除非有相反陳述,否則下列用在說明書和權利要求書中的術語具有下述含義。 在本文中使用的表示方式“Cx-y
”表示碳原子數的範圍,其中x和y均為整數,例如C3-8
環烷基表示具有3-8個碳原子的環烷基,即具有3、4、5、6、7或8個碳原子的環烷基。還應理解,“C3-8”還包含其中的任意亞範圍,例如C3-7、C3-6、C4-7、C4-6、C5-6等。 “烷基”指含有1至20個碳原子,例如1至18個碳原子、1至12個碳原子、1至8個碳原子、1至6個碳原子或1至4個碳原子的飽和的直鏈或支鏈的烴基基團。烷基的非限制性實例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、第三丁基、第二丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基和2-乙基丁基。所述烷基可以是取代的或未取代的。 “烯基”指含有至少一個碳碳雙鍵和通常2至20個碳原子,例如2至8個碳原子、2至6個碳原子或2至4個碳原子的直鏈或支鏈的烴基基團。烯基的非限制性實例包括乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、2-甲基-2-丙烯基、1,4-戊二烯基和1,4-丁二烯基。所述烯基可以是取代的或未取代的。 “炔基”指含有至少一個碳碳三鍵和通常2至20個碳原子,例如2至8個碳原子、2至6個碳原子或2至4個碳原子的直鏈或支鏈的烴基基團。炔基的非限制性實例包括乙炔基、1-丙炔基、2-丙炔基、1-丁炔基、2-丁炔基和3-丁炔基。所述炔基可以是取代的或未取代的。 “環烷基”指含有3至14個碳環原子的飽和環形烴基取代基。環烷基可以是單碳環,通常含有3至7個碳環原子。單環環烷基的非限制性實例包括環丙基、環丁基、環戊基、環己基和環庚基。環烷基可選擇地可以是稠合到一起的雙或三環,如十氫萘基。所述環烷基可以是取代的或未取代的。 “雜環或雜環基”指飽和或部分不飽和的單環或多環環狀基團,其包括3至20個環原子,例如可以是3至16個、3至14個、3至12個、3至10個、3至8個、3至6個或5至6個環原子,其中一個或多個環原子選自氮、氧或S(O)m
(其中m是整數0至2),但不包括-O-O-、-O-S-或-S-S-的環部分,其餘環原子為碳。較佳包括3至12個環原子,更佳3至10個環原子,最佳5或6個環原子,其中1~4個是雜原子,更佳1~3個是雜原子,最佳1~2個是雜原子。單環雜環基的非限制性實例包含吡咯烷基、呱啶基、呱嗪基、嗎啉基、硫代嗎啉基、高呱嗪基、氧雜環己烷基和氮雜環丁烷基。多環雜環基包括稠合、橋接或螺多環雜環基。所述雜環或雜環基可以是取代的或未取代的。 “芳基”指含有6至14個碳原子的芳香族單環或稠合多環基團,較佳為6至10元,例如苯基和萘基,最佳苯基。所述芳基環可以稠合於雜芳基、雜環基或環烷基環上,其中與母體結構連接在一起的環為芳基環,非限制性實例包括: 和, 所述芳基可以是取代的或未取代的。 “雜芳基或雜芳環”指包含5至14個環原子的雜芳族體系,其中1至4個環原子選自包括氧、硫和氮的雜原子。雜芳基較佳為5至10元。更佳雜芳基是5元或6元,例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、吡唑基、咪唑基、四唑基、噁唑基、異噁唑基、噻唑基、異噻唑基等。所述雜芳基環可以稠合於芳基、雜環基或環烷基環上,其中與母體結構連接在一起的環為雜芳基環,非限制性實例包括: 和, 所述雜芳基可以是取代的或未取代的。 “鹵素”指氟、氯、溴或碘。 “氰基”指-CN。 “任選”或“任選地”意味著隨後所描述的事件或環境可以但不必發生,該說明包括該事件或環境發生或不發生地場合。例如,“任選被烷基取代的雜環基團”意味著烷基可以但不必須存在,該說明包括雜環基團被烷基取代的情形和雜環基團不被烷基取代的情形。 “取代的”指基團中的一個或多個氫原子,較佳為5個,更佳為1~3個氫原子彼此獨立地被相應數目的取代基取代。不言而喻,取代基僅處在它們的可能的化學位置,本領域技術人員能夠在不付出過多努力的情況下確定(通過實驗或理論)可能或不可能的取代。例如,具有游離氫的胺基或羥基與具有不飽和(如烯屬)鍵的碳原子結合時可能是不穩定的。所述取代基包括但不限於羥基、胺基、鹵素、氰基、C1 - 6
烷基、C1 - 6
烷氧基、C2 - 6
烯基、C2 - 6
炔基、C3-8
環烷基等。 “藥物組合物”指含有一種或多種本文所述的化合物或其可藥用的鹽或前藥以及其他組分例如可藥用的載體和賦形劑的組合物。藥物組合物的目的是促進對生物體的給藥,利於活性成分的吸收進而發揮生物活性。 “異構體”指具有相同分子式但其原子結合的性質或順序或其原子的空間排列不同的化合物稱為“異構體”。其原子空間排列不同的異構體稱為“立體異構體”。立體異構體包括光學異構體、幾何異構體和構象異構體。 本發明的化合物可以以光學異構體形式存在。根據手性碳原子周圍取代基的構型,這些光學異構體是“R”或“S”構型。光學異構體包括對映異構體和非對映異構體。製備和分離光學異構體的方法是本領域中已知的。 本發明的化合物也可以存在幾何異構體。本發明考慮由碳-碳雙鍵、碳-氮雙鍵、環烷基或雜環基團周圍的取代基的分佈所產生的各種幾何異構體和其混合物。碳-碳雙鍵或碳-氮鍵周圍的取代基指定為Z或E構型,環烷基或雜環周圍的取代基指定為順式或反式構型。 本發明的化合物還可能顯示互變異構現象,例如酮-烯醇互變異構。 應該理解,本發明包括任何互變異構或立體異構形式和其混合物,並且不僅僅限於化合物的命名或化學結構式中所使用的任何一個互變異構或立體異構形式。 “同位素”是在本發明化合物中出現的原子的所有同位素。同位素包括具有相同原子序數但不同質量數的那些原子。適合併入本發明化合物中的同位素的實例是氫、碳、氮、氧、磷、氟和氯,分別例如但不限於2
H、3
H、13
C、14
C、15
N、18
O、17
O、31
P、32
P、35
S、18
F和36
Cl。本發明的同位素標記化合物通常可通過本領域技術人員已知的傳統技術或通過與所附實施例中描述的那些類似的方法使用適當的同位素標記的試劑代替非同位素標記的試劑來製備。這樣的化合物具有各種潛在用途,例如作為測定生物活性中的標樣和試劑。在穩定同位素的情況下,這樣的化合物具有有利地改變生物、藥理學或藥代動力學性質的潛力。 “前藥”是指本發明的化合物可以以前藥的形式給予。前藥是指在活體內的生理條件下例如通過氧化、還原、水解等(它們各自利用酶或在沒有酶參與下進行)轉化成本發明的生物活性化合物的衍生物。前藥的實例是下述化合物:其中本發明的化合物中的胺基被醯化、烷基化或磷酸化,例如二十烷醯基胺基、丙胺醯胺基、新戊醯氧基甲基胺基,或其中羥基被醯化、烷基化、磷酸化或轉化成硼酸鹽,例如乙醯氧基、棕櫚醯氧基、新戊醯氧基、琥珀醯氧基、富馬醯氧基、丙胺醯氧基,或其中羧基被酯化或醯胺化,或其中巰基與選擇性地向靶和/或向細胞的胞質溶膠遞送藥物的載體分子,例如肽形成二硫橋鍵。這些化合物可以由本發明的化合物根據公知方法製備。 “可藥用的鹽”或者“藥學上可接受的鹽”是指由可藥用的鹼或酸,包括無機鹼或酸和有機鹼或酸製成的鹽。在本發明的化合物含有一個或多個酸性或鹼性基團的情況下,本發明還包含它們相應的可藥用鹽。因此,含有酸性基團的本發明的化合物可以以鹽形式存在並可根據本發明使用,例如作為鹼金屬鹽、鹼土金屬鹽或作為銨鹽。這樣的鹽的更確切實例包括鈉鹽、鉀鹽、鈣鹽、鎂鹽或與胺或有機胺,例如乙胺、乙醇胺、三乙醇胺或胺基酸的鹽。含有鹼性基團的本發明的化合物可以以鹽形式存在並可根據本發明以它們與無機或有機酸的加成鹽的形式使用。合適的酸的實例包括鹽酸、氫溴酸、磷酸、硫酸、硝酸、甲磺酸、對甲苯磺酸、萘二磺酸、草酸、乙酸、酒石酸、乳酸、水楊酸、苯甲酸、甲酸、丙酸、特戊酸、丙二酸、琥珀酸、庚二酸、富馬酸、馬來酸、蘋果酸、胺基磺酸、苯基丙酸、葡糖酸、抗壞血酸、異煙酸、檸檬酸、己二酸和本領域技術人員已知的其它酸。如果本發明的化合物在分子中同時含有酸性和鹼性基團,本發明除所提到的鹽形式外還包括內鹽或內銨鹽。各鹽可通過本領域技術人員已知的常規方法獲得,例如通過在溶劑或分散劑中使這些與有機或無機酸或鹼接觸或通過與其它鹽陰離子交換或陽離子交換。 因此,在本申請中當提及“化合物”、“本發明化合物”或“本發明所述化合物”時,包括所有所述化合物形式,例如其前藥、穩定同位素衍生物、可藥用的鹽、異構體、內消旋體、外消旋體、對映異構體、非對映異構體及其混合物。 在本文中,術語“腫瘤”包括良性腫瘤和惡性腫瘤(例如癌症)。 在本文中,術語“癌症”包括FGFR參與其發生的各種惡性腫瘤,包括但不限於非小細胞肺癌、食管癌、黑色素瘤、橫紋肌肉瘤、腎細胞癌、多發性骨髓瘤、乳腺癌、卵巢癌、子宮內膜癌、宮頸癌、胃癌、結腸癌、膀胱癌、胰腺癌、肺癌、乳腺癌、前列腺癌和肝癌(例如肝細胞癌),更具體為肝癌、胃癌、非小細胞肺癌和膀胱癌。 在本文中,術語“炎症性疾病”是指FGFR參與其炎症發生的任何炎性疾病,例如骨關節炎。 在本文中,術語“治療有效量”是指包括可有效抑制FGFR的功能和/或治療或防止所述疾病的本發明化合物的量。 合成方法 本發明還提供製備所述化合物的方法。本發明通式(I)所述化合物的製備,可通過以下示例性方法和實施例完成,但這些方法和實施例不應以任何方式被認為是對本發明範圍的限制。也可通過本領域技術人員所知的合成技術合成本發明所述的化合物,或者綜合使用本領域已知方法和本發明所述方法。每步反應所得的產物用本領域已知的分離技術得到,包括但不限於萃取、過濾、蒸餾、結晶、色譜分離等。合成所需的起始原料和化學試劑可以根據文獻(可從SciFinder上查詢)常規合成或購買。 本發明通式(I)所述吡唑類化合物可按照方法A所述路線合成:1)起始物A1通過桑德邁爾(Sandmeyer)反應得到A2,也可被溴化得到A3,其中的R1
可以是-CN或酯(-COOR,其中R為烷基);2)A2或A3與前體X-L~N-P(其中X為離去基團、L~N-P為含有帶保護胺基的功能團、P為胺基的保護基)在鹼催化下發生取代反應生成A4,也可與一帶有羥基的前體(HO-L~N-P)通過光延反應(Mitsunobu反應)得到A4;3)當A4的R1
是-CN,在NaOH/H2
O2
條件下水解成醯胺A5;當A4的R1
是酯 (-COOR,其中R為烷基),先在鹼性條件(比如LiOH)下水解成羧酸,然後醯胺化得到A5;4)A5與炔通過Sonogashira偶聯得到A6;5)A6中胺基去保護得到A7;6)A7中的胺基被含有和激酶配體結合域內半胱胺酸殘基起反應的功能團的化學試劑(例如BrCN、烯丙醯氯等)衍生化得到目標化合物A8。 方法A:另外也可以按照方法B所述路線合成,在第二步引入吡唑NH保護基Q,在第五步脫保護得到共同的中間體B7,B7的吡唑NH再與含有帶保護胺基的不同前體反應取代反應,經過脫保護和衍生化,從而得到目標產物A8。 方法B:本發明通式(I)所述吡唑類化合物還可以按照方法C所述路線合成:1)A4先與炔通過Sonogashira偶聯得到C1;2)C1中的-NH2
在鹼催化下發生取代反應或通過還原胺化生成C2;3)C2的CN在NaOH/H2
O2
條件下水解成醯胺C3,在有些情況下需先用Boc保護-NH-,然後再水解;最後經過脫保護和衍生化,從而得到目標產物C5;C5也可通過對A8進行直接取代而得到。 方法C:實施例 化合物的結構是通過核磁共振(NMR)或質譜(MS)來確定的。NMR的測定是用Bruker AVANCE-400或Varian Oxford-300核磁儀,測定溶劑為氘代二甲基亞碸(DMSO-d6
)、氘代氯仿(CDC13
)、氘代甲醇(CD3
OD),內標為四甲基甲矽烷(TMS),化學位移是以10-6
(ppm)作為單位給出。 MS的測定用Agilent SQD(ESI)質譜儀(生產商:Agilent,型號:6120)。 HPLC的測定使用安捷倫1200 DAD高壓液相色譜儀(Sunfirc C18,150×4.6mm,5μm色譜柱)和Waters 2695-2996高壓液相色譜儀(Gimini C18 150×4.6mm,5μm色譜柱)。 薄層層析矽膠板使用青島海洋GF254矽膠板,薄層色譜法(TLC)使用的矽膠板採用的規格是0.15mm~0.2mm,薄層層析分離純化產品採用的規格是0.4mm~0.5mm矽膠板。 柱層析一般使用青島海洋200~300目矽膠為載體。 本發明的已知的起始原料可以採用或按照本領域已知的方法來合成,或可購買自ABCR GmbH&Co. KG、Acros Organics、Aldrich Chemical Company、韶遠化學科技(Accela ChemBio Inc.)、北京耦合化學品等公司。 實施例中如無特殊說明,反應均在氬氣氛或氮氣氛下進行。 氬氣氛或氮氣氛是指反應瓶連接一個約1L容積的氬氣或氮氣氣球。 氫氣氛是指反應瓶連接一個約1L容積的氫氣氣球。 加壓氫化反應使用北京佳維科創科技有限公司GCD-500G高純氫氣發生器和BLT-2000中壓氫化儀。 氫化反應通常抽真空,充入氫氣,反復操作3次。 微波反應使用CEM Discover-SP型微波反應器。 實施例中如無特殊說明,反應的溫度為室溫,溫度範圍是20°C - 30°C。 實施例中的反應進程的監測採用薄層色譜法(TLC),反應所使用的展開劑的體系有A:二氯甲烷和甲醇體系;B:石油醚和乙酸乙酯體系,溶劑的體積比根據化合物的極性不同而進行調節。 純化化合物採用的柱層析的洗脫劑的體系和薄層色譜法的展開劑的體系包括A:二氯甲烷和甲醇體系;B:石油醚和乙酸乙酯體系,溶劑的體積比根據化合物的極性不同而進行調節,也可以加入少量的三乙胺和酸性或鹼性試劑等進行調節。 實施例1 (S)-1-(1-丙烯醯基吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 (R)-3-(甲苯磺醯氧代)吡咯烷-1-甲酸第三丁基酯 將化合物(R)-3-羥基吡咯烷-1-甲酸第三丁基酯1a(3.5g,18.7mmol)、三乙胺(5.25mL,37.9mmol)、4-二甲胺基吡啶(0.35g,2.87mmol)溶於二氯甲烷(50mL),加入對甲苯磺醯氯(5.4g,28.1mmol),將反應混合物在室溫下攪拌12小時。加水(50mL)稀釋,用乙酸乙酯(100mL×3)萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 2/1),得到目標產物(R)-3-(甲苯磺醯氧代)吡咯烷-1-甲酸第三丁基酯1b(6.0g,黃色油狀物),產率:94%。 MS m/z (ESI): 364[M+23] 第二步 ((3,5-二甲氧基苯基)乙炔基)三甲基甲矽烷 將混合物1-溴-3,5-二甲氧基苯1c(6.51g,30mmol)、三甲基甲矽基乙炔(8.8g,90mmol)、二(三苯基膦)氯化鈀(1.05g,1.5mmol)、碘化亞銅(0.56g,3.0mmol)、三乙胺(80mL)和N,N-二甲基甲醯胺(150mL)加熱至80°C,並在氮氣保護下攪拌12小時。將反應混合物冷卻至室溫,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚),得到目標產物((3,5-二甲氧基苯基)乙炔基)三甲基甲矽烷1d(6.2g,棕色固體),產率:88%。 MS m/z (ESI): 235[M+1] 第三步 1-乙炔基-3,5-二甲氧基苯 將((3,5-二甲氧基苯基)乙炔基)三甲基甲矽烷1d(3.0g,12.8mmol)溶於甲醇(100mL),加入碳酸鉀(3.5g,25.6mmol),並在室溫下攪拌2小時。過濾,減壓濃縮濾液,殘餘物用矽膠柱層析純化(石油醚),得到目標產物1-乙炔基-3,5-二甲氧基苯1e(2g,黃色固體),產率:96%。 第四步 3-碘-1H-吡唑-4-甲酸乙酯 將3-胺基-1H-吡唑-4-甲酸乙酯1f(4.7g,30.3mmol)溶於濃鹽酸(12M,40mL),並冷卻至0°C,加入亞硝酸鈉(4.25g,60mmol)溶液(7.5mL),攪拌5分鐘,然後緩慢加入碘化鉀(12.5g,75mmol)溶液(17.5mL),繼續攪拌30分鐘。將上述反應混合物倒入飽和硫代硫酸鈉溶液(200mL),用乙酸乙酯(400mL×3)萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 2/1),得到目標產物3-碘-1H-吡唑-4-甲酸乙酯1g(6.4g,淺黃色固體),產率:80%。 MS m/z (ESI): 267[M+1] 第五步 (S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-碘-1H-吡唑-4-甲酸乙酯 將3-碘-1H-吡唑-4-甲酸乙酯1g(4.5g,17mmol)、(R)-3-(甲苯磺醯氧代)吡咯烷-1-甲酸第三丁基酯1b(6.1g,17.8mmol)、碳酸銫(7.5g,20.4mmol)和N,N-二甲基甲醯胺(50mL)的混合物加熱到80°C,攪拌3小時。將反應混合物冷卻至室溫,倒入飽和碳酸氫鈉溶液(200mL)中,用乙酸乙酯(300mL×3)萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 5/1到2/1),得到目標產物(S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-碘-1H-吡唑-4-甲酸乙酯1h(3.1g,淡黃色固體),產率:42%。 MS m/z (ESI): 458[M+23] 第六步 (S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲酸乙酯 將(S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-碘-1H-吡唑-4-甲酸乙酯1h(1g,2.25mmol)、1-乙炔基-3,5-二甲氧基苯1e(0.75g,4.5mmol)、二(三苯基膦)氯化鈀(175mg,0.25mmol)、碘化亞銅(95mg,0.5mmol)、三乙胺(12.5ml)和N,N-二甲基甲醯胺(12.5mL)的混合物加熱到80°C,攪拌12小時。將反應混合物冷卻至室溫,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 2/1),得到目標產物(S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲酸乙酯1i(0.95g,黃色油狀物),產率:90%。 MS m/z (ESI): 414[M+1-56] 第七步 (S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲酸 將(S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲酸乙酯1i(0.30g,0.64mmol)溶於四氫呋喃(3mL),加入氫氧化鈉溶液(4M,2mL),室溫攪拌1小時。將反應混合物減壓濃縮,殘餘物用鹽酸(6M,1mL)酸化,用乙酸乙酯(10mL×3)萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,得到目標產物(S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲酸1j(200mg,淡黃色油狀物),產率:71%。 MS m/z (ESI): 386[M+1-56] 第八步 (S)-3-(4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯 將(S)-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲酸1j(220mg,0.5mmol)、氯化銨(270mg,5mmol)、O-(7-氮雜苯並三唑-1-基)-N,N,N',N'-四甲基脲鎓六氟磷酸鹽(HATU)(228mg,0.6mmol)、N,N-二異丙基乙胺(129mg,1mmol)和N,N-二甲基甲醯胺(5mL)的反應混合物在室溫下攪拌過夜。加水稀釋,用乙酸乙酯萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用薄層矽膠製備色譜純化(二氯甲烷/甲醇 = 20/1),得到目標產物(S)-3-(4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯1k(140mg,白色固體),產率:64%。 MS m/z (ESI): 385[M+1-56] 第九步 (S)-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將(S)-3-(4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯1k(50mg,0.11mmol)、鹽酸(6M,5mL)和二氧六環(5mL)的反應混合物在室溫下攪拌1小時。在減壓下除去溶劑,得到目標產物(S)-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺1l(42mg,鹽酸鹽,粗品),產率:100%。 MS m/z (ESI): 341[M+1] 第十步 (S)-1-(1-丙烯醯基吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 向(S)-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽1l(30mg,0.08mmol)、N,N-二異丙基乙胺(31mg,0.24mmol)和四氫呋喃(15mL)的混合物中滴加丙烯醯氯(11mg,0.12mmol)的四氫呋喃(5mL)溶液,將反應混合物在室溫下攪拌30分鐘。加水(30mL)淬滅,用乙酸乙酯萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用薄層矽膠製備色譜純化(二氯甲烷/甲醇 = 20/1),得到目標產物(S)-1-(1-丙烯醯基吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺1(15mg,白色固體),產率:50%。 MS m/z (ESI): 395[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.10 (d,J
= 9.8 Hz, 1H), 6.96 (brs, 1H), 6.71 (d,J
= 2.3 Hz, 2H), 6.54 - 6.52 (m, 1H), 6.46 - 6.39 (m, 2H), 5.80 (brs, 1H), 5.76 - 5.72 (m, 1H), 5.01 - 4.92 (m, 1H), 4.13 - 4.00 (m, 2H), 3.90 - 3.75 (m, 8H), 2.62 - 2.44 (m, 2H)。 實施例2 1-(1-丙烯醯基呱啶-4-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲酸乙酯 將化合物3-碘-1H-吡唑-4-甲酸乙酯1g(2.01g,7.5mmol)溶於四氫呋喃(80mL)並冷卻至0°C,加入氫化鈉(60%礦物油分散體,0.42g,10.5mmol),室溫下攪拌1小時。向反應混合物中加入2-(三甲基甲矽烷基)乙氧基甲基氯(1.76g,10.5mmol),繼續攪拌15小時。向反應混合物中加入飽和食鹽水(100mL),用乙酸乙酯萃取(150mL×2)。有機相合併後用飽和食鹽水洗滌(100mL),在減壓下除去溶劑,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 5/1到1/2),得到目標產物3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲酸乙酯2a(2.6g,無色油狀物),產率:87%。 MS m/z (ESI): 397[M+1] 第二步 3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲酸 將化合物3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲酸乙酯2a(2.6g,6.5mmol)溶於四氫呋喃(40mL),加入氫氧化鋰水溶液(1M,13mL)並在室溫下攪拌15小時。加水(20mL)稀釋,用鹽酸(1M)酸化至pH = 4-5,用乙酸乙酯萃取(50mL×3)。有機相合併後用飽和食鹽水(100mL)洗滌,在減壓下除去溶劑,乾燥後得到目標產物3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲酸2b(2.03g,白色固體),產率:85%。 MS m/z (ESI): 391[M+23] 第三步 3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲醯胺 將化合物3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲酸2b(2.03g,5.5mmol)、二異丙基乙基胺(2.13g,16.5mmol)和N,N-二甲基甲醯胺(20mL)混合,依次加入O-(7-氮雜苯並三唑-1-基)-N,N,N',N'-四甲基脲鎓六氟磷酸鹽(HATU)(2.5g,6.6mmol)和1-羥基苯並三唑(890mg,6.6mmol),室溫下攪拌1小時後加入固體氯化銨(1.47g,27.5mmol),繼續攪拌15小時。向反應混合物物中加入飽和食鹽水(30mL),用乙酸乙酯萃取(50mL×3)。有機相合併後用飽和食鹽水洗滌(100mL),在減壓下除去溶劑後,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 20/1),得到目標產物3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲醯胺2c(2.3g,黃色油狀物),產率:100%。 MS m/z (ESI): 368[M+1] 第四步 3-((3,5-二甲氧基苯基)乙炔基)-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲醯胺 將化合物3-碘-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲醯胺2c(2.7g,7.3mmol)、1-乙炔基-3,5-二甲氧基苯(1.78g,11mmol)、三乙胺(2.2g,21.9mmol)、二(三苯基磷)氯化鈀(512mg,0.73mmol)和無水四氫呋喃(70mL)混合,除氧,在氬氣氣氛下室溫攪拌15小時。在減壓下除去溶劑,殘餘物用矽膠柱層析純化(乙酸乙酯/石油醚 = 10/1至2/1)得到目標產物3-((3,5-二甲氧基苯基)乙炔基)-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲醯胺2d(1.5g,黃色固體),產率:51%。 MS m/z (ESI): 402[M+1] 第五步 3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 將3-((3,5-二甲氧基苯基)乙炔基)-1-((2-(三甲基甲矽烷基)乙氧基)甲基)-1H-吡唑-4-甲醯胺2d(1.4g,3.5mmol)、乙二胺(525mg,8.75mmol)和四氫呋喃(30mL)混合,加入四丁基氟化銨的四氫呋喃溶液(1M,17.5mL,17.5mmol)。加熱回流15小時後,冷卻至室溫,加入飽和食鹽水(20mL),用乙酸乙酯(100mL×3)萃取。有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 20/1),得到目標產物3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺2e(600mg,白色固體),產率:63%。 MS m/z (ESI): 272[M+1] 第六步 4-(4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)呱啶-1-甲酸第三丁基酯 將化合物3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺2e(180mg,0.66mmol)、4-溴呱啶-1-甲酸第三丁基酯(264mg,0.99mmol)、碳酸鉀(182mg,1.32mmol)和N,N-二甲基甲醯胺(10mL)混合後,加熱到75°C,攪拌15小時。加入水(30mL),用乙酸乙酯(50mL×3)萃取。有機相合併後用飽和食鹽水洗滌,並用無水硫酸鈉乾燥。過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 20/1),得到目標產物4-(4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)呱啶-1-甲酸第三丁基酯2f(120mg,黃色固體,含區域異構體),產率:40%。 MS m/z (ESI): 477[M+23] 第七步 3-((3,5-二甲氧基苯基)乙炔基)-1-(呱啶-4-基)-1H-吡唑-4-甲醯胺 將化合物4-(4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)呱啶-1-甲酸第三丁基酯2f(120mg,0.26mmol,混合物)溶於乙醇(20mL),加入氯化氫的乙醇溶液(4M,1mL,4mmol),室溫下攪拌15小時。在減壓下除去溶劑,殘餘物溶於甲醇(20mL)後,用飽和碳酸氫鈉溶液調節至pH = 8-9。再次在減壓下除去溶劑後,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 10/1),得到目標產物3-((3,5-二甲氧基苯基)乙炔基)-1-(呱啶-4-基)-1H-吡唑-4-甲醯胺2g(25mg,白色固體),產率:27%。 MS m/z (ESI): 355[M+1] 第八步 1-(1-丙烯醯基呱啶-4-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 將化合物3-((3,5-二甲氧基苯基)乙炔基)-1-(呱啶-4-基)-1H-吡唑-4-甲醯胺2g(25mg,0.07mmol)、烯丙醯氯(10mg,0.11mmol)、固體碳酸氫鈉(18mg,0.21mmol)、水(2mL)和四氫呋喃(10mL)在0°C混合並在此溫度下攪拌10小時。用乙酸乙酯(20mL×3)萃取,有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,在減壓下除去溶劑,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 10/1),得到目標產物1-(1-丙烯醯基呱啶-4-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺2(17mg,白色固體),產率:60%。 MS m/z (ESI): 409[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.10 (s, 1H), 7.01 (brs, 1H), 6.72 (d,J
= 2.2 Hz, 2H), 6.62 (dd,J
= 16.8, 10.6 Hz, 1H), 6.55 (t,J
= 2.2 Hz, 1H), 6.33 (dd,J
= 16.8, 1.5 Hz, 1H), 5.80 (brs, 1H), 5.76 (dd,J
= 10.6, 1.6 Hz, 1H), 4.81 (brs, 1H), 4.40 (t,J
= 11.4 Hz, 1H), 4.18 (brs, 1H), 3.82 (s, 6H), 3.26 (brs, 1H), 2.89 (brs, 1H), 2.42 - 2.25 (m, 2H), 2.08 - 2.00 (m, 2H)。 參照實施例2的操作步驟合成實施例3-6: 實施例3 1-(1-丙烯醯基氮雜環丁烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 381[M+1]1
H NMR (400 MHz, DMSO-d6
) δ 8.43 (s, 1H), 7.30 (s, 2H), 6.73 (d,J
= 2.2 Hz, 2H), 6.60 (t,J
= 2.2 Hz, 1H), 6.38 (dd,J
= 17.0, 10.3 Hz, 1H), 6.16 (dd,J
= 17.0, 2.1 Hz, 1H), 5.73 (dd,J
= 10.3, 2.1 Hz, 1H), 5.41 - 5.28 (m, 1H), 4.71 (t,J
= 8.6 Hz, 1H), 4.50 (dd,J
= 9.2, 4.9 Hz, 1H), 4.46 - 4.36 (m, 1H), 4.20 (dd,J
= 10.7, 4.8 Hz, 1H), 3.78 (s, 6H)。 實施例4 1-((1-丙烯醯基呱啶-4-基)甲基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 423[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.00 (s, 1H), 6.98 (brs, 1H), 6.72 (s, 2H), 6.61 - 6.54 (m, 2H), 6.28 (d,J
= 16.8 Hz, 1H), 5.87 (brs, 1H), 5.70 (d,J
= 10.5 Hz, 1H), 4.72 (brs, 1H), 4.04 (brs, 3H), 3.82 (s, 6H), 3.05 (brs, 1H), 2.64 (brs, 1H), 2.27 (brs, 1H), 1.69 (brs, 2H), 1.24 (brs, 2H)。 實施例5 1-(4-丙烯醯基胺基環己基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 423[M+1]1
H NMR (400 MHz, CD3
OD) δ 8.25 (s, 1H), 6.77 (d,J
= 2.3 Hz, 2H), 6.58 (t,J
= 2.3 Hz, 1H), 6.38 (dd,J
= 17.1, 10.0 Hz, 1H), 6.26 (dd,J
= 17.1, 2.0 Hz, 1H), 5.68 (dd,J
= 10.1, 2.0 Hz, 1H), 4.38 - 4.33 (m, 1H), 4.13 - 4.11 (m, 1H), 3.82 (s, 6H), 2.28 - 2.18 (m, 2H), 2.07 - 2.02 (m, 2H), 1.96 - 1.80 (m, 4H)。 實施例6 3-((3,5-二甲氧基苯基)乙炔基)-1-(2-(N-甲基丙烯醯基胺基)乙基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 383[M+1]1
H NMR (300 MHz, DMSO-d6
) δ 8.24 (s, 1H), 7.10 - 6.90 (m, 2H), 6.76 (s, 2H), 6.69 - 6.54 (m, 2H), 6.07 (d,J
= 16.5 Hz, 1H), 5.64 (d,J
= 9.8 Hz, 1H), 4.37 (t,J
= 5.7 Hz, 2H), 3.89 - 3.80 (m, 8H), 2.94 (s, 3H)。 實施例7 (S)-1-(1-丙烯醯基吡咯烷-3-基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 5-胺基-3-溴-1H-吡唑-4-甲腈 將化合物5-胺基-1H-吡唑-4-甲腈7a(20g,185mmol)溶於N,N-二甲基甲醯胺(200mL),冷卻至0°C,分批加入N-溴代丁二醯亞胺(34g,190mmol),升至室溫攪拌2小時。將反應液倒入亞硫酸鈉溶液中,用乙酸乙酯(200mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 20/1),得到目標產物5-胺基-3-溴-1H-吡唑-4-甲腈7b(32g,黃色固體),產率:93%。 MS m/z (ESI): 187/189[M+1] 第二步 (S)-3-(5-胺基-3-溴-4-氰基-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯 將5-胺基-3-溴-1H-吡唑-4-甲腈7b(10g,53.8mmol)、3-(甲苯磺醯氧代)吡咯烷-1-甲酸第三丁基酯(22g,64.5mmol)、碳酸銫(58g,107.6mmol)和乙腈(250mL)的混合物加熱到90°C反應4小時。冷卻至室溫,過濾,濾餅用二氯甲烷洗滌,濾液合併後減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 5/1),得到目標產物(S)-3-(5-胺基-3-溴-4-氰基-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7c(5g,黃色油狀物),產率:26%。 MS m/z (ESI): 300/302[M+1-56] 第三步 (S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯 將(S)-3-(5-胺基-3-溴-4-氰基-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7c(5g,14.1mmol)、碘化亞銅(0.6g,2.8mmol)、三乙胺(9mL)、[1,1'-雙(二苯基膦基)二茂鐵]二氯化鈀(2g,2.8mmol)和N,N-二甲基甲醯胺(150mL)的混合物在氬氣保護下加熱至80°C,分批加入1-乙炔基-3,5-二甲氧基苯(14g,84.5mmol),攪拌2小時。冷卻至室溫,將反應液倒入水中,用乙酸乙酯(200mL×3)萃取。有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 5/1),得到目標產物(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(5g,棕色油狀物),產率:81%。 MS m/z (ESI): 382[M+1-56] 第四步 (S)-3-(5-胺基-4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯 將(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(5g,11.4mmol)、氫氧化鈉(1.5g,37.5mmol,溶於2mL水)、乙醇(50mL)和二甲基亞碸(10mL)的混合物冷卻至0°C,加入雙氧水(20mL),室溫攪拌2小時。將反應液倒入亞硫酸鈉溶液中,用乙酸乙酯(100mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 1/1),得到目標產物(S)-3-(5-胺基-4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7e(5g,棕色油狀物),產率:96%。 MS m/z (ESI): 400[M+1-56] 第五步 (S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將化合物(S)-3-(5-胺基-4-胺甲醯基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7e(5g,11mmol)溶於二氯甲烷(100mL)中,加入三氟乙酸(15mL),室溫攪拌2小時。減壓濃縮,得到目標產物(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(7.1g,棕色油狀物,三氟乙酸鹽,粗品),產率:>100%,產物不經純化直接用於下一步反應。 MS m/z (ESI): 356[M+1] 第六步 (S)-1-(1-丙烯醯基吡咯烷-3-基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 將化合物(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(7.1g,11mmol,三氟乙酸鹽,粗品)溶於四氫呋喃(50mL)中,冷卻至0°C,先後加入碳酸氫鈉飽和溶液(20mL)和丙烯醯氯(900mg,10mmol),攪拌30分鐘。將反應液倒入水(100mL)中,並用二氯甲烷(100mL×3)萃取。有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 1/2),得到目標產物(S)-1-(1-丙烯醯基吡咯烷-3-基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺7(1.9g,白色固體),產率:42%。 MS m/z (ESI): 410[M+1]1
H NMR (400 MHz, DMSO-d6
) δ 7.18 (brs, 1H), 6.75 (d,J
= 2.3 Hz, 2H), 6.69 - 6.55 (m, 3H), 6.20 - 6.14 (m, 1H), 5.72 - 5.67 (m, 1H), 5.03 - 4.91 (m, 1H), 4.01 - 3.96 (m, 1H), 3.84 - 3.70 (m, 7H), 3.66 - 3.60 (m, 1H), 3.55 - 3.48 (m, 1H), 2.36 - 2.21 (m, 2H)。 實施例8 (S,E)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(4-(二甲胺基)丁-2-烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺 第一步 (S,E)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(4-(二甲胺基)丁-2-烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將(E)-4-(二甲胺基)丁-2-烯酸(23mg,0.14mmol)、O-(7-氮雜苯並三唑-1-基)-N,N,N',N'-四甲基脲鎓六氟磷酸鹽(HATU)(64mg,0.17mmol)、(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(50mg,0.14mmol)、N,N-二異丙基乙胺(2mL)和二氯甲烷(3mL)的反應混合物在室溫下攪拌1小時。將反應液倒入水中,用二氯甲烷(20mL×3)萃取。有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用高效液相製備色譜純化,得到目標產物(S,E)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(4-(二甲胺基)丁-2-烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺8(2.4mg,白色固體,甲酸鹽),產率:4%。 MS m/z (ESI): 467[M+1]1
H NMR (400 MHz, DMSO-d6
) δ 8.27 (brs, 1H), 7.20 (brs, 1H), 6.75 (d,J
= 2.3 Hz, 2H), 6.70 - 6.61 (m, 3H), 6.44 - 6.35 (m, 1H), 5.01 - 4.93 (m, 1H), 4.01 - 3.93 (m,1H), 3.77 (s, 6H), 3.74 - 3.64 (m, 3H), 3.06 - 3.03 (m, 2H), 2.38 - 2.24 (m, 2H), 2.17 - 2.15 (m, 6H)。 實施例9 (S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(2-氟丙烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺 第一步 (S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(2-氟丙烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將化合物(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(50mg,0.14mmol)和2-氟丙烯酸(15mg,0.17mmol)溶於二氯甲烷,加入N,N-二異丙基乙胺(54mg,0.42mmol)和O-(7-氮雜苯並三唑-1-基)-N,N,N',N'-四甲基脲鎓六氟磷酸鹽(HATU)(69mg,0.18mmol),室溫攪拌2小時。加水(10mL)稀釋反應混合物,用二氯甲烷(10mL×3)萃取,有機相合併後減壓濃縮。殘餘物用薄層矽膠製備色譜純化(二氯甲烷/甲醇 = 20/1),得到目標產物(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(2-氟丙烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺9(3.6mg,白色固體),產率:6%。 MS m/z (ESI): 428 [M+1]1
H NMR (400 MHz, CD3
OD) δ 6.62 (t,J
= 2.5 Hz, 2H), 6.47 (t,J
= 2.3 Hz, 1H), 5.39 (dd,J
= 47.2, 3.5 Hz, 1H), 5.16 (ddd,J
= 16.6, 5.7, 3.5 Hz, 1H), 4.86 - 4.81 (m, 1H), 4.02 - 3.91 (m, 2H), 3.87 - 3.72 (m, 2H), 3.71 (s, 6H), 2.34 - 2.23 (m, 2H)。 實施例10 (S)-5-胺基-1-(1-(丁-2-炔醯基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 (S)-5-胺基-1-(1-(丁-2-炔醯基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 將(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(50mg,0.14mmol)和2-丁炔酸(14mg,0.17mmol)溶於二氯甲烷中,加入N,N-二異丙基乙胺(54mg,0.42mmol)和O-(7-氮雜苯並三唑-1-基)-N,N,N',N'-四甲基脲鎓六氟磷酸鹽(HATU)(69mg,0.18mmol),室溫攪拌2小時。反應混合物用水(10mL)稀釋,用二氯甲烷(10mL×3)萃取,有機相合併後減壓濃縮。殘餘物用薄層矽膠製備色譜純化(二氯甲烷/甲醇 = 20/1),得到目標產物(S)-5-胺基-1-(1-(丁-2-炔醯基)吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺10(5.1mg,淡黃色固體),產率:9%。 MS m/z (ESI): 422 [M+1]1
H NMR (400 MHz, CD3
OD) δ 6.62 (t,J
= 2.1 Hz, 2H), 6.47 (t,J
= 2.2 Hz, 1H), 4.85 - 4.81 (m, 1H), 4.01 - 3.86 (m, 2H), 3.77 - 3.62 (m, 7.5H), 3.54 - 3.46 (m, 0.5H), 2.32 - 2.27 (m, 2H), 1.95 - 1.93 (m, 3H)。 實施例11 (S,E)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(4-甲氧基丁-2-烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺 第一步 (E)-4-溴丁-2-烯酸 將(E)-4-溴丁-2-烯酸甲酯11a(3g,16.8mmol)、氫氧化鋰一水合物(1.1g,25.3mmol)、四氫呋喃(50mL)和水(50mL)在0°C混合並繼續攪拌2小時。反應結束後,用石油醚洗去四氫呋喃,水相用2M鹽酸調節至pH = 1,然後用乙酸乙酯(100mL×2)萃取。有機相合併後在減壓下除去溶劑,得到目標產物(E)-4-溴丁-2-烯酸11b(2.3g,黃色油狀物),產率:83%。 MS m/z (ESI): 163[M-1] 第二步 (E)-4-甲氧基丁-2-烯酸 將化合物(E)-4-溴丁-2-烯酸11b(100mg,0.61mmmol )溶於甲醇(5mL)後,加入甲醇鈉的甲醇溶液(30%,0.55mL,3.05mmol)並攪拌15小時。反應混合物在減壓下除去溶劑後溶於水,並用稀鹽酸調節至pH=1,然後用二氯甲烷萃取(10mL×3)。有機相合併後在減壓下除去溶劑,得到目標產物(E)-4-甲氧基丁-2-烯酸11c(50mg,黃色油狀物),產率:71%。1
H NMR (400 MHz, CDCl3
) δ 7.13 - 7.03 (m, 1H), 6.15 - 6.07 (m, 1H), 4.18 - 4.11 (m, 2H), 3.48 - 3.38 (s, 3H)。 第三步 (S,E)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(4-甲氧基丁-2-烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將化合物(E)-4-甲氧基丁-2-烯酸11c(22mg,0.19mmol)、二異丙基乙基胺(67mg,0.52mmol)、(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(50mg,0.13mmol)、2-(7-氧化苯並三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(72mg,0.19mmol)和N,N-二甲基甲醯胺(10mL)混合並攪拌2小時。在減壓下除去溶劑,殘餘物溶於乙酸乙酯(30mL)後依次用水和飽和食鹽水洗滌。在減壓下除去溶劑,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 20/1)得到目標產物(S,E)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(1-(4-甲氧基丁-2-烯醯基)吡咯烷-3-基)-1H-吡唑-4-甲醯胺11(30mg,白色固體),產率:51%。 MS m/z (ESI): 454[M+1]1
H NMR (400 MHz, CDCl3
) δ 6.98 (d,J
= 15.3 Hz, 1H), 6.86 (brs, 1H), 6.72 (d,J
= 2.1 Hz, 2H), 6.54 (s, 1H), 6.39 (dd,J
= 27.7, 16.0 Hz, 1H), 5.54 (brs, 1H), 4.73 - 4.70 (m, 1H), 4.14 - 4.12 (m, 2H), 4.05 - 4.00 (m, 2H), 3.95 - 3.93 (m, 1H), 3.82 (s, 6H), 3.77 - 3.68 (m, 1H), 3.43 (d,J
= 10.1 Hz, 3H), 2.72 (brs, 0.5H), 2.54 (brs, 0.5H), 2.43 - 2.35 (m, 1H)。 實施例12 (S)-5-胺基-1-(1-氰基吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 (S)-5-胺基-1-(1-氰基吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺 將化合物(S)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺7f(50mg,0.14mmol)溶於四氫呋喃(2mL),加入三乙胺(1mL),冷卻至0°C,加入溴化氰(17mg,0.15mmol),0°C攪拌2小時,升至室溫,繼續攪拌2小時。反應混合物減壓濃縮,殘餘物用薄層矽膠製備色譜純化(二氯甲烷/甲醇 = 15/1),得到目標產物(S)-5-胺基-1-(1-氰基吡咯烷-3-基)-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺12(18mg,白色固體),產率:34%。 MS m/z (ESI): 381[M+1]1
H NMR (400 MHz, CDCl3
) δ 6.77 (brs, 1H), 6.68 (d,J
= 1.9 Hz, 2H), 6.50 (s, 1H), 5.75 (s, 2H), 5.67 (brs, 1H), 4.79 - 4.73 (m, 1H), 3.84 - 3.73 (m, 9H), 3.61 - 3.53 (m, 1H), 2.53 - 2.43 (m, 1H), 2.37 - 2.26 (m, 1H)。 參照實施例7的操作步驟合成了實施例13-16: 實施例13 (R)-1-(1-丙烯醯基吡咯烷-3-基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 410[M+1]1
H NMR (400 MHz, CD3
OD) δ 6.73 (d,J
= 1.9 Hz, 2H), 6.71 - 6.60 (m, 1H), 6.58 (brs, 1H), 6.32 (dd,J
= 16.8, 1.7 Hz, 1H), 5.84 - 5.74 (m, 1H), 5.04 - 4.91 (m, 1H), 4.09 (m, 0.5H), 3.98 (td,J
= 11.1, 4.0 Hz, 1H), 3.91 (dd,J
= 7.8, 5.6 Hz, 1H), 3.86 (dd,J
= 9.9, 4.4 Hz, 1H), 3.81 (s, 6H), 3.73 - 3.63 (m, 0.5H), 2.47 (dd,J
= 13.2, 6.7 Hz, 1H), 2.38 (dd,J
= 13.6, 7.0 Hz, 1H)。 實施例14 1-(1-丙烯醯基氮雜環丁烷-3-基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 396[M+1]1
H NMR (400 MHz, CD3
OD) δ 6.75 (d,J
= 2.3 Hz, 2H), 6.60 (t,J
= 2.2 Hz, 1H), 6.45 - 6.28 (m, 2H), 5.80 (dd,J
= 10.1, 2.1 Hz, 1H), 5.29 - 5.21 (m, 1H), 4.79 - 4.64 (m, 2H), 4.54 - 4.47 (m, 1H), 4.46 - 4.39 (m, 1H), 3.82 (s, 6H)。 實施例15 1-(1-丙烯醯基呱啶-4-基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 424[M+1]1
H NMR (400 MHz, CD3
OD) δ 6.88 - 6.78 (m, 1H), 6.73 (d,J
= 2.2 Hz, 2H), 6.58 (t,J
= 2.2 Hz, 1H), 6.24 (dd,J
= 16.8, 1.7 Hz, 1H), 5.78 (dd,J
= 10.7, 1.7 Hz, 1H), 4.73 (d,J
= 13.2 Hz, 1H), 4.47 - 4.36 (m, 1H), 4.30 (d,J
= 13.3 Hz, 1H), 3.81 (s, 6H), 3.32 - 3.24 (m, 1H), 2.91 (t,J
= 9.9 Hz, 1H), 2.02 (d, J = 4.5 Hz, 4H)。 實施例16 1-((1-丙烯醯基吡咯烷-3-基)甲基)-5-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-4-甲醯胺MS m/z (ESI): 424[M+1]1
H NMR (400 MHz, CD3
OD) δ 6.73 (s, 2H), 6.66 - 6.55 (m, 2H), 6.28 (d,J
= 16.7 Hz, 1H), 5.75 (d,J
= 10.4 Hz, 1H), 4.13 - 3.99 (m, 2H), 3.82 (s, 6H), 3.78 - 3.61 (m, 2H), 3.48 (dd,J
= 14.8, 7.4 Hz, 1H), 3.39 - 3.34 (m, 1H), 2.94 - 2.75 (m, 1H), 2.20 - 2.02 (m, 1H), 1.94 - 1.71 (m, 1H)。 實施例17 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((2-氟-3,5-二甲氧苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 1-乙炔基-2-氟-3,5-二甲氧基苯 將混合物1-乙炔基-3,5-二甲氧基苯1e(2g,12.3mmol )溶於乙腈(15mL)中,降溫至0°C,並分批加入1-氯甲基-4-氟-1,4-重氮化二環2.2.2辛烷雙(四氟硼酸)鹽(6.6g,18.5mmol),然後室溫攪拌過夜。將反應液倒入水(50mL)中,並用二氯甲烷(30mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 30/1),得到目標產物1-乙炔基-2-氟-3,5-二甲氧基苯17a(800mg,黃色固體),產率:36%。1
H NMR (400 MHz, CDCl3
) δ 6.46 (dd,J
= 6.9, 2.9 Hz, 1H), 6.41 (dd,J
= 4.5, 3.0 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 3.22 (s, 1H)。 參照實施例7第一至第六步的操作步驟合成實施例17,但在第三步中用1-乙炔基-2-氟-3,5-二甲氧基苯取代1-乙炔基-3,5-二甲氧基苯。 MS m/z (ESI): 428[M+1]1
H NMR (400 MHz, CDCl3
) δ 7.00 (brs, 1H), 6.59 - 6.57 (m, 2H), 6.49 - 6.39 (m, 2H), 5.74 - 5.70 (m, 1H), 5.52 (d,J
= 8.5 Hz, 2H), 5.35 (brs, 1H), 4.73 - 4.64 (m, 1H), 4.07 - 3.90 (m, 3H), 3.88 (s, 3H), 3.78 (d,J
= 5.3 Hz, 3H), 3.75 - 3.67 (m, 1H), 2.72 - 2.67 (m, 0.5H), 2.54 - 2.31 (m, 1.5H)。 實施例18 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((5-氯-2-氟苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 ((2-氟-5-氯苯基)乙炔基)三甲基矽烷 將2-氟-5-氯溴苯18a(11.0g,52.8mmol)、乙炔基三甲基矽烷(7.7g,79mmol)和三乙胺(60mL)混合,然後加入碘化亞銅(100mg,0.53mmol)和二三苯基膦氯化鈀(1.86g,2.65mmol)。反應混合物在氮氣保護氣氛下加熱到80°C並繼續攪拌4小時。反應結束後,減壓脫溶,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 100/1)得到目標產物((2-氟-5-氯苯基)乙炔基)三甲基矽烷18b(11.0g,黃色油狀物),產率:90%。1
H NMR (400 MHz, CDCl3
) δ 7.45 (dd,J
= 6.0, 2.7 Hz, 1H), 7.28 - 7.22 (m, 1H), 7.02 (t,J
= 8.8 Hz, 1H), 0.29 (s, 9H)。 第二步 4-氯-2-乙炔基-1-氟苯 將((2-氟-5-氯苯基)乙炔基)三甲基矽烷18b(11.0g,48mmol)、碳酸鉀(8.1g,58mmol)、二氯甲烷(80mL)和甲醇(40mL)混合後,室溫下攪拌18小時。反應結束後,減壓脫溶,殘餘物用矽膠柱層析(石油醚/乙酸乙酯 = 100/1)純化得到目標產物4-氯-2-乙炔基-1-氟苯18c(5.5g,黃色固體),產率:74%。1
H NMR (400 MHz, CDCl3
) δ 7.45 (dd,J
= 6.0, 2.7 Hz, 1H), 7.31 - 7.27 (m, 1H), 7.04 (t,J
= 8.0, 1H), 3.35 (s, 1H)。 參照實施例7第一至第六步的操作步驟合成實施例18,但在第三步中用4-氯-2-乙炔基-1-氟苯取代1-乙炔基-3,5-二甲氧基苯。 MS m/z (ESI): 402[M+1]1
H NMR (400 MHz, CD3
OD) δ 7.62 - 7.61 (m, 1H), 7.47 - 7.45 (m, 1H), 7.24 (t,J
= 9.0 Hz, 1H), 6.69 - 6.56 (m, 1H), 6.30 (d,J
= 16.8 Hz, 1H), 5.77 (t,J
= 9.2 Hz, 1H), 5.02 - 1.91 (m, 1H), 4.09 - 3.95 (m, 2H), 3.84 - 3.78 (m, 2H), 2.46 (dd,J
= 13.1, 6.6 Hz, 1H), 2.37 (dd,J
= 13.6, 6.9 Hz, 1H)。 實施例19 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 4-氯-3-溴苯甲酸甲酯 將4-氯-3-溴苯甲酸19a(2g,8.5mmol)溶於甲醇(400mL)並冷卻至0°C,然後逐滴加入乙醯氯(2.3g,30mmol)並繼續攪拌18小時。反應結束後減壓脫溶,殘餘物用矽膠柱層析(石油醚/乙酸乙酯 = 10/1)純化得到目標產物4-氯-3-溴苯甲酸甲酯19b(1.2g,黃色固體),產率:57%。1
H NMR (400 MHz, CDCl3
) δ 8.31 (d,J
= 1.9 Hz, 1H), 7.93 (dd,J
= 8.3, 1.9 Hz, 1H), 7.55 (d,J
= 8.3 Hz, 1H), 3.95 (s, 3H)。 第二步 4-氯-3-((三甲基甲矽烷基)乙炔基)苯甲酸甲酯 將化合物4-氯-3-溴苯甲酸甲酯19b(1.2g,4.8mmol)、三甲基矽基乙炔(0.95g,9.7mmol)、醋酸鈀(108mg,0.48mmol)、三苯基膦(254mg,0.97mmol)、碘化亞銅(185mg,0.97mmol)和三乙胺(25mL)在封管中混合並在100°C下加熱並攪拌15小時。反應完成後減壓脫溶,殘餘物用柱層析矽膠色譜(石油醚/乙酸乙酯 = 10/1)純化得到目標產物4-氯-3-((三甲基甲矽烷基)乙炔基)苯甲酸甲酯19c(1g,黃色固體),產率:78%。1
H NMR (400 MHz, CDCl3
) δ 8.19 (d,J
= 2.0 Hz, 1H), 7.91 (dd,J
= 8.4, 2.1 Hz, 1H), 7.48 (d,J
= 8.4 Hz, 1H), 3.94 (s, 3H), 0.30 (s, 9H)。 第三步 4-氯-3-乙炔基苯甲酸甲酯 將4-氯-3-((三甲基甲矽烷基)乙炔基)苯甲酸甲酯19c(1g,3.76mmol)溶於甲醇(20mL),然後加入碳酸鉀(1.04g,7.52mmol)。室溫攪拌1小時後,減壓脫溶。殘餘物用水洗滌並過濾得到目標產品4-氯-3-乙炔基苯甲酸甲酯19d(380mg,黃色固體),產率:52%。1
H NMR (400 MHz, CDCl3
) δ 8.23 (d,J
= 2.1 Hz, 1H), 7.96 (dd,J
= 8.4, 2.0 Hz, 1H), 7.51 (d,J
= 8.4 Hz, 1H), 3.95 (s, 3H), 3.44 (s, 1H)。 第四步 (S)-3-(5-胺基-3-溴-4-胺基甲醯-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將化合物(S)-3-(5-胺基-3-溴-4-氰基-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯7c(2.20g,6.2mmol)、氫氧化鈉水溶液(0.5M,12.4mL,6.2mmol)、過氧化氫水溶液(30%,15mL)和二甲基亞碸(30mL)混合。室溫攪拌2小時後,反應物用飽和食鹽水(50mL)稀釋,並用乙酸乙酯(50mL×3)萃取。有機相合併後減壓脫溶,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 100/1至20/1)得到目標產物(S)-3-(5-胺基-3-溴-4-胺基甲醯-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19e(1.92g,淡黃色固體),產率:83%。 MS m/z (ESI): 374[M+1] 第五步 (S)-3-(5-胺基-4-胺基甲醯-3-((2-氯-5-(甲酯基<甲氧羰基>)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(5-胺基-3-溴-4-胺基甲醯-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19e(770mg,2.1mmol)、三乙胺(6mL)、1,1'-雙二苯基膦二茂鐵二氯化鈀(307mg,0.42mmol)、碘化亞銅(80mg,0.42mmol)和N,N-二甲基甲醯胺(20mL)混合,除氧,在氬氣氣氛下加熱至90°C,然後逐滴加入4-氯-3-乙炔基苯甲酸甲酯19d(3.20g,16.5mmol)的N,N-二甲基甲醯胺(2mL)溶液,並繼續攪拌12小時。減壓脫溶,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 20/1)得到目標產物(S)-3-(5-胺基-4-胺基甲醯-3-((2-氯-5-(甲酯基<甲氧羰基>)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19f(420mg,黃色固體),產率:41%。 MS m/z (ESI): 488[M+1] 第六步 (S)-3-((5-胺基-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-4-胺基甲醯-1H-吡唑-3-基)乙炔基)-4-氯苯甲酸 將(S)-3-(5-胺基-4-胺基甲醯-3-((2-氯-5-(甲酯基<甲氧羰基>)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19f(100mg,0.2mmol)溶於甲醇(4mL)和水(4mL)的混合溶劑中,然後加入氫氧化鈉(25mg,0.61mmol)並繼續攪拌2小時。反應完成後,在減壓條件下除去有機溶劑。殘餘物用鹽酸(1M)調節至pH = 4至5,然後用乙酸乙酯萃取(30mL×2)。有機相合併後用飽和食鹽水洗滌,無水硫酸鈉乾燥後過濾。濾液減壓脫溶得到目標產物(S)-3-((5-胺基-1-(1-(第三丁氧基羰基)吡咯烷-3-基)-4-胺基甲醯-1H-吡唑-3-基)乙炔基)-4-氯苯甲酸19g(80mg,棕色固體),產率:84%。 MS m/z (ESI): 418[M+H-56] 第七步 (S)-3-(5-胺基-4-胺基甲醯-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-((5-胺基-1-(1-(叔-丁氧基羰基)吡咯烷-3-基)-4-胺基甲醯-1H-吡唑-3-基)乙炔基)-4-氯苯甲酸19g(80mg,0.17mmol)溶於N,N-二甲基甲醯胺(2.5mL)中,然後依次加入甲胺鹽酸鹽(34mg,0.50mmol)、二異丙基乙基胺(129mg,1mmol)和2-(7-氧化苯並三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(64mg,0.17mmol)。反應物室溫攪拌2小時後用水淬滅,然後用乙酸乙酯(20mL×3)萃取。有機相合併後減壓脫溶,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 100/1至0/1)得到目標產物(S)-3-(5-胺基-4-胺基甲醯-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19h(41mg,棕色固體),產率:50%。 MS m/z (ESI): 387[M+H-Boc] 第八步 (S)-5-胺基-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽 將(S)-3-(5-胺基-4-胺基甲醯-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19h(40mg,0.08mmol)溶於乙酸乙酯(5mL)中,然後加入氯化氫的乙醇溶液(33%,3mL)並在室溫下攪拌1小時。反應完成後,減壓脫溶,得到目標產物(S)-5-胺基-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽19i(40mg,粗品,棕色固體),該產品未經進一步純化,直接用於下一步反應。 MS m/z (ESI): 387[M+H] 第九步 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-4-甲醯胺 將化合物(S)-5-胺基-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽19i(40mg,0.08mmol,粗品)、烯丙醯氯(7.5mg,0.08mmol)、碳酸鉀水溶液(0.4M,1.0mL,0.4mmol)和四氫呋喃(5mL)在0°C混合並在此溫度下攪拌0.5小時。用乙酸乙酯(20mL×2)萃取,有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,減壓脫溶,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 100/1至10/1),得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((2-氯-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-4-甲醯胺19(18mg,白色固體),產率:兩步51%。 MS m/z (ESI): 441[M+H]1
H NMR (400 MHz, DMSO-d6
) δ 8.65 (s, 1H), 8.18 (s, 1H), 7.90 (d,J
= 8.0 Hz, 1H), 7.72 (d,J
= 8.3 Hz, 1H),7.43 (s, 1H), 6.70 - 6.62 (m, 4H), 6.19 - 6.15 (m, 1H), 5.70 (t,J
= 10.2 Hz, 1H), 5.03 - 4.94 (m, 1H), 3.80 - 3.54 (m, 4H), 2.78 (d,J
= 3.8 Hz, 3H), 2.36 - 2.25 (m, 2H)。 實施例20 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 3-溴-5-甲氧基-N-甲基苯醯胺 將3-溴-5-甲氧基苯甲酸20a(500mg,2.17mmol)溶於N,N-二甲基甲醯胺(15mL)中,然後依次加入甲胺鹽酸鹽(291mg,4.35mmol)、二異丙基乙基胺(1.12g,8.68mmol)和2-(7-氧化苯並三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.24g,3.26mmol)。反應物室溫攪拌2小時後用水淬滅,減壓脫溶,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 1/1)得到目標產物3-溴-5-甲氧基-N-甲基苯醯胺20b(500mg,白色固體),產率:95%。 MS m/z (ESI): 244[M+H] 第二步 3-甲氧基-N-甲基-5-((三甲基甲矽烷基)乙炔基)苯醯胺 將化合物3-溴-5-甲氧基-N-甲基苯醯胺20b(500mg,2.1mmol)、三甲基矽基乙炔(302mg,3.1mmol)、醋酸鈀(47mg,0.21mmol)、三苯基膦(110mg,0.42mmol)、碘化亞銅(80mg,0.42mmol)和三乙胺(20mL)在封管中混合,加熱至100°C,攪拌15小時。反應完成後,減壓脫溶,殘餘物用矽膠柱層析(石油醚/乙酸乙酯 = 1/1)純化,得到目標產物3-甲氧基-N-甲基-5-((三甲基甲矽烷基)乙炔基)苯醯胺20c(220mg,黃色固體),產率:41%。 MS m/z (ESI): 262[M+H] 第三步 3-乙炔基-5-甲氧基-N-甲基苯醯胺 將3-甲氧基-N-甲基-5-((三甲基甲矽烷基)乙炔基)苯醯胺20c(220mg,0.84mmol)溶於甲醇(8mL),然後加入碳酸鉀(233mg,1.68mmol)。室溫攪拌1小時後,減壓脫溶。殘餘物用矽膠柱層析(石油醚/乙酸乙酯 = 1/1)純化,得到目標產物3-乙炔基-5-甲氧基-N-甲基苯醯胺20d(140mg,淡黃色固體),產率:88%。 MS m/z (ESI): 190[M+H] 第四步 (S)-3-(5-胺基-4-胺基甲醯-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(5-胺基-3-溴-4-胺基甲醯-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯19e(329mg,0.88mmol)、三乙胺(2mL)、1,1'-雙二苯基膦二茂鐵二氯化鈀(129mg,0.2mmol)、碘化亞銅(34mg,0.18mmol)和N,N-二甲基甲醯胺(8mL)混合,除氧,在氬氣氣氛下加熱至90°C。然後逐滴加入3-乙炔基-5-甲氧基-N-甲基苯醯胺20d(1.00g,5.3mmol)的N,N-二甲基甲醯胺(2mL)溶液並繼續攪拌12小時。減壓脫溶,殘餘物用矽膠柱層析(二氯甲烷/甲醇 = 20/1)純化,得到目標產物(S)-3-(5-胺基-4-胺基甲醯-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯20e(400mg,粗品,棕色固體)。 MS m/z (ESI): 383[M+H-100] 第五步 (S)-5-胺基-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽 將(S)-3-(5-胺基-4-胺基甲醯-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯20e(400mg,粗品)溶於二氯甲烷(5mL)中,然後加入氯化氫的乙醇溶液(30%,3mL),並在室溫下攪拌1小時。反應完成後,減壓脫溶,得到目標產物(S)-5-胺基-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽20f(300mg,粗品,棕色固體)。產品不經純化,直接用於下一步反應。 MS m/z (ESI): 383[M+H] 第六步 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-4-甲醯胺 將化合物(S)-5-胺基-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽20f(150mg,0.39mmol,粗品)、烯丙醯氯(42mg,0.47mmol)、碳酸氫鈉(131mg,1.56mmol)、水(4mL)和四氫呋喃(8mL)在0°C混合並在此溫度下攪拌0.5小時。用乙酸乙酯(20mL×2)萃取,有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,減壓脫溶,殘餘物用矽膠柱層析(二氯甲烷/甲醇 = 20/1)純化,得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((3-甲氧基-5-(甲基胺基甲醯)苯基)乙炔基)-1H-吡唑-4-甲醯胺20(60mg,白色固體),產率:兩步35%。 MS m/z (ESI): 437[M+H]1
H NMR (400 MHz, CD3
OD) δ 7.59 (s, 1H), 7.45 (s, 1H), 7.28 (s, 1H), 6.73 - 6.58 (m, 1H), 6.36 - 6.28 (m, 1H), 5.83 - 5.75 (m, 1H), 5.04 - 4.93 (m, 1H), 4.12 - 3.91 (m, 2H), 3.89 (s, 3H), 3.86 - 3.66 (m, 2H), 2.93 (s, 3H), 2.51 - 2.44 (m, 1H), 2.42 - 2.34 (m, 1H)。 實施例21 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-4-甲醯胺 第一步 (S)-3-(3-溴-4-氰基-5-(甲基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將化合物(S)-3-(5-胺基-3-溴-4-氰基-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯7c(178mg,0.5mmol)和對甲基苯磺酸一水和物(12mg,0.07mmol)溶於原甲酸三乙酯(4mL)中,加熱回流2小時。反應結束後減壓脫溶,殘餘物分散在水中,然後用乙酸乙酯(30mL×2)萃取。有機相合併後,用無水硫酸鈉乾燥。過濾後濾液減壓脫溶,殘餘物溶於乙醇(10mL)。冷卻至0°C後,加入硼氫化鈉(89mg,2.35mmol)並在室溫下攪拌2小時。反應完成後,用飽和食鹽水淬滅,然後用乙酸乙酯(30mL×2)萃取。有機相合併後,減壓脫溶,殘餘物用矽膠柱層析(石油醚/乙酸乙酯 = 100/1至1/1)純化得到目標產物(S)-3-(3-溴-4-氰基-5-(甲基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21a(178mg,白色固體),產率:100%。 MS m/z (ESI): 314[M+H-56] 第二步 (S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(3-溴-4-氰基-5-(甲基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21a(1.85g,5.0mmol)、三乙胺(20mL)、1,1'-雙二苯基膦二茂鐵二氯化鈀(816mg,1mmol)、碘化亞銅(190mg,1mmol)和N,N-二甲基甲醯胺(20mL)混合,除氧,在氬氣氣氛下加熱至90°C,然後逐滴加入1-乙炔基-3,5-二甲氧基苯(4.86g,30mmol)的N,N-二甲基甲醯胺(10mL)溶液並繼續攪拌12小時。減壓脫溶,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 50/1至0/1)得到目標產物(S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21b(2.1g,棕色固體),產率:80%。 MS m/z (ESI): 496[M+H-56] 第三步 (S)-3-(5-((第三丁氧基羰基)(甲基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21b(225mg,0.5mmol)溶於二氯甲烷(10mL),然後依次加入三乙胺(150mg,1.5mmol)、Boc酸酐(218mg,1mmol)和4-二甲胺基吡啶(6mg,0.05mmol)。室溫下攪拌2小時後,加入飽和食鹽水(10mL)並用乙酸乙酯(20mL×2)萃取。有機相合併後,減壓脫溶,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 50/1至1/1)得到目標產物(S)-3-(5-((第三丁氧基羰基)(甲基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21c(200mg,淡黃色固體),產率:72%。 MS m/z (ESI): 440[M+H-112] 第四步 (S)-3-(5-((第三丁氧基羰基)(甲基)胺基)-4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(5-((第三丁氧基羰基)(甲基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21c(55mg,0.1mmol)、氫氧化鈉水溶液(0.5M,0.1mL,0.05mmol)、過氧化氫水溶液(30%,0.5mL)和二甲基亞碸(1mL)混合,室溫攪拌2小時。反應物用飽和食鹽水(10mL)稀釋並用乙酸乙酯(20mL×2)萃取。有機相合併後減壓脫溶,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 100/1至1/100)得到(S)-3-(5-((第三丁氧基羰基)(甲基)胺基)-4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21d(30mg,棕色固體),產率:50%。 MS m/z (ESI): 414[M+H-156] 第五步 (S)-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽 將(S)-3-(5-((第三丁氧基羰基)(甲基)胺基)-4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯21d(570mg,1mmol)溶於乙酸乙酯(10mL)中,然後加入氯化氫的乙醇溶液(33%,5mL),並在室溫下攪拌1小時。反應完成後,減壓脫溶,得到目標產物(S)-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽21e(400mg,粗品,棕色固體),該產品未經進一步純化,直接用於下一步反應。 MS m/z (ESI): 370[M+H] 第六步 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-4-甲醯胺 將化合物(S)-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺鹽酸鹽21e(870mg,2.35mmol,粗品)、烯丙醯氯(254mg,2.82mmol)、碳酸鉀水溶液(2.5M,4.7mL,11.78mmol)和四氫呋喃(10mL)在0°C混合並在此溫度下攪拌0.5小時。用乙酸乙酯(50mL×2)萃取,有機相合併後用無水硫酸鈉乾燥,過濾除去乾燥劑,減壓脫溶。殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 100/1至10/1)得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-4-甲醯胺21(720mg,白色固體),產率:76%。 MS m/z (ESI): 424[M+H]1
HNMR (400 MHz, CDCl3
) δ 6.88 (s, 1H), 6.69 (d,J
= 2.3 Hz, 2H), 6.51 (t,J
= 2.2 Hz, 1H), 6.46 - 6.40 (m, 2H), 5.74 - 5.72 (m, 1H), 5.52 - 5.48 (m, 1H), 5.06 - 5.01 (m, 1H), 4.09 - 3.94 (m, 3H), 3.80 (s, 6H), 3.72 - 3.70 (m, 1H), 3.00 (s, 3H), 2.71 - 2.56 (m, 1H), 2.45 - 2.35 (m, 1H)。 實施例22 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((2-氟-3,5-二甲氧苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-4-甲醯胺參照實施例21的操作步驟合成實施例22,但在第二步中用1-乙炔基-2-氟-3,5-二甲氧基苯取代1-乙炔基-3,5-二甲氧基苯。 MS m/z (ESI): 442[M+H]1
H NMR (400 MHz, CDCl3
) δ 7.08 (s, 1H), 6.68 (d,J
= 7.2 Hz, 1H), 6.60 - 6.57 (m, 2H), 6.51 - 6.40 (m, 2H), 5.74 - 5.69 (m, 1H), 5.35 (s, 1H), 5.08 - 4.99 (m, 1H), 4.11 - 4.08 (m, 1H),4.05 - 3.94 (m, 2H), 3.88 (s, 3H), 3.79 (s, 3H), 3.75 - 3.65 (m, 1H), 3.00 (t,J
= 5.2 Hz, 3H), 2.72 - 2.58 (m, 1H), 2.44 - 2.33 (m, 1H)。 實施例23 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((5-氯-2-氟苯基)乙炔基)-5-(甲基胺基)-1H-吡唑-4-甲醯胺參照實施例21的操作步驟合成實施例23,但在第二步中用4-氯-2-乙炔基-1-氟苯取代1-乙炔基-3,5-二甲氧基苯。 MS m/z (ESI): 416[M+H]1
H NMR (400 MHz, CDCl3
) δ 7.56 - 7.52 (m, 1H), 7.35 - 7.33 (m, 1H), 7.08 (t,J
= 8.8 Hz, 1H), 7.02 - 6.92 (m, 1H), 6.51 - 6.39 (m, 2H), 5.74 (d,J
= 9.3 Hz, 1H), 5.55 - 5.44 (m, 1H), 5.09 - 4.98 (m, 1H), 4.14 - 3.90 (m, 3H), 3.80 - 3.65 (m, 1H), 3.01 (s, 3H), 2.74 - 2.55 (m, 1H), 2.49 - 2.34 (m, 1H)。 實施例24 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(乙基胺基)-1H-吡唑-4-甲醯胺 第一步 (S)-3-(5-乙基胺基-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將混合物(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(500mg,1.14mmol)和氫化鈉(91mg,2.28mmol,60%)加入到N,N-二乙基乙醯胺(5mL)中,攪拌10分鐘,加入碘乙烷(106mg,0.68mmol),攪拌0.5小時。將反應液倒入水中,減壓濃縮,殘餘物用反相高效液相製備色譜純化[乙腈/水(含0.1%甲酸):50%-90%],得到目標產物(S)-3-(5-乙基胺基-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯24a(70mg,白色固體),產率:22%。 MS m/z (ESI): 410[M+1-56] 第二步 (S)-3-(5-乙基胺基-4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(5-乙基胺基-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯24a(55mg,0.12mmol)溶於二甲基亞碸(3mL),加入雙氧水(2mL)和氫氧化鈉(300mg,7.5mmol),常溫攪拌10分鐘後,升溫至40o
C。待反應完成,冷卻後用水(20mL)稀釋,乙酸乙酯(30mL)萃取,並用水(20mL×3)洗滌,有機相減壓濃縮,殘餘物用反相高效液相製備色譜純化[乙腈/水(含0.1%甲酸):50%-90%],得到目標產物(S)-3-(5-乙基胺基-4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯24b(15mg),產率:26%。 MS m/z (ESI): 484 [M+1] 第三步 (S)-3-((3,5-二甲氧苯基)乙炔基)-5-(乙基胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將(S)-3-(5-乙基胺基-4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯24b(15mg,0.031mmol)溶於二氯甲烷(2mL),加入三氟乙酸(0.5mL),攪拌半小時。待反應完成,減壓濃縮,得到目標產物(S)-3-((3,5-二甲氧苯基)乙炔基)-5-(乙基胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺24c(20mg,粗品,棕色油狀物),產率:>100%。產物不經純化直接用於下一步反應。 MS m/z (ESI): 384[M+1] 第四步 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(乙基胺基)-1H-吡唑-4-甲醯胺 將化合物(S)-3-((3,5-二甲氧苯基)乙炔基)-5-(乙基胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺24c(20mg,0.031mmol,粗品)溶於四氫呋喃(5mL),加入飽和碳酸氫鈉溶液(2mL),再加入丙烯醯氯(2.7mg,0.03mmol)的四氫呋喃溶液,攪拌0.5小時。反應液減壓濃縮,殘餘物溶於乙酸乙酯(30mL),並用水(20mL×3)洗滌。有機相減壓濃縮,殘餘物用反相高效液相製備色譜純化[乙腈/水(含0.1%甲酸):20%-70%],得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(乙基胺基)-1H-吡唑-4-甲醯胺24(4.7mg,白色固體),產率:24%。 MS m/z (ESI): 438[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.87 (brs, 1H), 6.74 (s, 2H), 6.54 (s, 1H), 6.52 (s, 1H), 6.48 - 6.40 (m, 2H), 5.74 - 5.69 (m, 1H), 5.06 - 4.97 (m, 2H), 4.13 - 3.93 (m, 3H), 3.84 (s, 6H), 3.80 - 3.67 (m, 1H), 3.42 (brs, 2H), 2.75 - 2.35 (m, 2H), 1.31 - 1.25 (m, 3H)。 實施例25 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-(異丙基胺基)-1H-吡唑-4-甲醯胺 第一步 (S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-(異丙基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將混合物(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(600mg,1.37mmol)、碳酸銫(893mg,2.74mmol)和乙腈(25mL)攪拌10分鐘,迅速加入2-溴丙烷(186mg,1.51mmol),加熱至72o
C,攪拌6小時。冷卻至室溫,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 2/1),得到目標產物(S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-(異丙基胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯25a(600mg,淺黃色固體),產率:91%。 MS m/z (ESI): 424[M+1-56] 參照實施例24中第二步至第四步的操作步驟合成實施例25。 MS m/z (ESI): 452[M+1]1
H NMR (400 MHz, CDCl3
) δ 6.88 (brs, 1H), 6.70 (s, 2H), 6.54 (s, 1H), 6.51 - 6.39 (m, 2H), 6.03 (t,J
= 10.3 Hz, 1H), 5.74 - 5.69 (m, 1H), 5.49 (brs, 1H), 4.96 - 4.87 (m, 1H), 4.09 - 3.86 (m, 3H), 3.80 - 3.66 (m, 7H), 3.45 - 3.43 (m, 1H), 2.69 - 2.32 (m, 2H), 1.27 - 1.15 (m, 6H)。 實施例26 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-((環丙基甲基)胺基)-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-4-甲醯胺 第一步 (S)-1-(1-丙烯醯吡咯烷-3-基)-5-((環丙基甲基)胺基)-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-4-甲醯胺 將化合物(S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-4-甲醯胺7(50mg,0.12mmol)溶於乙腈(2mL)中,加入碳酸銫(80mg,0.24mmol)和(溴甲基)環丙烷(19mg,0.13mmol),加熱至70o
C,攪拌4小時。將反應液倒入水(30mL)中,並用乙酸乙酯(30mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用薄層矽膠製備色譜(二氯甲烷/甲醇 = 12/1)純化,得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-5-((環丙基甲基)胺基)-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-4-甲醯胺26(14mg,白色固體),產率:28%。 MS m/z (ESI): 464[M+1]1
H NMR (400 MHz, CDCl3
) δ 6.89 (brs, 1H), 6.71 (s, 2H), 6.54 (s, 1H), 6.51 - 6.37 (m, 2H), 5.76 - 5.71 (m, 1H), 5.40 (brs, 1H), 5.03 - 4.95 (m, 1H), 4.06 - 3.89 (m, 3H), 3.82 (s, 6H), 3.78 - 3.67 (m, 1H), 3.06 - 3.02 (m, 2H), 2.69 - 2.52 (m, 1H), 2.46 - 2.35 (m, 1H), 1.15 - 0.98 (m, 1H), 0.63 - 0.60 (m, 2H), 0.29 - 0.27 (m, 2H)。 實施例27 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2,2,2-三氟乙基)胺基)-1H-吡唑-4-甲醯胺 第一步 (S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-((2,2,2-三氟乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(430mg,0.98mmol)、三氟乙醛水溶液(75%)(304mg,1.96mmol)和鈦酸四乙酯(448mg,1.96mmol)加入到二氯甲烷(15mL)中,並攪拌2小時。待反應完全,向反應液中加入硼氫化鈉(75mg,1.96mmol),常溫下繼續攪拌1小時。將反應液倒入水中並用乙酸乙酯(20mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物經快速柱純化,得到目標產物(S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-((2,2,2-三氟乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯27a(120mg,黃色油狀物),產率:26%。 MS m/z (ESI): 464[M+1-56] 參照實施例24中第二步至第四步的操作步驟合成實施例27。 MS m/z (ESI): 492[M+1]1
H NMR (400 MHz, CDCl3
) δ 6.92 (brs, 1H), 6.70 (s, 2H), 6.52 (s, 1H), 6.47 - 6.39 (m, 2H), 6.31 - 6.25 (m, 1H), 5.75 - 5.65 (m, 1H), 5.65 (brs, 1H), 5.05 - 4.98 (m, 1H), 4.10 - 3.88 (m, 3H), 3.80 (s, 6H), 3.75 - 3.61 (m, 3H), 2.63 - 2.34 (m, 2H)。 實施例28 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-甲氧基乙基)胺基)-1H-吡唑-4-甲醯胺參照實施例25的操作步驟合成實施例28,但在第一步中用1-溴-2-甲氧基乙烷取代2-溴丙烷。 MS m/z (ESI): 468[M+1]1
H NMR (400 MHz, DMSO-d6
) δ 7.32 (brs, 1H), 6.74 (d,J
= 2.2 Hz, 2H), 6.64 (dd,J
= 16.8, 10.4 Hz, 1H), 6.60 (t,J
= 2.2 Hz, 1H), 6.50 (t,J
= 6.0 Hz, 1H), 6.16 (dd,J
= 16.8, 5.0 Hz, 1H), 5.68 (t,J
= 10.8 Hz, 1H), 5.15 - 5.05 (m, 1H), 4.05 - 4.01 (m, 0.5H), 3.86 - 3.81 (m, 1.5H), 3.77 (s, 6H), 3.70 - 3.61 (m, 1H), 3.59 - 3.50 (m, 1H), 3.46 (t,J
= 5.1 Hz, 2H), 3.39 - 3.34 (m, 2H), 3.26 (s, 3H), 2.42 - 2.23 (m, 2H)。 實施例29 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1H-吡唑-4-甲醯胺 第一步 (S)-3-(5-((2-乙醯氧基乙基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將混合物(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(300mg,0.685mmol)、2-溴乙基乙酸酯(126mg,0.753mmol)、碳酸銫(447mg,1.37mmol)和乙腈(4mL)加熱至90o
C,並攪拌2小時。將反應液冷卻至室溫,倒入水(50mL)中,並用乙酸乙酯(30mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇 = 15/1),得到目標產物(S)-3-(5-((2-乙醯氧基乙基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯29a(148mg,黃色固體),產率:41%。 MS m/z (ESI): 468[M+1-56] 第二步 (S)-3-(4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將混合物(S)-3-(5-((2-乙醯氧基乙基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯29a(68mg,0.146mmol)、乙醇(5mL)和二甲基亞碸(1mL),加入飽和氫氧化鈉溶液(3mL)和雙氧水(4mL),30o
C下攪拌1小時。待反應完成,將反應液倒入飽和亞硫酸鈉溶液(30mL)中,並用乙酸乙酯(30mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,得到目標產物(S)-3-(4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯29b(110mg,粗品,黃色油狀物),產率:>100%,產物不經純化直接用於下一步反應。 MS m/z (ESI): 444[M+1-56] 第三步 (S)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺 將化合物(S)-3-(4-胺基甲醯-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯29b(110mg,0.146mmol,粗品)溶於鹽酸的甲醇溶液(5mL),加熱至40o
C攪拌1小時。待反應完成後,減壓濃縮,得到目標產物(S)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺29c(160mg,粗品,白色固體),產率:>100%,產物不經純化直接用於下一步反應。 MS m/z (ESI): 400[M+1] 第四步 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1H-吡唑-4-甲醯胺 將化合物(S)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1-(吡咯烷-3-基)-1H-吡唑-4-甲醯胺29c(160mg,0.146mmol,粗品)溶於四氫呋喃(5mL)中,加入飽和碳酸氫鈉溶液(10mL),再加入丙烯醯氯(12mg,0.13mmol),室溫攪拌10分鐘。待反應完成後,將反應液倒入水(50mL)中,並用乙酸乙酯(30mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物通過反相高效液相製備色譜[乙腈/水(含0.2%甲酸):20%-60%]純化,得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-羥基乙基)胺基)-1H-吡唑-4-甲醯胺29(6mg,白色固體),產率:9%。 MS m/z (ESI): 454[M+1]1
H NMR (400 MHz, DMSO-d6
) δ 7.36 (brs, 1H), 6.76 (brs, 1H), 6.74 (s, 2H), 6.70 - 6.60 (m, 2H), 6.55 - 6.52 (m, 1H), 6.17 (d,J
= 16.9 Hz, 1H), 5.69 (t,J
= 10.9 Hz, 1H), 5.16 - 5.10 (m, 1H), 4.87 (s, 1H), 4.06 - 4.0 (m, 0.5H), 3.83 - 3.81 (m, 1.5H), 3.77 (s, 6H), 3.68 - 3.63 (m, 2H), 3.55 - 3.53 (m, 2H), 3.28 - 3.26 (m, 2H), 2.38 - 2.27 (m, 2H)。 實施例30 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((3-嗎啉代丙基)胺基)-1H-吡唑-4-甲醯胺 第一步 3-嗎啉代丙基4-甲基苯磺酸酯 將化合物3-嗎啉代丙烷-1-醇30a(500mg,3.45mmol)溶於二氯甲烷(100ml),加入4-二甲胺基吡啶(42mg,0.34mmol)、三乙胺(1.04g,10.3mmol)和對甲苯磺醯氯(988mg,5.17mmol),室溫攪拌過夜。待反應完成,將反應液倒入水(50mL)中,並用二氯甲烷(50mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 2/1,得到目標產物3-嗎啉代丙基4-甲基苯磺酸酯30b(660mg,黃色油狀物),產率:64%。 MS m/z (ESI): 300[M+1] 參照實施例25的操作步驟合成實施例30,但在第一步中用3-嗎啉代丙基4-甲基苯磺酸酯取代2-溴丙烷。 MS m/z (ESI): 537[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.23 (brs, 1H), 7.12 (brs, 1H), 6.94 (brs, 1H), 6.69 (s, 2H), 6.52 (s, 1H), 6.49 - 6.40 (m, 2H), 5.92 (brs, 1H), 5.74 - 5.70 (m, 1H), 5.03 - 4.96 (m, 1H), 4.09 - 3.90 (m, 3H), 3.80 - 3.68 (m, 11H), 3.28 (brs, 2H), 2.89 (brs, 6H), 2.69 - 2.33 (m, 2H), 1.93 (brs, 2H)。 實施例31 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-嗎啉代乙基)胺基)-1H-吡唑-4-甲醯胺參照實施例25的操作步驟合成實施例31,但在第一步中用4-(2-氯乙基)嗎啉取代2-溴丙烷。 MS m/z (ESI): 523[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.14 (s, 1H), 6.95 (brs, 1H), 6.69 (s, 2H), 6.63 (brs, 1H), 6.52 (s, 1H), 6.49 - 6.39 (m, 2H), 6.14 (brs, 1H), 5.75 - 5.70 (m, 1H), 5.06 - 4.98 (m, 1H), 4.11 - 3.85 (m, 3H), 3.80 - 3.72 (m, 11H), 3.37 - 3.33 (m, 2H), 2.80 - 2.73 (m, 2H), 2.65 (brs, 4H), 2.45 - 2.32 (m, 2H)。 實施例32 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((2-(吡咯烷-1-基)乙基)胺基)-1H-吡唑-4-甲醯胺 第一步 2-溴乙基4-甲基苯磺酸酯 將化合物2-溴乙醇32a(500mg,4.0mmol)、4-二甲胺基吡啶(246mg,2.02mmol)和三乙胺(1.22g,12.1mmol)溶於二氯甲烷(50mL),降溫至0o
C,再分批加對甲苯磺醯氯(1.15g,6.05mmol),加入完成後,升至室溫攪拌過夜。待反應完成,將反應液倒入水(50mL)中並用二氯甲烷(50mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(石油醚/乙酸乙酯 = 10/1),得到目標產物2-溴乙基4-甲基苯磺酸酯32b(600mg,黃色油狀物),產率:53%。 MS m/z (ESI): 277[M+1] 第二步 (S)-3-(5-((2-溴乙基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將混合物(S)-3-(5-胺基-4-氰基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁基酯7d(400mg,0.92mmol)、2-溴乙基4-甲基苯磺酸酯(380mg,1.37mmol)、碳酸銫(600mg,1.84mmol)和乙腈(10mL)加熱至70o
C,並攪拌2小時。將反應液倒入水(50mL)中,並用乙酸乙酯(50mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物通過快速柱分離,得到目標產物(S)-3-(5-((2-溴乙基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯32c(240mg,棕色油狀物),產率:48%。 MS m/z (ESI): 408[M+1-56-80] 第三步 (S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-((2-(吡咯烷-1-基)乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯 將混合(S)-3-(5-((2-溴乙基)胺基)-4-氰基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯32c(240mg,0.44mmol)、吡咯烷(47mg,0.66mmol)、碳酸銫(288mg,0.88mmol)和乙腈(5mL)加熱至70o
C,並攪拌1.5小時。將反應液倒入水(30mL)中,並用乙酸乙酯(30mL×3)萃取,有機相合併後經無水硫酸鈉乾燥,過濾除去乾燥劑,減壓濃縮,殘餘物用矽膠柱層析純化(二氯甲烷/甲醇= 10/1),得到目標產物(S)-3-(4-氰基-3-((3,5-二甲氧苯基)乙炔基)-5-((2-(吡咯烷-1-基)乙基)胺基)-1H-吡唑-1-基)吡咯烷-1-甲酸第三丁酯32d(200mg,黃色油狀物),產率:85%。 MS m/z (ESI): 479[M+1-56] 參照實施例24中第二步至第四步的操作步驟合成實施例32。 MS m/z (ESI): 507[M+1]1
H NMR (400 MHz, CDCl3
) δ 8.38 (s, 1H), 6.99 (brs, 1H), 6.69 (s, 2H), 6.51 (s, 1H), 6.47 - 6.36 (m, 2H), 5.72 - 5.67 m, 2H), 5.16 - 5.08 (m, 1H), 4.12 - 3.86 (m, 3H), 3.80 - 3.62 (m, 9H), 3.33 - 3.29 (m, 6H), 2.62 - 2.34 (m, 2H), 2.07 (brs, 4H)。 實施例33 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((四氫-2H-吡喃-4-基)胺基)-1H-吡唑-4-甲醯胺 第一步 4-碘四氫-2H-吡喃 將4-羥基四氫-2H-吡喃33a(2.04g,20mmol)、三苯基膦(6.81g,26)和咪唑(2.04g,30mmol)溶於二氯甲烷(100mL)中,冷卻至0o
C,然後加入碘(6.09g,24mmol),並在45o
C下攪拌14小時。反應用水淬滅並用乙酸乙酯(50mL×2)萃取,有機相合併後用無水硫酸鈉乾燥,過濾後濾液減壓脫溶。殘餘物用矽膠柱層析(石油醚/乙酸乙酯 = 1/1)純化得到目標產物4-碘四氫-2H-吡喃33b(2.12g,白色固體),產率:50%。1
H NMR (400 MHz, DMSO-d6
) δ 4.62 (dt,J
= 13.9, 4.5 Hz, 1H), 3.68 - 3.64 (m, 2H), 3.47 - 3.42 (m, 2H), 2.13 - 1.97 (m, 4H)。 參照實施例24的操作步驟合成實施例33,但在第一步中用4-碘四氫-2H-吡喃取代碘乙烷。 MS m/z (ESI): 494[M+H]1
H NMR (400 MHz, CD3
OD) δ 6.74 (t,J
= 2.2 Hz, 2H), 6.71 - 6.62 (m, 1H), 6.60 - 6.58(m, 1H), 6.36 - 6.30 (m, 1H), 5.82 - 5.77 (m, 1H), 5.18 - 5.12 (m, 1H), 4.04 - 3.94 (m, 6H), 3.81 (s, 6H), 3.52 - 3.46 (m, 3H), 2.55 - 2.39 (m, 2H), 1.94 - 1.92 (m, 2H), 1.60 - 1.55 (m, 2H)。 實施例34 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((1-甲基呱啶-4-基)胺基)-1H-吡唑-4-甲醯胺 第一步 4-碘-1-甲基呱啶 將4-羥基-1-甲基呱啶34a(2.3g,20mmol)、三苯基膦(6.81g,26mmol)、咪唑(2.04g,30mmol)和二氯甲烷(100mL)混合並冷卻至0o
C,然後加入碘(6.09g,24mmol)並繼續攪拌18小時。反應結束後用水淬滅,然後用二氯甲烷(50mL×2)萃取。有機相合併後用無水硫酸鈉乾燥並過濾,然後將濾液減壓脫溶。殘餘物用矽膠柱層析(二氯甲烷/甲醇 = 10/1)純化得到目標產物4-碘-1-甲基呱啶34b(2.25g,白色固體),產率:50%。 MS m/z (ESI): 226[M+H] 第二步 (S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((1-甲基呱啶-4-基)胺基)-1H-吡唑-4-甲醯胺 將化合物(S)-1-(1-丙烯醯吡咯烷-3-基)-5-胺基-3-((3,5-二甲氧苯基)乙炔基)-1H-吡唑-4-甲醯胺7(210mg,0.5mmmol)、4-碘-1-甲基呱啶34b(450mg,2mmol)、碳酸鉀(207mg,1.5mmol)和乙腈(10mL)混合並在90o
C下加熱並攪拌13小時。反應混合物減壓脫溶後溶於水,然後用乙酸乙酯萃取(50mL×2)。有機相合併後減壓脫溶,殘餘物用反向製備液相色譜純化得到目標產物(S)-1-(1-丙烯醯吡咯烷-3-基)-3-((3,5-二甲氧苯基)乙炔基)-5-((1-甲基呱啶-4-基)胺基)-1H-吡唑-4-甲醯胺34(8.1mg,白色固體),產率:3.2%。 MS m/z (ESI): 507[M+H]1
H NMR (400 MHz, CD3
OD) δ 6.77 (s, 2H), 6.68-6.65 (m, 1H), 6.59 (s, 1H), 6.34 - 6.30 (m, 1H), 5.81 - 5.78 (m, 1H), 5.69 - 5.67 (m, 1H), 5.03 - 5.00 (m, 1H), 4.98 - 5.95(m, 1H), 4.92 - 4.90 (m, 1H), 4.36 (s, 2H), 4.12 - 4.07 (m, 1H), 4.00 - 3.98 (m, 1H), 3.93 - 3.90 (m, 1H), 3.86 - 3.84 (m, 1H), 3.81 (s, 6H), 3.72 - 3.68 (m, 1H), 2.53 - 2.47 (m, 3H), 2.40 - 2.38 (m, 1H), 2.32 (s, 3H), 2.27 - 2.21 (m, 1H)。 生物學實驗 FGFR的活性抑制測試 使用HTRF激酶檢測試劑盒,通過檢測激酶反應中底物的磷酸化水準來評估本發明的化合物對FGFR體外活性的影響(表1)。 FGFR1的活性抑制測試 實驗方法概述如下: 反應緩衝液包含以下組分:5倍稀釋的enzymatic buffer/kinase 5X(Cisbio,貨號為62EZBFDD)(主要成分為50mM HEPES,pH7.0)、5mM MgCl2
和1mM DTT;人重組FGFR1催化結構域蛋白(胺基酸308-731)由公司自己純化,用反應緩衝液稀釋成0.6ng/uL的激酶溶液;底物反應溶液包括用反應緩衝液稀釋成400nM的生物素標記的酪胺酸激酶底物(Cisbio,貨號為62TK0PEC)和40uM ATP;檢測液包括用檢測緩衝液(Cisbio,貨號為62SDBRDF)稀釋成0.125ng/uL Eu3+
標記的籠狀抗體(Cisbio,貨號為61T66KLB)、25nM鏈黴親和素標記的XL665(Cisbio,貨號為610SAXLB)。 將化合物用DMSO溶解稀釋至1mM,然後用DMSO進行4倍的系列稀釋至最低濃度為0.061uM,每個濃度點再使用反應緩衝液稀釋40倍。如果化合物IC50
值非常低,可以降低化合物的起始濃度。 向384孔檢測板(Thermo,貨號為264706)中添加4uL化合物溶液和2uL的FGFR1激酶溶液,混合均勻後室溫孵育15分鐘;隨後加入4uL底物反應溶液,將反應混合物在室溫孵育60分鐘;隨後加入與反應等體積的10uL檢測液終止反應,混合均勻後室溫放置。60分鐘後,磷酸化的產物同時被Eu3+
標記的籠狀抗體(供體)和鏈黴親和素標記的XL665抗體(受體)識別,在鐳射激發後,靠近的供體和受體發生能量共振轉移,其從供體(620nm)轉移至受體(665nm)的能量用酶標儀EnVision(Perkin Elmer)檢測。665/620的比值與底物的磷酸化程度呈正相關,因此從而檢測FGFR1激酶的活性。 該實驗中未加酶組作為100%抑制組,加酶但是未加化合物組作為0%抑制組。化合物對FGFR1活性的抑制百分比用以下公式計算: 抑制百分比=100-100*(比值化合物
-比值100% 抑制
)/(比值0% 抑制
-比值100% 抑制
) 化合物的IC50
值由10個濃度點用Excel中XLfit軟體通過以下公式來計算: Y=Bottom+(Top-Bottom)/(1+10^((logIC50
-X)*slope factor)) 其中Y為抑制百分比,Bottom為S型曲線的底部平臺值,Top為S型曲線的頂部平臺值,X為待測化合物濃度的對數值,slope factor為曲線斜率係數。 FGFR2的活性抑制測試 實驗方法概述如下: 反應緩衝液包含以下組分:5倍稀釋的enzymatic buffer/kinase 5X(Cisbio,貨號為62EZBFDD)(主要成分為50mM HEPES,pH7.0)、5mM MgCl2
和1mM DTT;人重組FGFR2催化結構域蛋白(胺基酸400-821)購自義翹神州生物技術有限公司,用反應緩衝液稀釋成0.45ng/uL的激酶溶液;底物反應溶液包括用反應緩衝液稀釋成800nM的生物素標記的酪胺酸激酶底物(Cisbio,貨號為62TK0PEC)和50uM ATP;檢測液包括用檢測緩衝液(Cisbio,貨號為62SDBRDF)稀釋成0.125ng/uL Eu3+
標記的籠狀抗體(Cisbio,貨號為61T66KLB)、50nM鏈黴親和素標記的XL665(Cisbio,貨號為610SAXLB)。 將化合物用DMSO溶解稀釋至1mM,然後用DMSO進行4倍的系列稀釋至最低濃度為0.061uM,每個濃度點再使用反應緩衝液稀釋40倍。如果化合物IC50
值非常低,可以降低化合物的起始濃度。 向384孔檢測板(Thermo,貨號為264706)中添加4uL化合物溶液和2uL的FGFR2激酶溶液,混合均勻後室溫孵育15分鐘;隨後加入4uL底物反應溶液,將反應混合物在室溫孵育60分鐘;隨後加入與反應等體積的10uL檢測液終止反應,混合均勻後室溫放置。60分鐘後,磷酸化的產物同時被Eu3+
標記的籠狀抗體(供體)和鏈黴親和素標記的XL665抗體(受體)識別。在鐳射激發後,靠近的供體和受體發生能量共振轉移,其從供體(620nm)轉移至受體(665nm)的能量用酶標儀EnVision(Perkin Elmer)檢測。665/620的比值與底物的磷酸化程度呈正相關,因此從而檢測FGFR2激酶的活性。 該實驗中未加酶組作為100%抑制組,加酶但是未加化合物組作為0%抑制組。化合物對FGFR2活性抑制百分比用以下公式計算: 抑制百分比=100-100*(比值化合物
-比值100% 抑制
)/(比值0% 抑制
- 比值100% 抑制
) 化合物的IC50
值由10個濃度點用Excel中XLfit軟體通過以下公式來計算: Y=Bottom+(Top-Bottom)/(1+10^((logIC50
-X)*slope factor)) 其中Y為抑制百分比,Bottom為S型曲線的底部平臺值,Top為S型曲線的頂部平臺值,X為待測化合物濃度的對數值,slope factor為曲線斜率係數。 FGFR3的活性抑制測試 實驗方法概述如下: 反應緩衝液包含以下組分:5倍稀釋的enzymatic buffer/kinase 5X(Cisbio,貨號為62EZBFDD)(主要成分為50mM HEPES,pH7.0)、5mM MgCl2
和1mM DTT;人重組FGFR3催化結構域蛋白(胺基酸399-806)購自義翹神州生物技術有限公司,用反應緩衝液稀釋成0.3ng/uL的激酶溶液;底物反應溶液包括用反應緩衝液稀釋成1000nM的生物素標記的酪胺酸激酶底物(Cisbio,貨號為62TK0PEC)和90uM ATP;檢測液包括用檢測緩衝液(Cisbio,貨號為62SDBRDF)稀釋成0.125ng/uL Eu3+
標記的籠狀抗體(Cisbio,貨號為61T66KLB)、62.5nM鏈黴親和素標記的XL665(Cisbio,貨號為610SAXLB)。 將化合物用DMSO溶解稀釋至1mM,然後用DMSO進行4倍的系列稀釋至最低濃度為0.061uM,每個濃度點再使用反應緩衝液稀釋40倍。如果化合物IC50
值非常低,可以降低化合物的起始濃度。 向384孔檢測板(Thermo,貨號為264706)中添加4uL化合物溶液和2uL的FGFR3激酶溶液,混合均勻後室溫孵育15分鐘;隨後加入4uL底物反應溶液,將反應混合物在室溫孵育60分鐘;隨後加入與反應等體積的10uL檢測液終止反應,混合均勻後室溫放置。60分鐘後,磷酸化的產物同時被Eu3+
標記的籠狀抗體(供體)和鏈黴親和素標記的XL665抗體(受體)識別。在鐳射激發後,靠近的供體和受體發生能量共振轉移,其從供體(620nm)轉移至受體(665nm)的能量用酶標儀EnVision(Perkin Elmer)檢測。665/620的比值與底物的磷酸化程度呈正相關,因此從而檢測FGFR3激酶的活性。 該實驗中未加酶組作為100%抑制組,加酶但是未加化合物組作為0%抑制組。化合物對FGFR3活性抑制百分比用以下公式計算: 抑制百分比=100-100*(比值化合物
-比值100% 抑制
)/(比值0% 抑制
- 比值100% 抑制
) 化合物的IC50
值由10個濃度點用Excel中XLfit軟體通過以下公式來計算: Y=Bottom+(Top-Bottom)/(1+10^((logIC50
-X)*slope factor)) 其中Y為抑制百分比,Bottom為S型曲線的底部平臺值,Top為S型曲線的頂部平臺值,X為待測化合物濃度的對數值,slope factor為曲線斜率係數。 FGFR4的活性抑制測試 實驗方法概述如下: 反應緩衝液包含以下組分:5倍稀釋的enzymatic buffer/kinase 5X(Cisbio,貨號為62EZBFDD)(主要成分為50mM HEPES,pH7.0)、5mM MgCl2
和1mM DTT;人重組FGFR4催化結構域蛋白(胺基酸460-802)購自清華大學蛋白質研究技術中心,用反應緩衝液稀釋成0.5ng/uL的激酶溶液;底物反應溶液包括用反應緩衝液稀釋成500nM的生物素標記的酪胺酸激酶底物(Cisbio,貨號為62TK0PEC)和90uM ATP;檢測液包括用檢測緩衝液(Cisbio,貨號為62SDBRDF)稀釋成0.125ng/uL Eu3+
標記的籠狀抗體(Cisbio,貨號為61T66KLB)、31.25nM鏈黴親和素標記的XL665(Cisbio,貨號為610SAXLB)。 將化合物用DMSO溶解稀釋至1mM,然後用DMSO進行4倍的系列稀釋至最低濃度為0.061uM,每個濃度點再使用反應緩衝液稀釋40倍。如果化合物IC50
值非常低,可以降低化合物的起始濃度。 向384孔檢測板(Thermo,貨號為264706)中添加4uL化合物溶液和2uL的FGFR4激酶溶液,混合均勻後室溫孵育15分鐘;隨後加入4uL底物反應溶液,將反應混合物在室溫孵育60分鐘;隨後加入與反應等體積的10uL檢測液終止反應,混合均勻後室溫放置。60分鐘後,磷酸化的產物同時被Eu3+
標記的籠狀抗體(供體)和鏈黴親和素標記的XL665抗體(受體)識別。在鐳射激發後,靠近的供體和受體發生能量共振轉移,其從供體(620nm)轉移至受體(665nm)的能量用酶標儀EnVision(Perkin Elmer)檢測。665/620的比值與底物的磷酸化程度呈正相關,因此從而檢測FGFR4激酶的活性。 該實驗中未加酶組作為100%抑制組,加酶但是未加化合物組作為0%抑制組。化合物對FGFR4活性抑制百分比用以下公式計算: 抑制百分比=100-100*(比值化合物
-比值100% 抑制
)/(比值0% 抑制
- 比值100% 抑制
) 化合物的IC50
值由10個濃度點用Excel中XLfit軟體通過以下公式來計算: Y=Bottom+(Top-Bottom)/(1+10^((logIC50
-X)*slope factor)) 其中Y為抑制百分比,Bottom為S型曲線的底部平臺值,Top為S型曲線的頂部平臺值,X為待測化合物濃度的對數值,slope factor為曲線斜率係數。 表1
A﹤10nM;10 nM ≤ B < 100nM;100 nM ≤ C <1000nM 本發明的實施例化合物對FGFR的活性具有顯著抑制效應,較佳IC50
為100至1000nM,更佳IC50
小於100nM,最佳IC50
小於10nM。 Hep3B細胞增殖抑制的測定 使用發光細胞活力測試實驗評估本發明的化合物對Hep3B肝癌細胞系細胞增殖的影響(表2)。 實驗方法概述如下: CellTilter-Glo試劑(Promega,貨號為G7572)由CTG凍乾粉和CTG緩衝液組成,使用時將凍乾粉溶解到緩衝液中即可。 將化合物用DMSO(Sigma,貨號為D5879)溶解稀釋至5mM,然後用DMSO進行4倍的系列稀釋至最低濃度為0.31uM,每個濃度點再用不含FBS的DMEM培養基(ThermoFisher,貨號為11995073)稀釋50倍。如果化合物IC50值非常低,可以降低化合物的起始濃度。 Hep3B細胞(來自中國科學院上海生命科學研究院細胞資源中心)在含有10%FBS(GBICO,貨號為10099-141)和100U/mL青鏈黴素混合液(ThermoFisher,貨號為15140122)的DMEM完全培養基中培養,當細胞在培養容器中覆蓋率達80-90%時,用0.25%胰酶(含EDTA)(ThermoFisher,貨號為25200056)消化吹散後種植於白色384孔板(ThermoFisher,貨號為164610),每孔1000細胞(27uL DMEM完全培養基),然後把384孔板置於37ºC、5%CO2
的培養箱中培養過夜(18-20小時)。 過夜後每孔加入3uL DMEM稀釋後的化合物,輕輕離心混勻,然後把384孔板置於37ºC、5%CO2
的培養箱中繼續培養,72小時後取出於室溫放置。30分鐘,每孔加15uL平衡至室溫的CTG試劑,置於振盪器上輕輕震盪3分鐘以確保細胞裂解充分,放置10分鐘使冷光信號穩定,然後用EnVision(Perkin Elmer)讀取冷光信號。 其中,加10uM Blueprint的BLU9931(Cancer Discovery 2015, 5, 424)組的冷光信號作為signal100% 抑制
,加0.2%DMSO組的冷光信號作為signal0% 抑制
。 化合物對Hep3B細胞增殖抑制的百分比可以用以下公式計算: 抑制百分比=100-100*(signal化合物
-signal100% 抑制
)/ (signal0% 抑制
-signal100% 抑制
) 化合物IC50值由8個濃度點用XLfit(ID Business Solutions Ltd., UK)軟體通過以下公式計算: Y=Bottom+(Top-Bottom)/(1+10^((logIC50
-X)*slope factor)) 其中Y為抑制百分比,Bottom為S型曲線的底部平臺值,Top為S型曲線的頂部平臺值,X為待測化合物濃度的對數值,slope factor為曲線斜率係數。 RT4細胞增殖抑制的測定 使用發光細胞活力測試實驗評估本發明的化合物對RT4膀胱癌細胞系細胞增殖的影響(表2)。 實驗方法概述參照Hep3B細胞增殖抑制的測定方法,其中RT4細胞(來自中國科學院上海生命科學研究院細胞資源中心)、陽性對照物為Taiho專利申請WO2015008844A1中的實施例1((S)-1-(3-(4-胺基-3-((3,5-二甲氧基苯基)乙炔基)-1H-吡唑並[3,4-d]嘧啶-1-基)吡咯烷-1-基)丙-2-烯-1-酮)。 SNU-16細胞增殖抑制的測定 使用發光細胞活力測試實驗評估本發明的化合物對SNU-16胃癌細胞系細胞增殖的影響(表2)。 實驗方法概述參照Hep3B細胞增殖抑制的測定方法,其中SNU-16細胞(ATCC,HB-8064)、陽性對照物為Novartis的BJG398。 表2
說明:A﹤10nM;10nM≤B<100nM;100nM≤C<1000Nm N.D.: 未進行檢測 本發明的實施例化合物分別對Hep3B、RT4和SNU-16的細胞增殖具有顯著抑制效應,較佳IC50
為100至1000nM,更佳IC50
小於100nM。Unless stated to the contrary, the following terms used in the specification and claims have the following meanings.的 Notation used in this article "Cxy
"Means a range of carbon atoms, where x and y are integers, such as C3-8
Cycloalkyl means a cycloalkyl group having 3 to 8 carbon atoms, that is, a cycloalkyl group having 3, 4, 5, 6, 7, or 8 carbon atoms. It should also be understood that "C3-8" also includes any sub-range thereof, such as C3-7, C3-6, C4-7, C4-6, C5-6, and the like. "Alkyl" means a saturation of 1 to 20 carbon atoms, such as 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms Straight or branched chain hydrocarbyl group. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, third butyl, second butyl, n-pentyl, 1,1-dimethyl Propyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1- Ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl Radical, 1,3-dimethylbutyl and 2-ethylbutyl. The alkyl group may be substituted or unsubstituted. "Alkenyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond and usually 2 to 20 carbon atoms, such as 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Group. Non-limiting examples of alkenyl include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1 , 4-pentadienyl and 1,4-butadienyl. The alkenyl may be substituted or unsubstituted. "Alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond and usually 2 to 20 carbon atoms, such as 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Group. Non-limiting examples of alkynyl include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl. The alkynyl may be substituted or unsubstituted. "Cycloalkyl" refers to a saturated cyclic hydrocarbon substituent having 3 to 14 carbon ring atoms. A cycloalkyl group may be a single carbocyclic ring and typically contains 3 to 7 carbon ring atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl can optionally be bi or tricyclic fused together, such as decahydronaphthyl. The cycloalkyl group may be substituted or unsubstituted. "Heterocycle or heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic group, which includes 3 to 20 ring atoms, and may be 3 to 16, 3 to 14, 3 to 12, for example Three to ten, three to eight, three to six, or five to six ring atoms, one or more of which are selected from nitrogen, oxygen, or S (O)m
(Where m is an integer from 0 to 2), excluding the ring portion of -O-O-, -O-S-, or -S-S-, and the remaining ring atoms are carbon. It preferably includes 3 to 12 ring atoms, more preferably 3 to 10 ring atoms, most preferably 5 or 6 ring atoms, of which 1 to 4 are heteroatoms, more preferably 1 to 3 are heteroatoms, and most preferably 1 ~ 2 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, pyridinyl, fluorazinyl, morpholinyl, thiomorpholinyl, homoperazinyl, oxetanyl, and azetidine base. Polycyclic heterocyclyls include fused, bridged or spiro polycyclic heterocyclyls. The heterocyclic ring or heterocyclic group may be substituted or unsubstituted. "Aryl" means an aromatic monocyclic or fused polycyclic group containing 6 to 14 carbon atoms, preferably 6 to 10 members, such as phenyl and naphthyl, most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include: withThe aryl group may be substituted or unsubstituted. "Heteroaryl or heteroaryl ring" refers to a heteroaromatic system containing 5 to 14 ring atoms, where 1 to 4 ring atoms are selected from heteroatoms including oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10 members. More preferred heteroaryl groups are 5- or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, tetrazolyl, Oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. Non-limiting examples include: withThe heteroaryl group may be substituted or unsubstituted. "Halogen" means fluorine, chlorine, bromine or iodine. "Cyano" refers to -CN. "Optional" or "optionally" means that the event or environment described later can, but need not, occur, and the description includes situations where the event or environment occurs or does not occur. For example, "an optionally substituted heterocyclic group" means that an alkyl group may but need not exist, and this description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group . "Substituted" refers to one or more hydrogen atoms in a group, preferably five, more preferably one to three hydrogen atoms are independently substituted with each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amine or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated (eg, olefinic) bond. The substituent includes, but is not limited to, hydroxyl, amine, halogen, cyano, C1 - 6
Alkyl, C1 - 6
Alkoxy, C2 - 6
Alkenyl, C2 - 6
Alkynyl, C3-8
Cycloalkyl and the like. (2) "Pharmaceutical composition" means a composition containing one or more of the compounds described herein, or a pharmaceutically acceptable salt or prodrug thereof, and other components such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity. "Isomers" refer to compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or the arrangement of their atoms in space, and are called "isomers." Isomers whose atomic arrangement is different are called "stereoisomers". Stereoisomers include optical isomers, geometric isomers, and conformers.的 The compounds of the present invention can exist as optical isomers. Depending on the configuration of the substituents around the chiral carbon atom, these optical isomers are in the "R" or "S" configuration. Optical isomers include enantiomers and diastereomers. Methods for preparing and separating optical isomers are known in the art.几何 The compounds of the present invention may also exist as geometric isomers. The present invention contemplates various geometric isomers and mixtures thereof resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl or heterocyclic groups. The substituents around the carbon-carbon double bond or carbon-nitrogen bond are designated as the Z or E configuration, and the substituents around the cycloalkyl or heterocyclic ring are designated as the cis or trans configuration.化合物 The compounds of the present invention may also exhibit tautomerism, such as keto-enol tautomerism. It should be understood that the present invention includes any tautomeric or stereoisomeric form and mixtures thereof, and is not limited to any one of the tautomeric or stereoisomeric forms used in the nomenclature of a compound or the chemical structural formula. "Isotopes" are all isotopes of the atoms present in the compounds of the invention. Isotopes include those atoms that have the same atomic number but different mass numbers. Examples of isotopes suitable for incorporation into the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to, respectively2
H,3
H,13
C,14
C,15
N,18
O,17
O,31
P,32
P,35
S,18
F and36
Cl. The isotopically-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples using appropriate isotopically-labeled reagents instead of non-isotopically-labeled reagents. Such compounds have a variety of potential uses, such as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds have the potential to beneficially alter biological, pharmacological or pharmacokinetic properties. "Prodrug" means that a compound of the invention can be administered in the form of a prodrug. A prodrug refers to a derivative of a biologically active compound of the present invention that is transformed under physiological conditions in the living body, such as by oxidation, reduction, hydrolysis, etc. (they each use an enzyme or without the participation of an enzyme). An example of a prodrug is a compound in which the amine group in the compound of the present invention is tritiated, alkylated, or phosphorylated, such as eicosylamidoamino, propylaminoamido, neopentyloxymethyl Amino groups, or where hydroxyl groups are tritiated, alkylated, phosphorylated, or converted to borates, such as ethoxy, palmito, neopentyl, succinyl, fumarate, Alaninoxy, or in which the carboxyl group is esterified or amidated, or in which the sulfhydryl group forms a disulfide bridge with a carrier molecule, such as a peptide, that selectively delivers the drug to the target and / or to the cytosol of the cell. These compounds can be prepared from a compound of the present invention according to a known method. "Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt made from a pharmaceutically acceptable base or acid, including an inorganic base or acid and an organic base or acid. Where the compounds of the invention contain one or more acidic or basic groups, the invention also includes their corresponding pharmaceutically acceptable salts. Thus, the compounds of the invention containing acidic groups can exist in the form of salts and can be used according to the invention, for example as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium, potassium, calcium, magnesium, or salts with amines or organic amines, such as ethylamine, ethanolamine, triethanolamine, or amino acids. The compounds of the invention containing basic groups may exist in the form of salts and may be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propylene Acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid , Adipic acid, and other acids known to those skilled in the art. If the compounds of the invention contain both acidic and basic groups in the molecule, the invention includes, in addition to the salt forms mentioned, internal salts or internal ammonium salts. Each salt can be obtained by conventional methods known to those skilled in the art, such as by contacting these with organic or inorganic acids or bases in a solvent or dispersant or by anion exchange or cation exchange with other salts. Therefore, when referring to "compounds", "compounds of the invention" or "compounds of the invention" in this application, all such compound forms are included, such as their prodrugs, stable isotope derivatives, pharmaceutically acceptable salts , Isomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof.术语 As used herein, the term "tumor" includes both benign and malignant tumors (eg, cancer). As used herein, the term "cancer" includes various malignancies in which FGFR is involved, including but not limited to non-small cell lung cancer, esophageal cancer, melanoma, rhabdomyosarcoma, renal cell carcinoma, multiple myeloma, breast cancer, ovarian cancer , Endometrial cancer, cervical cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, lung cancer, breast cancer, prostate cancer, and liver cancer (such as hepatocellular carcinoma), more specifically liver cancer, gastric cancer, non-small cell lung cancer, and bladder cancer .术语 As used herein, the term "inflammatory disease" refers to any inflammatory disease in which FGFR is involved in its inflammation, such as osteoarthritis. As used herein, the term "therapeutically effective amount" means an amount that includes a compound of the invention that is effective to inhibit the function of FGFR and / or treat or prevent the disease. Synthetic method The present invention also provides a method for preparing the compound. The preparation of the compound of the general formula (I) of the present invention can be accomplished by the following exemplary methods and examples, but these methods and examples should not be considered as limiting the scope of the present invention in any way. The compounds of the present invention can also be synthesized by synthetic techniques known to those skilled in the art, or a combination of methods known in the art and methods of the present invention can be used in combination. The products obtained in each step are obtained by separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, chromatographic separation, and the like. The starting materials and chemical reagents required for the synthesis can be routinely synthesized or purchased according to the literature (available from SciFinder). The pyrazole compounds of the general formula (I) of the present invention can be synthesized according to the route described in Method A: 1) The starting material A1 can be obtained by Sandmeyer reaction A2, or can be brominated to obtain A3 R1
Can be -CN or ester (-COOR, where R is alkyl); 2) A2 or A3 and precursor XL ~ NP (where X is a leaving group, L ~ NP is a functional group containing a protected amine group, P is an amine-protecting group) A4 undergoes a substitution reaction under base catalysis to form A4, and A4 can also be obtained by a Mitsunobu reaction with a precursor with a hydroxyl group (HO-L ~ NP); 3) When R of A41
Yes -CN, in NaOH / H2
O2
Hydrolyzed to amidine A5 under the conditions; when R of A41
It is an ester (-COOR, where R is an alkyl group), which is firstly hydrolyzed to a carboxylic acid under basic conditions (such as LiOH), and then amidated to obtain A5; 4) A5 is coupled with alkyne to obtain A6 by Sonogashira; 5) A6 Deprotection of the amine group in the middle to obtain A7; 6) The amine group in A7 is derivatized with a chemical reagent (such as BrCN, allyl chloride, etc.) containing a functional group that reacts with a cysteine residue in the kinase ligand binding domain The target compound A8 was obtained. Method A:In addition, it can also be synthesized according to the route described in Method B. The pyrazole NH protecting group Q is introduced in the second step, and the common intermediate B7 is deprotected in the fifth step. The pyrazole NH of B7 is different from the one containing the protected amine group. The precursor reaction replaces the reaction, and after deprotection and derivatization, the target product A8 is obtained. Method B:The pyrazole compounds of the general formula (I) of the present invention can also be synthesized according to the route described in Method C: 1) A4 is firstly coupled with alkyne via Sonogashira to obtain C1; 2) -NH in C12
Substitution reaction occurs under base catalysis or C2 is generated by reductive amination; 3) CN of C2 is in NaOH / H2
O2
It is hydrolyzed to ammonium amine C3 under certain conditions. In some cases, it is necessary to first protect -NH- with Boc, and then hydrolyze. Finally, it is deprotected and derivatized to obtain the target product C5. C5 can also be obtained by directly replacing A8 . Method C:Example The structure of a europium compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR was measured using a Bruker AVANCE-400 or Varian Oxford-300 nuclear magnetic analyzer, and the solvent was DMSO-DMSO-d 6
), Deuterated chloroform (CDC13
), Deuterated methanol (CD3
OD), internal standard is tetramethylsilane (TMS), chemical shift is 10-6
(Ppm) is given as a unit. MS was measured using an Agilent SQD (ESI) mass spectrometer (manufacturer: Agilent, model: 6120). HPLC was measured using an Agilent 1200 DAD high pressure liquid chromatography (Sunfirc C18, 150 × 4.6mm, 5 μm column) and a Waters 2695-2996 high pressure liquid chromatography (Gimini C18 150 × 4.6mm, 5 μm column). The thin-layer chromatography silica gel plate uses Qingdao Ocean GF254 silicone plate. The thin-layer chromatography (TLC) silicon plate uses a size of 0.15mm ~ 0.2mm, and the thin-layer chromatography separation and purification product uses a size of 0.4mm ~ 0.5mm Silicone board. Column chromatography generally uses Qingdao Ocean 200-300 mesh silica gel as the carrier. The known starting materials of the present invention can be synthesized by or in accordance with methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Beijing Coupling companies such as chemicals.如 Unless otherwise specified in the examples, the reaction is performed under an argon atmosphere or a nitrogen atmosphere. Argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L. The tritium atmosphere refers to a hydrogen gas balloon with a volume of about 1L connected to the reaction flask. For the pressurized hydrogenation reaction, Beijing Jiawei Kechuang Technology Co., Ltd. GCD-500G high-purity hydrogen generator and BLT-2000 medium-pressure hydrogenation instrument were used. The tritium hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times. Microwave reaction uses CEM Discover-SP type microwave reactor.如 Unless otherwise specified in the examples, the reaction temperature is room temperature, and the temperature range is 20 ° C-30 ° C. The monitoring of the reaction progress in the examples uses thin layer chromatography (TLC). The developing systems used in the reaction are A: dichloromethane and methanol; B: petroleum ether and ethyl acetate. The volume ratio of the solvent is based on The polarity of the compounds is adjusted. The eluent system for column chromatography and the eluent system for thin-layer chromatography for purifying compounds include A: dichloromethane and methanol systems; B: petroleum ether and ethyl acetate systems. It can be adjusted by different polarities. It can also be adjusted by adding a small amount of triethylamine and acidic or alkaline reagents. Example 1 (S) -1- (1-propenylpyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-methyl Amidine (R) -3- (Toluenesulfonyloxo) pyrrolidine-1-carboxylic acid third butyl ester (R) -3-hydroxypyrrolidine-1-carboxylic acid third butyl ester 1a (3.5 g, 18.7 mmol), triethylamine (5.25 mL, 37.9 mmol), 4-dimethylaminopyridine (0.35 g, 2.87 mmol) were dissolved in dichloromethane (50 mL), and p-toluenesulfonyl chloride (5.4 g, 28.1 mmol), and the reaction mixture was stirred at room temperature for 12 hours. Dilute with water (50 mL) and extract with ethyl acetate (100 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2/1) to obtain the target product (R)- 3- (Toluenesulfonyloxo) pyrrolidine-1-carboxylic acid third butyl ester 1b (6.0 g, yellow oil), yield: 94%. MS m / z (ESI): 364 [M + 23] The second step ((3,5-dimethoxyphenyl) ethynyl) trimethylsilane is a mixture of 1-bromo-3,5-dimethyl Oxybenzene 1c (6.51g, 30mmol), trimethylsilylacetylene (8.8g, 90mmol), bis (triphenylphosphine) palladium chloride (1.05g, 1.5mmol), cuprous iodide (0.56g , 3.0 mmol), triethylamine (80 mL), and N, N-dimethylformamide (150 mL) were heated to 80 ° C. and stirred under nitrogen protection for 12 hours. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether) to obtain the target product ((3,5-dimethoxyphenyl) ethynyl) trimethylsilane 1d (6.2 g, brown solid), yield: 88%. MS m / z (ESI): 235 [M + 1] Step 1 1-Ethynyl-3,5-dimethoxybenzene will be ((3,5-dimethoxyphenyl) ethynyl) trimethyl Monosilane 1d (3.0 g, 12.8 mmol) was dissolved in methanol (100 mL), potassium carbonate (3.5 g, 25.6 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether) to obtain the target product 1-ethynyl-3,5-dimethoxybenzene 1e (2 g, yellow solid), yield: 96% . Step 4 3-Iodine-1H-pyrazole-4-carboxylic acid ethyl ester Dissolve 3-amino-1H-pyrazole-4-carboxylic acid ethyl ester 1f (4.7g, 30.3mmol) in concentrated hydrochloric acid (12M, 40mL) , And cooled to 0 ° C, added a solution of sodium nitrite (4.25g, 60mmol) (7.5mL), and stirred for 5 minutes, and then slowly added a solution of potassium iodide (12.5g, 75mmol) (17.5mL), and continued stirring for 30 minutes. The reaction mixture was poured into a saturated sodium thiosulfate solution (200 mL), and extracted with ethyl acetate (400 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2/1) to obtain the target product 3-iodine- 1 g of 1H-pyrazole-4-carboxylic acid ethyl ester (6.4 g, pale yellow solid), yield: 80%. MS m / z (ESI): 267 [M + 1] Step 5 (S) -1- (1- (Third-butoxycarbonyl) pyrrolidin-3-yl) -3-iodo-1H-pyrazole Ethyl-4-carboxylate 1 g (4.5 g, 17 mmol) of 3-iodo-1H-pyrazole-4-carboxylic acid ethyl ester, (R) -3- (tosylhydrazone oxo) pyrrolidine-1-carboxylic acid third A mixture of butyl ester 1b (6.1 g, 17.8 mmol), cesium carbonate (7.5 g, 20.4 mmol) and N, N-dimethylformamide (50 mL) was heated to 80 ° C and stirred for 3 hours. The reaction mixture was cooled to room temperature, poured into a saturated sodium bicarbonate solution (200 mL), and extracted with ethyl acetate (300 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5/1 to 2/1) to obtain the target product. (S) -1- (1- (Third-butoxycarbonyl) pyrrolidin-3-yl) -3-iodo-1H-pyrazole-4-carboxylic acid ethyl ester 1h (3.1 g, pale yellow solid), produced Rate: 42%. MS m / z (ESI): 458 [M + 23] Step 6 (S) -1- (1- (Third butoxycarbonyl) pyrrolidin-3-yl) -3-((3,5- Dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxylic acid ethyl ester (S) -1- (1- (third-butoxycarbonyl) pyrrolidin-3-yl) -3-iodine -1H-pyrazole-4-carboxylic acid ethyl ester 1h (1g, 2.25mmol), 1-ethynyl-3,5-dimethoxybenzene 1e (0.75g, 4.5mmol), bis (triphenylphosphine) chloride A mixture of palladium (175mg, 0.25mmol), cuprous iodide (95mg, 0.5mmol), triethylamine (12.5ml) and N, N-dimethylformamide (12.5mL) was heated to 80 ° C, Stir for 12 hours. The reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2/1) to obtain the target product (S) -1- (1- ( Tributoxycarbonyl) pyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxylic acid ethyl ester 1i (0.95 g, yellow oil Material), yield: 90%. MS m / z (ESI): 414 [M + 1-56] Step 7 (S) -1- (1- (third butoxycarbonyl) pyrrolidin-3-yl) -3-((3, 5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxylic acid (S) -1- (1- (third-butoxycarbonyl) pyrrolidin-3-yl) -3- ( (3,5-Dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxylic acid ethyl ester 1i (0.30 g, 0.64 mmol) was dissolved in tetrahydrofuran (3 mL), and a sodium hydroxide solution (4 M, 2 mL) was added. ), Stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, the residue was acidified with hydrochloric acid (6M, 1 mL), and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure to obtain the target product (S) -1- (1- (third-butoxycarbonyl) pyrrolidin-3-yl)- 3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxylic acid 1j (200 mg, pale yellow oil), yield: 71%. MS m / z (ESI): 386 [M + 1-56] Step 8 (S) -3- (4-aminomethylmethyl-3-((3,5-dimethoxyphenyl) ethynyl ) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -1- (1- (third-butoxycarbonyl) pyrrolidin-3-yl) -3- ((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxylic acid 1j (220 mg, 0.5 mmol), ammonium chloride (270 mg, 5 mmol), O- (7-azabenzene Benzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium hexafluorophosphate (HATU) (228 mg, 0.6 mmol), N, N-diisopropylethylamine (129 mg , 1 mmol) and N, N-dimethylformamide (5 mL) were stirred at room temperature overnight. Dilute with water and extract with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed under reduced pressure. The residue was purified by thin-layer silica gel preparative chromatography (dichloromethane / methanol = 20/1) to obtain the target product (S)- 3- (4-Aminomethylamino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 1k (140 mg, white solid), yield: 64%. MS m / z (ESI): 385 [M + 1-56] Step 9 (S) -3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidine-3- (Syl) -1H-pyrazole-4-carboxamide, (S) -3- (4-Aminomethoxo-3-((3,5-dimethoxyphenyl) ethynyl) -1H-py The reaction mixture of azole-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 1k (50mg, 0.11mmol), hydrochloric acid (6M, 5mL) and dioxane (5mL) was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure to give the target product (S) -3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H-pyrazole-4 -Formamidine 1 1 (42 mg, hydrochloride, crude), yield: 100%. MS m / z (ESI): 341 [M + 1] Step 10 (S) -1- (1-propenylpyrrolidin-3-yl) -3-((3,5-dimethoxybenzene (Yl) ethynyl) -1H-pyrazol-4-methylamidine (S) -3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3-yl) To a mixture of -1H-pyrazole-4-carboxamide hydrochloride 1 l (30 mg, 0.08 mmol), N, N-diisopropylethylamine (31 mg, 0.24 mmol) and tetrahydrofuran (15 mL) was added dropwise propylene arsine A solution of chlorine (11 mg, 0.12 mmol) in tetrahydrofuran (5 mL), and the reaction mixture was stirred at room temperature for 30 minutes. Add water (30 mL) to quench and extract with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed under reduced pressure. The residue was purified by thin-layer silica gel preparative chromatography (dichloromethane / methanol = 20/1) to obtain the target product (S)- 1- (1-propenylpyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 1 (15mg, white (Solid), yield: 50%. MS m / z (ESI): 395 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.10 (d,J
= 9.8 Hz, 1H), 6.96 (brs, 1H), 6.71 (d,J
= 2.3 Hz, 2H), 6.54-6.52 (m, 1H), 6.46-6.39 (m, 2H), 5.80 (brs, 1H), 5.76-5.72 (m, 1H), 5.01-4.92 (m, 1H), 4.13-4.00 (m, 2H), 3.90-3.75 (m, 8H), 2.62-2.44 (m, 2H). Example 2 1- (1-propenylpyridin-4-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide First step 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxylic acid ethyl ester Compound 3-iodo-1H-pyrazole- 1 g (2.01 g, 7.5 mmol) of ethyl 4-formate was dissolved in tetrahydrofuran (80 mL) and cooled to 0 ° C. Sodium hydride (60% mineral oil dispersion, 0.42 g, 10.5 mmol) was added and stirred at room temperature for 1 hour. . To the reaction mixture was added 2- (trimethylsilyl) ethoxymethyl chloride (1.76 g, 10.5 mmol), and stirring was continued for 15 hours. To the reaction mixture was added saturated saline (100 mL), and the mixture was extracted with ethyl acetate (150 mL × 2). The organic phases were combined and washed with saturated brine (100 mL), the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5/1 to 1/2) to obtain the target product 3- Iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxylic acid ethyl ester 2a (2.6 g, colorless oil), yield: 87% . MS m / z (ESI): 397 [M + 1] Second step 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4- Formic acid Dissolve the compound 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxylic acid ethyl ester 2a (2.6 g, 6.5 mmol) in tetrahydrofuran (40 mL), an aqueous lithium hydroxide solution (1M, 13 mL) was added and stirred at room temperature for 15 hours. Dilute with water (20 mL), acidify with hydrochloric acid (1M) to pH = 4-5, and extract with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), the solvent was removed under reduced pressure, and the target product 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) was obtained after drying. -1H-pyrazole-4-carboxylic acid 2b (2.03 g, white solid), yield: 85%. MS m / z (ESI): 391 [M + 23] Step 3 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4- Methylamine is a compound 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxylic acid 2b (2.03 g, 5.5 mmol), diiso Propylethylamine (2.13g, 16.5mmol) and N, N-dimethylformamide (20mL) were mixed, and O- (7-azabenzotriazol-1-yl) -N, N , N ', N'-tetramethylurenium hexafluorophosphate (HATU) (2.5 g, 6.6 mmol) and 1-hydroxybenzotriazole (890 mg, 6.6 mmol), stirred at room temperature for 1 hour and added solid Ammonium chloride (1.47 g, 27.5 mmol), stirring was continued for 15 hours. To the reaction mixture was added saturated brine (30 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine (100 mL). After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20/1) to obtain the target product 3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxamide 2c (2.3 g, yellow oil), yield: 100%. MS m / z (ESI): 368 [M + 1] Step 4 3-((3,5-dimethoxyphenyl) ethynyl) -1-((2- (trimethylsilyl) Ethoxy) methyl) -1H-pyrazole-4-carboxamide. Compound 3-iodo-1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole -4-formamidine 2c (2.7 g, 7.3 mmol), 1-ethynyl-3,5-dimethoxybenzene (1.78 g, 11 mmol), triethylamine (2.2 g, 21.9 mmol), di (tri Phenyl phosphorus) palladium chloride (512 mg, 0.73 mmol) and anhydrous tetrahydrofuran (70 mL) were mixed, deoxidized, and stirred at room temperature under an argon atmosphere for 15 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 10/1 to 2/1) to give the target product 3-((3,5-dimethoxyphenyl) acetylene Yl) -1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxamide 2d (1.5 g, yellow solid), yield: 51%. MS m / z (ESI): 402 [M + 1] Step 5 3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide will be 3- ( (3,5-dimethoxyphenyl) ethynyl) -1-((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carboxamide 2d ( 1.4g, 3.5mmol), ethylenediamine (525mg, 8.75mmol) and tetrahydrofuran (30mL) were mixed, and a tetrahydrofuran solution (1M, 17.5mL, 17.5mmol) of tetrabutylammonium fluoride was added. After heating under reflux for 15 hours, it was cooled to room temperature, saturated brine (20 mL) was added, and extraction was performed with ethyl acetate (100 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20/1) to obtain the target product 3-((3 , 5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-methylamidine 2e (600 mg, white solid), yield: 63%. MS m / z (ESI): 272 [M + 1] Step 6 4- (4-Aminomethylamido-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole 1-yl) piperidine-1-carboxylic acid third butyl ester compound 3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 2e (180mg , 0.66 mmol), 4-bromopyridine-1-carboxylic acid third butyl ester (264 mg, 0.99 mmol), potassium carbonate (182 mg, 1.32 mmol) and N, N-dimethylformamide (10 mL) , Heat to 75 ° C, and stir for 15 hours. Water (30 mL) was added, and extraction was performed with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20/1) to obtain the target product 4- (4-aminomethylamido-3-((3 , 5-Dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) piperidine-1-carboxylic acid third butyl ester 2f (120 mg, yellow solid, containing regioisomers), yield : 40%. MS m / z (ESI): 477 [M + 23] Step 7 3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyridin-4-yl) -1H-pyridine Azol-4-methylamidine is a compound 4- (4-aminomethylamido-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyridine- 1-formic acid third butyl ester 2f (120 mg, 0.26 mmol, mixture) was dissolved in ethanol (20 mL), hydrogen chloride in ethanol solution (4M, 1 mL, 4 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The solvent was removed under reduced pressure, and the residue was dissolved in methanol (20 mL), and then adjusted to pH = 8-9 with a saturated sodium bicarbonate solution. After the solvent was removed again under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane / methanol = 10/1) to obtain the target product 3-((3,5-dimethoxyphenyl) ethynyl) 1- (pyridin-4-yl) -1H-pyrazole-4-carboxamide 2 g (25 mg, white solid), yield: 27%. MS m / z (ESI): 355 [M + 1] Step 8 1- (1-propenylpyridin-4-yl) -3-((3,5-dimethoxyphenyl) ethynyl ) -1H-pyrazole-4-carboxamide will compound 3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyridin-4-yl) -1H-pyrazole-4 -Formamidine 2g (25mg, 0.07mmol), allyl chloride (10mg, 0.11mmol), solid sodium bicarbonate (18mg, 0.21mmol), water (2mL) and tetrahydrofuran (10mL) at 0 ° C and mixed at Stir at this temperature for 10 hours. Extract with ethyl acetate (20mL × 3), combine the organic phases and dry over anhydrous sodium sulfate, filter to remove the desiccant, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography (dichloromethane / methanol = 10 / 1), the target product 1- (1-propenylpyridin-4-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-methylpyrene is obtained Amine 2 (17 mg, white solid), yield: 60%. MS m / z (ESI): 409 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.10 (s, 1H), 7.01 (brs, 1H), 6.72 (d,J
= 2.2 Hz, 2H), 6.62 (dd,J
= 16.8, 10.6 Hz, 1H), 6.55 (t,J
= 2.2 Hz, 1H), 6.33 (dd,J
= 16.8, 1.5 Hz, 1H), 5.80 (brs, 1H), 5.76 (dd,J
= 10.6, 1.6 Hz, 1H), 4.81 (brs, 1H), 4.40 (t,J
= 11.4 Hz, 1H), 4.18 (brs, 1H), 3.82 (s, 6H), 3.26 (brs, 1H), 2.89 (brs, 1H), 2.42-2.25 (m, 2H), 2.08-2.00 (m, 2H). Example 3-6 was synthesized with reference to the operation steps of Example 2: Example 3 1- (1-propenylazetidin-3-yl) -3-((3,5-dimethoxyphenyl ) Ethynyl) -1H-pyrazole-4-carboxamideMS m / z (ESI): 381 [M + 1]1
H NMR (400 MHz, DMSO-d 6
) δ 8.43 (s, 1H), 7.30 (s, 2H), 6.73 (d,J
= 2.2 Hz, 2H), 6.60 (t,J
= 2.2 Hz, 1H), 6.38 (dd,J
= 17.0, 10.3 Hz, 1H), 6.16 (dd,J
= 17.0, 2.1 Hz, 1H), 5.73 (dd,J
= 10.3, 2.1 Hz, 1H), 5.41-5.28 (m, 1H), 4.71 (t,J
= 8.6 Hz, 1H), 4.50 (dd,J
= 9.2, 4.9 Hz, 1H), 4.46-4.36 (m, 1H), 4.20 (dd,J
= 10.7, 4.8 Hz, 1H), 3.78 (s, 6H). Example 4 1-((1-propenylpyridin-4-yl) methyl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-methyl AmidineMS m / z (ESI): 423 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.00 (s, 1H), 6.98 (brs, 1H), 6.72 (s, 2H), 6.61-6.54 (m, 2H), 6.28 (d,J
= 16.8 Hz, 1H), 5.87 (brs, 1H), 5.70 (d,J
= 10.5 Hz, 1H), 4.72 (brs, 1H), 4.04 (brs, 3H), 3.82 (s, 6H), 3.05 (brs, 1H), 2.64 (brs, 1H), 2.27 (brs, 1H), 1.69 (brs, 2H), 1.24 (brs, 2H). 5 Example 5 1- (4-propenylaminoaminocyclohexyl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamideMS m / z (ESI): 423 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 8.25 (s, 1H), 6.77 (d,J
= 2.3 Hz, 2H), 6.58 (t,J
= 2.3 Hz, 1H), 6.38 (dd,J
= 17.1, 10.0 Hz, 1H), 6.26 (dd,J
= 17.1, 2.0 Hz, 1H), 5.68 (dd,J
= 10.1, 2.0 Hz, 1H), 4.38-4.33 (m, 1H), 4.13-4.11 (m, 1H), 3.82 (s, 6H), 2.28-2.18 (m, 2H), 2.07-2.02 (m, 2H ), 1.96-1.80 (m, 4H). Example 6 3-((3,5-dimethoxyphenyl) ethynyl) -1- (2- (N-methacrylfluorenylamino) ethyl) -1H-pyrazole-4-methyl AmidineMS m / z (ESI): 383 [M + 1]1
H NMR (300 MHz, DMSO-d 6
) δ 8.24 (s, 1H), 7.10-6.90 (m, 2H), 6.76 (s, 2H), 6.69-6.54 (m, 2H), 6.07 (d,J
= 16.5 Hz, 1H), 5.64 (d,J
= 9.8 Hz, 1H), 4.37 (t,J
= 5.7 Hz, 2H), 3.89-3.80 (m, 8H), 2.94 (s, 3H). Example 7 (S) -1- (1-propenylpyrrolidin-3-yl) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyridine Azole-4-methylamidine First step 5-Amino-3-bromo-1H-pyrazole-4-carbonitrile Dissolve the compound 5-amino-1H-pyrazole-4-carbonitrile 7a (20 g, 185 mmol) in N, N-di Methylformamide (200 mL) was cooled to 0 ° C, and N-bromosuccinimide (34 g, 190 mmol) was added in portions, and the mixture was warmed to room temperature and stirred for 2 hours. The reaction solution was poured into a sodium sulfite solution, and extracted with ethyl acetate (200 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloro Methane / methanol = 20/1) to give the target product 5-amino-3-bromo-1H-pyrazole-4-carbonitrile 7b (32 g, yellow solid), yield: 93%. MS m / z (ESI): 187/189 [M + 1] Step (S) -3- (5-amino-3-bromo-4-cyano-1H-pyrazol-1-yl) pyrrole Alkane-1-carboxylic acid third butyl ester 5-amino-3-bromo-1H-pyrazole-4-carbonitrile 7b (10 g, 53.8 mmol), 3- (toluenesulfonyloxo) pyrrolidine-1 -A mixture of third butyl formate (22 g, 64.5 mmol), cesium carbonate (58 g, 107.6 mmol) and acetonitrile (250 mL) was heated to 90 ° C. and reacted for 4 hours. Cool to room temperature, filter, and wash the filter cake with dichloromethane. The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5/1) to obtain the target product (S)- 3- (5-amino-3-bromo-4-cyano-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7c (5g, yellow oil), yield: 26%. MS m / z (ESI): 300/302 [M + 1-56] The third step (S) -3- (5-amino-4-cyano-3-((3,5-dimethoxy) Phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester (S) -3- (5-amino-3-bromo-4-cyano-1H -Pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7c (5g, 14.1mmol), cuprous iodide (0.6g, 2.8mmol), triethylamine (9mL), [1,1 A mixture of '-bis (diphenylphosphino) ferrocene] palladium dichloride (2g, 2.8mmol) and N, N-dimethylformamide (150mL) was heated to 80 ° C under argon Then, 1-ethynyl-3,5-dimethoxybenzene (14 g, 84.5 mmol) was added in portions and stirred for 2 hours. After cooling to room temperature, the reaction solution was poured into water and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5/1) to obtain the target product (S) -3- ( 5-amino-4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (5 g, brown oil), yield: 81%. MS m / z (ESI): 382 [M + 1-56] Step 4 (S) -3- (5-Amino-4-aminomethylamido-3-((3,5-dimethoxy) Phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester (S) -3- (5-amino-4-cyano-3-((3 , 5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (5g, 11.4mmol), sodium hydroxide (1.5g, 37.5 mmol, dissolved in 2 mL of water), ethanol (50 mL) and dimethyl sulfene (10 mL) was cooled to 0 ° C, hydrogen peroxide (20 mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was poured into a sodium sulfite solution, and extracted with ethyl acetate (100 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether). / Ethyl acetate = 1/1) to give the target product (S) -3- (5-amino-4-aminomethylmethyl-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7e (5 g, brown oil), yield: 96%. MS m / z (ESI): 400 [M + 1-56] Step 5 (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- ( Pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide. Compound (S) -3- (5-amino-4-aminoformamyl-3-((3,5-dimethoxy) Phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7e (5 g, 11 mmol) was dissolved in dichloromethane (100 mL), and trifluoroacetic acid (15 mL) was added ), Stirred at room temperature for 2 hours. Concentrated under reduced pressure to give the target product (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H-pyrazole 4-methylamidine 7f (7.1 g, brown oil, trifluoroacetate, crude), yield:> 100%, the product was used in the next reaction without purification. MS m / z (ESI): 356 [M + 1] Step 6 (S) -1- (1-propenylpyrrolidin-3-yl) -5-amino-3-((3,5- Dimethoxyphenyl) ethynyl) -1H-pyrazole-4-methylamidine. Compound (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) 1- (Pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 7f (7.1 g, 11 mmol, trifluoroacetate, crude) was dissolved in tetrahydrofuran (50 mL) and cooled to 0 ° C. A saturated sodium bicarbonate solution (20 mL) and propylene chloride (900 mg, 10 mmol) were added successively, and stirred for 30 minutes. The reaction solution was poured into water (100 mL), and extracted with dichloromethane (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1/2) to obtain the target product (S) -1- ( 1-propenylpyrrolidin-3-yl) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 7 (1.9 g, white solid), yield: 42%. MS m / z (ESI): 410 [M + 1]1
H NMR (400 MHz, DMSO-d 6
) δ 7.18 (brs, 1H), 6.75 (d,J
= 2.3 Hz, 2H), 6.69-6.55 (m, 3H), 6.20-6.14 (m, 1H), 5.72-5.67 (m, 1H), 5.03-4.91 (m, 1H), 4.01-3.96 (m, 1H ), 3.84-3.70 (m, 7H), 3.66-3.60 (m, 1H), 3.55-3.48 (m, 1H), 2.36-2.21 (m, 2H). Example 8 (S, E) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (1- (4- (dimethylamino) butane-2 -Alkenyl) pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide First step (S, E) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (1- (4- (dimethylamino) butane-2 -Alkenyl) pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide (E) -4- (dimethylamino) but-2-enoic acid (23 mg, 0.14 mmol), O -(7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium hexafluorophosphate (HATU) (64 mg, 0.17 mmol), (S) -5 -Amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 7f (50mg, 0.14mmol ), A reaction mixture of N, N-diisopropylethylamine (2 mL) and dichloromethane (3 mL) was stirred at room temperature for 1 hour. The reaction solution was poured into water and extracted with dichloromethane (20 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. The residue was purified by high performance liquid chromatography to obtain the target product (S, E) -5-amino-3-((3,5 -Dimethoxyphenyl) ethynyl) -1- (1- (4- (dimethylamino) but-2-enylfluorenyl) pyrrolidin-3-yl) -1H-pyrazole-4-methyl Amidine 8 (2.4 mg, white solid, formate), yield: 4%. MS m / z (ESI): 467 [M + 1]1
H NMR (400 MHz, DMSO-d 6
) δ 8.27 (brs, 1H), 7.20 (brs, 1H), 6.75 (d,J
= 2.3 Hz, 2H), 6.70-6.61 (m, 3H), 6.44-6.35 (m, 1H), 5.01-4.93 (m, 1H), 4.01-3.93 (m, 1H), 3.77 (s, 6H), 3.74-3.64 (m, 3H), 3.06-3.03 (m, 2H), 2.38-2.24 (m, 2H), 2.17-2.15 (m, 6H). Example 9 (S) -5-Amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (1- (2-fluoropropenyl) pyrrolidin-3-yl ) -1H-pyrazole-4-carboxamide First step (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (1- (2-fluoropropenyl) pyrrolidin-3-yl ) -1H-pyrazole-4-carboxamide. Compound (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidine-3- Group) -1H-pyrazole-4-carboxamide 7f (50 mg, 0.14 mmol) and 2-fluoroacrylic acid (15 mg, 0.17 mmol) were dissolved in dichloromethane, and N, N-diisopropylethylamine (54 mg , 0.42 mmol) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium hexafluorophosphate (HATU) (69 mg, 0.18 mmol) And stirred at room temperature for 2 hours. Water (10 mL) was added to dilute the reaction mixture, and the mixture was extracted with dichloromethane (10 mL x 3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin-layer silica gel preparative chromatography (dichloromethane / methanol = 20/1) to give the target product (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl ) -1- (1- (2-fluoropropenyl) pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 9 (3.6 mg, white solid), yield: 6%. MS m / z (ESI): 428 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 6.62 (t,J
= 2.5 Hz, 2H), 6.47 (t,J
= 2.3 Hz, 1H), 5.39 (dd,J
= 47.2, 3.5 Hz, 1H), 5.16 (ddd,J
= 16.6, 5.7, 3.5 Hz, 1H), 4.86-4.81 (m, 1H), 4.02-3.91 (m, 2H), 3.87-3.72 (m, 2H), 3.71 (s, 6H), 2.34-2.23 (m , 2H). Example 10 (S) -5-Amino-1- (1- (but-2-ynylfluorenyl) pyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) acetylene ) -1H-pyrazole-4-carboxamide First step (S) -5-Amino-1- (1- (but-2-alkynyl) pyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) acetylene (Syl) -1H-pyrazole-4-carboxamide (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3- Group) -1H-pyrazole-4-carboxamide 7f (50 mg, 0.14 mmol) and 2-butynyl acid (14 mg, 0.17 mmol) were dissolved in dichloromethane, and N, N-diisopropylethylamine was added (54 mg, 0.42 mmol) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium hexafluorophosphate (HATU) (69 mg, 0.18 mmol), and stirred at room temperature for 2 hours. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (10 mL x 3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by thin-layer silica gel preparative chromatography (dichloromethane / methanol = 20/1) to give the target product (S) -5-amino-1- (1- (but-2-ynylidene) pyrrolidine- 3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 10 (5.1 mg, pale yellow solid), yield: 9%. MS m / z (ESI): 422 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 6.62 (t,J
= 2.1 Hz, 2H), 6.47 (t,J
= 2.2 Hz, 1H), 4.85-4.81 (m, 1H), 4.01-3.86 (m, 2H), 3.77-3.62 (m, 7.5H), 3.54-3.46 (m, 0.5H), 2.32-2.27 (m , 2H), 1.95-1.93 (m, 3H). Example 11 (S, E) -5-Amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (1- (4-methoxybut-2-enefluorene) Yl) pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide The first step (E) -4-bromobut-2-enoic acid is methyl (E) -4-bromobut-2-enoate 11a (3g, 16.8mmol), lithium hydroxide monohydrate (1.1g, 25.3 mmol), tetrahydrofuran (50 mL) and water (50 mL) were mixed at 0 ° C and stirring was continued for 2 hours. After the reaction, tetrahydrofuran was washed away with petroleum ether, and the aqueous phase was adjusted to pH = 1 with 2M hydrochloric acid, and then extracted with ethyl acetate (100 mL × 2). After the organic phases were combined, the solvent was removed under reduced pressure to obtain the target product (E) -4-bromobut-2-enoic acid 11b (2.3 g, yellow oil). Yield: 83%. MS m / z (ESI): 163 [M-1] The second step (E) -4-methoxybut-2-enoic acid will compound (E) -4-bromobut-2-enoic acid 11b (100mg After dissolving 0.61 mmmol in methanol (5 mL), a methanol solution of sodium methoxide (30%, 0.55 mL, 3.05 mmol) was added and stirred for 15 hours. After removing the solvent under reduced pressure, the reaction mixture was dissolved in water, adjusted to pH = 1 with dilute hydrochloric acid, and then extracted with dichloromethane (10 mL × 3). After the organic phases were combined, the solvent was removed under reduced pressure to obtain the target product (E) -4-methoxybut-2-enoic acid 11c (50 mg, yellow oil). Yield: 71%.1
H NMR (400 MHz, CDCl3
) δ 7.13-7.03 (m, 1H), 6.15-6.07 (m, 1H), 4.18-4.11 (m, 2H), 3.48-3.38 (s, 3H). The third step (S, E) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (1- (4-methoxybut-2-ene) Group) pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide The compound (E) -4-methoxybut-2-enoic acid 11c (22 mg, 0.19 mmol), diisopropylethyl (67mg, 0.52mmol), (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H- Pyrazole-4-carboxamide 7f (50mg, 0.13mmol), 2- (7-benzotriazole) -N, N, N ', N'-tetramethylurea hexafluorophosphate (72mg, 0.19 mmol) and N, N-dimethylformamide (10 mL) were mixed and stirred for 2 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (30 mL) and washed with water and saturated brine in this order. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20/1) to obtain the target product (S, E) -5-amino-3-((3,5-dimethyl) Oxyphenyl) ethynyl) -1- (1- (4-methoxybut-2-enylfluorenyl) pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 11 (30mg, White solid), yield: 51%. MS m / z (ESI): 454 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 6.98 (d,J
= 15.3 Hz, 1H), 6.86 (brs, 1H), 6.72 (d,J
= 2.1 Hz, 2H), 6.54 (s, 1H), 6.39 (dd,J
= 27.7, 16.0 Hz, 1H), 5.54 (brs, 1H), 4.73-4.70 (m, 1H), 4.14-4.12 (m, 2H), 4.05-4.00 (m, 2H), 3.95-3.93 (m, 1H ), 3.82 (s, 6H), 3.77-3.68 (m, 1H), 3.43 (d,J
= 10.1 Hz, 3H), 2.72 (brs, 0.5H), 2.54 (brs, 0.5H), 2.43-2.35 (m, 1H). Example 12 (S) -5-Amino-1- (1-cyanopyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole 4-formamidine First step (S) -5-amino-1- (1-cyanopyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole 4-Methylpyramine is compound (S) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H-pyridine Azole-4-methylamidine 7f (50mg, 0.14mmol) was dissolved in tetrahydrofuran (2mL), triethylamine (1mL) was added, cooled to 0 ° C, cyanogen bromide (17mg, 0.15mmol) was added, and stirred at 0 ° C After 2 hours, warm to room temperature and continue stirring for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by thin-layer silica gel preparative chromatography (dichloromethane / methanol = 15/1) to obtain the target product (S) -5-amino-1- (1-cyanopyrrolidine-3). -Yl) -3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 12 (18 mg, white solid), yield: 34%. MS m / z (ESI): 381 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 6.77 (brs, 1H), 6.68 (d,J
= 1.9 Hz, 2H), 6.50 (s, 1H), 5.75 (s, 2H), 5.67 (brs, 1H), 4.79-4.73 (m, 1H), 3.84-3.73 (m, 9H), 3.61-3.53 ( m, 1H), 2.53-2.43 (m, 1H), 2.37-2.26 (m, 1H). Example 13-16 was synthesized by referring to the operation steps of Example 7: Example 13 (R) -1- (1-propenylpyrrolidin-3-yl) -5-amino-3-((3,5 -Dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamideMS m / z (ESI): 410 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 6.73 (d,J
= 1.9 Hz, 2H), 6.71-6.60 (m, 1H), 6.58 (brs, 1H), 6.32 (dd,J
= 16.8, 1.7 Hz, 1H), 5.84-5.74 (m, 1H), 5.04-4.91 (m, 1H), 4.09 (m, 0.5H), 3.98 (td,J
= 11.1, 4.0 Hz, 1H), 3.91 (dd,J
= 7.8, 5.6 Hz, 1H), 3.86 (dd,J
= 9.9, 4.4 Hz, 1H), 3.81 (s, 6H), 3.73-3.63 (m, 0.5H), 2.47 (dd,J
= 13.2, 6.7 Hz, 1H), 2.38 (dd,J
= 13.6, 7.0 Hz, 1H). Example 14 1- (1-propenylazetidin-3-yl) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole 4-formamidineMS m / z (ESI): 396 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 6.75 (d,J
= 2.3 Hz, 2H), 6.60 (t,J
= 2.2 Hz, 1H), 6.45-6.28 (m, 2H), 5.80 (dd,J
= 10.1, 2.1 Hz, 1H), 5.29-5.21 (m, 1H), 4.79-4.64 (m, 2H), 4.54-4.47 (m, 1H), 4.46-4.39 (m, 1H), 3.82 (s, 6H ). Example 15 1- (1-propenylpyridin-4-yl) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4- FormamidineMS m / z (ESI): 424 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 6.88-6.78 (m, 1H), 6.73 (d,J
= 2.2 Hz, 2H), 6.58 (t,J
= 2.2 Hz, 1H), 6.24 (dd,J
= 16.8, 1.7 Hz, 1H), 5.78 (dd,J
= 10.7, 1.7 Hz, 1H), 4.73 (d,J
= 13.2 Hz, 1H), 4.47-4.36 (m, 1H), 4.30 (d,J
= 13.3 Hz, 1H), 3.81 (s, 6H), 3.32-3.24 (m, 1H), 2.91 (t,J
= 9.9 Hz, 1H), 2.02 (d, J = 4.5 Hz, 4H). Example 16 1-((1-propenylpyrrolidin-3-yl) methyl) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyridine Azole-4-methylamidineMS m / z (ESI): 424 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 6.73 (s, 2H), 6.66-6.55 (m, 2H), 6.28 (d,J
= 16.7 Hz, 1H), 5.75 (d,J
= 10.4 Hz, 1H), 4.13-3.99 (m, 2H), 3.82 (s, 6H), 3.78-3.61 (m, 2H), 3.48 (dd,J
= 14.8, 7.4 Hz, 1H), 3.39-3.34 (m, 1H), 2.94-2.75 (m, 1H), 2.20-2.02 (m, 1H), 1.94-1.71 (m, 1H). Example 17 (S) -1- (1-propenepyrrolidin-3-yl) -5-amino-3-((2-fluoro-3,5-dimethoxyphenyl) ethynyl) -1H -Pyrazole-4-carboxamide Step 1 1-Ethynyl-2-fluoro-3,5-dimethoxybenzene Dissolve the mixture 1-ethynyl-3,5-dimethoxybenzene 1e (2g, 12.3mmol) in acetonitrile (15mL) , Reduce the temperature to 0 ° C, and add 1-chloromethyl-4-fluoro-1,4-diazobicyclo 2.2.2 octane bis (tetrafluoroborate) salt (6.6g, 18.5mmol) in portions. And then stirred at room temperature overnight. The reaction solution was poured into water (50 mL) and extracted with dichloromethane (30 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography ( Petroleum ether / ethyl acetate = 30/1), the target product 1-ethynyl-2-fluoro-3,5-dimethoxybenzene 17a (800 mg, yellow solid) was obtained, yield: 36%.1
H NMR (400 MHz, CDCl3
) δ 6.46 (dd,J
= 6.9, 2.9 Hz, 1H), 6.41 (dd,J
= 4.5, 3.0 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 3.22 (s, 1H). Example 17 was synthesized with reference to the first to sixth steps of Example 7, but in the third step, 1-ethynyl-3 was substituted with 1-ethynyl-2-fluoro-3,5-dimethoxybenzene , 5-dimethoxybenzene. MS m / z (ESI): 428 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 7.00 (brs, 1H), 6.59-6.57 (m, 2H), 6.49-6.39 (m, 2H), 5.74-5.70 (m, 1H), 5.52 (d,J
= 8.5 Hz, 2H), 5.35 (brs, 1H), 4.73-4.64 (m, 1H), 4.07-3.90 (m, 3H), 3.88 (s, 3H), 3.78 (d,J
= 5.3 Hz, 3H), 3.75-3.67 (m, 1H), 2.72-2.67 (m, 0.5H), 2.54-2.31 (m, 1.5H). Example 18 (S) -1- (1-propenylpyrrolidin-3-yl) -5-amino-3-((5-chloro-2-fluorophenyl) ethynyl) -1H-pyrazole- 4-formamidine In the first step ((2-fluoro-5-chlorophenyl) ethynyl) trimethylsilane, 2-fluoro-5-chlorobromobenzene 18a (11.0g, 52.8mmol), ethynyltrimethylsilane (7.7g , 79 mmol) and triethylamine (60 mL) were mixed, then cuprous iodide (100 mg, 0.53 mmol) and ditriphenylphosphine palladium chloride (1.86 g, 2.65 mmol) were added. The reaction mixture was heated to 80 ° C under a nitrogen atmosphere and stirring was continued for 4 hours. After the reaction was completed, the solution was desolvated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100/1) to obtain the target product ((2-fluoro-5-chlorophenyl) ethynyl) trimethyl. Silane 18b (11.0 g, yellow oil), yield: 90%.1
H NMR (400 MHz, CDCl3
) δ 7.45 (dd,J
= 6.0, 2.7 Hz, 1H), 7.28-7.22 (m, 1H), 7.02 (t,J
= 8.8 Hz, 1H), 0.29 (s, 9H). The second step 4-chloro-2-ethynyl-1-fluorobenzene will be ((2-fluoro-5-chlorophenyl) ethynyl) trimethylsilane 18b (11.0 g, 48 mmol), potassium carbonate (8.1 g, 58 mmol), dichloromethane (80 mL) and methanol (40 mL) were mixed and stirred at room temperature for 18 hours. After the reaction, the solution was desolvated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100/1) to obtain the target product 4-chloro-2-ethynyl-1-fluorobenzene 18c (5.5 g, Yellow solid), yield: 74%.1
H NMR (400 MHz, CDCl3
) δ 7.45 (dd,J
= 6.0, 2.7 Hz, 1H), 7.31-7.27 (m, 1H), 7.04 (t,J
= 8.0, 1H), 3.35 (s, 1H). Example 18 was synthesized with reference to the first to sixth steps of Example 7, but in the third step, 4-chloro-2-ethynyl-1-fluorobenzene was substituted for 1-ethynyl-3,5-dimethyl Oxybenzene. MS m / z (ESI): 402 [M + 1]1
H NMR (400 MHz, CD3
OD) δ 7.62-7.61 (m, 1H), 7.47-7.45 (m, 1H), 7.24 (t,J
= 9.0 Hz, 1H), 6.69-6.56 (m, 1H), 6.30 (d,J
= 16.8 Hz, 1H), 5.77 (t,J
= 9.2 Hz, 1H), 5.02-1.91 (m, 1H), 4.09-3.95 (m, 2H), 3.84-3.78 (m, 2H), 2.46 (dd,J
= 13.1, 6.6 Hz, 1H), 2.37 (dd,J
= 13.6, 6.9 Hz, 1H). Example 19 (S) -1- (1-propenepyrrolidin-3-yl) -5-amino-3-((2-chloro-5- (methylaminomethyl) phenyl) ethynyl ) -1H-pyrazole-4-carboxamide First step 4-chloro-3-bromobenzoic acid methyl ester 4-chloro-3-bromobenzoic acid 19a (2g, 8.5mmol) was dissolved in methanol (400mL) and cooled to 0 ° C, and then acetamidine was added dropwise. Chlorine (2.3 g, 30 mmol) and stirring was continued for 18 hours. After the reaction was completed, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10/1) to obtain the target product, methyl 4-chloro-3-bromobenzoate 19b (1.2 g, yellow solid). Yield: 57%.1
H NMR (400 MHz, CDCl3
) δ 8.31 (d,J
= 1.9 Hz, 1H), 7.93 (dd,J
= 8.3, 1.9 Hz, 1H), 7.55 (d,J
= 8.3 Hz, 1H), 3.95 (s, 3H). The second step is methyl 4-chloro-3-((trimethylsilyl) ethynyl) benzoate. The compound methyl 4-chloro-3-bromobenzoate 19b (1.2 g, 4.8 mmol), trimethyl Silyl acetylene (0.95 g, 9.7 mmol), palladium acetate (108 mg, 0.48 mmol), triphenylphosphine (254 mg, 0.97 mmol), cuprous iodide (185 mg, 0.97 mmol) and triethylamine (25 mL) Mix in a tube and heat and stir at 100 ° C for 15 hours. After the reaction was completed, the solution was desolvated under reduced pressure, and the residue was purified by column chromatography silica gel chromatography (petroleum ether / ethyl acetate = 10/1) to obtain the target product 4-chloro-3-((trimethylsilyl) ethynyl). Methyl benzoate 19c (1 g, yellow solid), yield: 78%.1
H NMR (400 MHz, CDCl3
) δ 8.19 (d,J
= 2.0 Hz, 1H), 7.91 (dd,J
= 8.4, 2.1 Hz, 1H), 7.48 (d,J
= 8.4 Hz, 1H), 3.94 (s, 3H), 0.30 (s, 9H). The third step is methyl 4-chloro-3-ethynylbenzoate. Methyl 4-chloro-3-((trimethylsilyl) ethynyl) benzoate 19c (1 g, 3.76 mmol) is dissolved in methanol (20 mL). ), Then potassium carbonate (1.04 g, 7.52 mmol) was added. After stirring at room temperature for 1 hour, it was dissolved under reduced pressure. The residue was washed with water and filtered to give the target product, methyl 4-chloro-3-ethynylbenzoate 19d (380 mg, yellow solid), yield: 52%.1
H NMR (400 MHz, CDCl3
) δ 8.23 (d,J
= 2.1 Hz, 1H), 7.96 (dd,J
= 8.4, 2.0 Hz, 1H), 7.51 (d,J
= 8.4 Hz, 1H), 3.95 (s, 3H), 3.44 (s, 1H). Fourth step (S) -3- (5-Amino-3-bromo-4-aminoformamidine-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester -3- (5-amino-3-bromo-4-cyano-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7c (2.20g, 6.2mmol), aqueous sodium hydroxide solution (0.5M, 12.4 mL, 6.2 mmol), an aqueous hydrogen peroxide solution (30%, 15 mL), and dimethyl sulfene (30 mL) were mixed. After stirring at room temperature for 2 hours, the reaction was diluted with saturated brine (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined and desolvated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100/1 to 20/1) to obtain the target product (S) -3- (5-amino-3-bromo 4-Aminoformamidine-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19e (1.92 g, pale yellow solid), yield: 83%. MS m / z (ESI): 374 [M + 1] Step 5 (S) -3- (5-Amino-4-aminomethylformamidine-3-((2-chloro-5- (methyl ester) <Methoxycarbonyl>) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (5-amino-3-bromo-4 -Aminoformamidine-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19e (770mg, 2.1mmol), triethylamine (6mL), 1,1'-bisdiphenylphosphine Ferrocene palladium dichloride (307mg, 0.42mmol), cuprous iodide (80mg, 0.42mmol) and N, N-dimethylformamide (20mL) were mixed, deoxygenated, and heated under argon atmosphere to 90 ° C, then a solution of methyl 4-chloro-3-ethynylbenzoate 19d (3.20 g, 16.5 mmol) in N, N-dimethylformamide (2 mL) was added dropwise, and stirring was continued for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20/1) to obtain the target product (S) -3- (5-amino-4-aminoformamidine-3-(( 2-chloro-5- (methyl ester <methoxycarbonyl>) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19f (420 mg, yellow solid), Yield: 41%. MS m / z (ESI): 488 [M + 1] Step 6 (S) -3-((5-Amino-1- (1- (third-butoxycarbonyl) pyrrolidin-3-yl) 4-Aminoformamidine-1H-pyrazol-3-yl) ethynyl) -4-chlorobenzoic acid will be (S) -3- (5-amino-4-aminoformamidine-3-(( 2-Chloro-5- (methyl ester <methoxycarbonyl>) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19f (100 mg, 0.2 mmol) dissolved In a mixed solvent of methanol (4 mL) and water (4 mL), sodium hydroxide (25 mg, 0.61 mmol) was added and stirring was continued for 2 hours. After completion of the reaction, the organic solvent was removed under reduced pressure. The residue was adjusted to pH = 4 to 5 with hydrochloric acid (1M), and then extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was desolvated under reduced pressure to give the target product (S) -3-((5-amino-1- (1- (third-butoxycarbonyl) pyrrolidin-3-yl) -4-aminoformamidine-1H -Pyrazol-3-yl) ethynyl) -4-chlorobenzoic acid 19 g (80 mg, brown solid), yield: 84%. MS m / z (ESI): 418 [M + H-56] Step 7 (S) -3- (5-amino-4-aminomethylformamidine-3-((2-chloro-5- (formaldehyde) Methylaminomethyl)) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3-((5-amino-1- (1 -(Tert-butoxycarbonyl) pyrrolidin-3-yl) -4-aminoformamidine-1H-pyrazol-3-yl) ethynyl) -4-chlorobenzoic acid 19g (80mg, 0.17mmol) soluble In N, N-dimethylformamide (2.5mL), then methylamine hydrochloride (34mg, 0.50mmol), diisopropylethylamine (129mg, 1mmol) and 2- (7- Benzotriazole oxide) -N, N, N ', N'-tetramethylurea hexafluorophosphate (64 mg, 0.17 mmol). The reaction was stirred at room temperature for 2 hours, then quenched with water, and then extracted with ethyl acetate (20 mL × 3). The organic phases were combined and desolvated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100/1 to 0/1) to obtain the target product (S) -3- (5-amino-4- Aminoformamidine-3-((2-chloro-5- (methylaminoformamidine) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19h (41 mg, brown solid), Yield: 50%. MS m / z (ESI): 387 [M + H-Boc] Step 8 (S) -5-Amino-3-((2-chloro-5- (methylaminomethyl) phenyl) acetyl) acetylene (Syl) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamidine hydrochloride (2-Chloro-5- (methylaminomethane) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19h (40mg, 0.08mmol) dissolved in Ethyl acetate (5 mL), then a solution of hydrogen chloride in ethanol (33%, 3 mL) was added and stirred at room temperature for 1 hour. After the reaction was completed, the solution was desolvated under reduced pressure to obtain the target product (S) -5-amino-3-((2-chloro-5- (methylaminomethylamido) phenyl) ethynyl) -1- (pyrrole Alkyl-3-yl) -1H-pyrazole-4-carboxamide hydrochloride 19i (40 mg, crude, brown solid). This product was used in the next reaction without further purification. MS m / z (ESI): 387 [M + H] Step 9 (S) -1- (1-propenepyrrolidin-3-yl) -5-amino-3-((2-chloro-5 -(Methylaminomethylformamidine) phenyl) ethynyl) -1H-pyrazole-4-carboxamide. Compound (S) -5-amino-3-((2-chloro-5- (methyl Aminoformamidine) phenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamidine hydrochloride 19i (40mg, 0.08mmol, crude), allyl chloride (7.5 mg, 0.08 mmol), an aqueous potassium carbonate solution (0.4M, 1.0 mL, 0.4 mmol) and tetrahydrofuran (5 mL) were mixed at 0 ° C and stirred at this temperature for 0.5 hours. Extract with ethyl acetate (20mL × 2), combine the organic phases and dry over anhydrous sodium sulfate, filter to remove the desiccant, desolvate under reduced pressure, and purify the residue by silica gel column chromatography (dichloromethane / methanol = 100/1 to 10/1) to obtain the target product (S) -1- (1-propenepyrrolidin-3-yl) -5-amino-3-((2-chloro-5- (methylaminomethylformamidine) Phenyl) ethynyl) -1H-pyrazole-4-carboxamide 19 (18 mg, white solid), yield: 51% in two steps. MS m / z (ESI): 441 [M + H]1
H NMR (400 MHz, DMSO-d 6
) δ 8.65 (s, 1H), 8.18 (s, 1H), 7.90 (d,J
= 8.0 Hz, 1H), 7.72 (d,J
= 8.3 Hz, 1H), 7.43 (s, 1H), 6.70-6.62 (m, 4H), 6.19-6.15 (m, 1H), 5.70 (t,J
= 10.2 Hz, 1H), 5.03-4.94 (m, 1H), 3.80-3.54 (m, 4H), 2.78 (d,J
= 3.8 Hz, 3H), 2.36-2.25 (m, 2H). Example 20 (S) -1- (1-propenylpyrrolidin-3-yl) -5-amino-3-((3-methoxy-5- (methylaminoformyl) phenyl) (Ethynyl) -1H-pyrazole-4-carboxamide Step 1 3-Bromo-5-methoxy-N-methylbenzidine Dissolve 3-bromo-5-methoxybenzoic acid 20a (500mg, 2.17mmol) in N, N-dimethylformamidine Amine (15mL), then methylamine hydrochloride (291mg, 4.35mmol), diisopropylethylamine (1.12g, 8.68mmol) and 2- (7-benzotriazole) -N were added in that order. , N, N ', N'-tetramethylurea hexafluorophosphate (1.24 g, 3.26 mmol). The reaction was stirred at room temperature for 2 hours, then quenched with water, desolvated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1/1) to give the target product 3-bromo-5-methoxy- N-methylbenzidine 20b (500 mg, white solid), yield: 95%. MS m / z (ESI): 244 [M + H] The second step 3-methoxy-N-methyl-5-((trimethylsilyl) ethynyl) benzidine gives compound 3-bromo -5-methoxy-N-methylbenzidine 20b (500mg, 2.1mmol), trimethylsilylacetylene (302mg, 3.1mmol), palladium acetate (47mg, 0.21mmol), triphenylphosphine (110mg , 0.42 mmol), cuprous iodide (80 mg, 0.42 mmol) and triethylamine (20 mL) were mixed in a sealed tube, heated to 100 ° C, and stirred for 15 hours. After the reaction was completed, the solution was desolvated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1/1) to obtain the target product 3-methoxy-N-methyl-5-((trimethyl Methylsilyl) ethynyl) benzidine 20c (220 mg, yellow solid), yield: 41%. MS m / z (ESI): 262 [M + H] The third step 3-ethynyl-5-methoxy-N-methylbenzidine will be 3-methoxy-N-methyl-5- ( (Trimethylsilyl) ethynyl) benzidine 20c (220 mg, 0.84 mmol) was dissolved in methanol (8 mL), and potassium carbonate (233 mg, 1.68 mmol) was added. After stirring at room temperature for 1 hour, it was dissolved under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1/1) to obtain the target product 3-ethynyl-5-methoxy-N-methylbenzidine 20d (140 mg, pale yellow solid). Yield: 88%. MS m / z (ESI): 190 [M + H] Step 4 (S) -3- (5-Amino-4-aminomethylhydrazone-3-((3-methoxy-5- (methyl Methylaminomethyl)) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester (S) -3- (5-amino-3-bromo-4 -Aminoformamidine-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 19e (329mg, 0.88mmol), triethylamine (2mL), 1,1'-bisdiphenylphosphine Ferrocene palladium dichloride (129 mg, 0.2 mmol), cuprous iodide (34 mg, 0.18 mmol) and N, N-dimethylformamide (8 mL) were mixed, deoxidized, and heated to argon 90 ° C. Then a solution of 3-ethynyl-5-methoxy-N-methylbenzidine 20d (1.00 g, 5.3 mmol) in N, N-dimethylformamide (2 mL) was added dropwise and stirring was continued for 12 hours . The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20/1) to obtain the target product (S) -3- (5-amino-4-aminomethyl-3--3- ( (3-Methoxy-5- (methylaminomethane) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl 20e (400mg, crude, brown solid). MS m / z (ESI): 383 [M + H-100] Step 5 (S) -5-Amino-3-((3-methoxy-5- (methylaminoformamidine) phenyl) ) Ethynyl) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamidine hydrochloride (S) -3- (5-amino-4-aminoformamidine-3 -((3-methoxy-5- (methylaminomethane) phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 20e (400mg, crude ) Was dissolved in dichloromethane (5 mL), then a solution of hydrogen chloride in ethanol (30%, 3 mL) was added and stirred at room temperature for 1 hour. After the reaction was completed, the solution was desolvated under reduced pressure to obtain the target product (S) -5-amino-3-((3-methoxy-5- (methylaminomethylamido) phenyl) ethynyl) -1- (Pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide hydrochloride 20f (300 mg, crude, brown solid). The product was used without further purification in the next reaction. MS m / z (ESI): 383 [M + H] Step 6 (S) -1- (1-propenepyrrolidin-3-yl) -5-amino-3-((3-methoxy -5- (methylaminomethylformamidine) phenyl) ethynyl) -1H-pyrazole-4-carboxamide will compound (S) -5-amino-3-((3-methoxy-5 -(Methylaminoformyl) phenyl) ethynyl) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-formamidine hydrochloride 20f (150mg, 0.39mmol, crude), Allyl chloride (42 mg, 0.47 mmol), sodium bicarbonate (131 mg, 1.56 mmol), water (4 mL) and tetrahydrofuran (8 mL) were mixed at 0 ° C and stirred at this temperature for 0.5 hours. Extract with ethyl acetate (20mL × 2), combine the organic phases and dry over anhydrous sodium sulfate, remove the desiccant by filtration, desolvate under reduced pressure, and purify the residue by silica gel column chromatography (dichloromethane / methanol = 20/1) To give the target product (S) -1- (1-propenepyrrolidin-3-yl) -5-amino-3-((3-methoxy-5- (methylaminomethylformamidine) phenyl) ) Ethynyl) -1H-pyrazole-4-carboxamide 20 (60 mg, white solid), yield: 35% in two steps. MS m / z (ESI): 437 [M + H]1
H NMR (400 MHz, CD3
OD) δ 7.59 (s, 1H), 7.45 (s, 1H), 7.28 (s, 1H), 6.73-6.58 (m, 1H), 6.36-6.28 (m, 1H), 5.83-5.75 (m, 1H) , 5.04-4.93 (m, 1H), 4.12-3.91 (m, 2H), 3.89 (s, 3H), 3.86-3.66 (m, 2H), 2.93 (s, 3H), 2.51-2.44 (m, 1H) , 2.42-2.34 (m, 1H). Example 21 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (methylamino) -1H -Pyrazole-4-carboxamide First step (S) -3- (3-bromo-4-cyano-5- (methylamino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester S) -3- (5-amino-3-bromo-4-cyano-1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7c (178 mg, 0.5 mmol) and p-methyl The benzenesulfonic acid monohydrate (12 mg, 0.07 mmol) was dissolved in triethyl orthoformate (4 mL) and heated under reflux for 2 hours. After the reaction was completed, the solution was desolvated under reduced pressure, and the residue was dispersed in water and then extracted with ethyl acetate (30 mL × 2). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was desolvated under reduced pressure, and the residue was dissolved in ethanol (10 mL). After cooling to 0 ° C, sodium borohydride (89 mg, 2.35 mmol) was added and stirred at room temperature for 2 hours. After the reaction was completed, it was quenched with saturated brine, and then extracted with ethyl acetate (30 mL × 2). After the organic phases were combined and desolvated under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100/1 to 1/1) to obtain the target product (S) -3- (3-bromo-4- Cyano-5- (methylamino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl 21a (178 mg, white solid), yield: 100%. MS m / z (ESI): 314 [M + H-56] Second step (S) -3- (4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -5 -(Methylamino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (3-bromo-4-cyano-5- (methylamine Group) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl 21a (1.85 g, 5.0 mmol), triethylamine (20 mL), 1,1'-bisdiphenylphosphine dicene Iron palladium dichloride (816mg, 1mmol), cuprous iodide (190mg, 1mmol) and N, N-dimethylformamide (20mL) were mixed, deoxidized, and heated to 90 ° C in an argon atmosphere. A solution of 1-ethynyl-3,5-dimethoxybenzene (4.86 g, 30 mmol) in N, N-dimethylformamide (10 mL) was then added dropwise and stirring was continued for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50/1 to 0/1) to obtain the target product (S) -3- (4-cyano-3-((3, 5-Dimethoxyphenyl) ethynyl) -5- (methylamino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl 21b (2.1g, brown solid), produced Rate: 80%. MS m / z (ESI): 496 [M + H-56] The third step (S) -3- (5-((third butoxycarbonyl) (methyl) amino) -4-cyano- 3-((3,5-Dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (4-cyano- 3-((3,5-Dimethoxyphenyl) ethynyl) -5- (methylamino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl 21b (225mg, 0.5 mmol) was dissolved in dichloromethane (10 mL), and then triethylamine (150 mg, 1.5 mmol), Boc anhydride (218 mg, 1 mmol), and 4-dimethylaminopyridine (6 mg, 0.05 mmol) were added in this order. After stirring at room temperature for 2 hours, saturated brine (10 mL) was added and extracted with ethyl acetate (20 mL × 2). After the organic phases were combined and desolvated under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50/1 to 1/1) to obtain the target product (S) -3- (5-((third Butoxycarbonyl) (methyl) amino) -4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1- Third butyl formate 21c (200 mg, pale yellow solid), yield: 72%. MS m / z (ESI): 440 [M + H-112] Fourth step (S) -3- (5-((third butoxycarbonyl) (methyl) amino) -4-aminomethyl)醯 -3-((3,5-Dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (5- ( (Third butoxycarbonyl) (methyl) amino) -4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine Tertiary-butyl 1-formate 21c (55mg, 0.1mmol), aqueous sodium hydroxide solution (0.5M, 0.1mL, 0.05mmol), aqueous hydrogen peroxide solution (30%, 0.5mL), and dimethylsulfine (1mL) ) Mix and stir at room temperature for 2 hours. The reaction was diluted with saturated brine (10 mL) and extracted with ethyl acetate (20 mL × 2). The organic phases were combined and desolvated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100/1 to 1/100) to obtain (S) -3- (5-((third butoxy (Carbonyl) (methyl) amino) -4-aminomethylhydrazine-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid Third butyl ester 21d (30 mg, brown solid), yield: 50%. MS m / z (ESI): 414 [M + H-156] Step 5 (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (methylamino)- 1- (Pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide hydrochloride will be (S) -3- (5-((third butoxycarbonyl) (methyl) amino) 4-Aminoformamidine-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl 21d (570mg, 1mmol ) Was dissolved in ethyl acetate (10 mL), then a solution of hydrogen chloride in ethanol (33%, 5 mL) was added and stirred at room temperature for 1 hour. After the reaction was completed, the solution was desolvated under reduced pressure to obtain the target product (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (methylamino) -1- (pyrrolidine-3 -Yl) -1H-pyrazole-4-carboxamide hydrochloride 21e (400 mg, crude, brown solid). This product was used in the next reaction without further purification. MS m / z (ESI): 370 [M + H] Step 6 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) Ethynyl) -5- (methylamino) -1H-pyrazole-4-carboxamide will compound (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5- ( Methylamino) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide hydrochloride 21e (870 mg, 2.35 mmol, crude), allyl chloride (254 mg, 2.82 mmol) , Potassium carbonate aqueous solution (2.5M, 4.7mL, 11.78mmol) and tetrahydrofuran (10mL) were mixed at 0 ° C and stirred at this temperature for 0.5 hours. It was extracted with ethyl acetate (50 mL × 2), and the organic phases were combined and dried over anhydrous sodium sulfate. The drying agent was removed by filtration and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100/1 to 10/1) to obtain the target product (S) -1- (1-propenepyrrolidin-3-yl) -3-((3 , 5-Dimethoxyphenyl) ethynyl) -5- (methylamino) -1H-pyrazole-4-carboxamide 21 (720 mg, white solid), yield: 76%. MS m / z (ESI): 424 [M + H]1
HNMR (400 MHz, CDCl3
) δ 6.88 (s, 1H), 6.69 (d,J
= 2.3 Hz, 2H), 6.51 (t,J
= 2.2 Hz, 1H), 6.46-6.40 (m, 2H), 5.74-5.72 (m, 1H), 5.52-5.48 (m, 1H), 5.06-5.01 (m, 1H), 4.09-3.94 (m, 3H ), 3.80 (s, 6H), 3.72-3.70 (m, 1H), 3.00 (s, 3H), 2.71-2.56 (m, 1H), 2.45-2.35 (m, 1H). Example 22 (S) -1- (1-propenepyrrolidin-3-yl) -3-((2-fluoro-3,5-dimethoxyphenyl) ethynyl) -5- (methylamine ) -1H-pyrazole-4-carboxamideExample 22 was synthesized with reference to the procedure of Example 21, but in the second step, 1-ethynyl-2-fluoro-3,5-dimethoxybenzene was substituted for 1-ethynyl-3,5-dimethoxy Benzene. MS m / z (ESI): 442 [M + H]1
H NMR (400 MHz, CDCl3
) δ 7.08 (s, 1H), 6.68 (d,J
= 7.2 Hz, 1H), 6.60-6.57 (m, 2H), 6.51-6.40 (m, 2H), 5.74-5.69 (m, 1H), 5.35 (s, 1H), 5.08-4.99 (m, 1H), 4.11-4.08 (m, 1H), 4.05-3.94 (m, 2H), 3.88 (s, 3H), 3.79 (s, 3H), 3.75-3.65 (m, 1H), 3.00 (t,J
= 5.2 Hz, 3H), 2.72-2.58 (m, 1H), 2.44-2.33 (m, 1H). Example 23 (S) -1- (1-propenepyrrolidin-3-yl) -3-((5-chloro-2-fluorophenyl) ethynyl) -5- (methylamino) -1H -Pyrazole-4-carboxamideExample 23 was synthesized with reference to the procedures of Example 21, but in the second step, 4-chloro-2-ethynyl-1-fluorobenzene was used to replace 1-ethynyl-3,5-dimethoxybenzene. MS m / z (ESI): 416 [M + H]1
H NMR (400 MHz, CDCl3
) δ 7.56-7.52 (m, 1H), 7.35-7.33 (m, 1H), 7.08 (t,J
= 8.8 Hz, 1H), 7.02-6.92 (m, 1H), 6.51-6.39 (m, 2H), 5.74 (d,J
= 9.3 Hz, 1H), 5.55-5.44 (m, 1H), 5.09-4.98 (m, 1H), 4.14-3.90 (m, 3H), 3.80-3.65 (m, 1H), 3.01 (s, 3H), 2.74-2.55 (m, 1H), 2.49-2.34 (m, 1H). Example 24 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (ethylamino) -1H -Pyrazole-4-carboxamide (S) -3- (5-ethylamino-4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrole Alkane-1-carboxylic acid third butyl ester will mix (S) -3- (5-amino-4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyridine Azol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (500 mg, 1.14 mmol) and sodium hydride (91 mg, 2.28 mmol, 60%) were added to N, N-diethylacetamide (5 mL ), Stir for 10 minutes, add iodoethane (106 mg, 0.68 mmol), and stir for 0.5 hours. The reaction solution was poured into water and concentrated under reduced pressure. The residue was purified by reversed-phase high-performance liquid chromatography [acetonitrile / water (containing 0.1% formic acid): 50% -90%] to obtain the target product (S) -3- ( 5-ethylamino-4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 24a (70 mg, white solid), yield: 22%. MS m / z (ESI): 410 [M + 1-56] Second step (S) -3- (5-ethylamino-4-aminomethylhydrazine-3-((3,5-dimethyl Oxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (5-ethylamino-4-cyano-3- ( (3,5-Dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester 24a (55mg, 0.12mmol) was dissolved in dimethylsulfinium (3mL ), Add hydrogen peroxide (2mL) and sodium hydroxide (300mg, 7.5mmol), stir at room temperature for 10 minutes, then raise the temperature to 40o
C. After the reaction is completed, after cooling, it is diluted with water (20 mL), extracted with ethyl acetate (30 mL), and washed with water (20 mL × 3). The organic phase is concentrated under reduced pressure, and the residue is purified by reversed-phase high-performance liquid chromatography [acetonitrile / water (Containing 0.1% formic acid): 50% -90%] to obtain the target product (S) -3- (5-ethylamino-4-aminomethylhydrazine-3-((3,5-dimethoxybenzene Group) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester 24b (15 mg), yield: 26%. MS m / z (ESI): 484 [M + 1] Step 3 (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (ethylamino) -1- (Pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide (S) -3- (5-ethylamino-4-aminoformamidine-3-((3,5-di Methoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 24b (15mg, 0.031mmol) was dissolved in dichloromethane (2mL), and trifluoroacetic acid (0.5 mL) and stirred for half an hour. After the reaction is completed, the solution is concentrated under reduced pressure to obtain the target product (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (ethylamino) -1- (pyrrolidine-3- Yl) -1H-pyrazole-4-carboxamide 24c (20 mg, crude, brown oil), yield:> 100%. The product was used in the next reaction without purification. MS m / z (ESI): 384 [M + 1] Step 4 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) Ethynyl) -5- (ethylamino) -1H-pyrazole-4-carboxamide will compound (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5- ( Ethylamino) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 24c (20mg, 0.031mmol, crude) was dissolved in tetrahydrofuran (5mL), and a saturated sodium bicarbonate solution ( 2 mL), and then added a solution of propylene chloride (2.7 mg, 0.03 mmol) in tetrahydrofuran and stirred for 0.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (30 mL) and washed with water (20 mL × 3). The organic phase was concentrated under reduced pressure, and the residue was purified by reversed-phase high performance liquid chromatography [acetonitrile / water (containing 0.1% formic acid): 20% -70%] to obtain the target product (S) -1- (1-propenepyrrole Alk-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (ethylamino) -1H-pyrazole-4-carboxamide 24 (4.7 mg, white (Solid), yield: 24%. MS m / z (ESI): 438 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.87 (brs, 1H), 6.74 (s, 2H), 6.54 (s, 1H), 6.52 (s, 1H), 6.48-6.40 (m, 2H), 5.74-5.69 (m, 1H), 5.06- 4.97 (m, 2H), 4.13-3.93 (m, 3H), 3.84 (s, 6H), 3.80-3.67 (m, 1H), 3.42 (brs, 2H), 2.75-2.35 (m, 2H), 1.31- 1.25 (m, 3H). Example 25 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5- (isopropylamino)- 1H-pyrazole-4-carboxamide First step (S) -3- (4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -5- (isopropylamino) -1H-pyrazole-1- Propyl) pyrrolidine-1-carboxylic acid tert-butyl ester The mixture (S) -3- (5-amino-4-cyano-3-((3,5-dimethoxyphenyl) ethynyl)- 1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (600 mg, 1.37 mmol), cesium carbonate (893 mg, 2.74 mmol), and acetonitrile (25 mL) were stirred for 10 minutes, and 2-bromo was quickly added Propane (186mg, 1.51mmol), heated to 72o
C, stir for 6 hours. Cooled to room temperature, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2/1) to give the target product (S) -3- (4-cyano-3-((3 , 5-Dimethoxyphenyl) ethynyl) -5- (isopropylamino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl 25a (600mg, light yellow solid) Yield: 91%. MS m / z (ESI): 424 [M + 1-56] 合成 Example 25 was synthesized with reference to the second to fourth steps in Example 24. MS m / z (ESI): 452 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 6.88 (brs, 1H), 6.70 (s, 2H), 6.54 (s, 1H), 6.51-6.39 (m, 2H), 6.03 (t,J
= 10.3 Hz, 1H), 5.74-5.69 (m, 1H), 5.49 (brs, 1H), 4.96-4.87 (m, 1H), 4.09-3.86 (m, 3H), 3.80-3.66 (m, 7H), 3.45-3.43 (m, 1H), 2.69-2.32 (m, 2H), 1.27-1.15 (m, 6H). Example 26 (S) -1- (1-propenepyrrolidin-3-yl) -5-((cyclopropylmethyl) amino) -3-((3,5-dimethoxyphenyl) (Ethynyl) -1H-pyrazole-4-carboxamide First step (S) -1- (1-propenepyrrolidin-3-yl) -5-((cyclopropylmethyl) amino) -3-((3,5-dimethoxyphenyl) (Ethynyl) -1H-pyrazole-4-carboxamide will compound (S) -1- (1-propenylpyrrolidin-3-yl) -5-amino-3-((3,5-dimethyl Oxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 7 (50 mg, 0.12 mmol) was dissolved in acetonitrile (2 mL), and cesium carbonate (80 mg, 0.24 mmol) and (bromomethyl) cyclopropane were added. (19mg, 0.13mmol), heated to 70o
C, stir for 4 hours. The reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. Dichloromethane / methanol = 12/1) purification to give the target product (S) -1- (1-propenepyrrolidin-3-yl) -5-((cyclopropylmethyl) amino) -3- ((3,5-Dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 26 (14 mg, white solid), yield: 28%. MS m / z (ESI): 464 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 6.89 (brs, 1H), 6.71 (s, 2H), 6.54 (s, 1H), 6.51-6.37 (m, 2H), 5.76-5.71 (m, 1H), 5.40 (brs, 1H), 5.03- 4.95 (m, 1H), 4.06-3.89 (m, 3H), 3.82 (s, 6H), 3.78-3.67 (m, 1H), 3.06-3.02 (m, 2H), 2.69-2.52 (m, 1H), 2.46-2.35 (m, 1H), 1.15-0.98 (m, 1H), 0.63-0.60 (m, 2H), 0.29-0.27 (m, 2H). Example 27 (S) -1- (1-propenylpyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2,2,2- Trifluoroethyl) amino) -1H-pyrazole-4-carboxamide First step (S) -3- (4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -5-((2,2,2-trifluoroethyl) amino ) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester (S) -3- (5-amino-4-cyano-3-((3,5-dimethoxy Phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (430mg, 0.98mmol), trifluoroacetaldehyde aqueous solution (75%) (304mg, 1.96mmol ) And tetraethyl titanate (448 mg, 1.96 mmol) were added to dichloromethane (15 mL) and stirred for 2 hours. After the reaction was completed, sodium borohydride (75 mg, 1.96 mmol) was added to the reaction solution, and stirring was continued for 1 hour at normal temperature. The reaction solution was poured into water and extracted with ethyl acetate (20 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by a flash column to obtain the target product (S)- 3- (4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -5-((2,2,2-trifluoroethyl) amino) -1H-pyrazole- 1-yl) Pyrrolidine-1-carboxylic acid tert-butyl ester 27a (120 mg, yellow oil), yield: 26%. MS m / z (ESI): 464 [M + 1-56] 合成 Example 27 was synthesized by referring to the operation steps of the second step to the fourth step in Example 24. MS m / z (ESI): 492 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 6.92 (brs, 1H), 6.70 (s, 2H), 6.52 (s, 1H), 6.47-6.39 (m, 2H), 6.31-6.25 (m, 1H), 5.75-5.65 (m, 1H), 5.65 (brs, 1H), 5.05-4.98 (m, 1H), 4.10-3.88 (m, 3H), 3.80 (s, 6H), 3.75-3.61 (m, 3H), 2.63-2.34 (m, 2H). Example 28 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-methoxyethyl (Amino) amino) -1H-pyrazole-4-carboxamideExample 28 was synthesized with reference to the procedures of Example 25, but in the first step, 2-bromopropane was replaced with 1-bromo-2-methoxyethane. MS m / z (ESI): 468 [M + 1]1
H NMR (400 MHz, DMSO-d 6
) δ 7.32 (brs, 1H), 6.74 (d,J
= 2.2 Hz, 2H), 6.64 (dd,J
= 16.8, 10.4 Hz, 1H), 6.60 (t,J
= 2.2 Hz, 1H), 6.50 (t,J
= 6.0 Hz, 1H), 6.16 (dd,J
= 16.8, 5.0 Hz, 1H), 5.68 (t,J
= 10.8 Hz, 1H), 5.15-5.05 (m, 1H), 4.05-4.01 (m, 0.5H), 3.86-3.81 (m, 1.5H), 3.77 (s, 6H), 3.70-3.61 (m, 1H ), 3.59-3.50 (m, 1H), 3.46 (t,J
= 5.1 Hz, 2H), 3.39-3.34 (m, 2H), 3.26 (s, 3H), 2.42-2.23 (m, 2H). Example 29 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl) Amine group) -1H-pyrazole-4-carboxamide First step (S) -3- (5-((2-Ethyloxyethyl) amino) -4-cyano-3-((3,5-dimethoxyphenyl) ethynyl)- 1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester The mixture (S) -3- (5-amino-4-cyano-3-((3,5-dimethoxy) Phenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (300 mg, 0.685 mmol), 2-bromoethyl acetate (126 mg, 0.753 mmol), Cesium carbonate (447mg, 1.37mmol) and acetonitrile (4mL) were heated to 90o
C, and stirred for 2 hours. The reaction solution was cooled to room temperature, poured into water (50 mL), and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was subjected to silica gel. Purification by column chromatography (dichloromethane / methanol = 15/1) to obtain the target product (S) -3- (5-((2-ethylamidooxyethyl) amino) -4-cyano-3- ((3,5-Dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester 29a (148 mg, yellow solid), yield: 41%. MS m / z (ESI): 468 [M + 1-56] Second step (S) -3- (4-Aminoformamidine-3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl) amino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (5-((2-ethyl Ethoxyethyl) amino) -4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid Butyl ester 29a (68mg, 0.146mmol), ethanol (5mL) and dimethyl sulfene (1mL), add saturated sodium hydroxide solution (3mL) and hydrogen peroxide (4mL), 30o
Stir at C for 1 hour. After the reaction is completed, the reaction solution is poured into a saturated sodium sulfite solution (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases are combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain the target product. (S) -3- (4-Aminoformamidine-3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl) amino) -1H-pyrazole -1-yl) Pyrrolidine-1-carboxylic acid third butyl ester 29b (110 mg, crude, yellow oil), yield:> 100%, the product was used in the next reaction without purification. MS m / z (ESI): 444 [M + 1-56] The third step (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl ) Amino) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide. Compound (S) -3- (4-aminoformamidine-3-((3,5- Dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl) amino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 29b (110mg, 0.146mmol, Crude) dissolved in hydrochloric acid in methanol (5mL), heated to 40o
C for 1 hour. After the reaction is completed, it is concentrated under reduced pressure to obtain the target product (S) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl) amino) -1- (Pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 29c (160 mg, crude, white solid), yield:> 100%, the product was used in the next reaction without purification. MS m / z (ESI): 400 [M + 1] Fourth step (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) Ethynyl) -5-((2-hydroxyethyl) amino) -1H-pyrazole-4-carboxamide will compound (S) -3-((3,5-dimethoxyphenyl) ethynyl ) -5-((2-hydroxyethyl) amino) -1- (pyrrolidin-3-yl) -1H-pyrazole-4-carboxamide 29c (160 mg, 0.146 mmol, crude) was dissolved in tetrahydrofuran ( 5 mL), a saturated sodium bicarbonate solution (10 mL) was added, and then propylene chloride (12 mg, 0.13 mmol) was added, followed by stirring at room temperature for 10 minutes. After the reaction is completed, the reaction solution is poured into water (50mL) and extracted with ethyl acetate (30mL × 3). The organic phases are combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. -Phase high performance liquid chromatography [acetonitrile / water (containing 0.2% formic acid): 20% -60%] was purified to obtain the target product (S) -1- (1-propenepyrrolidin-3-yl) -3- ( (3,5-Dimethoxyphenyl) ethynyl) -5-((2-hydroxyethyl) amino) -1H-pyrazole-4-carboxamide 29 (6 mg, white solid), Yield: 9%. MS m / z (ESI): 454 [M + 1]1
H NMR (400 MHz, DMSO-d 6
) δ 7.36 (brs, 1H), 6.76 (brs, 1H), 6.74 (s, 2H), 6.70-6.60 (m, 2H), 6.55-6.52 (m, 1H), 6.17 (d,J
= 16.9 Hz, 1H), 5.69 (t,J
= 10.9 Hz, 1H), 5.16-5.10 (m, 1H), 4.87 (s, 1H), 4.06-4.0 (m, 0.5H), 3.83-3.81 (m, 1.5H), 3.77 (s, 6H), 3.68-3.63 (m, 2H), 3.55-3.53 (m, 2H), 3.28-3.26 (m, 2H), 2.38-2.27 (m, 2H). Example 30 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((3-morpholinopropane (Amino) amino) -1H-pyrazole-4-carboxamide Step 1 3-morpholinopropyl 4-methylbenzenesulfonate Dissolve the compound 3-morpholinopropane-1-ol 30a (500 mg, 3.45 mmol) in dichloromethane (100 ml) and add 4-di Methylaminopyridine (42 mg, 0.34 mmol), triethylamine (1.04 g, 10.3 mmol) and p-toluenesulfonyl chloride (988 mg, 5.17 mmol) were stirred overnight at room temperature. After the reaction is completed, the reaction solution is poured into water (50mL) and extracted with dichloromethane (50mL × 3). The organic phases are combined and dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. The residue is applied to a silica gel column. Chromatographic purification (petroleum ether / ethyl acetate = 2/1, the target product 3-morpholinopropyl 4-methylbenzenesulfonate 30b (660 mg, yellow oil) was obtained, yield: 64%. MS m / z (ESI): 300 [M + 1] 合成 Example 30 was synthesized with reference to the procedure of Example 25, but in the first step, 2-morpholinopropyl 4-methylbenzenesulfonate was used instead of Bromopropane. MS m / z (ESI): 537 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.23 (brs, 1H), 7.12 (brs, 1H), 6.94 (brs, 1H), 6.69 (s, 2H), 6.52 (s, 1H), 6.49-6.40 (m, 2H), 5.92 (brs, 1H), 5.74-5.70 (m, 1H), 5.03-4.96 (m, 1H), 4.09-3.90 (m, 3H), 3.80-3.68 (m, 11H), 3.28 (brs, 2H), 2.89 (brs, 6H), 2.69-2.33 (m, 2H), 1.93 (brs, 2H). Example 31 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2-morpholinoethyl (Amino) amino) -1H-pyrazole-4-carboxamideExample 31 was synthesized with reference to the procedures of Example 25, but in the first step, 4- (2-chloroethyl) morpholine was substituted for 2-bromopropane. MS m / z (ESI): 523 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.14 (s, 1H), 6.95 (brs, 1H), 6.69 (s, 2H), 6.63 (brs, 1H), 6.52 (s, 1H), 6.49-6.39 (m, 2H), 6.14 (brs, 1H), 5.75-5.70 (m, 1H), 5.06-4.98 (m, 1H), 4.11-3.85 (m, 3H), 3.80-3.72 (m, 11H), 3.37-3.33 (m, 2H), 2.80- 2.73 (m, 2H), 2.65 (brs, 4H), 2.45-2.32 (m, 2H). Example 32 (S) -1- (1-propenylpyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((2- (pyrrolidine- 1-yl) ethyl) amino) -1H-pyrazole-4-carboxamide In the first step, 2-bromoethyl 4-methylbenzenesulfonate compound 2-bromoethanol 32a (500 mg, 4.0 mmol), 4-dimethylaminopyridine (246 mg, 2.02 mmol) and triethylamine (1.22 g , 12.1 mmol) was dissolved in dichloromethane (50 mL), and the temperature was reduced to 0.o
C, p-toluenesulfonyl chloride (1.15 g, 6.05 mmol) was added in portions. After the addition was completed, the mixture was warmed to room temperature and stirred overnight. After the reaction is completed, the reaction solution is poured into water (50mL) and extracted with dichloromethane (50mL × 3). The organic phases are combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue is applied to a silica gel column layer. Analytical and purified (petroleum ether / ethyl acetate = 10/1) to obtain the target product 2-bromoethyl 4-methylbenzenesulfonate 32b (600 mg, yellow oil), yield: 53%. MS m / z (ESI): 277 [M + 1] Second step (S) -3- (5-((2-bromoethyl) amino) -4-cyano-3-((3,5 -Dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid tert-butyl ester (S) -3- (5-amino-4-cyano-3 -((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester 7d (400 mg, 0.92 mmol), 2-bromoethyl 4-methylbenzenesulfonate (380mg, 1.37mmol), cesium carbonate (600mg, 1.84mmol) and acetonitrile (10mL) were heated to 70o
C, and stirred for 2 hours. The reaction solution was poured into water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was separated through a flash column to obtain the target. Product (S) -3- (5-((2-bromoethyl) amino) -4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole- 1-yl) Pyrrolidine-1-carboxylic acid tert-butyl ester 32c (240 mg, brown oil), yield: 48%. MS m / z (ESI): 408 [M + 1-56-80] Third step (S) -3- (4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -5-((2- (pyrrolidin-1-yl) ethyl) amino) -1H-pyrazol-1-yl) pyrrolidine-1-carboxylic acid third butyl ester will be mixed with (S) -3- ( 5-((2-bromoethyl) amino) -4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazol-1-yl) pyrrolidine-1 -Third butyl formate 32c (240mg, 0.44mmol), pyrrolidine (47mg, 0.66mmol), cesium carbonate (288mg, 0.88mmol) and acetonitrile (5mL) heated to 70o
C and stirred for 1.5 hours. The reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography ( Dichloromethane / methanol = 10/1) to give the target product (S) -3- (4-cyano-3-((3,5-dimethoxyphenyl) ethynyl) -5-((2- (Pyrrolidin-1-yl) ethyl) amino) -1H-pyrazol-1-yl) third butyl pyrrolidine-1-carboxylic acid 32d (200 mg, yellow oil), yield: 85%. MS m / z (ESI): 479 [M + 1-56] 合成 Example 32 was synthesized with reference to the second to fourth steps in Example 24. MS m / z (ESI): 507 [M + 1]1
H NMR (400 MHz, CDCl3
) δ 8.38 (s, 1H), 6.99 (brs, 1H), 6.69 (s, 2H), 6.51 (s, 1H), 6.47-6.36 (m, 2H), 5.72-5.67 m, 2H), 5.16-5.08 (m, 1H), 4.12-3.86 (m, 3H), 3.80-3.62 (m, 9H), 3.33-3.29 (m, 6H), 2.62-2.34 (m, 2H), 2.07 (brs, 4H). Example 33 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((tetrahydro-2H-pyridine Alan-4-yl) amino) -1H-pyrazole-4-carboxamide Step 4 4-iodotetrahydro-2H-pyran 4-hydroxytetrahydro-2H-pyran 33a (2.04g, 20mmol), triphenylphosphine (6.81g, 26) and imidazole (2.04g, 30mmol) Dissolved in dichloromethane (100mL) and cooled to 0o
C, then add iodine (6.09g, 24mmol), and addo
Stir at C for 14 hours. The reaction was quenched with water and extracted with ethyl acetate (50 mL × 2). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was desolvated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1/1) to obtain the target product 4-iodotetrahydro-2H-pyran 33b (2.12 g, white solid), yield: 50%.1
H NMR (400 MHz, DMSO-d 6
) δ 4.62 (dt,J
= 13.9, 4.5 Hz, 1H), 3.68-3.64 (m, 2H), 3.47-3.42 (m, 2H), 2.13-1.97 (m, 4H).合成 Example 33 was synthesized with reference to the procedures of Example 24, but in the first step, 4-iodotetrahydro-2H-pyran was used instead of iodoethane. MS m / z (ESI): 494 [M + H]1
H NMR (400 MHz, CD3
OD) δ 6.74 (t,J
= 2.2 Hz, 2H), 6.71-6.62 (m, 1H), 6.60-6.58 (m, 1H), 6.36-6.30 (m, 1H), 5.82-5.77 (m, 1H), 5.18-5.12 (m, 1H ), 4.04-3.94 (m, 6H), 3.81 (s, 6H), 3.52-3.46 (m, 3H), 2.55-2.39 (m, 2H), 1.94-1.92 (m, 2H), 1.60-1.55 (m , 2H). Example 34 (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) ethynyl) -5-((1-methylpyridine -4-yl) amino) -1H-pyrazole-4-carboxamide The first step 4-iodo-1-methylpyridine is 4-hydroxy-1-methylpyridine 34a (2.3g, 20mmol), triphenylphosphine (6.81g, 26mmol), imidazole (2.04g, 30mmol) Mix with dichloromethane (100mL) and cool to 0o
C, then add iodine (6.09 g, 24 mmol) and continue stirring for 18 hours. After the reaction was completed, it was quenched with water and then extracted with dichloromethane (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate and filtered, and the filtrate was then dissolved under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10/1) to obtain the target product 4-iodo-1-methylpyridine 34b (2.25 g, white solid), yield: 50%. MS m / z (ESI): 226 [M + H] Step (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-dimethoxyphenyl) Ethynyl) -5-((1-methylpyridin-4-yl) amino) -1H-pyrazole-4-carboxamide will compound (S) -1- (1-propenepyrrolidine-3 -Yl) -5-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazole-4-carboxamide 7 (210mg, 0.5mmmol), 4-iodo-1 -Methylpyridine 34b (450 mg, 2 mmol), potassium carbonate (207 mg, 1.5 mmol) and acetonitrile (10 mL) were mixed and mixed at 90o
Heat and stir at C for 13 hours. The reaction mixture was dissolved under reduced pressure, dissolved in water, and then extracted with ethyl acetate (50 mL × 2). The organic phases were combined and desolvated under reduced pressure. The residue was purified by reverse preparative liquid chromatography to obtain the target product (S) -1- (1-propenepyrrolidin-3-yl) -3-((3,5-di Methoxyphenyl) ethynyl) -5-((1-methylpyridin-4-yl) amino) -1H-pyrazole-4-carboxamide 34 (8.1 mg, white solid), Yield: 3.2%. MS m / z (ESI): 507 [M + H]1
H NMR (400 MHz, CD3
OD) δ 6.77 (s, 2H), 6.68-6.65 (m, 1H), 6.59 (s, 1H), 6.34-6.30 (m, 1H), 5.81-5.78 (m, 1H), 5.69-5.67 (m, 1H), 5.03-5.00 (m, 1H), 4.98-5.95 (m, 1H), 4.92-4.90 (m, 1H), 4.36 (s, 2H), 4.12-4.07 (m, 1H), 4.00-3.98 ( m, 1H), 3.93-3.90 (m, 1H), 3.86-3.84 (m, 1H), 3.81 (s, 6H), 3.72-3.68 (m, 1H), 2.53-2.47 (m, 3H), 2.40- 2.38 (m, 1H), 2.32 (s, 3H), 2.27-2.21 (m, 1H).生物学 Biological experiments FGFR activity inhibition test Using the HTRF kinase detection kit to evaluate the effect of the compounds of the present invention on the in vitro activity of FGFR by detecting the level of substrate phosphorylation in the kinase reaction (Table 1). FGFR1 activity inhibition test The experimental method is summarized as follows: The reaction buffer contains the following components: 5-fold diluted enzyme buffer / kinase 5X (Cisbio, article number 62EZBFDD) (the main component is 50mM HEPES, pH7.0), 5mM MgCl2
And 1mM DTT; human recombinant FGFR1 catalytic domain protein (amino acid 308-731) was purified by the company itself and diluted with a reaction buffer to a 0.6ng / uL kinase solution; the substrate reaction solution included a 400nM dilution with a reaction buffer Biotin-labeled tyrosine kinase substrate (Cisbio, article number 62TK0PEC) and 40uM ATP; the test solution includes dilution with detection buffer (Cisbio, article number 62SDBRDF) to 0.125ng / uL Eu3+
Labeled cage antibody (Cisbio, Cat. No. 61T66KLB), 25nM streptavidin-labeled XL665 (Cisbio, Cat. No. 610SAXLB).化合物 The compound was dissolved and diluted to 1 mM with DMSO, and then serially diluted 4 times with DMSO to a minimum concentration of 0.061 uM, and each concentration point was diluted 40 times with reaction buffer. If the compound IC50
The values are very low, reducing the starting concentration of the compound. Add 4uL of compound solution and 2uL of FGFR1 kinase solution to a 384-well assay plate (Thermo, Cat. No. 264706), mix well and incubate at room temperature for 15 minutes; then add 4uL of substrate reaction solution, and incubate the reaction mixture at room temperature for 60 minutes ; Then add an equal volume of 10uL of detection solution to the reaction to stop the reaction, mix well and leave it at room temperature. After 60 minutes, the phosphorylated product was simultaneously Eu3+
Recognition of labeled cage antibodies (donors) and streptavidin-labeled XL665 antibodies (acceptors). After laser excitation, nearby donors and acceptors undergo energy resonance transfer, which is transferred from the donor (620nm) to The energy of the acceptor (665nm) was measured with a microplate reader EnVision (Perkin Elmer). The ratio of 665/620 is positively correlated with the degree of substrate phosphorylation, and thus the activity of FGFR1 kinase is detected. In this experiment, the group without enzyme was regarded as a 100% inhibition group, and the group without enzyme was added as a 0% inhibition group. The percentage inhibition of FGFR1 activity by the compound was calculated using the following formula: Percent inhibition = 100-100 * (ratioCompound
-ratio100% inhibition
)/(ratio0% inhibition
-ratio100% inhibition
) IC50
Values were calculated from 10 concentration points using the XLfit software in Excel using the following formula: Y = Bottom + (Top-Bottom) / (1 + 10 ^ ((logIC50
-X) * slope factor)) where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-shaped curve, Top is the top plateau value of the S-shaped curve, X is the logarithmic value of the concentration of the test compound, and slope factor is the slope coefficient of the curve . FGFR2 activity inhibition test The experimental method is summarized as follows: The reaction buffer contains the following components: 5-fold diluted enzyme buffer / kinase 5X (Cisbio, article number 62EZBFDD) (the main component is 50mM HEPES, pH7.0), 5mM MgCl2
And 1mM DTT; human recombinant FGFR2 catalytic domain protein (amino acid 400-821) was purchased from Yiqiao Shenzhou Biotechnology Co., Ltd. and diluted to 0.45ng / uL of kinase solution with reaction buffer; the substrate reaction solution included the reaction Buffer was diluted to 800nM biotin-labeled tyrosine kinase substrate (Cisbio, Cat. No. 62TK0PEC) and 50uM ATP; detection solution included diluted to 0.125ng / uL Eu with detection buffer (Cisbio, Cat. 62SDBRDF)3+
Labeled cage antibody (Cisbio, Cat. No. 61T66KLB), 50nM streptavidin-labeled XL665 (Cisbio, Cat. No. 610SAXLB).化合物 The compound was dissolved and diluted to 1 mM with DMSO, and then serially diluted 4 times with DMSO to a minimum concentration of 0.061 uM, and each concentration point was diluted 40 times with reaction buffer. If the compound IC50
The values are very low, reducing the starting concentration of the compound. Add 4uL of compound solution and 2uL of FGFR2 kinase solution to a 384-well detection plate (Thermo, Cat. No. 264706), mix well and incubate at room temperature for 15 minutes; then add 4uL of substrate reaction solution, and incubate the reaction mixture at room temperature for 60 minutes ; Then add an equal volume of 10uL of detection solution to the reaction to stop the reaction, mix well and leave it at room temperature. After 60 minutes, the phosphorylated product was simultaneously Eu3+
Recognition of labeled cage antibodies (donors) and streptavidin-labeled XL665 antibodies (acceptors). After laser excitation, the nearby donor and acceptor undergo energy resonance transfer, and the energy transferred from the donor (620nm) to the acceptor (665nm) is detected with a microplate reader EnVision (Perkin Elmer). The ratio of 665/620 is positively correlated with the degree of substrate phosphorylation, and thus the activity of FGFR2 kinase is detected. In this experiment, the group without enzyme was regarded as a 100% inhibition group, and the group without enzyme was added as a 0% inhibition group. The percentage inhibition of FGFR2 activity of the compound was calculated using the following formula: Percent inhibition = 100-100 * (ratioCompound
-ratio100% inhibition
)/(ratio0% inhibition
-Ratio100% inhibition
) IC50
Values were calculated from 10 concentration points using the XLfit software in Excel using the following formula: Y = Bottom + (Top-Bottom) / (1 + 10 ^ ((logIC50
-X) * slope factor)) where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-shaped curve, Top is the top plateau value of the S-shaped curve, X is the logarithmic value of the concentration of the test compound, and slope factor is the slope coefficient of the curve .测试 FGFR3 activity inhibition test The experimental method is summarized as follows: The reaction buffer contains the following components: 5-fold diluted enzyme buffer / kinase 5X (Cisbio, article number 62EZBFDD) (the main component is 50mM HEPES, pH7.0), 5mM MgCl2
And 1mM DTT; human recombinant FGFR3 catalytic domain protein (amino acid 399-806) was purchased from Yiqiao Shenzhou Biotechnology Co., Ltd. and diluted to 0.3 ng / uL of kinase solution with reaction buffer; the substrate reaction solution included the reaction Buffer was diluted to 1000nM biotin-labeled tyrosine kinase substrate (Cisbio, Cat. No. 62TK0PEC) and 90uM ATP; detection solution included diluted to 0.125ng / uL Eu with detection buffer (Cisbio, Cat. 62SDBRDF)3+
Labeled cage antibody (Cisbio, Cat. No. 61T66KLB), 62.5nM streptavidin-labeled XL665 (Cisbio, Cat. No. 610SAXLB).化合物 The compound was dissolved and diluted to 1 mM with DMSO, and then serially diluted 4 times with DMSO to a minimum concentration of 0.061 uM, and each concentration point was diluted 40 times with reaction buffer. If the compound IC50
The values are very low, reducing the starting concentration of the compound. Add 4uL of compound solution and 2uL of FGFR3 kinase solution to a 384-well detection plate (Thermo, Cat. No. 264706), mix well and incubate at room temperature for 15 minutes; then add 4uL of substrate reaction solution, and incubate the reaction mixture at room temperature for 60 minutes ; Then add an equal volume of 10uL of detection solution to the reaction to stop the reaction, mix well and leave it at room temperature. After 60 minutes, the phosphorylated product was simultaneously Eu3+
Recognition of labeled cage antibodies (donors) and streptavidin-labeled XL665 antibodies (acceptors). After laser excitation, the nearby donor and acceptor undergo energy resonance transfer, and the energy transferred from the donor (620nm) to the acceptor (665nm) is detected with a microplate reader EnVision (Perkin Elmer). The ratio of 665/620 is positively correlated with the degree of substrate phosphorylation, and thus the activity of FGFR3 kinase is detected. In this experiment, the group without enzyme was regarded as a 100% inhibition group, and the group without enzyme was added as a 0% inhibition group. The percentage inhibition of FGFR3 activity of the compound was calculated using the following formula: Percent inhibition = 100-100 * (ratioCompound
-ratio100% inhibition
)/(ratio0% inhibition
-Ratio100% inhibition
) IC50
Values were calculated from 10 concentration points using the XLfit software in Excel using the following formula: Y = Bottom + (Top-Bottom) / (1 + 10 ^ ((logIC50
-X) * slope factor)) where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-shaped curve, Top is the top plateau value of the S-shaped curve, X is the logarithmic value of the concentration of the test compound, and slope factor is the slope coefficient of the curve . FGFR4 activity inhibition test The experimental method is summarized as follows: The reaction buffer contains the following components: 5-fold diluted enzyme buffer / kinase 5X (Cisbio, article number 62EZBFDD) (the main component is 50mM HEPES, pH7.0), 5mM MgCl2
And 1mM DTT; human recombinant FGFR4 catalytic domain protein (amino acid 460-802) was purchased from the Protein Research and Technology Center of Tsinghua University, diluted with reaction buffer to a 0.5ng / uL kinase solution; the substrate reaction solution included the use of reaction buffer The solution was diluted to 500nM biotin-labeled tyrosine kinase substrate (Cisbio, article number 62TK0PEC) and 90uM ATP; the detection solution was diluted to 0.125ng / uL Eu with detection buffer (Cisbio, article number 62SDBRDF).3+
Labeled cage antibody (Cisbio, Cat. No. 61T66KLB), 31.25nM streptavidin-labeled XL665 (Cisbio, Cat. No. 610SAXLB).化合物 The compound was dissolved and diluted to 1 mM with DMSO, and then serially diluted 4 times with DMSO to a minimum concentration of 0.061 uM, and each concentration point was diluted 40 times with reaction buffer. If the compound IC50
The values are very low, reducing the starting concentration of the compound. Add 4uL of compound solution and 2uL of FGFR4 kinase solution to a 384-well assay plate (Thermo, Cat. No. 264706), mix well and incubate at room temperature for 15 minutes; then add 4uL of substrate reaction solution, and incubate the reaction mixture at room temperature for 60 minutes ; Then add an equal volume of 10uL of detection solution to the reaction to stop the reaction, mix well and leave it at room temperature. After 60 minutes, the phosphorylated product was simultaneously Eu3+
Recognition of labeled cage antibodies (donors) and streptavidin-labeled XL665 antibodies (acceptors). After laser excitation, the nearby donor and acceptor undergo energy resonance transfer, and the energy transferred from the donor (620nm) to the acceptor (665nm) is detected with a microplate reader EnVision (Perkin Elmer). The ratio of 665/620 is positively correlated with the degree of substrate phosphorylation, and thus the activity of FGFR4 kinase is detected. In this experiment, the group without enzyme was regarded as a 100% inhibition group, and the group without enzyme was added as a 0% inhibition group. The percentage inhibition of FGFR4 activity of the compound was calculated using the following formula: Percent inhibition = 100-100 * (ratioCompound
-ratio100% inhibition
)/(ratio0% inhibition
-Ratio100% inhibition
) IC50
Values were calculated from 10 concentration points using the XLfit software in Excel using the following formula: Y = Bottom + (Top-Bottom) / (1 + 10 ^ ((logIC50
-X) * slope factor)) where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-shaped curve, Top is the top plateau value of the S-shaped curve, X is the logarithmic value of the concentration of the test compound, and slope factor is the slope coefficient of the curve . Table 1
A ﹤ 10nM; 10 nM ≤ B <100nM; 100 nM ≤ C <1000nM The compounds of the embodiments of the present invention have a significant inhibitory effect on the activity of FGFR, preferably IC50
100 to 1000nM, better IC50
Less than 100nM, best IC50
Less than 10nM. Measurement of Hep3B Cell Proliferation Inhibition The effect of the compound of the present invention on the cell proliferation of Hep3B hepatocellular carcinoma cell line was evaluated using a luminescent cell viability test experiment (Table 2). The experimental method is summarized as follows: CellTilter-Glo reagent (Promega, article number G7572) consists of CTG lyophilized powder and CTG buffer, and the lyophilized powder can be dissolved in the buffer when used. The compound was dissolved and diluted to 5 mM in DMSO (Sigma, Cat. No. D5879), and then serially diluted 4 times with DMSO to a minimum concentration of 0.31 uM, and each concentration point was further treated with FMEM-free DMEM medium (ThermoFisher, Cat. ) Dilute 50 times. If the compound's IC50 value is very low, the starting concentration of the compound can be reduced. Hep3B cells (from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) in DMEM complete medium containing 10% FBS (GBICO, Cat. No. 10099-141) and 100U / mL penicillin-streptomycin mixed solution (ThermoFisher, Cat. No. 15140122) Medium culture, when the cell coverage in the culture container reaches 80-90%, digest with 0.25% trypsin (containing EDTA) (ThermoFisher, Cat. No. 25200056) and then disperse and plant it in a white 384-well plate (ThermoFisher, Cat. No. 164610) ), 1000 cells per well (27uL DMEM complete medium), and then place the 384-well plate at 37ºC, 5% CO2
Incubate overnight (18-20 hours) in the incubator. After overnight, add 3uL of DMEM diluted compound to each well, mix by centrifugation gently, and then place the 384-well plate at 37ºC, 5% CO.2
The culture was continued in the incubator, and after 72 hours, it was taken out and left at room temperature. For 30 minutes, add 15uL of CTG reagent equilibrated to room temperature to each well, and gently shake on the shaker for 3 minutes to ensure sufficient cell lysis. Leave for 10 minutes to stabilize the cold light signal, and then read the cold light signal with EnVision (Perkin Elmer) . Among them, add 10uM Blueprint's BLU9931 (Cancer Discovery 2015, 5, 424) group as the signal100% inhibition
, Plus the cold light signal of the 0.2% DMSO group as the signal0% inhibition
. The percentage of the inhibition of Hep3B cell proliferation by compounds can be calculated by the following formula:% inhibition = 100-100 * (signalCompound
-signal100% inhibition
) / (signal0% inhibition
-signal100% inhibition
) The IC50 value of the hydrazone compound is calculated from the 8 concentration points using XLfit (ID Business Solutions Ltd., UK) software through the following formula: Y = Bottom + (Top-Bottom) / (1 + 10 ^ ((logIC50
-X) * slope factor)) where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-shaped curve, Top is the top plateau value of the S-shaped curve, X is the logarithmic value of the concentration of the test compound, and slope factor is the slope coefficient of the curve .的 Measurement of RT4 cell proliferation inhibition The effect of the compound of the present invention on the proliferation of RT4 bladder cancer cell line cells was evaluated using a luminescent cell viability test experiment (Table 2). The summary of the experimental method refers to the method for measuring the inhibition of Hep3B cell proliferation, in which RT4 cells (from the Cell Resource Center of the Shanghai Academy of Life Sciences, Chinese Academy of Sciences) and the positive control are Example 1 ((S) -1- (1) in Taiho patent application WO2015008844A1 3- (4-amino-3-((3,5-dimethoxyphenyl) ethynyl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) pyrrolidine-1- Group) prop-2-en-1-one). Measurement of SNU-16 Cell Proliferation Inhibition The luminous cell viability test was used to evaluate the effect of the compound of the present invention on the cell proliferation of SNU-16 gastric cancer cell line (Table 2).概述 Summary of experimental methods Refer to the measurement method of Hep3B cell proliferation inhibition, in which SNU-16 cells (ATCC, HB-8064) and positive control are BJG398 of Novartis. Table 2
Note: A ﹤ 10nM; 10nM≤B <100nM; 100nM≤C <1000Nm N.D .: Not tested The compounds of the examples of the present invention have significant inhibitory effects on the cell proliferation of Hep3B, RT4 and SNU-16, respectively, and the preferred IC is50
100 to 1000nM, better IC50
Less than 100nM.