TW202313052A - Methods and treatment of viral infection with substituted pyrazolo-pyrimidines - Google Patents
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- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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Abstract
Description
本發明係關於以下之方法：利用含FYVE型指之磷酸肌醇激酶(「PIKfyve」)抑制劑之化合物來i)阻斷α-冠狀病毒、β-冠狀病毒譜系B及/或β-冠狀病毒譜系A進入宿主細胞；及ii)預防及治療由α-冠狀病毒、β-冠狀病毒譜系B及/或β-冠狀病毒譜系A引起的感染。The present invention relates to methods for i) blocking alpha-coronaviruses, beta-coronavirus lineage B and/or beta-coronaviruses using compounds containing FYVE-type fingered phosphoinositide kinase ("PIKfyve") inhibitors Lineage A enters the host cell; and ii) preventing and treating infections caused by alpha-coronaviruses, beta-coronavirus lineage B and/or beta-coronavirus lineage A.
根據世界衛生組織(World Health Organization)，截至2022年5月，全世界已確認超過5.17億由COVID-19所致之病例及約630萬死於COVID-19。SARS-CoV-2，其造成COVID-19 (2019年新冠狀病毒(2019-nCoV))疾病，為一種屬於β冠狀病毒屬( Betacoronavirusgenus)之有套膜的正義RNA病毒。Bouhaddou等人, Cell 182: 1-28 (2020)。對於病毒進入之關鍵步驟及破壞其之方式的理解增強可引起發展不僅針對COVID-19且亦針對未來病毒爆發的有效抗病毒藥物。非常需要針對COVID-19之治療。 According to the World Health Organization, as of May 2022, more than 517 million cases and approximately 6.3 million deaths from COVID-19 have been confirmed worldwide. SARS-CoV-2, which causes COVID-19 (2019 novel coronavirus (2019-nCoV)) disease, is an enveloped positive-sense RNA virus belonging to the Betacoronavirus genus. Bouhaddou et al., Cell 182: 1-28 (2020). Improved understanding of the critical steps of viral entry and the ways in which it is disrupted could lead to the development of effective antiviral drugs not only against COVID-19 but also against future viral outbreaks. There is a great need for treatment against COVID-19.
冠狀病毒進入易感細胞為一種複雜過程，其需要冠狀病毒S蛋白之受體結合及蛋白水解加工協同作用以促進病毒-細胞融合。Heald-Sargent及Gallagher, Viruses 4:557-580 (2012)；Millet及Whittaker, Virus Res. 202:120-134 (2015)。舉例而言，病毒進入細胞可由病毒醣蛋白(GP)介導，該醣蛋白將病毒粒子附著於細胞表面，將其遞送至內體且催化病毒與內體膜之間的融合。Murray等人, J. Virology 79:11742-11751 (2005)。在SARS-CoV-2之情況下，其刺突(S)蛋白與目標細胞上之ACE2受體結合，且隨後由裂解S蛋白且允許病毒與溶酶體膜融合之絲胺酸蛋白酶TMPRSS2激活(primed)。Hoffmann等人, Cell 181:271-280.e8 (2020)。The entry of coronavirus into susceptible cells is a complex process that requires the synergy of receptor binding and proteolytic processing of the coronavirus S protein to promote virus-cell fusion. Heald-Sargent and Gallagher, Viruses 4:557-580 (2012); Millet and Whittaker, Virus Res. 202:120-134 (2015). For example, viral entry into cells can be mediated by the viral glycoprotein (GP), which attaches the virion to the cell surface, delivers it to the endosome, and catalyzes fusion between the viral and endosomal membranes. Murray et al., J. Virology 79:11742-11751 (2005). In the case of SARS-CoV-2, its spike (S) protein binds to the ACE2 receptor on target cells, and is subsequently activated by the serine protease TMPRSS2, which cleaves the S protein and allows fusion of the virus with the lysosomal membrane ( primed). Hoffmann et al., Cell 181:271-280.e8 (2020).
類似於大部分病毒，冠狀病毒膜與宿主細胞膜融合發生在酸化的內體內。Kang等人, PNAS 117(34):20803-20813 (2020)。已知冠狀病毒與分佈在各種次細胞區室中之磷脂酸肌醇(PI)激酶相互作用。Beziau等人, Viruses 12, 1124; 數位物件識別碼：10.3390/v12101124 (2020)。參與內體酸化之PI激酶磷脂酸肌醇3-磷酸5激酶(PIKfyve)為SARS-CoV-2進入細胞所需的。Ou等人, Nat. Commun. 11(1620):1-12 (2020)。已顯示用小分子抑制劑抑制PIKfyve抑制SARS-CoV-2感染。Kang等人, PNAS 117(34):20803-20813 (2020)；Nelson等人, PLoS Negl. Trop. Dis. 11(4):e0005540 (2017)。去除PIKfyve遏制內體成熟(Ikonomov等人, J. Biol. Chem. 276(28):26141-26147 (2001)；Ikonomov等人, J. Biol. Chem. 277(11):9206-9211 (2002)；Jefferies等人, EMBO Rep. 9(2):164-170 (2008)；Rutherford等人, J. Cell Sci. 119(19):3944-3957 (2006)；Sbrissa等人, J. Biol. Chem. 277(8):6073-6079 (2002))，此使得PIKfyve成為針對利用內體路徑之病毒之新型藥物的候選目標。Similar to most viruses, fusion of the coronavirus membrane with the host cell membrane occurs within the acidified endosome. Kang et al., PNAS 117(34):20803-20813 (2020). Coronaviruses are known to interact with phosphatidylinositol (PI) kinases distributed in various subcellular compartments. Beziau et al., Viruses 12, 1124; Digital Object Identifier: 10.3390/v12101124 (2020). The PI kinase phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), involved in endosomal acidification, is required for entry of SARS-CoV-2 into cells. Ou et al., Nat. Commun. 11(1620):1-12 (2020). Inhibition of PIKfyve with small molecule inhibitors has been shown to suppress SARS-CoV-2 infection. Kang et al., PNAS 117(34):20803-20813 (2020); Nelson et al., PLoS Negl. Trop. Dis. 11(4):e0005540 (2017). Removal of PIKfyve suppresses endosome maturation (Ikonomov et al., J. Biol. Chem. 276(28):26141-26147 (2001); Ikonomov et al., J. Biol. Chem. 277(11):9206-9211 (2002) ; Jefferies et al., EMBO Rep. 9(2):164-170 (2008); Rutherford et al., J. Cell Sci. 119(19):3944-3957 (2006); Sbrissa et al., J. Biol. Chem . 277(8):6073-6079 (2002)), which makes PIKfyve a candidate target for novel drugs against viruses that utilize the endosomal pathway.
實施例1為一種阻斷α-冠狀病毒、β-冠狀病毒譜系B及/或β-冠狀病毒譜系A進入宿主細胞及預防由α-冠狀病毒、β-冠狀病毒譜系B及/或β-冠狀病毒譜系A引起之感染之方法，其包含向有需要之個體投與以下的化合物：
(i) 式(I)：
實施例2為如實施例1之方法，其中：
a.該α-冠狀病毒為HCoV 229E；
b.該β-冠狀病毒譜系B為SARS-CoV2；及
c.該β-冠狀病毒譜系A為HCoV OC43。
實施例3為如實施例2之方法，其中該感染係由SARS-CoV-2引起，且其中該感染為COVID-19。
實施例4為如前述實施例中任一項之方法，其中X為CH且Y為N。
實施例5為如實施例1至3中任一項之方法，其中X為N且Y為CR
a。
實施例6為如前述實施例中任一項之方法，其中R a為H、甲基、乙基、丙基、異丙基、丁基、異丁基、二級丁基或三級丁基。 Embodiment 6 is the method of any one of the preceding embodiments, wherein R is H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl or tertiary butyl .
實施例7為如實施例1至5中任一項之方法，其中R
a為H或甲基。
實施例8為如實施例1至5中任一項之方法，其中R
a為H。
實施例9為如實施例1至5中任一項之方法，其中R
a或R
1為視情況經取代之苯基。
實施例10為如實施例1至5中任一項之方法，其中R
a或R
1為視情況經取代之單環雜芳基。
實施例11為如實施例1至5中任一項之方法，其中R
a或R
1為視情況經取代之吡咯、咪唑、吡唑、***、四唑、呋喃、㗁唑、異㗁唑、噻唑、異噻唑、吡啶、嘧啶、吡𠯤或嗒𠯤。
Embodiment 11 is the method of any one of
實施例12為如實施例1至5中任一項之方法，其中R
a或R
1為視情況經取代之吡啶或嘧啶。
Embodiment 12 is the method of any one of
實施例13為如實施例1至5中任一項之方法，其中R a或R 1為視情況經取代之吡啶。 Embodiment 13 is the method of any one of embodiments 1-5, wherein R or R is optionally substituted pyridine.
實施例14為如前述實施例中任一項之方法，其中R a或R 1經一個R d取代，且該R d為C 1-4烷基、-NR gR h或鹵基。 Embodiment 14 is the method of any one of the preceding embodiments, wherein R a or R 1 is substituted with one R d , and the R d is C 1-4 alkyl, -NR g R h or halo.
實施例15為如實施例1至14中任一項之方法，其中R
a或R
1經一個R
d取代，且該R
d為甲基、NH
2、氟、氯或溴。
Embodiment 15 is the method of any one of
實施例16為如實施例1至5中任一項之方法，R
a或R
1為苯基或吡啶基，其各自視情況經一或兩個選自以下之取代基取代：C
1-4烷基、
-CF
3、氟、氯、溴、-OCH
3及-OCF
3。
Embodiment 16 is the method as any one of
實施例17為如實施例1至5中任一項之方法，其中R
a或R
1為未經取代之苯基或甲苯基。
實施例18為如實施例1至5中任一項之方法，其中R a或R 1為未經取代之苯基或間甲苯基。 Embodiment 18 is the method of any one of embodiments 1-5, wherein Ra or R is unsubstituted phenyl or m-tolyl.
實施例19為如實施例1至5中任一項之方法，其中R a或R 1為未經取代之吡啶基。 Embodiment 19 is the method of any one of embodiments 1-5, wherein R or R is unsubstituted pyridyl.
實施例20為如實施例1至5中任一項之方法，其中R
a或R
1為4-吡啶基。
實施例21為如前述實施例中任一項之方法，其中R 2及R 3與其所連接之氮一起形成吡咯啶、哌啶、哌𠯤、𠰌啉、硫代𠰌啉、硫代𠰌啉-1,1-二氧化物或2-氧雜-6-氮雜螺[3.3]庚烷，其各自視情況經一個、兩個或三個R j取代基取代。 Embodiment 21 is the method of any one of the preceding embodiments, wherein R and R together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperidine, thioline, thiophenoline, thiophenoline- 1,1-dioxide or 2-oxa-6-azaspiro[3.3]heptane, each optionally substituted with one, two or three Rj substituents.
實施例22為如實施例1至21中任一項之方法，其中R
2及R
3與其所連接之氮一起形成視情況經一或兩個R
j取代基取代之𠰌啉。
Embodiment 22 is the method of any one of
實施例23為如實施例1至22中任一項之方法，其中各R
j取代基獨立地為甲基、羥基、-OCH
3、側氧基、鹵基、-CF
3或-OCF
3。
Embodiment 23 is the method of any one of
實施例24為如前述實施例中任一項之方法，其中R k及R l各自獨立地為H或甲基。 Embodiment 24 is the method of any one of the preceding embodiments, wherein Rk and Rl are each independently H or methyl.
實施例25為如實施例1至24中任一項之方法，其中R
4為視情況經取代之苯基。
實施例26為如實施例1至24中任一項之方法，其中R 4為視情況經取代之雜芳基。 Embodiment 26 is the method of any one of embodiments 1-24, wherein R 4 is optionally substituted heteroaryl.
實施例27為如實施例1至24中任一項之方法，其中R 4為視情況經取代之吡唑、噻唑、㗁唑、吡啶或嘧啶。 Embodiment 27 is the method of any one of embodiments 1-24, wherein R is optionally substituted pyrazole, thiazole, oxazole, pyridine, or pyrimidine.
實施例28為如實施例1至24中任一項之方法，其中R 4為視情況經取代之吡啶。 Embodiment 28 is the method of any one of embodiments 1-24, wherein R 4 is optionally substituted pyridine.
實施例29為如實施例1至24中任一項之方法，其中R 4為吡啶。 Embodiment 29 is the method of any one of embodiments 1-24, wherein R 4 is pyridine.
實施例30為如實施例1至24中任一項之方法，其中R 4為4-吡啶基。 Embodiment 30 is the method of any one of embodiments 1-24, wherein R 4 is 4-pyridyl.
實施例31為如實施例1至24中任一項之方法，其中R 4視情況經一或兩個R z取代基取代。 Embodiment 31 is the method of any one of embodiments 1-24, wherein R4 is optionally substituted with one or two Rz substituents.
實施例32為如實施例1至24中任一項之方法，其中R
4為苯基或吡啶基，其各自視情況經一或兩個選自以下之取代基取代：C
1-4烷基、-CF
3、氟、氯、-OCH
3及-OCF
3。
Embodiment 32 is the method of any one of
實施例33為如實施例1至24中任一項之方法，其中R
4為
-C(O)NR
xR
y。
Embodiment 33 is the method of any one of
實施例34為如實施例1至33中任一項之方法，其中R x為H。 Embodiment 34 is the method of any one of embodiments 1-33, wherein Rx is H.
實施例35為如實施例1至33中任一項之方法，其中R
x為甲基或乙基，其視情況經一個、兩個或三個R
o取代基取代。
Embodiment 35 is the method of any one of
實施例36為如實施例1至33中任一項之方法，其中R x為甲基。 Embodiment 36 is the method of any one of embodiments 1-33, wherein R x is methyl.
實施例37為如前述實施例中任一項之方法，其中R y為H。 Embodiment 37 is the method of any one of the preceding embodiments, wherein Ry is H.
實施例38為如實施例1至36中任一項之方法，其中R
y為C
1-4烷基、-C
1-4烷基(單環環烷基)、單環環烷基、單環雜環烷基、單環雜環基、-O-單環雜環基、-O-C
1-4烷基、-SO
2-C
1-4烷基，其視情況經一個、兩個或三個R
o取代基取代。
Embodiment 38 is the method as any one of
實施例39為如實施例1至36中任一項之方法，其中R
y為視情況經一個、兩個或三個R
o取代基取代之C
1-4烷基。
Embodiment 39 is the method of any one of
實施例40為如實施例1至36中任一項之方法，其中R
y為甲基、乙基、丙基或異丙基，其各自視情況經一個、兩個或三個R
o取代基取代。
實施例41為如實施例1至36中任一項之方法，其中R
y為甲基、乙基、異丙基、甲氧基乙基、二甲氧基丙烷基、(二甲胺基)乙基或(二甲胺基)丁基。
Embodiment 41 is the method as any one of
實施例42為如實施例1至36中任一項之方法，其中R y為甲氧基。 Embodiment 42 is the method of any one of embodiments 1-36, wherein Ry is methoxy.
實施例43為如實施例1至36中任一項之方法，其中R
y為
-SO
2-甲基。
Embodiment 43 is the method of any one of
實施例44為如實施例1至36中任一項之方法，其中R
y為單環環烷基或-C
1-2烷基(單環環烷基)，其各自視情況經一個、兩個或三個R
o取代基取代。
Embodiment 44 is the method as any one of
實施例45為如實施例1至36中任一項之方法，其中R
y為視情況經一個、兩個或三個R
o取代基取代之單環環烷基。
Embodiment 45 is the method of any one of
實施例46為如實施例1至36中任一項之方法，其中R
y為環丙基、環丁基、環戊基或環己基，其各自視情況經一個、兩個或三個R
o取代基取代。
Embodiment 46 is the method of any one of
實施例47為如實施例1至36中任一項之方法，其中R y為環丙基。 Embodiment 47 is the method of any one of embodiments 1-36, wherein Ry is cyclopropyl.
實施例48為如實施例1至36中任一項之方法，其中R y為環戊基。 Embodiment 48 is the method of any one of embodiments 1-36, wherein Ry is cyclopentyl.
實施例49為如實施例1至36中任一項之方法，其中R
y為環丙基、環丁基、環戊基、環丙基甲基、1-環丙基乙基、2-環丙基乙基、環丁基甲基或環戊基甲基。
Embodiment 49 is the method as any one of
實施例50為如實施例1至36中任一項之方法，其中R
y為單環雜環基或-O-單環雜環基，其視情況經一個、兩個或三個R
o取代基取代。
實施例51為如實施例1至36中任一項之方法，其中R
y為視情況經取代之四氫呋喃基、四氫哌喃基、氧雜環丁基、吖呾基、吡咯啶基、哌啶基、𠰌啉基、哌𠯤基或氧雜環丁基氧基。
Embodiment 51 is the method of any one of
實施例52為如實施例1至36中任一項之方法，其中R y為氧雜環丁基或氧雜環丁基氧基。 Embodiment 52 is the method of any one of embodiments 1-36, wherein Ry is oxetanyl or oxetanyloxy.
實施例53為如實施例1至36中任一項之方法，其中R y為視情況經一個、兩個或三個R o取代基取代之單環雜環烷基。 Embodiment 53 is the method of any one of embodiments 1-36, wherein R y is monocyclic heterocycloalkyl optionally substituted with one, two, or three R o substituents.
實施例54為如實施例1至36中任一項之方法，其中R
y視情況經一或兩個R
o取代基取代，且R
o為甲基。
Embodiment 54 is the method of any one of
實施例55為如實施例1至33中任一項之方法，其中R x為甲基，且R y為甲基、乙基、環丙基、甲氧基或環戊基。 Embodiment 55 is the method of any one of embodiments 1-33, wherein R x is methyl, and R y is methyl, ethyl, cyclopropyl, methoxy, or cyclopentyl.
實施例56為如實施例1至33中任一項之方法，其中R x及R y與其所連接之氮一起形成視情況經C 1-4烷基取代之單環雜環烷基。 Embodiment 56 is the method of any one of embodiments 1-33, wherein R x and R y together with the nitrogen to which they are attached form an optionally C 1-4 alkyl substituted monocyclic heterocycloalkyl.
實施例57為如實施例1至33中任一項之方法，其中R x及R y與其所連接之氮一起形成視情況經C 1-4烷基取代之單環雜環基。 Embodiment 57 is the method of any one of embodiments 1-33, wherein R x and R y together with the nitrogen to which they are attached form an optionally C 1-4 alkyl substituted monocyclic heterocyclyl.
實施例58為如實施例1至33中任一項之方法，其中R
x及R
y與其所連接之氮一起形成吖呾基、吡咯啶基、哌啶基、哌𠯤基、𠰌啉基、硫代𠰌啉基、6-氧雜-1-氮雜螺[3.3]庚基或2-氧雜-6-氮雜螺[3.3]庚基，其各自視情況經甲基取代。
Embodiment 58 is the method of any one of
實施例59為如前述實施例中任一項之方法，其中各R z獨立地為C 1-4烷基、鹵基、-OH、-OC 1-4烷基、C 1-4烷基NR mR n或-NR mR n。 Embodiment 59 is the method of any one of the preceding embodiments, wherein each R z is independently C 1-4 alkyl, halo, -OH, -OC 1-4 alkyl, C 1-4 alkyl, NR m R n or -NR m R n .
實施例60為如實施例1至58中任一項之方法，其中各R
z獨立地為甲基、-OH、鹵基或-OCH
3。
實施例61為如實施例1至58中任一項之方法，其中R z為經 -NR mR n取代之C 2-3烷基。 Embodiment 61 is the method of any one of embodiments 1-58, wherein Rz is C2-3 alkyl substituted with -NRmRn .
實施例62為如前述實施例中任一項之方法，其中R m及R n各自獨立地為H或C 1-4烷基。 Embodiment 62 is the method of any one of the preceding embodiments, wherein R m and R n are each independently H or C 1-4 alkyl.
實施例63為如實施例1至61中任一項之方法，其中R m及R n各自為甲基。 Embodiment 63 is the method of any one of embodiments 1-61, wherein R m and R n are each methyl.
實施例64為如實施例1至61中任一項之方法，其中R
m及R
n與其所連接之氮一起形成視情況經一或兩個R
o取代基取代之單環雜環基。
Embodiment 64 is the method of any one of
實施例65為如實施例1至61中任一項之方法，其中R
m及R
n與其所連接之氮一起形成吡咯啶、哌啶、哌𠯤、𠰌啉、硫代𠰌啉或硫代𠰌啉-1,1-二氧化物，其各自視情況經一或兩個R
o取代基取代。
Embodiment 65 is the method of any one of
實施例66為如實施例1至61中任一項之方法，其中R
m及R
n與其所連接之氮一起形成吡咯啶、哌啶、哌𠯤或𠰌啉，其各自視情況經一或兩個R
o取代基取代。
Embodiment 66 is the method of any one of
實施例67為如前述實施例中任一項之方法，其中R o取代基為C 1-4烷基。 Embodiment 67 is the method of any one of the preceding embodiments, wherein the R substituent is C 1-4 alkyl.
實施例68為如實施例1至66中任一項之方法，其中R o取代基為-OH。 Embodiment 68 is the method of any one of embodiments 1-66, wherein the R o substituent is -OH.
實施例69為如實施例1至66中任一項之方法，其中R o取代基為-NR pR q。 Embodiment 69 is the method of any one of embodiments 1-66, wherein the R o substituent is -NR p R q .
實施例70為如前述實施例中任一項之方法，其中R p及R q各自獨立地為H或甲基。 Embodiment 70 is the method of any one of the preceding embodiments, wherein R p and R q are each independently H or methyl.
實施例71為如實施例1至69中任一項之方法，其中R p及R q與其所連接之氮一起形成雜環基。 Embodiment 71 is the method of any one of embodiments 1-69, wherein Rp and Rq together with the nitrogen to which they are attached form a heterocyclyl.
實施例72為如實施例1至69中任一項之方法，其中R
p及R
q與其所連接之氮一起形成吖呾基、吡咯啶基、哌啶基、哌𠯤基、𠰌啉基、硫代𠰌啉基、6-氧雜-1-氮雜螺[3.3]庚基或2-氧雜-6-氮雜螺[3.3]庚基。
Embodiment 72 is the method of any one of
實施例73為如前述實施例中任一項之方法，其中R 5為H、甲基、乙基、氯、溴、氟、-OH或-OCH 3。 Embodiment 73 is the method of any one of the preceding embodiments, wherein R5 is H, methyl, ethyl, chloro, bromo, fluoro, -OH, or -OCH3 .
實施例74為如實施例1至72中任一項之方法，其中R 5為H。 Embodiment 74 is the method of any one of embodiments 1-72, wherein R 5 is H.
實施例75為如前述實施例中任一項之方法，其中該化合物係選自表1之化合物或其醫藥學上可接受之鹽。
實施例76為如實施例1至75中任一項之方法，其中與該化合物之碳原子連接之一或多個氫原子經氘原子置換。Embodiment 76 is the method of any one of
實施例77為如前述實施例中任一項之方法，其中該化合物及/或該醫藥學上可接受之鹽係呈醫藥組合物形式。Embodiment 77 is the method of any one of the preceding embodiments, wherein the compound and/or the pharmaceutically acceptable salt is in the form of a pharmaceutical composition.
實施例78為如實施例77之方法，其中該醫藥組合物進一步包含醫藥學上可接受之賦形劑。Embodiment 78 is the method according to embodiment 77, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
相關申請案之交互參考Cross-references to related applications
本申請案主張2021年6月8日提交之美國臨時申請案第63/208,456號之優先權，該申請案之揭示內容以全文引用之方式併入本文中。This application claims priority to U.S. Provisional Application No. 63/208,456, filed June 8, 2021, the disclosure of which is incorporated herein by reference in its entirety.
本發明提供用於治療冠狀病毒感染之方法及組合物。在一些實施例中，本發明提供用呈磷酸肌醇激酶抑制劑，特定言之含FYVE型指之磷酸肌醇激酶(「PIKfyve」)抑制劑之化合物來阻斷α-冠狀病毒、β-冠狀病毒譜系B或β-冠狀病毒譜系A進入宿主細胞的方法及組合物。在一些實施例中，本發明提供用呈磷酸肌醇激酶抑制劑，特定言之PIKfyve抑制劑之化合物來預防及/或治療由α-冠狀病毒、β-冠狀病毒譜系B或β-冠狀病毒譜系A引起之感染的方法及組合物。The present invention provides methods and compositions for treating coronavirus infection. In some embodiments, the present invention provides for the use of compounds that are phosphoinositide kinase inhibitors, specifically phosphoinositide kinase ("PIKfyve") inhibitors containing the FYVE type designation, to block alpha-coronaviruses, beta-coronaviruses, Methods and compositions for virus lineage B or betacoronavirus lineage A entry into host cells. In some embodiments, the present invention provides compounds that are phosphoinositide kinase inhibitors, in particular PIKfyve inhibitors, for the prevention and/or treatment of infections caused by alpha-coronaviruses, beta-coronavirus lineage B or beta-coronavirus lineages Methods and compositions for infection caused by A.
現將詳細參考本發明之某些實施例。雖然將結合所描述之實施例描述本發明，但應理解，此類描述並不意欲將本發明限制於彼等實施例。相反，本發明意欲涵蓋所有替代方案、修改及等效物，其可如所附申請專利範圍所限定包括在本發明內。Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the described embodiments, it will be understood that such description is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the invention as defined by the appended claims.
在詳細描述本發明教示內容之前，應理解，本發明不限於特定組合物或方法步驟，因此可加以改變。應注意，除非上下文另外明確規定，否則如本說明書及所附申請專利範圍中所用，單數形式「一(a/an)」及「該(the)」包括複數個參考物。因此，舉例而言，提及「一界面活性劑」包括複數種界面活性劑及其類似者。Before the present teachings are described in detail, it is to be understood that this invention is not limited to particular compositions or method steps as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a/an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a surfactant" includes a plurality of surfactants and the like.
數值範圍包括限定該範圍之數字。考慮到有效數位及與量測有關之誤差，所量測及可量測值應理解為大致的。此外，「包含(comprise/comprises/comprising)」、「含有(contain/contains/ containing)」、「包括(include/includes/included/including)」之使用並不意欲為限制性的。應理解，前述一般描述與詳細描述僅為例示性及解釋性的且並不限制教示。Numerical ranges include the numbers defining the range. Measured and measurable values are to be understood as approximate, taking into account significant digits and errors associated with measurements. Furthermore, the use of "comprise/comprises/comprising", "contain/contains/containing", "include/includes/included/including" is not intended to be limiting. It is to be understood that the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive in teaching.
除非說明書中具體指出，否則本說明書中敍述「包含」各種組分之實施例亦設想為「由所敍述組分組成」或「基本上由所敍述組分組成」；本說明書中敍述「由各種組分組成」之實施例亦設想為「包含」所敍述組分或「基本上由所敍述組分組成」；且本說明書中敍述「基本上由各種組分組成」之實施例亦設想為「由所敍述組分組成」或「包含」所敍述組分(此互換性並不適用於此等術語在申請專利範圍中之使用)。Unless otherwise specified in the specification, the embodiments described in the specification as "comprising" various components are also contemplated as "consisting of the stated components" or "consisting essentially of the stated components"; The embodiments of "consisting of components" are also contemplated as "comprising" the recited components or "consisting essentially of the recited components"; Consists of" or "comprises" the stated components (this interchangeability does not apply to the use of these terms in the claims).
本文所用之章節標題僅出於組織目的而不應解釋為以任何方式限制所需主題。在以引用之方式併入之任何文獻與本說明書中定義之任何術語矛盾的情況下，以本說明書為准。雖然本發明教示係與各種實施例結合描述，但並不意欲使本發明教示限於此類實施例。相反，如熟習此項技術者應瞭解，本發明教示涵蓋各種替代方案、修改及等效物。 I.定義 The section headings used herein are for organizational purposes only and should not be construed as limiting the desired topic in any way. In the event that any term defined in this specification is contradicted by any document incorporated by reference, this specification controls. While the present teachings are described in connection with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings cover various alternatives, modifications and equivalents, as should be understood by those skilled in the art. I. Definition
除非本文中另外定義，否則本文所用之科學及技術術語具有一般熟習此項技術者通常所理解之含義。在發生任何潛在分岐之情況下，本文所提供之定義優先於任何辭典或外來定義。除非上下文另外需要，否則單數術語應包括複數且複數術語應包括單數。Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the event of any potential disagreement, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
除非另外陳述，否則在說明書及申請專利範圍中使用之以下術語係出於本發明之目的定義且具有以下含義。Unless otherwise stated, the following terms used in the specification and claims are defined for the purpose of the present invention and have the following meanings.
如本文所用，術語「約」係指在如一般熟習此項技術者所判定之特定值或組成之可接受的誤差範圍內之值或組成，該誤差範圍將部分地取決於如何量測或測定該值或組成，亦即，量測系統之侷限性。舉例而言，「約」或「大致」可意謂根據此項技術中之實踐在一個或超過一個標準差內。可替代地，「約」或「大致」可意謂高達10% (亦即，±10%)或更大的範圍，此視量測系統之侷限性而定。舉例而言，約5 mg可包括4.5 mg與5.5 mg之間的任何數值。此外，尤其在生物系統或過程方面，該術語可意謂值之至多一個數量級或至多5倍。當本發明提供特定值或組成時，除非另外說明，否則「約」或「大致」之含義應假定在該特定值或組成之可接受誤差範圍內。除非本文另有規定，否則「或」係依包括性意義使用，亦即，等效於「及/或」。As used herein, the term "about" refers to a value or composition within an acceptable error range for a particular value or composition as judged by one of ordinary skill in the art, which will depend in part on how measured or determined The value or composition, that is, the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more than one standard deviation, according to the practice in the art. Alternatively, "about" or "approximately" can mean up to 10% (ie, ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg can include any value between 4.5 mg and 5.5 mg. Furthermore, especially in terms of biological systems or processes, the term can mean up to an order of magnitude, or up to 5 times, a value. When a particular value or composition is provided herein, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition. Unless otherwise specified herein, "or" is used in an inclusive sense, ie, is equivalent to "and/or".
如本文所用，術語「及/或」應視為所規定特徵或組分中之各者及另一者或不存在另一者之特定揭示內容。舉例而言，如本文之諸如「A及/或B」之片語中所用，術語「及/或」意欲包括「A及B」、「A或B」、「A」(單獨)及「B」(單獨)。同樣，如在諸如「A、B及/或C」之片語中使用的術語「及/或」意欲涵蓋以下態樣中之各者：A、B及C；A、B或C；A或C；A或B；B或C；A及C；A及B；B及C；A (單獨)；B (單獨)；及C (單獨)。如本文所用，術語「或其組合(or a combination thereof/or combinations thereof)」係指在該術語前之所列舉術語之任何及所有排列與組合。舉例而言，「A、B、C或其組合」意欲包括以下中之至少一者：A、B、C、AB、AC、BC或ABC，且若在特定情況下順序為重要的，則亦包括BA、CA、CB、ACB、CBA、BCA、BAC或CAB。繼續此實例，明確地包括含有一或多個項目或術語之重複的組合，諸如BB、AAA、AAB、BBC、AAABCCCC、CBBAAA、CABABB等。熟習此項技術者應理解，除非另外自上下文顯而易見，否則通常不存在對任何組合中之項目或術語之數目的限制。As used herein, the term "and/or" shall be considered a specific disclosure of each of the stated features or components with the other or the absence of the other. For example, as used herein in phrases such as "A and/or B", the term "and/or" is intended to include "A and B", "A or B", "A" (alone) and "B "(alone). Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). As used herein, the term "or a combination thereof/or combinations thereof" refers to any and all permutations and combinations of the listed terms preceding that term. For example, "A, B, C, or a combination thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular case, then also Includes BA, CA, CB, ACB, CBA, BCA, BAC or CAB. Continuing with the example, combinations containing repetitions of one or more terms or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Those skilled in the art will appreciate that there is generally no limit to the number of items or terms in any combination, unless otherwise apparent from the context.
如本文所用，術語「個體」及「患者」係指人類及非人類動物，包括脊椎動物、哺乳動物及非哺乳動物。在一個實施例中，個體可為人類、非人類靈長類動物、猿猴、猿、鼠類(例如小鼠及大鼠)、牛科動物、豬科動物、馬科動物、犬科動物、貓科動物、山羊、狼科動物、蛙科動物或魚科動物。As used herein, the terms "individual" and "patient" refer to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject can be a human, non-human primate, simian, ape, murine (e.g., mouse and rat), bovine, porcine, equine, canine, cat goats, goats, wolves, frogs or fishes.
術語「投與(administering/administered)」及文法變化形式係指使用熟習此項技術者已知之多種方法及遞送系統中之任一者將藥劑以物理方式引入個體。本文所揭示之調配物之例示性投與途徑包括靜脈內、肌肉內、皮下、腹膜內、脊柱或其他非經腸投與途徑，例如藉由注射或輸注。如本文所用，片語「非經腸投與」意謂除經腸及局部投與以外之投與模式，通常藉由注射，且包括但不限於靜脈內、肌肉內、動脈內、鞘內、***內、病灶內、囊內、眶內、心內、皮內、腹膜內、經氣管、皮下、表皮下、關節內、囊下、蛛膜下、脊柱內、硬膜外及胸骨內注射及輸注，以及活體內電穿孔。在一個實施例中，調配物係經由非-非經腸途徑，例如經口投與。其他非-非經腸途徑包括局部、表皮或經黏膜投與途徑，例如鼻內、經***、經直腸、舌下或局部。投與亦可例如執行一次、複數次及/或經一或多個延長之時段。The terms "administering/administered" and grammatical variants refer to the physical introduction of an agent into an individual using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, eg, by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, Intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal and infusion, and in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route, eg, orally. Other non-parenteral routes include topical, topical or transmucosal administration routes, eg, intranasal, vaginal, rectal, sublingual or topical. Dosing can also be performed, for example, once, multiple times and/or over one or more extended periods of time.
如本文所用，術語「治療(treat/treating/treatment)」涵蓋對個體之疾病/病症之任何治療性投與或施加，且包括抑制疾病/病症、遏制其發展、緩解疾病/病症之一或多個症狀、或治癒疾病/病症。As used herein, the term "treat/treating/treatment" encompasses any therapeutic administration or application of a disease/condition in an individual, and includes inhibiting the disease/condition, arresting its development, alleviating one or more of the disease/condition a symptom, or cure a disease/disorder.
如本文所用，術語「預防(prevent/preventing)」意謂抑制或遏制視為無疾病/病症之個體發展疾病/病症。As used herein, the term "prevent/preventing" means inhibiting or suppressing the development of a disease/disorder in an individual considered to be free of the disease/disorder.
如本文所用，提及病毒進入宿主細胞，術語「阻斷(block/blocking)」係指終止一些或所有病毒(諸如冠狀病毒)進入一或多種宿主細胞。阻斷可為完全或部分的。部分阻斷包括預防至少一些病毒進入宿主細胞，例如足夠病毒以預防個體顯示至少一個與此類病毒感染相關之症狀。As used herein, in reference to virus entry into host cells, the term "blocking/blocking" refers to stopping entry of some or all viruses, such as coronaviruses, into one or more host cells. Blocking can be complete or partial. Partial blockade includes preventing at least some virus from entering a host cell, eg, enough virus to prevent an individual from displaying at least one symptom associated with such viral infection.
術語「有效量」、「治療有效量」或「有效劑量」或相關術語可互換使用，且係指當向個體投與時足夠影響與病毒感染相關之疾病或病症之可量測改善或預防之所描述的PIKfyve抑制劑的量。The terms "effective amount", "therapeutically effective amount" or "effective dose" or related terms are used interchangeably and refer to an amount sufficient to effect a measurable amelioration or prevention of a disease or condition associated with a viral infection when administered to a subject. The amount of PIKfyve inhibitor described.
出於治療目的之「醫藥學上可接受之媒劑」為可向個體投與之物理實施例。醫藥學上可接受之媒劑包括丸劑、膠囊、膠囊型錠劑、錠劑、口服液、注射液、噴霧劑、氣霧劑、糖衣錠、膳食補充劑、乳膏、洗劑、油劑、溶液、糊劑、散劑、蒸汽或液體，但不限於此等。醫藥學上可接受之媒劑之實例為緩衝等張溶液，諸如磷酸鹽緩衝鹽水(phosphate buffered saline；PBS)。A "pharmaceutically acceptable vehicle" for therapeutic purposes is the physical embodiment that can be administered to a subject. Pharmaceutically acceptable vehicles include pills, capsules, caplets, lozenges, oral liquids, injections, sprays, aerosols, dragees, dietary supplements, creams, lotions, oils, solutions , paste, powder, vapor or liquid, but not limited thereto. An example of a pharmaceutically acceptable vehicle is a buffered isotonic solution, such as phosphate buffered saline (PBS).
「烷基」意謂一至六個碳原子之直鏈飽和單價烴基或三至六個碳原子之分支鏈飽和單價烴基，例如甲基、乙基、丙基、2-丙基、丁基(包括所有異構形式)、戊基(包括所有異構形式)及其類似基團。"Alkyl" means a straight-chain saturated monovalent hydrocarbon group with one to six carbon atoms or a branched saturated monovalent hydrocarbon group with three to six carbon atoms, such as methyl, ethyl, propyl, 2-propyl, butyl (including all isomeric forms), pentyl (including all isomeric forms) and the like.
除非另外陳述，否則「伸烷基」意謂一至六個碳原子之直鏈飽和二價烴基或三至六個碳原子之分支鏈飽和二價烴基，例如亞甲基、伸乙基、伸丙基、1-甲基伸丙基、2-甲基伸丙基、伸丁基、伸戊基及其類似基團。Unless otherwise stated, "alkylene" means a straight chain saturated divalent hydrocarbon group of one to six carbon atoms or a branched chain saturated divalent hydrocarbon group of three to six carbon atoms, such as methylene, ethylene, propylene 1-methylpropylenyl, 2-methylpropylenyl, butyl, pentylene and the like.
「烷基磺醯基」意謂-SO 2R基團，其中R為如上文所定義之烷基；例如甲磺醯基、乙磺醯基及其類似基團。 "Alkylsulfonyl" means a -SO2R group where R is alkyl as defined above; for example methylsulfonyl, ethylsulfonyl and the like.
「胺基」意謂-NH 2。 "Amino" means -NH 2 .
「烷氧基」意謂-OR基團，其中R為如上文所定義之烷基；例如甲氧基、乙氧基、丙氧基或2-丙氧基、正丁氧基、異丁氧基或三級丁氧基及其類似基團。"Alkoxy" means an -OR group in which R is alkyl as defined above; for example methoxy, ethoxy, propoxy or 2-propoxy, n-butoxy, isobutoxy group or tertiary butoxy group and similar groups.
「烷氧基烷基」意謂經如上文所定義之烷氧基(在一個實施例中，一或兩個烷氧基)取代之一至六個碳原子之直鏈單價烴基或三至六個碳原子之分支鏈單價烴基，例如2-甲氧基乙基、1-甲氧基丙基、2-甲氧基丙基或3-甲氧基丙基、2-乙氧基乙基，及其類似基團。"Alkoxyalkyl" means a straight chain monovalent hydrocarbon group of one to six carbon atoms substituted by an alkoxy group as defined above (in one embodiment, one or two alkoxy groups) or three to six Branched chain monovalent hydrocarbon groups of carbon atoms, such as 2-methoxyethyl, 1-methoxypropyl, 2-methoxypropyl or 3-methoxypropyl, 2-ethoxyethyl, and its similar groups.
「烷氧羰基」意謂-C(O)OR基團，其中R為如上文所定義之烷基；例如甲氧羰基、乙氧羰基及其類似基團。"Alkoxycarbonyl" means a -C(O)OR group where R is alkyl as defined above; eg methoxycarbonyl, ethoxycarbonyl and the like.
「醯基」意謂-COR基團，其中R為烷基、鹵烷基或環烷基；例如乙醯基、丙醯基、環丙基羰基及其類似基團。當R為烷基時，該基團在本文中亦稱為烷基羰基。"Acyl" means a -COR group in which R is alkyl, haloalkyl, or cycloalkyl; for example, acetyl, propionyl, cyclopropylcarbonyl, and the like. When R is an alkyl group, the group is also referred to herein as an alkylcarbonyl group.
「環烷基」意謂三至十個碳原子之環狀飽和單價烴基，其中一或兩個碳原子可經側氧基置換；例如環丙基、環丁基、環戊基或環己基，及其類似基團。"Cycloalkyl" means a cyclic saturated monovalent hydrocarbon radical of three to ten carbon atoms, one or two of which may be replaced by a pendant oxygen group; for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and similar groups.
「羧基」意謂-COOH。"Carboxy" means -COOH.
「冠狀病毒」、「冠狀呼吸道病毒」或「CoV」在本文中可互換使用，係指屬於冠狀病毒科( Coronaviridae)之病毒。冠狀病毒為大致31 Kb之有套膜正義RNA病毒，使得此等病毒成為已知最大的RNA病毒。冠狀病毒感染多種宿主物種，包括人類及若干其他脊椎動物。此等病毒主要引起呼吸道及腸道感染且誘導廣泛範圍的臨床表現。大體而言，冠狀病毒可分類為低病原性CoV (包括人類CoV (hCoV))及高病原性CoV，諸如嚴重急性呼吸症候群CoV (SARS-CoV)及中東呼吸症候群CoV (MERS-CoV)。低病原性hCoV感染上呼吸道且在健康個體中引起季節性輕度至中度感冒狀呼吸道疾病。相比之下，高病原性hCoV (病原性hCoV)感染下呼吸道且引起嚴重肺炎，其有時導致致命性急性肺損傷(acute lung injury；ALI)及急性呼吸窘迫症候群(acute respiratory distress syndrome；ARDS)，導致高發病率及死亡率。 "Coronavirus", "coronavirus" or "CoV" are used interchangeably herein to refer to viruses belonging to the Coronaviridae family. Coronaviruses are enveloped positive-sense RNA viruses of approximately 31 Kb, making these viruses the largest known RNA viruses. Coronaviruses infect a variety of host species, including humans and several other vertebrates. These viruses mainly cause respiratory and intestinal infections and induce a wide range of clinical manifestations. In general, coronaviruses can be classified into low pathogenic CoVs, including human CoVs (hCoVs), and highly pathogenic CoVs, such as Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV). Low pathogenicity hCoVs infect the upper respiratory tract and cause seasonal mild to moderate cold-like respiratory illness in healthy individuals. In contrast, highly pathogenic hCoV (pathogenic hCoV) infects the lower respiratory tract and causes severe pneumonia, which sometimes leads to fatal acute lung injury (ALI) and acute respiratory distress syndrome (ARDS ), resulting in high morbidity and mortality.
「鹵基」意謂氟、氯、溴或碘。"Halo" means fluorine, chlorine, bromine or iodine.
「鹵烷基」意謂如上文所定義之烷基，其經一個或一至五個鹵素原子(在一個實施例中氟或氯)取代，包括經不同鹵素取代之彼等者，例如-CH 2Cl、-CF 3、-CHF 2、-CH 2CF 3、-CF 2CF 3、-CF(CH 3) 2及其類似基團。當烷基僅經氟取代時，其在本發明中可稱為氟烷基。 "Haloalkyl" means an alkyl group as defined above substituted with one or one to five halogen atoms (in one embodiment fluorine or chlorine), including those substituted with different halogens, for example -CH2 Cl, -CF 3 , -CHF 2 , -CH 2 CF 3 , -CF 2 CF 3 , -CF(CH 3 ) 2 and the like. When an alkyl group is substituted only with fluorine, it may be referred to as a fluoroalkyl group in the present invention.
「鹵烷氧基」意謂-OR基團，其中R為如上文所定義之鹵烷基；例如-OCF 3、-OCHF 2及其類似者。當R為其中烷基僅經氟基取代之鹵烷基時，其在本發明中可稱為氟烷氧基。 "Haloalkoxy" means an -OR group in which R is haloalkyl as defined above; eg -OCF3 , -OCHF2 and the like. When R is a haloalkyl group in which the alkyl group is substituted only with fluoro groups, it may be referred to as a fluoroalkoxy group in the present invention.
「羥烷基」意謂經一或兩個羥基取代之一至六個碳原子之直鏈單價烴基或三至六個碳原子之分支鏈單價烴基，其限制條件為若存在兩個羥基，則其不均在同一碳原子上。代表性實例包括但不限於羥甲基、2-羥乙基、2-羥丙基、3-羥丙基、1-(羥甲基)-2-甲基丙基、2-羥丁基、3-羥丁基、4-羥丁基、2,3-二羥丙基、1-(羥甲基)-2-羥乙基、2,3-二羥丁基、3,4-二羥丁基及2-(羥甲基)-3-羥丙基。其他實例包括但不限於2-羥乙基、2,3-二羥丙基及1-(羥甲基)-2-羥乙基。"Hydroxyalkyl" means a straight chain monovalent hydrocarbon radical of one to six carbon atoms or a branched chain monovalent hydrocarbon radical of three to six carbon atoms substituted by one or two hydroxyl groups, with the proviso that if two hydroxyl groups are present, the not even on the same carbon atom. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxy Butyl and 2-(hydroxymethyl)-3-hydroxypropyl. Other examples include, but are not limited to, 2-hydroxyethyl, 2,3-dihydroxypropyl, and 1-(hydroxymethyl)-2-hydroxyethyl.
「雜環基」意謂4至10個環原子之飽和或不飽和單價單環或雙環基團(稠合雙環或橋聯雙環或螺環化合物)，其中一或兩個環原子為選自N、O及S(O) n之雜原子，其中n為0至2之整數，其餘環原子為C。另外，雜環基環中之一或兩個環碳原子可視情況經-CO-基團置換。更具體言之，術語雜環基包括但不限於氧雜環丁基、吡咯啶基、哌啶基、高哌啶基、2-側氧基吡咯啶基、2-側氧基哌啶基、(N-𠰌啉基)、哌𠯤基、四氫哌喃基、N-硫代𠰌啉基、六氫吡咯并[1,2-a]吡𠯤-6(2H)-酮-基、四氫-1H-㗁唑并[3,4-a]吡𠯤-3(5H)-酮-基、5,6,7,8-四氫-[1,2,4]***并[4,3-a]吡𠯤-基、3-氧雜-8-氮雜雙環[3.2.1]辛烷-基、6-氧雜-1-氮雜螺[3.3]庚基、2-氧雜-6-氮雜螺[3.3]庚基及其類似基團。當雜環基環不飽和時，其可含有一或兩個環雙鍵，其限制條件為環不為芳香族環。 "Heterocyclyl" means a saturated or unsaturated monovalent monocyclic or bicyclic group (fused bicyclic or bridged bicyclic or spirocyclic compounds) of 4 to 10 ring atoms, one or two of which are selected from N , O and heteroatoms of S(O) n , wherein n is an integer from 0 to 2, and the remaining ring atoms are C. Additionally, one or both of the ring carbon atoms in the heterocyclyl ring can optionally be replaced by a -CO- group. More specifically, the term heterocyclyl includes, but is not limited to, oxetanyl, pyrrolidinyl, piperidinyl, homopiperidinyl, 2-oxopyrrolidinyl, 2-oxopiperidinyl, (N-𠰌-olinyl), piper-𠯤-yl, tetrahydropyranyl, N-thio-𠰌-linyl, hexahydropyrrolo[1,2-a]pyrrolo[1,2-a]pyrrolo-6(2H)-one-yl, tetrahydropyrrolo[1,2-a]pyrrolo-6(2H)-on-yl Hydrogen-1H-oxazolo[3,4-a]pyr-3(5H)-one-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4, 3-a] pyryrone-yl, 3-oxa-8-azabicyclo[3.2.1]octane-yl, 6-oxa-1-azaspiro[3.3]heptyl, 2-oxa- 6-azaspiro[3.3]heptyl and analogs thereof. When a heterocyclyl ring is unsaturated it may contain one or two ring double bonds, provided that the ring is not aromatic.
「雜環基烷基」或「雜環烷基」意謂-(伸烷基)-R基團，其中R為如上文所定義之雜環基環；例如四氫呋喃基甲基、哌𠯤基甲基、𠰌啉基乙基及其類似基團。"Heterocyclylalkyl" or "heterocycloalkyl" means a -(alkylene)-R group where R is a heterocyclyl ring as defined above; for example tetrahydrofuranylmethyl, piperoxymethyl group, 𠰌linylethyl group and similar groups.
「雜環胺基」意謂具有4至8個環原子之飽和或不飽和單價單環基團，其中一或兩個環原子為選自N、O或S(O) n之雜原子，其中n為0至2之整數，其餘環原子為C，其限制條件為環原子中之至少一者為N。另外，雜環胺基環中之一或兩個環碳原子可視情況經-CO-基團置換。當雜環胺基環不飽和時，其可含有一或兩個環雙鍵，其限制條件為環不為芳香族環。 "Heterocyclic amine" means a saturated or unsaturated monovalent monocyclic group having 4 to 8 ring atoms, one or two of which are heteroatoms selected from N, O or S(O) n , wherein n is an integer from 0 to 2, and the remaining ring atoms are C, provided that at least one of the ring atoms is N. In addition, one or both of the ring carbon atoms in the heterocyclic amino ring may optionally be replaced by a -CO- group. When the heterocyclic amine ring is unsaturated, it may contain one or two ring double bonds, provided that the ring is not aromatic.
「雜環胺基烷基」意謂-(伸烷基)-R基團，其中R為如上文所描述之雜環胺基。"Heterocycloaminoalkyl" means a -(alkylene)-R group where R is a heterocyclylamino group as previously described.
「雜芳基」意謂5至10個環原子之單價單環或雙環芳基，其中一或多個(在一個實施例中，一個、兩個或三個)環原子為選自N、O及S之雜原子，其餘環原子為碳。代表性實例包括但不限於吡咯基、噻吩基、噻唑基、咪唑基、呋喃基、吲哚基、異吲哚基、㗁唑基、異㗁唑基、苯并噻唑基、苯并㗁唑基、喹啉基、異喹啉基、吡啶基、嘧啶基、吡𠯤基、嗒𠯤基、***基、四唑基及其類似基團。"Heteroaryl" means a monovalent monocyclic or bicyclic aryl group of 5 to 10 ring atoms, wherein one or more (in one embodiment, one, two or three) ring atoms are selected from N, O and heteroatoms of S, and the remaining ring atoms are carbon. Representative examples include, but are not limited to, pyrrolyl, thienyl, thiazolyl, imidazolyl, furyl, indolyl, isoindolyl, oxazolyl, isoxazolyl, benzothiazolyl, benzoxazolyl , quinolinyl, isoquinolyl, pyridyl, pyrimidyl, pyridyl, pyridyl, triazolyl, tetrazolyl and the like.
「側氧基」意謂=(O)基團且「羰基」意謂＞C(O)基團。"Pendant oxy" means a =(O) group and "carbonyl" means a >C(O) group.
術語「視情況(optional/optionally)」意謂隨後描述之事件或情形可能發生但非必須發生，且該描述包括其中事件或情形發生之情況及不發生之情況。舉例而言，「視情況經烷基取代之雜環基」意謂烷基可能存在但非必須存在，且該描述包括雜環基經烷基取代之情況及雜環基未經烷基取代之情況。The terms "optional/optionally" mean that the subsequently described event or circumstance may but not necessarily occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "heterocyclyl optionally substituted with alkyl" means that an alkyl group may but need not be present, and the description includes both cases where the heterocyclyl is substituted with an alkyl group and where the heterocyclyl is not substituted with an alkyl group Condition.
本文所用之片語「醫藥學上可接受」係指彼等化合物、材料、組合物及/或劑型在合理醫學判斷之範疇內，適用於接觸人類及動物之組織而無過度毒性、刺激、過敏反應或其他問題或併發症，與合理的益處/風險比相稱。The phrase "pharmaceutically acceptable" as used herein means that those compounds, materials, compositions and/or dosage forms are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergy, within the scope of sound medical judgment reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
如本文所用，片語「醫藥學上可接受之賦形劑」或「醫藥學上可接受之載劑」意謂醫藥學上可接受之材料、組合物或媒劑，諸如液體或固體填充劑、稀釋劑、賦形劑、溶劑或包封材料。各載劑在與調配物之其他成分相容且對患者無害的意義上必須為「可接受的」。可充當醫藥學上可接受之載劑之材料之一些實例包括：(1)糖，諸如乳糖、葡萄糖及蔗糖；(2)澱粉，諸如玉米澱粉及馬鈴薯澱粉；(3)纖維素及其衍生物，諸如羧甲基纖維素鈉、乙基纖維素及乙酸纖維素；(4)粉末狀黃蓍膠；(5)麥芽；(6)明膠；(7)滑石；(8)賦形劑，諸如可可脂及栓劑蠟；(9)油，諸如花生油、棉籽油、紅花油、芝麻油、橄欖油、玉米油及大豆油；(10)二醇，諸如丙二醇；(11)多元醇，諸如甘油、山梨糖醇、甘露醇及聚乙二醇；(12)酯，諸如油酸乙酯及月桂酸乙酯；(13)瓊脂；(14)緩衝劑，諸如氫氧化鎂及氫氧化鋁；(15)褐藻酸；(16)無熱原水；(17)等張鹽水；(18)林格氏溶液(Ringer's solution)；(19)乙醇；(20)磷酸鹽緩衝液；及(21)醫藥調配物中採用之其他無毒相容性物質。As used herein, the phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler , diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives , such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, Sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) ) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) pharmaceutical formulations Other non-toxic compatible substances used in
「PIKfyve抑制劑」係指抑制磷脂酸肌醇3-磷酸5-激酶(PIKfyve)之分子。"PIKfyve inhibitor" refers to a molecule that inhibits phosphatidylinositol 3-phosphate 5-kinase (PIKfyve).
術語「鹽」或「醫藥學上可接受之鹽」係指衍生自此項技術中熟知的多種有機及無機相對離子之鹽。醫藥學上可接受之酸加成鹽可用無機酸及有機酸形成。可衍生鹽之無機酸包括例如鹽酸、氫溴酸、硫酸、硝酸、磷酸及其類似者。可衍生鹽之有機酸包括例如乙酸、丙酸、乙醇酸、丙酮酸、草酸、順丁烯二酸、丙二酸、丁二酸、反丁烯二酸、酒石酸、檸檬酸、苯甲酸、肉桂酸、杏仁酸、甲磺酸、乙磺酸、對甲苯磺酸、水楊酸及其類似者。醫藥學上可接受之鹼加成鹽可用無機鹼及有機鹼形成。可衍生鹽之無機鹼包括例如鈉、鉀、鋰、銨、鈣、鎂、鐵、鋅、銅、錳、鋁及其類似者。可衍生鹽之有機鹼包括例如一級胺、二級胺及三級胺、經取代之胺，包括天然存在之經取代之胺、環胺、鹼性離子交換樹脂以及其類似者，具體言之，諸如異丙胺、三甲胺、二乙胺、三乙胺、三丙胺及乙醇胺。在一些實施例中，醫藥學上可接受之鹼加成鹽係選自銨鹽、鉀鹽、鈉鹽、鈣鹽及鎂鹽。應理解，醫藥學上可接受之鹽為無毒的。關於合適的醫藥學上可接受之鹽的額外資訊可見於 Remington's Pharmaceutical Sciences, 第17版, Mack Publishing Company, Easton, PA,1985中，其以引用之方式併入本文中。 II. 治療方法、用途、投與及醫藥組合物 The term "salt" or "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamon acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, in particular, Such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium and magnesium salts. It is understood that pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences , 17th Edition, Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference. II. Methods of treatment, uses, administration and pharmaceutical compositions
本文提供治療由病毒引起之感染、抑制病毒進入宿主細胞或抑制病毒膜與宿主細胞膜融合之方法，其包含向有需要之個體投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。Provided herein are methods of treating an infection caused by a virus, inhibiting entry of a virus into a host cell, or inhibiting fusion of a viral membrane with a host cell membrane, comprising administering to an individual in need thereof a compound of formula (I) described herein, or any of its embodiments One (such as the compounds in Table 1).
在一個態樣中，本發明係關於一種阻斷α-冠狀病毒進入宿主細胞及預防由α-冠狀病毒引起之感染之方法，其包含向有需要之個體投與本文所描述之式(I)化合物或其實施例中之任一者。在一些實施例中，α-冠狀病毒為HCoV 229E。In one aspect, the present invention relates to a method of blocking α-coronavirus from entering host cells and preventing infection caused by α-coronavirus, which comprises administering the formula (I) described herein to an individual in need thereof Any one of the compound or its embodiments. In some embodiments, the alphacoronavirus is HCoV 229E.
在另一態樣中，本發明係關於一種阻斷β-冠狀病毒譜系B進入宿主細胞及預防由β-冠狀病毒譜系B引起之感染之方法，其包含向有需要之個體投與本文所描述之式(I)化合物或其實施例中之任一者。在一些實施例中，β-冠狀病毒譜系B為SARS-CoV2。在一些實施例中，由SARS-CoV-2引起之感染為COVID-19。在一些實施例中，該等方法治療或預防COVID-19感染。In another aspect, the invention relates to a method of blocking entry of betacoronavirus lineage B into a host cell and preventing infection caused by betacoronavirus lineage B comprising administering to an individual in need thereof a method described herein A compound of formula (I) or any one of its embodiments. In some embodiments, the lineage B betacoronavirus is SARS-CoV2. In some embodiments, the infection caused by SARS-CoV-2 is COVID-19. In some embodiments, the methods treat or prevent COVID-19 infection.
在另一態樣中，本發明係關於一種阻斷β-冠狀病毒譜系A進入宿主細胞及預防由β-冠狀病毒譜系A引起之感染之方法，其包含向有需要之個體投與本文所描述之式(I)化合物或其實施例中之任一者。在一些實施例中，β-冠狀病毒譜系A為HCoV OC43。In another aspect, the present invention relates to a method of blocking entry of β-coronavirus lineage A into a host cell and preventing infection caused by β-coronavirus lineage A comprising administering to an individual in need thereof a method described herein A compound of formula (I) or any one of its embodiments. In some embodiments, the betacoronavirus lineage A is HCoV OC43.
在一些實施例中，提供一種抑制冠狀病毒病毒膜與早期內體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與成熟化內體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與晚期內體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與內溶酶體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與溶酶體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與早期巨肌體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與巨肌體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與晚期巨肌體膜融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與內質網(ER)融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在一些實施例中，提供一種抑制冠狀病毒病毒膜與質膜直接融合之方法，其包含投與本文所描述之式(I)化合物或其實施例中之任一者(例如表1之化合物)。在各此類實施例中，冠狀病毒可為α-冠狀病毒、β-冠狀病毒譜系B或β-冠狀病毒譜系A。In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and an early endosomal membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as a compound in Table 1 ). In some embodiments, there is provided a method of inhibiting fusion of a coronavirus viral membrane with a mature endosomal membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as those in Table 1) compound). In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and a late endosomal membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as a compound in Table 1 ). In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and an endolysosomal membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as those in Table 1) compound). In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and a lysosomal membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as a compound in Table 1 ). In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and an early giant muscle membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as a compound in Table 1 ). In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and a giant muscle membrane, comprising administering any one of the compounds of formula (I) described herein or any of its embodiments (such as the compounds in Table 1) . In some embodiments, there is provided a method of inhibiting the fusion of a coronavirus viral membrane and a late giant muscle membrane, comprising administering a compound of formula (I) described herein or any one of its embodiments (such as a compound in Table 1 ). In some embodiments, there is provided a method of inhibiting the fusion of the coronavirus membrane and the endoplasmic reticulum (ER), which comprises administering any one of the compounds of formula (I) described herein or any of its embodiments (such as Table 1 compounds). In some embodiments, there is provided a method of inhibiting the direct fusion of the coronavirus membrane and the plasma membrane, which comprises administering the compound of formula (I) described herein or any one of its embodiments (such as the compound in Table 1) . In various such embodiments, the coronavirus can be an alpha-coronavirus, a beta-coronavirus lineage B, or a beta-coronavirus lineage A.
大體而言，本發明化合物將藉由提供類似效用之藥劑的任一可接受投與模式、以治療有效量投與。In general, compounds of the invention will be administered in therapeutically effective amounts by any acceptable mode of administration for agents that afford similar utility.
大體而言，本發明化合物將呈醫藥組合物形式，藉由以下途徑中之任一者投與：經口、全身性(例如經皮、鼻內或藉由栓劑)或非經腸(例如肌肉內、靜脈內或皮下)投與。在一些實施例中，投與方式為使用適宜日劑量方案之經鼻，該方案可根據病痛程度加以調節。組合物可採取錠劑、丸劑、膠囊、半固體、散劑、持續釋放調配物、溶液、懸浮液、酏劑、氣霧劑或任何其他適當組合物之形式。In general, the compounds of the invention will be in the form of pharmaceutical compositions administered by any of the following routes: oral, systemic (e.g., transdermally, intranasally, or via suppositories), or parenterally (e.g., intramuscularly). Intravenously, intravenously or subcutaneously). In some embodiments, administration is nasal using a suitable daily dosage regimen that can be adjusted according to the severity of the affliction. The compositions may take the form of tablets, pills, capsules, semi-solids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable composition.
可使用一或多種包含賦形劑及助劑之醫藥學上可接受之載劑調配醫藥組合物。調配物可視所選投與途徑而進行修改。醫藥組合物亦可包括呈游離鹼形式或醫藥學上可接受之鹽形式的本文所描述之化合物。Pharmaceutical compositions can be formulated using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries. The formulation can be modified depending on the chosen route of administration. Pharmaceutical compositions can also include compounds described herein in free base form or in pharmaceutically acceptable salt form.
用於調配醫藥組合物之方法可包括將本文所描述之化合物中之任一者與一或多種惰性、醫藥學上可接受之賦形劑或載劑調配以形成固體、半固體或液體組合物。固體組合物可包括例如散劑、錠劑、分散性顆粒及膠囊，且在一些態樣中，固體組合物進一步含有無毒性輔助物質，例如潤濕劑或乳化劑、pH緩衝劑及其他醫藥學上可接受之添加劑。可替代地，可以將本文所描述之組合物凍乾或使其呈粉末形式以供在使用前，用合適的媒劑(例如無菌無熱原水)復原。活性成分可包覆於例如藉由凝聚技術或藉由界面聚合製備之微膠囊(例如，分別為羥甲基纖維素或明膠微膠囊及聚-(甲基丙烯酸甲酯)微膠囊)、膠態藥物遞送系統(例如脂質體、白蛋白微球體、微乳液、奈米粒子及奈米膠囊)或***液中。Methods for formulating pharmaceutical compositions can include formulating any of the compounds described herein with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semi-solid or liquid compositions . Solid compositions may include, for example, powders, lozenges, dispersible granules, and capsules, and in some aspects, solid compositions further contain nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically useful substances. Acceptable additives. Alternatively, the compositions described herein can be lyophilized or presented in powder form for reconstitution with a suitable vehicle, eg sterile pyrogen-free water, before use. The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), colloidal Drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions.
可將醫藥組合物及調配物滅菌。可藉由經由無菌過濾進行過濾來實現滅菌。Pharmaceutical compositions and formulations can be sterilized. Sterilization can be achieved by filtration through sterile filtration.
本文所描述之醫藥組合物可經調配以作為注射液投與。注射用調配物之非限制性實例可包括於油性或水性媒劑中之無菌懸浮液、溶液或乳液。合適的油性媒劑可包括但不限於親脂性溶劑或媒劑(諸如脂肪油)、合成脂肪酸酯或脂質體。水性注射懸浮液可含有增加懸浮液之黏度的物質。懸浮液亦可含有合適的穩定劑。注射液可經調配以供彈丸注射或連續輸注。The pharmaceutical compositions described herein can be formulated for administration as injections. Non-limiting examples of formulations for injection may include sterile suspensions, solutions or emulsions in oily or aqueous vehicles. Suitable oily vehicles may include, but are not limited to, lipophilic solvents or vehicles such as fatty oils, synthetic fatty acid esters, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension. The suspension may also contain suitable stabilizers. Injectable solutions can be formulated for bolus injection or continuous infusion.
對於非經腸投與而言，可以單位劑量可注射形式(例如溶液、懸浮液、乳液)與醫藥學上可接受之非經腸媒劑結合來調配化合物。此類媒劑可為本質上無毒性的且非治療性的。媒劑可為水、鹽水、林格氏溶液、右旋糖溶液及5%人類血清白蛋白。亦可使用非水性媒劑，諸如不揮發性油及油酸乙酯。可使用脂質體作為載劑。媒劑可含有少量添加劑，諸如增強等張性及化學穩定性之物質(例如緩衝劑及防腐劑)。For parenteral administration, the compounds may be formulated in unit dosage injectable forms (eg, solutions, suspensions, emulsions) in combination with a pharmaceutically acceptable parenteral vehicle. Such vehicles can be non-toxic and non-therapeutic in nature. Vehicles can be water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles, such as fixed oils and ethyl oleate, can also be used. Liposomes can be used as carriers. The vehicle may contain minor amounts of additives such as substances which enhance isotonicity and chemical stability (eg buffers and preservatives).
亦可製備持續釋放製劑。持續釋放基質之實例可包括聚酯、水凝膠(例如聚(2-羥乙基-甲基丙烯酸酯)或聚(乙烯醇))、聚乳酸交酯、L-麩胺酸與γ乙基-L-麩胺酸酯之共聚物、不可降解的乙烯-乙酸乙烯酯、諸如LUPRON DEPO TM(亦即，由乳酸-乙醇酸共聚物及乙酸亮丙立德(leuprolide 乙酸酯)構成之可注射微球體)的可降解的乳酸-乙醇酸共聚物及聚-D-(-)-3-羥基丁酸。 Sustained release formulations may also be prepared. Examples of sustained release matrices may include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide, L-glutamic acid, and gamma ethyl - Copolymers of L-glutamate, non-degradable ethylene-vinyl acetate, such as LUPRON DEPO TM (i.e., polymers composed of lactic-co-glycolic acid and leuprolide acetate) Injectable microspheres) of degradable lactic-co-glycolic acid and poly-D-(-)-3-hydroxybutyric acid.
本文所描述之組合物之醫藥調配物可藉由將化合物與醫藥學上可接受之載劑、賦形劑及/或穩定劑混合來製備以供儲存。此調配物可為凍乾調配物或水溶液。可接受之載劑、賦形劑及/或穩定劑可在所用劑量及濃度下對接受者無毒。可接受載劑、賦形劑及/或穩定劑可包括緩衝劑，諸如磷酸鹽、檸檬酸鹽及其他有機酸；抗氧化劑，包括抗壞血酸及甲硫胺酸；防腐劑，多肽；蛋白質，諸如血清白蛋白或明膠；親水性聚合物；胺基酸；單醣、雙醣及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑，諸如EDTA；糖，諸如蔗糖、甘露醇、海藻糖或山梨糖醇；成鹽相對離子，諸如鈉；金屬錯合物；及/或非離子界面活性劑或聚乙二醇。Pharmaceutical formulations of the compositions described herein can be prepared for storage by mixing the compound with a pharmaceutically acceptable carrier, excipient and/or stabilizer. This formulation can be a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients and/or stabilizers can be nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers, excipients, and/or stabilizers may include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, polypeptides; proteins, such as serum Albumin or gelatin; hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or Sorbitol; a salt-forming counterion such as sodium; a metal complex; and/or a nonionic surfactant or polyethylene glycol.
本發明化合物可用於與一或多種其他組合藥劑(例如一種、兩種或三種其他藥物)組合治療之方法中，該等其他組合藥劑用於預防、治療、控制、改善或降低本發明化合物適用的疾病或病狀之風險。在一些實施例中，相比於任一單獨藥物，將藥物組合在一起更安全或更有效。在一些實施例中，本文所揭示之化合物及一或多種組合藥劑具有不會不利地影響彼此之互補活性。此類分子可以對預期目的有效之量存在於組合中。此類其他藥物可藉由因此常用之途徑且以因此常用之量與本發明化合物同時或依序投與。當本發明化合物與一或多種其他藥物同時使用時，在一些實施例中，藥劑在單一醫藥組合物中以單位劑型一起投與。因此，本發明之醫藥組合物亦包括除本發明化合物以外，含有一或多種其他活性成分之彼等醫藥組合物。本發明化合物與第二活性劑之重量比可以改變且將視各成分之有效劑量而定。大體而言，將使用各自之有效劑量。在一些實施例中，組合療法包括其中分別投與本發明化合物及一或多種其他藥物且在一些情況下，針對不同、重疊時程投與兩種或更多種藥劑之療法。亦考慮當與一或多種其他活性成分組合使用時，本發明化合物及其他活性成分可以比各自單獨使用時之劑量更低的劑量使用。The compounds of the present invention may be used in methods of combination therapy with one or more other combination agents (e.g., one, two or three other drugs) for preventing, treating, controlling, ameliorating or reducing the Risk of disease or condition. In some embodiments, the combination of drugs is safer or more effective than either drug alone. In some embodiments, a compound disclosed herein and one or more combination agents have complementary activities that do not adversely affect each other. Such molecules can be present in combination in amounts effective for the intended purpose. Such other drugs may be administered simultaneously or sequentially with the compounds of the invention by such usual routes and in such usual amounts. When a compound of the invention is used concomitantly with one or more other drugs, in some embodiments, the agents are administered together in unit dosage form in a single pharmaceutical composition. Accordingly, the pharmaceutical compositions of the present invention also include those containing one or more other active ingredients in addition to the compounds of the present invention. The weight ratio of the compound of the invention to the second active agent may vary and will depend on the effective dosage of each ingredient. In general, an effective dose of each will be used. In some embodiments, combination therapy includes therapy in which a compound of the invention and one or more other drugs are administered separately and, in some cases, two or more agents are administered on different, overlapping time courses. It is also contemplated that when used in combination with one or more other active ingredients, the compounds of the invention and the other active ingredients may be used in lower dosages than when each is used alone.
本發明之化合物、醫藥組合物及方法可適用於治療個體之感染及預防個體感染，該個體諸如但不限於哺乳動物、人類、非人類哺乳動物、馴養動物(例如實驗室動物、家養寵物或家畜)、非馴養動物(例如野生動物)、狗、貓、嚙齒動物、小鼠、倉鼠、牛、鳥、雞、魚、豬、馬、山羊、綿羊或兔。在較佳實施例中，本發明之化合物、醫藥組合物及方法用於治療人類。The compounds, pharmaceutical compositions and methods of the invention are useful for treating and preventing infections in subjects such as, but not limited to, mammals, humans, non-human mammals, domesticated animals (e.g., laboratory animals, domestic pets, or livestock) ), non-domesticated animals (such as wild animals), dogs, cats, rodents, mice, hamsters, cows, birds, chickens, fish, pigs, horses, goats, sheep or rabbits. In preferred embodiments, the compounds, pharmaceutical compositions and methods of the invention are used to treat humans.
本文所描述之化合物、醫藥組合物及方法可適用作為治療性或預防性的，例如可向有需要之個體投與之治療或預防性。治療性或預防性作用可藉由預防(完全或部分)、降低、抑制、緩解或根除疾病狀態(包括但不限於其症狀)而在個體中獲得。患有疾病或病狀或預處置以患有或開始患有疾病或病狀之個體中之治療作用可藉由降低、抑制、預防、緩解或根除病況或疾病或預病狀或預疾病狀態來獲得。The compounds, pharmaceutical compositions and methods described herein may be suitable as therapeutic or prophylactic, eg, may be administered to a subject in need thereof. A therapeutic or prophylactic effect may be obtained in an individual by preventing (completely or partially), reducing, inhibiting, alleviating or eradicating a disease state (including but not limited to its symptoms). Therapeutic effect in an individual having a disease or condition or preconditioning to have or begin to have a disease or condition may be by reducing, inhibiting, preventing, alleviating or eradicating the condition or disease or pre-condition or pre-disease state get.
在實踐本文所描述之方法中，可向有需要之個體投與治療有效量的本文所描述之化合物或醫藥組合物，通常用於治療及/或預防其病狀或進展。醫藥組合物可影響個體之生理，諸如免疫系統、發炎反應或其他生理影響。治療有效量可視疾病嚴重程度、個體之年齡及相對健康狀況、所用化合物之效力及其他因素而廣泛變化。In practicing the methods described herein, a therapeutically effective amount of a compound or pharmaceutical composition described herein can be administered to a subject in need thereof, generally for the treatment and/or prevention of the condition or progression thereof. Pharmaceutical compositions can affect the physiology of an individual, such as the immune system, inflammatory response, or other physiological effects. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the individual, the potency of the compound used, and other factors.
治療(Treat/treating)可以指在治療或改善疾病或病狀中成功之任何標誌。治療可包括例如降低、延緩或緩解疾病或病狀之一或多種症狀的嚴重程度，或其可包括降低患者經歷疾病、缺陷、病症或不利病狀及其類似者之症狀的頻率。治療可在本文中用於指引起疾病或病狀的一定程度之治療或改善之方法且可涵蓋一系列針對該目的之結果，包括但不限於完全預防病狀。Treating may refer to any indication of success in treating or ameliorating a disease or condition. Treatment may include, for example, reducing, delaying or alleviating the severity of one or more symptoms of a disease or condition, or it may include reducing the frequency with which a patient experiences symptoms of a disease, defect, disorder or adverse condition, and the like. Treatment may be used herein to refer to methods that result in some degree of treatment or amelioration of a disease or condition and may encompass a range of outcomes for that purpose, including but not limited to complete prevention of the condition.
預防(Prevent/preventing)及其類似者可以指預防患者之疾病或病狀。舉例而言，若處於感染疾病風險下之個體用本發明之方法治療且之後不感染該疾病，則在該個體中至少在一段時間內預防了該疾病。在一些實施例中，本文所描述之PIKfyve抑制劑可預防冠狀病毒感染。在一些實施例中，本文所描述之PIKfyve抑制劑可治療冠狀病毒感染。Prevent/preventing and the like can refer to preventing a disease or condition in a patient. For example, if an individual at risk of contracting a disease is treated with the methods of the invention and subsequently does not contract the disease, the disease is prevented in that individual, at least for a period of time. In some embodiments, the PIKfyve inhibitors described herein prevent coronavirus infection. In some embodiments, the PIKfyve inhibitors described herein treat coronavirus infection.
治療有效量可為足以向組合物所投與之個體提供有益作用或以其他方式減少有害的非有益事件的化合物或醫藥組合物或其活性組分之量。治療有效劑量可為產生一或多種其中劑量所投與之所需或合乎需要(例如有益)作用的劑量，此類投與在給定時間段內發生一或多次。精確劑量可視治療目的而定，且可由熟習此項技術者使用已知技術確定。A therapeutically effective amount may be that amount of a compound or pharmaceutical composition, or active ingredient thereof, sufficient to provide a beneficial effect to the subject to which the composition is administered or otherwise reduce deleterious non-beneficial events. A therapeutically effective dose may be that dose that produces one or more desired or desirable (eg, beneficial) effects of the dose in which such administration occurs one or more times within a given period of time. The precise dosage will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques.
可用於本文所描述之方法及用途中之本文所描述之化合物或醫藥組合物可考慮待治療之病症、個別患者之病狀、化合物或醫藥組合物之遞送部位、投與方法及醫師已知之其他因素，以與良好醫學實踐相符之方式調配且劑量確立。可根據本文所描述之製備描述來製備化合物或醫藥組合物。The compounds or pharmaceutical compositions described herein that are useful in the methods and uses described herein may take into account the condition to be treated, the condition of the individual patient, the site of delivery of the compound or pharmaceutical composition, the method of administration, and others known to the physician. factors, formulated and dosages established in a manner consistent with good medical practice. A compound or pharmaceutical composition can be prepared according to the preparative descriptions described herein.
一般熟習此項技術者應瞭解，向有需要之個體投與本文所描述之醫藥組合物或化合物的量、持續時間及頻率視若干因素而定，該等因素包括例如但不限於個體之健康狀況、患者之特定疾病或病狀、患者之特定疾病或病狀之級別或程度、個體正投與或已投與之額外療法及其類似者。Those of ordinary skill in the art will appreciate that the amount, duration, and frequency of administering a pharmaceutical composition or compound described herein to an individual in need thereof will depend on a number of factors, including, for example, but not limited to, the individual's health status , the patient's particular disease or condition, the grade or extent of the patient's particular disease or condition, additional therapy that the individual is administering or has been administered, and the like.
本文所描述之方法、用途、化合物及醫藥組合物可以用於向有需要之個體投與。通常，化合物或醫藥組合物之投與可包括投與途徑，投與途徑之非限制性實例包括靜脈內、動脈內、皮下、硬膜下、肌肉內、顱內、胸骨內、腫瘤內或腹膜內。另外，醫藥組合物或化合物可藉由額外投藥途徑，例如藉由吸入、經口、經皮、鼻內或鞘內投與而向個體投與。The methods, uses, compounds and pharmaceutical compositions described herein can be used for administration to individuals in need thereof. In general, administration of a compound or pharmaceutical composition may include a route of administration, non-limiting examples of which include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrasternal, intratumoral, or peritoneal Inside. Additionally, a pharmaceutical composition or compound may be administered to a subject by additional routes of administration, for example, by inhalation, oral, transdermal, intranasal or intrathecal administration.
本發明之醫藥組合物或化合物可在第一次投與及一或多次額外投與中向有需要之個體投與。一或多次額外投與可在第一次投與後數分鐘、數小時、數天、數週或數月向有需要之個體投與。額外投與中之任一者可在第一次投與之後少於21天，或少於14天、少於10天、少於7天、少於4天或少於1天向有需要之個體投與。一或多次投與可出現超過每天一次，超過每週一次或超過每月一次。化合物或醫藥組合物可以21天、14天、10天、7天、4天之週期或在一至七天之時段內每天向有需要之個體投與。A pharmaceutical composition or compound of the invention can be administered to an individual in need thereof in a first administration and one or more additional administrations. One or more additional administrations can be administered to individuals in need minutes, hours, days, weeks or months after the first administration. Any of the additional administrations may be given to those in need less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days, or less than 1 day after the first administration. individual investment. The one or more administrations may occur more than once a day, more than once a week or more than once a month. The compound or pharmaceutical composition can be administered daily to an individual in need thereof in cycles of 21 days, 14 days, 10 days, 7 days, 4 days or over a period of one to seven days.
本文所提供之化合物、醫藥組合物及方法可適用於治療複數種疾病或病狀或預防個體之疾病或病狀，或用於有需要之個體的其他治療性應用。 III.可用本發明組合物治療及預防之病毒 The compounds, pharmaceutical compositions, and methods provided herein may be useful for treating a plurality of diseases or conditions or preventing a disease or condition in an individual, or for other therapeutic applications in an individual in need thereof. III. Viruses that can be treated and prevented with the compositions of the present invention
可投與本文所描述之PIKfyve抑制劑以治療及/或預防某些冠狀病毒感染。The PIKfyve inhibitors described herein can be administered to treat and/or prevent certain coronavirus infections.
冠狀病毒屬於病毒目、冠狀病毒科及冠狀病毒亞科。其基因分類為四個屬：α冠狀病毒( Alphacoronavirus)、β冠狀病毒( Betacoronavirus)、γ冠狀病毒( Gammacoronavirus)及δ冠狀病毒( Deltacoronavirus)。Mahendra等人, Cureus, 12(3):e7423 (2020)。α冠狀病毒及β冠狀病毒通常感染哺乳動物，而γ冠狀病毒及δ冠狀病毒主要感染鳥類。在一些實施例中，冠狀病毒為α冠狀病毒、β冠狀病毒、γ冠狀病毒或δ冠狀病毒。 Coronaviruses belong to the order Viridae, the family Coronaviridae and the subfamily Coronaviridae. Its genes are classified into four genera: Alphacoronavirus , Betacoronavirus , Gammacoronavirus and Deltacoronavirus . Mahendra et al., Cureus, 12(3):e7423 (2020). Alphacoronaviruses and betacoronaviruses usually infect mammals, while gammacoronaviruses and deltacoronaviruses mainly infect birds. In some embodiments, the coronavirus is an alphacoronavirus, betacoronavirus, gammacoronavirus, or deltacoronavirus.
與人類疾病相關之α冠狀病毒株包括HCoV-229E及HCoV-NL63。(同上) α冠狀病毒株之其他實例包括貓科動物感染性腹膜炎病毒(FIPV)、犬科動物冠狀病毒(CCoV)、豬科動物呼吸道冠狀病毒(PRCV)、豬流行性下痢病毒(PEDV)、傳染性胃腸炎病毒(TGEV)、菊頭蝠屬( Rhinolophus)蝙蝠冠狀病毒(Rh-BatCoV) HKU2、長翼蝠屬( Miniopterus)蝙蝠冠狀病毒(Mi-BatCoV) HKU8、Mi-BatCoV 1A、Mi-BatCoV 1B、果蝠屬(Rousettus)蝙蝠冠狀病毒(Ro-BatCoV) HKU10183A、葉鼻蝠屬(Hipposideros)蝙蝠冠狀病毒(Hi-BatCoV) HKU10 LSH5A及黃蝠屬(Scotiphilus)蝙蝠冠狀病毒(Sc-BatCoV) 512。Lau等人, J. Virol. 86(21):11906-11918 (2012)；Woo等人, Viruses, 2(8):1804-1820 (2010)。在一些實施例中，冠狀病毒為HCoV-229E或HCoV-NL63。在其他實施例中，冠狀病毒為FIPV、CCoV、PRCV、PEDV、TGEV、Rh-BatCoV HKU2、Mi-BatCoV HKU8、Mi-BatCoV 1A、Mi-BatCoV 1B、Ro-BatCoV HKU10183A、Hi-BatCoV HKU10 LSH5A及Sc-BatCoV 512。 Alphacoronavirus strains associated with human disease include HCoV-229E and HCoV-NL63. (supra) Other examples of alphacoronavirus strains include feline infectious peritonitis virus (FIPV), canine coronavirus (CCoV), porcine respiratory coronavirus (PRCV), porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Rhinolophus bat coronavirus (Rh-BatCoV) HKU2, Miniopterus bat coronavirus (Mi-BatCoV) HKU8, Mi-BatCoV 1A, Mi- BatCoV 1B, Rousettus bat coronavirus (Ro-BatCoV) HKU10183A, leaf-nosed bat (Hi-BatCoV) HKU10 LSH5A, and yellow bat (Scotiphilus) bat coronavirus (Sc-BatCoV) ) 512. Lau et al., J. Virol. 86(21):11906-11918 (2012); Woo et al., Viruses, 2(8):1804-1820 (2010). In some embodiments, the coronavirus is HCoV-229E or HCoV-NL63. In other embodiments, the coronavirus is FIPV, CCoV, PRCV, PEDV, TGEV, Rh-BatCoV HKU2, Mi-BatCoV HKU8, Mi-BatCoV 1A, Mi-BatCoV 1B, Ro-BatCoV HKU10183A, Hi-BatCoV HKU10 LSH5A and Sc-BatCoV 512.
β冠狀病毒分成四個子組：埃貝冠狀病毒( Embecovirus) (譜系A)、薩貝冠狀病毒( Sarbecovirus) (譜系B)、莫貝冠狀病毒( Merbecovirus) (譜系C)及諾貝冠狀病毒( Nobecovirus) (譜系D)。Woo等人, Viruses, 2(8):1804-1820 (2010)。埃貝冠狀病毒(譜系A)之實例包括牛科動物冠狀病毒(BCoV)、人類冠狀病毒OC43 (HCoV-OC43)、HCoV-HKU1、豬科動物血球凝集腦脊髓炎病毒(PHEV)、長頸鹿冠狀病毒(GiCoV)及鼠類肝炎病毒(MHV)。同上。薩貝冠狀病毒(譜系B)之實例包括SARS-CoV-1及SARSr-Rh-BatCoV HKU3。同上。莫貝冠狀病毒(譜系C)之實例包括MERS-CoV、竹蝠屬(Tylonycteris)蝙蝠冠狀病毒(Ty-BatCoV) HKU4及家蝠屬(Pipistrellus)蝙蝠冠狀病毒(Pi-BatCoV) HKU5。同上。諾貝冠狀病毒(譜系D)之實例包括Ro-BatCoV HKU9。同上。與人類疾病相關之β冠狀病毒株包括SARS-CoV-1、HCoV-OC43、HCoV-HKU1及MERS-CoV。 Betacoronaviruses are divided into four subgroups: Embecovirus (lineage A), Sarbecovirus (lineage B), Merbecovirus (lineage C) and Nobecovirus (lineage C) ) (Pedigree D). Woo et al., Viruses, 2(8):1804-1820 (2010). Examples of Ebecoronavirus (lineage A) include bovine coronavirus (BCoV), human coronavirus OC43 (HCoV-OC43), HCoV-HKU1, porcine hemagglutinating encephalomyelitis virus (PHEV), giraffe coronavirus (GiCoV) and murine hepatitis virus (MHV). Ditto. Examples of Sabe coronaviruses (lineage B) include SARS-CoV-1 and SARSr-Rh-BatCoV HKU3. Ditto. Examples of Mobe coronaviruses (lineage C) include MERS-CoV, Tylonycteris bat coronavirus (Ty-BatCoV) HKU4 and Pipistrellus bat coronavirus (Pi-BatCoV) HKU5. Ditto. Examples of Norbecoronaviruses (lineage D) include Ro-BatCoV HKU9. Ditto. Betacoronavirus strains associated with human disease include SARS-CoV-1, HCoV-OC43, HCoV-HKU1, and MERS-CoV.
在一些實施例中，冠狀病毒引起上呼吸道疾病、下呼吸道疾病、發熱、喉嚨痛、腺腫脹、伴隨次要或主要症狀之感冒、不適、肌肉及關節疼痛、噁心、嘔吐、食慾不振、肺炎、繼發性細菌感染、支氣管炎、呼吸困難、腹瀉、呼吸短促、急性呼吸窘迫症候群、細胞介素風暴、多器官衰竭、敗血性休克、血凝塊、嗅覺喪失或味覺喪失。在一些實施例中，冠狀病毒不會引起任何症狀。In some embodiments, the coronavirus causes upper respiratory illness, lower respiratory illness, fever, sore throat, swollen glands, cold with secondary or primary symptoms, malaise, muscle and joint pain, nausea, vomiting, loss of appetite, pneumonia, Secondary bacterial infection, bronchitis, difficulty breathing, diarrhea, shortness of breath, acute respiratory distress syndrome, cytokine storm, multiple organ failure, septic shock, blood clots, loss of smell or taste. In some embodiments, the coronavirus does not cause any symptoms.
在一些實施例中，投與本發明化合物以阻斷α-冠狀病毒進入宿主細胞。在一些實施例中，投與本發明化合物以阻斷β-冠狀病毒譜系B進入宿主細胞。在一些實施例中，投與本發明化合物以阻斷β-冠狀病毒譜系A進入宿主細胞。在一些實施例中，投與本發明化合物以預防個體感染α-冠狀病毒。在一些實施例中，投與本發明化合物以預防個體感染β-冠狀病毒譜系B。在一些實施例中，投與本發明化合物以預防個體感染β-冠狀病毒譜系A。在一些實施例中，投與本發明化合物以阻斷α-冠狀病毒進入宿主細胞及預防個體感染α-冠狀病毒。在一些實施例中，投與本發明化合物以阻斷β-冠狀病毒譜系B進入宿主細胞，預防個體感染β-冠狀病毒譜系B。在一些實施例中，投與本發明化合物以阻斷β-冠狀病毒譜系A進入宿主細胞，預防個體感染β-冠狀病毒譜系A。在一些實施例中，α-冠狀病毒為HCoV 229E。在一些實施例中，β-冠狀病毒譜系B為SARS-CoV2。在一些實施例中，β-冠狀病毒譜系A為HCoV OC43。在一些實施例中，感染係由SARS-CoV-2引起，且感染為COVID-19。 IV. PIKfyve抑制劑化合物 In some embodiments, compounds of the invention are administered to block alphacoronavirus entry into host cells. In some embodiments, compounds of the invention are administered to block lineage B betacoronavirus entry into host cells. In some embodiments, compounds of the invention are administered to block lineage A betacoronavirus entry into host cells. In some embodiments, a compound of the invention is administered to prevent infection of an individual with an alphacoronavirus. In some embodiments, a compound of the invention is administered to prevent infection of an individual with lineage B betacoronaviruses. In some embodiments, a compound of the invention is administered to prevent infection of an individual with lineage A betacoronaviruses. In some embodiments, compounds of the invention are administered to block alphacoronavirus entry into host cells and prevent infection of an individual with alphacoronaviruses. In some embodiments, a compound of the invention is administered to block entry of a betacoronavirus lineage B into a host cell, preventing infection of an individual with a betacoronavirus lineage B. In some embodiments, a compound of the invention is administered to block entry of betacoronavirus lineage A into a host cell, preventing infection of a subject with betacoronavirus lineage A. In some embodiments, the alphacoronavirus is HCoV 229E. In some embodiments, the lineage B betacoronavirus is SARS-CoV2. In some embodiments, the betacoronavirus lineage A is HCoV OC43. In some embodiments, the infection is caused by SARS-CoV-2 and the infection is COVID-19. IV. PIKfyve Inhibitor Compounds
在一些情況下，本文所描述之化合物可以非鏡像異構物、鏡像異構物或其他立體異構形式存在。除非特定指示特定立體化學或異構形式，否則呈個別形式及其混合物之所有對掌性、非鏡像異構性、外消旋形式均在本發明之範疇內。含有經不對稱取代之原子的本發明化合物可以光學活性、光學增濃、光學純或外消旋形式分離。此項技術中熟知如何製備光學活性形式，諸如藉由材料之解析來製備。立體異構物之分離可藉由層析或藉由形成非鏡像異構物及藉由再結晶或層析分離或其任何組合來執行。(Jean Jacques, Andre Collet, Samuel H. Wilen, 「Enantiomers, Racemates and Resolutions」, John Wiley and Sons, Inc., 1981，關於該揭示內容，以引用的方式併入本文中)。立體異構物亦可藉由立體選擇性合成來獲得。In some cases, compounds described herein may exist as diastereomers, enantiomers, or other stereoisomeric forms. Unless a particular stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic forms, both individually and in mixtures thereof, are within the scope of the invention. Compounds of the invention containing asymmetrically substituted atoms may be isolated in optically active, optically enriched, optically pure or racemic forms. It is well known in the art how to prepare optically active forms, such as by analysis of materials. Separation of stereoisomers can be performed by chromatography or by formation of diastereomers and separation by recrystallization or chromatography or any combination thereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley and Sons, Inc., 1981, incorporated herein by reference for this disclosure). Stereoisomers may also be obtained by stereoselective synthesis.
某些式(I)化合物(或本文所描述之其實施例中之任一者)及/或其醫藥學上可接受之鹽可以互變異構物及/或幾何異構物之形式存在。呈個別形式及其混合物之所有可能互變異構物及順式及反式異構物均在本發明之範疇內。舉例而言，如下文所示之吡唑互變異構物為等效結構。一種此類結構之描述意欲涵蓋兩種結構。
另外，如本文所用，術語烷基包括該烷基之所有可能的異構形式，即使僅僅闡述了幾個實例。此外，當諸如雜芳基、雜環基之環狀基團經取代時，其包括所有位置異構物。Additionally, as used herein, the term alkyl includes all possible isomeric forms of the alkyl group, even though only a few examples are set forth. Furthermore, when a cyclic group such as heteroaryl, heterocyclyl is substituted, it includes all positional isomers.
式(I)化合物(或本文所描述之其實施例中之任一者)的醫藥學上可接受之鹽在本發明之範疇內。另外，本文所描述之化合物包括化合物或其醫藥學上可接受之鹽的水合物及溶劑合物。Pharmaceutically acceptable salts of compounds of formula (I) (or any of its embodiments described herein) are within the scope of this invention. In addition, the compounds described herein include hydrates and solvates of the compounds or pharmaceutically acceptable salts thereof.
本發明亦包括式(I)化合物(或本文所描述之其實施例中之任一者)之前藥及/或其醫藥學上可接受之鹽。術語前藥意欲代表共價鍵結載劑，當向哺乳動物個體投與前藥時其能夠釋放式(I) (或本文中描述之其實施例中之任一者)之活性成分。活體內發生活性成分之釋放。可藉由熟習此項技術者已知的技術製備前藥。此等技術一般修飾既定化合物中之適當官能基。然而，此等經修飾之官能基活體內或藉由常規操作再生原始官能基。式(I)化合物(或本文所描述之其實施例中之任一者)之前藥包括其中羥基、胺基、羧基或類似基團經修飾之化合物。前藥之實例包括但不限於酯(例如乙酸酯、甲酸酯及苯甲酸酯衍生物)、式(I)化合物中之羥基或胺基官能基之胺基甲酸酯(例如 N,N-二甲胺基羰基)、醯胺(例如三氟乙醯胺基、乙醯胺基及其類似基團)及其類似物。式(I)化合物(或本文所描述之其實施例中之任一者)之前藥及/或其醫藥學上可接受之鹽亦在本發明之範疇內。 The present invention also includes prodrugs of compounds of formula (I) (or any of its embodiments described herein) and/or pharmaceutically acceptable salts thereof. The term prodrug is intended to denote a covalently bonded carrier capable of releasing the active ingredient of formula (I) (or any of the embodiments thereof described herein) when administered to a mammalian subject. Release of the active ingredient occurs in vivo. Prodrugs can be prepared by techniques known to those skilled in the art. These techniques generally modify appropriate functional groups in a given compound. However, such modified functional groups regenerate the original functional groups in vivo or by routine manipulation. Prodrugs of compounds of formula (I) (or any of its embodiments described herein) include compounds in which the hydroxyl, amine, carboxyl or similar groups are modified. Examples of prodrugs include, but are not limited to, esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g. , N, N -dimethylaminocarbonyl), amides (such as trifluoroacetamide, acetamide and the like) and the like. Prodrugs of compounds of formula (I) (or any of its embodiments described herein) and/or pharmaceutically acceptable salts thereof are also within the scope of the present invention.
本發明亦包括式(I)化合物(或本文所描述之其實施例中之任一者)及/或其醫藥學上可接受之鹽之多晶型形式(非晶形以及結晶)及氘化形式。The present invention also includes polymorphic forms (amorphous and crystalline) and deuterated forms of the compound of formula (I) (or any of its embodiments described herein) and/or pharmaceutically acceptable salts thereof .
在一些實施例中，以不同的增濃同位素形式來使用本文所揭示之化合物，例如增濃 2H、 3H、 11C、 13C及/或 14C之含量。在一個特定實施例中，化合物在至少一個位置中經氘化。可藉由美國專利第5,846,514號及第6,334,997號中描述之步驟，來製備此類氘化形式。如美國專利第5,846,514號及第6,334,997號中所描述，氘化可改良代謝穩定性及/或功效，因此增加藥物作用之持續時間。 In some embodiments, the compounds disclosed herein are used in different isotopically enriched forms, such as enriched in 2 H, 3 H, 11 C, 13 C, and/or 14 C. In a particular embodiment, the compound is deuterated in at least one position. Such deuterated forms can be prepared by the procedures described in US Patent Nos. 5,846,514 and 6,334,997. As described in US Patent Nos. 5,846,514 and 6,334,997, deuteration can improve metabolic stability and/or efficacy, thus increasing the duration of drug action.
除非另外陳述，否則本文所描繪之結構意欲包括不同之處僅為存在一或多個同位素增濃原子的化合物。舉例而言，具有本發明結構，但其中氫經氘或氚置換或碳經 13C或 14C增濃碳置換的化合物在本發明之範疇內。 Unless otherwise stated, structures depicted herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structure of the present invention, but wherein the hydrogen is replaced by deuterium or tritium, or the carbon is replaced by a13C or14C enriched carbon are within the scope of the present invention.
本發明化合物視情況在構成此類化合物之一或多個原子處含有非天然比例之原子同位素。舉例而言，化合物可用同位素，諸如氘( 2H)、氚( 3H)、碘-125 ( 125I)或碳-14 ( 14C)標記。經 2H、 11C、 13C、 14C、 15C、 12N、 13N、 15N、 16N、 16O、 17O、 14F、 15F、 16F、 17F、 18F、 33S、 34S、 35S、 36S、 35Cl、 37Cl、 79Br、 81Br及 125I同位素取代均考慮在內。本發明化合物之所有同位素變體無論是否具放射性均涵蓋在本發明之範疇內。 The compounds of the present invention optionally contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, compounds can be labeled with isotopes such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C). 2 H, 11 C, 13 C, 14 C, 15 C, 12 N, 13 N, 15 N, 16 N, 16 O, 17 O, 14 F, 15 F, 16 F, 17 F, 18 F, 33 S, 34 S, 35 S, 36 S, 35 Cl, 37 Cl, 79 Br, 81 Br, and 125 I isotopic substitutions are all considered. All isotopic variations of the compounds of the invention, whether radioactive or not, are encompassed within the scope of the invention.
在某些實施例中，本文所揭示之化合物中之一些或全部 1H原子經 2H原子置換。合成含氘化合物之方法為此項技術中已知的，且包括(僅作為非限制性實例)以下合成方法。 In certain embodiments, some or all of the 1 H atoms in the compounds disclosed herein are replaced with 2 H atoms. Methods of synthesizing deuterium-containing compounds are known in the art and include, by way of non-limiting examples only, the following synthetic methods.
使用諸如描述於以下中之各種方法來合成氘取代的化合物：Dean, Dennis C.編. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000; 6(10)] 2000, 第110頁；George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21；及Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32。Deuterium-substituted compounds were synthesized using various methods such as described in: Dean, Dennis C. eds. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000; 6(10)] 2000, p. 110; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E. Anthony . Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.
氘化起始材料可容易地獲得，且經受本文所描述之合成方法以提供含氘化合物之合成。大量含氘試劑及建構嵌段可購自化學供應商，諸如Aldrich Chemical Co。Deuterated starting materials are readily available and subjected to the synthetic methods described herein to provide the synthesis of deuterium-containing compounds. A number of deuterium-containing reagents and building blocks are commercially available from chemical suppliers such as Aldrich Chemical Co.
在一個態樣中為一種式(I)化合物：
在一些實施例中，X為CH且Y為N。在一些實施例中，X為N且Y為CR a。在一些實施例中，R a為H、甲基、乙基、丙基、異丙基、丁基、異丁基、二級丁基或三級丁基。在一些實施例中，R a為H或甲基。在一些實施例中，R a為H。 In some embodiments, X is CH and Y is N. In some embodiments, X is N and Y is CR a . In some embodiments, Ra is H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, or tertiary butyl. In some embodiments, Ra is H or methyl. In some embodiments, Ra is H.
在一些實施例中，R a或R 1為視情況經取代之苯基。在一些實施例中，R a或R 1為甲苯基。在一些實施例中，R a或R 1為間甲苯基。在一些實施例中，R a或R 1為視情況經取代之單環雜芳基。在一些實施例中，R a或R 1為視情況經取代之吡咯、咪唑、吡唑、***、四唑、呋喃、㗁唑、異㗁唑、噻唑、異噻唑、吡啶、嘧啶、吡𠯤或嗒𠯤。在一些實施例中，R a或R 1為視情況經取代之吡啶或嘧啶。在一些實施例中，R a或R 1為視情況經取代之吡啶。在一些實施例中，R a或R 1為甲基吡啶。在一些實施例中，R a或R 1視情況經一或兩個R d取代基取代。 In some embodiments, R or R is optionally substituted phenyl. In some embodiments, R or R is tolyl. In some embodiments, R or R is m-tolyl. In some embodiments, R or R is an optionally substituted monocyclic heteroaryl. In some embodiments, R or R is optionally substituted pyrrole, imidazole, pyrazole, triazole, tetrazole, furan, oxazole, isoxazole, thiazole, isothiazole, pyridine, pyrimidine, pyridine Or click 𠯤. In some embodiments, R or R is optionally substituted pyridine or pyrimidine. In some embodiments, R or R is optionally substituted pyridine. In some embodiments, R or R is picoline. In some embodiments, R or R are optionally substituted with one or two R substituents.
在一些實施例中，各R d取代基獨立地為C 1-4烷基、C 1-4烯基、C 1-4炔基、-O-C 1-4烷基、鹵基、氰基、硝基、疊氮基、鹵基-C 1-4烷基、-O-C 1-4鹵烷基、-NR gR h、-NR gC(=O)R h、-NR gC(=O)NR gR h、-NR gC(=O)OR h、=NOR g、-NR gS(=O) 1-2R h、-NR gS(=O) 1-2NR gR h、=NSO 2R g、-C(=O)R g、-C(=O)OR g、-OC(=O)OR g、-OC(=O)R g、-C(=O)NR gR h、-OC(=O)NR gR h、-OR g、-SR g、-S(=O)R g、-S(=O) 2R g、-OS(=O) 1-2R g、-S(=O) 1-2OR g、-S(=O) 1-2NR gR h、苯基、-C 1-4烷基-苯基、單環環烷基、-C 1-4烷基-環烷基、單環雜環烷基或單環雜芳基，其中R d之苯基、單環環烷基、單環雜環烷基及單環雜芳基各自視情況經一或兩個取代基R e取代。在一些實施例中，各R d取代基獨立地為C 1-4烷基、鹵基-C 1-4烷基、苯基、-C 1-4烷基-苯基、吡啶基、苯硫基、環烷基或-C 1-4烷基-環烷基，其中苯基、吡啶基及苯硫基各自視情況經一或兩個取代基R e取代。在一些實施例中，各R d取代基獨立地為甲基、乙基、異丙基、-CF 3、-OCH 3、-OCF 3、苯基、吡啶基、苯硫基、苯甲基、環丙基、環丁基、環戊基、環丙基甲基、環丁基甲基或環戊基甲基，其中R d之苯基、環烷基及雜芳基各自視情況經一或兩個取代基R e取代。在一些實施例中，各R d係獨立地選自C 1-4烷基、-CF 3、氟、氯、-OCH 3及-OCF 3。在一些實施例中，R a或R 1經一個R d取代，且R d為C 1-4烷基。在一些實施例中，R a或R 1經一個R d取代且R d為甲基。在一些實施例中，R a或R 1為苯基或吡啶基，其各自視情況經一或兩個選自以下之取代基取代：C 1-4烷基、-CF 3、氟、氯、-OCH 3及-OCF 3。在一些實施例中，R a或R 1為未經取代之苯基或甲苯基。在一些實施例中，R a或R 1為未經取代之苯基或間甲苯基。在一些實施例中，R a或R 1為未經取代之吡啶基。在一些實施例中，R a或R 1為4-吡啶基。 In some embodiments, each R substituent is independently C 1-4 alkyl, C 1-4 alkenyl, C 1-4 alkynyl, -OC 1-4 alkyl, halo, cyano, nitro Base, azido, halo-C 1-4 alkyl, -OC 1-4 haloalkyl, -NR g R h , -NR g C(=O)R h , -NR g C(=O) NR g R h , -NR g C(=O)OR h , =NOR g , -NR g S(=O) 1-2 R h , -NR g S(=O) 1-2 NR g R h , =NSO 2 R g , -C(=O)R g , -C(=O)OR g , -OC(=O)OR g , -OC(=O)R g , -C(=O)NR g R h , -OC(=O)NR g R h , -OR g , -SR g , -S(=O)R g , -S(=O) 2 R g , -OS(=O) 1-2 R g , -S(=O) 1-2 OR g , -S(=O) 1-2 NR g R h , phenyl, -C 1-4 alkyl-phenyl, monocyclic cycloalkyl, - C 1-4 alkyl-cycloalkyl, monocyclic heterocycloalkyl or monocyclic heteroaryl, wherein the phenyl, monocyclic cycloalkyl, monocyclic heterocycloalkyl and monocyclic heteroaryl of R d are each Optionally substituted with one or two substituents Re . In some embodiments, each R substituent is independently C 1-4 alkyl, halo-C 1-4 alkyl, phenyl, -C 1-4 alkyl-phenyl, pyridyl, phenylthio Base, cycloalkyl or -C 1-4 alkyl-cycloalkyl, wherein phenyl, pyridyl and phenylthio are each optionally substituted by one or two substituents R e . In some embodiments, each R substituent is independently methyl, ethyl, isopropyl, -CF3 , -OCH3 , -OCF3 , phenyl, pyridyl, thiophenyl, benzyl, Cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl, wherein the phenyl, cycloalkyl and heteroaryl of R d are each optionally replaced by one or two The substituent R is substituted. In some embodiments, each R d is independently selected from C 1-4 alkyl, -CF 3 , fluorine, chlorine, -OCH 3 and -OCF 3 . In some embodiments, R a or R 1 is substituted with one R d , and R d is C 1-4 alkyl. In some embodiments, R or R is substituted with one R and R is methyl. In some embodiments, R a or R 1 is phenyl or pyridyl, each of which is optionally substituted with one or two substituents selected from: C 1-4 alkyl, —CF 3 , fluorine, chlorine, -OCH 3 and -OCF 3 . In some embodiments, R or R is unsubstituted phenyl or tolyl. In some embodiments, R or R is unsubstituted phenyl or m-tolyl. In some embodiments, R or R is unsubstituted pyridyl. In some embodiments, R or R is 4-pyridyl.
在一些實施例中，各R e取代基獨立地為C 1-4烷基、鹵基、鹵基-C 1-4烷基、-O-C 1-4烷基或-O-C 1-4鹵烷基。在一些實施例中，各R e取代基獨立地為甲基、-CF 3、氟、氯、-OCH 3或-OCF 3。在一些實施例中，各R d取代基獨立地為甲基、乙基、異丙基、-CF 3、苯基、吡啶基、苯硫基、苯甲基、環丙基、環丁基、環戊基、環丙基甲基、環丁基甲基或環戊基甲基，其中各R e獨立地為甲基、-CF 3、氟、氯、-OCH 3或-OCF 3。 In some embodiments, each R substituent is independently C 1-4 alkyl, halo, halo-C 1-4 alkyl, -OC 1-4 alkyl, or -OC 1-4 haloalkyl . In some embodiments, each R e substituent is independently methyl, —CF 3 , fluoro, chloro, —OCH 3 , or —OCF 3 . In some embodiments, each R substituent is independently methyl, ethyl, isopropyl, -CF3 , phenyl, pyridyl, thiophenyl, benzyl, cyclopropyl, cyclobutyl, Cyclopentyl, cyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl, wherein each R e is independently methyl, -CF 3 , fluorine, chlorine, -OCH 3 or -OCF 3 .
在一些實施例中，R g及R h各自獨立地為H或甲基。 In some embodiments, R g and Rh are each independently H or methyl.
在一些實施例中，R 2及R 3與其所連接之氮一起形成吡咯啶、哌啶、哌𠯤、𠰌啉、硫代𠰌啉或硫代𠰌啉-1,1-二氧化物，其各自視情況經一個、兩個或三個R j取代基取代。在一些實施例中，R 2及R 3與其所連接之氮一起形成視情況經一或兩個R j取代基取代之𠰌啉。 In some embodiments, R 2 and R 3 together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperidine, thioline, thiothioline or thiothioline-1,1-dioxide, each of which Optionally substituted with one, two or three R j substituents. In some embodiments, R 2 and R 3 together with the nitrogen to which they are attached form a phenoline optionally substituted with one or two R substituents.
在一些實施例中，各R j取代基獨立地為甲基、羥基、-OCH 3、鹵基、-CF 3或-OCF 3。 In some embodiments, each R j substituent is independently methyl, hydroxyl, -OCH 3 , halo, -CF 3 , or -OCF 3 .
在一些實施例中，R k及R l各自獨立地為H或甲基。 In some embodiments, Rk and Rl are each independently H or methyl.
在一些實施例中，R 4為視情況經取代之苯基。在一些實施例中，R 4為視情況經取代之雜芳基。在一些實施例中，R 4為視情況經取代之單環雜芳基。在一些實施例中，R 4為視情況經取代之吡咯、咪唑、吡唑、***、四唑、呋喃、㗁唑、異㗁唑、噻唑、異噻唑、吡啶、嘧啶、吡𠯤或嗒𠯤。在一些實施例中，R 4為視情況經取代之吡唑、吡啶或嘧啶。在一些實施例中，R 4為視情況經取代之吡啶。在一些實施例中，R 4為吡啶。在一些實施例中，R 4為4-吡啶基。在一些實施例中，R 4視情況經一或兩個R z取代基取代。在一些實施例中，R 4為苯基或吡啶基，其各自視情況經一或兩個選自以下之取代基取代：C 1-4烷基、-CF 3、氟、氯、-OCH 3及-OCF 3。 In some embodiments, R4 is optionally substituted phenyl. In some embodiments, R4 is optionally substituted heteroaryl. In some embodiments, R 4 is an optionally substituted monocyclic heteroaryl. In some embodiments, R is optionally substituted pyrrole, imidazole, pyrazole, triazole, tetrazole, furan, oxazole, isoxazole, thiazole, isothiazole, pyridine, pyrimidine, pyridine, or pyridine . In some embodiments, R4 is optionally substituted pyrazole, pyridine or pyrimidine. In some embodiments, R4 is optionally substituted pyridine. In some embodiments, R4 is pyridine. In some embodiments, R 4 is 4-pyridyl. In some embodiments, R4 is optionally substituted with one or two Rz substituents. In some embodiments, R 4 is phenyl or pyridyl, each of which is optionally substituted with one or two substituents selected from: C 1-4 alkyl, —CF 3 , fluoro, chloro, —OCH 3 and -OCF 3 .
在一些實施例中，R 4為-C(O)NR xR y。在一些實施例中，R x為H。在一些實施例中，R x為視情況經一個、兩個或三個R o取代基取代之甲基或乙基。在一些實施例中，R x為甲基。在一些實施例中，R y為H。在一些實施例中，R y為C 1-4烷基、-C 1-4烷基(單環環烷基)、單環環烷基、單環雜環烷基、單環雜環基、-O-單環雜環基、-O-C 1-4烷基、-SO 2-C 1-4烷基，其視情況經一個、兩個或三個R o取代基取代。在一些實施例中，R y為視情況經一個、兩個或三個R o取代基取代之C 1-4烷基。在一些實施例中，R y為甲基、乙基、丙基或異丙基，其各自視情況經一個、兩個或三個R o取代基取代。在一些實施例中，R y為甲基、乙基、異丙基、甲氧基乙基、二甲氧基丙烷基、(二甲胺基)乙基或(二甲胺基)丁基。在一些實施例中，R y為甲氧基。在一些實施例中，R y為-SO 2-甲基。 In some embodiments, R 4 is -C(O)NR x R y . In some embodiments, Rx is H. In some embodiments, Rx is methyl or ethyl optionally substituted with one, two, or three Ro substituents. In some embodiments, R x is methyl. In some embodiments, Ry is H. In some embodiments, R y is C 1-4 alkyl, -C 1-4 alkyl (monocyclic cycloalkyl), monocyclic cycloalkyl, monocyclic heterocycloalkyl, monocyclic heterocyclyl, -O-monocyclic heterocyclyl, -OC 1-4 alkyl, -SO 2 -C 1-4 alkyl, optionally substituted with one, two or three R o substituents. In some embodiments, R y is C 1-4 alkyl optionally substituted with one, two, or three R o substituents. In some embodiments, Ry is methyl, ethyl, propyl, or isopropyl, each of which is optionally substituted with one, two, or three Ro substituents. In some embodiments, Ry is methyl, ethyl, isopropyl, methoxyethyl, dimethoxypropyl, (dimethylamino)ethyl, or (dimethylamino)butyl. In some embodiments, Ry is methoxy. In some embodiments, Ry is -SO2 -methyl.
在一些實施例中，R y為單環環烷基或-C 1-2烷基(單環環烷基)，其各自視情況經一個、兩個或三個R o取代基取代。在一些實施例中，R y為視情況經一個、兩個或三個R o取代基取代之單環環烷基。在一些實施例中，R y為環丙基、環丁基、環戊基或環己基，其各自視情況經一個、兩個或三個R o取代基取代。在一些實施例中，R y為環丙基。在一些實施例中，R y為環戊基。在一些實施例中，R y為環丙基、環丁基、環戊基、環丙基甲基、1-環丙基乙基、2-環丙基乙基、環丁基甲基或環戊基甲基。在一些實施例中，R y為視情況經一個、兩個或三個R o取代基取代之單環雜環基或-O-單環雜環基。在一些實施例中，R y為視情況經取代之四氫呋喃基、四氫哌喃基、氧雜環丁基、吖呾基、吡咯啶基、哌啶基、𠰌啉基、哌𠯤基或氧雜環丁基氧基。 In some embodiments, R y is monocyclic cycloalkyl or -C 1-2 alkyl (monocyclic cycloalkyl), each of which is optionally substituted with one, two, or three R substituents. In some embodiments, Ry is a monocyclic cycloalkyl optionally substituted with one, two, or three Ro substituents. In some embodiments, R y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each of which is optionally substituted with one, two, or three R o substituents. In some embodiments, Ry is cyclopropyl. In some embodiments, Ry is cyclopentyl. In some embodiments, Ry is cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, 1-cyclopropylethyl, 2-cyclopropylethyl, cyclobutylmethyl, or cyclopentyl methyl. In some embodiments, Ry is monocyclic heterocyclyl or -O-monocyclic heterocyclyl optionally substituted with one, two, or three R o substituents. In some embodiments, Ry is optionally substituted tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, azithenyl, pyrrolidinyl, piperidinyl, oxolinyl, piperhexenyl, or oxo Heterocyclobutyloxy.
在一些實施例中，R y為氧雜環丁基或氧雜環丁基氧基。在一些實施例中，R y為視情況經一個、兩個或三個R o取代基取代之單環雜環烷基。在一些實施例中，其中R y為視情況經取代之氧雜環丁基甲基或(3-(羥甲基)氧雜環丁-3-基)甲基。在一些實施例中，R x為甲基，且R y為甲基、乙基、環丙基、甲氧基或環戊基。 In some embodiments, Ry is oxetanyl or oxetanyloxy. In some embodiments, Ry is monocyclic heterocycloalkyl optionally substituted with one, two, or three Ro substituents. In some embodiments, wherein Ry is optionally substituted oxetanylmethyl or (3-(hydroxymethyl)oxetan-3-yl)methyl. In some embodiments, Rx is methyl, and Ry is methyl, ethyl, cyclopropyl, methoxy, or cyclopentyl.
在一些實施例中，R x及R y與其所連接之氮一起形成視情況經C 1-4烷基取代之單環雜環烷基。在一些實施例中，R x及R y與其所連接之氮一起形成視情況經C 1-4烷基取代之單環雜環基。在一些實施例中，R x及R y與其所連接之氮一起形成吖呾基、吡咯啶基、哌啶基、哌𠯤基、𠰌啉基、硫代𠰌啉基、6-氧雜-1-氮雜螺[3.3]庚基或2-氧雜-6-氮雜螺[3.3]庚基，其各自視情況經甲基取代。 In some embodiments, R x and R y are taken together with the nitrogen to which they are attached to form an optionally C 1-4 alkyl substituted monocyclic heterocycloalkyl. In some embodiments, R x and R y are taken together with the nitrogen to which they are attached to form an optionally C 1-4 alkyl substituted monocyclic heterocyclyl. In some embodiments, R x and R y are taken together with the nitrogen to which they are attached to form acridyl, pyrrolidinyl, piperidinyl, piperoxyl, oxalinyl, thiolnyl, 6-oxa-1 - azaspiro[3.3]heptyl or 2-oxa-6-azaspiro[3.3]heptyl, each of which is optionally substituted with methyl.
在一些實施例中，各R z獨立地為C 1-4烷基、鹵基、-OH、-OC 1-4烷基、C 1-4烷基NR mR n或-NR mR n，其中各烷基視情況經-NR mR n取代。在一些實施例中，各R z獨立地為甲基、-OH、鹵基或-OCH 3。在一些實施例中，R z為經-NR mR n取代之C 2-3烷基。 In some embodiments, each R z is independently C 1-4 alkyl, halo, -OH, -OC 1-4 alkyl, C 1-4 alkyl NR m R n or -NR m R n , wherein each alkyl group is optionally substituted by -NR m R n . In some embodiments, each Rz is independently methyl, -OH, halo, or -OCH3 . In some embodiments , Rz is C2-3 alkyl substituted with -NRmRn .
在一些實施例中，R m及R n各自獨立地為H或C 1-4烷基。在一些實施例中，R m及R n各自為甲基。在一些實施例中，R m及R n與其所連接之氮一起形成視情況經一或兩個R o取代基取代之單環雜環烷基。在一些實施例中，R m及R n與其所連接之氮一起形成吡咯啶、哌啶、哌𠯤、𠰌啉、硫代𠰌啉或硫代𠰌啉-1,1-二氧化物，其各自視情況經一或兩個R o取代基取代。在一些實施例中，R m及R n與其所連接之氮一起形成吡咯啶、哌啶、哌𠯤或𠰌啉，其各自視情況經一或兩個R o取代基取代。 In some embodiments, R m and R n are each independently H or C 1-4 alkyl. In some embodiments, Rm and Rn are each methyl. In some embodiments, R and R taken together with the nitrogen to which they are attached form a monocyclic heterocycloalkyl optionally substituted with one or two R substituents. In some embodiments, R m and R n together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperidine, thioline, thiol or thiol-1,1-dioxide, each of which Optionally substituted with one or two R o substituents. In some embodiments, Rm and Rn are taken together with the nitrogen to which they are attached to form a pyrrolidine, piperidine, piperidine, or phenoline, each of which is optionally substituted with one or two R o substituents.
在一些實施例中，各R o取代基為C 1-4烷基。在一些實施例中，各R o取代基為甲基。在一些實施例中，各R o取代基為-OH。在一些實施例中，各R o取代基為-NR pR q。在一些實施例中，R p及R q各自獨立地為H或甲基。 In some embodiments, each R o substituent is C 1-4 alkyl. In some embodiments, each R o substituent is methyl. In some embodiments, each R o substituent is -OH. In some embodiments, each R o substituent is -NR p R q . In some embodiments, R p and R q are each independently H or methyl.
在一些實施例中，R p及R q與其所連接之氮一起形成雜環基。在一些實施例中，R p及R q與其所連接之氮一起形成吖呾基、吡咯啶基、哌啶基、哌𠯤基、𠰌啉基、硫代𠰌啉基、6-氧雜-1-氮雜螺[3.3]庚基或2-氧雜-6-氮雜螺[3.3]庚基。 In some embodiments, Rp and Rq together with the nitrogen to which they are attached form a heterocyclyl. In some embodiments, R p and R q are taken together with the nitrogen to which they are attached to form acridyl, pyrrolidinyl, piperidinyl, piperoneyl, oxalinyl, thiolnyl, 6-oxa-1 - azaspiro[3.3]heptyl or 2-oxa-6-azaspiro[3.3]heptyl.
在一些實施例中，R 5為H、甲基、乙基、氯、溴、氟、-OH或-OCH 3。在一些實施例中，R 5為H。 In some embodiments, R5 is H, methyl, ethyl, chloro, bromo, fluoro, -OH, or -OCH3 . In some embodiments, R 5 is H.
在一些實施例中，式(I)化合物或其醫藥學上可接受之鹽為式(II)化合物：
在一些實施例中，R 1a為各自視情況經甲基或-CF 3取代之苯基或吡啶基。在一些實施例中，R 1a為苯基或間甲苯基。在一些實施例中，R 1a為吡啶基。在一些實施例中，R 1a為4-吡啶基。在一些實施例中，R 4a為各自視情況經甲基或-CF 3取代之苯基或吡啶基。在一些實施例中，R 4a為苯基或間甲苯基。在一些實施例中，R 4a為吡啶基。在一些實施例中，R 4a為4-吡啶基。 In some embodiments, R 1a is phenyl or pyridyl, each optionally substituted with methyl or -CF 3 . In some embodiments, R 1a is phenyl or m-tolyl. In some embodiments, R 1a is pyridyl. In some embodiments, R 1a is 4-pyridyl. In some embodiments, R 4a is phenyl or pyridyl, each optionally substituted with methyl or -CF 3 . In some embodiments, R 4a is phenyl or m-tolyl. In some embodiments, R 4a is pyridyl. In some embodiments, R 4a is 4-pyridyl.
在一些實施例中，在式(I)化合物中：
在一些實施例中，一或多個與R 1、R 2、R 3、R 4、R 5、R 1a或R 4a之碳原子連接的氫原子經氘原子置換。 In some embodiments, one or more hydrogen atoms attached to carbon atoms of R 1 , R 2 , R 3 , R 4 , R 5 , R 1a or R 4a are replaced with deuterium atoms.
在一些實施例中，一或多個與R d、R e、R g、R h、R j、R k、R l、R m、R n、R o、R p、R q、R r、R x、R y或R z之碳原子連接的氫原子經氘原子置換。在一些實施例中，一或多個R d、R eR g、R h、R j、R k、R l、R m、R n、R o、R p、R q、R r、R x、R y或R z基團為C 1-4烷基，其中一或多個與碳原子連接之氫原子經氘原子置換。在一些實施例中，一或多個R d、R e、R g、R h、R j、R k、R l、R m、R n、R o、R p、R q、R r、R x、R y或R z基團為甲基，其中一或多個與碳原子連接之氫原子經氘原子置換。在一些實施例中，一或多個R d、R eR g、R h、R j、R k、R l、R m、R n、R o、R p、R q、R r、R x、R y或R z基團為-CD 3。 In some embodiments, one or more of R d , Re , R g , Rh , R j , R k , R l , R m , R n , R o , R p , R q , R r , The hydrogen atoms attached to the carbon atoms of R x , R y or R z are replaced by deuterium atoms. In some embodiments, one or more of R d , Re R g , Rh , R j , R k , R l , R m , R n , R o , R p , R q , R r , R x , R y or R z groups are C 1-4 alkyl groups, wherein one or more hydrogen atoms attached to carbon atoms are replaced by deuterium atoms. In some embodiments, one or more of R d , Re , R g , Rh , R j , R k , R l , R m , R n , R o , R p , R q , R r , R The x , Ry or Rz groups are methyl groups in which one or more hydrogen atoms bonded to carbon atoms are replaced by deuterium atoms. In some embodiments, one or more of R d , Re R g , Rh , R j , R k , R l , R m , R n , R o , R p , R q , R r , R x , R y or R z group is -CD 3 .
在一些實施例中，式(I)或式(II)之化合物包含-D替代至少一個-H，或-CD 3取代基替代至少一個CH 3。 In some embodiments, the compound of formula (I) or formula (II) comprises -D in place of at least one -H, or -CD 3 substituent in place of at least one CH 3 .
舉例而言，在4-[2-(1-甲基吡唑-3-基)-5-[3-[3-(三氘化甲基)苯基]吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉(化合物123)中，4-[2-(1-甲基吡唑-3-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉(化合物61)中之「(間甲苯基)吡唑-1-基」之苯環上的甲基經-CD 3基團置換。 For example, in 4-[2-(1-methylpyrazol-3-yl)-5-[3-[3-(trideuteromethyl)phenyl]pyrazol-1-yl]pyrazole And[1,5-a]pyrimidin-7-yl]𠰌line (compound 123), 4-[2-(1-methylpyrazol-3-yl)-5-[3-(m-tolyl) The form on the benzene ring of "(m-tolyl) pyrazol-1-yl" in pyrazol-1-yl]pyrazolo[1,5-a]pyrimidin-7-yl]𠰌line (Compound 61) The group is replaced by a -CD3 group.
在一些實施例中，化合物為選自表1之彼等者之化合物：
表 1. 
前述方法中任一者可經由套組實施以用於治療冠狀病毒感染。在各種實施例中，冠狀病毒為SARS-CoV-1、SARS-CoV-2、MERS-CoV、HCoV-OC43、HCoV-HKU1、HCoV-229E或HCoV-NL63。Any of the foregoing methods can be practiced via a kit for treating coronavirus infection. In various embodiments, the coronavirus is SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E or HCoV-NL63.
套組可含有式I化合物(或本文所描述之其實施例中之任一者)、醫藥學上可接受之載劑、生理學上可接受之載劑、使用說明書、容器、用於投與之器皿或其任何組合。A kit may contain a compound of formula I (or any of its embodiments described herein), a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a utensils or any combination thereof.
本發明進一步提供本文所揭示之任何化合物，其用於藉由療法來治療人類或動物身體之方法中。療法可為藉由本文所揭示之任何機制，諸如抑制、減少本文所揭示之疾病或減少其進展。本發明進一步提供本文所揭示之任何化合物，其用於預防或治療本文所揭示之任何病狀。本發明亦提供本文所揭示之任何化合物或其醫藥組合物，其用於獲得針對本文所揭示之任何病狀的本文所揭示之任何臨床結果。本發明亦提供一種本文所揭示之任何化合物的用途，其用於製造用於預防或治療本文所揭示之任何疾病或病狀之藥物。 實例 The invention further provides any of the compounds disclosed herein for use in a method of treating the human or animal body by therapy. Therapy can be by any of the mechanisms disclosed herein, such as inhibiting, reducing or reducing the progression of the diseases disclosed herein. The invention further provides any of the compounds disclosed herein for use in the prevention or treatment of any of the conditions disclosed herein. The invention also provides any compound disclosed herein, or a pharmaceutical composition thereof, for use in obtaining any clinical outcome disclosed herein for any condition disclosed herein. The invention also provides a use of any compound disclosed herein in the manufacture of a medicament for the prevention or treatment of any disease or condition disclosed herein. example
提供式(I)化合物及中間物之以下製備以使得熟習此項技術者能夠更清楚地理解及實踐本發明。其不應被視為限制本發明之範疇，而僅為本發明之說明及代表。The following preparations of compounds of formula (I) and intermediates are provided to enable those skilled in the art to understand and practice the present invention more clearly. They should not be considered as limiting the scope of the invention, but only as illustrative and representative of the invention.
在製備此等化合物中使用之起始材料及試劑可獲自商業供應商，諸如Aldrich Chemical Co. (Milwaukee, Wis.)、Bachem (Torrance, Calif.)或Sigma (St. Louis, Mo.)；或藉由熟習此項技術者已知之方法遵循在參考文獻中闡述之程序製備，該等參考文獻諸如Fieser及Fieser之Reagents for Organic Synthesis, 第1-17卷(John Wiley and Sons, 1991)；Rodd之Chemistry of Carbon Compounds, 第1-5卷及增刊(Elsevier Science Publishers, 1989)；Organic Reactions, 第1-40卷(John Wiley and Sons, 1991), March之Advanced Organic Chemistry, (John Wiley and Sons, 第4版)及Larock之Comprehensive Organic Transformations (VCH Publishers Inc., 1989)。此等流程僅說明可藉以合成本發明化合物之一些方法，且可作出對此等流程之各種修改且熟習此項技術者將參考本發明提出各種修改。可視需要使用習知技術，包括但不限於過濾、蒸餾、結晶、層析及其類似者來分離及純化反應之起始材料及中間物以及最終產物。可使用習知方式(包括物理常數及光譜資料)來表徵此類材料。Starting materials and reagents used in the preparation of these compounds are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), Bachem (Torrance, Calif.), or Sigma (St. Louis, Mo.); or by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Advanced Organic Chemistry in March, (John Wiley and Sons, 4th edition) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). These schemes illustrate only some of the methods by which the compounds of the invention may be synthesized, and various modifications to these schemes can be made and will be suggested by reference to the present disclosure by those skilled in the art. Starting materials and intermediates and final products of the reaction can be isolated and purified using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like, as desired. Such materials can be characterized using conventional means, including physical constants and spectral data.
除非相反地說明，否則本文所描述之反應係在大氣壓下在約-78℃至約150℃或約0℃至約125℃之溫度範圍內或在約室內(或環境)溫度，例如約20℃進行。Unless stated to the contrary, the reactions described herein are at atmospheric pressure in the temperature range from about -78°C to about 150°C or from about 0°C to about 125°C or at about room (or ambient) temperature, such as about 20°C conduct.
可如下文所說明及所描述來製備式(I)及子式之化合物及本文所描述之物種，包括其中取代基如本文所定義之彼等者。Compounds of formula (I) and subformulas and species described herein, including those wherein substituents are as defined herein, can be prepared as illustrated and described hereinafter.
除非另外指出，否則所有試劑均未經進一步純化即使用。
1H NMR譜係在室溫、在Bruker 300 MHz儀上在CDCl
3、DMSO-
d
6 或CD
3OD中獲得。當偵測到超過一個構形異構物時，報導最豐富構形異構物之化學位移。在來自殘餘溶劑之內標的
δ標度上記錄
1H NMR譜之化學位移，其以百萬分率(ppm)計。***圖案(Splitting pattern)經設計為：s，單峰；d，二重峰；t，三重峰；q，四重峰；m，多重峰；br，寬峰。LC-MS條件如下文所描述：
All reagents were used without further purification unless otherwise noted. 1 H NMR spectra were obtained at room temperature on a
LCMS管柱： Agilent Zorbax XDB C18 4.6×50 mm, 3.5µm a. 移動相， 溶劑A：水(+ 0.1%甲酸)；溶劑B：MeOH b. 流速： 1.0 mL/min c. 運行時間： 2分鐘梯度(20%-90% B)；接著3分鐘，90% B， d. 溫度： 30℃ LCMS column: Agilent Zorbax XDB C18 4.6×50 mm, 3.5µm a. Mobile phase, Solvent A: Water (+ 0.1% formic acid); Solvent B: MeOH b. Flow rate: 1.0 mL/min c. Running time: 2 minutes gradient (20%-90% B); then 3 minutes, 90% B, d. Temperature: 30℃
HPLC管柱： Agilent SB-C18 4.6×150 mm, 3.5µm a. 移動相， 溶劑A：水(+ 0.02% TFA)；溶劑B：MeOH b. 流速： 1.0 mL/min c. 運行時間： 0.5分鐘，10% B；9.5分鐘梯度(10%-90% B)；接 著10分鐘， 90% B d. 溫度： 30℃ HPLC column: Agilent SB-C18 4.6×150 mm, 3.5µm a. Mobile phase, Solvent A: Water (+ 0.02% TFA); Solvent B: MeOH b. Flow rate: 1.0 mL/min c. Run time: 0.5 minutes, 10% B; 9.5 minutes gradient (10%-90% B); followed by 10 minutes, 90% B d. Temperature: 30°C
製備型LC管柱：Phenomenex Luna 5u 100A, 21.2×250mm, 5µm a. 移動相，溶劑A：水；溶劑B：MeOH b. 流速： 10 mL/min c. 運行時間： 1分鐘，20% B；30分鐘梯度(20%-80% B)；接著 10分鐘， 90% B d. 溫度： 環境 Preparative LC column: Phenomenex Luna 5u 100A, 21.2×250mm, 5µm a. Mobile phase, solvent A: water; solvent B: MeOH b. Flow rate: 10 mL/min c. Run time: 1 minute, 20% B; 30 minutes gradient (20%-80% B); then 10 minutes, 90% B d. Temperature: environment
在本文中使用以下縮寫：PE =石油醚，EA或EtOAc=乙酸乙酯，DMSO=二甲亞碸，DMF = N,N-二甲基乙醯胺，MeOH=甲醇，EtOH=乙醇，Et
2O =***，EDCl =
N-(3-二甲胺基丙基)-
N′-乙基碳化二亞胺鹽酸鹽，MTBE =甲基三級丁基醚，DCM =二氯甲烷，TEA =三乙胺，DIPEA =二異丙基乙胺，TFA =三氟乙酸，TLC =薄層層析，(BPin)2 = 雙(
中間物A：4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉
製備 A.2 ：在25℃，向 A.1(200.0 g，1.46 mol，172.4 mL，1.0 eq)於甲苯(1800 mL)中之溶液中添加NaH (116.6 g，2.92 mol，60%純度，2.0 eq)。將混合物加熱至90℃，且接著逐滴添加MeCN (280.1 g，6.83 mol，359.2 mL，4.68 eq)。在90℃攪拌此混合物8小時，之後藉由TLC (石油醚/乙酸乙酯= 5/1)進行之分析指示耗盡大部分起始材料。將反應混合物過濾，得到黃色固體，接著將黃色固體濾餅分配於水(1000 mL)與DCM (800 mL)之間。用1 M HCl溶液將混合物之pH調整至pH = 5-6，接著分離有機層，且水相用DCM (3 × 600 mL)萃取。濃縮經合併之有機層，得到呈黃色固體狀之A. 2(160.0 g，粗物質)。 Preparation A.2 : To a solution of A.1 (200.0 g, 1.46 mol, 172.4 mL, 1.0 eq ) in toluene (1800 mL) was added NaH (116.6 g, 2.92 mol, 60% purity, 2.0 eq ). The mixture was heated to 90 °C, and then MeCN (280.1 g, 6.83 mol, 359.2 mL, 4.68 eq ) was added dropwise. The mixture was stirred at 90°C for 8 hours, after which analysis by TLC (petroleum ether/ethyl acetate=5/1) indicated that most of the starting material was consumed. The reaction mixture was filtered to give a yellow solid, which was then partitioned between water (1000 mL) and DCM (800 mL). The pH of the mixture was adjusted to pH = 5-6 with 1 M HCl solution, then the organic layer was separated and the aqueous phase was extracted with DCM (3 x 600 mL). The combined organic layers were concentrated to afford A.2 (160.0 g, crude) as a yellow solid.
製備 A.3 ：向 A.2(150.0 g，1.03 mol，1.0 eq)於EtOH (800 mL)中之溶液中添加NH 2NH 2-H 2O (78.6 g，1.54 mol，76.3 mL，98%純度，1.5 eq)。在90℃攪拌混合物。TLC (二氯甲烷/甲醇= 10/1，R f= 0.3)顯示，在2小時之後耗盡大部分起始材料。濃縮反應混合物，得到粗產物，接著此產物自乙醇(200 mL)中結晶，得到黃色固體。在分離黃色固體之後，將其維持在減壓下，以移除最後痕量之溶劑。得到呈黃色固體狀之純化的 A.3(85.0 g，530.6 mmol，51.7%產率)。 Preparation A.3 : To a solution of A.2 (150.0 g, 1.03 mol, 1.0 eq ) in EtOH (800 mL) was added NH 2 NH 2 -H 2 O (78.6 g, 1.54 mol, 76.3 mL, 98% Purity, 1.5 eq ). The mixture was stirred at 90°C. TLC (dichloromethane/methanol = 10/1, Rf = 0.3) showed that most of the starting material was consumed after 2 hours. The reaction mixture was concentrated to give a crude product which was then crystallized from ethanol (200 mL) to give a yellow solid. After isolation of the yellow solid, it was maintained under reduced pressure to remove the last traces of solvent. Purified A.3 (85.0 g, 530.6 mmol, 51.7% yield) was obtained as a yellow solid.
製備 A.4 ：向 A.3(80.0 g，499.4 mmol，1.0 eq)及丙二酸二乙酯(80.0 g，499.4 mmol，75.4 mL，1.0 eq)於EtOH (480 mL)中之溶液中添加乙醇鈉(84.9 g，1.25 mol，2.5 eq)至25℃之混合物中。在90℃攪拌混合物8小時，隨即藉由TLC (二氯甲烷/甲醇= 10/1，R f= 0.3)進行之分析顯示起始材料耗盡。過濾此混合物，得到呈黃色固體狀之 A.4(80.0 g，粗物質)。LC-MS (ESI+)：m/z 229 (MH +)。 Preparation A.4 : To a solution of A.3 (80.0 g, 499.4 mmol, 1.0 eq ) and diethyl malonate (80.0 g, 499.4 mmol, 75.4 mL, 1.0 eq ) in EtOH (480 mL) was added Sodium ethoxide (84.9 g, 1.25 mol, 2.5 eq ) was added to the mixture at 25°C. The mixture was stirred at 90 °C for 8 h, then analysis by TLC (dichloromethane/methanol = 10/1, Rf = 0.3) showed consumption of the starting material. This mixture was filtered to afford A.4 (80.0 g, crude) as a yellow solid. LC-MS (ESI+): m/z 229 (MH + ).
製備 A.5 ：向在25℃之 A.4(20.0 g，87.6 mmol，1.0 eq)於POCl 3(322.5 g，2.10 mol，195.4 mL，24.0 eq)中之溶液中添加N,N-二甲基苯胺(13.8 g，113.9 mmol，14.4 mL，1.3 eq)。在攪拌下在110℃加熱此混合物。TLC (二氯甲烷/甲醇= 10/1，R f= 0.8)顯示在12小時之後起始材料耗盡。真空濃縮混合物，得到黃色固體。將此固體倒入冰水(200 mL)中且過濾，得到呈黃色固體狀之 A.5(14.0 g)；LC-MS (ESI+)：m/z 265/267 (MH +)。 1HNMR (300 MHz, DMSO-d 6) δ: 8.74 (d, J = 5.4 Hz, 2H), 8.03 (d, J = 5.7 Hz, 2H), 7.77 (s, 1H)及7.62 (s, 1H) ppm。 Preparation A.5 : To a solution of A.4 (20.0 g, 87.6 mmol, 1.0 eq ) in POCl 3 (322.5 g, 2.10 mol, 195.4 mL, 24.0 eq ) at 25°C was added N,N-dimethyl Aniline (13.8 g, 113.9 mmol, 14.4 mL, 1.3 eq). The mixture was heated at 110°C with stirring. TLC (dichloromethane/methanol = 10/1, Rf = 0.8) showed the starting material was consumed after 12 hours. The mixture was concentrated in vacuo to give a yellow solid. This solid was poured into ice water (200 mL) and filtered to afford A.5 (14.0 g) as a yellow solid; LC-MS (ESI+): m/z 265/267 (MH + ). 1 HNMR (300 MHz, DMSO-d 6 ) δ: 8.74 (d, J = 5.4 Hz, 2H), 8.03 (d, J = 5.7 Hz, 2H), 7.77 (s, 1H) and 7.62 (s, 1H) ppm.
中間物 A ：向在25℃之A. 5(2.0 g，7.54 mmol，1.0 eq)於Et 3N (1.53 g，15.0 mmol，2.10 mL，2.0 eq)及1,4-二㗁烷(12 mL)中之溶液中添加𠰌啉(1.31 g，15.0 mmol，1.33 mL，2.0 eq)。在25℃攪拌此混合物；在3小時之後，TLC (石油醚/乙酸乙酯= 1/1，R f=0.5)顯示起始材料耗盡。過濾反應混合物以分離固體產物，接著其藉由自MeOH (30 mL)中再結晶來純化，得到呈黃色固體狀之中間物A，4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(1.80 g，5.70 mmol，75.5%產率)；LC-MS (ESI+)：m/z 316/318 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.70 (d, J = 3.9 Hz, 2H), 7.99 (d, J = 4.8 Hz, 2H), 7.25 (s, 1H), 6.53 (s, 1H)及3.92-3.85 (m, 8H) ppm。 Intermediate A : Add A.5 (2.0 g, 7.54 mmol, 1.0 eq ) in Et 3 N (1.53 g, 15.0 mmol, 2.10 mL, 2.0 eq ) and 1,4-dioxane (12 mL ) was added 𠰌line (1.31 g, 15.0 mmol, 1.33 mL, 2.0 eq ). The mixture was stirred at 25°C; after 3 hours, TLC (petroleum ether/ethyl acetate=1/1, Rf =0.5) showed the starting material was consumed. The reaction mixture was filtered to isolate a solid product, which was then purified by recrystallization from MeOH (30 mL) to afford Intermediate A, 4-(5-chloro-2-(pyridin-4-yl) as a yellow solid Pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (1.80 g, 5.70 mmol, 75.5% yield); LC-MS (ESI+): m/z 316/318 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.70 (d, J = 3.9 Hz, 2H), 7.99 (d, J = 4.8 Hz, 2H), 7.25 (s, 1H), 6.53 (s, 1H) and 3.92 -3.85 (m, 8H) ppm.
中間物B：5-胺基-N,N-二甲基-3-(4-甲基苯基)-1H-吡唑-1-磺醯胺
向在0℃之3-(4-甲基苯基)-1H-吡唑并-5-胺(250 mg，1.57 mmol)於THF (5 mL)中之溶液中添加NaH (94 mg，2.35 mmol)。在0℃攪拌1 h之後，將二甲基胺磺醯氯(270 mg，1.88 mmol)添加至溶液中。藉由TLC來監測反應。當其完成時，反應混合物用飽和NH 4Cl溶液淬滅。水溶液用乙酸乙酯(3 × 50 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。所得殘餘物藉由用20% EtOAc/PE至33% EtOAc/PE之梯度溶離之矽膠管柱層析純化，得到5-胺基-N,N-二甲基-3-苯基-1H-吡唑-1-磺醯胺(中間物B，212 mg，0.79 mmol)；LC-MS：m/z 281 (MH +)。 1HNMR (300 MHz, DMSO-d 6) δ: 7.67 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 5.7 (s, 1H), 4.82 (s, 2H), 3.02 (s, 6H)及2.37 (s, 3H) ppm。 To a solution of 3-(4-methylphenyl)-1H-pyrazolo-5-amine (250 mg, 1.57 mmol) in THF (5 mL) at 0 °C was added NaH (94 mg, 2.35 mmol) ). After stirring at 0 °C for 1 h, dimethylsulfamoyl chloride (270 mg, 1.88 mmol) was added to the solution. The reaction was monitored by TLC. When it was complete, the reaction mixture was quenched with saturated NH4Cl solution. The aqueous solution was extracted with ethyl acetate (3 x 50 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with gradient elution from 20% EtOAc/PE to 33% EtOAc/PE to give 5-amino-N,N-dimethyl-3-phenyl-1H-pyridine Azole-1-sulfonamide (Intermediate B, 212 mg, 0.79 mmol); LC-MS: m/z 281 (MH + ). 1 HNMR (300 MHz, DMSO-d 6 ) δ: 7.67 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 5.7 (s, 1H), 4.82 (s, 2H) , 3.02 (s, 6H) and 2.37 (s, 3H) ppm.
實例1：7-(N-𠰌啉基)-2-(吡啶-4-基)- N-(3-(對甲苯基)-1 H-吡唑-5-基)吡唑并[1,5- a]嘧啶-5-胺鹽酸鹽 Example 1: 7-(N-𠰌linyl)-2-(pyridin-4-yl) -N- (3-(p-tolyl) -1H -pyrazol-5-yl)pyrazolo[1, 5- a ]pyrimidin-5-amine hydrochloride
步驟 1 ：製備 3-(4- 氟苯基 )-N,N- 二甲基 -5-((7-(N- 𠰌 啉基 )-2-( 吡啶 -4- 基 ) 吡唑并 [1,5-a] 嘧啶 -5- 基 ) 胺基 )-1H- 吡唑 -1- 磺醯胺 
在微波條件下，將4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(中間物A，60 mg，0.19 mmol)、5-胺基-3-(4-氟苯基)-N,N-二甲基-1H-吡唑-1-磺醯胺(中間物B，76 mg，0.28 mmol)、Cs 2CO 3(142 mg，0.44 mmol)、Pd(OAc) 2(4.2 mg，0.019 mmol)及Xantphos (10.2 mg，0.019 mmol)於DMF/1,4-二㗁烷(7:1，5 mL)中之溶液加熱至90℃持續30 min。將反應混合物直接濃縮，且所得殘餘物藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之不純3-(4-氟苯基)-N,N-二甲基-5-((7-(N-𠰌啉基)-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-5-基)胺基)-1H-吡唑-1-磺醯胺(50 mg，0.09 mmol)；LC-MS (ESI+)：m/z 560 (MH +)。 Under microwave conditions, 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (Intermediate A, 60 mg, 0.19 mmol ), 5-amino-3-(4-fluorophenyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide (intermediate B, 76 mg, 0.28 mmol), Cs 2 CO 3 (142 mg, 0.44 mmol), Pd(OAc) 2 (4.2 mg, 0.019 mmol) and Xantphos (10.2 mg, 0.019 mmol) in DMF/1,4-dioxane (7:1, 5 mL) The solution was heated to 90 °C for 30 min. The reaction mixture was directly concentrated and the resulting residue was purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to give impure 3-(4-fluorophenyl as a yellow solid )-N,N-Dimethyl-5-((7-(N-𠰌linyl)-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amine yl)-1H-pyrazole-1-sulfonamide (50 mg, 0.09 mmol); LC-MS (ESI+): m/z 560 (MH + ).
步驟2：製備7-(N-𠰌啉基)-2-(吡啶-4-基)-
N-(3-(對甲苯基)-1
H-吡唑-5-基)吡唑并[1,5-
a]嘧啶-5-胺鹽酸鹽
向N,N-二甲基-5-((7-(N-𠰌啉基)-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-5-基) 胺基)-3-苯基-1H-吡唑-1-磺醯胺(50 mg，0.09 mmol)於DCM中之溶液中添加HCl/Et 2O (1 mL)。在環境溫度攪拌反應物2 h。沈澱出大量固體。在濃縮之後，使殘餘物在MeOH/Et 2O (1/20，2 mL)中濕磨；得到呈白色固體狀之7-(N-𠰌啉基)-2-(吡啶-4-基)- N-(3-(對甲苯基)-1 H-吡唑-5-基)吡唑并[1,5- a]嘧啶-5-胺鹽酸鹽(實例1, 18.6 mg，0.04 mmol)；LC-MS (ESI+)：m/z 453 (MH+)。 1HNMR (300 MHz, DMSO-d 6) δ: 10.06 (s, 1H), 8.95 (d, J = 6.6 Hz, 2H), 8.50 (d, J = 6.3 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.16 (s, 1H), 6.97 (s, 1H), 6.32 (s, 1H), 3.94-3.86 (m, 4H), 3.69-3.63 (m, 4H)及2.39 (s, 3H) ppm。 To N,N-dimethyl-5-((7-(N-𠰌linyl)-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino To a solution of )-3-phenyl-1H-pyrazole-1-sulfonamide (50 mg, 0.09 mmol) in DCM was added HCl/ Et2O (1 mL). The reaction was stirred at ambient temperature for 2 h. A large amount of solid precipitated out. After concentration, the residue was triturated in MeOH/Et 2 O (1/20, 2 mL); 7-(N-𠰌linyl)-2-(pyridin-4-yl) was obtained as a white solid -N- (3-(p-tolyl) -1H -pyrazol-5-yl)pyrazolo[1,5- a ]pyrimidin-5-amine hydrochloride (Example 1, 18.6 mg, 0.04 mmol) ; LC-MS (ESI+): m/z 453 (MH+). 1 HNMR (300 MHz, DMSO-d 6 ) δ: 10.06 (s, 1H), 8.95 (d, J = 6.6 Hz, 2H), 8.50 (d, J = 6.3 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.16 (s, 1H), 6.97 (s, 1H), 6.32 (s, 1H), 3.94-3.86 (m, 4H), 3.69- 3.63 (m, 4H) and 2.39 (s, 3H) ppm.
實例2：7-(N-𠰌啉基)-N-(5-苯基-1H-吡唑-3-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺
化合物2係根據實例1使用之相同兩步驟程序，自中間物A及受保護的吡唑製備。根據用於製備中間物B之程序，自3-苯基-1H-吡唑-5-胺製備受保護的吡唑。化合物2，7-(N-𠰌啉基)-N-(5-苯基-1H-吡唑-3-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺提供以下資料：LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, DMSO-d
6) δ: 12.84 (s, 1H), 9.89 (s, 1H), 8.70 (d, J = 5.7 Hz, 2H), 7.99 (d, J = 5.7 Hz, 2H), 7.77 (d, J = 7.5 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H), 7.38-7.33 (m, 1H), 7.09 (s, 1H), 6.92 (s, 1H), 6.27 (s, 1H), 3.89-3.78 (m, 4H)及3.63-3.57 (m, 4H) ppm。
實例3：7-(N-𠰌啉基)-N-[5-(鄰甲苯基)-1H-吡唑-3-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺
化合物3係根據實例1使用之相同兩步驟程序，自中間物A及受保護的吡唑製備。根據用於製備中間物B之程序，自3-(2-甲基苯基)-1H-吡唑-5-胺製備受保護的吡唑。得到化合物3，7-(N-𠰌啉基)-N-[5-(鄰甲苯基)-1H-吡唑-3-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺；LC-MS (ESI+)：m/z 453 (MH
+)。1HNMR (300 MHz, CD
3OD) δ: 8.62 (d, J = 6.3 Hz, 2H), 8.03 (d, J = 6.3 Hz, 2H), 7.46 (d, J = 6.6 Hz, 1H), 7.32-7.24 (m, 3H), 6.81 (s, 1H), 6.54 (s, 1H), 6.06 (s, 1H), 4.04-3.97 (m, 4H), 3.75-3.65 (m, 4H)及2.45 (s, 3H) ppm。
實例4：7-(N-𠰌啉基)-N-[3-(間甲苯基)-1H-吡唑-5-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺鹽酸鹽
化合物4係根據實例1使用之相同兩步驟程序，自中間物A及受保護的吡唑製備。根據用於製備中間物B之程序，自3-(3-甲基苯基)-1H-吡唑-5-胺製備受保護的吡唑。得到化合物4，7-(N-𠰌啉基)-N-[3-(間甲苯基)-1H-吡唑-5-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺鹽酸鹽；LC-MS (ESI+)：m/z 453 (MH
+)。
1HNMR (300 MHz, DMSO-d
6) δ: 10.03 (s, 1H), 8.94 (d, J = 6.0 Hz, 2H), 8.47 (d, J = 5.1 Hz, 2H), 7.59-7.54 (m, 2H), 7.47-7.32 (m, 1H), 7.19-7.15 (m, 2H), 6.98 (s, 1H), 6.30 (s, 1H), 3.95- 3.86 (m, 4H), 3.71-3.63 (m, 4H)及2.38 (s, 3H) ppm。
實例5：N-(5-甲基-1H-吡唑-3-基)-7-(N-𠰌啉基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺
化合物5係根據實例1使用之相同兩步驟程序，自中間物A及受保護的吡唑製備。根據用於製備中間物B之程序，自3-(3-甲基)-1H-吡唑-5-胺製備受保護的吡唑。得到化合物5，N-(5-甲基-1H-吡唑-3-基)-7-(N-𠰌啉基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-5-胺；LC-MS (ESI+)：m/z 377 (MH
+)。
1HNMR (300 MHz, CD
3OD) δ: 8.60 (dd, J = 3.6, 1.2 Hz, 2H), 7.99 (dd, J = 3.6, 1.2 Hz, 2H), 6.69 (s, 1H), 6.21 (s, 1H), 6.02 (s, 1H), 3.97-3.95 (m, 4H), 3.49-3.47 (m, 4H)及2.29 (s, 3H) ppm。
實例6：7-(N-𠰌啉基)-2-(4-吡啶基)-N-[5-(4-吡啶基)-1H-吡唑-3-基]吡唑并[1,5-a]嘧啶-5-胺
化合物6係根據實例1使用之相同兩步驟程序，自中間物A及受保護的吡唑製備。根據用於製備中間物B之程序，自3-(吡啶-4-基)-1H-吡唑-5-胺製備受保護的吡唑。得到化合物6，7-(N-𠰌啉基)-2-(4-吡啶基)-N-[5-(4-吡啶基)-1H-吡唑-3-基]吡唑并[1,5-a]嘧啶-5-胺：LC-MS (ESI+)：m/z 440 (MH +)。 1HNMR (300 MHz, CD 3OD) δ: 8.89 (d, J = 5.1 Hz, 2H), 8.34 (d, J = 5.4 Hz, 2H), 8.63 (d, J = 5.1 Hz, 2H), 8.48 (d, J = 5.1 Hz, 2H), 7.22 (s, 1H), 6.91 (s, 1H), 6.02 (s, 1H), 4.05-3.95 (m, 4H)及3.82-3.80 (m, 4H) ppm。 Compound 6 was prepared from Intermediate A and the protected pyrazole according to the same two-step procedure used in Example 1 . According to the procedure used for the preparation of intermediate B, the protected pyrazole was prepared from 3-(pyridin-4-yl)-1 H-pyrazol-5-amine. To obtain compound 6, 7-(N-𠰌linyl)-2-(4-pyridyl)-N-[5-(4-pyridyl)-1H-pyrazol-3-yl]pyrazolo[1, 5-a] Pyrimidin-5-amine: LC-MS (ESI+): m/z 440 (MH + ). 1 HNMR (300 MHz, CD 3 OD) δ: 8.89 (d, J = 5.1 Hz, 2H), 8.34 (d, J = 5.4 Hz, 2H), 8.63 (d, J = 5.1 Hz, 2H), 8.48 ( d, J = 5.1 Hz, 2H), 7.22 (s, 1H), 6.91 (s, 1H), 6.02 (s, 1H), 4.05-3.95 (m, 4H) and 3.82-3.80 (m, 4H) ppm.
實例7：1-(7-(N-𠰌啉基)-2-(吡啶-4-基)吡唑并[1,5-
a]嘧啶-5-基)-3-苯基-1
H-吡唑-5-胺
在微波條件下，將4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(中間物A，30 mg，0.095 mmol)、3-苯基-1H-吡唑-5-胺(22.5 mg，0.143 mmol)、三級丁醇鈉(18.3 mg，0.191 mmol)、Pd
2(dba)
3(4.2 mg，0.005 mmol)及Xantphos (2.1 mg，0.005 mmol)於DMF (3 mL)中之溶液加熱至110℃持續30分鐘。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之1-(7-(N-𠰌啉基)-2-(吡啶-4-基)吡唑并[1,5-
a]嘧啶-5-基)-3-苯基-1
H-吡唑-5-胺(化合物7，14.2 mg，0.03 mmol)；LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, DMSO-d
6) δ: 8.72 (d, J = 5.7 Hz, 2H), 7.98 (d, J = 5.7 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 7.45-7.40 (m, 3H), 7.19 (s, 1H), 7.12 (s, 2H), 7.03 (s, 1H), 5.92 (s, 1H)及3.94-3.83 (m, 8H) ppm。
Under microwave conditions, 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (Intermediate A, 30 mg, 0.095 mmol ), 3-phenyl-1H-pyrazol-5-amine (22.5 mg, 0.143 mmol), sodium tertiary butoxide (18.3 mg, 0.191 mmol), Pd 2 (dba) 3 (4.2 mg, 0.005 mmol) and A solution of Xantphos (2.1 mg, 0.005 mmol) in DMF (3 mL) was heated to 110° C. for 30 minutes. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford 1-(7-(N-𠰌linyl) as a white solid -2-(pyridin-4-yl)pyrazolo[1,5- a ]pyrimidin-5-yl)-3-phenyl- 1H -pyrazol-5-amine (
實例8：1-(7-(N-𠰌啉基)-2-(吡啶-4-基)吡唑并[1,5-
a]嘧啶-5-基)-3-(鄰甲苯基)-1
H-吡唑-5-胺
化合物8係根據實例7所使用之程序，自中間物A及3-(2-甲基苯基)-1H-吡唑-5-胺製備；LC-MS (ESI+)：m/z 453 (MH
+)。
1HNMR (300 MHz, CDCl
3) δ: 8.73 (d, J = 3.9 Hz, 2H), 7.85 (d, J = 4.8 Hz, 2H), 7.60 (d, J = 4.5 Hz, 1H), 7.31-7.26 (m, 3H), 7.09 (s, 1H), 6.82 (s, 1H), 6.08 (s, 2H), 5.74 (s, 1H), 4.03-3.98 (m, 4H), 3.87-3.82 (m, 4H)及2.56 (s, 3H) ppm。
實例9：4-[5-(4-苯基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
向在0℃之4-苯基-1H-吡唑(29.4 mg，0.20 mmol)於DMF (5 mL)中之溶液中添加NaH (14 mg，0.34 mmol)。攪拌混合物30分鐘。接著向混合物中添加4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(中間物A，62 mg，0.19 mmol)。將反應混合物加熱至80℃且攪拌過夜。藉由TLC監測反應進程。反應混合物用水(10 mL)淬滅。水溶液用乙酸乙酯(3 × 10 mL)萃取。經合併之有機相經無水Na
2SO
4乾燥，過濾且減壓濃縮。所得殘餘物藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈灰白色固體狀之4-[5-(4-苯基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉(化合物9，17.5 mg，0.04 mmol)；LC-MS (ESI+)：m/z 424 (MH
+)。
1HNMR (300 MHz, CDCl
3) δ 8.87 (s, 1H), 8.73 (d, J = 6.0 Hz, 2H), 8.05 (s, 1H), 7.85 (d, J = 5.4 Hz, 2H), 7.62 (d, J = 7.8 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.33-7.28 (m, 1H), 7.03 (s, 1H), 6.88 (s, 1H), 4.06-4.03 (m, 4H)及3.95-3.92 (m, 4H) ppm。
To a solution of 4-phenyl-1H-pyrazole (29.4 mg, 0.20 mmol) in DMF (5 mL) at 0 °C was added NaH (14 mg, 0.34 mmol). The mixture was stirred for 30 minutes. Then 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (Intermediate A, 62 mg, 0.19 mmol) was added to the mixture . The reaction mixture was heated to 80 °C and stirred overnight. The progress of the reaction was monitored by TLC. The reaction mixture was quenched with water (10 mL). The aqueous solution was extracted with ethyl acetate (3 x 10 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford 4-[5-(4-phenylpyrazol-1-yl as an off-white solid )-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7-yl]𠰌line (
中間物CIntermediate C
步驟 1 ：合成 4-(3- 甲基苯基 )-1H- 吡唑 -1- 甲酸三級丁酯 
將4-(4,4,5,5-四甲基-1,3,2-二氧雜硼雜環戊烷-2-基)-1H-吡唑-1-甲酸三級丁酯(1 g，3.4 mmol)、1-溴-3-甲基苯(581 mg，3.4 mmol)、CsF (775 mg，5.1 mmol)、Pd 2(PPh 3) 2Cl 2(392 mg，0.34 mmol)於1,4-二㗁烷/H 2O (30 mL，2/1)中之溶液加熱至80℃過夜。藉由TLC確認反應完成。反應混合物用水(50 mL)淬滅。水溶液用乙酸乙酯(3 × 20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。所得殘餘物藉由用15% EtOAc/PE至33% EtOAc/PE之梯度溶離之矽膠管柱層析純化。得到4-(3-甲基苯基)-1H-吡唑-1-甲酸三級丁酯(117 mg，0.45 mmol)；LC- MS (ESI+)：m/z 259 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.29 (s, 1H), 7.99 (s, 1H), 7.34-7.28 (m, 3H), 7.13-7.10 (m, 1H), 2.39 (s, 3H)及1.68 (s, 9H) ppm。 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylic acid tertiary butyl ester (1 g, 3.4 mmol), 1-bromo-3-methylbenzene (581 mg, 3.4 mmol), CsF (775 mg, 5.1 mmol), Pd 2 (PPh 3 ) 2 Cl 2 (392 mg, 0.34 mmol) in 1 , A solution in 4-dioxane/H 2 O (30 mL, 2/1) was heated to 80° C. overnight. Reaction completion was confirmed by TLC. The reaction mixture was quenched with water (50 mL). The aqueous solution was extracted with ethyl acetate (3 x 20 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with gradient elution from 15% EtOAc/PE to 33% EtOAc/PE. This gave ter-butyl 4-(3-methylphenyl)-1H-pyrazole-1-carboxylate (117 mg, 0.45 mmol); LC-MS (ESI+): m/z 259 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.29 (s, 1H), 7.99 (s, 1H), 7.34-7.28 (m, 3H), 7.13-7.10 (m, 1H), 2.39 (s, 3H) and 1.68 (s, 9H) ppm.
步驟2：合成4-(3-甲基苯基)-1H-吡唑鹽酸鹽，中間物C
向4-(3-甲基苯基)-1H-吡唑-1-甲酸三級丁酯(117 mg，0.45 mmol)於DCM中之溶液中添加HCl/Et 2O (1 mL)。在環境溫度攪拌反應物過夜。沈澱出大量固體。在濃縮之後，使殘餘物在MeOH/Et 2O (1/20，2 mL)中濕磨；得到呈白色固體狀之4-(3-甲基苯基)-1H-吡唑鹽酸鹽(中間物C，90 mg，0.46 mmol)；LC-MS (ESI+)：m/z 159 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.13 (s, 2H), 7.38-7.20 (m, 4H)及2.42 (s, 3H) ppm。 To a solution of tert-butyl 4-(3-methylphenyl)-1H-pyrazole-1-carboxylate (117 mg, 0.45 mmol) in DCM was added HCl/ Et2O (1 mL). The reaction was stirred overnight at ambient temperature. A large amount of solid precipitated out. After concentration, the residue was triturated in MeOH/ Et2O (1/20, 2 mL); 4-(3-methylphenyl)-1H-pyrazole hydrochloride was obtained as a white solid ( Intermediate C, 90 mg, 0.46 mmol); LC-MS (ESI+): m/z 159 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.13 (s, 2H), 7.38-7.20 (m, 4H) and 2.42 (s, 3H) ppm.
實例 10 ： 4-[5-[4-( 間甲苯基 ) 吡唑 -1- 基 ]-2-(4- 吡啶基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ] 𠰌 啉 
向在0℃之4-(3-甲基苯基)-1H-吡唑鹽酸鹽(中間物C，40 mg，0.21 mmol)於DMF (5 mL)中之溶液中添加NaH (16.8 mg，0.42 mmol)。攪拌混合物30 min。向此混合物中添加4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(中間物A，65 mg，0.20 mmol)。將所得反應混合物加熱至80℃過夜。藉由TLC確認反應完成。反應混合物用水(10 mL)淬滅，且水溶液用乙酸乙酯(3 × 10 mL)萃取。經合併之有機相經無水Na
2SO
4乾燥，過濾且減壓濃縮。所得殘餘物藉由用30% EtOAc/PE至EtOAc之梯度溶離之矽膠管柱層析純化，得到呈灰白色固體狀之4-[5-[4-(間甲苯基)吡唑-1-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉(化合物10，15.5 mg，0.035 mmol)；LC-MS (ESI+)：m/z 438 (MH
+)。
1HNMR (300 MHz, CDCl
3) δ: 8.86 (s, 1H), 8.72 (d, J = 6.0 Hz, 2H), 8.04 (s, 1H), 7.85 (d, J = 6.0 Hz, 2H), 7.44-7.41 (m, 2H), 7.35-7.32 (m, 1H), 7.15-7.09 (m, 1H), 7.02 (s, 1H), 6.88 (s, 1H), 4.06-4.03 (m, 4H), 3.95-3.92 (m, 4H)及2.42 (s, 3H) ppm。
To a solution of 4-(3-methylphenyl)-1H-pyrazole hydrochloride (Intermediate C, 40 mg, 0.21 mmol) in DMF (5 mL) at 0 °C was added NaH (16.8 mg, 0.42 mmol). The mixture was stirred for 30 min. To this mixture was added 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (Intermediate A, 65 mg, 0.20 mmol) . The resulting reaction mixture was heated to 80 °C overnight. Reaction completion was confirmed by TLC. The reaction mixture was quenched with water (10 mL), and the aqueous solution was extracted with ethyl acetate (3 x 10 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel with a gradient elution from 30% EtOAc/PE to EtOAc to afford 4-[5-[4-(m-tolyl)pyrazol-1-yl] as an off-white solid -2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7-yl]𠰌line (
實例11：4-[2-(4-吡啶基)-5-[4-(4-吡啶基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物11係根據實例9及10所使用之條件，藉由中間物A與4-(4-吡啶基)-1H-吡唑之反應製備。得到化合物11，4-[2-(4-吡啶基)-5-[4-(4-吡啶基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉；LC-MS (ESI+)：m/z 425 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 9.01 (s, 1H), 8.73 (d, J = 6.0 Hz, 2H), 8.64 (d, J = 6.0 Hz, 2H), 8.11 (s, 1H), 7.85 (d, J = 6.0 Hz, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.02 (s, 1H), 6.90 (s, 1H), 4.06-4.03 (m, 4H)及3.98-3.96 (m, 4H) ppm。 Compound 11 was prepared by reaction of Intermediate A with 4-(4-pyridyl)-1H-pyrazole according to the conditions used in Examples 9 and 10. Compound 11 was obtained, 4-[2-(4-pyridyl)-5-[4-(4-pyridyl)pyrazol-1-yl]pyrazolo[1,5-a]pyrimidin-7-yl] 𠰌line; LC-MS (ESI+): m/z 425 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 9.01 (s, 1H), 8.73 (d, J = 6.0 Hz, 2H), 8.64 (d, J = 6.0 Hz, 2H), 8.11 (s, 1H), 7.85 (d, J = 6.0 Hz, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.02 (s, 1H), 6.90 (s, 1H), 4.06-4.03 (m, 4H) and 3.98-3.96 ( m, 4H) ppm.
實例12：4-[5-(4-甲基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物12係根據實例9及10所使用之條件，藉由中間物A與4-甲基吡唑之反應製備。得到化合物12，4-[5-(4-甲基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉；LC-MS (ESI+)：m/z 362 (MH +)。 1HNMR (300 MHz, DMSO-d 6) δ: 8.70 (d, J = 5.7 Hz, 2H), 8.45 (s, 1H), 7.98 (d, J = 6.0 Hz, 2H), 7.72 (s, 1H), 7.17 (s, 1H), 6.91 (s, 1H), 3.89 (s, 8H),及2.13 (s, 3H) ppm。 Compound 12 was prepared by reaction of Intermediate A with 4-methylpyrazole according to the conditions used in Examples 9 and 10. To obtain compound 12, 4-[5-(4-methylpyrazol-1-yl)-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7-yl]𠰌line; LC - MS (ESI+): m/z 362 (MH + ). 1 HNMR (300 MHz, DMSO-d 6 ) δ: 8.70 (d, J = 5.7 Hz, 2H), 8.45 (s, 1H), 7.98 (d, J = 6.0 Hz, 2H), 7.72 (s, 1H) , 7.17 (s, 1H), 6.91 (s, 1H), 3.89 (s, 8H), and 2.13 (s, 3H) ppm.
實例13：4-[5-(3-苯基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
向在0℃之3-苯基吡唑(29.4 mg，0.20 mmol)於DMF (5 mL)中之溶液中添加NaH (14 mg，0.34 mmol)。攪拌混合物30分鐘。向混合物中添加4-(5-氯-2-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(中間物A，62 mg，0.19 mmol)。將反應混合物加熱至80℃且攪拌過夜。藉由TLC監測反應進程。反應混合物用水(10 mL)淬滅。水溶液用乙酸乙酯(3 × 10 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。所得殘餘物藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈灰白色固體狀之4-[5-(3-苯基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉(化合物13，17.5 mg，0.04 mmol)；LC-MS (ESI+)：m/z 424 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.72 (d, J = 6.0 Hz, 2H), 8.65 (d, J = 2.7 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.85 (dd, J = 4.8, 1.5 Hz, 2H), 7.50-7.39 (m, 3H), 7.12 (s, 1H), 6.87-6.84 (m, 2H), 4.08-4.05 (m, 4H)及3.96-3.93 (m, 4H) ppm。 To a solution of 3-phenylpyrazole (29.4 mg, 0.20 mmol) in DMF (5 mL) at 0 °C was added NaH (14 mg, 0.34 mmol). The mixture was stirred for 30 minutes. To the mixture was added 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (Intermediate A, 62 mg, 0.19 mmol). The reaction mixture was heated to 80 °C and stirred overnight. The progress of the reaction was monitored by TLC. The reaction mixture was quenched with water (10 mL). The aqueous solution was extracted with ethyl acetate (3 x 10 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford 4-[5-(3-phenylpyrazol-1-yl as an off-white solid )-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7-yl]𠰌line (Compound 13, 17.5 mg, 0.04 mmol); LC-MS (ESI+): m/z 424 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.72 (d, J = 6.0 Hz, 2H), 8.65 (d, J = 2.7 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.85 (dd , J = 4.8, 1.5 Hz, 2H), 7.50-7.39 (m, 3H), 7.12 (s, 1H), 6.87-6.84 (m, 2H), 4.08-4.05 (m, 4H) and 3.96-3.93 (m , 4H) ppm.
實例14：4-[5-[3-(間甲苯基)吡唑-1-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉4-[5-[3-(3-甲基苯基)吡唑-1-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物14係根據實例13所使用之條件，藉由中間物A與3-(3-甲基苯基)吡唑之反應製備。得到化合物14，4-[5-[3-(間甲苯基)吡唑-1-基]-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉；LC-MS (ESI+)：m/z 438 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.72 (d, J = 6.0 Hz, 2H), 7.64 (d, J = 2.4 Hz, 1H), 7.84 (d, J = 5.7 Hz, 2H), 7.77-7.71 (m, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.26-7.23 (m, 1H), 7.12 (s, 1H), 6.86-6.83 (m, 2H), 4.08-4.05 (m, 4H), 3.96-3.93 (m, 4H),及2.45 (s, 3H) ppm。 Compound 14 was prepared by reaction of Intermediate A with 3-(3-methylphenyl)pyrazole according to the conditions used in Example 13. To obtain compound 14, 4-[5-[3-(m-tolyl)pyrazol-1-yl]-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7-yl]𠰌 morphine; LC-MS (ESI+): m/z 438 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.72 (d, J = 6.0 Hz, 2H), 7.64 (d, J = 2.4 Hz, 1H), 7.84 (d, J = 5.7 Hz, 2H), 7.77-7.71 (m, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.26-7.23 (m, 1H), 7.12 (s, 1H), 6.86-6.83 (m, 2H), 4.08-4.05 (m, 4H ), 3.96-3.93 (m, 4H), and 2.45 (s, 3H) ppm.
實例15：4-[2-(4-吡啶基)-5-[3-(4-吡啶基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物15係根據實例13所使用之條件，藉由中間物A與3-(4-吡啶基)吡唑之反應製備。得到化合物15，4-[2-(4-吡啶基)-5-[3-(4-吡啶基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉；LC-MS (ESI+)：m/z 425 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.74-8.70 (m, 5H), 7.86-7.81 (m, 4H), 7.09 (s, 1H), 6.93-6.89 (m, 2H), 4.09-4.06 (m, 4H)及3.98-3.96 (m, 4H) ppm。 Compound 15 was prepared by reaction of Intermediate A with 3-(4-pyridyl)pyrazole according to the conditions used in Example 13. Compound 15 was obtained, 4-[2-(4-pyridyl)-5-[3-(4-pyridyl)pyrazol-1-yl]pyrazolo[1,5-a]pyrimidin-7-yl] 𠰌line; LC-MS (ESI+): m/z 425 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.74-8.70 (m, 5H), 7.86-7.81 (m, 4H), 7.09 (s, 1H), 6.93-6.89 (m, 2H), 4.09-4.06 (m , 4H) and 3.98-3.96 (m, 4H) ppm.
實例16：4-[5-(3-甲基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物16係根據實例13所使用之條件，藉由中間物A與3-(4-吡啶基)吡唑之反應製備。得到化合物16，4-[5-(3-甲基吡唑-1-基)-2-(4-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉；LC-MS (ESI+)：m/z 362 (MH +)。 1HNMR (300 MHz, CDCl 3) δ: 8.70 (dd, J= 4.5, 1.5 Hz, 2H), 8.49 (d, J= 2.4 Hz, 1H), 7.83 (dd, J= 4.5, 1.5 Hz, 2H), 6.97 (s, 1H), 6.83 (s, 1H), 6.31 (d, J= 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.92-3.89 (m, 4H)及2.39 (s, 3H) ppm。 Compound 16 was prepared by reaction of Intermediate A with 3-(4-pyridyl)pyrazole according to the conditions used in Example 13. Obtain compound 16, 4-[5-(3-methylpyrazol-1-yl)-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7-yl]𠰌line; LC - MS (ESI+): m/z 362 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.70 (dd, J = 4.5, 1.5 Hz, 2H), 8.49 (d, J = 2.4 Hz, 1H), 7.83 (dd, J = 4.5, 1.5 Hz, 2H) , 6.97 (s, 1H), 6.83 (s, 1H), 6.31 (d, J = 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.92-3.89 (m, 4H) and 2.39 (s, 3H ) ppm.
中間物D：4-(5-氯-2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉
D.2 ：合成 3-( 吡啶 -2- 基 )-1H- 吡唑 -5- 胺 
向3-側氧基-3-(吡啶-2-基)丙腈(2.5 g，17.1 mmol)於EtOH (75 mL)中之溶液中添加NH 2NH 2.H 2O (1.71 g，34.2 mmol)。將反應混合物加熱至回流過夜。在經TLC分析指示反應完成後，將反應混合物直接濃縮，且藉由用1% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈棕色固體狀之3-(吡啶-2-基)-1H-吡唑-5-胺(1.34 g，8.37 mmol)。LC-MS (ESI+)：m/z 161 (MH +)。 1HNMR (300 MHz, CDCl 3) δ:8.58 (d, J= 4.2 Hz, 1H), 7.75-7.69 (m, 1H), 7.52 (d, J= 8.1 Hz, 1H), 7.24-7.20 (m, 1H), 6.09 (s, 1H)。 To a solution of 3-oxo-3-(pyridin-2-yl)propionitrile (2.5 g, 17.1 mmol) in EtOH (75 mL) was added NH 2 NH 2 .H 2 O (1.71 g, 34.2 mmol ). The reaction mixture was heated to reflux overnight. After the reaction was complete as indicated by TLC analysis, the reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 1% MeOH/DCM to 3% MeOH/DCM to afford 3-( Pyridin-2-yl)-1H-pyrazol-5-amine (1.34 g, 8.37 mmol). LC-MS (ESI+): m/z 161 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ: 8.58 (d, J = 4.2 Hz, 1H), 7.75-7.69 (m, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.24-7.20 (m, 1H), 6.09 (s, 1H).
D.3：合成2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-5,7(4H,6H)-二酮
在環境溫度，向無水EtOH (10 mL)之溶液中小心地添加小塊鈉(580 mg，25.2 mmol)。在溶解所有鈉之後，濃縮溶液，得到呈白色固體狀之新鮮NaOEt。將新鮮製備之NaOEt添加至丙二酸二乙酯(40 mL)及3-(吡啶-2-基)-1H-吡唑-5-胺(1.34 g，8.37 mmol)之混合物中。將混合物加熱至110℃，且在該溫度攪拌過夜。在將反應混合物冷卻至環境溫度之後，沈澱出大量固體。過濾之後，濾餅用***洗滌兩次，得到呈黃色固體狀之粗物質2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-5,7(4H,6H)-二酮(3.89 g，17.1 mg)。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：229 (MH +)。 To a solution of anhydrous EtOH (10 mL) was carefully added a small piece of sodium (580 mg, 25.2 mmol) at ambient temperature. After all the sodium was dissolved, the solution was concentrated to give fresh NaOEt as a white solid. Freshly prepared NaOEt was added to a mixture of diethylmalonate (40 mL) and 3-(pyridin-2-yl)-1H-pyrazol-5-amine (1.34 g, 8.37 mmol). The mixture was heated to 110 °C and stirred at this temperature overnight. After cooling the reaction mixture to ambient temperature, a large amount of solid precipitated out. After filtration, the filter cake was washed twice with diethyl ether to give crude 2-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-di Ketones (3.89 g, 17.1 mg). The crude product was used directly in the next step without further purification. LC-MS (ESI+): 229 (MH + ).
D.4：合成5,7-二氯-2-(吡啶-2-基)吡唑并[1,5-a]嘧啶
將粗物質2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-5,7(4H,6H)-二酮(3.89 g，17.1 mmol)於苯膦醯二氯(20 mL)中之溶液加熱至110℃過夜。反應混合物用NaHCO
3飽和溶液淬滅且使其鹼化至pH 8。水溶液用DCM/MeOH (15:1，6 × 50 mL)萃取。經合併之有機相經無水Na
2SO
4乾燥，過濾且減壓濃縮。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 265/267 (MH
+)。
The crude material 2-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (3.89 g, 17.1 mmol) was dissolved in phenylphosphonyl dichloride (20 mL) was heated to 110°C overnight. The reaction mixture was quenched with saturated NaHCO 3 solution and allowed to basify to
中間物D：合成4-(5-氯-2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉
向粗物質5,7-二氯-2-(吡啶-2-基)吡唑并[1,5-a]嘧啶(0.78 g，2.83 mmol)於1,4-二㗁烷(50 mL)中之溶液中添加𠰌啉(0.49 g，5.65 mmol)。在環境溫度攪拌反應物1 h。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之4-(5-氯-2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(400 mg，1.27 mmol)。LC-MS (ESI+)：m/z 316/318 (MH
+)。
1HNMR (300 MHz, CDCl
3) δ 8.71 (d,
J= 4.5 Hz, 1H), 8.12 (d,
J= 7.8 Hz, 1H), 7.82-7.77 (m, 1H), 7.33-7.26 (m, 1H), 7.17 (s, 1H), 6.12 (s, 1H), 4.03-3.99 (m, 4H), 3.88-3.85 (m, 4H)。
To
中間物E：4-(5-氯-2-(吡啶-3-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉
中間物E係藉由用於製備中間物D之相同方法製備，不同之處在於第一步驟中所使用之起始材料為3-側氧基-3-(吡啶-3-基)丙腈而非3-側氧基-3-(吡啶-2-基)丙腈。Intermediate E was prepared by the same method used to prepare Intermediate D, except that the starting material used in the first step was 3-oxo-3-(pyridin-3-yl)propionitrile and Non-3-oxo-3-(pyridin-2-yl)propionitrile.
實例17：4-(2-(吡啶-3-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉。
化合物17係根據實例18所使用之條件，藉由中間物E與3-(間甲苯基)-1H-吡唑之反應製備。LC-MS (ESI+)：m/z 438 (MH
+)。
1HNMR (300 MHz, CDCl
3) δ 9.23 (s, 1H), 8.65 (d, J = 2.7 Hz, 2H), 8.24 (d, J = 8.1 Hz, 1H), 7.77-7.72 (m, 2H), 7.43-7.33 (m, 2H), 7.26-7.20 (m, 1H), 7.10 (s, 1H), 6.84-6.82 (m, 2H), 4.06-4.04 (m, 4H), 3.96-3.94 (m, 4H), 2.45 (s, 3H)。
實例18：4-(2-(吡啶-2-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉。
將4-(5-氯-2-(吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 中間物 D ，150 mg，0.48 mmol)、3-(間甲苯基)-1H-吡唑(82.7 mg，0.52 mmol)、Cs2CO3 (312 mg，0.96 mmol)及CuI (18 mg，0.014 mmol)於DMF (10 mL)中之懸浮液加熱至120℃過夜。在經TLC分析指示反應完成後，反應混合物用水(20 mL)淬滅且用DCM/MeOH (15:1，3 × 20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用1% MeOH/DCM至5% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之4-(2-(吡啶-2-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 18 ，40.6 mg，0.093 mmol)。LC-MS (ESI+)：m/z 438 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.72 (d, J = 4.8 Hz, 1H), 8.68 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.80-7.72 (m, 3H), 7.38-7.26 (m, 2H), 7.22-7.20 (m, 1H), 7.14 (s, 1H), 7.11 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.07- 4.06 (m, 4H), 3.96-3.94 (m, 4H), 2.45 (s, 3H)。 4-(5-chloro-2-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line ( intermediate D , 150 mg, 0.48 mmol), 3-( A suspension of m-tolyl)-1H-pyrazole (82.7 mg, 0.52 mmol), Cs2CO3 (312 mg, 0.96 mmol) and CuI (18 mg, 0.014 mmol) in DMF (10 mL) was heated to 120 °C overnight. After TLC analysis indicated the reaction was complete, the reaction mixture was quenched with water (20 mL) and extracted with DCM/MeOH (15:1, 3 x 20 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with gradient elution from 1% MeOH/DCM to 5% MeOH/DCM to afford 4-(2-(pyridin-2-yl)-5-(3 -(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line ( Compound 18 , 40.6 mg, 0.093 mmol). LC-MS (ESI+): m/z 438 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.72 (d, J = 4.8 Hz, 1H), 8.68 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.80-7.72 ( m, 3H), 7.38-7.26 (m, 2H), 7.22-7.20 (m, 1H), 7.14 (s, 1H), 7.11 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.07 - 4.06 (m, 4H), 3.96-3.94 (m, 4H), 2.45 (s, 3H).
中間物F：7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸
F.2 ： 5,7- 二側氧基 -4,5,6,7- 四氫吡唑并 [1,5-a] 嘧啶 -2- 甲酸乙酯 
在環境溫度，向無水EtOH (10 mL)之溶液中小心地添加小塊鈉(890 mg，38.7 mmol)。在溶解所有鈉之後，濃縮溶液，得到呈白色固體狀之新鮮NaOEt。將新鮮製備之NaOEt添加至丙二酸二乙酯(40 mL)及5-胺基-1H-吡唑-3-甲酸乙酯(2 g，12.9 mmol)之混合物中。將混合物加熱至120℃，且在該溫度攪拌過夜。在將反應混合物冷卻至室溫之後，沈澱出大量固體。過濾之後，濾餅用***洗滌兩次，得到呈黃色固體狀之5,7-二側氧基-4,5,6,7-四氫吡唑并[1,5-a]嘧啶-2-甲酸乙酯(5.92 g，26.5 mmol)。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 224 (MH +)。 To a solution of anhydrous EtOH (10 mL) was carefully added a small piece of sodium (890 mg, 38.7 mmol) at ambient temperature. After all the sodium was dissolved, the solution was concentrated to give fresh NaOEt as a white solid. Freshly prepared NaOEt was added to a mixture of diethylmalonate (40 mL) and ethyl 5-amino-1H-pyrazole-3-carboxylate (2 g, 12.9 mmol). The mixture was heated to 120 °C and stirred at this temperature overnight. After cooling the reaction mixture to room temperature, a large amount of solid precipitated out. After filtration, the filter cake was washed twice with diethyl ether to give 5,7-dioxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2- Ethyl formate (5.92 g, 26.5 mmol). The crude product was used directly in the next step without further purification. LC-MS (ESI+): m/z 224 (MH + ).
F.3合成5,7-二氯吡唑并[1,5-a]嘧啶-2-甲酸乙酯
將粗物質5,7-二側氧基-4,5,6,7-四氫吡唑并[1,5-a]嘧啶-2-甲酸乙酯(5.92 g，26.5 mmol)於苯膦醯二氯(15 mL)中之溶液加熱至120℃過夜。反應混合物用NaHCO
3飽和溶液淬滅直至其呈鹼性(pH 8)。水溶液用DCM/MeOH (15:1，6 × 40 mL)萃取。經合併之有機相經無水Na
2SO
4乾燥，過濾且減壓濃縮。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 260/262 (MH+)。
The
F.4)合成5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯
向粗物質5,7-二氯吡唑并[1,5-a]嘧啶-2-甲酸乙酯(4.83 g，18.6 mmol)於1,4-二㗁烷(60 mL)中之溶液中添加𠰌啉(3.2 g，37.3 mmol)。在環境溫度攪拌反應物1 h。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.3 g，10.6 mmol)。LC-MS (ESI+)：m/z 311/313 (MH+)。
1HNMR (300 MHz, CDCl
3) δ 6.98 (s, 1H), 6.18 (s, 1H), 4.49-4.41 (m, 2H), 3.98-3.96 (m, 4H), 3.92-3.82 (m, 4H), 1.33 (t, J = 8.4 Hz, 3H)。
To a solution of
F.5：7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯
將5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.2 g，10.6 mmol)、3-(間甲苯基)-1H-吡唑(2.0 g，12.8 mmol)、Cs 2CO 3(6.9 g，21.3 mmol)及CuI (400 mg，2.1 mmol)於DMF (120 mL)中之懸浮液加熱至110℃過夜。在經TLC分析指示反應完成後，反應混合物用水(20 mL)淬滅且用DCM/MeOH (15:1，3 × 30 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用1% MeOH/DCM至5% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.8 g，8.8 mmol)。LC-MS (ESI+)：m/z 433 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J = 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.38-7.33 (m, 1H), 7.22-7.20 (m, 1H), 7.16 (s, 1H), 6.97 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 4.50-4.43 (m, 2H), 4.03-4.01 (m, 4H), 3.93-3.91 (m, 4H), 2.45 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H)。 5-Chloro-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (3.2 g, 10.6 mmol), 3-(m-tolyl)-1H-pyridine A suspension of azole (2.0 g, 12.8 mmol), Cs2CO3 (6.9 g, 21.3 mmol) and CuI (400 mg , 2.1 mmol) in DMF (120 mL) was heated to 110 °C overnight. After TLC analysis indicated the reaction was complete, the reaction mixture was quenched with water (20 mL) and extracted with DCM/MeOH (15:1, 3 x 30 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with a gradient elution from 1% MeOH/DCM to 5% MeOH/DCM to afford 7-(N-𠰌linyl)-5-(3-(m- Tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (3.8 g, 8.8 mmol). LC-MS (ESI+): m/z 433 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.38-7.33 (m, 1H), 7.22-7.20 (m, 1H), 7.16 (s, 1H), 6.97 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 4.50-4.43 (m, 2H), 4.03-4.01 (m, 4H), 3.93-3.91 (m, 4H ), 2.45 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).
中間物F：7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸
向7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.8 g，8.8 mmol)於THF (160 mL)中之溶液中添加2 N NaOH水溶液(30 mL)。將反應物加熱至40℃持續3 h。在經TLC分析指示反應完成後，向反應混合物中添加1 M HCl水溶液直至pH 4。所得溶液用EtOAc (3 × 10 mL)萃取。經合併之有機相經無水Na
2SO
4乾燥，過濾且減壓濃縮，得到呈白色固體狀之乙基 7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸(
中間物 F ，3.5 g，8.7 mmol)。LC-MS (ESI+)：m/z 405 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ8.73 (s, 1H), 7.83-7.80 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.24 (d,
J= 7.2 Hz, 1H), 7.15 (s, 1H), 7.10 (s, 1H), 6.90 (s, 1H), 3.89 (s, 8H), 2.40 (s, 3H)。
To 7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (3.8 g, 8.8 mmol) in THF (160 mL) was added 2 N aqueous NaOH (30 mL). The reaction was heated to 40 °C for 3 h. After the reaction was complete as indicated by TLC analysis, 1 M aqueous HCl was added to the reaction mixture until
實例19：N-乙基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺。
在環境溫度攪拌7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸(中間物F，100 mg，0.25 mmol)、乙胺鹽酸鹽(22.3 mg，0.27 mmol)、EDCl (118.8 mg，0.62 mmol)及DMAP (76 mg，0.62 mmol)於DCM中之溶液過夜。在經TLC分析指示反應完成後，將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之乙基 N-乙基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺(化合物 19 ，58 mg，0.13 mmol)。LC-MS (ESI+)：m/z 432 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J = 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.18 (s, 1H), 7.02 (s, 1H), 6.94-6.83 (m, 1H), 6.82 (d, J = 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.92-3.83 (m, 4H), 3.60-3.49 (m, 2H), 2.45 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H)。 Stirring 7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ( Intermediate F, 100 mg, 0.25 mmol), ethylamine hydrochloride (22.3 mg, 0.27 mmol), EDCl (118.8 mg, 0.62 mmol) and DMAP (76 mg, 0.62 mmol) in DCM overnight. After the reaction was complete as indicated by TLC analysis, the reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford ethyl N as a yellow solid. -Ethyl-7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-formyl Amine (Compound 19 , 58 mg, 0.13 mmol). LC-MS (ESI+): m/z 432 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.18 (s, 1H), 7.02 (s, 1H), 6.94-6.83 (m, 1H), 6.82 (d, J = 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.92-3.83 ( m, 4H), 3.60-3.49 (m, 2H), 2.45 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H).
實例20：N-環丙基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺。
化合物20係根據實例19所使用之方法，自中間物F及胺基環丙烷製備。LC-MS (ESI+)：m/z 444 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.64 (d,
J= 2.4 Hz, 1H), 7.75-7.71 (m, 2H), 7.35 (t,
J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.18 (s, 1H), 7.02 (s, 2H), 6.83 (d,
J= 2.7 Hz, 1H), 4.03-4.01 (m, 4H), 3.84-3.82 (m, 4H), 2.94-2.90 (m, 1H), 2.45 (s, 3H), 0.94-0.88 (m, 2H), 0.72-0.69 (m, 2H)。
實例21：(
R)-
N-(1-環丙基乙基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物21係根據實例19所使用之程序，自中間物F及(R)-1-環丙基乙胺製備。LC-MS (ESI+)：m/z 472 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.64 (d, J= 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.18 (s, 1H), 7.01 (s, 1H), 6.92 (d, J= 7.8 Hz, 1H), 6.83 (d, J= 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.87-3.85 (m, 4H), 3.65-3.63 (m, 1H), 2.45 (s, 3H), 1.35 (d, J= 6.6 Hz, 3H), 0.98-0.96 (m, 1H), 0.59-0.49 (m, 3H), 0.49-0.46 (m, 1H)。 Compound 21 was prepared according to the procedure used in Example 19 from Intermediate F and (R)-1-cyclopropylethylamine. LC-MS (ESI+): m/z 472 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.18 (s, 1H), 7.01 (s, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.87 -3.85 (m, 4H), 3.65-3.63 (m, 1H), 2.45 (s, 3H), 1.35 (d, J = 6.6 Hz, 3H), 0.98-0.96 (m, 1H), 0.59-0.49 (m , 3H), 0.49-0.46 (m, 1H).
實例22：(
S)-
N-(1-環丙基乙基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物22係根據實例19所使用之程序，自中間物F及(S)-1-環丙基乙胺製備。LC-MS (ESI+)：m/z 472 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.64 (d, J= 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.18 (s, 1H), 7.01 (s, 1H), 6.92 (d, J= 7.8 Hz, 1H), 6.83 (d, J= 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.87-3.85 (m, 4H), 3.65-3.63 (m, 1H), 2.45 (s, 3H), 1.35 (d, J= 6.6 Hz, 3H), 0.98-0.96 (m, 1H), 0.59-0.49 (m, 3H), 0.49-0.46 (m, 1H)。 Compound 22 was prepared according to the procedure used in Example 19 from Intermediate F and (S)-1-cyclopropylethylamine. LC-MS (ESI+): m/z 472 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.18 (s, 1H), 7.01 (s, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.87 -3.85 (m, 4H), 3.65-3.63 (m, 1H), 2.45 (s, 3H), 1.35 (d, J = 6.6 Hz, 3H), 0.98-0.96 (m, 1H), 0.59-0.49 (m , 3H), 0.49-0.46 (m, 1H).
實例23：
N-(2-甲氧基乙基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物23係根據實例19所使用之程序，自中間物F及3-(甲氧基)乙胺製備。LC-MS (ESI+)：m/z 462 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 HZ, 2H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.01 (s, 1H), 6.83 (d, J= 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.87-3.84 (m, 4H), 3.72-3.67 (m, 2H), 3.62-3.58 (m, 2H), 3.42 (s, 3H), 2.45 (s, 3H)。 Compound 23 was prepared according to the procedure used in Example 19 from Intermediate F and 3-(methoxy)ethylamine. LC-MS (ESI+): m/z 462 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 HZ, 2H), 7.22-7.20 (m, 1H ), 7.17 (s, 1H), 7.01 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.87-3.84 (m, 4H), 3.72-3.67 ( m, 2H), 3.62-3.58 (m, 2H), 3.42 (s, 3H), 2.45 (s, 3H).
實例24：
N,
N-二甲基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物24係根據實例19所使用之程序，自中間物F及二甲胺製備。LC-MS (ESI+)：m/z 432 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.63 (d, J= 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 HZ, 1H), 7.22-7.20 (m, 1H), 7.11 (s, 1H), 6.82 (d, J= 2.7 Hz, 1H), 6.77 (s, 1H), 4.00-3.98 (m, 4H), 3.89-3.88 (m, 4H), 3.31 (s, 3H), 3.18 (s, 3H), 2.45 (s, 3H)。 Compound 24 was prepared according to the procedure used in Example 19 from Intermediate F and dimethylamine. LC-MS (ESI+): m/z 432 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 HZ, 1H), 7.22-7.20 (m, 1H ), 7.11 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 6.77 (s, 1H), 4.00-3.98 (m, 4H), 3.89-3.88 (m, 4H), 3.31 (s, 3H), 3.18 (s, 3H), 2.45 (s, 3H).
實例25：
N-乙基-
N-甲基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物25係根據實例37所使用之程序，藉由化合物19之N-甲基化製備。LC-MS (ESI+)：m/z 446 (MH
+)。
1HNMR (300 MHz, CDCl
3) δ 8.63 (d,
J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t,
J= 7.5 HZ, 1H), 7.22-7.20 (m, 1H), 7.11 (s, 1H), 6.82 (d,
J= 2.7 Hz, 1H), 6.78 (s, 1H), 4.02-3.98 (m, 4H), 3.92-3.88 (m, 4H), 3.69-3.64 (m, 2H), 3.28 (s, 1H), 3.14 (s, 2H), 2.45 (s, 3H), 1.32-1.28 (m, 3H)。
實例26：
N-環丙基-
N-甲基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物26係根據實例19所使用之方法，自中間物F及N-甲基(環丙基)胺製備。LC-MS (ESI+)：m/z 458 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 HZ, 1H), 7.21 (d, J= 7.2 HZ, 1H), 7.10 (s, 1H), 6.82 (d, J= 2.4 Hz, 1H), 6.74 (s, 1H), 3.99-3.98 (m, 4H), 3.89-3.88 (m, 4H), 3.17 (s, 3H), 3.03-2.96 (m, 1H), 2.45 (s, 3H), 0.88-0.82 (m, 1H), 0.80-0.59 (m, 3H)。 Compound 26 was prepared according to the method used in Example 19 from Intermediate F and N-methyl(cyclopropyl)amine. LC-MS (ESI+): m/z 458 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 HZ, 1H), 7.21 (d, J = 7.2 HZ, 1H), 7.10 (s, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.74 (s, 1H), 3.99-3.98 (m, 4H), 3.89-3.88 (m, 4H), 3.17 (s, 3H), 3.03-2.96 (m, 1H), 2.45 (s, 3H), 0.88-0.82 (m, 1H), 0.80-0.59 (m, 3H).
實例27：
N-(環丙基甲基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物27係根據實例19所使用之偶合程序，藉由中間物F與N-(環丙基甲基)胺之反應製備。LC-MS (ESI+)：m/z 458 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J= 7.5 HZ, 1H), 7.22-7.20 (m, 1H), 7.18 (s, 1H), 7.08-7.06 (m, 1H), 7.02 (s, 1H), 6.83 (d, J= 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.88-3.85 (m, 4H), 3.37 (t, J= 6.6 HZ, 1H), 2.45 (s, 3H), 1.11-1.09 (m, 1H), 0.59-0.57 (m, 2H), 0.33-0.31 (m, 2H)。 Compound 27 was prepared according to the coupling procedure used in Example 19 by reaction of intermediate F with N-(cyclopropylmethyl)amine. LC-MS (ESI+): m/z 458 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J = 7.5 HZ, 1H), 7.22-7.20 (m, 1H ), 7.18 (s, 1H), 7.08-7.06 (m, 1H), 7.02 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.88-3.85 ( m, 4H), 3.37 (t, J = 6.6 HZ, 1H), 2.45 (s, 3H), 1.11-1.09 (m, 1H), 0.59-0.57 (m, 2H), 0.33-0.31 (m, 2H) .
實例28：吖呾-1-基(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)甲酮
化合物28係根據實例19所使用之偶合程序，自吖呾及中間物F製備。LC-MS (ESI+)：m/z 444 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.83-7.66 (m, 2H), 7.35 (t, J= 7.5 HZ, 1H), 7.22-7.20 (m, 1H), 7.13 (s, 1H), 6.98 (s, 1H), 6.82 (d, J= 2.4 Hz, 1H), 4.65 (t, J= 7.5 HZ, 2H), 4.28 (t, J= 7.5 HZ, 1H), 4.01-3.98 (m, 4H), 3.88-3.85 (m, 4H), 2.45-2.37 (m, 5H)。 Compound 28 was prepared from acridine and Intermediate F according to the coupling procedure used in Example 19. LC-MS (ESI+): m/z 444 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.83-7.66 (m, 2H), 7.35 (t, J = 7.5 HZ, 1H), 7.22-7.20 (m, 1H ), 7.13 (s, 1H), 6.98 (s, 1H), 6.82 (d, J = 2.4 Hz, 1H), 4.65 (t, J = 7.5 HZ, 2H), 4.28 (t, J = 7.5 HZ, 1H ), 4.01-3.98 (m, 4H), 3.88-3.85 (m, 4H), 2.45-2.37 (m, 5H).
實例29：(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)(吡咯啶-1-基)甲酮
化合物29係藉由實例19所使用之相同程序，藉由中間物F與吡咯啶之反應製備。LC-MS (ESI+)：m/z 458 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 HZ, 1H), 7.22-7.20 (m, 1H), 7.12 (s, 1H), 6.94 (s, 1H), 6.82 (d, J= 2.4 Hz, 1H), 4.00-3.88 (m, 10H), 3.76-3.71 (m, 2H), 2.45 (s, 3H), 1.98-1.94 (m, 4H)。 Compound 29 was prepared by the same procedure used in Example 19, by reaction of intermediate F with pyrrolidine. LC-MS (ESI+): m/z 458 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 HZ, 1H), 7.22-7.20 (m, 1H ), 7.12 (s, 1H), 6.94 (s, 1H), 6.82 (d, J = 2.4 Hz, 1H), 4.00-3.88 (m, 10H), 3.76-3.71 (m, 2H), 2.45 (s, 3H), 1.98-1.94 (m, 4H).
實例30：(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)(哌啶-1-基)甲酮
化合物30係藉由實例19所使用之相同程序，藉由中間物F與哌啶之反應製備。LC-MS (ESI+)：m/z 472 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 HZ, 1H), 7.21 (t, J= 7.5 HZ, 1H), 7.10 (s, 1H), 6.82 (d, J= 2.4 Hz, 1H), 6.71 (s, 1H), 4.00-3.98 (m, 4H), 3.90-3.88 (m, 4H), 3.80-3.76 (m, 4H), 2.45 (s, 3H), 1.80-1.60 (m, 6H)。 Compound 30 was prepared by the same procedure used in Example 19, by reaction of intermediate F with piperidine. LC-MS (ESI+): m/z 472 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 HZ, 1H), 7.21 (t, J = 7.5 HZ, 1H), 7.10 (s, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.71 (s, 1H), 4.00-3.98 (m, 4H), 3.90-3.88 (m, 4H), 3.80 -3.76 (m, 4H), 2.45 (s, 3H), 1.80-1.60 (m, 6H).
實例31：(N-𠰌啉基)(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)甲酮
化合物31係藉由實例19所使用之相同程序，藉由中間物F與𠰌啉之反應製備。LC-MS (ESI+)：m/z 474 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J= 7.5 HZ, 1H), 7.23-7.20 (m, 1H), 7.13 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 6.77 (s, 1H), 4.00-3.97 (m, 6H), 3.90-3.86 (m, 8H), 3.80-3.76 (m, 2H), 2.45 (s, 3H)。 Compound 31 was prepared by the same procedure used in Example 19, by reaction of intermediate F with oxoline. LC-MS (ESI+): m/z 474 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.4 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J = 7.5 HZ, 1H), 7.23-7.20 (m, 1H ), 7.13 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.77 (s, 1H), 4.00-3.97 (m, 6H), 3.90-3.86 (m, 8H), 3.80-3.76 ( m, 2H), 2.45 (s, 3H).
實例32：(4-甲基哌𠯤-1-基)(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)甲酮
化合物32係藉由實例19所使用之相同程序，藉由中間物F與N-(甲基)哌啶之反應製備。LC-MS (ESI+)：m/z 487 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 HZ, 1H), 7.21 (t, J= 7.5 HZ, 1H), 7.11 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 6.75 (s, 1H), 3.99-3.96 (m, 4H), 3.92-3.87 (m, 8H), 2.60-2.54 (m, 2H), 2.50-2.45 (m, 5H), 2.35 (s, 3H)。 Compound 32 was prepared by the same procedure used in Example 19, by reaction of Intermediate F with N-(methyl)piperidine. LC-MS (ESI+): m/z 487 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 HZ, 1H), 7.21 (t, J = 7.5 HZ, 1H), 7.11 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.75 (s, 1H), 3.99-3.96 (m, 4H), 3.92-3.87 (m, 8H), 2.60 -2.54 (m, 2H), 2.50-2.45 (m, 5H), 2.35 (s, 3H).
實例33：
N-甲氧基-
N-甲基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物33係藉由實例19所使用之相同程序，藉由中間物F與N-甲氧基-N-甲胺之反應製備。LC-MS (ESI+)：m/z 448 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J= 7.5 HZ, 1H), 7.21 (t, J= 7.2 HZ, 1H), 7.14 (s, 1H), 6.92 (s, 1H), 6.82 (d, J= 2.7 Hz, 1H), 4.13-4.11 (m, 4H), 4.00-3.99 (m, 4H), 3.82 (s, 3H), 3.48 (s, 3H), 2.45 (s, 3H)。 Compound 33 was prepared by the same procedure used in Example 19, by reaction of Intermediate F with N-methoxy-N-methylamine. LC-MS (ESI+): m/z 448 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J = 7.5 HZ, 1H), 7.21 (t, J = 7.2 Hz, 1H), 7.14 (s, 1H), 6.92 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 4.13-4.11 (m, 4H), 4.00-3.99 (m, 4H), 3.82 (s, 3H), 3.48 (s, 3H), 2.45 (s, 3H).
實例34：
N-甲氧基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物34係藉由實例19所使用之相同程序，藉由中間物F與N-甲氧胺之反應製備。LC-MS (ESI+)：m/z 434 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 9.39 (s, 1H), 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J= 7.5 HZ, 1H), 7.23-7.19 (m, 2H), 7.04 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 4.05-4.02 (m, 4H), 3.96 (s, 3H), 3.84-3.83 (m, 4H), 2.45 (s, 3H)。 Compound 34 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with N-methoxyamine. LC-MS (ESI+): m/z 434 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 9.39 (s, 1H), 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.36 (t, J = 7.5 HZ, 1H), 7.23-7.19 (m, 2H), 7.04 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 4.05-4.02 (m, 4H), 3.96 (s, 3H), 3.84-3.83 (m, 4H), 2.45 (s, 3H).
實例35：
N-(甲磺醯基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物35係藉由實例19所使用之相同程序，藉由中間物F與甲基磺醯胺之反應製備。LC-MS (ESI+)：m/z 482 (MH +). 1HNMR (300 MHz, CDCl 3) δ 9.09 (s, 1H), 8.62 (d, J= 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.36 (t, J= 7.5 HZ, 1H), 7.25-7.21 (m, 2H), 7.09 (s, 1H), 6.84 (d, J= 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.83-3.82 (m, 4H), 3.47 (s, 3H), 2.45 (s, 3H)。 Compound 35 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with methylsulfamide. LC-MS (ESI+): m/z 482 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 9.09 (s, 1H), 8.62 (d, J = 2.7 Hz, 1H), 7.75-7.71 (m , 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.25-7.21 (m, 2H), 7.09 (s, 1H), 6.84 (d, J = 2.7 Hz, 1H), 4.05-4.02 (m, 4H), 3.83-3.82 (m, 4H), 3.47 (s, 3H), 2.45 (s, 3H).
實例36：
N-環戊基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物36係藉由實例19所使用之相同程序，藉由中間物F與環戊胺之反應製備。LC-MS (ESI+)：m/z 472 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.89 (d, J= 7.5 Hz, 1H), 6.83 (d, J= 2.4 Hz, 1H), 4.45-4.43 (m, 1H), 4.03-4.02 (m, 4H), 3.85-3.84 (m, 4H), 2.45 (s, 3H), 2.14-2.09 (m, 2H), 1.76-1.71 (m, 4H), 1.60-1.56 (m, 2H)。 Compound 36 was prepared by the same procedure used in Example 19, by reaction of Intermediate F with cyclopentylamine. LC-MS (ESI+): m/z 472 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.17 (s, 1H), 7.00 (s, 1H), 6.89 (d, J = 7.5 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H), 4.45-4.43 (m, 1H), 4.03 -4.02 (m, 4H), 3.85-3.84 (m, 4H), 2.45 (s, 3H), 2.14-2.09 (m, 2H), 1.76-1.71 (m, 4H), 1.60-1.56 (m, 2H) .
實例37：
N-環戊基-
N-甲基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
向在0℃之N-環戊基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺(化合物36，55 mg，0.11 mmol)於DMF (5 mL)中之溶液中添加NaH (10 mg，0.24 mmol)。在該溫度攪拌0.5 h之後，將碘甲烷(25 mg，0.17 mmol)添加至上述溶液中。在經TLC分析指示反應完成後，反應混合物用水(20 mL)淬滅且用DCM/MeOH (15:1，3 × 10 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用1% MeOH/DCM至5% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之N-環戊基-N-甲基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺(化合物37，30 mg，0.062 mmol)。LC-MS (ESI+)：m/z 486 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.21 (d, J= 7.5 Hz, 1H), 7.10 (s, 1H), 6.82 (d, J= 2.7 Hz, 1H), 6.71 (s, 1H), 4.76-7.71 (m, 1H), 4.03-4.02 (m, 4H), 3.89-3.87 (m, 4H), 3.11 (s,1H), 3.03 (s, 2H), 2.45 (s, 3H), 2.02-1.52 (m, 8H)。 To N-cyclopentyl-7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a at 0°C ] To a solution of pyrimidine-2-carboxamide (Compound 36, 55 mg, 0.11 mmol) in DMF (5 mL) was added NaH (10 mg, 0.24 mmol). After stirring at this temperature for 0.5 h, iodomethane (25 mg, 0.17 mmol) was added to the above solution. After TLC analysis indicated the reaction was complete, the reaction mixture was quenched with water (20 mL) and extracted with DCM/MeOH (15:1, 3 x 10 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with gradient elution from 1% MeOH/DCM to 5% MeOH/DCM to afford N-cyclopentyl-N-methyl-7-(N-𠰌 Linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (Compound 37, 30 mg, 0.062 mmol ). LC-MS (ESI+): m/z 486 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.10 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 6.71 (s, 1H), 4.76-7.71 (m, 1H), 4.03-4.02 (m, 4H), 3.89 -3.87 (m, 4H), 3.11 (s,1H), 3.03 (s, 2H), 2.45 (s, 3H), 2.02-1.52 (m, 8H).
實例38：
N-異丙基-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物38係藉由實例19所使用之相同程序，藉由中間物F與異丙胺之反應製備。LC-MS (ESI+)：m/z 446 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.01 (s, 1H), 6.83 (d, J= 2.7 Hz, 1H), 6.75 (d, J= 8.1 Hz, 1H), 4.36-4.27 (m, 1H), 4.04-4.03 (m, 4H), 3.86-3.84 (m, 4H), 2.45 (s, 3H), 1.31 (d, J = 6.6 Hz, 6H)。 Compound 38 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with isopropylamine. LC-MS (ESI+): m/z 446 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.17 (s, 1H), 7.01 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 4.36-4.27 (m, 1H), 4.04 -4.03 (m, 4H), 3.86-3.84 (m, 4H), 2.45 (s, 3H), 1.31 (d, J = 6.6 Hz, 6H).
實例39：
N-(1,3-二甲氧基丙-2-基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物39係藉由實例19所使用之相同程序，藉由中間物F與N-(1,3-二甲氧基丙-2-基)胺之反應製備。LC-MS (ESI+)：m/z 506 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.44 (d, J= 8.4 Hz, 1H), 7.35 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 4.42-4.40 (m, 1H), 4.04-4.02 (m, 4H), 3.87-3.85 (m, 4H), 3.69-3.64 (m, 2H), 3.57-3.52 (m, 2H), 3.43 (s, 6H), 2.45 (s, 3H)。 Compound 39 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with N-(1,3-dimethoxypropan-2-yl)amine. LC-MS (ESI+): m/z 506 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 4.42-4.40 (m, 1H), 4.04 -4.02 (m, 4H), 3.87-3.85 (m, 4H), 3.69-3.64 (m, 2H), 3.57-3.52 (m, 2H), 3.43 (s, 6H), 2.45 (s, 3H).
實例40：
N-(2-(二甲胺基)乙基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物40係藉由實例19所使用之相同程序，藉由中間物F與
N-(2-(二甲胺基)乙基)胺之反應製備。LC-MS (ESI+)：m/z 475 (MH
+)。
1HNMR (300 MHz, CD
3OD) δ 8.66 (d,
J= 2.7 Hz, 1H), 7.79 (s, 1H), 7.40 (d,
J= 7.5 Hz, 1H), 7.34 (t,
J= 7.5 Hz, 1H), 7.23-7.20 (m, 2H), 6.97 (d,
J= 2.7 Hz, 1H), 6.92 (s, 1H), 3.98-3.95 (m, 8H), 3.83-3.79 (m, 2H), 3.37-3.35 (m, 2H), 2.95 (s, 6H), 2.43 (s, 3H)。
實例41：
N-(4-(二甲胺基)丁基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物41係藉由實例19所使用之相同程序，藉由中間物F與 N-(4-(二甲胺基)丁基)胺之反應製備。LC-MS (ESI+)：m/z 503 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.41-7.33 (m, 2H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.83 (d, J= 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.88-3.86 (m, 4H), 3.58-3.52 (m, 2H), 2.82-2.80 (m, 2H), 2.60 (s, 6H), 2.43 (s, 3H), 1.81-1.79 (m, 4H)。 Compound 41 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with N- (4-(dimethylamino)butyl)amine. LC-MS (ESI+): m/z 503 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.41-7.33 (m, 2H), 7.22-7.20 (m, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 4.05-4.03 (m, 4H), 3.88-3.86 (m, 4H), 3.58-3.52 (m, 2H ), 2.82-2.80 (m, 2H), 2.60 (s, 6H), 2.43 (s, 3H), 1.81-1.79 (m, 4H).
實例42：7-(N-𠰌啉基)-
N-(氧雜環丁-3-基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物42係藉由實例19所使用之相同程序，藉由中間物F與 N-(氧雜環丁-3-基)胺之反應製備。LC-MS (ESI+)：m/z 460 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.63 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.42-7.33 (m, 2H), 7.22-7.20 (m, 2H), 7.01 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 5.36-5.26 (m, 1H), 5.06 (t, J= 7.2 Hz, 2H), 4.68 (t, J= 6.6 Hz, 2H), 4.07-4.04 (m, 4H), 3.86-3.85 (m, 4H), 2.45 (s, 3H)。 Compound 42 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with N- (oxetan-3-yl)amine. LC-MS (ESI+): m/z 460 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.42-7.33 (m, 2H), 7.22-7.20 (m, 2H), 7.01 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 5.36-5.26 (m, 1H), 5.06 (t, J = 7.2 Hz, 2H), 4.68 (t, J = 6.6 Hz, 2H) , 4.07-4.04 (m, 4H), 3.86-3.85 (m, 4H), 2.45 (s, 3H).
實例43：7-(N-𠰌啉基)-
N-(氧雜環丁-3-基甲基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物43係藉由實例19所使用之相同程序，藉由中間物F與 N-(氧雜環丁-3-基甲基)胺之反應製備。LC-MS (ESI+)：m/z 474 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 3H), 7.01 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 4.87 (t, J= 7.5 Hz, 2H), 4.52 (t, J= 6.0 Hz, 2H), 4.03-4.01 (m, 4H), 3.85-3.78 (m, 6H), 3.40-3.30 (m, 1H), 2.45 (s, 3H)。 Compound 43 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with N- (oxetan-3-ylmethyl)amine. LC-MS (ESI+): m/z 474 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 3H ), 7.01 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 4.87 (t, J = 7.5 Hz, 2H), 4.52 (t, J = 6.0 Hz, 2H), 4.03-4.01 (m , 4H), 3.85-3.78 (m, 6H), 3.40-3.30 (m, 1H), 2.45 (s, 3H).
實例44：
N-((3-(羥基甲基)氧雜環丁-3-基)甲基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
化合物44係藉由實例19所使用之相同程序，藉由中間物F與 N-((3-(羥基甲基)氧雜環丁-3-基)甲基)胺之反應製備。LC-MS (ESI+)：m/z 504 (MH +)。 1HNMR (300 MHz, DMSO- d 6) δ 8.73 (d, J= 2.7 Hz, 1H), 8.67-8.64 (m, 1H), 7.83-7.80 (m, 2H), 7.38 (t, J= 7.5 Hz, 1H), 7.23 (d, J= 7.8 Hz, 1H), 7.15 (d, J= 2.7 Hz, 1H), 7.08 (s, 1H), 6.84 (s, 1H), 5.12 (t, J= 7.8 HZ, 1H), 4.42 (d, J= 5.7 Hz, 2H), 4.30 (d, J= 6.0 Hz, 2H), 3.89 (s, 8H), 3.68 (d, J = 5.1 Hz, 2H), 3.60 (d, J = 5.4 Hz, 2H), 2.40 (s, 3H)。 Compound 44 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with N -((3-(hydroxymethyl)oxetan-3-yl)methyl)amine. LC-MS (ESI+): m/z 504 (MH + ). 1 HNMR (300 MHz, DMSO- d 6 ) δ 8.73 (d, J = 2.7 Hz, 1H), 8.67-8.64 (m, 1H), 7.83-7.80 (m, 2H), 7.38 (t, J = 7.5 Hz , 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 2.7 Hz, 1H), 7.08 (s, 1H), 6.84 (s, 1H), 5.12 (t, J = 7.8 Hz , 1H), 4.42 (d, J = 5.7 Hz, 2H), 4.30 (d, J = 6.0 Hz, 2H), 3.89 (s, 8H), 3.68 (d, J = 5.1 Hz, 2H), 3.60 (d , J = 5.4 Hz, 2H), 2.40 (s, 3H).
實例45：(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)(2-氧雜-6-氮雜螺[3.3]庚烷-6-基)甲酮
化合物45係藉由實例19所使用之相同程序，藉由中間物F與2-氧雜-6-氮雜螺[3.3]庚烷之反應製備。LC-MS (ESI+)：m/z 486 (MH +)。 1HNMR (300 MHz, CDCl 3) δ 8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.15 (s, 1H), 6.97 (s, 1H), 6.83 (d, J= 2.4 Hz, 1H), 4.91-4.83 (m, 4H), 4.78 (s, 2H), 4.39 (s, 2H), 4.04-4.02 (m, 4H), 3.88-3.87 (m, 4H), 2.45 (s, 3H)。 Compound 45 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with 2-oxa-6-azaspiro[3.3]heptane. LC-MS (ESI+): m/z 486 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.15 (s, 1H), 6.97 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 4.91-4.83 (m, 4H), 4.78 (s, 2H), 4.39 (s, 2H) , 4.04-4.02 (m, 4H), 3.88-3.87 (m, 4H), 2.45 (s, 3H).
實例46：(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-基)(6-氧雜-1-氮雜螺[3.3]庚烷-1-基)甲酮
化合物46係藉由實例19所使用之相同程序，藉由中間物F與6-氧雜-1-氮雜螺[3.3]庚烷之反應製備。LC-MS (ESI+)：m/z 486 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.64 (d, J= 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.22-7.20 (m, 1H), 7.15 (s, 1H), 7.02 (s, 1H), 6.83 (d, J= 2.7 Hz, 1H), 5.69 (d, J= 6.6 Hz, 2H), 4.69 (d, J= 6.6 Hz, 2H), 4.50 (t, J= 7.5 Hz, 2H), 3.99-3.97 (m, 4H), 3.88-3.84 (m, 4H), 2.69 (t, J= 7.2 Hz, 2H), 2.45 (s, 3H)。 Compound 46 was prepared by the same procedure used in Example 19, by the reaction of Intermediate F with 6-oxa-1-azaspiro[3.3]heptane. LC-MS (ESI+): m/z 486 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.76-7.71 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.22-7.20 (m, 1H ), 7.15 (s, 1H), 7.02 (s, 1H), 6.83 (d, J = 2.7 Hz, 1H), 5.69 (d, J = 6.6 Hz, 2H), 4.69 (d, J = 6.6 Hz, 2H ), 4.50 (t, J = 7.5 Hz, 2H), 3.99-3.97 (m, 4H), 3.88-3.84 (m, 4H), 2.69 (t, J = 7.2 Hz, 2H), 2.45 (s, 3H) .
實例47：7-(N-𠰌啉基)-
N-(氧雜環丁-3-基氧基)-5-(3-(間甲苯基)-1
H-吡唑-1-基)吡唑并[1,5-
a]嘧啶-2-甲醯胺
合成2-(氧雜環丁-3-基氧基)異吲哚啉-1,3-二酮
向在0℃之2-羥基異吲哚啉-1,3-二酮(4.84 g，27 mmol)於THF (120 mL)中之溶液中添加PPh 3(8.5 g，32.4 mmol)、DEAD (6.56 g，32.4 mmol)及氧雜環丁-3-醇(2 g，29.7 mmol)。將反應物加熱至30℃過夜。在經TLC分析指示反應完成後，反應混合物用NH 4Cl水溶液淬滅且用EtOAc (3 × 30 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用25% EtOAc/PE至33% EtOAc/PE之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之不純2-(氧雜環丁-3-基氧基)異吲哚啉-1,3-二酮(2.3 g，10.5 mmol)。純度為約10%。LC-MS (ESI+)：m/z 220 (MH +)。 To a solution of 2-hydroxyisoindoline-1,3-dione (4.84 g, 27 mmol) in THF (120 mL) at 0 °C was added PPh (8.5 g, 32.4 mmol), DEAD (6.56 g, 32.4 mmol) and oxetan-3-ol (2 g, 29.7 mmol). The reaction was heated to 30°C overnight. After TLC analysis indicated the reaction was complete, the reaction mixture was quenched with aqueous NH4Cl and extracted with EtOAc (3 x 30 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with gradient elution from 25% EtOAc/PE to 33% EtOAc/PE to give impure 2-(oxetan-3-yloxy)isoind as a white solid Indoline-1,3-dione (2.3 g, 10.5 mmol). The purity is about 10%. LC-MS (ESI+): m/z 220 (MH + ).
合成O-(氧雜環丁-3-基)羥胺
向不純的2-(氧雜環丁-3-基氧基)異吲哚啉-1,3-二酮(1 g，0.5 mmol)於MeOH (75 mL)中之溶液中添加NH 2NH 2.H 2O (229 mg，4.58 mmol)。將反應混合物加熱至65℃且在該溫度攪拌1.5 h。直接濃縮反應混合物。向殘餘物中添加EtOAc (10 mL)且沈澱出大量固體。過濾之後，濃縮濾液，得到粗物質O-(氧雜環丁-3-基)羥胺(400 mg，4.49 mmol)。粗產物未經進一步純化即直接用於下一步驟中。 To a solution of impure 2-(oxetan-3 - yloxy)isoindoline-1,3-dione (1 g, 0.5 mmol) in MeOH (75 mL) was added NH2NH2 .H2O (229 mg, 4.58 mmol). The reaction mixture was heated to 65 °C and stirred at this temperature for 1.5 h. The reaction mixture was directly concentrated. EtOAc (10 mL) was added to the residue and a large amount of solid precipitated out. After filtration, the filtrate was concentrated to afford crude O-(oxetan-3-yl)hydroxylamine (400 mg, 4.49 mmol). The crude product was used directly in the next step without further purification.
合成7-(N-𠰌啉基)-N-(氧雜環丁-3-基氧基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
在室溫攪拌乙基 7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸(100 mg，0.25 mmol)、粗物質O-(氧雜環丁-3-基)羥胺(400 mg，4.49 mmol)、EDCl (154 mg，0.80 mmol)及DMAP (98 mg，0.80 mmol)於DCM中之溶液過夜。在經TLC分析指示反應完成後，將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到7-(N-𠰌啉基)-N-(氧雜環丁-3-基氧基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺(化合物47，19 mg，0.04 mmol)。LC-MS (ESI+)：m/z 476 (MH +)。 1HNMR (300 MHz, DMSO- d 6) δ 12.08 (s, 1H), 8.72 (d, J= 2.7 Hz, 1H), 7.82-7.80 (m, 2H), 7.38 (t, J= 6.9 Hz, 1H), 7.23 (d, J= 7.8 Hz, 1H), 7.15 (d, J= 2.7 Hz, 1H), 7.08 (s, 1H), 6.85 (s, 1H), 5.11-5.08 (m, 1H), 4.78-4.74 (m, 2H), 4.69-4.65 (m, 2H), 3.89 (s, 8H), 2.40 (s, 3H)。 Ethyl 7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2- Formic acid (100 mg, 0.25 mmol), crude O-(oxetan-3-yl) hydroxylamine (400 mg, 4.49 mmol), EDCl (154 mg, 0.80 mmol) and DMAP (98 mg, 0.80 mmol) in Solution in DCM overnight. After the reaction was complete as indicated by TLC analysis, the reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford 7-(N-alphalinyl )-N-(oxetan-3-yloxy)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2 - Formamide (compound 47, 19 mg, 0.04 mmol). LC-MS (ESI+): m/z 476 (MH + ). 1 HNMR (300 MHz, DMSO- d 6 ) δ 12.08 (s, 1H), 8.72 (d, J = 2.7 Hz, 1H), 7.82-7.80 (m, 2H), 7.38 (t, J = 6.9 Hz, 1H ), 7.23 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 2.7 Hz, 1H), 7.08 (s, 1H), 6.85 (s, 1H), 5.11-5.08 (m, 1H), 4.78 -4.74 (m, 2H), 4.69-4.65 (m, 2H), 3.89 (s, 8H), 2.40 (s, 3H).
通用程序1
化合物 48 ： N-[(3S)-1- 甲基吡咯啶 -3- 基 ]-7-(N- 𠰌 啉基 )-5-[3-( 間甲苯基 ) 吡唑 -1- 基 ] 吡唑并 [1,5-a] 嘧啶 -2- 甲醯胺 
合成 5,7- 二側氧基 -4,5,6,7- 四氫吡唑并 [1,5-a] 嘧啶 -2- 甲酸乙酯 
在室溫，向無水EtOH (10 mL)之溶液中小心地添加小塊鈉(890 mg，38.7 mmol)。在溶解所有鈉之後，濃縮溶液，得到呈白色固體狀之新鮮NaOEt。將新鮮製備之NaOEt添加至丙二酸二乙酯(40 mL)及5-胺基-1H-吡唑-3-甲酸乙酯(2 g，12.9 mmol)之混合物中。將混合物加熱至120℃且在該溫度攪拌過夜。在將反應混合物冷卻至室溫之後，沈澱出大量固體。過濾之後，濾餅用***洗滌兩次，得到呈黃色固體狀之5,7-二側氧基-4,5,6,7-四氫吡唑并[1,5-a]嘧啶-2-甲酸乙酯(5.92 g，26.5 mmol)。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 224 (MH +)。 To a solution of anhydrous EtOH (10 mL) was carefully added a small piece of sodium (890 mg, 38.7 mmol) at room temperature. After all the sodium was dissolved, the solution was concentrated to give fresh NaOEt as a white solid. Freshly prepared NaOEt was added to a mixture of diethylmalonate (40 mL) and ethyl 5-amino-1H-pyrazole-3-carboxylate (2 g, 12.9 mmol). The mixture was heated to 120 °C and stirred at this temperature overnight. After cooling the reaction mixture to room temperature, a large amount of solid precipitated out. After filtration, the filter cake was washed twice with diethyl ether to give 5,7-dioxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-2- Ethyl formate (5.92 g, 26.5 mmol). The crude product was used directly in the next step without further purification. LC-MS (ESI+): m/z 224 (MH + ).
合成 5,7- 二氯吡唑并 [1,5-a] 嘧啶 -2- 甲酸乙酯 
將粗物質5,7-二側氧基-4,5,6,7-四氫吡唑并[1,5-a]嘧啶-2-甲酸乙酯(5.92 g，26.5 mmol)於苯膦醯二氯(15 mL)中之溶液加熱至120℃過夜。反應混合物用NaHCO
3飽和溶液淬滅直至pH = 8。水溶液用DCM/MeOH (15:1，6 × 40 mL)萃取。經合併之有機相經無水Na
2SO
4乾燥，過濾且減壓濃縮。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 260/262 (MH
+)。
The
合成 5- 氯 -7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 甲酸乙酯 
向粗物質5,7-二氯吡唑并[1,5-a]嘧啶-2-甲酸乙酯(4.83 g，18.6 mmol)於1,4-二㗁烷(60 mL)中之溶液中添加𠰌啉(3.2 g，37.3 mmol)。在室溫攪拌反應物1 h。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.3 g，10.6 mmol)。LC-MS (ESI+)：m/z 311/313 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ6.98 (s, 1H), 6.18 (s, 1H), 4.49-4.41 (m, 2H), 3.98-3.96 (m, 4H), 3.92-3.82 (m, 4H), 1.33 (t,
J= 8.4 Hz, 3H)。
To a solution of
合成 7-(N- 𠰌 啉基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 甲酸乙酯 
將5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.2 g，10.6 mmol)、3-(間甲苯基)-1H-吡唑(2.0 g，12.8 mmol)、Cs 2CO 3(6.9 g，21.3 mmol)及CuI (400 mg，2.1 mmol)於DMF (120 mL)中之懸浮液加熱至110℃過夜。藉由TLC監測反應完成。反應混合物用水(20 mL)淬滅且用DCM/MeOH (15:1，3 × 30 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用1% MeOH/DCM至5% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.8 g，8.8 mmol)。LC-MS (ESI+)：m/z 433 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.63 (d, J= 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.38-7.33 (m, 1H), 7.22-7.20 (m, 1H), 7.16 (s, 1H), 6.97 (s, 1H), 6.82 (d, J= 2.7 Hz, 1H), 4.50-4.43 (m, 2H), 4.03-4.01 (m, 4H), 3.93-3.91 (m, 4H), 2.45 (s, 3H), 1.44 (t, J= 7.2 Hz, 3H)。 5-Chloro-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (3.2 g, 10.6 mmol), 3-(m-tolyl)-1H-pyridine A suspension of azole (2.0 g, 12.8 mmol), Cs2CO3 (6.9 g, 21.3 mmol) and CuI (400 mg , 2.1 mmol) in DMF (120 mL) was heated to 110 °C overnight. The completion of the reaction was monitored by TLC. The reaction mixture was quenched with water (20 mL) and extracted with DCM/MeOH (15:1, 3 x 30 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with a gradient elution from 1% MeOH/DCM to 5% MeOH/DCM to afford 7-(N-𠰌linyl)-5-(3-(m- Tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (3.8 g, 8.8 mmol). LC-MS (ESI+): m/z 433 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.75-7.71 (m, 2H), 7.38-7.33 (m, 1H), 7.22-7.20 (m, 1H), 7.16 (s, 1H), 6.97 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 4.50-4.43 (m, 2H), 4.03-4.01 (m, 4H), 3.93-3.91 (m, 4H ), 2.45 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).
合成 7-(N- 𠰌 啉基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 甲酸 
向7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸乙酯(3.8 g，8.8 mmol)於THF (160 mL)中之溶液中添加2 M NaOH水溶液(30 mL)。將反應物加熱至40℃持續3 h。藉由TLC監測反應完成。向反應混合物中添加1 M HCl水溶液直至pH = 4。所得溶液用EtOAc (3 × 10 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮，得到呈白色固體狀之7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸(3.5 g，8.7 mmol)。LC-MS (ESI+)：m/z 405 (MH +)。 1HNMR (300 MHz, DMSO- d 6) δ8.73 (s, 1H), 7.83-7.80 (m, 2H), 7.38 (t, J= 7.5 Hz, 1H), 7.24 (d, J= 7.2 Hz, 1H), 7.15 (s, 1H), 7.10 (s, 1H), 6.90 (s, 1H), 3.89 (s, 8H), 2.40 (s, 3H)。 To 7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (3.8 g, 8.8 mmol) in THF (160 mL) was added 2 M aqueous NaOH (30 mL). The reaction was heated to 40 °C for 3 h. The completion of the reaction was monitored by TLC. Aqueous 1 M HCl was added to the reaction mixture until pH=4. The resulting solution was extracted with EtOAc (3 x 10 mL). The combined organic phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure to afford 7-(N-?olinyl)-5-(3-(m-tolyl)-1H-pyrazole as a white solid -1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid (3.5 g, 8.7 mmol). LC-MS (ESI+): m/z 405 (MH + ). 1 HNMR (300 MHz, DMSO- d 6 ) δ 8.73 (s, 1H), 7.83-7.80 (m, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H ), 7.15 (s, 1H), 7.10 (s, 1H), 6.90 (s, 1H), 3.89 (s, 8H), 2.40 (s, 3H).
合成 (S)-N-(1- 甲基吡咯啶 -3- 基 )-7-(N- 𠰌 啉基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 甲醯胺 
在室溫攪拌7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸(60 mg，0.15 mmol)、(S)-1-甲基吡咯啶-3-胺(16 mg，0.16 mmol)、EDCl (70.8 mg，0.37 mmol)及DMAP (46 mg，0.37 mmol)於DCM中之溶液過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用1% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之(S)-N-(1-甲基吡咯啶-3-基)-7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺( 化合物 48 ，32 mg，0.07 mmol)。LC-MS (ESI+)：m/z 487 (MH +)。 1HNMR (300 MHz, DMSO- d 6) δ8.72 (d, J= 2.4 Hz, 1H), 8.35 (d, J= 7.5 Hz, 1H), 7.82-7.79 (m, 2H), 7.38 (t, J= 7.5 Hz, 1H), 7.23 (d, J= 7.5 Hz, 1H), 7.13 ( J= 2.4 Hz, 1H), 7.07 (s, 1H), 6.85 (s, 1H), 4.50-4.40 (m, 1H), 3.90 (s, 8H), 2.82-2.72 (m, 2H), 2.50-2.45 (m, 1H), 2.40 (s, 3H), 2.31 (s, 3H), 2.27-2.19 (m, 1H), 1.87-1.82 (m, 2H)。 7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid ( 60 mg, 0.15 mmol), (S)-1-methylpyrrolidin-3-amine (16 mg, 0.16 mmol), EDCl (70.8 mg, 0.37 mmol) and DMAP (46 mg, 0.37 mmol) in DCM solution overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 1% MeOH/DCM to 3% MeOH/DCM to afford (S)-N-(1-methylpyrrole as a white solid Pyridin-3-yl)-7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2 - Formamide ( Compound 48 , 32 mg, 0.07 mmol). LC-MS (ESI+): m/z 487 (MH + ). 1 HNMR (300 MHz, DMSO- d 6 ) δ 8.72 (d, J = 2.4 Hz, 1H), 8.35 (d, J = 7.5 Hz, 1H), 7.82-7.79 (m, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 7.13 ( J = 2.4 Hz, 1H), 7.07 (s, 1H), 6.85 (s, 1H), 4.50-4.40 (m, 1H ), 3.90 (s, 8H), 2.82-2.72 (m, 2H), 2.50-2.45 (m, 1H), 2.40 (s, 3H), 2.31 (s, 3H), 2.27-2.19 (m, 1H), 1.87-1.82 (m, 2H).
通用程序2
化合物 91 ： N,N- 二甲基 -2-[5- 甲基 -3-[7-(N- 𠰌 啉基 )-5-[3-( 間甲苯基 ) 吡唑 -1- 基 ] 吡唑并 [1,5-a] 嘧啶 -2- 基 ] 吡唑 -1- 基 ] 乙胺 
合成 2- 溴吡唑并 [1,5-a] 嘧啶 -5,7(4H,6H)- 二酮 
向丙二酸二乙酯(50 mL)及3-溴-1H-吡唑-5-胺(5 g，30.86 mmol)之溶液中添加NaOEt (20%於無水EtOH中，26 g)。將混合物加熱至120℃以藉由迪安-斯塔克分離器(Dean-Stark trap)移除EtOH。接著在該溫度攪拌反應混合物過夜。在將反應混合物冷卻至室溫之後，沈澱出大量固體。過濾之後，濾餅用***洗滌兩次，得到呈黃色固體狀之粗物質2-溴吡唑并[1,5-a]嘧啶-5,7(4H,6H)-二酮(13.5 g，59.2 mmol)。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 230/232 (MH +)。 To a solution of diethylmalonate (50 mL) and 3-bromo-1H-pyrazol-5-amine (5 g, 30.86 mmol) was added NaOEt (20% in anhydrous EtOH, 26 g). The mixture was heated to 120 °C to remove EtOH by Dean-Stark trap. The reaction mixture was then stirred overnight at this temperature. After cooling the reaction mixture to room temperature, a large amount of solid precipitated out. After filtration, the filter cake was washed twice with diethyl ether to give crude 2-bromopyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (13.5 g, 59.2 mmol). The crude product was used directly in the next step without further purification. LC-MS (ESI+): m/z 230/232 (MH + ).
合成 2- 溴 -5,7- 二氯吡唑并 [1,5-a] 嘧啶 
將粗物質2-溴吡唑并[1,5-a]嘧啶-5,7(4H,6H)-二酮(13.8 g，56.7 mmol)於苯膦醯二氯(100 mL)中之溶液加熱至120℃過夜。反應混合物在冰浴中用NaHCO 3飽和溶液淬滅直至pH = 8。水溶液用DCM/MeOH (15:1，6 × 40 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮，得到粗物質2-溴-5,7-二氯吡唑并[1,5-a]嘧啶(10 g，37.8 mmol)。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 266/268 (MH +)。 A solution of crude 2-bromopyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (13.8 g, 56.7 mmol) in phenylphosphonic dichloride (100 mL) was heated to 120°C overnight. The reaction mixture was quenched with a saturated solution of NaHCO 3 in an ice bath until pH=8. The aqueous solution was extracted with DCM/MeOH (15:1, 6 x 40 mL). The combined organic phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure to give crude 2-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine (10 g, 37.8 mmol) . The crude product was used directly in the next step without further purification. LC-MS (ESI+): m/z 266/268 (MH + ).
合成 4-(2- 溴 -5- 氯吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
向粗物質2-溴-5,7-二氯吡唑并[1,5-a]嘧啶(10 g，37.8 mmol)於1,4-二㗁烷(120 mL)中之溶液中添加𠰌啉(6.58 g，75.6 mmol)。在室溫攪拌反應物1 h。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之4-(2-溴-5-氯吡唑并[1,5-a]嘧啶-7-基)𠰌啉(8.6 g，27.2 mmol)。LC-MS (ESI+)：m/z 317/319 (MH +)。 1HNMR (300 MHz, CDCl 3) δ6.52 (s, 1H), 6.08 (s, 1H), 3.98-3.93 (m, 4H), 3.82-3.76 (m, 4H)。 To a solution of crude 2-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine (10 g, 37.8 mmol) in 1,4-dioxane (120 mL) was added (6.58 g, 75.6 mmol). The reaction was stirred at room temperature for 1 h. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to give 4-(2-bromo-5-chloropyrazole as a yellow solid and[1,5-a]pyrimidin-7-yl)𠰌line (8.6 g, 27.2 mmol). LC-MS (ESI+): m/z 317/319 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 6.52 (s, 1H), 6.08 (s, 1H), 3.98-3.93 (m, 4H), 3.82-3.76 (m, 4H).
合成 4-(5-( 苯甲氧基 )-2- 溴吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
向苯甲醇(1.71 g，15.8 mmol)於DMF (60 mL)中之溶液中添加NaH (1.27 g，31.6 mmol)。在0℃攪拌混合物30 min。向反應物中添加4-(2-溴-5-氯吡唑并[1,5-a]嘧啶-7-基)𠰌啉(5 g，15.8 mmol)於DMF (20 mL)中之溶液。在0℃攪拌反應混合物2 h。藉由TLC監測反應完成。將水(200 mL)添加至上述溶液中且沈澱出大量固體。過濾之後，濾餅用***洗滌兩次，得到呈白色固體狀之4-(5-(苯甲氧基)-2-溴吡唑并[1,5-a]嘧啶-7-基)𠰌啉(7.5 g，19.3 mmol)。LC-MS (ESI+)：m/z 389/391 (MH +)。 1HNMR (300 MHz, CDCl 3) δ7.61-7.32 (m, 5H), 6.34 (s, 1H), 5.66 (s, 1H), 5.34 (s, 2H), 3.98-3.92 (m, 4H), 3.66-3.59 (m, 4H)。 To a solution of benzyl alcohol (1.71 g, 15.8 mmol) in DMF (60 mL) was added NaH (1.27 g, 31.6 mmol). The mixture was stirred at 0 °C for 30 min. To the reaction was added a solution of 4-(2-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (5 g, 15.8 mmol) in DMF (20 mL). The reaction mixture was stirred at 0 °C for 2 h. The completion of the reaction was monitored by TLC. Water (200 mL) was added to the above solution and a large amount of solid precipitated out. After filtration, the filter cake was washed twice with diethyl ether to give 4-(5-(benzyloxy)-2-bromopyrazolo[1,5-a]pyrimidin-7-yl)methanol as a white solid (7.5 g, 19.3 mmol). LC-MS (ESI+): m/z 389/391 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 7.61-7.32 (m, 5H), 6.34 (s, 1H), 5.66 (s, 1H), 5.34 (s, 2H), 3.98-3.92 (m, 4H), 3.66 -3.59 (m, 4H).
合成 3-(5-( 苯甲氧基 )-7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-5- 甲基 -1H- 吡唑 -1- 甲酸三級丁酯 
將4-(5-(苯甲氧基)-2-溴吡唑并[1,5-a]嘧啶-7-基)𠰌啉(7.5 g，19.3 mmol)、(1-(三級丁氧基羰基)-5-甲基-1H-吡唑-3-基)硼酸(4.78 g，21.2 mmol)、Pd(PPh) 2Cl 2(1.35 g，1.92 mmol)及CsF (8.77 g，57.7 mmol)於1,4-二㗁烷/H 2O (330 mL，10:1)中之懸浮液加熱至95℃過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用25% EtOAc/己烷至33% EtOAc/己烷之梯度溶離之矽膠管柱層析純化，得到呈淺黃色油狀之3-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-甲酸三級丁酯(4.2 g，8.57 mmol)。LC-MS (ESI+)：m/z 491 (MH +)。 1HNMR (300 MHz, CDCl 3) δ7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.50 (s, 1H), 6.44 (s, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 3.94-3.88 (m, 4H), 3.68-3.63 (m, 4H), 2.36 (s, 3H), 1.49 (s, 9H)。 4-(5-(Benzyloxy)-2-bromopyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (7.5 g, 19.3 mmol), (1-(tertiary butoxy ylcarbonyl)-5-methyl-1H-pyrazol-3-yl)boronic acid (4.78 g, 21.2 mmol), Pd(PPh) 2 Cl 2 (1.35 g, 1.92 mmol) and CsF (8.77 g, 57.7 mmol) The suspension in 1,4-dioxane/H 2 O (330 mL, 10:1) was heated to 95° C. overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 25% EtOAc/hexanes to 33% EtOAc/hexanes to afford 3-(5-(benzyloxy) as a light yellow oil. Base)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-5-methyl-1H-pyrazole-1-carboxylic acid tertiary butyl ester (4.2 g, 8.57 mmol). LC-MS (ESI+): m/z 491 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.50 (s, 1H), 6.44 (s, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 3.94-3.88 (m, 4H), 3.68-3.63 (m, 4H), 2.36 (s, 3H), 1.49 (s, 9H).
合成 4-(5-( 苯甲氧基 )-2-(5- 甲基 -1H- 吡唑 -3- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
向3-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-甲酸三級丁酯(4.2 g，8.57 mmol)於DCM (40 mL)中之溶液中添加TFA (5 mL)。在室溫攪拌混合物2 h。藉由TLC監測反應完成。反應混合物用NaHCO 3飽和溶液淬滅直至pH = 8。沈澱出大量固體。過濾之後，濾餅用***洗滌兩次，得到呈白色固體狀之粗物質4-(5-(苯甲氧基)-2-(5-甲基-1H-吡唑-3-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(4.5 g，11.5 mmol)。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 391 (MH +)。 1HNMR (300 MHz, CDCl 3) δ7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.58 (s, 1H), 6.51 (s, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 3.98-3.92 (m, 4H), 3.68-3.61 (m, 4H), 2.38 (s, 3H)。 To 3-(5-(benzyloxy)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-5-methyl-1H-pyrazole-1 - To a solution of tert-butyl formate (4.2 g, 8.57 mmol) in DCM (40 mL) was added TFA (5 mL). The mixture was stirred at room temperature for 2 h. The completion of the reaction was monitored by TLC. The reaction mixture was quenched with NaHCO 3 saturated solution until pH=8. A large amount of solid precipitated out. After filtration, the filter cake was washed twice with diethyl ether to give crude 4-(5-(benzyloxy)-2-(5-methyl-1H-pyrazol-3-yl)pyrazole as a white solid and[1,5-a]pyrimidin-7-yl)𠰌line (4.5 g, 11.5 mmol). The crude product was used directly in the next step without further purification. LC-MS (ESI+): m/z 391 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.58 (s, 1H), 6.51 (s, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 3.98-3.92 (m, 4H), 3.68-3.61 (m, 4H), 2.38 (s, 3H).
合成 2-(3-(5-( 苯甲氧基 )-7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-5- 甲基 -1H- 吡唑 -1- 基 )-N,N- 二甲基乙胺及 2-(5-(5-( 苯甲氧基 )-7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-3- 甲基 -1H- 吡唑 -1- 基 )-N,N- 二甲基乙胺 
向粗物質4-(5-(苯甲氧基)-2-(5-甲基-1H-吡唑-3-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(4.5 g，11.5 mmol)於DMF中之溶液中添加Cs 2CO 3(11.28g, 34.62mmol)及2-氯-N,N-二甲基乙胺鹽酸鹽(2 g，13.85 mmol)。在80℃攪拌混合物過夜。TLC顯示兩個新墨點。LC-MS顯示所需產物。在冷卻至室溫之後，反應混合物用水(50 mL)稀釋且用DCM (3 × 70 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用5% MeOH/DCM至10% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之2-(5-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-3-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(上部墨點，2.1 g，4.5 mmol)及2-(3-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(下部墨點，0.6 g，1.3 mmol)。 To crude material 4-(5-(benzyloxy)-2-(5-methyl-1H-pyrazol-3-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line ( 4.5 g, 11.5 mmol) in DMF were added Cs2CO3 (11.28 g, 34.62 mmol) and 2-chloro-N,N-dimethylethylamine hydrochloride (2 g, 13.85 mmol). The mixture was stirred overnight at 80°C. TLC showed two new ink spots. LC-MS showed the desired product. After cooling to room temperature, the reaction mixture was diluted with water (50 mL) and extracted with DCM (3 x 70 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with gradient elution from 5% MeOH/DCM to 10% MeOH/DCM to afford 2-(5-(5-(benzyloxy)-7- (N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-3-methyl-1H-pyrazol-1-yl)-N,N-dimethylethylamine (upper ink dot, 2.1 g, 4.5 mmol) and 2-(3-(5-(benzyloxy)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl) -5-methyl-1H-pyrazol-1-yl)-N,N-dimethylethylamine (lower blot, 0.6 g, 1.3 mmol).
2-(5-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-3-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(上部墨點)。LC-MS (ESI+)：m/z 462 (MH +)。 1HNMR (300 MHz, CDCl 3) δ7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.48 (s, 1H), 6.38 (s, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 4.66 (t, J= 7.5 Hz, 2H), 3.98-3.92 (m, 4H), 3.70-3.64 (m, 4H), 2.76 (t, J= 7.5 Hz, 2H), 2.35 (s, 3H), 2.26 (s, 6H)。2-(3-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(下部墨點)。LC-MS (ESI+)：m/z 462 (MH +)。 1HNMR (300 MHz, CDCl 3) δ7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.64 (s, 1H), 6.50 (s, 1H), 5.65 (s, 1H), 5.42 (s, 2H), 4.33 (t, J= 6.9 Hz, 2H), 3.98-3.93 (m, 4H), 3.68-3.63 (m, 4H), 3.08 (t, J= 6.9 Hz, 2H), 2.46 (s, 6H), 2.36 (s, 3H)。 2-(5-(5-(Benzyloxy)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-3-methyl-1H-pyrazole -1-yl)-N,N-dimethylethylamine (upper blot). LC-MS (ESI+): m/z 462 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.48 (s, 1H), 6.38 (s, 1H), 5.70 (s, 1H), 5.42 (s, 2H), 4.66 (t, J = 7.5 Hz, 2H), 3.98-3.92 (m, 4H), 3.70-3.64 (m, 4H), 2.76 (t, J = 7.5 Hz, 2H), 2.35 (s, 3H), 2.26 (s, 6H). 2-(3-(5-(Benzyloxy)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-5-methyl-1H-pyrazole -1-yl)-N,N-dimethylethylamine (lower blot). LC-MS (ESI+): m/z 462 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 7.48-7.42 (m, 2H), 7.39-7.32 (m, 3 H), 6.64 (s, 1H), 6.50 (s, 1H), 5.65 (s, 1H), 5.42 (s, 2H), 4.33 (t, J = 6.9 Hz, 2H), 3.98-3.93 (m, 4H), 3.68-3.63 (m, 4H), 3.08 (t, J = 6.9 Hz, 2H), 2.46 (s, 6H), 2.36 (s, 3H).
合成 2-(1-(2-( 二甲胺基 ) 乙基 )-5- 甲基 -1H- 吡唑 -3- 基 )-7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -5- 醇 
在H 2氣球下，在室溫向2-(3-(5-(苯甲氧基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(0.6 g，1.3 mmol)於MeOH中之溶液中添加Pd/C持續4 h。藉由TLC監測反應完成。過濾之後，直接濃縮濾液，得到呈白色固體狀之粗物質2-(1-(2-(二甲胺基)乙基)-5-甲基-1H-吡唑-3-基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-5-醇(0.4 g，1.01 mmol)。LC-MS (ESI+)：m/z 372 (H +)。 2- (3-(5-(Benzyloxy)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl) - A solution of 5-methyl-1H-pyrazol-1-yl)-N,N-dimethylethylamine (0.6 g, 1.3 mmol) in MeOH was added Pd/C for 4 h. The completion of the reaction was monitored by TLC. After filtration, the filtrate was directly concentrated to give crude 2-(1-(2-(dimethylamino)ethyl)-5-methyl-1H-pyrazol-3-yl)-7- (N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-5-ol (0.4 g, 1.01 mmol). LC-MS (ESI+): m/z 372 (H + ).
合成 2-(3-(5- 氯 -7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-5- 甲基 -1H- 吡唑 -1- 基 )-N,N- 二甲基乙胺 
將2-(1-(2-(二甲胺基)乙基)-5-甲基-1H-吡唑-3-基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-5-醇(0.4 g，1.01 mmol)於苯膦醯二氯(15 mL)中之溶液加熱至110℃持續4 h。反應混合物用NaHCO 3飽和溶液淬滅直至pH = 8。水溶液用DCM/MeOH (15:1，2 × 30 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。粗產物藉由製備型TLC來純化，得到呈白色固體狀之2-(3-(5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(250 mg，0.64 mmol)。LC-MS (ESI+)：m/z 390/392 (MH +)。 2-(1-(2-(Dimethylamino)ethyl)-5-methyl-1H-pyrazol-3-yl)-7-(N-𠰌linyl)pyrazolo[1,5 -a] A solution of pyrimidin-5-ol (0.4 g, 1.01 mmol) in phenylphosphonic dichloride (15 mL) was heated to 110° C. for 4 h. The reaction mixture was quenched with NaHCO 3 saturated solution until pH=8. The aqueous solution was extracted with DCM/MeOH (15:1, 2 x 30 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The crude product was purified by prep-TLC to afford 2-(3-(5-chloro-7-(N-alphalinyl)pyrazolo[1,5-a]pyrimidin-2-yl as a white solid )-5-methyl-1H-pyrazol-1-yl)-N,N-dimethylethylamine (250 mg, 0.64 mmol). LC-MS (ESI+): m/z 390/392 (MH + ).
合成 N,N- 二甲基 -2-(5- 甲基 -3-(7-(N- 𠰌 啉基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-1H- 吡唑 -1- 基 ) 乙胺 
將2-(3-(5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(39 mg，0.10 mmol)、4-(間甲苯基)-1H-吡唑(17 mg，0.11 mmol)、Cs
2CO
3(65 mg，0.20 mmol)及Cu
2O (2.8 mg，0.02 mmol)於DMF (5 mL)中之懸浮液加熱至110℃過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用5% MeOH/DCM至10% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之N,N-二甲基-2-(5-甲基-3-(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基)-1H-吡唑-1-基)乙胺(
化合物 91 ，9 mg，0.018 mmol)。LC-MS (ESI+)：m/z 512 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.4 Hz, 1H), 7.76-7.61 (m, 2H), 7.35 (t,
J= 7.5 Hz, 1H), 7.19 (d,
J= 7.5 Hz, 1H), 7.03 (s, 1H), 6.81-6.75 (m, 2H), 6.53 (s, 1H), 4.35-4.25 (m, 2H), 4.10-4.00 (m, 4H), 3.95-3.85 (m, 4H), 3.00-2.85 (m, 2H), 2.45 (s, 3H), 2.39 (s, 9H)。
2-(3-(5-chloro-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-5-methyl-1H-pyrazol-1-yl )-N,N-Dimethylethylamine (39 mg, 0.10 mmol), 4-(m-tolyl)-1H-pyrazole (17 mg, 0.11 mmol), Cs 2 CO 3 (65 mg, 0.20 mmol) and a suspension of Cu2O (2.8 mg, 0.02 mmol) in DMF (5 mL) was heated to 110 °C overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with gradient elution from 5% MeOH/DCM to 10% MeOH/DCM to afford N,N-dimethyl-2-(5 -Methyl-3-(7-(N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2 -yl)-1H-pyrazol-1-yl)ethanamine (
通用程序3
化合物 97 ： 2-[3-[5-[3-(3- 氯苯基 ) 吡唑 -1- 基 ]-7-(N- 𠰌 啉基 )- 吡唑并 [1,5-a] 嘧啶 -2- 基 ]-5- 甲基 - 吡唑 -1- 基 ]-N,N- 二甲基 - 乙胺 
合成 5-(3- 氯苯基 )-1-( 四氫 -2H- 哌喃 -2- 基 )-1H- 吡唑 
將1-(四氫-2H-哌喃-2-基)-5-(4,4,5,5-四甲基-1,3,2-二氧雜硼雜環戊烷-2-基)-1H-吡唑(500 mg，1.18 mmol)、1-氯-3-碘苯(280 mg，1.18 mmol)、CsF (488 mg，3.21 mmol)及Pd(dppf)Cl 2(225 mg，0.30 mmol)於1,4-二㗁烷/H 2O (44 mL，10:1)中之懸浮液加熱至60℃過夜。將反應混合物直接濃縮，且藉由用10% EtOAc/己烷至20% EtOAc/己烷之梯度溶離之矽膠管柱層析純化，得到呈黃色油狀之不純5-(3-氯苯基)-1-(四氫-2H-哌喃-2-基)-1H-吡唑(377 mg，1.44 mmol)。LC-MS (ESI+)：m/z 263/265 (MH +)。 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl )-1H-pyrazole (500 mg, 1.18 mmol), 1-chloro-3-iodobenzene (280 mg, 1.18 mmol), CsF (488 mg, 3.21 mmol) and Pd(dppf)Cl 2 (225 mg, 0.30 mmol) in 1,4-dioxane/H 2 O (44 mL, 10:1) was heated to 60° C. overnight. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 10% EtOAc/hexanes to 20% EtOAc/hexanes to give impure 5-(3-chlorophenyl) as a yellow oil -1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (377 mg, 1.44 mmol). LC-MS (ESI+): m/z 263/265 (MH + ).
合成 3-(3- 氯苯基 )-1H- 吡唑 
向不純的5-(3-氯苯基)-1-(四氫-2H-哌喃-2-基)-1H-吡唑(377 mg，1.44 mmol)於DCM中之溶液中添加TFA (3 mL)。在室溫攪拌反應物2 h。藉由TLC監測反應完成。反應混合物用NaHCO 3飽和溶液淬滅直至pH = 8。水溶液用DCM/MeOH (15:1，3 × 20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。粗產物未經進一步純化即直接用於下一步驟中。LC-MS (ESI+)：m/z 179/181 (MH +)。 To a solution of impure 5-(3-chlorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (377 mg, 1.44 mmol) in DCM was added TFA (3 mL). The reaction was stirred at room temperature for 2 h. The completion of the reaction was monitored by TLC. The reaction mixture was quenched with NaHCO 3 saturated solution until pH=8. The aqueous solution was extracted with DCM/MeOH (15:1, 3 x 20 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The crude product was used directly in the next step without further purification. LC-MS (ESI+): m/z 179/181 (MH + ).
合成 2-(3-(5-(3-(3- 氯苯基 )-1H- 吡唑 -1- 基 )-7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-5- 甲基 -1H- 吡唑 -1- 基 )-N,N- 二甲基乙胺 
將3-(3-氯苯基)-1H-吡唑(22 mg，0.12 mmol)、2-(3-(5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(40 mg，0.10 mmol)、Cs 2CO 3(68 mg，0.20 mmol)及Cu 2O (3 mg，0.02 mmol)於DMF (10 mL)中之懸浮液加熱至110℃過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之2-(3-(5-(3-(3-氯苯基)-1H-吡唑-1-基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺( 化合物 97 ，15.2 mg，0.03 mmol)。LC-MS (ESI+)：m/z 532/534 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.67 (d, J= 2.7 Hz, 1H), 7.96 (s, 1H), 7.78 (d, J= 7.2 Hz, 1H), 7.46-7.33 (m, 2H), 7.00 (s, 1H), 6.81-6.78 (m, 2H), 6.53 (s, 1H), 4.26-4.22 (m, 2H), 4.06-4.00 (m, 4H), 3.95-3.89 (m, 4H), 2.84-2.80 (m, 2H), 2.38 (s, 3H), 2.33 (s, 6H)。 3-(3-Chlorophenyl)-1H-pyrazole (22 mg, 0.12 mmol), 2-(3-(5-chloro-7-(N- a] pyrimidin-2-yl)-5-methyl-1H-pyrazol-1-yl)-N,N-dimethylethylamine (40 mg, 0.10 mmol), Cs 2 CO 3 (68 mg, 0.20 mmol) and Cu2O (3 mg, 0.02 mmol) in DMF (10 mL) was heated to 110 °C overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford 2-(3-(5-(3-( 3-Chlorophenyl)-1H-pyrazol-1-yl)-7-(N-𠰌linyl)pyrazolo[1,5-a]pyrimidin-2-yl)-5-methyl-1H- Pyrazol-1-yl)-N,N-dimethylethylamine ( Compound 97 , 15.2 mg, 0.03 mmol). LC-MS (ESI+): m/z 532/534 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.67 (d, J = 2.7 Hz, 1H), 7.96 (s, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.46-7.33 (m, 2H), 7.00 (s, 1H), 6.81-6.78 (m, 2H), 6.53 (s, 1H), 4.26-4.22 (m, 2H), 4.06-4.00 (m, 4H), 3.95-3.89 (m, 4H), 2.84-2.80 (m, 2H), 2.38 (s, 3H), 2.33 (s, 6H).
通用程序4
化合物 96 ： N,N- 二甲基 -2-[5- 甲基 -3-[5-[3-(2- 甲基 -4- 吡啶基 ) 吡唑 -1- 基 ]-7-(N- 𠰌 啉基 )- 吡唑并 [1,5-a] 嘧啶 -2- 基 ] 吡唑 -1- 基 ] 乙胺 
合成 (E)-3-( 二甲胺基 )-1-(2- 甲基吡啶 -4- 基 ) 丙 -2- 烯 -1- 酮 
將1-(2-甲基吡啶-4-基)乙酮(100 mg，0.75 mmol) 於1,1-二甲氧基-N,N-二甲基甲胺中之溶液加熱至回流持續3 h。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色油狀之(E)-3-(二甲胺基)-1-(2-甲基吡啶-4-基)丙-2-烯-1-酮(135 mg，0.71 mmol)。LC-MS (ESI+)：m/z 191 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.68 (d, J= 5.1 Hz, 1H), 7.83 (d, J= 12.3 Hz, 1H), 7.59 (s, 1H), 7.47 (d, J= 5.1 Hz, 1H), 5.63 (d, J= 12.3 Hz, 1H), 3.18 (s, 3H), 2.96 (s, 3H), 2.41 (s, 3H)。 A solution of 1-(2-methylpyridin-4-yl)ethanone (100 mg, 0.75 mmol) in 1,1-dimethoxy-N,N-dimethylmethylamine was heated to reflux for 3 h. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford (E)-3-(dimethylamino) as a yellow oil -1-(2-Methylpyridin-4-yl)prop-2-en-1-one (135 mg, 0.71 mmol). LC-MS (ESI+): m/z 191 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.68 (d, J = 5.1 Hz, 1H), 7.83 (d, J = 12.3 Hz, 1H), 7.59 (s, 1H), 7.47 (d, J = 5.1 Hz, 1H), 5.63 (d, J = 12.3 Hz, 1H), 3.18 (s, 3H), 2.96 (s, 3H), 2.41 (s, 3H).
合成 2- 甲基 -4-(1H- 吡唑 -3- 基 ) 吡啶 
向(E)-3-(二甲胺基)-1-(2-甲基吡啶-4-基)丙-2-烯-1-酮(135 mg，0.71 mmol)於乙醇(2 mL)中之溶液中添加肼(0.3 mL)。將反應混合物加熱至60℃持續30 min。藉由TLC監測反應完成。反應混合物用水(10 mL)淬滅且用DCM (3 × 20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮，得到呈黃色固體狀之2-甲基-4-(1H-吡唑-3-基)吡啶(109 mg，0.68 mmol)。LC-MS (ESI+)：m/z 160 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.53 (d, J= 3.9 Hz, 1H), 7.67 (d, J= 2.4 Hz, 1H), 7.60 (d, J= 3.6 Hz, 1H), 7.51-7.49 (m, 1H), 6.73 (d, J= 2.4 Hz, 1H), 2.64 (s, 3H)。 To (E)-3-(dimethylamino)-1-(2-methylpyridin-4-yl)prop-2-en-1-one (135 mg, 0.71 mmol) in ethanol (2 mL) To the solution was added hydrazine (0.3 mL). The reaction mixture was heated to 60 °C for 30 min. The completion of the reaction was monitored by TLC. The reaction mixture was quenched with water (10 mL) and extracted with DCM (3 x 20 mL). The combined organic phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure to give 2-methyl-4-(1H-pyrazol-3-yl)pyridine (109 mg, 0.68 mmol) as a yellow solid . LC-MS (ESI+): m/z 160 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.53 (d, J = 3.9 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 7.60 (d, J = 3.6 Hz, 1H), 7.51-7.49 ( m, 1H), 6.73 (d, J = 2.4 Hz, 1H), 2.64 (s, 3H).
合成 N,N- 二甲基 -2-(5- 甲基 -3-(5-(3-(2- 甲基吡啶 -4- 基 )-1H- 吡唑 -1- 基 )-7-(N- 𠰌 啉基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-1H- 吡唑 -1- 基 ) 乙胺 
將2-甲基-4-(1H-吡唑-3-基)吡啶(20 mg，0.12 mmol)、2-(3-(5-氯-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-5-甲基-1H-吡唑-1-基)-N,N-二甲基乙胺(40 mg，0.10 mmol)、Cs 2CO 3(68 mg，0.20 mmol)及Cu 2O (2 mg，0.01 mmol)於DMF (4 mL)中之懸浮液加熱至110℃過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之N,N-二甲基-2-(5-甲基-3-(5-(3-(2-甲基吡啶-4-基)-1H-吡唑-1-基)-7-(N-𠰌啉基)吡唑并[1,5-a]嘧啶-2-基)-1H-吡唑-1-基)乙胺( 化合物 96 ，15.6 mg，0.03 mmol)。LC-MS (ESI+)：m/z 513 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.70 (d, J= 2.7 Hz, 1H), 8.57 (d, J= 5.4 Hz, 1H), 7.68 (s, 1H), 7.62-7.60 (m, 1H), 7.00 (s, 1H), 6.87 (d, J= 2.7 Hz, 1H), 6.82 (s, 1H), 6.53 (s, 1H), 4.27-4.22 (m, 2H), 4.05-4.00 (m, 4H), 3.95-3.91 (m, 4H), 2.85-2.81 (m, 2H), 2.65 (s, 3H), 2.38 (s, 3H), 2.34 (s, 6H)。 2-Methyl-4-(1H-pyrazol-3-yl)pyridine (20 mg, 0.12 mmol), 2-(3-(5-chloro-7-(N-𠰌linyl)pyrazolo[ 1,5-a]pyrimidin-2-yl)-5-methyl-1H-pyrazol-1-yl)-N,N-dimethylethylamine (40 mg, 0.10 mmol), Cs 2 CO 3 ( 68 mg, 0.20 mmol) and Cu 2 O (2 mg, 0.01 mmol) in DMF (4 mL) was heated to 110° C. overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to afford N,N-dimethyl-2-(5 -Methyl-3-(5-(3-(2-methylpyridin-4-yl)-1H-pyrazol-1-yl)-7-(N-𠰌linyl)pyrazolo[1,5 -a] pyrimidin-2-yl)-1H-pyrazol-1-yl)ethanamine ( Compound 96 , 15.6 mg, 0.03 mmol). LC-MS (ESI+): m/z 513 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.70 (d, J = 2.7 Hz, 1H), 8.57 (d, J = 5.4 Hz, 1H), 7.68 (s, 1H), 7.62-7.60 (m, 1H), 7.00 (s, 1H), 6.87 (d, J = 2.7 Hz, 1H), 6.82 (s, 1H), 6.53 (s, 1H), 4.27-4.22 (m, 2H), 4.05-4.00 (m, 4H) , 3.95-3.91 (m, 4H), 2.85-2.81 (m, 2H), 2.65 (s, 3H), 2.38 (s, 3H), 2.34 (s, 6H).
通用程序5
化合物compound 8484 ：: 4-[2-(2,5-4-[2-(2,5- 二甲基吡唑Dimethylpyrazole -3--3- 基base )-5-[3-()-5-[3-( 間甲苯基m-tolyl )) 吡唑pyrazole -1--1- 基base ]] 吡唑并Pyrazolo [1,5-a][1,5-a] 嘧啶pyrimidine -7--7- 基base ]] 𠰌𠰌 啉及morphine and
化合物 87 ： 4-[2-(1,5- 二甲基吡唑 -3- 基 )-5-[3-( 間甲苯基 ) 吡唑 -1- 基 ] 吡唑并 [1,5-a] 嘧啶 -7- 基 ] 𠰌 啉 
合成 4-(2- 溴 -5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
將4-(2-溴-5-氯吡唑并[1,5-a]嘧啶-7-基)𠰌啉(5 g，15.8 mmol)、3-苯基-1H-吡唑(2.75 g，17.4 mmol)、Cs 2CO 3(10.3 g，31.6 mmol)及Cu 2O (453 mg，3.2 mmol)於DMF (80 mL)中之懸浮液加熱至110℃過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用20% EtOAc/己烷至33% EtOAc/己烷之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之4-(2-溴-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(5.0 g，11.4 mmol)。LC-MS (ESI+)：m/z 439/441 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.59 (d, J= 2.7 Hz, 1H), 7.80-7.62 (m, 2H), 7.35 (t, J= 7.5 Hz, 1H), 7.19-7.10 (m, 1H), 6.91 (s, 1H), 6.82 (d, J= 2.7 Hz, 1H), 6.48 (s, 1H), 4.05-3.95 (m, 4H), 3.85-3.80 (m, 4H), 2.44 (s, 3H)。 4-(2-Bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (5 g, 15.8 mmol), 3-phenyl-1H-pyrazole (2.75 g, 17.4 mmol), Cs 2 CO 3 (10.3 g, 31.6 mmol) and Cu 2 O (453 mg, 3.2 mmol) in DMF (80 mL) was heated to 110° C. overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 20% EtOAc/hexanes to 33% EtOAc/hexanes to afford 4-(2-bromo-5-( 3-(m-Tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (5.0 g, 11.4 mmol). LC-MS (ESI+): m/z 439/441 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.59 (d, J = 2.7 Hz, 1H), 7.80-7.62 (m, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.19-7.10 (m, 1H ), 6.91 (s, 1H), 6.82 (d, J = 2.7 Hz, 1H), 6.48 (s, 1H), 4.05-3.95 (m, 4H), 3.85-3.80 (m, 4H), 2.44 (s, 3H).
合成 5- 甲基 -3-(7-(N- 𠰌 啉基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 )-1H- 吡唑 -1- 甲酸三級丁酯 
將4-(2-溴-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(200 mg，0.46 mmol)、(1-(三級丁氧基羰基)-3-甲基-1H-吡唑-5-基)硼酸(114 mg，0.50 mmol)、CsF (208 mg，1.37 mmol)及Pd(dppf)Cl 2(32 mg，0.05 mmol)於1,4-二㗁烷/H 2O (22 mL，10:1)中之懸浮液加熱至100℃持續1 h。將反應混合物直接濃縮，且藉由用20% EtOAc/己烷至33% EtOAc/己烷之梯度溶離之矽膠管柱層析純化，得到呈無色油狀之5-甲基-3-(7-(N-𠰌啉基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基)-1H-吡唑-1-甲酸酯(220 mg，0.41 mmol)。LC-MS (ESI+)：m/z 541 (MH +)。 4-(2-Bromo-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (200 mg, 0.46 mmol), (1-(tertiary butoxycarbonyl)-3-methyl-1H-pyrazol-5-yl)boronic acid (114 mg, 0.50 mmol), CsF (208 mg, 1.37 mmol) and Pd( dppf) A suspension of Cl 2 (32 mg, 0.05 mmol) in 1,4-dioxane/H 2 O (22 mL, 10:1) was heated to 100° C. for 1 h. The reaction mixture was directly concentrated and purified by silica gel column chromatography with gradient elution from 20% EtOAc/hexanes to 33% EtOAc/hexanes to afford 5-methyl-3-(7- (N-𠰌linyl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-2-yl)-1H-pyrazole- 1-Carboxylate (220 mg, 0.41 mmol). LC-MS (ESI+): m/z 541 (MH + ).
合成 4-(2-(5- 甲基 -1H- 吡唑 -3- 基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
向4-(2-(5-甲基-1H-吡唑-3-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(220 mg，0.41 mmol)於DCM (5 mL)中之溶液中添加HCl/Et 2O溶液(2 mL)。藉由TLC監測反應完成。反應混合物用NaHCO 3飽和溶液淬滅直至pH = 8。水溶液用DCM/MeOH (15:1，2×20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮，得到呈淺黃色固體狀之4-(2-(5-甲基-1H-吡唑-3-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(138 mg，0.31 mmol)。LC-MS (ESI+)：m/z 441 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.63 (d, J= 2.7 Hz, 1H), 7.76-7.61 (m, 2H), 7.38-7.32 (m, 2H), 7.22-7.15 (m, 1H), 7.06 (s, 1H), 6.81 (d, J= 2.7 Hz, 1H), 6.73 (s, 1H), 6.56 (brs, 1H), 4.05-4.00 (m, 4H), 3.95-3.85 (m, 4H), 2.45 (s, 3H), 2.40 (s, 3H)。 To 4-(2-(5-methyl-1H-pyrazol-3-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a] To a solution of pyrimidin-7-yl)𠰌line (220 mg, 0.41 mmol) in DCM (5 mL) was added HCl/ Et2O solution (2 mL). The completion of the reaction was monitored by TLC. The reaction mixture was quenched with NaHCO 3 saturated solution until pH=8. The aqueous solution was extracted with DCM/MeOH (15:1, 2 x 20 mL). The combined organic phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure to afford 4-(2-(5-methyl-1H-pyrazol-3-yl)-5-( 3-(m-Tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (138 mg, 0.31 mmol). LC-MS (ESI+): m/z 441 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.63 (d, J = 2.7 Hz, 1H), 7.76-7.61 (m, 2H), 7.38-7.32 (m, 2H), 7.22-7.15 (m, 1H), 7.06 (s, 1H), 6.81 (d, J = 2.7 Hz, 1H), 6.73 (s, 1H), 6.56 (brs, 1H), 4.05-4.00 (m, 4H), 3.95-3.85 (m, 4H), 2.45 (s, 3H), 2.40 (s, 3H).
合成 4-(2-(1,5- 二甲基 -1H- 吡唑 -3- 基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉及 4-(2-(1,3- 二甲基 -1H- 吡唑 -5- 基 )-5-(3-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
向在室溫之4-(2-(5-甲基-1H-吡唑-3-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(100 mg，0.23 mmol)及Cs 2CO 3(222 mg，0.68 mmol)於DMF (5 mL)中之懸浮液中添加CH 3I (48 mg，0.34 mmol)。藉由TLC監測反應完成。反應物用水(50 ml)淬滅，且該溶液用DCM (3 × 20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。殘餘物藉由用5% MeOH/DCM至8% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到4-(2-(1,5-二甲基-1H-吡唑-3-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 87 ，下部墨點，21 mg，0.046 mmol)及4-(2-(1,3-二甲基-1H-吡唑-5-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 84 ，上部墨點，42 mg，0.092 mmol)。 To 4-(2-(5-methyl-1H-pyrazol-3-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[ To a suspension of 1,5-a]pyrimidin-7-yl)𠰌line (100 mg, 0.23 mmol) and Cs 2 CO 3 (222 mg, 0.68 mmol) in DMF (5 mL) was added CH 3 I (48 mg, 0.34 mmol). The completion of the reaction was monitored by TLC. The reaction was quenched with water (50 ml), and the solution was extracted with DCM (3 x 20 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with gradient elution from 5% MeOH/DCM to 8% MeOH/DCM to give 4-(2-(1,5-dimethyl-1H-pyrazol-3-yl )-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line ( compound 87 , lower ink spot, 21 mg , 0.046 mmol) and 4-(2-(1,3-dimethyl-1H-pyrazol-5-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyridine Azolo[1,5-a]pyrimidin-7-yl)𠰌line ( compound 84 , upper blot, 42 mg, 0.092 mmol).
4-(2-(1,5-二甲基-1H-吡唑-3-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 87 ，下部墨點)。LC-MS (ESI+)：m/z 455 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.67 (d, J= 2.7 Hz, 1H), 7.79-7.73 (m, 2H), 7.36 (t, J= 7.8 Hz, 1H), 7.22 (d, J= 7.5 Hz, 1H), 7.05 (s, 1H), 6.83-6.82 (m, 2H), 6.56 (s, 1H), 4.07-4.02 (m, 4H), 3.95-3.90 (m, 4H), 3.89 (s, 3H), 2.47 (s, 3H), 2.36 (s, 3H)。 4-(2-(1,5-Dimethyl-1H-pyrazol-3-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1, 5-a] pyrimidin-7-yl) 𠰌line ( compound 87 , lower blot). LC-MS (ESI+): m/z 455 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.67 (d, J = 2.7 Hz, 1H), 7.79-7.73 (m, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.05 (s, 1H), 6.83-6.82 (m, 2H), 6.56 (s, 1H), 4.07-4.02 (m, 4H), 3.95-3.90 (m, 4H), 3.89 (s, 3H), 2.47 (s, 3H), 2.36 (s, 3H).
4-(2-(1,3-二甲基-1H-吡唑-5-基)-5-(3-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 84 ，上部墨點)。LC-MS (ESI+)：m/z 455 (MH +). 1HNMR (300 MHz, CDCl 3) δ8.60 (d, J= 2.7 Hz, 1H), 7.75-7.70 (m, 2H), 7.33 (t, J= 7.5 Hz, 1H), 7.20 (d, J= 7.5 Hz, 1H), 7.04 (s, 1H), 6.79 (d, J= 2.7 Hz, 1H), 6.59 (s, 1H), 6.44 (s, 1H), 4.17 (s, 3H), 4.02-3.90 (m, 4H), 3.88-3.85 (m, 4H), 2.43 (s, 3H), 2.32 (s, 3H)。 4-(2-(1,3-Dimethyl-1H-pyrazol-5-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1, 5-a] pyrimidin-7-yl) 𠰌line ( compound 84 , upper blot). LC-MS (ESI+): m/z 455 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.60 (d, J = 2.7 Hz, 1H), 7.75-7.70 (m, 2H), 7.33 (t , J = 7.5 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.04 (s, 1H), 6.79 (d, J = 2.7 Hz, 1H), 6.59 (s, 1H), 6.44 (s , 1H), 4.17 (s, 3H), 4.02-3.90 (m, 4H), 3.88-3.85 (m, 4H), 2.43 (s, 3H), 2.32 (s, 3H).
通用程序6General procedure 6
化合物 57 ： 4-[5-[4-( 間甲苯基 ) 吡唑 -1- 基 ]-2- 嘧啶 -4- 基 - 吡唑并 [1,5-a] 嘧啶 -7- 基 ] 𠰌 啉 
合成 4-(2- 溴 -5-(4-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
將4-(2-溴-5-氯吡唑并[1,5-a]嘧啶-7-基)𠰌啉(3 g，9.45 mmol)、4-(間甲苯基)-1H-吡唑(1.5 g，9.45 mmol)、Cs 2CO 3(6.16 g，18.9 mmol)及Cu 2O (135 mg，0.945 mmol)於DMF (150 mL)中之懸浮液加熱至110℃過夜。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈淺黃色固體狀之4-(2-溴-5-(4-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(4.5 g，10.27 mmol)。LC-MS (ESI+)：m/z 439/441 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.81 (s, 1H), 8.02 (s, 1H), 7.47-7.33 (m, 2H), 7.30-7.26 (m, 1H), 7.13-7.11 (m, 1H), 6.94 (s, 1H), 6.50 (s, 1H), 3.98-3.92 (m, 4H), 3.85-3.78 (m, 4H), 2.41 (s, 3H)。 4-(2-Bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (3 g, 9.45 mmol), 4-(m-tolyl)-1H-pyrazole ( A suspension of 1.5 g , 9.45 mmol), Cs2CO3 (6.16 g, 18.9 mmol) and Cu2O (135 mg, 0.945 mmol) in DMF (150 mL) was heated to 110 °C overnight. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to give 4-(2-bromo-5-(4 -(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (4.5 g, 10.27 mmol). LC-MS (ESI+): m/z 439/441 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.81 (s, 1H), 8.02 (s, 1H), 7.47-7.33 (m, 2H), 7.30-7.26 (m, 1H), 7.13-7.11 (m, 1H) , 6.94 (s, 1H), 6.50 (s, 1H), 3.98-3.92 (m, 4H), 3.85-3.78 (m, 4H), 2.41 (s, 3H).
合成 4-(2-( 嘧啶 -4- 基 )-5-(4-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
在N 2下，將4-(2-溴-5-(4-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(100 mg，0.23 mmol)、4-(三丁基錫烷基)嘧啶(168 mg，0.45 mmol)、CuI (13 mg，0.068 mmol)及Pd(PPh) 2Cl 2於DMF (3 mL)中之懸浮液加熱至100℃持續3 h。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至3% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈淺黃色固體狀之4-(2-(嘧啶-4-基)-5-(4-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 57 ，24 mg，0.055 mmol)。LC-MS (ESI+)：m/z 439 (MH +). 1HNMR (300 MHz, CDCl 3) δ9.33 (s, 1H), 8.89 (s, 1H), 8.86 (s, 1H), 8.09 (s, 1H), 8.05 (s, 1H), 7.53-7.42 (m, 2H), 7.34-7.29 (m, 2H), 7.14-7.12 (m, 1H), 7.06 (s, 1H), 4.10-4.00 (m, 4H), 3.92-3.81 (m, 4H), 2.42 (s, 3H)。 4-( 2 -Bromo-5-(4-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌 Phenyl (100 mg, 0.23 mmol), 4-(tributylstannyl)pyrimidine (168 mg, 0.45 mmol), CuI (13 mg, 0.068 mmol) and Pd(PPh) 2 Cl 2 in DMF (3 mL) The suspension was heated to 100 °C for 3 h. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 3% MeOH/DCM to give 4-(2-(pyrimidin-4-yl) as a pale yellow solid )-5-(4-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line ( compound 57 , 24 mg, 0.055 mmol) . LC-MS (ESI+): m/z 439 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 9.33 (s, 1H), 8.89 (s, 1H), 8.86 (s, 1H), 8.09 (s , 1H), 8.05 (s, 1H), 7.53-7.42 (m, 2H), 7.34-7.29 (m, 2H), 7.14-7.12 (m, 1H), 7.06 (s, 1H), 4.10-4.00 (m , 4H), 3.92-3.81 (m, 4H), 2.42 (s, 3H).
通用程序7
化合物 58 ： 4-[5-[4-( 間甲苯基 ) 吡唑 -1- 基 ]-2- 嘧啶 -5- 基 - 吡唑并 [1,5-a] 嘧啶 -7- 基 ] 𠰌 啉 
合成 4-(2-( 嘧啶 -5- 基 )-5-(4-( 間甲苯基 )-1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -7- 基 ) 𠰌 啉 
將4-(2-溴-5-(4-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉(100 mg，0.23 mmol)、嘧啶-5-基硼酸(34 mg，0.28 mmol)、CsF (52 mg，0.34 mmol)及Pd(dppf)Cl 2(16 mg，0.023 mmol)於1,4-二㗁烷/H 2O (7.5 mL，2:1)中之懸浮液加熱至80℃過夜。將反應混合物直接濃縮，且藉由用1% MeOH/DCM至2% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈黃色固體狀之4-(2-(嘧啶-5-基)-5-(4-(間甲苯基)-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基)𠰌啉( 化合物 58 ，21 mg，0.048 mmol)。LC-MS (ESI+)：m/z 439 (MH +)。 1HNMR (300 MHz, CDCl 3) δ9.38-9.25 (m, 2H), 8.85 (s, 1H), 8.04 (s, 1H), 7.55-7.41 (m, 2H), 7.34-7.29 (m, 2H), 7.13 (d, J= 6.9 Hz, 1H), 7.06 (s, 1H), 6.85 (s, 1H), 4.10-4.00 (m, 4H), 3.95-3.86 (m, 4H), 2.42 (s, 3H)。 4-(2-Bromo-5-(4-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line (100 mg, 0.23 mmol), pyrimidin-5-ylboronic acid (34 mg, 0.28 mmol), CsF (52 mg, 0.34 mmol) and Pd(dppf)Cl 2 (16 mg, 0.023 mmol) in 1,4-dioxane/H The suspension in 2 O (7.5 mL, 2:1) was heated to 80 °C overnight. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 1% MeOH/DCM to 2% MeOH/DCM to afford 4-(2-(pyrimidin-5-yl) as a yellow solid -5-(4-(m-Tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)𠰌line ( Compound 58 , 21 mg, 0.048 mmol). LC-MS (ESI+): m/z 439 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 9.38-9.25 (m, 2H), 8.85 (s, 1H), 8.04 (s, 1H), 7.55-7.41 (m, 2H), 7.34-7.29 (m, 2H) , 7.13 (d, J = 6.9 Hz, 1H), 7.06 (s, 1H), 6.85 (s, 1H), 4.10-4.00 (m, 4H), 3.95-3.86 (m, 4H), 2.42 (s, 3H ).
通用程序8
化合物 89 ： N-[[7-(N- 𠰌 啉基 )-5-(3- 苯基吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 ] 甲基 ] 胺基甲酸甲酯 
合成 7-(N- 𠰌 啉基 )-5-(3- 苯基 -1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 甲醯胺 
向7-(N-𠰌啉基)-5-(3-苯基-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲酸(370 mg，0.94 mmol)於DCM (50 mL)中之溶液中添加氯甲酸甲酯(107 mg，1.12 mmol)。在室溫攪拌混合物10 min，且接著向混合物中添加氫氧化銨(143 mg，1.44 mmol)。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至10% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之7-(N-𠰌啉基)-5-(3-苯基-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺(326 mg，0.84 mmol)。LC-MS (ESI+)：m/z 390 (MH +)。 1HNMR (300 MHz, DMSO- d 6) δ8.73 (d, J= 2.7 Hz, 1H), 8.10-8.00 (m, 3H), 7.63 (s, 1H), 7.53-7.30 (m, 3H), 7.16 (d, J= 2.7 Hz, 1H), 7.04 (s, 1H), 6.83 (s, 1H), 3.95-3.85 (m, 8H)。 To 7-(N-𠰌linyl)-5-(3-phenyl-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid (370 mg, 0.94 mmol) To a solution in DCM (50 mL) was added methyl chloroformate (107 mg, 1.12 mmol). The mixture was stirred at room temperature for 10 min, and then ammonium hydroxide (143 mg, 1.44 mmol) was added to the mixture. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with gradient elution from 2% MeOH/DCM to 10% MeOH/DCM to afford 7-(N-𠰌linyl)-5- as a white solid (3-Phenyl-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (326 mg, 0.84 mmol). LC-MS (ESI+): m/z 390 (MH + ). 1 HNMR (300 MHz, DMSO- d 6 ) δ 8.73 (d, J = 2.7 Hz, 1H), 8.10-8.00 (m, 3H), 7.63 (s, 1H), 7.53-7.30 (m, 3H), 7.16 (d, J = 2.7 Hz, 1H), 7.04 (s, 1H), 6.83 (s, 1H), 3.95-3.85 (m, 8H).
合成 (7-(N- 𠰌 啉基 )-5-(3- 苯基 -1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 ) 甲胺 
向7-(N-𠰌啉基)-5-(3-苯基-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺(200 mg，0.26 mmol)於THF (50 mL)中之溶液中添加硼烷-四氫呋喃錯合物(2 mL，1.02 mmol)。在60℃攪拌混合物過夜。藉由TLC監測反應完成。反應混合物用1 M HCl水溶液淬滅且接著使用NaHCO 3溶液將PH調整至8。水溶液用DCM/MeOH (10:1，3 × 20 mL)萃取。經合併之有機相經無水Na 2SO 4乾燥，過濾且減壓濃縮。粗產物藉由用2% MeOH/DCM至5% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之(7-(N-𠰌啉基)-5-(3-苯基-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基)甲胺(110 mg，0.29 mmol)。LC-MS (ESI+)：m/z 376 (MH +)。 To 7-(N-𠰌linyl)-5-(3-phenyl-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (200 mg, 0.26 mmol) in THF (50 mL) was added borane-tetrahydrofuran complex (2 mL, 1.02 mmol). The mixture was stirred overnight at 60°C. The completion of the reaction was monitored by TLC. The reaction mixture was quenched with 1 M aqueous HCl and then the pH was adjusted to 8 using NaHCO 3 solution. The aqueous solution was extracted with DCM/MeOH (10:1, 3 x 20 mL). The combined org. phases were dried over anhydrous Na2SO4 , filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel with a gradient elution from 2% MeOH/DCM to 5% MeOH/DCM to afford (7-(N-𠰌linyl)-5-(3-benzene) as a white solid yl-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-2-yl)methanamine (110 mg, 0.29 mmol). LC-MS (ESI+): m/z 376 (MH + ).
合成 ((7-(N- 𠰌 啉基 )-5-(3- 苯基 -1H- 吡唑 -1- 基 ) 吡唑并 [1,5-a] 嘧啶 -2- 基 ) 甲基 ) 胺基甲酸甲酯 
向(7-(N-𠰌啉基)-5-(3-苯基-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基)甲胺(55 mg，0.013 mmol)於DCM (50 mL)中之溶液中添加氯甲酸甲酯(13.8 mg，0.016 mmol)。在室溫攪拌混合物10 min，且接著向混合物中添加Et 3N (22 mg，0.026 mmol)。藉由TLC監測反應完成。將反應混合物直接濃縮，且藉由用2% MeOH/DCM至10% MeOH/DCM之梯度溶離之矽膠管柱層析純化，得到呈白色固體狀之((7-(N-𠰌啉基)-5-(3-苯基-1H-吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基)甲基)胺基甲酸甲酯( 化合物 89 ，16 mg，0.037 mmol)。LC-MS (ESI+)：m/z 434 (MH +)。 1HNMR (300 MHz, CDCl 3) δ8.64 (d, J= 2.7 Hz, 1H), 7.93 (d, J= 6.9 Hz, 2H), 7.49-7.36 (m, 3H), 7.05 (s, 1H), 6.82 (d, J= 2.4 Hz, 1H), 6.42 (s, 1H), 5.24 (brs, 1H), 4.59 (d, J= 6.0 Hz, 2H), 4.13-4.02 (m, 4H), 3.86-3.80 (m, 4H), 3.74 (s, 3H)。 To (7-(N-𠰌linyl)-5-(3-phenyl-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-2-yl)methanamine (55 mg , 0.013 mmol) in DCM (50 mL) was added methyl chloroformate (13.8 mg, 0.016 mmol). The mixture was stirred at room temperature for 10 min, and then Et3N (22 mg, 0.026 mmol) was added to the mixture. The completion of the reaction was monitored by TLC. The reaction mixture was directly concentrated and purified by silica gel column chromatography with a gradient elution from 2% MeOH/DCM to 10% MeOH/DCM to afford ((7-(N-𠰌linyl)- 5-(3-Phenyl-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-2-yl)methyl)carbamate ( Compound 89 , 16 mg, 0.037 mmol ). LC-MS (ESI+): m/z 434 (MH + ). 1 HNMR (300 MHz, CDCl 3 ) δ 8.64 (d, J = 2.7 Hz, 1H), 7.93 (d, J = 6.9 Hz, 2H), 7.49-7.36 (m, 3H), 7.05 (s, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.42 (s, 1H), 5.24 (brs, 1H), 4.59 (d, J = 6.0 Hz, 2H), 4.13-4.02 (m, 4H), 3.86-3.80 (m, 4H), 3.74 (s, 3H).
實例48：N-[(3S)-1-甲基吡咯啶-3-基]-7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物48係藉由通用程序1製備。LC-MS (ESI+)：m/z 487 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ8.72 (d,
J= 2.4 Hz, 1H), 8.35 (d,
J= 7.5 Hz, 1H), 7.82-7.79 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.23 (d,
J= 7.5 Hz, 1H), 7.13 (
J= 2.4 Hz, 1H), 7.07 (s, 1H), 6.85 (s, 1H), 4.50-4.40 (m, 1H), 3.90 (s, 8H), 2.82-2.72 (m, 2H), 2.50-2.45 (m, 1H), 2.40 (s, 3H), 2.31 (s, 3H), 2.27-2.19 (m, 1H), 1.87-1.82 (m, 2H)。
Compound 48 was prepared by
實例49：N-[(3R)-1-甲基吡咯啶-3-基]-7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物49係藉由通用程序1製備。LC-MS (ESI+)：m/z 487 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ8.72 (d,
J= 2.7 Hz, 1H), 8.40 (d,
J= 7.5 Hz, 1H), 7.83-7.80 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.23 (d,
J= 7.8 Hz, 1H), 7.13 (d,
J= 2.4 Hz, 1H), 7.07 (s, 1H), 6.85 (s, 1H), 4.46-4.42 (m, 1H), 3.90 (s, 8H), 2.83-2.73 (m, 2H), 2.69-2.63 (m, 1H), 2.50 (s, 3H), 2.40 (s, 3H), 2.28-2.23 (m, 1H), 1.98-1.85 (m, 2H)。
Compound 49 was prepared by
實例50：7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]-N-[(3S)-四氫呋喃-3-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物50係藉由通用程序1製備。LC-MS (ESI+)：m/z 474 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.72 (d,
J= 2.4 Hz, 1H), 7.75-7.71 (m, 2H), 7.35 (t,
J= 7.5 Hz, 1H), 7.22-7.19 (m, 2H), 7.07 (d,
J= 7.8 Hz, 1H), 7.00 (s, 1H), 6.82 (d,
J= 2.4 Hz, 1H), 4.78-4.70 (m, 1H), 4.07-3.94 (m, 6H), 3.90-3.78 (m, 6H), 2.45 (s, 3H), 2.41-2.39 (m, 1H), 1.98-1.85 (m, 1H)。
實例51：7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]-N-[(3R)-四氫呋喃-3-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物51係藉由通用程序1製備。LC-MS (ESI+)：m/z 474 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.63 (d,
J= 2.4 Hz, 1H), 7.75-7.71 (m, 2H), 7.35 (t,
J= 7.5 Hz, 1H), 7.22-7.18 (m, 2H), 7.07 (d,
J= 7.5 Hz, 1H), 7.00 (s, 1H), 6.82 (d,
J= 2.4 Hz, 1H), 4.78-4.70 (m, 1H), 4.04-3.94 (m, 6H), 3.90-3.78 (m, 6H), 2.45 (s, 3H), 2.41-2.34 (m, 1H), 1.98-1.85 (m, 1H)。
Compound 51 was prepared by
實例52：N-[(3R)-1-甲基-3-哌啶基]-7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物52係藉由通用程序1製備。LC-MS (ESI+)：m/z 501 (MH+)。1HNMR (300 MHz, DMSO-d6)
δ8.72 (d,
J= 2.7 Hz, 1H), 8.05 (d,
J= 8.1 Hz, 1H), 7.82-7.79 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.23 (d,
J= 7.2 Hz, 1H), 7.13 (d,
J= 2.7 Hz, 1H), 7.07 (s, 1H), 6.83 (s, 1H), 4.02-3.95 (m, 1H), 3.90 (s, 8H), 2.70-2.61 (m, 1H), 2.50-2.40 (m, 4H), 2.21 (s, 3H), 2.11-2.07 (m, 2H), 1.80-1.60 (m, 2H), 1.58-1.40 (m, 2H)。
Compound 52 was prepared by
實例53：N-[(3S)-1-甲基-3-哌啶基]-7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物53係藉由通用程序1製備。LC-MS (ESI+)：m/z 501 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ8.72 (d,
J= 2.4 Hz, 1H), 8.06 (d,
J= 8.7 Hz, 1H), 7.83-7.80 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.24 (d,
J= 7.5 Hz, 1H), 7.14 (d,
J= 2.4 Hz, 1H), 7.08 (s, 1H), 6.84 (s, 1H), 4.09-3.99 (m, 1H), 3.90 (s, 8H), 2.70-2.61 (m, 1H), 2.50-2.40 (m, 4H), 2.21 (s, 3H), 2.11-2.07 (m, 2H), 1.80-1.60 (m, 2H), 1.58-1.40 (m, 2H)。
Compound 53 was prepared by
實例54：N-(1-甲基-4-哌啶基)-7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物54係藉由通用程序1製備。LC-MS (ESI+)：m/z 501 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ8.72 (s, 1H), 8.16-8.06 (m, 1H), 7.83-7.80 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.23 (d,
J= 6.9 Hz, 1H), 7.14 (s, 1H), 7.08 (s, 1H), 6.84 (s, 1H), 3.90-3.80 (m, 9H), 2.95-2.86 (m, 2H), 2.40 (s, 3H), 2.32-2.21 (m, 5H), 1.91-1.73 (m, 4H)。
Compound 54 was prepared by
實例55：N-(1-甲基-4-哌啶基)-7-(N-𠰌啉基)-5-[4-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物55係藉由通用程序1製備。LC-MS (ESI+)：m/z 501 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.87 (s, 1H), 8.06 (s, 1H), 7.49-7.43 (m, 2H), 7.33-7.28 (m, 1H), 7.16-7.12 (m, 2H), 6.99-6.94 (m, 2H), 4.32-4.21 (m, 1H), 4.10-4.05 (m, 4H), 3.86-3.79 (m, 4H), 3.65-3.60 (m, 2H), 2.98-2.90 (m, 2H), 2.85 (s, 3H), 2.55-2.47 (m, 2H), 2.43 (s, 3H), 2.30-2.25 (m, 2H)。
Compound 55 was prepared by
實例56：7-(N-𠰌啉基)-5-[4-(間甲苯基)吡唑-1-基]-N-四氫哌喃-4-基-吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物56係藉由通用程序1製備。LC-MS (ESI+)：m/z 488 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.86 (s, 1H), 8.04 (s, 1H), 7.44-7.41 (m, 2H), 7.37-7.31 (m, 1H), 7.14-7.09 (m, 2H), 7.03 (s, 1H), 6.86-6.83 (m, 1H), 4.30-4.21 (m, 1H), 4.10-4.00 (m, 6H), 3.85-3.75 (m, 4H), 3.61-3.54 (m, 2H), 2.41 (s, 3H), 2.08-2.03 (m, 2H), 1.71-1.58 (m, 2H)。
Compound 56 was prepared by
實例57：4-[5-[4-(間甲苯基)吡唑-1-基]-2-嘧啶-4-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物57係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.33 (s, 1H), 8.89 (s, 1H), 8.86 (s, 1H), 8.09 (s, 1H), 8.05 (s, 1H), 7.53-7.42 (m, 2H), 7.34-7.29 (m, 2H), 7.14-7.12 (m, 1H), 7.06 (s, 1H), 4.10-4.00 (m, 4H), 3.92-3.81 (m, 4H), 2.42 (s, 3H)。
Compound 57 was prepared by
實例58：4-[5-[4-(間甲苯基)吡唑-1-基]-2-嘧啶-5-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物58係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.38-9.25 (m, 2H), 8.85 (s, 1H), 8.04 (s, 1H), 7.55-7.41 (m, 2H), 7.34-7.29 (m, 2H), 7.13 (d,
J= 6.9 Hz, 1H), 7.06 (s, 1H), 6.85 (s, 1H), 4.10-4.00 (m, 4H), 3.95-3.86 (m, 4H), 2.42 (s, 3H)。
Compound 58 was prepared by
實例59：3-[7-(N-𠰌啉基)-5-[4-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-基]吡啶-2-胺
化合物59係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 453 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ9.09 (s, 1H), 8.40 (s, 1H), 8.16 (d,
J= 6.3 Hz, 1H), 8.06 (d,
J= 4.2 Hz, 1H), 7.68 (s, 1H), 7.62 (d,
J= 7.5 Hz, 1H), 7.31 (t,
J= 7.5 Hz, 1H), 7.12-7.07 (m, 4H), 6.96 (s, 1H), 6.74-6.70 (m, 1H), 3.95-3.85 (m, 4H), 3.85-3.80 (m, 4H), 2.36 (s, 3H)。
Compound 59 was prepared by
實例60：5-[7-(N-𠰌啉基)-5-[4-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-基]嘧啶-2-胺
化合物60係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 454 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ9.07 (s, 1H), 8.88 (s, 2H), 8.37 (s, 1H), 7.67 (s, 1H), 7.61 (d,
J= 8.7 Hz, 1H), 7.30 (t,
J= 7.5 Hz, 1H), 7.10 (d,
J= 7.2 Hz, 1H), 6.99 (s, 2H), 6.92 (d,
J= 9.0 Hz, 2H), 3.89 (s, 8H), 2.36 (s, 3H)。
實例61：4-[2-(1-甲基吡唑-3-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物61係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 441 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.66 (d,
J= 2.7 Hz, 1H), 7.77-7.61 (m, 2H), 7.42 (d,
J= 2.1 Hz, 1H), 7.35 (t,
J= 7.5 Hz, 1H), 7.22-7.15 (m, 1H), 7.05 (s, 1H), 6.83-6.81 (m, 2H), 6.76 (d,
J= 2.1 Hz, 1H), 4.13-4.08 (m, 4H), 4.02 (s, 3H), 3.95-3.90 (m, 4H), 2.45 (s, 3H)。
Compound 61 was prepared by
實例62：4-[5-[3-(間甲苯基)吡唑-1-基]-2-(1H-吡唑-3-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物62係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 427 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6+D
2O)
δ8.71 (d,
J= 2.4 Hz, 1H), 7.82-7.79 (m, 3H), 7.39 (t,
J= 7.5 Hz, 1H), 7.24 (d,
J= 7.2 Hz, 1H), 7.12 (d,
J= 2.4 Hz, 1H), 6.99 (s, 1H), 6.85-6.80 (m, 2H), 4.05 (s, 8H), 2.41 (s, 3H)。
Compound 62 was prepared by
實例63：4-[5-(3-苯基吡唑-1-基)-2-嘧啶-2-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物63係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 425 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.90 (d,
J= 4.8 Hz, 2H), 8.69 (d,
J= 2.7 Hz 1H), 7.95 (d,
J= 2.7 Hz, 2H), 7.50-7.31 (m, 5H), 7.15 (s, 1H), 6.85 (d,
J= 2.7 Hz, 1H), 4.13-4.08 (m, 4H), 4.07-3.95 (m, 4H)。
Compound 63 was prepared by
實例64：4-[5-(3-苯基吡唑-1-基)-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物64係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 424 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.23 (s, 1H), 8.65 (d,
J= 2.7 Hz, 2H), 8.25 (d,
J= 7.8 Hz, 1H), 7.95 (d,
J= 7.2 Hz, 2H), 7.50-7.30 (m, 4H), 7.11 (s, 1H), 6.85 (d,
J= 2.7 Hz, 1H), 6.82 (s, 1H), 4.10-4.04 (m, 4H), 3.96-3.90 (m, 4H)。
Compound 64 was prepared by
實例65：4-[2-(5-甲基-1H-吡唑-3-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物65係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 441 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.63 (d,
J= 2.7 Hz, 1H), 7.76-7.61 (m, 2H), 7.38-7.32 (m, 2H), 7.22-7.15 (m, 1H), 7.06 (s, 1H), 6.81 (d,
J= 2.7 Hz, 1H), 6.73 (s, 1H), 6.56 (brs, 1H), 4.05-4.00 (m, 4H), 3.95-3.85 (m, 4H), 2.45 (s, 3H), 2.40 (s, 3H)。
Compound 65 was prepared by
實例66：4-[2-(1-甲基吡唑-3-基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物66係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 427 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.66 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 6.9 Hz, 2H), 7.48- 7.32 (m, 4H), 7.05 (s, 1H), 6.82 (s, 2H), 6.73 (d,
J= 2.1 Hz, 1H), 4.15-4.05 (m, 4H), 4.02 (s, 3H), 3.95-3.88 (m, 4H)。
Compound 66 was prepared by
實例67：4-[5-(3-苯基吡唑-1-基)-2-(1H-吡唑-3-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物67係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 413 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6+D
2O)
δ8.73 (d,
J= 2.7 Hz, 1H), 8.02 (d,
J= 7.2 Hz, 2H), 7.78 (d,
J= 2.1 Hz, 1H), 7.53-7.40 (m, 3H), 7.15 (d,
J= 2.7 Hz, 1H), 6.99 (s, 1H), 6.80-6.78 (m, 2H), 3.92 (s, 8H)。
Compound 67 was prepared by
實例68：4-[5-(3-苯基吡唑-1-基)-2-嘧啶-4-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物68係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 425 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.90 (d,
J= 2.7 Hz, 1H), 8.84 (d,
J= 4.8 Hz 1H), 8.68 (d,
J= 2.7 Hz, 1H), 8.10 (d,
J= 4.8 Hz, 1H), 7.94 (d,
J= 7.2 Hz, 2H), 7.50-7.37 (m, 3H), 7.23 (s, 1H), 7.17 (s, 1H), 6.85 (d,
J= 2.7 Hz, 1H), 4.13-4.06 (m, 4H), 3.95-3.90 (m, 4H)。
Compound 68 was prepared by
實例69：4-[5-(3-苯基吡唑-1-基)-2-嘧啶-5-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物69係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 425 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.32 (s, 2H), 9.25 (s, 1H), 8.65 (d,
J= 2.7 Hz, 1H), 7.95 (d,
J= 6.9 Hz, 2H), 7.63-7.37 (m, 3H), 7.17 (s, 1H), 6.86-6.85 (m, 2H), 4.06-4.01 (m, 4H), 3.95-3.90 (m, 4H)。
Compound 69 was prepared by
實例70：3-[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]吡啶-2-胺
化合物70係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 8.11-8.09 (m, 1H), 7.80-7.94 (m, 3H), 7.50-7.33 (m, 3H), 7.11 (s, 1H), 6.86-6.77 (m, 3H), 6.63 (s, 2H), 4.04-4.00 (m, 4H), 3.86-3.83 (m, 4H)。
Compound 70 was prepared by
實例71：5-[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]嘧啶-2-胺
化合物71係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 440 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.67 (s, 2H), 8.64 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 7.2 Hz, 2H), 7.50-7.33 (m, 3H), 7.09 (s, 1H), 6.83 (d,
J= 2.7 Hz, 1H), 6.67 (s, 1H), 5.20 (s, 2H), 4.04-4.00 (m, 4H), 3.96-3.87 (m, 4H)。
Compound 71 was prepared by
實例72：4-[5-(3-苯基吡唑-1-基)-2-(2-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物72係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 424 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.73 (d,
J= 4.2 Hz, 1H), 8.69 (d,
J= 2.7 Hz, 1H), 8.17 (d,
J= 8.1 Hz, 1H), 7.95 (d,
J= 8.1 Hz, 2H), 7.84-7.80 (m, 1H), 7.49-7.30 (m, 4H), 7.16 (s, 1H), 7.11 (s, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 4.11-4.03 (m, 4H), 3.96-3.87 (m, 4H)。
Compound 72 was prepared by
實例73：N-異丙基-7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物73係藉由通用程序1製備。LC-MS (ESI+)：m/z 432 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 6.9 Hz, 2H), 7.49-7.37 (m, 3H), 7.18 (s, 1H), 7.01 (s, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 6.77-6.74 (m, 1H), 4.36-4.27 (m, 1H), 4.09-4.00 (m, 4H), 3.90-3.80 (m, 4H), 1.31 (d,
J= 6.6 Hz, 6H)。
Compound 73 was prepared by
實例74：N-[(1R)-1-環丙基乙基]-7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物74係藉由通用程序1製備。LC-MS (ESI+)：m/z 458 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 6.9 Hz, 2H), 7.49-7.37 (m, 3H), 7.18 (s, 1H), 7.01 (s, 1H), 6.93-6.85 (m, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 4.09-4.00 (m, 4H), 3.90-3.80 (m, 4H), 3.71-3.60 (m, 1H), 1.35 (d,
J= 6.6 Hz, 3H), 1.00-0.90 (m, 1H), 0.57-0.40 (m, 3H), 0.34-0.31 (m, 1H)。
Compound 74 was prepared by
實例75：N-[(1S)-1-環丙基乙基]-7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物75係藉由通用程序1製備。LC-MS (ESI+)：m/z 458 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 6.9 Hz, 2H), 7.49-7.37 (m, 3H), 7.18 (s, 1H), 7.01 (s, 1H), 6.93-6.85 (m, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 4.06-4.00 (m, 4H), 3.88-3.80 (m, 4H), 3.70-3.60 (m, 1H), 1.35 (d,
J= 6.6 Hz, 3H), 1.00-0.90 (m, 1H), 0.57-0.40 (m, 3H), 0.34-0.31 (m, 1H)。
實例76：7-(N-𠰌啉基)-N-(氧雜環丁-3-基甲基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物76係藉由通用程序1製備。LC-MS (ESI+)：m/z 460 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 6.9 Hz, 2H), 7.49-7.37 (m, 3H), 7.25-7.15 (m, 1H), 7.18 (s, 1H), 7.01 (s, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 4.89-4.80 (m, 2H), 4.54-4.45 (m, 2H), 4.06-4.00 (m, 4H), 3.88-3.70 (m, 6H), 3.39-3.30 (m, 1H)。
Compound 76 was prepared by
實例77：N-環戊基-7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物77係藉由通用程序1製備。LC-MS (ESI+)：m/z 458 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 7.2 Hz, 2H), 7.49-7.37 (m, 3H), 7.18 (s, 1H), 7.01 (s, 1H), 6.91-6.88 (m, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 4.50-4.40 (m, 1H), 4.06-4.00 (m, 4H), 3.88-3.75 (m, 4H), 2.16-2.08 (m, 2H), 1.80-1.60 (m, 4H), 1.60-1.50 (m, 2H)。
Compound 77 was prepared by
實例78：4-[5-(4-苯基吡唑-1-基)-2-(2-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物78係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 424 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.90 (s, 1H), 8.73 (d,
J= 3.6 Hz, 1H), 8.17 (d,
J= 7.8 Hz, 1H), 8.04 (s, 1H), 7.80 (t,
J= 7.5 Hz, 1H), 7.63 (d,
J= 8.4 Hz, 2H), 7.45 (t,
J= 7.5 Hz, 2H), 7.33-7.20 (m, 2H), 7.16 (s, 1H), 7.01 (s, 1H), 4.11-4.03 (m, 4H), 3.96-3.88 (m, 4H)。
Compound 78 was prepared by
實例79：4-[5-(4-苯基吡唑-1-基)-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物79係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 424 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ9.28 (d,
J= 1.5 Hz, 1H), 9.12 (s, 1H), 8.68-8.60 (m, 1H), 8.43-8.41 (m, 2H), 7.83 (d,
J= 7.5 Hz, 2H), 7.57-7.53 (m, 1H), 7.45 (t,
J= 7.5 Hz, 2H), 7.32-7.29 (m, 1H), 7.16 (s, 1H), 6.97 (s, 1H), 3.92 (s, 8H)。
Compound 79 was prepared by
實例80：N-異丙基-7-(N-𠰌啉基)-5-(4-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-甲醯胺
化合物80係藉由通用程序1製備。LC-MS (ESI+)：m/z 432 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.87 (s, 1H), 8.04 (s, 1H), 7.61 (d,
J= 7.2 Hz, 2H), 7.33 (t,
J= 7.5 Hz, 2H), 7.32-7.29 (m, 1H), 7.06 (s, 1H), 7.03 (s, 1H), 6.76-6.70 (m, 1H), 4.39-4.27 (m, 1H), 4.08-4.00 (m, 4H), 3.85-3.80 (m, 4H), 1.31 (d,
J= 6.6 Hz, 6H)。
實例81：4-[2-(5-甲基-1H-吡唑-3-基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物81係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 427 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6+D
2O)
δ8.73 (d,
J= 2.7 Hz, 1H), 8.02 (d,
J= 7.2 Hz, 2H), 7.60-7.40 (m, 3H), 7.15 (d,
J= 2.7 Hz, 1H), 6.98 (s, 1H), 6.74 (s, 1H), 6.54 (s, 1H), 3.91 (s, 8H), 2.39 (s, 3H)。
Compound 81 was prepared by
實例82：4-[5-(3-苯基吡唑-1-基)-2-吡𠯤-2-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物82係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 440 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.41 (s, 1H), 8.68 (d,
J= 2.7 Hz, 2H), 8.58 (s, 1H), 7.95 (d,
J= 7.2 Hz, 2H), 7.50-7.33 (m, 4H), 7.15 (s, 2H), 6.85 (d,
J= 2.7 Hz, 1H), 4.11-4.00 (m, 4H), 3.96-3.83 (m, 4H)。
Compound 82 was prepared by
實例83：N,N-二甲基-2-[3-甲基-5-[7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物83係藉由通用程序2製備。LC-MS (ESI+)：m/z 512 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ8.71 (d,
J= 2.7 Hz, 1H), 7.83-7.80 (m, 2H), 7.38 (t,
J= 7.5 Hz, 1H), 7.23 (d,
J= 7.5 Hz, 1H), 7.15 (d,
J= 2.7 Hz, 1H), 7.02 (s, 1H), 6.88 (s, 1H), 6.65 (s, 1H), 4.65-4.60 (m, 2H), 3.88 (s, 8H), 2.70-2.60 (m, 2H), 2.35 (s, 3H), 2.21 (s, 3H), 2.18 (s, 6H)。
Compound 83 was prepared by
實例84：4-[2-(2,5-二甲基吡唑-3-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物84係藉由通用程序2及5製備。LC-MS (ESI+)：m/z 455 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.60 (d,
J= 2.7 Hz, 1H), 7.75-7.70 (m, 2H), 7.33 (t,
J= 7.5 Hz, 1H), 7.20 (d,
J= 7.5 Hz, 1H), 7.04 (s, 1H), 6.79 (d,
J= 2.7 Hz, 1H), 6.59 (s, 1H), 6.44 (s, 1H), 4.17 (s, 3H), 4.02-3.90 (m, 4H), 3.88-3.85 (m, 4H), 2.43 (s, 3H), 2.32 (s, 3H)。
Compound 84 was prepared by
實例85：4-[2-(2,5-二甲基吡唑-3-基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物85係藉由通用程序2及5製備。LC-MS (ESI+)：m/z 441 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.63 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 7.2 Hz, 2H), 7.49- 7.35 (m, 3H), 7.09 (s, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 6.63 (s, 1H), 6.46 (s, 1H), 4.19 (s, 3H), 4.12-3.99 (m, 4H), 3.90-3.85 (m, 4H), 2.32 (s, 3H)。
Compound 85 was prepared by
實例86：4-[2-(1,5-二甲基吡唑-3-基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物86係藉由通用程序2及5製備。LC-MS (ESI+)：m/z 441 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.63 (d,
J= 2.7 Hz, 1H), 7.93 (d,
J= 7.2 Hz, 2H), 7.49- 7.35 (m, 3H), 7.04 (s, 1H), 6.79 (d,
J= 3.9 Hz, 2H), 6.53 (s, 1H), 4.12-3.99 (m, 4H), 3.90-3.85 (m, 4H), 3.78 (s, 3H), 2.32 (s, 3H)。
Compound 86 was prepared by
實例87：4-[2-(1,5-二甲基吡唑-3-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物87係藉由通用程序2及5製備。LC-MS (ESI+)：m/z 455 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.67 (d,
J= 2.7 Hz, 1H), 7.79-7.73 (m, 2H), 7.36 (t,
J= 7.8 Hz, 1H), 7.22 (d,
J= 7.5 Hz, 1H), 7.05 (s, 1H), 6.83-6.82 (m, 2H), 6.56 (s, 1H), 4.07-4.02 (m, 4H), 3.95-3.90 (m, 4H), 3.89 (s, 3H), 2.47 (s, 3H), 2.36 (s, 3H)。
Compound 87 was prepared by
實例88：4-[5-(3-苯基吡唑-1-基)-2-嗒𠯤-3-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物88係藉由通用程序2及6製備。LC-MS (ESI+)：m/z 425 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.21 (d,
J= 3.3 Hz, 1H), 8.70 (d,
J= 2.7 Hz, 1H), 8.30 (d,
J= 8.4 Hz, 1H), 7.95 (d,
J= 8.4 Hz, 2H), 7.61 (t,
J= 4.5 Hz, 1H), 7.50-7.37 (m, 4H), 7.15 (s, 1H), 6.85 (d,
J= 2.7 Hz, 1H), 4.11-4.02 (m, 4H), 3.96-3.85 (m, 4H)。
Compound 88 was prepared by
實例89：N-[[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]甲基]胺基甲酸甲酯
化合物89係藉由通用程序8製備。LC-MS (ESI+)：m/z 434 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.64 (d,
J= 2.7 Hz, 1H), 7.93 (d,
J= 6.9 Hz, 2H), 7.49-7.36 (m, 3H), 7.05 (s, 1H), 6.82 (d,
J= 2.4 Hz, 1H), 6.42 (s, 1H), 5.24 (brs, 1H), 4.59 (d,
J= 6.0 Hz, 2H), 4.13-4.02 (m, 4H), 3.86-3.80 (m, 4H), 3.74 (s, 3H)。
Compound 89 was prepared by
實例90：N-[[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]甲基]丙烯醯胺
化合物90係藉由通用程序8製備。LC-MS (ESI+)：m/z 432 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.61 (d,
J= 2.7 Hz, 1H), 7.93 (d,
J= 6.9 Hz, 2H), 7.49-7.36 (m, 3H), 7.05 (s, 1H), 6.82 (d,
J= 2.7 Hz, 1H), 6.39 (s, 1H), 6.01 (brs, 1H), 4.66 (d,
J= 5.4 Hz, 2H), 4.10-3.99 (m, 4H), 3.87-3.80 (m, 4H), 2.31 (q,
J= 7.5 Hz, 2 H), 1.16 (t,
J= 7.5 Hz, 3H)。
Compound 90 was prepared by
實例91：N,N-二甲基-2-[5-甲基-3-[7-(N-𠰌啉基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物91係藉由通用程序2製備。LC-MS (ESI+)：m/z 512 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.4 Hz, 1H), 7.76-7.61 (m, 2H), 7.35 (t,
J= 7.5 Hz, 1H), 7.19 (d,
J= 7.5 Hz, 1H), 7.03 (s, 1H), 6.81-6.75 (m, 2H), 6.53 (s, 1H), 4.35-4.25 (m, 2H), 4.10-4.00 (m, 4H), 3.95-3.85 (m, 4H), 3.00-2.85 (m, 2H), 2.45 (s, 3H), 2.39 (s, 9H)。
Compound 91 was prepared by
實例92：N,N-二甲基-2-[5-甲基-3-[7-(N-𠰌啉基)-5-[3-(2-吡啶基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物92係藉由通用程序2製備。LC-MS (ESI+)：m/z 499 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.71-8.68 (m, 2H), 8.13 (d,
J= 8.1 Hz, 1H), 7.82-7.79 (m, 1H), 7.30-7.27 (m, 1H), 7.14 (d,
J= 2.7 Hz, 1H), 7.04 (s, 1H), 6.82 (s, 1H), 6.53 (s, 1H), 4.35-4.20 (m, 2H), 4.10-4.00 (m, 4H), 3.95-3.85 (m, 4H), 2.92-2.80 (m, 2H), 2.39 (s, 3H), 2.36 (s, 6H)。
Compound 92 was prepared by
實例93：N,N-二甲基-2-[5-甲基-3-[7-(N-𠰌啉基)-5-[3-(4-吡啶基)吡唑-1-基]吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物93係藉由通用程序2製備。LC-MS (ESI+)：m/z 499 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.72-8.69 (m, 3H), 7.81 (d,
J= 6.0 Hz, 2H), 7.00 (s, 1H), 6.90 (d,
J= 6.6 Hz, 1H), 6.82 (s, 1H), 6.53 (s, 1H), 4.30-4.20 (m, 2H), 4.10-4.00 (m, 4H), 3.95-3.85 (m, 4H), 2.91-2.80 (m, 2H), 2.38 (s, 3H), 2.35 (s, 6H)。
Compound 93 was prepared by
實例94：N,N-二甲基-2-[5-甲基-3-[5-[3-(6-甲基-2-吡啶基)吡唑-1-基]-7-(N-𠰌啉基)-吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物94係藉由通用程序2製備。LC-MS (ESI+)：m/z 513 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.69 (s, 1H), 7.99 (d,
J= 7.2 Hz, 1H), 7.67 (t,
J= 7.5 Hz, 1H), 7.20-7.10 (m, 2H), 7.05 (s, 1H), 6.80 (s, 1H), 6.53 (s, 1H), 4.40-4.20 (m, 2H), 4.10-4.00 (m, 4H), 3.95-3.85 (m, 4H), 3.07-2.89 (m, 2H), 2.58 (s, 3H), 2.40 (s, 9H)。
Compound 94 was prepared by
實例95：N,N-二甲基-2-[5-甲基-3-[5-[3-(4-甲基-2-吡啶基)吡唑-1-基]-7-(N-𠰌啉基)-吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物95係藉由通用程序2及4製備。LC-MS (ESI+)：m/z 513 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.69 (d,
J= 2.7 Hz, 1H), 8.54 (d,
J= 5.1 Hz, 1H), 7.95 (s, 1H), 7.14-7.10 (m, 2H), 7.04 (s, 1H), 6.81 (s, 1H), 6.46 (s, 1H), 4.35-4.20 (m, 2H), 4.10-4.00 (m, 4H), 3.95-3.85 (m, 4H), 3.01-2.85 (m, 2H), 2.46 (s, 3H), 2.40 (s, 9H)。
Compound 95 was prepared by
實例96：N,N-二甲基-2-[5-甲基-3-[5-[3-(2-甲基-4-吡啶基)吡唑-1-基]-7-(N-𠰌啉基)-吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物96係藉由通用程序2及4製備。LC-MS (ESI+)：m/z 513 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.70 (d,
J= 2.7 Hz, 1H), 8.57 (d,
J= 5.4 Hz, 1H), 7.68 (s, 1H), 7.62-7.60 (m, 1H), 7.00 (s, 1H), 6.87 (d,
J= 2.7 Hz, 1H), 6.82 (s, 1H), 6.53 (s, 1H), 4.27-4.22 (m, 2H), 4.05-4.00 (m, 4H), 3.95-3.91 (m, 4H), 2.85-2.81 (m, 2H), 2.65 (s, 3H), 2.38 (s, 3H), 2.34 (s, 6H)。
Compound 96 was prepared by
實例97：2-[3-[5-[3-(3-氯苯基)吡唑-1-基]-7-(N-𠰌啉基)-吡唑并[1,5-a]嘧啶-2-基]-5-甲基-吡唑-1-基]-N,N-二甲基-乙胺
化合物97係藉由通用程序2及3製備。LC-MS (ESI+)：m/z 532/534 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.67 (d,
J= 2.7 Hz, 1H), 7.96 (s, 1H), 7.78 (d,
J= 7.2 Hz, 1H), 7.46-7.33 (m, 2H), 7.00 (s, 1H), 6.81-6.78 (m, 2H), 6.53 (s, 1H), 4.26-4.22 (m,
2H), 4.06-4.00 (m, 4H), 3.95-3.89 (m, 4H), 2.84-2.80 (m, 2H), 2.38 (s, 3H), 2.33 (s, 6H)。
Compound 97 was prepared by
實例98：5-[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]吡啶-2-胺
化合物98係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 432 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.64 (d,
J= 2.7 Hz, 2H), 8.05 (dd,
J= 8.1 Hz, 2.1 Hz, 1H), 7.94 (d,
J= 7.2 Hz, 2H), 7.49-7.36 (m, 3H), 7.05 (s, 1H), 6.82 (d,
J= 2.7 Hz, 1H), 6.67 (s, 1H), 6.62 (d,
J= 8.7 Hz, 1H), 4.96 (brs, 2H), 4.10-4.01 (m, 4H), 3.90-3.80 (m, 4H)。
Compound 98 was prepared by
實例99：N,N-二甲基-2-[5-甲基-3-[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]吡唑-1-基]乙胺
化合物99係藉由通用程序2製備。LC-MS (ESI+)：m/z 498 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.66 (d,
J= 2.7 Hz, 1H), 7.94 (d,
J= 6.9 Hz, 2H), 7.49-7.38 (m, 3H), 7.04 (s, 1H), 6.81 (d,
J= 2.7 Hz, 1H), 6.80 (s, 1H), 6.53 (s, 1H), 4.30-4.25 (m, 2H), 4.04-4.00 (m, 4H), 3.95-3.90 (m, 4H), 2.92-2.80 (m, 2H), 2.38 (s, 3H), 2.35 (s, 6H)。
Compound 99 was prepared by
實例100：2-[3-[5-[3-(3-甲氧基苯基)吡唑-1-基]-7-(N-𠰌啉基)-吡唑并[1,5-a]嘧啶-2-基]-5-甲基-吡唑-1-基]-N,N-二甲基-乙胺
化合物100係藉由通用程序2製備。LC-MS (ESI+)：m/z 528 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.65 (d,
J= 2.7 Hz, 1H), 7.61-7.52 (m, 2H), 7.37 (t,
J= 8.1 Hz, 1H), 7.02 (s, 1H), 6.95-6.90 (m, 1H), 6.81-6.78 (m, 2H), 6.53 (s, 1H), 4.28-4.23 (m, 2H), 4.04-4.00 (m, 4H), 3.95-3.85 (m, 7H), 2.90-2.82 (m, 2H), 2.38 (s, 3H), 2.35 (s, 6H)。
實例101：4-[5-[3-(3-氯苯基)吡唑-1-基]-2-(1-甲基吡唑-3-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物101係藉由通用程序2及3製備。LC-MS (ESI+)：m/z 461/463 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.67 (d,
J= 2.7 Hz, 1H), 7.97 (s, 1H), 7.79 (d,
J= 4.8 Hz, 1H), 7.47-7.33 (m, 3H), 7.02 (s, 1H), 6.84-6.77 (m, 3H), 4.10-4.00 (m, 7H), 3.95-3.91 (m, 4H)。
Compound 101 was prepared by
實例102：4-[5-[3-(5-甲基-3-吡啶基)吡唑-1-基]-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物102係藉由通用程序2製備。LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.23 (s, 1H), 8.98 (s, 1H), 8.69 (d,
J= 2.7 Hz, 1H), 8.66-8.64 (m, 1H), 8.46 (s, 1H), 8.25 (d,
J= 7.8 Hz, 1H), 8.07 (s, 1H), 7.50-7.40 (m, 1H), 7.07 (s, 1H), 6.87 (d,
J= 2.7 Hz, 1H), 6.84 (s, 1H), 4.13-4.03 (m, 4H), 3.98-3.91 (m, 4H), 2.46 (s, 3H)。
Compound 102 was prepared by
實例103：4-[5-[3-(2-甲基-4-吡啶基)吡唑-1-基]-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物103係藉由通用程序2及4製備。LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.23 (s, 1H), 8.98 (s, 1H), 8.72 (d,
J= 2.7 Hz, 1H), 8.66-8.64 (m, 1H), 8.25 (d,
J= 7.8 Hz, 1H), 7.75-7.71 (m, 2H), 7.43-7.26 (m, 1H), 7.07 (s, 1H), 6.92-6.87 (m, 1H), 6.84 (s, 1H), 4.10-4.03 (m, 4H), 3.98-3.93 (m, 4H), 2.72 (s, 3H)。
實例104：4-[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]嘧啶-2-胺
化合物104係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 440 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ8.67 (d,
J= 2.7 Hz, 1H), 8.40 (d,
J= 5.4 Hz, 1H), 7.95 (d,
J= 6.9 Hz, 2H), 7.49-7.36 (m, 4H), 7.14 (s, 1H), 7.11 (s, 1H), 6.84 (d,
J= 2.7 Hz, 1H), 5.32 (brs, 2H), 4.10-4.01 (m, 4H), 3.95-3.85 (m, 4H)。
實例105：4-[5-[3-(3-氯苯基)吡唑-1-基]-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物105係藉由通用程序2及3製備。LC-MS (ESI+)：m/z 458/460 (MH
+)。
1HNMR (300 MHz, DMSO-
d 6)
δ9.23 (s, 1H), 8.76 (d,
J= 2.7 Hz, 1H), 8.65 (d,
J= 4.2 Hz, 1H), 8.41 (d,
J= 7.8 Hz, 1H), 8.08 (s, 1H), 8.00 (d,
J= 7.2 Hz, 1H), 7.57-7.40 (m, 3H), 7.26-7.25 (m, 1H), 7.18 (s, 1H), 7.05 (s, 1H), 3.98-3.87 (m, 8H)。
實例106：4-[5-[3-(3-溴苯基)吡唑-1-基]-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物106係藉由通用程序2及7製備。LC-MS (ESI+)：m/z 502/504 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.24 (s, 1H), 8.67-8.65 (m, 2H), 8.27 (d,
J= 7.8 Hz, 1H), 8.13 (t,
J= 1.5 Hz, 1H), 7.83 (d,
J= 8.1 Hz, 1H), 7.52 (d,
J= 8.1 Hz, 1H), 7.47-7.43 (m, 1H), 7.34 (t,
J= 8.1 Hz, 1H), 7.08 (s, 1H), 6.83 (s, 1H), 6.82 (d,
J= 6.6 Hz, 1H), 4.08-4.02 (m, 4H), 3.98-3.91 (m, 4H)。
實例107：4-[5-[3-(3-甲氧基苯基)吡唑-1-基]-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物107係藉由通用程序2及3製備。LC-MS (ESI+)：m/z 454 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.23 (s, 1H), 8.65 (d,
J= 2.7 Hz, 2H), 8.26 (d,
J= 7.8 Hz, 1H), 7.61-7.50 (m, 2H), 7.46-7.36 (m, 2H), 7.10 (s, 1H), 6.96-6.91 (m, 1H), 6.84-6.80 (m, 2H), 4.10-4.03 (m, 4H), 3.98-3.90 (m, 4H), 3.89 (s, 3H)。
實例108：4-[5-[3-(6-甲基-2-吡啶基)吡唑-1-基]-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物108係藉由通用程序2製備。LC-MS (ESI+)：m/z 439 (MH
+)。
1HNMR (300 MHz, CDCl
3)
δ9.25 (s, 1H), 8.71-8.62 (m, 2H), 8.26 (d,
J= 7.8 Hz, 1H), 7.91 (d,
J= 7.5 Hz, 1H), 7.68 (t,
J= 7.5 Hz, 1H), 7.43-7.36 (m, 1H), 7.17-7.13 (m, 3H), 6.83 (s, 1H), 4.10-4.03 (m, 4H), 3.98-3.90 (m, 4H), 2.66 (s, 3H)。
Compound 108 was prepared by
實例109：6-[7-(N-𠰌啉基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-2-基]吡啶-2-胺
化合物109係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 438 (MH +)。 Compound 109 was prepared by the method described above. LC-MS (ESI+): m/z 438 (MH + ).
實例110：4-[2-(3-甲基異㗁唑-5-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物110係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 441 (MH +)。 Compound 110 was prepared by the method described above. LC-MS (ESI+): m/z 441 (MH + ).
實例111：4-[5-[3-(間甲苯基)吡唑-1-基]-2-噻唑-2-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物111係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 443 (MH +)。 Compound 111 was prepared by the method described above. LC-MS (ESI+): m/z 443 (MH + ).
實例112：4-[2-(1-甲基吡唑-4-基)-5-[3-(間甲苯基)吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物112係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 440 (MH +)。 Compound 112 was prepared by the method described above. LC-MS (ESI+): m/z 440 (MH + ).
實例113：4-[5-[3-(間甲苯基)吡唑-1-基]-2-(1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物113係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 426 (MH +)。 Compound 113 was prepared by the method described above. LC-MS (ESI+): m/z 426 (MH + ).
實例114：4-[5-(3-苯基吡唑-1-基)-2-噻唑-2-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物114係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 429 (MH +)。 Compound 114 was prepared by the method described above. LC-MS (ESI+): m/z 429 (MH + ).
實例115：4-[2-(1-甲基吡唑-4-基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物115係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 426 (MH +)。 Compound 115 was prepared by the method described above. LC-MS (ESI+): m/z 426 (MH + ).
實例116：4-[5-(3-苯基吡唑-1-基)-2-(1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物116係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 412 (MH +)。 Compound 116 was prepared by the method described above. LC-MS (ESI+): m/z 412 (MH + ).
實例117：3-[1-[7-(N-𠰌啉基)-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-5-基]吡唑-3-基]苯甲酸甲酯
化合物117係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 481 (MH +)。 Compound 117 was prepared by the method described above. LC-MS (ESI+): m/z 481 (MH + ).
實例118：3-[1-[7-(N-𠰌啉基)-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-5-基]吡唑-3-基]苯甲醯胺
化合物118係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 482 (MH +)。 Compound 118 was prepared by the method described above. LC-MS (ESI+): m/z 482 (MH + ).
實例119：3-[1-[7-(N-𠰌啉基)-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-5-基]吡唑-3-基]苯甲腈
化合物119係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 448 (MH +)。 Compound 119 was prepared by the method described above. LC-MS (ESI+): m/z 448 (MH + ).
實例120：4-[5-[3-(間甲苯基)吡唑-1-基]-2-㗁唑-2-基-吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物120係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 427 (MH
+)。
實例121：4-[2-(3-甲基異㗁唑-5-基)-5-(3-苯基吡唑-1-基)吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物121係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 427 (MH +)。 Compound 121 was prepared by the method described above. LC-MS (ESI+): m/z 427 (MH + ).
實例122：3-[1-[7-(N-𠰌啉基)-2-(3-吡啶基)吡唑并[1,5-a]嘧啶-5-基]吡唑-3-基]苯甲酸
化合物122係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 467 (MH +)。 Compound 122 was prepared by the method described above. LC-MS (ESI+): m/z 467 (MH + ).
實例123：4-[2-(1-甲基吡唑-3-基)-5-[3-[3-(三氘甲基)苯基]吡唑-1-基]吡唑并[1,5-a]嘧啶-7-基]𠰌啉
化合物123係藉由上文描述之方法製備。LC-MS (ESI+)：m/z 443。 生物學實例1：PIKfyve之抑制 Compound 123 was prepared by the method described above. LC-MS (ESI+): m/z 443. Biological Example 1: Inhibition of PIKfyve
在桿狀病毒表現系統中作為N端GST-融合蛋白(265 kDa)表現之全長人類重組PIKFYVE係獲自Carna Biosciences (Kobe, Japan)。藉由在50 mM HEPES緩衝液pH 7.5中以1:10比率混合且音波處理螢光標記之磷脂醯肌醇3-磷酸(PI3P)與磷酸-L-絲胺酸(PS)來製備激酶受質。Full-length human recombinant PIKFYVE expressed as an N-terminal GST-fusion protein (265 kDa) in a baculovirus expression system was obtained from Carna Biosciences (Kobe, Japan). Kinase substrates were prepared by mixing and sonicating fluorescently labeled phosphatidylinositol 3-phosphate (PI3P) and phospho-L-serine (PS) at a ratio of 1:10 in 50 mM HEPES buffer pH 7.5 .
將激酶反應物以20 mL之總容積如下裝配於384孔培養盤(Greiner)中。將激酶蛋白質預稀釋於包含25 mM HEPES (pH 7.5)、1 mM DTT、2.5 mM MgCl 2及2.5 mM MnCl 2以及0.005% Triton X-100之分析緩衝液中，且分配至384孔盤(每孔10 µL)中。將測試化合物連序預稀釋於DMSO中且藉由聲學分配(Labcyte Echo)添加至蛋白樣品中。DMSO之濃度在所有樣品中等同於1%。以12個濃度測試所有測試化合物。阿匹莫德(Apilimod)用作參考化合物且在各分析盤中以相同方式進行測試。將對照樣品(在不存在抑制劑之情況下，0%抑制，僅DMSO)及100%抑制(在不存在酶之情況下)組裝於四個重複中且用於計算在化合物存在下之抑制%。藉由添加10 µL補充有ATP之2× PI3P/PS受質來起始反應。酶之最終濃度為2 nM，ATP之最終濃度為10 mM，且PI3P/PS受質之最終濃度為1 µM (PI3P)。使激酶反應在室溫進行3 h。在培育之後，藉由添加50 mL終止緩衝液(100 mM HEPES，pH 7.5，0.01% Triton X-100，20 mM EDTA)來淬滅反應。在微流體電泳儀(Caliper LabChip® 3000, Caliper Life Sciences/Perkin Elmer)上分析終止的盤。量測PI(3)P受質及PI(3,5)P產物峰之相對螢光強度的變化。各測試樣品中之活性測定為產物與總和之比(PSR)：P/(S+P)，其中P為產物之峰高且S為受質之峰高。使用以下等式判定抑制百分比(P inh)： P inh= (PSR 0%inh- PSR compound)/(PSR 0%inh- PSR 100%inh)×100 其中PSR compound為在化合物之存在下產物/總和之比率，PSR0 %inh為在不存在化合物之情況下產物/總和之比率，且PSR 100%inh為在不存在酶之情況下產物/總和之比率。為了測定測試化合物之IC 50(50%抑制)，使用XLfit軟體(IDBS)藉由四參數S型劑量-反應模型來擬合抑制%濃度資料(%-inh cdata) (相比於化合物濃度之P inh)。 Kinase reactions were assembled in 384-well plates (Greiner) in a total volume of 20 mL as follows. Kinase proteins were pre-diluted in assay buffer containing 25 mM HEPES (pH 7.5), 1 mM DTT, 2.5 mM MgCl 2 and 2.5 mM MnCl 2 and 0.005% Triton X-100 and dispensed into 384-well plates (per well 10 µL). Test compounds were serially pre-diluted in DMSO and added to protein samples by acoustic dispensing (Labcyte Echo). The concentration of DMSO was equal to 1% in all samples. All test compounds were tested at 12 concentrations. Apilimod was used as a reference compound and tested in the same manner in each assay plate. Control samples (0% inhibition in the absence of inhibitor, DMSO only) and 100% inhibition (in absence of enzyme) were assembled in quadruplicate and used to calculate % inhibition in the presence of compound . Reactions were initiated by adding 10 µL of 2x PI3P/PS substrate supplemented with ATP. The final concentration of enzyme was 2 nM, the final concentration of ATP was 10 mM, and the final concentration of PI3P/PS substrate was 1 µM (PI3P). The kinase reaction was allowed to proceed for 3 h at room temperature. After incubation, the reaction was quenched by adding 50 mL of stop buffer (100 mM HEPES, pH 7.5, 0.01% Triton X-100, 20 mM EDTA). Terminated discs were analyzed on a microfluidic electrophoresis instrument (Caliper LabChip® 3000, Caliper Life Sciences/Perkin Elmer). Changes in the relative fluorescence intensity of the PI(3)P substrate and PI(3,5)P product peaks were measured. Activity in each test sample was determined as the product-to-sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the substrate. The percent inhibition (P inh ) was determined using the following equation: P inh = (PSR 0%inh - PSR compound )/(PSR 0%inh - PSR 100%inh ) x 100 where PSR compound is the product/sum in the presence of compound PSR0 %inh is the ratio of product/sum in the absence of compound and PSR 100%inh is the ratio of product/sum in the absence of enzyme. In order to determine the IC50 (50% inhibition) of the test compound, XLfit software (IDBS) was used to fit the inhibition % concentration data (%-inh cdata) (compared to the p inh ).
本發明之某些化合物的IC
50值提供於下表2中。
表2
材料及方法Materials and methods
基於在Vero(非洲綠猴腎臟上皮細胞(African green monkey kidney epithelial))細胞中之細胞病變效應(CPE)，基本上根據Ivens等人(2005) Journal of Virological Methods 129, 56-63之方法，設計且運行活體外抗病毒分析以測試化合物針對SARS-CoV-2之作用。Based on the cytopathic effect (CPE) in Vero (African green monkey kidney epithelial cells (African green monkey kidney epithelial)) cells, basically according to the method of people such as Ivens (2005) Journal of Virological Methods 129, 56-63, design And an in vitro antiviral assay was run to test the effect of compounds against SARS-CoV-2.
使VeroE6-EGFP細胞(由Lab of Virology & Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium提供) (在本文中有時稱為VeroE6、Vero-E6或Vero-E6-GFP)在生長培養基中繁殖，該生長培養基係藉由對DMEM (Gibco 41965-039)補充10% v/v熱滅活FCS及5 mL碳酸氫鈉7.5% (Gibco 25080-060)來製備。在T150瓶子中培養細胞，且拆分1/4，一週兩次。以1/100稀釋度將青黴素-鏈黴素(Pen-strep)直接添加至T150瓶子中。VeroE6-EGFP cells (provided by Lab of Virology & Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium) (sometimes referred to herein as VeroE6, Vero-E6 or Vero-E6-GFP) were propagated in growth medium, The growth medium was prepared by supplementing DMEM (Gibco 41965-039) with 10% v/v heat-inactivated FCS and 5 mL of sodium bicarbonate 7.5% (Gibco 25080-060). Cells were cultured in T150 bottles and split 1/4 twice a week. Pen-strep was added directly to the T150 bottle at a 1/100 dilution.
分析培養基係藉由對DMEM (Gibco 41965-039)補充2% v/v熱滅活FCS及5 mL碳酸氫鈉7.5% (Gibco 25080-060)來製備。Assay medium was prepared by supplementing DMEM (Gibco 41965-039) with 2% v/v heat-inactivated FCS and 5 mL of sodium bicarbonate 7.5% (Gibco 25080-060).
在向其中添加細胞之96孔盤中製備化合物之連續稀釋液(25,000個細胞/孔)，之後培育盤過夜(37℃；5% CO
2)。次日，添加病毒接種液(SARS-CoV-2臨床分離株，比利時株(Belgian strain)：BetaCov/Belgium/GHB-03021/2020)，且培育盤4天(37℃；5% CO
2)，直至在未處理的感染(病毒對照)條件下可觀測到最大細胞病變效應(CPE)。在第4天，使用高通量成像測定GFP訊號。
Serial dilutions of compounds (25,000 cells/well) were prepared in 96-well plates to which cells were added, after which plates were incubated overnight (37°C; 5% CO2 ). The next day, add the virus inoculum (SARS-CoV-2 clinical isolate, Belgian strain: BetaCov/Belgium/GHB-03021/2020), and incubate the plate for 4 days (37°C; 5% CO 2 ), Until a maximal cytopathic effect (CPE) was observed under untreated infection (virus control) conditions. On
抗病毒活性係以EC 50或使病毒誘導之細胞病變之GFP訊號回復50%時所需的濃度表示。該訊號係以覆蓋有螢光像素(其與活細胞相關)之孔表面的對數提供。 Antiviral activity is expressed as EC50 , or the concentration required to restore 50% of the GFP signal to virus-induced cytopathic effects. The signal is provided as a logarithm of the well surface covered with fluorescent pixels associated with living cells.
平行且為了避免假陽性，藉由未感染細胞在所測試濃度之測試化合物存在下之生長來評定細胞毒性。在4天培育之後，使用市售套組來量測細胞存活力。In parallel and to avoid false positives, cytotoxicity is assessed by the growth of uninfected cells in the presence of the test compound at the concentration tested. After 4 days of incubation, cell viability was measured using a commercial kit.
使用高通量成像器讀取抗病毒。使用5×物鏡，幾乎一次即捕捉到384孔盤之全部孔(對於96孔盤，由4個視野涵蓋培育孔)。可以由位於細胞質與核兩者中之GFP標記來計算(仍)被細胞覆蓋之孔表面(SpotTotalAreaCh2)。將資料輸入.csv檔案，且在Dotmatics中處理劑量反應曲線。Antiviral readout using a high-throughput imager. Using a 5× objective, almost all wells of a 384-well plate were captured at once (for a 96-well plate, 4 fields of view covered the wells). The pore surface (still) covered by cells (SpotTotalAreaCh2) can be calculated from the GFP label located both in the cytoplasm and in the nucleus. Data were imported into .csv files and dose response curves were processed in Dotmatics.
結果及討論Results and Discussion
測試代表性PYKfyve抑制劑以測定其針對SARS-CoV2之活性。測試所有化合物，重複兩次，其中最高濃度為10 µM (8個濃度；稀釋階梯1/3)。結果之概述顯示於圖1A至圖1C及表3中。圖1A至圖1C顯示，在Vero-E6細胞中，化合物22、化合物18及化合物17以治療可接受之安全性指數產生可再現且穩固的抗病毒活性。圖1A顯示化合物22之資料。圖1B顯示化合物18之資料。圖1C顯示化合物17之資料。安全性指數為CC
50與EC
50之比率。
表 3
材料及方法Materials and methods
使用由Institut Pasteur, Paris, France提供之經轉導以表現人類血管收縮素轉化酶2 (ACE2)之A549-ACE2細胞(腺癌人類肺泡基底上皮細胞)，評定PIKfyve抑制劑針對SARS-CoV-2之抗病毒活性。使A549-ACE2細胞在96孔盤中生長，且測試各所測試PIKfyve抑制劑之十個濃度，重複三次。所測試化合物之濃度包括：0.001 - 1 μM (0、0.001、0.003、0.01、0.03、0.1、0.3、1、3、10)。最終分析中之DMSO濃度＜ 1% (在培養基之連續稀釋液中製備之儲備液稀釋液)。Evaluation of PIKfyve inhibitors against SARS-CoV-2 using A549-ACE2 cells (adenocarcinoma human alveolar basal epithelial cells) transduced to express human angiotensin-converting enzyme 2 (ACE2) provided by the Institut Pasteur, Paris, France of antiviral activity. A549-ACE2 cells were grown in 96-well plates and ten concentrations of each PIKfyve inhibitor tested were tested in triplicate. Concentrations of the compounds tested included: 0.001 - 1 μM (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10). The DMSO concentration in the final assay was <1% (dilution of stock prepared in serial dilutions of culture medium).
在用SARS-CoV-2病毒(分離株BetaCoV/France /IDF0372/2020 C2)感染之前，預處理細胞2小時。在病毒感染之後，在37℃培育細胞1小時。在48小時培育之後，洗滌所有細胞。Cells were pretreated for 2 h prior to infection with SARS-CoV-2 virus (isolate BetaCoV/France/IDF0372/2020 C2). Following virus infection, cells were incubated at 37°C for 1 hour. After 48 hours of incubation, all cells were washed.
在存在及不存在所測試化合物之情況下，藉由定量RT-PCR來量測病毒複製。Viral replication was measured by quantitative RT-PCR in the presence and absence of the compounds tested.
平行且為了避免假陽性，藉由在以所測試濃度之三種所測試化合物存在下生長未感染細胞來評定細胞毒性。在48小時培育之後，使用市售套組來量測細胞存活力。In parallel and to avoid false positives, cytotoxicity was assessed by growing uninfected cells in the presence of the three tested compounds at the tested concentrations. After 48 hours of incubation, cell viability was measured using commercially available kits.
三種測試化合物：化合物14、化合物17及化合物43係以於100 μL DMSO中之10 mM溶液形式提供。Three test compounds: Compound 14,
結果result
例示性結果顯示於圖2A至圖2C中。所測試化合物顯示具有極小毒性之強效抗病毒作用。病毒效價顯示為左軸上之每mL空斑形成單位(PFU/mL)之Log
10(資料由圓圈表示)。在右軸上顯示百分比細胞存活力(資料由方塊表示)。圖2A顯示化合物14之資料，其IC
50= 0.009345 µM。圖2B顯示化合物17之資料，其IC
50= 0.032 µM。圖2C顯示化合物43之資料，其IC
50= 0.08925 µM。其他所測試化合物之IC
50值顯示於下表4中。化合物61、65、81及114之CC
50均＞ 10 µM。
表 4
材料及方法Materials and methods
Vero 76 (非洲綠猴腎上皮細胞)細胞之匯合或幾乎匯合細胞培養單層係由Institute for Antiviral Research, Utah State University, Utah, USA提供，且在測試前一天在96孔一次性微量盤中製備。將細胞維持在補充有5% FBS之MEM中。對於抗病毒分析而言，使用相同培養基，但FBS降至2%且補充有50 µg/ml建它黴素(gentamicin)。將化合物溶解於DMSO中。以四個連續log10濃度：0.1、1.0、10及100 µg/ml或µM製備測試化合物。每稀釋使用五個微孔：三個用於經感染培養物且兩個用於未感染毒性培養物。實驗之對照由每盤上之經感染且未處理(病毒對照)之六個微孔以及未處理且未感染(細胞對照)之六者組成使用與對於測試化合物進行之相同方法，平行測試已知活性藥物蛋白酶抑制劑M128533作為陽性對照藥物。Confluent or nearly confluent cell culture monolayers of Vero 76 (African green monkey kidney epithelial cells) cells were provided by the Institute for Antiviral Research, Utah State University, Utah, USA and prepared in 96-well disposable microplates the day before testing . Cells were maintained in MEM supplemented with 5% FBS. For antiviral assays, the same medium was used but with FBS reduced to 2% and supplemented with 50 µg/ml gentamicin. Compounds were dissolved in DMSO. Test compounds were prepared at four consecutive log10 concentrations: 0.1, 1.0, 10 and 100 µg/ml or µM. Five microwells were used per dilution: three for infected cultures and two for non-infected virulent cultures. Controls for the experiment consisted of six microwells per plate that were infected and untreated (virus control) and six wells that were untreated and uninfected (cell control). Active drug protease inhibitor M128533 was used as positive control drug.
自細胞移除生長培養基，且以0.1 ml體積、以2×濃度將測試化合物施加至各孔中。將SARS-CoV-2、USA_WA1/2020株(通常在~60 CCID50 (50%細胞培養感染劑量)，0.1 ml體積)添加至經指定用於病毒感染之孔中。將不含病毒之培養基置放於毒性對照孔及細胞對照孔中。在37℃與5% CO 2下培育盤，直至在病毒對照孔中觀測到經標記之CPE (對於大部分病毒菌株，＞80% CPE)。接著，在5% CO 2培育箱中在37℃各盤用0.011%中性紅染色約兩小時。藉由完全抽吸移除中性紅培養基，且視情況用磷酸鹽緩衝溶液(phosphate buffered solution；PBS)沖洗細胞1×以移除殘餘染料。完全移除PBS，且用50%索倫森氏檸檬酸鹽緩衝液(Sorensen's citrate buffer)/50%乙醇溶離所併入之中性紅至少30分鐘。中性紅染料滲透至活細胞中，因此，紅色愈強，孔中所存在之活細胞數愈多。在540 nm波長下使用分光光度計定量各孔中之染料含量。使用基於Microsoft Excel電腦之試算表將各組孔中之染料含量轉化成存在於未處理對照孔中的染料之百分比，且基於病毒對照標準化。接著藉由回歸分析計算50%有效(EC 50，病毒抑制)濃度及50%細胞毒性(CC 50，細胞抑制)濃度。CC 50除以EC 50之商得到選擇性指數(selectivity index；SI)值。顯示SI值＞10之化合物視為活性的。 The growth medium was removed from the cells and the test compound was applied to each well in a volume of 0.1 ml at 2x concentration. Add SARS-CoV-2, USA_WA1/2020 strain (typically at ~60 CCID50 (50% cell culture infectious dose), 0.1 ml volume) to wells designated for virus infection. Virus-free medium was placed in toxicity control wells and cell control wells. Plates were incubated at 37°C with 5% CO2 until labeled CPE was observed in virus control wells (>80% CPE for most virus strains). Next, each plate was stained with 0.011% neutral red for about two hours at 37°C in a 5% CO 2 incubator. Neutral red medium was removed by complete aspiration, and cells were optionally washed 1× with phosphate buffered solution (PBS) to remove residual dye. The PBS was completely removed and the infused neutral red was eluted with 50% Sorensen's citrate buffer/50% ethanol for at least 30 minutes. Neutral red dye penetrates into living cells, therefore, the stronger the red color, the greater the number of living cells present in the well. The dye content in each well was quantified using a spectrophotometer at a wavelength of 540 nm. The dye content in each set of wells was converted to the percentage of dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet and normalized to the virus control. Then, the 50% effective (EC 50 , virus inhibition) concentration and the 50% cytotoxicity (CC 50 , cell inhibition) concentration were calculated by regression analysis. The quotient of CC50 divided by EC50 gives selectivity index (SI) value. Compounds showing SI values >10 were considered active.
結果result
結果顯示於表5中。所有三種測試化合物呈現具有治療可接受之安全性指數之抗病毒活性。
材料及方法Materials and methods
測試化合物17及18針對HCoV 229E及HCoV OC43之抑制病毒或殺病毒特性。製備儲備液濃度為10 mM之各化合物之儲備溶液以用於立即使用。
EC
50(
半最大有效濃度 )- 使用適當濃度病毒，此研究評定化合物17及18之抑制病毒或殺病毒特性。將細胞接種至96孔盤中。將化合物17及18連續稀釋(8點，3倍劑量滴定)，且預防性添加至細胞一小時。接著，以單一感染性劑量(100×中位數組織培養感染劑量；TCID50)，使細胞經病毒感染一小時。將額外培養基添加至孔中，其中在研究期間，化合物之濃度相等。設定媒劑及陽性對照孔來控制對於細胞存活力之任何影響。每天視覺測試細胞之任何細胞病變性(cytophathic/cytopathogenic)作用(CPE)之出現。一旦CPE完成，則進行比色哺乳動物細胞存活力分析(噻唑基藍四唑鎓溴化物(MTT)分析；Sigma目錄號M5655-1G；批次號MKCL1832)。如下計算病毒抑制百分比：
抑制% = [(A−B)/ (C−B)]×100，其中：
A：測試之平均光密度，B：病毒對照之平均光密度，C：細胞對照之平均光密度。
EC50 ( half maximal effective concentration ) - Using appropriate concentrations of virus, this study assesses the virustatic or virucidal properties of
歸因於天然變化或化合物作用，當A＞C時，存在高於100%抑制之值。Values above 100% inhibition exist when A > C due to natural variation or compound action.
歸因於天然變化或化合物毒性，當A＜B時，存在低於0%抑制之值。Values below 0% inhibition exist when A<B due to natural variation or compound toxicity.
使用非線性回歸(log(促效劑)與反應可變斜率(四參數))，使用log(濃度)，計算EC 50值。 EC50 values were calculated using non-linear regression (log(agonist) vs. variable slope of response (four parameters)) using log(concentration).
CC 50( 半最大細胞毒性濃度 )- 以上文針對EC 50所描述之方式測定CC 50。各盤逐漸(developed to)顯示在不存在病毒感染下化合物對於細胞之任何細胞毒性作用。一旦CPE完成，則進行MTT分析。如下計算細胞存活力百分比： 存活力% = (A/B)×100，其中： A：測試之平均光密度，B：細胞對照之平均光密度。 歸因於天然變化或化合物作用，當A＞B時，存在高於100%抑制之值。 CC50 ( half maximal cytotoxic concentration ) - CC50 was determined in the manner described above for EC50 . Plates were developed to show any cytotoxic effect of the compound on the cells in the absence of viral infection. Once CPE is complete, MTT analysis is performed. Percent cell viability was calculated as follows: % Viability = (A/B) x 100, where: A: mean optical density of test, B: mean optical density of cell control. Values above 100% inhibition exist when A > B due to natural variation or compound action.
使用如上針對EC 50之非線性回歸計算CC 50值。 CC50 values were calculated using nonlinear regression for EC50 as above.
實驗條件及讀數- 所使用之病毒株/血清型為人類α冠狀病毒229E (HCoV 229E；ATCC
®CVR-740™)及β冠狀病毒1 OC-4 (HCoV OC43；ATCC
®VR-1558™)。用人類支氣管上皮細胞(16BHE)測試HCoV 229E。用人類肺黏液表皮樣(H292)細胞測試HCoV OC43。瑞德西韋(Remdesivir)用作對照化合物。
Experimental Conditions and Readouts - The virus strains/serotypes used were human alphacoronavirus 229E (HCoV 229E; ATCC ® CVR-740™) and
結果result
在 16BHE 細胞中 ， 針對 HCoV 229E 之 功效-化合物17顯示針對HCoV 229E之功效，其中在大致1 µM下百分比病毒抑制達到50% (圖3A)。歸因於在10 µM及3.16 µM下觀測到之細胞毒性，吾人計算~1 µM之大致EC
50。
Efficacy against HCoV 229E in 16BHE cells -
化合物18顯示針對HCoV 229E之功效，其中在大致1 µM下百分比病毒抑制達到50% (圖3B)。歸因於在10 µM及3.16 µM下觀測到之細胞毒性，吾人計算~1 µM之大致EC 50及CC 50= 1.76 µM。 Compound 18 showed efficacy against HCoV 229E with a percent viral inhibition reaching 50% at approximately 1 µM (Figure 3B). Due to the cytotoxicity observed at 10 µM and 3.16 µM, we calculated an approximate EC 50 and CC 50 = 1.76 µM for ~1 µM.
在 H292 細胞中 ， 針對 HCoV OC43 之 功效-化合物17顯示針對HCoV OC43之功效，其EC
50為0.22 µM (圖4A)及CC50 ≥ 10 µM。化合物17提高細胞存活力至比未感染細胞更高的水準，從而產生高於100%之百分比。感染細胞中之存活力在兩個最高濃度10 µM及3.16 µM下降低，但未降低至在未感染細胞中觀測到之水準。為了計算更具代表性之EC
50，吾人排除此等濃度下之資料點。
Efficacy against HCoV OC43 in H292 cells -
化合物18顯示針對HCoV OC43之功效，其EC 50為0.33 µM (圖4B)。化合物18提高細胞存活力至比未感染細胞更高的水準，從而產生高於100%之百分比。感染細胞中之存活力在10 µM下降低至約0%。為了計算更具代表性之EC 50，吾人排除10 µM資料點。該化合物之CC 50≥ 10 µM。 生物學實例 6 ： 在 Vero E6 細胞中 ， SARS CoV2 誘導之細胞病變效應 (CPE) 之 PIKfyve 抑制 Compound 18 showed potency against HCoV OC43 with an EC 50 of 0.33 µM ( FIG. 4B ). Compound 18 increased cell viability to a higher level than uninfected cells, resulting in percentages above 100%. Viability in infected cells was reduced to approximately 0% at 10 µM. In order to calculate a more representative EC 50 , we excluded the 10 µM data point. The compound has a CC 50 ≥ 10 µM. Biological Example 6 : PIKfyve Inhibition of SARS CoV2- Induced Cytopathic Effect (CPE) in Vero E6 Cells
材料及方法Materials and methods
使用Vero E6及Vero 6細胞，根據Severson等人(2007) Journal of Biomolecular Screening 33-40之方法，評定五種PIKfyve抑制劑針對SARS-CoV-2株USA_WA1/2020之抗病毒活性。Using Vero E6 and Vero 6 cells, according to the method of Severson et al. (2007) Journal of Biomolecular Screening 33-40, the antiviral activity of five PIKfyve inhibitors against SARS-CoV-2 strain USA_WA1/2020 was evaluated.
結果result
結果之概述顯示於表6中。所測試化合物顯示具有極小毒性之強效抗病毒作用。
表 6.
製備儲備溶液Prepare stock solutions
在二甲亞碸(DMSO)中製備在10 mM濃度下之測試化合物之儲備溶液，接著用DMSO稀釋至2 mM。測試化合物之最終濃度為10 μM。Stock solutions of test compounds were prepared at a concentration of 10 mM in dimethylsulfoxide (DMSO) and then diluted to 2 mM with DMSO. The final concentration of test compound was 10 μM.
製備陽性抑制劑Preparation of Positive Inhibitors
陽性抑制劑之濃度在表7中列出。對於儲備溶液製備而言，若陽性對照在最高濃度下無法充分溶解於DMSO與乙腈(1:4)之混合物中，則使用乙腈與DMSO之另一種混合物、乙腈與H
2O或DMSO之混合物來溶解該化合物。
表7.
製備受質儲備溶液Prepare substrate stock solution
此等受質之製備詳情在表8中給出。在使用之前，將儲存在-20℃之受質溶液升溫至室溫。
表 8.
製備磷酸鹽緩衝液Prepare Phosphate Buffer (100 mM(100 mM ，, pH 7.4)pH 7.4)
溶液A係藉由如下來製備：添加7.098 g磷酸氫二鈉至500 mL純水中，隨後音波處理。溶液B係藉由如下來製備：添加3.400 g磷酸二氫鉀至250 mL純水中，隨後音波處理。將溶液A置放於攪拌器上，且將溶液B緩慢添加至溶液A中，直至pH達到7.4。Solution A was prepared by adding 7.098 g of disodium hydrogen phosphate to 500 mL of pure water followed by sonication. Solution B was prepared by adding 3.400 g potassium dihydrogen phosphate to 250 mL pure water followed by sonication. Solution A was placed on a stirrer, and solution B was slowly added to solution A until the pH reached 7.4.
在使用之前，藉由以8.334 mg/mL將菸鹼醯胺腺二核苷酸磷酸酯(NADPH)溶解於磷酸鹽緩衝液中，來新鮮製備10 mM NADPH溶液。Prior to use, a 10 mM NADPH solution was freshly prepared by dissolving nicotinamide adenonucleotide phosphate (NADPH) at 8.334 mg/mL in phosphate buffered saline.
製備主溶液Prepare master solution
根據表9製備主溶液。在96深孔盤中進行培育。將以下體積分配至培育盤之各孔中，179 μL含受質及人類肝微粒體(HLM)混合物之磷酸鹽緩衝液，1 μL化合物工作溶液或媒劑(DMSO與乙腈(1:4)之混合物)。在藉由添加20 μL於磷酸鹽緩衝液中之10 mM NADPH溶液起始反應之前，將培育盤置放於水浴中且在37℃預溫熱15分鐘。在添加NADPH之後，在37℃培育培育盤對應時間。重複進行兩次分析。
表 9.
藉由添加1.5體積(300 μL)含有3%甲酸及內標物(200 nM拉貝洛爾(Labetalol)、200 nM阿普唑侖(Alprazolam)及200 nM甲苯磺丁尿(tolbutamide))之冷乙腈來淬滅反應物。以3,220 g離心盤40分鐘，將100 μL上清液轉移至新盤中。若適當，則上清液用100 μL純水稀釋。在混合之後，使用超效液相層析-串聯質譜(UPLC/MS/MS)分析樣品。By adding 1.5 volumes (300 μL) of cold water containing 3% formic acid and internal standards (200 nM Labetalol, 200 nM Alprazolam, and 200 nM tolbutamide) Acetonitrile was used to quench the reaction. Centrifuge the plate at 3,220 g for 40 min and transfer 100 μL of the supernatant to a new plate. If appropriate, the supernatant was diluted with 100 μL of pure water. After mixing, samples were analyzed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS).
資料分析ANALYSE information
檢查所有樣品之自動峰積分面積。將分析物峰面積及內標峰面積導至excel試算表中。人類肝微粒體中各P450酶之抑制量測為相較於非抑制對照(= 100%活性)，標記代謝物形成之活性的降低百分比。Check the automatic peak integration area for all samples. Import the peak area of the analyte and the peak area of the internal standard into the excel spreadsheet. Inhibition of each P450 enzyme in human liver microsomes was measured as the percentage decrease in the activity of labeled metabolite formation compared to non-inhibited controls (= 100% activity).
如下計算剩餘活性百分比： 面積比率=分析物峰面積/內標峰面積 剩餘活性(%) =測試化合物面積比率/媒劑面積比率×100% 抑制% = 100-剩餘活性(%) The percent activity remaining was calculated as follows: Area ratio = analyte peak area / internal standard peak area Residual activity (%) = area ratio of test compound/area ratio of vehicle × 100% Inhibition % = 100 - remaining activity (%)
此實例之結果顯示於表10中。
表10.
製備MDCK-MDR1細胞Preparation of MDCK-MDR1 cells
將50 μL及25 mL細胞培養基分別添加至Transwell ®***件及儲集器之各孔中。在細胞接種之前，在37℃、5% CO 2下培育HTS Transwell ®盤1小時。 Add 50 μL and 25 mL of cell culture medium to each well of the Transwell® Insert and Reservoir, respectively. Before cell seeding, incubate the HTS Transwell® dish at 37°C, 5% CO 2 for 1 hour.
用培養基將MDCK-MDR1細胞稀釋至1.56×10 6個細胞/毫升，且將50 µL細胞懸浮液分配至96孔HTS Transwell盤之過濾孔中。在37℃、5% CO 2及95%相對濕度下，在細胞培養培育箱中培養細胞4-8天。每隔一天更換細胞培養基，在初始塗鋪之後不晚於24小時開始。 MDCK-MDR1 cells were diluted to 1.56×10 6 cells/ml with medium, and 50 µL of the cell suspension was dispensed into filter wells of a 96-well HTS Transwell plate. Cells were cultured in a cell culture incubator at 37°C, 5% CO 2 and 95% relative humidity for 4-8 days. Cell culture medium was changed every other day, starting no later than 24 hours after the initial plating.
製備儲備溶液Prepare stock solutions
在二甲亞碸(DMSO)中製備濃度為10 mM之測試化合物及陽性對照的儲備溶液。美托洛爾(Metoprolol)及地高辛(Digoxin)用作對照化合物。Stock solutions of test compounds and positive controls were prepared at a concentration of 10 mM in dimethylsulfoxide (DMSO). Metoprolol and Digoxin were used as control compounds.
評定細胞單層完整性Assessing Cell Monolayer Integrity
自儲集器及各Transwell***件移除培養基，且用經預熱之新鮮培養基更換。使用Millicell®上皮細胞伏特-歐姆量測系統(Millipore®, USA)來量測跨單層之跨上皮電阻(TEER)。一旦量測完成，則將盤放回培育箱，且根據以下等式計算TEER值： TEER量測值(歐姆) × 膜面積(cm 2) = TEER值(ohm•cm 2) Media was removed from the reservoir and each Transwell insert and replaced with pre-warmed fresh media. Transepithelial electrical resistance (TEER) across the monolayer was measured using the Millicell® Epithelial Cell Volt-Ohm Measurement System (Millipore®, USA). Once the measurement is complete, return the dish to the incubator and calculate the TEER value according to the following equation: TEER measurement (ohms) x membrane area (cm 2 ) = TEER value (ohm cm 2 )
TEER值應高於42 ohm•cm 2，其指示充分合格的MDCK-MDR1單層。 TEER values should be higher than 42 ohm·cm 2 , which indicates a well qualified MDCK-MDR1 monolayer.
分析程序analysis program
自培育箱移出MDCK-MDR1盤，且用預溫熱的漢克氏平衡鹽溶液(Hanks' Balanced Salt solution；HBSS) (10 mM HEPES，pH 7.4)洗滌兩次，且接著在37℃培育30分鐘。將對照化合物之儲備溶液稀釋於DMSO中，得到200 μM溶液，且接著用HBSS (10 mM HEPES，pH 7.4)稀釋，得到1 μM工作溶液。將測試化合物稀釋於DMSO中，得到200 μM溶液，且接著用HBSS (具有4% BSA之10 mM HEPES，pH 7.4)稀釋，得到1 μM工作溶液。培育系統中之DMSO之最終濃度為0.5%。MDCK-MDR1 plates were removed from the incubator and washed twice with pre-warmed Hanks' Balanced Salt solution (HBSS) (10 mM HEPES, pH 7.4) and then incubated at 37°C for 30 minutes . Stock solutions of control compounds were diluted in DMSO to give 200 μM solutions and then diluted with HBSS (10 mM HEPES, pH 7.4) to give 1 μM working solutions. Test compounds were diluted in DMSO to give 200 μM solutions and then diluted with HBSS (10 mM HEPES with 4% BSA, pH 7.4) to give 1 μM working solutions. The final concentration of DMSO in the incubation system was 0.5%.
為了測定在頂部至底側方向上藥物運輸之速率，將125 μL工作溶液添加至Transwell***件中(頂部區室)，且立即將來自頂部區室之50 μL樣品移轉至新96孔盤中之200 μL含有內標物(IS) (100 nM阿普唑侖、200 nM咖啡因及100 nM甲苯磺丁尿)的乙腈(ACN)中作為初始供體樣品(A-B)。在以1000 rpm渦旋10分鐘之後，用235 μL運輸緩衝液填充接受體盤中之各孔(底側區室)。To determine the rate of drug transport in the top-to-bottom direction, 125 μL of working solution was added to the Transwell insert (top compartment) and 50 μL of sample from the top compartment was immediately transferred to a new 96-
為了測定在底側至頂部方向上藥物運輸之速率，將285 μL工作溶液添加至接受體盤孔(底側區室)中，且立即將來自底側區室之50 μL樣品移轉至新96孔盤中之含有IS (100 nM阿普唑侖、200 nM咖啡因及100 nM甲苯磺丁尿)的200 μL乙腈中作為初始供體樣品(B-A)。在以1000 rpm渦旋10分鐘之後，用75 μL運輸緩衝液填充Transwell***件(頂部區室)，其中頂部至底側方向及底側至頂部方向係在同時量測。To determine the rate of drug transport in the bottom-to-top direction, 285 μL of working solution was added to the receptor plate wells (bottom compartment) and 50 μL of sample from the bottom compartment was immediately transferred to a new 96 200 μL of acetonitrile containing IS (100 nM alprazolam, 200 nM caffeine, and 100 nM tolbutidine) in well plates served as initial donor samples (B-A). After vortexing at 1000 rpm for 10 minutes, the Transwell insert (top compartment) was filled with 75 μL of transport buffer, with top-to-bottom and bottom-to-top directions being measured simultaneously.
在37℃培育盤2小時之後，將來自供體側(對於Ap→Bl流為頂部區室，且對於Bl→Ap為底側區室)及接受體側(對於Ap→Bl流為底側區室，且對於Bl→Ap為頂部區室)之各50 μL樣品轉移至新96孔盤之各孔中，隨後添加4體積含有IS (100 nM阿普唑侖、200 nM咖啡因及100 nM甲苯磺丁尿)的乙腈。將樣品渦旋10分鐘，且接著以3,220 g離心40分鐘。在LC-MS/MS分析之前，將一等分試樣之100 µL上清液與適當體積之超純水混合。After incubating the plate for 2 hours at 37°C, the samples from the donor side (top compartment for Ap→Bl flow and bottom side compartment for Bl→Ap) and acceptor side (bottom side compartment for Ap→Bl flow) were separated. , and for Bl→Ap is the top compartment), each 50 μL sample was transferred to each well of a new 96-well plate, followed by the addition of 4 volumes containing IS (100 nM alprazolam, 200 nM caffeine, and 100 nM tosylate butyl urine) in acetonitrile. Samples were vortexed for 10 minutes and then centrifuged at 3,220 g for 40 minutes. Prior to LC-MS/MS analysis, an aliquot of 100 µL of the supernatant was mixed with an appropriate volume of ultrapure water.
為了測定2小時運輸時段後之螢光黃滲漏率，在DMSO中製備螢光黃之儲備溶液且用HBSS (10 mM HEPES，pH 7.4)稀釋以達到100 μM之最終濃度。將100 μL螢光黃溶液添加至各Transwell***件(頂部區室)中，隨後用300 µL HBSS (10 mM HEPES，pH 7.4)填充接受體盤(底側區室)中之各孔。在37℃培育盤30 min。自頂部及底側孔(使用底側進入孔)直接移出80 μL樣品，且轉移至新96孔盤之各孔中。在螢光盤讀取器中在480 nM激發及530 nM發射下量測螢光黃螢光(以監測單層完整性)訊號。To determine the lucifer yellow leakage rate after a 2 hour transport period, a stock solution of lucifer yellow was prepared in DMSO and diluted with HBSS (10 mM HEPES, pH 7.4) to reach a final concentration of 100 μΜ. 100 μL of Luciferin solution was added to each Transwell insert (top compartment), followed by filling each well in the acceptor disc (bottom compartment) with 300 μL of HBSS (10 mM HEPES, pH 7.4). Plates were incubated at 37°C for 30 min. 80 μL of sample was removed directly from the top and bottom side wells (using the bottom side access wells) and transferred to each well of a new 96-well plate. Fluorescent yellow fluorescence (to monitor monolayer integrity) signal was measured in a fluorescent disc reader at 480 nM excitation and 530 nM emission.
資料分析ANALYSE information
使用以下等式計算以厘米/秒為單位之表觀滲透係數(Papp)，以用於MDCK-MDR1藥物運輸分析： Papp = (VA ×[藥物]受體)/(面積×時間×[藥物]初始，供體) 其中VA為受體孔中之體積(以mL為單位)，面積為膜之表面積(對於Transwell-96孔可滲透性支撐物為0.143 cm 2)，且時間為以秒為單位之總運輸時間。 The apparent permeability coefficient (Papp) in cm/s was calculated for the MDCK-MDR1 drug transport analysis using the following equation: Papp = (VA x [drug] receptor)/(area x time x [drug] Initial, Donor) where VA is the volume in the acceptor pore (in mL), area is the surface area of the membrane (0.143 cm 2 for a Transwell-96 pore permeable support), and time is in seconds total transit time.
可使用以下等式計算以百分比(%)為單位之螢光黃滲漏率： LY滲漏% = 100×[LY]受體/([LY]供體+[LY]受體) LY滲漏率＜1%為可接受的，以指示充分合格的MDCK-MDR1單層。 Fluorescent Yellow leakage rate in percent (%) can be calculated using the following equation: LY leakage % = 100×[LY]acceptor/([LY]donor+[LY]acceptor) A LY leakage rate of <1% is acceptable to indicate a well-qualified MDCK-MDR1 monolayer.
此實例之結果顯示於表11中。 生物學實例 9 ： 肝細胞藥物代謝研究 The results of this example are shown in Table 11. Biological Example 9 : Drug Metabolism Study in Hepatocytes
製備工作溶液Prepare working solution
在DMSO中製備測試化合物及陽性對照之10 mM儲備溶液。在獨立錐形管中，藉由合併198 µL 50%乙腈/50%水及2 µL 10 mM儲備液，將10 mM測試化合物及陽性對照物稀釋至100 µM。10 mM stock solutions of test compounds and positive controls were prepared in DMSO. In separate conical tubes, dilute 10 mM test compound and positive control to 100 µM by combining 198 µL of 50% acetonitrile/50% water and 2 µL of 10 mM stock solution.
製備肝細胞preparation of hepatocytes
將培育培養基(補充有GlutaMAX TM之威廉姆氏E培養基(William's E Medium))及肝細胞解凍培養基置放於37℃水浴中，且在使用之前使其升溫至少15分鐘。 Incubation medium (William's E Medium supplemented with GlutaMAX ™ ) and hepatocyte thawing medium were placed in a 37°C water bath and allowed to warm for at least 15 minutes before use.
自儲存移轉一小瓶之冷凍保存的肝細胞，確保小瓶保持在低溫溫度直至隨即進行解凍過程。藉由將小瓶置放在37℃水浴中且平緩地振盪小瓶2分鐘來解凍細胞。完成解凍後，小瓶用70%乙醇噴射且轉移至生物安全櫃中。Transfer a vial of cryopreserved hepatocytes from storage, ensuring that the vial remains at cryogenic temperature until immediately following the thawing process. Cells were thawed by placing the vial in a 37°C water bath and shaking the vial gently for 2 minutes. After complete thawing, the vials were sparged with 70% ethanol and transferred to a biosafety cabinet.
將肝細胞轉移至含有解凍培養基之50 mL錐形管中。將50 mL錐形管置放於離心機中且以100 g旋轉10分鐘。旋轉完成後，抽吸解凍培養基，且將肝細胞再懸浮於足夠的培育培養基中，得到~1.5×10 6個細胞/毫升。 Transfer hepatocytes to a 50 mL conical tube containing thawed medium. Place the 50 mL conical tube in a centrifuge and spin at 100 g for 10 minutes. After the spin is complete, the thawed medium is aspirated, and the hepatocytes are resuspended in sufficient incubation medium to yield ~1.5 x 106 cells/ml.
使用AO/PI染色來計數細胞且測定活細胞密度，之後細胞用培育培養基稀釋至0.5×10 6個活細胞/毫升之工作細胞密度。 AO/PI staining was used to count cells and determine viable cell density, after which cells were diluted with culture medium to a working cell density of 0.5 x 106 viable cells/ml.
穩定性測定之程序Procedure for Stability Determination
將198 μL肝細胞吸入96孔未經塗佈之盤之各孔中，接著將其置放於培育箱中以使肝細胞升溫10分鐘。將2 μL 100 μM測試化合物或陽性對照吸入96孔未經塗佈之盤之各別孔中以起始反應，且將盤放回培育箱中持續設計的時間點。198 μL of hepatocytes were pipetted into each well of a 96-well uncoated dish, which was then placed in an incubator to warm the hepatocytes for 10 minutes. Reactions were initiated by pipetting 2 μL of 100 μM test compound or positive control into individual wells of a 96-well uncoated plate, and the plate was returned to the incubator for the programmed time points.
在0、15、30、60、90及120分鐘之時間點，將孔內容物移轉至25 μL等分試樣中，接著與6體積(150 μL)含有內標物IS (100 nM阿普唑侖、200 nM咖啡因及100 nM甲苯磺丁尿)之乙腈混合以終止反應。在5分鐘渦旋之後，以3,220 g離心樣品45分鐘。各等分試樣之100 µL上清液用100 µL超純水稀釋，且該混合物用於液相層析(LC)串聯質譜(MS) (LC/MS/MS)分析。所有培育均重複進行兩次。At time points of 0, 15, 30, 60, 90 and 120 minutes, the contents of the wells were transferred to 25 μL aliquots and then mixed with 6 volumes (150 μL) containing the internal standard IS (100 nM Azolam, 200 nM caffeine and 100 nM tolbutidine) in acetonitrile were mixed to stop the reaction. After vortexing for 5 minutes, samples were centrifuged at 3,220 g for 45 minutes. 100 µL of the supernatant from each aliquot was diluted with 100 µL of ultrapure water, and the mixture was used for liquid chromatography (LC) tandem mass spectrometry (MS) (LC/MS/MS) analysis. All incubations were performed in duplicate.
資料分析ANALYSE information
使用Microsoft Excel進行所有計算。由萃取離子層析圖判定峰面積。藉由對母體消失百分比相對於時間曲線之回歸分析來測定母體化合物之活體外半衰期(t 1/2)。 All calculations were performed using Microsoft Excel. The peak area was determined from the extracted ion chromatogram. The in vitro half-life (t 1/2 ) of the parent compound was determined by regression analysis of the percent disappearance of the parent versus time curve.
根據斜率值測定活體外半衰期(活體外t 1/2)： 活體外t 1/2= 0.693/k Determination of the in vitro half-life (in vitro t 1/2 ) from the slope value: in vitro t 1/2 = 0.693/k
使用以下等式將活體外t 1/2(以min為單位)轉化為活體外固有清除率(活體外CLint，以µL/min/1×10 6個細胞為單位) (兩次重複測定之平均值)： 活體外CLint = kV/N 其中V =培育體積(0.2 mL)；且N =每孔肝細胞數目(0.1 × 10 6個細胞)。 Convert in vitro t 1/2 (in min) to in vitro intrinsic clearance (in vitro CLint in µL/min/1× 106 cells) using the following equation (average of duplicate determinations value): In vitro CLint = kV/N where V = incubation volume (0.2 mL); and N = number of hepatocytes per well (0.1 x 10 6 cells).
使用以下等式計算按比例增加的CLint (mL/min/kg)、預測CLH (mL/min/kg)及EH：
按比例增加的CLint = (0.693/T1/2) × (1/(肝細胞濃度(0.5 × 10
6個細胞/毫升))) × 比例因數(參見表9)；
預測CLH = (QH ×按比例增加的CLint × f
ub) / (QH +按比例增加的CLint × f
ub)；且EH =預測CLH/QH
其中QH為肝血流(mL/min/kg) (表9)，f
ub為血漿中未結合藥物之分數，其假定為1。表11顯示人類、猴、狗、大鼠及小鼠肝細胞中固有清除預測之比例因數。
表11.
用於資料處理之規則顯示於表12中。
表12.
此實例之結果顯示於表13中。
表13.
已研發且驗證SARS-CoV-2敍利亞金色(SG)倉鼠感染模型(Boudewijns BioRxiv 2020, doi.org/10.1101/2020.04.23.056838)。此模型用於評估化合物之潛在抗病毒活性及選擇性。A SARS-CoV-2 Syrian golden (SG) hamster infection model has been developed and validated (Boudewijns BioRxiv 2020, doi.org/10.1101/2020.04.23.056838). This model was used to evaluate the potential antiviral activity and selectivity of compounds.
150 cm
2Vero E6細胞培養燒瓶用在1/1000最終稀釋度下之先前低繼代SARS-CoV-2儲備液[BetaCoV/Belgium/GHB-03021/2020]感染。在感染後第3天，在完全CPE後，收集含有病毒之上清液，等分且儲存在-80℃。藉由空斑檢定測定儲備液之感染性病毒負荷。
150 cm Vero E6 cell culture flasks were infected with a previous low passage SARS-CoV-2 stock [BetaCoV/Belgium/GHB-03021/2020] at a final dilution of 1/1000. On
對90-120 g且6-8週齡之雌性SG倉鼠進行耳標記，且隨機分組至三個不同處理組中。各組由6隻倉鼠組成。第一組媒劑對照組經感染但未接受處理。第二組(組2)及第三組(組3)根據下表14中之時程，藉由經口管飼每天一次接受20 mg/kg及50 mg/kg劑量之化合物17。
表14.
在第0天，根據以上時程對未感染倉鼠給藥。在處理之後，各倉鼠用2.4 × 106 PFU (空斑形成單位)之SARS-CoV-2感染。On
額外處理在第1、2及3天進行。Additional treatments were performed on
在第1-4天中之各天，對經刺激之動物稱重，一天一次，且觀測運動性、自我維持、行為及人道終點(後肢麻痹、駝背、眼睛發酸)。On each of days 1-4, stimulated animals were weighed once a day and observed for motility, self-sustainment, behavior, and humane endpoints (hind limb paralysis, hunching, eye soreness).
在第4天，將動物處死，且收集肺及血液並用於藉由即時定量RT-qPCR定量病毒負荷、藉由(終點)滴定、組織檢查及PK分析定量感染性病毒含量。收集血液且儲存。On
結果：result:
相較於媒劑對照，在化合物17處理之兩個組之肺中未看見病毒RNA或感染性病毒負荷降低。表15顯示獲自組2及組3個體之病毒RNA複本及病毒負荷量測。圖5A及圖5B以圖形方式顯示此資料。在用50 mg/kg處理之組中觀測到重量損失，如下表15中所示。
表15.
表16顯示使用以下組織病理學標記量測之各個體及對照之累積肺分數：充血(A欄)、胞內血紅素(B欄)、淋巴濾泡(C欄)、支氣管壁中之凋亡體(D欄)、壞死性細支氣管炎(E欄)、血管周水腫(F欄)、支氣管肺炎(G欄)、血管周發炎(H欄)、氣管周發炎(I欄)及血管炎(J欄)。Table 16 shows cumulative lung fractions for each individual and control measured using the following histopathological markers: hyperemia (column A), intracellular hemoglobin (column B), lymphoid follicles (column C), apoptosis in the bronchial wall body (column D), necrotizing bronchiolitis (column E), perivascular edema (column F), bronchopneumonia (column G), perivascular inflammation (column H), peritracheal inflammation (column I), and vasculitis (column Column J).
圖6為各劑量組及對照之累積肺分數之圖。表16中顯示之個體肺分數用於計算累積肺分數。表中之空白框指示0之分數。在經處理之倉鼠肺病理組織學中看見累積肺分數之降低。
表16.
材料及方法Materials and methods
為了測定化合物針對SARS-CoV-2之半最大抑制濃度(IC 50)，針對兩種(2) SARS-CoV-2株：野生型(WT)及δ (D)進行抗病毒篩選。 To determine the half-maximal inhibitory concentration (IC 50 ) of compounds against SARS-CoV-2, antiviral screening was performed against two (2) SARS-CoV-2 strains: wild type (WT) and delta (D).
將附著性Vero-E6細胞接種於多孔盤中。在不存在及存在連續化合物稀釋液之情況下，對細胞培養物接種標準化量之病毒，隨後18-24小時培育及適當病毒偵測方法(例如免疫染色)。在分別用各SARS-CoV-2株感染之前，將八個3.16倍化合物稀釋液(最低濃度1 nM)添加至分析細胞中1 h。使用在各化合物濃度存在下偵測到之病毒訊號來測定IC
50。在感染之後，將細胞固定且針對病毒抗原進行免疫染色，且使用高通量成像相對於感染的未處理對照定量感染細胞之百分比。平行地，藉由MTT分析，測試在不存在病毒感染下相同濃度之各化合物的細胞毒性。包括瑞德西韋作為陽性對照。
Adherent Vero-E6 cells were seeded in multiwell dishes. Cell cultures are inoculated with standardized amounts of virus in the absence and presence of serial compound dilutions, followed by 18-24 hour incubation and appropriate virus detection methods (eg, immunostaining). Eight 3.16-fold compound dilutions (
結果及討論Results and Discussion
測試代表性PYKfyve抑制劑以測定其針對兩種SARS-CoV2株：野生型(WT)及δ (D)之活性。結果之概述顯示於表17中。圖7A及圖7B以圖形方式顯示此資料。
表17.
前述揭示內容已經藉助於說明及實例，出於清晰性及理解的目的相當詳細地描述。因此，應理解以上描述意欲為說明性而非限定性的。因此，本發明之範疇不應參考以上描述判定，但應替代地參考以下所附申請專利範圍，以及該等申請專利範圍授權之等效物之完整範疇判定。The foregoing disclosure has been described in some detail, by way of illustration and examples, for purposes of clarity and understanding. Accordingly, it should be understood that the foregoing description is intended to be illustrative rather than restrictive. Accordingly, the scope of the invention should not be judged with reference to the above description, but should instead be judged with reference to the following appended claims, and the full scope of equivalents to which such claims are entitled.
圖1A至圖1C顯示在Vero-E6細胞中，化合物22、化合物18及化合物17之抗病毒作用及細胞毒性資料。濃度依賴性抗病毒作用(亦即，抗病毒活性)顯示為左軸上之細胞存活百分比(亦即，抑制百分比) (資料由圓圈與實線表示)。濃度依賴性細胞毒性顯示為右軸上之細胞存活百分比(資料由方塊與虛線表示)。圖1A顯示化合物22之資料。圖1B顯示化合物18之資料。圖1C顯示化合物17之資料。Figures 1A to 1C show the antiviral effects and cytotoxicity data of Compound 22, Compound 18 and
圖2A至圖2C顯示在A549-ACE2細胞中，化合物14、化合物17及化合物43之抗病毒作用。病毒效價顯示為左軸上之每mL空斑形成單位(PFU/mL)之Log
10(資料由圓圈表示)。在右軸上顯示百分比細胞存活力(資料由方塊表示)。圖2A顯示化合物14之資料。圖2B顯示化合物17之資料。圖2C顯示化合物43之資料。
Figures 2A to 2C show the antiviral effects of Compound 14,
圖3A至圖3B顯示化合物17及18對於人類α冠狀病毒株229E (HCoV 229E)之作用。將感染HCoV 229E之人類支氣管上皮細胞(16BHE)與未感染的16HBE細胞進行比較。資料呈現為平均百分比病毒抑制(EC
50；圓圈)或細胞存活力(CC
50；方塊) ± SEM (n=3)。圖3A顯示化合物17之資料；圖3B顯示化合物18之資料。
Figures 3A-3B show the effects of
圖4A至圖4B顯示化合物17及18對於人類β冠狀病毒株OC43 (HCoV OC43)之作用。將感染HCoV OC43之人類肺黏液表皮樣(H292)細胞與未感染的H292細胞進行比較。資料呈現為平均百分比病毒抑制(EC
50；圓圈)或細胞存活力(CC
50；方塊) ± SEM (n=3)。圖4A顯示化合物17之資料；圖4B顯示化合物18之資料。
Figures 4A-4B show the effects of
圖5A至圖5B顯示在用化合物17治療後，倉鼠肺組織中之感染水準。圖5A顯示相較於媒劑對照，在用20及50 mg/kg劑量之化合物17治療倉鼠後，每mg肺組織之病毒RNA複本數。圖5B顯示相較於媒劑對照，在用20及50 mg/kg劑量之化合物17治療倉鼠後，每mg肺組織之病毒負荷。Figures 5A-5B show the level of infection in hamster lung tissue after treatment with
圖6顯示相較於媒劑對照，在用20及50 mg/kg劑量之化合物17治療後，倉鼠中之累積肺分數改善。Figure 6 shows improvement in cumulative lung fraction in hamsters following treatment with
圖7A至圖7B顯示在Vero-E6-SARS-CoV-2細胞病變分析中，化合物17 (圖17A)及瑞德西韋(remdesivir)對照(圖17B)針對兩種SARS-CoV2株：野生型(WT)及δ (D)之活性。Figures 7A to 7B show that compound 17 (Figure 17A) and remdesivir control (Figure 17B) are effective against two SARS-CoV2 strains in Vero-E6-SARS-CoV-2 cytopathic assays: wild type (WT) and δ (D) activities.
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