TW202320750A - Tetrahydrofuran-containing polycyclic derivative and pharmaceutically acceptable salt thereof, preparation method therefor and use thereof - Google Patents
Tetrahydrofuran-containing polycyclic derivative and pharmaceutically acceptable salt thereof, preparation method therefor and use thereof Download PDFInfo
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Abstract
Description
本申請主張申請日為2021年10月25日的中國專利申請2021112420210的優先權，本申請主張申請日為2022年8月25日的中國專利申請2022110367844的優先權，本申請主張申請日為2022年10月14日的中國專利申請2022112838508的優先權。本申請引用上述中國專利申請的全文。This application claims the priority of Chinese patent application 2021112420210 with a filing date of October 25, 2021. This application claims priority of Chinese patent application 2022110367844 with a filing date of August 25, 2022. This application claims that its filing date is 2022 Priority of Chinese patent application 2022112838508 dated October 14. This application cites the full text of the above-mentioned Chinese patent application.
本發明屬於藥物合成領域。具體涉及含四氫呋喃多環類衍生物、其藥學上可接受的鹽及其製備方法，以及其作為CDK2抑制劑在製備治療癌症的藥物中的應用。The invention belongs to the field of medicine synthesis. It specifically relates to tetrahydrofuran-containing polycyclic derivatives, their pharmaceutically acceptable salts and their preparation methods, and their use as CDK2 inhibitors in the preparation of medicines for treating cancer.
不受控制的持續的增殖是癌症和其他增殖紊亂的標誌，因此誘導細胞週期阻滯可有效抑制腫瘤的生長。細胞週期是細胞連續***之間的一段時間，在此期間，細胞透過對多種酵素反應的精確控制實現細胞的精確複製和***。在真核生物中，細胞週期有4個主要階段，G1、S、G2、M期，細胞週期轉換主要被CDKs家族激酶調控。Uncontrolled, sustained proliferation is a hallmark of cancer and other proliferative disorders, so inducing cell cycle arrest is effective in inhibiting tumor growth. The cell cycle is the period of time between successive cell divisions during which cells replicate and divide precisely through the precise control of the reactions of a variety of enzymes. In eukaryotes, the cell cycle has four main phases, G1, S, G2, and M phases, and cell cycle transitions are mainly regulated by CDKs family kinases.
細胞週期蛋白依賴性激酶(CDK)屬於絲/蘇胺酸蛋白激酶家族，是一種由細胞週期催化激酶次單元和調節次單元組成的異二聚體複合物，是參與細胞週期調節的關鍵激酶，目前已發現11個CDK成員。根據CDK功能的不同，可以將其主要分為兩大類。一類CDK參與細胞週期調控，主要包括CDK1、CDK2、CDK4、CDK6等；另一大類CDK參與轉錄調節，主要包括CDK7、CDK8、CDK9、CDK10、CDK11等。CDK1參與M期活動；CDK2參與G1、S、M期活動；CDK4/6參與G1期活動。其餘的CDK 5/7/8/9/19最近也被發現在調控週期活動中有重要作用。Cyclin-dependent kinase (CDK) belongs to the silk/threonine protein kinase family, is a heterodimeric complex composed of a cell cycle catalytic kinase subunit and a regulatory subunit, and is a key kinase involved in cell cycle regulation, Eleven CDK members have been discovered so far. According to the different functions of CDK, it can be mainly divided into two categories. One type of CDK is involved in cell cycle regulation, mainly including CDK1, CDK2, CDK4, CDK6, etc.; another large type of CDK is involved in transcriptional regulation, mainly including CDK7, CDK8, CDK9, CDK10, CDK11, etc. CDK1 participates in M phase activities; CDK2 participates in G1, S, M phase activities; CDK4/6 participates in G1 phase activities. The remaining CDKs 5/7/8/9/19 were also recently found to be important in regulating cycle activity.
細胞週期蛋白依賴激酶(CDK)構成了一個異二聚體家族的絲胺酸/蘇胺酸蛋白激酶參與細胞週期和轉錄。它們包括兩大類：細胞週期CDK和轉錄CDK。CDK的功能依賴於與調控蛋白細胞週期蛋白(cyclins)的特定相互作用，cyclins與CDKs形成異二聚體而活化調節細胞週期過程。Cyclin-dependent kinases (CDKs) constitute a heterodimeric family of serine/threonine protein kinases involved in the cell cycle and transcription. They include two broad categories: cell cycle CDKs and transcriptional CDKs. The function of CDK depends on the specific interaction with regulatory protein cyclins (cyclins), and cyclins and CDKs form heterodimers to activate and regulate cell cycle process.
CDK2的過量表現與細胞週期的異常調節有關。細胞週期蛋白E/CDK2複合物在調控G1/S轉換、組蛋白生物合成和中心體複製方面起著重要作用。細胞週期蛋白D/CDK4/6和細胞週期蛋白E/CDK2對視網膜母細胞瘤(Rb)進行逐步的磷酸化，釋放G1轉錄因子E2F，促進進入S期。在S期早期，細胞週期蛋白A/CDK2的活化促進了允許DNA複製的內源性受質的磷酸化和E2F的失活，以完成S期。Excessive expression of CDK2 is associated with abnormal regulation of the cell cycle. The cyclin E/CDK2 complex plays an important role in the regulation of G1/S transition, histone biosynthesis and centrosome duplication. Cyclin D/CDK4/6 and cyclin E/CDK2 progressively phosphorylate retinoblastoma (Rb), release the G1 transcription factor E2F, and promote entry into S phase. Early in S phase, activation of cyclin A/CDK2 promotes phosphorylation of endogenous substrates that allow DNA replication and inactivation of E2F to complete S phase.
Cyclin E是CDK2的調節性週期蛋白，在癌症中經常過度表現。Cyclin E的擴增或過度表現長期以來一直與乳腺癌的不良後果有關。細胞週期蛋白E2(CCNE2)的過度表現與下列因素有關。細胞週期蛋白E1(CCNE1)的擴增或過度表現也與卵巢癌、胃癌、子宮內膜癌和其他癌症的不良後果有關。Cyclin E過度表現或擴增也引起乳腺癌對CDK4/6的抑制劑抗藥性。抑制CDK2能夠抑制Cyclin E過度表現的癌症細胞生長，此外抑制CDK2也能夠抑制MYCN相關的癌症，CDK2抑制劑具有良好的癌症治療前景。Cyclin E, a regulatory cyclin of CDK2, is often overexpressed in cancer. Amplification or overexpression of cyclin E has long been associated with adverse outcomes in breast cancer. Overexpression of cyclin E2 (CCNE2) is associated with the following factors. Amplification or overexpression of cyclin E1 (CCNE1) has also been associated with adverse outcomes in ovarian, gastric, endometrial and other cancers. Cyclin E overexpression or amplification also causes breast cancer resistance to CDK4/6 inhibitors. Inhibiting CDK2 can inhibit the growth of cancer cells with excessive expression of Cyclin E. In addition, inhibiting CDK2 can also inhibit MYCN-related cancers. CDK2 inhibitors have good prospects for cancer treatment.
CDK1在細胞M期轉換時起著關鍵作用，抑制CDK1活性會帶來比較大的毒性。CDK1和CDK2在激酶結構區域極為相似，因此開發高度選擇性的CDK2抑制劑有著極大的難度。目前已經有多種CDK抑制劑被發現，包括CDK4/6抑制劑、CDK7抑制劑、CDK9抑制劑、CDK2/4/6抑制劑及CDK2/9抑制劑，但是選擇性較好的CDK2特異性抑制劑仍然具有潛在的優勢。CDK1 plays a key role in cell M-phase transition, and inhibition of CDK1 activity will bring relatively large toxicity. CDK1 and CDK2 are very similar in the kinase structure region, so it is extremely difficult to develop highly selective CDK2 inhibitors. A variety of CDK inhibitors have been discovered, including CDK4/6 inhibitors, CDK7 inhibitors, CDK9 inhibitors, CDK2/4/6 inhibitors and CDK2/9 inhibitors, but CDK2-specific inhibitors with better selectivity There are still potential advantages.
本發明發現了一系列新型的CDK2激酶抑制劑化合物，且對CDK2特異性抑制，具有良好的選擇性。The present invention discovers a series of novel CDK2 kinase inhibitor compounds, which specifically inhibit CDK2 and have good selectivity.
本發明的目的在於提供一種通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其中通式( I )所示的結構如下：
本發明還提供了一種優選方案，所述通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，某些基團的定義可如下所述，其他基團的定義可如本發明任一方案所述(以下簡稱“本發明還提供了一種優選方案”)，R中，所述C 1-6烷基獨立地為C 1-4烷基，例如甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred scheme, the compound represented by the general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, the definition of some groups can be as follows, other The definition of the group can be as described in any scheme of the present invention (hereinafter referred to as "the present invention also provides a preferred scheme"), in R, the C 1-6 alkyl is independently a C 1-4 alkyl, for example Methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R中，所述C 1-6鹵代烷基獨立地為C 1-4鹵代烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基；所述鹵代可為F、Cl、Br或I，例如F。 The present invention also provides a preferred scheme, in R, the C 1-6 haloalkyl is independently a C 1-4 haloalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; the halo can be F, Cl, Br or I, such as F.
本發明還提供了一種優選方案，R中，所述C 1-6羥烷基獨立地為C 1-4羥烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred scheme, in R, the C 1-6 hydroxyalkyl is independently a C 1-4 hydroxyalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, isopropyl base, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R中，所述C 2-6烯基獨立地為C 2-4烯基，例如為乙烯基、丙烯基或丁烯基。 The present invention also provides a preferred solution, in R, the C 2-6 alkenyl is independently a C 2-4 alkenyl, such as vinyl, propenyl or butenyl.
本發明還提供了一種優選方案，R中，所述C 2-6炔基獨立地為C 2-4炔基，例如為乙炔基、丙炔基或丁炔基。 The present invention also provides a preferred scheme, in R, the C 2-6 alkynyl is independently a C 2-4 alkynyl, such as ethynyl, propynyl or butynyl.
本發明還提供了一種優選方案，R中，所述C
3-12環烷基獨立地為C
3-6環烷基，例如為環丙基、環丁基、環戊基、雙環[1.1.1]戊烷基或環己基，還例如環丙基、環丁基或
本發明還提供了一種優選方案，R中，所述3-12員雜環基獨立地為3-8員雜環基(例如7員)；其中的3-8員雜環基中雜原子個數獨立地可為1或2個(例如1個)；其中的3-8員雜環基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。The present invention also provides a preferred scheme. In R, the 3-12 membered heterocyclic group is independently a 3-8 membered heterocyclic group (for example, 7 members); wherein the heteroatoms in the 3-8 membered heterocyclic group are The number can be independently 1 or 2 (eg 1); the heteroatoms in the 3-8 membered heterocyclic group can be independently selected from one or both of N and O (eg N).
本發明還提供了一種優選方案，R中，所述C 6-14芳基獨立地為C 6-10芳基，例如苯基或萘基。 The present invention also provides a preferred scheme, in R, the C 6-14 aryl is independently a C 6-10 aryl, such as phenyl or naphthyl.
本發明還提供了一種優選方案，R中，所述5-14員雜芳基獨立地為5-6員雜芳基(例如6員)，其中的5-6員雜芳基中雜原子個數獨立地可為1或2個(例如1個)；其中的5-6員雜芳基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。The present invention also provides a preferred scheme. In R, the 5-14 membered heteroaryl group is independently a 5-6 membered heteroaryl group (for example, 6 members), wherein the heteroatoms in the 5-6 membered heteroaryl group are The number can be independently 1 or 2 (eg 1); the heteroatoms in the 5-6 membered heteroaryl group can be independently selected from one or both of N and O (eg N).
本發明還提供了一種優選方案，環A中，所述C 3-12環烷基為C 3-6環烷基，例如為環丙基、環丁基、環戊基或環己基。 The present invention also provides a preferred solution, in the ring A, the C 3-12 cycloalkyl is a C 3-6 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
本發明還提供了一種優選方案，環A中，所述3-12員雜環基為C 3-8員雜環基；其中的3-8員雜環基中雜原子個數獨立地可為1或2個(例如1個)；其中的3-8員雜環基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。 The present invention also provides a preferred scheme, in the ring A, the 3-12 membered heterocyclic group is a C 3-8 membered heterocyclic group; the number of heteroatoms in the 3-8 membered heterocyclic group can be independently 1 or 2 (eg 1); wherein the heteroatoms in the 3-8 membered heterocyclic group can be independently selected from one or both of N and O (eg N).
本發明還提供了一種優選方案，環A中，所述C 6-14芳基為C 6-10芳基，例如苯基或萘基。 The present invention also provides a preferred solution, in the ring A, the C 6-14 aryl is a C 6-10 aryl, such as phenyl or naphthyl.
本發明還提供了一種優選方案，環A中，所述5-14員雜芳基為5-10員雜芳基獨立地為5-6員雜芳基(例如6員)，其中的5-6員雜芳基中雜原子個數獨立地可為1或2個(例如2個)；其中的5-6員雜芳基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。The present invention also provides a preferred scheme, in Ring A, the 5-14 membered heteroaryl is 5-10 membered heteroaryl independently 5-6 membered heteroaryl (for example 6 members), wherein the 5- The number of heteroatoms in the 6-membered heteroaryl group can be independently 1 or 2 (for example, 2); the heteroatoms in the 5-6 membered heteroaryl group can be independently selected from one or both of N and O species (eg N).
本發明還提供了一種優選方案，環A中，所述C 1-6烷基，例如甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基，還例如甲基、乙基或丙基。 The present invention also provides a preferred scheme, in ring A, the C 1-6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert- Butyl, also eg methyl, ethyl or propyl.
本發明還提供了一種優選方案，環A中，所述C 1-6鹵代烷基獨立地為C 1-4鹵代烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基；所述鹵代可為F、Cl、Br或I，例如F。 The present invention also provides a preferred scheme, in Ring A, the C 1-6 haloalkyl is independently a C 1-4 haloalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl, sec-butyl or tert-butyl; the halo can be F, Cl, Br or I, such as F.
本發明還提供了一種優選方案，環A中，所述C 1-6烷氧基獨立地為C 1-4烷氧基，例如甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、異丁氧基、仲丁氧基或叔丁氧基。 The present invention also provides a preferred scheme, in Ring A, the C 1-6 alkoxy group is independently a C 1-4 alkoxy group, such as methoxy, ethoxy, n-propoxy, isopropoxy group, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.
本發明還提供了一種優選方案，環A中，所述C 1-6烷硫基獨立地為C 1-4烷硫基，例如甲硫基、乙硫基、正丙硫基、異丙硫基、正丁硫基、異丁硫基、仲丁硫基或叔丁硫基。 The present invention also provides a preferred scheme, in Ring A, the C 1-6 alkylthio group is independently a C 1-4 alkylthio group, such as methylthio, ethylthio, n-propylthio, isopropylthio , n-butylthio, isobutylthio, sec-butylthio or tert-butylthio.
本發明還提供了一種優選方案，環A中，所述C 1-6羥烷基獨立地為C 1-4羥烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred scheme, in Ring A, the C 1-6 hydroxyalkyl is independently a C 1-4 hydroxyalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, iso Propyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，環A中，所述C 2-6烯基獨立地為C 2-4烯基，例如為乙烯基、丙烯基或丁烯基。 The present invention also provides a preferred solution, in the ring A, the C 2-6 alkenyl is independently a C 2-4 alkenyl, such as vinyl, propenyl or butenyl.
本發明還提供了一種優選方案，環A中，所述C 2-6炔基獨立地為C 2-4炔基，例如為乙炔基、丙炔基或丁炔基。 The present invention also provides a preferred solution, in the ring A, the C 2-6 alkynyl is independently a C 2-4 alkynyl, such as ethynyl, propynyl or butynyl.
本發明還提供了一種優選方案，R 1中，所述鹵素獨立地為F、Cl、Br或I，例如F。 The present invention also provides a preferred solution, in R 1 , the halogen is independently F, Cl, Br or I, such as F.
本發明還提供了一種優選方案，R 1中，所述C 1-6烷基獨立地為C 1-4烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基，例如甲基或乙基。 The present invention also provides a preferred scheme, in R , the C 1-6 alkyl is independently a C 1-4 alkyl, and the alkyl is preferably methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl, sec-butyl or tert-butyl, eg methyl or ethyl.
本發明還提供了一種優選方案，R 1中，所述C 1-6鹵代烷基獨立地為C 1-4鹵代烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基；所述鹵代可為F、Cl、Br或I，例如F。 The present invention also provides a preferred scheme, in R , the C 1-6 haloalkyl is independently a C 1-4 haloalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl, sec-butyl or tert-butyl; the halo can be F, Cl, Br or I, such as F.
本發明還提供了一種優選方案，R 1中，所述C 1-6烷氧基獨立地為C 1-4烷氧基，例如甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、異丁氧基、仲丁氧基或叔丁氧基。 The present invention also provides a preferred scheme, in R , the C 1-6 alkoxy group is independently a C 1-4 alkoxy group, such as methoxy, ethoxy, n-propoxy, isopropoxy group, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.
本發明還提供了一種優選方案，R 1中，所述C 1-6羥烷基獨立地為C 1-4羥烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred solution. In R , the C 1-6 hydroxyalkyl group is independently a C 1-4 hydroxyalkyl group, and the alkyl group is preferably methyl, ethyl, n-propyl, iso Propyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R 1中，所述C 2-6烯基獨立地為C 2-4烯基，例如為乙烯基、丙烯基或丁烯基。 The present invention also provides a preferred solution, in R 1 , the C 2-6 alkenyl is independently a C 2-4 alkenyl, such as vinyl, propenyl or butenyl.
本發明還提供了一種優選方案，R 1中，所述C 2-6炔基獨立地為C 2-4炔基，例如為乙炔基、丙炔基或丁炔基。 The present invention also provides a preferred solution, in R 1 , the C 2-6 alkynyl is independently a C 2-4 alkynyl, such as ethynyl, propynyl or butynyl.
本發明還提供了一種優選方案，R 1中，所述C 3-12環烷基獨立地為C 3-6環烷基，例如為環丙基、環丁基、環戊基、雙環[1.1.1]戊烷基或環己基，還例如環丙基。 The present invention also provides a preferred solution. In R , the C 3-12 cycloalkyl group is independently a C 3-6 cycloalkyl group, such as cyclopropyl, cyclobutyl, cyclopentyl, bicyclo[1.1 .1] Pentyl or cyclohexyl, also eg cyclopropyl.
本發明還提供了一種優選方案，R 1中，所述3-12員雜環基獨立地為3-8員雜環基(例如7員)；其中的3-8員雜環基中雜原子個數獨立地可為1或2個(例如1個)；其中的3-8員雜環基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。 The present invention also provides a preferred scheme, in R 1 , the 3-12 membered heterocyclic group is independently a 3-8 membered heterocyclic group (for example, 7 members); the heteroatom in the 3-8 membered heterocyclic group is The number can be 1 or 2 (eg 1) independently; the heteroatoms in the 3-8 membered heterocyclic group can be independently selected from one or both of N and O (eg N).
本發明還提供了一種優選方案，R 1中，所述C 6-14芳基獨立地為C 6-10芳基，例如苯基或萘基。 The present invention also provides a preferred solution, in R 1 , the C 6-14 aryl group is independently a C 6-10 aryl group, such as phenyl or naphthyl.
本發明還提供了一種優選方案，R 1中，所述5-14員雜芳基立地為5-6員雜芳基或5員并6員雜芳基，其中的5-6員雜芳基和5員并6員雜芳基中雜原子個數獨立地可為1或2個；其中的5-6員雜芳基和5員并6員雜芳基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。 The present invention also provides a preferred scheme, in R , the 5-14 membered heteroaryl is 5-6 membered heteroaryl or 5-6 membered heteroaryl, wherein the 5-6 membered heteroaryl The number of heteroatoms in the 5-membered and 6-membered heteroaryl can be 1 or 2 independently; the heteroatoms in the 5-6-membered heteroaryl and the 5-membered and 6-membered heteroaryl can be independently selected from One or both of N and O (eg N).
本發明還提供了一種優選方案，R 1中，所述C 3-12環烷基氧基獨立地為C 3-6環烷基氧基，例如為環丙基氧基、環丁基氧基、環戊基氧基或環己基氧基，還例如環丙基氧基。 The present invention also provides a preferred scheme, in R 1 , the C 3-12 cycloalkyloxy group is independently a C 3-6 cycloalkyloxy group, such as cyclopropyloxy, cyclobutyloxy , cyclopentyloxy or cyclohexyloxy, also eg cyclopropyloxy.
本發明還提供了一種優選方案，R y中，所述C 1-6烷基獨立地為C 1-4烷基，例如甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred scheme, in Ry , the C 1-6 alkyl is independently C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R y中，所述C 1-6鹵代烷基獨立地為C 1-4鹵代烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基；所述鹵代可為F、Cl、Br或I，例如F。 The present invention also provides a preferred scheme, in Ry , the C 1-6 haloalkyl is independently a C 1-4 haloalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl, sec-butyl or tert-butyl; the halo can be F, Cl, Br or I, such as F.
本發明還提供了一種優選方案，R y中，所述C 1-6烷氧基獨立地為C 1-4烷氧基，例如甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、異丁氧基、仲丁氧基或叔丁氧基。 The present invention also provides a preferred scheme, in R y , the C 1-6 alkoxy group is independently a C 1-4 alkoxy group, such as methoxy, ethoxy, n-propoxy, isopropoxy group, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.
本發明還提供了一種優選方案，R y中，所述C 1-6烷硫基獨立地為C 1-4烷硫基，例如甲硫基、乙硫基、正丙硫基、異丙硫基、正丁硫基、異丁硫基、仲丁硫基或叔丁硫基。 The present invention also provides a preferred scheme, in R y , the C 1-6 alkylthio group is independently a C 1-4 alkylthio group, such as methylthio, ethylthio, n-propylthio, isopropylthio , n-butylthio, isobutylthio, sec-butylthio or tert-butylthio.
本發明還提供了一種優選方案，R y中，所述C 1-6羥烷基獨立地為C 1-4羥烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred solution. In Ry , the C 1-6 hydroxyalkyl is independently a C 1-4 hydroxyalkyl, and the alkyl is preferably methyl, ethyl, n-propyl, iso Propyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R y中，所述C 2-6烯基獨立地為C 2-4烯基，例如為乙烯基、丙烯基或丁烯基。 The present invention also provides a preferred solution, in R y , the C 2-6 alkenyl is independently a C 2-4 alkenyl, such as vinyl, propenyl or butenyl.
本發明還提供了一種優選方案，R y中，所述C 2-6炔基獨立地為C 2-4炔基，例如為乙炔基、丙炔基或丁炔基。 The present invention also provides a preferred solution, in R y , the C 2-6 alkynyl is independently a C 2-4 alkynyl, such as ethynyl, propynyl or butynyl.
本發明還提供了一種優選方案，R y中，所述C 3-12環烷基獨立地為C 3-6環烷基，例如為環丙基、環丁基、環戊基、雙環[1.1.1]戊烷基或環己基，還例如環丙基。 The present invention also provides a preferred solution, in R y , the C 3-12 cycloalkyl is independently a C 3-6 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, bicyclo[1.1 .1] Pentyl or cyclohexyl, also eg cyclopropyl.
本發明還提供了一種優選方案，R y中，所述3-12員雜環基獨立地為3-8員雜環基；其中的3-8員雜環基中雜原子個數獨立地可為1或2個；其中的3-8員雜環基中的雜原子可獨立地選自N和O中的一種或兩種。 The present invention also provides a preferred scheme, in R y , the 3-12 membered heterocyclic group is independently a 3-8 membered heterocyclic group; the number of heteroatoms in the 3-8 membered heterocyclic group can be independently 1 or 2; the heteroatoms in the 3-8 membered heterocyclic group can be independently selected from one or both of N and O.
本發明還提供了一種優選方案，R y中，所述C 6-14芳基獨立地為C 6-10芳基，例如苯基或萘基。 The present invention also provides a preferred solution, in R y , the C 6-14 aryl is independently a C 6-10 aryl, such as phenyl or naphthyl.
本發明還提供了一種優選方案，R y中，所述5-14員雜芳基立地為5-6員雜芳基基，其中的5-6員雜芳基中雜原子個數獨立地可為1或2個；其中的5-6員雜芳基中的雜原子可獨立地選自N和O中的一種或兩種。 The present invention also provides a preferred scheme, in R y , the 5-14 membered heteroaryl group is a 5-6 membered heteroaryl group, wherein the number of heteroatoms in the 5-6 membered heteroaryl group can be independently 1 or 2; the heteroatoms in the 5-6 membered heteroaryl group can be independently selected from one or both of N and O.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 1-6烷基獨立地為C 1-4烷基，例如甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred solution, in R a , R b , R c and R d , the C 1-6 alkyl is independently a C 1-4 alkyl, such as methyl, ethyl, n-propyl , isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 1-6鹵代烷基獨立地為C 1-4鹵代烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基；所述鹵代可為F、Cl、Br或I，例如F。 The present invention also provides a preferred solution, in R a , R b , R c and R d , the C 1-6 haloalkyl is independently a C 1-4 haloalkyl, and the alkyl is preferably methyl, ethyl Base, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; said halo can be F, Cl, Br or I, such as F.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 1-6烷氧基獨立地為C 1-4烷氧基，例如甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、異丁氧基、仲丁氧基或叔丁氧基。 The present invention also provides a preferred scheme, in R a , R b , R c and R d , the C 1-6 alkoxy group is independently a C 1-4 alkoxy group, such as methoxy, ethoxy , n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 1-6烷硫基獨立地為C 1-4烷硫基，例如甲硫基、乙硫基、正丙硫基、異丙硫基、正丁硫基、異丁硫基、仲丁硫基或叔丁硫基。 The present invention also provides a preferred solution, in R a , R b , R c and R d , the C 1-6 alkylthio group is independently a C 1-4 alkylthio group, such as methylthio, ethylthio , n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio or tert-butylthio.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 1-6羥烷基獨立地為C 1-4羥烷基，所述烷基優選為甲基、乙基、正丙基、異丙基、正丁基、異丁基、仲丁基或叔丁基。 The present invention also provides a preferred scheme, in R a , R b , R c and R d , the C 1-6 hydroxyalkyl is independently a C 1-4 hydroxyalkyl, and the alkyl is preferably methyl , ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 2-6烯基獨立地為C 2-4烯基，例如為乙烯基、丙烯基或丁烯基。 The present invention also provides a preferred solution, in R a , R b , R c and R d , the C 2-6 alkenyl is independently a C 2-4 alkenyl, such as vinyl, propenyl or butene base.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 2-6炔基獨立地為C 2-4炔基，例如為乙炔基、丙炔基或丁炔基。 The present invention also provides a preferred scheme, in R a , R b , R c and R d , the C 2-6 alkynyl is independently a C 2-4 alkynyl, such as ethynyl, propynyl or butyl Alkynyl.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 3-12環烷基獨立地為C 3-6環烷基，例如為環丙基、環丁基、環戊基、雙環[1.1.1]戊烷基或環己基，還例如環丙基。 The present invention also provides a preferred solution, in R a , R b , R c and R d , the C 3-12 cycloalkyl is independently a C 3-6 cycloalkyl, such as cyclopropyl, cyclobutyl radical, cyclopentyl, bicyclo[1.1.1]pentyl or cyclohexyl, also eg cyclopropyl.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述3-12員雜環基獨立地為3-8員雜環基(例如7員)；其中的3-8員雜環基中雜原子個數獨立地可為1或2個(例如1個)；其中的3-8員雜環基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。 The present invention also provides a preferred scheme, among R a , R b , R c and R d , the 3-12 membered heterocyclic group is independently a 3-8 membered heterocyclic group (for example, 7 members); wherein 3 The number of heteroatoms in the -8-membered heterocyclic group can be independently 1 or 2 (for example, 1); the heteroatoms in the 3-8-membered heterocyclic group can be independently selected from one of N and O or Two (e.g. N).
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述C 6-14芳基獨立地為C 6-10芳基，例如苯基或萘基。 The present invention also provides a preferred solution, in R a , R b , R c and R d , the C 6-14 aryl groups are independently C 6-10 aryl groups, such as phenyl or naphthyl.
本發明還提供了一種優選方案，R a、R b、R c和R d中，所述5-14員雜芳基獨立地為5-6員雜芳基(例如6員)，其中的5-6員雜芳基中雜原子個數獨立地可為1或2個(例如1個)；其中的5-6員雜芳基中的雜原子可獨立地選自N和O中的一種或兩種(例如N)。 The present invention also provides a preferred scheme, among R a , R b , R c and R d , the 5-14 membered heteroaryl is independently a 5-6 membered heteroaryl (for example, 6 members), wherein 5 The number of heteroatoms in the -6-membered heteroaryl group can be independently 1 or 2 (for example, 1); the heteroatoms in the 5-6-membered heteroaryl group can be independently selected from one of N and O or Two (e.g. N).
本發明還提供了一種優選方案，所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其具體結構如通式( I-A )或通式( I-B )所示：
本發明還提供了一種優選方案，所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其具體結構如通式( II )所示：
本發明還提供了一種優選方案，所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其具體結構如通式( II-A )或通式( II-B )所示：
在本發明的一個優選實施方式中，所述環A選自5-6員單環雜芳基、8-10員雙環稠雜環基或8-10員雙環稠雜芳基。In a preferred embodiment of the present invention, the ring A is selected from a 5-6 membered monocyclic heteroaryl group, an 8-10 membered bicyclic fused heterocyclic group or an 8-10 membered bicyclic fused heteroaryl group.
在本發明的進一步優選實施方式中，所述環A選自吡唑基、吡啶基或吡唑并5-6員雜芳基。In a further preferred embodiment of the present invention, the ring A is selected from pyrazolyl, pyridyl or pyrazolo 5-6 membered heteroaryl.
在本發明的進一步優選實施方式中，所述環A選自吡唑基、吡啶基或吡唑并嘧啶基。In a further preferred embodiment of the present invention, the ring A is selected from pyrazolyl, pyridyl or pyrazolopyrimidinyl.
在本發明的進一步優選實施方式中，所述環A選自
在本發明的一個優選實施方式中，所述L為鍵、-C(O)-或-C(O)CH 2。 In a preferred embodiment of the present invention, said L is a bond, -C(O)- or -C(O)CH 2 .
本發明還提供了一種優選方案，所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其具體結構如通式( III )所示：
本發明還提供了一種優選方案，所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其具體結構如通式( IV )所示：
本發明還提供了一種優選方案，所述R a選自氫或甲基； 所述R b選自甲基、乙基、叔丁基、異丁基、環丙基、環丁基或雙環[1.1.1]戊烷基，所述的甲基、乙基、異丙基、叔丁基、異丁基、環丙基、環丁基和雙環[1.1.1]戊烷基，任選地進一步被甲基、乙基、異丙基或叔丁基中的一個或多個取代基所取代； 或者，R a和R b以及它們所在的原子一起形成氮雜環丁烷基或7-氮雜雙環[2.2.1]庚烷基，所述的氮雜環丁烷基和7-氮雜雙環[2.2.1]庚烷基，任選地進一步被一個或多個甲基所取代。 The present invention also provides a preferred scheme, the R a is selected from hydrogen or methyl; the R b is selected from methyl, ethyl, tert-butyl, isobutyl, cyclopropyl, cyclobutyl or bicyclo[ 1.1.1] Pentyl, said methyl, ethyl, isopropyl, tert-butyl, isobutyl, cyclopropyl, cyclobutyl and bicyclo[1.1.1]pentyl, optionally Further substituted by one or more substituents in methyl, ethyl, isopropyl or tert-butyl; or, R a and R b and their atoms together form azetidinyl or 7-nitrogen Heterobicyclo[2.2.1]heptyl, said azetidinyl and 7-azabicyclo[2.2.1]heptanyl, optionally further substituted by one or more methyl groups.
本發明還提供了一種優選方案，
本發明還提供了一種優選方案，
本發明還提供了一種優選方案，R 1獨立地選自氫、氘、鹵素、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基或C 3-12環烷基，所述的C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基和C 3-8環烷基，進一步被氘、鹵素、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基和C 3-12環烷基中的一個或多個取代基所取代。 The present invention also provides a preferred scheme, R independently selected from hydrogen, deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 3-12 cycloalkyl , the C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 3-8 cycloalkyl, further deuterium, halogen, C 1-6 alkyl, C 1- One or more substituents in 6 haloalkyl, C 1-6 alkoxy and C 3-12 cycloalkyl.
本發明還提供了一種優選方案，R 1獨立地選自氘、鹵素、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基或C 3-8環烷基，所述的C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基和C 3-8環烷基，進一步被氘、鹵素、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基和C 3-8環烷基中的一個或多個取代基所取代。 The present invention also provides a preferred scheme, R 1 is independently selected from deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 3-8 cycloalkyl, so The C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy and C 3-8 cycloalkyl, further deuterium, halogen, C 1-6 alkyl, C 1-6 haloalkane One or more substituents in radical, C 1-6 alkoxy and C 3-8 cycloalkyl.
本發明還提供了一種優選方案，R 1獨立地選自C 1-6烷基，所述的C 1-6烷基任選地進一步被C 1-6烷基和C 1-6烷氧基中的一個或多個取代基所取代。 The present invention also provides a preferred scheme, R 1 is independently selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally further replaced by C 1-6 alkyl and C 1-6 alkoxy Substituted by one or more substituents.
本發明還提供了一種優選方案，所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，其具體結構如通式( IV-A )和通式( IV-B )所示：
在本發明的一個優選實施方式中，所述R a和R b各自獨立地選自氫、氘、鹵素、羥基、巰基、硝基、氰基、胺基、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基、C 1-6烷硫基、C 1-6羥烷基、C 2-6烯基、C 2-6炔基、C 3-8環烷基、3-10員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基或5-10員雜芳基氧基，所述的胺基、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基、C 1-6烷硫基、C 1-6羥烷基、C 2-6烯基、C 2-6炔基、C 3-8環烷基、3-10員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基和5-10員雜芳基氧基，任選地，進一步被氫、氘、鹵素、羥基、巰基、硝基、氰基、胺基、氧代基、硫代基、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基、C 1-6烷硫基、C 1-6羥烷基、C 2-6烯基、C 2-6炔基、C 3-8環烷基、3-8員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基和5-10員雜芳基氧基中的一個或多個取代基所取代； 或者，R a和R b以及它們所在的原子一起形成3-10員雜環基或5-10員雜芳基，所述的3-10員雜環基和5-10員雜芳基，任選地，進一步被氘、鹵素、羥基、巰基、硝基、氰基、胺基、氧代基、硫代基、C 1-6烷基、C 1-6鹵代烷基、C 1-6烷氧基、C 1-6烷硫基、C 1-6羥烷基、C 2-6烯基、C 2-6炔基、C 3-8環烷基、3-8員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基和5-10員雜芳基氧基中的一個或多個取代基所取代。 In a preferred embodiment of the present invention, said R a and R b are each independently selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, nitro, cyano, amino, C 1-6 alkyl, C 1 -6 haloalkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 hydroxyalkyl , C 2-6 alkenyl, C 2-6 alkynyl , C 3-8 cycloalkyl , 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryl Oxygen or 5-10 membered heteroaryloxy, the amino, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 hydroxyalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl Base, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy and 5-10 membered heteroaryloxy, optionally, further hydrogen, deuterium , halogen, hydroxyl, mercapto, nitro, cyano, amino, oxo, thio, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 Alkylthio, C 1-6 hydroxyalkyl , C 2-6 alkenyl, C 2-6 alkynyl , C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, 5 -One or more of 10 membered heteroaryl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy and 5-10 membered heteroaryloxy Substituents; Or, R a and R b and their atoms together form a 3-10 membered heterocyclic group or a 5-10 membered heteroaryl group, the 3-10 membered heterocyclic group and 5-10 membered heterocyclic group Member heteroaryl, optionally, further replaced by deuterium, halogen, hydroxyl, mercapto, nitro, cyano, amino, oxo, thio, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 hydroxyalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, 3-8 members Heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy and 5- One or more substituents in the 10-membered heteroaryloxy group are substituted.
在本發明進一步優選實施方式中，所述R a選自氫或甲基； 所述R b選自甲基、乙基、異丙基、叔丁基、異丁基、環丙基、環丁基或雙環[1.1.1]戊烷基，所述的甲基、乙基、異丙基、叔丁基、異丁基、環丙基、環丁基和雙環[1.1.1]戊烷基，任選地進一步被甲基、乙基、異丙基或叔丁基中的一個或多個取代基所取代； 或者，R a和R b以及它們所在的原子一起形成氮雜環丁烷基或7-氮雜雙環[2.2.1]庚烷基，所述的氮雜環丁烷基和7-氮雜雙環[2.2.1]庚烷基，任選地進一步被一個或多個甲基所取代。 In a further preferred embodiment of the present invention, the R a is selected from hydrogen or methyl; the R b is selected from methyl, ethyl, isopropyl, tert-butyl, isobutyl, cyclopropyl, cyclobutyl base or bicyclo[1.1.1]pentyl, the methyl, ethyl, isopropyl, tert-butyl, isobutyl, cyclopropyl, cyclobutyl and bicyclo[1.1.1]pentyl , optionally further substituted by one or more substituents in methyl, ethyl, isopropyl or tert-butyl; or, R a and R b and the atoms where they are located together form an azetidinyl group Or 7-azabicyclo[2.2.1]heptyl, said azetidinyl and 7-azabicyclo[2.2.1]heptanyl, optionally further surrounded by one or more methyl groups replaced.
在本發明進一步優選實施方式中，所述
在本發明的一個優選實施方式中，所述R 1獨立地選自氫、氘、氰基、胺基、C 1-3烷基、C 1-3鹵代烷基、C 1-3烷氧基、C 1-3羥烷基、-CH 2OR c、-(CH 2) 2OR c、-CH 2NR dR c、-(CH 2) 2NR dR c、-O(CH 2) 2NR dR c、-CH 2S(O)(=NR d)R c、-S(O)(=NR d)R c、-CH 2N=S(=O)R cR d或-C(O)NR dR c，所述的胺基、C 1-3烷基、C 1-3鹵代烷基、C 1-3烷氧基和C 1-3羥烷基，任選地進一步被氘、鹵素、羥基、巰基、硝基、氰基、胺基、氧代基、硫代基、C 1-3烷基、C 1-3鹵代烷基、C 1-3烷氧基、C 1-3烷硫基、C 1-3羥烷基、C 2-3烯基、C 2-3炔基、C 3-8環烷基、3-8員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基和5-10員雜芳基氧基中的一個或多個取代基所取代； R c和R d各自獨立地選自氫、氘、鹵素、羥基、巰基、硝基、氰基、胺基、C 1-3烷基、C 1-3鹵代烷基、C 1-3烷氧基、C 1-3烷硫基、C 1-3羥烷基、C 2-3烯基、C 2-3炔基、C 3-8環烷基、3-8員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基或5-10員雜芳基氧基，所述的胺基、C 1-3烷基、C 1-3鹵代烷基、C 1-3烷氧基、C 1-3烷硫基、C 1-3羥烷基、C 2-3烯基、C 2-3炔基、C 3-8環烷基、3-8員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基和5-10員雜芳基氧基，任選地，進一步被氫、氘、鹵素、羥基、巰基、硝基、氰基、胺基、氧代基、硫代基、C 1-3烷基、C 1-3鹵代烷基、C 1-3烷氧基、C 1-3烷硫基、C 1-3羥烷基、C 2-3烯基、C 2-3炔基、C 3-8環烷基、3-8員雜環基、C 6-10芳基、5-10員雜芳基、C 3-8環烷基氧基、3-8員雜環基氧基、C 6-10芳基氧基和5-10員雜芳基氧基中的一個或多個取代基所取代。 In a preferred embodiment of the present invention, said R 1 is independently selected from hydrogen, deuterium, cyano, amino, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 hydroxyalkyl, -CH 2 OR c , -(CH 2 ) 2 OR c , -CH 2 NR d R c , -(CH 2 ) 2 NR d R c , -O(CH 2 ) 2 NR d R c , -CH 2 S(O)(=NR d )R c , -S(O)(=NR d )R c , -CH 2 N=S(=O)R c R d or -C( O) NR d R c , the amino group, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy and C 1-3 hydroxyalkyl, optionally further replaced by deuterium, Halogen, hydroxyl, mercapto, nitro, cyano, amino, oxo, thio, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 alkane Thio, C 1-3 hydroxyalkyl, C 2-3 alkenyl, C 2-3 alkynyl, C 3-8 cycloalkyl , 3-8 membered heterocyclyl, C 6-10 aryl, 5- One or more of 10-membered heteroaryl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy and 5-10 membered heteroaryloxy Substituents are substituted; R c and R d are each independently selected from hydrogen, deuterium, halogen, hydroxyl, mercapto, nitro, cyano, amino, C 1-3 alkyl, C 1-3 haloalkyl, C 1 -3 alkoxy, C 1-3 alkylthio, C 1-3 hydroxyalkyl, C 2-3 alkenyl, C 2-3 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocycle Base, C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy or 5-10 membered Heteroaryloxy, the amino group, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 alkylthio, C 1-3 hydroxyalkyl, C 2-3 alkenyl, C 2-3 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclic group, C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 ring Alkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy and 5-10 membered heteroaryloxy, optionally, further replaced by hydrogen, deuterium, halogen, hydroxyl, mercapto, Nitro, cyano, amino, oxo, thio, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 alkylthio, C 1- 3 hydroxyalkyl, C 2-3 alkenyl, C 2-3 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclic group, C 6-10 aryl, 5-10 membered heteroaryl, One or more substituents in C 3-8 cycloalkyloxy, 3-8 membered heterocyclyloxy, C 6-10 aryloxy and 5-10 membered heteroaryloxy.
在本發明的進一步優選實施方式中，所述R 1獨立地選自-CN、-NH 2、-OH、-CH 3、-CF 3、-OCH 3、-CH 2OCH 3、-CH 2NH 2、-CH(OH)CH 3、-C(O)NH 2、-CH 2N(CH 3) 2、-C(O)N(CH 3) 2、-(CH 2) 2OCH 3、-OCH(CH 3) 2、-O(CH 2) 2OCH 3或-O(CH 2) 2N(CH 3) 2。 In a further preferred embodiment of the present invention, said R 1 is independently selected from -CN, -NH 2 , -OH, -CH 3 , -CF 3 , -OCH 3 , -CH 2 OCH 3 , -CH 2 NH 2 , -CH(OH)CH 3 , -C(O)NH 2 , -CH 2 N(CH 3 ) 2 , -C(O)N(CH 3 ) 2 , -(CH 2 ) 2 OCH 3 , - OCH(CH 3 ) 2 , —O(CH 2 ) 2 OCH 3 , or —O(CH 2 ) 2 N(CH 3 ) 2 .
在本發明的進一步優選實施方式中，所述R
1獨立地選自H、D、F、Cl、-CN、-NH
2、-OH、-CH
3、-CF
3、-CD
3、-OCH
3、-OCF
3、-CH
2OCH
3、-(CH
2)
2OCH
3、
在本發明的進一步優選實施方式中，R為
在本發明的進一步優選實施方式中，R為
在本發明的進一步優選實施方式中，-L-R
x為
在本發明的進一步優選實施方式中，R為
在本發明的進一步優選實施方式中，-L-R
x為
在本發明的進一步優選實施方式中，R為
在本發明的進一步優選實施方式中，-L-R
x為
本發明還提供了一種優選方案，當本發明沒有特別說明是取代還是未取代，通常理解為未取代。The present invention also provides a preferred solution. When the present invention does not specify whether it is substituted or unsubstituted, it is generally understood as unsubstituted.
本發明還提供了一種優選方案，如上所示的各通式化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，具體選自如下化合物：
本發明還涉及一種製備如前所述所述的通式( I )所示的化合物、其前驅藥或立體異構體及其藥學上可接受鹽的方法，其特徵在於，包含以下步驟：
式( Ia )與式( Ib )透過單步或多步親核取代、偶聯反應、Mitsunobu反應、酯化反應等常見有機反應引入R基團，得到式( Ic )所示化合物或立體異構體及其藥學上可接受鹽； 式( Ic )所示的化合物透過還原反應得到如式( Id )所示的化合物或立體異構體及其藥學上可接受鹽； 式( Id )與式( Ie )透過醯胺化反應、親核取代或者偶聯反應等常見有機反應得到式( If )所示的化合物或其立體異構體； 式( If )所示的化合物脫去保護基團R 1a得到式( Ig )所示化合物、其前驅藥或立體異構體及其藥學上可接受鹽； 式( Ig )透過分離純化得到式( I )所示的化合物、其前驅藥或立體異構體及其藥學上可接受鹽； 任選地，式( I )所示的化合物進一步透過手性拆分得到單一構型化合物、其前驅藥或立體異構體及其藥學上可接受鹽； 其中： X 1選自但不限於羥基、鹵素或三氟甲磺酸酯基(OTf)等； X 2選自但不限於羥基、鹵素或三氟甲磺酸酯基(OTf)等； R 1a為吡唑氮上的保護基，選自但不限於叔丁基( t-Bu)、四氫-2 H-吡喃-2-基(THP)、(三甲基矽烷基)乙氧甲基(SEM)、對甲氧基苄基(PMB)等； L、R和R x如通式( I )所定義。 Formula (Ia) and formula (Ib) introduce R groups through common organic reactions such as single-step or multi-step nucleophilic substitution, coupling reaction, Mitsunobu reaction, esterification reaction, etc., to obtain the compound or stereoisomerism of formula (Ic) body and its pharmaceutically acceptable salt; the compound represented by formula (Ic) can be obtained by reduction reaction as compound or stereoisomer and its pharmaceutically acceptable salt as represented by formula (Id); formula (Id) and formula ( Ie) Obtain the compound represented by formula (If) or its stereoisomers through common organic reactions such as amidation reaction, nucleophilic substitution or coupling reaction; the compound represented by formula (If) removes the protecting group R 1a Obtain the compound represented by formula (Ig), its prodrug or stereoisomer and pharmaceutically acceptable salt thereof; Formula (Ig) obtains the compound represented by formula (I), its prodrug or stereoisomer through separation and purification And its pharmaceutically acceptable salt; Optionally, the compound shown in formula (I) is further obtained through chiral resolution to obtain a single configuration compound, its prodrug or stereoisomer and its pharmaceutically acceptable salt; wherein: X1 is selected from but not limited to hydroxyl, halogen or trifluoromethanesulfonate (OTf) etc.; X2 is selected from but not limited to hydroxyl, halogen or trifluoromethanesulfonate (OTf) etc.; R 1a is pyridine The protecting group on the azole nitrogen is selected from but not limited to tert-butyl ( t- Bu), tetrahydro- 2H -pyran-2-yl (THP), (trimethylsilyl) ethoxymethyl (SEM ), p-methoxybenzyl (PMB), etc.; L, R and R x are as defined in general formula (I).
本發明還涉及一種製備如前所述的通式( II )所示的化合物、其前驅藥或立體異構體及其藥學上可接受鹽的方法，其特徵在於，包含以下步驟：
本發明涉及一種式( IIa )所示的中間體化合物或其立體異構體及其鹽，其具體結構如下：
本發明涉及一種式( IIa )所示的中間體化合物或其立體異構體及其藥學上可接受鹽，其具體結構如下：
本發明的一個優選實施方式，所述式( IIa )選自如下化合物：
本發明進一步涉及一種製備所述式( IIa )所示的中間體化合物或其立體異構體及其藥學上可接受鹽的方法，其特徵在於，包含以下步驟：
本發明涉及一種式( IIa-1 )所示的中間體化合物或其立體異構體及其鹽，其具體結構如下：
本發明的一個優選實施方式，所述式( IIa-1 )選自如下化合物：
本發明進一步涉及一種製備所述式( IIa-1 )所示的中間體化合物或其立體異構體及其藥學上可接受鹽的方法，其特徵在於，包含以下步驟：
本發明還涉及一種藥用組成物，其包含有治療疾病效劑量的如上所述各通式化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及一種或多種藥學上可接受的載劑或賦形劑。The present invention also relates to a pharmaceutical composition, which contains the above-mentioned compounds of the general formulas, their prodrugs, stereoisomers or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable acceptable carrier or excipient.
本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及上述藥用組成物在製備治療CDK2媒介的疾病的藥物中的用途。The present invention further relates to compounds represented by general formula (I), their prodrugs, stereoisomers or pharmaceutically acceptable salts thereof, and the use of the above pharmaceutical compositions in the preparation of drugs for treating CDK2-mediated diseases.
本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及上述藥用組成物在製備治療細胞異常生長的藥物中的用途。The present invention further relates to the compound represented by the general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating abnormal cell growth.
本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及上述藥用組成物在製備治療癌症的藥物中的用途。The present invention further relates to the compound represented by the general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating cancer.
另一方面，本發明進一步涉及通式( I )所示的化合物、其立體異構體或其藥學上可接受的鹽，以及其藥用組成物治療由CDK2媒介的疾病的方法。On the other hand, the present invention further relates to a compound represented by general formula (I), its stereoisomer or a pharmaceutically acceptable salt thereof, and a method for treating diseases mediated by CDK2 with its pharmaceutical composition.
另一方面，本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及其藥用組成物治療細胞異常生長的方法。On the other hand, the present invention further relates to the compound represented by general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and a method for treating abnormal cell growth with its pharmaceutical composition.
另一方面，本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及其藥用組成物治療癌症的用途。On the other hand, the present invention further relates to the use of the compound represented by the general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and its pharmaceutical composition for treating cancer.
本發明的一個優選實施方式，所述癌症可為卵巢癌。In a preferred embodiment of the present invention, the cancer may be ovarian cancer.
另一方面，本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及其藥用組成物治療由CDK2媒介的疾病的用途。On the other hand, the present invention further relates to the use of the compound represented by general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and its pharmaceutical composition for treating diseases mediated by CDK2.
另一方面，本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及其藥用組成物治療細胞異常生長的用途。On the other hand, the present invention further relates to the use of the compound represented by the general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and its pharmaceutical composition for treating abnormal cell growth.
另一方面，本發明進一步涉及通式( I )所示的化合物、其前驅藥、立體異構體或其藥學上可接受的鹽，以及其藥用組成物治療癌症的用途。 發明的詳細說明 On the other hand, the present invention further relates to the use of the compound represented by the general formula (I), its prodrug, stereoisomer or pharmaceutically acceptable salt thereof, and its pharmaceutical composition for treating cancer. Detailed Description of the Invention
除非有相反陳述，在說明書和申請專利範圍中使用的技術術語有下述含義。Unless stated to the contrary, the technical terms used in the specification and claims have the following meanings.
術語“烷基”指飽和脂肪鏈烴中缺少一個氫的烴基，其為包含1至20個碳原子的直鏈或支鏈基團，優選含有1至8個碳原子的烷基，更優選1至6個碳原子的烷基，最優選1至3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、叔丁基、仲丁基、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。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，所述取代基優選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、氧代基、羧基或羧酸酯基，本發明優選甲基、乙基、異丙基、叔丁基、鹵代烷基、氘代烷基、烷氧基取代的烷基和羥基取代的烷基。The term "alkyl" refers to a hydrocarbon group lacking one hydrogen in a saturated aliphatic chain hydrocarbon, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably 1 An alkyl group of 1 to 6 carbon atoms, most preferably an alkyl group of 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-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-dimethyl Pentyl, 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 ylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethyl 2,2-diethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being preferably one or more of the following groups independently selected from alkyl Alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, ring Alkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate, preferably methyl, ethyl, isopropyl, tert-butyl, haloalkane in the present invention radical, deuterated alkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl.
術語“亞烷基”是指烷基的一個氫進一步被取代的基團，即飽和脂肪鏈烴中缺少兩個氫的烴基，其為包含1至20個碳原子的直鏈或支鏈基團，優選含有1至8個碳原子的亞烷基，更優選1至6個碳原子的亞烷基，最優選1至3個碳原子的亞烷基。非限制性實例包括“亞甲基”(-CH 2-)、“亞乙基”(-(CH 2) 2-)、“亞正丙基”(-(CH 2) 3-)、“亞異丙基”(-(CH)(CH 3)(CH 2)-)、“亞正丁基”(-(CH 2) 4-)等。 The term "alkylene" refers to a group in which one hydrogen of an alkyl group is further substituted, that is, a hydrocarbon group lacking two hydrogens in a saturated aliphatic chain hydrocarbon, which is a straight-chain or branched chain group containing 1 to 20 carbon atoms , preferably an alkylene group having 1 to 8 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, and most preferably an alkylene group having 1 to 3 carbon atoms. Non-limiting examples include "methylene" (-CH 2 -), "ethylene" (-(CH 2 ) 2 -), "n-propylene" (-(CH 2 ) 3 -), "ethylene "Isopropyl" (-(CH)(CH 3 )(CH 2 )-), "n-butylene" (-(CH 2 ) 4 -), etc.
術語“烯基”指至少含有兩個碳原子和至少包含一個碳-碳雙鍵組成的不飽和脂肪族烴基團，其為包含2至20個碳原子的直鏈或支鏈基團，優選含有2至8個碳原子的烯基，更優選2至6個碳原子的烯基，最優選2至4個碳原子的烯基。例如乙烯基、1-丙烯基、2-丙烯基、1-、2-或3-丁烯基等。烯基可以是取代的或非取代的，當被取代時，取代基優選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。The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group containing at least two carbon atoms and at least one carbon-carbon double bond, which is a straight or branched chain group containing 2 to 20 carbon atoms, preferably containing An alkenyl group of 2 to 8 carbon atoms, more preferably an alkenyl group of 2 to 6 carbon atoms, most preferably an alkenyl group of 2 to 4 carbon atoms. For example vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3-butenyl and the like. Alkenyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero Cycloalkylthio.
術語“炔基”指至少含有兩個碳原子和至少包含一個碳-碳三鍵組成的不飽和脂肪族炔基團，其為包含2至20個碳原子的直鏈或支鏈基團，優選含有2至8個碳原子的炔基，更優選2至6個碳原子的炔基，最優選2至4個碳原子的炔基。例如乙炔基、1-丙炔基、2-丙炔基、1-、2-或3-丁炔基等。炔基可以是取代的或非取代的，當被取代時，取代基優選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。The term "alkynyl" refers to an unsaturated aliphatic alkynyl group containing at least two carbon atoms and at least one carbon-carbon triple bond, which is a straight-chain or branched group containing 2 to 20 carbon atoms, preferably An alkynyl group containing 2 to 8 carbon atoms, more preferably an alkynyl group of 2 to 6 carbon atoms, most preferably an alkynyl group of 2 to 4 carbon atoms. For example, ethynyl, 1-propynyl, 2-propynyl, 1-, 2- or 3-butynyl and the like. Alkynyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero Cycloalkylthio.
術語“環烷基”指飽和或部分不飽和單環或多環非芳香環狀烴取代基，環烷基環原子包含3至20個碳原子，優選包含3至12個碳原子，更優選包含3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，優選環丙基、環丁基、環己基、環戊基和環庚基；多環環烷基包括螺環烷基、稠環烷基和橋環烷基等。The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic non-aromatic cyclic hydrocarbon substituent, the cycloalkyl ring atoms containing 3 to 20 carbon atoms, preferably containing 3 to 12 carbon atoms, more preferably containing 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Cyclopropyl, cyclooctyl, etc., preferably cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl and cycloheptyl; polycyclic cycloalkyl includes spirocycloalkyl, fused cycloalkyl and bridged cycloalkyl, etc.
術語“螺環烷基”指含有5至20個碳原子，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，其整體不具有芳香性(即整體不能形成共軛的π電子系統)，但可以有一個環或多個環具有共軛的π電子系統。螺環烷基環原子優選為6至14個碳原子，更優選為7至10個碳原子。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，優選為單螺環烷基和雙螺環烷基。更優選為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“橋環烷基”指含有5至20個碳原子，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，其整體不具有芳香性(即整體不能形成共軛的π電子系統)，但可以有一個環或多個環具有共軛的π電子系統。橋環烷基環原子優選為6至14員個碳原子，更優選為7至10個碳原子。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，優選為雙環、三環或四環，更有選為雙環或三環。橋環烷基的非限制性實例包括：
所述環烷基環可以稠合於環烷基或芳基環上，其中與母體結構連接在一起的環可以為環烷基或芳基環，非限制性實例包括茚滿基、四氫萘基、苯并環庚烷基等。環烷基可以是任選取代的或非取代的，當被取代時，取代基優選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、氧代基、羧基或羧酸酯基。The cycloalkyl ring may be fused to a cycloalkyl or aryl ring, where the ring attached to the parent structure may be a cycloalkyl or aryl ring, non-limiting examples include indanyl, tetrahydronaphthalene base, benzocycloheptyl, etc. Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio group, heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.
術語“雜環基”指飽和或部分不飽和單環或多環非芳香環狀烴取代基，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、磷、硼或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。雜環基優選包含3至12個環原子，其中1～4個是雜原子；更優選包含3至10個環原子；進一步優選包含3至8個環原子；最優選包含3至8個環原子。單環雜環基的非限制性實例包括氧雜環丁烷、四氫吡喃基、氮雜環庚烷基、吡咯烷基、咪唑烷基、四氫呋喃基、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、呱啶基、吡啶酮基、呱嗪基、嗎啉基、硫代嗎啉基、高呱嗪基、吡喃基等，優選氧雜環丁烷、四氫呋喃基、四氫吡喃基、氮雜環庚烷基、呱啶基、吡啶酮基和呱嗪基。多環雜環基包括螺環、稠環和橋環的雜環基等；其中涉及到的螺環、稠環和橋環的雜環基任選與其他基團透過單鍵相連接，或者透過環上的任意兩個或者兩個以上的原子與其他環烷基、雜環基、芳基和雜芳基進一步併環連接。 The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic non-aromatic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen, phosphorus, Heteroatoms of boron or S(O) m (where m is an integer from 0 to 2), excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Heterocyclyl preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably contains 3 to 10 ring atoms; further preferably contains 3 to 8 ring atoms; most preferably contains 3 to 8 ring atoms . Non-limiting examples of monocyclic heterocyclyl groups include oxetane, tetrahydropyranyl, azepanyl, pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl , dihydrofuryl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, pyridonyl, piperazinyl, morpholinyl, thiomorpholinyl, homopirazinyl, pyranyl, etc., preferably Oxetane, tetrahydrofuranyl, tetrahydropyranyl, azepanyl, piperidinyl, pyridinonyl and piperazinyl. Polycyclic heterocyclic groups include spiro rings, fused rings and bridged ring heterocyclic groups, etc.; wherein the spiro rings, condensed rings and bridged ring heterocyclic groups are optionally connected to other groups through single bonds, or through Any two or more atoms on the ring are further linked with other cycloalkyl groups, heterocyclic groups, aryl groups and heteroaryl groups.
術語“螺雜環基”指含有5至20個環原子，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選自氮、氧、磷、硼或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，其整體不具有芳香性(即整體不能形成共軛的π電子系統)，但可以有一個環或多個環具有共軛的π電子系統。螺雜環基環原子優選為6至14員，更優選為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，優選為單螺雜環基和雙螺雜環基。更優選為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”指含有5至20個環原子，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，其中一個或多個環原子選自氮、氧、磷、硼或S(O)
m(其中m是整數0至2)的雜原子，其餘環原子為碳。其一個或多個環可以含有一個或多個雙鍵，其整體不具有芳香性(即整體不能形成共軛的π電子系統)，但可以有一個環或多個環具有共軛的π電子系統。稠雜環基環原子優選為6至14員，更優選為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，優選為雙環或三環，更優選為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”指含有5至20個環原子，任意兩個環共用兩個不直接連接的原子的多環雜環基團，其中一個或多個環原子為選自氮、氧、磷、硼或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，其整體不具有芳香性(即整體不能形成共軛的π電子系統)，但可以有一個環或多個環具有共軛的π電子系統。橋雜環基環原子優選為6至14員，更優選為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，優選為雙環、三環或四環，更有選為雙環或三環。橋雜環基的非限制性實例包括：
所述雜環基環可以稠合於環烷基、雜環基、芳基或雜芳基環上，其中與母體結構連接在一起的環可以為環烷基、雜環基、芳基環或雜芳基環，其非限制性實例包括：
雜環基可以是任選取代的或非取代的，當被取代時，取代基優選為一個或多個以下基團，其獨立地選自氧代基(=O)、硫代基(=S)、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。The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from oxo (=O), thioxo (=S ), alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, carboxyl group or carboxylate group.
術語“芳基”指具有共軛的π電子體系的6至20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，芳基環原子優選為6至14員，更優選為6至10員，例如苯基和萘基。更優選苯基。The term "aryl" refers to a 6 to 20 membered full carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group having a conjugated pi electron system, preferably 6 to 14 aryl ring atoms members, more preferably 6 to 10 members, such as phenyl and naphthyl. Phenyl is more preferred.
芳基可以是取代的或非取代的，當被取代時，取代基優選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。Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero Cycloalkylthio, carboxyl or carboxylate.
術語“雜芳基”指包含1至4個雜原子、5至20個環原子的雜芳族體系，其中雜原子選自氧、硫或氮等。雜芳基分為單環雜芳基和多環雜芳基；雜芳基環原子優選為5至14員，更優選為5至10員；單環雜芳基環原子優選為5員或6員，例如咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基、異噁唑基、吡咯基、***基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基、噠嗪基、噁二唑基等，優選吡啶基、噁唑基、異噁唑基、吡啶基、嘧啶基、吡唑基、噻唑基、噻二唑基和噁二唑基。多環雜芳基一般指所述雜芳基環稠合於芳基或雜芳基形成多環稠雜芳基，其中與母體結構連接在一起的環可以為芳基環或雜芳基環，多環稠雜芳基優選雙環稠雜芳基，雙環稠雜芳基非限制性實例包括：
雜芳基可以是任選取代的或非取代的，當被取代時，取代基優選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 groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio group, heterocycloalkylthio group, carboxyl group or carboxylate group.
術語“鹵代烷基”指烷基中的氫被一個或多個鹵素取代的基團，其中烷基的定義如上所述。鹵代烷基的非限制性實例包括：一氟甲基、二氟甲基、三氟甲基、一氯甲基、二氯甲基、三氯甲基、1-氟乙基、2-氟乙基、1,1-二氟乙基、1,2-二氟乙基等，鹵代烷基可以是任選取代的或非取代的，當被取代時，取代基優選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。The term "haloalkyl" refers to a group in which the hydrogens of an alkyl group are replaced by one or more halogens, wherein alkyl is as defined above. Non-limiting examples of haloalkyl include: monofluoromethyl, difluoromethyl, trifluoromethyl, monochloromethyl, dichloromethyl, trichloromethyl, 1-fluoroethyl, 2-fluoroethyl , 1,1-difluoroethyl, 1,2-difluoroethyl, etc., the haloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, It is independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl , heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.
“羥烷基”指烷基中的氫被一個或多個羥基取代的基團，其中烷基如上所定義。羥烷基的非限制性實例包括：羥甲基、1-羥基乙基、2-羥基乙基、1,2-二羥基乙基、1-羥基丙基、1-羥基丁基等，羥烷基可以是任選取代的或非取代的，當被取代時，取代基優選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。"Hydroxyalkyl" means an alkyl group in which a hydrogen is replaced by one or more hydroxy groups, wherein alkyl is as defined above. Non-limiting examples of hydroxyalkyl groups include: hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl, 1-hydroxypropyl, 1-hydroxybutyl, etc., hydroxyalkyl The group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio , alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, carboxyl or carboxylate.
術語“烷氧基”指-O-(烷基)，其中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、丙氧基或丁氧基，烷氧基可以是任選取代的或非取代的，當被取代時，取代基優選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。The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy or butoxy, alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or a plurality of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, Heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.
術語“烷硫基”指-S-(烷基)，其中烷基的定義如上所述。烷硫基的非限制性實例包括：甲硫基、乙硫基、丙硫基、丁硫基，烷硫基可以是任選取代的或非取代的，當被取代時，取代基優選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。The term "alkylthio" refers to -S-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkylthio include: methylthio, ethylthio, propylthio, butylthio, alkylthio may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or a plurality of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, Heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.
術語“環烷基氧基”指-O-(環烷基)，其中環烷基的定義如上所述。The term "cycloalkyloxy" refers to -O-(cycloalkyl), wherein cycloalkyl is as defined above.
術語“雜環基氧基” 指-O-(雜環基)，其中雜環基的定義如上所述。The term "heterocyclyloxy" refers to -O-(heterocyclyl), wherein heterocyclyl is as defined above.
術語“C 6-14芳基氧基” 指-O-(C 6-14芳基)，其中C 6-14芳基的定義如上所述。 The term "C 6-14 aryloxy" refers to -O-(C 6-14 aryl), wherein C 6-14 aryl is as defined above.
術語“5-14員雜芳基氧基” 指-O-(5-14員雜芳基氧基)，其中5-14員雜芳基氧基的定義如上所述。The term "5-14 membered heteroaryloxy" refers to -O-(5-14 membered heteroaryloxy), wherein 5-14 membered heteroaryloxy is as defined above.
“羥基”指-OH。"Hydroxy" means -OH.
“鹵素”指氟(F)、氯(Cl)、溴(Br)或碘(I)。"Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
“胺基”指-NH 2。 "Amino" refers to -NH2 .
“氰基”指-CN。"Cyano" refers to -CN.
“硝基”指-NO 2。 "Nitro" refers to -NO2 .
“羧基”指-C(O)OH。"Carboxy" refers to -C(O)OH.
“THF”指四氫呋喃。"THF" means tetrahydrofuran.
“PE”指石油醚。"PE" means petroleum ether.
“EA”指乙酸乙酯。"EA" means ethyl acetate.
“IPA”指異丙醇。"IPA" means isopropanol.
“MeOH”指甲醇。"MeOH" means methanol.
“DMF”指N,N-二甲基甲醯胺。"DMF" refers to N,N-dimethylformamide.
“TFA”指三氟乙酸。"TFA" means trifluoroacetic acid.
“ACN”指乙腈。"ACN" means acetonitrile.
“DMA”指N,N-二甲基乙醯胺。"DMA" refers to N,N-dimethylacetamide.
“Et 2O”指***。 " Et2O " means diethyl ether.
“DCM”指二氯甲烷。"DCM" means dichloromethane.
“DCE”指1,2二氯乙烷。"DCE" means 1,2 dichloroethane.
“DIPEA”指N,N-二異丙基乙胺。"DIPEA" refers to N,N-diisopropylethylamine.
“NBS”指N-溴代丁二醯亞胺。"NBS" refers to N-bromosuccinimide.
“NIS”指N-碘代丁二醯亞胺。"NIS" refers to N-iodosuccinimide.
“Cbz-Cl”指氯甲酸苄酯。"Cbz-Cl" refers to benzyl chloroformate.
“Pd 2(dba) 3”指三(二亞苄基丙酮)二鈀。 "Pd 2 (dba) 3 " refers to tris(dibenzylideneacetone)dipalladium.
“Dppf”指1,1’-雙二苯基膦二茂鐵。"Dppf" refers to 1,1'-bisdiphenylphosphinoferrocene.
“HATU”指2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯。"HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
“KHMDS”指雙三甲基二矽基胺基鉀。"KHMDS" refers to potassium bistrimethyldisilazide.
“LiHMDS”指雙三甲基二矽基胺基鋰。"LiHMDS" refers to lithium bistrimethyldisilazide.
“MeLi”指甲基鋰。"MeLi" means methyllithium.
“n-BuLi”指正丁基鋰。"n-BuLi" refers to n-butyllithium.
“NaBH(OAc) 3”指三乙醯氧基硼氫化鈉。 "NaBH(OAc) 3 " refers to sodium triacetyloxyborohydride.
“X選自A、B、或C”、“X選自A、B和C”、“X為A、B或C”、“X為A、B和C”等不同用語均表達了相同的意義，即表示X可以是A、B、C中的任意一種或幾種。Different terms such as "X is selected from A, B, or C", "X is selected from A, B, and C", "X is A, B, or C", "X is A, B, and C" all express the same The meaning means that X can be any one or several of A, B, and C.
本發明所述的氫均可被其同位素氘所取代，本發明涉及的實施例化合物中的任一氫也均可被氘取代。The hydrogen described in the present invention can be replaced by its isotope deuterium, and any hydrogen in the example compounds involved in the present invention can also be replaced by deuterium.
本發明的所有化合物，如所示化合物結構與名稱有衝突，以結構表示為准。For all compounds of the present invention, if there is a conflict between the structure and the name of the compound shown, the structural representation shall prevail.
“任選”或“任選地”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的場合。例如，“任選被烷基取代的雜環基團”意味著烷基可以但不是必須存在，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。"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, a "heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes cases where the heterocycle group is substituted with an alkyl group and cases where the heterocycle group is not substituted with an alkyl group .
“取代的”指基團中的一個或多個氫，優選為5個，更優選為1～3個氫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(透過實驗或理論)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" means that one or more hydrogens in a group, preferably 5, more preferably 1 to 3 hydrogens are independently substituted by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions and that a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amine or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (eg, ethylenic) bond.
“藥用組成物”表示含有一種或多種本文所述化合物或其生理學上/可藥用的鹽或前驅藥物與其他化學組分的混合物，以及其他組分例如生理學/可藥用的載劑和賦形劑。藥用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 compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, and other components such as physiologically/pharmaceutically acceptable agents and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert 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 the salt of the compound of the present invention, which is safe and effective when used in mammals, and has proper biological activity.
在不違背本領域常識的基礎上，上述各優選條件，可任意組合，即得本發明各較佳實例。On the basis of not violating common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本發明所用試劑和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本發明的積極進步效果在於：本發明實施例化合物對CDK2/CycE1激酶具有良好的抑制活性，且對CDK2/CDK1激酶抑制具有良好的選擇性。本發明實施例化合物對於OVCAR-3細胞增殖具有良好的抑制活性。本發明實施例化合物在Caco-2細胞模型上表現出更好的滲透性和更低的外排活性。本發明實施例明顯優化了產品藥物代謝動力學性質。The positive progress effect of the present invention lies in: the compound of the embodiment of the present invention has good inhibitory activity to CDK2/CycE1 kinase, and has good selectivity to CDK2/CDK1 kinase inhibition. The compounds of the examples of the present invention have good inhibitory activity on the proliferation of OVCAR-3 cells. The compounds of the examples of the present invention showed better permeability and lower efflux activity on the Caco-2 cell model. The embodiment of the present invention obviously optimizes the pharmacokinetic properties of the product.
具體實施方式化學合成方法： The specific embodiment chemical synthesis method:
在此描述的化合物可以利用商業化可獲得的或者由已知實驗方法可製備的起始原料，透過有機合成領域技術人員掌握的多種方法進行合成。本發明的化合物可以採用下述方法，或合成有機化學領域中已知的合成方法，結合本領域技術人員所掌握的方法變化來合成。優選的合成方法包括但不限於以下描述。有關各個反應步驟的更詳細說明，請參閱下面的“實施例”部分。The compounds described herein can be synthesized by a variety of methods well known to those skilled in the art of organic synthesis, using starting materials that are commercially available or can be prepared by known experimental procedures. The compounds of the present invention can be synthesized by using the following methods, or synthetic methods known in the field of synthetic organic chemistry, combined with method changes mastered by those skilled in the art. Preferred synthetic methods include, but are not limited to, those described below. For a more detailed description of the individual reaction steps, see the "Examples" section below.
如無特殊說明，合成步驟中的起始原料可以採用或按照本領域已知的方法來合成，或可購買自西格瑪奧德里奇(Sigma-Aldrich Co. Ltd)、畢得醫藥(Bide Pharmatech Ltd.)、韶遠化學(Accela ChemBio Co. Ltd)等公司。Unless otherwise specified, the starting materials in the synthesis steps can be synthesized using or according to methods known in the art, or can be purchased from Sigma-Aldrich Co. Ltd, Bide Pharmatech Ltd. ), Shaoyuan Chemical (Accela ChemBio Co. Ltd) and other companies.
在無特殊說明的情況下，本發明的所有反應均在連續的磁力攪拌下，在乾燥氮氣或氬氣氛下進行，溶劑為乾燥溶劑，反應溫度單位為攝氏度。 分析方法和儀器設備： Unless otherwise specified, all the reactions in the present invention are carried out under a dry nitrogen or argon atmosphere under continuous magnetic stirring, the solvent is a dry solvent, and the unit of the reaction temperature is Celsius. Analytical methods and equipment:
用於實施例特徵分析的LCMS數據是透過帶有DAD檢測器的Agilent 1260-6120/6125 MSD系統獲得的。測試方法包括：LCMS data used for the characterization of the examples were obtained through an Agilent 1260-6120/6125 MSD system with DAD detector. Test methods include:
LCMS方法A： 管柱：HALO C18 4.6 × 30 mm, 2.7 μm 柱溫：45℃ 流動相：溶劑A為0.025%三氟乙酸+99.975%水；溶劑B為0.025%三氟乙酸+99.975%乙腈 流速：1.8 mL/min 梯度：在0.8分鐘內，溶劑B由5%至95%線性遞增，然後在95%下保持0.8分鐘，最後溶劑B在5%下保持到2.0分鐘 LCMS Method A: Column: HALO C18 4.6 × 30 mm, 2.7 μm Column temperature: 45°C Mobile phase: solvent A is 0.025% trifluoroacetic acid + 99.975% water; solvent B is 0.025% trifluoroacetic acid + 99.975% acetonitrile Flow rate: 1.8 mL/min Gradient: In 0.8 minutes, solvent B was increased linearly from 5% to 95%, then held at 95% for 0.8 minutes, and finally solvent B was held at 5% for 2.0 minutes
LCMS方法B： 管柱：HALO C18 4.6 × 30 mm, 2.7 μm 柱溫：45℃ 流動相：溶劑A為0.1%甲酸+99.9%水；溶劑B為0.1%甲酸+99.9%乙腈 流速：1.8 mL/min 梯度：在0.8分鐘內，溶劑B由5%至95%線性遞增，然後在95%下保持0.8分鐘，最後溶劑B在5%下保持到2.0分鐘 LCMS Method B: Column: HALO C18 4.6 × 30 mm, 2.7 μm Column temperature: 45°C Mobile phase: solvent A is 0.1% formic acid + 99.9% water; solvent B is 0.1% formic acid + 99.9% acetonitrile Flow rate: 1.8 mL/min Gradient: In 0.8 minutes, solvent B was increased linearly from 5% to 95%, then held at 95% for 0.8 minutes, and finally solvent B was held at 5% for 2.0 minutes
LCMS方法C： 管柱：Xbrigde® C18 4.6 × 50 mm, 2.5 μm 柱溫：40℃ 流動相：溶劑A為0.05%氨水+99.5%水；溶劑B為100%乙腈 流速：1.8 mL/min 梯度：在2.5分鐘內，溶劑B由5%至95%線性遞增到1分鐘，然後在95%下保持到2分鐘，最後溶劑B從2.05分鐘開始保持5%到2.5分鐘 LCMS Method C: Column: Xbrigde® C18 4.6 × 50 mm, 2.5 μm Column temperature: 40°C Mobile phase: solvent A is 0.05% ammonia + 99.5% water; solvent B is 100% acetonitrile Flow rate: 1.8 mL/min Gradient: Solvent B linearly increased from 5% to 95% over 2.5 minutes to 1 minute, then held at 95% for 2 minutes, and finally solvent B was held at 5% to 2.5 minutes from 2.05 minutes
用於實施例特徵分析的NMR數據是透過Bruker傅立葉變換光譜儀( 1H NMR: 400 MHz)獲得的。數據以化學位移(多重性、氫原子數目)的形式提供。化學位移以內部標準四甲基矽烷( δ 四甲基矽烷＝0 ppm)和/或參考溶劑峰來進行指定，所述參考溶劑峰在 1H NMR光譜中對於氘代二甲基亞碸(DMSO- d 6 )為2.49 ppm，對於氘代甲醇(CD 3OD)為3.30 ppm，對於氘代乙腈(CD 3CN)為1.94 ppm，對於氘代氯仿(CDCl 3)為7.24 ppm。 純化方法： The NMR data used for the characteristic analysis of the examples were obtained through a Bruker Fourier transform spectrometer ( 1 H NMR: 400 MHz). Data are presented as chemical shifts (multiplicity, number of hydrogen atoms). Chemical shifts are assigned in terms of internal standard tetramethylsilane (δTetramethylsilane = 0 ppm) and/or reference solvent peaks that appear in 1 H NMR spectra for deuterated dimethylsulfone (DMSO - d 6 ) is 2.49 ppm, for deuterated methanol (CD 3 OD ) is 3.30 ppm, for deuterated acetonitrile (CD 3 CN) is 1.94 ppm, and for deuterated chloroform (CDCl 3 ) is 7.24 ppm. Purification method:
實施例和中間體的純化透過矽膠管柱層析法、反相矽膠管柱層析法和/或超臨界流體層析法(SFC)進行。矽膠管柱層析法一般是以矽膠或預裝矽膠管柱為載體、石油醚/乙酸乙酯或二氯甲烷/甲醇等體系為洗提劑；反相矽膠管柱層析法一般是以C18矽膠管柱為載體，使用UV檢測器(214 nm和254 nm)和製備型LCMS進行檢測，流動相包括乙腈/水(0.1%甲酸)、乙腈/水(0.1%三氟乙酸)和乙腈/水(0.1%氨水)等體系。超臨界流體層析法(SFC)一般是採用不同類型的管柱作為載體，以CO
2/含0.2% NH
3的甲醇(7M氨甲醇溶液)等體系為流動相。
中間體的製備：
中間體 A ：5-(3-(3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-甲醯胺基)-1
H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯
在0℃將氯化亞碸(98.5 g, 0.83 mol)加入到5-硝基-1氫-吡唑-3-甲酸(50.0 g, 0.32 mol)的甲醇溶液(500 mL)中，反應液在70℃下攪拌16小時。反應結束後真空濃縮除去溶劑。冰浴冷卻下，加水稀釋並用碳酸氫鈉飽和水溶液將pH調至8，然後用乙酸乙酯萃取，飽和食鹽水洗滌後，有機相真空濃縮並乾燥，得到化合物 A-2(粗產物，白色固體，48.0 g)。LC-MS (ESI), m/z: [M+H] +=170.1 At 0°C, argonoxide (98.5 g, 0.83 mol) was added to 5-nitro-1 hydrogen-pyrazole-3-carboxylic acid (50.0 g, 0.32 mol) in methanol solution (500 mL), and the reaction solution was Stir at 70°C for 16 hours. After the reaction was completed, the solvent was removed by vacuum concentration. Under cooling in an ice bath, dilute with water and adjust the pH to 8 with a saturated aqueous solution of sodium bicarbonate, then extract with ethyl acetate, wash with saturated brine, and concentrate the organic phase in vacuo and dry to obtain compound A-2 (crude product, white solid , 48.0 g). LC-MS (ESI), m/z: [M+H] + =170.1
1H NMR (400 MHz, DMSO- d 6 ) δ 15.23 (s, 1H), 7.53 (s, 1H), 3.90 (s, 3H). 步驟2：1-(叔-丁基)-3-硝基-1 H-吡唑-5-甲酸甲酯( A-3) 1 H NMR (400 MHz, DMSO- d 6 ) δ 15.23 (s, 1H), 7.53 (s, 1H), 3.90 (s, 3H). Step 2: 1-(tert-butyl)-3-nitro -1 H -pyrazole-5-carboxylic acid methyl ester ( A-3 )
將5-硝基-1 H-吡唑-3-甲酸甲酯(15.0 g, 87.7 mmol)、三苯基膦(69.1 g, 263 mmol)和叔丁醇(19.5 g, 263 mmol)溶於四氫呋喃(150 mL)中。於0℃下逐滴向以上溶液中加入偶氮二甲酸二乙酯(45.8 g, 263 mmol)，並將混合物於70℃下攪拌16小時。混合物經乙酸乙酯萃取，飽和食鹽水洗滌後，有機相真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(PE: DCM=3:1)純化得到化合物 A-3(粉紅色油狀物，10.0 g，產率：50%)。LC-MS (ESI), m/z: [M+H] +=227.2 步驟3：(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)甲醇( A-4) 5-Nitro- 1H -pyrazole-3-carboxylic acid methyl ester (15.0 g, 87.7 mmol), triphenylphosphine (69.1 g, 263 mmol) and tert-butanol (19.5 g, 263 mmol) were dissolved in THF (150 mL). To the above solution was added diethyl azodicarboxylate (45.8 g, 263 mmol) dropwise at 0°C, and the mixture was stirred at 70°C for 16 hours. The mixture was extracted with ethyl acetate, washed with saturated brine, and the organic phase was concentrated in vacuo and dried. The obtained crude product was purified by silica gel column chromatography (PE: DCM=3:1) to obtain compound A-3 (pink oily material, 10.0 g, yield: 50%). LC-MS (ESI), m/z: [M+H] + =227.2 Step 3: (1-(tert-butyl)-3-nitro-1 H -pyrazol-5-yl)methanol ( A -4 )
0℃下向1-(叔-丁基)-3-硝基-1 H-吡唑-5-甲酸甲酯(11.0 g, 48.4 mmol)的四氫呋喃溶液(55.0 mL)中分批加入硼氫化鋰(3.20 g, 31.7 mmol)，並將混合物於室溫中攪拌16小時。加水稀釋並用乙酸乙酯萃取，飽和食鹽水洗滌後，有機相真空濃縮並乾燥。所得粗產物透過矽膠管柱層析法(PE: EA＝4:1)純化得到化合物 A-4(黃色油狀物，7.3 g, 產率：72%)。LC-MS (ESI), m/z: [M+H] +=199.2 步驟4：1-(叔-丁基)-3-硝基-1 H-吡唑-5-甲醛( A-5) To a solution of methyl 1-(tert-butyl)-3-nitro- 1H -pyrazole-5-carboxylate (11.0 g, 48.4 mmol) in THF (55.0 mL) was added portionwise lithium borohydride at 0°C (3.20 g, 31.7 mmol), and the mixture was stirred at room temperature for 16 hours. It was diluted with water and extracted with ethyl acetate. After washing with saturated brine, the organic phase was concentrated in vacuo and dried. The obtained crude product was purified by silica gel column chromatography (PE:EA=4:1) to obtain compound A-4 (yellow oil, 7.3 g, yield: 72%). LC-MS (ESI), m/z: [M+H] + =199.2 Step 4: 1-(tert-butyl)-3-nitro-1 H -pyrazole-5-carbaldehyde ( A-5 )
(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)甲醇(6.0 g, 30.1 mmol)的乙酸乙酯溶液(55.0 mL)中分批加入二氧化錳(78.5 g, 903 mmol)，將反應混合物於80℃下攪拌16小時。過濾並用乙酸乙酯洗滌濾餅，有機相真空濃縮。所得粗產物透過矽膠管柱層析法(PE: EA＝10:1)純化得到化合物 A-5(淡黃色油狀物，4.0 g, 產率：67%)。LC-MS (ESI), m/z: [M+H] +=197.2. Manganese dioxide ( 78.5 g, 903 mmol), the reaction mixture was stirred at 80°C for 16 hours. Filter and wash the filter cake with ethyl acetate, and the organic phase is concentrated in vacuo. The obtained crude product was purified by silica gel column chromatography (PE:EA=10:1) to obtain compound A-5 (light yellow oil, 4.0 g, yield: 67%). LC-MS (ESI), m/z: [M+H] + =197.2.
1H NMR (400 MHz, DMSO- d 6 ) δ 10.04 (s, 1H), 7.94 (s, 1H), 1.69 (s, 9H). 步驟5：1-(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)丁-3-烯-1-醇( A-6) 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.04 (s, 1H), 7.94 (s, 1H), 1.69 (s, 9H). Step 5: 1-(1-(tert-butyl)-3 -Nitro- 1H -pyrazol-5-yl)but-3-en-1-ol ( A-6 )
室溫下，向1-(叔-丁基)-3-硝基-1 H-吡唑-5-甲醛(1.80 g, 9.1 mmol)和四丁基碘化銨(0.34 g, 0.91 mmol)的二氯甲烷和水混合溶液(36.0 mL)中加入烯丙基三氟硼酸鉀(2.69 g, 18.2 mmol)，混合物於室溫下攪拌2小時。混合物經二氯甲烷萃取，飽和食鹽水洗滌後，真空濃縮並乾燥，所得殘留物透過矽膠管柱層析法(PE:EA=3:1)純化得到化合物 A-6(黃色油狀物，1.60 g, 產率：74%)。LC-MS (ESI), m/z: [M+H] +=239.3. At room temperature, to 1-(tert-butyl)-3-nitro-1 H -pyrazole-5-carbaldehyde (1.80 g, 9.1 mmol) and tetrabutylammonium iodide (0.34 g, 0.91 mmol) Potassium allyl trifluoroborate (2.69 g, 18.2 mmol) was added to a mixed solution of dichloromethane and water (36.0 mL), and the mixture was stirred at room temperature for 2 hours. The mixture was extracted with dichloromethane, washed with saturated brine, concentrated in vacuo and dried, and the resulting residue was purified by silica gel column chromatography (PE:EA=3:1) to obtain compound A-6 (yellow oil, 1.60 g, yield: 74%). LC-MS (ESI), m/z: [M+H] + =239.3.
1H NMR (400 MHz, DMSO- d 6 ) δ 7.09 (s, 1H), 5.82 (ddt, J=17.2, 10.2, 6.8 Hz, 1H), 5.69 (s, 1H), 5.19 – 5.02 (m, 2H), 5.00 (s, 1H), 2.59 (td, J=6.8, 4.0 Hz, 2H), 1.65 (s, 9H). 步驟6：1-(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)-2-(噁丙環-2-基)乙烷-1-醇( A-7) 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.09 (s, 1H), 5.82 (ddt, J =17.2, 10.2, 6.8 Hz, 1H), 5.69 (s, 1H), 5.19 – 5.02 (m, 2H ), 5.00 (s, 1H), 2.59 (td, J= 6.8, 4.0 Hz, 2H), 1.65 (s, 9H). Step 6: 1-(1-(tert-butyl)-3-nitro- 1 H -pyrazol-5-yl)-2-(oxapropane-2-yl)ethan-1-ol ( A-7 )
室溫下向1-(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)丁-3-烯-1-醇(4.50 g, 18.8 mmol)的二氯甲烷溶液(50.0 mL)中分批加入3-氯過氧苯甲酸(9.75 g, 56.5 mmol)，混合物於40℃下攪拌16小時。混合物經二氯甲烷萃取，飽和食鹽水洗滌後，有機相真空濃縮並乾燥，所得殘留物透過矽膠管柱層析法(PE: EA＝3:2)純化得到化合物 A-7(無色油狀物，4.00 g, 產率：83%)。LC-MS (ESI), m/z: [M+H] +=255.3. 步驟7：5-(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)四氫呋喃-3-醇( A-8) To 1-(1-(tert-butyl)-3-nitro- 1H -pyrazol-5-yl)but-3-en-1-ol (4.50 g, 18.8 mmol) dichloro 3-Chloroperoxybenzoic acid (9.75 g, 56.5 mmol) was added in portions to the methane solution (50.0 mL), and the mixture was stirred at 40°C for 16 hours. The mixture was extracted with dichloromethane, washed with saturated brine, the organic phase was concentrated in vacuo and dried, and the resulting residue was purified by silica gel column chromatography (PE:EA=3:2) to obtain compound A-7 (colorless oil , 4.00 g, yield: 83%). LC-MS (ESI), m/z: [M+H] + =255.3. Step 7: 5-(1-(tert-butyl)-3-nitro- 1H -pyrazol-5-yl) Tetrahydrofuran-3-ol ( A-8 )
室溫下向1-(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)-2-(噁丙環-2-基)乙烷-1-醇(1.00 g, 3.90 mmol)的1,4-二噁烷溶液(50.0 mL)中逐滴加入濃硫酸(380 mg, 3.90 mmol)，並將混合物於50℃下攪拌16小時。冰浴下，向反應體系加水稀釋並用碳酸氫鈉飽和水溶液將pH調至8，然後用乙酸乙酯萃取，飽和食鹽水洗滌後，真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(PE: EA＝1:1)純化得到化合物 A-8(無色油狀物，400 mg, 產率：41%)。 To 1-(1-(tert-butyl)-3-nitro-1 H -pyrazol-5-yl)-2-(oxapropane-2-yl)ethan-1-ol ( 1.00 g, 3.90 mmol) in 1,4-dioxane (50.0 mL) was added dropwise concentrated sulfuric acid (380 mg, 3.90 mmol), and the mixture was stirred at 50°C for 16 hours. Under ice bath, dilute the reaction system with water and adjust the pH to 8 with a saturated aqueous solution of sodium bicarbonate, then extract with ethyl acetate, wash with saturated brine, concentrate in vacuo and dry, and the obtained crude product is purified by silica gel column chromatography ( PE:EA=1:1) was purified to obtain compound A-8 (colorless oil, 400 mg, yield: 41%).
LC-MS (ESI), m/z: [M+H] +=255.3. 步驟8：5-(3-胺基-1-(叔-丁基)-1 H-吡唑-5-基)四氫呋喃-3-醇( A-9) LC-MS (ESI), m/z: [M+H] + =255.3. Step 8: 5-(3-Amino-1-(tert-butyl)-1 H -pyrazol-5-yl) Tetrahydrofuran-3-ol ( A-9 )
室溫下向5-(1-(叔-丁基)-3-硝基-1 H-吡唑-5-基)四氫呋喃-3-醇(600 mg, 2.35 mmol) 的四氫呋喃溶液(15.0 mL)中加入鈀碳(300 mg)，混合物於氫氣氛圍中50℃下攪拌16小時。待反應完成後，反應混合物經二氯甲烷萃取，飽和食鹽水洗滌後，真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(PE: EA＝20:1)純化得到化合物 A-9(淡黃色油狀物，300 mg, 產率：57%)。LC-MS (ESI), m/z: [M+H] +=226.1. 步驟9：N-(1-(叔-丁基)-5-(4-羥基四氫呋喃-2-基)-1 H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺( A-10) Add 5-(1-(tert-butyl)-3-nitro- 1H -pyrazol-5-yl)tetrahydrofuran-3-ol (600 mg, 2.35 mmol) in tetrahydrofuran (15.0 mL) at room temperature Palladium carbon (300 mg) was added thereto, and the mixture was stirred at 50° C. for 16 hours in a hydrogen atmosphere. After the reaction was completed, the reaction mixture was extracted with dichloromethane, washed with saturated brine, concentrated in vacuo and dried, and the obtained crude product was purified by silica gel column chromatography (PE:EA=20:1) to obtain compound A-9 ( Pale yellow oil, 300 mg, yield: 57%). LC-MS (ESI), m/z: [M+H] + =226.1. Step 9: N-(1-(tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1 H -pyrazol-3-yl)-3-(methoxymethyl)-1-methyl-1 H -pyrazole-5-carboxamide ( A-10 )
將3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-羧酸(230 mg, 1.35 mmol)溶於N,N-二甲基甲醯胺溶液(10.0 mL)中，室溫下向以上溶液中加入2-(7-偶氮苯并三氮唑)- N,N,N',N'-四甲基脲六氟磷酸酯(668 mg, 1.76 mmol)，混合物於室溫下攪拌30分鐘。然後下向以上溶液中分別加入N,N-二異丙基乙胺(349 mg, 2.70 mmol)，5-(3-胺基-1-(叔-丁基)-1氫-吡唑-5-基)四氫呋喃-3-醇(335 mg, 1.49 mmol)，並將反應混合物於70℃下攪拌16小時。待反應完成後，反應混合物經乙酸乙酯萃取，飽和食鹽水洗滌後，有機相真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(DCM: MeOH=20:1)純化得到化合物 A-10(黃色固體，280 mg, 產率：55%)。LC-MS (ESI), m/z: [M+H] +=378.2. 步驟10：5-(1-(叔-丁基)-3-(3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺基)-1 H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( A-11) Dissolve 3-(methoxymethyl)-1-methyl- 1H -pyrazole-5-carboxylic acid (230 mg, 1.35 mmol) in N,N-dimethylformamide solution (10.0 mL) 2-(7-azobenzotriazole) -N,N,N',N' -tetramethyluronium hexafluorophosphate (668 mg, 1.76 mmol) was added to the above solution at room temperature, The mixture was stirred at room temperature for 30 minutes. Then, N,N-diisopropylethylamine (349 mg, 2.70 mmol), 5-(3-amino-1-(tert-butyl)-1 hydrogen-pyrazole-5 -yl)tetrahydrofuran-3-ol (335 mg, 1.49 mmol), and the reaction mixture was stirred at 70°C for 16 hours. After the reaction was completed, the reaction mixture was extracted with ethyl acetate, washed with saturated brine, the organic phase was concentrated in vacuo and dried, and the obtained crude product was purified by silica gel column chromatography (DCM: MeOH=20:1) to obtain compound A- 10 (yellow solid, 280 mg, yield: 55%). LC-MS (ESI), m/z: [M+H] + =378.2. Step 10: 5-(1-(tert-butyl)-3-(3-(methoxymethyl)-1- Methyl- 1H -pyrazole-5-carboxamido) -1H -pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( A-11 )
將 N-(1-(叔-丁基)-5-(4-羥基四氫呋喃-2-基)-1 H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺(300 mg, 0.79 mmol)、4-二甲胺基吡啶(10 mg, 0.082 mmol)和吡啶(189 mg, 2.38 mmol)溶於四氫呋喃溶液(150 mL)中，並在0℃下向以上溶液中加入4-硝基苯基氧基甲醯氯(104 mg, 0.52 mmol)，並將反應混合物於室溫下攪拌16小時。待反應完成後，將混合物用乙酸乙酯萃取，飽和食鹽水洗滌後，真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(PE: EA=1:2)純化得到化合物 A-11(白色油狀物，300 mg, 產率：70%)。LC-MS (ESI), m/z: [M+H] +=543.20. 步驟11：5-(3-(3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺基)-1 H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 中間體 A) N- (1-(tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1 H -pyrazol-3-yl)-3-(methoxymethyl)-1-methyl 1 H -pyrazole-5-carboxamide (300 mg, 0.79 mmol), 4-dimethylaminopyridine (10 mg, 0.082 mmol) and pyridine (189 mg, 2.38 mmol) were dissolved in THF solution (150 mL), and to the above solution was added 4-nitrophenyloxyformyl chloride (104 mg, 0.52 mmol) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was extracted with ethyl acetate, washed with saturated brine, concentrated in vacuo and dried, and the obtained crude product was purified by silica gel column chromatography (PE: EA=1:2) to obtain compound A-11 ( White oil, 300 mg, yield: 70%). LC-MS (ESI), m/z: [M+H] + =543.20. Step 11: 5-(3-(3-(methoxymethyl)-1-methyl-1 H -pyrazole- 5-Formamido) -1H -pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( Intermediate A )
5-(1-(叔-丁基)-3-(3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-甲醯胺基)-1
H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯(600 mg, 2.35 mmol)的甲酸溶液(5.0 mL)於75℃下攪拌16小時。待反應完成後，將反應混合物減壓濃縮得到
中間體 A(淡黃色油狀物，粗產物，600 mg)，直接用於下一步。LC-MS (ESI), m/z: [M+H]
+=487.1.
中間體 B:順式-5-(3-胺基-1-(4-叔丁基)-1H-吡唑-5-基)四氫呋喃-3-醇
室溫氮氣保護下，向5-(1-(叔丁基)-3-硝基-1H-吡唑-5-基)四氫呋喃-3-醇(3.30 g, 12.9 mmol)的二氯甲烷(50 mL)溶液中分批加入氯鉻酸吡啶(2.80 g, 25.9 mmol)，並將反應混合物在室溫攪拌過夜。隨後向反應體系中補加氯鉻酸吡啶(1.40 g, 12.9 mmol)並繼續攪拌2小時。待反應完成後，將反應混合物減壓濃縮，所的殘留物透過矽膠管柱層析法(石油醚：乙酸乙酯=10：1 – 1：1)純化得到化合物 B-1(2.0 g, 產率：61 %)。LC-MS (ESI), m/z: [M+H]+=254.1 步驟2：順式-5-(1-(叔丁基)-3-硝基-1H-吡唑-5-基)四氫呋喃-3-醇( B-2) Under nitrogen protection at room temperature, 5-(1-(tert-butyl)-3-nitro-1H-pyrazol-5-yl)tetrahydrofuran-3-ol (3.30 g, 12.9 mmol) in dichloromethane (50 mL) was added in portions to pyridinium chlorochromate (2.80 g, 25.9 mmol), and the reaction mixture was stirred at room temperature overnight. Then pyridinium chlorochromate (1.40 g, 12.9 mmol) was added to the reaction system and stirring was continued for 2 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1-1:1) to obtain compound B-1 (2.0 g, yield rate: 61 %). LC-MS (ESI), m/z: [M+H]+=254.1 Step 2: cis-5-(1-(tert-butyl)-3-nitro-1H-pyrazol-5-yl) Tetrahydrofuran-3-ol ( B-2 )
氮氣保護，在-60℃下，向化合物 B-1(3.7 g, 15 mmol)的四氫呋喃(20 mL)溶液中，緩慢加入三乙基硼氫化鋰(1 M的四氫呋喃溶液，18.3 mL, 36.5 mmol)。滴加完畢後，將反應混合物在-60℃氮氣保護下攪拌2小時。待反應完成後，將反應體系中用飽和氯化銨水溶液(50 mL)淬滅，乙酸乙酯萃取(20 mL×3)，合併有機相，再用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾除去固體並減壓濃縮。所得殘留物透過矽膠管柱層析法(二氯甲烷：甲醇=20：1)分離純化得到化合物 B-2(2.8 g, 產率68%)。LC-MS (ESI), m/z: [M+H] +=256.0 步驟3：順式-5-(3-胺基-1-(4-叔丁基)-1H-吡唑-5-基)四氫呋喃-3-醇( 中間體 B) Under nitrogen protection, at -60°C, to a solution of compound B-1 (3.7 g, 15 mmol) in tetrahydrofuran (20 mL), slowly add triethyllithium borohydride (1 M solution in tetrahydrofuran, 18.3 mL, 36.5 mmol ). After the dropwise addition was complete, the reaction mixture was stirred at -60°C under nitrogen protection for 2 hours. After the reaction was completed, the reaction system was quenched with saturated aqueous ammonium chloride (50 mL), extracted with ethyl acetate (20 mL×3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered The solids were removed and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound B-2 (2.8 g, yield 68%). LC-MS (ESI), m/z: [M+H] + =256.0 Step 3: cis-5-(3-amino-1-(4-tert-butyl)-1H-pyrazole-5- base) tetrahydrofuran-3-ol ( intermediate B )
在氫氣氛圍下，向化合物
B-2(600 mg, 2.35 mmol)的四氫呋喃溶液(15 mL)中加入鈀/碳(300 mg)，並將該反應混合物在50℃攪拌16小時。待反應完成後，將反應混合物過濾，並用二氯甲烷/甲醇混合溶液(二氯甲烷：甲醇=10：1)洗滌濾餅。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(二氯甲烷：甲醇=20：1)得到中間體
B(300 mg，產率：57%)。LC-MS (ESI), m/z: [M+H]
+=226.1
中間體 C ：順式-5-(3-胺基-1-(4-甲氧基苄基)-1H-吡唑-5-基)四氫呋喃-3-醇
氮氣氛圍下，向0℃下的5-硝基-1 H-吡唑-3-羧酸(100 g, 0.637 mol)的甲醇(1000 mL)溶液中緩慢滴加氯化亞碸(348 g, 2.93 mol)。滴加完成後，將該反應混合物在0℃下攪拌10分鐘，然後升溫至70℃並在此溫度下攪拌3小時。待反應完成後，將反應混合物冷卻至室溫，減壓濃縮。將所得殘留物溶解於乙酸乙酯(1000 mL)後，用飽和碳酸氫鈉水溶液和飽和食鹽水洗滌，無水硫酸鈉乾燥後過濾除去固體。將濾液減壓濃縮後得到粗產物化合物 C-1(107 g)，直接用於下一步。LC-MS (ESI), m/z: [M+H] +=172.0 步驟2：1-(4-甲氧苄基)-3-硝基-1H-吡唑-5-羧酸甲酯( C-2) Under a nitrogen atmosphere, to a solution of 5-nitro-1 H -pyrazole-3-carboxylic acid (100 g, 0.637 mol) in methanol (1000 mL) at 0°C, was slowly added dropwise phosphide chloride (348 g, 2.93 mol). After the addition was complete, the reaction mixture was stirred at 0° C. for 10 minutes, then warmed to 70° C. and stirred at this temperature for 3 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate (1000 mL), washed with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure to obtain the crude compound C-1 (107 g), which was directly used in the next step. LC-MS (ESI), m/z: [M+H] + =172.0 Step 2: 1-(4-Methoxybenzyl)-3-nitro-1H-pyrazole-5-carboxylic acid methyl ester ( C-2 )
氮氣氛圍下，向化合物 C-1(30 g, 0.18 mol)的 N,N-二甲基甲醯胺(300 mL)的溶液中加入對甲氧基苄溴(52 g, 0.26 mol)和無水碳酸鉀(48 g, 0.35 mol)。將反應混合物在50℃攪拌15小時。待反應完成後，將反應混合物冷卻到室溫，過濾除去固體並將濾液減壓濃縮，所得殘留物經過打漿(石油醚：乙酸乙酯=10：1)收集固體。將固體烘乾後得到化合物 C-2(36 g, 71%)。LC-MS (ESI), m/z: [M+H] +=309.0 步驟3：1-(4-甲氧基苄基)-3-硝基-1H-吡唑-5-甲醇( C-3) Under nitrogen atmosphere, add p - methoxybenzyl bromide (52 g, 0.26 mol) and anhydrous Potassium carbonate (48 g, 0.35 mol). The reaction mixture was stirred at 50°C for 15 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, the solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was beaten (petroleum ether:ethyl acetate=10:1) to collect the solid. Compound C-2 (36 g, 71%) was obtained after drying the solid. LC-MS (ESI), m/z: [M+H] + =309.0 Step 3: 1-(4-methoxybenzyl)-3-nitro-1H-pyrazole-5-methanol ( C- 3 )
氮氣氛圍下，向0℃的化合物 C-2(130 g, 0.421 mol)的四氫呋喃(1.3 L)溶液中分批加入硼氫化鈉(39 g, 1.8 mol)。加入完成後，讓反應混合物自然恢復到室溫，並在室溫下攪拌15小時。待反應完成後，向反應混合物中緩慢加入冰水混合物(1 L)並攪拌30分鐘。將水相用乙酸乙酯(700 mL × 3)萃取，合併有機相並用飽和食鹽水洗滌，有機相用無水硫酸鈉乾燥後過濾除去固體。將所得濾液減壓濃縮得到粗產物化合物 C-3(120 g)，直接用於下一步反應。LC-MS (ESI), m/z: [M+H] +=285.9 步驟4：1-(4-甲氧基苄基)-3-硝基-1H-吡唑-5-甲醛( C-4) Under nitrogen atmosphere, to a solution of compound C-2 (130 g, 0.421 mol) in tetrahydrofuran (1.3 L) at 0°C was added sodium borohydride (39 g, 1.8 mol) in portions. After the addition was complete, the reaction mixture was allowed to come to room temperature naturally and stirred at room temperature for 15 hours. After the reaction was complete, ice-water mixture (1 L) was slowly added to the reaction mixture and stirred for 30 minutes. The aqueous phase was extracted with ethyl acetate (700 mL × 3), the organic phases were combined and washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and filtered to remove the solid. The obtained filtrate was concentrated under reduced pressure to obtain the crude product compound C-3 (120 g), which was directly used in the next reaction. LC-MS (ESI), m/z: [M+H] + =285.9 Step 4: 1-(4-Methoxybenzyl)-3-nitro-1H-pyrazole-5-carbaldehyde ( C- 4 )
氮氣氛圍下，將草醯氯(32 mL, 0.38 mol)的二氯甲烷(500 mL)溶液冷卻至-78℃，並向反應中緩慢加入二甲基亞碸(34 mL, 0.48 mol)，並將反應混合物在-78℃攪拌1小時。向反應中滴加化合物 C-3(50 g, 0.19 mol)的二氯甲烷溶液(200 mL)，在滴加完成後將反應混合物在-78℃攪拌2小時。向反應中緩慢滴加三乙胺(131 g, 1.30 mol)，滴加完成後，將反應體系的溫度自然恢復至-20℃，並在此溫度下將反應混合物攪拌1小時。待反應完成後，向反應混合物中加水(300 mL)淬滅，並用二氯甲烷萃取(200 mL × 3)。有機相合併後使用飽和食鹽水洗滌，無水硫酸鈉乾燥後過濾除去固體。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(石油醚：乙酸乙酯=2：1)純化，得到化合物 C-4(40 g，產率：81%)。 1H NMR (400 MHz, DMSO- d 6) δ 9.96 (s, 1H), 7.85 (s, 1H), 7.30 – 7.22 (m, 3H), 6.96 – 6.88 (m, 3H), 5.73 (s, 2H), 3.73 (s, 3H). 步驟5：1-(1-(4-甲氧苄基)-3-硝基-1 H-吡唑-5-基)丁-3-炔-1-醇( C-5) Under a nitrogen atmosphere, a solution of oxalyl chloride (32 mL, 0.38 mol) in dichloromethane (500 mL) was cooled to -78°C, and dimethylsulfoxide (34 mL, 0.48 mol) was slowly added to the reaction, and The reaction mixture was stirred at -78°C for 1 hour. A dichloromethane solution (200 mL) of compound C-3 (50 g, 0.19 mol) was added dropwise to the reaction, and the reaction mixture was stirred at -78°C for 2 hours after the dropwise addition was complete. Triethylamine (131 g, 1.30 mol) was slowly added dropwise to the reaction. After the dropwise addition, the temperature of the reaction system was naturally returned to -20°C, and the reaction mixture was stirred at this temperature for 1 hour. After the reaction was completed, water (300 mL) was added to the reaction mixture to quench, and extracted with dichloromethane (200 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound C-4 (40 g, yield: 81%). 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.96 (s, 1H), 7.85 (s, 1H), 7.30 – 7.22 (m, 3H), 6.96 – 6.88 (m, 3H), 5.73 (s, 2H) ), 3.73 (s, 3H). Step 5: 1-(1-(4-methoxybenzyl)-3-nitro-1 H -pyrazol-5-yl)but-3-yn-1-ol ( C-5 )
氮氣氛圍下，將含有鋅粉(18 g, 0.28 mol)的四氫呋喃(240 mL)懸浮液冷卻至0℃，並向上述懸浮液中緩慢滴加炔丙基溴(16 g, 0.14 mol)。滴加完成後，將反應混合物在0℃攪拌1小時。隨後，向反應體系中加入化合物 C-4(24 g, 0.92 mol)的四氫呋喃(100 mL)溶液。滴加完成後，將反應體系的溫度緩慢恢復到室溫，並將反應混合物在室溫攪拌16小時。待反應完成後，向反應混合物中加水(300 mL)淬滅，過濾除去固體，並將濾餅用乙酸乙酯洗滌。所得濾液用乙酸乙酯(300 mL × 3)萃取後將有機相合併，使用飽和食鹽水洗滌，無水硫酸鈉乾燥後過濾除去固體。將濾液減壓濃縮，所的殘留物用矽膠管柱層析(石油醚：乙酸乙酯=2：1)純化，得到化合物 C-5(20 g，產率：74%)。LC-MS (ESI), m/z: [M+Na] +=324.1 步驟6：5-(1-((4-甲氧基苯基)甲基)-3-硝基吡唑-5-基)四氫呋喃-3-酮( C-6) Under nitrogen atmosphere, the tetrahydrofuran (240 mL) suspension containing zinc powder (18 g, 0.28 mol) was cooled to 0 °C, and propargyl bromide (16 g, 0.14 mol) was slowly added dropwise to the above suspension. After the dropwise addition was complete, the reaction mixture was stirred at 0 °C for 1 hour. Subsequently, a solution of compound C-4 (24 g, 0.92 mol) in tetrahydrofuran (100 mL) was added to the reaction system. After the dropwise addition was completed, the temperature of the reaction system was slowly returned to room temperature, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (300 mL) was added to the reaction mixture to quench, the solid was removed by filtration, and the filter cake was washed with ethyl acetate. The resulting filtrate was extracted with ethyl acetate (300 mL × 3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound C-5 (20 g, yield: 74%). LC-MS (ESI), m/z: [M+Na] + =324.1 Step 6: 5-(1-((4-methoxyphenyl)methyl)-3-nitropyrazole-5- Base) Tetrahydrofuran-3-one ( C-6 )
氮氣氛圍下，向化合物 C-5(50 g, 0.17 mol)的1,2-二氯乙烷溶液(500 mL)中緩慢加入二溴吡啶氮氧化物(57 g, 0.33 mol)的1,2-二氯乙烷溶液(500 mL)、甲磺酸(19 g, 0.20 mol) 的1,2-二氯乙烷溶液(0.2 mol/L, 1000 mL)以及三苯基膦金(I)雙三氟甲烷磺醯亞胺(3.68 g, 4.98 mmol)的1,2-二氯乙烷(36 mL)溶液。將該反應混合物在室溫下攪拌16小時。待反應完成後，向反應混合物中加入水(1000 mL)稀釋，並用二氯甲烷(200 mL × 2)萃取。有機相合併後用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾除去固體。將濾液減壓濃縮，所得殘留物用矽膠管柱層析(石油醚：乙酸乙酯=2：1)純化，得到化合物 C-6(29.0 g，產率：55 %)。LC-MS (ESI), m/z: [M+Na] +=340.1 步驟7：順式-5-(1-((4-甲氧基苯基)甲基)-3-硝基吡唑-5-基)四氫呋喃-3-醇( C-7) Under nitrogen atmosphere, slowly add dibromopyridine nitroxide (57 g, 0.33 mol) in 1,2 -Dichloroethane solution (500 mL), methanesulfonic acid (19 g, 0.20 mol) in 1,2-dichloroethane solution (0.2 mol/L, 1000 mL) and triphenylphosphine gold (I) bis A solution of trifluoromethanesulfonimide (3.68 g, 4.98 mmol) in 1,2-dichloroethane (36 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was diluted with water (1000 mL), and extracted with dichloromethane (200 mL × 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound C-6 (29.0 g, yield: 55%). LC-MS (ESI), m/z: [M+Na] + =340.1 Step 7: cis-5-(1-((4-methoxyphenyl)methyl)-3-nitropyrazole -5-yl)tetrahydrofuran-3-ol ( C-7 )
氮氣氛圍下，將化合物 C-6(16.7 g, 52.7 mmol)的四氫呋喃(170 mL)溶液冷卻至-70℃，並向反應中緩慢加入三乙基硼氫化鋰(1.0 mol/L, 130 mL, 130 mmol)，並將反應混合物在-60℃攪拌2小時。待反應完成後，向反應混合物中加入飽和氯化銨水溶液(300 mL)淬滅，並用乙酸乙酯(200 mL × 3)萃取。有機相合併後用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾除去固體。將濾液減壓濃縮，所的殘留物用矽膠管柱層析(石油醚：乙酸乙酯=2：1)純化，得到化合物 C-7(12.4 g，產率：78 %)。LC-MS (ESI), m/z: [M+Na] +=342.0 步驟8：順式-5-(1-(4-甲氧基苄基)-3-胺基吡唑-5-基)四氫呋喃-3-醇( 中間體 C) Under a nitrogen atmosphere, a solution of compound C-6 (16.7 g, 52.7 mmol) in tetrahydrofuran (170 mL) was cooled to -70°C, and lithium triethylborohydride (1.0 mol/L, 130 mL, 130 mmol), and the reaction mixture was stirred at -60°C for 2 hours. After the reaction was complete, the reaction mixture was quenched by adding saturated aqueous ammonium chloride (300 mL), and extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound C-7 (12.4 g, yield: 78%). LC-MS (ESI), m/z: [M+Na] + =342.0 Step 8: cis-5-(1-(4-methoxybenzyl)-3-aminopyrazol-5-yl ) Tetrahydrofuran-3-ol ( Intermediate C )
在氫氣氛圍下，向化合物
C-7(50 g, 0.16 mol)的四氫呋喃溶液(500 mL)中加入鈀/碳(10 g)，並將該反應混合物在室溫下攪拌16小時。待反應完成後，將反應混合物過濾，並將濾液減壓濃縮，得到粗產物化合物
中間體 C(40 g)，直接用於下一步反應。LC-MS (ESI), m/z: [M+H]
+=290.1
中間體 D：順式-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯
向化合物A-10 (25.80 g, 68.25 mmol)的二氯甲烷溶液(387 mL)中加入吡啶氯鉻酸鹽(22.00 g, 102.4 mmol)，並將該反應混合物在室溫下攪拌16小時。待反應結束後，向反應體系中加入水(400 mL)，並將混合物用二氯甲烷萃取(400 mL × 2)。將有機相合併後用飽和食鹽水(400 mL × 2)洗滌，無水硫酸鈉乾燥，過濾除去固體。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(二氯甲烷：甲醇=50：1)純化得到化合物 D-1(15.0 g, 58%)。LC-MS (ESI), m/z: [M+H] +=376.1 步驟2：順式- N-(1-叔丁基-5-((2 R,4 R)-4-羥基四氫呋喃-2-基)-1 H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺( D-2) To a dichloromethane solution (387 mL) of compound A-10 (25.80 g, 68.25 mmol) was added pyridinium chlorochromate (22.00 g, 102.4 mmol), and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, water (400 mL) was added to the reaction system, and the mixture was extracted with dichloromethane (400 mL × 2). The combined organic phases were washed with saturated brine (400 mL × 2), dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane:methanol=50:1) to obtain compound D-1 (15.0 g, 58%). LC-MS (ESI), m/z: [M+H] + =376.1 Step 2: cis- N- (1-tert-butyl-5-((2 R ,4 R )-4-hydroxytetrahydrofuran- 2-yl) -1H -pyrazol-3-yl)-3-(methoxymethyl)-1-methyl- 1H -pyrazole-5-carboxamide ( D-2 )
在-65℃下，向化合物D-1 (11.00 g, 29.26 mmol)的四氫呋喃溶液(440 mL)中加入三乙基硼鋰(58.5 mL, 1.0 mol/L的四氫呋喃溶液, 58.5 mmol)，並將該反應混合物在-65℃下攪拌2小時。待反應結束後，在0℃下向反應體系中加水(100 mL)淬滅反應。然後，向反應體系中加入水(300 mL)稀釋，並將混合物用乙酸乙酯(400 mL × 2)萃取。將有機相合併後用飽和食鹽水(400 mL × 2)洗滌，無水硫酸鈉乾燥，過濾除去固體。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(二氯甲烷：甲醇=100：1到1：1)純化得到化合物 D-2(10.0 g, 90%)。LC-MS (ESI), m/z: [M+H] +=378.2 步驟3：順式-5-(1-(叔-丁基)-3-(3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺基)-1 H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 中間體 D) At -65°C, triethylboron lithium (58.5 mL, 1.0 mol/L solution in tetrahydrofuran, 58.5 mmol) was added to compound D-1 (11.00 g, 29.26 mmol) in tetrahydrofuran (440 mL), and The reaction mixture was stirred at -65°C for 2 hours. After the reaction was completed, water (100 mL) was added to the reaction system at 0 °C to quench the reaction. Then, water (300 mL) was added to the reaction system for dilution, and the mixture was extracted with ethyl acetate (400 mL×2). The combined organic phases were washed with saturated brine (400 mL × 2), dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane:methanol=100:1 to 1:1) to obtain compound D-2 (10.0 g, 90%). LC-MS (ESI), m/z: [M+H] + =378.2 Step 3: cis-5-(1-(tert-butyl)-3-(3-(methoxymethyl)- 1-Methyl- 1H -pyrazole-5-carboxamido) -1H -pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( Intermediate D )
將化合物
D-2(10.00 g, 26.46 mmol)和4-二甲胺基吡啶(323 mg, 2.64 mmol)溶於二氯甲烷(200 mL)中，並在室溫下向以上溶液中加入吡啶(6.28 g, 79.4 mmol)和4-硝基苯基氧基甲醯氯(7.98 mg, 39.7 mmol)，並將反應混合物於室溫下攪拌16小時。待反應完成後，將混合物用乙酸乙酯萃取，飽和食鹽水洗滌後，真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(石油醚：乙酸乙酯=100：1到3：7)純化得到
中間體 D(11.20 g, 78%)。LC-MS (ESI), m/z: [M+H]
+=543.20
實施例的製備：
實施例 1 ：順式-5-(3-(3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-甲醯胺基)-1
H-吡唑-5-基)四氫呋喃-3-基叔-丁基胺基甲酸酯
室溫下向5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯(600 mg, 1.23 mmol)的四氫呋喃溶液(20 mL)中逐滴加入叔丁基胺(902 mg, 12.3 mmol)，混合物於室溫下攪拌1小時。待反應結束後，反應混合物經乙酸乙酯萃取，合併有機相。用飽和食鹽水洗滌後，真空濃縮並乾燥，所得殘餘物透過製備HPLC (管柱：Gemini 5u C18 150 × 21.2 mm; 流動相：乙腈(30-40%)/水(0.1% 甲酸)；流速：20 mL/min)得到順式產物
實施例 1(HPLC前峰, t
R=4.43 min, 150 mg，LC-MS (ESI), m/z: [M+H]
+=421.20)和反式產物
實施例 1’(HPLC後峰, t
R=4.581 min, 140 mg，產率：33%，LC-MS (ESI), m/z: [M+H]
+=421.20)。
實施例 1A 和實施例 1B ：(3R,5R)-5-(3-(3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-甲醯胺基)-1
H-吡唑-5-基)四氫呋喃-3-基叔-丁基胺基甲酸酯(
實施例 1A)和(3S,5S)-5-(3-(3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-甲醯胺基)-1
H-吡唑-5-基)四氫呋喃-3-基叔-丁基胺基甲酸酯(
實施例 1B)
順式-5-(3-(3-(甲氧基甲基)-1-甲基-1 H-吡唑-5-甲醯胺基)-1 H-吡唑-5-基)四氫呋喃-3-基叔-丁基胺基甲酸酯( 實施例 1)經手性製備HPLC (管柱：CHIRALPAK AD-H 250 mm 20 mm, 5 μm，流動相：35% IPA (NH 4OH 0.2%)，流速：40 mL/min)拆分得到 實施例 1A(62.1 mg, 產率：11%)和 實施例 1B(66.4 mg, 產率：12%)。 實施例 1A ： cis-5-(3-(3-(methoxymethyl)-1-methyl-1 H -pyrazole-5-carboxamido)-1 H -pyrazol-5-yl)tetrahydrofuran- 3-yl tert-butyl carbamate ( Example 1 ) was prepared by chiral preparative HPLC (column: CHIRALPAK AD-H 250 mm 20 mm, 5 μm, mobile phase: 35% IPA (NH 4 OH 0.2%) , flow rate: 40 mL/min) were resolved to obtain Example 1A (62.1 mg, yield: 11%) and Example 1B (66.4 mg, yield: 12%). Example 1A :
t R=2.35 min. t R =2.35 min.
LC-MS (ESI), m/z: [M+H] +=421.2. LC-MS (ESI), m/z: [M+H] + =421.2.
1H NMR (400 MHz, DMSO- d 6 ) δ 12.49 (s, 1H), 10.81 (s, 1H), 7.13 (s, 1H), 6.95 (s, 1H), 6.58 (s, 1H), 5.15 (br, 1H), 4.85 (br, 1H), 4.34 (s, 2H), 4.06 (s, 3H), 3.86 (br, 2H), 3.27 (s, 3H), 2.75 – 2.64 (m, 1H), 1.93 (br, 1H), 1.22 (s, 9H). 實施例 1B ： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.49 (s, 1H), 10.81 (s, 1H), 7.13 (s, 1H), 6.95 (s, 1H), 6.58 (s, 1H), 5.15 ( br, 1H), 4.85 (br, 1H), 4.34 (s, 2H), 4.06 (s, 3H), 3.86 (br, 2H), 3.27 (s, 3H), 2.75 – 2.64 (m, 1H), 1.93 (br, 1H), 1.22 (s, 9H). Embodiment 1B :
t R=4.64 min. t R =4.64 min.
LC-MS (ESI), m/z: [M+H] +=421.2. LC-MS (ESI), m/z: [M+H] + =421.2.
1H NMR (400 MHz, DMSO- d 6 ) δ 12.49 (s, 1H), 10.81 (s, 1H), 7.13 (s, 1H), 6.95 (s, 1H), 6.58 (s, 1H), 5.15 (br, 1H), 4.85 (br, 1H), 4.34 (s, 2H), 4.05 (s, 3H), 3.85 (br, 2H), 3.27 (s, 3H), 2.75 – 2.64 (m, 1H), 1.93 (br, 1H), 1.22 (s, 9H). 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.49 (s, 1H), 10.81 (s, 1H), 7.13 (s, 1H), 6.95 (s, 1H), 6.58 (s, 1H), 5.15 ( br, 1H), 4.85 (br, 1H), 4.34 (s, 2H), 4.05 (s, 3H), 3.85 (br, 2H), 3.27 (s, 3H), 2.75 – 2.64 (m, 1H), 1.93 (br, 1H), 1.22 (s, 9H).
實施例3~11參考實施例1、1A和1B的合成方法製備得到，具體特徵分析數據如下：
上表中實施例3~7和9~10透過手性製備(CHIRALPAK IC 250mm x 20 mm，5 μm， 40% MeOH(0.2% NH
4OH), 流速：40 g/min)分離得到對應的單一構型化合物；實施例11透過手性製備(CHIRALPAK OJ-H 250mm x 20 mm, 5 μm， 40% MeOH(0.2% NH
4OH), 流速：40 g/min)分離得到對應的單一構型化合物。
實施例 12 ：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
氮氣保護下，向化合物 12-1(0.135 g, 1.07 mmol)的 N,N-二甲基甲醯胺(5 mL)溶液中，加入HATU (0.446 g, 1.17 mmol)，並將反應混合物在25℃下攪拌0.5小時。然後向體系中加入 N,N-二異丙基乙基胺(0.252 g, 1.95 mmol)和 中間體 B(0.22 g, 0.98 mmol)。將反應體系升溫至50℃並攪拌2小時。反應完全後，加水(20 mL)稀釋，混合物用乙酸乙酯(15 mL × 3)萃取。合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到化合物 12-2(0.24 g, 產率：73%)。LC-MS (ESI), m/z: [M+H] +=333.9 步驟2：順式-5-(1-(叔丁基)-3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 12-3) Under nitrogen protection, to a solution of compound 12-1 (0.135 g, 1.07 mmol) in N,N -dimethylformamide (5 mL), HATU (0.446 g, 1.17 mmol) was added, and the reaction mixture was heated at 25 Stir at 0.5°C for 0.5 hours. Then N,N -diisopropylethylamine (0.252 g, 1.95 mmol) and Intermediate B (0.22 g, 0.98 mmol) were added to the system. The reaction system was warmed up to 50°C and stirred for 2 hours. After the reaction was complete, it was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL×3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 12-2 (0.24 g, yield: 73%). LC-MS (ESI), m/z: [M+H] + =333.9 Step 2: cis-5-(1-(tert-butyl)-3-(1-methyl-1H-pyrazole-5 -formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 12-3 )
氮氣保護下，向化合物 12-2(0.24 g, 0.72 mmol)的二氯甲烷(3 mL)和四氫呋喃(3 mL)混合溶液中，加入氯甲酸-4-硝基苯酯(0.218 g, 1.08 mmol)、吡啶(0.171 g, 2.16 mmol)和4-二甲胺基吡啶(9.0 mg, 0.070 mmol)，並將反應混合物於室溫下攪拌16小時。待反應完成後，向反應體系加水(20 mL)稀釋，並將混合物用乙酸乙酯(15 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾除掉固體然後減壓濃縮。所得殘留物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到化合物 12-3(0.20 g, 產率：55 %)。LC-MS (ESI), m/z: [M+H] +=499.2 步驟3：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 12-4) Under nitrogen protection, to a mixed solution of compound 12-2 (0.24 g, 0.72 mmol) in dichloromethane (3 mL) and tetrahydrofuran (3 mL), was added 4-nitrophenyl chloroformate (0.218 g, 1.08 mmol ), pyridine (0.171 g, 2.16 mmol) and 4-dimethylaminopyridine (9.0 mg, 0.070 mmol), and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction system was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove the solid and then Concentrate under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 12-3 (0.20 g, yield: 55%). LC-MS (ESI), m/z: [M+H] + =499.2 Step 3: cis-5-(3-(1-methyl-1H-pyrazole-5-carboxamido)-1H -pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 12-4 )
將化合物 12-3(0.20 g, 0.40 mmol)溶於甲酸(4 mL)溶液中，並將反應混合物升溫至85℃，然後攪拌2小時。反應結束後將反應液減壓濃縮，所得殘餘物為粗產物化合物 12-4(0.15 g)，直接用於下一步反應。LC-MS (ESI), m/z: [M+H] +=442.8 步驟4：順式-5-(3-(1-甲基-1 H-吡唑-5-甲醯胺基)-1 H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 12) Compound 12-3 (0.20 g, 0.40 mmol) was dissolved in formic acid (4 mL) solution, and the reaction mixture was warmed up to 85° C., and then stirred for 2 hours. After the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was the crude compound 12-4 (0.15 g), which was directly used in the next reaction. LC-MS (ESI), m/z: [M+H] + =442.8 Step 4: cis-5-(3-(1-methyl-1 H -pyrazole-5-carboxamido)- 1 H -pyrazol-5-yl)tetrahydrofuran-3-yl tert-butyl carbamate ( Example 12 )
室溫下，向化合物 12-4(0.15 g, 0.34 mmol)的四氫呋喃(3 mL)溶液中加入叔丁胺(0.248 g, 3.39 mmol)，並將反應混合物於室溫下攪拌16小時。待反應結束後，將反應液直接減壓濃縮，所得殘餘物透過製備HPLC分離純化(管柱：Gemini-C18 150 × 21.2 mm，5 µm；流動相：乙腈/水(0.1%三氟乙酸)；梯度：30%-40%)，得到 實施例 12。LC-MS (ESI), m/z: [M+H] +=376.9 實施 12A和 實施例 12B： To a solution of compound 12-4 (0.15 g, 0.34 mmol) in tetrahydrofuran (3 mL) was added tert-butylamine (0.248 g, 3.39 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the obtained residue was separated and purified by preparative HPLC (column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% trifluoroacetic acid); Gradient: 30%-40%), to obtain Example 12 . LC-MS (ESI), m/z: [M+H] = 376.9 Example 12A and Example 12B :
(3
S,5
S)-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯(
實施例 12A)和(3
R,5
R)-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯(
實施例 12B)
實施例12經手性製備HPLC (管柱：CHIRALPAK WHELK-01 250 mm x 20 mm, 5 μm；流動相：40%異丙醇(0.2%氨水)；流速：50 mL/min)拆分得到 實施例 12A(11.5 mg, 產率：16%)和 實施例 12B(14.2 mg, 產率：20%)。 實施例 12A： Example 12 was resolved by chiral preparative HPLC (column: CHIRALPAK WHELK-01 250 mm x 20 mm, 5 μm; mobile phase: 40% isopropanol (0.2% ammonia water); flow rate: 50 mL/min) to obtain the example 12A (11.5 mg, yield: 16%) and Example 12B (14.2 mg, yield: 20%). Example 12A :
t R=3.08 min t R =3.08 min
LC-MS (ESI), m/z: [M+H] +=376.9 LC-MS (ESI), m/z: [M+H] + =376.9
1H NMR (400 MHz, CD 3OD) δ7.54 (d, J=2.1 Hz, 1H), 6.98 (d, J=2.1 Hz, 1H), 6.55 (s, 1H), 5.25 – 5.19 (m, 1H), 5.08 – 5.01 (m, 1H), 4.19 (s, 3H), 4.08 – 4.04 (m, 1H), 3.98 – 3.94 (m, 1H), 2.79 – 2.70 (m, 1H), 2.20 – 2.12 (m, 1H), 1.30 (s, 9H). 實施例 12B ： 1 H NMR (400 MHz, CD 3 OD) δ 7.54 (d, J =2.1 Hz, 1H), 6.98 (d, J =2.1 Hz, 1H), 6.55 (s, 1H), 5.25 – 5.19 (m, 1H ), 5.08 – 5.01 (m, 1H), 4.19 (s, 3H), 4.08 – 4.04 (m, 1H), 3.98 – 3.94 (m, 1H), 2.79 – 2.70 (m, 1H), 2.20 – 2.12 (m , 1H), 1.30 (s, 9H). Embodiment 12B :
tR=3.31 mintR=3.31min
LC-MS (ESI), m/z: [M+H]+=376.9LC-MS (ESI), m/z: [M+H]+=376.9
1H NMR (400 MHz, CD 3OD) δ7.48 (d, J=2.1 Hz, 1H), 6.92 (d, J=2.1 Hz, 1H), 6.51 (s, 1H), 5.21 – 5.14 (m, 1H), 5.02 – 4.95 (m, 1H), 4.13 (s, 3H), 4.01 (d, J=10.2 Hz, 1H), 3.91 (dd, J=10.3, 4.5 Hz, 1H), 2.75 – 2.63 (m, 1H), 2.16 – 2.06 (m, 1H), 1.25 (s, 9H). 1 H NMR (400 MHz, CD 3 OD) δ 7.48 (d, J =2.1 Hz, 1H), 6.92 (d, J =2.1 Hz, 1H), 6.51 (s, 1H), 5.21 – 5.14 (m, 1H ), 5.02 – 4.95 (m, 1H), 4.13 (s, 3H), 4.01 (d, J =10.2 Hz, 1H), 3.91 (dd, J =10.3, 4.5 Hz, 1H), 2.75 – 2.63 (m, 1H), 2.16 – 2.06 (m, 1H), 1.25 (s, 9H).
實施例14-15參考實施例1、1A和1B的合成方法製備得到，具體特徵分析數據如下：
上表中實施例14透過手性製備(CHIRALPAK WHELK-01 250 mm x 20 mm, 5 μm m，流動相：40%異丙醇(0.2%氨水)；流速：50 mL/min)分離得到對應的單一構型化合物；實施例15透過手性製備(CHIRALPAK AD-H 250 mm 20 mm, 5 μm，流動相：35% 異丙醇(0.2%氨水)，流速：40 g/min)分離得到對應的單一構型化合物。
實施例 18 ：順式-
N-(5-(4-((4-異丙基吡啶-2-基)四氫呋喃-2-基)-1H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺
合成途徑：
在室溫下，向化合物 18-1(3.35 g, 19.0 mmol)和4,4,5,5-四甲基-2-(丙-1-烯-2-基)-1,3-二氧雜硼烷(9.58 g, 56.9 mmol)的1,4-二氧六環/水(24 mL)溶液中加入碳酸鉀(7.88 g, 56.9 mmol)和1,1'-雙二苯基膦二茂鐵二氯化鈀(0.70 g, 0.95 mmol)。氬氣氛圍下，將該反應混合物在70℃攪拌16小時。待反應完成後，將反應混合物冷卻至室溫，加入水(150 mL)稀釋，並用乙酸乙酯(150 mL × 2)萃取。將有機相合併後用飽和食鹽水(200 mL × 2)洗滌，無水硫酸鈉乾燥，並過濾除去固體。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(乙酸乙酯：石油醚=1：9)純化得到化合物 18-2(1.1 g，產率：42%)。LC-MS (ESI), m/z: [M+H] +=138.1 步驟2：2-((5-(1-(叔丁基)-5-硝基-1H-吡唑-3-基)四氫呋喃-3-基)氧基)-4-(丙-1-烯-2-基)吡啶( 18-3) At room temperature, to compound 18-1 (3.35 g, 19.0 mmol) and 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3-dioxo To a solution of borane (9.58 g, 56.9 mmol) in 1,4-dioxane/water (24 mL) was added potassium carbonate (7.88 g, 56.9 mmol) and 1,1'-bisdiphenylphosphine Iron palladium dichloride (0.70 g, 0.95 mmol). The reaction mixture was stirred at 70°C for 16 hours under argon atmosphere. After the reaction was complete, the reaction mixture was cooled to room temperature, diluted with water (150 mL), and extracted with ethyl acetate (150 mL×2). The combined organic phases were washed with saturated brine (200 mL × 2), dried over anhydrous sodium sulfate, and filtered to remove the solid. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:9) to obtain compound 18-2 (1.1 g, yield: 42%). LC-MS (ESI), m/z: [M+H] + =138.1 Step 2: 2-((5-(1-(tert-butyl)-5-nitro-1H-pyrazol-3-yl )tetrahydrofuran-3-yl)oxy)-4-(prop-1-en-2-yl)pyridine ( 18-3 )
在0℃下，向化合物 18-2(250 mg, 1.82 mmol)和5-(1-叔丁基-5-硝基吡唑-3-基)四氫呋喃-3-醇( A-8, 512 mg, 2.00 mmol)的 N,N-二甲基甲醯胺(10 mL)溶液中加入氫化鈉(60%, 1.83 g, 4.56 mmol)。將該反應混合物在80℃攪拌16小時。待反應完成後，在0℃下用水淬滅反應。將淬滅後的混合物加水(100 mL)稀釋，並用乙酸乙酯(100 mL × 2)萃取。將有機相合併後用水(100 mL × 2)洗滌，無水硫酸鈉乾燥，並過濾除去固體。將濾液減壓濃縮，得到化合物 18-3(284 mg，產率：42%)。LC-MS (ESI), m/z: [M+H] +=373.2 步驟3：1-(叔丁基)-3-(-4-((異丙基吡啶-2-基)氧基)四氫呋喃-2-基)-1H-吡咯-5-胺(化合物 18-4) At 0°C, compound 18-2 (250 mg, 1.82 mmol) and 5-(1-tert-butyl-5-nitropyrazol-3-yl) tetrahydrofuran-3-ol ( A-8 , 512 mg , 2.00 mmol) in N,N -dimethylformamide (10 mL) was added sodium hydride (60%, 1.83 g, 4.56 mmol). The reaction mixture was stirred at 80°C for 16 hours. After the reaction was complete, the reaction was quenched with water at 0 °C. The quenched mixture was diluted with water (100 mL), and extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with water (100 mL x 2), dried over anhydrous sodium sulfate, and filtered to remove solids. The filtrate was concentrated under reduced pressure to obtain compound 18-3 (284 mg, yield: 42%). LC-MS (ESI), m/z: [M+H] + =373.2 Step 3: 1-(tert-Butyl)-3-(-4-((isopropylpyridin-2-yl)oxy) Tetrahydrofuran-2-yl)-1H-pyrrol-5-amine (compound 18-4 )
在氫氣氛圍下，向化合物 18-3(300 mg, 0.804 mmol)的四氫呋喃/水(5 mL)溶液中加入鈀/碳(300 mg, 2.82 mmol)和氫氧化鈀/碳(300 mg, 2.09 mmol)。將該反應混合物在100℃攪拌16小時。待反應完成後，將反應混合物過濾，並用二氯甲烷/甲醇混合溶液(二氯甲烷：甲醇=10：1)洗滌濾餅。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(乙酸乙酯：石油醚=1：9)純化得到化合物 18-4(220 mg，產率：79%)。LC-MS (ESI), m/z: [M+H] +=345.3 步驟4：順式- N-(1-(叔丁基)-5-(4-((4-異丙基吡啶-2-基)氧基)四氫呋喃-2-基)-1H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺( 18-5) To a solution of compound 18-3 (300 mg, 0.804 mmol) in THF/water (5 mL) was added palladium/carbon (300 mg, 2.82 mmol) and palladium hydroxide/carbon (300 mg, 2.09 mmol) under hydrogen atmosphere. ). The reaction mixture was stirred at 100°C for 16 hours. After the reaction was completed, the reaction mixture was filtered, and the filter cake was washed with a dichloromethane/methanol mixed solution (dichloromethane:methanol=10:1). The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:9) to obtain compound 18-4 (220 mg, yield: 79%). LC-MS (ESI), m/z: [M+H] + =345.3 Step 4: cis- N- (1-(tert-butyl)-5-(4-((4-isopropylpyridine- 2-yl) oxy)tetrahydrofuran-2-yl)-1H-pyrazol-3-yl)-3-(methoxymethyl)-1-methyl-1H-pyrazole-5-carboxamide ( 18-5 )
向5-(甲氧基甲基)-2-甲基吡唑-3-羧酸(50 mg, 0.29 mmol)的 N,N-二甲基甲醯胺(5 mL)溶液中加入2-(7-氮雜苯并三氮唑)- N,N,N',N'-四甲基脲六氟磷酸酯(145 mg, 0.382 mmol)，將反應混合物在室溫下攪拌30分鐘。向反應體系中加入 N,N-二異丙基乙胺(76 mg, 0.59 mmol)和化合物 18-4(111 mg, 0.322 mmol)，並將反應混合物在室溫下攪拌2小時。待反應完成後，將反應混合物冷卻至室溫，加入水(100 mL)稀釋，並用乙酸乙酯(100 mL × 2)萃取。將有機相合併後用飽和食鹽水(150 mL × 2)洗滌，無水硫酸鈉乾燥，並過濾除去固體。將濾液減壓濃縮，所得殘留物經過矽膠管柱層析(乙酸乙酯：石油醚=3：2)純化得到化合物 18-5(90 mg，產率：62%)。LC-MS (ESI), m/z: [M+H] +=497.2 步驟5：順式- N-(5-(4-((4-異丙基吡啶-2-基)四氫呋喃-2-基)-1H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺( 實施例 18) To a solution of 5-(methoxymethyl)-2-methylpyrazole-3-carboxylic acid (50 mg, 0.29 mmol) in N,N -dimethylformamide (5 mL) was added 2-( 7-azabenzotriazole) -N,N,N',N' -tetramethyluronium hexafluorophosphate (145 mg, 0.382 mmol), and the reaction mixture was stirred at room temperature for 30 minutes. N,N -diisopropylethylamine (76 mg, 0.59 mmol) and compound 18-4 (111 mg, 0.322 mmol) were added to the reaction system, and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated brine (150 mL × 2), dried over anhydrous sodium sulfate, and filtered to remove solids. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=3:2) to obtain compound 18-5 (90 mg, yield: 62%). LC-MS (ESI), m/z: [M+H] + =497.2 Step 5: cis- N- (5-(4-((4-isopropylpyridin-2-yl)tetrahydrofuran-2- Base)-1H-pyrazol-3-yl)-3-(methoxymethyl)-1-methyl-1H-pyrazole-5-carboxamide ( Example 18 )
將化合物 18-5(80 mg, 0.16 mmol)加入甲酸(3 mL)中，將該反應混合物在40℃攪拌16小時。待反應完成後，將反應混合物冷卻至室溫，加入水(100 mL)稀釋，並用乙酸乙酯(100 mL × 2)萃取。將有機相合併後用飽和食鹽水(150 mL × 2)洗滌，無水硫酸鈉乾燥，並過濾除去固體。將濾液減壓濃縮，所得殘留物經製備HPLC純化(管柱：Gemini-C18 150 × 21.2 mm，5 µm；流動相：乙腈/水(0.1%甲酸)；梯度：0-35%)，得到化合物 實施例 18。LC-MS (ESI), m/z: [M + H] +=441.2 實施例 18A 和 實施例 18B ： Compound 18-5 (80 mg, 0.16 mmol) was added to formic acid (3 mL), and the reaction mixture was stirred at 40° C. for 16 hr. After the reaction was complete, the reaction mixture was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL×2). The combined organic phases were washed with saturated brine (150 mL × 2), dried over anhydrous sodium sulfate, and filtered to remove solids. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% formic acid); gradient: 0-35%) to obtain the compound Example 18 . LC-MS (ESI), m/z: [M+H] + =441.2 Example 18A and Example 18B :
N-(5-((2R,4R)-4-((4-異丙基吡啶-2-基)氧基)四氫呋喃-2-基)-1
H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-胺基甲酸酯和
N-(5-((2S,4S)-4-((4-異丙基吡啶-2-基)氧基)四氫呋喃-2-基)-1
H-吡唑-3-基)-3-(甲氧基甲基)-1-甲基-1
H-吡唑-5-胺基甲酸酯
將實施例18經手性製備HPLC (管柱：CHIRALPAK OJ-H 250 mm x 20 mm, 5 μm；流動相：40%乙醇/0.2%氨水；流速：50 mL/min)進一步拆分得到 實施例 18A(27 mg, 產率：36%)和 實施例 18B(25 mg, 產率：34 %)。 實施例 18A ： Example 18 was further resolved by chiral preparative HPLC (column: CHIRALPAK OJ-H 250 mm x 20 mm, 5 μm; mobile phase: 40% ethanol/0.2% ammonia; flow rate: 50 mL/min) to obtain Example 18A (27 mg, yield: 36%) and Example 18B (25 mg, yield: 34%). Example 18A :
t R= 2.22 min t R = 2.22 min
LC-MS (ESI), m/z: [M + H] +=441.2 LC-MS (ESI), m/z: [M + H] + =441.2
1H NMR (400 MHz, DMSO- d 6 ) δ 12.55 (s, 1H), 10.80 (s, 1H), 8.05 (d, J=5.3 Hz, 1H), 7.13 (s, 1H), 6.90 (dd, J=5.3, 1.2 Hz, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 5.56 (s, 1H), 4.98 (t, J=6.8 Hz, 1H), 4.34 (s, 2H), 4.06 (s, 3H), 4.03 – 3.96 (m, 2H), 3.27 (s, 3H), 2.89 – 2.75 (m, 2H), 2.15 – 2.05 (m, 1H), 1.17 (d, J=6.9 Hz, 6H). 實施例 18B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.55 (s, 1H), 10.80 (s, 1H), 8.05 (d, J =5.3 Hz, 1H), 7.13 (s, 1H), 6.90 (dd, J =5.3, 1.2 Hz, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 5.56 (s, 1H), 4.98 (t, J =6.8 Hz, 1H), 4.34 (s, 2H), 4.06 (s, 3H), 4.03 – 3.96 (m, 2H), 3.27 (s, 3H), 2.89 – 2.75 (m, 2H), 2.15 – 2.05 (m, 1H), 1.17 (d, J =6.9 Hz, 6H). Example 18B :
t R= 2.90 min t R = 2.90 min
LC-MS (ESI), m/z: [M + H] +=441.2 LC-MS (ESI), m/z: [M + H] + =441.2
1H NMR (400 MHz, DMSO-
d
6 ) δ 12.55 (s, 1H), 10.80 (s, 1H), 8.05 (d,
J=5.3 Hz, 1H), 7.13 (s, 1H), 6.90 (dd,
J=5.3, 1.2 Hz, 1H), 6.69 (s, 1H), 6.63 (s, 1H), 5.56 (s, 1H), 4.98 (t,
J=6.8 Hz, 1H), 4.34 (s, 2H), 4.06 (s, 3H), 4.03 – 3.96 (m, 2H), 3.27 (s, 3H), 2.88 – 2.74 (m, 2H), 2.15 – 2.06 (m, 1H), 1.17 (d,
J=6.9 Hz, 6H).
實施例 20:順式-5-(3-(1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)叔丁基胺基甲酸酯
室溫下，向化合物 20-1(1.0 g, 6.4 mmol)的1,4-二氧六環(20 mL)溶液中，加入氫化鈉(0.384 g , 9.61 mmol)。反應混合物在25℃下攪拌30分鐘後，加入2-溴-2,2-二氟乙酸鈉(1.89 g , 9.61 mmol)，將混合物升溫至80℃攪拌16小時。待反應完全後，加入1,4-二氧六環(50 mL)稀釋。將混合物抽濾，濾液減壓濃縮。得到的固體中加入鹽酸二氧六環溶液(4.0 mol/L)，室溫攪拌2分鐘，然後減壓濃縮除去溶劑，所得殘餘物用水(50 mL)稀釋，並用乙酸乙酯(50 mL × 2)萃取，將合併的有機層用無水硫酸鈉乾燥並濃縮。殘餘物透過製備HPLC分離得到產物化合物 20-3(1.10 g，產率：68%)。LC-MS (ESI), m/z: [M+H] +=251.1 步驟2：1-甲基-3-(三氟甲氧基)-1H-吡唑-5-羧酸甲酯( 20-4) To a solution of compound 20-1 (1.0 g, 6.4 mmol) in 1,4-dioxane (20 mL) was added sodium hydride (0.384 g, 9.61 mmol) at room temperature. After the reaction mixture was stirred at 25°C for 30 minutes, sodium 2-bromo-2,2-difluoroacetate (1.89 g, 9.61 mmol) was added, and the mixture was heated to 80°C and stirred for 16 hours. After the reaction was complete, 1,4-dioxane (50 mL) was added for dilution. The mixture was filtered with suction, and the filtrate was concentrated under reduced pressure. Dioxane hydrochloride solution (4.0 mol/L) was added to the obtained solid, stirred at room temperature for 2 minutes, and then concentrated under reduced pressure to remove the solvent, and the obtained residue was diluted with water (50 mL) and washed with ethyl acetate (50 mL × 2 ) extraction, the combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was separated by preparative HPLC to obtain the product compound 20-3 (1.10 g, yield: 68%). LC-MS (ESI), m/z: [M+H] + =251.1 Step 2: 1-Methyl-3-(trifluoromethoxy)-1H-pyrazole-5-carboxylic acid methyl ester ( 20 -4 )
室溫和氮氣保護條件下，向化合物 20-3(0.50 g, 2.0 mol)的氘代氯仿(10 mL)溶液中加入二氟化氙(1.0 g, 6.0 mol)。反應體系在超聲下室溫反應2分鐘，然後在25℃攪拌下20分鐘。反應結束後，向反應體系中加水(50 mL)稀釋，並將混合物用氯仿(50 mL × 2)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=5：1)分離純化得到化合物 20-4(0.30 g，產率：67%)。LC-MS (ESI), m/z: [M+H] +=225.0 步驟3：1-甲基-3-(三氟甲氧基)-1H-吡唑-5-羧酸( 20-5) To a solution of compound 20-3 (0.50 g, 2.0 mol) in deuterated chloroform (10 mL) was added xenon difluoride (1.0 g, 6.0 mol) at room temperature under nitrogen protection. The reaction system was ultrasonically reacted at room temperature for 2 minutes, and then stirred at 25° C. for 20 minutes. After the reaction, the reaction system was diluted with water (50 mL), and the mixture was extracted with chloroform (50 mL × 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 20-4 (0.30 g, yield: 67%). LC-MS (ESI), m/z: [M+H] + =225.0 Step 3: 1-Methyl-3-(trifluoromethoxy)-1H-pyrazole-5-carboxylic acid ( 20-5 )
於25℃下，向化合物 20-4(0.30 g, 1.34 mmol)的甲醇(5 mL)和水(5 mL)混合溶液中，加入氫氧化鈉(0.16 g, 4.0 mmol)。將反應混合物於25℃攪拌2小時。待反應完成後，向體系中加入稀鹽酸(1.0 mol/L)調整pH=5，並將混合物用乙酸乙酯(20 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並濃縮，得粗產物化合物 20-5(0.25 g)。LC-MS (ESI), m/z: [M+H] +=211.1 步驟4：順式- N-(1-(叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺( 20-6) To a mixed solution of compound 20-4 (0.30 g, 1.34 mmol) in methanol (5 mL) and water (5 mL) was added sodium hydroxide (0.16 g, 4.0 mmol) at 25°C. The reaction mixture was stirred at 25°C for 2 hours. After the reaction was completed, dilute hydrochloric acid (1.0 mol/L) was added to the system to adjust the pH=5, and the mixture was extracted with ethyl acetate (20 mL × 3), the combined organic phases were washed with saturated brine, anhydrous sodium sulfate Drying, filtration and concentration gave crude compound 20-5 (0.25 g). LC-MS (ESI), m/z: [M+H] + =211.1 Step 4: cis- N- (1-(tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H -pyrazol-3-yl)-1-methyl-3-(trifluoromethoxy)-1H-pyrazole-5-carboxamide ( 20-6 )
氮氣保護下，向化合物 20-5(0.25 g, 1.2 mmol)的 N,N-二甲基甲醯胺(5 mL)溶液中，加入HATU(0.547 g, 1.44 mmol)，反應混合物在25℃下攪拌0.5小時。然後，向反應體系中加入 N,N-二異丙基乙基胺(0.464 g, 3.60 mmol)和中 間體 B(0.268 g, 1.19 mmol)。升溫至50℃並攪拌2小時。反應結束後，向反應體系中加水(20 mL)稀釋，並將混合物用乙酸乙酯(15 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得化合物 20-6(0.35 g, 產率：71%)。LC-MS (ESI), m/z: [M+H] +=418.2 步驟5：順式-5-(1-(叔丁基)-3-(1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基))碳酸鹽( 20-7) Under the protection of nitrogen, to the solution of compound 20-5 (0.25 g, 1.2 mmol) in N,N -dimethylformamide (5 mL), HATU (0.547 g, 1.44 mmol) was added, and the reaction mixture was heated at 25°C Stir for 0.5 hours. Then, N,N -diisopropylethylamine (0.464 g, 3.60 mmol) and intermediate B (0.268 g, 1.19 mmol) were added to the reaction system. Warm to 50°C and stir for 2 hours. After the reaction, the reaction system was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 20-6 (0.35 g, yield: 71%). LC-MS (ESI), m/z: [M+H] + =418.2 Step 5: cis-5-(1-(tert-butyl)-3-(1-methyl-3-(trifluoroform Oxygen)-1H-pyrazole-5-formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl))carbonate ( 20-7 )
氮氣保護下，向化合物 20-6(0.30 g, 0.72 mmol)的二氯甲烷(5 mL)和四氫呋喃(5 mL)混合溶液中，加入4-硝基苯氯甲酸酯(0.29 g, 1.44 mmol)，吡啶(0.17 g, 2.16 mmol)和4-二甲胺基吡啶(9.0 mg, 0.07 mmol)，並將反應混合物在25℃下攪拌16小時。待反應結束後，向反應體系中加水(20 mL)稀釋，並將混合物用乙酸乙酯(15 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到化合物 20-7(0.30 g, 產率：68%)。LC-MS (ESI), m/z: [M+H] +=583.2 步驟6：順式-5-(3-(1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 20-8) Under nitrogen protection, to a mixed solution of compound 20-6 (0.30 g, 0.72 mmol) in dichloromethane (5 mL) and tetrahydrofuran (5 mL), was added 4-nitrobenzene chloroformate (0.29 g, 1.44 mmol ), pyridine (0.17 g, 2.16 mmol) and 4-dimethylaminopyridine (9.0 mg, 0.07 mmol), and the reaction mixture was stirred at 25°C for 16 hours. After the reaction was completed, water (20 mL) was added to the reaction system to dilute, and the mixture was extracted with ethyl acetate (15 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and reduced pressure concentrate. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 20-7 (0.30 g, yield: 68%). LC-MS (ESI), m/z: [M+H] + =583.2 Step 6: cis-5-(3-(1-methyl-3-(trifluoromethoxy)-1H-pyrazole -5-formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 20-8 )
將化合物 20-7(0.28 g, 0.48 mmol)溶於甲酸(10 mL)中，升溫至90℃，攪拌2小時。待反應結束後，將反應液減壓濃縮，得粗產物 20-8(0.30 g)，直接用於下一步反應。LC-MS (ESI), m/z: [M+H] +=527.1 步驟7：順式-5-(3-(1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 20) Compound 20-7 (0.28 g, 0.48 mmol) was dissolved in formic acid (10 mL), heated to 90°C, and stirred for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product 20-8 (0.30 g), which was directly used in the next reaction. LC-MS (ESI), m/z: [M+H] + =527.1 Step 7: cis-5-(3-(1-methyl-3-(trifluoromethoxy)-1H-pyrazole -5-formamide)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butyl carbamate ( Example 20 )
室溫下，向化合物 20-8(0.10 g, 0.20 mmol)的四氫呋喃(3 mL)溶液中，加入叔丁胺(0.16 mg, 2.0 mmol)。將反應混合物繼續攪拌16小時。反應完成後將反應液減壓濃縮，所得殘餘物透過製備HPLC分離純化(管柱：Gemini-C18, 150 × 21.2 mm，5 µm；流動相：乙腈/水（0.1%甲酸）；梯度：30%-34%)，得到 實施例 20。LC-MS (ESI), m/z: [M+H] +=460.9 實施例 20A和 實施例 20B： To a solution of compound 20-8 (0.10 g, 0.20 mmol) in tetrahydrofuran (3 mL) was added tert-butylamine (0.16 mg, 2.0 mmol) at room temperature. The reaction mixture was stirred for an additional 16 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the obtained residue was separated and purified by preparative HPLC (column: Gemini-C18, 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% formic acid); gradient: 30% -34%), resulting in Example 20 . LC-MS (ESI), m/z: [M+H] + =460.9 Example 20A and Example 20B :
(3
R,5
R)-5-(3-(1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3,S5
S)-5-(3-(1-甲基-3-(三氟甲氧基)-1H-吡唑-5-甲醯胺)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
實施例20透過手性分離(層析管柱型號：CHIRALPAK AD-H 250 mm x 20 mm，5 μm；流動相：40%異丙醇(0.2%氨水)；流速：40 mL/min)進一步純化得到 實施例 20A(28.1 mg, 產率：31%)和 實施例 20B(25.8 mg, 產率：28%)。 實施例 20A： Example 20 was further purified by chiral separation (column model: CHIRALPAK AD-H 250 mm x 20 mm, 5 μm; mobile phase: 40% isopropanol (0.2% ammonia water); flow rate: 40 mL/min) Example 20A (28.1 mg, yield: 31%) and Example 20B (25.8 mg, yield: 28%) were obtained. Example 20A :
t R=1.57 min t R =1.57min
LC-MS (ESI), m/z: [M+H] +=460.9 LC-MS (ESI), m/z: [M+H] + =460.9
1H NMR (400 MHz, CD 3OD) δ6.80 (s, 1H), 6.55 (s, 1H), 5.20 – 5.14 (m, 1H), 5.01 – 4.95 (m, 1H), 4.11 (s, 3H), 4.06 – 4.01 (m, 1H), 3.97 – 3.91 (m, 1H), 2.76 – 2.67 (m, 1H), 2.16 – 2.10 (m, 1H), 1.28 (s, 9H). 實施例 20B： 1 H NMR (400 MHz, CD 3 OD) δ 6.80 (s, 1H), 6.55 (s, 1H), 5.20 – 5.14 (m, 1H), 5.01 – 4.95 (m, 1H), 4.11 (s, 3H) , 4.06 – 4.01 (m, 1H), 3.97 – 3.91 (m, 1H), 2.76 – 2.67 (m, 1H), 2.16 – 2.10 (m, 1H), 1.28 (s, 9H). Embodiment 20B :
t R=2.62 min t R =2.62 min
LC-MS (ESI), m/z: [M+H] +=460.8 LC-MS (ESI), m/z: [M+H] + =460.8
1H NMR (400 MHz, CD
3OD)
δ6.77 (s, 1H), 6.53 (s, 1H), 5.20 – 5.14 (m, 1H), 5.01 – 4.95 (m, 1H), 4.08 (s, 3H), 4.01 (d,
J=10.3 Hz, 1H), 3.91 (dd, J=10.4, 4.3 Hz, 1H), 2.75 – 2.63 (m, 1H), 2.14 – 2.06 (m, 1H), 1.26 (s, 9H).
實施例 21：順式-5-(3-(6-甲氧基甲基-4-甲基煙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
氮氣保護下，將硼烷-四氫呋喃錯合物(0.074 L, 0.074 mol)中滴入化合 21-1(4.0 g, 0.019 mol)的四氫呋喃溶液(30 mL)中。室溫下攪拌12小時後，向反應混合物中加入飽和氯化銨水溶液(100 mL)淬滅，用二氯甲烷萃取(30 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析法(石油醚：乙酸乙酯=5：1) 純化，得到化合物 21-2(2.0 g, 產率：52%)。LC-MS (ESI), m/z: [M+H] +=202.1, 204.1 步驟2：5-溴-2-甲氧基甲基-4-甲基吡啶( 21-3) Under nitrogen protection, borane-tetrahydrofuran complex (0.074 L, 0.074 mol) was added dropwise into a solution of compound 21-1 (4.0 g, 0.019 mol) in tetrahydrofuran (30 mL). After stirring at room temperature for 12 hours, the reaction mixture was quenched by adding saturated aqueous ammonium chloride (100 mL), extracted with dichloromethane (30 mL × 3), combined organic phases, washed with saturated brine, and dried over anhydrous sodium sulfate , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 21-2 (2.0 g, yield: 52%). LC-MS (ESI), m/z: [M+H] + =202.1, 204.1 Step 2: 5-Bromo-2-methoxymethyl-4-methylpyridine ( 21-3 )
氮氣保護和0℃下，將氫化鈉(0.48 g, 0.012 mol)分批加入到化合物 21-2(2.00 g, 9.90 mmol)的四氫呋喃溶液(30 mL)中，將反應混合物在0℃攪拌0.5小時後，將碘甲烷(2.81 g, 0.012 mol)加入到反應液中，隨後移除冷浴，將反應混合物在室溫下攪拌2小時。反應完畢後，向反應體系中加入飽和氯化銨水溶液(100 mL)淬滅反應，用二氯甲烷萃取(30 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析(石油醚：乙酸乙酯=5：1)純化，得到化合物 21-3(0.50 g, 產率：23 %)。LC-MS (ESI), m/z: [M+H] +=216.1, 218.1. 步驟3：6-甲氧基甲基-4-甲基煙酸叔丁酯( 21-4) Under nitrogen protection and at 0°C, sodium hydride (0.48 g, 0.012 mol) was added in portions to a solution of compound 21-2 (2.00 g, 9.90 mmol) in tetrahydrofuran (30 mL), and the reaction mixture was stirred at 0°C for 0.5 hours Finally, methyl iodide (2.81 g, 0.012 mol) was added to the reaction solution, then the cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, saturated aqueous ammonium chloride (100 mL) was added to the reaction system to quench the reaction, extracted with dichloromethane (30 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered Concentrate under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 21-3 (0.50 g, yield: 23%). LC-MS (ESI), m/z: [M+H] + =216.1, 218.1. Step 3: tert-butyl 6-methoxymethyl-4-methylnicotinate ( 21-4 )
氮氣保護下，將化合物 21-3(0.30 g, 1.39 mmol)的四氫呋喃溶液(10 mL)降溫至-78℃，滴加丁基鋰(1.20 mL, 2.78 mmol)。反應液在-78℃下攪拌0.5小時，然後滴加碳酸酐二叔丁酯(0.606 g, 2.78 mmol)的四氫呋喃溶液(5 mL)，滴加完畢後繼續在-78℃下反應2小時。待反應完成後，向反應體系中加入飽和氯化銨水溶液(15 mL)，用二氯甲烷萃取(10 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(石油醚：乙酸乙酯=5：1)，得到化合物 21-4(0.130 g, 產率：39%)。LC-MS (ESI), m/z: [M+H] +=238.1 步驟4：6-甲氧基甲基-4-甲基煙酸( 21-5) Under the protection of nitrogen, the tetrahydrofuran solution (10 mL) of compound 21-3 (0.30 g, 1.39 mmol) was cooled to -78°C, and butyllithium (1.20 mL, 2.78 mmol) was added dropwise. The reaction solution was stirred at -78°C for 0.5 hours, then a tetrahydrofuran solution (5 mL) of di-tert-butyl carbonate anhydride (0.606 g, 2.78 mmol) was added dropwise, and the reaction was continued at -78°C for 2 hours after the addition was complete. After the reaction is complete, add saturated aqueous ammonium chloride (15 mL) to the reaction system, extract with dichloromethane (10 mL × 3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter and reduce pressure concentrate. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 21-4 (0.130 g, yield: 39%). LC-MS (ESI), m/z: [M+H] + =238.1 Step 4: 6-methoxymethyl-4-methylnicotinic acid ( 21-5 )
室溫下，將化合物 21-4(0.13 g, 0.55 mmol)溶於二氯甲烷(2.0 mL)中，降溫至0℃，滴加鹽酸/二氧六環溶液(4 mol/L, 5.0 ml)。將反應液保持在0℃下攪拌3小時。待反應完畢後，向反應體系中加入飽和碳酸氫鈉水溶液(10 mL)，並將反應混合物用二氯甲烷萃取(10 mL × 3)，合併有機相，飽和食鹽水洗滌，水相加鹽酸(2 mol/L)調至pH=5，再用二氯甲烷萃取 (10 mL × 3)。合併的有機相用無水硫酸鈉乾燥，過濾後減壓濃縮，得到化合物 21-5(95.0 mg, 產率：95%)。LC-MS (ESI), m/z: [M+H] +=182.0 步驟5：順式- N-(1-叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-6-甲氧基甲基-4-甲基煙醯胺( 21-6) At room temperature, compound 21-4 (0.13 g, 0.55 mmol) was dissolved in dichloromethane (2.0 mL), cooled to 0°C, and hydrochloric acid/dioxane solution (4 mol/L, 5.0 ml) was added dropwise . The reaction solution was kept stirring at 0°C for 3 hours. After the reaction was complete, saturated aqueous sodium bicarbonate solution (10 mL) was added to the reaction system, and the reaction mixture was extracted with dichloromethane (10 mL × 3), the organic phases were combined, washed with saturated brine, and the aqueous phase was added with hydrochloric acid ( 2 mol/L) to pH=5, and extracted with dichloromethane (10 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 21-5 (95.0 mg, yield: 95%). LC-MS (ESI), m/z: [M+H] + =182.0 Step 5: cis- N- (1-tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H- Pyrazol-3-yl)-6-methoxymethyl-4-methylnicotinamide ( 21-6 )
氮氣保護下，向化合物 21-5(0.301 g, 1.33 mmol)的 N, N二甲基甲醯胺溶液(8 mL)中加入二環己基碳二亞胺(0.18 g, 0.87 mmol)和4-二甲胺基吡啶(81 mg, 0.67 mmol)。常溫下攪拌0.5小時，隨後加入 中間體 B(0.15 g, 0.67 mmol)，加料完成後繼續攪拌2小時。待反應完畢後，向反應體系中加入水(30 mL)淬滅反應，並用二氯甲烷萃取(10 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(石油醚：乙酸乙酯=1：9)，得到化合物 21-6(0.14 g, 產率：54 %)。LC-MS (ESI), m/z: [M+H] +=389.0 步驟6：順式-5-(1-叔丁基)-3-(6-甲氧基甲基)-4-甲基煙醯胺基-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 21-7) Under nitrogen protection , dicyclohexylcarbodiimide (0.18 g, 0.87 mmol) and 4- Dimethylaminopyridine (81 mg, 0.67 mmol). Stir at room temperature for 0.5 hours, then add Intermediate B (0.15 g, 0.67 mmol), and continue stirring for 2 hours after the addition is complete. After the reaction was complete, water (30 mL) was added to the reaction system to quench the reaction, and extracted with dichloromethane (10 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure . The resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:9) to obtain compound 21-6 (0.14 g, yield: 54%). LC-MS (ESI), m/z: [M+H] + =389.0 Step 6: cis-5-(1-tert-butyl)-3-(6-methoxymethyl)-4-methanol Nicotinylamino-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 21-7 )
氮氣保護下，將化合物 21-6(0.15 g, 0.77 mmol)、吡啶(0.122 g, 1.54 mmol)、氯甲酸對硝基苯酯(0.19 mg, 0.93 mmol)和4-二甲胺基吡啶(9.44 mg, 0.0774 mmol)溶於二氯甲烷(10 mL)中，將反應混合物在室溫攪拌5小時。待反應完畢後，向反應體系中加入水(10 mL)淬滅反應。反應混合物用二氯甲烷萃取(10 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析(石油醚：乙酸乙酯=5：1)分離純化，得到化合物 21-7(0.115 g, 產率：27%)。LC-MS (ESI), m/z: [M+H] +=553.8 步驟7：順式-5-(3-(6-甲氧基甲基)-4-甲基煙醯胺基-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 21-8) Under nitrogen protection, compound 21-6 (0.15 g, 0.77 mmol), pyridine (0.122 g, 1.54 mmol), p-nitrophenyl chloroformate (0.19 mg, 0.93 mmol) and 4-dimethylaminopyridine (9.44 mg, 0.0774 mmol) was dissolved in dichloromethane (10 mL), and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, water (10 mL) was added to the reaction system to quench the reaction. The reaction mixture was extracted with dichloromethane (10 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 21-7 (0.115 g, yield: 27%). LC-MS (ESI), m/z: [M+H] + =553.8 Step 7: cis-5-(3-(6-methoxymethyl)-4-methylnicotinylamino-1H -pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 21-8 )
室溫下，將化合物 21-7(0.11 g, 0.20 mmol)溶於甲酸(3 mL)中，升溫至90℃，攪拌2小時。反應完畢後將反應液減壓濃縮，得到粗產物化合物 21-8(80 mg)。LC-MS (ESI), m/z: [M+H] +=497.8 步驟8：順式-5-(3-(6-甲氧基甲基-4-甲基煙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯（ 實施例 21） Compound 21-7 (0.11 g, 0.20 mmol) was dissolved in formic acid (3 mL) at room temperature, heated to 90°C, and stirred for 2 hours. After the completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain the crude compound 21-8 (80 mg). LC-MS (ESI), m/z: [M+H] + =497.8 Step 8: cis-5-(3-(6-methoxymethyl-4-methylnicotinylamino)-1H -pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 21 )
氮氣保護下，向化合物 21-8(80 mg, 0.16 mmol)的四氫呋喃(5 mL)溶液中加入 N, N-二異丙基乙胺(21 mg, 0.16 mmol)和叔丁胺(59 mg, 0.80 mmol)。反應液在室溫下繼續攪拌5小時。反應結束後，加入水(10 mL)淬滅反應，並用二氯甲烷萃取(5 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過製備HPLC(管柱: Gemini-C18 150 × 21.2 mm，5 µm；流動相：乙腈/水(0.1%甲酸)；梯度：30-95%)分離純化得到 實施例 21(41.0 mg)。LC-MS (ESI), m/z: [M+H] +=432.0 實施例 21A和 實施例 21B： Under nitrogen protection, N , N -diisopropylethylamine (21 mg, 0.16 mmol) and tert-butylamine (59 mg, 0.80 mmol) were added to a solution of compound 21-8 (80 mg, 0.16 mmol) in tetrahydrofuran (5 mL). ). The reaction solution was stirred at room temperature for 5 hours. After the reaction was completed, water (10 mL) was added to quench the reaction, and extracted with dichloromethane (5 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by preparative HPLC (column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% formic acid); gradient: 30-95%) to obtain Example 21 (41.0 mg) . LC-MS (ESI), m/z: [M+H] + =432.0 Example 21A and Example 21B :
(3
R,5
R)-5-(3-(6-甲氧基甲基-4-甲基煙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3
S,5
S)-5-(3-(6-甲氧基甲基-4-甲基煙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
實施例21透過手性製備SFC(儀器：SFC Thar prep 80；管柱：CHIRALPAK AD-H 250mm x 20 mm, 5 μm；流動相：40%乙醇(0.2%氨水)；流速：40 g/min)分離純化得到 實施例 21A(20.0 mg, 產率：29%)和 實施例 21B(19.5 mg, 產率：28%)。 實施例 21A： Example 21 Preparation of SFC by chirality (instrument: SFC Thar prep 80; column: CHIRALPAK AD-H 250mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2% ammonia water); flow rate: 40 g/min) Example 21A (20.0 mg, yield: 29%) and Example 21B (19.5 mg, yield: 28%) were obtained by separation and purification. Example 21A :
t R=5.04 min t R =5.04 min
LC-MS (ESI), m/z: [M+H] +=431.9 LC-MS (ESI), m/z: [M+H] + =431.9
1H NMR (400 MHz, DMSO- d 6 ) δ12.43 (s, 1H), 10.91 (s, 1H), 8.52 (s, 1H), 7.34 (s, 1H), 6.96 (s, 1H), 6.62 (brs, 1H), 5.18 – 5.10 (m, 1H), 4.90 – 4.79 (m, 1H), 4.50 (s, 2H), 91 – 3.83 (m, 2H), 3.36 (s, 3H), 2.74 – 2.67 (m, 1H), 2.41 (s, 3H), 2.01 – 1.94 (m, 1H), 1.22 (s, 9H). 實施例 21B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.43 (s, 1H), 10.91 (s, 1H), 8.52 (s, 1H), 7.34 (s, 1H), 6.96 (s, 1H), 6.62 ( brs, 1H), 5.18 – 5.10 (m, 1H), 4.90 – 4.79 (m, 1H), 4.50 (s, 2H), 91 – 3.83 (m, 2H), 3.36 (s, 3H), 2.74 – 2.67 ( m, 1H), 2.41 (s, 3H), 2.01 – 1.94 (m, 1H), 1.22 (s, 9H). Example 21B :
t R=7.14 min t R =7.14 min
LC-MS (ESI), m/z: [M+H] +=432.0 LC-MS (ESI), m/z: [M+H] + =432.0
1H NMR (400 MHz, DMSO-
d 6)
δ12.43 (s, 1H), 10.91 (s, 1H), 8.52 (s, 1H), 7.35 (s, 1H), 6.94 (s, 1H), 6.62 (s, 1H), 5.20 – 5.09 (m, 1H), 4.91 – 4.81 (s, 1H), 4.50 (s, 2H), 3.90 – 3.76 (m, 2H), 3.38 (s, 3H), 2.76 – 2.65 (m, 1H), 2.41 (s, 3H), 2.01 – 1.93 (m, 1H), 1.22 (s, 9H).
實施例 22：順式-5-(3-(2-(2-甲氧基吡啶-4-基)乙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-叔丁基胺基甲酸酯
氮氣保護下，向化合物 22-1(0.10 g, 0.60 mmol)的二氯甲烷(8 mL)中加入三乙胺(61 mg, 0.60 mmol)、1-乙基-(3-二甲基胺基丙基)碳二亞胺鹽酸鹽(0.14 g, 0.60 mmol)和4-二甲胺基吡啶(73 mg, 0.60 mmol)。反應液在室溫下攪拌0.5小時後，將 中間體 B(0.14 g, 0.60 mmol)加入到反應液中，室溫下繼續反應2小時。待反應完畢後，向反應體系中加入水(15 mL)淬滅反應，並將反應混合物用二氯甲烷萃取(5 mL × 3)，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(石油醚：乙酸乙酯=1：9)得到化合物 22-2(0.12 g, 產率：53%)。LC-MS (ESI), m/z: [M+H] +=375.1 步驟2：順式-5-(1-叔丁基)-3-(2-(2-甲氧基吡啶-4-基)乙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 22-3) Under nitrogen protection, triethylamine ( 61 mg, 0.60 mmol), 1-ethyl-(3-dimethylamino Propyl) carbodiimide hydrochloride (0.14 g, 0.60 mmol) and 4-dimethylaminopyridine (73 mg, 0.60 mmol). After the reaction solution was stirred at room temperature for 0.5 hours, intermediate B (0.14 g, 0.60 mmol) was added to the reaction solution, and the reaction was continued at room temperature for 2 hours. After the reaction was complete, water (15 mL) was added to the reaction system to quench the reaction, and the reaction mixture was extracted with dichloromethane (5 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure . The resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:9) to obtain compound 22-2 (0.12 g, yield: 53%). LC-MS (ESI), m/z: [M+H] + =375.1 Step 2: cis-5-(1-tert-butyl)-3-(2-(2-methoxypyridine-4- Base) acetamido) -1H-pyrazol-5-yl) tetrahydrofuran -3-yl (4-nitrophenyl) carbonate ( 22-3 )
氮氣保護下，將化合物 22-2(0.27 g, 0.72 mmol)，吡啶(0.114 g, 1.44 mmol)，氯甲酸對硝基苯酯(0.17 g, 0.86 mmol)和4-二甲胺基吡啶(10 mg，0.070 mmol)溶於二氯甲烷(10 mL)中，室溫攪拌5小時。待反應完畢後，向反應體系加入水(10 mL)淬滅反應，反應混合物用二氯甲烷萃取(10 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(石油醚：乙酸乙酯=5：1)分離純化，得到產物化合物 22-3(0.180 g, 產率：46%)。LC-MS (ESI), m/z: [M+H] +=539.8 步驟3：順式-5-(3-(2-(2-甲氧基吡啶-4-基)乙醯胺基) -1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 22-4) Under nitrogen protection, compound 22-2 (0.27 g, 0.72 mmol), pyridine (0.114 g, 1.44 mmol), p-nitrophenyl chloroformate (0.17 g, 0.86 mmol) and 4-dimethylaminopyridine (10 mg, 0.070 mmol) was dissolved in dichloromethane (10 mL), and stirred at room temperature for 5 hours. After the reaction was completed, water (10 mL) was added to the reaction system to quench the reaction, the reaction mixture was extracted with dichloromethane (10 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and reduced pressure concentrate. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain the product compound 22-3 (0.180 g, yield: 46%). LC-MS (ESI), m/z: [M+H] + =539.8 Step 3: cis-5-(3-(2-(2-methoxypyridin-4-yl)acetamido) -1H-pyrazol-5-yl) tetrahydrofuran-3-yl (4-nitrophenyl) carbonate ( 22-4 )
室溫下，將化合物 22-3(0.16 g, 0.30 mmol)溶於甲酸(3 mL)中，升溫至90℃然後攪拌2小時。反應完畢後減壓濃縮反應液，得到粗產物化合物 22-4(0.120 g)，直接用於下一步反應。LC-MS (ESI), m/z: [M+H] +=484.1 步驟4：順式-5-(3-(2-(2-甲氧基吡啶-4-基)乙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-叔丁基胺基甲酸酯( 實施例 22) Compound 22-3 (0.16 g, 0.30 mmol) was dissolved in formic acid (3 mL) at room temperature, heated to 90°C and stirred for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product compound 22-4 (0.120 g), which was directly used in the next reaction. LC-MS (ESI), m/z: [M+H] + =484.1 Step 4: cis-5-(3-(2-(2-methoxypyridin-4-yl)acetamido) -1H-pyrazol-5-yl)tetrahydrofuran-3-tert-butylcarbamate ( Example 22 )
室溫下，向化合物 22-4(90 mg, 0.19 mmol)的四氫呋喃(5 mL)溶液中加入 N,N-二異丙基乙胺(48 mg, 0.37 mmol)和叔丁胺(68 mg, 0.93 mmol)。將反應混合物在室溫下攪拌5小時。待反應結束後，向反應體系中加入水(10 mL)淬滅反應，並將混合物用二氯甲烷萃取(5 mL × 3)，合併有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過製備HPLC(層析管柱：Gemini-C18 150 × 21.2 mm，5 µm；流動相：乙腈-水(0.1%甲酸)； 梯度：10-95%)分離純化得到 實施例 22(16.0 mg, 產率：20%)。LC-MS (ESI), m/z: [M+H] +=418.2. 實施例 22A和 實施例 22B： Add N,N -diisopropylethylamine (48 mg, 0.37 mmol) and tert-butylamine (68 mg, 0.93 mmol) to a solution of compound 22-4 (90 mg, 0.19 mmol) in tetrahydrofuran (5 mL) at room temperature ). The reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, water (10 mL) was added to the reaction system to quench the reaction, and the mixture was extracted with dichloromethane (5 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered Concentrate under reduced pressure. The resulting residue was separated and purified by preparative HPLC (chromatographic column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile-water (0.1% formic acid); gradient: 10-95%) to obtain Example 22 (16.0 mg, yield: 20%). LC-MS (ESI), m/z: [M+H] + =418.2. Example 22A and Example 22B :
(3
R,5
R)-5-(3-(2-(2-甲氧基吡啶-4-基)乙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-叔丁基胺基甲酸酯和(3
S,5
S)-5-(3-(2-(2-甲氧基吡啶-4-基)乙醯胺基)-1H-吡唑-5-基)四氫呋喃-3-叔丁基胺基甲酸酯
實施例22透過手性製備SFC(儀器：SFC Thar prep 80；管柱: CHIRALPAK AD-H 250mm x 20 mm, 5 μm；流動相：40%乙醇(0.2%氨水)；流速：40 g/min)分離純化得到 實施例 22A(6.3 mg, 產率：8 %)和 實施例 22B(8.9 mg, 產率：11%)。 實施例 22A： Example 22 Preparation of SFC by chirality (instrument: SFC Thar prep 80; column: CHIRALPAK AD-H 250mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2% ammonia water); flow rate: 40 g/min) Separation and purification gave Example 22A (6.3 mg, yield: 8%) and Example 22B (8.9 mg, yield: 11%). Example 22A :
t R=3.17 min t R =3.17min
LC-MS (ESI), m/z: [M+H] +=418.2 LC-MS (ESI), m/z: [M+H] + =418.2
1H NMR (400 MHz, CD 3OD) δ7.95 (d, J=5.3 Hz, 1H), 6.85 (dd, J=5.3, 1.3 Hz, 1H), 6.70 (s, 1H), 6.34 (brs, 1H), 5.07 (s, 1H), 4.94 – 4.83 (m, 1H), 3.94 – 3.89 (m, 1H), 3.83 (d, J=4.5 Hz, 1H), 3.79 (s, 3H), 3.58 (s, 2H), 2.62 – 2.52 (m, 1H), 2.04 – 1.96 (m, 1H), 1.14 (s, 9H). 實施例 22B： 1 H NMR (400 MHz, CD 3 OD) δ 7.95 (d, J =5.3 Hz, 1H), 6.85 (dd, J= 5.3, 1.3 Hz, 1H), 6.70 (s, 1H), 6.34 (brs, 1H ), 5.07 (s, 1H), 4.94 – 4.83 (m, 1H), 3.94 – 3.89 (m, 1H), 3.83 (d, J =4.5 Hz, 1H), 3.79 (s, 3H), 3.58 (s, 2H), 2.62 – 2.52 (m, 1H), 2.04 – 1.96 (m, 1H), 1.14 (s, 9H). Embodiment 22B :
t R=5.82 min t R =5.82 min
LC-MS (ESI), m/z: [M+H] +=418.2 LC-MS (ESI), m/z: [M+H] + =418.2
1H NMR (400 MHz, CD
3OD) δ 8.03 (d,
J=5.3 Hz, 1H), 6.92 (dd,
J=5.3, 0.9 Hz, 1H), 6.77 (s, 1H), 6.43 (brs, 1H), 5.14 (s, 1H), 5.00 – 4.91 (m, 1H), 3.98 (d,
J=10.3 Hz, 1H), 3.90 (d, J=3.6 Hz, 1H), 3.87 (s, 3H),3.65 (s, 2H), 2.70 – 2.59 (m, 1H), 2.07 (d,
J=9.0 Hz, 1H), 1.22 (s, 9H).
實施例 23：順式-5-(3-(3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
氮氣保護下，將化合物 23-1(5.0 g, 0.029 mol)、碘甲烷(8.35 g, 5.90 mmol)和碳酸鉀(8.13 g, 5.90 mmol)溶於乙腈(50 mL)中。將反應體系升溫至80℃並攪拌10小時。待反應完成後，將反應混合物降至室溫，過濾，濾餅用乙腈(20 mL)淋洗。將濾液減壓濃縮，所得殘留物透過矽膠管柱層析 (石油醚：乙酸乙酯=2：1)純化得到化合物 23-2(1.30 g, 產率：20%)。LC-MS (ESI), m/z: [M+H] +=185.1 步驟2：3-溴甲基-1-甲基吡唑-5-甲酸乙酯( 23-3) Under nitrogen protection, compound 23-1 (5.0 g, 0.029 mol), iodomethane (8.35 g, 5.90 mmol) and potassium carbonate (8.13 g, 5.90 mmol) were dissolved in acetonitrile (50 mL). The reaction system was warmed up to 80°C and stirred for 10 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, filtered, and the filter cake was rinsed with acetonitrile (20 mL). The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound 23-2 (1.30 g, yield: 20%). LC-MS (ESI), m/z: [M+H] + =185.1 Step 2: Ethyl 3-bromomethyl-1-methylpyrazole-5-carboxylate ( 23-3 )
氮氣保護下，將化合物 23-2(0.750 g, 4.07 mmol)溶於乙腈(25 mL)中，隨後加入三溴化磷(1.65 g, 6.01 mmol)，並將反應體系升溫至80℃攪拌2小時。待反應結束後，將反應混合物冷卻至室溫，加水(100 mL)淬滅反應。繼續攪拌5分鐘後，向反應體系中加入乙酸乙酯(100 mL)。將反應混合物分液，水相用乙酸乙酯萃取(50 mL × 2)。合併的有機相飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(石油醚：乙酸乙酯=2：1)得到化合物 23-3(0.83 g, 產率：78%)。LC-MS (ESI), m/z: [M+H] +=247.1, 249.1 步驟3：3-環丙氧基甲基-1-甲基吡唑-5-羧酸乙酯( 23-4) Under nitrogen protection, compound 23-2 (0.750 g, 4.07 mmol) was dissolved in acetonitrile (25 mL), then phosphorus tribromide (1.65 g, 6.01 mmol) was added, and the reaction system was heated to 80°C and stirred for 2 hours . After the reaction was completed, the reaction mixture was cooled to room temperature, and water (100 mL) was added to quench the reaction. After continuing to stir for 5 minutes, ethyl acetate (100 mL) was added to the reaction system. The reaction mixture was separated, and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to obtain compound 23-3 (0.83 g, yield: 78%). LC-MS (ESI), m/z: [M+H] + =247.1, 249.1 Step 3: Ethyl 3-cyclopropoxymethyl-1-methylpyrazole-5-carboxylate ( 23-4 )
室溫下，將環丙醇(0.390 g, 6.71 mol)溶於四氫呋喃(30 mL)中。將反應體系降溫至0℃後分批加入氫化鈉(269 mg, 60%, 6.71 mol)，加完後繼續攪拌0.5小時。向反應中加入化合物 23-3(0.830 g, 3.35 mol)，在室溫下攪拌10小時。反應結束後加水(100 mL)淬滅反應。攪拌5分鐘後，反應混合物用乙酸乙酯萃取(50 mL × 3)。合併的有機相飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(石油醚：乙酸乙酯=1：1)純化得到產物化合物 23-5(0.468 g, 產率：55%)。LC-MS (ESI), m/z: [M+H] +=225.0 步驟4：3-環丙氧基甲基-1-甲基吡唑-5-羧酸( 23-5) Cyclopropanol (0.390 g, 6.71 mol) was dissolved in tetrahydrofuran (30 mL) at room temperature. After the reaction system was cooled to 0°C, sodium hydride (269 mg, 60%, 6.71 mol) was added in batches, and stirring was continued for 0.5 hours after the addition was complete. Compound 23-3 (0.830 g, 3.35 mol) was added to the reaction and stirred at room temperature for 10 hours. After the reaction was completed, water (100 mL) was added to quench the reaction. After stirring for 5 minutes, the reaction mixture was extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the product compound 23-5 (0.468 g, yield: 55%). LC-MS (ESI), m/z: [M+H] + =225.0 Step 4: 3-Cyclopropoxymethyl-1-methylpyrazole-5-carboxylic acid ( 23-5 )
室溫下，將化合物 23-4(0.20 g, 0.89 mmol)和一水合氫氧化鋰(74.8 mg, 1.78 mmol)溶於四氫呋喃和水(10：1, 20 mL)中。將反應混合物在室溫下攪拌過夜。反應結束後，向反應體系中加入水(50 ml)，隨後用稀鹽酸(1 mol/L)調整體系的pH至3-4，反應混合物用乙酸乙酯萃取(50 mL × 3)，合併的有機相，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(二氯甲烷：甲醇=20：1)純化得到化合物 23-5(0.130 g, 產率：67%)。LC-MS (ESI), m/z: [M+H] +=197.1 步驟5：順式- N-(1-(叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺( 23-6) Compound 23-4 (0.20 g, 0.89 mmol) and lithium hydroxide monohydrate (74.8 mg, 1.78 mmol) were dissolved in tetrahydrofuran and water (10:1, 20 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. After the reaction, water (50 ml) was added to the reaction system, and then the pH of the system was adjusted to 3-4 with dilute hydrochloric acid (1 mol/L), and the reaction mixture was extracted with ethyl acetate (50 mL × 3), and the combined The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound 23-5 (0.130 g, yield: 67%). LC-MS (ESI), m/z: [M+H] + =197.1 Step 5: cis- N- (1-(tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H -pyrazol-3-yl)-3-(cyclopropoxymethyl)-1-methyl-1H-pyrazole-5-carboxamide ( 23-6 )
氮氣保護下，向化合物 23-5(0.218 g, 1.11 mmol)的 N,N-二甲基甲醯胺(5 mL)溶液中加入HATU(0.586 g, 1.33 mmol)，將反應混合物攪拌0.5小時。然後向體系中加入 N,N-二異丙基乙基胺(0.287 g, 2.22 mmol)和 中間體 B(0.250 g, 1.11 mmol)。將反應體系升溫至50℃並攪拌2小時。反應結束後，向反應體系中加水(30 mL)稀釋，然後用乙酸乙酯(20 mL × 3)萃取。合併的有機相飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(石油醚：乙酸乙酯=1：1)得到化合物 23-6(0.250 g, 產率：56%)。LC-MS (ESI), m/z: [M+H] +=404.1 步驟6：順式-5-(1-(叔丁基)-3-(3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 23-7) Under nitrogen protection, HATU (0.586 g, 1.33 mmol) was added to a solution of compound 23-5 (0.218 g, 1.11 mmol) in N,N -dimethylformamide (5 mL), and the reaction mixture was stirred for 0.5 hours. Then N,N -diisopropylethylamine (0.287 g, 2.22 mmol) and Intermediate B (0.250 g, 1.11 mmol) were added to the system. The reaction system was warmed up to 50°C and stirred for 2 hours. After the reaction, the reaction system was diluted with water (30 mL), and then extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 23-6 (0.250 g, yield: 56%). LC-MS (ESI), m/z: [M+H] + =404.1 Step 6: cis-5-(1-(tert-butyl)-3-(3-(cyclopropoxymethyl)- 1-Methyl-1H-pyrazole-5-formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 23-7 )
氮氣保護下，向化合物 23-6(0.25 g, 0.62 mmol)的二氯甲烷(5 mL)和四氫呋喃(5 mL)溶液中，加入4-硝基苯氯甲酸酯(0.19 g, 0.93 mmol)、吡啶(0.147 g, 1.86 mmol)和4-二甲胺基吡啶(8.0 mg, 0.066 mmol)。反應混合物於25℃下攪拌16小時。反應完成後加水(20 mL)稀釋，混合物用乙酸乙酯(15 mL × 3)萃取。合併的有機相飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(石油醚：乙酸乙酯=1：1)得到產物 23-7(0.280 g, 產率：79%)。LC-MS (ESI), m/z: [M+H] +=569.2. 步驟7：順式-5-(3-(3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 23-8) Under nitrogen protection, to a solution of compound 23-6 (0.25 g, 0.62 mmol) in dichloromethane (5 mL) and tetrahydrofuran (5 mL), was added 4-nitrobenzene chloroformate (0.19 g, 0.93 mmol) , pyridine (0.147 g, 1.86 mmol) and 4-dimethylaminopyridine (8.0 mg, 0.066 mmol). The reaction mixture was stirred at 25°C for 16 hours. After the reaction was completed, it was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL×3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain product 23-7 (0.280 g, yield: 79%). LC-MS (ESI), m/z: [M+H] + =569.2. Step 7: cis-5-(3-(3-(cyclopropoxymethyl)-1-methyl-1H- Pyrazole-5-carboxamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 23-8 )
將化合物 23-7(0.28 g, 0.49 mmol)溶於四氫呋喃(3 mL)和甲酸(3 mL)中，並將反應混合物在70℃攪拌72小時。反應結束後將反應混合物減壓濃縮，所得殘留物透過矽膠管柱層析法(二氯甲烷：甲醇=20：1)純化得到化合物 23-8(0.100 g, 產率：40%)。LC-MS (ESI), m/z: [M+H] +=523.1 步驟8：順式-5-(3-(3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯（ 實施例 23） Compound 23-7 (0.28 g, 0.49 mmol) was dissolved in tetrahydrofuran (3 mL) and formic acid (3 mL), and the reaction mixture was stirred at 70° C. for 72 hr. After the reaction, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound 23-8 (0.100 g, yield: 40%). LC-MS (ESI), m/z: [M+H] + =523.1 Step 8: cis-5-(3-(3-(cyclopropoxymethyl)-1-methyl-1H-pyridine Azole-5-carboxamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 23 )
室溫下，向化合物 23-8(0.10 g, 0.20 mmol)的四氫呋喃溶液(3 mL)中，加入叔丁胺(0.143 g, 1.96 mmol)，並將反應混合物繼續攪拌16小時。反應完全後將反應體系濃縮，所得殘餘物透過製備HPLC純化(管柱：Gemini-C18, 150 × 21.2 mm，5 µm；流動相：乙腈/水(0.1%三氟乙酸)；梯度：30%-32%)，得到 實施例 23(30.0 mg, 產率：34%)。LC-MS (ESI), m/z: [M+H] +=446.9. 實施例 23A和 實施例 23B： To a solution of compound 23-8 (0.10 g, 0.20 mmol) in tetrahydrofuran (3 mL) was added tert-butylamine (0.143 g, 1.96 mmol) at room temperature, and the reaction mixture was further stirred for 16 hours. After the reaction was complete, the reaction system was concentrated, and the resulting residue was purified by preparative HPLC (column: Gemini-C18, 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% trifluoroacetic acid); gradient: 30%- 32%) to obtain Example 23 (30.0 mg, yield: 34%). LC-MS (ESI), m/z: [M+H] + =446.9. Example 23A and Example 23B :
(3
S,5
S)-5-(3-(3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3
R,5
R)-5-(3-(3-(環丙氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
實施例 23透過手性製備SFC (管柱：CHIRALPAK IC, 250mm x 20 mm，5 μm；流動相：40%乙醇(0.2%氨水)；流速：40 mL/min)進一步純化得到 實施例 23A(6.4 mg, 產率：7 %)和 實施例 23B(7.1 mg, 產率：8%)。 實施例 23A： Example 23 was further purified by chiral preparative SFC (column: CHIRALPAK IC, 250mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2% ammonia water); flow rate: 40 mL/min) to obtain Example 23A (6.4 mg, yield: 7%) and Example 23B (7.1 mg, yield: 8%). Example 23A :
t R=2.18 min t R =2.18 min
LC-MS (ESI), m/z: [M+H]+=446.9LC-MS (ESI), m/z: [M+H]+=446.9
1H NMR (400 MHz, CD 3OD) δ6.96 (s, 1H), 6.71 – 6.54 (m, 1H), 5.27 – 5.17 (m, 1H), 5.08 – 5.00 (m, 1H), 4.56 (s, 2H), 4.15 (s, 3H), 4.08 – 4.04 (m, 1H), 3.98 – 3.94 (m, 1H), 3.46 – 3.42 (m, 1H), 2.80 – 2.70 (m, 1H), 2.20 – 2.10 (m, 1H), 1.30 (s, 9H), 0.64 – 0.58 (m, 2H), 0.56 – 0.50 (m, 2H). 實施例 23B： 1 H NMR (400 MHz, CD 3 OD) δ 6.96 (s, 1H), 6.71 – 6.54 (m, 1H), 5.27 – 5.17 (m, 1H), 5.08 – 5.00 (m, 1H), 4.56 (s, 2H), 4.15 (s, 3H), 4.08 – 4.04 (m, 1H), 3.98 – 3.94 (m, 1H), 3.46 – 3.42 (m, 1H), 2.80 – 2.70 (m, 1H), 2.20 – 2.10 ( m, 1H), 1.30 (s, 9H), 0.64 – 0.58 (m, 2H), 0.56 – 0.50 (m, 2H). Embodiment 23B :
t R=2.35 min t R =2.35 min
LC-MS (ESI), m/z: [M+H] +=446.9 LC-MS (ESI), m/z: [M+H] + =446.9
1H NMR (400 MHz, CD
3OD)
δ6.96 (s, 1H), 6.63 (s, 1H), 5.25 – 5.20 (m, 1H), 5.10 – 5.00 (m, 1H), 4.56 (s, 2H), 4.15 (s, 3H), 4.08 – 4.04 (m, 1H), 3.98 – 3.94 (m, 1H), 3.46 – 3.40 (m, 1H), 2.79 – 2.69 (m, 1H), 2.20 – 2.10 (m, 1H), 1.30 (s, 9H), 0.64 – 0.58 (m, 2H), 0.56 – 0.50 (m, 2H).
實施例 24：順式-5-(3-(4-氟-1-甲基-1
H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
室溫下，向化合物 24-1(2.5 g, 0.020 mol)的 N, N二甲基甲醯胺(30 mL)溶液中加入碳酸鉀(5.5 g, 0.040 mol)和碘甲烷(3.4 g, 0.020 mol)，然後在室溫下反應6小時。反應完畢後加入水(80 mL)淬滅反應。反應混合物用二氯甲烷(20 mL × 3)萃取。合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(石油醚：乙酸乙酯=5：1)得到化合物 24-2(1.80 g, 產率：58%)。LC-MS (ESI), m/z: [M+H] +=141.1 步驟2：4-氟-1-甲基吡唑-5-羧酸甲酯( 24-3) At room temperature, potassium carbonate (5.5 g, 0.040 mol) and methyl iodide (3.4 g, 0.020 mol), and then reacted at room temperature for 6 hours. After the reaction was complete, water (80 mL) was added to quench the reaction. The reaction mixture was extracted with dichloromethane (20 mL×3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 24-2 (1.80 g, yield: 58%). LC-MS (ESI), m/z: [M+H] + =141.1 Step 2: Methyl 4-fluoro-1-methylpyrazole-5-carboxylate ( 24-3 )
室溫下，向化合物 24-2(1.8 g, 0.013 mol)的硝基甲烷(20 mL)溶液中加入碳酸鈉(0.551 g, 5.20 mmol)和1-氯甲基-4-氟-1,4-氮鎓雙環[2.2.2]辛烷雙(四氟硼酸鹽)(5.1 g, 0.014 mol)。反應體系升溫至80℃並攪拌12小時。反應完畢後，加入水(30 mL)淬滅反應。反應混合物用二氯甲烷萃取(20 mL × 3)。合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(石油醚：乙酸乙酯=5：1)得到化合物 24-3(0.30 g, 產率：15%)。LC-MS (ESI), m/z: [M+H] +=159.1 步驟3：4-氟-1-甲基-1H-吡唑-5-羧酸( 24-4) At room temperature, sodium carbonate ( 0.551 g, 5.20 mmol) and 1-chloromethyl-4-fluoro-1,4 - Azoniabicyclo[2.2.2]octanebis(tetrafluoroborate) (5.1 g, 0.014 mol). The reaction system was warmed up to 80°C and stirred for 12 hours. After the reaction was complete, water (30 mL) was added to quench the reaction. The reaction mixture was extracted with dichloromethane (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 24-3 (0.30 g, yield: 15%). LC-MS (ESI), m/z: [M+H] + =159.1 Step 3: 4-fluoro-1-methyl-1H-pyrazole-5-carboxylic acid ( 24-4 )
室溫下，將化合物 24-3(0.460 g, 2.91 mmol)和一水合氫氧化鋰 (0.244 g, 5.82 mmol)溶於四氫呋喃和水的混合液(10：1, 15 mL)中。反應液室溫攪拌過夜。反應完全後，加入水50 mL，隨後用稀鹽酸(1 mol/L)調整pH至3-4，混合物用乙酸乙酯萃取(50 mL × 2)。合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(二氯甲烷：甲醇=20：1)得到化合物 24-4(0.230 g, 產率：49%)。LC-MS (ESI), m/z: [M+H] +=145.1 步驟4：順式- N-(1-叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-4-氟-1-甲基-1H-吡唑-5-甲醯胺( 24-5) Compound 24-3 (0.460 g, 2.91 mmol) and lithium hydroxide monohydrate (0.244 g, 5.82 mmol) were dissolved in a mixture of tetrahydrofuran and water (10:1, 15 mL) at room temperature. The reaction solution was stirred overnight at room temperature. After the reaction was complete, 50 mL of water was added, then the pH was adjusted to 3-4 with dilute hydrochloric acid (1 mol/L), and the mixture was extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound 24-4 (0.230 g, yield: 49%). LC-MS (ESI), m/z: [M+H] + =145.1 Step 4: cis- N- (1-tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H- Pyrazol-3-yl)-4-fluoro-1-methyl-1H-pyrazole-5-carboxamide ( 24-5 )
氮氣保護下，向化合物 24-4(96 mg, 0.66 mmol)的 N,N-二甲基甲醯胺(8 mL)溶液中加入HATU (0.38 g, 0.99 mmol)和 N,N-二異丙基乙基胺(0.172 g, 1.33 mmol)。將反應混合物攪拌0.5小時後向體系中加入 中間體 B(0.15 g, 0.66 mmol)。將反應體系繼續攪拌2小時。反應結束後，向反應體系中加水(30 mL)稀釋，反應混合物用二氯甲烷萃取(20 mL × 3)。合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘留物透過矽膠管柱層析純化(二氯甲烷：甲醇=20：1)得到產物化合物 24-5(54 mg, 產率：23%)。LC-MS (ESI), m/z: [M+H] +=517.2 步驟5：順式-5-(1-叔丁基)-3-(4-氟-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 24-6) Under nitrogen protection, HATU ( 0.38 g, 0.99 mmol) and N,N - diisopropyl ethylethylamine (0.172 g, 1.33 mmol). The reaction mixture was stirred for 0.5 hours and Intermediate B (0.15 g, 0.66 mmol) was added to the system. The reaction was stirred for an additional 2 hours. After the reaction was completed, water (30 mL) was added to the reaction system for dilution, and the reaction mixture was extracted with dichloromethane (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain the product compound 24-5 (54 mg, yield: 23%). LC-MS (ESI), m/z: [M+H] + =517.2 Step 5: cis-5-(1-tert-butyl)-3-(4-fluoro-1-methyl-1H-pyridine Azole-5-carboxamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 24-6 )
氮氣保護下，將化合物 24-5(40 mg, 0.11 mmol)、吡啶(27 mg, 0.34 mmol)、氯甲酸對硝基苯酯(28 mg, 0.13 mmol)和4-二甲胺基吡啶(2.0 mg, 0.016 mmol)溶於二氯甲烷(5 mL)中，室溫下攪拌5小時。反應完畢後，加入水(10 mL)淬滅反應，反應混合物用二氯甲烷萃取(10 mL × 3)。合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(二氯甲烷：甲醇=20：1)得到產物化合物 24-6(46 mg, 產率：78%)。LC-MS (ESI), m/z: [M+H] +=517.2 步驟6：順式-5-(4-氟-1-甲基吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 24-7) Under nitrogen protection, compound 24-5 (40 mg, 0.11 mmol), pyridine (27 mg, 0.34 mmol), p-nitrophenyl chloroformate (28 mg, 0.13 mmol) and 4-dimethylaminopyridine (2.0 mg, 0.016 mmol) was dissolved in dichloromethane (5 mL), and stirred at room temperature for 5 hours. After the reaction was complete, water (10 mL) was added to quench the reaction, and the reaction mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain the product compound 24-6 (46 mg, yield: 78%). LC-MS (ESI), m/z: [M+H] + =517.2 Step 6: cis-5-(4-fluoro-1-methylpyrazole-5-carboxamido)-1H-pyridine Azol-5-yl) tetrahydrofuran-3-yl (4-nitrophenyl) carbonate ( 24-7 )
室溫下，將化合物 24-6(46 mg, 0.089 mmol)溶於甲酸(3 mL)中，室溫攪拌2小時。反應混合物減壓濃縮得到粗產物 24-7(50 mg)，直接用於下一步反應。LC-MS (ESI), m/z: [M+H] +=461.1. 步驟7：順式-5-(3-(4-氟-1-甲基-1 H-吡唑-5-甲醯胺基)-1 H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 24) Compound 24-6 (46 mg, 0.089 mmol) was dissolved in formic acid (3 mL) at room temperature and stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product 24-7 (50 mg), which was directly used in the next reaction. LC-MS (ESI), m/z: [M+H] + =461.1. Step 7: cis-5-(3-(4-fluoro-1-methyl-1 H -pyrazole-5-carba Amino) -1H -pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 24 )
室溫下，向化合物 24-7(50 mg, 0.11 mmol)的四氫呋喃溶液(5 mL)中加入叔丁胺(40 mg, 0.54 mmol)和 N, N-二異丙基乙胺(28 mg, 0.21 mmol)，攪拌5小時。反應完全後，向反應體系中加入水(10 mL)淬滅反應，反應混合物用二氯甲烷萃取(5 mL × 3)。合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得粗產品透過矽膠管柱層析純化(二氯甲烷：甲醇=20：1)得到 實施例 24(7.0 mg)。LC-MS (ESI), m/z: [M+H] +=395.1. 實施例 24A和 實施例 24B： To compound 24-7 (50 mg, 0.11 mmol) in tetrahydrofuran (5 mL) was added tert-butylamine (40 mg, 0.54 mmol) and N , N -diisopropylethylamine (28 mg, 0.21 mmol) at room temperature ), stirred for 5 hours. After the reaction was complete, water (10 mL) was added to the reaction system to quench the reaction, and the reaction mixture was extracted with dichloromethane (5 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain Example 24 (7.0 mg). LC-MS (ESI), m/z: [M+H] + =395.1. Example 24A and Example 24B :
(3
R,5
R)-5-(3-(4-氟-1-甲基-1
H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3
S,5
S)-5-(3-(4-氟-1-甲基-1
H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
實施例24透過手性SFC (管柱：CHIRALPAK AD-H, 250 mm x 20 mm，5 μm；流動相：40%乙醇(0.2%氨水)；流速：40 g/min)進一步純化得到 實施例 24A(3.3 mg, 產率：9%)和 實施例 24B(3.3 mg, 產率：9%)。 實施例 24A： Example 24 was further purified by chiral SFC (column: CHIRALPAK AD-H, 250 mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2% ammonia); flow rate: 40 g/min) to obtain Example 24A (3.3 mg, yield: 9%) and Example 24B (3.3 mg, yield: 9%). Example 24A :
tR=2.76 mintR=2.76min
LC-MS (ESI), m/z: [M+H] +=395.1 LC-MS (ESI), m/z: [M+H] + =395.1
1H NMR (400 MHz, CD 3OD) δ7.50 – 7.43 (m, 1H), 6.58 – 6.49 (m, 1H), 5.21 (s, 1H), 5.06 – 4.96 (m, 1H), 4.08 (s, 3H), 4.05 (s, 1H), 3.98 – 3.92 (m, 1H), 2.75 – 2.65 (m, 1H), 2.20-2.10 (m, 1H), 1.30 (s, 9H). 實施例 24B： 1 H NMR (400 MHz, CD 3 OD) δ 7.50 – 7.43 (m, 1H), 6.58 – 6.49 (m, 1H), 5.21 (s, 1H), 5.06 – 4.96 (m, 1H), 4.08 (s, 3H), 4.05 (s, 1H), 3.98 – 3.92 (m, 1H), 2.75 – 2.65 (m, 1H), 2.20-2.10 (m, 1H), 1.30 (s, 9H). Embodiment 24B :
t R=6.23 min t R =6.23 min
LC-MS (ESI), m/z: [M+H] +=395.1 LC-MS (ESI), m/z: [M+H] + =395.1
1H NMR (400 MHz, CD
3OD)
δ7.50 – 7.43 (m, 1H), 6.58 – 6.49 (m, 1H), 5.21 (s, 1H), 5.06 – 4.96 (m, 1H), 4.08 (s, 3H), 4.05 (s, 1H), 3.98 – 3.92 (m, 1H), 2.75 – 2.65 (m, 1H), 2.20-2.10 (m, 1H), 1.30 (s, 9H).
實施例 25 ：順式-5-(3-(3-氟-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
室溫下，向化合物 25-1(1.00 g, 6.30 mmol)的 N,N-二甲基甲醯胺(15 mL)溶液中加入碳酸鉀(1.74 g, 12.6 mmol)和碘甲烷(1.34 g, 9.50 mmol)，將反應混合物攪拌2小時。反應結束後加水(150 mL)稀釋，並將混合物用乙酸乙酯萃取(100 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=10：1)純化得到化合物 25-2(0.65 g, 產率：60%)。 At room temperature , potassium carbonate (1.74 g, 12.6 mmol) and methyl iodide (1.34 g, 9.50 mmol), the reaction mixture was stirred for 2 hours. After the reaction was completed, it was diluted with water (150 mL), and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain compound 25-2 (0.65 g, yield: 60%).
1H NMR (400 MHz, CDCl 3) δ6.29 (d, J=6.0 Hz, 1H), 4.32 (q, J= 7.2 Hz, 2H), 4.04 (s, 3H), 1.35 (t, J= 7.2 Hz, 3H). 步驟2：3-氟-1-甲基-1H-吡唑-5-羧酸( 25-3) 1 H NMR (400 MHz, CDCl 3 ) δ 6.29 (d, J = 6.0 Hz, 1H), 4.32 (q, J = 7.2 Hz, 2H), 4.04 (s, 3H), 1.35 (t, J = 7.2 Hz , 3H). Step 2: 3-fluoro-1-methyl-1H-pyrazole-5-carboxylic acid ( 25-3 )
室溫下，向化合物 25-2(0.65 g, 38 mmol)的四氫呋喃(5 mL)和水(5 mL)混合溶液中，加入一水合氫氧化鋰(0.48 g, 11 mmol)，並將反應混合物攪拌1小時。反應結束後向體系中加入稀鹽酸(1 mol/L)調整pH至5，然後混合物用乙酸乙酯萃取(20 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物為粗產物化合物 25-3(0.48 g)。 At room temperature, to a mixed solution of compound 25-2 (0.65 g, 38 mmol) in tetrahydrofuran (5 mL) and water (5 mL), was added lithium hydroxide monohydrate (0.48 g, 11 mmol), and the reaction mixture Stir for 1 hour. After the reaction, dilute hydrochloric acid (1 mol/L) was added to the system to adjust the pH to 5, then the mixture was extracted with ethyl acetate (20 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered and concentrated under reduced pressure. The obtained residue was crude compound 25-3 (0.48 g).
1H NMR (400 MHz, DMSO- d 6) δ13.7 (brs, 1H), 6.50 (d, J= 6.0 Hz, 1H), 3.98 (s, 3H). 步驟3：順式- N-(1-(叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-3-氟-1-甲基-1H-吡唑-5-甲醯胺( 25-4) 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.7 (brs, 1H), 6.50 (d, J = 6.0 Hz, 1H), 3.98 (s, 3H). Step 3: cis- N -(1- (tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H-pyrazol-3-yl)-3-fluoro-1-methyl-1H-pyrazole-5-carboxamide ( 25 -4 )
室溫下，向化合物 25-3(0.12 g, 0.83 mmol)的 N,N-二甲基甲醯胺(3 mL)溶液中加入HATU(0.380 g, 1.00 mmol)，並將反應混合物攪拌0.5小時。然後向體系中加入 N, N-二異丙基乙基胺(0.323 g, 2.50 mmol)和 中間體 B(0.19 g, 0.83 mmol)。加料完畢後將反應體系升溫至50℃並攪拌2小時。反應結束後，向反應中加水(20 mL)稀釋，並將混合物用乙酸乙酯萃取(15 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到產物化合物 25-4(0.15 g, 產率：51%)。LC-MS (ESI), m/z: [M+H] +=352.2 步驟4：順式-5-(1-(叔丁基)-3-(3-氟-1-甲基-1H-吡唑-5-甲醯胺)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 25-5) To a solution of compound 25-3 (0.12 g, 0.83 mmol) in N,N -dimethylformamide (3 mL) was added HATU (0.380 g, 1.00 mmol) at room temperature, and the reaction mixture was stirred for 0.5 h . Then N , N -diisopropylethylamine (0.323 g, 2.50 mmol) and intermediate B (0.19 g, 0.83 mmol) were added to the system. After the addition was complete, the reaction system was heated to 50° C. and stirred for 2 hours. After the reaction was completed, water (20 mL) was added to the reaction to dilute, and the mixture was extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the product compound 25-4 (0.15 g, yield: 51%). LC-MS (ESI), m/z: [M+H] + =352.2 Step 4: cis-5-(1-(tert-butyl)-3-(3-fluoro-1-methyl-1H- Pyrazole-5-carboxamide)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 25-5 )
室溫下，向化合物 25-4(0.15 g, 0.43 mmol)的二氯甲烷(3 mL)和四氫呋喃(3 mL)混合溶液中，加入4-硝基苯氯甲酸酯(0.17 g, 0.85 mmol)、吡啶(0.101 g, 1.28 mmol)和4-二甲胺基吡啶(5.0 mg, 0.041 mmol)，並將反應混合物繼續攪拌16小時。反應結束後，向反應體系中加水(20 mL)稀釋，並將混合物用乙酸乙酯萃取(15 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到化合物 25-5(0.120 g, 產率：54%)。LC-MS (ESI), m/z: [M+H] +=516.8 步驟5：順式-5-(3-(3-氟-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 25-6) At room temperature, to a mixed solution of compound 25-4 (0.15 g, 0.43 mmol) in dichloromethane (3 mL) and tetrahydrofuran (3 mL), was added 4-nitrobenzene chloroformate (0.17 g, 0.85 mmol ), pyridine (0.101 g, 1.28 mmol) and 4-dimethylaminopyridine (5.0 mg, 0.041 mmol), and the reaction mixture was stirred for an additional 16 hours. After the reaction, the reaction system was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure . The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 25-5 (0.120 g, yield: 54%). LC-MS (ESI), m/z: [M+H] + =516.8 Step 5: cis-5-(3-(3-fluoro-1-methyl-1H-pyrazole-5-carboxamide Base) -1H-pyrazol-5-yl) tetrahydrofuran -3-yl (4-nitrophenyl) carbonate ( 25-6 )
室溫下，將化合物 25-5(0.12 g, 0.23 mmol)溶於甲酸(4 mL)中，隨後將反應體系升溫至90℃，並繼續攪拌1小時。反應結束後，將反應混合物冷卻至室溫，然後減壓濃縮，所得殘餘物為粗產物化合物 25-6(0.100 g)，直接用於下一步。LC-MS (ESI), m/z: [M+H] +=460.7 步驟6：順式-5-(3-(3-氟-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 25) Compound 25-5 (0.12 g, 0.23 mmol) was dissolved in formic acid (4 mL) at room temperature, and then the reaction system was heated to 90° C. and stirred for 1 hour. After the reaction, the reaction mixture was cooled to room temperature, then concentrated under reduced pressure, and the obtained residue was the crude product compound 25-6 (0.100 g), which was directly used in the next step. LC-MS (ESI), m/z: [M+H] + =460.7 Step 6: cis-5-(3-(3-fluoro-1-methyl-1H-pyrazole-5-carboxamide Base)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 25 )
室溫下，向化合物 25-6(0.10 g, 0.22 mmol)的四氫呋喃溶液(3 mL)中，加入叔丁胺(0.158 g, 2.17 mmol)，並將反應混合物攪拌16小時。反應結束後，將反應混合物減壓濃縮，所得殘餘物透過製備HPLC純化(管柱：Gemini-C18, 150 × 21.2 mm，5 µm；流動相：乙腈/水(0.1%甲酸)；梯度：30%-34%)，得到 實施例 25(50.0 mg)。LC-MS (ESI), m/z: [M+H] +=394.9 實施例 25A和 實施例 25B： To a solution of compound 25-6 (0.10 g, 0.22 mmol) in tetrahydrofuran (3 mL) was added tert-butylamine (0.158 g, 2.17 mmol) at room temperature, and the reaction mixture was stirred for 16 hr. After the reaction, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (column: Gemini-C18, 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% formic acid); gradient: 30% -34%), yielding Example 25 (50.0 mg). LC-MS (ESI), m/z: [M+H] + =394.9 Example 25A and Example 25B :
(3
R,5
R)-5-(3-(3-氟)-1-甲基-1
H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3
S,5
S)-5-(3-(3-氟-1-甲基-1
H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
實施例25透過手性SFC分離(管柱：CHIRALPAK AD-H, 250 mm x 20 mm，5 μm；流動相：40%乙醇(0.2%氨水)；流速：40 mL/min)進一步純化得到 實施例 25A(19.4 mg, 產率：23%)和 實施例 25B(19.3 mg, 產率：23%)。 實施例 25A： Example 25 was further purified by chiral SFC separation (column: CHIRALPAK AD-H, 250 mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2% ammonia water); flow rate: 40 mL/min) to obtain Example 25A (19.4 mg, yield: 23%) and Example 25B (19.3 mg, yield: 23%). Example 25A :
t R=2.88 min t R =2.88 min
LC-MS (ESI), m/z: [M+H] +=394.9 LC-MS (ESI), m/z: [M+H] + =394.9
1H NMR (400 MHz, CD 3OD) δ6.62 (brs, 1H), 6.53 (d, J=5.8 Hz, 1H), 5.25 – 5.20 (m, 1H), 5.10 – 5.00 (m, 1H), 4.06 (s, 3H), 4.05 – 4.03 (m, 1H), 3.98 – 3.93 (m, 1H), 2.80 – 2.70 (m, 1H), 2.20 – 2.10 (m, 1H), 1.30 (s, 9H). 實施例 25B： 1 H NMR (400 MHz, CD 3 OD) δ 6.62 (brs, 1H), 6.53 (d, J =5.8 Hz, 1H), 5.25 – 5.20 (m, 1H), 5.10 – 5.00 (m, 1H), 4.06 (s, 3H), 4.05 – 4.03 (m, 1H), 3.98 – 3.93 (m, 1H), 2.80 – 2.70 (m, 1H), 2.20 – 2.10 (m, 1H), 1.30 (s, 9H). Implementation Example 25B :
t R=7.10 min t R =7.10 min
LC-MS (ESI), m/z: [M+H] +=394.9 LC-MS (ESI), m/z: [M+H] + =394.9
1H NMR (400 MHz, CD
3OD)
δ6.62 (brs, 1H), 6.53 (d,
J=5.8 Hz, 1H), 5.25 – 5.20 (m, 1H), 5.10 – 5.00 (m, 1H), 4.06 (s, 3H), 4.05 – 4.03 (s, 1H), 3.98 – 3.93 (m, 1H), 2.80 – 2.70 (m, 1H), 2.20 – 2.10 (m, 1H), 1.30 (s, 9H).
實施例 26 ：順式-5-(3-(1-環丙基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
氮氣保護下，向化合物 26-1(1.00 g, 7.10 mmol)的1,2-二氯乙烷(15 mL)溶液中加入環丙基硼酸(1.22 g, 14.2 mmol)、2, 2-聯吡啶(1.29 g, 7.10 mmol)、醋酸銅(1.29 g, 7.10 mmol)和碳酸鈉(1.51 g, 14.20 mmol)。隨後將反應混合物升溫至70℃並攪拌16小時。反應結束後，向反應體系加水(100 mL)稀釋，反應液過濾，濾液用乙酸乙酯(100 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=10：1)分離純化得到化合物 26-2(0.28 g, 產率：22%)。LC-MS (ESI), m/z: [M+H] +=181.0 步驟2：1-環丙基-1H-吡唑-5-羧酸( 26-3) Under nitrogen protection, cyclopropylboronic acid (1.22 g, 14.2 mmol), 2, 2-bipyridyl (1.29 g, 7.10 mmol), copper acetate (1.29 g, 7.10 mmol) and sodium carbonate (1.51 g, 14.20 mmol). The reaction mixture was then warmed to 70 °C and stirred for 16 hours. After the reaction, add water (100 mL) to the reaction system to dilute, filter the reaction solution, extract the filtrate with ethyl acetate (100 mL × 3), wash the combined organic phase with saturated brine, dry over anhydrous sodium sulfate, filter and reduce pressure concentrate. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain compound 26-2 (0.28 g, yield: 22%). LC-MS (ESI), m/z: [M+H] + =181.0 Step 2: 1-Cyclopropyl-1H-pyrazole-5-carboxylic acid ( 26-3 )
室溫下，向化合物 26-2(0.20 g, 1.1 mmol)的四氫呋喃(5 mL)和水(5 mL)混合溶液中，加入一水合氫氧化鋰(0.14 g, 3.3 mmol)，將反應混合物攪拌1小時。反應結束後，向體系中加入稀鹽酸(1 mol/L)調整pH至5，然後將混合物用乙酸乙酯萃取(10 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘留物為粗產物化合物 26-3(0.13 g)。LC-MS (ESI), m/z: [M+H] +=153.1 步驟3：順式- N-(1-(叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-1-環丙基-1H-吡唑-5-甲醯胺( 26-4) At room temperature, to a mixed solution of compound 26-2 (0.20 g, 1.1 mmol) in tetrahydrofuran (5 mL) and water (5 mL), was added lithium hydroxide monohydrate (0.14 g, 3.3 mmol), and the reaction mixture was stirred 1 hour. After the reaction was completed, dilute hydrochloric acid (1 mol/L) was added to the system to adjust the pH to 5, then the mixture was extracted with ethyl acetate (10 mL × 3), the combined organic phases were washed with saturated brine, and dried over anhydrous sodium sulfate. , filtered and concentrated under reduced pressure. The obtained residue was crude compound 26-3 (0.13 g). LC-MS (ESI), m/z: [M+H] + =153.1 Step 3: cis- N- (1-(tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H -pyrazol-3-yl)-1-cyclopropyl-1H-pyrazole-5-carboxamide ( 26-4 )
室溫下，向化合物 26-3(0.12 g, 0.79 mmol)的 N, N-二甲基甲醯胺(3 mL)溶液中加入HATU (0.36 g, 0.95 mmol)，並將反應混合物繼續攪拌0.5小時。然後向體系中加入 N, N-二異丙基乙基胺(0.306 g, 2.37 mmol)和 中間體 B(0.18 g, 0.79 mmol)。加料完畢後將反應體系升溫至50℃並攪拌2小時。反應結束後加水(20 mL)稀釋，並將混合物用乙酸乙酯萃取(15 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=10：1)分離純化得到產物化合物 26-4(0.15 g, 產率：53%)。LC-MS (ESI), m/z: [M+H] +=360.2 步驟4：順式-5-(1-(叔丁基)-3-(1-環丙基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 26-5) At room temperature, HATU (0.36 g, 0.95 mmol) was added to a solution of compound 26-3 (0.12 g, 0.79 mmol) in N , N -dimethylformamide (3 mL), and the reaction mixture was stirred continuously for 0.5 Hour. Then N , N -diisopropylethylamine (0.306 g, 2.37 mmol) and Intermediate B (0.18 g, 0.79 mmol) were added to the system. After the addition was complete, the reaction system was heated to 50° C. and stirred for 2 hours. After the reaction was completed, it was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain the product compound 26-4 (0.15 g, yield: 53%). LC-MS (ESI), m/z: [M+H] + =360.2 Step 4: cis-5-(1-(tert-butyl)-3-(1-cyclopropyl-1H-pyrazole- 5-formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 26-5 )
室溫下，向化合物 26-4(0.15 g, 0.42 mmol)的二氯甲烷(3 mL)和四氫呋喃(3 mL)混合溶液中加入4-硝基苯氯甲酸酯(0.17 g, 0.83 mmol)、吡啶(0.099 g, 1.2 mmol)和4-二甲胺基吡啶(5.0 mg, 0.041 mmol)，將反應混合物繼續攪拌16小時。反應結束後，向反應體系中加水(20 mL)稀釋，並將混合物用乙酸乙酯萃取(15 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到產物化合物 26-5(0.160 g, 產率：73%)。LC-MS (ESI), m/z: [M+H] +=525.1 步驟5：順式-5-(3-(1-環丙基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 26-6) To a mixed solution of compound 26-4 (0.15 g, 0.42 mmol) in dichloromethane (3 mL) and tetrahydrofuran (3 mL) was added 4-nitrobenzene chloroformate (0.17 g, 0.83 mmol) at room temperature , pyridine (0.099 g, 1.2 mmol) and 4-dimethylaminopyridine (5.0 mg, 0.041 mmol), the reaction mixture was stirred for an additional 16 hours. After the reaction, the reaction system was diluted with water (20 mL), and the mixture was extracted with ethyl acetate (15 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure . The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the product compound 26-5 (0.160 g, yield: 73%). LC-MS (ESI), m/z: [M+H] + =525.1 Step 5: cis-5-(3-(1-cyclopropyl-1H-pyrazole-5-carboxamido)- 1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 26-6 )
室溫下，將化合物 26-5(0.16 g, 0.31 mmol)溶於甲酸(4 mL)中，隨後將反應體系升溫至85℃，繼續攪拌1小時。反應結束後將反應體系冷卻至室溫，然後將反應混合物減壓濃縮，所得殘餘物為粗產物化合物 26-6(0.120 g)，直接用於下一步。LC-MS (ESI), m/z: [M+H] +=469.1 步驟6：順式-5-(3-(1-環丙基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 26) Compound 26-5 (0.16 g, 0.31 mmol) was dissolved in formic acid (4 mL) at room temperature, then the reaction system was warmed up to 85°C and stirred for 1 hour. After the reaction, the reaction system was cooled to room temperature, and then the reaction mixture was concentrated under reduced pressure, and the obtained residue was the crude product Compound 26-6 (0.120 g), which was directly used in the next step. LC-MS (ESI), m/z: [M+H] + =469.1 Step 6: cis-5-(3-(1-cyclopropyl-1H-pyrazole-5-carboxamido)- 1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butyl carbamate ( Example 26 )
室溫下，向化合物
26-6(0.10 g, 0.21 mmol)的四氫呋喃溶液(3 mL)中加入叔丁胺(0.156 g, 2.14 mmol)，攪拌16小時。反應結束後將，反應混合物減壓濃縮，所得殘餘物透過製備液相層析法分離純化(層析管柱型號：Gemini-C18, 150 x 21.2 mm，5 µm，流動相：乙腈/水(0.1%甲酸)，梯度：29%-31%)，得到
實施例 26(50.0 mg)。LC-MS (ESI), m/z: [M+H]
+=402.9.
實施例 26A和
實施例 26B：
實施例26透過SFC手性分離(層析管柱型號：CHIRALPAK AD-H, 250 mm x 20 mm，5μm; 流動相：40%異丙醇(0.2%氨水)；流速：40 g/min)進一步純化得到 實施例 26A(21.8 mg, 產率：25%)和 實施例 26B(20.4 mg, 產率：24%)。 實施例 26A： Example 26 was further separated by SFC chiral separation (chromatographic column model: CHIRALPAK AD-H, 250 mm x 20 mm, 5 μm; mobile phase: 40% isopropanol (0.2% ammonia water); flow rate: 40 g/min) Purification afforded Example 26A (21.8 mg, yield: 25%) and Example 26B (20.4 mg, yield: 24%). Example 26A :
t R=2.51 min t R =2.51 min
LC-MS (ESI), m/z: [M+H] +=402.9 LC-MS (ESI), m/z: [M+H] + =402.9
1H NMR (400 MHz, CD 3OD) δ7.42 (d, J=2.1 Hz, 1H), 6.83 (d, J=1.7 Hz, 1H), 6.61 (brs, 1H), 5.25 – 5.12 (m, 1H), 5.05 – 4.95 (m, 1H), 4.31 – 4.19 (m, 1H), 4.04 – 3.99 (m, 1H), 3.94 – 3.89 (m, 1H), 2.75 – 2.65 (m, 1H), 2.17 – 2.05 (m, 1H), 1.25 (s, 9H), 1.15 – 1.10 (m, 2H), 1.04 – 0.98 (m, 2H). 實施例 26B： 1 H NMR (400 MHz, CD 3 OD) δ 7.42 (d, J =2.1 Hz, 1H), 6.83 (d, J =1.7 Hz, 1H), 6.61 (brs, 1H), 5.25 – 5.12 (m, 1H ), 5.05 – 4.95 (m, 1H), 4.31 – 4.19 (m, 1H), 4.04 – 3.99 (m, 1H), 3.94 – 3.89 (m, 1H), 2.75 – 2.65 (m, 1H), 2.17 – 2.05 (m, 1H), 1.25 (s, 9H), 1.15 – 1.10 (m, 2H), 1.04 – 0.98 (m, 2H). Embodiment 26B :
t R=5.34 min t R =5.34 min
LC-MS (ESI), m/z: [M+H] +=402.9 LC-MS (ESI), m/z: [M+H] + =402.9
1H NMR (400 MHz, CD
3OD)
δ7.42 (d,
J=2.1 Hz, 1H), 6.83 (d,
J=1.7 Hz, 1H), 6.61 (brs, 1H), 5.25 – 5.20 (m, 1H), 5.05 – 4.95 (m, 1H), 4.31 – 4.19 (m, 1H), 4.04 – 3.99 (m, 1H), 3.94 – 3.89 (m, 1H), 2.75 – 2.65 (m, 1H), 2.15 – 2.05 (m, 1H), 1.25 (s, 9H), 1.15 – 1.10 (m, 2H), 1.04 – 0.98 (m, 2H).
實施例 27：順式-5-(3-(1-(環丙基甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
室溫下，向化合物 27-1(2.0 g, 0.016 mol)的 N, N二甲基甲醯胺溶液(20 mL)溶液中加入碳酸銫(10 g, 0.032 mol)和溴甲基環丙烷(2.58 g, 0.019 mol)，將反應混合物室溫下攪拌6小時。反應完畢後，向反應體系中加入水(80 mL)淬滅反應，反應混合物用二氯甲烷萃取(20 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=5：1)分離純化得到產物化合物 27-2(0.850 g, 產率：29%)。 At room temperature, cesium carbonate (10 g, 0.032 mol) and bromomethylcyclopropane ( 2.58 g, 0.019 mol), the reaction mixture was stirred at room temperature for 6 hours. After the reaction was complete, water (80 mL) was added to the reaction system to quench the reaction, the reaction mixture was extracted with dichloromethane (20 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and reduced Concentrate under pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain the product compound 27-2 (0.850 g, yield: 29%).
1H NMR (400 MHz, CDCl 3) δ7.49 (d, J= 2.0 Hz, 1H), 6.84 (d, J= 2.0 Hz, 1H), 4.44 (d, J= 7.2 Hz, 2H), 3.88 (s, 3H), 1.42 – 1.31 (m, 1H), 0.53 – 0.48 (m, 2H), 0.43 – 0.39 (m, 2H). 步驟2：1-環丙基甲基吡唑-5-羧酸( 27-3) 1 H NMR (400 MHz, CDCl 3 ) δ 7.49 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 4.44 (d, J = 7.2 Hz, 2H), 3.88 (s , 3H), 1.42 – 1.31 (m, 1H), 0.53 – 0.48 (m, 2H), 0.43 – 0.39 (m, 2H). Step 2: 1-cyclopropylmethylpyrazole-5-carboxylic acid ( 27 -3 )
室溫下，向化合物 27-2(0.85 g, 4.7 mmol)的四氫呋喃(20 mL)和水(14 mL)混合溶液中，加入一水合氫氧化鋰(0.594 g, 14.2 mmol)，將反應混合物在室溫攪拌2小時。反應結束後，向體系中加入稀鹽酸(1 mol/L)調整pH至5，然後混合物用二氯甲烷萃取(20 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物為粗產物化合物 27-3(0.58 g)。LC-MS (ESI), m/z: [M+H] +=167.1 步驟3：順式- N-(1-叔丁基)-5-(4-羥基四氫呋喃-2-基)-1H-吡唑-3-基)-1-環丙基甲基-1H-吡唑-5-甲醯胺( 27-4) At room temperature, to a mixed solution of compound 27-2 (0.85 g, 4.7 mmol) in tetrahydrofuran (20 mL) and water (14 mL), was added lithium hydroxide monohydrate (0.594 g, 14.2 mmol), and the reaction mixture was Stir at room temperature for 2 hours. After the reaction was completed, dilute hydrochloric acid (1 mol/L) was added to the system to adjust the pH to 5, then the mixture was extracted with dichloromethane (20 mL × 3), the combined organic phases were washed with saturated brine, and dried over anhydrous sodium sulfate. Filter and concentrate under reduced pressure. The obtained residue was crude compound 27-3 (0.58 g). LC-MS (ESI), m/z: [M+H] + =167.1 Step 3: cis- N- (1-tert-butyl)-5-(4-hydroxytetrahydrofuran-2-yl)-1H- Pyrazol-3-yl)-1-cyclopropylmethyl-1H-pyrazole-5-carboxamide ( 27-4 )
室溫下，向化合物 27-3(0.50 g, 3.1 mmol)的 N,N-二甲基甲醯胺(10 mL)溶液中加入 N, N-二異丙基乙基胺(0.778 g, 6.02 mmol)和HATU(1.70 g, 4.52 mmol)，將反應混合物攪拌0.5小時，然後向體系中加入 中間體 B(0.678 g, 3.09 mmol)。加料完畢後反應繼續攪拌2小時。反應結束後，向反應體系中加水(30 mL)稀釋，並將混合物用二氯甲烷萃取(10 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：9)分離純化得到產物化合物 27-4(0.40 g, 產率：35%)。LC-MS (ESI), m/z: [M+H] +=374.2 步驟4：順式-5-(1-叔丁基)-3-(1-環丙基甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 27-5) To a solution of compound 27-3 (0.50 g, 3.1 mmol) in N,N -dimethylformamide (10 mL) was added N , N -diisopropylethylamine (0.778 g, 6.02 mmol) and HATU (1.70 g, 4.52 mmol), the reaction mixture was stirred for 0.5 hours, and then Intermediate B (0.678 g, 3.09 mmol) was added to the system. The reaction was stirred for an additional 2 hours after the addition was complete. After the reaction, the reaction system was diluted with water (30 mL), and the mixture was extracted with dichloromethane (10 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure . The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:9) to obtain the product compound 27-4 (0.40 g, yield: 35%). LC-MS (ESI), m/z: [M+H] + =374.2 Step 4: cis-5-(1-tert-butyl)-3-(1-cyclopropylmethyl)-1H-pyridine Azole-5-carboxamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 27-5 )
室溫下，向化合物 27-4(0.40 g, 1.07 mmol)的二氯甲烷(10 mL)溶液中加入4-硝基苯氯甲酸酯(0.258 g, 1.29 mmol)、吡啶(0.170 g, 2.14 mmol)和4-二甲胺基吡啶(13.1 mg, 0.11 mmol)，反應混合物攪拌5小時。反應結束後加水(10 mL)稀釋，並將混合物用二氯甲烷萃取(10 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=5：1)分離純化得到產物化合物 27-5(0.33 g, 產率：61%)。LC-MS (ESI), m/z: [M+H] +=538.8 步驟5：順式-5-(3-(1-環丙基甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 27-6) At room temperature, to a solution of compound 27-4 (0.40 g, 1.07 mmol) in dichloromethane (10 mL) was added 4-nitrobenzene chloroformate (0.258 g, 1.29 mmol), pyridine (0.170 g, 2.14 mmol) and 4-dimethylaminopyridine (13.1 mg, 0.11 mmol), the reaction mixture was stirred for 5 hours. After the reaction was completed, it was diluted with water (10 mL), and the mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain the product compound 27-5 (0.33 g, yield: 61%). LC-MS (ESI), m/z: [M+H] + =538.8 Step 5: cis-5-(3-(1-cyclopropylmethyl)-1H-pyrazole-5-carboxamide Base)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 27-6 )
室溫下，將化合物 27-5(0.24 g, 0.45 mmol)溶於甲酸(3 mL)中，升溫至90 ℃，攪拌2小時。反應結束後，將反應體系冷卻至室溫，然後減壓濃縮，所得殘餘物為粗產物化合物 27-6(0.180 g)，直接用於下一步。LC-MS (ESI), m/z: [M+H] +=483.0 步驟6：順式(5-(3-(1-(環丙基甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 27) Compound 27-5 (0.24 g, 0.45 mmol) was dissolved in formic acid (3 mL) at room temperature, heated to 90 °C, and stirred for 2 hours. After the reaction, the reaction system was cooled to room temperature, and then concentrated under reduced pressure. The obtained residue was the crude product Compound 27-6 (0.180 g), which was directly used in the next step. LC-MS (ESI), m/z: [M+H] + =483.0 Step 6: cis(5-(3-(1-(cyclopropylmethyl)-1H-pyrazole-5-formyl Amino)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 27 )
氮氣保護下，向化合物 27-6(0.15 g, 0.31 mmol)的四氫呋喃(5 mL)溶液中加入 N, N-二異丙基乙胺(60 mg, 0.47 mmol)和叔丁胺(0.40 g, 0.78 mmol)。室溫下攪拌5小時。反應結束後，向反應體系中加入水(10 mL)淬滅反應。反應混合物用二氯甲烷萃取(5 mL × 3)，合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾後減壓濃縮。所得殘餘物透過製備HPLC(管柱: Gemini-C18 150 × 21.2 mm，5 µm；流動相: 乙腈/水(0.1%甲酸); 梯度：10-95%)純化得到 實施例 27(80.0 mg)。LC-MS (ESI), m/z: [M+H] +=417.1. 實施例 27A和 實施例 27B： Under nitrogen protection, N , N -diisopropylethylamine (60 mg, 0.47 mmol) and tert-butylamine (0.40 g, 0.78 mmol) were added to a solution of compound 27-6 (0.15 g, 0.31 mmol) in tetrahydrofuran (5 mL). ). Stir at room temperature for 5 hours. After the reaction, water (10 mL) was added to the reaction system to quench the reaction. The reaction mixture was extracted with dichloromethane (5 mL × 3), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% formic acid); gradient: 10-95%) to obtain Example 27 (80.0 mg). LC-MS (ESI), m/z: [M+H] + =417.1. Example 27A and Example 27B :
(3
R,5
R)-5-(3-(1-(環丙基甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3
S,5
S)-5-(3-(1-(環丙基甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
實施例27透過手性SFC (儀器：SFC Thar prep 80；管柱: CHIRALPAK AD-H 250mm x 20 mm, 5 μm；流動相：40%乙醇(0.2%氨水)；流速：40 g/min)分離純化得到 實施例 27A(37.7 mg, 產率：29%)和 實施例 27B(30.5 mg, 產率：24%)。 實施例 27A： Example 27 was separated by chiral SFC (instrument: SFC Thar prep 80; column: CHIRALPAK AD-H 250mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2% ammonia); flow rate: 40 g/min) Purification afforded Example 27A (37.7 mg, yield: 29%) and Example 27B (30.5 mg, yield: 24%). Example 27A :
t R=2.61 min t R =2.61 min
LC-MS (ESI), m/z: [M+H] +=417.1 LC-MS (ESI), m/z: [M+H] + =417.1
1H NMR (400 MHz, DMSO- d 6) δ12.50 (s, 1H), 10.81 (s, 1H), 7.47 (d, J= 1.6 Hz, 1H), 7.12 (s, 1H), 6.89 (s, 1H), 6.55 (s, 1H), 5.16 – 5.06 (m, 1H), 4.87 – 4.76 (m, 1H), 4.35 (d, J= 7.1 Hz, 2H), 3.86 – 3.73 (m, 2H), 2.72 – 2.63 (m, 1H), 1.97 – 1.88 (m, 1H), 1.28 – 1.20 (m,1H), 1.18 (s, 9H), 0.47 – 0.35 (m, 2H), 0.33 – 0.26 (m, 2H). 實施例 27B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.50 (s, 1H), 10.81 (s, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.12 (s, 1H), 6.89 (s, 1H), 6.55 (s, 1H), 5.16 – 5.06 (m, 1H), 4.87 – 4.76 (m, 1H), 4.35 (d, J = 7.1 Hz, 2H), 3.86 – 3.73 (m, 2H), 2.72 – 2.63 (m, 1H), 1.97 – 1.88 (m, 1H), 1.28 – 1.20 (m,1H), 1.18 (s, 9H), 0.47 – 0.35 (m, 2H), 0.33 – 0.26 (m, 2H) . Embodiment 27B :
t R=4.75 min t R =4.75 min
LC-MS (ESI), m/z: [M+H] +=417.2 LC-MS (ESI), m/z: [M+H] + =417.2
1H NMR (400 MHz, DMSO-
d 6)
δ12.50 (s, 1H), 10.81 (s, 1H), 7.47 (d,
J= 1.6 Hz, 1H), 7.12 (s, 1H), 6.89 (s, 1H), 6.55 (s, 1H), 5.16 – 5.06 (m, 1H), 4.87 – 4.76 (m, 1H), 4.35 (d,
J= 7.1 Hz, 2H), 3.86 – 3.73 (m, 2H), 2.72 – 2.63 (m, 1H), 1.97 – 1.88 (m, 1H), 1.28 – 1.20 (m,1H), 1.18 (s, 9H), 0.47 – 0.35 (m, 2H), 0.33 – 0.26 (m, 2H).
實施例 28:順式-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-7-氮雜二環[2.2.1]庚烷-7-甲酸酯
室溫下，將 中間體 D(150 mg, 0.276 mmol)、7-氮雜雙環[2.2.1]庚烷鹽酸鹽(111 mg, 0.830 mmol)和DIEA(214 mg, 1.66 mmol)加入THF (2mL)中。將該混合物於40℃攪拌2小時之後冷卻至室溫。混合物經乙酸乙酯萃取，合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥後，過濾除去固體。將濾液真空濃縮並乾燥，所得粗產物透過矽膠管柱層析法(二氯甲烷：甲醇=15：1)純化得到化合物 28-1(110 mg，產率：80%)。LC-MS (ESI), m/z: [M+H] +=501.3 步驟2：順式-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-7-氮雜二環[2.2.1]庚烷-7-甲酸酯( 實施例 28) Intermediate D (150 mg, 0.276 mmol), 7-azabicyclo[2.2.1]heptane hydrochloride (111 mg, 0.830 mmol) and DIEA (214 mg, 1.66 mmol) were added to THF ( 2mL). The mixture was stirred at 40°C for 2 hours and then cooled to room temperature. The mixture was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove solids. The filtrate was concentrated and dried in vacuo, and the obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol=15:1) to obtain compound 28-1 (110 mg, yield: 80%). LC-MS (ESI), m/z: [M+H] + =501.3 Step 2: cis-5-(3-(3-(methoxymethyl)-1-methyl-1H-pyrazole -5-formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl-7-azabicyclo[2.2.1]heptane-7-carboxylate ( Example 28 )
室溫下，化合物 28-1(110 mg, 0.220 mmol)加入甲酸(2mL)中。將該混合物於75℃攪拌12小時之後冷卻至室溫。將反應混合物真空濃縮並乾燥，所得殘餘物透過製備HPLC (管柱：Gemini 5u C18 150 × 21.2 mm；流動相：乙腈-水(0.1%甲酸)；梯度：40-60%；流速：20 mL/min)得到 實施例 28(45 mg，產率：46%)。 LC-MS (ESI), m/z: [M+H] +=445.1. 實施例 28A和 實施例 28B： Compound 28-1 (110 mg, 0.220 mmol) was added to formic acid (2 mL) at room temperature. The mixture was stirred at 75°C for 12 hours and then cooled to room temperature. The reaction mixture was concentrated in vacuo and dried, and the obtained residue was analyzed by preparative HPLC (column: Gemini 5u C18 150 × 21.2 mm; mobile phase: acetonitrile-water (0.1% formic acid); gradient: 40-60%; flow rate: 20 mL/ min) to obtain Example 28 (45 mg, yield: 46%). LC-MS (ESI), m/z: [M+H] + =445.1. Example 28A and Example 28B :
(3
R,5
R)-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-7-氮雜二環[2.2.1]庚烷-7-甲酸酯和(3
S,5
S)-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-7-氮雜二環[2.2.1]庚烷-7-甲酸酯
實施例28經手性製備SFC (管柱：CHIRALPAK-OJ，流動相：二氧化碳/甲醇(三乙胺)，流速：12.5 mL/min)拆分得到 實施例 28A(19.7 mg, 產率：88%)和 實施例 28B(23.0 mg, 產率：98%) 實施例 28A： Example 28 was resolved by chiral preparative SFC (column: CHIRALPAK-OJ, mobile phase: carbon dioxide/methanol (triethylamine), flow rate: 12.5 mL/min) to obtain Example 28A (19.7 mg, yield: 88%) and Example 28B (23.0 mg, yield: 98%) Example 28A :
t R=2.09 min. t R =2.09 min.
LC-MS (ESI), m/z: [M+H] +=445.1 LC-MS (ESI), m/z: [M+H] + =445.1
1H NMR (400 MHz, DMSO- d 6 ) δ 12.48 (s, 1H), 10.80 (s, 1H), 7.13 (s, 1H), 6.61 (s, 1H), 5.19 (s, 1H), 5.04 – 5.00 (m, 1H), 4.33 (s, 2H), 4.08 (s, 2H), 4.05 (s, 3H), 3.94 – 3.86 (m, 2H), 3.27 (s, 2H), 2.69 – 2.57 (m, 1H), 2.16 – 2.02 (m, 1H), 1.60 – 1.57 (m, 4H), 1.34 (d, J= 6.7 Hz, 4H). 實施例 28B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.48 (s, 1H), 10.80 (s, 1H), 7.13 (s, 1H), 6.61 (s, 1H), 5.19 (s, 1H), 5.04 – 5.00 (m, 1H), 4.33 (s, 2H), 4.08 (s, 2H), 4.05 (s, 3H), 3.94 – 3.86 (m, 2H), 3.27 (s, 2H), 2.69 – 2.57 (m, 1H), 2.16 – 2.02 (m, 1H), 1.60 – 1.57 (m, 4H), 1.34 (d, J = 6.7 Hz, 4H). Example 28B :
t R=2.73 min. t R =2.73 min.
LC-MS (ESI), m/z: [M+H] +=445.1 LC-MS (ESI), m/z: [M+H] + =445.1
1H NMR (400 MHz, DMSO-
d
6 ) δ 12.47 (s, 1H), 10.81 (s, 1H), 7.12 (s, 1H), 6.60 (s, 1H), 5.24 – 5.13 (m, 1H), 5.01 (s, 1H), 4.34 (s, 2H), 4.08 (s, 2H), 4.05 (s, 3H), 3.94 – 3.86 (m, 2H), 3.27 (s, 3H), 2.61 (dd,
J= 14.2, 6.4 Hz, 1H), 2.08 (dd,
J=13.6, 3.4 Hz, 1H), 1.60 – 1.58 (m, 4H), 1.34 (d,
J =6.8 Hz, 4H).
實施例 29：順式-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基二環[1.1.1]戊烷-1-基胺基甲酸酯
室溫下，將 中間體 D(100 mg, 0.184 mmol)和 N,N-二異丙基乙胺(71 mg, 0.55 mmol)加入THF(5 mL)中。將該混合物於室溫下攪拌0.5小時之後，加入雙環[1.1.1]戊-1-胺鹽酸鹽(33 mg, 0.28 mmol)。將該混合物於室溫下攪拌5小時。反應混合物經乙酸乙酯萃取，合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥後，過濾除去固體。將濾液真空濃縮並乾燥，所得粗產物透過製備薄層層析法(二氯甲烷：甲醇=20：1)純化得到化合物 29-1(80 mg，產率：89%). LC-MS (ESI), m/z: [M+H] +=487.2 步驟2：順式-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基二環[1.1.1]戊烷-1-基胺基甲酸酯( 實施例 29) Intermediate D (100 mg, 0.184 mmol) and N,N -diisopropylethylamine (71 mg, 0.55 mmol) were added to THF (5 mL) at room temperature. After the mixture was stirred at room temperature for 0.5 h, bicyclo[1.1.1]pentan-1-amine hydrochloride (33 mg, 0.28 mmol) was added. The mixture was stirred at room temperature for 5 hours. The reaction mixture was extracted with ethyl acetate, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove solids. The filtrate was concentrated in vacuo and dried, and the resulting crude product was purified by preparative thin-layer chromatography (dichloromethane:methanol=20:1) to obtain compound 29-1 (80 mg, yield: 89%). LC-MS (ESI ), m/z: [M+H] + =487.2 Step 2: cis-5-(3-(3-(methoxymethyl)-1-methyl-1H-pyrazole-5-formyl Amino)-1H-pyrazol-5-yl)tetrahydrofuran-3-ylbicyclo[1.1.1]pentan-1-ylcarbamate ( Example 29 )
室溫下，將化合物 29-1(60 mg, 0.087 mmol)加入甲酸(5 mL)中。將該混合物於75℃攪拌2小時之後冷卻至室溫。將反應混合物真空濃縮並乾燥，所得殘餘物透過製備HPLC (管柱：Gemini 5u C18 150 × 21.2 mm；流動相：乙腈-水(0.1%甲酸)；梯度：40-60%；流速：20 mL/min)得到 實施例 29(28 mg，產率：44%). LC-MS (ESI), m/z: [M+H] +=431.2 實施例 29A和 實施例 29B： Compound 29-1 (60 mg, 0.087 mmol) was added to formic acid (5 mL) at room temperature. The mixture was stirred at 75°C for 2 hours and then cooled to room temperature. The reaction mixture was concentrated in vacuo and dried, and the obtained residue was analyzed by preparative HPLC (column: Gemini 5u C18 150 × 21.2 mm; mobile phase: acetonitrile-water (0.1% formic acid); gradient: 40-60%; flow rate: 20 mL/ min) to obtain Example 29 (28 mg, yield: 44%). LC-MS (ESI), m/z: [M+H] + =431.2 Example 29A and Example 29B :
(3
R,5
R)-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基二環[1.1.1]戊烷-1-基胺基甲酸酯和(3
S,5S)-5-(3-(3-(甲氧基甲基)-1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基二環[1.1.1]戊烷-1-基胺基甲酸酯
實施例 29經手性製備HPLC (管柱：CHIRALPAK AD-H 250 mm x 20 mm, 5 μm；流動相：40%異丙醇(0.2%氨水)；流速：40 mL/min)拆分得到 實施例 29A(9.1 mg, 產率：65%)和 實施例 29B(9.9 mg, 產率：71%)。 實施例 29A： Example 29 was resolved by chiral preparative HPLC (column: CHIRALPAK AD-H 250 mm x 20 mm, 5 μm; mobile phase: 40% isopropanol (0.2% ammonia water); flow rate: 40 mL/min) to obtain the example 29A (9.1 mg, yield: 65%) and Example 29B (9.9 mg, yield: 71%). Example 29A :
t R=1.61 min t R =1.61 min
LC-MS (ESI), m/z: [M+H] +=431.2 LC-MS (ESI), m/z: [M+H] + =431.2
1H NMR (400 MHz, DMSO- d 6 ) δ 12.51 (s, 1H), 10.79 (s, 1H), 7.93 (s, 1H), 7.13 (s, 1H), 6.57 (s, 1H), 5.16 (dd, J=6.8, 3.4 Hz, 1H), 4.87 (s, 1H), 4.35 (s, 2H), 4.06 (s, 3H), 3.86 (s, 2H), 3.28 (s, 3H), 2.76 – 2.67 (m, 1H), 2.36 (s, 1H), 1.91 (m, 7H). 實施例 29B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.51 (s, 1H), 10.79 (s, 1H), 7.93 (s, 1H), 7.13 (s, 1H), 6.57 (s, 1H), 5.16 ( dd, J =6.8, 3.4 Hz, 1H), 4.87 (s, 1H), 4.35 (s, 2H), 4.06 (s, 3H), 3.86 (s, 2H), 3.28 (s, 3H), 2.76 – 2.67 (m, 1H), 2.36 (s, 1H), 1.91 (m, 7H). Example 29B :
t R=2.81 min t R =2.81min
LC-MS (ESI), m/z: [M+H] +=431.2 LC-MS (ESI), m/z: [M+H] + =431.2
1H NMR (400 MHz, DMSO-
d
6 ) δ 12.51 (s, 1H), 10.79 (s, 1H), 7.93 (s, 1H), 7.13 (s, 1H), 6.57 (s, 1H), 5.16 (dd,
J=6.8, 3.4 Hz, 1H), 4.87 (s, 1H), 4.35 (s, 2H), 4.06 (s, 3H), 3.86 (s, 2H), 3.28 (s, 3H), 2.76 – 2.67 (m, 1H), 2.36 (s, 1H), 1.91 (m, 7H).
實施例 30：順式-5-(3-(1-(
S)-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(1-(
S)-1-甲基-1-環丙基甲基)胺基甲酸酯
將2-甲基吡唑-3-羧酸(140 mg, 1.11 mmol)和 中間體 C(321 mg, 1.11 mmol)溶於二氯乙烷中，在室溫下向攪拌中的反應混合物中加入三乙胺(337 mg, 3.33 mmol)和1-丙基磷酸酐(706 mg, 2.22 mmol)。將反應混合物在室溫下攪拌5小時。反應結束後，用二氯甲烷萃取，合併的有機相用無水硫酸鈉乾燥。過濾後，減壓濃縮濾液。殘餘物透過矽膠管柱層析純化(二氯甲烷：甲醇=92：8)，得到化合物 30-2(170 mg, 產率：38%)。LC-MS (ESI), m/z: [M+H] +=398.1 步驟2：順式-5-(1-(4-甲氧苄基)-3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(4-硝基苯基)碳酸酯( 30-3) 2-Methylpyrazole-3-carboxylic acid (140 mg, 1.11 mmol) and intermediate C (321 mg, 1.11 mmol) were dissolved in dichloroethane and added to the stirring reaction mixture at room temperature Triethylamine (337 mg, 3.33 mmol) and 1-propylphosphoric anhydride (706 mg, 2.22 mmol). The reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, it was extracted with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=92:8) to obtain compound 30-2 (170 mg, yield: 38%). LC-MS (ESI), m/z: [M+H] + =398.1 Step 2: cis-5-(1-(4-methoxybenzyl)-3-(1-methyl-1H-pyridine Azole-5-carboxamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl-(4-nitrophenyl)carbonate ( 30-3 )
室溫下，向化合物 30-2(160 mg, 0.403 mmol)和4-二甲胺基吡啶(4.0 mg, 0.033 mmol)在二氯甲烷(5 mL)中的懸濁液中加入吡啶(96 mg, 1.2 mmol)和對硝基苯基氯甲酸酯(162 mg, 0.806 mmol)。將反應混合物在室溫下攪拌16小時。反應結束後，將反應液直接旋乾，殘餘物透過矽膠管柱層析純化(石油醚：乙酸乙酯=0~3：7)，得到化合物 30-3(200 mg，產率：88%)。LC-MS (ESI), m/z: [M+H] +=563.0 步驟三：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基 (4-硝基苯基)碳酸酯( 30-4) Add pyridine (96 mg , 1.2 mmol) and p-nitrophenyl chloroformate (162 mg, 0.806 mmol). The reaction mixture was stirred at room temperature for 16 hours. After the reaction, the reaction solution was directly spin-dried, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0~3:7) to obtain compound 30-3 (200 mg, yield: 88%) . LC-MS (ESI), m/z: [M+H] + =563.0 Step 3: cis-5-(3-(1-methyl-1H-pyrazole-5-carboxamido)-1H -pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 30-4 )
將化合物 30-3(200 mg, 0.357 mmol)溶於三氟乙酸(3 mL)中，並將反應混合物在75℃下攪拌16小時。反應混合物冷卻至室溫後，減壓濃縮得到化合物 30-4(120 mg，產率：76%)。LC-MS (ESI), m/z: [M+H] +=443.1 步驟四：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基 (1-( S)-甲基環丁基)胺基甲酸酯( 實施例 30) Compound 30-3 (200 mg, 0.357 mmol) was dissolved in trifluoroacetic acid (3 mL), and the reaction mixture was stirred at 75° C. for 16 hr. After the reaction mixture was cooled to room temperature, it was concentrated under reduced pressure to obtain compound 30-4 (120 mg, yield: 76%). LC-MS (ESI), m/z: [M+H] + =443.1 Step 4: cis-5-(3-(1-methyl-1H-pyrazole-5-carboxamido)-1H -pyrazol-5-yl)tetrahydrofuran-3-yl (1-( S )-methylcyclobutyl)carbamate ( Example 30 )
將(1S)-1-環丙基乙胺(58 mg, 0.68 mmol)溶於四氫呋喃中，加入 N,N-二異丙基乙胺(175 mg, 1.36 mmol)。在室溫下攪拌反應半小時後加入化合物 30-4(60 mg, 0.14 mmol)，繼續攪拌16小時。反應結束後，加入水，用乙酸乙酯萃取，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，有機相減壓濃縮濾液，殘餘物經製備HPLC純化得到 實施例 30(15 mg，29%)。LC-MS (ESI), m/z: [M+H] +=389.1. 實施例 30 A和 實施例 30B： (1S)-1-Cyclopropylethylamine (58 mg, 0.68 mmol) was dissolved in tetrahydrofuran, and N,N -diisopropylethylamine (175 mg, 1.36 mmol) was added. After stirring the reaction at room temperature for half an hour, compound 30-4 (60 mg, 0.14 mmol) was added, and stirring was continued for 16 hours. After the reaction, water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the organic phase was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain Example 30 (15 mg, 29%). LC-MS (ESI), m/z: [M+H] + = 389.1 . Example 30A and Example 30B :
(3
R,5
R)-5-(3-(1-(
S)-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(1-(
S)-1-甲基-1-環丙基甲基)胺基甲酸酯和(3
S,5
S)-5-(3-(1-(
S)-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(1-(
S)-1-甲基-1-環丙基甲基)胺基甲酸酯
實施例 30經手性製備SFC (管柱：CHIRALPAK-AD；流動相：二氧化碳/異丙醇(甲酸)；流速：1.8 mL/min)拆分得到 實施例 30A(5.0 mg, 產率：67%)和 實施例 30B(4.9 mg, 產率：65%)。 實施例 30A： Example 30 was resolved by chiral preparative SFC (column: CHIRALPAK-AD; mobile phase: carbon dioxide/isopropanol (formic acid); flow rate: 1.8 mL/min) to obtain Example 30A (5.0 mg, yield: 67%) and Example 30B (4.9 mg, yield: 65%). Example 30A :
t R=11.61 min. t R =11.61 min.
LC-MS (ESI), m/z: [M+H] +=389.1 LC-MS (ESI), m/z: [M+H] + =389.1
1H NMR (400 MHz, DMSO- d 6 ) δ 12.53 (s, 1H), 10.81 (s, 1H), 7.50 (s, 1H), 7.17 (d, J= 13.8 Hz, 2H), 6.56 (s, 1H), 5.16 (s, 1H), 4.84 (s, 1H), 4.09 (s, 3H), 3.86 (s, 2H), 3.03 – 2.94 (m, 1H), 2.74 – 2.66 (m, 1H), 1.97 (s, 1H), 1.10 (d, J= 6.6 Hz, 3H), 0.86 – 0.79 (m, 1H), 0.41 – 0.29 (m, 2H), 0.28 – 0.21 (m, 1H), 0.14 – 0.06 (m, 1H). 實施例 30B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.53 (s, 1H), 10.81 (s, 1H), 7.50 (s, 1H), 7.17 (d, J = 13.8 Hz, 2H), 6.56 (s, 1H), 5.16 (s, 1H), 4.84 (s, 1H), 4.09 (s, 3H), 3.86 (s, 2H), 3.03 – 2.94 (m, 1H), 2.74 – 2.66 (m, 1H), 1.97 (s, 1H), 1.10 (d, J = 6.6 Hz, 3H), 0.86 – 0.79 (m, 1H), 0.41 – 0.29 (m, 2H), 0.28 – 0.21 (m, 1H), 0.14 – 0.06 (m , 1H). Embodiment 30B :
t R=14.13 min. t R =14.13 min.
LC-MS (ESI), m/z: [M+H] +=389.1 LC-MS (ESI), m/z: [M+H] + =389.1
1H NMR (400 MHz, DMSO-
d
6 ) δ 12.52 (s, 1H), 10.81 (s, 1H), 7.49 (d,
J= 1.8 Hz, 1H), 7.17 (d,
J= 11.3 Hz, 2H), 6.58 (s, 1H), 5.16 (s, 1H), 4.85 (s, 1H), 4.09 (s, 3H), 3.86 (s, 2H), 3.03 – 2.94 (m, 1H), 2.75 – 2.66 (m, 1H), 2.01 – 1.91 (m, 1H), 1.10 (d,
J=6.6 Hz, 3H), 0.87 – 0.77 (m, 1H), 0.41 – 0.28 (m, 2H), 0.28 – 0.21 (m, 1H), 0.14 – 0.06 (m, 1H).
實施例 31：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(1-甲基環丁基)胺基甲酸酯
將1-甲基環丁烷-1-胺(115 mg, 1.36 mmol)溶於四氫呋喃後加入 N,N-二異丙基乙胺(263 mg, 2.03 mmol)。在室溫下攪拌反應半小時後加入化合物 12-4(60 mg, 0.14 mmol)，並將反應混合物在室溫下攪拌16小時。反應結束後，向反應體系和中加入水，用乙酸乙酯萃取，飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，有機相減壓濃縮濾液，殘餘物經得到化合物 實施例 31(10 mg，19%)。LC-MS (ESI), m/z: [M+H] +=389.1. 實施例 31A和 實施例 31B： 1-Methylcyclobutane-1-amine (115 mg, 1.36 mmol) was dissolved in tetrahydrofuran and N,N -diisopropylethylamine (263 mg, 2.03 mmol) was added. After the reaction was stirred at room temperature for half an hour, compound 12-4 (60 mg, 0.14 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, water was added to the reaction system, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the organic phase was concentrated under reduced pressure, and the residue was obtained by compound Example 31 (10 mg, 19 %). LC-MS (ESI), m/z: [M+H] = 389.1. Example 31A and Example 31B :
(3
R,5
R)-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(1-甲基環丁基)胺基甲酸酯和(3
S,5
S)-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基-(1-甲基環丁基)胺基甲酸酯
實施例31經手性製備SFC (管柱：CHIRALPAK-AD；流動相：二氧化碳/異丙醇(二乙胺)；流速：0.8 mL/min)拆分得到 實施例 31A(3.5 mg, 產率：70%)和 實施例 31B(3.5 mg, 產率：70%)。 實施例 31A： Example 31 was resolved by chiral preparative SFC (column: CHIRALPAK-AD; mobile phase: carbon dioxide/isopropanol (diethylamine); flow rate: 0.8 mL/min) to obtain Example 31A (3.5 mg, yield: 70 %) and Example 31B (3.5 mg, yield: 70%). Example 31A :
t R=3.44 min. t R =3.44 min.
LC-MS (ESI), m/z: [M+H] +=389.1 LC-MS (ESI), m/z: [M+H] + =389.1
1H NMR (400 MHz, DMSO- d 6 ) δ 12.52 (s, 1H), 10.81 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 7.16 (s, 1H), 6.59 (s, 1H), 5.16 (s, 1H), 4.86 (s, 1H), 4.09 (s, 3H), 3.85 (s, 2H), 2.74 – 2.65 (m, 1H), 2.27 – 2.18 (m, 2H), 1.95 (s, 1H), 1.85 – 1.77 (m, 2H), 1.75 – 1.67 (m, 2H), 1.32 (s, 3H). 實施例 31B： 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.52 (s, 1H), 10.81 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 7.16 (s, 1H), 6.59 ( s, 1H), 5.16 (s, 1H), 4.86 (s, 1H), 4.09 (s, 3H), 3.85 (s, 2H), 2.74 – 2.65 (m, 1H), 2.27 – 2.18 (m, 2H) , 1.95 (s, 1H), 1.85 – 1.77 (m, 2H), 1.75 – 1.67 (m, 2H), 1.32 (s, 3H). Embodiment 31B :
t R=14.65 min. t R =14.65 min.
LC-MS (ESI), m/z: [M+H] +=389.1 LC-MS (ESI), m/z: [M+H] + =389.1
1H NMR (400 MHz, DMSO-
d
6 ) δ 12.52 (s, 1H), 10.81 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 7.16 (s, 1H), 6.59 (s, 1H), 5.16 (s, 1H), 4.85 (s, 1H), 4.09 (s, 3H), 3.86 (s, 2H), 2.74 – 2.65 (m, 1H), 2.28 – 2.17 (m, 2H), 1.95 (s, 1H), 1.85 – 1.76 (m, 2H), 1.75 – 1.65 (m, 2H), 1.32 (s, 3H) .
實施例 32：順式(3-甲基-1H-吡唑-5-甲醯胺基-3,4-二氘)-1H-吡唑-5-基四氫呋喃-3-基叔丁基胺基甲酸酯
於室溫下，將化合物1H-吡唑-5-甲酸乙酯(1.1 g，7.9 mmol)溶於 N,N-二甲基甲醯胺(10 mL)中。向反應中加入碳酸鉀(2.1 g, 16 mmol)和碘甲烷(1.4 g, 9.4 mmol)並將反應混合物在室溫下攪拌12小時。液質檢測反應完畢後，向反應體系中加水(30 mL)稀釋，並將混合物用乙酸乙酯萃取(10 mL × 3)，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析純化(石油醚：乙酸乙酯=5：1)得到化合物 32-2(560 mg，產率：46%)。LC-MS (ESI) m/z: [M+H] +=155.1 步驟2：3,4-二碘-1-甲基-1H-吡唑-5-羧酸乙酯( 32-3) Compound 1H-pyrazole-5-carboxylic acid ethyl ester (1.1 g, 7.9 mmol) was dissolved in N,N- dimethylformamide (10 mL) at room temperature. Potassium carbonate (2.1 g, 16 mmol) and iodomethane (1.4 g, 9.4 mmol) were added to the reaction and the reaction mixture was stirred at room temperature for 12 hours. After the liquid mass detection reaction was completed, water (30 mL) was added to the reaction system to dilute, and the mixture was extracted with ethyl acetate (10 mL × 3), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and Concentrate under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain compound 32-2 (560 mg, yield: 46%). LC-MS (ESI) m/z: [M+H] + =155.1 Step 2: Ethyl 3,4-diiodo-1-methyl-1H-pyrazole-5-carboxylate ( 32-3 )
於室溫下，將化合物 32-2(500 mg, 3.24 mmol)溶於四氯化碳(8 mL)中。向反應中加入碘(1.65 g, 6.48 mmol)、碘酸(570 mg, 3.24 mmol)、醋酸(19 mg, 0.32 mmol)和硫酸(106 mg, 0.32 mmol)，並將反應混合物在80℃下攪拌2小時。反應完畢後，向反應體系加水(30 mL)稀釋，並將混合物用乙酸乙酯萃取(10 mL × 3)，合併的有機相經飽和食鹽水(10 mL)洗滌，減壓濃縮。所得殘餘物透過矽膠管柱層析法純化(石油醚：乙酸乙酯=1：1)得到化合物 32-3(1.3 g，產率：98%)。LC-MS (ESI) m/z: [M+H] +=406.8 步驟3：1-甲基吡唑-3,4-二氘-5-羧酸( 32-4) Compound 32-2 (500 mg, 3.24 mmol) was dissolved in carbon tetrachloride (8 mL) at room temperature. Iodine (1.65 g, 6.48 mmol), iodic acid (570 mg, 3.24 mmol), acetic acid (19 mg, 0.32 mmol) and sulfuric acid (106 mg, 0.32 mmol) were added to the reaction, and the reaction mixture was stirred at 80 °C 2 hours. After the reaction was complete, the reaction system was diluted with water (30 mL), and the mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL), and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 32-3 (1.3 g, yield: 98%). LC-MS (ESI) m/z: [M+H] + =406.8 Step 3: 1-Methylpyrazole-3,4-dideutero-5-carboxylic acid ( 32-4 )
於室溫下，將化合物 32-3(300 mg, 0.739 mmol)溶於氘代氫氧化鈉(99.5%氘代)的氘水溶液(40%濃度，5 mL)中，加入鋅粉(4.7 mg, 0.073 mmol)，並將混合物在80℃下攪拌1小時。待反應完成後，將反應混合物減壓濃縮，得到粗產物化合物 32-4(80 mg)。 Compound 32-3 (300 mg, 0.739 mmol) was dissolved in deuterium aqueous solution (40% concentration, 5 mL) of deuterated sodium hydroxide (99.5% deuterated) at room temperature, and zinc powder (4.7 mg, 0.073 mmol), and the mixture was stirred at 80°C for 1 hour. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to obtain crude compound 32-4 (80 mg).
1H NMR (400 MHz, CDCl 3) δ 4.21 (s, 3H). 步驟4：順式- N-(5-羥基四氫呋喃-2-基)-1-(4-甲氧基苄基)-1H-吡唑-3-基)-1-甲基-1H-吡唑-3,4-二氘-5-甲醯胺( 32-5) 1 H NMR (400 MHz, CDCl 3 ) δ 4.21 (s, 3H). Step 4: cis- N- (5-hydroxytetrahydrofuran-2-yl)-1-(4-methoxybenzyl)-1H -pyrazol-3-yl)-1-methyl-1H-pyrazole-3,4-dideuterium-5-carboxamide ( 32-5 )
氮氣保護下，在室溫下將化合物 32-4(80 mg, 0.70 mmol) 的 N,N-二甲基甲醯胺溶液(5 mL)中加入 N,N-二異丙基乙胺(181 mg, 1.40 mmol)和2-(7-氮雜苯并三氮唑)- N,N ， N' ， N'-四甲基脲六氟磷酸酯(533 mg, 1.40 mmol)，反應液在室溫下攪拌0.5小時後，將順式-5-(3-胺基-1-(4-甲氧基苄基)-1H-吡唑-5-基)四氫呋喃-3-醇( 中間體 C，203 mg, 0.702 mmol)加入。將該反應混合物在室溫下攪拌2小時。液質檢測反應完畢後，向反應體系和中加水(15 mL)稀釋，並將混合物用二氯甲烷萃取(10 mL × 3)，合併的有機相經飽和食鹽水(10 mL)洗滌，減壓濃縮。所得殘餘物透過矽膠管柱層析純化(石油醚：乙酸乙酯=5：1)得到化合物 32-5(120 mg，產率：43 %)。LC-MS (ESI) m/z: [M+H] +=400.0 步驟5：順式-5-(1-(4-甲氧基苄基)-3-(1-甲基-1H-吡唑-5-甲醯胺基-3,4-二氘)-1H-吡唑-5-基)四氫呋喃-3-基-(4-硝基苯基)碳酸酯( 32-6) Under nitrogen protection, N, N -diisopropylethylamine (181 mg, 1.40 mmol) and 2-(7-azabenzotriazole)-N ,N , N' , N' -tetramethyluronium hexafluorophosphate (533 mg, 1.40 mmol), the reaction solution was in room After stirring at room temperature for 0.5 hours, cis-5-(3-amino-1-(4-methoxybenzyl)-1H-pyrazol-5-yl)tetrahydrofuran-3-ol ( intermediate C , 203 mg, 0.702 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the liquid mass detection reaction was completed, water (15 mL) was added to the reaction system to dilute, and the mixture was extracted with dichloromethane (10 mL × 3), and the combined organic phase was washed with saturated brine (10 mL), and depressurized concentrate. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 32-5 (120 mg, yield: 43%). LC-MS (ESI) m/z: [M+H] + =400.0 Step 5: cis-5-(1-(4-methoxybenzyl)-3-(1-methyl-1H-pyridine Azole-5-carboxamido-3,4-dideuterium)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl-(4-nitrophenyl)carbonate ( 32-6 )
氮氣保護下，在室溫下將化合物 32-5(120 mg, 0.30 mmol)、吡啶(72 mg，0.65 mmol)，氯甲酸對硝基苯酯(74 mg, 0.39 mmol)和4-二甲胺基吡啶(4.0 mg, 0.033 mmol)溶於二氯甲烷(10 mL)中。將反應混合物在室溫攪拌5小時。液質監控反應完畢後，加入水(10 mL)稀釋，並用二氯甲烷萃取(10 mL × 3)，合併的有機相用飽和食鹽水(10 mL)洗滌，減壓濃縮，所得殘留物經過矽膠管柱層析純化(石油醚：乙酸乙酯=2：1)純化得到化合物 32-6(150 mg，產率：89 %)。LC-MS (ESI) m/z: [M+H] +=565.0 步驟6：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基-3,4-二氘)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 32-7) Under nitrogen protection, compound 32-5 (120 mg, 0.30 mmol), pyridine (72 mg, 0.65 mmol), p-nitrophenyl chloroformate (74 mg, 0.39 mmol) and 4-dimethylamine were mixed at room temperature Dipyridine (4.0 mg, 0.033 mmol) was dissolved in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 5 hours. After the liquid mass monitoring reaction was completed, add water (10 mL) to dilute, and extract with dichloromethane (10 mL × 3), the combined organic phase was washed with saturated brine (10 mL), concentrated under reduced pressure, and the obtained residue was filtered through silica gel. Purified by column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound 32-6 (150 mg, yield: 89%). LC-MS (ESI) m/z: [M+H] + =565.0 Step 6: cis-5-(3-(1-methyl-1H-pyrazole-5-carboxamido-3,4 -Dideuterium)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 32-7 )
在室溫下，將化合物 32-6(150 mg, 0.266 mmol)溶於三氟乙酸(3 mL)中，90℃下攪拌2小時，反應完畢後將反應液減壓濃縮得到粗產物化合物 32-7(100 mg)。LC-MS (ESI) m/z: [M+H] +=445.0 步驟7：順式-5-(3-(1-甲基-1H-吡唑-5-甲醯胺基-3,4-二氘)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 32) Compound 32-6 (150 mg, 0.266 mmol) was dissolved in trifluoroacetic acid (3 mL) at room temperature and stirred at 90°C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product compound 32- 7 (100 mg). LC-MS (ESI) m/z: [M+H] + =445.0 Step 7: cis-5-(3-(1-methyl-1H-pyrazole-5-carboxamido-3,4 -Dideuterium)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 32 )
在室溫下，向化合物 32-7(100 mg, 0.225 mmol)的四氫呋喃溶液(5 mL)中，加入 N， N-二異丙基乙胺(60 mg，0.47 mmol)和叔丁胺(82.3 mg, 1.13 mmol)。將反應混合物在室溫下攪拌5小時。待反應完成後，將反應混合物加入水(10 mL)稀釋，並用二氯甲烷萃取(5 mL × 3)，飽和食鹽水(5 mL)洗滌，減壓濃縮。所得殘餘物透過製備HPLC (管柱： Gemini-C18 150 × 21.2 mm，5 µm；流動相：10 – 95%乙腈-水(0.1%甲酸)；流速：20 mL/min) 純化得到 實施例 32(30 mg，產率：34%)。LC-MS (ESI) m/z: [M+H] +=379.1. 實施例 32A和 實施例 32B： To compound 32-7 (100 mg, 0.225 mmol) in tetrahydrofuran (5 mL), add N , N -diisopropylethylamine (60 mg, 0.47 mmol) and tert-butylamine (82.3 mg, 1.13 mmol). The reaction mixture was stirred at room temperature for 5 hours. After the reaction was complete, the reaction mixture was diluted with water (10 mL), extracted with dichloromethane (5 mL × 3), washed with saturated brine (5 mL), and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: 10-95% acetonitrile-water (0.1% formic acid); flow rate: 20 mL/min) to obtain Example 32 ( 30 mg, yield: 34%). LC-MS (ESI) m/z: [M+H] + =379.1. Example 32A and Example 32B :
(3
R,5
R)-(3-甲基-1H-吡唑-5-甲醯胺基-3,4-二氘)-1H-吡唑-5-基四氫呋喃-3-基叔丁基胺基甲酸酯和(3
S,5
S)-(3-甲基-1
H-吡唑-5-甲醯胺基-3,4-二氘)-1H-吡唑-5-基四氫呋喃-3-基叔丁基胺基甲酸酯
實施例32(30 mg, 0.079 mmol)經手性SFC(管柱：CHIRALPAK AD-H 250mm x 20 mm, 5 μm；流動相：40%乙醇(0.2%)氨水；流速：40 g/min)拆分得到 實施例 32A( 9.4 mg，產率：31%)和 實施例 32B( 10 mg，產率：33%)。 實施例 32A: Example 32 (30 mg, 0.079 mmol) was resolved by chiral SFC (column: CHIRALPAK AD-H 250mm x 20 mm, 5 μm; mobile phase: 40% ethanol (0.2%) ammonia; flow rate: 40 g/min) Example 32A (9.4 mg, yield: 31%) and Example 32B (10 mg, yield: 33%) were obtained. Example 32A :
t R=2.7 min t R =2.7 min
LC-MS (ESI), m/z: [M+H] +=379.1 LC-MS (ESI), m/z: [M+H] + =379.1
1H NMR (400 MHz, MeOD) δ 6.52 (s, 1H), 5.10 (s, 1H), 4.93 (s, 1H), 4.06 (s, 3H), 3.94 (d, J=10.2 Hz, 1H), 3.87 – 3.81 (m, 1H), 2.62 (dt, J=14.5, 7.6 Hz, 1H), 2.08 – 1.98 (m, 1H), 1.18 (s, 9H). 實施例 32B: 1 H NMR (400 MHz, MeOD) δ 6.52 (s, 1H), 5.10 (s, 1H), 4.93 (s, 1H), 4.06 (s, 3H), 3.94 (d, J =10.2 Hz, 1H), 3.87 – 3.81 (m, 1H), 2.62 (dt, J =14.5, 7.6 Hz, 1H), 2.08 – 1.98 (m, 1H), 1.18 (s, 9H). Example 32B :
t R=6.1 min t R =6.1 min
LC-MS (ESI), m/z: [M+H] +=379.1 LC-MS (ESI), m/z: [M+H] + =379.1
1H NMR (400 MHz, MeOD) δ 6.64 (s, 1H), 5.22 (s, 1H), 5.05 (s, 1H), 4.18 (s, 3H), 4.06 (d,
J=10.2 Hz, 1H), 3.98 – 3.94 (m, 1H), 2.74 (dt,
J=14.5, 7.6 Hz, 1H), 2.19 – 2.11 (m, 1H), 1.30 (s, 9H).
實施例 33：順式-5-(3-(1-(三氘代甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
在氮氣保護下，室溫下，向1 H-吡唑-5-甲酸乙酯(2.00 g, 14.3 mmol)的 N,N-二甲基甲醯胺(20 mL)溶液中加入氘代碘甲烷(4.15 g, 28.6 mmol)和碳酸鉀(3.95 g, 28.6 mmol)。將反應混合物在室溫下攪拌2小時。待反應完成後，向反應混合物加水(100 mL)稀釋後過濾。濾液用乙酸乙酯(100 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析(石油醚：乙酸乙酯=5：1)分離純化得到化合物 33-2(1.00 g, 產率：45%)。LC-MS (ESI), m/z: [M+H] +=158.1 步驟2：1-(三氘代甲基)-1H-吡唑-5-羧酸( 33-3) Under nitrogen protection, add deuteroiodomethane to a solution of ethyl 1 H -pyrazole-5-carboxylate (2.00 g, 14.3 mmol) in N,N -dimethylformamide (20 mL) at room temperature (4.15 g, 28.6 mmol) and potassium carbonate (3.95 g, 28.6 mmol). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was diluted with water (100 mL) and filtered. The filtrate was extracted with ethyl acetate (100 mL × 3), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 33-2 (1.00 g, yield: 45%). LC-MS (ESI), m/z: [M+H] + =158.1 Step 2: 1-(trideuteromethyl)-1H-pyrazole-5-carboxylic acid ( 33-3 )
在室溫下，向四氫呋喃(5 mL)和水(5 mL)混合溶液中加入化合物 33-2(500 mg, 3.18 mmol)，並將反應混合物是室溫下攪拌1小時。待反應完成後，向體系中加入稀鹽酸(1 mol/L)調整pH=5，並將酸化後的混合物用乙酸乙酯(20 mL × 3)萃取。將合併的有機相用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮後得到化合物 33-3(0.36 g，產率：87%)。LC-MS (ESI) m/z: [M+H] +=130.1 步驟3：順式- N-(5-(4-羥基四氫呋喃-2-基)-1-(4-甲氧基苄基)-1H-吡唑-3-基)-1-(三氘代甲基)-1H-吡唑-5-甲醯胺( 33-4) To a mixed solution of tetrahydrofuran (5 mL) and water (5 mL) was added compound 33-2 (500 mg, 3.18 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 1 hr. After the reaction was complete, dilute hydrochloric acid (1 mol/L) was added to the system to adjust the pH to 5, and the acidified mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 33-3 (0.36 g, yield: 87%). LC-MS (ESI) m/z: [M+H] + =130.1 Step 3: cis- N- (5-(4-hydroxytetrahydrofuran-2-yl)-1-(4-methoxybenzyl )-1H-pyrazol-3-yl)-1-(trideuteromethyl)-1H-pyrazole-5-carboxamide ( 33-4 )
在氮氣保護下，室溫下，向化合物 33-3(100 mg, 0.769 mmol)的 N,N-二甲基甲醯胺(3 mL)溶液中加入2-(7-偶氮苯并三氮唑)- N,N,N',N'-四甲基脲六氟磷酸酯(354 mg, 0.931 mmol)。將該混合物在室溫下攪拌0.5小時後，加入 N,N-二異丙基乙基胺(301 mg, 2.32 mmol)和 中間體 C(224 mg, 0.775 mmol)。將反應混合物在室溫繼續攪拌2小時。待反應完成後，將反應混合物加水(20 mL)稀釋，並用乙酸乙酯(15 mL × 3)萃取。合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)純化得到化合物 33-4(220 mg，產率：70%)。 Under nitrogen protection, at room temperature, to compound 33-3 (100 mg, 0.769 mmol) in N,N -dimethylformamide (3 mL) was added 2-(7-azobenzotriazol azole) -N,N,N',N' -tetramethyluronium hexafluorophosphate (354 mg, 0.931 mmol). After the mixture was stirred at room temperature for 0.5 hours, N,N -diisopropylethylamine (301 mg, 2.32 mmol) and Intermediate C (224 mg, 0.775 mmol) were added. The reaction mixture was stirred for an additional 2 hours at room temperature. After the reaction was complete, the reaction mixture was diluted with water (20 mL), and extracted with ethyl acetate (15 mL×3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 33-4 (220 mg, yield: 70%).
LC-MS (ESI) m/z: [M+H] +=401.1 步驟4：順式-5-(1-(4-甲氧基苄基)-3-(1-(三氘代甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 33-5) LC-MS (ESI) m/z: [M+H] + =401.1 Step 4: cis-5-(1-(4-methoxybenzyl)-3-(1-(trideuteromethyl )-1H-pyrazole-5-formamido)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 33-5 )
在室溫下，向化合物 33-4(220 mg, 0.549 mmol)的二氯甲烷(3 mL)和四氫呋喃(3 mL)混合溶液中加入4-硝基苯氯甲酸酯(167 mg, 0.829 mmol)、吡啶(131 mg, 1.65 mmol)和4-二甲胺基吡啶(7 mg, 0.06 mmol)。反應混合物於室溫下攪拌16小時後，將反應混合物加水(20 mL)稀釋，並用乙酸乙酯(15 mL × 3)萃取，合併的有機相經飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾並減壓濃縮。所得殘餘物透過矽膠管柱層析法(石油醚：乙酸乙酯=1：1)分離純化得到化合物 33-5(150 mg，產率：50%)。LC-MS (ESI), m/z: [M+H] +=566.1 步驟5：順式-5-(3-(1-(三氘代甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基(4-硝基苯基)碳酸酯( 33-6) To a mixed solution of compound 33-4 (220 mg, 0.549 mmol) in dichloromethane (3 mL) and tetrahydrofuran (3 mL) was added 4-nitrobenzene chloroformate (167 mg, 0.829 mmol ), pyridine (131 mg, 1.65 mmol) and 4-dimethylaminopyridine (7 mg, 0.06 mmol). After the reaction mixture was stirred at room temperature for 16 hours, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and Concentrate under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 33-5 (150 mg, yield: 50%). LC-MS (ESI), m/z: [M+H] + =566.1 Step 5: cis-5-(3-(1-(trideuteromethyl)-1H-pyrazole-5-formyl Amino)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl(4-nitrophenyl)carbonate ( 33-6 )
室溫下，將化合物 33-5(150 mg, 0.265 mmol)溶於甲酸(3 mL)中並將該反應混合物於85℃攪拌1小時。反應結束後，將反應混合物減壓濃縮，濃縮所得殘餘物即為粗產物化合物 33-6(100 mg)。該粗產物直接用於下一步反應。LC-MS (ESI) m/z: [M+H] +=445.8 步驟6：順式-5-(3-(1-(三氘代甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯( 實施例 33) Compound 33-5 (150 mg, 0.265 mmol) was dissolved in formic acid (3 mL) at room temperature and the reaction mixture was stirred at 85°C for 1 hr. After the reaction, the reaction mixture was concentrated under reduced pressure, and the residue obtained from the concentration was the crude product compound 33-6 (100 mg). The crude product was directly used in the next reaction. LC-MS (ESI) m/z: [M+H] + =445.8 Step 6: cis-5-(3-(1-(trideuteromethyl)-1H-pyrazole-5-carboxamide Base)-1H-pyrazol-5-yl)tetrahydrofuran-3-yl tert-butylcarbamate ( Example 33 )
室溫下，向化合物 33-6(100 mg, 0.225 mmol)的四氫呋喃溶液(3 mL)中加入叔丁胺(164 mg, 2.25 mmol)，並將該反應混合物於室溫攪拌16小時。反應結束後，將反應體系減壓濃縮，濃縮所得殘餘物透過製備HPLC純化(管柱：Gemini-C18 150 × 21.2 mm，5 µm；流動相：乙腈/水(0.1%三氟乙酸)；梯度：30%-34%；流速：20 mL/min)，得到 實施例 33(50 mg，產率：58%)。LC-MS (ESI), m/z: [M+H] +=380.2. 實施例 33A和 實施例 33B ： To a solution of compound 33-6 (100 mg, 0.225 mmol) in tetrahydrofuran (3 mL) was added tert-butylamine (164 mg, 2.25 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 16 hr. After the reaction, the reaction system was concentrated under reduced pressure, and the concentrated residue was purified by preparative HPLC (column: Gemini-C18 150 × 21.2 mm, 5 µm; mobile phase: acetonitrile/water (0.1% trifluoroacetic acid); gradient: 30%-34%; flow rate: 20 mL/min), to obtain Example 33 (50 mg, yield: 58%). LC-MS (ESI), m/z: [M+H] + =380.2. Example 33A and Example 33B :
(3
R,5
R)-5-(3-(1-(三氘代甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯和(3
S,5
S)-5-(3-(1-(三氘代甲基)-1H-吡唑-5-甲醯胺基)-1H-吡唑-5-基)四氫呋喃-3-基叔丁基胺基甲酸酯
將實施例33 (50 mg, 0.13 mmol)透過手性SFC層析管柱分離(管柱：CHIRALPAK AD-H 250mm x 20 mm，5 μm；流動相：40%異丙醇(0.2%氨水)；流速：40 g/min)得到 實施例 33A(24.1 mg，產率：48%)和 實施例 33B(22.8 mg，產率：46%)。 實施例 33A： Example 33 (50 mg, 0.13 mmol) was separated through a chiral SFC chromatography column (column: CHIRALPAK AD-H 250mm x 20 mm, 5 μm; mobile phase: 40% isopropanol (0.2% ammonia water); Flow rate: 40 g/min) gave Example 33A (24.1 mg, yield: 48%) and Example 33B (22.8 mg, yield: 46%). Example 33A :
t R=2.38 min t R =2.38 min
LC-MS (ESI) m/z: [M+H] +=380.1 LC-MS (ESI) m/z: [M+H] + =380.1
1H NMR (400 MHz, CD 3OD) δ 7.53 (d, J=2.1 Hz, 1H), 6.97 (d, J=2.1 Hz, 1H), 6.60 (d, J=16.6 Hz, 1H), 5.22 (s, 1H), 5.03 (t, J=6.3 Hz, 1H), 4.06 (d, J=10.2 Hz, 1H), 3.98 – 3.94 (m, 1H), 2.77 – 2.70 (m, 1H), 2.16 (dd, J=6.1, 5.0 Hz, 1H), 1.30 (s, 9H). 實施例 33B： 1 H NMR (400 MHz, CD 3 OD) δ 7.53 (d, J =2.1 Hz, 1H), 6.97 (d, J =2.1 Hz, 1H), 6.60 (d, J =16.6 Hz, 1H), 5.22 ( s, 1H), 5.03 (t, J =6.3 Hz, 1H), 4.06 (d, J =10.2 Hz, 1H), 3.98 – 3.94 (m, 1H), 2.77 – 2.70 (m, 1H), 2.16 (dd , J =6.1, 5.0 Hz, 1H), 1.30 (s, 9H). Embodiment 33B :
t R=5.39 min t R =5.39 min
LC-MS (ESI) m/z: [M+H]+=380.1LC-MS (ESI) m/z: [M+H]+=380.1
1H NMR (400 MHz, CD 3OD) δ 7.53 (d, J=2.1 Hz, 1H), 6.96 (d, J=2.1 Hz, 1H), 6.58 (d, J=16.6 Hz, 1H), 5.22 (s, 1H), 5.03 (t, J=6.3 Hz, 1H), 4.06 (d, J=10.2 Hz, 1H), 3.98 – 3.94 (m, 1H), 2.77 – 2.70 (m, 1H), 2.16 (dd, J=6.1, 5.0 Hz, 1H), 1.30 (s, 9H). 生物評價 1 H NMR (400 MHz, CD 3 OD) δ 7.53 (d, J =2.1 Hz, 1H), 6.96 (d, J =2.1 Hz, 1H), 6.58 (d, J =16.6 Hz, 1H), 5.22 ( s, 1H), 5.03 (t, J =6.3 Hz, 1H), 4.06 (d, J =10.2 Hz, 1H), 3.98 – 3.94 (m, 1H), 2.77 – 2.70 (m, 1H), 2.16 (dd , J =6.1, 5.0 Hz, 1H), 1.30 (s, 9H). Biological evaluation
以下結合測試進一步描述解釋本發明，但這些實施例並非意味著限制本發明的範圍。 一、酵素活性實驗(一)CDK1/CDK2酵素活性測試 1. 實驗目的： The following further describes and explains the present invention in combination with tests, but these examples are not meant to limit the scope of the present invention. 1. Enzyme activity test (1) CDK1/CDK2 enzyme activity test 1. Experimental purpose:
測試本發明化合物CDK2/CycE1及CDK1/cyclinB的體外酵素活性。
2. 實驗儀器和試劑：
所有的化合物最初在DMSO中製備成10 mM的母液，隨後在DMSO中稀釋到50×其最終濃度，將每個DMSO稀釋液的10 µL轉移到含有90 µL 1×激酶緩衝液(50 mM HEPES pH 7.5, 10 mM MgCl 2, 2 mM DTT和0.01% Brij-35)的新96孔集束板的一個孔中，然後混合10分鐘。隨後將5µL每個化合物稀釋液加入384孔盤中。在每個孔中加入10 µL含有CDK2/CycE1或CDK1/cyclinB(終濃度3 nM)的酵素溶液，混合後在室溫下培育10分鐘。隨後在每個孔中加入10 μL含有FAM標記的胜肽(最終濃度為3000nM FAM-P18(5-FAM-QSPKKG-CONH2))、ATP(最終濃度分別為77 μM或20 μM)的1×激酶緩衝液的胜肽溶液。所有反應在20-30℃培育30分鐘，然後加入25 μL終止緩衝液(100mM HEPES pH 7.5，50mM EDTA，0.2% Coating Reagent #3和0.015% Brij-35)終止。 4. 數據處理方法： All compounds were initially prepared as 10 mM stock solutions in DMSO, subsequently diluted in DMSO to 50× their final concentrations, and 10 µL of each DMSO dilution was transferred to 90 µL of 1× kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl 2 , 2 mM DTT and 0.01% Brij-35) in one well of a new 96-well cluster plate, and then mixed for 10 minutes. 5 µL of each compound dilution was then added to the 384-well plate. Add 10 µL of enzyme solution containing CDK2/CycE1 or CDK1/cyclinB (final concentration 3 nM) to each well, mix and incubate at room temperature for 10 minutes. Then, 10 μL of 1× kinase containing FAM-labeled peptide (final concentration 3000 nM FAM-P18 (5-FAM-QSPKKG-CONH2)), ATP (final concentration 77 μM or 20 μM, respectively) was added to each well. buffered peptide solution. All reactions were incubated at 20-30°C for 30 minutes and then terminated by adding 25 μL of stop buffer (100mM HEPES pH 7.5, 50mM EDTA, 0.2% Coating Reagent #3 and 0.015% Brij-35). 4. Data processing method:
反應結束後使用Caliper EZ ReaderⅡ檢測，抑制率=[(MA-X)/(MA-MI)] ×100%，其中MA=僅DMSO對照的值，MI=無酵素對照的值，X=任何特定化合物劑量下的值。然後透過繪製劑量-反應曲線，使用XLfit計算IC 50值。 5. 實驗結果： Use Caliper EZ Reader II to detect after the reaction, inhibition rate=[(MA-X)/(MA-MI)]×100%, where MA=the value of DMSO control only, MI=the value of no enzyme control, X=any specific Values at compound doses. IC50 values were then calculated using XLfit by plotting dose-response curves. 5. Experimental results:
透過以上方案得出，本發明所示的化合物在CDK2/CycE1酵素活性實驗中，顯示出約0.01 nM至10 nM的IC 50抑制活性；本發明所示的化合物在CDK1/cyclinB酵素活性實驗中，顯示出約1 nM至1000 nM的IC 50抑制活性。 Through the above scheme, the compounds shown in the present invention show an IC50 inhibitory activity of about 0.01 nM to 10 nM in the CDK2/CycE1 enzyme activity test; the compounds shown in the present invention in the CDK1/cyclinB enzyme activity test, Shows IC 50 inhibitory activity of about 1 nM to 1000 nM.
本發明實施例化合物對CDK2/CDK1抑制活性的選擇性倍數為2~1000倍；有些實施例化合物的選擇性倍數為10~30；有些實施例化合物的選擇性倍數為30~50；有些實施例化合物的選擇性倍數甚至大於50倍。
表1 本發明化合物對CDK2/CycE1和CDK1/cyclinB的抑制活性及選擇性倍數
以上數據顯示，本發明實施例化合物對CDK2/CycE1激酶具有良好的抑制活性，且對CDK2/CDK1激酶抑制具有良好的選擇性。 (二)CDK4/CDK6/CDK7酵素活性測試 1. 實驗目的： The above data show that the compounds of the examples of the present invention have good inhibitory activity on CDK2/CycE1 kinase and good selectivity on CDK2/CDK1 kinase inhibition. (2) CDK4/CDK6/CDK7 Enzyme Activity Test 1. Purpose of the experiment:
測試本發明化合物CDK4/CycD1、CDK6/cycD3以及CDK7/cycH/MAT1的體外酵素活性。
2. 實驗儀器和試劑：
本實驗透過利用螢光微流體遷移率檢測技術(Mobility-Shift Assay)，檢測小分子抑制劑對CDK4/CycD1, CDK6/cycD3和CDK7/cyclinH/MAT1激酶的抑制作用。激酶催化ATP脫去一個磷酸基團生成ADP，並將該磷酸基團轉移到基質胜肽上，該基質胜肽帶有螢光標記，其產物因增加了一個磷酸基團，所帶的電荷發生了變化，在電泳泳動過程中，受質和磷酸化的產物因遷移率不同被分開，並分別被檢測到，其量與螢光信號成正比。利用Caliper儀器測定受質與產物的量，並計算出產物的轉化率，進而計算出抑制率。In this experiment, the inhibitory effects of small molecule inhibitors on CDK4/CycD1, CDK6/cycD3 and CDK7/cyclinH/MAT1 kinases were detected by using the fluorescent microfluidic mobility assay technology (Mobility-Shift Assay). Kinase catalyzes the removal of a phosphate group from ATP to generate ADP, and transfers the phosphate group to the substrate peptide, which is fluorescently labeled, and the product is charged due to the addition of a phosphate group. During electrophoresis, substrates and phosphorylated products are separated due to different mobility and detected separately, and the amount is proportional to the fluorescent signal. Use the Caliper instrument to measure the amount of the substrate and the product, and calculate the conversion rate of the product, and then calculate the inhibition rate.
所有的化合物，用100%DMSO配製成初始濃度的50倍，轉移50 µL到384孔Echo盤中；按照客戶的需求用100%DMSO進行稀釋，轉移50 µL 100% DMSO到兩個空的孔中作為不加化合物和不加酵素的對照。使用Echo 550轉移400 nL化合物到384孔反應盤中。轉移10µL 的用1倍激酶緩衝液(50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT and 0.01% Brij-35)配置的CDK4/CycD1, CDK6/cycD3和CDK7/cyclinH/MAT1 2倍激酶溶液到384孔盤反應盤中，陰性對照孔加入1倍激酶緩衝液。混勻後室溫下培育10分鐘；將FAM標記的多肽(CDK4/CycD1, CDK6/cycD3上終濃度均是3000 nM FAM-P8 ，CDK7/cyclinH/MAT1上終濃度均是3000 nM FAM-CTD3)，ATP (CDK4/CycD1, CDK6/cycD3和CDK7/cyclinH/MAT1上終濃度分別是672 µM，800 µM和70µM)加入1倍激酶緩衝液，形成2倍受質溶液；轉移10µL上述2倍受質溶液到384孔盤反應盤中起始反應；28℃下培育60分鐘，向384孔盤反應盤中加25 µL終止液(100 mM HEPES pH 7.5, 50 mM EDTA, 0.2% Coating Reagent #3 and 0.015% Brij-35)終止反應。 4. 數據處理方法： For all compounds, prepare 50 times the initial concentration with 100% DMSO, transfer 50 µL to a 384-well Echo plate; dilute with 100% DMSO according to customer needs, transfer 50 µL 100% DMSO to two empty wells As the control without compound and without enzyme. Use the Echo 550 to transfer 400 nL of compounds into a 384-well reaction plate. Transfer 10 µL of CDK4/CycD1, CDK6/cycD3 and CDK7/cyclinH/MAT1 2x Kinase Solution in 1x Kinase Buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT and 0.01% Brij-35) to In the 384-well plate reaction plate, add 1 times kinase buffer to the negative control well. After mixing, incubate at room temperature for 10 minutes; add FAM-labeled peptides (the final concentration of CDK4/CycD1, CDK6/cycD3 is 3000 nM FAM-P8, and the final concentration of CDK7/cyclinH/MAT1 is 3000 nM FAM-CTD3) , ATP (final concentrations of CDK4/CycD1, CDK6/cycD3 and CDK7/cyclinH/MAT1 are 672 µM, 800 µM and 70 µM, respectively) were added to 1X kinase buffer to form a 2X substrate solution; transfer 10µL of the above 2X substrate Add the solution to a 384-well plate to initiate the reaction; incubate at 28°C for 60 minutes, add 25 µL of stop solution (100 mM HEPES pH 7.5, 50 mM EDTA, 0.2% Coating Reagent #3 and 0.015 % Brij-35) to terminate the reaction. 4. Data processing method:
在CaliperEZ Reader II(下游電壓：-500V，上游電壓：-2250V，基準壓力：-0.5 PSI，篩選壓力：-1.2 PSI)上讀取轉化率數據。把轉化率轉化成抑制率數據。抑制百分比=[(MA–X)/(MA–MI)]×100%。“MI”為不加酵素進行反應的對照樣孔讀數；“MA”為加入DMSO作為對照孔讀數；“X”為化合物不同孔讀數。使用Excel軟體中的XLFit(excel add-in version 5.4.0.8)擬合計算IC 50值。 5. 實驗結果： Conversion data was read on a CaliperEZ Reader II (Downstream Voltage: -500V, Upstream Voltage: -2250V, Base Pressure: -0.5 PSI, Screening Pressure: -1.2 PSI). Convert conversion rate to inhibition rate data. Inhibition percentage = [(MA–X)/(MA–MI)]×100%. "MI" is the reading of the control well without adding enzyme; "MA" is the reading of the control well adding DMSO; "X" is the reading of different wells of the compound. IC 50 values were calculated using XLFit (excel add-in version 5.4.0.8) in Excel software. 5. Experimental results:
透過以上方案得出，本發明所示的化合物對CDK4/CycD1、CDK6/CycD3和CDK7/cycH/MAT1的激酶抑制的IC
50值如表2所示。
表2 本發明化合物對CDK4/CycD1、CDK6/CycD3和CDK7/cycH/MAT1的抑制活性
以上數據顯示，本發明實施例化合物對CDK4/CycD1、CDK6/cycD3和CDK7/cycH/MAT1的激酶抑制活性較弱；本發明實施例化合物對CDK2/CDK4、CDK2/CDK6以及CDK2/CDK7激酶抑制具有良好的選擇性。 二、細胞活性實驗1. 實驗目的： The above data show that the compounds of the embodiments of the present invention have weak kinase inhibitory activity on CDK4/CycD1, CDK6/cycD3 and CDK7/cycH/MAT1; good selectivity. 2. Cell activity experiment 1. Experiment purpose:
測試本發明化合物對OVCAR-3細胞的增殖抑制活性。
2. 實驗儀器和試劑
酵素標示讀取儀：TECAN Spark 10M (TECAN)
OVCAR-3培養條件：RPMI1640, 20% FBS, 1% 青黴素和鏈黴素，0.01 mg/mL牛胰島素。OVCAR-3 culture conditions: RPMI1640, 20% FBS, 1% penicillin and streptomycin, 0.01 mg/mL bovine insulin.
第0天：將細胞種入96孔盤中，加入120 μL培養基培養過夜。Day 0: Seed the cells into a 96-well plate, add 120 μL medium and culture overnight.
第1天：將化合物溶於DMSO中至10 mM的儲備溶液。將參考和測試化合物在DMSO中稀釋至200倍終濃度。在96孔盤中對DMSO中的化合物進行3倍稀釋至8個不同濃度，然後用細胞培養基稀釋試驗化合物40倍，在每孔細胞中加入30 μL(5×)化合物溶液。在1000 rpm下離心細胞培養盤1分鐘，然後在37°C和5%CO 2下培養細胞培養盤7天。0.5%DMSO培養基作為空白對照，0.5% DMSO培養基溶液培養的細胞作為100%對照。 Day 1: Dissolve compounds in DMSO to 10 mM stock solution. Reference and test compounds were diluted in DMSO to 200-fold final concentrations. The compound in DMSO was diluted 3-fold to 8 different concentrations in a 96-well plate, then the test compound was diluted 40-fold with cell culture medium, and 30 μL (5×) compound solution was added to each well of cells. Centrifuge the cell culture dish at 1,000 rpm for 1 min, then incubate the cell culture dish at 37 °C and 5% CO for 7 days. 0.5% DMSO medium was used as blank control, and cells cultured in 0.5% DMSO medium solution were used as 100% control.
第8天：細胞使用CellTiter-Glo®試劑進行檢測。 4. 數據處理： Day 8: Cells are detected using CellTiter-Glo® Reagent. 4. Data processing:
抑制率按照以下公式計算：The inhibition rate was calculated according to the following formula:
抑制率=100-100*(化合物藥物處理的讀數-低對照的讀數)/(高對照的讀數-低對照的讀數)，根據公式計算IC 50。 5. 實驗結果： Inhibition rate=100-100*(compound drug-treated reading-low control reading)/(high control reading-low control reading), IC 50 was calculated according to the formula. 5. Experimental results:
透過以上方案得出，本發明實施例化合物對OVCAR-3細胞的增值抑制的IC
50如表3所示。
表3 本發明化合物對OVCAR-3細胞的增值抑制活性
以上數據顯示，本發明實施例化合物對於OVCAR-3細胞增殖具有良好的抑制活性。 三、 Caco-2 細胞滲透性實驗1. 實驗目的： The above data show that the compounds of the examples of the present invention have good inhibitory activity on the proliferation of OVCAR-3 cells. 3. Caco-2 cell permeability experiment 1. Experimental purpose:
透過Caco-2細胞模型對發明化合物在給藥濃度下的滲透性進行研究。
2. 實驗儀器：
第一步：將Caco-2細胞(ATCC)用加入10%FBS、1%PS和1%NEAA的DMEM培養液，於37°C，5% CO 2條件下在培養瓶中培養。細胞生長融合至70%~80%，將細胞用0.25%EDTA-胰蛋白酶消化，計數，用DMEM培養液稀釋至80,000個細胞/孔，細胞接種於Transwell-24孔盤，置於CO 2培養箱中培養，每隔兩到三天將培養基棄去，加入新鮮培養基，繼續於37°C，5% CO 2條件下培養，連續培養18~22天。 Step 1: Caco-2 cells (ATCC) were cultured in culture flasks at 37°C and 5% CO 2 in DMEM medium supplemented with 10% FBS, 1% PS and 1% NEAA. The cells grow confluent to 70%~80%, the cells are digested with 0.25% EDTA-trypsin, counted, diluted with DMEM culture medium to 80,000 cells/well, the cells are seeded in Transwell-24 well plates, and placed in a CO 2 incubator Medium culture, discard the medium every two to three days, add fresh medium, continue to culture at 37 ° C, 5% CO 2 conditions, continuous culture for 18 to 22 days.
第二步：將TC儲備液用預熱至37°C的轉運緩衝液1000倍稀釋，混合均勻，即為受試化合物給藥溶液，給藥濃度為10 μM。將細胞培養液棄去，加入預熱至37°C的轉運緩衝液潤洗，對於頂膜側至基底側的轉運研究(轉運方向為A→B)，A側加入500 μL含對照化合物或受試化合物的給藥溶液，B側加入1300 μL轉運緩衝液。對於基底側至頂膜側的轉運研究(轉運方向為B→A)，A側加入500 μL轉運緩衝液，B側加入1300 μL含對照化合物或受試化合物的給藥溶液。細胞培養盤培育90 min。培育結束後，取樣品加入一定體積沉澱劑。振盪混勻1 min，4000 rpm/分鐘離心15 min。取上清復溶後LC-MS/MS檢測分析。Step 2: Dilute the TC stock solution 1000 times with the transport buffer preheated to 37°C, and mix well to obtain the test compound dosing solution with a dosing concentration of 10 μM. Discard the cell culture medium, and add transport buffer preheated to 37°C to rinse. For the transport study from the apical membrane side to the basal side (the transport direction is A→B), add 500 μL of the control compound or the subject to the A side. For the dosing solution of the test compound, add 1300 μL of transport buffer to the B side. For the transport study from the basolateral side to the apical membrane side (transport direction is B→A), 500 μL of transport buffer was added to side A, and 1300 μL of dosing solution containing control compound or test compound was added to side B. Cell culture plates were incubated for 90 min. After the incubation, take a sample and add a certain volume of precipitant. Shake and mix for 1 min, and centrifuge at 4000 rpm/min for 15 min. The supernatant was reconstituted and analyzed by LC-MS/MS.
第三步：透過測定細胞單層的跨膜電阻值(TEER)來評價細胞單層的完整性，要求每個細胞孔的跨膜電阻值不小於600 Ohms。Step 3: Evaluate the integrity of the cell monolayer by measuring the transmembrane resistance value (TEER) of the cell monolayer, which requires that the transmembrane resistance value of each cell well is not less than 600 Ohms.
第四步：透過LC/MS/MS方法檢測樣品中受試化合物TC和陽性對照納多洛爾、普萘洛爾、P-gp受質紫杉醇的濃度 5. 數據處理： Step 4: Detect the concentration of the test compound TC and positive controls nadolol, propranolol, and P-gp substrate paclitaxel in the sample by LC/MS/MS method 5. Data processing:
利用以下公式計算出TC以及陽性化合物的表觀滲透係數(Papp)以及表觀滲透係數的比率Efflux ratio(ER)。Use the following formula to calculate the apparent permeability coefficient (Papp) of TC and the positive compound and the ratio Efflux ratio (ER) of the apparent permeability coefficient.
Papp(cm/sec)=( υ•∂ C/∂ t)/(A•C) Papp(cm/sec)= ( υ• ∂ C/ ∂ t )/(A•C)
流出比率(Efflux ratio)= Papp （ B→A ）/ Papp （ A→B ） Outflow ratio (Efflux ratio)= Papp ( B→A ) / Papp ( A→B )
註：v為接收池的體積；A為轉運膜的面積；C為培育初始濃度；∂C/∂t為單位時間藥物轉運量 6. 實驗結果： Note: v is the volume of the receiving pool; A is the area of the transport membrane; C is the initial concentration of incubation; ∂C/∂t is the amount of drug transported per unit time 6. Experimental results:
透過以上方案得出，本發明實施例化合物的滲透性實驗數據如表4所示：
表4
以上數據顯示，本發明實施例化合物在Caco-2細胞模型上表現出更好的滲透性和更低的外排活性。 四、 代謝穩定性測定1. 實驗目的： The above data show that the compounds of the examples of the present invention exhibit better permeability and lower efflux activity on the Caco-2 cell model. 4. Determination of metabolic stability 1. Experimental purpose:
測試化合物在肝細胞中的穩定性。
使用台盼藍測定冷凍保存的肝細胞的活力，並用緩衝液將細胞濃度調節至10 6個細胞/mL。在24孔盤中，將400 μL陽性對照/測試化合物溶液(培養介質中含2 μM化合物)與400 μL肝細胞(每毫升200萬個細胞)一起培養。在不同時間點(0、15、30、60、90和120分鐘)，透過將300 μL人硫酸吲哚酚(IS)添加到30 μL反應混合物中並在4°C以4000 rpm離心15分鐘，來停止反應。取100 μL上清液用LC-MS/MS分析。體外肝細胞清除率是根據化合物從其初始濃度的消除半衰期(t 1/2)來估計的。計算每種化合物（測試或對照）與IS的峰面積比。藥物消除速率常數k(min-1）、t 1/2(min)和體外固有清除率CL int(微升/分鐘/百萬細胞)根據以下等式計算： Assay the viability of the cryopreserved hepatocytes using trypan blue and adjust the cell concentration to 106 cells/mL with buffer. Incubate 400 µL of positive control/test compound solution (2 µM compound in culture medium) with 400 µL of hepatocytes (2 million cells per mL) in a 24-well dish. At different time points (0, 15, 30, 60, 90, and 120 min), by adding 300 μL of human indoxyl sulfate (IS) to a 30 μL reaction mixture and centrifuging at 4000 rpm for 15 min at 4 °C, to stop the reaction. 100 μL of the supernatant was analyzed by LC-MS/MS. In vitro hepatocyte clearance was estimated based on the elimination half-life (t 1/2 ) of the compound from its initial concentration. Calculate the peak area ratio of each compound (test or control) to IS. Drug elimination rate constants k (min-1), t 1/2 (min) and intrinsic clearance CL int in vitro (μl/min/million cells) were calculated according to the following equation:
K=-斜率K=-slope
t1/2 = 0.693/kt1/2 = 0.693/k
CL int= (0.693/t 1/2)×(培養體積(μL)/細胞數(百萬)) CL int = (0.693/t 1/2 )×(culture volume (μL)/cell number (million))
CL in=體外CL int× (肝重(g)/體重(Kg)) × (細胞數(百萬)/肝重(g)) CL in = in vitro CL int × (liver weight (g)/body weight (Kg)) × (cell number (million)/liver weight (g))
CL hepatic= (Q hep× F u× CL int)/(Q hep+ F u× CL int) CL hepatic = (Q hep × F u × CL int )/(Q hep + F u × CL int )
提取率= CL hepatic/Q hep4.實驗結果： Extraction rate = CL hepatic /Q hep 4. Experimental results:
透過以上方案得出本發明所示的化合物在人肝穩定性試驗實驗的結果如下表5所示：
表 5
以上數據顯示，本發明實施例化合物在人肝細胞穩定性試驗實驗中顯示出較好的代謝穩定性。 五、藥代動力學研究 The above data show that the compounds of the examples of the present invention show better metabolic stability in the human hepatocyte stability test. 5. Pharmacokinetic study
實驗目的：測試化合物在比格犬體內實驗的藥代動力學The purpose of the experiment: to test the pharmacokinetics of the compound in the beagle dog experiment
實驗材料：比格犬(雄性，Marshall Bio)Experimental material: Beagle (male, Marshall Bio)
實驗操作： 3條雄性比格犬，Crossover給藥。在給藥前一天下午17點30分對比格犬進行禁食處理，給藥後4小時恢復餵食。給藥第一週期，單次前肢頭靜脈注射待測化合物，給藥劑量為1 mg/kg；洗提7天後，進行第二週期，單次口服待測化合物，給藥劑量為10 mg/kg。前肢頭靜脈注射組於給藥前，給藥結束後5 min，15 min，30 min，1 h，2 h，4 h，8 h，12 h及24 h透過頸靜脈取血。單次口服組於給藥前，給藥結束後15 min，30 min，1 h，2 h，4 h，8 h，12 h及24 h透過頸靜脈取血。取血置於EDTA-K2抗凝管中，每個時間點取800 µL全血。血樣採集後，立即放在濕冰上等待離心。樣品將在30分鐘內離心 (設置保持4度，10分鐘，3000轉/分) ，以獲得血漿。將血漿樣本分別放入2支試管 (目標為150µL/支)。取50 µL血漿加入5 µL MeOH, 然後加入200µL 含有5 ng/mL ISTD (Terfenadine)的MeOH/ACN溶液，振盪混合均勻後4000 rpm離心15 min，取上清液用MeOH/水 (1:1, v/v, 0.1% FA)稀釋5倍，使用LC-MS/MS進樣，以分析方法定量分析血藥濃度，並計算藥代參數，如達峰濃度，達峰時間，清除率，半衰期，藥時曲線下面積，生物利用度等。 Experimental operation: 3 male beagle dogs, Crossover administration. Beagle dogs were fasted at 17:30 p.m. one day before the administration, and the feeding was resumed 4 hours after the administration. In the first cycle of administration, the compound to be tested was injected into the cephalic vein of the forelimb once, and the dosage was 1 mg/kg; after 7 days of elution, the second cycle was performed, and the compound to be tested was administered orally once, and the dosage was 10 mg/kg. kg. In the forelimb cephalic vein injection group, blood was collected from the jugular vein before administration, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after the administration. In the single oral administration group, blood was collected from the jugular vein at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after the administration. Blood was collected and placed in EDTA-K2 anticoagulant tubes, and 800 μL whole blood was taken at each time point. Immediately after blood samples were collected, they were placed on wet ice and awaited centrifugation. Samples will be centrifuged within 30 minutes (set to hold 4 degrees, 10 minutes, 3000 rpm) to obtain plasma. Put plasma samples into 2 test tubes (target 150µL/tube). Take 50 µL plasma and add 5 µL MeOH, then add 200 µL MeOH/ACN solution containing 5 ng/mL ISTD (Terfenadine), shake and mix well, then centrifuge at 4000 rpm for 15 min, take the supernatant and wash with MeOH/water (1:1, v/v, 0.1% FA) diluted 5 times, using LC-MS/MS to inject samples, quantitatively analyze blood drug concentration by analytical method, and calculate pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, Area under the drug-time curve, bioavailability, etc.
實驗結果：
透過以上方案得出，本發明實施例化合物的藥代動力學數據如表6所示：
表6
實驗結論： 本發明化合物在犬中具有良好的藥代動力學參數；尤其是實施例12B，相比參比化合物PF-07104091，清除率要低9倍，暴露量高近24倍，生物利用度也是要高1倍。 六、體內藥效研究 Experimental conclusion: The compound of the present invention has good pharmacokinetic parameters in dogs; especially Example 12B, compared with the reference compound PF-07104091, the clearance rate is 9 times lower, the exposure is nearly 24 times higher, and the bioavailability It is also twice as high. 6. In vivo efficacy research
實驗目的 評價化合物在OVCAR-3腫瘤小鼠移植模型上的體內藥效。 Purpose The in vivo efficacy of the compounds was evaluated on the OVCAR-3 tumor xenograft mouse model.
動物與模型
動物資訊
Balb/c裸小鼠，6-9周，雌性，購於江蘇集萃藥康生物科技有限公司。小鼠飼養於12小時光照12小時黑暗環境。接種前用小鼠耳標標記小鼠。
細胞資訊
實驗操作Experimental operation
細胞培養 OVCAR-3腫瘤細胞在含20%牛血清及10 µ/mL胰島素的RPMI1640培養液中進行培養。細胞置於培養箱內，溫度37ºC，CO 2濃度5%。細胞生長進入對數期時收集並計數細胞。 Cell culture OVCAR-3 tumor cells were cultured in RPMI1640 medium containing 20% bovine serum and 10 µ/mL insulin. Cells were placed in an incubator at a temperature of 37ºC and a CO 2 concentration of 5%. Cells were harvested and counted when cell growth entered logarithmic phase.
細胞接種 收集1x 10 7OVCAR-3細胞重新懸浮於0.2毫升的PBS與Matrigel(1：1)的懸浮液中，並接種於每只小鼠右側背部靠右肩位置。 Cell inoculation 1x 10 7 OVCAR-3 cells were collected and resuspended in 0.2 ml of PBS and Matrigel (1:1) suspension, and inoculated on the right back of each mouse near the right shoulder.
分組 當荷瘤小鼠腫瘤平均腫瘤體積達到150-200 mm 3時，對小鼠進行隨機分組並開始給藥。 Grouping When the average tumor volume of the tumor-bearing mice reached 150-200 mm 3 , the mice were randomly divided into groups and drug administration began.
小鼠觀察於數據採集 細胞接種後，每天檢查小鼠發病於死亡。每週測量腫瘤與小鼠體重兩次。 腫瘤體積按以下公式計算： 腫瘤體積(mm 3)=長(mm)x寬(mm)x高(mm)/2。 給藥方式：口服，每天給藥兩次，給藥週期28天。 實驗結束後安樂死動物。 用Excel等軟體處理數據，並計算化合物抑瘤率TGI(%)。 TGI(%)=(1-藥物處理組平均腫瘤體積/對照組平均腫瘤體積）x 100% Mice were observed for data collection. After cell inoculation, mice were checked daily for morbidity and mortality. Tumor and mouse body weights were measured twice a week. The tumor volume was calculated according to the following formula: tumor volume (mm 3 )=length (mm)×width (mm)×height (mm)/2. Administration method: oral administration, twice a day, administration cycle 28 days. Euthanize the animals after the experiment. The data was processed with software such as Excel, and the tumor inhibition rate TGI (%) of the compound was calculated. TGI(%)=(1-average tumor volume of drug treatment group/average tumor volume of control group) x 100%
試驗結果如下表
表7 化合物的移植瘤藥效參數
實驗結果 以上結果顯示，在OVCAR-3小鼠移植瘤模型中，實施例12B在75 mg/kg劑量下顯示出良好的腫瘤增長抑制效果；並且，顯示出比PF-07104091在175 mg/kg劑量下更好的腫瘤增長抑制效果。 Experimental results The above results show that in the OVCAR-3 mouse xenograft tumor model, Example 12B shows a good tumor growth inhibitory effect at a dose of 75 mg/kg; Good tumor growth inhibitory effect.
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