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WO2019114741A1 - 作为Akt抑制剂的盐型及其晶型 - Google Patents

作为Akt抑制剂的盐型及其晶型 Download PDF

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WO2019114741A1
WO2019114741A1 PCT/CN2018/120531 CN2018120531W WO2019114741A1 WO 2019114741 A1 WO2019114741 A1 WO 2019114741A1 CN 2018120531 W CN2018120531 W CN 2018120531W WO 2019114741 A1 WO2019114741 A1 WO 2019114741A1
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compound
formula
added
akt
crystal form
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French (fr)
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李刚
卢伦
陆青青
胡利红
丁照中
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Medshine Discovery Inc
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Medshine Discovery Inc
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Priority to JP2020532703A priority Critical patent/JP7101781B2/ja
Priority to KR1020207020007A priority patent/KR102394934B1/ko
Priority to ES18887717T priority patent/ES2943092T3/es
Priority to CN201880078387.9A priority patent/CN111433213B/zh
Priority to US16/772,322 priority patent/US11261196B2/en
Priority to EP18887717.9A priority patent/EP3725791B1/en
Publication of WO2019114741A1 publication Critical patent/WO2019114741A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D495/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention relates to a salt form and a crystal form thereof as an Akt inhibitor, in particular to a salt form of a compound of the formula (I) and a crystal form thereof, and also to the preparation of a tumor, a diabetes and a rheumatoid Application in arthritis drugs.
  • Akt also known as protein kinase B (PKB)
  • PPKB protein kinase B
  • Akt also known as protein kinase B (PKB)
  • PKI protein kinase B
  • Akt signaling pathway is closely related to the occurrence and development of various diseases such as malignant tumor, diabetes and rheumatoid arthritis, and it is receiving more and more attention.
  • Akt consists of a N-terminal regulatory region, an intermediate enzyme active region, a C-terminal regulatory region, and a hinge region linking the PH region to the kinase active region.
  • the N-terminal regulatory region has a platelet-leukocyte c kinase homology region, the PH region, and its exact mechanism during signal transduction is unclear;
  • the intermediate enzyme active region has catalytic filament/threonine residue phosphorylation activity, wherein Phosphorylation at the Thr308 site in the variable peptide loop (T-loop) is required for Akt activation; a proline-rich hydrophobic domain (HM) at the C-terminus, which contains the essentials for complete activation of Akt Two phosphorylation sites Ser473.
  • Akt subtypes are currently found in mammals: Aktl (PKB ⁇ ), Akt2 (PKB ⁇ ) and Akt3 (PKB ⁇ ), encoded by three different genes located on chromosomes 14q32, 19q13 and 1q43, respectively, with 85% sequence Homology, belonging to the AGC protein kinase family.
  • Mouse embryonic fibroblast studies have found that different Akt subtype-deficient strains eventually lead to different phenotypes: Akt1 deficiency manifests as placental malnutrition, growth delay and weight loss, Akt2 deficiency is characterized by insulin and blood glucose abnormalities, and Akt3 deficiency is present. The size of the brain is reduced. In human diseases, the expression and effects of the three subtypes of Akt are also significantly different.
  • Akt can be activated by a variety of substances in the cell such as hormones, growth factors, cytokines, intercellular matrix and the like. Akt is at the center of the PI3K/Akt pathway and is a direct target gene for PI3K. Many cytokines, growth factors, and physical stimuli can phosphorylate Akt by activating PI3K. Akt phosphorylation is often used as an indicator of PI3K activity.
  • Akt Akt-like protein kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase, kinase-like kinase-like kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kin
  • the Akt pathway intersects with many signal pathways to form a complex signal network.
  • the Akt pathway activates NF- ⁇ B pro-survival signaling or inhibits JNK/p38 apoptosis signaling, but the mode of action between p38 and Akt is inconclusive.
  • the interaction between the PI3K/Akt pathway and the Ras/MAPK pathway is evident in the different phases of the two pathways, but in different cell types, different cell differentiation stages, and different experimental conditions, but the Akt pathway Whether the Ras/MAPK pathway is positively or negatively regulated and which regulation is dominant in certain cells has not yet been fully revealed.
  • Akt signaling pathway is closely related to the occurrence and development of diseases such as tumor and rheumatoid arthritis. The interaction and influence between the pathways may play an important role in the occurrence of diseases.
  • Different solid forms of the pharmaceutically active ingredient may have different properties. Changes in properties of different solid forms can provide improved formulations, for example, ease of synthesis or handling, increased stability and shelf life. Changes in properties caused by different solid forms can also improve the final dosage form. Different solid forms of the pharmaceutically active ingredient can also produce polycrystalline or other crystalline forms, thereby providing more opportunities to assess changes in the properties of a solid active pharmaceutical ingredient.
  • the preparation method of the salt form of the compound of the formula (I) and the crystal form thereof is simple, and the crystal form is relatively stable, less affected by heat and humidity, and is convenient for preparation.
  • the invention provides a compound of formula (I),
  • the invention provides a crystalline form of a compound of formula (I), the X-ray powder diffraction pattern comprising characteristic peaks at 2 ⁇ values of 12.25° ⁇ 0.2°, 14.46° ⁇ 0.2°, and 21.83° ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the crystalline form of the compound of formula (I) is included in the 2 ⁇ values of 8.50 ° ⁇ 0.2 °, 10.20 ° ⁇ 0.2 °, 12.25 ° ⁇ 0.2 °, 14.46 ° ⁇ 0.2 ° Characteristic peaks of 15.30 ° ⁇ 0.2 °, 21.83 ° ⁇ 0.2 °, 27.31 ° ⁇ 0.2 ° and 31.41 ° ⁇ 0.2 °.
  • the X-ray powder diffraction pattern of the crystalline form of the compound of formula (I) is shown in Figure 1.
  • the analytical data for the X-ray powder diffraction pattern of the crystalline form of the compound of formula (I) is shown in Table 1.
  • the DSC curve for the crystalline form of the compound of formula (I) has an onset of an endothermic peak at 195.10 °C ⁇ 3 °C and an onset of an exothermic peak at 209.23 °C ⁇ 3 °C.
  • the DSC curve for the crystalline form of the compound of formula (I) is shown in Figure 2.
  • the TGA curve for the crystalline form of the compound of formula (I) at 100.00 ° C ⁇ 3 ° C has a weight loss of 0.295%.
  • the TGA profile of the crystalline form of the compound of formula (I) is shown in Figure 3.
  • the infrared spectrum of crystalline form of the compound of formula (I) comprises at 3316cm -1 ⁇ 5cm -1, 3079cm -1 ⁇ 5cm -1, 3058cm -1 ⁇ 5cm -1, 3016cm ⁇ 5cm -1 , 2954cm -1 ⁇ 5cm -1 , 2935cm -1 ⁇ 5cm -1 , 2915cm -1 ⁇ 5cm -1 , 2902cm -1 ⁇ 5cm -1 , 2864cm -1 ⁇ 5cm -1 , 1704cm -1 ⁇ 2cm -1 1633cm -1 ⁇ 2cm -1 , 1623cm -1 ⁇ 2cm -1 , 1589cm -1 ⁇ 2cm -1 , 1530cm -1 ⁇ 2cm -1 , 1514cm -1 ⁇ 2cm -1 , 1486cm -1 ⁇
  • intermediate 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, combinations thereof with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalents, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations:
  • TBTU O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate
  • DIEA N,N-diisopropylethylamine
  • DMF N,N-dimethyl Carboxamide
  • MeOH methanol
  • THF tetrahydrofuran
  • Pd(dppf)Cl 2 [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride
  • Pd 2 (dba) 3 three (dibenzylideneacetone) dipalladium
  • x-PHOS 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl
  • Test method Approximately 10-20 mg samples were used for XRPD detection.
  • Test method sample (0.5 ⁇ 1mg) was placed in a DSC aluminum pot for testing, heating the sample from room temperature (25 ° C) to 300 ° C or 350 at a heating rate of 10 ° C / min under 50 mL / min N 2 conditions °C.
  • Test method sample (2 ⁇ 5mg) was placed in a TGA platinum pot for testing, heating the sample from room temperature (25 ° C) to 300 ° C at a heating rate of 10 ° C / min under 25 mL / min N 2 conditions, 350 °C or 20% weight loss.
  • Test conditions Approximately 10-15 mg of sample was used for DVS detection.
  • Very hygroscopic Wet weight gain is not less than 15%
  • Humidity Wet weight gain is less than 15% but not less than 2%
  • Slightly hygroscopic Wet weight gain is less than 2% but not less than 0.2%
  • No or almost no hygroscopicity Wet weight gain is less than 0.2%
  • the analysis method is as follows:
  • Figure 1 is an XRPD spectrum of the crystalline form of the compound of formula (I).
  • Figure 2 is a DSC chart of the crystalline form of the compound of formula (I).
  • Figure 3 is a TGA spectrum of the crystalline form of the compound of formula (I).
  • Figure 4 is a graph showing the infrared absorption spectrum of the crystal form of the compound of the formula (I).
  • Figure 5 is a graph showing the ultraviolet absorption spectrum of the crystal form of the compound of the formula (I).
  • compound 1-B (2.0Kg, 12.03mol) was dissolved in anhydrous tetrahydrofuran (12L). The solution was added to a 50L spherical kettle, cooled to -40 ° C ⁇ -20 ° C, and n-butyl was added dropwise. Lithium (5.3 L, 13.24 mol), after completion of the dropwise addition, stirring was continued at this temperature for 1 hour, followed by dropwise addition of isopropyl pinacol borate (2.351 Kg, 12.63 mol), and the addition was completed, and the cooling bath was removed. The reaction was stirred for 40 min, and the reaction was completed by HPLC.
  • N,N-dimethylformamide (11.3 L), 1-J (1417.92 g, 5.69 mol), 1-K (2007.29 g, 5.92 mol), and N,N-di, at 20-25 ° C
  • Isopropylethylamine (2.487 L, 14.24 mol) was added to a 50 L high and low temperature reaction kettle and stirred until the solid was completely dissolved.
  • the mixture was cooled to 0 to 5 ° C, and TBTU (2200.23 g, 6.85 mol) was added in portions, and the temperature was controlled to not exceed 20 ° C when added. After the addition, the temperature was raised to 20 to 25 ° C, and the mixture was stirred for 2 hours.
  • the reaction was stopped by HPLC, when the 1-J content was ⁇ 0.5%.
  • Ethyl acetate (3 L) and dilute HCl (0.25 N, 34 L) were successively added thereto at 20 to 25 ° C under stirring, and a large amount of solid was precipitated, and stirring was continued for 10 minutes.
  • Filtration the filter cake was washed with water (7 L ⁇ 2), the filter cake was added to a 50 L reaction kettle, and methanol (14 L) was added to be beaten, filtered with a centrifuge, and the filter cake was naturally dried for 3 days.
  • the filter cake was detected by HPLC, and the pulp was repeatedly beaten when the purity was ⁇ 98.0%.
  • Step 11 Synthesis of the crystalline form of the compound of formula (I)
  • the mixture was stirred at 40 to 50 ° C for 1 hour, and stirred at 20 to 25 ° C for 13.5 hours. Filtration, washing of the filter cake with methanol (500 mL) and drying of the filter cake in vacuo to give a crude compound of formula (I).
  • methanol (13L) and the compound of formula (I) (1680g) were added to a 50L high and low temperature reactor, stirred for about 3 hours, filtered, and the filter cake was washed with methanol (2 L) and dried under vacuum to give the formula ( I)
  • the crystalline form of the compound is a white powder.
  • Example 1 Stability test of the crystalline form of the compound of formula (I)
  • 100 mL of pure water and 100 mL of pure acetonitrile were mixed in a glass bottle, degassed by ultrasonic for 10 min, and cooled to room temperature.
  • Acetonitrile was taken as mobile phase B.
  • the reference solution STD-1 (0 day sample) was diluted 2000 times and recorded as LOQ.
  • RRT relative main peak retention time
  • TRS total impurity content
  • RH relative humidity
  • a crystal form of 10 mg of the compound of the formula (I) was accurately weighed and placed in a glass bottle, and 1 mL of a solvent was added thereto, and vigorously shaken at 25 ⁇ 2 ° C for 5 seconds every 5 minutes to observe the dissolution in 30 minutes. Record the corresponding data.
  • Step 2.1 Insoluble sample, accurately weigh 1mg into a glass bottle, add appropriate solvent, shake vigorously for 30 seconds at 25 ⁇ 2°C every 5 minutes, observe the dissolution phenomenon within 30 minutes. Record the corresponding data.
  • Table 6 shows the approximate solubility of the crystalline form of the compound of formula (I) in different solvents
  • Solvent Solubility classification Solvent Solubility classification Dimethyl sulfoxide Soluble water Almost insoluble or insoluble Methanol Slightly soluble 0.1N HCl Very slightly soluble N-methylpyrrolidone Soluble 0.1N NaOH Almost insoluble or insoluble Acetonitrile Almost insoluble or insoluble
  • Table 7 shows the wettability of the crystalline form of the compound of formula (I)
  • the wettability average value of the crystal form of the compound of the formula (I) is 0.07% ⁇ 0.2%, so that the crystal form of the compound of the formula (I) has no or almost no hygroscopicity.
  • Table 8 shows the results of infrared absorption spectrometry of the crystal form of the compound of formula (I)
  • s indicates a strong peak
  • m indicates a medium intensity peak
  • w indicates a weak peak
  • the structure of the product contains an amide, a benzene ring, an aromatic heterocycle, an alkenyl group, a -CH-, -CH 2 -, -CH 3 , an amine salt, a carboxylate, and a CF bond structure.
  • the above IR spectrum results are consistent with the crystal structure of the compound of formula (I).
  • ⁇ max 208 nm, which is the characteristic absorption of the E-band of the aromatic heterocyclic ring
  • Example 1 AKT enzyme activity test of the crystalline form of the compound of formula (I)
  • AKT also known as protein kinase B (PKB)
  • PKA protein kinase B
  • AKT signaling pathway plays an important role in various cellular processes such as glucose metabolism, apoptosis, cell proliferation transcription and cell migration. This study is intended to analyze the inhibition of the kinase activity of AKT family members by the crystal form of the compound of formula (I).
  • This experiment utilizes homogeneous time-resolved fluorescence conjugate energy transfer (LANCE
  • the enzyme, the biotin-labeled polypeptide substrate and the test compound are mixed, and ATP is added to initiate the reaction and incubate.
  • the reaction was terminated by the addition of EDTA, and an Eu-labeled antibody was simultaneously added to carry out the reaction and detection.
  • the assay plate was analyzed using PE company's Envision, and the analysis mode was TR-FRET.
  • the data were represented by readings of fluorescent signals of 665 nm and 615 nm, respectively. Among them, a high ratio of 665 nm / 615 nm indicates a higher activity, and a low ratio of 665 nm / 615 nm indicates that the activity is inhibited.
  • test compound preparation The test compound and the reference compound were diluted with 100% DMSO at a starting concentration of 10 ⁇ M, and a 10-fold gradient of 10 concentrations. The compound was transferred to a cell plate and a double duplicate well experiment was set up.
  • Aktl, Akt2, and Akt3 kinase assay The enzyme and substrate were mixed with different concentrations of pre-diluted compounds. The final concentrations of Aktl, Akt2, and Akt3 kinase were 1.25 nM, 2 nM, and 2 nM, respectively. Leave at room temperature for 15 minutes, double well at each concentration. ATP was added, and the final concentrations were 36 ⁇ M, 360 ⁇ M, and 160 ⁇ M in the Aktl, Akt2, and Akt3 kinase reactions, respectively, and the reaction was carried out for 90 minutes at room temperature (in which a negative positive control was set). After the reaction was completed, the antibody was added for detection. After incubation at room temperature for 60 minutes, Evnvision was detected and data was collected. Data analysis and mapping according to *Lfit5 software.
  • Example 2 Cellular activity assay of the crystalline form of the compound of formula (I)
  • This experiment is intended to investigate the inhibitory effect of the crystalline form of the compound of formula (I) on AKT overexpressing prostate cancer cell line LNCaP.
  • Cell culture RPMI-1640 medium, fetal bovine serum, Accutase, DPBS
  • Detection reagent live cell detection kit CellTiter-Glo
  • the amount of ATP directly reflects the number of cells and the state of the cells, and the number of living cells can be detected by quantitatively measuring ATP.
  • the live cell assay kit contains luciferase and its substrate. Through the participation of ATP, luciferase can catalyze the substrate, emit a stable optical signal, and determine the amount of ATP in the cell by detecting the intensity of the signal. The light signal is proportional to the amount of ATP in the cell, and ATP is positively correlated with the number of living cells, so that the cell proliferation can be detected.
  • the assay plate was analyzed using PE company's Envision.
  • LNCaP cells were separately seeded in 384-well plates containing 1000 cells per well. The cell plates were placed in a carbon dioxide incubator for overnight culture.
  • a double-fold experiment was performed using a 3 fold dilution with Bravo and 10 compound concentrations.
  • Compounds were transferred to cell plates at a starting concentration of 10 [mu]M.
  • the cell plates were incubated in a carbon dioxide incubator for 3 days.
  • the Promega CellTiter-Glo reagent was added to the cell plate and incubated for 10 minutes at room temperature to stabilize the luminescence signal. Readings were performed using a PerkinElmer Envision multi-label analyzer.

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Abstract

一种作为Akt抑制剂的盐型及其晶型，具体涉及式(I)化合物的盐型及其晶型。

Description

作为Akt抑制剂的盐型及其晶型
相关申请的交叉引用
本申请主张如下优先权：CN201711331448.1，申请日2017年12月13日。
技术领域
本发明涉及一种作为Akt抑制剂的盐型及其晶型，具体涉及式(I)化合物的盐型及其晶型，还包括所述盐型和晶型在制备治疗肿瘤、糖尿病和类风湿关节炎药物中的应用。
背景技术
Akt又被称为蛋白激酶B(protein kinase B,PKB)，是一种分子量约为60kDa的丝/苏氨酸蛋白激酶，它处于多条信号通路的重要交叉点,可调节细胞因子、生长因子和癌基因Ras激活的细胞生存信号，在真核生物的调控网络中普遍存在。Akt信号通路与恶性肿瘤、糖尿病、类风湿关节炎等多种疾病的发生发展密切相关,也因此越来越受关注。近年来，不断深入的研究使人们不仅对Akt生物学作用有了更深的了解，对Akt信号通路调控机制的研究也有了很多重大突破，它在人类一些疾病中发挥的具体作用及其分子调节机制正逐渐被阐明。
Akt由N末端的调节区、中间酶活性区、C末端的调节区和连接PH区与激酶活性区的铰链区四部分组成。N末端调节区有一个血小板-白细胞c激酶同源区即PH区，它在信号转导过程中的确切机制还不清楚；中间酶活性区具有催化丝/苏氨酸残基磷酸化活性，其中位于可变肽环(T环)中的Thr308位点的磷酸化是Akt活化所必需的；C末端有一个富含脯氨酸的疏水结构域(HM)，其中含有Akt完全活化所必需的第2个磷酸化位点Ser473。近年来，其各个结构域相应的晶体结构陆续得以解析，对关键磷酸化位点、ATP结合位点和蛋白质底物结合位点也有了更深刻的认识，为开发Akt/PKB特异性的小分子抑制剂奠定了基础。
目前在哺乳动物中发现至少3种Akt亚型:Aktl(PKBα)、Akt2(PKBβ)和Akt3(PKBγ)，分别由位于染色体14q32、19q13和1q43上的三个不同基因编码，有85％的序列同源性，同属AGC蛋白激酶家族。小鼠胚胎成纤维细胞研究发现，不同的Akt亚型缺陷株最后导致不同的表型：Akt1缺陷表现为胎盘营养不良、生长延迟和体重下降，Akt2缺陷表现为胰岛素和血糖的异常，Akt3缺陷呈现出大脑体积的缩小。在人类疾病中,Akt三种亚型的表达和作用也显著不同。
Akt能被细胞内多种物质如激素、生长因子、细胞因子、细胞间基质等刺激活化。Akt处于PI3K/Akt通路的中心环节，是PI3K的直接靶基因。许多细胞因子、生长因子和物理刺激等都可通过激活PI3K而使Akt磷酸化。通常情况下Akt磷酸化可作为衡量PI3K活性的指标。
对Akt生理功能的研究离不开对其底物的发现。到目前为止，已发现了100多种Akt的底物。Akt的三种亚型以及多样化的底物，是其发挥多种功能的结构基础。活化的Akt通过多种信号途径影响下游一系列效应分子的活化状态，在细胞内发挥着抑制凋亡、促进增殖等生物学效应。
Akt通路与许多信号途径相互交叉，形成复杂的信号网络。在特定情况下，Akt通路能激活NF-κB促生存信号或抑制JNK/p38凋亡信号，但关于p38与Akt之间的作用方式尚没有定论。无论是在不同的细胞类型、不同的细胞分化时期、不同的实验条件下，PI3K/Akt通路和Ras/MAPK通路之间的相互作用都会十分明显地表现在这两条通路的不同阶段，但Akt通路究竟对Ras/MAPK通路是正性调节还是负性调节作用 以及在某种细胞中何种调节方式占优势至今仍未完全地揭示出来。Akt信号通路与肿瘤、类风湿关节炎等疾病的发生发展密切相关，各通路间的相互联系和影响，可能共同对疾病的发生起重要作用。
参考文献：WO/2008/098104，WO2009/158371A1。
药物活性成分的不同固态形式可能具有不同的性质。不同固态形式在性质方面的改变可以提供改良配方，例如，易于合成或处理、提高稳定性和保质期。不同固态形式所导致的性质改变也可以改进最终的剂型。药物活性成分的不同固态形态还可以产生多晶或其他晶型，从而提供更多的机会来评估一个固体的活性药物成分的性质变化。
技术效果
本发明式(I)化合物的盐型及其晶型的制备工艺简单，并且所述晶型相对比较稳定、受热和湿度影响较小，便于制剂。
发明内容
一方面，本发明提供了式(I)化合物，
Figure PCTCN2018120531-appb-000001
另一方面，本发明提供了式(I)化合物的晶型，其X-射线粉末衍射图谱包含在2θ值为12.25°±0.2°、14.46°±0.2°和21.83°±0.2°的特征峰。
本发明的一些方案中，式(I)化合物的晶型的X-射线粉末衍射图谱包含在2θ值为8.50°±0.2°、10.20°±0.2°、12.25°±0.2°、14.46°±0.2°、15.30°±0.2°、21.83°±0.2°、27.31°±0.2°和31.41°±0.2°的特征峰。
本发明的一些方案中，式(I)化合物的晶型的X-射线粉末衍射图谱如图1所示。
本发明的一些方案中，式(I)化合物的晶型的X-射线粉末衍射图谱解析数据如表1所示。
表1式(I)化合物的晶型的XRPD解析数据
Figure PCTCN2018120531-appb-000002
Figure PCTCN2018120531-appb-000003
本发明的一些方案中，式(I)化合物的晶型的DSC曲线在195.10℃±3℃处有一个吸热峰的起始点，且在209.23℃±3℃有一个放热峰的起始点。
本发明的一些方案中，式(I)化合物的晶型的DSC曲线如图2所示。
本发明的一些方案中，式(I)化合物的晶型的TGA曲线在100.00℃±3℃时，失重为0.295％。
本发明的一些方案中，式(I)化合物的晶型的TGA曲线如图3所示。
本发明的一些方案中，式(I)化合物的晶型的红外光谱图包含在3316cm -1±5cm -1、3079cm -1±5cm -1、3058cm -1±5cm -1、3016cm -1±5cm -1、2954cm -1±5cm -1、2935cm -1±5cm -1、2915cm -1±5cm -1、2902cm -1±5cm -1、2864cm -1±5cm -1、1704cm -1±2cm -1、1633cm -1±2cm -1、1623cm -1±2cm -1、1589cm -1±2cm -1、1530cm -1±2cm -1、1514cm -1±2cm -1、1486cm -1±2cm -1、1445cm -1±2cm -1、1429cm -1±2cm -1、1408cm -1±2cm -1、1395cm -1±2cm -1、1373cm -1±2cm -1、1359cm -1±2cm -1、1345cm -1±2cm -1、1332cm -1±2cm -1、1282cm -1±2cm -1、1247cm -1±2cm -1、1214cm -1±2cm -1、1173cm -1±2cm -1、1135cm -1±2cm -1、1114cm -1±2cm -1、1054cm -1±2cm -1、1030cm -1±2cm -1、1000cm -1±2cm -1、952cm -1±2cm -1、914cm -1±2cm -1、902cm -1±2cm -1、883cm -1±2cm -1、861cm -1±2cm -1、839cm -1±2cm -1、825cm -1±2cm -1、789cm -1±2cm -1、763cm -1±2cm -1、736cm -1±2cm -1、720cm -1±2cm -1和704cm -1±2cm -1处的特征吸收峰。
本发明的一些方案中，式(I)化合物的晶型的红外光谱图如图4所示。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名时，旨在指代其对应的商品或其活性成分。
本发明的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词：
TBTU：O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸；DIEA：N,N-二异丙基乙基胺；DMF：N,N-二甲基甲酰胺；MeOH：甲醇；THF：四氢呋喃；Pd(dppf)Cl 2：[1,1'-双(二苯基膦基)二茂铁]二氯化钯；Pd 2(dba) 3：三(二亚苄基丙酮)二钯；x-PHOS：2-二环己基磷-2,4,6-三异丙基联苯
仪器及分析方法
1.1粉末X-射线衍射(X-ray powder diffractometer,XRPD)
仪器型号：布鲁克D8advance X-射线衍射仪
测试方法：大约10～20mg样品用于XRPD检测。
详细的XRPD参数如下：
光管：Cu,kα,
Figure PCTCN2018120531-appb-000004
光管电压：40kV，光管电流：40mA
发散狭缝：0.60mm
探测器狭缝：10.50mm
防散射狭缝：7.10mm
扫描范围：3或4-40deg
步径：0.02deg
步长：0.12秒
样品盘转速：15rpm
1.2差式扫描量热法(Differential Scanning Calorimeter,DSC)
仪器型号：TA DSC Q2000差示扫描量热仪
测试方法：取样品(0.5～1mg)置于DSC铝锅内进行测试，在50mL/min N 2条件下，以10℃/min的升温速率，加热样品从室温(25℃)到300℃或350℃。
1.3热重分析(Thermal Gravimetric Analyzer,TGA)
仪器型号：TA Q5000热重分析仪
测试方法：取样品(2～5mg)置于TGA铂金锅内进行测试，在25mL/min N 2条件下，以10℃/min的升温速率，加热样品从室温(25℃)到300℃，350℃或失重20％。
1.4动态气体吸附仪(DVS)
仪器型号：DVS Advantage-1(SMS)
测试条件：大约10-15mg样品用于DVS检测。
平衡dm/dt：0.01％/min：(时间：10min最大180min)
干燥：0％RH，120min
RH(％)测量梯度：10％
RH(％)测量梯度范围：0％～90％～0％
下列表2为引湿性的判断标准：
表2引湿性的判断标准
引湿性分类 引湿增重*
潮解 吸收足量水分形成液体
极具引湿性 引湿增重不小于15％
有引湿性 引湿增重小于15％但不小于2％
略有引湿性 引湿增重小于2％但不小于0.2％
无或几乎无引湿性 引湿增重小于0.2％
*在25℃/80％RH下的引湿增重
1.5高效液相色谱(High Performance Liquid Chromatograph,HPLC)
仪器型号：安捷伦1200高效液相色谱仪
分析方法如下：
表3有关物质含量测试的HPLC分析方法
Figure PCTCN2018120531-appb-000005
附图说明
图1为式(I)化合物的晶型的XRPD谱图。
图2为式(I)化合物的晶型的DSC谱图。
图3为式(I)化合物的晶型的TGA谱图。
图4为式(I)化合物的晶型的红外吸收光谱图。
图5为式(I)化合物的晶型的紫外吸收光谱图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
制备实施例
Figure PCTCN2018120531-appb-000006
步骤1：中间体1-B的合成
将化合物1-A(5.0Kg,60.9mol)溶于无水甲苯(7L)，所得溶液加入到50L反应釜中，随后依次将二氢吡喃(5.6Kg,60.9mol)和三氟乙酸(50mL)加入其中，将反应釜外温设定至80℃，搅拌反应过夜，HPLC检测反应完成，加入乙酸乙酯(10L)，水(2L)，搅拌后静置，分液，有机相用水(1L×2)洗涤，旋干得中间体1-B，为淡黄色油状液体。LCMS(ESI)m/z:167(M+1)； 1H NMR(400MHz,CDCl 3):δppm1.66-1.67(m,3H),2.02-2.08(m,6H),3.66-3.72(m,1H),4.03-4.07(m,1H),5.30-5.33(dd,J＝10.0,2.8Hz,1H),7.37-7.38(d,J＝6.0Hz,1H)。
步骤2：中间体1-C的合成
氮气保护下，将化合物1-B(2.0Kg,12.03mol)溶于无水四氢呋喃(12L)中，所得溶液加入到50L球形釜中，降温至-40℃～-20℃，滴加正丁基锂(5.3L,13.24mol)，滴加完毕，继续在此温度下搅拌反应1小时，随后滴加异丙基频那醇硼酸酯(2.351Kg,12.63mol)，滴加完毕，撤去冷却浴，继续搅拌反应40min， HPLC检测反应完成，滴加饱和氯化铵溶液(2L)淬灭，随后滴加6N HCl调节pH为5～6，加入水20L，加入乙酸乙酯(5L)萃取一次，随后再加入乙酸乙酯(3L)萃取一次，合并有机相，旋干得中间体1-C，为黄色固体。LCMS(ESI)m/z:293(M+1)； 1H NMR(400MHz,CDCl 3):δppm 1.356(s,12H),1.54-1.57(m,1H),1.70-1.72(m,2H),1.99-2.00(m,1H),2.08-2.09(m,1H),2.23(s,3H),2.45-2.51(m,1H),3.63-3.70(m,1H),4.02-4.06(m,1H),5.81-5.84(dd,J＝10.0,2.4Hz,1H),7.40(s,1H)。
步骤3：中间体1-D的合成
0～20℃，搅拌下，将1-C(3.75Kg,10.64mol)加入到氯化氢甲醇溶液(4N,16L,64mol)中20℃左右搅拌反应过夜，HPLC检测反应完毕，将反应液浓缩至无溶剂蒸出，往残物中加入4升石油醚，搅拌，静置后倒出上层清液，下层残物稍微旋干，固体析出，过滤，滤饼用1.5L乙酸乙酯打浆，过滤，滤饼再次用1.5L乙酸乙酯打浆，收集滤饼，合并滤液，滤液重复以上操作，再次收集滤饼，合并滤饼减压干燥得中间体1-D，为白色固体。LCMS(ESI)m/z:209(M+1)； 1H NMR(CD 3OD):δppm 1.22-1.42(d,12H),2.27-2.34(d,3H),8.03-7.14(d,1H)。
步骤4：中间体1-F的合成
20～25℃下，依次往50L反应釜中加入1-E(2502.05g,10.64mol)、DMF(12.5L)、三乙胺(2.24L,16.09mol)和Pd(dppf)Cl 2(84.00g,0.115mol)，随后将氮气鼓入反应液中，鼓气约10分钟，将丙炔醇(943mL,15.95mol)滴加到反应釜中，氮气保护下升温至60℃，保温反应1小时，随后升温至80℃，保温反应约6.5小时，HPLC检测反应完成，降温至室温，加入乙酸乙酯(20L)和盐酸(2N,1L)，搅拌后分液，水相再用乙酸乙酯(5L)萃取一次，合并的有机相加入水(5L×2)洗涤，旋干有机相，残余物用硅胶柱(石油醚至石油醚/乙酸乙酯＝3/1)洗脱，旋干洗脱液得中间体1-F，为黄色固体。LCMS(ESI)m/z:211(M+1)； 1H NMR(400MHz,CDCl 3):δppm 1.39(t,J＝7.15Hz,3H),1.78-1.88(m,1H),4.30-4.42(m,2H),4.50-4.59(m,2H),7.13-7.20(m,1H),7.61-7.70(m,1H)。
步骤5：中间体1-G的合成
将1-F(4039.55g,19.1mol)溶于24L乙醇(质量分数95％)中，加入三乙胺(20mL,0.144mol)，混匀，将此混合物均匀分置于20个2L氢化瓶中，氮气保护下将湿钯碳(419.89g，10％)均匀分置于以上氢化瓶中，置换氢气2次，设定氢气压力50PSI，温度25℃，反应约3小时，HPLC检测反应完成，过滤，合并滤液，旋干，残物加入0.1N盐酸(3L)和乙酸乙酯(6L)，搅拌后分液，有机相加入无水硫酸钠干燥，过滤，旋干有机相得中间体1-G，为黄色油状液体。LCMS(ESI)m/z:215(M+1)； 1H NMR(400MHz,CDCl 3):δppm 1.38(t,J＝7.15Hz,3H),1.90-2.06(m,2H),2.97(t,J＝7.59Hz,2H),3.73(s,2H),4.34(d,J＝7.03Hz,2H),6.81-6.89(m,1H),7.61-7.70(m,1H)。
步骤6：中间体1-H的合成
20～25℃下，依次往50L反应釜中加入二氯甲烷(19.6L)、1-G(3916.73g,18.3mol)和三氯化铁(296.83g,1.83mol)，随后在10℃以下，将液溴(1415mL,27.5mol)滴加到反应釜中，随后在15～25℃，反应过夜，HPLC检测反应完成，加入水(20L)，搅拌后分液，有机相加入饱和硫代硫酸钠溶液(20L)，搅拌分液，有机相加入水(20L)，搅拌分液，有机相再次加入水(20L)，搅拌分液，旋干有机相，残物用硅胶柱(石油醚至石油醚/乙酸乙酯＝3/1)洗脱，旋干洗脱液得中间体1-H，为黄色油状液体。LCMS(ESI)m/z:295(M+1)； 1H NMR(400MHz,CDCl 3):δppm 1.36(t, J＝7.15Hz,3H),1.88-2.00(m,2H),2.93(t,J＝7.59Hz, 2H),3.66-3.79(m,2H),4.33(d,J＝7.03Hz,2H),7.57-7.66(s,1H)。
步骤7：中间体1-I的合成
20～25℃下，依次往50L反应釜中加入1,4-二氧六环(26L)、水(5.2L)、1-H(3277.79g,11.18mol)、1-D(3300.00g,13.50mol)和无水碳酸氢钠(2751.12g,32.75mol)，随后将氮气鼓入反应液中，鼓气约15分钟，然后加入x-PHOS(1044.07g,2.19mol)和Pd 2(dba) 3(1000.00g,1.09mol)，继续鼓气约20分钟，氮气保护下，在110℃下反应过夜，HPLC检测反应完成，降温至室温，将反应液过滤，滤液加入乙酸乙酯(22L)和水(22L)，搅拌后分液，水相用乙酸乙酯(20L)萃取一次，合并的有机相旋干，残余物加入甲基叔丁基醚(20L)和2N盐酸(20L)，搅拌分液，有机相加入2N盐酸(20L)搅拌分液，合并水相用甲基叔丁基醚(5L)和石油醚(2.5L)混合液洗涤，水相在10～20℃下用饱和碳酸钠调节pH至7-8，大量固体析出，过滤，收集固体，将固体溶于乙醇(5L)中，加入活性炭(300g)，加热回流半小时，趁热过滤，滤液浓缩干，残物加入乙酸异丙酯(2L)，加热至60℃，打浆，冷却至室温，过滤，收集固体，加压干燥得中间体1-I，为淡黄色固体。LCMS(ESI)m/z:295(M+1)； 1H NMR(CDCl 3):δppm 1.39-1.42(m,3H),1.98-2.04(m,2H),2.17(s,2H),3.16-3.20(m,2H),3.64-3.67(m,2H),4.35-4.41(m,2H),7.43(s,1H),7.79(s,1H)。
步骤8：中间体1-J的合成
20～25℃下，依次往50L反应釜中加入二氯甲烷(18L)、1-I(1890.24g,6.42mol)、三乙胺(2700mL,19.26mol)和甲烷磺酰氯(1238mL,16.05mol)，搅拌反应约0.5小时，HPLC检测反应完毕，加入4.5L饱和氯化铵溶液，搅拌分液，水相加入4.5L二氯甲烷萃取，合并有机相，无水硫酸钠干燥，过滤，旋干有机相，残余物转移到50L反应釜中，接着加入无水乙醇(18L)、碘化钾(213.34g,1.28mol)和甲醇钠(1041.01g,19.28mol)，升温至90℃，反应过夜，HPLC检测反应完毕，降温至50～60℃，加入水(18L)和氢氧化钠固体(771.12g,19.28mol)，搅拌反应约半小时，HPLC检测反应完毕，减压浓缩至20L左右，加入甲基叔丁基醚(9L)，搅拌分液，水相加入甲基叔丁基醚(9L)搅拌分液，水相加入浓盐酸调节pH至3～4，大量固体析出，过滤，收集固体，滤饼依次用水(1L×2)和丙酮(2.25L×2)洗涤，过滤，减压干燥得中间体1-J，为淡黄色固体。LCMS(ESI)m/z:249(M+1)； 1H NMR(400MHz,DMSO-d 6):δppm 2.14(s,3H),2.17-2.21(m,2H),3.07-3.11(m,2H),4.23-4.25(m,2H),7.28(s,1H)7.80(s,1H)。
步骤9：中间体1-L的合成
20～25℃下，依次将N,N-二甲基甲酰胺(11.3L)，1-J(1417.92g,5.69mol)，1-K(2007.29g,5.92mol)，以及N,N-二异丙基乙基胺(2.487L,14.24mol)加入50L高低温反应釜，搅拌至固体完全溶解。混合液冷却至0～5℃，将TBTU(2200.23g,6.85mol)分批加入，加入时控温不超过20℃。加毕，升温至20～25℃，保温搅拌2小时。HPLC监测，当1-J含量≤0.5％时，停止反应。搅拌下，于20～25℃依次加入乙酸乙酯(3L)和稀HCl(0.25N,34L)，大量固体析出，继续保温搅拌10分钟。过滤，滤饼用水(7L×2)洗涤，滤饼加入50L反应釜，加入甲醇(14L)打浆，用离心机过滤，滤饼自然晾干3天。HPLC检测滤饼，当纯度≤98.0％时重复打浆，滤饼加入50L反应釜，加入甲醇(30L)打浆，用离心机过滤，滤饼真空干燥得中间体1-L，为白色固体。LCMS(ESI)m/z:529(M+1)； 1H NMR(CDCl 3):δppm 2.21(s,3H),2.24-2.28(m,2H),2.91-2.93(m,1H),2.98-3.02(m,2H),3.06-3.07(m,1H),3.76-3.78(m,1H),3.83-3.84(m,1H),4.21-4.24(m,2H),4.51-4.52(m,1H),6.82-6.93(m,2H),6.97-7.01(m,1H),7.04-7.08(m,1H),7.21-7.25(m,1H),7.48(s,1H), 7.67-7.68(m,2H),7.76-7.78(dd,J＝5.2,3.0Hz,2H)。
步骤10：中间体1-M的合成
20～25℃，依次将甲醇(20L)和1-L(2233.25g,4.19mol)加入50L高低温反应釜中，搅拌，加入98％水合肼(1050mL，98％，24.54mol)，升温至80～90℃，回流反应1.7小时，大量固体析出。HPLC监测，当1-L含量≤0.5％时，停止反应。趁热过滤，滤液冷却至20～25℃，大量固体析出，过滤，滤饼用甲醇(5L)洗涤。滤液浓缩至原体积1/5左右(约5L)后，搅拌下将20L水缓慢倾倒入上述浓缩液中，大量固体析出，过滤，滤饼用水(5L)洗涤。20～25℃，将稀盐酸(1N,20L)加入滤饼中，搅拌，过滤，滤饼用水(5L)洗涤。滤液转入50L分液器，用乙酸乙酯和甲醇混合溶剂(10/1(v/v)，11L×2)反萃。水相降温至0～5℃，搅拌下，用碳酸氢钠固体调节pH至8～9，大量固体析出，过滤，滤饼用水(2L)洗涤，真空干燥得到中间体1-M为白色固体。LCMS(ESI)m/z:399(M+1)； 1H NMR(CD 3OD):δppm 2.23(s,3H),2.24-2.34(m,2H),2.89-2.98(m,4H),3.06-3.09(m,2H),4.23-4.25(m,2H),4.25-4.35(m,1H),6.93-7.02(m,1H),7.04-7.11(m,2H),7.27-7.29(m,1H),7.33(s,1H),7.83(s,1H)。
步骤11：式(I)化合物的晶型的合成
20～25℃下，依次将甲醇(13.2L)和1-M(1654.31g,3.93mol)加入50L高低温反应釜，升温至80～90℃，保温搅拌0.5小时。趁热过滤，滤液再次转入50L高低温反应釜中，升温至80～90℃，加入马来酸甲醇溶液(马来酸505.94g溶解于1L甲醇中,4.36mol)，保温搅拌1小时。梯度降温，外温60～70℃保温搅拌2小时，固体析出。40～50℃保温搅拌1小时，20～25℃搅拌13.5小时。过滤，用甲醇(500mL)洗涤滤饼，滤饼真空干燥得式(I)化合物的粗品。20～25℃下，将甲醇(13L)和式(I)化合物(1680g)加入50L高低温反应釜中，保温搅拌约3小时，过滤，滤饼用甲醇(2L)洗涤，真空干燥得到式(I)化合物的晶型为白色粉末。LCMS(ESI)m/z:399(M+1)； 1H NMR(CD 3OD):δppm 2.23(s,3H),2.28-2.39(m,2H),2.97-3.13(m,4H),3.13-3.29(m,2H),4.25(br d,J＝6.0Hz,2H),4.47-4.59(m,1H),6.25(s,2H),6.92-7.02(m,1H),7.03-7.18(m,2H),7.33(s,2H),7.80(s,1H)。
表征实施例
实施例1：式(I)化合物的晶型的稳定性试验
1.1实验步骤
准确称重式(I)化合物的晶型约10mg置于干燥洁净的玻璃瓶中，摊成薄薄一层，作为正式供试样品，放置于加速试验条件下(40℃/75％RH和60℃/75％RH)，其样品为完全暴露放样，用铝箔纸盖上，扎上小孔。试验在10天，1个月，2个月取样分析。结果见表4。
1.2稀释剂及流动相的制备
稀释剂：乙腈：水(1:1)作为稀释剂
如：取100mL纯水和100mL纯乙腈相混合于玻璃瓶中，超声脱气10min，冷至室温。
流动相A：0.1％磷酸水溶液
如：量取2.0mL磷酸加入到2000mL水中，超声10分钟，混匀，放冷至室温，作为流动相A。
流动相B：乙腈
取乙腈作为流动相B。
1.3对照品溶液的制备(0.25mg/mL,以游离碱计)
以0天的样品为对照品样品，加入稀释剂16mL，超声5min，混合均匀。再稀释一倍，分别标记为STD-1和STD-2。
1.4灵敏度样品溶液的制备
取对照品溶液STD-1(0天的样品)稀释2000倍，记作LOQ。
1.5样品溶液的制备(0.25mg/mL,以游离碱计)
取出样品后，恢复至室温后加入16mL稀释剂溶解。再稀释一倍作为检测有关物质含量的样品溶液。
表4式(I)化合物的晶型的稳定性试验
Figure PCTCN2018120531-appb-000007
RRT：相对主峰保留时间；TRS：总杂质含量；RH：相对湿度。
从实验结果可以看出，该式(I)化合物的晶型在高温和高湿条件下均稳定，杂质含量基本无变化。
实施例2：式(I)化合物的晶型的溶解度实验
2.1实验步骤
精密称取10mg式(I)化合物的晶型放入玻璃瓶，加入1mL溶剂，于25±2℃下，每隔5分钟强力振摇30秒，观察30分钟内的溶解现象。记录相应数据。
步骤2.1不溶解的样品，精密称取1mg放入玻璃瓶，加入适当溶剂，于25±2℃下，每隔5分钟强力振摇30秒，观察30分钟内的溶解现象。记录相应数据。
表5溶解度判断标准
Figure PCTCN2018120531-appb-000008
表6式(I)化合物的晶型在不同溶剂中的大致溶解度情况
溶剂 溶解度分类 溶剂 溶解度分类
二甲亚砜 易溶 水 几乎不溶或不溶
甲醇 微溶 0.1N HCl 极微溶解
N-甲基吡咯烷酮 易溶 0.1N NaOH 几乎不溶或不溶
乙腈 几乎不溶或不溶    
从实验结果可以看出，式(I)化合物的晶型在二甲基亚砜或N-甲基吡咯烷酮中易溶，在甲醇中微溶，在乙腈或水中几乎不溶或不溶；在0.1N盐酸或0.1N氢氧化钠溶液中几乎不溶或不溶。
实施例3：式(I)化合物的晶型的引湿性试验
实验过程
取3只干燥的具塞玻璃称量瓶(外径为50mm,高为30mm)置于下部放置氯化铵饱和溶液的干燥器内，称量瓶敞口放置，盖好干燥器盖子，然后将干燥器置于25℃的恒温箱内，放置过夜。
称量瓶放置过夜后取出精密称定重量，分别为m 1 1,m 1 2,m 1 3
取式(I)化合物的晶型适量，平铺于已称定重量的称量瓶中(样品厚度约为1mm)，然后精密称量重量，分别为m 2 1,m 2 2,m 2 3。
将称量瓶敞口放置，并于瓶盖一起置于下部放置氯化铵饱和溶液的干燥器内，称量瓶敞口放置，盖好干燥器盖子，然后将干燥器置于25℃的恒温箱内，放置24小时。
放置24小时后，盖好称量瓶盖，然后取出精密称定重量，分别记为m 3 1,m 3 2,m 3 3。
引湿性增重计算，计算公式如下：
增重百分率＝100％×(m 3-m 2)/(m 2-m 1)
表7式(I)化合物的晶型的引湿情况表
Figure PCTCN2018120531-appb-000009
根据引湿性测试结果，式(I)化合物的晶型的引湿平均值0.07％<0.2％，故式(I)化合物的晶型无或几乎无引湿性。
实施例4：式(I)化合物的晶型的红外吸收试验
仪器：Nicolet 6700FT-IR型红外光谱仪
方法：ATR衰减全反射法
式(I)化合物的晶型样品的红外吸收光谱图见附图4。
表8式(I)化合物的晶型的红外吸收光谱测定结果
Figure PCTCN2018120531-appb-000010
Figure PCTCN2018120531-appb-000011
s：表示强峰；m：表示中等强度峰；w：表示弱峰。
红外光谱解析：
1)3316cm -1：N-H伸缩振动；1704～1623cm -1：C＝O伸缩振动；1359～1000cm -1：C-N伸缩振动；952～704cm -1：N-H面外弯曲振动；证明本品结构中含有酰胺；
2)3079，3058，3016cm -1：＝C-H伸缩振动；1704～1445cm -1：C＝C和C＝N伸缩振动；1359～1000cm -1：C-H面内弯曲振动；952～704cm -1：＝C-H面外弯曲振动；证明本品结构中含有苯环，芳杂环，烯基；
3)2954～2864cm -1：C-H对称和不对称伸缩振动；1486～1373cm -1：C-H变形振动；证明本品结构中含有-CH-、-CH 2-和-CH 3的结构；
4)3316～2864cm -1：NH 3 +的伸缩振动；1359～1000cm -1：C-N伸缩振动；952～704cm -1：N-H伸缩振动；证明本品结构中含有胺盐；1633～1530cm -1：羧酸盐的反对称伸缩振动；1445～1395cm -1：羧酸盐的对称伸缩振动；本品结构中含有羧酸盐；
5)1359～1000cm -1：C-F伸缩振动；证明本品中含有C-F键；
误差范围符合《中国药典》的规定，即3000cm -1附近可存在±5cm -1的误差容限，1000cm -1附近可存在±2cm -1的误差容限。
由红外光谱可知，本品结构中含酰胺、苯环、芳杂环、烯基、-CH-、-CH 2-、-CH 3、胺盐、羧酸盐和C-F键结构等。上述IR光谱结果与式(I)化合物的晶型结构相符。
实施例5：式(I)化合物的晶型的紫外吸收试验
仪器：Agilent 8453紫外可见光分光光度计
溶剂：甲醇/纯水＝1/1(v/v)
母液：称取式(I)化合物的晶型的样品10.198mg至100mL容量瓶中，用稀释剂(甲醇/纯水＝1/1(v/v))充分溶解并定容至刻度，摇匀。
供试液：移取母液2.2mL于10mL容量瓶中，用稀释剂(甲醇/纯水＝1/1(v/v))定容至刻度，摇匀，即得浓度为4.3601×10 -5mol/L的样品溶液。
测定波长：200～700nm
样品在甲醇中的紫外光谱图见附图5。
表9式(I)化合物的晶型的紫外吸收光谱测定结果
Figure PCTCN2018120531-appb-000012
在甲醇溶液中，λ max＝208nm，为芳杂环的E带特征吸收，λ max＝248nm，为芳环的B带特征吸收，λ max＝300nm为芳环的K带特征吸收。
活性测试
实施例1：式(I)化合物的晶型的AKT酶活性测试
AKT，亦被称为蛋白激酶B(PKB)，是一种丝氨酸/苏氨酸特异性蛋白激酶，主要包括AKT1，AKT2及AKT3。AKT信号通路在葡萄糖代谢、凋亡、细胞增殖转录及细胞迁移等多种细胞过程中起到重要的作用。本研究拟分析式(I)化合物的晶型对AKT家族成员激酶活性的抑制作用。
试剂和耗材:
ULight-CREBtide(PerkinElmer TRF0107-M,批次2035700)
Europium-Anti-P-CREB(Ser-133)(PerkinElmer TRF0200-M,批次2002391)
LANCE Detection Buffer,10*(PerkinElmer CR97-100)
ATP(Invitrogen PV3227)
Akt1protein(life technology批次11629928C)
Akt2protein(life technology批次28870GG)
Akt3protein(life technology批次1715050B)
384孔板_检测板(PerkinElmer 6007299)
实验原理:
本实验利用均相时间分辨的荧光共轭能量转移(LANCE
Figure PCTCN2018120531-appb-000013
)方法，在检测板中，将酶、生物素标记的多肽底物及检测化合物混合，加入ATP启动反应并孵育。反应后，加入EDTA终止反应，并同时加入Eu标记的抗体进行反应并检测。检测板使用PE公司的Envision进行分析，分析模式为TR-FRET，数据分别用荧光信号665nm和615nm的读数来表示。其中665nm/615nm的高比值表示活性较高，而665nm/615nm的低比值则表示活性受到抑制。
实验方法:
化合物配制：将受试化合物及参考化合物用100％DMSO稀释，化合物起始浓度为10μM，3倍梯度稀释10个浓度。将化合物转移到细胞板中，设置双复孔实验。
激酶检测：将酶和底物与预先稀释配制的不同浓度化合物混合，Akt1、Akt2、Akt3激酶终浓度分别为1.25nM、2nM及2nM。室温放置15分钟，每个浓度双复孔。加入ATP，在Akt1、Akt2、Akt3激酶反应中，终浓度分别为36μM、360μM及160μM，室温反应90分钟(其中设置阴阳性对照)。反应完毕加入抗体检测，室温孵育60分钟后Evnvision检测，采集数据。根据*Lfit5软件进行数据分析及拟图。
实验结果:见表10。
表10式(I)化合物的晶型对激酶活性抑制的IC 50值
Figure PCTCN2018120531-appb-000014
本实验结果显示：式(I)化合物的晶型对AKT1、AKT2和AKT3激酶活性均具有较强的抑制作用，其中对AKT1激酶活性的抑制作用最显著。
实施例2：式(I)化合物的晶型的细胞活性测试
本实验拟研究式(I)化合物的晶型对AKT过度表达的前列腺癌细胞LNCaP的抑制作用。
试剂和耗材:
1.细胞培养：RPMI-1640培养基、胎牛血清、Accutase、DPBS
2.细胞系：LNCaP
3.检测试剂：活细胞检测试剂盒CellTiter-Glo
4.其他主要耗材及试剂：化合物稀释板，中间板，检测板，DMSO
实验原理:
ATP的含量直接反应了细胞的数量及细胞状态，通过对ATP进行定量测定可以检测活细胞的数目。活细胞检测试剂盒含有萤光素酶及其底物，通过ATP的参与，荧光素酶可以催化底物，发出稳定的光学信号，通过检测信号的强度来测定细胞中ATP的数量。其中光信号和细胞中ATP量成正比，而ATP又和活细胞数正相关，从而可以检测出细胞的增殖情况。检测板使用PE公司的Envision进行分析。
实验方法:
1.制备细胞板
将LNCaP细胞分别种于384孔板中，每孔包含1000个细胞。细胞板置于二氧化碳培养箱中过夜培养。
2.准备化合物
用Bravo进行3倍稀释，10个化合物浓度，设置双复孔实验。
3.化合物处理细胞
将化合物转移到细胞板中，化合物起始浓度为10μM。细胞板置于二氧化碳培养箱中培养3天。
4.检测
向细胞板中加入Promega CellTiter-Glo试剂，室温孵育10分钟使发光信号稳定。采用PerkinElmer Envision多标记分析仪读数。
实验结果:见表11。
表11式(I)化合物的晶型对细胞增殖抑制的IC 50值
Figure PCTCN2018120531-appb-000015
本实验结果显示：式(I)化合物的晶型对AKT过度表达的细胞LNCaP的生长有明显的抑制效果。

Claims (10)

  1. 式(I)化合物，
    Figure PCTCN2018120531-appb-100001
  2. 式(I)化合物的晶型，其X-射线粉末衍射图谱包含在2θ值为12.25°±0.2°、14.46°±0.2°和21.83°±0.2°的特征峰。
  3. 根据权利要求2所述的晶型，其X-射线粉末衍射图谱包含在2θ值为8.50°±0.2°、10.20°±0.2°、12.25°±0.2°、14.46°±0.2°、15.30°±0.2°、21.83°±0.2°、27.31°±0.2°和31.41°±0.2°的特征峰。
  4. 根据权利要求3所述的晶型，其X-射线粉末衍射图谱如图1所示。
  5. 根据权利要求2～4任一项所述的晶型，其中所述晶型的DSC曲线在195.10℃±3℃处有一个吸热峰的起始点，且在209.23℃±3℃有一个放热峰的起始点。
  6. 根据权利要求5所述的晶型，其中所述晶型的DSC曲线如图2所示。
  7. 根据权利要求2～4任一项所述的晶型，其中所述晶型的TGA曲线在100.00℃±3℃时，失重为0.295％。
  8. 根据权利要求7所述的晶型，其中所述晶型的TGA曲线如图3所示。
  9. 根据权利要求2～4任一项所述的晶型，其中所述晶型的红外光谱图包含在3316cm -1±5cm -1、3079cm -1±5cm -1、3058cm -1±5cm -1、3016cm -1±5cm -1、2954cm -1±5cm -1、2935cm -1±5cm -1、2915cm -1±5cm -1、2902cm -1±5cm -1、2864cm -1±5cm -1、1704cm -1±2cm -1、1633cm -1±2cm -1、1623cm -1±2cm -1、1589cm -1±2cm -1、1530cm -1±2cm -1、1514cm -1±2cm -1、1486cm -1±2cm -1、1445cm -1±2cm -1、1429cm -1±2cm -1、1408cm -1±2cm -1、1395cm -1±2cm -1、1373cm -1±2cm -1、1359cm -1±2cm -1、1345cm -1±2cm -1、1332cm -1±2cm -1、1282cm -1±2cm -1、1247cm -1±2cm -1、1214cm -1±2cm -1、1173cm -1±2cm -1、1135cm -1±2cm -1、1114cm -1±2cm -1、1054cm -1±2cm -1、1030cm -1±2cm -1、1000cm -1±2cm -1、952cm -1±2cm -1、914cm -1±2cm -1、902cm -1±2cm -1、883cm -1±2cm -1、861cm -1±2cm -1、839cm -1±2cm -1、825cm -1±2cm -1、789cm -1±2cm -1、763cm -1±2cm -1、736cm -1±2cm -1、720cm -1±2cm -1和704cm -1±2cm -1处的特征吸收峰。
  10. 根据权利要求9所述的晶型，其红外光谱图如图4所示。
PCT/CN2018/120531 2017-12-13 2018-12-12 作为Akt抑制剂的盐型及其晶型 Ceased WO2019114741A1 (zh)

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