合物可含有一種或多種選自以下的成分：甜味劑、矯味劑、著色劑和防腐劑，以提供悅目和可口的藥用製劑。片劑含有活性成分和用於混合的適宜製備片劑的無毒的可藥用的賦形劑。這些賦形劑可以是惰性賦形劑，造粒劑、崩解劑，黏合劑，和潤滑劑。這些片劑可以不包衣或可藉由掩蓋藥物的味道或在胃腸道中延遲崩解和吸收，因而在較長時間內提供緩釋作用的已知技術將其包衣。 The active ingredient-containing pharmaceutical composition may be in a form suitable for oral administration, such as tablets, dragees, lozenges, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Tincture. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives to provide pleasing looks And delicious medicinal preparations. Tablets contain the active ingredients and non-toxic pharmaceutically acceptable excipients suitable for the preparation of tablets for mixing. These excipients can be inert excipients, granulating agents, disintegrating agents, binders, and lubricants. These tablets can be uncoated or they can be coated by known techniques that mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained release over a longer period.
也可用其中活性成分與惰性固體稀釋劑或其中活性成分與水溶性載體或油溶媒混合的軟明膠膠囊提供口服製劑。 Oral formulations may also be provided in soft gelatin capsules in which the active ingredient is mixed with an inert solid diluent or in which the active ingredient is mixed with a water-soluble carrier or an oil vehicle.
水懸浮液含有活性物質和用於混合的適宜製備水懸浮液的賦形劑。此類賦形劑是懸浮劑，分散劑或濕潤劑。水混懸液也可以含有一種或多種防腐劑、一種或多種著色劑、一種或多種矯味劑和一種或多種甜味劑。 Aqueous suspensions contain the active substance and excipients suitable for the preparation of the aqueous suspension for mixing. Such excipients are suspending, dispersing or wetting agents. The aqueous suspension may also contain one or more preservatives, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents.
油混懸液可藉由使活性成分懸浮於植物油，或礦物油配製而成。油懸浮液可含有增稠劑。可加入上述的甜味劑和矯味劑，以提供可口的製劑。可藉由加入抗氧化劑保存這些組合物。 Oil suspensions can be formulated by suspending the active ingredient in a vegetable or mineral oil. The oil suspension may contain a thickener. The sweeteners and flavoring agents described above can be added to provide a palatable formulation. These compositions can be preserved by the addition of antioxidants.
本發明的醫藥組成物也可以是水包油乳劑的形式。油相可以是植物油，或礦物油或其混合物。適宜的乳化劑可 以是天然產生的磷脂，乳劑也可以含有甜味劑、矯味劑、防腐劑和抗氧劑。此類製劑也可含有緩和劑、防腐劑、著色劑和抗氧劑。 The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, or a mineral oil or a mixture thereof. Suitable emulsifiers can be naturally occurring phospholipids, and emulsions can also contain sweeteners, flavoring agents, preservatives and antioxidants. Such formulations may also contain a demulcent, a preservative, a colorant, and an antioxidant.
本發明的醫藥組成物可以是無菌注射水溶液形式。可以使用的可接受的溶媒或溶劑有水、林格氏液和等滲氯化鈉溶液。無菌注射製劑可以是其中活性成分溶於油相的無菌注射水包油微乳可藉由局部大量注射，將注射液或微乳注入患者的血流中。或者，最好按可保持本發明化合物恒定循環濃度的方式給予溶液和微乳。為保持這種恒定濃度，可使用連續靜脈內遞藥裝置。這種裝置的實例是Deltec CADD-PLUS.TM.5400型靜脈注射泵。 The pharmaceutical composition of the present invention may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase, and the injection solution or microemulsion may be injected into the bloodstream of the patient by local large-volume injection. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of a compound of the invention. To maintain this constant concentration, continuous intravenous drug delivery devices can be used. An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.
醫藥組成物可以是用於肌內和皮下給藥的無菌注射水或油混懸液的形式。可按已知技術，用上述那些適宜的分散劑或濕潤劑和懸浮劑配製該混懸液。無菌注射製劑也可以是在腸胃外可接受的無毒稀釋劑或溶劑中製備的無菌注射溶液或混懸液。此外，可方便地用無菌固定油作為溶劑或懸浮介質。 The pharmaceutical composition may be in the form of a sterile injectable water or oily suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a parenterally acceptable non-toxic diluent or solvent. In addition, a sterile fixed oil can be conveniently used as a solvent or suspension medium.
可按用於直腸給藥的栓劑形式給予本發明化合物。可藉由將藥物與在普通溫度下為固體但在直腸中為液體，因而在直腸中會溶化而釋放藥物的適宜的無刺激性賦形劑混合來製備這些醫藥組成物。此類物質包括可可脂、甘油明膠、氫化植物油、各種分子量的聚乙二醇和聚乙二醇的脂肪酸酯的混合物。 The compounds of the invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum and therefore will dissolve in the rectum to release the drug. Such materials include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.
如本領域技術人員所熟知的，藥物的給藥劑量依賴於 多種因素，包括但並非限定於以下因素：所用具體化合物的活性、患者的年齡、患者的體重、患者的健康狀況、患者的行為、患者的飲食、給藥時間、給藥方式、***的速率、藥物的組合等；另外，最佳的治療方式如治療的模式、通式化合物(I)的日用量或可藥用的鹽的種類可以根據傳統的治療方案來驗證。 As known to those skilled in the art, the dosage of a drug depends on a number of factors, including but not limited to the following: the activity of the specific compound used, the age of the patient, the weight of the patient, the patient's health, the patient's behavior, Patient's diet, time of administration, mode of administration, rate of excretion, combination of drugs, etc .; In addition, the best treatment such as the mode of treatment, the daily dosage of the general compound (I) or the type of pharmaceutically acceptable salt Can be verified according to the traditional treatment plan.
除非有相反陳述，在說明書和權利要求書中使用的術語具有下述含義。 Unless stated to the contrary, terms used in the specification and claims have the following meanings.
術語“烷基”指飽和脂肪族烴基團，其為包含1至20個碳原子的直鏈或支鏈基團，較佳含有1至12個碳原子的烷基，更佳含有1至6個碳原子的烷基。非限制性實例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、第三丁基、第二丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2- 乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各種支鏈異構體等。更佳的是含有1至6個碳原子的低級烷基，非限制性實施例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、第三丁基、第二丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，當被取代時，取代基可以在任何可使用的連接點上被取代，該取代基較佳為一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基、羧基或羧酸酯基。 The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably 1 to 6 Carbon atom alkyl. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, third butyl, second butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl- 2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1 , 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl , 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-di Methylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl , 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4 -Ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3 -Ethylhexyl , 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, third butyl, Dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl Methyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Pentyl, 2,3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups, which are independently selected from alkane Alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, ring Alkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendant oxygen, carboxyl or carboxylate groups.
術語“伸烷基”指飽和的直鏈或支鏈脂肪族烴基，其具有2個從母體烷的相同碳原子或兩個不同的碳原子上除去兩個氫原子所衍生的殘基，其為包含1至20個碳原子的直鏈或支鏈基團，較佳含有1至12個碳原子，更佳含有1至6個碳原子的伸烷基。伸烷基的非限制性實例包括但不限於伸甲基(-CH2-)、1,1-伸乙基(-CH(CH3)-)、1,2-伸乙基(-CH2CH2)-、1,1-伸丙基(-CH(CH2CH3)-)、1,2-伸丙基 (-CH2CH(CH3)-)、1,3-伸丙基(-CH2CH2CH2-)、1,4-伸丁基(-CH2CH2CH2CH2-)和1,5-伸丁基(-CH2CH2CH2CH2CH2-)等。伸烷基可以是取代的或非取代的，當被取代時，取代基可以在任何可使用的連接點上被取代，該取代基較佳獨立地視需要選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基、-C(O)R5、-C(O)OR5、-S(O)mR5、-NR6R7和-C(O)NR6R7中的一個或多個取代基所取代。 The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the same carbon atom or two different carbon atoms of the parent alkane by removing two hydrogen atoms, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methyl (-CH 2- ), 1,1-ethyl (-CH (CH 3 )-), 1,2-ethyl (-CH 2 CH 2 )-, 1,1-propane (-CH (CH 2 CH 3 )-), 1,2-propane (-CH 2 CH (CH 3 )-), 1,3-propane (-CH 2 CH 2 CH 2- ), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2- ), and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -)Wait. The alkylene may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from the group consisting of alkyl, alkenyl, and alkynyl. , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy Group, cycloalkylthio group, heterocycloalkylthio group, pendant oxygen group, -C (O) R 5 , -C (O) OR 5 , -S (O) m R 5 , -NR 6 R 7 and -C (O) NR 6 R 7 is substituted with one or more substituents.
術語“烯基”指分子中含有碳碳雙鍵的烷基化合物，其中烷基的定義如上所述。烯基可以是取代的或非取代的，當被取代時，取代基較佳為一個或多個以下基團，其獨立地選自氫原子、烷基、烷氧基、鹵素、鹵烷基、羥基、羥烷基、氰基、胺基、硝基、環烷基、雜環基、芳基、雜芳基、-C(O)R5、-C(O)OR5、-S(O)mR5、-NR6R7和-C(O)NR6R7中的一個或多個取代基所取代。 The term "alkenyl" refers to an alkyl compound containing a carbon-carbon double bond in a molecule, wherein an alkyl group is as defined above. An alkenyl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a haloalkyl group, Hydroxyl, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R 5 , -C (O) OR 5 , -S (O ) m R 5 , -NR 6 R 7 and -C (O) NR 6 R 7 are substituted with one or more substituents.
術語“炔基”指分子中含有碳碳三鍵的烷基化合物，其中烷基的定義如上所述。炔基可以是取代的或非取代的，當被取代時，取代基較佳為一個或多個以下基團，其獨立地選自氫原子、烷基、烷氧基、鹵素、鹵烷基、羥基、羥烷基、氰基、胺基、硝基、環烷基、雜環基、芳基、雜芳基、-C(O)R5、-C(O)OR5、-S(O)mR5、-NR6R7和-C(O)NR6R7中的一個或多個取代基所取代。 The term "alkynyl" refers to an alkyl compound containing a carbon-carbon triple bond in a molecule, wherein the definition of an alkyl group is as described above. The alkynyl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from a hydrogen atom, an alkyl group, an alkoxy group, a halogen, a haloalkyl group, Hydroxyl, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R 5 , -C (O) OR 5 , -S (O ) m R 5 , -NR 6 R 7 and -C (O) NR 6 R 7 are substituted with one or more substituents.
術語“環烷基”指飽和或部分不飽和單環或多環環狀烴 取代基，環烷基環包含3至20個碳原子，較佳包含3至12個碳原子，更佳包含3至6個碳原子，最佳包含5至6個碳原子。單環環烷基的非限制性實例包括環丙基、環丁基、環戊基、環戊烯基、環己基、環己烯基、環己二烯基、環庚基、環庚三烯基、環辛基等；多環環烷基包括螺環、稠環和橋環的環烷基。 The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably contains 3 to 12 carbon atoms, and more preferably contains 3 to 6 carbon atoms, preferably 5 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl.
術語“螺環烷基”指5至20員的單環之間共用一個碳原子(稱螺原子)的多環基團，其可以含有一個或多個雙鍵，但沒有一個環具有完全共軛的π電子系統。較佳為6至14員，更佳為7至10員。根據環與環之間共用螺原子的數目將螺環烷基分為單螺環烷基、雙螺環烷基或多螺環烷基，較佳為單螺環烷基和雙螺環烷基。更佳為4員/4員、4員/5員、4員/6員、5員/5員或5員/6員單螺環烷基。螺環烷基的非限制性實例包括：
術語“稠環烷基”指5至20員，系統中的每個環與體系中的其他環共享毗鄰的一對碳原子的全碳多環基團，其中一個或多個環可以含有一個或多個雙鍵，但沒有一個環具有完全共軛的π電子系統。較佳為6至14員，更佳為7至10員。根據組成環的數目可以分為雙環、三環、四環或多環稠環烷基，較佳為雙環或三環，更佳為5員/5員或5員/6員雙環烷基。稠環烷基的非限制性實例包括：
術語“橋環烷基”指5至20員，任意兩個環共用兩個不直接連接的碳原子的全碳多環基團，其可以含有一個或多個雙鍵，但沒有一個環具有完全共軛的π電子系統。較佳為6至14員，更佳為7至10員。根據組成環的數目可以分為雙環、三環、四環或多環橋環烷基，較佳為雙環、三環或四環，更佳為雙環或三環。橋環烷基的非限制性實例包括：
該環烷基環可以稠合於芳基、雜芳基或雜環烷基環上，其中與母體結構連接在一起的環為環烷基，非限制性實例包括茚滿基、四氫萘基、苯并環庚烷基等；較佳為苯基并環戊基、四氫萘基。環烷基可以是視需要取代的或非取代的，當被取代時，取代基較佳為一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基、羧基或羧酸酯基。 The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl , Benzocycloheptyl and the like; phenylcyclopentyl and tetrahydronaphthyl are preferred. A cycloalkyl group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane A thio group, a heterocycloalkylthio group, a pendant oxygen group, a carboxyl group, or a carboxylate group.
術語“雜環基”指飽和或部分不飽和單環或多環環狀烴取代基，其包含3至20個環原子，其中一個或多個環原子 為選自氮、氧或S(O)m(其中m是整數0至2)的雜原子，但不包括-O-O-、-O-S-或-S-S-的環部分，其餘環原子為碳。較佳包含3至12個環原子，其中1至4個是雜原子；最佳包含3至8個環原子，其中1至3個是雜原子；最佳包含5至6個環原子，其中1至2或1至3個是雜原子。單環雜環基的非限制性實例包括吡咯烷基、咪唑烷基、四氫呋喃基、四氫吡喃基、四氫噻吩基、二氫咪唑基、二氫呋喃基、二氫吡唑基、二氫吡咯基、哌啶基、哌嗪基、嗎啉基、硫嗎啉基、高哌嗪基等，較佳為四氫吡喃基、哌啶基、吡咯烷基。多環雜環基包括螺環、稠環和橋環的雜環基。 The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent that contains 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S (O) A heteroatom of m (where m is an integer from 0 to 2), excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; most preferably contains 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; most preferably contains 5 to 6 ring atoms, of which 1 From 2 to 1 or 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuryl, dihydropyrazolyl, di Hydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl and the like are preferably tetrahydropyranyl, piperidinyl, pyrrolidinyl. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.
術語“螺雜環基”指5至20員的單環之間共用一個原子(稱螺原子)的多環雜環基團，其中一個或多個環原子為選自氮、氧或S(O)m(其中m是整數0至2)的雜原子，其餘環原子為碳。其可以含有一個或多個雙鍵，但沒有一個環具有完全共軛的π電子系統。較佳為6至14員，更佳為7至10員。根據環與環之間共用螺原子的數目將螺雜環基分為單螺雜環基、雙螺雜環基或多螺雜環基，較佳為單螺雜環基和雙螺雜環基。更佳為4員/4員、4員/5員、4員/6員、5員/5員或5員/6員單螺雜環基。螺雜環基的非限制性實例包括：
術語“稠雜環基”指5至20員，系統中的每個環與體系 中的其他環共享毗鄰的一對原子的多環雜環基團，一個或多個環可以含有一個或多個雙鍵，但沒有一個環具有完全共軛的π電子系統，其中一個或多個環原子為選自氮、氧或S(O)m(其中m是整數0至2)的雜原子，其餘環原子為碳。較佳為6至14員，更佳為7至10員。根據組成環的數目可以分為雙環、三環、四環或多環稠雜環基，較佳為雙環或三環，更佳為5員/5員或5員/6員雙環稠雜環基。稠雜環基的非限制性實例包括：
術語“橋雜環基”指5至14員，任意兩個環共用兩個不直接連接的原子的多環雜環基團，其可以含有一個或多個雙鍵，但沒有一個環具有完全共軛的π電子系統，其中一個或多個環原子為選自氮、氧或S(O)m(其中m是整數0至2)的雜原子，其餘環原子為碳。較佳為6至14員，更佳為7至10員。根據組成環的數目可以分為雙環、三環、四環或多環橋雜環基，較佳為雙環、三環或四環，更佳為雙環或三環。橋雜環基的非限制性實例包括：
該雜環基環可以稠合於芳基、雜芳基或環院基環上，其中與母體結構連接在一起的環為雜環基，其非限制性實例包括： 和等。 The heterocyclyl ring may be fused to an aryl, heteroaryl, or cyclohexyl ring. The ring connected to the parent structure is a heterocyclyl. Non-limiting examples include: with Wait.
雜環基可以是視需要取代的或非取代的，當被取代時，取代基較佳為一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基、羧基或羧酸酯基。 The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane A thio group, a heterocycloalkylthio group, a pendant oxygen group, a carboxyl group, or a carboxylate group.
術語“芳基”指具有共軛的π電子體系的6至14員全碳單環或稠合多環(也就是共享毗鄰碳原子對的環)基團，較佳為6至10員，例如苯基和萘基。該芳基環可以稠合於雜芳基、雜環基或環烷基環上，其中與母體結構連接在一起的環為芳基環，其非限制性實例包括：
術語“雜芳基”指包含1至4個雜原子、5至14個環原子的雜芳族體系，其中雜原子選自氧、硫和氮。雜芳基較佳為5至10員，含1至3個雜原子；更佳為5員或6員，含1至2個雜原子；較佳例如咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基等，較佳為咪唑基、吡唑基或嘧啶基、噻唑基；更佳為吡唑基。所述雜芳基環可以稠合於芳基、雜環基或環烷基環上，其中與母體結構連接在一起的環為雜芳基環，其非限制性實例包括：
雜芳基可以是視需要取代的或非取代的，當被取代時，取代基較佳為一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰 基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、羧基或羧酸酯基。 Heteroaryl may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio, carboxyl or carboxylate.
術語“烷氧基”指-O-(烷基)和-O-(非取代的環烷基)，其中烷基和環烷基的定義如上所述。烷氧基的非限制性實例包括：甲氧基、乙氧基、丙氧基、丁氧基、環丙氧基、環丁氧基、環戊氧基、環己氧基。烷氧基可以是視需要取代的或非取代的，當被取代時，取代基較佳為一個或多個以下基團，其獨立地選自氫原子、鹵素、烷基、烷氧基、鹵烷基、羥基、羥烷基、氰基、胺基、硝基、環烷基、雜環基、芳基、雜芳基中的一個或多個取代基所取代。 The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl and cycloalkyl are as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from a hydrogen atom, halogen, alkyl, alkoxy, halogen It is substituted with one or more substituents of alkyl, hydroxy, hydroxyalkyl, cyano, amine, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.
術語“胺基保護基”是為了使分子其它部位進行反應時胺基保持不變，用易於脫去的基團對胺基進行保護。非限制性實施例包含第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基等。這些基團可視需要地被選自鹵素、烷氧基或硝基中的1至3個取代基所取代。該胺基保護基較佳為對甲氧苄基。 The term "amino-protecting group" is to protect the amine group with an easily removable group in order to keep the amine group unchanged during the reaction of other parts of the molecule. Non-limiting examples include tertiary butoxycarbonyl, ethenyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy or nitro. The amine-protecting group is preferably p-methoxybenzyl.
術語“雜環基烷基”指被雜環基取代的烷基，其中烷基和雜環基如上所定義。 The term "heterocyclylalkyl" refers to an alkyl substituted with a heterocyclyl, wherein alkyl and heterocyclyl are as defined above.
術語“鹵烷基”指烷基被一個或多個鹵素取代，其中烷基如上所定義。 The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above.
術語“羥基”指-OH基團。 The term "hydroxy" refers to the -OH group.
術語“羥烷基”指被羥基取代的烷基，其中烷基如上所定義。 The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein the alkyl group is as defined above.
術語“鹵素”指氟、氯、溴或碘。 The term "halogen" refers to fluorine, chlorine, bromine or iodine.
術語“胺基”指-NH2。 The term "amino" refers to -NH 2.
術語“氰基”指-CN。 The term "cyano" refers to -CN.
術語“硝基”指-NO2。 The term "nitro" refers to -NO 2.
術語“側氧基”指=O。 The term "lateral oxygen" refers to = O.
“視需要”或“視需要地”意味著隨後所描述的事件或環境可以但不必發生，該說明包括該事件或環境發生或不發生地場合。例如，“視需要被烷基取代的雜環基團”意味著烷基可以但不必須存在，該說明包括雜環基團被烷基取代的情形和雜環基團不被烷基取代的情形。 "As needed" or "as needed" means that the event or environment described later can, but does not have to occur, and the description includes situations where the event or environment occurs or does not occur. For example, "heterocyclic group substituted with an alkyl group as necessary" means that the alkyl group may but need not exist, and the description includes a case where the heterocyclic group is substituted with an alkyl group and a case where the heterocyclic group is not substituted with an alkyl group .
“取代的”指基團中的一個或多個氫原子，較佳為最多5個，更佳為1至3個氫原子彼此獨立地被相應數目的取代基取代。不言而喻，取代基僅處在它們的可能的化學位置，本領域技術人員能夠在不付出過多努力的情況下確定(藉由實驗或理論)可能或不可能的取代。例如，具有游離氫的胺基或羥基與具有不飽和(如烯屬)鍵的碳原子結合時可能是不穩定的。 "Substituted" refers to one or more hydrogen atoms in a group, preferably up to 5 and more preferably 1 to 3 hydrogen atoms independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amine or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (eg, olefinic) bond.
“醫藥組成物”表示含有一種或多種本文所述化合物或其生理學上/可藥用的鹽或前體藥物與其他化學組分的混合物，以及其他組分例如生理學/可藥用的載體和賦形劑。醫藥組成物的目的是促進對生物體的給藥，利於活性成分的吸收進而發揮生物活性。 "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein, or a physiological / pharmaceutically acceptable salt or prodrug thereof, with other chemical components, and other components such as physiological / pharmaceutically acceptable carriers And excipients. The purpose of the pharmaceutical composition is to promote the administration to the living body, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.
“可藥用鹽”是指本發明化合物的鹽，這類鹽用於哺乳動物體內時具有安全性和有效性，且具有應有的生物活 性。 "Pharmaceutically acceptable salt" refers to a salt of a compound of the present invention. Such salts are safe and effective when used in mammals, and have due biological activity.
m和R5至R7如通式(I)化合物中所定義。 m and R 5 to R 7 are as defined in the compound of general formula (I).
為了完成本發明的目的，本發明採用如下技術方案： In order to achieve the object of the present invention, the present invention adopts the following technical solutions:
本發明通式(I)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
通式(IA)的化合物在酸性條件下，脫去保護基得到通式(I)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；G1、L1、X1、R1和R2如通式(I)中所定義。 The compound of the general formula (IA) is deprotected under acidic conditions to obtain a compound of the general formula (I); wherein: W is an amine-protecting group, and is selected from the group consisting of a third butoxycarbonyl group, an ethylfluorenyl group, a benzyl group, and an alkenyl group. and propyl p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is a hydrogen atom or an amino protecting group, the amino protecting group is selected from tertiary-butoxycarbonyl, acetyl, benzyl, alkenyl Propyl and p-methoxybenzyl, preferably p-methoxybenzyl; G 1 , L 1 , X 1 , R 1 and R 2 are as defined in general formula (I).
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluene Sulfonic acid and TMSOTf.
上述反應較佳在溶劑中進行，所用溶劑包括但不限於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
本發明通式(II)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
第一步，通式(II-1)的化合物在還原劑存在下，在酸性條件下，反應得到通式(IIB)的化合物；第二步，通式(IIB)的化合物在鹼性條件下，發生消除反應得到通式(IIA)的化合物；第三步，通式(IIA)的化合物在酸性條件下，脫去保護基得到通式(II)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基； Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Rb為烷基，較佳為乙基或甲基；G1、L1、X1、R1和R2如通式(II)中所定義。 In the first step, the compound of the general formula (II-1) is reacted in the presence of a reducing agent under acidic conditions to obtain the compound of the general formula (IIB); in the second step, the compound of the general formula (IIB) is under basic conditions , A elimination reaction occurs to obtain a compound of the general formula (IIA); in a third step, the compound of the general formula (IIA) is deprotected under acidic conditions to obtain a compound of the general formula (II); wherein: W is an amine protecting group And is selected from the group consisting of a third butoxycarbonyl group, an ethanoyl group, a benzyl group, an allyl group, and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is an amine protecting group or a hydrogen atom, and the amine protecting group The group is selected from the group consisting of a third butoxycarbonyl group, an ethenyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R b is an alkyl group, preferably an ethyl group or a methyl group; G 1 , L 1 , X 1 , R 1 and R 2 are as defined in the general formula (II).
提供鹼性條件的試劑包括有機鹼和無機鹼類，該有機鹼類包括但不限於三乙胺、吡啶、4-二甲胺基吡啶、N,N-二異丙基乙胺、1,8-二氮雜二環[5.4.0]十一碳-7-烯(DBU)、1,5-二氮雜雙環壬-5-烯(DBN)、正丁基鋰、二異丙基胺基鋰、雙三甲基矽基胺基鋰、醋酸鉀、第三丁醇鈉、第三丁醇鉀和正丁醇鈉，該無機鹼類包括但不限於氫化鈉、磷酸鉀、碳酸鈉、碳酸鉀、醋酸鉀、碳酸銫、氫氧化鈉、氫氧化鉀和氫氧化鋰。 Reagents that provide basic conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, 1,8 -Diazabicyclo [5.4.0] undec-7-ene (DBU), 1,5-diazabicyclonon-5-ene (DBN), n-butyllithium, diisopropylamino Lithium, bistrimethylsilyl lithium, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide, the inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate , Potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluene Sulfonic acid and TMSOTf.
還原試劑包括但不限於：鐵粉、鋅粉、硫化鈉、硫代硫酸鈉、二硫化鈉、氯化亞錫、Pd/C/H2、Pt/C/H2、雷尼鎳/H2、氫化鋁鋰、硼氫化鈉、DIBAL-H、NaAlH(O-t-Bu)3、AlH3、NaCNBH3、Na(AcO)3BH和Li(Et)3BH。 Reducing reagents include, but are not limited to: iron powder, zinc powder, sodium sulfide, sodium thiosulfate, sodium disulfide, stannous chloride, Pd / C / H 2 , Pt / C / H 2 , Raney nickel / H 2 , Lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (Ot-Bu) 3 , AlH 3 , NaCNBH 3 , Na (AcO) 3 BH, and Li (Et) 3 BH.
上述反應較佳在溶劑中進行，所用溶劑包括但不限於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
本發明通式(IIAa)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
第一步，通式(IIAa-1)的化合物在酸性條件下，加熱反應得到通式(IIAa-2)的化合物；第二步，通式(IIAa-2)的化合物在酸性條件下，脫去保護基得到通式(IIAa)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Rb為烷基，較佳為乙基或甲基；G1、L1、X1、R1和R2如通式(IIAa)中所定義。 In the first step, the compound of the general formula (IIAa-1) is heated under acidic conditions to obtain a compound of the general formula (IIAa-2); in the second step, the compound of the general formula (IIAa-2) is dehydrated under acidic conditions. Deprotection to obtain a compound of the general formula (IIAa); wherein: W is an amine protecting group, selected from the group consisting of a third butoxycarbonyl group, an ethyl fluorenyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a methoxybenzyl group; R a is a hydrogen atom or an amino protecting group, the amino protecting group is selected from tertiary-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl group, preferably for the Methoxybenzyl; R b is alkyl, preferably ethyl or methyl; G 1 , L 1 , X 1 , R 1 and R 2 are as defined in general formula (IIAa).
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、濃鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, 1,4-dioxane solution of hydrogen chloride, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, concentrated hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-benzene Methanesulfonic acid and TMSOTf.
上述反應較佳在溶劑中進行，所用溶劑包括但不限 於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
本發明通式(III)或通式(IV)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
通式(IIIA)的化合物在酸性條件下，脫去保護基得到通式(III)的化合物；或通式(IVA)的化合物在酸性條件下，脫去保護基得到通式(IV)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基； Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；環A、G1、L1、X1、X2、R1、R6~R8和s如通式(III)中所定義。 Deprotection of a compound of general formula (IIIA) under acidic conditions to obtain a compound of general formula (III); or deprotection of a compound of general formula (IVA) under acidic conditions to obtain a compound of general formula (IV) Wherein: W is an amine protecting group, selected from the group consisting of a third butoxycarbonyl group, an ethyl fluorenyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is an amine group protecting group Or a hydrogen atom, the amine protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethyl fluorenyl group, a benzyl group, an allyl group, and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; ring A, G 1 , L 1 , X 1 , X 2 , R 1 , R 6 to R 8 and s are as defined in the general formula (III).
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸、Me3SiCl和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluene Sulfonic acid, Me 3 SiCl and TMSOTf.
上述反應較佳在溶劑中進行，所用溶劑包括但不限於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
本發明通式(V)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
第一步，通式(V-1)的化合物在還原劑存在下，在酸性條件下，反應得到通式(V-2)的化合物；第二步，通式(V-2)的化合物在鹼性條件下，發生消除反應得到通式(VA)的化合物；第三步，通式(VA)的化合物在酸性條件下，脫去保護基得到通式(V)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Rb為烷基，較佳為乙基或甲基；環A、L1、X2、R1、R6和R7如通式(V)中所定義。 In the first step, the compound of the general formula (V-1) is reacted in the presence of a reducing agent under acidic conditions to obtain the compound of the general formula (V-2); in the second step, the compound of the general formula (V-2) is Under basic conditions, a elimination reaction occurs to obtain a compound of the general formula (VA); in the third step, the compound of the general formula (VA) is deprotected to obtain a compound of the general formula (V) under acidic conditions; wherein: W is An amino protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethenyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is an amine protecting group or a hydrogen atom, The amine protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethanoyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R b is an alkyl group, preferably an ethyl group. Or methyl; rings A, L 1 , X 2 , R 1 , R 6 and R 7 are as defined in general formula (V).
提供鹼性條件的試劑包括有機鹼和無機鹼類，該有機鹼類包括但不限於三乙胺、吡啶、4-二甲胺基吡啶、N,N- 二異丙基乙胺、1,8-二氮雜二環[5.4.0]十一碳-7-烯(DBU)、1,5-二氮雜雙環壬-5-烯(DBN)、正丁基鋰、二異丙基胺基鋰、雙三甲基矽基胺基鋰、醋酸鉀、第三丁醇鈉、第三丁醇鉀和正丁醇鈉，該無機鹼類包括但不限於氫化鈉、磷酸鉀、碳酸鈉、碳酸鉀、醋酸鉀、碳酸銫、氫氧化鈉、氫氧化鉀和氫氧化鋰。 Reagents that provide basic conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, 1,8 -Diazabicyclo [5.4.0] undec-7-ene (DBU), 1,5-diazabicyclonon-5-ene (DBN), n-butyllithium, diisopropylamino Lithium, bistrimethylsilyl lithium, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide, the inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate , Potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluene Sulfonic acid and TMSOTf.
還原試劑包括但不限於：鐵粉、鋅粉、硫化鈉、硫代硫酸鈉、二硫化鈉、氯化亞錫、Pd/C/H2、Pt/C/H2、雷尼鎳/H2、氫化鋁鋰、硼氫化鈉、DIBAL-H、NaAlH(O-t-Bu)3、AlH3、NaCNBH3、Na(AcO)3BH和Li(Et)3BH。 Reducing reagents include, but are not limited to: iron powder, zinc powder, sodium sulfide, sodium thiosulfate, sodium disulfide, stannous chloride, Pd / C / H 2 , Pt / C / H 2 , Raney nickel / H 2 , Lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (Ot-Bu) 3 , AlH 3 , NaCNBH 3 , Na (AcO) 3 BH, and Li (Et) 3 BH.
上述反應較佳在溶劑中進行，所用溶劑包括但不限於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
本發明通式(VI)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
第一步，通式(VI-1)的化合物在還原劑存在下，在酸性條件下，反應得到通式(V-2)的化合物；第二步，通式(VI-2)的化合物在鹼性條件下，發生消除反應得到通式(VIA)的化合物；第三步，通式(VIA)的化合物在酸性條件下，脫去保護基得到通式(VI)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Rb為烷基，較佳為乙基或甲基；L1、R1、n、R6和R7如通式(VI)中所定義。 In the first step, the compound of the general formula (VI-1) is reacted in the presence of a reducing agent under acidic conditions to obtain the compound of the general formula (V-2); in the second step, the compound of the general formula (VI-2) is Under basic conditions, a elimination reaction occurs to obtain a compound of the general formula (VIA); in the third step, the compound of the general formula (VIA) is removed under acidic conditions to obtain a compound of the general formula (VI); where: W is An amino protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethenyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is an amine protecting group or a hydrogen atom, The amine protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethanoyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R b is an alkyl group, preferably an ethyl group. Or methyl; L 1 , R 1 , n, R 6 and R 7 are as defined in general formula (VI).
提供鹼性條件的試劑包括有機鹼和無機鹼類，該有機鹼類包括但不限於三乙胺、吡啶、4-二甲胺基吡啶、N,N-二異丙基乙胺、1,8-二氮雜二環[5.4.0]十一碳-7-烯(DBU)、 1,5-二氮雜雙環壬-5-烯(DBN)、正丁基鋰、二異丙基胺基鋰、雙三甲基矽基胺基鋰、醋酸鉀、第三丁醇鈉、第三丁醇鉀和正丁醇鈉，該無機鹼類包括但不限於氫化鈉、磷酸鉀、碳酸鈉、碳酸鉀、醋酸鉀、碳酸銫、氫氧化鈉、氫氧化鉀和氫氧化鋰。 Reagents that provide basic conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, 1,8 -Diazabicyclo [5.4.0] undec-7-ene (DBU), 1,5-diazabicyclonon-5-ene (DBN), n-butyllithium, diisopropylamino Lithium, bistrimethylsilyl lithium, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide, the inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate , Potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-benzoyl Sulfonic acid and TMSOTf.
還原試劑包括但不限於：鐵粉、鋅粉、硫化鈉、硫代硫酸鈉、二硫化鈉、氯化亞錫、Pd/C/H2、Pt/C/H2、雷尼鎳/H2、氫化鋁鋰、硼氫化鈉、DIBAL-H、NaAlH(O-t-Bu)3、AlH3、NaCNBH3、Na(AcO)3BH和Li(Et)3BH。 Reducing reagents include, but are not limited to: iron powder, zinc powder, sodium sulfide, sodium thiosulfate, sodium disulfide, stannous chloride, Pd / C / H 2 , Pt / C / H 2 , Raney nickel / H 2 , Lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (Ot-Bu) 3 , AlH 3 , NaCNBH 3 , Na (AcO) 3 BH, and Li (Et) 3 BH.
上述反應較佳在溶劑中進行，所用溶劑包括但不限於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
本發明通式(VII)所示的化合物或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式，或其可藥用的鹽的製備方法，包括以下步驟：
第一步，通式(VII-1)的化合物在還原劑存在下，在酸性條件下，反應得到通式(VII-2)的化合物；第二步，通式(VII-2)的化合物在鹼性條件下，發生消除反應得到通式(VIIA)的化合物；第三步，通式(VIIA)的化合物在酸性條件下，脫去保護基得到通式(VII)的化合物；其中：W為胺基保護基，選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Ra為胺基保護基或氫原子，該胺基保護基選自第三丁氧羰基、乙醯基、苄基、烯丙基和對甲氧苄基，較佳為對甲氧苄基；Rb為烷基，較佳為乙基或甲基；L1、X2、R1、R6和R7如通式(VII)中所定義。 In the first step, the compound of the general formula (VII-1) is reacted in the presence of a reducing agent under acidic conditions to obtain the compound of the general formula (VII-2); in the second step, the compound of the general formula (VII-2) is Under basic conditions, a elimination reaction occurs to obtain a compound of the general formula (VIIA); in a third step, the compound of the general formula (VIIA) is deprotected to obtain a compound of the general formula (VII) under acidic conditions; wherein: W is An amino protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethenyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R a is an amine protecting group or a hydrogen atom, The amine protecting group is selected from the group consisting of a third butoxycarbonyl group, an ethanoyl group, a benzyl group, an allyl group and a p-methoxybenzyl group, preferably a p-methoxybenzyl group; R b is an alkyl group, preferably an ethyl group. Or methyl; L 1 , X 2 , R 1 , R 6 and R 7 are as defined in general formula (VII).
提供鹼性條件的試劑包括有機鹼和無機鹼類，該有機 鹼類包括但不限於三乙胺、吡啶、4-二甲胺基吡啶、N,N-二異丙基乙胺、1,8-二氮雜二環[5.4.0]十一碳-7-烯(DBU)、1,5-二氮雜雙環壬-5-烯(DBN)、正丁基鋰、二異丙基胺基鋰、雙三甲基矽基胺基鋰、醋酸鉀、第三丁醇鈉、第三丁醇鉀和正丁醇鈉，該無機鹼類包括但不限於氫化鈉、磷酸鉀、碳酸鈉、碳酸鉀、醋酸鉀、碳酸銫、氫氧化鈉、氫氧化鉀和氫氧化鋰。 Reagents that provide basic conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, 4-dimethylaminopyridine, N , N -diisopropylethylamine, 1,8 -Diazabicyclo [5.4.0] undec-7-ene (DBU), 1,5-diazabicyclonon-5-ene (DBN), n-butyllithium, diisopropylamino Lithium, bistrimethylsilyl lithium, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide, the inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate , Potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.
提供酸性的條件的試劑包括但不限於氯化氫、氯化氫的1,4-二噁烷溶液、氯化銨、三氟乙酸、甲酸、乙酸、鹽酸、硫酸、甲磺酸、硝酸、磷酸、對苯甲磺酸和TMSOTf。 Reagents that provide acidic conditions include, but are not limited to, hydrogen chloride, a solution of hydrogen chloride in 1,4-dioxane, ammonium chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluene Sulfonic acid and TMSOTf.
還原試劑包括但不限於：鐵粉、鋅粉、硫化鈉、硫代硫酸鈉、二硫化鈉、氯化亞錫、Pd/C/H2、Pt/C/H2、雷尼鎳/H2、氫化鋁鋰、硼氫化鈉、DIBAL-H、NaAlH(O-t-Bu)3、AlH3、NaCNBH3、Na(AcO)3BH和Li(Et)3BH。 Reducing reagents include, but are not limited to: iron powder, zinc powder, sodium sulfide, sodium thiosulfate, sodium disulfide, stannous chloride, Pd / C / H 2 , Pt / C / H 2 , Raney nickel / H 2 , Lithium aluminum hydride, sodium borohydride, DIBAL-H, NaAlH (Ot-Bu) 3 , AlH 3 , NaCNBH 3 , Na (AcO) 3 BH, and Li (Et) 3 BH.
上述反應較佳在溶劑中進行，所用溶劑包括但不限於：醋酸、甲醇、乙醇、正丁醇、甲苯、四氫呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亞碸、1,4-二噁烷、水、N,N-二甲基甲醯胺及其混合物。 The above reaction is preferably performed in a solvent. The solvents used include, but are not limited to, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfene, 1,4-dioxane, water, N , N -dimethylformamide and mixtures thereof.
化合物的結構是藉由核磁共振(NMR)或/和質譜(MS)來確定的。NMR位移(δ)以10-6(ppm)的單位給出。NMR的 測定是用Bruker AVANCE-400核磁儀，測定溶劑為氘代二甲基亞碸(DMSO-d 6 )、氘代氯仿(CDCl3)、氘代甲醇(CD3OD)，內標為四甲基矽烷(TMS)。 The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The NMR shift (δ) is given in units of 10 -6 (ppm). The NMR measurement was performed using Bruker AVANCE-400 nuclear magnetic analyzer. The measurement solvents were deuterated dimethylsulfine (DMSO- d 6 ), deuterated chloroform (CDCl 3 ), and deuterated methanol (CD 3 OD). The internal standard was four. Methylsilane (TMS).
MS的測定用FINNIGAN LCQAd(ESI)質譜儀(生產商：Thermo，型號：Finnigan LCQ advantage MAX)。 MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
高效液相色譜法(HPLC)分析使用Agilent HPLC 1200DAD、Agilent HPLC 1200VWD和Waters HPLC e2695-2489高壓液相色譜儀。 High performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLC e2695-2489 high pressure liquid chromatograph.
手性HPLC分析測定使用Agilent 1260 DAD高效液相色譜儀。 Chiral HPLC analysis was performed using an Agilent 1260 DAD high performance liquid chromatography.
高效液相製備使用Waters 2767、Waters 2767-SQ Detecor2、Shimadzu LC-20AP和Gilson-281製備型色譜儀。 HPLC preparation was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatography.
手性製備使用Shimadzu LC-20AP製備型色譜儀。 Chiral preparation was performed using a Shimadzu LC-20AP preparative chromatograph.
CombiFlash快速製備儀使用Combiflash Rf200(TELEDYNE ISCO)。 The CombiFlash rapid preparation instrument uses a Combiflash Rf200 (TELEDYNE ISCO).
薄層層析矽膠板使用煙臺黃海HSGF254或青島GF254矽膠板，薄層色譜法(TLC)使用的矽膠板採用的規格是0.15mm~0.2mm，薄層層析分離純化產品採用的規格是0.4mm~0.5mm。 The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silicone plate. The thin-layer chromatography (TLC) silicon plate uses a size of 0.15mm ~ 0.2mm. The thin-layer chromatography separation and purification product uses a size of 0.4mm. ~ 0.5mm.
矽膠管柱色譜法一般使用煙臺黃海矽膠200~300目矽膠為載體。 Silicone column chromatography generally uses Yantai Huanghai Silicone 200 ~ 300 mesh silica gel as the carrier.
激酶平均抑制率及IC50值的測定用NovoStar酶標儀(德國BMG公司)。 The average inhibition rate of the kinase and the IC 50 value were measured using a NovoStar microplate reader (BMG, Germany).
本發明的已知的起始原料可以採用或按照本領域已知的方法來合成，或可購買自ABCR GmbH & Co.KG，Acros Organics，Aldrich Chemical Company，韶遠化學科技(Accela ChemBio Inc)、達瑞化學品等公司。 The known starting materials of the present invention can be synthesized by or in accordance with methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Dari Chemicals and other companies.
實施例中無特殊說明，反應能夠均在氬氣氛或氮氣氛下進行。 There is no special description in the examples, and the reaction can be performed under an argon atmosphere or a nitrogen atmosphere.
氬氣氛或氮氣氛是指反應瓶連接一個約1L容積的氬氣或氮氣氣球。 An argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.
氫氣氛是指反應瓶連接一個約1L容積的氫氣氣球。 The hydrogen atmosphere means that a reaction balloon is connected to a hydrogen gas balloon with a volume of about 1 L.
加壓氫化反應使用Parr 3916EKX型氫化儀和清藍QL-500型氫氣發生器或HC2-SS型氫化儀。 Pressurized hydrogenation reaction uses Parr 3916EKX type hydrogenation instrument and clear blue QL-500 type hydrogen generator or HC2-SS type hydrogenation instrument.
氫化反應通常抽真空，充入氫氣，重複操作3次。 The hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
微波反應使用CEM Discover-S 908860型微波反應器。 For the microwave reaction, a CEM Discover-S 908860 microwave reactor was used.
實施例中無特殊說明，溶液是指水溶液。 There is no special description in the examples, and the solution means an aqueous solution.
實施例中無特殊說明，反應的溫度為室溫，為20℃~30℃。 There is no special description in the examples, and the reaction temperature is room temperature, which is 20 ° C to 30 ° C.
實施例中的反應進程的監測採用薄層色譜法(TLC)，反應所使用的展開劑，純化化合物採用的管柱層析的沖提劑的體系和薄層色譜法的展開劑體系包括：A：二氯甲烷/甲醇體系，B：正己烷/乙酸乙酯體系，溶劑的體積比根據化合物的極性不同而進行調節，也可以加入少量的三乙胺和醋酸等鹼性或酸性試劑進行調節。 The monitoring of the reaction progress in the examples uses thin layer chromatography (TLC), a developing agent used in the reaction, a column chromatography eluent system for purifying compounds, and a thin layer chromatography developing system including: A : Methylene chloride / methanol system, B: n-hexane / ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.
將2-丁氧基-4,6-二氯-5-硝基嘧啶1a(4.62g，17.43mmol，採用公知的方法“Journal of Medicinal Chemistry,2012,55(23),10387-10404”製備而得)溶於50mL四氫呋喃溶劑中，加入三乙胺(2.64g，26.14mmol)和N,N-雙(4-甲氧基苄基)胺(4.49g，17.43mmol)，攪拌反應4小時。反 應液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系B純化殘餘物，得到標題化合物1b(6.20g，產率：73.8%)。 2-butoxy-4,6-dichloro-5-nitropyrimidine 1a (4.62 g, 17.43 mmol, prepared by a known method " Journal of Medicinal Chemistry , 2012, 55 (23), 10387-10404" and (Acquired) was dissolved in 50 mL of tetrahydrofuran solvent, and triethylamine (2.64 g, 26.14 mmol) and N , N -bis (4-methoxybenzyl) amine (4.49 g, 17.43 mmol) were added, and the reaction was stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified with the eluent system B using a CombiFlash rapid preparation instrument to obtain the title compound 1b (6.20 g, yield: 73.8%).
MS m/z(ESI)：487.5[M+1] MS m / z (ESI): 487.5 [M + 1]
將乙酸乙酯(1.09g，12.32mmol)溶於40mL四氫呋喃中，冷卻至-70℃，滴加入1M雙(三甲基矽基)胺基鋰的四氫呋喃溶液(12.3mL，12.3mmol)，-70℃下攪拌反應0.5小時，加入化合物1b(4g，8.21mmol)，-70℃下攪拌反應5小時。反應液中加入50mL飽和氯化銨溶液，用乙酸乙酯萃取(300mL×1)，有機相用水洗滌(50mL×1)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系B純化所得殘餘物，得到標題化合物1c(2.1g，產率：47%)。 Ethyl acetate (1.09 g, 12.32 mmol) was dissolved in 40 mL of tetrahydrofuran, cooled to -70 ° C, and a 1 M solution of bis (trimethylsilyl) lithium lithium in tetrahydrofuran (12.3 mL, 12.3 mmol) was added dropwise,- The reaction was stirred at 70 ° C for 0.5 hours. Compound 1b (4 g, 8.21 mmol) was added, and the reaction was stirred at -70 ° C for 5 hours. 50 mL of saturated ammonium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (300 mL × 1). The organic phase was washed with water (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by the eluent system B to obtain the title compound 1c (2.1 g, yield: 47%).
MS m/z(ESI)：539.5[M+1] MS m / z (ESI): 539.5 [M + 1]
將化合物1c(355mg，0.65mmol)溶於5mL N,N-二甲基甲醯胺中，加入1-(4-(氯甲基)苄基)-吡咯烷鹽酸鹽1d(240mg，0.97mmol，採用專利申請“WO2002012224”公開的方法製備而得)和碳酸銫(1.06g，3.25mmol)，50℃下攪拌反應5小時。反應液冷卻至室溫，加入20mL飽和氯化 鈉溶液，用二氯甲烷萃取(100mL×1)，有機相用水洗滌(30mL×1)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物1e(270mg，產率：58%)。 Compound 1c (355mg, 0.65mmol) was dissolved in 5mL N, N -dimethylformamide, and 1- (4- (chloromethyl) benzyl) -pyrrolidine hydrochloride 1d (240mg, 0.97mmol) was added. , Prepared by the method disclosed in the patent application "WO2002012224") and cesium carbonate (1.06 g, 3.25 mmol), and the reaction was stirred at 50 ° C for 5 hours. The reaction solution was cooled to room temperature, 20 mL of a saturated sodium chloride solution was added, and the mixture was extracted with dichloromethane (100 mL × 1). The organic phase was washed with water (30 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by a flash prep with eluent system A to give the title compound 1e (270 mg, yield: 58%).
MS m/z(ESI)：712.4[M+1] MS m / z (ESI): 712.4 [M + 1]
將化合物1e(270mg，0.38mmol)和氫氧化鋰一水合物(159.5mg，3.8mmol)溶於10mL四氫呋喃和5mL水的混合溶劑中，70℃攪拌反應16小時。反應液冷卻至室溫，加入20mL飽和氯化鈉溶液，用二氯甲烷萃取(50mL×1)，有機相用飽和氯化鈉溶液洗滌(20mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物1f(220mg，產率：90%)。 Compound 1e (270 mg, 0.38 mmol) and lithium hydroxide monohydrate (159.5 mg, 3.8 mmol) were dissolved in a mixed solvent of 10 mL of tetrahydrofuran and 5 mL of water, and the reaction was stirred at 70 ° C for 16 hours. The reaction solution was cooled to room temperature, 20 mL of a saturated sodium chloride solution was added, and extracted with dichloromethane (50 mL × 1). The organic phase was washed with a saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate The organic layer was concentrated under reduced pressure, and the resulting residue was purified with the eluent system A using a CombiFlash rapid preparation instrument to obtain the title compound 1f (220 mg, yield: 90%).
MS m/z(ESI)：640.6[M+1] MS m / z (ESI): 640.6 [M + 1]
將化合物1f(220mg，0.34mmol)溶於5mL N,N-二甲基甲醯胺中，加入第三丁醇鉀(116mg，1.03mmol)，再加入50%乙醛酸乙酯的甲苯溶液1g(210mg，1.03mmol)，攪拌反應0.5小時。反應中加入20mL飽和氯化銨溶液， 用二氯甲烷萃取(50mL×1)，有機相用水洗滌(20mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗品標題化合物1h(250mg)，產品不經純化直接用於下一步反應。 Compound 1f (220 mg, 0.34 mmol) was dissolved in 5 mL of N, N -dimethylformamide, potassium tert-butoxide (116 mg, 1.03 mmol) was added, and 1 g of a 50% ethyl glyoxylate solution in toluene was added. (210 mg, 1.03 mmol) and stirred for 0.5 hours. To the reaction was added 20 mL of a saturated ammonium chloride solution, and the mixture was extracted with dichloromethane (50 mL × 1). The organic phase was washed with water (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude title compound 1h ( 250 mg), and the product was used in the next reaction without purification.
MS m/z(ESI)：742.7[M+1] MS m / z (ESI): 742.7 [M + 1]
將粗品化合物1h(250mg，0.34mmol)溶於5mL醋酸中，加入鋅粉(219mg，3.4mmol)，攪拌反應0.5小時。反應液過濾，濾液減壓濃縮，用飽和碳酸鉀溶液調節所得殘餘物的pH為7後，用二氯甲烷萃取(50mL×1)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗品標題化合物1i(220mg)，產品不經純化直接用於下一步反應。 The crude compound was dissolved in 5 mL of acetic acid for 1 h (250 mg, 0.34 mmol), zinc powder (219 mg, 3.4 mmol) was added, and the reaction was stirred for 0.5 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The pH of the obtained residue was adjusted to 7 with a saturated potassium carbonate solution, and then extracted with dichloromethane (50 mL × 1). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude title compound 1i (220 mg) was obtained and the product was used in the next reaction without purification.
MS m/z(ESI)：666.2[M+1] MS m / z (ESI): 666.2 [M + 1]
將粗品化合物1i(220mg，0.34mmol)溶於5mL乙腈中，加入1,8-二氮雜二環[5.4.0]十一碳-7-烯(155mg，1.02mmol)，80℃攪拌反應0.5小時。反應液冷卻至室溫，減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物1j(100mg，產率：45%)。 The crude compound 1i (220 mg, 0.34 mmol) was dissolved in 5 mL of acetonitrile, 1,8-diazabicyclo [5.4.0] undec-7-ene (155 mg, 1.02 mmol) was added, and the reaction was stirred at 80 ° C for 0.5 hour. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified with the extractant system A using a CombiFlash rapid preparation instrument to obtain the title compound 1j (100 mg, yield: 45%).
MS m/z(ESI)：648.7[M+1] MS m / z (ESI): 648.7 [M + 1]
將化合物1j(100mg，0.15mmol)溶於10mL三氟乙酸中，密閉加熱至100℃攪拌反應16小時。反應液冷卻至室溫，減壓濃縮，用高效液相色譜法(Waters-2767，沖提體系：10mmol/L碳酸氫銨，水，乙腈)純化所得殘餘物，得到標題化合物1(20mg，產率：32%)。 Compound 1j (100 mg, 0.15 mmol) was dissolved in 10 mL of trifluoroacetic acid, and the mixture was heated to 100 ° C. with stirring and stirred for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified by high performance liquid chromatography (Waters-2767, extraction system: 10 mmol / L ammonium bicarbonate, water, acetonitrile) to obtain the title compound 1 (20 mg, product Rate: 32%).
MS m/z(ESI)：408.5[M+1] MS m / z (ESI): 408.5 [M + 1]
1H NMR(400MHz,DMSO-d 6 )δ 11.18(s,1H),7.40(s,2H),7.25(d,2H),7.20(d,2H),6.58(s,1H),4.21(t,2H),4.09(s,2H),3.51(s,2H),2.39(s,4H),1.66(s,6H),1.41-1.36(m,2H),0.91(t,3H)。 1 H NMR (400MHz, DMSO- d 6 ) δ 11.18 (s, 1H), 7.40 (s, 2H), 7.25 (d, 2H), 7.20 (d, 2H), 6.58 (s, 1H), 4.21 (t , 2H), 4.09 (s, 2H), 3.51 (s, 2H), 2.39 (s, 4H), 1.66 (s, 6H), 1.41-1.36 (m, 2H), 0.91 (t, 3H).
將化合物1c(2.10g，3.90mmol)和1,5-二溴戊烷(2.67g，11.70mmol)溶於30mL N,N-二甲基甲醯胺中，加入碳酸銫(3.80g，11.70mmol)，攪拌反應6小時。反應液中加入60mL飽和氯化銨溶液，用乙酸乙酯萃取(100mL×1)，有機相用水洗滌(50mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系B純化所得殘餘物，得到標題化合物2a(1.16g，產率：43%)。 Compound 1c (2.10 g, 3.90 mmol) and 1,5-dibromopentane (2.67 g, 11.70 mmol) were dissolved in 30 mL of N, N -dimethylformamide, and cesium carbonate (3.80 g, 11.70 mmol) was added. ), Stirred for 6 hours. 60 mL of saturated ammonium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 1), and the organic phase was washed with water (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified with the eluent system B to obtain the title compound 2a (1.16 g, yield: 43%).
MS m/z(ESI)：687.5[M+1] MS m / z (ESI): 687.5 [M + 1]
將化合物2a(1.16g，1.69mmol)、吡咯烷(360mg，5.06mmol)和三乙胺(511mg，5.06mmol)溶於15mL N,N-二甲基甲醯胺中，80℃攪拌反應1小時。反應液冷卻至室 溫，加入30mL飽和氯化銨溶液，用二氯甲烷萃取(100mL×1)，有機相用水洗滌(50mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物2b(1.01g，產率：87%)。 Compound 2a (1.16 g, 1.69 mmol), pyrrolidine (360 mg, 5.06 mmol) and triethylamine (511 mg, 5.06 mmol) were dissolved in 15 mL of N, N -dimethylformamide, and the reaction was stirred at 80 ° C for 1 hour. . The reaction solution was cooled to room temperature, 30 mL of a saturated ammonium chloride solution was added, and the mixture was extracted with dichloromethane (100 mL × 1). The organic phase was washed with water (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The CombiFlash rapid preparation device was used to purify the obtained residue with eluent system A to obtain the title compound 2b (1.01 g, yield: 87%).
MS m/z(ESI)：678.4[M+1] MS m / z (ESI): 678.4 [M + 1]
將化合物2b(900mg，1.33mmol)和氫氧化鋰一水合物(167.3mg，3.98mmol)溶於20mL四氫呋喃和10mL水的混合溶劑中，70℃攪拌反應16小時。反應液冷卻至室溫，加入40mL飽和氯化鈉水溶液，用二氯甲烷萃取(100mL×1)，有機相用飽和氯化鈉溶液洗滌(30mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物2c(700mg，產率：87%)。 Compound 2b (900 mg, 1.33 mmol) and lithium hydroxide monohydrate (167.3 mg, 3.98 mmol) were dissolved in a mixed solvent of 20 mL of tetrahydrofuran and 10 mL of water, and the reaction was stirred at 70 ° C for 16 hours. The reaction solution was cooled to room temperature, 40 mL of a saturated sodium chloride aqueous solution was added, and extracted with dichloromethane (100 mL × 1). The organic phase was washed with a saturated sodium chloride solution (30 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate The organic layer was concentrated under reduced pressure, and the resulting residue was purified with the eluent system A using a CombiFlash rapid preparation instrument to obtain the title compound 2c (700 mg, yield: 87%).
MS m/z(ESI)：606.4[M+1] MS m / z (ESI): 606.4 [M + 1]
將化合物2c(220mg，0.36mmol)溶於5mL N,N-二甲基甲醯胺中，加入第三丁醇鉀(122mg，1.09mmol)，再加入50%乙醛酸乙酯的甲苯溶液1g(223mg，1.09mmol)， 攪拌反應0.5小時。反應液中加入20mL飽和氯化銨溶液，用二氯甲烷萃取(50mL×1)，有機相用水洗滌(20mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗品標題化合物2d(250mg)，產品不經純化直接用於下一步反應。 Compound 2c (220mg, 0.36mmol) was dissolved in 5mL N, N -dimethylformamide, potassium tert-butoxide (122mg, 1.09mmol) was added, and 1g of a 50% ethyl glyoxylate solution in toluene was added. (223 mg, 1.09 mmol) and stirred for 0.5 hours. To the reaction solution was added 20 mL of a saturated ammonium chloride solution, and the mixture was extracted with dichloromethane (50 mL × 1). The organic phase was washed with water (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 2d. (250 mg), the product was used in the next reaction without purification.
MS m/z(ESI)：708.3[M+1] MS m / z (ESI): 708.3 [M + 1]
將粗品化合物2d(250mg，0.36mmol)溶於5mL醋酸中，加入鋅粉(234mg，3.6mmol)，反應0.5小時。反應液過濾，濾液減壓濃縮，用飽和碳酸鉀溶液調節所得殘餘物pH為7，用二氯甲烷萃取(50mL×1)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗品標題化合物2e(220mg)，產品不經純化直接用於下一步反應。 The crude compound 2d (250 mg, 0.36 mmol) was dissolved in 5 mL of acetic acid, zinc powder (234 mg, 3.6 mmol) was added, and the reaction was performed for 0.5 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was adjusted to pH 7 with a saturated potassium carbonate solution, and extracted with dichloromethane (50 mL × 1). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The title compound 2e (220 mg) was used in the next reaction without purification.
MS m/z(ESI)：632.3[M+1] MS m / z (ESI): 632.3 [M + 1]
將粗品化合物2e(220mg，0.36mmol)溶於5mL乙腈中，加入1,8-二氮雜二環[5.4.0]十一碳-7-烯(164mg，1.08mmol)，80℃攪拌反應0.5小時。反應液冷卻至室溫，減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物2f(100mg，產率：45%)。 The crude compound 2e (220 mg, 0.36 mmol) was dissolved in 5 mL of acetonitrile, 1,8-diazabicyclo [5.4.0] undec-7-ene (164 mg, 1.08 mmol) was added, and the reaction was stirred at 80 ° C for 0.5 hour. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified with the extractant system A using a CombiFlash rapid preparation instrument to obtain the title compound 2f (100 mg, yield: 45%).
MS m/z(ESI)：614.4[M+1] MS m / z (ESI): 614.4 [M + 1]
將化合物2f(100mg，0.16mmol)溶於10mL三氟乙酸中，密閉加熱至100℃攪拌反應16小時。反應液冷卻至室溫，減壓濃縮，用高效液相色譜法(Waters-2767，沖提體系：10mmol/L碳酸氫銨，水，乙腈)純化所得殘餘物，得到標題化合物2(30mg，產率：50%)。 Compound 2f (100 mg, 0.16 mmol) was dissolved in 10 mL of trifluoroacetic acid, and the mixture was heated to 100 ° C. with stirring and stirred for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified by high performance liquid chromatography (Waters-2767, extraction system: 10 mmol / L ammonium bicarbonate, water, acetonitrile) to obtain the title compound 2 (30 mg, product Rate: 50%).
MS m/z(ESI)：374.3[M+1] MS m / z (ESI): 374.3 [M + 1]
1H NMR(400MHz,CD3OD)：δ 6.76(s,1H),4.33(t,2H),2.88(t,2H),2.71(t,4H),2.63(t,2H),1.90-1.86(m,4H),1.82-1.74(m,4H),1.70-1.62(m,2H),1.55-1.45(m,4H),0.99(t,3H)。 1 H NMR (400MHz, CD 3 OD): δ 6.76 (s, 1H), 4.33 (t, 2H), 2.88 (t, 2H), 2.71 (t, 4H), 2.63 (t, 2H), 1.90-1.86 (m, 4H), 1.82-1.74 (m, 4H), 1.70-1.62 (m, 2H), 1.55-1.45 (m, 4H), 0.99 (t, 3H).
將7-溴庚酸甲酯3a(1.12g，5mmol，採用公知的方法“Journal of Natural Products,79(1),244-247；2016”製備而得)、吡咯烷(710mg，10mmol)和三乙胺(1.01mg，10mmol)溶於15mL N,N-二甲基甲醯胺中，80℃攪拌反應1小時。反應液冷卻至室溫，加入30mL飽和氯化銨溶液，用二氯甲烷萃取(100mL×1)，有機相用水洗滌(50mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物3b(920mg，產率：86%)。 Methyl 7-bromoheptanoate 3a (1.12 g, 5 mmol, prepared by a known method " Journal of Natural Products , 79 (1), 244-247; 2016"), pyrrolidine (710 mg, 10 mmol), and three Ethylamine (1.01 mg, 10 mmol) was dissolved in 15 mL of N, N -dimethylformamide, and the reaction was stirred at 80 ° C for 1 hour. The reaction solution was cooled to room temperature, 30 mL of a saturated ammonium chloride solution was added, and the mixture was extracted with dichloromethane (100 mL × 1). The organic phase was washed with water (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The CombiFlash rapid preparation device was used to purify the obtained residue with eluent system A to obtain the title compound 3b (920 mg, yield: 86%).
將化合物1b(4g，8.21mmol)溶於30mL乙醇、30mL四氫呋喃和15mL水的混合溶劑中，加入鋅粉(2.67g，41.07mmol)和氯化銨(2.18g，41.07mmol)，攪拌反應2 小時。反應液中加入100mL飽和氯化鈉溶液，用乙酸乙酯萃取(200mL×1)，有機相用水洗滌(60mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系B純化所得殘餘物，得到標題化合物3c(2g，產率：53%)。 Compound 1b (4g, 8.21mmol) was dissolved in a mixed solvent of 30mL ethanol, 30mL tetrahydrofuran and 15mL water, zinc powder (2.67g, 41.07mmol) and ammonium chloride (2.18g, 41.07mmol) were added, and the reaction was stirred for 2 hours. . Add 100 mL of saturated sodium chloride solution to the reaction solution, extract with ethyl acetate (200 mL × 1), wash the organic phase with water (60 mL × 1), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The obtained residue was purified with an eluent system B to obtain the title compound 3c (2 g, yield: 53%).
MS m/z(ESI)：457.5[M+1] MS m / z (ESI): 457.5 [M + 1]
將化合物3c(1g，2.19mmol)、[1,1'-雙(二苯基膦基)二茂鐵]二氯化鈀(160mg，0.22mmol)和三乙胺(442mg，4.38mmol)溶於20mL甲醇和10mL N,N-二甲基甲醯胺的混合溶劑中，用氮氣置換空氣，再用一氧化碳置換氮氣後，密閉加熱至70℃攪拌反應7小時。反應液冷卻至室溫，減壓濃縮，用CombiFlash快速製備儀以沖提劑體系B純化所得殘餘物，得到標題化合物3d(1g，產率：95%)。 Compound 3c (1 g, 2.19 mmol), [1,1'-bis (diphenylphosphino) ferrocene] palladium dichloride (160 mg, 0.22 mmol) and triethylamine (442 mg, 4.38 mmol) were dissolved in In a mixed solvent of 20 mL of methanol and 10 mL of N, N -dimethylformamide, the air was replaced with nitrogen, and the nitrogen was replaced with carbon monoxide, and the reaction was stirred and heated to 70 ° C. for 7 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified with the eluent system B using a CombiFlash rapid preparation instrument to obtain the title compound 3d (1 g, yield: 95%).
MS m/z(ESI)：481.2[M+1] MS m / z (ESI): 481.2 [M + 1]
將化合物3d(760mg，1.58mmol)溶於10mL二氯甲烷中，冷卻至-70℃，加入1M二異丁基氫化鋁的正己烷溶液(5.54mL，5.54mmol)，在-70℃氮氣保護下攪拌反應3小時。反應液升溫至0℃，加入20mL飽和氯化銨溶液 淬滅反應，用二氯甲烷萃取(50mL×1)，有機相用水洗滌(30mL×1)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系B純化所得殘餘物，得到標題化合物3e(200mg，產率：28%)。 Compound 3d (760 mg, 1.58 mmol) was dissolved in 10 mL of dichloromethane, cooled to -70 ° C, and a 1 M solution of diisobutylaluminum hydride in n-hexane (5.54 mL, 5.54 mmol) was added and protected at -70 ° C under nitrogen. The reaction was stirred for 3 hours. The reaction solution was heated to 0 ° C, and 20 mL of a saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with dichloromethane (50 mL × 1), and the organic phase was washed with water (30 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was decompressed. Concentrate, and purify the resulting residue with the eluent system B using a CombiFlash rapid preparation instrument to obtain the title compound 3e (200 mg, yield: 28%).
MS m/z(ESI)：451.5[M+1] MS m / z (ESI): 451.5 [M + 1]
將化合物3b(142mg，0.67mmol)溶於5mL四氫呋喃中，冷卻至-70℃，加入2M二異丙胺基鋰的四氫呋喃/乙苯/庚烷溶液(0.45mL，0.9mmol)，在-70℃氮氣保護下攪拌反應0.5小時，加入化合物3e(200mg，0.45mmol)，將反應液溫度緩緩升至室溫，攪拌反應1小時。反應液用10mL飽和氯化銨溶液淬滅，用二氯甲烷萃取(50mL×1)，有機相用飽和氯化鈉溶液洗滌(20mL×1)後，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用CombiFlash快速製備儀以沖提劑體系A純化所得殘餘物，得到標題化合物3f(40mg，產率：14%)。 Compound 3b (142 mg, 0.67 mmol) was dissolved in 5 mL of tetrahydrofuran, cooled to -70 ° C, and a 2 M solution of lithium diisopropylamino tetrahydrofuran / ethylbenzene / heptane (0.45 mL, 0.9 mmol) was added at -70 ° C. The reaction was stirred for 0.5 hours under the protection of nitrogen, compound 3e (200 mg, 0.45 mmol) was added, the temperature of the reaction solution was gradually raised to room temperature, and the reaction was stirred for 1 hour. The reaction solution was quenched with 10 mL of a saturated ammonium chloride solution, extracted with dichloromethane (50 mL × 1), and the organic phase was washed with a saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was decompressed. Concentrated, and the resulting residue was purified with the eluent system A using a CombiFlash rapid preparation instrument to obtain the title compound 3f (40 mg, yield: 14%).
MS m/z(ESI)：664.4[M+1] MS m / z (ESI): 664.4 [M + 1]
將化合物3f(40mg，0.06mmol)溶於5mL1,4-二噁烷中，加入0.5mL濃鹽酸和0.5mL水，加熱至85℃攪拌 反應2小時。反應液冷卻至室溫，用飽和碳酸氫鈉溶液調節pH為7，減壓濃縮，得到粗品標題化合物3g(31mg)，產品不經純化直接用於下一步反應。 Compound 3f (40 mg, 0.06 mmol) was dissolved in 5 mL of 1,4-dioxane, 0.5 mL of concentrated hydrochloric acid and 0.5 mL of water were added, and the reaction was heated to 85 ° C with stirring for 2 hours. The reaction solution was cooled to room temperature, adjusted to pH 7 with a saturated sodium bicarbonate solution, and concentrated under reduced pressure to obtain 3 g (31 mg) of the crude title compound. The product was used in the next reaction without purification.
MS m/z(ESI)：494.3[M+1] MS m / z (ESI): 494.3 [M + 1]
將粗品化合物3g(31mg，0.06mmol)溶於5mL三氟乙酸中，密閉加熱至100℃攪拌反應16小時。反應液冷卻至室溫，減壓濃縮，用高效液相色譜法(Gilson-281，沖提體系：水，乙腈)純化所得殘餘物，得到標題化合物3(3mg，產率：13%)。 3 g (31 mg, 0.06 mmol) of the crude compound was dissolved in 5 mL of trifluoroacetic acid, and the mixture was heated to 100 ° C. with stirring and stirred for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified by high performance liquid chromatography (Gilson-281, extraction system: water, acetonitrile) to obtain the title compound 3 (3 mg, yield: 13%).
MS m/z(ESI)：374.3[M+1] MS m / z (ESI): 374.3 [M + 1]
1H NMR(400MHz,CD3OD)δ 7.62(s,1H),4.52(s,2H),3.65(s,2H),3.20(t,2H),3.08(s,2H),2.75(s,2H),2.15(s,2H),2.03(s,2H),1.83-1.74(m,6H),1.54-1.49(m,4H),1.00(t,3H)。 1 H NMR (400MHz, CD 3 OD) δ 7.62 (s, 1H), 4.52 (s, 2H), 3.65 (s, 2H), 3.20 (t, 2H), 3.08 (s, 2H), 2.75 (s, 2H), 2.15 (s, 2H), 2.03 (s, 2H), 1.83-1.74 (m, 6H), 1.54-1.49 (m, 4H), 1.00 (t, 3H).
測試例1、本發明化合物對人源TLR7激動活性的測定 Test Example 1. Determination of agonistic activity of the compound of the present invention on human TLR7
本發明化合物對HEK-BlueTM hTLR7穩轉株細胞表達的hTLR7激活作用採用如下實驗方法測定： The hTLR7 activation effect of the compound of the present invention on the expression of HEK-Blue TM hTLR7 stable transfected cells is determined by the following experimental method:
一、實驗材料及儀器 I. Experimental materials and instruments
1. DMEM(Gibco，10564-029), 2. 胎牛血清(GIBCO,10099), 3. 台盼藍溶液(Sigma,T8154-100ML), 4. Flexstation 3多功能酶標儀(Molecμlar Devices), 5. HEK-BlueTM hTLR7細胞系(InvivoGen,hkb-hTLR7), 6. HEK-Blue檢測試劑(InvivoGen,hb-det3), 7. 磷酸鹽緩衝液(PBS)pH7.4(上海源培生物科技股份有限公司，B320)。 1. DMEM (Gibco, 10564-029), 2. Fetal Bovine Serum (GIBCO, 10099), 3. Trypan Blue Solution (Sigma, T8154-100ML), 4. Flexstation 3 Multifunctional Microplate Reader (Molec μlar Devices), 5. HEK-Blue TM hTLR7 cell line (InvivoGen, hkb-hTLR7), 6. HEK-Blue detection reagent (InvivoGen, hb-det3), 7. Phosphate buffer (PBS) pH7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).
二、實驗步驟 Experimental steps
配置HEK-Blue檢測培養基，取HEK-Blue檢測乾粉一袋，加入50ml去內毒素水溶解，再放入37℃培養箱，10分鐘後無菌過濾。化合物先配製成20mM的原液；再用純DMSO稀釋至最高濃度為6 x 106nM，經3倍梯度稀釋，共10個點。 Configure HEK-Blue detection medium, take a bag of HEK-Blue detection dry powder, add 50ml of endotoxin-free water to dissolve it, put it into a 37 ° C incubator, and filter it for 10 minutes. The compound was first prepared into a 20 mM stock solution; it was then diluted with pure DMSO to a maximum concentration of 6 x 10 6 nM, and diluted by a 3-fold gradient for a total of 10 points.
用培養基先把上述配製好的化合物稀釋20倍，然後每孔加入20μl稀釋後的化合物。 Dilute the compound prepared above 20-fold with culture medium, and then add 20 μl of the diluted compound to each well.
取HEK-BlueTM hTLR7細胞，先去掉上清，再加入2至5ml預熱的PBS，放入培養箱1-2分鐘，輕輕吹打細胞，台盼藍染色計數。用HEK-Blue檢測培養基重新懸浮細胞調整濃度為2.2 x 105個細胞/ml，加180μl細胞至上述已加入20μl藥物的96孔細胞培養板中，37℃，培養6-16h。 Take HEK-Blue TM hTLR7 cells, remove the supernatant first, then add 2 to 5 ml of pre-warmed PBS, put in the incubator for 1-2 minutes, gently blow the cells, and trypan blue staining to count. HEK-Blue with assay medium and resuspended cells were adjusted to a concentration of 2.2 x 10 5 cells / ml, 180μl added to cells in 96 well cell culture plate above is added to 20μl of the drug, 37 ℃, culture 6-16h.
酶標儀讀數，波長為620nm。可獲得相應的OD值，經Graphpad Prism計算得到藥物的EC50值。 Microplate reader reads at 620nm. Corresponding OD values obtained by Graphpad Prism calculated EC 50 value of the drug.
本發明化合物對人源TLR7激活作用可藉由以上的試驗進行測定，測得的EC50值見表1。 The activation of human-derived TLR7 by the compounds of the present invention can be determined by the above test. The measured EC 50 values are shown in Table 1.
結論：本發明化合物對人源TLR7具有較好的激活作用。 Conclusion: The compound of the present invention has a better activation effect on human TLR7.
測試例2、本發明化合物對人源TLR8激動活性的測定 Test example 2. Determination of the agonistic activity of the compound of the present invention on human TLR8
本發明化合物對HEK-BlueTM hTLR8穩轉株細胞表達的hTLR8激活作用採用如下實驗方法測定： The hTLR8 activation effect of the compound of the present invention on the expression of HEK-Blue TM hTLR8 stable transfected cells is determined by the following experimental method:
一、實驗材料及儀器 I. Experimental materials and instruments
1. DMEM(Gibco，10564-029),2. 胎牛血清(GIBCO,10099),3. 台盼藍溶液(Sigma,T8154-100ML),4. Flexstation 3多功能酶標儀(Molecμlar Devices),5. HEK-BlueTM hTLR8細胞系(InvivoGen,hkb-hTLR8),6. HEK-Blue檢測試劑(InvivoGen,hb-det3),7. 磷酸鹽緩衝液(PBS)pH7.4(上海源培生物科技股份有限公司，B320)。 1. DMEM (Gibco, 10564-029), 2. Fetal bovine serum (GIBCO, 10099), 3. Trypan blue solution (Sigma, T8154-100ML), 4. Flexstation 3 multifunctional microplate reader (Molec μlar Devices), 5. HEK-Blue TM hTLR8 cell line (InvivoGen, hkb-hTLR8), 6. HEK-Blue detection reagent (InvivoGen, hb-det3), 7. Phosphate buffer (PBS) pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320).
二、實驗步驟 Experimental steps
配置HEK-Blue檢測培養基，取HEK-Blue檢測乾粉一袋，加入50ml去內毒素水溶解，再放入37℃培養箱，10分鐘後無菌過濾。化合物先配製成20mM的原液；再用純DMSO稀釋至最高濃度為6 x 106nM，然後3倍梯度稀釋，共10個點；用培養基先把化合物稀釋20倍，然後每孔加入20μl稀釋後的化合物。 Configure HEK-Blue detection medium, take a bag of HEK-Blue detection dry powder, add 50ml of endotoxin-free water to dissolve it, put it into a 37 ° C incubator, and filter it for 10 minutes. The compound was first prepared into a 20 mM stock solution; it was then diluted with pure DMSO to a maximum concentration of 6 x 10 6 nM, followed by a three-fold gradient of 10 points; the compound was first diluted 20-fold with the medium, and then 20 μl was added to each well for dilution. After the compound.
取HEK-BlueTM hTLR8細胞，先去掉上清，加入2-5ml預熱的PBS，放入培養箱1-2分鐘，輕輕吹打細胞，台盼藍染色計數。用HEK-Blue檢測培養基重新懸浮細胞調整濃度為2.2 x 105個細胞/ml，加180μl細胞至上述已加入20μl藥物的96孔細胞培養板中，37℃，培養6-16h。 Take HEK-Blue TM hTLR8 cells, remove the supernatant first, add 2-5ml pre-warmed PBS, put in the incubator for 1-2 minutes, gently blow the cells, and trypan blue staining counts. HEK-Blue with assay medium and resuspended cells were adjusted to a concentration of 2.2 x 10 5 cells / ml, 180μl added to cells in 96 well cell culture plate above is added to 20μl of the drug, 37 ℃, culture 6-16h.
酶標儀讀數，波長為620nm。可獲得相應的OD值，經Graphpad Prism計算得到藥物的EC50值。 Microplate reader reads at 620nm. Corresponding OD values obtained by Graphpad Prism calculated EC 50 value of the drug.
本發明化合物對人源TLR8激活作用可藉由以上的試驗進行測定，測得的EC50值見表2。 The activation of human-derived TLR8 by the compounds of the present invention can be determined by the above test. The measured EC 50 values are shown in Table 2.
結論：本發明化合物對人源TLR8激活作用較弱，說明本發明化合物對TLR7具有選擇性。 Conclusion: The compounds of the present invention have relatively weak activation of human TLR8, which indicates that the compounds of the present invention are selective for TLR7.
測試例3、本發明中化合物刺激外周血單個核細胞(PBMC)分泌IFN-α能力的測定 Test Example 3. Determination of the ability of the compounds of the present invention to stimulate IFN-α secretion from peripheral blood mononuclear cells (PBMC)
本發明中化合物刺激PBMC分泌IFN-α能力採用如下實驗方法測定： The ability of the compounds in the present invention to stimulate PBMC to secrete IFN-α is determined by the following experimental methods:
一、實驗材料及儀器 I. Experimental materials and instruments
1. RPMI 1640(Invitrogen,11875),2. FBS(Gibco,10099-141),3. Ficoll-Paque PREMIUM(GE,17-5442-02),4. 台盼藍溶液(Sigma,T8154-100ML),5. SepMateTM-50(Stemcell,15460),6. Bright-LineTM血細胞計數儀(Sigma,Z359629-1EA),7. 96孔平底板(Corning,3599),8. 96孔v底板(Corning,3894),9. 人源IFN-α試劑盒(cisbio,6FHIFPEB),10. PHERAStar多功能酶標儀(BMG，PHERAStar)。 1. RPMI 1640 (Invitrogen, 11875), 2. FBS (Gibco, 10099-141), 3. Ficoll-Paque PREMIUM (GE, 17-5442-02), 4. Trypan blue solution (Sigma, T8154-100ML) , 5. SepMateTM-50 (Stemcell, 15460), 6. Bright-Line TM blood cell counter (Sigma, Z359629-1EA), 7. 96-well flat bottom plate (Corning, 3599), 8. 96-well v bottom plate (Corning, 3894), 9. Human-derived IFN-α kit (cisbio, 6FHIFPEB), 10. PHERAStar multifunctional microplate reader (BMG, PHERAStar).
二、實驗步驟 Experimental steps
化合物用純DMSO稀釋，最高濃度為5mM，4倍梯度稀釋，共9個點。然後取4μl化合物，加入到196μl含10%FBS的RMPI 1640培養基中，混勻。每孔取50μl至新的96孔細胞培養板。 The compound was diluted with pure DMSO, with a maximum concentration of 5 mM and a 4-fold gradient dilution for a total of 9 points. Then 4 μl of the compound was added to 196 μl of RMPI 1640 medium containing 10% FBS, and mixed. Take 50 μl of each well to a new 96-well cell culture plate.
所有試劑平衡到室溫，取250ml培養瓶，將60ml血液和PBS+2% FBS加入其中，輕輕吹打混勻稀釋。取50ml PBMC分離管SepMateTM-50，加入15ml淋巴細胞分離液Ficoll-Paque PREMIUM，然後加入30ml稀釋後血液。1200g離心10分鐘，室溫。取上清，然後300g，離心8分鐘。用含10%FBS的RMPI 1640培養基重新懸浮並計數，調整 PBMC數量至3.33×106個細胞/ml，取150μl至已加入化合物的細胞培養板中，37℃，5.0% CO2的培養箱中培養24h。 All reagents were equilibrated to room temperature. A 250 ml culture flask was taken. 60 ml of blood and PBS + 2% FBS were added to it. Take 50ml PBMC separation tube SepMateTM-50, add 15ml Ficoll-Paque PREMIUM, and then add 30ml diluted blood. Centrifuge at 1200g for 10 minutes at room temperature. The supernatant was taken, then 300g, and centrifuged for 8 minutes. Resuspend and count in RMPI 1640 medium containing 10% FBS, adjust the number of PBMCs to 3.33 × 10 6 cells / ml, take 150 μl into the cell culture plate with the compound, 37 ° C, 5.0% CO 2 incubator Cultivate for 24h.
將細胞培養板放入離心機中，1200rpm，室溫離心10分鐘。每孔取出150μl上清。先平衡人源IFN-α試劑盒中的試劑至常溫，在避光條件下根據試劑盒說明書配製抗-IFN-α-Eu3+-穴狀結合物(Cryptate conjugate)和抗-IFN-α-d2-結合物，兩者均以1：40的比例與結合緩衝液(conjugate Buffer)混勻。然後每孔加入16μl的離心取得的上清液。再每孔加入2μl剛配好的抗-IFN-α-Eu3+-穴狀結合物和抗-IFN-α-d2-結合物，震盪混勻，室溫避光孵育3h。 The cell culture plate was placed in a centrifuge at 1200 rpm and centrifuged at room temperature for 10 minutes. Remove 150 μl of supernatant from each well. First balance the reagents in the human-derived IFN-α kit to room temperature, and prepare anti-IFN-α-Eu 3+ -Cryptate conjugate and anti-IFN-α- d2-conjugate, both are mixed with conjugate buffer at a ratio of 1:40. Then add 16 μl of the supernatant obtained by centrifugation to each well. Then add 2 μl of freshly prepared anti-IFN-α-Eu 3+ -hole conjugate and anti-IFN-α-d2-conjugate to each well, mix by shaking, and incubate at room temperature for 3 hours in the dark.
在PHERAStar上用HTRF模式讀數。我們將刺激產生最低檢測限至少3倍以上細胞因子水平的最低藥物濃度，定義為該化合物在該細胞因子刺激實驗上的MEC(最小有效濃度Minimal Effective Concentration)值。 Read on PHERAStar in HTRF mode. We define the minimum drug concentration at which the stimulus produces a minimum detection limit of at least three times the cytokine level, as the compound's MEC (Minimal Effective Concentration) value on the cytokine stimulation experiment.
本發明化合物刺激PBMC分泌IFN-α的能力藉由以上的試驗進行測定，測得的MEC值見表3。 The ability of the compounds of the present invention to stimulate PBMC to secrete IFN-α was determined by the above test. The measured MEC values are shown in Table 3.
結論：從刺激PBMC分泌IFN-α的活性的數據上看，本發明化合物能夠較好的引起IFN-α釋放。 Conclusion: From the data of stimulating the activity of PBMC to secrete IFN-α, the compound of the present invention can better induce the release of IFN-α.
測試例4、本發明化合物對人肝微粒體CYP3A4咪達唑侖代謝位點的酶活性的抑制作用 Test Example 4. Inhibitory effect of the compound of the present invention on the enzyme activity of human liver microsome CYP3A4 midazolam metabolism site
本發明化合物對人肝微粒體CYP3A4咪達唑侖代謝位點的酶活性採用如下實驗方法測定： The enzyme activity of the compound of the present invention on the human liver microsome CYP3A4 midazolam metabolism site is determined by the following experimental method:
一、實驗材料及儀器 I. Experimental materials and instruments
1. 磷酸緩衝液(PBS),2. NADPH(Sigma N-1630),3. 人肝微粒體(Corning Gentest),4. ABI QTrap 4000液質兩用儀(AB Sciex),5. Inertsil C8-3管柱，4.6×50mm,5μm(美國迪馬公司)，6. CYP探針受質(15μM的咪達唑侖，SIGMA UC429)和陽性對照抑制劑(酮康唑，SIGMA K1003)。 1. Phosphate buffer (PBS), 2. NADPH (Sigma N-1630), 3. Human liver microsomes (Corning Gentest), 4. ABI QTrap 4000 dual-use instrument (AB Sciex), 5. Inertsil C8- 3 columns, 4.6 × 50 mm, 5 μm (Dima Corporation), 6. CYP probe substrate (15 μM midazolam, SIGMA UC429) and positive control inhibitor (ketoconazole, SIGMA K1003).
二、實驗步驟 Experimental steps
配置100mM的PBS緩衝液，用該緩衝液配製2.5mg/ml的微粒體溶液和5mM的NADPH溶液，用PBS梯度稀釋5X濃度的化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS梯度稀釋5X濃度的酮康唑工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS稀釋至15μM濃度的咪達唑侖工作液。 Configure 100 mM PBS buffer, prepare 2.5 mg / ml microsome solution and 5 mM NADPH solution with this buffer, and dilute 5X concentration of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015) with PBS. , 0 μM). Ketoconazole working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted with PBS gradient. Midazolam working solution diluted to a concentration of 15 μM with PBS.
分別取2.5mg/ml的微粒體溶液、15μM的咪達唑侖工作液、MgCl2溶液和化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM，每個濃度設置不同的反應體系)各20μl，混合均勻。陽性對照組用相同濃度的酮康唑代替化合物。同時將5mM的NADPH溶液一起在37℃預孵育5分鐘。5分鐘之後取20μl NADPH加入到各孔中，啟動反應，孵育30分鐘。所有孵育樣品設雙樣本。30分鐘後向所有樣本中 加入250μl含內標的乙腈，混勻，800rpm搖10分鐘，然後3700rpm離心10分鐘。取80μl的上清液，轉移至LC-MS/MS分析。 Take 2.5mg / ml microsome solution, 15μM midazolam working solution, MgCl 2 solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0μM, each concentration setting is different). Reaction system) 20 μl each, mixed well. The positive control group replaced the compound with ketoconazole at the same concentration. At the same time, 5mM NADPH solutions were pre-incubated together at 37 ° C for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was started, and the reaction was incubated for 30 minutes. All samples were incubated in duplicate. After 30 minutes, 250 μl of acetonitrile containing internal standard was added to all samples, mixed, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. Take 80 μl of the supernatant and transfer to LC-MS / MS for analysis.
數值經Graphpad Prism計算得到藥物對CYP3A4咪達唑侖代謝位點的IC50值見表4。 The values are calculated by Graphpad Prism and the IC 50 values of the drug on the CYP3A4 midazolam metabolism site are shown in Table 4.
結論：本發明化合物對人肝微粒體CYP3A4的咪達唑侖代謝位點沒有抑制作用，表現出更好的安全性，提示不會發生基於CYP3A4代謝咪達唑侖代謝位點的代謝性藥物相互作用。 Conclusion: The compound of the present invention has no inhibitory effect on the midazolam metabolism site of human liver microsome CYP3A4, and shows better safety, suggesting that metabolic drugs based on the CYP3A4 metabolism midazolam metabolism site will not occur with each other effect.
測試例5、本發明化合物對人肝微粒體CYP2D6酶活性的抑制作用 Test Example 5. Inhibitory effect of the compound of the present invention on human liver microsomal CYP2D6 enzyme activity
本發明化合物對人肝微粒體CYP2D6酶活性採用如下實驗方法測定： The enzyme activity of the compound of the present invention on human liver microsomes CYP2D6 is determined by the following experimental method:
一、實驗材料及儀器 I. Experimental materials and instruments
1. 磷酸緩衝液(PBS),2. NADPH(Sigma N-1630),3. 人肝微粒體(Corning Gentest),4. ABI QTrap 4000液質兩用儀(AB Sciex), 5. Inertsil C8-3管柱，4.6×50mm,5μm(美國迪馬公司)，6. CYP探針受質(20μM的右美沙芬，SIGMA Q0750)和陽性對照抑制劑(奎尼丁，SIGMA D9684)。 1. Phosphate buffer (PBS), 2. NADPH (Sigma N-1630), 3. Human liver microsomes (Corning Gentest), 4. ABI QTrap 4000 dual-use instrument (AB Sciex), 5. Inertsil C8- 3 columns, 4.6 × 50 mm, 5 μm (Dima Corporation), 6. CYP probe substrate (20 μM dextromethorphan, SIGMA Q0750) and positive control inhibitor (quinidine, SIGMA D9684).
二、實驗步驟 Experimental steps
配置100mM的PBS緩衝液，用該緩衝液配製2.5mg/ml的微粒體溶液和5mM的NADPH溶液，用PBS梯度稀釋5X濃度的化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS梯度稀釋5X濃度的奎尼丁工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS稀釋至20μM濃度的右美沙芬工作液。 Configure 100 mM PBS buffer, prepare 2.5 mg / ml microsome solution and 5 mM NADPH solution with this buffer, and dilute 5X concentration of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015) with PBS. , 0 μM). Quinine working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted 5X in PBS. Dextromethorphan working solution diluted to a concentration of 20 μM with PBS.
分別取2.5mg/ml的微粒體溶液、20μM的右美沙芬工作液、MgCl2溶液和化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM，每個濃度設置不同的反應體系)各20μl，混合均勻。陽性對照組用相同濃度的奎尼丁代替化合物。同時將5mM的NADPH溶液一起在37℃預孵育5分鐘，5分鐘之後取20μl NADPH加入到各孔中，啟動反應，孵育30分鐘。所有孵育樣品設雙樣本。30分鐘後向所有樣本中加入250μl含內標的乙腈，混勻，800rpm搖10分鐘。3700rpm離心10分鐘。取80μl的上清液，轉移至LC-MS/MS分析。 Take 2.5mg / ml microsome solution, 20μM dextromethorphan working solution, MgCl 2 solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, respectively, and set different reactions for each concentration System) 20 μl each, mixed well. The positive control group replaced the compound with quinidine at the same concentration. At the same time, 5 mM NADPH solution was pre-incubated together at 37 ° C for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was started, and the reaction was incubated for 30 minutes. All samples were incubated in duplicate. After 30 minutes, add 250 μl of acetonitrile with internal standard to all samples, mix well, and shake at 800 rpm for 10 minutes. Centrifuge at 3700 rpm for 10 minutes. Take 80 μl of the supernatant and transfer to LC-MS / MS for analysis.
數值經Graphpad Prism計算得到藥物對CYP2D6代謝位點的IC50值見表5。 The IC 50 value of the drug on the CYP2D6 metabolic site was calculated by Graphpad Prism, as shown in Table 5.
結論：本發明化合物對人肝微粒體CYP2D6的酶活性沒有抑制作用，表現出更好的安全性，提示不會發生基於CYP2D6發生代謝性藥物相互作用。 Conclusion: The compounds of the present invention have no inhibitory effect on the enzyme activity of human liver microsomes CYP2D6, and show better safety, suggesting that metabolic drug interactions based on CYP2D6 will not occur.
測試例6、本發明化合物對人肝微粒體CYP3A4睾酮代謝位點的酶活性的抑制作用 Test Example 6. Inhibitory effect of the compound of the present invention on the enzyme activity of testosterone metabolism sites of human liver microsomes CYP3A4
本發明化合物對人肝微粒體CYP3A4睾酮代謝位點的酶活性採用如下實驗方法測定： The enzyme activity of the compound of the present invention on human liver microsome CYP3A4 testosterone metabolism site is determined by the following experimental method:
一、實驗材料及儀器 I. Experimental materials and instruments
1. 磷酸緩衝液(PBS),2. NADPH(Sigma N-1630),3. 人肝微粒體(Corning Gentest),4. ABI QTrap 4000液質兩用儀(AB Sciex),5. Inertsil C8-3管柱，4.6×50mm,5μm(美國迪馬公司)，6. CYP探針受質(睾酮/100μM，SIGMA K1003)和陽性對照抑制劑(酮康唑，Dr.Ehrenstorfer GmbH,C17322500)。 1. Phosphate buffer (PBS), 2. NADPH (Sigma N-1630), 3. Human liver microsomes (Corning Gentest), 4. ABI QTrap 4000 dual-use instrument (AB Sciex), 5. Inertsil C8- 3 columns, 4.6 × 50 mm, 5 μm (Dima Corporation), 6. CYP probe substrate (testosterone / 100 μM, SIGMA K1003) and positive control inhibitor (ketoconazole, Dr. Ehrenstorfer GmbH, C17322500).
二、實驗步驟 Experimental steps
配置100mM的PBS緩衝液，用該緩衝液配製2.5mg/ml的微粒體溶液和5mM的NADPH溶液，用PBS梯度稀釋 5X濃度的化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS梯度稀釋5X濃度的酮康唑工作液(150、50、15、5、1.5、0.15、0.015、0μM)。用PBS稀釋至50μM濃度的右美沙芬工作液。 Configure 100 mM PBS buffer, prepare 2.5 mg / ml microsome solution and 5 mM NADPH solution with this buffer, and dilute 5X concentration of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015) with PBS. , 0 μM). Ketoconazole working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM) was diluted with PBS gradient. Dextromethorphan working solution diluted to a concentration of 50 μM with PBS.
分別取2.5mg/ml的微粒體溶液、50μM的睾酮工作液、MgCl2溶液和化合物工作液(150、50、15、5、1.5、0.15、0.015、0μM，每個濃度設置不同的反應體系)各20μl，混合均勻。陽性對照組用相同濃度的酮康唑代替化合物。同時將5mM的NADPH溶液一起在37℃預孵育5分鐘。5分鐘之後取20μl NADPH加入到各孔中，啟動反應，孵育30分鐘。所有孵育樣品設雙樣本。30分鐘後向所有樣本中加入250μl含內標的乙腈，混勻，800rpm搖10分鐘。3700rpm離心10分鐘。取80μl的上清液，轉移至LC-MS/MS分析。 Take 2.5mg / ml microsomal solution, 50μM testosterone working solution, MgCl 2 solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0μM, each concentration is set different reaction system) 20 μl each, mix well. The positive control group replaced the compound with ketoconazole at the same concentration. At the same time, 5mM NADPH solutions were pre-incubated together at 37 ° C for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was started, and the reaction was incubated for 30 minutes. All samples were incubated in duplicate. After 30 minutes, add 250 μl of acetonitrile with internal standard to all samples, mix well, and shake at 800 rpm for 10 minutes. Centrifuge at 3700 rpm for 10 minutes. Take 80 μl of the supernatant and transfer to LC-MS / MS for analysis.
數值經Graphpad Prism計算得到藥物對CYP3A4睾酮代謝位點的IC50值見表6。 The values are calculated by Graphpad Prism and the IC 50 values of the drug on the CYP3A4 testosterone metabolism site are shown in Table 6.
結論：本發明化合物對對人肝微粒體CYP3A4的睾酮代謝位點沒有抑制作用，表現出更好的安全性，提示不會發生基於CYP3A4的睾酮代謝位點的代謝性藥物相互作用。 Conclusion: The compound of the present invention has no inhibitory effect on testosterone metabolism sites of human liver microsomes CYP3A4, and shows better safety, suggesting that no metabolic drug interaction based on CYP3A4 testosterone metabolism sites will occur.
測試例7、本發明化合物對hERG鉀電流的阻斷作用 Test Example 7. Blocking effect of the compound of the present invention on hERG potassium current
1、實驗目的 1.Experimental purpose
應用全自動膜片鉗在轉染hERG鉀通道的穩定細胞株 上測試本發明化合物對hERG鉀電流的阻斷作用。 The blocking effect of the compound of the present invention on hERG potassium current was tested on a stable cell line transfected with hERG potassium channel using a fully automatic patch clamp.
2、實驗方法 2.Experimental methods
2.1 實驗材料與儀器 2.1 Experimental materials and instruments
2.1.1 實驗材料：
2.1.2 實驗儀器：
2.2 全自動膜片鉗實驗步驟 2.2 Fully automatic patch clamp experimental steps
HEK293-hERG穩定細胞株按照1：4的密度在MEM/EBSS培養基(10%FBS，400μg/ml G418,1% MEM非必需胺基酸溶液(100×)，1%丙酮酸鈉溶液)中進行傳代培養，培養48-72小時之內進行全自動膜片鉗實驗。實驗當天將細胞用0.25%胰酶消化後，離心收集細胞，用細胞外液(140mM NaCl，4mM KCl，1mM MgCl2，2mM CaCl2，5mMD一水葡萄糖，10mM Hepes,pH7.4,298mOsmol)重新懸浮細胞製成細胞懸液。將細胞懸液放置在Patchliner儀器的細胞庫上，Patchliner儀器利用負壓控制器將細胞加到芯片(NPC-16)上，負壓將單個細胞吸引在芯片的小孔上。當形成全細胞模式後，儀器將按照設定的hERG電流電壓程序得到hERG電流，然後儀器自動的由低濃度到高濃度，進行化合物灌流。藉由HEAK Patchmaster,HEAK EPC10膜片鉗放大器(Nanion)和Pathlinersoftware以及Pathcontrol HTsoftware提供的數據分析軟件，對化合物各濃度下的電流以及空白對照電流進行分析。 HEK293-hERG stable cell line was performed at a density of 1: 4 in MEM / EBSS medium (10% FBS, 400 μg / ml G418, 1% MEM non-essential amino acid solution (100 ×), 1% sodium pyruvate solution). Subculture, fully automatic patch clamp experiments within 48-72 hours. After the cells were digested with 0.25% trypsin on the day of the experiment, the cells were collected by centrifugation and resuspended with extracellular fluid (140 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 5 mMD glucose monohydrate, 10 mM Hepes, pH 7.4, 298 mOsmol) The cells are made into a cell suspension. The cell suspension was placed on the cell bank of the Patchliner instrument. The Patchliner instrument used a negative pressure controller to add cells to the chip (NPC-16). The negative pressure attracted individual cells to the small holes of the chip. When the whole-cell mode is formed, the instrument will obtain hERG current according to the set hERG current voltage program, and then the instrument will automatically perfuse the compound from low concentration to high concentration. Data analysis software provided by HEAK Patchmaster, HEAK EPC10 patch clamp amplifier (Nanion) and Pathlinersoftware and Pathcontrol HTsoftware were used to analyze the current at each concentration of the compound and the blank control current.
2.3 測試結果 2.3 Test results
本發明化合物對hERG鉀電流的阻斷作用藉由以上的試驗進行測定，測得的IC50值見表7。 The blocking effect of the compound of the present invention on hERG potassium current was measured by the above test. The measured IC 50 values are shown in Table 7.
結論：本發明化合物對hERG的抑制作用弱，由hERG通路引起的副作用可能性小。 Conclusion: The compounds of the present invention have a weak inhibitory effect on hERG, and the possibility of side effects caused by the hERG pathway is small.
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