TW202321221A - Crystal form of compound that inhibits the function of adamts-5 and/or adamts-4, and preparation method and application thereof - Google Patents
Crystal form of compound that inhibits the function of adamts-5 and/or adamts-4, and preparation method and application thereof Download PDFInfo
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Abstract
Description
本公開涉及一種抑制ADAMTS-5和/或ADAMTS-4功能的化合物的晶型,具體地涉及式I化合物的A晶型及製備方法和應用。 The present disclosure relates to a crystal form of a compound that inhibits the function of ADAMTS-5 and/or ADAMTS-4, in particular to the crystal form A of the compound of formula I, its preparation method and application.
本申請要求申請日為2021年8月3日的中國專利申請2021108844766的優先權。本申請引用上述中國專利申請的全文。 This application claims the priority of Chinese patent application 2021108844766 with a filing date of August 3, 2021. This application cites the full text of the above-mentioned Chinese patent application.
軟骨是關節高度特化的結締組織。其主要功能是為關節提供承載和抗壓的能力。軟骨細胞是關節軟骨的細胞成分,僅占組織體積的5%左右。軟骨的主要成分是細胞外基質,包括蛋白聚糖和膠原蛋白。在生理條件下,軟骨穩態是通過聚集蛋白聚糖和膠原蛋白的產生(合成代謝)和降解(分解代謝)之間的平衡來維持的。然而,在骨關節炎等疾病中,平衡就偏移到分解代謝。 Cartilage is a highly specialized connective tissue in joints. Its main function is to provide load-bearing and compressive capacity to the joints. Chondrocytes are the cellular components of articular cartilage, accounting for only about 5% of the tissue volume. The main component of cartilage is the extracellular matrix, including proteoglycans and collagen. Under physiological conditions, cartilage homeostasis is maintained by a balance between the production (anabolism) and degradation (catabolism) of aggrecan and collagen. However, in diseases such as osteoarthritis, the balance is shifted towards catabolism.
骨關節炎是最常見的慢性關節疾病,也是疼痛和殘疾的主要原因。它可能發生在臀部、膝蓋、脊柱、手和其他部位的關節上。據估計,目前全球有2.5億人受到骨關節炎的影響,而且患病率正在逐步上升。疼痛和功能喪失伴隨著糖尿病、癌症或心血管疾病等其他疾病的風險增加。骨關 節炎是一種全關節疾病,其結構變化包括關節軟骨退化、滑膜炎以及軟骨下骨和其他關節周圍組織的改變。骨關節炎的發病機制尚不十分清楚,涉及機械損傷、炎症、衰老、代謝等因素。骨關節炎不是被動退行性疾病,而是由關節組織修復和破壞之間的不平衡引起的主動動態改變。目前,可用於骨關節炎的藥物治療僅限於緩解疼痛和炎症的症狀。阻止或減緩疾病進展的藥物目前尚不可用。 Osteoarthritis is the most common chronic joint disease and a leading cause of pain and disability. It can occur in the hips, knees, spine, hands, and other joints. Osteoarthritis currently affects an estimated 250 million people worldwide, and the prevalence is steadily increasing. Pain and loss of function are accompanied by an increased risk of other diseases such as diabetes, cancer or cardiovascular disease. bones Arthritis is a general joint disorder with structural changes including degeneration of articular cartilage, synovitis, and changes in the subchondral bone and other periarticular tissues. The pathogenesis of osteoarthritis is still not very clear, involving mechanical damage, inflammation, aging, metabolism and other factors. Osteoarthritis is not a passive degenerative disease, but an active dynamic change caused by an imbalance between joint tissue repair and destruction. Currently, the drug treatments available for osteoarthritis are limited to symptomatic relief of pain and inflammation. Drugs that stop or slow disease progression are not currently available.
關節軟骨的進行性喪失目前被視為骨關節炎的早期標誌。聚集蛋白聚糖可能具有保護膠原蛋白損失的作用。這些研究表明聚集蛋白聚糖在骨關節炎和其他關節疾病中具有關鍵作用。聚集蛋白聚糖是一種蛋白聚糖,其核心蛋白具有共價連接的硫酸化糖胺聚糖(GAG)鏈。其核心蛋白具有三個球狀結構域,即N端的G1和G2結構域,以及C端的G3結構域。G2和G3結構域之間的廣泛區域被GAG硫酸角質素(KS)和硫酸軟骨素(CS)嚴重修飾。根據氨基酸序列的不同,CS域進一步分為兩個亞域,CS1和CS2。GAG鏈為蛋白聚糖提供高陰離子電荷。多個聚集蛋白聚糖單體通過G1結構域與透明質酸(HA)結合,該結構域由鏈接蛋白穩定,形成大的超分子聚集體。大的聚集蛋白聚糖的聚集體吸收水分並為軟骨提供彈性。軟骨的正常功能需要高濃度的聚集蛋白聚糖、高度硫酸化和形成大量聚集的能力。 Progressive loss of articular cartilage is currently considered an early sign of osteoarthritis. Aggrecan may have a protective effect against collagen loss. These studies suggest that aggrecan has a key role in osteoarthritis and other joint diseases. Aggrecan is a proteoglycan whose core protein has covalently linked sulfated glycosaminoglycan (GAG) chains. Its core protein has three globular domains, the N-terminal G1 and G2 domains, and the C-terminal G3 domain. The extensive region between the G2 and G3 domains is heavily modified by the GAGs keratan sulfate (KS) and chondroitin sulfate (CS). Depending on the amino acid sequence, the CS domain is further divided into two subdomains, CS1 and CS2. GAG chains provide a high anionic charge to proteoglycans. Multiple aggrecan monomers associate with hyaluronic acid (HA) through the G1 domain, which is stabilized by connexins, forming large supramolecular aggregates. Aggregates of large aggrecan absorb water and provide elasticity to cartilage. Proper function of cartilage requires high concentrations of aggrecan, a high degree of sulfation, and the ability to form large aggregates.
蛋白水解酶可以裂解聚集蛋白聚糖的擴展結構,導致軟骨的正常功能受損。ADAMTS(具有血小板反應蛋白基序的去整合素和金屬蛋白酶)是鋅離子依賴性金屬蛋白酶家族。ADAMTS-4和-5,也稱為「聚集蛋白聚糖酶」,在IGD和CS2域中的幾個特定位置降解聚集蛋白聚糖,(Glasson et al.,Nature.2005,434:644-648;Stanton et al.,Nature.2005,434:648-652)結果表明,ADAMTS-5缺乏可防止手術誘導的小鼠骨關節炎模型中的蛋白聚 糖損失和軟骨損傷,這表明ADAMTS-5與驅動軟骨損失和骨關節炎疾病嚴重程度有關。然而,人類軟骨移植培養中的一些研究表明,不僅ADAMTS-5,而且ADAMTS-4對人類骨關節炎也很重要(Verma et al.,Journal of Cellular Biochemistry.2011,112..3507-3514)。這些研究強烈表明抑制ADAMTS-5和ADAMT-4的酶促功能可能在骨關節炎中發揮保護作用。 Proteolytic enzymes can cleave the extended structure of aggrecan, resulting in impairment of the normal function of cartilage. ADAMTS (disintegrins and metalloproteases with thrombospondin motifs) are a family of zinc ion-dependent metalloproteases. ADAMTS-4 and -5, also known as "aggrecanase", degrade aggrecan at several specific positions in the IGD and CS2 domains, ( Glasson et al., Nature.2005, 434:644-648 ; Stanton et al., Nature.2005,434:648-652 ) showed that ADAMTS-5 deficiency could prevent proteoglycan loss and cartilage damage in a surgically induced mouse osteoarthritis model, suggesting that ADAMTS-5 is associated with Driving cartilage loss is associated with osteoarthritic disease severity. However, some studies in human cartilage graft cultures have shown that not only ADAMTS-5 but also ADAMTS-4 is important for human osteoarthritis ( Verma et al., Journal of Cellular Biochemistry. 2011, 112..3507-3514 ). These studies strongly suggest that inhibition of the enzymatic function of ADAMTS-5 and ADAMT-4 may play a protective role in osteoarthritis.
總之,ADAMTS-5和/或ADAMTS-4在軟骨降解中的作用已得到公認。因此,能夠抑制ADAMTS-5和/或ADAMTS-4的化合物在關節炎的治療中可能具有治療價值。 In conclusion, the role of ADAMTS-5 and/or ADAMTS-4 in cartilage degradation has been recognized. Therefore, compounds capable of inhibiting ADAMTS-5 and/or ADAMTS-4 may have therapeutic value in the treatment of arthritis.
PCT/US2021/016364提供了一種新的抑制ADAMTS-5和/或ADAMTS-4功能的化合物(結構如式I所示),該化合物具有較好的藥學活性。 PCT/US2021/016364 provides a new compound (structure shown in formula I) for inhibiting the function of ADAMTS-5 and/or ADAMTS-4, which has better pharmaceutical activity.
藥物的活性成分的晶型結構往往影響到該藥物的化學穩定性,結晶條件及儲存條件的不同有可能導致化合物的晶型結構的變化,有時還會伴隨著產生其他形態的晶型。一般來說,無定形的藥物產品沒有規則的晶型結構,往往具有其它缺陷,比如產物穩定性較差,析晶較細,過濾較難,易結塊,流動性差等。因此,我們需要深入研究找到純度較高並且具備良好化學穩定的晶型。 The crystal structure of the active ingredient of a drug often affects the chemical stability of the drug. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by the production of other crystal forms. Generally speaking, amorphous pharmaceutical products have no regular crystal structure and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, and poor fluidity. Therefore, we need to conduct in-depth research to find crystal forms with high purity and good chemical stability.
本公開提供了一種式I所示化合物的A晶型,其具備良好的穩定性,可更好地應用於臨床。 The present disclosure provides a crystal form A of the compound represented by formula I, which has good stability and can be better applied clinically.
本公開提供的式I所示化合物的A晶型,其X-射線粉末繞射圖譜在2θ角為11.2、13.8、16.6、21.0、27.6處有特徵峰。 The crystal form A of the compound represented by formula I provided by the present disclosure has characteristic peaks at 2θ angles of 11.2, 13.8, 16.6, 21.0, and 27.6 in its X-ray powder diffraction pattern.
本公開提供的式II所示化合物的A晶型,其X-射線粉末繞射圖譜在2θ角為8.4、11.2、13.8、14.9、16.6、17.5、18.5、21.0、22.9、27.6處有特徵峰。 The crystal form A of the compound represented by formula II provided by the present disclosure has characteristic peaks at 2θ angles of 8.4, 11.2, 13.8, 14.9, 16.6, 17.5, 18.5, 21.0, 22.9, and 27.6 in the X-ray powder diffraction pattern.
本公開提供的式II所示化合物的A晶型,其X-射線粉末繞射圖譜在2θ角為8.4、11.2、13.8、14.9、15.4、16.6、17.5、18.5、21.0、22.9、27.6處有特徵峰。 The crystal form A of the compound represented by formula II provided by the present disclosure has X-ray powder diffraction patterns at 2θ angles of 8.4, 11.2, 13.8, 14.9, 15.4, 16.6, 17.5, 18.5, 21.0, 22.9, and 27.6. peak.
本公開提供的式I所示化合物的A晶型,其X-射線粉末繞射圖譜如圖2所示。 The crystal form A of the compound represented by formula I provided by the present disclosure has an X-ray powder diffraction pattern as shown in FIG. 2 .
本公開提供的式I所示化合物的A晶型,其中所述2θ角的誤差範圍為±0.2。 The present disclosure provides the crystal form A of the compound represented by formula I, wherein the error range of the 2θ angle is ±0.2.
本公開進一步提供一種製備式I所示化合物的A晶型的方法,所述方法包括:將式I所示化合物與適量的溶劑混合,所述溶劑為有機溶劑,優選溶劑為四氫呋喃、乙醇、異丙醇、丙酮、乙酸乙酯。 The present disclosure further provides a method for preparing crystal form A of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, the solvent being an organic solvent, preferably tetrahydrofuran, ethanol, iso Propanol, acetone, ethyl acetate.
通過X-射線粉末繞射圖譜(XRPD)、差示掃描量熱分析(DSC)對本公開所得到晶型進行結構測定、晶型研究。 The structure determination and crystal form research of the crystal form obtained in the present disclosure are carried out by X-ray powder diffraction pattern (XRPD) and differential scanning calorimetry (DSC).
本公開中晶型的析晶方法是常規的,例如揮發析晶、降溫析晶或室溫下析晶。 The crystallization method of the crystal form in the present disclosure is conventional, such as volatilization crystallization, cooling crystallization or room temperature crystallization.
本公開晶型製備方法中所用的起始原料可以是任意形式的式I所示化合物,具體形式包括但不限於:無定形、任意晶型、水合物、溶劑合物等。 The starting material used in the preparation method of the disclosed crystal form can be any form of the compound represented by formula I, and the specific form includes but not limited to: amorphous, any crystal form, hydrate, solvate, etc.
本公開進一步提供一種藥物組合物,包含式I所示化合物的A晶型,以及一種或多種藥學上可接受的載體或賦形劑。 The present disclosure further provides a pharmaceutical composition, comprising the crystal form A of the compound represented by formula I, and one or more pharmaceutically acceptable carriers or excipients.
本公開進一步提供一種藥物組合物,其通過式I所示化合物的A晶型,與一種或多種藥學上可接受的載體或賦形劑製備得到。 The present disclosure further provides a pharmaceutical composition, which is prepared by the crystal form A of the compound represented by formula I, and one or more pharmaceutically acceptable carriers or excipients.
本公開進一步提供一種藥物組合物的製備方法,包括將式I所示化合物的A晶型與一種或多種藥學上可接受的載體或賦形劑混合的步驟。 The present disclosure further provides a preparation method of a pharmaceutical composition, comprising the step of mixing the crystal form A of the compound represented by formula I with one or more pharmaceutically acceptable carriers or excipients.
本公開進一步提供前述的式I所示化合物的A晶型、或由前述方法製備得到的組合在製備用於抑制ADAMTS-5和/或AIDAMTS-4的藥物的用途。 The present disclosure further provides the use of the crystal form A of the aforementioned compound represented by formula I, or the combination prepared by the aforementioned method, in the preparation of a medicament for inhibiting ADAMTS-5 and/or AIDAMTS-4.
本公開進一步提供前述的式I所示化合物的A晶型、或由前述方法製備得到的組合在製備用於預防或治療涉及軟骨降解和/或軟骨穩態破壞的炎性病症或疾病的藥物中的用途。 The present disclosure further provides the aforementioned crystal form A of the compound represented by formula I, or the combination prepared by the aforementioned method in the preparation of a medicament for preventing or treating inflammatory disorders or diseases involving cartilage degradation and/or cartilage homeostasis disruption the use of.
本公開進一步提供前述的式I所示化合物的A晶型、或由前述方法製備得到的組合在製備用於預防或治療關節炎的藥物中的用途,所述關節炎優選類風濕性關節炎、乾癬性關節炎、骨關節炎和肥大性關節炎。 The present disclosure further provides the use of the crystal form A of the compound represented by the aforementioned formula I, or the combination prepared by the aforementioned method in the preparation of medicines for the prevention or treatment of arthritis, the arthritis is preferably rheumatoid arthritis, Psoriatic arthritis, osteoarthritis and hypertrophic arthritis.
本公開進一步提供前述的式I所示化合物的A晶型、或由前述方法製備得到的組合在製備用於治療與ADAMTS-5和/或ADAMTS-4活性相關的疾病或病症的藥物中的用途,其中與ADAMTS-5和/或ADAMTS-4活 性相關的疾病或病症是關節炎,優選類風濕性關節炎、乾癬性關節炎、骨關節炎或肥大性關節炎。 The present disclosure further provides the use of crystal form A of the compound represented by the aforementioned formula I, or the combination prepared by the aforementioned method in the preparation of a drug for treating diseases or disorders related to ADAMTS-5 and/or ADAMTS-4 activity , which live with ADAMTS-5 and/or ADAMTS-4 The sex-related disease or condition is arthritis, preferably rheumatoid arthritis, psoriatic arthritis, osteoarthritis or hypertrophic arthritis.
在本公開的說明書和發明申請專利範圍中,除非另有說明,否則本文中使用的科學和技術名詞具有本發明所屬技術領域具有通常知識者所通常理解的含義。然而,為了更好地理解本公開,下面提供了部分相關術語的定義和解釋。另外,當本申請所提供的術語的定義和解釋與本發明所屬技術領域具有通常知識者所通常理解的含義不一致時,以本申請所提供的術語的定義和解釋為準。 In the specification and patent scope of the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the technical field to which the present invention belongs. However, for a better understanding of the present disclosure, definitions and explanations of some related terms are provided below. In addition, when the definitions and explanations of the terms provided in this application are inconsistent with the meanings commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs, the definitions and explanations of the terms provided in this application shall prevail.
除另有說明,當一個位置被特別地指定為氘(D)時,該位置應理解為具有大於氘的天然豐度(其為0.015%)至少1000倍的豐度的氘(即,至少10%的氘摻入)。示例中化合物的具有大於氘的天然豐度可以是至少1000倍的豐度的氘、至少2000倍的豐度的氘、至少3000倍的豐度的氘、至少4000倍的豐度的氘、至少5000倍的豐度的氘、至少6000倍的豐度的氘或更高豐度的氘。 Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (i.e., at least 10 % deuterium incorporation). Exemplary compounds having a natural abundance greater than deuterium can be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium.
本公開所述的「X-射線粉末繞射圖譜或XRPD」是指根據布拉格公式2d sin θ=nλ(式中,λ為X射線的波長,繞射的級數n為任何正整數,一般取一級繞射峰,n=1),當X射線以掠角θ(入射角的餘角,又稱為布拉格角)入射到晶體或部分晶體樣品的某一具有d點陣平面間距的原子面上時,就能滿足布拉格方程,從而測得了這組X射線粉末繞射圖。 The "X-ray powder diffraction pattern or XRPD" described in the present disclosure means that according to the Bragg formula 2d sin θ=nλ (wherein, λ is the wavelength of X-rays, and the order n of diffraction is any positive integer, generally taken as The first-order diffraction peak, n=1), when the X-ray is incident on an atomic surface with a d-lattice plane spacing of a crystal or a part of the crystal sample at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle) , the Bragg equation can be satisfied, and thus this group of X-ray powder diffraction patterns has been measured.
本公開所述的「X-射線粉末繞射圖譜或XRPD」是通過在X-射線粉末繞射儀中使用Cu-Kα輻射得到的圖譜。 An "X-ray powder diffraction pattern or XRPD" as used in this disclosure is a pattern obtained by using Cu-Kα radiation in an X-ray powder diffractometer.
本公開所述的「差示掃描量熱分析或DSC」是指在樣品升溫或恒溫過程中,測量樣品與參考物之間的溫度差、熱流差,以表徵所有與熱效應有關的物理變化和化學變化,得到樣品的相變信息。 "Differential scanning calorimetry or DSC" in this disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference object during the heating or constant temperature process of the sample, so as to characterize all the physical changes and chemical reactions related to thermal effects. change to obtain the phase transition information of the sample.
本公開所述的「熱重分析或TGA」是指在程序控制溫度下,連續測量樣品的質量隨溫度或時間的變化。 "Thermogravimetric analysis or TGA" in this disclosure refers to the continuous measurement of the mass change of a sample with temperature or time at a programmed temperature.
本公開所述的「2θ或2θ角度」是指繞射角,θ為布拉格角,單位為°或度,2θ的誤差範圍為±0.3或±0.2或±0.1。 The “2θ or 2θ angle” mentioned in this disclosure refers to the diffraction angle, θ is the Bragg angle, the unit is ° or degree, and the error range of 2θ is ±0.3 or ±0.2 or ±0.1.
本公開所述的「晶面間距或晶面間距(d值)」是指空間點陣選擇3個不相平行的連結相鄰兩個點陣點的單位矢量a,b,c,它們將點陣劃分成並置的平行六面體單位,稱為晶面間距。空間點陣按照確定的平行六面體單位連線劃分,獲得一套直線網格,稱為空間格子或晶格。點陣和晶格是分別用幾何的點和線反映晶體結構的週期性,不同的晶面,其面間距(即相鄰的兩個平行晶面之間的距離)各不相同;單位為Å或埃。 The "interplanar spacing or interplanar spacing (d value)" in this disclosure means that the space lattice selects 3 non-parallel unit vectors a, b, and c that connect two adjacent lattice points. The matrix is divided into juxtaposed parallelepiped units called interplanar spacing. The spatial lattice is divided according to the determined parallelepiped unit connection lines to obtain a set of linear grids, which are called spatial lattices or lattices. Lattice and lattice reflect the periodicity of the crystal structure with geometric points and lines respectively. Different crystal planes have different interplanar spacing (that is, the distance between two adjacent parallel crystal planes); the unit is Å or Angstroms.
圖1為實施例2-2中式I化合物A晶型的XRPD圖譜; Fig. 1 is the XRPD spectrum of formula I compound A crystal form in embodiment 2-2;
圖2為實施例2-2中式I化合物A晶型的DSC圖譜。 Fig. 2 is the DSC spectrum of the crystal form of compound A of formula I in Example 2-2.
以下將結合實施例更詳細地解釋本公開,本公開的實施例僅用於說明本公開的技術方案,並非限定本公開的實質和範圍。如果本公開實施例中未對實驗方法的具體條件進行說明,則一般按照原料和產品製造商的常規條件或推薦條件進行。未注明具體來源的試劑均為市售常規試劑。 The following will explain the present disclosure in more detail in conjunction with the embodiments, and the embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the essence and scope of the present disclosure. If the specific conditions of the experimental method are not described in the examples of the present disclosure, it is generally carried out according to the conventional conditions or recommended conditions of the raw materials and product manufacturers. Reagents whose specific sources were not indicated were commercially available routine reagents.
化合物的結構通過核磁共振(NMR)和/或質譜(MS)鑒定。NMR由Bruker AVANCE II(或III)-400MHz測定。溶劑是氘代二甲基亞碸 (DMSO-d 6 )、氘代氯仿(CDCl3)和氘代甲醇(CD3OD),以四甲基矽烷(TMS)作為內標。NMR化學位移(δ)以10-6(ppm)的單位給出。 The structures of the compounds were identified by nuclear magnetic resonance (NMR) and/or mass spectroscopy (MS). NMR was determined by Bruker AVANCE II (or III)-400 MHz. The solvents were deuterated dimethylsulfoxide (DMSO- d 6 ), deuterated chloroform (CDCl 3 ) and deuterated methanol (CD 3 OD), with tetramethylsilane (TMS) as internal standard. NMR chemical shifts (δ) are given in units of 10 -6 (ppm).
LC/MS(ESI)在下列儀器上分析測定:配備Sunfire C18(5μm 50×4.6mm)色譜柱的Shimadzu LCMS2020、配備ACQUITY UPLC® BEH(2.1*50mm 1.7μm)色譜柱的Waters UPLC-QDa、配備Xbridge C18(5μm 50×4.6mm)色譜柱的Agilent Agilent6120。 LC/MS (ESI) was analyzed and determined on the following instruments: Shimadzu LCMS2020 equipped with Sunfire C18 (5μm 50×4.6mm) chromatographic column, Waters UPLC-QDa equipped with ACQUITY UPLC® BEH (2.1*50mm 1.7μm) chromatographic column, equipped with Agilent Agilent6120 of Xbridge C18 (5μm 50×4.6mm) chromatographic column.
HPLC在下列儀器上分析測定:配備Sunfire C18(5μm 150×4.6mm)色譜柱的Agilent 1200DAD和配備Xbridge C18(5μm 150×4.6mm)色譜柱的Shimadzu UFLC。 HPLC was analyzed and determined on the following instruments: Agilent 1200DAD equipped with Sunfire C18 (5 μm 150×4.6 mm) chromatographic column and Shimadzu UFLC equipped with Xbridge C18 (5 μm 150×4.6 mm) chromatographic column.
手性HPLC在Waters-UPC2儀器上分析測定。 Chiral HPLC was analyzed and determined on a Waters-UPC 2 instrument.
本公開中使用的已知原料採用本領域常規合成方法製備,或購自Aldrich Chemical、Fisher Scientific或Combi-Blocks等公司。 Known materials used in this disclosure are prepared by conventional synthetic methods in the art, or purchased from companies such as Aldrich Chemical, Fisher Scientific, or Combi-Blocks.
除非實施例中另有說明,反應可在氮氣氛下進行。 Unless otherwise stated in the examples, the reactions can be carried out under nitrogen atmosphere.
除非實施例中另有說明,反應中的反應溫度均指室溫,溫度範圍為20℃~30℃。 Unless otherwise stated in the examples, the reaction temperature in the reaction refers to room temperature, and the temperature range is 20° C. to 30° C.
實施例中的反應進程通過LC-MS或薄層色譜(TLC)監測,展開劑體系包括:A:二氯甲烷和甲醇,B:己烷和乙酸乙酯。溶劑的體積比根據化合物的極性不同而進行調節。柱層析、薄層層析和CombiFlash快速製備儀純化化合物的洗脫系統包括:A:二氯甲烷和甲醇,B:己烷和乙酸乙酯。溶劑的體積比例根據化合物的極性進行調整,有時可以加入少量鹼性試劑如氨水或酸性試劑如乙酸進行調節。 The progress of the reaction in the examples is monitored by LC-MS or thin-layer chromatography (TLC), and the developer system includes: A: dichloromethane and methanol, B: hexane and ethyl acetate. The volume ratio of the solvent is adjusted according to the polarity of the compound. The elution systems of column chromatography, thin-layer chromatography and CombiFlash rapid preparation instrument for purifying compounds include: A: dichloromethane and methanol, B: hexane and ethyl acetate. The volume ratio of the solvent is adjusted according to the polarity of the compound, and sometimes it can be adjusted by adding a small amount of basic reagent such as ammonia water or acidic reagent such as acetic acid.
Prep-HPLC分析測定使用Shimadzu(LC-20AD,SPD20A)Preparative HPLC(Phenomenex Gemini-NX 5μm C18 21.2×100mm柱)、配備Sunfire Pre C18(10μm 19×250mm)柱的Waters 2767和配備Xbridge Pre C18(10μm 19×250mm)柱的Waters 2767-QDa。 Prep-HPLC analysis and determination using Shimadzu (LC-20AD, SPD20A) Preparative HPLC (Phenomenex Gemini-NX 5μm C18 21.2×100mm column), equipped with Sunfire Pre Waters 2767 with C18 (10 μm 19×250 mm) column and Waters 2767-QDa with Xbridge Pre C18 (10 μm 19×250 mm) column.
Pre-SFC分析測定使用配備Daciel AD/OD/OJ/IC/IA/ID(10μm 20×250mm)柱的Waters-SFC80。
Waters-SFC80 equipped with Daciel AD/OD/OJ/IC/IA/ID (10
CombiFlash在Teledyne ISCO或Agela Technologies的系統上運行。 CombiFlash runs on systems from Teledyne ISCO or Agela Technologies.
實施例中使用到的試劑的縮寫如下: The abbreviation of the reagent used in the embodiment is as follows:
AIBN是2,2'-偶氮雙(2-甲基丙腈), AIBN is 2,2'-azobis(2-methylpropionitrile),
EDCI是N-(3-二甲基氨基丙基)-N'-乙基碳二亞胺鹽酸鹽, EDCI is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride,
HATU是O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-四甲基脲鎓六氟磷酸鹽, HATU is O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate,
LDA是二異丙基氨基鋰, LDA is lithium diisopropylamide,
NBS是N-溴代琥珀醯亞胺, NBS is N-bromosuccinimide,
Pd(dppf)Cl2為[1,1'-雙(二苯基膦)二茂鐵]二氯鈀(II), Pd(dppf)Cl is [1,1'- bis (diphenylphosphino)ferrocene]dichloropalladium(II),
DCE是1,2-二氯乙烷, DCE is 1,2-dichloroethane,
DCM是二氯甲烷, DCM is dichloromethane,
DMF是N,N-二甲基甲醯胺, DMF is N,N-dimethylformamide,
MeCN或ACN是乙腈, MeCN or ACN is acetonitrile,
THF是四氫呋喃, THF is tetrahydrofuran,
HEPES是4-羥乙基哌乙磺酸, HEPES is 4-Hydroxyethylpiperene ethanesulfonic acid,
CHAPS是3-[3-(膽醯胺丙基)二甲氨基]丙磺酸內鹽, CHAPS is the inner salt of 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid,
FBS是胎牛血清。 FBS is fetal bovine serum.
試驗所用儀器的測試條件: The test conditions of the instruments used in the test:
1、差示掃描量熱儀(Differential Scanning Calorimeter,DSC) 1. Differential Scanning Calorimeter (DSC)
儀器型號:Mettler Toledo DSC 3+
Instrument model:
吹掃氣:氮氣,50mL/min Purge gas: nitrogen, 50mL/min
升溫速率:10.0℃/min Heating rate: 10.0°C/min
溫度範圍:30-300℃ Temperature range: 30-300°C
2、X-射線粉末繞射儀(X-ray Powder Diffraction,XRPD) 2. X-ray Powder Diffraction (XRPD)
儀器型號:BRUKER D8 DISCOVER Instrument model: BRUKER D8 DISCOVER
射線:單色Cu-Kα射線 Rays: monochromatic Cu-Kα rays
(Cu-Kα1波長為1.5406Å,Cu-Kα2波長為1.54439Å,Cu-Kα波長取Kα1與Kα2的加權平均值λ=1.5418Å) (The wavelength of Cu-Kα1 is 1.5406Å, the wavelength of Cu-Kα2 is 1.54439Å, and the wavelength of Cu-Kα is the weighted average of Kα1 and Kα2 λ=1.5418Å)
掃描方式:Coupled θ/2θ,掃描模式:step,掃描時間:45s,掃描步數:3,2θ起始:10,2θ結束:48°,每步增量:19°; Scanning method: Coupled θ/2θ, scanning mode: step, scanning time: 45s, scanning steps: 3, 2θ start: 10, 2θ end: 48°, each step increment: 19°;
電壓:40KV,電流:40mA Voltage: 40KV, Current: 40mA
3、X-射線粉末繞射儀(X-ray Powder Diffraction,XRPD) 3. X-ray Powder Diffraction (XRPD)
儀器型號:BRUKER D8 Advance Instrument model: BRUKER D8 Advance
射線:單色Cu-Kα射線 Rays: monochromatic Cu-Kα rays
(Cu-Kα1波長為1.5406Å,Cu-Kα2波長為1.54439Å,Cu-Kα波長取Kα1與Kα2的加權平均值λ=1.5418Å) (The wavelength of Cu-Kα1 is 1.5406Å, the wavelength of Cu-Kα2 is 1.54439Å, and the wavelength of Cu-Kα is the weighted average of Kα1 and Kα2 λ=1.5418Å)
掃描方式:Coupled θ/2θ,掃描模式:Continuous PSD fast,掃描速率:0.1s/step,掃描範圍:3-50°,掃描步數:2286,每步增量:0.02° Scanning method: Coupled θ/2θ, scanning mode: Continuous PSD fast, scanning rate: 0.1s/step, scanning range: 3-50°, scanning steps: 2286, each step increment: 0.02°
電壓:40KV,電流:40mA Voltage: 40KV, Current: 40mA
4、熱重分析儀(Thermogravimetric Analysis,TGA) 4. Thermogravimetric Analysis (TGA)
儀器型號:METTLER TOLEDO TGA 2 Instrument model: METTLER TOLEDO TGA 2
吹掃氣:氮氣,50mL/min Purge gas: nitrogen, 50mL/min
升溫速率:10.0℃/min Heating rate: 10.0°C/min
溫度範圍:30-350℃ Temperature range: 30-350°C
5、動態蒸汽吸附儀(Dynamic Vapour Sorption,DVS) 5. Dynamic Vapor Sorption (DVS)
儀器型號:DVS Intrinsic Instrument model: DVS Intrinsic
載氣:氮氣,200sccm Carrier gas: nitrogen, 200sccm
溫度:25℃ Temperature: 25°C
濕度程度:以10%RH步長從50%RH-90% RH-95%RH-90% RH-0% RH,為循環1;再以10%RH步長從0%RH-90% RH-95%RH-90% RH-50% RH,為循環2;
Humidity level:
終止參數:dM/dT=0.002%/min,Maxtime=360min,Mintime=5min。 Termination parameters: dM/dT=0.002%/min, Maxtime=360min, Mintime=5min.
實施例1:式I化合物的製備 Embodiment 1: the preparation of formula I compound
式I化合物的合成路線如下: The synthetic route of formula I compound is as follows:
第一步: first step:
將LDA(15.28g,142.66mmol,71.43mL)溶於50mL四氫呋喃,冷卻至-78℃,滴加溶於10mL THF的Int-1-1溶液(10g,118.88mmol)。產生的液體升溫至20℃,攪拌30分鐘。反應混合物再次冷卻至-78℃,將溶於10ML THF的2-溴乙酸三級丁酯緩慢加入。反應液在室溫下攪拌過夜。反應完成後,反應液中加入飽和氯化銨(50mL,aq.)淬滅,用乙酸乙酯(50mL×3)萃取,有機相用100mL濃鹽水洗滌,硫酸鈉乾燥,濃縮,得到粗品化合物Int-1-2(22g,110.97mmol,產率93.34%)。 LDA (15.28g, 142.66mmol, 71.43mL) was dissolved in 50mL of tetrahydrofuran, cooled to -78°C, and Int-1-1 solution (10g, 118.88mmol) dissolved in 10mL of THF was added dropwise. The resulting liquid was warmed to 20°C and stirred for 30 minutes. The reaction mixture was cooled to -78°C again, and tert-butyl 2-bromoacetate dissolved in 10 mL THF was added slowly. The reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was quenched by adding saturated ammonium chloride (50mL, aq.), extracted with ethyl acetate (50mL×3), the organic phase was washed with 100mL concentrated brine, dried over sodium sulfate, and concentrated to obtain the crude compound Int -1-2 (22 g, 110.97 mmol, yield 93.34%).
1H NMR(400MHz,CDCl3):δ 2.83(t,2H),2.50(t,2H),1.97-1.92(m,1H),1.45(s,9H),1.06-1.01(m,2H),0.91-0.86(m,2H)。 1 H NMR (400MHz, CDCl 3 ): δ 2.83(t,2H),2.50(t,2H),1.97-1.92(m,1H),1.45(s,9H),1.06-1.01(m,2H), 0.91-0.86 (m, 2H).
第二步: Step two:
將化合物Int-1-2(8.2g,41.36mmol),碳酸銨(33.78g,351.56mmol),氰化鈉(5.07g,103.40mmol),50mL乙醇和50mL水混合的液體密封,加熱至80℃,加熱18小時。反應混合液冷卻,倒入100mL乙酸乙酯和100mL水的混合液中,各層是分離的,水層用乙酸乙酯(100mL×3)提取。合併有機溶液,用濃鹽水洗滌,硫酸鈉乾燥,濃縮。所得殘餘物用矽膠色譜法純化(乙酸乙酯/正己烷=1/2),得到化合物Int-1-3(5.7g,21.24mmol,產率51.36%)。 Seal the mixture of compound Int-1-2 (8.2g, 41.36mmol), ammonium carbonate (33.78g, 351.56mmol), sodium cyanide (5.07g, 103.40mmol), 50mL ethanol and 50mL water, and heat to 80°C , heated for 18 hours. The reaction mixture was cooled, poured into a mixture of 100 mL ethyl acetate and 100 mL water, the layers were separated, and the aqueous layer was extracted with ethyl acetate (100 mL×3). The organic solutions were combined, washed with concentrated brine, dried over sodium sulfate, and concentrated. The resulting residue was purified by silica gel chromatography (ethyl acetate/n-hexane=1/2) to obtain compound Int-1-3 (5.7 g, 21.24 mmol, yield 51.36%).
1H NMR(400MHz,DMSO):δ 10.61(s,1H),7.66(s,1H),2.29-2.08(m,2H),1.93-1.88(m,2H),1.29(s,9H),1.09-1.02(m,1H),0.47-0.26(m,3H),0.11-0.04(m,1H)。 1 H NMR (400MHz, DMSO): δ 10.61(s,1H),7.66(s,1H),2.29-2.08(m,2H),1.93-1.88(m,2H),1.29(s,9H),1.09 -1.02(m,1H),0.47-0.26(m,3H),0.11-0.04(m,1H).
第三步和第四步: The third and fourth steps:
將化合物Int-1-3(7.2g,26.83mmol)溶於鹽酸/二氧己環溶液(4M,50mL),室溫攪拌4小時並濃縮。產生的固體用30mL的乙腈研磨1小時,過濾得到的純外消旋物體為白色固體。用超臨界流體色譜SFC(使用CHIRALPAK AD-H 10μm 2.5*25cm手性柱;流速/監測:70g/min;監測器波長:214nm;流動相A:超臨界二氧化碳;流動相B:甲醇)分離手性固體,獲得化合物Int-1(2g,9.42mmol,產率35.12%)。
Compound Int-1-3 (7.2 g, 26.83 mmol) was dissolved in hydrochloric acid/dioxane solution (4M, 50 mL), stirred at room temperature for 4 hours and concentrated. The resulting solid was triturated with 30 mL of acetonitrile for 1 hour and filtered to give a pure racemic mass as a white solid. With supercritical fluid chromatography SFC (using CHIRALPAK AD-
1H NMR(400MHz,DMSO):δ 12.20(s,1H),10.63(s,1H),7.71(s,1H),2.32-2.09(m,2H),1.99-1.87(m,2H),1.11-1.03(m,1H),0.48-0.27(m,3H),0.12-0.05(m,1H). 1 H NMR (400MHz, DMSO): δ 12.20(s,1H),10.63(s,1H),7.71(s,1H),2.32-2.09(m,2H),1.99-1.87(m,2H),1.11 -1.03(m,1H),0.48-0.27(m,3H),0.12-0.05(m,1H).
Chiral HPLC:98.04% ee,Rt:2.918min。 Chiral HPLC: 98.04% ee, Rt: 2.918min.
LCMS:MS m/z(ESI):213.1[M+1]+。 LCMS: MS m/z (ESI): 213.1 [M+1] + .
第五步: the fifth step:
將化合物40a(1g,4.87mmol)溶於20mL DMF中,加入NBS(870mg,4.89mmol)。將混合物在室溫下攪拌2小時,將所得的混合物倒入20mL的冰水中,用乙酸乙酯(20mL×2)萃取混合物。合併有機相用20mL水,20mL濃鹽水洗滌,硫酸鈉乾燥,過濾。過濾物濃縮得到粗品40b(1g,3.52mmol,產率72.22%)。 Compound 40a (1 g, 4.87 mmol) was dissolved in 20 mL of DMF, and NBS (870 mg, 4.89 mmol) was added. The mixture was stirred at room temperature for 2 hours, the resulting mixture was poured into 20 mL of ice water, and the mixture was extracted with ethyl acetate (20 mL×2). The combined organic phases were washed with 20 mL of water, 20 mL of concentrated brine, dried over sodium sulfate, and filtered. The filtrate was concentrated to give crude 40b (1 g, 3.52 mmol, 72.22% yield).
第六步: Step six:
將化合物40b(1g,3.52mmol)溶於10mL甲醇中,滴加硫酸(18M,0.7mL)。混合液體75℃攪拌過夜,降溫至室溫,倒入20mL冰水中,混合液用50rmL乙酸乙酯萃取,有機成分用硫酸鈉乾燥,過濾。過濾物濃縮得到粗品40c(1g,3.36mmol,產率95.29%)。 Compound 40b (1 g, 3.52 mmol) was dissolved in 10 mL of methanol, and sulfuric acid (18M, 0.7 mL) was added dropwise. The mixed liquid was stirred overnight at 75°C, cooled to room temperature, poured into 20 mL of ice water, the mixed liquid was extracted with 50 mL of ethyl acetate, the organic components were dried over sodium sulfate, and filtered. The filtrate was concentrated to give crude 40c (1 g, 3.36 mmol, 95.29% yield).
1H NMR(400MHz,DMSO-d 6):δ 7.57(s,1H),7.21(s,1H),6.11(brs,2H),3.85(s,3H)。 1 H NMR (400 MHz, DMSO- d 6 ): δ 7.57 (s, 1H), 7.21 (s, 1H), 6.11 (brs, 2H), 3.85 (s, 3H).
第七步: Step seven:
將化合物40c(5.45g,18.29mmol)和乙烯三氟硼酸鉀(2.45g,18.29mmol)溶液溶於50mL二氧己環和10mL水中,加入Pd(dppf)Cl2(1.34g,1.83mmol)和碳酸鉀(6.35g,45.71mmol)。將所得混合物抽真空並用N2重新填充3次。將所得的混合物在80℃下攪拌16小時。用100mL乙酸乙酯稀釋混合物,合併有機相,用100mL濃鹽水洗滌,硫酸鈉乾燥,過濾,減壓濃縮。所得殘餘物用矽膠柱色譜法純化,得到化合物41b(3.56g,14.52mmol,產率79.40%)。 A solution of compound 40c (5.45g, 18.29mmol) and potassium ethylene trifluoroborate (2.45g, 18.29mmol) was dissolved in 50mL of dioxane and 10mL of water, and Pd(dppf)Cl 2 (1.34g, 1.83mmol) was added and Potassium carbonate (6.35 g, 45.71 mmol). The resulting mixture was evacuated and refilled 3 times with N2 . The resulting mixture was stirred at 80°C for 16 hours. The mixture was diluted with 100 mL of ethyl acetate, and the combined organic phases were washed with 100 mL of concentrated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain compound 41b (3.56 g, 14.52 mmol, yield 79.40%).
LCMS:MS m/z(ESI):246.1[M+H]+。 LCMS: MS m/z (ESI): 246.1 [M+H] + .
第八步: Step Eight:
將化合物41b(3.56g,14.52mmol)溶於20mL甲醇,加入鈀碳(1.55g,1.45mmol,285.48uL,10%純度)。將所得的混合物用氫氣抽真空和充氣。所 得的混合物在室溫下攪拌16小時,液相色譜法顯示反應結束。過濾混合物,塊狀物用甲醇洗滌,過濾物減壓濃縮,得到化合物41c(3.45g,13.96mmol,產率96.12%)。 Compound 41b (3.56 g, 14.52 mmol) was dissolved in 20 mL of methanol, and palladium on carbon (1.55 g, 1.45 mmol, 285.48 uL, 10% purity) was added. The resulting mixture was evacuated and gassed with hydrogen. Place The resulting mixture was stirred at room temperature for 16 hours, liquid chromatography showed the reaction was complete. The mixture was filtered, the block was washed with methanol, and the filtrate was concentrated under reduced pressure to obtain compound 41c (3.45 g, 13.96 mmol, yield 96.12%).
LCMS:MS m/z(ESI):248.1[M+H]+。 LCMS: MS m/z (ESI): 248.1 [M+H] + .
第九步: Step Nine:
將化合物41c(3.36g,13.59mmol)溶於34mL丙酮,加入3.36mL鹽酸。所得的混合物在室溫下攪拌20分鐘。混合物降溫至0℃,加入溶于5mL水的亞硝酸鈉(1.88g,27.18mmol)。在0℃下少量加入氯化銅(1.48g,14.95mmol)。所得的混合物在室溫下攪拌1小時。混合物倒入1M鹽酸(60mL)中,水相用乙酸乙酯(100mL×3)萃取。合併的有機相用100mL濃鹽水洗滌,硫酸鈉乾燥,過濾,減壓濃縮。殘餘物用矽膠柱色譜法純化(用正乙烷/乙酸乙酯=50/1洗脫),得到化合物41d(2.23g,8.36mmol,產率61.53%)。 Compound 41c (3.36 g, 13.59 mmol) was dissolved in 34 mL of acetone, and 3.36 mL of hydrochloric acid was added. The resulting mixture was stirred at room temperature for 20 minutes. The mixture was cooled to 0 °C and sodium nitrite (1.88 g, 27.18 mmol) dissolved in 5 mL of water was added. Copper chloride (1.48 g, 14.95 mmol) was added in small amounts at 0 °C. The resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into 1M hydrochloric acid (60 mL), and the aqueous phase was extracted with ethyl acetate (100 mL×3). The combined organic phases were washed with 100 mL of concentrated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluted with n-ethane/ethyl acetate=50/1) to obtain compound 41d (2.23 g, 8.36 mmol, yield 61.53%).
1H NMR(400MHz,DMSO-d 6):δ 7.99(s,1H),7.87(s,1H),3.88(s,3H),2.92(q,2H),1.17(t,3H)。 1 H NMR (400 MHz, DMSO- d 6 ): δ 7.99 (s, 1H), 7.87 (s, 1H), 3.88 (s, 3H), 2.92 (q, 2H), 1.17 (t, 3H).
第十步: Step ten:
將化合物41d(2.23g,8.36mmol)溶於35mL四氯化碳,加入AIBN(412.00mg,2.51mmol)和NBS(1.64g,9.20mmol)。所得的混合物在80℃下攪拌16小時。過濾混合物。用DCM洗滌固體,過濾物在真空下濃縮,得到粗品化合物41e(2.5g,7.24mmol,產率86.51%)。 Compound 41d (2.23 g, 8.36 mmol) was dissolved in 35 mL of carbon tetrachloride, and AIBN (412.00 mg, 2.51 mmol) and NBS (1.64 g, 9.20 mmol) were added. The resulting mixture was stirred at 80°C for 16 hours. Filter the mixture. The solid was washed with DCM, and the filtrate was concentrated in vacuo to give crude compound 41e (2.5 g, 7.24 mmol, 86.51% yield).
1H NMR(400MHz,DMSO-d 6):δ 8.17(s,1H),8.04(s,1H),6.08(q,1H),3.92(s,3H),2.05(d,3H)。 1 H NMR (400 MHz, DMSO- d 6 ): δ 8.17 (s, 1H), 8.04 (s, 1H), 6.08 (q, 1H), 3.92 (s, 3H), 2.05 (d, 3H).
第十一步: Eleventh step:
將化合物41e(2.5g,7.24mmol)溶於10mL甲醇,加入NH3/MeOH(7M,30mL)。所得的混合物在室溫下攪拌16小時。用prep-HPLC純化混合物,得到化合物41f(1.18g,4.73mmol,產率65.34%)。 Compound 41e (2.5 g, 7.24 mmol) was dissolved in 10 mL of methanol, and NH 3 /MeOH (7M, 30 mL) was added. The resulting mixture was stirred at room temperature for 16 hours. The mixture was purified by prep-HPLC to obtain compound 41f (1.18 g, 4.73 mmol, yield 65.34%).
1H NMR(400MHz,DMSO-d 6):δ 9.11(brs,1H),8.20(s,1H),7.91(s,1H),4.71(q,1H),1.42(d,3H)。 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.11 (brs, 1H), 8.20 (s, 1H), 7.91 (s, 1H), 4.71 (q, 1H), 1.42 (d, 3H).
19F NMR(376.5MHz,DMSO-d 6):δ -60.99。 19 F NMR (376.5 MHz, DMSO- d 6 ): δ -60.99.
LCMS:MS m/z(ESI):250.0[M+H]+。 LCMS: MS m/z (ESI): 250.0 [M+H] + .
第十二步: Step twelve:
小規模 small scale
將化合物41f(50mg,0.2mmol)溶於THF(2mL),加入硼烷-d3-THF多元溶液(6mmol,6mL)。反應液60℃下攪拌18小時。滴加2mL甲醇,接著加入鹽酸(6M,2mL)。反應液80℃下攪拌2小時。接著,加入5M氫氧化鈉調節混合物pH值至7,液體乾燥,濃縮。殘餘物用矽膠色譜法純化(DCM:MeOH=20:1),得到化合物71b(39mg,0.147mmol,產率70%)。 Compound 41f (50 mg, 0.2 mmol) was dissolved in THF (2 mL), and borane-d3-THF multiple solution (6 mmol, 6 mL) was added. The reaction solution was stirred at 60°C for 18 hours. 2 mL of methanol was added dropwise, followed by hydrochloric acid (6M, 2 mL). The reaction solution was stirred at 80°C for 2 hours. Next, 5M sodium hydroxide was added to adjust the pH of the mixture to 7, and the liquid was dried and concentrated. The residue was purified by silica gel chromatography (DCM:MeOH=20:1) to obtain compound 71b (39 mg, 0.147 mmol, yield 70%).
大規模 massive
將化合物41f(800mg,3.20mmol)溶於10mLTHF中,加入BD3(1M in THF,64ml,64mmol)。加入之後,反應液在60℃下(放置在一個封閉管裡)攪拌10小時。用10mL甲醇淬滅反應,加入鹽酸(6M,20mL)。之後在80℃下攪拌8小時。加入2N氫氧化鈉調節pH值到7,用乙酸乙酯萃取,合併的有機相用50mL濃鹽水洗滌,硫酸鈉乾燥,過濾和降壓濃縮。殘餘物用矽膠柱色譜法純化,用5%甲醇的DMC溶液洗脫,得到化合物71b,供下一步驟中使用。 Compound 41f (800 mg, 3.20 mmol) was dissolved in 10 mL THF, and BD 3 (1M in THF, 64 ml, 64 mmol) was added. After the addition, the reaction was stirred at 60°C (in a closed tube) for 10 hours. The reaction was quenched with 10 mL of methanol and hydrochloric acid (6M, 20 mL) was added. It was then stirred at 80° C. for 8 hours. 2N sodium hydroxide was added to adjust the pH value to 7, extracted with ethyl acetate, the combined organic phases were washed with 50 mL concentrated brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 5% methanol in DMC to afford compound 71b which was used in the next step.
LCMS:MS m/z(ESI):238.1[M+H]+。 LCMS: MS m/z (ESI): 238.1 [M+H] + .
第十三步: Step Thirteen:
小規模 small scale
將化合物71b(20mg,0.084mmol)溶於2mL的DMF,加入三乙胺(40mg,0.28mmol),化合物Int-1(23mg,0.108mmol)和HATU(57mg,0.13mmol)。反應液在室溫下攪拌18小時。加入3mL水,用乙酸乙酯(20mL×3)萃取混合物。合併的有機層用濃鹽水洗滌,硫酸鈉乾燥,濃縮。殘餘物用prep-HPLC純化,得到化合物71(24mg,0.055mol,產率51%)。 Compound 71b (20 mg, 0.084 mmol) was dissolved in 2 mL of DMF, triethylamine (40 mg, 0.28 mmol), compound Int-1 (23 mg, 0.108 mmol) and HATU (57 mg, 0.13 mmol) were added. The reaction was stirred at room temperature for 18 hours. 3 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by prep-HPLC to obtain compound 71 (24 mg, 0.055 mol, yield 51%).
大規模 massive
將化合物Int-1(680mg,3.2mmol)溶於10ml DMF中,加入EDCI(920mg,4.8mmol)和HATU(1.83g,4.8mmol)。攪拌10分鐘後,加入之前步驟收集的化合物71b。反應液在室溫下攪拌3小時。LCMS顯示反應完成。直接用反相液相色譜法純化,得到化合物71(1.10g,兩步後產率79.6%)。 Compound Int-1 (680 mg, 3.2 mmol) was dissolved in 10 ml of DMF, EDCI (920 mg, 4.8 mmol) and HATU (1.83 g, 4.8 mmol) were added. After stirring for 10 minutes, compound 71b collected from the previous step was added. The reaction solution was stirred at room temperature for 3 hours. LCMS showed the reaction was complete. Direct purification by reverse phase liquid chromatography afforded compound 71 (1.10 g, 79.6% yield after two steps).
1H NMR(400MHz,CD3OD,):7.76(s,1 H),7.63-7.60(m,1 H),5.57-5.53(m,1 H),2.59-2.40(m,2 H),2.28-2.19(m,2 H),1.56-1.50(m,3 H),1.28-1.21(m,1 H),0.62-0.58(m,1 H),0.49-0.41(m,3 H)。 1 H NMR (400MHz, CD 3 OD,): 7.76 (s, 1 H), 7.63-7.60 (m, 1 H), 5.57-5.53 (m, 1 H), 2.59-2.40 (m, 2 H), 2.28-2.19(m,2H),1.56-1.50(m,3H),1.28-1.21(m,1H),0.62-0.58(m,1H),0.49-0.41(m,3H).
LCMS:MS m/z(ESI):432[M+H]+。 LCMS: MS m/z (ESI): 432 [M+H] + .
第十四步: Step fourteen:
化合物71(1.10g)用超臨界流體色譜法分離,得到兩個異構體(分別是325mg和415mg)。 Compound 71 (1.10 g) was separated by supercritical fluid chromatography to obtain two isomers (325 mg and 415 mg, respectively).
對映體式I化合物(71-1)(保留時間較短): Enantiomer formula I compound (71-1) (shorter retention time):
1H NMR(500MHz,DMSO-d 6)δ 10.63(s,1H),7.89(d,1H),7.75(t,2H),5.30-5.16(m,1H),4.23(d,1H),2.37-2.27(m,2H),1.99(dq,2H),1.53(s,1H),1.43(dd,3H),1.11(td,1H),0.49-0.30(m,3H),0.11(dt,1H)。 1 H NMR (500MHz,DMSO- d 6 )δ 10.63(s,1H),7.89(d,1H),7.75(t,2H),5.30-5.16(m,1H),4.23(d,1H),2.37 -2.27(m,2H),1.99(dq,2H),1.53(s,1H),1.43(dd,3H),1.11(td,1H),0.49-0.30(m,3H),0.11(dt,1H ).
LCMS:MS m/z(ESI):432.3[M+H]+。 LCMS: MS m/z (ESI): 432.3 [M+H] + .
ChirHPLC(1% DEA in乙醇/己烷60/40,1.0mL/min,35℃,CHIRALPAK IG,150*4.6mm,5μm):Rt:4.594min,de:100%。 ChirHPLC (1% DEA in ethanol/hexane 60/40, 1.0 mL/min, 35°C, CHIRALPAK IG, 150*4.6mm, 5μm): Rt: 4.594min, de: 100%.
對映體化合物71-2(保留時間較長): Enantiomer compound 71-2 (longer retention time):
1H NMR(500MHz,DMSO-d 6)δ 10.53(s,1H),7.89(d,1H),7.80-7.66(m,2H),5.30-5.12(m,1H),4.23(d,1H),2.44-2.36(m,1H),2.31-2.20(m,1H),2.05-1.95(m,2H),1.44(dd,3H),1.11(td,1H),0.50-0.29(m,3H),0.16-0.08(m,1H)。 1 H NMR (500MHz,DMSO- d 6 )δ 10.53(s,1H),7.89(d,1H),7.80-7.66(m,2H),5.30-5.12(m,1H),4.23(d,1H) ,2.44-2.36(m,1H),2.31-2.20(m,1H),2.05-1.95(m,2H),1.44(dd,3H),1.11(td,1H),0.50-0.29(m,3H) ,0.16-0.08(m,1H).
LCMS:MS m/z(ESI):432.3[M+H]+。 LCMS: MS m/z (ESI): 432.3 [M+H] + .
ChirHPLC(1% DEA in乙醇/己烷60/40,1.0mL/min,35℃,CHIRALPAK IG,150*4.6mm,5μm):Rt:10.931min,de:100%。 ChirHPLC (1% DEA in ethanol/hexane 60/40, 1.0 mL/min, 35° C., CHIRALPAK IG, 150*4.6 mm, 5 μm): Rt: 10.931 min, de: 100%.
生物學測試 biological test
本公開將參照以下測試案例進一步描述,但是這些測試案例不應該被視為限制本公開的範圍。 The present disclosure will be further described with reference to the following test cases, but these test cases should not be considered as limiting the scope of the present disclosure.
測試例1:體外螢光測定ADAMTS-4或ADAMTS-5活性 Test Example 1: In Vitro Fluorescence Determination of ADAMTS-4 or ADAMTS-5 Activity
FRET(螢光共振能量轉移)肽被重組的ADAMTS-4或ADAMTS-5蛋白分割成兩個獨立的片段,導致被定量的螢光信號增加。多肽為5-FAM-TEGEARGSVILLK(5-TAMRA)K-NH2,從ANASPEC定制。ADAMTS-4重組蛋白(目錄號4307-AD)和ADAMTS-5重組蛋白(目錄號2198-AD)購自R&D Systems。 FRET (fluorescence resonance energy transfer) peptides are split into two separate fragments by recombinant ADAMTS-4 or ADAMTS-5 proteins, resulting in an increase in the quantified fluorescent signal. The polypeptide is 5-FAM-TEGEARGSVILLK(5-TAMRA)K-NH 2 , customized from ANASPEC. ADAMTS-4 recombinant protein (Cat. No. 4307-AD) and ADAMTS-5 recombinant protein (Cat. No. 2198-AD) were purchased from R&D Systems.
製備含有50mM的HEPES pH值為7.5、100mM氯化鈉、5mM氯化鈣、0.1% CHAPS和5%甘油的測定緩衝液。將2.5μL的含化合物的測定緩衝液分配到384孔板中,並加入2.5μL ADAMTS-4或ADAMTS-5蛋白(反應中的最終濃度為10nM)。將化合物和蛋白質在室溫下預培養15分鐘。然後,向每個孔中加入5μL底物。ADAMTS-4和ADAMTS-5的最終 底物濃度分別為15μM和8μM。在37℃培養3小時後,在TECAN板讀數器上測定每個孔中的螢光信號(激發,490nm;散發,520nm)。 Prepare assay buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl, 0.1% CHAPS, and 5% glycerol. 2.5 μL of compound-containing assay buffer was dispensed into a 384-well plate and 2.5 μL of ADAMTS-4 or ADAMTS-5 protein was added (final concentration in the reaction was 10 nM). Compounds and proteins were pre-incubated for 15 min at room temperature. Then, 5 μL of substrate was added to each well. Final ADAMTS-4 and ADAMTS-5 Substrate concentrations were 15 μM and 8 μM, respectively. After 3 hours of incubation at 37°C, the fluorescent signal (excitation, 490nm; emission, 520nm) in each well was measured on a TECAN plate reader.
數據分析 data analysis
數據錄入GraphPad Prism軟件,使用「log(inhibitor)vs.response--Variable slope(four parameters)」功能計算IC50值(見表1) Enter the data into the GraphPad Prism software, and use the "log(inhibitor)vs.response--Variable slope(four parameters)" function to calculate the IC 50 value (see Table 1)
結論:本公開的式I化合物對ADAMTS-4和ADAMTS-5的酶活性具有顯著的抑制作用。 Conclusion: The compound of formula I disclosed in the present disclosure has significant inhibitory effect on the enzymatic activities of ADAMTS-4 and ADAMTS-5.
測試例2. ADAMTS-5活性的體外ELISA測試(酶聯免疫吸附法測定) Test example 2. In vitro ELISA test of ADAMTS-5 activity (enzyme-linked immunosorbent assay)
在該測定中,用蛋白質底物,聚集蛋白聚糖IGD蛋白測定重組ADAMTS-5蛋白(目錄號2198-AD,R&D Systems)的酶活性。聚集蛋白聚糖IGD蛋白是連接人蛋白聚糖球狀結構域1和2(T331-G458)的多肽,其在C端帶有組氨酸標簽(目錄號30411000,BIOTEZ),在大腸桿菌中表達產生。使用BioTEZ的ELISA試劑盒(目錄號30510111)檢測酶促產物ARGSVIL-肽。
In this assay, the enzymatic activity of recombinant ADAMTS-5 protein (Cat. No. 2198-AD, R&D Systems) was determined using the protein substrate, aggrecan IGD protein. The aggrecan IGD protein is a polypeptide linking human aggrecan
製備含有50mM的HEPES pH值為7.5、100mM NaCl、5mM CaCl2、0.1% CHAPS和5%甘油的測定緩衝液。將重組ADAMTS-5蛋白在測定緩衝液中稀釋至0.3nM。將10μL緩衝液和10μL化合物溶液轉移到96孔板的每個孔中,並在室溫下培養15分鐘。用測定緩衝液將底物蛋白聚糖-IGD稀釋至100nM,並向每個孔中加入20μL。孔板在37℃下培養45分鐘。培養後,按照生產商的說明書,使用蛋白聚糖酶活性ELISA測定試劑盒測量新產生的抗原表位ARGSVIL-肽。然後,加入100μL終止溶液,並在TECAN讀板器上使用620nM作為參考,在450nM處讀取每個孔的吸光度。
Prepare assay buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS, and 5% glycerol. Recombinant ADAMTS-5 protein was diluted to 0.3 nM in assay buffer.
數據分析: data analysis:
在GraphPad Prism軟件中使用Sigmoidal 4PL功能,得到ELISA測定的標準曲線,並基於標準曲線計算相應的肽濃度。使用「log(inhibitor)vs.response--Variable slope(four parameters)」功能計算IC50值(見表2)。 Use the Sigmoidal 4PL function in GraphPad Prism software to obtain the standard curve determined by ELISA, and calculate the corresponding peptide concentration based on the standard curve. IC 50 values were calculated using the "log(inhibitor) vs. response--Variable slope(four parameters)" function (see Table 2).
結論:式I化合物對ADAMTS-5的酶活性具有顯著的抑制作用 Conclusion: the compound of formula I has significant inhibitory effect on the enzymatic activity of ADAMTS-5
測試例3.小鼠外植體測試 Test Example 3. Mouse Explant Test
在該測試中,用培養基中的IL-1α蛋白(Sigma-Aldrich,目錄號I2778)處理新鮮的小鼠股骨頭軟骨,誘導軟骨分解代謝。然後,通過糖胺聚糖檢測試劑盒(Chondrex,目錄號6022)中的二甲基亞甲基藍染料測定附著於裂解的蛋白聚糖片段上的GAG和附著於完整蛋白聚糖的GAG。 In this test, fresh mouse femoral head cartilage is treated with IL-1α protein (Sigma-Aldrich, catalog number I2778) in culture medium to induce cartilage catabolism. GAGs attached to cleaved proteoglycan fragments and GAGs attached to intact proteoglycans were then measured by dimethylmethylene blue dye in the Glycosaminoglycan Detection Kit (Chondrex, cat# 6022).
從小鼠(25天齡,雄性,C57BL/6,來自Charles River Lab)分離股骨頭軟骨樣品,並放入2.0ml裝滿培養基(細胞培養基、10% FBS、4mM麩醯胺酸、青黴素-鏈黴素、20mM HEPES)的管中。將200μL不含胎牛血清的培養基添加到48孔板的每個孔中,並將一塊軟骨轉移到孔板中的一個孔中。然後吸出培養基,並將式I化合物和IL-1α蛋白以總體積400μl且不含FBS的新鮮培養基添加到平板中。IL-1α的最終濃度為1ng/mL。將孔板在37℃下,同時有5%二氧化碳供應的濕潤培養箱中培養72小時。 Femoral head cartilage samples were isolated from mice (25 days old, male, C57BL/6, from Charles River Lab) and placed in 2.0 ml refill medium (cell culture medium, 10% FBS, 4 mM glutamic acid, penicillin-streptavidin plain, 20mM HEPES). Add 200 µL of FBS-free medium to each well of the 48-well plate and transfer a piece of cartilage to one well of the well plate. The medium was then aspirated and the compound of formula I and IL-la protein were added to the plate in a total volume of 400 μl of fresh medium without FBS. The final concentration of IL-la was 1 ng/mL. The plate was incubated at 37°C for 72 hours in a humidified incubator with 5% carbon dioxide supply.
將上清液轉移到一個1.5mL試管中並保持在-20℃。將每個軟骨樣品轉移到另外的含有400μL新鮮製備的木瓜蛋白酶溶液的1.5mL管中。木瓜蛋白酶溶液含有125μg/mL木瓜蛋白酶(Sigma-Aldrich,目錄號P3125)、0.1M乙酸鈉(Sigma-Aldrich,目錄號S7899)、pH 5.5和5mM EDTA和5mM L-半胱氨酸鹽酸鹽(Sigma-Aldrich,目錄號C7880)。將軟骨樣品在60℃水浴中保持搖動24小時。 The supernatant was transferred to a 1.5 mL tube and kept at -20°C. Transfer each cartilage sample to an additional 1.5 mL tube containing 400 µL of freshly prepared papain solution. Papain solution contained 125 μg/mL papain (Sigma-Aldrich, catalog number P3125), 0.1 M sodium acetate (Sigma-Aldrich, catalog number S7899), pH 5.5 and 5 mM EDTA and 5 mM L-cysteine hydrochloride ( Sigma-Aldrich, catalog number C7880). Cartilage samples were kept shaking in a 60 °C water bath for 24 hours.
將溶解產物渦旋10秒並以10,000rpm旋轉2分鐘。用磷酸鹽緩衝液稀釋上清液和溶解產物樣品,並與100μL來自糖胺聚糖檢測試劑盒的染料混合。用設定為525nm波長的TECAN讀板儀測定來自每個孔的光密度。 The lysate was vortexed for 10 seconds and spun at 10,000 rpm for 2 minutes. Dilute the supernatant and lysate samples with phosphate buffer and mix with 100 µL of the dye from the Glycosaminoglycan Detection Kit. The optical density from each well was measured with a TECAN plate reader set to a wavelength of 525 nm.
數據分析: data analysis:
基於標準曲線用試劑盒中提供的硫酸軟骨素的劑量範圍確定上清液和溶解產物中GAGs的濃度。 Concentrations of GAGs in supernatants and lysates were determined based on standard curves using the dose range of chondroitin sulfate provided in the kit.
GAG釋放的比例計算如下: The proportion of GAG release was calculated as follows:
測試的化合物效果表示為用以下公式計算的抑制百分比: The tested compound effects are expressed as percent inhibition calculated using the following formula:
在2μM和20μM濃度下式I化合物的抑制數據列於表3中。 Inhibition data for compounds of formula I are presented in Table 3 at concentrations of 2 μM and 20 μM.
前述實施例和示例僅用於說明,並不旨在限制本公開的範圍。基於本公開,對所公開的實施例的各種改變和修改對於本發明所屬技術領域具有通常知識者將是顯而易見的,並且可以在不脫離本公開的精神和範圍的情況下進行這樣的改變和修改。所有引用的文獻通過引用整體併入本文,而不承認它們作為現有技術。 The foregoing embodiments and examples are for illustration only, and are not intended to limit the scope of the present disclosure. Various changes and modifications to the disclosed embodiments will become apparent to those having ordinary skill in the art to which this invention pertains based on the present disclosure, and such changes and modifications can be made without departing from the spirit and scope of the present disclosure. . All cited documents are hereby incorporated by reference in their entirety, without admission that they are prior art.
實施例2-1:式I化合物A晶型的製備 Example 2-1: Preparation of Formula I Compound A Crystal Form
將式I化合物(10mg,23.16μmol)溶於1mL THF中,溶清,過濾除去不溶物,靜置過夜。有晶體析出,過濾,收集濾餅,真空乾燥,得到式I化合物的A晶型(2mg,產率:20.0%)。 The compound of formula I (10 mg, 23.16 μmol) was dissolved in 1 mL THF, dissolved, filtered to remove insoluble matter, and left to stand overnight. Crystals were precipitated, filtered, and the filter cake was collected and dried in vacuo to obtain Form A of the compound of formula I (2 mg, yield: 20.0%).
實施例2-2:式I化合物A晶型的製備 Embodiment 2-2: Preparation of Formula I Compound A Crystal Form
將式I化合物(876mg,2.03mmol)溶於10mL無水乙醇中,未溶清,室溫攪拌,反應液過濾,收集濾餅,真空乾燥,得到式I化合物的A晶型(578mg,產率:66.0%)。 The compound of formula I (876mg, 2.03mmol) was dissolved in 10mL of absolute ethanol, the undissolved liquid was stirred at room temperature, the reaction solution was filtered, the filter cake was collected, and dried in vacuum to obtain the crystal form A of the compound of formula I (578mg, yield: 66.0%).
其特徵峰位置如表4所示,X-射線粉末繞射譜圖如圖1所示。 The characteristic peak positions are shown in Table 4, and the X-ray powder diffraction spectrum is shown in Figure 1.
DSC圖譜如圖2所示,吸熱峰峰值280.16℃。 The DSC spectrum is shown in Figure 2, and the endothermic peak is at 280.16°C.
實施例2-3:式I化合物A晶型的製備 Embodiment 2-3: Preparation of Formula I Compound A Crystal Form
將式I所示化合物(1.5g,3.47mmol)溶於15mL無水乙醇中,未溶清,室溫攪拌,反應液過濾,收集濾餅,真空乾燥,得到產物(1188mg,產率:79.2%)。經X-射線粉末繞射檢測,該產物為式I化合物的A晶型。 The compound shown by formula I (1.5g, 3.47mmol) was dissolved in 15mL of absolute ethanol, the undissolved liquid was stirred at room temperature, the reaction solution was filtered, the filter cake was collected, and vacuum-dried to obtain the product (1188mg, yield: 79.2%) . Through X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.
實施例2-4:式I化合物A晶型的製備 Embodiment 2-4: Preparation of formula I compound A crystal form
將式I所示化合物(10.8g,25.01mmol)溶於60mL異丙醇中,攪拌未溶清,50℃下攪拌,降溫至室溫,繼續攪拌,反應液過濾,收集濾餅,將上述濕品溶於60mL異丙醇中,攪拌未溶清,50℃下攪拌,降溫至室溫,繼 續攪拌過夜,反應液過濾,收集濾餅,真空乾燥,得到產物(6.4g,產率:59.3%)。經X-射線粉末繞射檢測,該產物為式I化合物的A晶型。 Dissolve the compound shown by formula I (10.8g, 25.01mmol) in 60mL of isopropanol, stir the undissolved liquid, stir at 50°C, cool down to room temperature, continue stirring, filter the reaction solution, collect the filter cake, and dissolve the above wet Dissolve the product in 60mL isopropanol, stir the undissolved liquid, stir at 50°C, cool to room temperature, and then Stirring was continued overnight, the reaction solution was filtered, and the filter cake was collected and vacuum-dried to obtain the product (6.4 g, yield: 59.3%). Through X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.
實施例2-5:式I化合物A晶型的製備 Embodiment 2-5: Preparation of Formula I Compound A Crystal Form
將式I所示化合物A晶型(實施例4)(30mg,69.5μmol)溶於0.5mL丙酮中,攪拌未溶清,室溫攪拌,反應液過濾,收集濾餅,得到產物(12mg,產率:40.0%)。經X-射線粉末繞射檢測,該產物為式I化合物的A晶型。 Dissolve the crystal form of Compound A (Example 4) (30 mg, 69.5 μmol) shown in formula I in 0.5 mL of acetone, stir the undissolved solution, stir at room temperature, filter the reaction solution, collect the filter cake, and obtain the product (12 mg, produced rate: 40.0%). Through X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.
實施例2-6:式I化合物A晶型的製備 Embodiment 2-6: Preparation of formula I compound A crystal form
將式I所示化合物A晶型(實施例4)(30mg,69.5μmol)溶於0.5mL乙酸乙酯中,未溶清,室溫攪拌,反應液過濾,收集濾餅,真空乾燥,得到產物(17mg,產率:56.7%)。經X-射線粉末繞射檢測,該產物為式I化合物的A晶型。 Dissolve the crystal form of Compound A (Example 4) (30 mg, 69.5 μmol) shown in formula I in 0.5 mL of ethyl acetate, stir at room temperature, filter the reaction solution, collect the filter cake, and dry in vacuo to obtain the product (17 mg, yield: 56.7%). Through X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.
實施例3、A晶型影響因素實驗
將式I所示化合物A晶型(實施例2-3製備所得)敞口平攤放置,考察在加熱(40℃、60℃)、紫外光照(4500Lux)、高濕(RH 75%、RH 90%)條件下樣品的穩定性,取樣考察期為30天。實驗結果見表5。 The compound A crystal form shown in formula I (prepared in Example 2-3) was placed open and flat, and examined under heating (40°C, 60°C), ultraviolet light (4500 Lux), high humidity (RH 75%, RH 90 %), the stability of the sample under the conditions, the sampling investigation period is 30 days. The experimental results are shown in Table 5.
實驗結論: Experimental results:
化合物在光照、高溫、高濕條件下,物理化學穩定性良好,濕度條件對樣品有影響,建議樣品在乾燥、密封條件下保存。 The compound has good physical and chemical stability under light, high temperature, and high humidity conditions. Humidity conditions will affect the sample. It is recommended that the sample be stored in a dry and sealed condition.
實施例4、A晶型長期加速穩定性實驗 Embodiment 4, long-term accelerated stability experiment of crystal form A
式I化合物A晶型(實施例2-3製備所得)進行3個月的長期加速穩定性考察。 The crystal form of compound A of formula I (prepared in Example 2-3) was subjected to a 3-month long-term accelerated stability investigation.
實驗結果見表6。 The experimental results are shown in Table 6.
實驗結論: Experimental results:
在避光、密封條件下(內包裝為密封袋,外用鋁箔包裝),化合物在長期(25℃、60%RH),加速(40℃、75%RH)條件下放置3個月,物理化學穩定性好。 Under the condition of avoiding light and sealing (inner packaging is sealed bag, external packaging with aluminum foil), the compound is stored for 3 months under long-term (25°C, 60%RH) and accelerated (40°C, 75%RH) conditions, and the compound is physically and chemically stable Good sex.
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