WO2022213335A1 - Procédé de préparation d'un intermédiaire d'un inhibiteur de bcl-2 - Google Patents

Procédé de préparation d'un intermédiaire d'un inhibiteur de bcl-2 Download PDF

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Publication number
WO2022213335A1
WO2022213335A1 PCT/CN2021/086044 CN2021086044W WO2022213335A1 WO 2022213335 A1 WO2022213335 A1 WO 2022213335A1 CN 2021086044 W CN2021086044 W CN 2021086044W WO 2022213335 A1 WO2022213335 A1 WO 2022213335A1
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WIPO (PCT)
Prior art keywords
fluoro
pyrrolo
bcl
pyridin
oxy
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PCT/CN2021/086044
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English (en)
Inventor
Weihua Sun
Si CHEN
Hai Xue
Yunhang GUO
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Beigene (Beijing) Co., Ltd.
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Priority to PCT/CN2021/086044 priority Critical patent/WO2022213335A1/fr
Priority to CN202180095624.4A priority patent/CN116981668A/zh
Publication of WO2022213335A1 publication Critical patent/WO2022213335A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to a method for preparing an intermediate of a Bcl-2 inhibitor, in particular to a method for preparing a compound of formula (II) which is methyl 4-fluoro-2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate.
  • BCL-2 The B cell lymphoma 2 (BCL-2) gene family, a group of proteins homologous to the Bcl-2 protein, encodes more than 20 proteins that regulate the intrinsic apoptosis pathway. Targeting to Bcl-2, a number of small-molecule BH3 mimetics have been reported.
  • the compound of Formula (I) is also described as a Bcl-2 wild type and Bcl-2 G101V mutation inhibitor.
  • the present invention is aim at providing a new preparation method that can increase the yield of the intermediate of a Bcl-2 inhibitor and the yield of the Bcl-2 inhibitor.
  • the method comprises the step of reacting 5- (5-fluoro-2- (methoxycarbonyl) phenoxy) -1H-pyrrolo [2, 3-b] pyridine-3-carboxylic acid with a fluorine reagent in a first solvent at a first reaction temperature to obtain the intermediate of formula (II) .
  • the method further comprises the steps of reacting methyl 4-fluoro-2- ( (3- (2, 2, 2-trichloroacetyl) -1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate with an alkali metal hydroxide in a second solvent at a second reaction temperature, and acidifying to obtain the 5- (5-fluoro-2- (methoxycarbonyl) phenoxy) -1H-pyrrolo [2, 3-b] pyridine-3-carboxylic acid.
  • the method even further comprises the step of reacting methyl 2- ( (1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) -4-fluorobenzoate, aluminum halide and 2, 2, 2-trichloroacetyl chloride in a third solvent at a third reaction temperature to obtain the methyl 4-fluoro-2- ( (3- (2, 2, 2-trichloroacetyl) -1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate.
  • sodium carbonate is added to the 5- (5-fluoro-2- (methoxycarbonyl) phenoxy) -1H-pyrrolo [2, 3-b] pyridine-3-carboxylic acid.
  • TEA was added during the preparation of 5- (5-fluoro-2- (methoxycarbonyl) phenoxy) -1H-pyrrolo [2, 3-b] pyridine-3-carboxylic acid.
  • the first solvent contains ethyl acetate and water.
  • the first temperature is -10°C to 10°C. In preferred embodiments, the first temperature is -5°Cto 5°C. In more preferred embodiments, the first temperature is about 0°C.
  • the fluorine reagent is 1-chloromethyl-4-fluoro-1, 4-diazoniabicyclo [2.2.2] octane bis (tetrafluoroborate) . In some embodiments, the fluorine reagent is added portion wise.
  • the first solvent is consisted of ethyl acetate and water. In preferred embodiments, the volume ratio of ethyl acetate and water is 2: 1.
  • the aluminum halide is AlCl 3 ⁇ AlBr 3 or AlI 3 . In preferred embodiments, the aluminum halide is AlCl 3 .
  • the alkali metal hydroxide is NaOH or KOH. In preferred embodiments, the alkali metal hydroxide is NaOH.
  • the second solvent comprises tetrahydrofuran and water. In some embodiments, the second temperature is room temperature.
  • the third solvent contains dichloromethane. In some embodiments, the third temperature is -10°C to 10°C. In preferred embodiments, the third temperature is -5°Cto 5°C. In more preferred embodiments, the third temperature is about 0°C.
  • the first solvent contains ethyl acetate and water.
  • the first temperature is -10°C to 10°C. In preferred embodiments, the first temperature is -5°Cto 5°C. In more preferred embodiments, the first temperature is about 0°C.
  • the fluorine reagent is 1-chloromethyl-4-fluoro-1, 4-diazoniabicyclo [2.2.2] octane bis (tetrafluoroborate) . In some embodiments, the fluorine reagent is added portion wise.
  • the first solvent is consisted of ethyl acetate and water. In preferred embodiments, the volume ratio of ethyl acetate and water is 2: 1.
  • the aluminum halide is AlCl 3 ⁇ AlBr 3 or AlI 3 . In preferred embodiments, the aluminum halide is AlCl 3 .
  • the alkali metal hydroxide is NaOH or KOH. In preferred embodiments, the alkali metal hydroxide is NaOH.
  • the second solvent comprises tetrahydrofuran and water. In some embodiments, the second temperature is room temperature.
  • the third solvent contains dichloromethane. In some embodiments, the third temperature is -10°C to 10°C. In preferred embodiments, the third temperature is -5°Cto 5°C. In more preferred embodiments, the third temperature is about 0°C.
  • the method of the present invention adopts a new synthesis process (reaction conditions and reaction raw materials as described above) , thereby obtaining the intermediate product of the compound of formula (II) and the final product with a higher yield.
  • the whole preparation method has good repeatability, is suitable for industrialized large-scale production, and has good economic value.
  • the temperature is in °C.
  • Reagents are purchased from commercial providers such as Sigma-Aldrich, Alfa Aesar or TCI, and can be used without further purification unless otherwise stated. If the specific conditions are not specified in the following, it shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For example, the room temperature described herein is 25°C.
  • Compounds not specifically synthesized below are all synthesized according to conventional methods in the art or methods disclosed in existing documents.
  • Step 1 synthesis of tert-butyl (R) -2- (2- (2-isopropylphenyl) -4- (3, 4-dimethoxybenzyl) piperazin-1-yl) -7-azaspiro [3.5] nonane-7-carboxylate
  • Step 2 synthesis of (R) -2- (4- (3, 4-dimethoxybenzyl) -2- (2-isopropylphenyl) piperazin-1-yl) -7-azaspiro [3.5] nonane
  • Step 3 synthesis of methyl 4-fluoro-2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate
  • Step 3-1 methyl 4-fluoro-2- ( (3- (2, 2, 2-trichloroacetyl) -1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate
  • Step 3-2 5- (5-fluoro-2- (methoxycarbonyl) phenoxy) -1H-pyrrolo [2, 3-b] pyridine-3-carboxylic acid
  • Step 3-3 methyl 4-fluoro-2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate
  • Step 4 synthesis of methyl (R) -4- (2- (4- (3, 4-dimethoxybenzyl) -2- (2-isopropylphenyl) piperazin-1-yl) -7-azaspiro [3.5] nonan-7-yl) -2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate
  • Step 5 synthesis of (R) -4- (2- (4- (3, 4-dimethoxybenzyl) -2- (2-isopropylphenyl) piperazin-1-yl) -7-azaspiro [3.5] nonan-7-yl) -2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoic acid
  • methyl (R) -4- (2- (4- (3, 4-dimethoxybenzyl) -2- (2-isopropylphenyl) piperazin-1-yl) -7-azaspiro [3.5] nonan-7-yl) -2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) benzoate (4.2g, 5.191 mmol) in CH 3 OH (50 mL) and THF (100 mL) was added aq.
  • Step 6 synthesis of 4- (2- ( (R) -4- (3, 4-dimethoxybenzyl) -2- (2-isopropylphenyl) piperazin-1-yl) -7-azaspiro [3.5] nonan-7-yl) -2- ( (3-fluoro-1H-pyrrolo [2, 3-b] pyridin-5-yl) oxy) -N- ( (4- ( ( ( (1r, 4r) -4-hydroxy-4-methylcyclohexyl) methyl) amino) -3-nitrophenyl) sulfonyl) benzamide
  • Recombinant human 0.05nM Bcl-2 protein was pre-incubated with a serial dilution of the compound disclosed herein (top final concentration is 1uM or 0.1uM or 0.02uM or 0.01uM, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 mM potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05%Tween-20, 0.01%BSA.
  • Compound disclosed herein was tested for blocking of Bcl-2-G101V protein with its ligand in an assay based on time-resolved fluorescence resonance energy transfer methodology.
  • 0.1nM recombinant human Bcl-2-G101V protein was pre-incubated with a serial dilution of the compound disclosed herein (top final concentration is 10uM or 1uM or 0.1uM, 4-fold or 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 mM potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05%Tween-20, 0.01%BSA.
  • the cells (ATCC, CRL-1873) were cultured in RPMI-1640 complete medium (RPMI-1640 medium, HEPES (Gibco, 22400-105) supplemented with 10%fetal bovine serum (FBS) (Gibco, 10099-1441) , 100 unit/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco, 15140122) ) and maintained in a humidified chamber at 37°C containing 5%CO 2 .
  • FBS fetal bovine serum
  • streptomycin (Gibco, 15140122)
  • Compound of formula (I) was serially diluted with 1 ⁇ M as the maximum concentration.
  • the cells were seeded at 50,000 in 180 ⁇ l per well in 96-well plates and treated with 10-point dilution series of compound of formula (I) for 48 hrs at 37°C.
  • Cell viability was assessed after the treatment using CellTiter-GLO luminescent assay (Promega) according to the manufacturer’s recommendations. Briefly, 30 ⁇ l of CellTiter-GLO reagent was added into 200 ⁇ l of cell culture. Mixture was agitated on an orbital shaker for 5 min to ensure cell lysis followed by 7 min incubation at room temperature to allow development and stabilization of luminescent signals, which corresponded to the quantity of ATP and thus the quantity of metabolically active cells. Luminescent signals were measured using PHERAstar FS reader (BMG) . Mean IC 50 values were determined with GraphPad Prism software. Data are shown in Table 1.
  • RS4; 11 BCL2-G101V knock-in cell pool was generated using Crisper/Cas9 gene editing system and the BCL2-G101V knock-in single clone H96 was picked from the knock-in cell pool and validated by WES (whole exome-sequencing) and RNA-seq.
  • the BCL-2 dependent acute lymphoblastic leukemia (ALL) cell line, RS4; 11 was cultured in RPMI-1640 complete medium (RPMI-1640 medium, HEPES (Gibco, 22400-105) supplemented with 10%fetal bovine serum (FBS) (Gibco, 10099-1441) , 100 unit/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco, 15140122) ) and maintained in a humidified chamber at 37°C containing 5%CO 2 .
  • FBS fetal bovine serum
  • BCL2-G101V knock-in cell pool RS4; 11 was co-transfected with the Cas9-gRNA which also expressed GFP label, and the Donor gene which contained the BCL2-G101V mutant sequence. After 48hrs of electroporation, the GFP positive cells were collected through FACSAriaIII cell sorting system. Cell pool was recovered for 3 days and then cultured under 2nM ABT-199 stress for 4 weeks. TA clone sequencing results showed 9%knock-in rate in the pool after ABT-199 treatment. Then cells were plated in 96 well U-plate with 1 cell/well for 10000 wells for single clone selection. After 3-5 weeks growing, clones were successively screened by PCR sequencing. And three BCL2-G101V knock-in clones: H96, H142 and H233, have been picked out.
  • the BCL-2 G101V knock-in cell line H96 (derive from RS4; 11) were used to study the cellular potency of BCL-2 inhibitors.
  • the cells were cultured in RPMI-1640 complete medium (RPMI-1640 medium, HEPES (Gibco, 22400-105) supplemented with 10%fetal bovine serum (FBS) (Gibco, 10099-1441) , 100 unit/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco, 15140122) ) and maintained in a humidified chamber at 37°C containing 5%CO 2 .
  • Compound of formula (I) was serially diluted with 1 ⁇ M or 10 ⁇ M as the maximum concentration.
  • the cells were seeded at 50,000 in 90 ⁇ l per well in 96-well plates and treated with 10-point dilution series of compound of formula (I) for 48 hrs at 37°C.
  • Cell viability was assessed after the treatment using CellTiter-GLO luminescent assay (Promega) according to the manufacturer’s recommendations. Briefly, 30 ⁇ l of CellTiter-GLO reagent was added into 100 ⁇ l of cell culture. Mixture was agitated on an orbital shaker for 5 min to ensure cell lysis followed by 7 min incubation at room temperature to allow development and stabilization of luminescent signals, which corresponded to the quantity of ATP and thus the quantity of metabolically active cells. Luminescent signals were measured using PHERAstar FS reader (BMG) . Mean IC 50 values were determined with GraphPad Prism software. Data are shown in Table 1.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

L'invention concerne un procédé de préparation d'un intermédiaire d'un inhibiteur de Bcl-2, en particulier un procédé de préparation d'un composé de formule (II) : le 4-fluoro-2-((3-fluoro-lH-pyrrolo [2,3-b] pyridin-5-yl) oxy) benzoate de méthyle. Le procédé est stable, a une bonne répétabilité et un rendement élevé, et est approprié pour une production industrialisée à grande échelle.
PCT/CN2021/086044 2021-04-09 2021-04-09 Procédé de préparation d'un intermédiaire d'un inhibiteur de bcl-2 WO2022213335A1 (fr)

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PCT/CN2021/086044 WO2022213335A1 (fr) 2021-04-09 2021-04-09 Procédé de préparation d'un intermédiaire d'un inhibiteur de bcl-2
CN202180095624.4A CN116981668A (zh) 2021-04-09 2021-04-09 制备bcl-2抑制剂的中间体的方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4136084A4 (fr) * 2020-04-15 2023-11-01 BeiGene, Ltd. Inhibiteur de bcl -2

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012072200A1 (fr) * 2010-12-03 2012-06-07 Merck Patent Gmbh Dérivés de pyrrolo[2,3-b]pyridine 3-hétaryl-substitués utilisés comme inhibiteurs de pdk1
CN106397377A (zh) * 2016-09-05 2017-02-15 中南大学 一种富电子五元杂环酸及其衍生物脱羧上氟的方法
WO2020140005A2 (fr) * 2018-12-29 2020-07-02 Newave Pharmaceutical Inc. Inhibiteurs de bcl-2
CN111434661A (zh) * 2019-01-11 2020-07-21 爱科诺生物医药股份有限公司 具有细胞坏死抑制活性的芳香杂环化合物及其应用
WO2020252240A1 (fr) * 2019-06-14 2020-12-17 Ifm Due, Inc. Composés et compositions pour traiter des états associés à une activité de sting
WO2021138434A1 (fr) * 2019-12-31 2021-07-08 Ifm Due, Inc. Composés et compositions pour traiter des états associés à une activité de sting
WO2021208963A1 (fr) * 2020-04-15 2021-10-21 Beigene, Ltd. Inhibiteur de bcl -2

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012072200A1 (fr) * 2010-12-03 2012-06-07 Merck Patent Gmbh Dérivés de pyrrolo[2,3-b]pyridine 3-hétaryl-substitués utilisés comme inhibiteurs de pdk1
CN106397377A (zh) * 2016-09-05 2017-02-15 中南大学 一种富电子五元杂环酸及其衍生物脱羧上氟的方法
WO2020140005A2 (fr) * 2018-12-29 2020-07-02 Newave Pharmaceutical Inc. Inhibiteurs de bcl-2
CN111434661A (zh) * 2019-01-11 2020-07-21 爱科诺生物医药股份有限公司 具有细胞坏死抑制活性的芳香杂环化合物及其应用
WO2020252240A1 (fr) * 2019-06-14 2020-12-17 Ifm Due, Inc. Composés et compositions pour traiter des états associés à une activité de sting
WO2021138434A1 (fr) * 2019-12-31 2021-07-08 Ifm Due, Inc. Composés et compositions pour traiter des états associés à une activité de sting
WO2021208963A1 (fr) * 2020-04-15 2021-10-21 Beigene, Ltd. Inhibiteur de bcl -2

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4136084A4 (fr) * 2020-04-15 2023-11-01 BeiGene, Ltd. Inhibiteur de bcl -2

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