WO2017050224A1 - Salt form and crystal form of quinoline derivative, preparation method therefor, and intermediate - Google Patents

Salt form and crystal form of quinoline derivative, preparation method therefor, and intermediate Download PDF

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WO2017050224A1
WO2017050224A1 PCT/CN2016/099537 CN2016099537W WO2017050224A1 WO 2017050224 A1 WO2017050224 A1 WO 2017050224A1 CN 2016099537 W CN2016099537 W CN 2016099537W WO 2017050224 A1 WO2017050224 A1 WO 2017050224A1
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compound
crystal form
solvent
crystalline form
crystal
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PCT/CN2016/099537
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French (fr)
Chinese (zh)
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吴颢
龙超峰
林军
陈小新
廖勇刚
刘卓伟
韦昌青
陈俐娟
陈曙辉
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广东众生药业股份有限公司
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Priority to CN201680054765.0A priority Critical patent/CN108290859B/en
Publication of WO2017050224A1 publication Critical patent/WO2017050224A1/en

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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings

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  • the present invention relates to a crystalline form, a salt form, a salt form of a quinoline derivative, and a process for the preparation thereof.
  • the Hedgehog protein was originally a secreted signaling protein found in Drosophila, a highly hydrophobic protein that plays a crucial role in embryonic development.
  • Three homologous hedgehog proteins have been identified in humans, namely Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh).
  • Shh is not only important in embryonic development, but also has many evidences that it plays an important role in the carcinogenic mechanisms of some cancers including basal cell carcinoma (Caro, I. and Jalow, Clin Cancer Res, 2010. 16(13): 3335 -9).
  • Shh synthesizes a 45kDa precursor protein in vivo and produces a 20kDa N-terminal fragment by self-resection.
  • This N-terminal fragment has all the biological activities known in the body, including activation of intracellular hedgehog information pathway.
  • the major members of the pathway include patched (PTCH), G-like protein and receptor oncogene smoothened (SMO), and transcription factor Gli (Bale, AE and KPYu, Hum Mol Genet, 2001.10(7): 757-62) .
  • PTCH-1 is a membrane protein with 12 transmembrane structures that is a direct acting receptor for Shh.
  • PTCH-1 In the absence of Shh, PTCH-1 interacts with SMO, which is a 7-transmembrane structural protein that inhibits the biological viability of SMO.
  • SMO which is a 7-transmembrane structural protein that inhibits the biological viability of SMO.
  • the combination of Shh and PTCH-1 causes PTCH-1 to detach from SMO, freeing SMO from the suppressed state.
  • the Gli transcription factor is controlled by SMO, which acts as a switch for gene transcription, with the major members including Glil, Gli2 and Gli3.
  • the entire hedgehog pathway plays an important role in the normal development of the embryo. Disturbing this information pathway will lead to severe malformations, such as the natural teratogenic compound cyclopamine is a hedgehog inhibitor. Under normal conditions, the concentration of hedgehog protein in adults is very low.
  • PTCH-1 In the case of a low concentration of hedgehog protein, PTCH-1 binds to SMO and inhibits its biological activity, so the entire pathway is in an inactive or low-activity state.
  • the binding of the hedgehog protein to the PTCH-1 receptor causes it to detach from the SMO, thereby losing the inhibition of SMO.
  • SMO further activates the transcription factor Gli-1 to regulate gene transcription and cell growth.
  • the cause of most basal cell carcinomas is the excessive activity of the hedgehog signaling pathway due to mutations or other causes. Therefore, the activity of the excessively high hedgehog information transduction pathway is inhibited, and the growth of cancer cells can be inhibited to achieve treatment of basal cell carcinoma or other cancers caused by the same mechanism.
  • SMO inhibitors already exist in the prior art, they need to be improved in terms of activity, solubility, pharmacokinetics, drug-forming properties and the like.
  • CN201410110890.1 describes a new class of quinoline derivatives which act as inhibitors of the hedgehog pathway, particularly as SMO inhibitors. It can be used to treat conditions related to the hedgehog pathway including cancer.
  • the present invention provides Form A of Compound 1, the XRPD pattern of which is shown in Figure 1.
  • the A crystal form XRPD analytical data is shown in Table 1.
  • the A crystalline form of Compound 1 above has a differential scanning calorimetry curve having an end point of endothermic peak at 111.41 °C ⁇ 2 °C, 126.08 °C ⁇ 2 °C, and 146.06 °C ⁇ 2 °C.
  • the A crystal form of the above compound 1 has a DSC pattern as shown in FIG.
  • the A crystal form of the above compound 1 has a thermogravimetric analysis curve weight loss of 0.7658% ⁇ 0.2% at 135.65 ⁇ 2 °C.
  • the T crystal of Form A of Compound 1 above is shown in Figure 3.
  • the present invention also provides Form B of Compound 1, the XRPD pattern of which is shown in Figure 4.
  • the B-form XRPD analytical data is shown in Table 2.
  • the B crystal form of Compound 1 above has a differential scanning calorimetry curve having a starting point of endothermic peak at 165.73 °C ⁇ 2 °C.
  • the B crystal form of the above Compound 1 has a DSC pattern as shown in FIG.
  • the B crystal form of the above compound 1 has a thermogravimetric analysis curve having a weight loss of 0.2383% ⁇ 0.2% at 120.00 ⁇ 2 ° C and a loss of 0.2005% ⁇ 0.2% at 247.60 ⁇ 2 ° C.
  • the T crystal of Form B of Compound 1 above is shown in Figure 6.
  • the invention also provides a preparation method of the B crystal form, which comprises adding the crystal form of the compound A to the solvent of the ester solvent, the solvent of the alcohol or the solvent of the ester and the solvent of the alcohol solvent, heating to reflux to dissolve, and then slowly Cooled and crystallized.
  • the ester is dissolved in the group consisting of propyl formate, isopropyl formate, butyl formate, isobutyl formate, ethyl acetate, isopropyl acetate, and isopropyl acetate.
  • the alcohol solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, and tert-butanol.
  • the volume ratio of the ester solvent to the alcohol solvent is from 0.1:1 to 12.
  • the volume ratio of the ester solvent to the alcohol solvent is 1:2 to 8.
  • the present invention provides a compound 2 of the following formula,
  • the present invention provides a crystalline form C of Compound 2, the XRPD pattern of which is shown in FIG.
  • the C-form XRPD pattern analysis data is shown in Table 3.
  • the C crystal form of Compound 2 above has a differential scanning calorimetry curve having an end point of endothermic peak at 57.33 ° C ⁇ 5 ° C and 212.56 ° C ⁇ 5 ° C.
  • the C crystal form of the above Compound 2 has a DSC spectrum as shown in FIG.
  • the C crystal form of the above compound 2 has a thermogravimetric analysis curve having a weight loss of 5.10% ⁇ 0.5% at 120.00 ⁇ 3 °C.
  • the C crystal form of the above compound 2 has a TGA pattern as shown in FIG.
  • the invention provides a preparation method of the C crystal form, which comprises the compound 1 dissolved in an organic solvent, slowly adding a sulfuric acid aqueous solution, and stirring and crystallization.
  • the organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, and cyclohexanone.
  • the molar ratio of Compound 1 to sulfuric acid is selected from 1:2 to 4.
  • the invention also provides the use of the above A crystal form, B crystal form, compound 2 and C crystal form in the preparation of a medicament for treating diseases related to SMO receptors.
  • 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.
  • DCM dichloromethane
  • PE petroleum ether
  • EA ethyl acetate
  • DMF N,N-dimethylformamide
  • DMAC N,N-dimethylacetamide
  • DMSO dimethyl sulfoxide
  • EtOAc ethyl acetate
  • tol stands for toluene
  • THF tetrahydrofuran
  • EtOH stands for ethanol
  • MeOH stands for methanol
  • NMP stands for N-methylpyrrolidone
  • 2-METHF stands for 2-methyltetrahydrofuran
  • Bn stands for benzyl
  • Cbz stands for benzyloxycarbonyl and is an amine protecting group
  • Boc stands for t-butylcarbonyl which is an amine protecting group
  • Fmoc fluorenylmethoxycarbonyl and is an amine a protecting group
  • XRPD X-ray powder diffractometer
  • Tube voltage 40kV
  • tube current 40mA
  • DSC Differential Scanning Calorimeter
  • Test conditions The sample ( ⁇ 1 mg) was placed in a DSC aluminum pan for testing at 25 ° C - 350 ° C and a heating rate of 10 ° C / min.
  • TGA Thermal Gravimetric Analyzer
  • Test conditions Samples (2 to 5 mg) were placed in a TGA platinum pot for testing at room temperature - 350 ° C and a heating rate of 10 ° C / min.
  • Figure 1 is an XRPD spectrum of Cu-K ⁇ radiation of Form A.
  • Figure 2 is a DSC pattern of Form A.
  • Figure 3 is a TGA pattern of Form A.
  • Figure 4 is an XRPD spectrum of Cu-K ⁇ radiation of Form B.
  • Figure 5 is a DSC pattern of Form B.
  • Figure 6 is a TGA pattern of Form B.
  • Figure 7 is an XRPD spectrum of Cu-K ⁇ radiation of Form C.
  • Figure 8 is a DSC pattern of Form C.
  • Figure 9 is a TGA pattern of Form C.
  • the third step 8-bromo-4-hydroxyquinoline (2 kg, 8.93 mol), (4-cyanophenyl)boronic acid (1.31 kg, 8.93 mol), Pd(dppf)Cl 2 (130.68 g, 178.60 m
  • tetrahydrofuran 10 L
  • DMF 2 L
  • water 2 L
  • the mixture was heated to 78 °C. After 3 hours, 4 liters of water was added and the mixture was filtered.
  • Step 4 In a 30 L reactor, add DMF (10 L) and 4-(4-hydroxyquinolin-8-yl)benzonitrile (2.2 kg, 8.93 mol), stir at room temperature, then slowly in 5 minutes. NIS (2.01 kg, 8.93 mol) was added. After half an hour, the samples were detected by LCMS and the reaction was completed. Add 20 L of ice water and stir and filter. The solid was washed with water (5 L x 3). The product was dried in a vacuum oven at 50 ° C for three days to give 4-(4-hydroxy-3-iodo-8-quinolinyl)benzonitrile (2.55 kg, yield 76.73%) as a yellow solid.
  • Step 6 4-(4-Bromo-3-iodo-8-quinoline)benzonitrile (3.5 kg, 8.04 mol), (2S,6R)-2,6-dimethyl-4-(5(5) 4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]morpholine (3.07 kg, 9.65 mol) in tetrahydrofuran (30.00 L) Heat to 65 ° C. After all the reactants were dissolved, add Na 2 CO 3 (1.70 kg, 16.08 mol) and Pd (dppf) Cl 2 (294.14 g, 402.00 mmol, 0.05 eq), then add DMF (5 L) and water (L). 4) mixture.
  • Step 7 4-(4-Bromo-3-(6-((2S,6R)-2,6-dimethylmorphinolin-4-yl)-3-pyridyl)-8-quinoline)
  • the benzonitrile (2.68 kg, 5.37 mol) solvent was heated to 60 ° C in toluene (25 L) to be completely dissolved.
  • the free base compound 1 (1.19 kg, 2.58 mol, 1 eq.) was dissolved in acetone (12 L), sulfuric acid (3M, 1.72 liters, 2 eq) was slowly added for about 30 minutes, and then the mixture was stirred at 25 ° C for 1.5 hours. The mixture was filtered, dried and dried under vacuum at 35 ° C.
  • the target product salt (1.56 kg, yield 92.07%) in 48 hours was a yellow solid, which was crystal form.
  • the prepared samples were placed at 60 ° C, 92.5% RH (relative humidity), 25 ° C / 60% RH and 40 ° C / 75% RH, respectively, and the physical stability of the samples was examined for 10 days. On day 10, all samples were taken, returned to room temperature, the appearance change of the samples was observed, and the crystal form of the samples was examined by XRPD. The solid physical stability of the C crystal form was judged by comparison with the crystal form of the 0 day sample. Table 8 shows the results of solid physical stability experiments of Form C.
  • the luciferase (Gli-Luc) reporter gene with Gli response element was stably transfected into C3H10T1/2 cells, and the expression of Hedgehog signaling pathway inhibitor was determined by detecting the expression of intracellular reporter gene under Shh-N stimulation conditions. active.
  • the IC50 value of the compound was used as an indicator to evaluate the inhibitory effect of the compound on the Hedgehog signaling pathway.
  • C3H10T1/2/Gli-Luc cell culture medium (DMEM/high glucose HyClone, #SH30022.01B; 10% serum Hyclone, #SV30087;
  • the logarithmic growth phase C3H10T1/2/Gli-Luc cells were plated in a 96-well plate at 20,000 cells per well, and the cells were cultured overnight at 37 ° C in a 5% CO 2 incubator. On the next day, the compounds were diluted as follows: the positive compound GDC0449 (1 mM) and the test compound (1 mM) were serially diluted to a concentration of 7 compounds in DMSO at 1:3 and 1:10, respectively, and the 8th was DMSO. Control, and then diluted 100 times with fresh medium.
  • the cells were cultured overnight, the medium was removed, 80 ⁇ L of fresh medium was added to each well, and then 20 ⁇ L of the gradient-diluted positive compound and the test compound were added, and 100 ⁇ L of conditioned medium containing 30 nM Shh-N, 2 for each concentration.
  • Double wells, set both positive and negative reference wells ie Shh Ctrl: 80 ⁇ L fresh medium + 20 ⁇ L containing 1% DMSO medium + 100 ⁇ L conditioned medium containing 30 nM Shh-N; Unstimulated Ctrl: 180 ⁇ L fresh medium + 20 ⁇ L containing 1
  • the cells were cultured for 24 hours in a CO 2 incubator.
  • luciferase activity 96-well plate cells were removed, the medium was discarded, and the cells were washed twice with PBS, followed by addition of 20 ⁇ L of lysate (Promega E1531) per well, and cleavage at room temperature for 30 min. Transfer 5 ⁇ L of cell lysate to a 384-well plate (Greiner 781074), then add 25 ⁇ L of luciferase reaction solution (Promega E1501) to each well, and mix it immediately into the VarioSkan Flash multi-reader to read the relative light unit. (RLU) value.
  • RLU relative light unit.

Abstract

Disclosed are a crystal form and salt form of a quinoline derivative, a crystal form of the salt form, and a preparation method therefor.

Description

喹啉衍生物的盐型、晶型及其制备方法和中间体Salt type and crystal form of quinoline derivative, preparation method and intermediate thereof 技术领域Technical field
本发明涉及一种喹啉衍生物的晶型、盐型、盐型的晶型及其制备方法。The present invention relates to a crystalline form, a salt form, a salt form of a quinoline derivative, and a process for the preparation thereof.
背景技术Background technique
Hedgehog蛋白最初是在果蝇中发现的分泌的信号蛋白，它们是高度疏水的蛋白质，其在胚胎发育中起着至关重要的作用。已在人类中鉴别了三种同源hedgehog蛋白，分别为Sonic hedgehog(Shh)、Indian hedgehog(Ihh)和Desert hedgehog(Dhh)。其中Shh不仅在胚胎发育中至关重要，许多证据显示它在包括基底细胞癌等一些癌症的致癌机制上也有重要的作用(Caro，I.and J.A.Low,Clin Cancer Res,2010.16(13):3335-9)。Shh在体内合成一个分子量为45kDa的前体蛋白，通过自切除作用产生一个分子量为20kDa的N一端片断，这个N一端片断具各了体内所知的所有生物活性，包括激活细胞内的hedgehog信息通路，其通路主要成员包括patched(PTCH)，类G蛋白偶和受体致癌基因smoothened(SMO)以及转录因子Gli等(Bale,A.E.and K.P.Yu,Hum Mol Genet,2001.10(7):757-62)。基底细胞癌hedgehog信息通路的变异分析结果显示大多数变异发生在PTCH-1和SMO上(Von Hoff,D.D.,et al.,N Engl J Med,2009.361(12):1164-72)。PTCH-1是个有着12次跨膜结构的膜蛋白，它是Shh的直接作用受体。在没有Shh的情况下PTCH-1与SMO相作用，SMO为7次跨膜结构蛋白，抑制SMO的生物活力。Shh与PTCH-1的结合导致PTCH-1脱离SMO，使SMO摆脱受抑制状态。Gli转录因子受控于SMO，它起着基因转录的开关作用，其中主要的成员包括Glil，Gli2和Gli3。整个hedgehog通路对胚胎正常发育起着重要的作用。扰乱了这一信息通路将会导致严重的畸形，比如天然致畸化合物cyclopamine就是一个hedgehog抑制剂。在通常条件下，成人体内hedgehog蛋白的浓度非常低。在hedgehog蛋白浓度很低的情况下，PTCH-1与SMO相结合并抑制其生物活力，因而整个通路处于没有活力，或活力很低的状态。当细胞分泌hedgehog蛋白后，hedgehog蛋白与PTCH-1受体的结合使其脱离SMO，从而失去对SMO的抑制作用。SMO进一步激活转录因子Gli-1从而调控基因转录和细胞生长。越来越多的证据表明，大部分基底细胞癌的病因是由于突变或其他原因导致过高的hedgehog信息传导通路活力。因此抑制过高的hedgehog信息传导通路的活力，刚一能抑制癌细胞的生长从而达到治疗基底细胞癌或由相同机制引起的其他癌症。SMO的组成性活化导致癌症(例如BCC)以及解除Ptch对其的抑制可以使SMO致癌的证据说明了SMO拮杭剂作为治疗剂在治疗所述病症中的用途(Stone等人，(1996)Nature 384:129)。一系列科学和临床试验结果显示hedgehog抑制剂能有效地治疗多种癌症。最新临床试验数据显示hedgehog抑制剂GDC-0449能有效地治疗基底细胞癌和髓母细胞癌(LorussoPM.et al.Clin Cancer Res.2011；17(8):2502-11)，并于2012年1月得到FDA认证，或由相同机制引起的其他癌症，例如基底细胞痣综合征(BCNS)(Goldberg LH.et al.Arch Dermatol.2011Mar 21.)。生物化学研究表明GDC-0449的抑制点是在SMO上，抑制了SMO的活力就抑制了整个hedgehog通路的活力，从而达到抗癌的目的。 除了基底细胞癌和髓母细胞癌两种癌症，还有许多其他癌症也和hedgehog信息传导通路的超高活力有关系，包括胰腺癌、肠胃癌、直肠癌、卵巢癌及前列腺癌，还有部分血癌等(De Smaele E.et al.Curr Opin Investig Drugs.2010；11(6):707-18)。因此研发hedgehog抑制剂作为新型抗癌药物的前景非常广泛。The Hedgehog protein was originally a secreted signaling protein found in Drosophila, a highly hydrophobic protein that plays a crucial role in embryonic development. Three homologous hedgehog proteins have been identified in humans, namely Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Among them, Shh is not only important in embryonic development, but also has many evidences that it plays an important role in the carcinogenic mechanisms of some cancers including basal cell carcinoma (Caro, I. and Jalow, Clin Cancer Res, 2010. 16(13): 3335 -9). Shh synthesizes a 45kDa precursor protein in vivo and produces a 20kDa N-terminal fragment by self-resection. This N-terminal fragment has all the biological activities known in the body, including activation of intracellular hedgehog information pathway. The major members of the pathway include patched (PTCH), G-like protein and receptor oncogene smoothened (SMO), and transcription factor Gli (Bale, AE and KPYu, Hum Mol Genet, 2001.10(7): 757-62) . Mutation analysis of the hedgehog signaling pathway of basal cell carcinoma showed that most of the variation occurred on PTCH-1 and SMO (Von Hoff, D.D., et al., N Engl J Med, 2009. 361(12): 1164-72). PTCH-1 is a membrane protein with 12 transmembrane structures that is a direct acting receptor for Shh. In the absence of Shh, PTCH-1 interacts with SMO, which is a 7-transmembrane structural protein that inhibits the biological viability of SMO. The combination of Shh and PTCH-1 causes PTCH-1 to detach from SMO, freeing SMO from the suppressed state. The Gli transcription factor is controlled by SMO, which acts as a switch for gene transcription, with the major members including Glil, Gli2 and Gli3. The entire hedgehog pathway plays an important role in the normal development of the embryo. Disturbing this information pathway will lead to severe malformations, such as the natural teratogenic compound cyclopamine is a hedgehog inhibitor. Under normal conditions, the concentration of hedgehog protein in adults is very low. In the case of a low concentration of hedgehog protein, PTCH-1 binds to SMO and inhibits its biological activity, so the entire pathway is in an inactive or low-activity state. When the cell secretes the hedgehog protein, the binding of the hedgehog protein to the PTCH-1 receptor causes it to detach from the SMO, thereby losing the inhibition of SMO. SMO further activates the transcription factor Gli-1 to regulate gene transcription and cell growth. There is increasing evidence that the cause of most basal cell carcinomas is the excessive activity of the hedgehog signaling pathway due to mutations or other causes. Therefore, the activity of the excessively high hedgehog information transduction pathway is inhibited, and the growth of cancer cells can be inhibited to achieve treatment of basal cell carcinoma or other cancers caused by the same mechanism. The constitutive activation of SMO leads to cancer (eg, BCC) and the inhibition of Ptch inhibition thereof. Evidence that SMO is carcinogenic illustrates the use of SMO antagonists as therapeutic agents in the treatment of such conditions (Stone et al., (1996) Nature 384:129). A series of scientific and clinical trials have shown that hedgehog inhibitors are effective in treating a variety of cancers. Recent clinical trial data show that the hedgehog inhibitor GDC-0449 is effective in the treatment of basal cell carcinoma and medulloblastoma (Lorusso PM. et al. Clin Cancer Res. 2011; 17(8): 2502-11), and in 2012 1 Other cancers that are FDA-approved or caused by the same mechanism, such as basal cell nevus syndrome (BCNS) (Goldberg LH. et al. Arch Dermatol. 2011 Mar 21.). Biochemical studies have shown that the inhibition point of GDC-0449 is on SMO, and inhibiting the activity of SMO inhibits the activity of the entire hedgehog pathway, thereby achieving anti-cancer purposes. In addition to basal cell carcinoma and medulloblastic cancer, many other cancers are also associated with the ultra-high activity of the hedgehog signaling pathway, including pancreatic cancer, intestinal cancer, rectal cancer, ovarian cancer, and prostate cancer. Blood cancer, etc. (De Smaele E. et al. Curr Opin Investig Drugs. 2010; 11(6): 707-18). Therefore, the prospect of developing hedgehog inhibitors as novel anticancer drugs is very broad.
Figure PCTCN2016099537-appb-000001
Figure PCTCN2016099537-appb-000001
尽管现有技术中已经存在一些SMO抑制剂，但是它们在活性、溶解性、药代动力学、成药性的等方面有待改进。Although some SMO inhibitors already exist in the prior art, they need to be improved in terms of activity, solubility, pharmacokinetics, drug-forming properties and the like.
申请号CN201410110890.1记载了一类新的喹啉衍生物，其可作为hedgehog通路的抑制剂、特别是作为SMO抑制剂。可用于治疗包括癌症在内的hedgehog通路有关病症。Application No. CN201410110890.1 describes a new class of quinoline derivatives which act as inhibitors of the hedgehog pathway, particularly as SMO inhibitors. It can be used to treat conditions related to the hedgehog pathway including cancer.
其结构如式(B-1)所示：Its structure is as shown in formula (B-1):
Figure PCTCN2016099537-appb-000002
Figure PCTCN2016099537-appb-000002
发明内容Summary of the invention
本发明提供化合物1的A晶型，其XRPD图谱如图1所示，The present invention provides Form A of Compound 1, the XRPD pattern of which is shown in Figure 1.
Figure PCTCN2016099537-appb-000003
Figure PCTCN2016099537-appb-000003
本发明的一些方案中，A晶型XRPD解析数据如表1所示。In some aspects of the invention, the A crystal form XRPD analytical data is shown in Table 1.
表1A晶型XRPD解析数据Table 1A crystal form XRPD analytical data
NO.NO. 2-Theta2-Theta d(A)d(A) I％I% NO.NO. 2-Theta2-Theta d(A)d(A) I％I%
11 3.8553.855 22.900522.9005 100.0100.0 88 14.38414.384 6.15286.1528 1.41.4
22 7.7377.737 11.416711.4167 5.85.8 99 14.73914.739 6.00516.0051 3.53.5
33 9.6979.697 9.11329.1132 0.80.8 1010 19.49019.490 4.55074.5507 14.814.8
44 10.16110.161 8.69818.6981 8.58.5 1111 20.04320.043 4.42654.4265 4.04.0
55 10.61810.618 8.32498.3249 2.02.0 1212 20.40120.401 4.34974.3497 20.920.9
66 12.03712.037 7.34677.3467 12.812.8 1313 22.74522.745 3.90633.9063 2.12.1
77 12.78512.785 6.91816.9181 1.21.2 1414 23.14023.140 3.84053.8405 2.42.4
本发明的一些方案中，上述化合物1的A晶型，其差示扫描量热曲线在111.41℃±2℃、126.08℃±2℃和146.06℃±2℃处具有吸热峰的起始点。In some aspects of the invention, the A crystalline form of Compound 1 above has a differential scanning calorimetry curve having an end point of endothermic peak at 111.41 °C ± 2 °C, 126.08 °C ± 2 °C, and 146.06 °C ± 2 °C.
本发明的一些方案中，上述化合物1的A晶型，其DSC图谱如图2所示。In some aspects of the invention, the A crystal form of the above compound 1 has a DSC pattern as shown in FIG.
本发明的一些方案中，上述化合物1的A晶型，其热重分析曲线在135.65±2℃失重0.7658％±0.2％。In some aspects of the invention, the A crystal form of the above compound 1 has a thermogravimetric analysis curve weight loss of 0.7658% ± 0.2% at 135.65 ± 2 °C.
本发明的一些方案中，上述化合物1的A晶型，其TGA图谱如图3所示。In some aspects of the invention, the T crystal of Form A of Compound 1 above is shown in Figure 3.
本发明还提供了化合物1的B晶型，其XRPD图谱如图4所示。The present invention also provides Form B of Compound 1, the XRPD pattern of which is shown in Figure 4.
本发明的一些方案中，B晶型XRPD解析数据如表2所示。In some aspects of the invention, the B-form XRPD analytical data is shown in Table 2.
表2 B晶型XRPD解析数据Table 2 B crystal form XRPD analytical data
NO.NO. 2-Theta2-Theta d(A)d(A) I％I% NO.NO. 2-Theta2-Theta d(A)d(A) I％I%
11 5.8535.853 15.087815.0878 67.067.0 1919 22.93422.934 3.87463.8746 18.118.1
22 9.3079.307 9.49469.4946 4.74.7 2020 23.42523.425 3.79453.7945 80.280.2
33 10.54710.547 8.38078.3807 100.0100.0 21twenty one 23.95623.956 3.71153.7115 26.326.3
44 11.41511.415 7.74517.7451 47.947.9 22twenty two 24.31324.313 3.65793.6579 29.729.7
55 11.67111.671 7.57617.5761 54.954.9 23twenty three 25.04025.040 3.55333.5533 8.78.7
66 12.45912.459 7.09897.0989 2.32.3 24twenty four 25.83225.832 3.44613.4461 9.39.3
77 13.89913.899 6.36646.3664 26.826.8 2525 27.47027.470 3.24423.2442 2.62.6
88 14.27514.275 6.19956.1995 88.388.3 2626 28.12028.120 3.17073.1707 2.92.9
99 15.12015.120 5.85475.8547 99.699.6 2727 28.71428.714 3.10653.1065 6.46.4
1010 15.55615.556 5.69155.6915 10.010.0 2828 29.36429.364 3.03913.0391 13.113.1
1111 17.05317.053 5.19535.1953 6.86.8 2929 29.77729.777 2.99792.9979 1.91.9
1212 18.65218.652 4.75334.7533 15.415.4 3030 30.46230.462 2.93212.9321 1.81.8
1313 19.08519.085 4.64634.6463 11.711.7 3131 31.09931.099 2.87342.8734 5.95.9
1414 20.06820.068 4.42094.4209 20.520.5 3232 32.71732.717 2.73492.7349 3.53.5
1515 20.60220.602 4.30764.3076 10.710.7 3333 33.63833.638 2.66212.6621 1.81.8
1616 21.15621.156 4.19614.1961 18.018.0 3434 37.32537.325 2.40722.4072 1.81.8
1717 21.74721.747 4.08334.0833 75.575.5 3535 38.86338.863 2.31542.3154 2.02.0
1818 22.31822.318 3.98013.9801 38.938.9 -- -- -- --
本发明的一些方案中，上述化合物1的B晶型，其差示扫描量热曲线在165.73℃±2℃处具有吸热峰的起始点。In some aspects of the invention, the B crystal form of Compound 1 above has a differential scanning calorimetry curve having a starting point of endothermic peak at 165.73 °C ± 2 °C.
本发明的一些方案中，上述化合物1的B晶型，其DSC图谱如图5所示。In some aspects of the invention, the B crystal form of the above Compound 1 has a DSC pattern as shown in FIG.
本发明的一些方案中，上述化合物1的B晶型，其热重分析曲线在120.00±2℃失重达0.2383％±0.2％，在247.60±2℃失重达0.2005％±0.2％。In some aspects of the present invention, the B crystal form of the above compound 1 has a thermogravimetric analysis curve having a weight loss of 0.2383% ± 0.2% at 120.00 ± 2 ° C and a loss of 0.2005% ± 0.2% at 247.60 ± 2 ° C.
本发明的一些方案中，上述化合物1的B晶型，其TGA图谱如图6所示。 In some aspects of the invention, the T crystal of Form B of Compound 1 above is shown in Figure 6.
本发明还提供了B晶型的制备方法，其包括将化合物1的A晶型加入到酯类溶剂、醇类溶剂或酯类溶解和醇类溶剂的混合溶剂中，加热至回流溶解，然后缓慢降温结晶制得。The invention also provides a preparation method of the B crystal form, which comprises adding the crystal form of the compound A to the solvent of the ester solvent, the solvent of the alcohol or the solvent of the ester and the solvent of the alcohol solvent, heating to reflux to dissolve, and then slowly Cooled and crystallized.
本发明的一些方案中，上述酯类溶解选自甲酸丙酯、甲酸异丙酯、甲酸丁酯、甲酸异丁酯、乙酸乙酯、乙酸异丙酯、乙酸异丙酯。In some embodiments of the invention, the ester is dissolved in the group consisting of propyl formate, isopropyl formate, butyl formate, isobutyl formate, ethyl acetate, isopropyl acetate, and isopropyl acetate.
本发明的一些方案中，上述醇类溶剂选自甲醇、乙醇、正丙醇、异丙醇、正丁醇、叔丁醇。In some aspects of the invention, the alcohol solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, and tert-butanol.
本发明的一些方案中，上述酯类溶剂和醇类溶剂的混合溶剂中，酯类溶剂和醇类溶剂的体积比为0.1:1～12。In some aspects of the invention, in the mixed solvent of the ester solvent and the alcohol solvent, the volume ratio of the ester solvent to the alcohol solvent is from 0.1:1 to 12.
本发明的一些方案中，上述酯类溶剂和醇类溶剂的混合溶剂中，酯类溶剂和醇类溶剂的体积比为1:2～8。In some aspects of the invention, in the mixed solvent of the ester solvent and the alcohol solvent, the volume ratio of the ester solvent to the alcohol solvent is 1:2 to 8.
本发明提供了下式所示化合物2，The present invention provides a compound 2 of the following formula,
Figure PCTCN2016099537-appb-000004
Figure PCTCN2016099537-appb-000004
本发明提供了化合物2的C晶型，其XRPD图谱如图7所示。The present invention provides a crystalline form C of Compound 2, the XRPD pattern of which is shown in FIG.
本发明的一些方案中，C晶型XRPD图谱解析数据如表3所示。In some aspects of the invention, the C-form XRPD pattern analysis data is shown in Table 3.
表3 C晶型XRPD解析数据Table 3 C crystal form XRPD analytical data
NO.NO. 2-Theta2-Theta d(A)d(A) I％I% NO.NO. 2-Theta2-Theta d(A)d(A) I％I%
11 5.7755.775 15.290615.2906 48.548.5 1717 20.05620.056 4.42364.4236 25.125.1
22 6.0126.012 14.689314.6893 100.0100.0 1818 20.39120.391 4.35174.3517 7.27.2
33 9.8389.838 8.98288.9828 5.75.7 1919 20.80420.804 4.26614.2661 16.016.0
44 10.29210.292 8.58768.5876 5.65.6 2020 21.30121.301 4.16784.1678 8.08.0
55 10.78410.784 8.19738.1973 6.56.5 21twenty one 21.95021.950 4.04604.0460 4.54.5
66 11.33611.336 7.79947.7994 11.411.4 22twenty two 22.63822.638 3.92463.9246 10.410.4
77 11.87211.872 7.44857.4485 21.521.5 23twenty three 23.42923.429 3.79383.7938 9.79.7
88 12.36612.366 7.15207.1520 2.12.1 24twenty four 23.78123.781 3.73853.7385 20.120.1
99 14.41314.413 6.14036.1403 15.515.5 2525 24.59124.591 3.61713.6171 5.65.6
1010 14.84914.849 5.96105.9610 9.89.8 2626 25.77625.776 3.45353.4535 3.73.7
1111 16.28616.286 5.43815.4381 6.56.5 2727 26.63926.639 3.34343.3434 2.32.3
1212 16.86016.860 5.25445.2544 8.08.0 2828 27.47327.473 3.24393.2439 4.54.5
1313 17.82417.824 4.97214.9721 3.83.8 2929 27.92627.926 3.19233.1923 3.93.9
1414 18.73318.733 4.73294.7329 9.59.5 3030 28.68928.689 3.10903.1090 3.23.2
1515 19.34519.345 4.58464.5846 12.912.9 3131 29.72129.721 3.00353.0035 4.64.6
1616 19.66119.661 4.51164.5116 4.44.4 3232 30.27130.271 2.95012.9501 3.63.6
本发明的一些方案中，上述化合物2的C晶型，其差示扫描量热曲线在57.33℃±5℃和212.56℃±5℃处具有吸热峰的起始点。In some aspects of the invention, the C crystal form of Compound 2 above has a differential scanning calorimetry curve having an end point of endothermic peak at 57.33 ° C ± 5 ° C and 212.56 ° C ± 5 ° C.
本发明的一些方案中，上述化合物2的C晶型，其DSC图谱如图8所示。In some embodiments of the present invention, the C crystal form of the above Compound 2 has a DSC spectrum as shown in FIG.
本发明的一些方案中，上述化合物2的C晶型，其热重分析曲线在120.00±3℃失重达5.110％±0.5％。In some aspects of the present invention, the C crystal form of the above compound 2 has a thermogravimetric analysis curve having a weight loss of 5.10% ± 0.5% at 120.00 ± 3 °C.
本发明的一些方案中，上述化合物2的C晶型，其TGA图谱如图9所示。In some aspects of the invention, the C crystal form of the above compound 2 has a TGA pattern as shown in FIG.
本发明提供了C晶型的制备方法，其包括化合物1溶解到有机溶剂中，缓慢滴加硫酸水溶液，搅拌析晶制得。The invention provides a preparation method of the C crystal form, which comprises the compound 1 dissolved in an organic solvent, slowly adding a sulfuric acid aqueous solution, and stirring and crystallization.
本发明的一些方案中，上述机溶剂选自丙酮、甲乙酮、环己酮。In some aspects of the invention, the organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, and cyclohexanone.
本发明的一些方案中，化合物1与硫酸的摩尔比选自1:2～4。In some aspects of the invention, the molar ratio of Compound 1 to sulfuric acid is selected from 1:2 to 4.
本发明还提供了上述A晶型、B晶型、化合物2及C晶型在制备治疗与SMO受体相关疾病药物上的应用。The invention also provides the use of the above A crystal form, B crystal form, compound 2 and C crystal form in the preparation of a medicament for treating diseases related to SMO receptors.
定义和说明：Definition and description:
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。Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered undefined or unclear without a particular definition, but should be understood in the ordinary sense. When a trade name appears in this document, it is intended to refer to its corresponding commodity or its active ingredient.
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。The 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.
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。The chemical reaction of a particular embodiment of the invention is carried out in a suitable solvent which is suitable for the chemical changes of the invention and the reagents and materials required thereof. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction schemes based on the prior embodiments.
本领域任何合成路线规划中的一个重要考量因素是为反应性官能团(如本发明中的氨基)选择合适的保护基。对于经过训练的从业者来说，Greene and Wuts的(Protective Groups In Organic Synthesis,Wiley and Sons,1991)是这方面的权威。本发明引用的所有参考文献整体上并入本发明。An important consideration in any synthetic route planning in the art is the selection of a suitable protecting group for a reactive functional group, such as an amino group in the present invention. For trained practitioners, Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991) is the authority in this regard. All references cited in the present invention are incorporated by reference in their entirety.
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。The invention is specifically described by the following examples, which are not intended to limit the invention.
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。反应一般是在惰性氮气下、无水溶剂中进行的。质子核磁共振数据记录在Bruker Avance III 400(400MHz)分光仪上，化学位移以四甲基硅烷低场处的(ppm)表示。质谱是在安捷伦1200系列加6110(&1956A)上测定。LC/MS或Shimadzu MS包含一个DAD：SPD-M20A(LC)和Shimadzu Micromass 2020检测器。质谱仪配备有一个正或负模式下操作的电喷雾离子源(ESI)。All solvents used in the present invention are commercially available and can be used without further purification. The reaction is generally carried out under an inert nitrogen atmosphere in an anhydrous solvent. Proton nuclear magnetic resonance data was recorded on a Bruker Avance III 400 (400 MHz) spectrometer with chemical shifts expressed in ppm at the low field of tetramethylsilane. Mass spectra were measured on an Agilent 1200 Series Plus 6110 (&1956A). LC/MS or Shimadzu MS contains a DAD: SPD-M20A (LC) and Shimadzu Micromass 2020 detector. The mass spectrometer is equipped with an electrospray ionization source (ESI) operating in either positive or negative mode.
本发明采用下述缩略词：DCM代表二氯甲烷；PE代表石油醚；EA代表乙酸乙酯；DMF代表 N,N-二甲基甲酰胺；DMAC代表N,N-二甲基乙酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；tol代表甲苯；THF代表四氢呋喃；EtOH代表乙醇；MeOH代表甲醇；NMP代表N-甲基吡咯烷酮；2-METHF代表2-甲基四氢呋喃；i-PrOH代表2-丙醇；Bn代表苄基；Cbz代表苄氧羰基，是一种胺保护基团；Boc代表叔丁基羰基是一种胺保护基团；Fmoc代表笏甲氧羰基，是一种胺保护基团；Alloc代表烯丙氧羰基，是一种胺保护基团；Teoc代表三甲基硅乙氧羰基，是一种胺保护基团；Boc2O代表二-叔丁基二碳酸酯；HCl(g)代表氯化氢气体；H2SO4代表硫酸；HOAc代表乙酸；TFA代表三氟乙酸；DIPEA代表二异丙基乙基胺；DIEA代表二异丙基乙基胺；NMM代表N-甲基吗啡啉；DBU代表1,8-二氮杂二环十一碳-7-烯；Et3N代表三乙胺；LDA代表二异丙基胺锂；NaHMDS代表双(三甲基硅基)氨基钠；KHMDS代表双(三甲基硅基)氨基钾；LiAlH4代表四氢铝锂；t-BuOK代表叔丁醇钾；H2O2代表过氧化氢；NH4Cl代表氯化铵；BaSO4代表硫酸钡；CaCO3代表碳酸钙；SnCl2代表氯化亚锡；Zn(BH4)2代表硼氢化锌；PPh3代表三苯基膦；HMDS代表六甲基二硅胺烷；Pd/C代表钯碳；PtO2代表二氧化铂；Pd(OH)2代表氢氧化钯；Pd2(dba)3代表三(二亚苄基丙酮)二钯；Pd(PPh3)4代表四三苯基膦钯；Pd(dppf)Cl2代表1,1'-双(二苯基磷)二茂铁氯化钯；Pd(PPh3)2Cl2代表二氯双(三苯基膦)钯(II)；Pd(OAc)2代表醋酸钯；PdCl2代表氯化钯；CuI代表碘化亚铜；CuBr代表溴化亚铜；CuCl代表氯化亚铜；Cu代表铜粉；Cu2O代表氧化亚铜；Xantphos代表4,5-双(二苯基磷)-9,9-二甲基氧杂蒽；Sphos代表2-二环己基亚膦基-2',6'-二甲氧基联苯；Xphos代表2-二环己基磷-2',4',6'-三异丙基联苯；Ruphos代表2-双环己基膦-2',6'-二异丙氧基-,1,1'-联苯；Brettphos代表2-(二环己基膦基)-3,6-二甲氧基-2'-4'-6'-三异丙基-1,1'-联苯。The present invention employs the following abbreviations: DCM stands for dichloromethane; PE stands for petroleum ether; EA stands for ethyl acetate; DMF stands for N,N-dimethylformamide; DMAC stands for N,N-dimethylacetamide; DMSO stands for dimethyl sulfoxide; EtOAc stands for ethyl acetate; tol stands for toluene; THF stands for tetrahydrofuran; EtOH stands for ethanol; MeOH stands for methanol; NMP stands for N-methylpyrrolidone; 2-METHF stands for 2-methyltetrahydrofuran; i-PrOH Represents 2-propanol; Bn stands for benzyl; Cbz stands for benzyloxycarbonyl and is an amine protecting group; Boc stands for t-butylcarbonyl which is an amine protecting group; Fmoc stands for fluorenylmethoxycarbonyl and is an amine a protecting group; Alloc represents an allyloxycarbonyl group, which is an amine protecting group; Teoc represents a trimethylsilyloxycarbonyl group, which is an amine protecting group; and Boc 2 O represents a di-tert-butyl dicarbonate; HCl (g) represents hydrogen chloride gas; H 2 SO 4 represents sulfuric acid; HOAc represents acetic acid; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; DIEA represents diisopropylethylamine; NMM represents N-A Benzomorpholine; DBU stands for 1,8-diazabicycloundec-7-ene; Et 3 N stands for triethylamine; LDA stands for Lithium diisopropylamide; NaHMDS represents sodium bis(trimethylsilyl)amide; KHMDS represents potassium bis(trimethylsilyl)amide; LiAlH 4 represents lithium tetrahydroaluminum; t-BuOK represents potassium t-butoxide; H 2 O 2 represents hydrogen peroxide; NH 4 Cl represents ammonium chloride; BaSO 4 represents barium sulfate; CaCO 3 represents calcium carbonate; SnCl 2 represents stannous chloride; Zn(BH 4 ) 2 represents zinc borohydride; PPh 3 Represents triphenylphosphine; HMDS stands for hexamethyldisilazane; Pd/C stands for palladium carbon; PtO 2 stands for platinum dioxide; Pd(OH) 2 stands for palladium hydroxide; Pd 2 (dba) 3 stands for three (two) Phenylacetone) dipalladium; Pd(PPh 3 ) 4 represents tetrakistriphenylphosphine palladium; Pd(dppf)Cl 2 represents 1,1'-bis(diphenylphosphino)ferrocene palladium chloride; Pd( PPh 3 ) 2 Cl 2 represents dichlorobis(triphenylphosphine)palladium(II); Pd(OAc) 2 represents palladium acetate; PdCl 2 represents palladium chloride; CuI represents cuprous iodide; CuBr represents cuprous bromide CuCl stands for cuprous chloride; Cu stands for copper powder; Cu 2 O stands for cuprous oxide; Xantphos stands for 4,5-bis(diphenylphosphino)-9,9-dimethyloxaxan; Sphos stands for 2- Dicyclohexylphosphinyl-2',6'-dimethoxybiphenyl; Xphos stands for 2-dicyclohexylphos-2',4',6'-triiso Base biphenyl; Ruphos stands for 2-biscyclohexylphosphine-2',6'-diisopropoxy-,1,1'-biphenyl; Brettphos stands for 2-(dicyclohexylphosphino)-3,6-di Methoxy-2'-4'-6'-triisopropyl-1,1'-biphenyl.
化合物经手工或者
Figure PCTCN2016099537-appb-000005
软件命名，市售化合物采用供应商目录名称。Compound by hand or
Figure PCTCN2016099537-appb-000005
Software naming, commercially available compounds using the supplier catalog name.
本发明粉末X-射线衍射(X-ray powder diffractometer,XRPD)方法X-ray powder diffractometer (XRPD) method of the present invention
仪器型号：布鲁克D8advance X-射线衍射仪Instrument model: Brooke D8advance X-ray diffractometer
测试条件：详细的XRPD参数如下：Test conditions: Detailed XRPD parameters are as follows:
X-ray发生器：Cu,kα,
Figure PCTCN2016099537-appb-000006
X-ray generator: Cu, kα,
Figure PCTCN2016099537-appb-000006
管电压：40kV，管电流：40mA.Tube voltage: 40kV, tube current: 40mA.
发射狭缝：1deg.Launch slit: 1deg.
限高狭缝：10mmHeight limit slit: 10mm
散射狭缝：1deg.Scattering slit: 1deg.
接受狭缝：0.15mmAccept the slit: 0.15mm
单色器：固定的单色器Monochromator: fixed monochromator
扫描范围：2-40deg.Scanning range: 2-40deg.
扫描速度：10deg/minScanning speed: 10deg/min
本发明差热分析(Differential Scanning Calorimeter,DSC)方法 Differential Scanning Calorimeter (DSC) method of the present invention
仪器型号：TA Q2000差示扫描量热仪Instrument model: TA Q2000 Differential Scanning Calorimeter
测试条件：取样品(～1mg)置于DSC铝锅内进行测试，方法为：25℃-350℃，升温速率为10℃/min。Test conditions: The sample (~1 mg) was placed in a DSC aluminum pan for testing at 25 ° C - 350 ° C and a heating rate of 10 ° C / min.
本发明热重分析(Thermal Gravimetric Analyzer,TGA)方法Thermal Gravimetric Analyzer (TGA) method of the present invention
仪器型号：TA Q5000IR热重分析仪Instrument model: TA Q5000IR thermogravimetric analyzer
测试条件：取样品(2～5mg)置于TGA铂金锅内进行测试，方法为：室温-350℃，升温速率为10℃/min。Test conditions: Samples (2 to 5 mg) were placed in a TGA platinum pot for testing at room temperature - 350 ° C and a heating rate of 10 ° C / min.
附图说明DRAWINGS
图1为A晶型的Cu-Kα辐射的XRPD谱图。Figure 1 is an XRPD spectrum of Cu-Kα radiation of Form A.
图2为A晶型的DSC图谱。Figure 2 is a DSC pattern of Form A.
图3为A晶型的TGA图谱。Figure 3 is a TGA pattern of Form A.
图4为B晶型的Cu-Kα辐射的XRPD谱图。Figure 4 is an XRPD spectrum of Cu-Kα radiation of Form B.
图5为B晶型的DSC图谱。Figure 5 is a DSC pattern of Form B.
图6为B晶型的TGA图谱。Figure 6 is a TGA pattern of Form B.
图7为C晶型的Cu-Kα辐射的XRPD谱图。Figure 7 is an XRPD spectrum of Cu-Kα radiation of Form C.
图8为C晶型的DSC图谱。Figure 8 is a DSC pattern of Form C.
图9为C晶型的TGA图谱。Figure 9 is a TGA pattern of Form C.
具体实施方式detailed description
为了更好的理解本发明的内容，下面结合具体实施例来做进一步的说明，但具体的实施方式并不是对本发明的内容所做的限制。For a better understanding of the content of the present invention, the following detailed description is made in conjunction with the specific embodiments, but the specific embodiments are not limited to the content of the present invention.
实施例1Example 1
化合物1及A晶型的制备 Preparation of Compound 1 and Form A
Figure PCTCN2016099537-appb-000007
Figure PCTCN2016099537-appb-000007
第一步：将原甲酸三甲酯(55kg，137.11mol)和2,2-二甲基-1,3-二氧六环-4,6-二酮(4kg，27.75mol)加入到30L反应釜中，加热到100℃。体系回流三小时后加入2-溴代苯胺(3.80kg，22.09摩尔)。整个反应回流15小时，LCMS检测显示反应完成。蒸出多余的溶剂，将所得固体用乙醇(1L)洗涤，烘干。得到目标产物5-((2-溴苯胺基)亚甲基)-2,2-二甲基-1,3-二氧六环-4,6-二酮(48kg，产率89.94％)为黄色固体。First step: Trimethyl orthoformate (55 kg, 137.11 mol) and 2,2-dimethyl-1,3-dioxol-4,6-dione (4 kg, 27.75 mol) were added to the 30 L reaction. In a kettle, heat to 100 °C. After refluxing the system for three hours, 2-bromoaniline (3.80 kg, 22.09 mol) was added. The entire reaction was refluxed for 15 hours and LCMS showed the reaction was completed. Excess solvent was distilled off, and the obtained solid was washed with ethanol (1 L) and dried. The target product 5-((2-bromoanilino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (48 kg, yield 89.94%) was obtained. Yellow solid.
第二步：将5-((2-溴苯胺基)亚甲基)-2,2-二甲基-1,3-二氧六环-4,6-二酮(1kg，3.07mol)溶剂在二苯醚(4升)中，然后加热到185℃，在这个温度下搅拌10分钟。然后将混合物加热到250℃并继续搅拌30分钟。停止加热，冷却至75℃，TLC显示反应完成，将混合物过滤，固体用正庚烷(5L)洗涤得到8-溴-4-羟基喹啉(580g，2.59mol，收率84.32％)为黄色固体。Second step: 5-((2-bromoanilino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (1 kg, 3.07 mol) solvent In diphenyl ether (4 liters), it was then heated to 185 ° C and stirred at this temperature for 10 minutes. The mixture was then heated to 250 ° C and stirring was continued for 30 minutes. The heating was stopped, cooled to 75 ° C, TLC showed the reaction was completed, the mixture was filtered, and the solid was washed with n-heptane (5 L) to give 8-bromo-4-hydroxyquinoline (580 g, 2.59 mol, yield 84.32%) as a yellow solid. .
第三步：将8-溴-4-羟基喹啉(2kg，8.93mol)，(4-氰基苯基)硼酸(1.31kg，8.93mol)，Pd(dppf)Cl2(130.68克，178.60毫摩尔)和碳酸钠(1.89kg，17.86mol)的混合物中加入四氢呋喃(10L)，DMF(2升)和水(2升)。将混合物加热到78℃。3小时后，加入4升水然后将混合物过滤。固体用水(2L)洗涤，在50℃真空干燥箱烘干三天得到产物4-(4-羟基喹啉-8-基)苯甲腈(1.90kg，收率86.40％)为黄色固体。The third step: 8-bromo-4-hydroxyquinoline (2 kg, 8.93 mol), (4-cyanophenyl)boronic acid (1.31 kg, 8.93 mol), Pd(dppf)Cl 2 (130.68 g, 178.60 m To a mixture of sodium and sodium carbonate (1.89 kg, 17.86 mol) was added tetrahydrofuran (10 L), DMF (2 L) and water (2 L). The mixture was heated to 78 °C. After 3 hours, 4 liters of water was added and the mixture was filtered. The solid was washed with water (2 L) and dried in a vacuum oven at 50 ° C for three days to give the product 4-(4-hydroxyquinolin-8-yl)benzonitrile (1.90 kg, yield 86.40%) as a yellow solid.
第四步：在30L的反应釜里，加入DMF(10L)和4-(4-羟基喹啉-8-基)苯甲腈(2.2kg，8.93摩尔)，室温搅拌,然后在5分钟内缓慢加入NIS(2.01kg，8.93摩尔)。半小时后采样用LCMS检测，显示反应完成。 加入20L冰水搅拌后过滤。固体用水洗(5L×3)。产物用50℃是真空干燥箱烘三天得4-(4-羟基-3-碘-8-喹啉基)苯甲腈(2.55kg，收率76.73％)为黄色固体。Step 4: In a 30 L reactor, add DMF (10 L) and 4-(4-hydroxyquinolin-8-yl)benzonitrile (2.2 kg, 8.93 mol), stir at room temperature, then slowly in 5 minutes. NIS (2.01 kg, 8.93 mol) was added. After half an hour, the samples were detected by LCMS and the reaction was completed. Add 20 L of ice water and stir and filter. The solid was washed with water (5 L x 3). The product was dried in a vacuum oven at 50 ° C for three days to give 4-(4-hydroxy-3-iodo-8-quinolinyl)benzonitrile (2.55 kg, yield 76.73%) as a yellow solid.
第五步：在30L反应釜中，将4-(4-羟基-3-碘-8-喹啉基)苯甲腈(2.55kg，6.85mol)溶解在DMF(13L)中，于20℃在氮气保护下滴加三溴化磷(1.85kg,6.85mol)。混合物在20℃下搅拌1小时。LCMS检测反应完成。用饱和碳酸钠溶液将体系中和至pH＝7～8。减压过滤后将固体用水(10L)洗涤。得到固体用50℃是真空干燥箱烘三天得4-(4-溴-3-碘-8-喹啉)苯甲腈(2.95kg，6.78mol)为黄色固体。Step 5: Dissolve 4-(4-hydroxy-3-iodo-8-quinolinyl)benzonitrile (2.55 kg, 6.85 mol) in DMF (13 L) in a 30 L reactor at 20 ° C Phosphorus tribromide (1.85 kg, 6.85 mol) was added dropwise under nitrogen. The mixture was stirred at 20 ° C for 1 hour. The reaction was completed by LCMS. The system was neutralized to pH = 7-8 with a saturated sodium carbonate solution. After filtration under reduced pressure, the solid was washed with water (10L). The solid was obtained by drying in a vacuum oven at 50 ° C for three days to give 4-(4-bromo-3-iodo-8-quinoline) benzonitrile (2.95 kg, 6.78 mol) as a yellow solid.
第六步：4-(4-溴-3-碘-8-喹啉)苯甲腈(3.5kg，8.04摩尔)，(2S,6R)-2,6-二甲基-4-(5(4,4,5,5-四甲基-1,3,2-二氧硼烷-2-基)-2-吡啶基]吗啉(3.07kg，9.65mol)，在四氢呋喃(30.00L)中加热到65℃。所有反应物全部溶解后,加入Na2CO3(1.70kg，16.08摩尔)和Pd(dppf)Cl2(294.14g，402.00mmol，0.05eq)，然后再加入DMF(5L)和水(L 4)的混合物.整个反应在在65℃反应6小时。LCMS检测反应完全,后冷至室温。用乙酸乙酯(10L)提取,水相用二氯甲烷DCM(5L×2)提取。有机相用硫酸钠干燥后浓缩。粗品用硅胶柱纯化(乙酸乙酯：二氯甲烷：正庚烷比例为1∶1∶1)后得到4-(4-溴-3-(6-((2S,6R)-2,6-二甲基吗啡啉-4-基)-3-吡啶基)-8-喹啉)苯甲腈(2.68kg，收率66.75％)为黄色固体。Step 6: 4-(4-Bromo-3-iodo-8-quinoline)benzonitrile (3.5 kg, 8.04 mol), (2S,6R)-2,6-dimethyl-4-(5(5) 4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]morpholine (3.07 kg, 9.65 mol) in tetrahydrofuran (30.00 L) Heat to 65 ° C. After all the reactants were dissolved, add Na 2 CO 3 (1.70 kg, 16.08 mol) and Pd (dppf) Cl 2 (294.14 g, 402.00 mmol, 0.05 eq), then add DMF (5 L) and water (L). 4) mixture. The whole reaction was carried out at 65 ° C for 6 hours. The reaction was completed by LCMS and then cooled to room temperature. extracted with ethyl acetate (10 L), and the aqueous phase was extracted with dichloromethane DCM (5L×2). After drying over sodium sulfate and concentrating, the crude material was purified using silica gel column (ethyl acetate: dichloromethane: n-heptane ratio: 1:1) to give 4-(4-bromo-3-(6-((2S, 6R)-2,6-Dimethylmorpholine-4-yl)-3-pyridyl)-8-quinoline)benzonitrile (2.68 kg, yield 66.75%) was obtained as a yellow solid.
第七步：将4-(4-溴-3-(6-((2S,6R)-2,6-二甲基吗啡啉-4-基)-3-吡啶基)-8-喹啉)苯甲腈(2.68kg,5.37mol)溶剂在甲苯(25L)中加热至60℃待溶解完全.将环丙基硼酸(1.84kg，21.47摩尔)，正丁基二(1-金刚烷基)膦(192.41g，536.64mmol)，碳酸铯(3.5kg，10.73mol)和Pd(OAc)2(60.24g，268.32mmol)加入反应体系后升温至80℃反应1小时。LCMS检测反应完全，将体系冷却后加入水(15L)稀释.用乙酸乙酯(20L*2)提取.有机相用盐水(5L×2)洗涤，用硫酸钠干燥，然后浓缩.粗产物由柱层析(正庚烷：乙酸乙酯＝5：1)纯化后得到目标产物(2kg，收率80.86％)的白色固体，为A晶型。Step 7: 4-(4-Bromo-3-(6-((2S,6R)-2,6-dimethylmorphinolin-4-yl)-3-pyridyl)-8-quinoline) The benzonitrile (2.68 kg, 5.37 mol) solvent was heated to 60 ° C in toluene (25 L) to be completely dissolved. Cyclopropylboronic acid (1.84 kg, 21.47 mol), n-butylbis(1-adamantyl)phosphine (192.41 g, 536.64 mmol), cesium carbonate (3.5 kg, 10.73 mol) and Pd(OAc) 2 (60.24 g, 268.32 mmol) were added to the reaction system, and the mixture was heated to 80 ° C for 1 hour. The reaction was completed by LCMS. The mixture was cooled and then diluted with water (15L). The mixture was extracted with ethyl acetate (20L*2). The organic phase was washed with brine (5L×2), dried over sodium sulfate and then concentrated. After purification by chromatography (n-heptane:ethyl acetate = 5:1), the title product (2g, yield: 80.86%) was obtained as white crystal.
B晶型制备B crystal form preparation
将1kg A晶型溶于2L乙酸乙酯，加热溶清后，回流下缓慢加入12L乙醇，加完后保持溶液澄清,搅拌下自然冷却过夜，有大量黄色固体析出，过滤,滤饼用2L 0℃乙醇淋洗，抽干后得到850g黄色固体，即为B晶型。1kg A crystal form was dissolved in 2L ethyl acetate, heated and dissolved, and 12L ethanol was slowly added under reflux. After the addition, the solution was kept clear. After cooling, it was naturally cooled overnight. A large amount of yellow solid was precipitated and filtered. The filter cake was used 2L 0 The ethanol was rinsed at °C, and after drying, 850 g of a yellow solid was obtained, which was a crystal form of B.
C晶型制备C crystal form preparation
将游离碱化合物1(1.19kg，2.58mol，1当量)溶解在丙酮(12L)中，缓慢加入硫酸(3M，1.72升，2eq)持续约30分钟，然后将混合物在25℃下搅拌1.5小时。将混合物过滤，固体干燥后在35℃下真空干燥 48小时的目标产物盐(1.56kg，收率92.07％)为黄色固体，即为C晶型。The free base compound 1 (1.19 kg, 2.58 mol, 1 eq.) was dissolved in acetone (12 L), sulfuric acid (3M, 1.72 liters, 2 eq) was slowly added for about 30 minutes, and then the mixture was stirred at 25 ° C for 1.5 hours. The mixture was filtered, dried and dried under vacuum at 35 ° C. The target product salt (1.56 kg, yield 92.07%) in 48 hours was a yellow solid, which was crystal form.
A晶型在不同溶剂中的溶解度试验Solubility test of crystal form A in different solvents
称取约2mg样品到1.5mL的液相小瓶中，分别用移液枪逐级加入如下溶剂，手动振摇溶解。该测试是在室温下进行，通过肉眼来判断溶解情况，溶解度结果见表4。Approximately 2 mg of the sample was weighed into a 1.5 mL liquid phase vial, and the following solvents were added stepwise with a pipette, and shaken by hand. The test was carried out at room temperature and the dissolution was judged by the naked eye. The solubility results are shown in Table 4.
表4 A晶型在不同溶剂中的溶解度结果Table 4 Solubility results of Form A in different solvents
编号Numbering 溶剂Solvent 溶解度(mg/mL)Solubility (mg/mL) 编号Numbering 溶剂Solvent 溶解度(mg/mL)Solubility (mg/mL)
11 甲醇Methanol 3.5～4.53.5～4.5 1414 甲苯Toluene 51.0～68.551.0~68.5
22 乙醇Ethanol <2<2 1515 正庚烷N-heptane <2<2
33 异丙醇Isopropanol <2<2 1616 环己烷Cyclohexane <2<2
44 正丁醇N-butanol 2.5～3.02.5 to 3.0 1717 二氧六环Dioxane 25.0～33.525.0~33.5
55 乙腈Acetonitrile 20.0～25.020.0 to 25.0 1818 水water <2<2
66 丙酮acetone 20.0～25.520.0 to 25.5 1919 甲醇-水(1:1)Methanol-water (1:1) <2<2
77 甲基乙基酮Methyl ethyl ketone 53.5～71.553.5~71.5 2020 甲醇-水(3:1)Methanol-water (3:1) <2<2
88 4-甲基-2-戊酮4-methyl-2-pentanone 50.0～66.550.0~66.5 21twenty one 乙醇-水(1:1)Ethanol-water (1:1) <2<2
99 乙酸乙酯Ethyl acetate 52.5～70.052.5~70.0 22twenty two 乙醇-水(3:1)Ethanol-water (3:1) <2<2
1010 乙酸异丙酯Isopropyl acetate 34.0～41.534.0~41.5 23twenty three 乙腈-水(1:1)Acetonitrile-water (1:1) <2<2
1111 甲基叔丁基醚Methyl tert-butyl ether 6.5～8.06.5~8.0 24twenty four 丙酮-水(1:2)Acetone-water (1:2) <2<2
1212 四氢呋喃Tetrahydrofuran 33.5～40.533.5~40.5 2525 异丙醇-水(1:1)Isopropyl alcohol-water (1:1) <2<2
1313 2-甲基四氢呋喃2-methyltetrahydrofuran 53.0～71.053.0~71.0 -- -- --
A晶型在不同溶剂中的稳定性试验Stability test of crystal form A in different solvents
取50mg的A晶型多份，分别加入下表中的单一或混合溶剂，25℃条件下搅拌。如果化合物全部溶解，自然挥发除去溶剂；若为混悬液，则持续搅拌3天。收集所有样品中的固体，XRPD检测其晶型状态。结果见表5。Take 50 mg of the A crystal form and add to the single or mixed solvent in the table below, and stir at 25 °C. If the compound is completely dissolved, the solvent is naturally volatilized; if it is a suspension, stirring is continued for 3 days. The solids in all samples were collected and XRPD was tested for crystal form. The results are shown in Table 5.
表5 A晶型在不同溶剂中的稳定性实验Table 5 Stability of A crystal form in different solvents
序号Serial number 溶剂Solvent 溶剂加入量(mL)Solvent addition amount (mL) 外观(2天)Appearance (2 days) 结果result
11 甲醇Methanol 0.60.6 混悬液Suspension B晶型B crystal form
22 正丁醇N-butanol 0.60.6 混悬液Suspension B晶型B crystal form
33 乙腈Acetonitrile 0.50.5 混悬液Suspension B晶型B crystal form
44 丙酮acetone 0.50.5 混悬液Suspension A晶型 A crystal form
55 甲基乙基酮Methyl ethyl ketone 0.30.3 自然挥发除去溶剂后析出固体Naturally volatile to remove solvent and precipitate solid B晶型B crystal form
66 乙酸乙酯Ethyl acetate 0.20.2 混悬液Suspension B晶型B crystal form
77 甲基叔丁基醚Methyl tert-butyl ether 0.60.6 混悬液Suspension A/B混晶A/B mixed crystal
88 四氢呋喃Tetrahydrofuran 0.20.2 混悬液Suspension A晶型A crystal form
99 甲苯Toluene 0.30.3 自然挥发除去溶剂后析出固体Naturally volatile to remove solvent and precipitate solid B晶型 B crystal form
1010 1,4-二氧六环1,4-dioxane 0.20.2 混悬液Suspension A晶型A crystal form
1111 乙腈:水＝4:1Acetonitrile: water = 4:1 0.50.5 混悬液Suspension B晶型B crystal form
1212 丙酮:水＝4:1Acetone: water = 4:1 0.50.5 混悬液Suspension A晶型A crystal form
C晶型在不同溶剂中的稳定性试验Stability test of crystal form C in different solvents
取适量的C晶型多份，分别加入0.2mL的下表中的单一或混合溶剂，40℃条件下搅拌过夜。搅拌16小时后，若样品为溶液状态，自然挥发除去溶剂；若样品仍为混悬液，则离心样品。收集所有样品中的固体，XRPD检测其晶型状态。结果见表6。An appropriate amount of the C crystal form was added in portions, and 0.2 mL of the single or mixed solvent in the following table was added, and the mixture was stirred at 40 ° C overnight. After stirring for 16 hours, if the sample is in a solution state, the solvent is naturally volatilized; if the sample is still a suspension, the sample is centrifuged. The solids in all samples were collected and XRPD was tested for crystal form. The results are shown in Table 6.
表6 C晶型在不同溶剂中的稳定性实验Table 6 Stability of C crystal form in different solvents
序号Serial number 溶剂Solvent 外观(16hrs)Appearance (16hrs) 结果result
11 甲醇Methanol 自然挥发除去溶剂后析出固体Naturally volatile to remove solvent and precipitate solid C晶型C crystal form
22 正丁醇N-butanol 混悬液Suspension C晶型C crystal form
33 乙腈Acetonitrile 混悬液Suspension C晶型C crystal form
44 乙酸乙酯Ethyl acetate 混悬液Suspension C晶型 C crystal form
55 乙腈:水＝4:1Acetonitrile: water = 4:1 自然挥发除去溶剂后析出固体Naturally volatile to remove solvent and precipitate solid C晶型C crystal form
A晶型在高温，高湿及强光照条件下的固体稳定性试验Solid stability test of A crystal form under high temperature, high humidity and strong light conditions
称取A晶型样品约10mg，置于玻璃样品瓶的底部，摊成薄薄一层。60℃及92.5％相对湿度条件下放置的样品用铝箔纸封瓶口，并在铝箔纸上扎些小孔，保证样品能与环境空气充分接触；强光照(5Klux)条件下放置的样品用螺纹瓶盖密封。不同条件下放置的样品于第10天取样检测，检测结果与0 天的初始检测结果进行比较，试验结果见下表7所示：Approximately 10 mg of the A crystal form sample was weighed and placed on the bottom of the glass sample vial to form a thin layer. Samples placed at 60 ° C and 92.5% relative humidity were sealed with aluminum foil paper and small holes were placed on the aluminum foil to ensure that the sample was in full contact with ambient air; samples placed under strong light (5Klux) were threaded. The cap is sealed. Samples placed under different conditions were sampled and tested on the 10th day, and the test results were 0. The initial test results of the day are compared, and the test results are shown in Table 7 below:
表7化合物晶型I的固体稳定性试验Table 7 Solid Stability Test of Compound Form I
试验条件Test conditions 取样时间(天)Sampling time (days) 外观Exterior 含量(％)content(%) 总杂质(％)Total impurities (%)
-- 00 白色粉末White powder 98.998.9 0.100.10
60℃(敞口)60 ° C (open) 1010 白色粉末White powder 98.598.5 0.110.11
92.5％RH(敞口)92.5% RH (open) 1010 白色粉末White powder 99.599.5 0.100.10
强光照(密闭)Strong light (closed) 1010 白色粉末White powder 99.499.4 0.110.11
C晶型在不同温度和湿度条件下的物理稳定性试验Physical stability test of C crystal form under different temperature and humidity conditions
取C晶型置于玻璃样品瓶的底部，摊成薄薄一层，铝箔纸封瓶口，并在铝箔纸上扎些小孔，保证样品能与环境空气充分接触。制备的样品分别置于60℃，92.5％RH(相对湿度)，25℃/60％RH及40℃/75％RH的条件下，考察样品10天的物理稳定性。在第10天，取出所有样品，恢复至室温，观察样品外观变化，并用XRPD检测样品晶型。通过与0天样品的晶型进行比较，判断C晶型的固体物理稳定性。表8为C晶型的固体物理稳定性实验结果。Place the C crystal form on the bottom of the glass vial, spread it into a thin layer, seal the aluminum foil paper, and place some small holes on the aluminum foil to ensure that the sample can fully contact the ambient air. The prepared samples were placed at 60 ° C, 92.5% RH (relative humidity), 25 ° C / 60% RH and 40 ° C / 75% RH, respectively, and the physical stability of the samples was examined for 10 days. On day 10, all samples were taken, returned to room temperature, the appearance change of the samples was observed, and the crystal form of the samples was examined by XRPD. The solid physical stability of the C crystal form was judged by comparison with the crystal form of the 0 day sample. Table 8 shows the results of solid physical stability experiments of Form C.
表8 C晶型在不同温度和湿度条件下固体物理稳定性试验(10天)Table 8 Solid physical stability test of C crystal form under different temperature and humidity conditions (10 days)
Figure PCTCN2016099537-appb-000008
Figure PCTCN2016099537-appb-000008
C晶型在高温、高湿及强光照条件下的固体稳定性试验Solid stability test of C crystal form under high temperature, high humidity and strong light conditions
称取C晶型样品约18.75mg，置于玻璃样品瓶的底部，摊成薄薄一层。60℃及92.5％RH条件下放置的样品用铝箔纸封瓶口，并在铝箔纸上扎些小孔，保证样品能与环境空气充分接触，强光照(5Klux)条件下放置的样品用螺纹瓶盖密封。不同条件下放置的样品于第5和10天取样检测，检测结果与第0天的初始检测结果进行比较，试验结果见下表9所示：Approximately 18.75 mg of the C crystal form sample was weighed and placed on the bottom of the glass vial to form a thin layer. The samples placed at 60 ° C and 92.5% RH were sealed with aluminum foil paper, and small holes were placed on the aluminum foil paper to ensure that the sample could be in full contact with ambient air. The sample was placed under strong light (5Klux). The lid is sealed. Samples placed under different conditions were sampled on days 5 and 10, and the results were compared with the initial test results on day 0. The test results are shown in Table 9 below:
表9 C晶型固体稳定性试验Table 9 C crystal solid stability test
Figure PCTCN2016099537-appb-000009
Figure PCTCN2016099537-appb-000009
Figure PCTCN2016099537-appb-000010
Figure PCTCN2016099537-appb-000010
体外活性评价In vitro activity evaluation
实验目的：Purpose:
将具有Gli反应元件的萤光素酶(Gli-Luc)报告基因稳定转染到C3H10T1/2细胞，在Shh-N刺激条件下，通过检测细胞内报告基因表达情况从而判断Hedgehog信号通路抑制剂的活性。本实验以化合物的IC50值为指标，来评价化合物对Hedgehog信号通路的抑制作用。The luciferase (Gli-Luc) reporter gene with Gli response element was stably transfected into C3H10T1/2 cells, and the expression of Hedgehog signaling pathway inhibitor was determined by detecting the expression of intracellular reporter gene under Shh-N stimulation conditions. active. In this experiment, the IC50 value of the compound was used as an indicator to evaluate the inhibitory effect of the compound on the Hedgehog signaling pathway.
实验材料:Experimental Materials:
细胞系：C3H10T1/2/Gli-Luc稳转细胞株Cell line: C3H10T1/2/Gli-Luc stable cell line
C3H10T1/2/Gli-Luc细胞培养基(DMEM/高糖HyClone,#SH30022.01B；10％血清Hyclone,#SV30087；C3H10T1/2/Gli-Luc cell culture medium (DMEM/high glucose HyClone, #SH30022.01B; 10% serum Hyclone, #SV30087;
0.4％Hygromycin B Roche,#13398200)0.4% Hygromycin B Roche, #13398200)
0.25％Trypsin-EDTA(Gibco,#25200)0.25% Trypsin-EDTA (Gibco, #25200)
PBS(KH2PO4 0.24g，Na2HPO4 1.44g，NaCl 8.0g，KCl 0.2g，加水至1L调PH 7.4)PBS (KH 2 PO 4 0.24g, Na 2 HPO 4 1.44g, NaCl 8.0g, KCl 0.2g, add water to 1L to adjust pH 7.4)
Shh-N，HEK293/SHH-N稳转细胞培养上清Shh-N, HEK293/SHH-N stable cell culture supernatant
裂解液(Promega,#E1531)Lysate (Promega, #E1531)
反应液(Promega,#E1501)Reaction solution (Promega, #E1501)
384孔板，Greiner#781074384-well plate, Greiner #781074
96孔培养板，Greiner#65518096-well culture plate, Greiner #655180
96孔微孔板，碧云天#FPT01996-well microplate, Biyuntian #FPT019
CO2培养箱，Thermo#3423CO2 incubator, Thermo#3423
洁净工作台，AIRTECH#A10051560Clean workbench, AIRTECH#A10051560
倒置显微镜，Nikon#TS100Inverted microscope, Nikon#TS100
离心机，湘仪#L530Centrifuge, Xiangyi #L530
Therom VarioSkan Flash多功能读数仪Therom VarioSkan Flash Multi-Function Reader
实验步骤和方法Experimental steps and methods
取对数生长期C3H10T1/2/Gli-Luc细胞，在96孔板中按照每孔20000个细胞铺板，将细胞置37℃， 5％CO2的培养箱中培养过夜。次日，将化合物按下列方式进行稀释：阳性化合物GDC0449(1mM)和待测化合物(1mM)按1:3和1:10分别用DMSO进行连续系列稀释至7个化合物浓度，第8个为DMSO对照，再用新鲜培养基将其稀释100倍。取培养过夜的细胞，除去培养基，每孔加入80μL新鲜培养基，然后加入20μL梯度稀释过的阳性化合物和待测化合物，及100μL含30nM Shh-N的条件培养基，每个浓度做2个复孔，同时设置阳性和阴性参照孔(即Shh Ctrl:80μL新鲜培基+20μL含1％DMSO培基+100μL含30nM Shh-N的条件培养基；Unstimulated Ctrl:180μL新鲜培基+20μL含1％DMSO培基),细胞置CO2培养箱继续培养24小时。The logarithmic growth phase C3H10T1/2/Gli-Luc cells were plated in a 96-well plate at 20,000 cells per well, and the cells were cultured overnight at 37 ° C in a 5% CO 2 incubator. On the next day, the compounds were diluted as follows: the positive compound GDC0449 (1 mM) and the test compound (1 mM) were serially diluted to a concentration of 7 compounds in DMSO at 1:3 and 1:10, respectively, and the 8th was DMSO. Control, and then diluted 100 times with fresh medium. The cells were cultured overnight, the medium was removed, 80 μL of fresh medium was added to each well, and then 20 μL of the gradient-diluted positive compound and the test compound were added, and 100 μL of conditioned medium containing 30 nM Shh-N, 2 for each concentration. Double wells, set both positive and negative reference wells (ie Shh Ctrl: 80 μL fresh medium + 20 μL containing 1% DMSO medium + 100 μL conditioned medium containing 30 nM Shh-N; Unstimulated Ctrl: 180 μL fresh medium + 20 μL containing 1 The cells were cultured for 24 hours in a CO 2 incubator.
检测细胞内荧光素酶活性：取出96孔板细胞，弃去培养基，用PBS清洗细胞两遍，随后每孔加入20μL裂解液(Promega E1531)，室温振荡裂解30min。取5μL细胞裂解液转移至384孔板(Greiner 781074)中，随后每孔加25μL荧光素酶反应液(Promega E1501)，经快速混合后立即放进VarioSkan Flash多功能读数仪中读取相对光单位(RLU)值。Detection of intracellular luciferase activity: 96-well plate cells were removed, the medium was discarded, and the cells were washed twice with PBS, followed by addition of 20 μL of lysate (Promega E1531) per well, and cleavage at room temperature for 30 min. Transfer 5 μL of cell lysate to a 384-well plate (Greiner 781074), then add 25 μL of luciferase reaction solution (Promega E1501) to each well, and mix it immediately into the VarioSkan Flash multi-reader to read the relative light unit. (RLU) value.
数据分析：使用GraphPad公司的Prism 5进行数据分析。以对数化的化合物浓度对代表荧光素酶活性的RLU读数作图，并用下列方程进行曲线方程拟合得出IC50值，Y(RLU读数)＝最小读数+(最大读数-最小读数)/(1+10^(对数化化合物浓度-LogIC50))。Data analysis: Data analysis was performed using Prith 5 from GraphPad. The logarithmic concentration of the compound is plotted against the RLU reading representing luciferase activity and the curve equation is fitted to the IC 50 value, Y (RLU reading) = minimum reading + (maximum reading - minimum reading) / (1+10^ (logarithmic compound concentration - LogIC 50 )).
表10.化合物对hedgehog通路的抑制活力的IC50值或化合物在某浓度下的抑制率Table 10. IC50 values of inhibitory activity of compounds on the hedgehog pathway or inhibition of compounds at a certain concentration
受试化合物Test compound 生物活性Biological activity
化合物1Compound 1 AA
注：A≤50nM。Note: A ≤ 50nM.
结论：化合物1对hedgehog通路的抑制作用显著。 Conclusion: Compound 1 has a significant inhibitory effect on the hedgehog pathway.

Claims (22)

  1. 化合物1的A晶型，其XRPD图谱如图1所示，The crystal form of Compound A has an XRPD pattern as shown in Figure 1.
    Figure PCTCN2016099537-appb-100001
    Figure PCTCN2016099537-appb-100001
  2. 根据权利要求1所述化合物1的A晶型，其差示扫描量热曲线在111.41℃±2℃、126.08℃±2℃和146.06℃±2℃处具有吸热峰的起始点。The crystalline form A of Compound 1 according to claim 1, wherein the differential scanning calorimetry curve has a starting point of an endothermic peak at 111.41 ° C ± 2 ° C, 126.08 ° C ± 2 ° C and 146.06 ° C ± 2 ° C.
  3. 根据权利要求2所述化合物1的A晶型，其DSC图谱如图2所示。A crystalline form of Compound A according to claim 2, the DSC pattern of which is shown in Figure 2.
  4. 根据权利要求1所述化合物1的A晶型，其热重分析曲线在135.65±2℃失重0.7658％±0.2％。A crystal form of Compound 1 according to claim 1, wherein the thermogravimetric analysis curve has a weight loss of 0.7658% ± 0.2% at 135.65 ± 2 °C.
  5. 根据权利要求4所述化合物1的A晶型，其TGA图谱如图3所示。A crystal form of Compound 1 according to claim 4, the TGA spectrum of which is shown in FIG.
  6. 化合物1的B晶型，其XRPD图谱如图4所示。The B crystal form of Compound 1 has an XRPD pattern as shown in FIG.
  7. 根据权利要求6所述化合物1的B晶型，其差示扫描量热曲线在165.73℃±2℃处具有吸热峰的起始点。The crystalline form B of Compound 1 according to claim 6, wherein the differential scanning calorimetry curve has a starting point of an endothermic peak at 165.73 ° C ± 2 ° C.
  8. 根据权利要求7所述化合物1的B晶型，其DSC图谱如图5所示。The crystalline form B of Compound 1 according to claim 7, the DSC spectrum of which is shown in FIG.
  9. 根据权利要求6所述化合物1的B晶型，其热重分析曲线在120.00±2℃失重达0.2383％±0.2％，在247.60±2℃失重达0.2005％±0.2％。According to the crystal form B of the compound 1 according to claim 6, the thermogravimetric analysis curve has a weight loss of 0.2383%±0.2% at 120.00±2°C and a loss of 0.2005%±0.2% at 247.60±2°C.
  10. 根据权利要求6所述化合物1的B晶型，其TGA图谱如图6所示。The crystalline form B of Compound 1 according to claim 6, the TGA spectrum of which is shown in FIG.
  11. 根据权利要求6所述B晶型的制备方法，其包括将化合物1的A晶型加入到酯类溶剂、醇类溶剂或酯类溶解和醇类溶剂的混合溶剂中，加热至回流溶解，然后缓慢降温结晶制得。The method for preparing Form B according to claim 6, which comprises adding Form A of Compound 1 to a solvent mixture of an ester solvent, an alcohol solvent or an ester, and an alcohol solvent, heating to reflux to dissolve, and then Slowly cooled and crystallized.
  12. 根据权利要求11所述制备方法，其中所述酯类溶解选自甲酸丙酯、甲酸异丙酯、甲酸丁酯、甲酸异丁酯、乙酸乙酯、乙酸异丙酯、乙酸异丙酯；The preparation method according to claim 11, wherein the ester is dissolved and selected from the group consisting of propyl formate, isopropyl formate, butyl formate, isobutyl formate, ethyl acetate, isopropyl acetate, and isopropyl acetate;
    醇类溶剂选自甲醇、乙醇、正丙醇、异丙醇、正丁醇、叔丁醇。The alcohol solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, and tert-butanol.
  13. 根据权利要求11所述制备方法，其中，酯类溶剂和醇类溶剂的混合溶剂中，酯类溶剂和醇类溶剂的体积比为0.1:1～12，优选自1:2～8。The process according to claim 11, wherein the volume ratio of the ester solvent to the alcohol solvent in the mixed solvent of the ester solvent and the alcohol solvent is from 0.1:1 to 12, preferably from 1:2 to 8.
  14. 下式所示化合物2，Compound 2 shown in the following formula,
    Figure PCTCN2016099537-appb-100002
    Figure PCTCN2016099537-appb-100002
  15. 化合物2的C晶型，其XRPD图谱如图7所示。The C crystal form of Compound 2 has an XRPD pattern as shown in FIG.
  16. 根据权利要求15所述化合物2的C晶型，其差示扫描量热曲线在57.33℃±5℃和212.56℃±5℃处具有吸热峰的起始点。A crystalline form C of Compound 2 according to claim 15 having a differential scanning calorimetry curve having an onset of endotherms at 57.33 ° C ± 5 ° C and 212.56 ° C ± 5 ° C.
  17. 根据权利要求16所述化合物2的C晶型，其DSC图谱如图8所示。The crystalline form C of Compound 2 according to claim 16, the DSC spectrum of which is shown in FIG.
  18. 根据权利要求15所述化合物2的C晶型，其热重分析曲线在120.00±3℃失重达5.110％±0.5％。The crystal form of Compound 2 according to claim 15, wherein the thermogravimetric analysis curve has a weight loss of 5.10% ± 0.5% at 120.00 ± 3 °C.
  19. 根据权利要求18所述化合物2的C晶型，其TGA图谱如图9所示。The C crystal form of Compound 2 according to claim 18, the TGA spectrum of which is shown in FIG.
  20. 根据权利要求15所述C晶型的制备方法，其包括化合物1溶解到有机溶剂中，缓慢滴加硫酸水溶液，搅拌析晶制得。The method for preparing Form C according to claim 15, which comprises dissolving Compound 1 in an organic solvent, slowly adding an aqueous solution of sulfuric acid, and stirring and crystallization.
  21. 根据权利要求20所述制备方法，其中有机溶剂选自丙酮、甲乙酮、环己酮；The preparation method according to claim 20, wherein the organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, and cyclohexanone;
    化合物1与硫酸的摩尔比选自1:2～4。The molar ratio of Compound 1 to sulfuric acid is selected from 1:2 to 4.
  22. 根据权利要求1所述A晶型、权利要求6所述B晶型、权利要求15所述化合物2及权利要求15所述C晶型在制备治疗与SMO受体相关疾病药物上的应用。 The use of the crystalline form A according to claim 1, the crystalline form B according to claim 6, the compound 2 of claim 15 and the crystalline form C according to claim 15 for the preparation of a medicament for treating a disease associated with SMO receptors.
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