WO2022194225A1 - FXIa抑制剂化合物杂质及其制备方法和用途FXIa抑制剂化合物杂质及其制备方法和用途 - Google Patents
FXIa抑制剂化合物杂质及其制备方法和用途FXIa抑制剂化合物杂质及其制备方法和用途 Download PDFInfo
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- WO2022194225A1 WO2022194225A1 PCT/CN2022/081291 CN2022081291W WO2022194225A1 WO 2022194225 A1 WO2022194225 A1 WO 2022194225A1 CN 2022081291 W CN2022081291 W CN 2022081291W WO 2022194225 A1 WO2022194225 A1 WO 2022194225A1
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- inhibitor compound
- fxia inhibitor
- fxia
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- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229940068918 polyethylene glycol 400 Drugs 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
- 238000001303 quality assessment method Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/50—Pyridazines; Hydrogenated pyridazines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
- C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
- C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
- C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D237/14—Oxygen atoms
- C07D237/16—Two oxygen atoms
-
- 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
Definitions
- the invention belongs to the technical field of chemical medicines, and relates to FXIa inhibitor compound impurities and a preparation method and application thereof.
- FXIa is currently a key target for thrombus inhibition
- patent applications disclosing compounds with FXIa inhibitory activity include WO9630396, WO9941276, WO2013093484, WO2004002405, WO2013056060, WO2017005725, WO2017/023992, WO2018041122, etc.
- the present invention preliminarily establishes a detection method for the target impurities by preparing targeted impurities, and effectively controls the quality of the raw materials of the FXIa inhibitor compound and the pharmaceutical compositions.
- the present invention provides FXIa inhibitor compound impurities and preparation methods and uses thereof. Specifically, first the present invention provides FXIa inhibitor compound impurities, including impurity 1 and impurity 2:
- the invention provides a kind of FXIa inhibitor compound impurity 1, and its structure is as follows:
- the present invention also provides a kind of FXIa inhibitor compound impurity 2, and its structure is as follows:
- the present invention also provides a kind of FXIa inhibitor compound impurity 4, and its structure is as follows:
- impurity 2 and impurity 4 are a pair of diastereomers, and are specifically selected from:
- the present invention further provides a method for preparing the FXIa inhibitor compound impurity 1, which includes the following steps: synthesizing through the synthesis process of compound A, or obtaining through liquid phase separation of compound A raw materials, preferably through the preparation method of the embodiment.
- the present invention further provides a method for preparing the FXIa inhibitor compound impurity 2, which includes the following steps: synthesizing through the synthesis process of compound A, or obtaining through liquid phase separation of compound A raw materials, preferably through the preparation method of the embodiment.
- the present invention further provides a method for preparing the FXIa inhibitor compound impurity 4, comprising the following steps: synthesizing through the synthesis process of compound A, or obtaining through liquid phase separation of compound A raw materials, preferably through the preparation method of the embodiment.
- Another aspect of the present invention provides the use of the FXIa inhibitor compound impurities (including impurity 1, impurity 2, and impurity 4) as a reference substance for quality research of FXIa inhibitor compound APIs and pharmaceutical compositions thereof. It provides a basis for effective quality control of FXIa inhibitor compound APIs and pharmaceutical compositions.
- Beneficial results of the present invention include:
- New substances FXIa inhibitor compound impurity 1, impurity 2 and impurity 4 were prepared for the first time.
- the purity of the impurities met the requirements of quality research, and the related substances of compound A were accurately and quantitatively controlled, which can be used as reference substances for Quality research on APIs and pharmaceutical compositions containing them.
- Figure 1 shows the mass spectrum of FXIa inhibitor compound impurity 1
- FIG. 1 shows the hydrogen spectrum of FXIa inhibitor compound impurity 1
- FIG. 3 shows the carbon spectrum of FXIa inhibitor compound impurity 1
- FIG. 4 shows the mass spectrum of FXIa inhibitor compound impurity 2
- FIG. 5 shows the hydrogen spectrum of FXIa inhibitor compound impurity 2
- Figure 6 shows the carbon spectrum of the FXIa inhibitor compound Impurity 2.
- FIG. 7 shows the mass spectrum of FXIa inhibitor compound impurity 4.
- FIG. 8 shows the hydrogen spectrum of FXIa inhibitor compound impurity 4.
- FIG. 9 shows the carbon spectrum of FXIa inhibitor compound Impurity 4.
- Figure 10 shows the HPLC chromatograms of FXIa inhibitor compounds impurity 1, impurity 2 and impurity 4 used for quality assessment of compound A API after high temperature degradation.
- Step B Synthesis of 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3(2H)-one
- 2-bromo-4-chloroacetophenone (5.00 g, 21.41 mmol), pinacol biboronate (8.16 g, 32.12 mmol) and potassium acetate (4.20 g, 42.82 mmol) were added to three In the neck flask, nitrogen was replaced, 1,4-dioxane (60.0 mL) was added, nitrogen was replaced, and 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (1.75 g, 2.14 mmol) was added. , replaced nitrogen, and heated to 80 °C for 3 hours.
- Step D Synthesis of 5-(2-Acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3(2H)-one
- Step F Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- 3-Phenylpropionamido) tert-butyl benzoate
- Step G Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- 3-Phenylpropionamido)benzoic acid
- Enzyme Human Factor XIa (ENZYME RESEARCH, Cat. No. HFXIa 1111a)
- Buffer 145mM NaCl, 5mM KCl, 1mg/mL PEG 8000, 30mM HEPES, pH7.4
- 10 mM test compound in 100% DMSO was diluted with 100% DMSO to 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0.00128 ⁇ M; /mL) of FXIa enzyme solution, the blank wells were replaced by 98 ⁇ L of buffer, then 2 ⁇ L of compounds of different concentrations were added, the blank and control wells were replaced by DMSO, mixed with a shaker, and incubated at 37°C for 20 min.
- Plasma Human blood was collected in a vacuum blood collection tube containing 3.2% sodium citrate (volume ratio 1:9), centrifuged at 3000 rpm for 10 min at room temperature, plasma was collected, packaged in EP tubes, and stored at -80°C.
- APTT assay kit activated partial thromboplastin time assay kit, mindray
- calcium chloride solution calcium chloride solution
- hFXa Human Factor Xa: 71nkat.
- hFIIa HT5146L.
- hFVIIa Human Factor VIIa: hFVIIa 4591L.
- kallikrein LOT180223.
- Substrate S-2222 TM : CHROMOGENIX, NO864682.
- S-2238 TM CHROMOGENIX, NO770996.
- S-2288 TM CHROMOGENIX, NO378902. ADG302.
- hFXa buffer 100 mM NaCl, 5 mM CaCl2, 33% ethylene glycol, 50 mM Tris (pH 7.5).
- hFIIa buffer 0.145M NaCl, 0.005M KCl, 1 mg/ml PEG-8000, 0.030M HEPES (pH 7.4).
- hFVIIa buffer 0.145M NaCl, 0.005M KCl, 1 mg/ml PEG-8000, 0.030M HEPES (pH 7.4).
- kallikrein buffer 50 mM Tris, 50 mM imidazole and 150 mM NaCl (pH 8.2).
- 10mM test compound dissolved in 100% DMSO was diluted with 100% DMSO to 1000, 200, 40, 8, 1.6 ⁇ M; 98 ⁇ L of enzyme solution was added to each well in a 96-well plate, and 98 ⁇ L of buffer was added to blank wells, and then Add 2 ⁇ L of compounds of different concentrations, replace the blank and control wells with DMSO, mix with a shaker, and incubate at 37°C for 20 min.
- the concentrations of hFXa and S-2222 TM were FXa (1:28) and 800 ⁇ mol/L, respectively.
- the concentrations of hFIIa and S-2238 TM were hFIIa (0.06 U/ml) and 500 ⁇ mol/L, respectively.
- the concentrations of hFVIIa and S-2288 TM were hFVIIa (80 nM) and 1600 ⁇ mol/L, respectively.
- the concentrations of kallikrein and substrate were kallikrein (20 nM) and 1600 ⁇ mol/L, respectively.
- SD rats male, 180-250 g, purchased from Guangdong Medical Laboratory Animal Center.
- Cynomolgus monkey male, 4-6kg, purchased from Guangzhou Chunsheng Biological Research Institute Co., Ltd.
- Beagle male, 8-12kg, developed in Kanglong Chemical (Ningbo) New Drug Technology Co., Ltd.
- DMSO dimethyl sulfoxide
- PEG-400 polyethylene glycol 400
- physiological saline physiological saline
- heparin acetonitrile
- formic acid formic acid
- propranolol internal standard
- the compound was weighed and dissolved in DMSO-PEG-400-physiological saline (5:60:35, v/v/v) system, after intravenous or intragastric administration to rats/monkeys, 5min (gastrically not collected), After 15min, 30min, 1h, 2h, 4h, 6h, 8h, and 24h, 200 ⁇ L of venous blood was collected in a heparinized EP tube, centrifuged at 12000 rpm for 2 min, and the plasma was frozen at -80°C for testing. Precisely weigh a certain amount of the test sample and dissolve it in DMSO to 1 mg/mL as a stock solution.
- Chromatographic column Thermo Scientific HYPERSIL GOLD C-18 UPLC column, 100*2.1mm, 1.9 ⁇ m.
- WinNonlin 6.1 software was used to calculate pharmacokinetic parameters by non-compartmental model method. The results are shown in Tables 4, 5 and 6.
- CONCLUSION Compound A has a certain absorption in rats and monkeys orally. It is well absorbed in dogs, and its clearance rate is moderately slow. Most of the compounds have a long oral half-life and have good pharmacokinetic characteristics.
- DMEM (Corning), FBS (Corning), double antibody (Solarbio), 96-well HTS transwell plate (Corning), Caco-2 cells.
- HEK293-hERG stably transfected cell line invitrogen.
- DMEM medium Gabco
- HEPES invitrogen
- Blasticidin invitrogen
- HEK293-hERG stably transfected cells were cultured to a degree of polymerization of 40%-80% for experiments.
- Peak current inhibition (1-Peak tail current compound/Peak tail current vehicle)*100
- Compound A has a higher IC50 for hERG current and better cardiac safety.
- the compound of Example 143 of CN201680058331 refer to the preparation method of Example 143 of CN201680058331 to obtain the corresponding target compound.
- Example 1 The crude product obtained in Example 1 was prepared and separated using reverse-phase HPLC, and two enrichments of compound A impurity 1_1stS and compound A impurity 2_1stS were obtained respectively according to the retention time.
- retention time RT 20.947min is target compound A impurity 1
- RT 25.355min is target compound A impurity 2.
- Post-processing the fractions collected after separation are removed by rotary evaporator to remove acetonitrile, and then freeze-dried to obtain enrichment compound A impurity 1_1stS and enrichment compound A impurity 2_1stS.
- Reverse-phase HPLC was used to further purify the enriched compound A impurity 1_1stS of Example 2, and finally obtain the target compound A impurity 1 with qualified purity.
- Chromatographic column ChiralCel OJ, 300 ⁇ 50mm I.D., 10 ⁇ m
- Post-processing the fractions collected after separation are subjected to a rotary evaporator to obtain a compound A impurity 2_2ndS enrichment.
- Compound A impurity 2_2ndS was further purified using the SFC method to obtain compound A impurity 2_3rdS enrichment.
- Chromatographic column ChiralCel OJ, 300 ⁇ 50mm I.D., 10 ⁇ m
- Post-processing the fractions collected after separation are subjected to rotary evaporator to obtain compound A impurity 2_3rdS enrichment.
- the compound A impurity 2_3rdS enrichment was further purified by reverse-phase HPLC, and finally the target compound A impurity 2 with qualified purity was obtained.
- test sample compound A impurity 2 For the confirmation of the molecular structure of the test sample compound A impurity 2, high-resolution mass spectrometry, hydrogen nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy and other analytical methods were used for identification.
- Synthesis method Take 100g of the raw material drug in an oven, heat it to 100°C, and carry out separation and purification by liquid phase after 48 hours.
- Post-processing After separation, the obtained two target fractions were collected respectively, and rt0.98_1stS and rt1.15_1stS enrichment were obtained after freeze-drying.
- the enrichment rt0.98_1stS was further purified by reverse-phase HPLC, and finally the target impurity rt0.98 with qualified purity was obtained.
- Post-processing the fractions collected after separation are subjected to freeze-drying treatment to finally obtain the target impurity rt0.98.
- the enrichment rt1.15_1stS was further purified by reverse-phase HPLC, and finally the target impurity rt1.15 with qualified purity was obtained.
- Impurity 2 and Impurity 4 are a pair of diastereomers, and are specifically selected from one of the following structures:
- Embodiment 6 the application of the impurity obtained by the present invention in the research on the quality of the bulk drug
- Operation method Determination by high performance liquid chromatography in accordance with the 2020 edition of the Chinese Pharmacopoeia, general rule 0512.
- Test solution configuration Precisely weigh 250 mg of Compound A, put it in a 20 ml headspace bottle, add 2 mL of DMSO, dissolve, shake well, and seal with a cap.
- the detection wavelength was 254 nm
- the column temperature was 30 °C
- the flow rate was 0.4 ml/min
- the injection volume was 10 ⁇ l.
- the content of impurities was calculated based on the peak area of the FXIa inhibitor compound according to the external standard method.
- Example 6 Using the analytical method of Example 6, the compound A obtained by the method of Example 1 was placed at 40°C and 60°C for 10 days and 30 days to determine the content of impurity 1 and impurity 2.
- the HPLC spectrum is shown in Figure 10, and the results are as follows:
- Example 6 Using the analysis method of Example 6, the compound A obtained by the method of Example 1 was placed at 40 ° C and 60 ° C for 15 days and 30 days, and the content of impurity 4 was measured. The results are as follows:
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Abstract
FXIa抑制剂化合物杂质及其制备方法和用途,包括FXIa抑制剂化合物杂质1、杂质2、杂质4,还公开了杂质的制备方法和用途,提供的FXIa抑制剂化合物相关杂质及其制备方法为FXIa抑制剂化合物原料药及其药物组合物的质量研究提供了基础。
Description
本发明属于化学药物技术领域,涉及FXIa抑制剂化合物杂质及其制备方法和用途。
FXIa作为目前抑制血栓的重点靶点,公开具有FXIa抑制活性的化合物的专利申请有WO9630396、WO9941276、WO2013093484、WO2004002405、WO2013056060、WO2017005725、WO2017/023992、WO2018041122等。
申请人在前期申请PCT/CN2020/117257中申请了包括下式所示的化合物A(本发明所述FXIa抑制剂化合物)的一系列FXIa抑制剂化合物:
本发明预通过定向目标杂质制备,建立目标杂质的检测方法,对FXIa抑制剂化合物原料药及药物组合物的质量进行有效控制。
发明内容
鉴于现有技术的需求,本发明提供了FXIa抑制剂化合物杂质及其制备方法和用途。具体地,首先本发明提供了FXIa抑制剂化合物杂质,包括杂质1和杂质2:
本发明提供了一种FXIa抑制剂化合物杂质1,其结构如下:
本发明还提供了一种FXIa抑制剂化合物杂质2,其结构如下:
本发明还提供了一种FXIa抑制剂化合物杂质4,其结构如下:
注:±5°为旋光度的测定误差。
本发明进一步提供了所述FXIa抑制剂化合物杂质1的制备方法,包括如下步骤:通过化合物A的合成工艺合成得到,或者通过化合物A原料进行液相分离得到优选通过实施例的制备方法获得。
本发明进一步提供了所述FXIa抑制剂化合物杂质2的制备方法,包括如下步骤:通过化合物A的合成工艺合成得到,或者通过化合物A原料进行液相分离得到优选通过实施例的制备方法获得。
本发明进一步提供了所述FXIa抑制剂化合物杂质4的制备方法,包括如下步骤:通过化合物A的合成工艺合成得到,或者通过化合物A原料进行液相分离得到优选通过实施例的制备方法获得。
本发明另一方面提供了所述FXIa抑制剂化合物杂质(包括杂质1和杂质2、杂质4)作为FXIa抑制剂化合物原料药及其药物组合物的质量研究的对照品的用途。为FXIa抑制剂化合物原料药和药物组合物的质量进行有效控制提供了基础。
本发明的有益结果包括:
(1)首次获得了FXIa抑制剂化合物杂质1、杂质2、杂质4,并且高纯度的杂质1、杂质2、杂质4的制备具有相当难度,本发明通过多级分离方法,不断调整分离手段,才最终获得高纯度的杂质1、杂质2、杂质4;
(2)首次制备得到了新物质FXIa抑制剂化合物杂质1、杂质2、杂质4,杂质的纯度符合质量研究的要求,对化合物A的有关物质进行了准确定量控制,可以作为对照品用于的原料药及含有该药物的药物组合物的质量研究。
图1表示的是FXIa抑制剂化合物杂质1的质谱图;
图2表示的是FXIa抑制剂化合物杂质1的氢谱图;
图3表示的是FXIa抑制剂化合物杂质1的碳谱图;
图4表示的是FXIa抑制剂化合物杂质2的质谱图;
图5表示的是FXIa抑制剂化合物杂质2的氢谱图;
图6表示的是FXIa抑制剂化合物杂质2的碳谱图。
图7表示的是FXIa抑制剂化合物杂质4的质谱图;
图8表示的是FXIa抑制剂化合物杂质4的氢谱图;
图9表示的是FXIa抑制剂化合物杂质4的碳谱图。
图10表示的是FXIa抑制剂化合物杂质1、杂质2和杂质4用于化合物A原料药高温降解后质量评估的HPLC谱图。
下面通过本发明实施例和附图进行具体描述本发明的实施方案,但本发明不局限于此。
实施例1
合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
具体合成路线如下:
步骤A:合成5-溴-6-羟基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮
室温下,将溴马来酸酐(2.00克,11.3毫摩尔)和4-甲氧基苄基肼盐酸盐(2..13克,11.3毫摩尔)加入冰醋酸(50.0毫升)中,100℃反应3小时。
反应结束,冷却至室温,将反应液倒入水中,析出大量固体,搅拌一段时间后抽滤,滤饼用水洗,滤饼烘干得1.50克淡黄色固体5-溴-6-羟基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮,无需纯化,直接用于下步反应。LCMS:RT=3.44min,[M+H]
+=311.03。
步骤B:合成5-溴-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮
室温下,将5-溴-6-羟基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(1.50克,4.82毫摩尔)和碳酸钾(2.66克,19.29毫摩尔)加入N,N-二甲基甲酰胺(15.0毫升)中,80℃搅拌15分钟,在该温度下,加入碘甲烷(1.2毫升),继续反应30分钟。
反应结束,加水淬灭,混合液用乙酸乙酯(50毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(50毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂:乙酸乙酯/正己烷=1/3)。得到1.10克白色固体5-溴-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(收率:70.3%)。LCMS:RT=3.87min,[M+H]
+=325.01。
步骤C:合成6-乙酰基-3-氯苯硼酸频哪醇酯
室温下,将2-溴-4-氯苯乙酮(5.00克,21.41毫摩尔)、联硼酸频哪醇酯(8.16克,32.12毫摩尔)和醋酸钾(4.20克,42.82毫摩尔)加入三颈瓶中,置换氮气,加入1,4-二氧六环(60.0毫升),置换氮气,加入1,1'-双二苯基膦二茂铁二氯化钯(1.75克,2.14毫摩尔),置换氮气,升温至80℃反应3小时。
反应结束,加水淬灭,垫硅藻土抽滤,乙酸乙酯洗涤滤饼,滤液用乙酸乙酯(80毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(50毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂:乙酸乙酯/正己烷=1/50)。得到2.1克黄色固体6-乙酰基-3-氯苯硼酸频哪醇酯(收率:35.0%)。LCMS:RT=4.26min,[M-H]
-=279.08。
步骤D:合成5-(2-乙酰基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮
室温下,将5-溴-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(1.10克,3.39毫摩尔)、6-乙酰基-3-氯苯硼酸频哪醇酯(949毫克,3.39毫摩尔)和碳酸钠(718毫克,6.78毫摩尔)加入三颈瓶中,置换氮气,加入混合溶剂(10毫升,1,2-二甲氧基乙烷:乙醇:水=8:1:1),置换氮气,加入1,1'-双二苯基膦二茂铁二氯化钯(249毫克,0.34毫摩尔),置换氮气,升温至90℃反应1小时。
反应结束,加水淬灭,混合液用乙酸乙酯(50毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(50毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂:乙酸乙酯/正己烷=1/2)。得到676毫克黄色固体5-(2-乙酰基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(收率:50.2%)。LCMS:RT=3.99min,[M+H]
+=399.07。
步骤E:合成5-(2-乙酰基-5-氯苯基)-6-甲氧基哒嗪-3(2H)-酮
0℃下,将5-(2-乙酰基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(676毫克,1.70毫摩尔)加入混合溶剂(4毫升,乙腈:水=3:1)中,再缓慢加入硝酸铈铵(7.46克,13.60毫摩尔),加毕,室温下反应30分钟。
反应结束,加水淬灭,混合液用乙酸乙酯(30毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(30毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂:乙酸乙酯/正己烷=1/1)。得到238毫克黄色固体5-(2-乙酰基-5-氯苯基)-6-甲氧基哒嗪-3(2H)-酮(收率:50.0%)。LCMS:RT=3.23min,[M+H]
+=279.08。
步骤F:合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯
室温下,将5-(2-乙酰基-5-氯苯基)-6-甲氧基哒嗪-3(2H)-酮(50毫克,0.18毫摩尔)、(R)-4-(2-(((4-硝基苯基)磺酰基)氧基)-3-苯基丙酰胺基)苯甲酸叔丁酯(113毫克,0.22毫摩尔)和碳酸钾(50毫克,0.36毫摩尔)加入N,N-二甲基甲酰胺(2.0毫升)中,室温反应过夜。
反应结束,加水淬灭,混合液用乙酸乙酯(10毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(10毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂:乙酸乙酯/正己烷=1/2)。得到75毫克淡黄色固体(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(收率:66.7%)。LCMS:RT=4.53min,[M+H]
+=602.13。
步骤G:合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
室温下,将(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(75毫克,0.12毫摩尔)加入二氯甲烷(2.0毫升)中,滴加三氟乙酸(0.25毫升),室温反应3小时。
反应结束,蒸干二氯甲烷并用油泵抽干三氟乙酸,所得残余物用溶于二氯甲烷(1.0毫升)中,将其滴加入正己烷(10.0毫升)中,析出白色固体,抽滤,滤饼用正己烷洗涤,干燥得到50毫克白色固体(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3- 甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸(收率:76.5%,化合物A)。
LCMS:RT=3.98min,[M-H]
-=544.10。
1H NMR(500MHz,DMSO)δ12.79(s,1H),10.52(s,1H),7.99(d,J=8.4Hz,1H),7.91(d,J=8.7Hz,2H),7.72(d,J=8.7Hz,2H),7.69(dd,J=8.3,2.1Hz,1H),7.50(d,J=2.1Hz,1H),7.37–7.23(m,4H),7.19(t,J=7.1Hz,1H),6.91(s,1H),5.74(dd,J=10.2,4.9Hz,1H),3.67(s,3H),3.52(dd,J=14.1,10.3Hz,1H),3.41(dd,J=14.1,4.7Hz,1H),2.53(s,3H)。
化合物A的生物活性考察:
(A):吸收光法检测化合物A对人凝血因子XIa抑制的生物活性
1、实验材料
酶:Human Factor XIa(ENZYME RESEARCH,货号HFXIa 1111a)
底物:S-2366
TM:(CHROMOGENIX,货号82109039)
缓冲液:145mM NaCl,5mM KCl,1mg/mL PEG 8000,,30mM HEPES,pH7.4
2、实验步骤
将溶于100%DMSO的10mM受试化合物用100%DMSO稀释至1000、200、40、8、1.6、0.32、0.064、0.0128、0.00256、0.00128μM;在96孔板中每孔加入98μL(77.7ng/mL)的FXIa酶溶液,空白孔加入98μL缓冲液代替,再加入2μL不同浓度的化合物,空白和对照孔用DMSO代替,用振荡器混匀,37℃孵育20min。
最后每孔加入800μM的底物100μL,在405nm处测其吸光度。
3、数据处理
用GraphPad Prism软件进行曲线拟合,计算IC
50值,见表一。
表一.化合物A的对人FXIa抑制的IC
50
hFXIa IC 50(nM) | |
化合物A | 7.61 |
结论:化合物A的对人FXIa具有明显的抑制活性。
(B):化合物A对人血浆体外抗凝血作用的测定
1、实验材料
血浆:人血收集于含3.2%柠檬酸钠(体积比1:9)的真空采血管中,室温3000rpm离心10min,收集血浆,分装在EP管中,-80℃保存。
试剂:APTT测定试剂盒(活化部分凝血活酶时间检测定剂盒,mindray)、氯化钙溶液。
仪器:凝血仪(mindray,C2000-A)
2、实验方法
取分装的冻存人血浆室温融化后,混合均匀。将溶于100%DMSO的10mM受试化合物用100%DMSO稀释至1500、750、375、187.5、93.75、46.88、23.44、11.72μM;在1.5mL EP管中加入98μL人血浆,再加入2μL不同浓度的化合物,空白组加入2μL 100%DMSO,37℃水浴孵育10min,将样品放入凝血仪中对应的位置,进行化合物的APTT测定。
3、数据处理
用GraphPad Prism软件进行曲线拟合,分别计算EC1.5×和EC2×值,即1.5倍和2倍空白对照组的APTT所对应的化合物的浓度,结果见表二。
表二.化合物A的对人血浆体外抗凝血作用
结论:从表二中可以看出化合物A的对人血浆具有明显的抗凝血作用。
(C):化合物A对凝血因子选择性考察
1、实验材料
酶:hFXa:Human Factor Xa:71nkat。hFIIa:HT5146L。hFVIIa:Human Factor VIIa:hFVIIa 4591L。kallikrein:LOT180223。
底物:S-2222
TM:CHROMOGENIX,NO864682。S-2238
TM:CHROMOGENIX,NO770996。S-2288
TM:CHROMOGENIX,NO378902。ADG302。
缓冲液:
hFXa缓冲液:100mM NaCl,5mM CaCl2,33%ethylene glycol,50mM Tris(pH 7.5)。
hFIIa缓冲液:0.145M NaCl,0.005M KCl,1mg/ml PEG-8000,0.030M HEPES(pH 7.4)。
hFVIIa缓冲液:0.145M NaCl,0.005M KCl,1mg/ml PEG-8000,0.030M HEPES(pH 7.4)。
kallikrein缓冲液:50mM Tris,50mMimidazole and 150mM NaCl(pH 8.2)。
2、实验步骤
将溶于100%DMSO的10mM受试化合物用100%DMSO稀释至1000、200、40、8、1.6μM;在96孔板中每孔加入98μL的酶溶液,空白孔加入98μL缓冲液代替,再加入2μL不同浓度的化合物,空白和对照孔用DMSO代替,用振荡器混匀,37℃孵育20min。
hFXa和S-2222
TM的浓度分别为FXa(1:28)和800μmol/L。hFIIa和S-2238
TM的浓度分别为hFIIa(0.06U/ml)和500μmol/L。hFVIIa和S-2288
TM的浓度分别为hFVIIa(80nM)和1600μmol/L。kallikrein和底物的浓度分别为kallikrein(20nM)和1600μmol/L。
最后每孔加入底物100μL,在405nm处测其吸光度。
3、数据处理
用GraphPad Prism软件进行曲线拟合,计算IC
50值,见表三。
表三.化合物A的对凝血因子选择性考察
结论:化合物A的对其它凝血因子选择性较好。
(D):化合物A的药代动力学特征考察
1、实验材料
SD大鼠:雄性,180-250g,购于广东省医学实验动物中心。食蟹猴:雄性,4-6kg,购于广州春盛生物研究院有限公司。比格犬:雄性,8-12kg,在康龙化成(宁波)新药技术股份有限公司开展。
试剂:DMSO(二甲亚砜),PEG-400(聚乙二醇400),生理盐水,肝素,乙腈,甲酸,普萘洛尔(内标)均为市售可得。
仪器:赛默飞LC-MS(U300UPLC,TSQ QUANTUMN ULTRA三重四级杆质谱)。
2、实验方法
称取化合物溶于DMSO-PEG-400-生理盐水(5:60:35,v/v/v)体系中,大鼠/猴静脉或灌胃给药后,于5min(灌胃不采)、15min、30min、1h、2h、4h、6h、8h、24h采集静脉血200μL于肝素化EP管中,12000rpm离心2min,取血浆-80℃冻存待测。精密称取一定量供试品用DMSO溶解至1mg/mL,作为储备液。准确吸取适量的化合物储备液,加入乙腈稀释制成标准系列溶液。准确吸取上述标准系列溶液各20μL,加入空白血浆180μL,涡旋混匀,配制成相当于血浆浓度为1、3、10、30、100、300、1000、3000和5000ng/mL的血浆样品,每一浓度进行双样本分析,建立标准曲线。取20μL血浆,加入内标普萘洛尔(5ng/mL)的乙腈溶液200μL,涡旋混匀后4000rpm离心5min,取上清LC-MS分析。LC-MS检测条件如下:
色谱柱:赛默飞HYPERSIL GOLD C-18 UPLC柱,100*2.1mm,1.9μm。
流动相:水(0.1%甲酸)-乙腈按下表进行梯度洗脱
时间(min) | 水(含0.1%甲酸) | 乙腈 |
0 | 90% | 10% |
0.6 | 90% | 10% |
1 | 10% | 90% |
2.6 | 10% | 90% |
2.61 | 90% | 10% |
4 | 90% | 10% |
3、数据处理
LC-MS检测血药浓度后,采用WinNonlin 6.1软件,非房室模型法计算药动学参数。结果见表四、五、六。
表四.化合物A的的大鼠药代动力学参数
表五.化合物A的的食蟹猴药代动力学参数
表六.化合物A的的比格犬药代动力学参数
结论:化合物A的在大鼠和猴口服均有一定的吸收,犬口服吸收较好,体内清除速率中等偏慢,多数化合物口服半衰期较长,具有良好的药代动力学特征。
(E):化合物A的caco-2数据考察
实验材料:
培养基:DMEM(Corning),FBS(Corning),双抗(Solarbio),96-well HTS transwell plate(Corning),Caco-2细胞。
实验方法:Caco-2细胞在96-well HTS transwell plate培养14-18天后,检测每孔TEER值确保每孔细胞形成完整单层,加药物孵育2h,检测A-B和B-A药物浓度。
数据处理:计算PappA-B和PappB-A值,Papp=(VA×[drug]acceptor)/(Area×Time×[drug]initial,donor),计算Efflux Ratio,Efflux Ratio=Papp(B-A)/Papp(A-B)。
表七.化合物A的caco-2数据
结论:化合物A的膜通透性良好。
(F):化合物A的CYP酶抑制考察
实验材料:
肝微粒体(150-donor,Corning,Cat.452117;Lot.38292),NADPH.
实验方法:
先配制0.2mg/mL微粒体体系,加入各测试物及底物,测试物终浓度为50μM,预孵8min后,加入10mM NADPH启动反应,NADPH最终浓度为1mM,孵育一段时间后加如甲醇内标终止反应。检测各反应孔中底物代谢物生成量。
数据处理:以空白孔代谢物生成量为100%,计算每个测试物孔中代谢物生成减少量,并计算抑制率。
表八.化合物A的CYP酶抑制数据
结论:化合物A的对主要CYP酶无抑制,DDI风险较小。
(G):化合物A的hERG考察
实验材料:
HEK293-hERG稳转细胞系(invitrogen)。DMEM培养基(Gibco),HEPES(invitrogen),Blasticidin(invitrogen)
实验方法:
HEK293-hERG稳转细胞培养至40%-80%聚合度时用于实验,首先用空白溶媒应用到细胞中,建立基线。一旦发现hERG电流稳定5分钟后,开始测试化合物。在测试化合物存在下,记录大约5分钟的hERG电流以达到稳定状态,然后捕获5个扫频。为了保证培养细胞和操作的良好性能,同样使用阳性对照多非利酮对同一批次细胞进行检测。
数据处理:
Peak current inhibition=(1-Peak tail current compound/Peak tail current vehicle)*100
表九.化合物A的hERG实验数据
实施例 | hERG IC50[μM] | Comment |
1 | >10 | 10μM下抑制率为1.17% |
结论:化合物A对hERG电流IC50较高,心脏安全性较好。
注:化合物A1:(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
化合物B:(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2H)-基)-3-苯基丙酰胺基)苯甲酸
CN201680058331实施例143化合物:参照CN201680058331实施例143的制备方法获得相应目标化合物。
实施例2
使用反相HPLC制备分离实施例1得到粗品,根据保留时间分别得到化合物A杂质1_1stS和化合物A杂质2_1stS两部分富集物。
其中,保留时间RT 20.947min为目标物化合物A杂质1,RT 25.355min为目标物化合物A杂质2。
仪器:Gilson GX-281制备收集器,Gilson 322液相泵,Gilson 156紫外检测器
色谱柱:YMC-Actus Triart C18,150×30.0mmI.D.S-5μm
流动相:A:水(0.225%甲酸(v/v)),B:乙腈
流速:25mL/min
柱温:25℃
波长:254nm&220nm
梯度:
时间(min) | B% |
0 | 45 |
2.0 | 45 |
20.0 | 63 |
20.2 | 95 |
24.2 | 100 |
24.4 | 45 |
样品溶解:将化合物A粗品用DMF溶解
进样体积:0.5mL/每针
后处理:分离后收集得到的馏份经旋转蒸发仪除去乙腈,再冷冻干燥后得到富集物化合物A杂质1_1stS和富集物化合物A杂质2_1stS。
实施例3 FXIa抑制剂化合物杂质1的进一步制备
使用反相HPLC进一步纯化实施例2富集物化合物A杂质1_1stS,最终获得纯度合格的化合物A杂质1目标物。
仪器:Gilson GX-281制备收集器,Gilson 322液相泵,Gilson 156紫外检测器
色谱柱:YMC-Actus Triart C18,150×30.0mmI.D.S-5μm
流动相:A:水(0.225%甲酸(v/v)),B:乙腈
流速:25mL/min
柱温:25℃
波长:254nm&220nm
梯度:
时间(min) | B% |
0 | 45 |
2.0 | 45 |
20.0 | 63 |
20.2 | 95 |
24.2 | 100 |
24.4 | 45 |
样品溶解:将富集物化合物A杂质1_1stS用DMF溶解
进样体积:1.0mL/每针
后处理:分离后收集得到的馏份经冷冻干燥后,得到目标物化合物A杂质1。
对于供试样品化合物A杂质1的分子结构确证,采用了高分辨质谱、核磁共振氢谱、核磁共振碳谱等分析方法鉴别。
样品结构式:
分子式:C
28H
22ClN
3O
6
分子量:531.95
a)质谱
质谱解析:
供试样品化合物A杂质1+ESI高分辨质谱图,如图1所示,所显示的m/z=554.1264元素组成为C
28H
22ClN
3O
6Na与供试样品的相对分子量理论值554.1095(M+Na)相符。
b)核磁共振波谱
溶剂:DMSO-d
6
送检样品化合物A杂质1的核磁共振氢谱、核磁共振碳谱分别如图2和图3所示。
实施例4 FXIa抑制剂化合物杂质2的进一步制备
4.1使用SFC方法进一步纯化实施例2化合物A杂质2_1stS,得到化合物A杂质2_2ndS富集物。
仪器:Thar 350 preparative SFC(SFC-7)
色谱柱:ChiralCel OJ,300×50mm I.D.,10μm
流动相:A:二氧化碳,B:乙醇
流速:200mL/min
柱温:38℃
波长:220nm
梯度:40%乙醇等度
样品溶解:将富集物化合物A杂质2_1stS用TEE/DCM溶解
进样体积:5.0mL/每针
后处理:分离后收集得到的馏份经旋转蒸发仪后,得到化合物A杂质2_2ndS富集物。
4.2杂质2的进一步制备分离
使用SFC方法进一步纯化化合物A杂质2_2ndS,得到化合物A杂质2_3rdS富集物。
仪器:Thar 350 preparative SFC(SFC-7)
色谱柱:ChiralCel OJ,300×50mm I.D.,10μm
流动相:A:二氧化碳,B:乙醇
流速:200mL/min
柱温:38℃
波长:220nm
梯度:40%乙醇等度
样品溶解:将富集物化合物A杂质2_2ndS用TEE/DCM溶解
进样体积:5.0mL/每针
后处理:分离后收集得到的馏份经旋转蒸发仪后,得到化合物A杂质2_3rdS富集物。
4.3杂质2的进一步制备分离
使用反相HPLC进一步纯化化合物A杂质2_3rdS富集物,最终获得纯度合格的化合物A杂质2目标物。
仪器:Gilson GX-281制备收集器,Gilson 322液相泵,Gilson 156紫外检测器
色谱柱:YMC-Actus Triart C18,150×30.0mmI.D.S-5μm
流动相:A:水(0.225%甲酸(v/v)),B:乙腈
流速:25mL/min
柱温:25℃
波长:254nm&220nm
梯度:
时间(min) | B% |
0 | 45 |
2.0 | 45 |
20.0 | 63 |
20.2 | 95 |
24.2 | 100 |
24.4 | 45 |
样品溶解:将富集物化合物A杂质2_3rdS用DMF溶解
进样体积:1.0mL/每针
后处理:分离后收集得到的馏份经冷冻干燥后,得到化合物A杂质2目标物。
对于供试样品化合物A杂质2的分子结构确证,采用了高分辨质谱、核磁共振氢谱、核磁共振碳谱等分析方法鉴别。
样品结构式:
分子式:C
29H
24ClN
3O
6
分子量:545.98
a)质谱
质谱解析:
供试样品化合物A杂质2+ESI高分辨质谱图,谱图如图4所示,所显示的m/z=568.1202元素组成为C
29H
24ClN
3O
6Na与供试样品的相对分子量理论值568.1251(M+Na)相符。
b)核磁共振波谱
溶剂:DMSO-d
6
送检样品化合物A杂质1的核磁共振氢谱、核磁共振碳谱分别如图5和图6所示。
实施例5杂质4的分离制备
合成方法:取100g原料药至于烘箱中,加热至100℃,48h后通过液相进行分离纯化。
5.1粗品的制备分离
使用反相HPLC制备分离粗品,获得rt0.98_1stS和rt1.15_1stS富集物。
仪器:吉尔森215系列,322溶剂泵,156检测器
色谱柱:Xtimate C18,150×25.0mm,5μm
流动相:A:水(0.05%氨水,v/v),B:乙腈
流速:25mL/min
柱温:25℃
波长:220nm&254nm
梯度:
时间(min) | B% |
0 | 5 |
2 | 5 |
22 | 35 |
22.2 | 95 |
25.2 | 100 |
25.4 | 5 |
26.4 | 5 |
样品溶解:将粗品用纯水溶解
进样体积:5.0mL/每针
后处理:分离后分别收集得到的两部分目标馏份,经冷冻干燥后得到rt0.98_1stS和rt1.15_1stS富集物。
5.2第二遍制备分离
5.2.1 rt0.98的第二遍制备分离
使用反相HPLC进一步纯化富集物rt0.98_1stS,最终获得纯度合格的目标杂质rt0.98。
仪器:吉尔森215系列,322溶剂泵,156检测器
色谱柱:Xtimate C18,150×25.0mm,5μm
流动相:A:水(0.05%氨水,v/v),B:乙腈
流速:25mL/min
柱温:25℃
波长:220nm&254nm
梯度:
时间(min) | B% |
0 | 5 |
2 | 5 |
18 | 29 |
18.2 | 95 |
20.2 | 100 |
20.4 | 5 |
21.1 | 5 |
样品溶解:将富集物rt0.98_1stS用10%乙腈-水(0.5%氨水,v/v)溶解
进样体积:5.0mL/每针
后处理:分离后收集得到的馏份经冷冻干燥处理后,最终得到目标杂质rt0.98。
5.2.2 rt1.15的第二遍制备分离
使用反相HPLC进一步纯化富集物rt1.15_1stS,最终获得纯度合格的目标杂质rt1.15。
仪器:吉尔森215系列,322溶剂泵,156检测器
色谱柱:Gemini NX-C18 C18,150×30.0mm,5μm
流动相:A:水(0.075%三氟乙酸,v/v),B:乙腈
流速:25mL/min
柱温:25℃
波长:220nm&254nm
梯度:
时间(min) | B% |
0 | 53 |
2 | 53 |
12 | 77 |
12.2 | 95 |
14.2 | 100 |
14.4 | 5 |
15.4 | 5 |
样品溶解:将富集物rt1.15_1stS用10%乙腈-水(0.5%氨水,v/v)溶解
进样体积:1.5mL/每针
后处理:分离后收集得到的馏份经冷冻干燥处理后,最终得到目标杂质rt1.15。
5.3结构鉴定
对于供试样品杂质4的分子结构确证,采用了高分辨质谱、核磁共振氢谱、核磁共振碳谱等分析方法鉴别。
样品结构式:
分子式:C
29H
24ClN
3O
6
分子量:545.98
a)质谱
质谱解析:
供试样品杂质4-ESI高分辨质谱图(图10)所显示的m/z=544.2341元素组成为C
29H
23ClN
3O
6与供试样品的相对分子量理论值544.1275(M-H)相符。
b)核磁共振波谱
溶剂:DMSO-d
6
核磁共振谱图解析:
送检样品化合物A杂质4的核磁共振氢谱、核磁共振碳谱分别如图8和图9所示。
杂质2和杂质4为一对非对映异构体,具体选自以下结构之一:
实施例6,本发明获得的杂质在原料药质量研究中的应用
操作方法:按照《中国药典》2020年版,通则0512高效液相色谱法测定。
供试品溶液配置:精密称取化合物A 250mg,置20ml顶空瓶,加入DMSO 2mL,溶解,摇匀,压盖密封。
色谱条件:
检测波长254nm,柱温:30℃,流速0.4ml/min,进样量10μl。
流动相:流动相A:甲酸-乙腈-水体积比=1:50:950,流动相B:四氢呋喃:乙腈体积比=1:4,梯度洗脱条件:
按外标法以FXIa抑制剂化合物峰面积计算杂质含量。
按上述色谱条件,FXIa抑制剂化合物与杂质之间分离度好,各杂质理论板数和拖尾因子符合要求,分析时间短,效率高,节约成本,满足《中国药典》的要求,可以有效确定FXIa抑制剂化合物原料和药物组合物的质量。
实施例7原料药稳定性考察
采用实施例6的分析方法,将实施例1方法获得的化合物A在40℃和60℃放置10天和30天后进行杂质1和杂质2含量测定,HPLC谱图如图10所示,结果如下:
通过本实施例考察发现,化合物A的原料药在保存过程中,杂质1和杂质2受高温影响,含量存在一定量的增加,所以,对于该两杂质进行分析检测,对于控制化合物A原料药的质量至关重要。
实施例8原料药稳定性考察
采用实施例6的分析方法,将实施例1方法获得的化合物A在40℃和60℃放置15天和30天后进行杂质4含量测定,结果如下:
通过本实施例考察发现,化合物A的原料药在保存过程中,杂质4受高温影响,含量存在一定量的增加,所以,对于该两杂质进行分析检测,对于控制化合物A原料药的质量至关重要。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作为各种各样的改变,而不偏离所附权利要求所限定的本发明的精神和范围。
Claims (10)
- 根据权利要求1所述FXIa抑制剂化合物杂质1的制备方法,其特征在于,包括如下步骤:通过化合物A的合成工艺合成得到,或者通过化合物A原料进行液相分离得到。
- 根据权利要求2所述FXIa抑制剂化合物杂质2的制备方法,其特征在于,包括如下步骤:通过化合物A的合成工艺合成得到,或者通过化合物A原料进行液相分离得到。
- 根据权利要求3所述FXIa抑制剂化合物杂质4的制备方法,其特征在于,包括如下步骤:通过化合物A的合成工艺合成得到,或者通过化合物A原料进行液相分离得到。
- 如权利要求1所述FXIa抑制剂化合物杂质1作为FXIa抑制剂化合物原料药及其药物组合物的质量研究的对照品的用途。
- 如权利要求2所述FXIa抑制剂化合物杂质2作为FXIa抑制剂化合物原料药及其药物组合物的质量研究的对照品的用途。
- 如权利要求3所述FXIa抑制剂化合物杂质4作为FXIa抑制剂化合物原料药及其药物组合物的质量研究的对照品的用途。
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