TW202227079A - Pharmaceutical use of FXIa inhibitor compound or salt thereof - Google Patents
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Abstract
Description
本發明屬於藥物技術領域,提供了一系列的FXIa抑制劑化合物或其鹽的醫藥用途,具體涉及其在製備預防和/或治療動脈和靜脈血栓藥物中的用途。The invention belongs to the technical field of medicine, and provides a series of medicinal uses of FXIa inhibitor compounds or salts thereof, in particular to their use in preparing medicines for preventing and/or treating arterial and venous thrombosis.
全球每年腦血管、腦梗塞、心肌梗塞、冠心病、動脈硬化等心腦血管疾病奪走近1200萬人的生命,接近世界總死亡人數的1/4,成為人類健康的頭號大敵。中國每年死於心血管疾病的人數達到260萬人以上,存活的患者75%致殘,其中40%以上重殘。由心腦血管疾病和糖尿病及其併發症引起的血栓問題,成為當今要解決的刻不容緩的問題。Every year, cardiovascular and cerebrovascular diseases such as cerebrovascular disease, cerebral infarction, myocardial infarction, coronary heart disease and arteriosclerosis kill nearly 12 million people in the world, which is close to 1/4 of the total number of deaths in the world, and has become the number one enemy of human health. More than 2.6 million people die of cardiovascular disease in China every year, and 75% of the surviving patients are disabled, of which more than 40% are severely disabled. The thrombosis caused by cardiovascular and cerebrovascular diseases and diabetes and its complications has become an urgent problem to be solved today.
人體血液凝固過程由內源性途徑(intrinsic pathway)、外源性途徑(extrinsic pathway)和共同通路組成(Annu.Rev.Med.2011.62:41–57),是通過多種酶原被順序啟動而過程不斷得到加強和放大的一種連鎖反應。凝血級聯反應由內源性途徑(又稱接觸啟動途徑)及外源性途徑(又稱組織因子途徑)啟動生成FXa,再經共同途徑生成凝血酶(FIIa),最終形成纖維蛋白。The human blood coagulation process is composed of intrinsic pathway, extrinsic pathway and common pathway (Annu.Rev.Med.2011.62:41–57), which is initiated by multiple zymogens in sequence. A chain reaction that is continuously strengthened and amplified. The coagulation cascade is initiated by the endogenous pathway (also known as the contact initiation pathway) and the exogenous pathway (also known as the tissue factor pathway) to generate FXa, and then through the common pathway to generate thrombin (FIIa), and finally form fibrin.
內源性途徑是指由XII因子被啟動形XIa-VIIIa-Ca 2+-P L複合物、並啟動X因子的過程,外源性凝血途徑則是從組織因子(TF)釋放到TF-VIIa-Ca 2+複合物形成並啟動因子Ⅹ的過程。共同通路是指因子Xa形成後,兩條途徑合二為一,啟動凝血酶原並最終生成纖維蛋白的過程,其中FXI是維持內源性途徑所必需的,而且在凝血級聯反應放大過程中發揮關鍵作用。在凝血級聯反應中,凝血酶可回饋啟動FXI,活化的FXI(FXIa)又促使凝血酶的大量產生,從而使凝血級聯反應放大。因此,FXI的拮抗劑被廣泛開發,用於各種血栓的治療。 The intrinsic pathway refers to the process in which factor XII is activated to form XIa-VIIIa-Ca 2+ -PL complex and initiate factor X, while the extrinsic coagulation pathway is released from tissue factor (TF) to TF-VIIa- The Ca 2+ complex forms and initiates the factor X process. The common pathway means that after the formation of factor Xa, the two pathways are combined into one, starting the process of prothrombin and finally generating fibrin, of which FXI is necessary to maintain the endogenous pathway, and in the process of coagulation cascade amplification play a key role. In the coagulation cascade reaction, thrombin can feed back to initiate FXI, and the activated FXI (FXIa) promotes the massive production of thrombin, thereby amplifying the coagulation cascade reaction. Therefore, antagonists of FXI have been widely developed for the treatment of various thrombi.
傳統的抗凝藥物,如華法林、肝素、低分子量肝素(LMWH),以及近年上市的新藥,如FXa抑制劑(利伐沙班、阿呱沙班等)和凝血酶抑制劑(達比加群酯、水蛭素等),對減少血栓形成均具有較好效果,以其顯著有效性佔據廣大心腦血管市場,然而其副作用也越來越顯著,其中“出血風險(bleeding risk)”是首當其衝 最為嚴峻的問題之一(N Engl J Med 1991;325:153-8、Blood.2003;101:4783-4788)。Traditional anticoagulant drugs, such as warfarin, heparin, low molecular weight heparin (LMWH), and new drugs launched in recent years, such as FXa inhibitors (rivaroxaban, araguaxaban, etc.) and thrombin inhibitors (Darby Gatran etexilate, hirudin, etc.), all have good effects on reducing thrombosis, and occupy the vast cardiovascular and cerebrovascular market with their significant effectiveness. However, their side effects are becoming more and more significant. Among them, the "bleeding risk" is Bear the brunt of one of the most serious problems (N Engl J Med 1991; 325: 153-8, Blood. 2003; 101: 4783-4788).
研究發現,在血栓模型中,抑制FXIa因子可以有效抑制血栓的形成,但在更為嚴重的血栓情況下,FXIa的作用微乎其微(Blood.2010;116(19):3981-3989)。臨床統計顯示,提高FXIa的量會增加VTE的患病率(Blood 2009;114:2878-2883),而FXIa嚴重不足者其患有DVT的風險性減少(Thromb Haemost 2011;105:269–273)。Studies have found that in the thrombosis model, inhibition of FXIa factor can effectively inhibit the formation of thrombus, but in more severe thrombosis, the effect of FXIa is minimal (Blood. 2010; 116(19): 3981-3989). Clinical statistics show that increasing the amount of FXIa increases the prevalence of VTE (Blood 2009;114:2878-2883), while those with severe FXIa deficiency have a reduced risk of DVT (Thromb Haemost 2011;105:269–273) .
FXIa作為目前抑制血栓的新興靶點,公開具有FXIa抑制活性的化合物的專利申請有WO9630396、WO9941276、WO2013093484、WO2004002405、WO2013056060、WO2017005725、WO2017/023992、WO2018041122等。其中,目前僅拜耳公司的反義寡核苷酸BAY-2306001進入了臨床二期研究。FXIa is currently an emerging target for inhibiting thrombosis, and patent applications for compounds with FXIa inhibitory activity are disclosed in WO9630396, WO9941276, WO2013093484, WO2004002405, WO2013056060, WO2017005725, WO2017/023992, WO2018041122, etc. Among them, only Bayer's antisense oligonucleotide BAY-2306001 has entered the Phase II clinical study.
本發明化合物具有更高的活性。特別是本發明化合物表現出優異的對人血液的抗凝血作用,並具有良好的藥代活性,可用於有效治療和/或預防心腦血管疾病及血栓症狀。The compounds of the present invention have higher activity. In particular, the compound of the present invention exhibits excellent anticoagulant effect on human blood, and has good pharmacokinetic activity, and can be used for effective treatment and/or prevention of cardiovascular and cerebrovascular diseases and thrombosis symptoms.
本發明提供了一系列的氧代噠嗪醯胺類衍生物在醫藥上的應用。The present invention provides a series of oxopyridazinamide derivatives used in medicine.
具體而言,FXIa抑制劑化合物或其鹽的醫藥用途,所述用途包括所述在FXIa抑制劑化合物或其鹽製備預防和/或治療動脈血栓、靜脈血栓、動靜脈旁路血栓藥物中的用途藥物中的用途,所述化合物結構式如下: 。 Specifically, the pharmaceutical use of the FXIa inhibitor compound or its salt, including the use of the FXIa inhibitor compound or its salt in the preparation of a drug for preventing and/or treating arterial thrombosis, venous thrombosis, and arteriovenous bypass thrombosis Use in medicine, the compound structural formula is as follows: .
作為本發明的一種實施方案,所述FXIa抑制劑化合物的鹽結構式如下: 其中: n為0.5-3; M與羧基成鹽,所述鹽選自鋰鹽、鈉鹽、鉀鹽、鈣鹽、鎂鹽、鋁鹽、鐵鹽、鋅鹽或銨鹽中的至少一種;或所述鹽選自甲胺鹽、二甲胺鹽、三甲胺鹽、乙胺鹽、二乙胺鹽、三乙胺鹽、異丙胺鹽、2-乙氨基乙醇鹽、吡啶鹽、甲基吡啶鹽、乙醇胺鹽、二乙醇胺鹽、銨鹽、四甲基銨鹽、四乙基銨鹽、三乙醇胺鹽、呱啶鹽、呱嗪鹽、嗎啉鹽、賴氨酸鹽、精氨酸鹽、L-精氨酸鹽、組氨酸鹽、L-組氨酸鹽、葡甲胺鹽、二甲基葡糖胺鹽、乙基葡糖胺鹽、二環己基胺鹽、1,6-己二胺鹽、葡糖胺鹽、肌氨酸鹽、絲氨醇鹽、三羥基甲基氨基甲烷鹽、氨基丙二醇鹽、1-氨基-2,3,4-丁三醇鹽、L-賴氨酸鹽、鳥氨酸鹽或膽鹼鹽中的至少一種。 As an embodiment of the present invention, the salt structural formula of the FXIa inhibitor compound is as follows: Wherein: n is 0.5-3; M forms a salt with a carboxyl group, and the salt is selected from at least one of lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, iron salts, zinc salts or ammonium salts; Or the salt is selected from methylamine salt, dimethylamine salt, trimethylamine salt, ethylamine salt, diethylamine salt, triethylamine salt, isopropylamine salt, 2-ethylaminoethanol salt, pyridine salt, picoline salts, ethanolamine salts, diethanolamine salts, ammonium salts, tetramethylammonium salts, tetraethylammonium salts, triethanolamine salts, oxidine salts, oxazine salts, morpholine salts, lysine salts, arginine salts, L-arginine salt, histidine salt, L-histidine salt, meglumine salt, dimethylglucamine salt, ethyl glucamine salt, dicyclohexylamine salt, 1,6-hexylamine salt Diamine salt, glucosamine salt, sarcosinate, serine alcohol salt, trihydroxymethyl aminomethane salt, amino propanediol salt, 1-amino-2,3,4-butanetriol salt, L-lysine at least one of acid salts, ornithine salts or choline salts.
作為本發明的一種實施方案,n為0.5、1、1.5、2、2.5或3。As an embodiment of the present invention, n is 0.5, 1, 1.5, 2, 2.5 or 3.
作為本發明的一種實施方案,所述的鹽選自鈉鹽、鉀鹽、葡甲胺鹽、鈣鹽、鎂鹽、膽鹼鹽。As an embodiment of the present invention, the salt is selected from the group consisting of sodium salt, potassium salt, meglumine salt, calcium salt, magnesium salt, and choline salt.
作為本發明的一種實施方案,所述的鹽選自鈉鹽、n=1;鉀鹽、n=1;膽鹼鹽、n=1;葡甲胺鹽、n=1;鈣鹽、n=0.5;鎂鹽、n=0.5。As an embodiment of the present invention, the salt is selected from sodium salt, n=1; potassium salt, n=1; choline salt, n=1; meglumine salt, n=1; calcium salt, n=1 0.5; magnesium salt, n=0.5.
作為本發明的一種實施方案,所述化合物或其鹽為晶型、或者無定型,或其混合物。As an embodiment of the present invention, the compound or its salt is in crystalline form, or amorphous form, or a mixture thereof.
作為本發明的一種實施方案,所述化合物或其鹽的一個以上的氫原子上被同位素氘取代。As an embodiment of the present invention, more than one hydrogen atom of the compound or its salt is substituted with the isotope deuterium.
作為本發明的一種實施方案,所述化合物或其鹽,和一種以上藥學上可接受的載體組成藥物組合物。As an embodiment of the present invention, the compound or its salt, and one or more pharmaceutically acceptable carriers form a pharmaceutical composition.
作為本發明的一種實施方案,所述動脈血栓導致動脈栓塞性疾病,包括冠心病、心肌梗死、缺血性腦中風、外周動脈疾病、房顫和心瓣膜病;所述靜脈血栓導致靜脈血栓栓塞性疾病,包括關節置換術後深靜脈血栓,肺栓塞,深靜脈血栓形成;以及透析後的動靜脈血栓。As an embodiment of the present invention, the arterial thrombus causes arterial embolic diseases, including coronary heart disease, myocardial infarction, ischemic stroke, peripheral arterial disease, atrial fibrillation and valvular heart disease; the venous thrombus causes venous thromboembolism Sexual diseases, including deep vein thrombosis after joint replacement, pulmonary embolism, deep vein thrombosis; and arteriovenous thrombosis after dialysis.
作為本發明的一種實施方案,關節置換術後深靜脈血栓包括全膝關節置換術後靜脈血栓,髖關節置換術後靜脈血栓等。As an embodiment of the present invention, deep venous thrombosis after joint replacement includes venous thrombosis after total knee replacement, venous thrombosis after hip replacement, and the like.
心房顫動為一種較常見的心律失常,可加重心肌缺血及惡化心功能,並引起血栓形成,已確定為腦中風及全身性栓塞性疾病的危險因素。房顫患者中血栓形成機制較為複雜,現有證據表明房顫中血栓形成和潛在病理改變相關:1.左心房血流瘀滯、2.血管壁完整性破壞、3.血液成分異常(凝血因子和血小板啟動等),這些改變完全符合Virchow’s三聯徵(靜脈血栓形成三要素)。所以,房顫患者常伴隨有多種類型的血栓,如深靜脈血栓、動靜脈旁路血栓等。Atrial fibrillation is a common arrhythmia, which can aggravate myocardial ischemia, worsen cardiac function, and cause thrombosis. It has been identified as a risk factor for stroke and systemic embolic disease. The mechanism of thrombosis in patients with atrial fibrillation is complex. The existing evidence shows that thrombosis in atrial fibrillation is related to underlying pathological changes: 1. Left atrial blood flow stasis, 2. Damage to the integrity of the vessel wall, 3. Abnormal blood components (coagulation factors and platelet activation, etc.), these changes are fully consistent with Virchow's triad (three elements of venous thrombosis). Therefore, patients with atrial fibrillation are often accompanied by various types of thrombosis, such as deep vein thrombosis, arteriovenous bypass thrombosis, etc.
人體動靜脈內瘻具有血流量充足、併發症少、能長期反復使用的優點,是血液透析患者最常用的血管通道。隨著透析品質的提高,維持性血液透析患者的生存時間延長,但反復穿刺損傷血管內膜,再加上壓迫止血不當、大量脫水致低血壓、血流緩慢、內瘻血流量下降及血液黏度增加等可促進血栓形成,所以,透析患者常見動靜脈內瘻血栓等血栓疾病。Human arteriovenous fistula has the advantages of sufficient blood flow, few complications, and long-term repeated use. It is the most commonly used vascular channel for hemodialysis patients. With the improvement of dialysis quality, the survival time of maintenance hemodialysis patients is prolonged, but repeated puncture damages the vascular intima, coupled with improper compression and hemostasis, massive dehydration resulting in hypotension, slow blood flow, decreased blood flow in the internal fistula and blood viscosity Increased thrombosis can promote thrombosis, so thrombosis diseases such as arteriovenous fistula thrombosis are common in dialysis patients.
作為本發明的一種實施方案,本發明動物實驗表明本發明化合物對家兔靜脈血栓和動靜脈旁路血栓均有很好預防/治療效果,所以,本發明化合物用於動靜脈血栓的治療包括房顫患者和腎透析患者的血栓疾病。As an embodiment of the present invention, animal experiments of the present invention show that the compounds of the present invention have good preventive/therapeutic effects on venous thrombosis and arteriovenous bypass thrombosis in rabbits. Therefore, the compounds of the present invention are used for the treatment of arteriovenous thrombosis including atrial Thrombotic disease in patients with fibrillation and renal dialysis.
本發明進一步提供了一種預防和/或治療動脈、靜脈血栓的藥物組合物,含有下式化合物或其鹽,和一種以上藥學上可接受的載體, 。 The present invention further provides a pharmaceutical composition for preventing and/or treating arterial and venous thrombosis, comprising a compound of the following formula or a salt thereof, and one or more pharmaceutically acceptable carriers, .
本發明化合物或其鹽、或含有所述化合物或其鹽的藥物組合物,對FXIa活性較好,對其他凝血酶選擇性高;具有顯著的延長APTT的效果,對人血漿的作用最強;對家兔靜脈血栓模型可達到利伐沙班藥效水平,且無出血風險。The compound of the present invention or a salt thereof, or a pharmaceutical composition containing the compound or a salt thereof, has good activity on FXIa and high selectivity to other thrombin; has a significant effect of prolonging APTT, and has the strongest effect on human plasma; The rabbit model of venous thrombosis can reach the level of rivaroxaban's efficacy without the risk of bleeding.
除非另有說明,本文所用的下列術語和短語旨在具有下列含義。一個特定的術語或短語在沒有特別定義的情況下不應該被認為是不確定的或不清楚的,而應該按照普通的含義去理解。當本文中出現商品名時,意在指代其對應的商品或其活性成分。這裡所採用的術語“藥學上可接受的”,是針對那些化合物、材料、組合物和/或劑型而言,它們在可靠的醫學判斷的範圍之內,適用於與人類和動物的組織接觸使用,而沒有過多的毒性、刺激性、過敏性反應或其它問題或併發症,與合理的利益/風險比相稱。Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without specific definitions, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
本發明化合物的鹽是指 “藥學上可接受的鹽”,由本發明發現的具有特定取代基的化合物與藥學上可接受的酸或鹼製備。Salts of the compounds of the present invention are referred to as "pharmaceutically acceptable salts", which are prepared from compounds with specific substituents discovered in the present invention and a pharmaceutically acceptable acid or base.
本發明的某些化合物的鹽可以以非溶劑化形式或者溶劑化形式存在,包括水合物形式。一般而言,溶劑化形式與非溶劑化的形式相當,都包含在本發明的範圍之內。Salts of certain compounds of the present invention may exist in unsolvated as well as solvated forms, including hydrated forms. In general, solvated and unsolvated forms are equivalent and are intended to be included within the scope of the present invention.
本發明的化合物可以存在特定的幾何或立體異構體形式。本發明設想所有的這類化合物,包括順式和反式異構體、(-)- 和 (+)-對對映體、(R)- 和 (S)-對映體、非對映異構體、(D)-異構體、(L)-異構體,及其外消旋混合物和其他混合物,例如對映異構體或非對映體富集的混合物,所有這些混合物都屬於本發明的範圍之內。烷基等取代基中可存在另外的不對稱碳原子。所有這些異構體以及它們的混合物,均包括在本發明的範圍之內。The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
可以通過的手性合成或手性試劑或者其他常規技術製備光學活性的(R)-和(S)-異構體,以及D和L異構體。如果想得到本發明某化合物的一種對映體,可以通過不對稱合成或者具有手性助劑的衍生作用來製備,其中將所得非對映體混合物分離,並且輔助基團裂開以提供純的所需對映異構體。或者,當分子中含有鹼性官能團(如氨基)或酸性官能團(如羧基)時,與適當的光學活性的酸或鹼形成非對映異構體的鹽,然後通過本領域所公知的常規方法進行非對映異構體拆分,然後回收得到純的對映體。此外,對映異構體和非對映異構體的分離通常是通過使用色譜法完成的,所述色譜法採用手性固定相,並任選地與化學衍生法相結合(例如由胺生成氨基甲酸鹽)。Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure Enantiomers are required. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
本發明化合物分子的原子是同位素,通過同位素衍生化通常可以延長半衰期、降低清除率、代謝穩定和提高體內活性等效果。並且,包括一個實施方案,其中至少一個原子被具有相同原子數(質子數)和不同質量數(質子和中子和)的原子取代。本發明化合物中包括的同位素的實例包括氫原子、碳原子、氮原子、氧原子、磷原子、硫原子、氟原子、氯原子,其分別包括 2H、 3H、 13C、 14C、 15N、 17O、 18O、 31P、 32P、 35S、 18F、 36Cl。特別的是,隨其衰退而發射輻射的放射性同位素例如 3H或 14C可用於藥物製劑或者體內化合物的局部解剖學檢驗。穩定的同位素既不隨其量衰減或變化,也不具有放射性,因此其可以安全使用。當構成本發明化合物分子的原子是同位素時,通過用包含相應同位素的試劑替代合成中所用的試劑,可以根據通用方法轉化同位素。 The atoms of the molecules of the compounds of the present invention are isotopes, and the isotope derivatization can usually prolong the half-life, reduce the clearance rate, stabilize the metabolism and improve the activity in vivo. Also, an embodiment is included in which at least one atom is replaced by an atom having the same atomic number (number of protons) and a different mass number (sum of protons and neutrons). Examples of isotopes included in the compounds of the present invention include hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, chlorine atom, which respectively include 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl. In particular, radioisotopes that emit radiation as they decay, such as 3 H or 14 C, are useful in the topological examination of pharmaceutical formulations or compounds in vivo. Stable isotopes neither decay or change with their amount nor are they radioactive, so they are safe to use. When the atoms making up the molecules of the compounds of the present invention are isotopes, the isotopes can be converted according to general methods by substituting the reagents used in the synthesis with reagents containing the corresponding isotopes.
本發明的化合物可以在一個或多個構成該化合物的原子上包含非天然比例的原子同位素。例如,可用放射性同位素標記化合物,比如氘( 2H),碘-125( 125I)或C-14( 14C)。本發明的化合物的所有同位素組成的變換,無論放射性與否,都包括在本發明的範圍之內。 The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as deuterium ( 2 H), iodine-125 ( 125 I) or C-14 ( 14 C). All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.
進一步地,本發明的化合物一個或多個氫原子上被同位素氘( 2H)取代,本發明化合物氘代後,具有延長半衰期、降低清除率、代謝穩定和提高體內活性等效果。 Further, one or more hydrogen atoms of the compounds of the present invention are substituted by the isotope deuterium ( 2 H). After deuteration, the compounds of the present invention have the effects of prolonging half-life, reducing clearance rate, stabilizing metabolism and improving in vivo activity.
所述同位素衍生物的製備方法通常包括:相轉移催化方法。例如,較佳的氘化方法採用相轉移催化劑(例如,四烷基銨鹽,NBu 4HSO 4)。使用相轉移催化劑交換二苯基甲烷化合物的亞甲基質子,導致比在酸(例如,甲磺酸)存在下用氘化矽烷(例如三乙基氘化甲矽烷)或用路易士酸如三氯化鋁採用氘化硼酸鈉還原而引入較高的氘。 The preparation methods of the isotopic derivatives generally include: a phase transfer catalysis method. For example, preferred methods of deuteration employ phase transfer catalysts (eg, tetraalkylammonium salts, NBu4HSO4 ) . The methylene protons of diphenylmethane compounds are exchanged using a phase transfer catalyst, resulting in higher ratios than with deuterated silanes (eg triethyl deuterated silane) or with Lewis acids such as trisulfuric acid in the presence of an acid (eg, methanesulfonic acid) Aluminum chloride is reduced with sodium deuteroborate to introduce higher deuterium.
術語“藥學上可接受的載體”是指能夠遞送本發明有效量活性物質、不干擾活性物質的生物活性並且對宿主或者患者無毒副作用的任何製劑載體或介質,代表性的載體包括水、油、蔬菜和礦物質、膏基、洗劑基質、軟膏基質等。這些基質包括懸浮劑、增黏劑、透皮促進劑等。它們的製劑為化妝品領域或局部藥物領域的技術人員所周知。關於載體的其他資訊,可以參考Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005),該文獻的內容通過引用的方式併入本文。The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active substance of the present invention, without interfering with the biological activity of the active substance, and without toxic side effects to the host or patient, and representative carriers include water, oil, Vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, skin penetration enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical field. For additional information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.
術語“賦形劑”通常是指配製有效的藥物組合物所需要載體、稀釋劑和/或介質。The term "excipient" generally refers to the carrier, diluent and/or medium required to formulate an effective pharmaceutical composition.
針對藥物或藥理學活性劑而言,術語“有效量”或“治療有效量”是指無毒的但能達到預期效果的藥物或藥劑的足夠用量。對於本發明中的口服劑型,組合物中一種活性物質的“有效量”是指與該組合物中另一種活性物質聯用時為了達到預期效果所需要的用量。有效量的確定因人而異,取決於受體的年齡和一般情況,也取決於具體的活性物質,個案中合適的有效量可以由本領域技術人員根據常規試驗確定。The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a nontoxic but sufficient amount of the drug or agent to achieve the desired effect. For oral dosage forms of the present invention, an "effective amount" of one active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depends on the age and general condition of the recipient, and also depends on the specific active substance, and the appropriate effective amount in individual cases can be determined by those skilled in the art based on routine experiments.
術語 “治療”是指一種化學實體,它可以有效地治療目標紊亂、疾病或病症。The term "treatment" refers to a chemical entity that is effective in treating the target disorder, disease, or condition.
“任選”或“任選地”指的是隨後描述的事件或狀況可能但不是必需出現的,並且該描述包括其中所述事件或狀況發生的情況以及所述事件或狀況不發生的情況。"Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
本發明的化合物可以通過本領域技術人員所熟知的多種合成方法來製備,包括下面列舉的具體實施方式、其與其他化學合成方法的結合所形成的實施方式以及本領域技術上人員所熟知的等同替換方式,較佳的實施方式包括但不限於本發明的實施例。The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other chemical synthesis methods, and equivalents known to those skilled in the art Alternatively, preferred embodiments include, but are not limited to, the embodiments of the present invention.
下面結合實施例對本發明作進一步詳細的描述,但發明的實施方式不限於此。The present invention will be further described in detail below with reference to the examples, but the embodiments of the invention are not limited thereto.
化合物的結構是通過核磁共振(NMR)或質譜(MS)來確定的。NMR位移(δ)以10-6(ppm)的單位給出。NMR的測定是用Bruker AVANCE-III核磁儀,測定溶劑為氘代二甲基亞碸(DMSO-d6),氘代氯仿(CDCl 3),內標為四甲基矽烷(TMS)。 The structures of compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR shifts ([delta]) are given in units of 10-6 (ppm). NMR was measured by Bruker AVANCE-III nuclear magnetic instrument, and the solvent was deuterated dimethylsulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), and the internal standard was tetramethylsilane (TMS).
MS的測定用ISQ EC質譜儀(生產商: Thermo, 型號: ISQ EC)。The MS was measured using an ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).
高效液相色譜法(HPLC)分析使用Thermo U3000 HPLC DAD高效液相色譜儀。High performance liquid chromatography (HPLC) analysis was performed using a Thermo U3000 HPLC DAD high performance liquid chromatograph.
CombiFlash快速製備儀使用CombiFlash Rf+ LUMEN (TELEDYNE ISCO)。The CombiFlash Rapid Preparation System uses the CombiFlash Rf+ LUMEN (TELEDYNE ISCO).
薄層層析矽膠板使用煙臺銀龍HSGF254或GF254矽膠板,薄層色譜法(TLC)使用的矽膠板採用的規格是0.17mm~0.23mm,薄層層析分離純化產品採用的規格是0.4mm~0.5mm。The thin layer chromatography silica gel plate uses Yantai Yinlong HSGF254 or GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.17mm~0.23mm, and the size of the TLC separation and purification products is 0.4mm ~0.5mm.
矽膠柱色譜法一般使用乳山上邦矽膠100~200目矽膠為載體。Silica gel column chromatography generally uses Rushan Shangbang silica gel 100~200 mesh silica gel as the carrier.
實施例1Example 1
合成 (S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸 Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3 -Phenylpropionamido)benzoic acid
具體合成路線如下。The specific synthetic route is as follows.
步驟A:合成5-溴-6-羥基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮 Step A: Synthesis of 5-bromo-6-hydroxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one
室溫下,將溴馬來酸酐(2.00克,11.3毫莫耳)和4-甲氧基苄基肼鹽酸鹽(2..13克,11.3毫莫耳)加入冰醋酸(50.0毫升)中,100℃反應3小時。Bromomaleic anhydride (2.00 g, 11.3 mmol) and 4-methoxybenzylhydrazine hydrochloride (2..13 g, 11.3 mmol) were added to glacial acetic acid (50.0 mL) at room temperature , 100 ℃ reaction for 3 hours.
反應結束,冷卻至室溫,將反應液倒入水中,析出大量固體,攪拌一段時間後抽濾,濾餅用水洗,濾餅烘乾得1.50克淡黃色固體5-溴-6-羥基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮,無需純化,直接用於下步反應。LCMS: RT = 3.44 min, [M+H] += 311.03。 The reaction was completed, cooled to room temperature, the reaction solution was poured into water, and a large amount of solid was precipitated. After stirring for a period of time, suction filtration, the filter cake was washed with water, and the filter cake was dried to obtain 1.50 grams of light yellow solid 5-bromo-6-hydroxy-2 -(4-Methoxybenzyl)pyridazin-3( 2H )-one was used in the next step without purification. LCMS: RT = 3.44 min, [M+H] + = 311.03.
步驟B:合成5-溴-6-甲氧基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮 Step B: Synthesis of 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one
室溫下,將5-溴-6-羥基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮(1.50克,4.82毫莫耳)和碳酸鉀(2.66克,19.29毫莫耳)加入 N,N-二甲基甲醯胺(15.0毫升)中,80℃攪拌15 分鐘,在該溫度下,加入碘甲烷(1.2毫升),繼續反應30分鐘。 At room temperature, 5-bromo-6-hydroxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (1.50 g, 4.82 mmol) and potassium carbonate (2.66 g, 19.29 mmol) was added to N,N -dimethylformamide (15.0 mL), and the mixture was stirred at 80°C for 15 minutes. At this temperature, methyl iodide (1.2 mL) was added and the reaction was continued for 30 minutes.
反應結束,加水淬滅,混合液用乙酸乙酯(50毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(50毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化(洗脫劑:乙酸乙酯/正己烷=1/3)。得到1.10克白色固體5-溴-6-甲氧基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮(收率:70.3%)。LCMS: RT = 3.87 min, [M+H] += 325.01。 The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (50 ml × 3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (50 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/3). 1.10 g of white solid 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one was obtained (yield: 70.3%). LCMS: RT = 3.87 min, [M+H] + = 325.01.
步驟C:合成6-乙醯基-3-氯苯硼酸頻哪醇酯 Step C: Synthesis of 6-Acetyl-3-chlorophenylboronic acid pinacol ester
室溫下,將2-溴-4-氯苯乙酮(5.00克,21.41毫莫耳)、聯硼酸頻哪醇酯(8.16克,32.12 毫莫耳)和醋酸鉀(4.20克,42.82毫莫耳)加入三頸瓶中,置換氮氣,加入1,4-二氧六環(60.0毫升),置換氮氣,加入1,1'-雙二苯基膦二茂鐵二氯化鈀(1.75克,2.14毫莫耳),置換氮氣,升溫至80℃反應3小時。At room temperature, combine 2-bromo-4-chloroacetophenone (5.00 g, 21.41 mmol), pinacol diboronate (8.16 g, 32.12 mmol) and potassium acetate (4.20 g, 42.82 mmol) ear) into a three-necked flask, replacing nitrogen, adding 1,4-dioxane (60.0 ml), replacing nitrogen, adding 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (1.75 g, 2.14 mmol), nitrogen was replaced, and the temperature was raised to 80 °C to react for 3 hours.
反應結束,加水淬滅,墊矽藻土抽濾,乙酸乙酯洗滌濾餅,濾液用乙酸乙酯(80毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(50毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化(洗脫劑:乙酸乙酯/正己烷=1/50)。得到2.1克黃色固體6-乙醯基-3-氯苯硼酸頻哪醇酯 (收率:35.0%)。LCMS: RT = 4.26 min, [M-H] -= 279.08。 The reaction was completed, quenched by adding water, suction filtration through celite, washing the filter cake with ethyl acetate, extracting the filtrate with ethyl acetate (80 ml × 3 times), combining the organic phases, first adding saturated brine (50 ml × 3 times) to the organic phase. 2 times), then dried over anhydrous sodium sulfate, and finally concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/50). 2.1 g of 6-acetyl-3-chlorophenylboronic acid pinacol ester was obtained as a yellow solid (yield: 35.0%). LCMS: RT = 4.26 min, [MH] - = 279.08.
步驟D:合成5-(2-乙醯基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮 Step D: Synthesis of 5-(2-Acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one
室溫下,將5-溴-6-甲氧基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮(1.10克,3.39毫莫耳)、6-乙醯基-3-氯苯硼酸頻哪醇酯(949毫克,3.39毫莫耳)和碳酸鈉(718毫克,6.78毫莫耳)加入三頸瓶中,置換氮氣,加入混合溶劑(10毫升,1,2-二甲氧基乙烷:乙醇:水=8:1:1),置換氮氣,加入1,1'-雙二苯基膦二茂鐵二氯化鈀(249毫克,0.34毫莫耳),置換氮氣,升溫至90℃反應1小時。 At room temperature, 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (1.10 g, 3.39 mmol), 6-acetyl Alkyl-3-chlorophenylboronic acid pinacol ester (949 mg, 3.39 mmol) and sodium carbonate (718 mg, 6.78 mmol) were added to the three-necked flask, nitrogen was replaced, and mixed solvent (10 mL, 1, 2-dimethoxyethane:ethanol:water=8:1:1), replace nitrogen, add 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (249 mg, 0.34 mmol) , replaced nitrogen gas, heated to 90 °C and reacted for 1 hour.
反應結束,加水淬滅,混合液用乙酸乙酯(50毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(50毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化(洗脫劑:乙酸乙酯/正己烷=1/2)。得到676毫克黃色固體5-(2-乙醯基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮 (收率:50.2%)。LCMS: RT = 3.99 min, [M+H] += 399.07。 The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (50 ml × 3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (50 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/2). 676 mg of yellow solid 5-(2-acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (acquired rate: 50.2%). LCMS: RT = 3.99 min, [M+H] + = 399.07.
步驟E:合成5-(2-乙醯基-5-氯苯基)-6-甲氧基噠嗪-3(2 H)-酮 Step E: Synthesis of 5-(2-Acetyl-5-chlorophenyl)-6-methoxypyridazin-3( 2H )-one
0℃下,將5-(2-乙醯基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)噠嗪-3(2 H)-酮(676毫克,1.70毫莫耳)加入混合溶劑(4毫升,乙腈: 水=3:1)中,再緩慢加入硝酸鈰銨(7.46克,13.60毫莫耳),加畢,室溫下反應30分鐘。 5-(2-Acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (676 mg, 1.70 mmol) was added to the mixed solvent (4 ml, acetonitrile: water = 3:1), and then slowly added ceric ammonium nitrate (7.46 g, 13.60 mmol), the addition was completed, and the reaction was carried out at room temperature for 30 minutes.
反應結束,加水淬滅,混合液用乙酸乙酯(30毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(30毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化(洗脫劑:乙酸乙酯/正己烷=1/1)。得到238毫克黃色固體5-(2-乙醯基-5-氯苯基)-6-甲氧基噠嗪-3(2 H)-酮(收率:50.0%)。LCMS: RT = 3.23 min, [M+H] += 279.08。 The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (30 ml × 3 times), the organic phases were combined, and the organic phase was first washed with saturated brine (30 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/1). 238 mg of 5-(2-acetyl-5-chlorophenyl)-6-methoxypyridazin-3( 2H )-one were obtained as a yellow solid (yield: 50.0%). LCMS: RT = 3.23 min, [M+H] + = 279.08.
步驟F:合成 (S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯 Step F: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1( 6H )-yl )-3-Phenylpropionamido) tert-butyl benzoate
室溫下,將5-(2-乙醯基-5-氯苯基)-6-甲氧基噠嗪-3(2 H)-酮(50毫克,0.18毫莫耳)、(R)-4-(2-(((4-硝基苯基)磺醯基)氧基)-3-苯基丙醯胺基)苯甲酸叔丁酯(113毫克,0.22毫莫耳)和碳酸鉀(50毫克,0.36毫莫耳)加入 N,N-二甲基甲醯胺(2.0毫升)中,室溫反應過夜。 5-(2-Acetyl-5-chlorophenyl)-6-methoxypyridazin-3( 2H )-one (50 mg, 0.18 mmol), (R)- tert-butyl 4-(2-(((4-nitrophenyl)sulfonyl)oxy)-3-phenylpropionamido)benzoate (113 mg, 0.22 mmol) and potassium carbonate ( 50 mg, 0.36 mmol) was added to N,N -dimethylformamide (2.0 mL) and reacted overnight at room temperature.
反應結束,加水淬滅,混合液用乙酸乙酯(10毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(10毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化(洗脫劑:乙酸乙酯/正己烷=1/2)。得到75毫克淡黃色固體(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯(收率:66.7%)。LCMS: RT = 4.53 min, [M+H] += 602.13。 The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (10 ml × 3 times), the organic phases were combined, the organic phase was first washed with saturated brine (10 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/2). 75 mg of pale yellow solid (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazine -1(6H) was obtained -yl)-3-phenylpropionamido) tert-butyl benzoate (yield: 66.7%). LCMS: RT = 4.53 min, [M+H] + = 602.13.
步驟G:合成 (S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸 化合物A Step G: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1( 6H )-yl )-3-Phenylpropionamido)benzoic acid Compound A
室溫下,將(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯(75毫克,0.12毫莫耳)加入二氯甲烷(2.0毫升)中,滴加三氟乙酸(0.25毫升),室溫反應3小時。 At room temperature, (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazine -1(6H)- tert-butyl)-3-phenylpropionamido)benzoate (75 mg, 0.12 mmol) was added to dichloromethane (2.0 mL), trifluoroacetic acid (0.25 mL) was added dropwise, and the reaction was carried out at room temperature for 3 Hour.
反應結束,蒸乾二氯甲烷並用油泵抽乾三氟乙酸,所得殘餘物用溶於二氯甲烷(1.0毫升)中,將其滴加入正己烷(10.0毫升)中,析出白色固體,抽濾,濾餅用正己烷洗滌,乾燥得到50毫克白色固體(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸(收率:76.5%)。LCMS: RT = 3.98 min, [M-H] -= 544.10。 1H NMR (500 MHz, DMSO) δ 12.79 (s, 1H), 10.52 (s, 1H), 7.99 (d, J= 8.4 Hz, 1H), 7.91 (d, J= 8.7 Hz, 2H), 7.72 (d, J= 8.7 Hz, 2H), 7.69 (dd, J= 8.3, 2.1 Hz, 1H), 7.50 (d, J= 2.1 Hz, 1H), 7.37–7.23 (m, 4H), 7.19 (t, J= 7.1 Hz, 1H), 6.91 (s, 1H), 5.74 (dd, J= 10.2, 4.9 Hz, 1H), 3.67 (s, 3H), 3.52 (dd, J= 14.1, 10.3 Hz, 1H), 3.41 (dd, J= 14.1, 4.7 Hz, 1H), 2.53 (s, 3H)。 After the reaction was completed, dichloromethane was evaporated to dryness and trifluoroacetic acid was dried with an oil pump. The obtained residue was dissolved in dichloromethane (1.0 mL) and added dropwise to n-hexane (10.0 mL) to precipitate a white solid, which was filtered off with suction. The filter cake was washed with n-hexane and dried to give 50 mg of white solid (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridine) oxazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid (yield: 76.5%). LCMS: RT = 3.98 min, [MH] - = 544.10. 1 H NMR (500 MHz, DMSO) δ 12.79 (s, 1H), 10.52 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.72 ( d, J = 8.7 Hz, 2H), 7.69 (dd, J = 8.3, 2.1 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.37–7.23 (m, 4H), 7.19 (t, J = 7.1 Hz, 1H), 6.91 (s, 1H), 5.74 (dd, J = 10.2, 4.9 Hz, 1H), 3.67 (s, 3H), 3.52 (dd, J = 14.1, 10.3 Hz, 1H), 3.41 (dd, J = 14.1, 4.7 Hz, 1H), 2.53 (s, 3H).
實施列2Implement column 2
(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鈉鹽 化合物A鈉鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid sodium salt Compound A sodium salt
零攝氏度下,向含有(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6H)-基)-3-苯基丙醯胺基)苯甲酸(150.0毫克,0.28毫莫耳)的甲醇(10.0毫升)中,滴加氫氧化鈉水溶液(氫氧化鈉;6.72毫克,0.28毫莫耳;水:2.0毫升),保持該溫度反應5小時。At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( 6H)-yl)-3-phenylpropionamido)benzoic acid (150.0 mg, 0.28 mmol) in methanol (10.0 mL) was added dropwise aqueous sodium hydroxide (sodium hydroxide; 6.72 mg, 0.28 mmol) Molar; water: 2.0 ml), and the reaction was maintained at this temperature for 5 hours.
反應結束,蒸除甲醇,所得水溶液低溫凍乾得到155.0毫克Form A白色固體(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6H)-基)-3-苯基丙醯胺基)苯甲酸鈉Form A (收率:97.5%)。LCMS: RT = 2.00 min, [M+H]+ = 546.31。 1H NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 7.99 (d, J= 8.4 Hz, 1H), 7.86 (d, J= 8.6 Hz, 2H), 7.68 (dd, J= 8.3, 2.2 Hz, 1H), 7.59 (d, J= 8.6 Hz, 2H), 7.50 (d, J= 2.1 Hz, 1H), 7.36–7.24 (m, 4H), 7.18 (t, J= 7.1 Hz, 1H), 6.90 (s, 1H), 5.75 (dd, J= 10.2, 4.8 Hz, 1H), 3.68 (s, 3H), 3.47–3.37 (m, 2H), 2.53 (s, 3H)。 The reaction was completed, methanol was evaporated, and the obtained aqueous solution was freeze-dried at low temperature to obtain 155.0 mg of Form A white solid (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3- Sodium Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoate Form A (Yield: 97.5%). LCMS: RT = 2.00 min, [M+ H]+ = 546.31. 1 H NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.68 (dd , J = 8.3, 2.2 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 2.1 Hz, 1H), 7.36–7.24 (m, 4H), 7.18 (t, J = 7.1 Hz, 1H), 6.90 (s, 1H), 5.75 (dd, J = 10.2, 4.8 Hz, 1H), 3.68 (s, 3H), 3.47–3.37 (m, 2H), 2.53 (s, 3H).
取40毫克Form A樣品加入到1 毫升丙酮中,加熱至50攝氏度,加入20微升水,再加入320毫克Form A樣品,固體完全溶解,50攝氏度攪拌24小時後有固體析出,離心得化合物A鈉鹽結晶性固體Type A。Take 40 mg of Form A sample and add it to 1 ml of acetone, heat it to 50 degrees Celsius, add 20 microliters of water, and then add 320 mg of Form A sample, the solid is completely dissolved, and after 24 hours of stirring at 50 degrees Celsius, a solid precipitates out, and centrifugation to obtain Compound A sodium Salt crystalline solid Type A.
實施例3Example 3
(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鉀鹽 化合物A鉀鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid potassium salt Compound A Potassium Salt
零攝氏度下,向含有(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸(100.0毫克,0.18毫莫耳)的甲醇(10.0毫升)中,滴加氫氧化鉀水溶液(氫氧化鉀;10.3毫克,0.18毫莫耳;水:2.0毫升),保持該溫度反應5小時。 At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( 6H)-yl)-3-phenylpropionamido)benzoic acid (100.0 mg, 0.18 mmol) in methanol (10.0 mL) was added dropwise aqueous potassium hydroxide (potassium hydroxide; 10.3 mg, 0.18 mmol; water: 2.0 ml), and the reaction was maintained at this temperature for 5 hours.
反應結束,蒸除甲醇,所得水溶液低溫凍乾得到98.0毫克白色固體(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鉀鹽(收率:93.4%)。LCMS: RT = 2.00 min, [M+H] += 546.22。 After the reaction was completed, methanol was evaporated, and the obtained aqueous solution was freeze-dried at low temperature to obtain 98.0 mg of white solid (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy) yl-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid potassium salt (yield: 93.4%). LCMS: RT = 2.00 min, [M+H ] + = 546.22.
1H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.98 (d, J= 8.4 Hz, 1H), 7.77 (d, J= 8.6 Hz, 2H), 7.68 (dd, J= 8.3, 2.2 Hz, 1H), 7.50 (d, J= 2.1 Hz, 1H), 7.46 (d, J= 8.5 Hz, 2H), 7.38–7.24 (m, 4H), 7.18 (t, J= 7.1 Hz, 1H), 6.89 (s, 1H), 5.75 (dd, J= 10.3, 4.7 Hz, 1H), 3.68 (s, 3H), 3.56–3.41 (m, 2H), 2.52 (s, 3H)。 1 H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.68 (dd, J = 8.3, 2.2 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.38–7.24 (m, 4H), 7.18 (t, J = 7.1 Hz, 1H), 6.89 (s, 1H), 5.75 (dd, J = 10.3, 4.7 Hz, 1H), 3.68 (s, 3H), 3.56–3.41 (m, 2H), 2.52 (s, 3H).
實施列4Implement column 4
(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸葡甲胺鹽 化合物A葡甲胺鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido) benzoic acid meglumine salt Compound A Meglumine Salt
將(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸(1.0克)和葡甲胺(358毫克)以1:1當量比加入20毫升丙酮中製成混懸液,在溫度迴圈下(1000 rpm,50 ºC~5 ºC,0.1 ºC/min,2個迴圈)混懸攪拌3天,得到(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6H)-基)-3-苯基丙醯胺基)苯甲酸葡甲胺鹽晶型A。 (S)-4-(2-(4-(2-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl )-3-Phenylpropionamido)benzoic acid (1.0 g) and meglumine (358 mg) were added to 20 mL of acetone in a 1:1 equiv. ratio to make a suspension, under temperature cycling (1000 rpm , 50 ºC~5 ºC, 0.1 ºC/min, 2 cycles) suspension and stirring for 3 days to obtain (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)- 3-Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid meglumine salt form A.
1H NMR (400 MHz, DMSO) δ 10.42 (s, 1H), 8.00 (d, J= 8.4 Hz, 1H), 7.88 (d, J= 8.7 Hz, 2H), 7.69 (dd, J= 8.3, 2.2 Hz, 1H), 7.66 (d, J= 8.7 Hz, 2H), 7.51 (d, J= 2.1 Hz, 1H), 7.34–7.25 (m, 4H), 7.22-7.18 (m, 1H), 6.91 (s, 1H), 5.75 (dd, J= 10.2, 4.9 Hz, 1H), 3.79-3.74 (m, 1H), 3.68 (s, 3H), 3.67–3.65 (m, 1H), 3.60 (dd, J= 10.8, 3.5 Hz, 1H), 3.56–3.46 (m, 2H), 3.43-3.33 (m, 3H), 2.80–2.66 (m, 1H), 2.55 (s, 1H), 2.53 (s, 3H), 2.39 (s, 3H)。 1 H NMR (400 MHz, DMSO) δ 10.42 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.69 (dd, J = 8.3, 2.2 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 2.1 Hz, 1H), 7.34–7.25 (m, 4H), 7.22-7.18 (m, 1H), 6.91 (s , 1H), 5.75 (dd, J = 10.2, 4.9 Hz, 1H), 3.79-3.74 (m, 1H), 3.68 (s, 3H), 3.67–3.65 (m, 1H), 3.60 (dd, J = 10.8 , 3.5 Hz, 1H), 3.56–3.46 (m, 2H), 3.43-3.33 (m, 3H), 2.80–2.66 (m, 1H), 2.55 (s, 1H), 2.53 (s, 3H), 2.39 ( s, 3H).
實施例5Example 5
(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鎂鹽 化合物A鎂鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido) benzoic acid magnesium salt Compound A magnesium salt
零攝氏度下,向含有(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲鈉(100.0毫克,0.18毫莫耳)的甲醇(10.0毫升)中,滴加氯化鎂水溶液(氯化鎂;16.8毫克,0.18毫莫耳;水:2.0毫升),保持該溫度反應5小時。 At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( To sodium 6H )-yl)-3-phenylpropionamido)benzyl (100.0 mg, 0.18 mmol) in methanol (10.0 mL) was added aqueous magnesium chloride (MgCl; 16.8 mg, 0.18 mmol) dropwise ; water: 2.0 ml), and kept the temperature for 5 hours.
反應結束,蒸除甲醇,析出白色固體,抽濾,乾燥得到62.0毫克白色固體(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鎂鹽(收率:30.9%)。LCMS: RT = 2.00 min, [M+H] += 546.20。 1H NMR (500 MHz, DMSO) δ 10.33 (s, 1H), 7.98 (d, J= 8.4 Hz, 1H), 7.93 (s, 2H), 7.67 (dd, J= 8.3, 2.1 Hz, 1H), 7.59 (d, J= 8.2 Hz, 2H), 7.49 (d, J= 1.9 Hz, 1H), 7.36–7.22 (m, 4H), 7.17 (t, J= 7.2 Hz, 1H), 6.88 (s, 1H), 5.73 (dd, J= 10.2, 4.8 Hz, 1H), 3.66 (s, 3H), 3.41 (dd, J= 14.3, 4.7 Hz, 2H), 2.51 (s, 3H)。 After the reaction was completed, methanol was evaporated to precipitate a white solid, which was filtered off with suction and dried to obtain 62.0 mg of a white solid (S)-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3) -Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid magnesium salt (yield: 30.9%). LCMS: RT = 2.00 min, [ M+H] + = 546.20. 1 H NMR (500 MHz, DMSO) δ 10.33 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.93 (s, 2H), 7.67 (dd, J = 8.3, 2.1 Hz, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 1.9 Hz, 1H), 7.36–7.22 (m, 4H), 7.17 (t, J = 7.2 Hz, 1H), 6.88 (s, 1H), 5.73 (dd, J = 10.2, 4.8 Hz, 1H), 3.66 (s, 3H), 3.41 (dd, J = 14.3, 4.7 Hz, 2H), 2.51 (s, 3H) ).
實施例6Example 6
(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鈣鹽 化合物A鈣鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid calcium salt Compound A calcium salt
零攝氏度下,向含有(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲鈉(100.0毫克,0.18毫莫耳)的甲醇(10.0毫升)中,滴加氯化鈣水溶液(氯化鈣;20.0毫克,0.18毫莫耳;水:2.0毫升),保持該溫度反應5小時。 At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( Aqueous calcium chloride (calcium chloride; 20.0 mg, 0.18 mmol; water: 2.0 mL), and the reaction was maintained at this temperature for 5 hours.
反應結束,蒸除甲醇,析出白色固體,抽濾,水洗,乾燥得到58.0毫克白色固體(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸鈣鹽(收率:28.5%)。LCMS: RT = 2.00 min, [M+H] += 546.17。 After the reaction was completed, methanol was evaporated to precipitate a white solid, which was filtered off with suction, washed with water, and dried to obtain 58.0 mg of white solid (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)) -3-Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid calcium salt (yield: 28.5%). LCMS: RT = 2.00 min , [M+H] + = 546.17.
實施例7Example 7
(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸膽鹼鹽 化合物A膽鹼鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid choline salt Compound A Choline Salt
將(S)-4-(2-(4-(2-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧并噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸和膽鹼以1:1當量比加入丙酮中,在溫度迴圈下(50 oC ~ 5 oC,0.1 oC /min,2迴圈)攪拌3天得到膠狀樣品,膠狀樣品在室溫下真空乾燥8小時得固體粉末(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-甲氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸膽鹼鹽晶型A。 (S)-4-(2-(4-(2-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl )-3-Phenylpropionamido)benzoic acid and choline were added to acetone in an equivalent ratio of 1:1, under the temperature cycle (50 o C ~ 5 o C, 0.1 o C /min, 2 loops) Stir for 3 days to obtain a colloidal sample, and the colloidal sample is vacuum-dried at room temperature for 8 hours to obtain a solid powder (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3- Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid choline salt form A.
對比實施例1化合物A1Comparative Example 1 Compound A1
合成 (S)-4-(2-(4-(5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1 (6 H)-基)-3-苯基丙醯胺基)苯甲酸 化合物A1 Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidine- 1(6H)-yl)-3-phenylpropionamido)benzoic acid Compound A1
具體合成路線如下:The specific synthetic route is as follows:
步驟A:合成 4-氯-2-(四甲基-1,3,2-二氧雜硼雜環戊烷-2-基)苯胺 Step A: Synthesis of 4-chloro-2-(tetramethyl-1,3,2-dioxaborol-2-yl)aniline
將2-溴-4-氯苯胺(3.1克,14.5毫莫耳)加入2-溴-4-氯苯胺(3.0克,15.0毫莫耳),4,4,5,5-四甲基-2-(四甲基-1,3,2-二氧雜硼雜環戊烷-2-基)-1,3,2-二氧雜硼雜環戊烷(38克,150.0毫莫耳),醋酸鉀(2.9克,30.0毫莫耳), [1,1'-雙(二苯基膦)二茂鐵]二氯化鈀二氯甲烷絡合物(1.1克,1.5毫莫耳)溶解於二甲基亞碸(75毫升)。氮氣保護後,在80℃加熱5小時。將反應冷卻至室溫。加入水溶解鹽,然後過濾反應。將剩餘的固體懸浮於二氯甲烷中並過濾不溶固體。濃縮濾液,然後通過矽膠柱層析純化,得到5.2克白色固體4-氯-2-(四甲基-1,3,2-二氧雜硼雜環戊烷-2-基)苯胺(收率:100%)。LCMS:RT = 4.40 min, [M+H] += 254.10。 2-Bromo-4-chloroaniline (3.1 g, 14.5 mmol) was added to 2-bromo-4-chloroaniline (3.0 g, 15.0 mmol), 4,4,5,5-tetramethyl-2 -(Tetramethyl-1,3,2-dioxaborol-2-yl)-1,3,2-dioxaborolane (38 g, 150.0 mmol), Potassium acetate (2.9 g, 30.0 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.1 g, 1.5 mmol) was dissolved in Dimethyl sulfite (75 mL). After nitrogen protection, it was heated at 80 °C for 5 hours. The reaction was cooled to room temperature. Water was added to dissolve the salt, and the reaction was filtered. The remaining solids were suspended in dichloromethane and the insoluble solids were filtered. The filtrate was concentrated and then purified by silica gel column chromatography to give 5.2 g of white solid 4-chloro-2-(tetramethyl-1,3,2-dioxaborol-2-yl)aniline (yield : 100%). LCMS: RT = 4.40 min, [M+H] + = 254.10.
步驟B:合成 4-氯-2-(6-甲氧基嘧啶-4-基)苯胺 Step B: Synthesis of 4-chloro-2-(6-methoxypyrimidin-4-yl)aniline
將4-氯-6-甲氧基嘧啶(3.9克,15.4毫莫耳)碳酸鈉(3.2克,30.8毫莫耳),乙二醇二甲醚(16毫升),乙醇(2毫升)和水(2毫升)置於三口瓶中。氮氣保護後,加入[1,1'-雙(二苯基膦)二茂鐵]二氯化鈀二氯甲烷絡合物(1.3克,1.5毫莫耳)。將4-氯-2-(四甲基-1,3,2-二氧雜硼雜環戊烷-2-基)苯胺(3.31克,23.1毫莫耳)的乙二醇二甲醚(8毫升),將反應在90℃加熱2小時。LCMS監測,反應完全後,冷卻至室溫,墊矽藻土過濾,濾餅用乙酸乙酯(30毫升)洗滌3次,合併濾液及洗滌液,水洗一次,飽和氯化銨洗滌兩次,有機相經無水硫酸鈉乾燥,過濾,旋乾,殘渣經矽膠柱層析純化得到1.0克黃色固體4-氯-2-(6-甲氧基嘧啶-4-基)苯胺(收率:28%)。LCMS:RT = 3.95 min, [M+H] += 236.04。 Combine 4-chloro-6-methoxypyrimidine (3.9 g, 15.4 mmol), sodium carbonate (3.2 g, 30.8 mmol), ethylene glycol dimethyl ether (16 mL), ethanol (2 mL) and water (2 ml) in a three-necked bottle. After a nitrogen blanket, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.3 g, 1.5 mmol) was added. 4-Chloro-2-(tetramethyl-1,3,2-dioxaborol-2-yl)aniline (3.31 g, 23.1 mmol) in ethylene glycol dimethyl ether (8 mL), the reaction was heated at 90 °C for 2 h. LCMS monitoring, after the reaction was completed, cooled to room temperature, filtered through celite, the filter cake was washed three times with ethyl acetate (30 ml), the filtrate and washings were combined, washed once with water, washed twice with saturated ammonium chloride, organic The phase was dried over anhydrous sodium sulfate, filtered, and spin-dried. The residue was purified by silica gel column chromatography to obtain 1.0 g of yellow solid 4-chloro-2-(6-methoxypyrimidin-4-yl)aniline (yield: 28%) . LCMS: RT = 3.95 min, [M+H] + = 236.04.
步驟C:合成 4-{5-氯-2-[4-(三甲基甲矽烷基)-1 H-1,2,3-三唑-1-基]-苯基}-6-甲氧基-嘧啶 Step C : Synthesis of 4-{5-Chloro-2-[4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl]-phenyl}-6-methoxy base-pyrimidine
將4-氯-2-(6-甲氧基嘧啶-4-基)苯胺(0.9克,3.8毫莫耳)溶於乙腈(60毫升)中,在0℃下加入3-甲基丁基亞硝酸酯(0.6毫升,5.8毫莫耳),然後滴加疊氮基三甲基矽烷(0.6毫升,5.8毫莫耳)。觀察到氣體產生。10分鐘後,除去冰浴,讓反應溫熱至室溫。1小時後,加入乙炔基三甲基甲矽烷(1.8毫升,11.4毫莫耳)和氧化亞銅(0.06g,0.36毫莫耳),將反應再攪拌1小時。向反應液中加乙酸乙酯和飽和氯化銨水溶液分層。有機相用鹽水洗滌,經無水硫酸鈉乾燥,過濾並濃縮。經矽膠柱層析進一步純化得到730毫克黃色固體4-{5-氯-2-[4-(三甲基-甲矽烷基)-1 H-1,2,3-三唑-1-基]苯基}-6-甲氧基嘧啶(收率:45%)。LCMS:RT = 2.04 min, [M+H] += 360.10。 4-Chloro-2-(6-methoxypyrimidin-4-yl)aniline (0.9 g, 3.8 mmol) was dissolved in acetonitrile (60 mL) and 3-methylbutylidene was added at 0 °C Nitrate (0.6 mL, 5.8 mmol), followed by azidotrimethylsilane (0.6 mL, 5.8 mmol) dropwise. Gas evolution was observed. After 10 minutes, the ice bath was removed and the reaction was allowed to warm to room temperature. After 1 hour, ethynyltrimethylsilane (1.8 mL, 11.4 mmol) and cuprous oxide (0.06 g, 0.36 mmol) were added and the reaction was stirred for an additional 1 hour. Ethyl acetate and saturated aqueous ammonium chloride were added to the reaction solution to separate the layers. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Further purification by silica gel column chromatography gave 730 mg of yellow solid 4-{5-chloro-2-[4-(trimethyl-silyl)-1H- 1,2,3 -triazol-1-yl] Phenyl}-6-methoxypyrimidine (yield: 45%). LCMS: RT = 2.04 min, [M+H] + = 360.10.
步驟D:合成 4-[5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基]-6-甲氧基嘧啶 Step D: Synthesis of 4-[5-Chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]-6-methoxypyrimidine
將4-{5-氯-2-[4-(三甲基甲矽烷基)-1 H-1,2,3-三唑-1-基]苯基}-6-甲氧基嘧啶(700毫克,1.94毫莫耳)溶於乙腈(20毫升),溶液中加入 N-氯代丁二醯亞胺(0.9克,7.2毫莫耳)和矽膠(2.9克,50.44毫莫耳)。反應在80℃攪拌1小時。然後將反應過濾以除去矽膠,將收集的矽膠用乙酸乙酯洗滌。濾液用水洗,鹽水洗滌,濃縮。殘渣經矽膠柱層析進一步純化得到450毫克黃色固體4-[5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基]-6-甲氧基嘧啶(收率:72%)。LCMS:RT = 2.00 min, [M+H] += 322.05。 4-{5-Chloro-2-[4-(trimethylsilyl)-1H- 1,2,3 -triazol-1-yl]phenyl}-6-methoxypyrimidine (700 mg, 1.94 mmol) was dissolved in acetonitrile (20 mL), and N -chlorobutanediimide (0.9 g, 7.2 mmol) and silica gel (2.9 g, 50.44 mmol) were added to the solution. The reaction was stirred at 80°C for 1 hour. The reaction was then filtered to remove the silica gel and the collected silica gel was washed with ethyl acetate. The filtrate was washed with water, brine and concentrated. The residue was further purified by silica gel column chromatography to obtain 450 mg of yellow solid 4-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]-6-methane oxypyrimidine (yield: 72%). LCMS: RT = 2.00 min, [M+H] + = 322.05.
步驟E:合成 6-[5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基]嘧啶-4-醇 Step E: Synthesis of 6-[5-Chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]pyrimidin-4-ol
向4-[5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基]-6-甲氧基嘧啶(450毫克,1.4毫莫耳)在醋酸(3毫升)中的溶液中加入48%氫溴酸水溶液(1.5毫升,13.3毫莫耳)。混合物在95℃攪拌1小時。將反應濃縮至乾,然後用乙酸乙酯和飽和碳酸氫鈉溶液分液。有機相濃縮,殘渣經矽膠柱層析純化得到190毫克黃色固體6-[5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基]嘧啶-4-醇(收率:44%)。LCMS:RT = 1.74 min, [M-H] -= 305.97。 To 4-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]-6-methoxypyrimidine (450 mg, 1.4 mmol) To a solution in acetic acid (3 mL) was added 48% aqueous hydrobromic acid (1.5 mL, 13.3 mmol). The mixture was stirred at 95°C for 1 hour. The reaction was concentrated to dryness, then partitioned with ethyl acetate and saturated sodium bicarbonate solution. The organic phase was concentrated, and the residue was purified by silica gel column chromatography to obtain 190 mg of yellow solid 6-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]pyrimidine -4-ol (yield: 44%). LCMS: RT = 1.74 min, [MH] - = 305.97.
步驟F:合成 (S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯 Step F: Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxo tert-Butyl pyrimidin-1( 6H )-yl)-3-phenylpropionamido)benzoate
室溫下,將6-[5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基]嘧啶-4-醇(45毫克,0.15毫莫耳)和(R)-4-(2-(((4-硝基苯基)磺醯基)氧基)-3-苯基丙醯胺基)苯甲酸叔丁酯(93毫克,0.18毫莫耳)以及碳酸鉀(40毫克,0.3毫莫耳)加入N,N-二甲基甲醯胺(3.0毫升)中,室溫反應過夜。向反應液中加水淬滅,混合液用乙酸乙酯(40毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(30毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化得到150毫克黃色液體(S)-4-(2-(4-(5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯(收率:59%)。LCMS:RT =2.00min, [M+H] +=631.18。 At room temperature, 6-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]pyrimidin-4-ol (45 mg, 0.15 mM ear) and (R)-tert-butyl 4-(2-(((4-nitrophenyl)sulfonyl)oxy)-3-phenylpropionamido)benzoate (93 mg, 0.18 mg) mol) and potassium carbonate (40 mg, 0.3 mmol) were added to N,N-dimethylformamide (3.0 mL) and reacted overnight at room temperature. Water was added to the reaction solution to quench, the mixture was extracted with ethyl acetate (40 mL × 3 times), the organic phases were combined, and the organic phases were first washed with saturated brine (30 mL × 2 times), then dried over anhydrous sodium sulfate, and finally Concentrate under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain 150 mg of yellow liquid (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazole-) 1-yl)phenyl)-6- oxopyrimidine -1(6H)-yl)-3-phenylpropionamido)benzoic acid tert-butyl ester (yield: 59%). LCMS: RT =2.00min, [M+H] + =631.18.
步驟F:合成 (S)-4-(2-(4-(5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1 (6 H)-基)-3-苯基丙醯胺基)苯甲酸 Step F: Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl)-6- Oxopyrimidine -1(6H)-yl)-3-phenylpropionamido)benzoic acid
將(S)-4-(2-(4-(5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯(150毫克,0.25毫莫耳)溶於二氯甲烷(2.0毫升)中。隨後,向上述溶液中加入三氟乙酸(0.5毫升),在室溫下攪拌1小時。將反應液空氣浴中減壓濃縮。將所得殘餘物經製備純化得到70毫克白色固體(S)-4-(2-(4-(5-氯-2-(4-氯-1 H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸(收率:59%)。 (S)-4-(2-(4-(5-Chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidine- tert-Butyl 1( 6H )-yl)-3-phenylpropionamido)benzoate (150 mg, 0.25 mmol) was dissolved in dichloromethane (2.0 mL). Subsequently, trifluoroacetic acid (0.5 mL) was added to the above solution, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure in an air bath. The resulting residue was preparatively purified to give 70 mg of white solid (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazole-1-) ( yield : 59%).
LCMS:RT = 2.00min, [M+H] += 573.16。 1H NMR (400 MHz, CD 3OD) δ 10.36 (s, 1H), 8.36 (s, 1H), 8.18 (s, 1H), 7.87 (dd, J= 12.0, 5.1 Hz, 2H), 7.72 (d, J= 2.3 Hz, 1H), 7.66–7.47 (m, 4H), 7.28–7.07 (m, 5H), 6.22 (d, J= 0.8 Hz, 1H), 5.74 (dd, J= 10.5, 6.2 Hz, 1H), 3.49 (dd, J= 14.1, 6.3 Hz, 1H), 3.34–3.24 (m, 1H)。 LCMS: RT = 2.00 min, [M+H] + = 573.16. 1 H NMR (400 MHz, CD 3 OD) δ 10.36 (s, 1H), 8.36 (s, 1H), 8.18 (s, 1H), 7.87 (dd, J = 12.0, 5.1 Hz, 2H), 7.72 (d , J = 2.3 Hz, 1H), 7.66–7.47 (m, 4H), 7.28–7.07 (m, 5H), 6.22 (d, J = 0.8 Hz, 1H), 5.74 (dd, J = 10.5, 6.2 Hz, 1H), 3.49 (dd, J = 14.1, 6.3 Hz, 1H), 3.34–3.24 (m, 1H).
對比實施例2化合物BComparative Example 2 Compound B
合成 (S)-4-(2-(4-(2-乙醯基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2 H)-基)-3-苯基丙醯胺基)苯甲酸 化合物B Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl)-3- Phenylpropionamido)benzoic acid Compound B
具體合成路線如下:The specific synthetic route is as follows:
步驟A:合成 (2,5-二甲氧基吡啶-4-基)硼酸 Step A: Synthesis of (2,5-dimethoxypyridin-4-yl)boronic acid
將2,5-二甲氧基吡啶(10.0克,71.9毫莫耳)溶於乾燥四氫呋喃(40毫升)中,置於乾燥三口燒瓶中,氮氣保護後,於乾冰/乙醇浴中攪拌15 分鐘後,將二異丙基氨基鋰(20毫升,2.0 M in THF)緩慢滴加到反應液中, 30分鐘後滴加完畢, 乾冰/乙醇浴中攪拌3 h後,將硼酸三異丙酯(33.0毫升,143.8毫莫耳)加入混合液中,然後自然升溫到室溫並恆溫攪拌18小時.LCMS監測,反應完全後,向反應液中加稀鹽酸調節pH至3~4,攪拌15分鐘後,旋蒸除去溶劑,殘渣加乙腈打漿得到10.6克白色固體(2,5-二甲氧基吡啶-4-基)硼酸(收率:80%)。LCMS:RT = 1.73min, [M+H] += 184.08。 2,5-Dimethoxypyridine (10.0 g, 71.9 mmol) was dissolved in dry tetrahydrofuran (40 mL), placed in a dry three-necked flask, under nitrogen protection, and stirred in a dry ice/ethanol bath for 15 minutes. , lithium diisopropylamide (20 mL, 2.0 M in THF) was slowly added dropwise to the reaction solution, the dropwise addition was completed after 30 minutes, and after stirring in a dry ice/ethanol bath for 3 h, triisopropyl borate (33.0 milliliter, 143.8 mmol) was added in the mixed solution, then naturally warmed up to room temperature and stirred at constant temperature for 18 hours. LCMS monitoring, after the reaction was complete, add dilute hydrochloric acid to the reaction solution to adjust pH to 3 ~ 4, after stirring for 15 minutes, The solvent was removed by rotary evaporation, and the residue was slurried with acetonitrile to obtain 10.6 g of white solid (2,5-dimethoxypyridin-4-yl)boronic acid (yield: 80%). LCMS: RT = 1.73 min, [M+H] + = 184.08.
步驟B:合成 1-(4-氯-2-(2,5-二甲氧基吡啶-4-基)苯基)乙-1-酮 Step B: Synthesis of 1-(4-Chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethan-1-one
將2-溴-4-氯苯乙酮(14.8克,63.6毫莫耳)和(2,5-二甲氧基吡啶-4-基)硼酸(9.7克,53.0毫莫耳)溶於1,4-二氧六環 (40毫升),碳酸鉀(14.6克,106毫莫耳)溶於水(10毫升),置於乾燥三口燒瓶中,氮氣保護後,將[1,1'-雙(二苯基膦)二茂鐵]二氯化鈀二氯甲烷絡合物(3.87克,5.3毫莫耳)加入反應液中, 氮氣保護後,升溫到100℃並恆溫攪拌18小時.LCMS監測,反應完全後,冷卻至室溫,墊矽藻土過濾,濾餅用EA(30毫升)洗滌3次,合併濾液及洗滌液,水洗一次,飽和氯化銨洗滌兩次,有機相經無水硫酸鈉乾燥,過濾,旋乾,殘渣經矽膠柱層析純化得到8.2克黃色固體1-(4-氯-2-(2,5-二甲氧基吡啶-4-基)苯基)乙-1-酮(收率:53%)。LCMS:RT = 4.03 min, [M+H] += 292.03。 2-Bromo-4-chloroacetophenone (14.8 g, 63.6 mmol) and (2,5-dimethoxypyridin-4-yl)boronic acid (9.7 g, 53.0 mmol) were dissolved in 1, 4-Dioxane (40 mL), potassium carbonate (14.6 g, 106 mmol) were dissolved in water (10 mL), placed in a dry three-necked flask, under nitrogen protection, [1,1'-bis( Diphenylphosphine) ferrocene] dichloropalladium dichloromethane complex (3.87 g, 5.3 mmol) was added to the reaction solution, after nitrogen protection, the temperature was raised to 100 ° C and stirred at constant temperature for 18 hours. LCMS monitoring, After the reaction was completed, it was cooled to room temperature, filtered through a pad of celite, the filter cake was washed three times with EA (30 mL), the filtrate and washings were combined, washed once with water, and washed twice with saturated ammonium chloride, and the organic phase was washed with anhydrous sodium sulfate. Dry, filter, spin dry, and the residue was purified by silica gel column chromatography to obtain 8.2 g of yellow solid 1-(4-chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethyl-1- Ketone (yield: 53%). LCMS: RT = 4.03 min, [M+H] + = 292.03.
步驟C:合成 4-(2-乙醯基-5-氯苯基)-5-甲氧基吡啶-2(1 H)-酮 Step C: Synthesis of 4-(2-Acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one
將1-(4-氯-2-(2,5-二甲氧基吡啶-4-基)苯基)乙-1-酮(8.2 克,28毫莫耳), 吡啶氫溴酸鹽(22 g,140毫莫耳)溶於N,N-二甲基甲醯胺(20 毫升),置於乾燥燒瓶中,氮氣保護後,升溫到110℃並恆溫攪拌4h.LCMS監測,反應完全後,冷卻至室溫,將反應液滴加到100毫升水中,加5%碳酸鈉調節pH至10~11,DCM(40 毫升×4)萃取四次,合併有機相,有機相經無水硫酸鈉乾燥,過濾,旋乾,殘渣用DCM (10 毫升)溶解,然後滴加到正己烷(120 毫升)中,析出大量固體,過濾,收集濾餅,即產物粗品, 經矽膠柱層析進一步純化得到6.4 g黃色固體4-(2-乙醯基-5-氯苯基)-5-甲氧基吡啶-2(1 H)-酮(收率:82%)。LCMS:RT = 3.81 min, [M-H] -= 277.04。 1-(4-Chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethan-1-one (8.2 g, 28 mmol), pyridine hydrobromide (22 g, 140 mmol) was dissolved in N,N-dimethylformamide (20 mL), placed in a dry flask, under nitrogen protection, heated to 110 °C and stirred at constant temperature for 4 h. LCMS monitoring, after the reaction was complete, Cool to room temperature, drop the reaction into 100 ml of water, add 5% sodium carbonate to adjust the pH to 10-11, extract four times with DCM (40 ml × 4), combine the organic phases, and dry the organic phases over anhydrous sodium sulfate, Filtration, spin-dried, the residue was dissolved in DCM (10 mL), then added dropwise to n-hexane (120 mL), a large amount of solid was precipitated, filtered, and the filter cake was collected, that is, the crude product, which was further purified by silica gel column chromatography to obtain 6.4 g 4-(2-Acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one (yield: 82%) as a yellow solid. LCMS: RT = 3.81 min, [MH] - = 277.04.
步驟D:合成 (S)-4-(2-(4-(2-乙醯基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯 Step D: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl) -3-Phenylpropionamido) tert-butyl benzoate
室溫下,將4-(2-乙醯基-5-氯苯基)-5-甲氧基吡啶-2(1 H)-酮(1.5克,5.4毫莫耳)和(R)-4-(2-(((4-硝基苯基)磺醯基)氧基)-3-苯基丙醯胺基)苯甲酸叔丁酯(4.0克,7.6毫莫耳)以及碳酸鉀(1.5克,10.8毫莫耳)加入N,N-二甲基甲醯胺(20.0毫升)中,室溫反應過夜。向反應液中加水淬滅,混合液用乙酸乙酯(40毫升×3次)萃取,合併有機相,有機相先用飽和食鹽水(30毫升×2次),然後用無水硫酸鈉乾燥,最後減壓濃縮。所得殘餘物用矽膠柱層析純化(洗脫劑:乙酸乙酯/正己烷=1/2)得到1.9克黃色固體(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯(收率:59%)。LCMS:RT = 4.42 min, [M+H] += 601.18。 At room temperature, 4-(2-acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one (1.5 g, 5.4 mmol) and (R)-4 tert-butyl-(2-(((4-nitrophenyl)sulfonyl)oxy)-3-phenylpropionamido)benzoate (4.0 g, 7.6 mmol) and potassium carbonate (1.5 g, 10.8 mmol) was added to N,N-dimethylformamide (20.0 mL) and reacted at room temperature overnight. Water was added to the reaction solution to quench, the mixture was extracted with ethyl acetate (40 mL×3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (30 mL×2 times), then dried over anhydrous sodium sulfate, and finally Concentrate under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane = 1/2) to obtain 1.9 g of yellow solid (S)-4-(2-(4-(2-acetyl-5) -Chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl)-3-phenylpropionamido)benzoic acid tert-butyl ester (yield: 59%). LCMS: RT = 4.42 min, [M+H] + = 601.18.
步驟E:合成 (S)-4-(2-(4-(2-乙醯基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2 H)-基)-3-苯基丙醯胺基)苯甲酸 Step E: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl) -3-Phenylpropionamido)benzoic acid
將(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-3-乙氧基-6-氧代噠嗪-1(6 H)-基)-3-苯基丙醯胺基)苯甲酸叔丁酯(1.9克,3.2毫莫耳)溶於二氯甲烷(12.0毫升)中。隨後,向上述溶液中加入三氟乙酸(3毫升),在室溫下攪拌1小時。將反應液空氣浴中減壓濃縮。將所得殘餘物用甲醇打漿純化,得到1.0克黃色固體(S)-4-(2-(4-(2-乙醯基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2 H)-基)-3-苯基丙醯胺基)苯甲酸 (收率:59%)。LCMS:RT = 3.88 min, [M-H] -=543.06。 1H NMR (400 MHz, DMSO) δ 10.82 (s, 1H), 7.92 (d, J= 8.8 Hz, 2H), 7.82 (d, J= 8.3 Hz, 1H), 7.76 (d, J= 8.8 Hz, 2H), 7.61 (dd, J= 8.4, 2.3 Hz, 2H), 7.42 (s, 1H), 7.38 (s, 1H),7.33–7.23 (m, 4H), 7.22–7.14 (m, 1H), 6.30 (s, 1H), 6.02 (dd, J= 9.5, 6.6 Hz, 1H), 3.53 (s, 3H), 3.49-3.44 (m, 2H), 2.36 (s, 3H)。 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-ethoxy-6- oxopyridazin -1(6H)-yl)-3 - tert-butyl phenylpropionamido)benzoate (1.9 g, 3.2 mmol) was dissolved in dichloromethane (12.0 mL). Subsequently, trifluoroacetic acid (3 mL) was added to the above solution, followed by stirring at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure in an air bath. The resulting residue was purified by beating with methanol to give 1.0 g of yellow solid (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridine) Onium-1( 2H )-yl)-3-phenylpropionamido)benzoic acid (yield: 59%). LCMS: RT = 3.88 min, [MH] - = 543.06. 1 H NMR (400 MHz, DMSO) δ 10.82 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.61 (dd, J = 8.4, 2.3 Hz, 2H), 7.42 (s, 1H), 7.38 (s, 1H), 7.33–7.23 (m, 4H), 7.22–7.14 (m, 1H), 6.30 (s, 1H), 6.02 (dd, J = 9.5, 6.6 Hz, 1H), 3.53 (s, 3H), 3.49-3.44 (m, 2H), 2.36 (s, 3H).
對比實施例3 CN201680058331實施例143化合物Comparative Example 3 CN201680058331 Example 143 Compound
參照CN201680058331實施例143的製備方法獲得相應目標化合物。The corresponding target compound was obtained with reference to the preparation method of Example 143 of CN201680058331.
實施例8:吸收光法檢測本發明化合物對人凝血因子XIa抑制的生物活性Example 8: Detection of the biological activity of the compounds of the present invention on the inhibition of human coagulation factor XIa by absorptiometry
1、實驗材料1. Experimental materials
酶:Human Factor XIa(ENZYME RESEARCH,貨號HFXIa 1111a)Enzyme: Human Factor XIa (ENZYME RESEARCH, Cat. No. HFXIa 1111a)
底物:S-2366 TM:(CHROMOGENIX,貨號82109039) Substrate: S- 2366TM : (CHROMOGENIX, Cat. No. 82109039)
緩衝液:145mM NaCl,5 mM KCl,1mg/mL PEG 8000,,30 mM HEPES ,pH7.4Buffer: 145 mM NaCl, 5 mM KCl, 1 mg/mL PEG 8000, 30 mM HEPES, pH 7.4
2、實驗步驟2. Experimental steps
將溶於100%DMSO的10mM受試化合物用100%DMSO稀釋至1000、200、40、8、1.6、0.32、0.064、0.0128、0.00256、0.00128μM;在96孔板中每孔加入98μL(77.7ng/mL)的FXIa酶溶液,空白孔加入98μL緩衝液代替,再加入2μL不同濃度的化合物,空白和對照孔用DMSO代替, 用振盪器混勻,37℃培養20min。10 mM test compound in 100% DMSO was diluted with 100% DMSO to 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0.00128 μM; /mL) of FXIa enzyme solution, the blank wells were replaced by 98 μL of buffer solution, and then 2 μL of compounds of different concentrations were added. The blank and control wells were replaced by DMSO, mixed with a shaker, and incubated at 37°C for 20 min.
最後每孔加入800μM的底物100μL,在405nm處測其吸光度。Finally, 100 μL of 800 μM substrate was added to each well, and its absorbance was measured at 405 nm.
3、資料處理3. Data processing
用GraphPad Prism軟體進行曲線擬合,計算IC
50值,見表一。
[表一] 本發明化合物對人FXIa抑制的IC
50
結論:本發明化合物對人FXIa具有明顯的抑制活性。Conclusion: The compounds of the present invention have obvious inhibitory activity on human FXIa.
實施例9:本發明化合物對人血漿體外抗凝血作用的測定Example 9: Determination of the anticoagulant effect of the compounds of the present invention on human plasma in vitro
1、實驗材料1. Experimental materials
血漿:人血收集於含3.2%檸檬酸鈉(體積比1:9)的真空採血管中,室溫3000rpm離心10min,收集血漿,分裝在EP管中,-80℃保存。Plasma: Human blood was collected in a vacuum blood collection tube containing 3.2% sodium citrate (volume ratio 1:9), centrifuged at 3000 rpm for 10 min at room temperature, and the plasma was collected, divided into EP tubes, and stored at -80 °C.
試劑:APTT測定試劑盒(活化部分凝血活酶時間檢測定劑盒,mindray)、氯化鈣溶液。Reagents: APTT assay kit (activated partial thromboplastin time assay kit, mindray), calcium chloride solution.
儀器:凝血儀(mindray,C2000-A)Instrument: coagulation meter (mindray, C2000-A)
2、實驗方法2. Experimental method
取分裝的凍存人血漿室溫融化後,混合均勻。將溶於100%DMSO的10mM受試化合物用100%DMSO稀釋至1500、750、375、187.5、93.75、46.88、23.44、11.72μM;在1.5mL EP管中加入98μL人血漿,再加入2μL不同濃度的化合物,空白組加入2μL 100%DMSO,37℃水浴培養10min,將樣品放入凝血儀中對應的位置,進行化合物的APTT測定。Thaw the frozen human plasma in aliquots at room temperature and mix well. 10mM test compound dissolved in 100% DMSO was diluted with 100% DMSO to 1500, 750, 375, 187.5, 93.75, 46.88, 23.44, 11.72 μM; 98 μL of human plasma was added to a 1.5 mL EP tube, followed by 2 μL of different concentrations 2 μL of 100% DMSO was added to the blank group, incubated in a water bath at 37°C for 10 min, and the samples were placed in the corresponding position in the coagulation instrument to perform APTT determination of the compounds.
3、資料處理3. Data processing
用GraphPad Prism軟體進行曲線擬合,分別計算EC1.5×和EC2×值,即1.5倍和2倍空白對照組的APTT所對應的化合物的濃度,結果見表二。
[表二] 本發明化合物對人血漿體外抗凝血作用
結論:從表二中可以看出本發明化合物對人血漿具有明顯的抗凝血作用。Conclusion: It can be seen from Table 2 that the compound of the present invention has obvious anticoagulant effect on human plasma.
實施例10: 本發明化合物的藥代動力學特徵考察Example 10: Investigation of the pharmacokinetic characteristics of the compounds of the present invention
1、實驗材料1. Experimental materials
SD大鼠:雄性,180-250g,購於廣東省醫學實驗動物中心、北京維通利華。食蟹猴:雄性,4-6kg,購於廣州春盛生物研究院有限公司。比格犬:雄性,8-12kg,在康龍化成(寧波)新藥技術股份有限公司開展。SD rats: male, 180-250 g, purchased from Guangdong Provincial Medical Laboratory Animal Center, Beijing Weitonglihua. Cynomolgus monkey: male, 4-6kg, purchased from Guangzhou Chunsheng Biological Research Institute Co., Ltd. Beagle: male, 8-12kg, developed in Kanglong Chemical (Ningbo) New Drug Technology Co., Ltd.
試劑:DMSO(二甲亞碸),PEG-400(聚乙二醇400),生理鹽水,肝素,乙腈,甲酸,普萘洛爾(內標)均為市售可得。Reagents: DMSO (dimethyl sulfoxide), PEG-400 (polyethylene glycol 400), normal saline, heparin, acetonitrile, formic acid, and propranolol (internal standard) are commercially available.
儀器:賽默飛LC-MS(U300 UPLC,TSQ QUANTUMN ULTRA三重四級杆質譜)。Instrumentation: Thermo Fisher Scientific LC-MS (U300 UPLC, TSQ QUANTUMN ULTRA triple quadrupole mass spectrometer).
2、實驗方法2. Experimental method
稱取化合物溶於DMSO-PEG-400-生理鹽水(5:60:35,v/v/v)體系中,大鼠/猴靜脈或灌胃給藥後,於5min(灌胃不採)、15min、30min、1h、2h、4h、6h、8h、24h採集靜脈血200μL於肝素化EP管中,12000rpm離心2min,取血漿-80℃凍存待測。精密稱取一定量供試品用DMSO溶解至1mg/mL,作為儲備液。準確吸取適量的化合物儲備液,加入乙腈稀釋製成標準系列溶液。準確吸取上述標準系列溶液各20μL,加入空白血漿180μL,渦旋混勻,配製成相當於血漿濃度為1、3、10、30、100、300、1000、3000和5000 ng/mL的血漿樣品,每一濃度進行雙樣本分析,建立標準曲線。取20μL血漿,加入內標普萘洛爾(5ng/mL)的乙腈溶液200μL,渦旋混勻後4000rpm離心5min,取上清LC-MS分析。LC-MS檢測條件如下:The compound was weighed and dissolved in DMSO-PEG-400-physiological saline (5:60:35, v/v/v) system, and after intravenous or intragastric administration to rats/monkeys, 5 min (gastrically not taken), After 15min, 30min, 1h, 2h, 4h, 6h, 8h, and 24h, 200 μL of venous blood was collected in a heparinized EP tube, centrifuged at 12000 rpm for 2 min, and the plasma was frozen at -80°C for testing. Precisely weigh a certain amount of the test sample and dissolve it in DMSO to 1 mg/mL as a stock solution. Accurately draw an appropriate amount of compound stock solution, add acetonitrile and dilute to make standard series solutions. Accurately aspirate 20 μL of each of the above standard series solutions, add 180 μL of blank plasma, vortex and mix to prepare plasma samples with plasma concentrations of 1, 3, 10, 30, 100, 300, 1000, 3000 and 5000 ng/mL. , a double-sample analysis was performed for each concentration, and a standard curve was established. Take 20 μL of plasma, add 200 μL of internal standard propranolol (5 ng/mL) in acetonitrile solution, vortex and mix, centrifuge at 4000 rpm for 5 min, and take the supernatant for LC-MS analysis. LC-MS detection conditions are as follows:
色譜柱:賽默飛HYPERSIL GOLD C-18 UPLC柱,100*2.1mm,1.9μm。Chromatographic column: Thermo Scientific HYPERSIL GOLD C-18 UPLC column, 100*2.1mm, 1.9μm.
流動相:水(0.1%甲酸)-乙腈按下表進行梯度洗脫
3、資料處理3. Data processing
LC-MS檢測血藥濃度後,採用WinNonlin 6.1軟體,非房室模型法計算藥動學參數。結果見表三、四、五。
[表三] 本發明化合物的大鼠藥代動力學參數
結論:本發明化合物在大鼠和猴口服均有一定的吸收,犬口服吸收較好,體內清除速率中等偏慢,多數化合物口服半衰期較長,具有良好的藥代動力學特徵。CONCLUSION: The compounds of the present invention have certain absorption in rats and monkeys orally. The oral absorption in dogs is good, and the clearance rate in vivo is moderately slow. Most of the compounds have a long oral half-life and have good pharmacokinetic characteristics.
實施例11:本發明化合物的體內藥效學徵考察Example 11: Investigation of in vivo pharmacodynamics of the compounds of the present invention
本實施例參考新藥(西藥)臨床前研究指導原則彙編(藥學 藥理學 毒理學)要求進行以下試驗:This example refers to the compilation of preclinical research guidelines for new drugs (western medicines) (Pharmaceutics, Pharmacology, Toxicology) and requires the following tests:
11.1 實施例4化合物對電刺激誘導的家兔下腔靜脈血栓藥效考察11.1 Investigating the efficacy of the compound of Example 4 on electrical stimulation-induced thrombus in the inferior vena cava in rabbits
試驗方法:家兔禁食不禁水24 h,按體重分為模型對照組、實施例4化合物30 mg/kg/h(以游離酸計)劑量組、陽性藥1利伐沙班3 mg/kg/h劑量組、陽性藥2依諾肝素鈉20 mg/kg/h劑量組,均靜脈輸注給藥一次,以1.4 ml/kg/h的速率持續2 h,模型對照組給予等體積溶媒。Test method: The rabbits were fasted for 24 hours and could not help but water. According to their body weight, they were divided into a model control group, the compound of Example 4 at a dose of 30 mg/kg/h (calculated as free acid), and the positive drug 1 Rivaroxaban at a dose of 3 mg/kg. /h dose group and positive drug 2 enoxaparin sodium 20 mg/kg/h dose group were administered by intravenous infusion once, at a rate of 1.4 ml/kg/h for 2 hours, and the model control group was given an equal volume of vehicle.
於給藥後1 h進行電刺激,電刺激前15 min,家兔40mg/kg戊巴比妥鈉耳緣靜脈注射麻醉,仰臥固定,腹部備皮、消毒,經腹中線、腹白線開腹,鹽水紗布保護腸管並推向一側,剪開後腹膜,游離下腔靜脈(範圍自左腎靜脈下至髂腰靜脈開口以上),結紮近心端,間隔2 cm處狹窄遠心端(使用直徑2 mm的塑膠管平行血管放置,一同結紮,結紮後緩慢抽出塑膠管),結紮此段間隔處的各分支靜脈。將針狀電極放置於結紮段下腔靜脈內皮上,使用1.5 mA的電流刺激30 min,電刺激時動脈夾夾閉狹窄部位阻斷靜脈。電刺激結束後鬆開動脈夾,家兔放置至電刺激停止後30 min。Electrical stimulation was performed 1 hour after administration, and 15 minutes before electrical stimulation, the rabbits were anesthetized by injection of 40 mg/kg sodium pentobarbital into the ear marginal vein, supine immobilized, skin preparation and disinfection of the abdomen, laparotomy through the midline of the abdomen and linea alba, and saline. The gauze protects the bowel and pushes it to one side, cut the retroperitoneum, dissociate the inferior vena cava (ranging from the left renal vein below the opening of the iliolumbar vein), ligate the proximal end, and narrow the distal end at an interval of 2 cm (use a diameter of 2 mm). The plastic tube is placed parallel to the blood vessels, ligated together, and the plastic tube is slowly drawn out after ligation), and each branch vein at this interval is ligated. A needle electrode was placed on the endothelium of the inferior vena cava in the ligated segment, and a current of 1.5 mA was used to stimulate it for 30 min. During electrical stimulation, the stenotic site was clamped with an arterial clip and the vein was blocked. After the electrical stimulation was over, the arterial clips were released, and the rabbits were placed until 30 min after the electrical stimulation stopped.
電刺激結束後,使用手術刀在腎臟做長度2 mm,深度1 mm的切口,測定出血時間。試驗結束後,取刺激部位血栓,稱量血栓重量,計算血栓形成抑制率。試驗結果:實施例4化合物和陽性對照藥靜脈滴注給藥能顯著降低家兔下腔靜脈血栓重量,實施例4化合物30mg/kg/h血栓抑制率為54.3%;利伐沙班3mg/kg/h血栓抑制率為59.8%;依諾肝素20mg/kg/h血栓抑制率為54.4%,試驗結果見下表六。
[表六] 實施例4化合物家兔下腔靜脈血栓藥效結果
試驗結論:實施例4化合物達到和利伐沙班及依諾肝素相當的藥效水平時,出血風險明顯降低。Test conclusion: When the compound of Example 4 reaches the same efficacy level as rivaroxaban and enoxaparin, the bleeding risk is significantly reduced.
11.2 實施例2化合物對三氯化鐵誘導的家兔頸動脈血栓藥效考察11.2 Investigation of the efficacy of the compound of Example 2 on ferric chloride-induced carotid artery thrombosis in rabbits
試驗方法:家兔禁食不禁水24h,分為模型對照組、陽性藥氯吡格雷3mg/kg/h和10mg/kg/h劑量組,實施例2化合物10mg/kg/h(以游離酸計)劑量組,每組3只家兔。Test method: The rabbits were fasted for 24 hours and were divided into model control group, positive drug clopidogrel 3mg/kg/h and 10mg/kg/h dose groups, Example 2 compound 10mg/kg/h (calculated as free acid) ) dose group, 3 rabbits in each group.
給藥前先對家兔稱重,耳緣靜脈40%烏拉坦1.5mL/kg麻醉,固定,手術分離右股動、靜脈和頸動脈,右股靜脈給藥,右股動脈採血,均靜滴給藥2h,滴注體積為1.4 mL/kg/h,模型對照組給予等體積溶媒。靜滴1h後開始造模:用10mm*10mm濾紙上加入20μL 50%FeCl3溶液, 敷在頸動脈周圍5min,去除濾紙片,靜置55min,剪取頸動脈分離血栓稱重。。The rabbits were weighed before administration, 40% urethane 1.5mL/kg was anesthetized in the marginal ear vein, fixed, and the right femoral artery, vein and carotid artery were surgically separated. After administration for 2 h, the instillation volume was 1.4 mL/kg/h, and the model control group was given an equal volume of vehicle. After intravenous infusion for 1 hour, start modeling: add 20 μL of 50% FeCl3 solution on 10mm*10mm filter paper, apply it around the carotid artery for 5 minutes, remove the filter paper, let it stand for 55 minutes, cut the carotid artery to separate the thrombus and weigh it. .
兔子血栓實驗結束後,剪開腹部皮膚、腹肌,然後剝離腎包膜,將腎放入37℃預熱的生理鹽水中,用一次性1ml注射器刺破腎組織,保證5mm的深度,引起腎出血,在生理鹽水中觀察腎出血時間。After the rabbit thrombus experiment, cut the abdominal skin and abdominal muscle, then peel off the renal capsule, put the kidney into pre-warmed normal saline at 37°C, and use a disposable 1ml syringe to puncture the kidney tissue to ensure a depth of 5mm to cause renal failure. Bleeding, renal bleeding time was observed in normal saline.
試驗結果:氯吡格雷單藥3mg/kg/h劑量組血栓抑制率約49.1%,腎出血時間無明顯變化,10mg/kg/h劑量組血栓抑制率達到72.7%時,腎出血時間有明顯延長。實施例2化合物10mg/kg/h劑量組血栓抑制率約47.9%,腎出血時間無明顯變化。試驗結果見表七。
[表七] 實施例2化合物家兔頸動脈血栓藥效結果
試驗結論:氯吡格雷對家兔頸動脈血栓有較好的效果,且有明顯的量效關係, 氯吡格雷隨著給藥劑量增加腎出血時間會明顯變長。實施例2化合物10mg/kg/h給藥時表現出一定的預防血栓形成效果,同時降低了出血風險。Conclusion: Clopidogrel has a good effect on carotid artery thrombosis in rabbits, and there is an obvious dose-effect relationship. With the increase of the dose of clopidogrel, the renal bleeding time will be significantly longer. When the compound of Example 2 was administered at 10 mg/kg/h, it showed a certain effect of preventing thrombosis, and at the same time reduced the risk of bleeding.
11.3實施例2化合物對三氯化鐵誘導的家兔動靜脈旁路血栓藥效考察11.3 Investigation of the efficacy of the compound of Example 2 on ferric chloride-induced arteriovenous bypass thrombosis in rabbits
試驗方法:家兔按體重分為模型對照組、實施例2化合物30mg/kg/h劑量組,手術分離右頸動脈和左頸靜脈,右股動、靜脈。右頸動脈和左頸靜脈用於連接動靜脈旁路裝置用於動靜脈血栓模型製作;右股靜脈插管用於靜脈輸注給藥。均採用靜脈輸注的方式給藥,輸注容積1.4ml/kg/h,模型對照組給予等體積溶媒。微量輸液泵勻速輸注60min 後,接通動靜脈旁路裝置,裝置內充滿生理鹽水,兩端連接右頸動脈和左頸靜脈,靜置15min。Test method: The rabbits were divided into a model control group and a 30 mg/kg/h dose group of the compound of Example 2 according to their body weight. The right carotid artery and left jugular vein, and the right femoral artery and vein were surgically separated. The right carotid artery and left jugular vein were used to connect the arteriovenous bypass device for arteriovenous thrombosis modeling; the right femoral vein was cannulated for intravenous infusion. All patients were administered by intravenous infusion, with an infusion volume of 1.4 ml/kg/h, and the model control group was given an equal volume of vehicle. After the microinfusion pump was infused at a constant rate for 60 minutes, the arteriovenous bypass device was connected, the device was filled with normal saline, and the two ends were connected to the right carotid artery and the left jugular vein, and left for 15 minutes.
造模前,家兔40mg/kg戊巴比妥鈉耳緣靜脈注射麻醉,仰臥固定,頸部備皮、消毒,用手術剪刀剪開頸部皮膚,分離右頸動脈和左頸靜脈,首先結紮右頸動脈遠心端,用血管夾夾閉近心端,靠近遠心端結紮線內側剪開血管,將充滿生理鹽水的動靜脈旁路裝置接入,用兩道真絲非吸收縫線結紮固定,暫不打開血管夾;左頸靜脈用同樣的方法接入動靜脈旁路裝置,用兩道真絲非吸收縫線結紮固定,暫不打開血管夾。輸注時間結束後首先打開動靜脈旁路裝置靜脈端血管夾,再打開動脈端血管夾,計時15min。取下動靜脈旁路裝置,利用減量法計算血栓重量。同時檢測腎出血時間。使用1ml一次性注射器針頭,在剝離腎包膜的腎臟表面,紮一深度3mm的針孔,測定出血時間。Before modeling, the rabbits were anesthetized by injection of 40 mg/kg sodium pentobarbital into the ear vein, supine and fixed, the neck skin was prepared and disinfected, the neck skin was cut with surgical scissors, the right carotid artery and left jugular vein were separated, and the right carotid artery and left jugular vein were separated first. At the distal end of the carotid artery, clamp the proximal end with a vascular clip, cut the blood vessel near the inner side of the ligation line at the distal end, connect the arteriovenous bypass device filled with normal saline, and ligate and fix it with two silk non-absorbable sutures. Open the vascular clip; the left jugular vein is connected to the arteriovenous bypass device in the same way, and is ligated and fixed with two silk non-absorbable sutures, and the vascular clip is not opened temporarily. After the end of the infusion time, first open the venous vascular clip of the arteriovenous bypass device, and then open the arterial vascular clip for 15 minutes. The arteriovenous bypass device was removed, and the weight of the thrombus was calculated by the decrement method. At the same time, the renal bleeding time was detected. Using a 1 ml disposable syringe needle, a needle hole with a depth of 3 mm was pierced on the surface of the kidney from which the renal capsule was peeled off, and the bleeding time was measured.
試驗結果:實施例2化合物給藥劑量30mg/kg/h能明顯減少家兔動靜脈旁路血栓重量,血栓抑制率為61.9%,試驗結果見表八。
[表八] 實施例2化合物家兔動靜脈旁路血栓藥效結果
試驗結論:實施例2化合物在家兔動靜脈旁路血栓模型中均有較好的預防血栓形成效果,實施例2化合物腎出血時間仍無明顯增加,表明實施例2化合物在達到較好藥效水平時出血風險較小。Test conclusion: The compound of Example 2 has a good effect of preventing thrombosis in the rabbit arteriovenous bypass thrombosis model, and the compound of Example 2 still does not significantly increase the renal bleeding time, indicating that the compound of Example 2 can achieve better efficacy. There is less bleeding risk at the level.
上述實施例為本發明較佳的實施方式,但本發明的實施方式並不受上述實施例的限制, 其他的任何未背離本發明的精神實質與原理下所作的改變、修飾、替代、組合、簡化,均應 為等效的置換方式,都包含在本發明的保護範圍之內。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.
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