CN114402198A - Impurity detection method for 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonane-2-yl) acetic acid benzene sulfonate or composition thereof - Google Patents
Impurity detection method for 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonane-2-yl) acetic acid benzene sulfonate or composition thereof Download PDFInfo
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- CN114402198A CN114402198A CN202080030217.0A CN202080030217A CN114402198A CN 114402198 A CN114402198 A CN 114402198A CN 202080030217 A CN202080030217 A CN 202080030217A CN 114402198 A CN114402198 A CN 114402198A
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- China
- Prior art keywords
- solution
- diluent
- acetonitrile
- tricyclo
- aminomethyl
- Prior art date
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- 238000001514 detection method Methods 0.000 title claims abstract description 61
- 239000000203 mixture Substances 0.000 title claims abstract description 49
- PQWTVFMYKNMDNY-GMAIVYIGSA-N C1(=CC=CC=C1)S(=O)(=O)O.NC[C@@]1([C@@H]2[C@H]3C[C@H](CC[C@@H]13)C2)CC(=O)O Chemical compound C1(=CC=CC=C1)S(=O)(=O)O.NC[C@@]1([C@@H]2[C@H]3C[C@H](CC[C@@H]13)C2)CC(=O)O PQWTVFMYKNMDNY-GMAIVYIGSA-N 0.000 title claims abstract description 30
- 239000012535 impurity Substances 0.000 title claims description 76
- -1 nonane-2-yl Chemical group 0.000 claims abstract description 77
- 239000000126 substance Substances 0.000 claims abstract description 55
- CVBYVUQVPQRITK-UHFFFAOYSA-N acetic acid;benzenesulfonic acid Chemical compound CC(O)=O.OS(=O)(=O)C1=CC=CC=C1 CVBYVUQVPQRITK-UHFFFAOYSA-N 0.000 claims abstract description 52
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims abstract description 51
- 238000000034 method Methods 0.000 claims abstract description 44
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 14
- 238000013353 HPLC-CAD method Methods 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 288
- 239000000243 solution Substances 0.000 claims description 110
- 239000003085 diluting agent Substances 0.000 claims description 100
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 90
- 239000013558 reference substance Substances 0.000 claims description 67
- 239000012085 test solution Substances 0.000 claims description 60
- GRTOGORTSDXSFK-XJTZBENFSA-N ajmalicine Chemical compound C1=CC=C2C(CCN3C[C@@H]4[C@H](C)OC=C([C@H]4C[C@H]33)C(=O)OC)=C3NC2=C1 GRTOGORTSDXSFK-XJTZBENFSA-N 0.000 claims description 57
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 46
- 238000002360 preparation method Methods 0.000 claims description 45
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- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 13
- 238000000889 atomisation Methods 0.000 claims description 13
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- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 claims description 10
- 150000002500 ions Chemical class 0.000 claims description 9
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
- 230000003287 optical effect Effects 0.000 claims description 6
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 108
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 34
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- 239000007787 solid Substances 0.000 description 30
- 238000003756 stirring Methods 0.000 description 29
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 25
- 238000005160 1H NMR spectroscopy Methods 0.000 description 24
- 238000001914 filtration Methods 0.000 description 23
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 21
- 239000000706 filtrate Substances 0.000 description 20
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- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 18
- 239000003208 petroleum Substances 0.000 description 17
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 14
- 238000010898 silica gel chromatography Methods 0.000 description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 13
- 239000008346 aqueous phase Substances 0.000 description 13
- 239000012065 filter cake Substances 0.000 description 13
- 239000007789 gas Substances 0.000 description 13
- 239000005457 ice water Substances 0.000 description 13
- 230000035945 sensitivity Effects 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 12
- 230000006378 damage Effects 0.000 description 12
- 238000001035 drying Methods 0.000 description 12
- 238000000605 extraction Methods 0.000 description 12
- 238000010438 heat treatment Methods 0.000 description 12
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 12
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical class OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 11
- 239000003921 oil Substances 0.000 description 11
- XJPBWXJOMSEWFS-GJMOJQLCSA-N BrCC[C@H]1C([C@H]2CC=C[C@@H]12)=O Chemical compound BrCC[C@H]1C([C@H]2CC=C[C@@H]12)=O XJPBWXJOMSEWFS-GJMOJQLCSA-N 0.000 description 10
- 235000019270 ammonium chloride Nutrition 0.000 description 10
- 229940092714 benzenesulfonic acid Drugs 0.000 description 10
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 10
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- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
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- 208000021722 neuropathic pain Diseases 0.000 description 1
- LAIZPRYFQUWUBN-UHFFFAOYSA-L nickel chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ni+2] LAIZPRYFQUWUBN-UHFFFAOYSA-L 0.000 description 1
- BKIMMITUMNQMOS-UHFFFAOYSA-N nonane Chemical compound CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 description 1
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- NFEGNISFSSLEGU-UHFFFAOYSA-N tert-butyl 2-diethoxyphosphorylacetate Chemical compound CCOP(=O)(OCC)CC(=O)OC(C)(C)C NFEGNISFSSLEGU-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/28—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and containing rings
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate or the content of the composition thereof, related substances and a chiral isomer detection method. Detecting the content and related substances I by an HPLC-CAD method, and detecting related substances II and chiral isomers by LC-MS. The method can realize 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]The nonane-2-yl) acetic acid benzene sulfonate or the composition thereof is effectively separated and quantitatively detected from related substances I and II and chiral isomers thereof, and the separation degree is more than 1.5; the specificity, the repeatability and the stability are good, and the medicine quality and the medicine safety of patients are ensured; the detection cost is greatly reduced, and the method is favorable for industrial production.
Description
The invention relates to the field of medicaments, in particular to analysis and detection of medicaments, and particularly relates to 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0%3,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition containing the same, related substances and a method for detecting chiral isomers.
PCT/CN2017/101364 discloses a fused tricyclic gamma-amino acid derivative which is a brand new medicine for treating diabetic peripheral neuralgia, postherpetic neuralgia and fibromyalgia by oral administration. Preclinical tests show that the 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]The nonane-2-yl) acetic acid benzene sulfonate has strong and long-acting analgesic effect and small central side effect, and the compound is prompted to have obvious clinical advantages of obvious drug effect, good safety, convenient medication, good patient compliance and the like in clinical application. If the composition is successfully put into the market, the monopoly status of pregabalin and gabapentin can be effectively broken, and a better medication choice is provided for a plurality of neuropathic pain patients in China, so that the composition has important clinical significance and social significance.
2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The main structure body of the nonane-2-yl) acetic acid benzene sulfonate is that 5 benzene sulfonates are containedThe four-five-six-membered fused ring of the chiral carbon atom has a rigid structure which enables 4 chiral isomers to actually exist, the chiral stereo structure is complex, the stereo structure difference of the enantiomer and the diastereoisomer is complex, and meanwhile, the chiral stereo structure has amino and carboxylic group functional groups, the whole structure is unconjugated, and the ultraviolet absorption is very weak.
2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzenesulfonate is related to several related substances, which are likewise weak UV or non-UV-absorbing compounds, some of which are related to 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate structures are highly similar.
Conventional general-purpose detectors include a differential Refractive Index Detector (RID) and an Evaporative Light Scattering Detector (ELSD), wherein the RID has low detection sensitivity, is not suitable for gradient elution, and is greatly influenced by fluctuations of ambient temperature, mobile phase composition and the like; the ELSD has poor detection sensitivity and reproducibility, the used mobile phase is required to be a volatile solution, and the response peak area and the concentration of the substance to be detected are not directly in a linear relationship.
To sum up, 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The non-conjugated structural characteristics of nonane-2-yl) acetate benzene sulfonate determine that the nonane-2-yl) acetate benzene sulfonate cannot be detected by a conventional ultraviolet detector, and the detector is greatly limited; the multi-handedness center makes the heterogeneous detection analysis difficult. Thus, 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The establishment of the nonane-2-yl) acetic acid benzene sulfonate analysis and detection method faces huge challenges, and needs to be comprehensively researched and developed.
Disclosure of Invention
The low sensitivity RID, ELSD is not suitable for 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The quality research of nonane-2-yl) acetic acid benzene sulfonate or the composition thereof can not reach the detection requirement of the impurity report limit.
For effective and accurate monitoring of 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03, 8)]Nonan-2-yl) acetic acidQuality of benzenesulfonate or its composition, an object of the present invention is to find a suitable detector, develop reasonable chromatographic conditions, and establish 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ]3,8]The nonane-2-yl) acetic acid benzene sulfonate or the related substance and content analysis and detection method of the composition thereof obviously improves the detection sensitivity.
2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate has 5 chiral carbon atoms, a rigid structure of a condensed ring enables 4 chiral isomers to actually exist, and the benzene sulfonate has both amino and carboxylic acid functional groups, and a common chiral column and most of special chiral columns are paired with 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate and isomers thereof can not be effectively separated, and separation and detection are carried out after derivatization treatment. Another objective of the invention is to find a suitable chiral column, develop reasonable chromatographic conditions and establish 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ]3,8]Method for detecting nonane-2-yl) acetic acid benzene sulfonate isomer by using 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate and isomers thereof can be directly and effectively separated without derivatization treatment.
It is another object of the present invention to provide a reference for detection assays of compounds that are free of uv absorption.
2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzenesulfonate of the formula:
according to one embodiment of the invention, the 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ] is performed using HPLC-CAD method3,8]Detecting the content of nonane-2-yl) acetic acid benzene sulfonate and related substances I.
An electrospray detector (CAD) is a novel general detection technology and is widely applied to the industries of medicine, chemical industry, food and the like. The detection principle is as follows: CAD first atomizes the eluent into droplets and then dries them into particles, the size of which increases with the content of the analyte; then the solute particles collide with the positively charged nitrogen, the charge is transferred to the particles, and the larger the solute particles, the more charged; finally, the solute particles transfer the charges of the solute particles to a collector, the charge quantity of the solute particles is measured by a high-sensitivity electrometer, and the signal current generated by the charge quantity is in direct proportion to the content of the solute.
The invention uses HPLC-CAD method, and related substances I and 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The nonane-2-yl) acetic acid benzene sulfonate can be effectively separated, the sensitivity is higher, and the detection limit is lower.
The method parameters are as follows:
high performance liquid chromatograph: siemer femier (Thermo) Ultimate 3000 UHPLC, binary pump, CAD;
a chromatographic column: octadecylsilane bonded silica gel column (4.6X 250mm, 5 μm), e.g., Zishengtang CAPCELL PAK ADME;
mobile phase: a: 10mM ammonium formate solution B: acetonitrile;
gradient elution procedure: 0-5 min, A90%; 5-25 min, A90% → 40%; 25-29 min, A40% → 20%; 29-35 min, A20%; 35-36 min, A20% → 90%; 36-40 min, A90%;
flow rate: 1.0 mL/min;
column temperature: 35 ℃;
CAD parameters: the atomization temperature is 50 ℃, and the acquisition frequency is 5.0 Hz.
Under the chromatographic conditions, the related substances I and 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate can be effectively separated, and the separation degree is more than 1.5; under the CAD parameters of the detector, the related substance I has good response, and the detection sensitivity can reach 0.02 percent or lower.
According to one embodiment of the invention, the substance of interest I is of the formulaAndthe method comprises the following steps:
1) preparation of test solution of related substance I: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Adding a diluent into nonane-2-yl) acetic acid benzene sulfonate to prepare a related substance I test solution;
2) preparation of control solutions for substance i of interest: adding a diluent into the related substance I test solution obtained in the step 1) to dilute by 100 times to obtain the test solution;
3) injecting the test solution of the related substance I obtained in the step 1) and the reference solution of the related substance I obtained in the step 2) into a liquid chromatograph under the chromatographic conditions that:
high performance liquid chromatograph: siemer femier (Thermo) Ultimate 3000 UHPLC, binary pump, CAD;
a chromatographic column: octadecylsilane bonded silica gel column (4.6X 250mm, 5 μm); such as the seniority house CAPCELL PAK ADME;
mobile phase: a: 10mM ammonium formate solution, B: acetonitrile;
gradient elution procedure: 0-5 min, A90%; 5-25 min, A90% → 40%; 25-29 min, A40% → 20%; 29-35 min, A20%; 35-36 min, A20% → 90%; 36-40 min, A90%;
flow rate: 1.0 mL/min;
column temperature: 35 ℃;
CAD parameters: the atomization temperature is 50 ℃, the acquisition frequency is 5.0Hz, and the optical filter is 5.
According to an embodiment of the present invention, the diluent in the step 1) or the step 2) is a mixed solvent of ammonium formate and acetonitrile, preferably a mixed solvent of 10mM ammonium formate and acetonitrile, more preferably 10mM ammonium formate: acetonitrile volume ratio of 80: 20, or a mixed solvent thereof.
According to the bookIn one embodiment of the present invention, the above step 1) of preparing a test solution of the substance I comprises: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is dissolved and diluted to scale by adding a diluent in a volumetric flask with the volume of 50mg to 10mL to obtain the product; the diluent is a mixed solvent of ammonium formate and acetonitrile, preferably a mixed solvent of 10mM ammonium formate and acetonitrile, more preferably 10mM ammonium formate: acetonitrile volume ratio of 80: 20, or a mixed solvent thereof.
According to one embodiment of the present invention, the concentration of the test solution of the substance I obtained in the above step 1) is 5 mg/mL.
According to one embodiment of the present invention, the above step 2) of preparing a control solution of substance I comprises: precisely measuring a 1.0-100 mL volumetric flask of the related substance I test solution obtained in the step 1), adding a diluent to dilute to a scale, and shaking up to obtain the test solution; the diluent is a mixed solvent of ammonium formate and acetonitrile, preferably a mixed solvent of 10mM ammonium formate and acetonitrile, more preferably 10mM ammonium formate: acetonitrile volume ratio of 80: 20, or a mixed solvent thereof.
According to another embodiment of the invention, LC-MS can be used for the detection of substance II, with the following method parameters:
a chromatographic column: octadecylsilane chemically bonded silica gel column (4.6X 250mm, 3.5 μm); such as Waters Xselect HSS T3;
mobile phase: a: 10mM ammonium formate solution B: acetonitrile;
gradient elution procedure: 0-15 min, A70% → 30%; 15-16 min, A30% → 10%; 16-18 min, A10%; 18-20 min, 10% → 70%; 20-25 min, A70%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+).
Under the chromatographic conditions, the related substance II is 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) ethanesThe acid benzene sulfonate can be effectively separated, and the separation degree is more than 1.5; under the detector MS parameters, the related substance II has very good response, and the detection sensitivity can reach 0.001 percent or lower.
According to one embodiment of the invention, related substance ii comprises impurity A, B, C, D, E:
the method comprises the following steps:
1) preparation of control solutions for related substance II: respectively dissolving appropriate amount of reference substance II with diluent to obtain reference substance solution of reference substance II;
2) preparation of related substance II test solution: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Preparing a related substance II test sample solution from a nonane-2-yl) acetic acid benzene sulfonate test sample;
3) injecting the related substance II reference substance solution obtained in the step 1) and the related substance II test solution obtained in the step 2) into a liquid chromatograph under the chromatographic conditions that:
LC-MS (liquid chromatography-mass spectrometer): agilent (Agilent) LCMS 6120, binary pump, MS;
a chromatographic column: waters XSelect HST 3 (4.6X 250mm, 3.5 μm);
mobile phase: a: 10mM ammonium formate solution, B: acetonitrile;
gradient elution procedure: 0-15 min, A70% → 30%; 15-16 min, A30% → 10%; 16-18 min, A10%; 18-20 min, 10% → 70%; 20-25 min, A70%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+).
According to one embodiment of the present invention, the above step 1) of preparing a reference solution of substance II comprises: adding a related substance II reference substance into a diluent to dissolve and dilute to obtain a related substance II reference substance solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water 5:1, v/v.
According to one embodiment of the present invention, the above step 1) of preparing a reference solution of substance II comprises: precisely weighing a reference substance II of a related substance II in a volumetric flask with the volume of 25mg to 10mL, adding a diluent to dissolve and dilute the reference substance II to a scale mark to be used as a reference substance stock solution of the related substance II; precisely measuring related substance II in a volumetric flask with a volume of 100 μ L to 100mL as reference substance stock solution, adding diluent to dilute to scale, and shaking up to obtain the final product.
According to one embodiment of the present invention, the above step 1) of preparing a reference solution of substance II comprises: respectively weighing 10mg of reference substances of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E, precisely weighing, placing in a 10mL measuring flask, dissolving and diluting with a diluent (acetonitrile: water is 5:1) to prepare a solution containing 1mg per 1mL, and taking the solution as a reference substance stock solution; precisely measuring 25 mu L of each of reference stock solutions of impurities A, D and E, 50 mu L of reference stock solution of impurity C and 200 mu L to 100mL of reference stock solution of impurity B, and diluting with a diluent to obtain a mixed solution containing about 0.25 mu g of impurity A, D and E, about 2 mu g of impurity B and about 0.5 mu g of impurity C per 1mL of reference solution.
The diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water 5:1, v/v.
According to one embodiment of the invention, the concentration of the reference substance II obtained in step 1) above is 2.5. mu.g/mL.
According to one embodiment of the present invention, the preparation of the test solution of substance II of interest in step 2) above comprises: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Adding and diluting nonane-2-yl) acetic acid benzene sulfonate or composition thereofDissolving and diluting the reagent to obtain a related substance II test solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water 5:1, v/v.
According to one embodiment of the present invention, the step 2) of preparing the test solution of substance II comprises: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Adding a diluent into a volumetric flask with 25 mg-25 mL of nonane-2-yl) acetic acid benzene sulfonate or a composition thereof for dissolving, diluting to scale, and shaking up to obtain the nonane-2-yl) acetic acid benzene sulfonate; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water 5:1, v/v.
According to one embodiment of the present invention, the concentration of the test solution of related substance II obtained in the above step 2) is 1 mg/mL.
According to another embodiment of the invention, 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]The chiral isomer of nonane-2-yl) acetic acid benzene sulfonate is analyzed and detected by adopting a special chiral column which is screened and suitable, and the method parameters are as follows:
a chromatographic column: CROWNPAK CR-I (+) (3X 150mm, 5 μm);
mobile phase: a: perchloric acid solution (ph1.3) B: acetonitrile;
elution procedure: mobile phase A: mobile phase B80: 20 isocratic elution for 35 minutes;
flow rate: 0.2 mL/min;
column temperature: 20 ℃;
MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+).
Under the chromatographic conditions, enantiomers, diastereomers and 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonane-2-yl) acetic acid benzene sulfonate can be effectively separated, and the separation degree is more than 1.5; under the MS parameters of the detector, the isomer has good response, and the detection sensitivity can reach below 0.05 percent.
According to one embodiment of the invention, the chiral isomer comprises impurity F, G, H, impurity F, G, H is as follows:
the method comprises the following steps:
1) preparation of chiral isomer control solution: taking a proper amount of chiral isomer reference substance, dissolving by using a diluent and preparing an isomer reference substance solution;
2) preparation of chiral isomer test solution: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is used for preparing a chiral isomer test solution;
3) injecting the chiral isomer reference solution obtained in the step 1) and the chiral isomer test solution obtained in the step 2) into a liquid chromatograph, wherein the chromatographic conditions are as follows:
LC-MS (liquid chromatography-mass spectrometer): agilent (Agilent) LCMS 6120, binary pump, MS;
a chromatographic column: CROWNPAK CR-I (+) (3X 150mm, 5 μm);
mobile phase: a: perchloric acid solution (ph1.3), B: acetonitrile;
elution procedure: mobile phase A: mobile phase B80: 20 isocratic elution for 35 minutes;
flow rate: 0.2 mL/min;
column temperature: 20 ℃;
MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+).
According to one embodiment of the present invention, the step 1) of preparing the chiral isomer control solution comprises: adding a diluent into a chiral isomer reference substance to dissolve and dilute the chiral isomer reference substance to obtain a chiral isomer reference substance solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferred, acetonitrile: 20 parts of water: 80, v/v.
According to one embodiment of the present invention, the step 1) of preparing the chiral isomer control solution comprises: precisely weighing a chiral isomer reference substance 10 mg-10 mL volumetric flask, adding a diluent to dissolve and dilute to a scale, and taking the reference substance as a reference substance stock solution; precisely measuring the reference substance stock solution in a volumetric flask with the volume of 100 mu L to 100mL, adding a diluent to dilute to a scale, and shaking up to obtain the product.
According to one embodiment of the present invention, the step 1) of preparing the chiral isomer control solution comprises: respectively weighing 10mg of impurity F and impurity G (or impurity H) reference substances, precisely weighing, placing in a 10mL measuring flask, dissolving and diluting with diluent (acetonitrile: water: 1:4) to prepare 1mg solution per 1mL, and using the solution as reference substance stock solution; precisely measuring 100 μ L to 100mL reference stock solutions containing impurity F and impurity G (or impurity H), and diluting with diluent to obtain 1 μ G impurity F and 1 μ G impurity G (or impurity H) per 1mL reference solution.
The diluent is preferably a mixed solvent of acetonitrile and water; further preferred, acetonitrile: 20 parts of water: 80, v/v.
According to one embodiment of the invention, the chiral isomer control solution obtained in step 1) has a concentration of 1. mu.g/mL.
According to one embodiment of the present invention, the step 2) of preparing the chiral isomer test solution comprises: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Adding a diluent into nonane-2-yl) acetic acid benzene sulfonate or a composition thereof to obtain a chiral isomer test solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the molar ratio of acetonitrile: 20 parts of water: 80, v/v.
According to one embodiment of the present invention, the step 2) of preparing the chiral isomer test solution comprises: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]10mg of nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is added into a 10mL volumetric flask, and a diluent is added for dissolving and diluting to a scale to be used as a stock solution of a test sample; precisely measuring a volumetric flask with 1-10 mL of the stock solution of the sample, adding a diluent to a constant volume to a scale, and shaking up to obtain the test solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the molar ratio of acetonitrile: 20 parts of water: 80, v/v.
According to one embodiment of the invention, the chiral isomer test solution obtained in step 2) has a concentration of 0.1 mg/mL.
Another object of the present invention is to provide a method for detecting impurities in 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonan-2-yl) acetic acid benzenesulfonate or a composition thereof, which can realize effective separation and accurate quantitative detection of 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonan-2-yl) acetic acid benzenesulfonate from related substances i, ii and isomers thereof, wherein the degree of separation is greater than 1.5; the repeatability and the stability are good, and the medicine quality and the medicine safety of patients are ensured; the detection cost is greatly reduced, and the method is favorable for industrial production.
The method adopts CAD to detect 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]The content of nonane-2-yl) acetic acid benzene sulfonate and related substances I have the following advantages compared with the conventional universal detectors RID and ELSD:
1. detection of 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0) using CAD3,8]The detection limit of nonane-2-yl) acetic acid benzene sulfonate and related substances can reach 0.02 percent, and the sensitivity is far higher than that of RID and ELSD (the detection limit is about 0.5 to 1 percent);
2. the response is independent of the chemical structure, and has response consistency;
3. compared with ELSD, logarithm taking is not needed, a wider linear range can be realized, and quantitative calculation is facilitated;
4. broad applicability (all semi-volatile, non-volatile compounds can be measured by the detector-including materials that do not contain chromophores);
5. the reproducibility and the stability are better, and the influence by a mobile phase and the environment is small.
The invention adopts MS to carry out 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]The nonane-2-yl) acetate benzene sulfonate related substance II and isomer detection not only obviously improves the impurity detection sensitivity, but also better guarantees the detection specificity by fixed ion mode detection, and quickly and effectively realizes the separation detection of the related substance II.
The invention adopts a CROWNPAK CR-I (+) column to separate isomers, and 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0%3,8]Nonane-2-yl) acetic acid benzene sulfonate and isomers thereof can be directly and effectively separated without derivatization, and the detection is intuitive and simple.
FIG. 1 is a chromatogram for detection of substance I.
FIG. 2 is a chromatogram for detection of substance II.
FIG. 3 is a chromatogram for detection of chiral isomers.
FIG. 4 is a chromatogram for content detection.
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the scope of the present invention includes but is not limited thereto.
2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0378]Nonan-2-yl) acetic acid benzenesulfonic acid was prepared according to the method disclosed in PCT/CN 2017/101364.
The content reference substance, the related substance I, the related substance II and the chiral isomer reference substance are prepared by self. The rest raw materials and reagents are all purchased from the market.
Example 1: 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetate benzenesulfonate, related to substance I and content determination.
Preparation of test solution of related substance I: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonane-2-yl) acetic acid benzenesulfonate test sample 50mg to 10mL plastic volumetric flasks, diluent (10mM ammonium formate: acetonitrile 80: 20, v/v) and diluting to the scale as a test solution.
Preparation of control solutions for substance i of interest: precisely measuring a related substance I test solution in a plastic volumetric flask with the volume of 1.0mL to 100mL, adding a diluent (10mM ammonium formate: acetonitrile: 80: 20, v/v) to dilute to a scale, and shaking up to obtain the test solution.
Preparing a content test sample solution: self-made 2- ((1) is precisely weighedS,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzenesulfonate test sample 10mg to 10mL plastic volumetric flasks, diluent (10mM ammonium formate: acetonitrile 80: 20, v/v) are dissolved and diluted to the scale as a test stock. Precisely measuring a test sample stock solution in a plastic volumetric flask with the volume of 0.5mL to 10mL, adding the diluent (10mM ammonium formate: acetonitrile: 80: 20, v/v) to dilute to a scale, and shaking up to obtain the test sample.
Preparation of content reference solution: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonane-2-yl) acetic acid benzenesulfonate control 10mg to 10mL plastic volumetric flasks, diluent (10mM ammonium formate: acetonitrile 80: 20, v/v) were dissolved and diluted to the scale as control stock. Precisely measuring a plastic volumetric flask with 0.5mL to 10mL of reference stock solution, adding the diluent to dilute to a scale, and shaking up to obtain the product.
Liquid chromatography conditions:
high performance liquid chromatograph: siemer femier (Thermo) Ultimate 3000 UHPLC, binary pump, CAD;
a chromatographic column: senecio CAPCELL PAK ADME (4.6X 250mm, 5 μm);
mobile phase: a: 10mM ammonium formate solution B: acetonitrile;
gradient elution procedure: 0-5 min, A90%; 5-25 min, A90% → 40%; 25-29 min, A40% → 20%; 29-35 min, A20%; 35-36 min, A20% → 90%; 36-40 min, A90%;
flow rate: 1.0 mL/min;
column temperature: 35 ℃;
CAD parameters: the atomization temperature is 50 ℃, and the acquisition frequency is 5.0 Hz;
sample introduction amount: 20 μ L.
And (3) testing: respectively and precisely measuring a related substance I test solution, a related substance I reference solution, a content test solution and a content reference solution by 20 mu L, injecting into a liquid chromatograph, recording a chromatogram for analysis and calculation, calculating the content by an external standard method, and detecting the chromatogram as shown in figure 4; the related substance I is calculated by a self-contrast method, and the detection chromatogram is shown in figure 1.
Example 2: 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzenesulfonate related to substance II.
Preparation of a reference solution: accurately weighing related substance II reference substances (impurity A, impurity B, impurity C, impurity D and impurity E) 25mg to 10mL volumetric flasks respectively, adding a diluent (acetonitrile: water ═ 5:1, v/v) to dissolve and dilute to a scale mark, and taking the reference substance II reference substance as a reference substance stock solution. Precisely measuring the reference substance stock solution in a volumetric flask with the volume of 100 mu L to 100mL, adding a diluent to dilute to a scale, and shaking up to obtain the product.
A second preparation method of the reference solution: weighing 10mg of reference substances of the impurity A, the impurity B, the impurity C, the impurity D and the impurity E respectively, precisely weighing, placing in a 10mL measuring flask, dissolving and diluting with a diluent (acetonitrile: water: 5:1) to prepare a solution containing 1mg of the reference substances per 1mL, and taking the solution as a reference substance stock solution. Precisely measuring 25 mu L of each of reference stock solutions of impurities A, D and E, 50 mu L of reference stock solution of impurity C and 200 mu L to 100mL of reference stock solution of impurity B, and diluting with a diluent to obtain a mixed solution containing about 0.25 mu g of impurity A, D and E, about 2 mu g of impurity B and about 0.5 mu g of impurity C per 1mL of reference solution.
Preparing a test solution: precisely weighing self-made 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzenesulfonate test sample 25mg to 25mL volumetric flasks, the aforementioned diluent (acetonitrile: water 5:1, v/v) and diluting until the scales are evenly shaken to obtain the product.
Liquid chromatography conditions:
LC-MS (liquid chromatography-mass spectrometer): agilent (Agilent) LCMS 6120, binary pump, MS;
a chromatographic column: waters XSelect HST 3 (4.6X 250mm, 3.5 μm);
mobile phase: a: 10mM ammonium formate solution B: acetonitrile;
gradient elution procedure: 0-15 min, A70% → 30%; 15-16 min, A30% → 10%; 16-18 min, A10%; 18-20 min, 10% → 70%; 20-25 min, A70%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+);
sample introduction amount: 10 μ L.
And (3) determination: precisely measuring 10 μ L of each of the sample solution and the reference solution, injecting into a liquid chromatograph, recording chromatogram for analysis and calculation, and calculating the content of related substance II by external standard method. The detection chromatogram is shown in FIG. 2.
Example 3: 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate isomers.
Preparation of a reference solution: chiral isomer controls (impurity F, impurity G (or impurity H)) were weighed precisely, each 10mg to 10mL volumetric flasks, dissolved and diluted to the scale with a diluent (acetonitrile: water ═ 20: 80, v/v) as control stock solutions. Precisely measuring a reference substance stock solution in a volumetric flask with the volume ranging from 100 mu L to 100mL, adding a diluent (acetonitrile: water: 20: 80, v/v) to dilute to a scale, and shaking up to obtain the compound.
A second preparation method of the reference solution: 10mg of impurity F and impurity G (or impurity H) as reference substances are weighed respectively, precisely weighed, placed in a 10mL measuring flask, dissolved and diluted by a diluent (acetonitrile: water: 1:4) to prepare a solution containing 1mg of impurity F and impurity G per 1mL, and used as a reference substance stock solution. Precisely measuring 100 μ L to 100mL reference stock solutions containing impurity F and impurity G (or impurity H), and diluting with diluent to obtain 1 μ G impurity F and 1 μ G impurity G (or impurity H) per 1mL reference solution.
Preparing a test solution: self-made 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0) is precisely weighed3,8]Nonane-2-yl) acetic acid benzenesulfonate test sample 10mg to a 10mL volumetric flask, diluent (acetonitrile: 20 parts of water: 80, v/v) ultrasonic dissolving and diluting to a scale as a test article stock solution. Precisely measuring a 1-10 mL volumetric flask of the stock solution of the sample, adding the diluent (acetonitrile: water: 20: 80, v/v) to a constant volume to a scale, and shaking up to obtain the test solution.
Liquid chromatography conditions:
LC-MS (liquid chromatography-mass spectrometer): agilent (Agilent) LCMS 6120, binary pump, MS;
a chromatographic column: CROWNPAK CR-I (+) (3X 150mm, 5 μm);
mobile phase: a: perchloric acid solution (ph1.3) B: acetonitrile;
elution procedure: mobile phase A: mobile phase B80: 20 isocratic elution for 35 minutes;
flow rate: 0.2 mL/min;
column temperature: 20 ℃;
MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+);
sample introduction amount: 5 μ L.
And (3) determination: precisely measuring 5 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, recording chromatogram for analysis, and calculating chiral isomer content by external standard method. The detection chromatogram is shown in FIG. 3.
EXAMPLE 4 forced degradation test of substance I
Weighing 6 parts of 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The nonane-2-yl) acetic acid benzene sulfonate samples, 125mg each, were placed in 10ml measuring flasks and, after each treatment with non-destructive, acid, base, high temperature, oxidation, light, etc., diluents (10mM ammonium formate: acetonitrile 80: 20, v/v) is dissolved and prepared into a solution containing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ] in each 1mL3,8]Nonan-2-yl) acetic acid benzenesulfonate about 5mg of sample solution; the sample solutions after the non-destruction and destruction treatments were subjected to HPLC-CAD and HPLC-MS detection, and the destruction conditions and test results are shown in Table 1 below.
TABLE 1 destructive test results for substances I
And (4) analyzing results: no new impurity is detected in the method under each forced degradation condition, and the mass spectrum shows that the m/z of a main peak under each condition is 210(2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonane-2-yl) acetic acid benzene sulfonate); ② under each condition, the mass spectrum can detect the peak with m/z of 192(RT is the quasi-molecular ion peak of impurity C in 24min, RT is the fragment peak of impurity D in 22 min), the detection sensitivity of CAD to impurity D, C is weaker than that of MS; ③ the strong peak with m/z 192(RT 24min) is detected by the mass spectrum under the condition of alkali destruction, which indicates that the alkali destruction is one of the degradation paths of the impurity C. The main peak is overloaded, the CAD belongs to a general-purpose and high-sensitivity detector, and the probability of missing detection of impurities is low, so that the material balance statistics is not carried out.
By integrating the results of impurity positioning and destruction tests, the method has good specificity in detecting the related substance I. The result of methodology verification shows that the method has good sensitivity, repeatability, linear range, accuracy and stability.
EXAMPLE 5 forced degradation test of related substance II
Taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Appropriate amount of nonane-2-yl) acetic acid benzene sulfonate to be tested is precisely weighed, and after being respectively treated by non-destruction, acid, alkali, high temperature, oxidation, illumination, high humidity and the like, a diluent (10mM ammonium formate: acetonitrile 80: 20, v/v) was formulated to contain about 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ] per 1mL3,8]1mg of nonane-2-yl) acetic acid benzene sulfonate sample solution, and preparing a blank solution by the same method; the sample solutions after non-destruction and destruction treatments were injected and tested and the results compared, and the specific destruction conditions and test results are shown in Table 2 below.
TABLE 2 destructive test results for related substances II
And (4) analyzing results: the impurity E is obviously increased under the alkaline condition, and the alkali is2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0) under sexual conditions3,8]Nonane-2-yl) acetic acid benzenesulfonate may be degraded to produce impurity C; ② the impurity B is obviously reduced under the conditions of acid, alkali and high temperature, and the impurity B is possibly degraded to generate 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ] under the conditions of acid, alkali and high temperature3,8]Nonan-2-yl) acetic acid benzenesulfonate; no new impurity is produced under each damage condition.
By integrating the results of impurity positioning and destruction tests, the method has good specificity in detecting related substances II; the result of methodology verification shows that the method has ultrahigh sensitivity and good repeatability, linear range, accuracy and stability.
The preparation process comprises the following steps:
DMF: n, N-dimethylformamide
DBU: 1, 8-diazabicycloundec-7-enes
DCM: methylene dichloride
PE: petroleum ether
EA: ethyl acetate
DEA: diethylamine
INT1 preparation method:
the first step is as follows: 3- (cyclohexyl-3-en-1-yl) propanoic acid (1B)
3-(cyclohex-3-en-1-yl)propanoic acid
Anhydrous formic acid (18.82Kg,409.09moL) was pumped into a 100 liter reactor and the pumping was complete. Starting stirring and refrigerating equipment, and cooling to 10 ℃. When the internal temperature was 10 ℃, triethylamine (16.53Kg,163.64moL) was added dropwise to the reaction mixture, and the mixture was stirred for 20 minutes after the addition. When the internal temperature is 10 ℃, adding the cyclopropane dicarboxylic acid cyclo (methylene) isopropyl ester (7.86Kg,54.55moL) into the reaction kettle, and then slowly dripping the 3-cyclohexene-1-formaldehyde (6.00Kg,54.55moL) into the reaction solution at 40 ℃ after the addition. Heating to 140 ℃ and 150 ℃ to react until no gas exists. The pH of the aqueous phase was adjusted to between 1 and 2 with 6N HCl (24.0L). The aqueous phase was extracted with dichloromethane (12L X2), and the organic phases were combined and washed with saturated brine (10L X2). The organic phase was dried over anhydrous sodium sulfate (2.0Kg) for 1 hour, filtered, and the filtrate was concentrated to dryness to give 3- (cyclohexyl-3-en-1-yl) propionic acid (1B) as a yellow oil (8.80Kg, 100% yield).
1H NMR(400MHz,CDCl 3)δ10.23(s,1H),5.73–5.55(m,2H),2.46–2.30(m,2H),2.09–1.96(m,2H),1.81–1.53(m,6H),1.35–1.17(m,1H)。
LCMS m/z=153.1[M-1]。
The second step is that: 3- (cyclohexyl-3-en-1-yl) -1- (pyrrolidinyl-1-yl) propyl-1-one (1C)
3-(cyclohex-3-en-1-yl)-1-(pyrrolidin-1-yl)propan-1-one
3- (cyclohexyl-3-en-1-yl) propanoic acid (1B) (11.20Kg,72.727moL) was dissolved in methylene chloride (60.0L) and then pumped into a 100L reactor. With stirring, DMF (3.0mL) was added and oxalyl chloride (9.046Kg,71.272moL) was added dropwise to the reaction. After the addition, the mixture was stirred at room temperature for 2.0 hours. TLC detects that the reaction is complete, and then pyrrolidine (5.689Kg,79.999moL) and triethylamine (8.814Kg,87.272moL) are added into the reaction kettle in a dropwise manner. Controlling the internal temperature to be less than 10 ℃, and stopping cooling. Stir at room temperature overnight. After completion of the TLC detection reaction, the reaction solution was cooled to 10 ℃. 3N hydrochloric acid (20.0L) was added dropwise to adjust the pH of the reaction mixture to 1-2. After standing and separating, the aqueous phase was extracted with dichloromethane (10.0L. times.1). The combined organic phases were washed successively with 5% sodium hydroxide solution (10.0 L.times.1) and saturated ammonium chloride solution (20.0 L.times.1). The organic phase was dried over anhydrous sodium sulfate (2.0Kg) for 30 minutes, filtered, and the filtrate was concentrated to give 3- (cyclohexyl-3-en-1-yl) -1- (pyrrolidinyl-1-yl) propyl-1-one (1C) as a brown liquid (15.00Kg, 99.6% yield).
1H NMR(400MHz,CDCl 3)δ5.73–5.56(m,2H),3.43(dd,4H),2.37–2.22(m,2H),2.16–2.01(m,4H),1.90(dt,4H),1.81–1.51(m,6H),1.30–1.15(m,2H)。
LCMS m/z=208.1[M+1]。
The third step: tricyclic [4.2.1.03,8]Nonanyl-2-ones (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (INT1)
tricyclo[4.2.1.0 3,8]nonan-2-one(1R,3S,6R,8R and 1S,3R,6S,8S racemate)
The intermediate 3- (cyclohex-3-en-1-yl) -1- (pyrrolidin-1-yl) propyl-1-one (1C) (5.64Kg, 27.22moL) was dissolved in dichloromethane (40.0L) and then pumped into a 100L reactor. Stirring, cooling to-10 deg.C, and adding 2, 4, 6-trimethylpyridine (4.94Kg, 40.83 mol). A solution of trifluoromethanesulfonic anhydride (11.50Kg, 40.83mol) in dichloromethane (16.0L) was added dropwise to the reaction mixture, and after the addition, the mixture was refluxed for 12 hours. After completion of the reaction was checked, an aqueous solution (23.0L) of sodium hydroxide (3.10Kg, 77.5mol) was added dropwise to the reaction solution, and the pH of the reaction solution was adjusted to 10 to 11. The reaction exothermed and continued to reflux for 5-6 hours after the addition was complete. The mixture was allowed to stand for separation, the aqueous phase was extracted with methylene chloride (5.0L. times.1), and the organic phases were combined. The organic phase was pumped into a reaction kettle, stirred and cooled to 10 ℃. A 2.0M hydrochloric acid solution (20.0L) was added dropwise to adjust the pH of the reaction solution to 1-2. The mixture was allowed to stand for liquid separation, and the organic phase was washed with saturated brine (20L. times.1). The organic phase was directly concentrated and the residue was dissolved in acetone (20.0L) and pumped into a 100L reactor. Stirring is started, concentrated sulfuric acid (4.0L) and water (20.0L) are added dropwise to prepare a solution, the reaction releases heat, and the solution is refluxed for 2 hours after the addition. The temperature was reduced to 15 ℃ and a saturated saline solution (20.0L) was added to the reaction mixture, followed by extraction with n-hexane (15.0L. times.2). The organic phases were combined, washed with saturated brine (20.0L. times.1), and dried over anhydrous sodium sulfate overnight. Filtering, concentrating the filtrate under reduced pressure to obtain yellow solid crude tricyclo [ 4.2.1.0%3,8]Nonanyl-2-one (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (INT1) (3.00Kg, 81% yield).
1H NMR(400MHz,CDCl 3)δ3.39(m,1H),3.19(m,1H),2.77(m,1H),2.38(m,1H),2.05(m,1H),1.93(d,1H),1.77(m,1H),1.45(m,4H),1.20(m,1H)。
LCMS m/z=137.1[M+1]。
Refining:
dissolving anhydrous sodium bisulfite (5.735Kg, 55.147moL) in 66L of purified water, adding into a 100L reaction kettle, stirring at room temperature, dropwise adding ethanol (3.0L) solution of (INT1) crude product (3.00Kg,22.059moL), stirring at room temperature overnight, extracting with ethyl acetate (20 Lx 2), adding water phase into the reaction kettle, stirring, cooling to 10 deg.C, dropwise adding water (10L) solution of sodium hydroxide (2.250Kg,56.250moL), adjusting pH to 10-12, stirring at room temperature for 2 hr, extracting with n-hexane (20 Lx 2), mixing organic phases, washing with purified water (20 Lx 1), drying organic phase with anhydrous sodium sulfate 2Kg for 1 hr, filtering, and evaporating filtrate to obtain colorless crystalline tricyclic [ 4.2.1.0.0 ]3,8]Nonanyl-2-one (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (INT1) (2.7Kg, yield 90%).
LCMS m/z=137.1[M+1]。
INT2 preparation method:
the first step is as follows: tert-butyl 2- (tricyclo [4.2.1.0 ]3,8]Nonyl-2-yliden) acetate (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (INT2)
tert-butyl 2-tricyclo[4.2.1.0 3,8]nonan-2-ylidene)acetate(1R,3S,6R,8Rand 1S,3R,6S,8S racemate)
Potassium tert-butoxide (4.94g,44.0mmol) and tetrahydrofuran (30mL) were added to a 100mL reaction flask. The temperature is reduced to 5 ℃, tert-butyl dimethoxyphosphonoacetate (9.86g,44.0mmol,1.1eq) is added into the reaction liquid, the reaction temperature is controlled to be 10-15 ℃, and the dropwise addition is finished within about 20 minutes. The temperature is controlled to be 10-15 ℃ and the mixture is stirred for 20 minutes. Then (1R,6R,8R) -tricyclo [4.2.1.03,8]A solution of nonanyl-2-one (INT1) (5.40g,40.0mmol,1.0eq) in tetrahydrofuran (30mL) was added dropwise over about 0.3 hour,after the addition, the temperature naturally rises to room temperature for reaction for 3 hours. The reaction was quenched by the sequential addition of saturated ammonium chloride (200mL) and purified water (200 mL). After stirring for 20 minutes, the mixture was allowed to stand for separation, and the aqueous phase was extracted with methylene chloride (30 mL. times.1). The organic phases were combined, washed with saturated brine (30 mL. times.1), and dried over anhydrous sodium sulfate. Filtering, concentrating, and purifying to obtain 5.0 crude product to obtain yellow liquid tert-butyl 2- (tricyclo [4.2.1.0 ]3,8]Nonanyl-2-yliden) acetate (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (INT2) (4.0g, yield: 80%).
LCMS m/z=235.3[M+1]。
δH(ppm)(400MHz,CD3OD) | Multiplicity of properties |
1.86,1.28/1.26 | 2,m |
1.47,1.45 | 9,s |
1.71,1.53/1.58,1.32 | 2,m |
1.87,1.51/1.85,1.38 | 2,m |
1.99,1.33 | 2,m |
2.30 | 1,m |
2.81,2.76 | 1,m |
3.41,2.99 | 1,m |
3.66,3.20 | 1,m |
5.45,5.36 | 1,dd |
INT3 preparation method:
tert-butyl 2- (2- (nitromethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (1R,2R,3S,6R,8R and 1S,2S,3R,6S,8S racemate) (INT3)
tert-butyl 2-(2-(nitromethyl)tricyclo[4.2.1.03,8]nonan-2-yl)acetate(1R,2R,3S,6R,8R and 1S,2S,3R,6S,8S racemate)
Tert-butyl 2- (tricyclo [4.2.1.0 ] was added to the reaction flask in sequence3,8]Nonyl-2-yliden) acetate (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (INT2) (2.34g,10.0mmol), nitromethane (1.68g,25.0mmol), 1, 8-diazabicycloundec-7-ene (1.83g,12.0mmol) and dimethyl sulfoxide (5.0 mL). Stirring, heating to 80-85 deg.C, and keeping the temperature for 6 hr. Cooled to room temperature, purified water (100.0mL, aqueous phase with dichloromethane (40mL) was added to the kettleX 3) extraction. The organic phases were combined, washed with saturated brine (30 mL. times.1), and dried over anhydrous sodium sulfate. Filtering, and concentrating to obtain colorless liquid tert-butyl 2- (2- (nitromethyl) tricyclo [ 4.2.1.0%3,8]Nonanyl-2-yl) acetate (1R,2R,3S,6R,8R and 1S,2S,3R,6S,8S racemate) (INT 3).
LCMS m/z=318.1[M+Na]。
INT4 preparation method:
tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (INT4)
tert-butyl2-((1S,2S,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.03,8]nonan-2-yl)acetate(S)-2-acetoxy-2-phenylacetate
To a 50L reactor was added tert-butyl 2- (2- (nitromethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (1R,2R,3S,6R,8R and 1S,2S,3R,6S,8S racemate) (INT3) (730.0g,2.47mol) and methanol (7.3L). Starting stirring, adding nickel chloride hexahydrate (118g,0.49mol,0.2eq) into a reaction kettle, cooling the reaction liquid to 5 ℃, adding sodium borohydride (374g,9.88mol,4.0eq) in batches, keeping the temperature of the reaction liquid system at 20-30 ℃ for about 3 hours, and finishing the addition. The reaction was stirred for 2 hours after the addition. The reaction was quenched by adding ice water (16.4L) to the reaction kettle and filtered through celite at 2-3 cm. The filtrate was extracted with dichloromethane (3.0L X2), and the organic phases were combined, washed with saturated brine (4L X1), and dried over anhydrous sodium sulfate. After filtration, O-acetyl-L-mandelic acid (384g,1.97mol,0.8eq) was added to the filtrate, and the mixture was stirred for 20 minutes after the addition. The organic phase was concentrated by distillation until no solvent was distilled off to give a crude product of tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0%3,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (1H) crude product. The crude product was slurried with isopropanol (5.9L) for 2 hours under stirring, cooled to 5 deg.C and stirred for 1 hour. Filtering, washing filter cake with isopropanol (0.4L is multiplied by 1), drying to obtain white solid product of tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0%3,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (INT4) (422g, yield: 34.96%). The ee value of the sampled derivatization assay was 48.35%.
First crystallization: tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (INT4) (420.0g,0.92mol), isopropanol (4.20L) and water (0.21L) were added to the kettle in that order. The temperature is raised to 82 ℃ to completely dissolve the solid, and the temperature is kept for 0.5 hour. Cooling to 20 ℃ for crystallization for about 6 hours. When the internal temperature reached 20 ℃ it was filtered and the filter cake was washed with isopropanol (0.40 L.times.1). The solids are combined and dried by blowing at 60-65 ℃ for 4 hours until the weight is constant. The ee value after sampling and derivatization was 81.25%. To obtain a white solid product, namely tert-butyl 2- ((1R,2R,3S,6R,8R) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (1H) crude-1 (260g, yield: 62%). The ee value of the sampled derivatization assay was 81.00%.
And (3) second crystallization: tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (INT4) (177g,0.39mol), isopropanol (2.53L) and water (0.126L) were added sequentially to the kettle. The temperature is programmed to 82 ℃ to completely dissolve the solid, and the temperature is kept for 0.5 hour. Cooling to 20 ℃ for crystallization and about 4.5 hours. When the internal temperature reached 30 ℃ it was filtered and the filter cake was washed with isopropanol (0.10L. times.1). The solids are combined and dried by blowing at 60-65 ℃ for 4 hours until the weight is constant. The ee value after sampling and derivatization was 99.73%. To obtain a white solid product, namely tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (INT4) (128g, yield: 72%).
LCMS m/z=266.3[M+1]。
INT5 preparation method:
2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetic acid benzenesulfonic acid compound (1:1) (INT5)
2-((1S,2S,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid compound with benzenesulfonic acid(1:1)
Tert-butyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (S) -2-acetoxy-2-phenylacetic acid (INT4) (150.0g, 0.326mol) and purified water (1.5L) were added to the reaction kettle in sequence, stirred and cooled to 0-10 ℃. And maintaining the temperature at 0-10 ℃, dropwise adding 1mol/L NaOH (360mL) aqueous solution into the reaction kettle, and adjusting the pH of the aqueous phase to 9-10. The layers were separated by settling and the aqueous phase was extracted with dichloromethane (0.80L. times.2). The organic phases were combined and washed successively with 1mol/L NaOH (0.30L. times.1) solution and saturated brine (0.15L. times.1). The organic phase was decolorized by adding activated carbon (5.0g) and dried over anhydrous sodium sulfate. Filtration, concentration of the filtrate and dissolution of the concentrated residue with acetonitrile (328mL) gave an acetonitrile solution. In a suitable container, benzenesulfonic acid monohydrate (114g, 0.359mol) was added to purified water (328mL), and the mixture was stirred until it was dissolved and clarified to prepare an aqueous solution of benzenesulfonic acid. And dropwise adding the prepared benzenesulfonic acid aqueous solution into the acetonitrile solution, and finishing the addition. The temperature is increased to 80-85 ℃. Keeping the temperature for reacting for 4-6 hours, and stopping heating after detecting that the raw materials are completely reacted. Cooling to 10-20 deg.C, and crystallizing for 4-6 hr. When the internal temperature reached 10-20 ℃, the mixture was filtered, and the filter cake was washed with water (30 mL. times.1) and acetonitrile (30 mL. times.1) in this order. Drying to obtain white solid 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonanyl-2-yl) acetic acid benzenesulfonic acid compound (1:1) (INT5) (104 g)Yield, yield: 86.9%).
δH(ppm)(400MHz,CD3OD) | Multiplicity of properties |
7.87~7.78 | 2H,m |
7.47~7.36 | 3H,m |
3.34 | 2H,A-B system,J*=13.62Hz |
2.55 | 2H,A-Bsystem,J*=16.4Hz |
2.51~2.44 | 1H,m |
2.36~2.27 | 1H,m |
2.19~2.10 | 1H,m |
2.09~1.96 | 1H,m |
1,83~1.71 | 1H,m |
1.71~1.64 | 1H,m |
1.66~1.59 | 1H,m |
1.59~1.51 | 2H,m |
1.49~1.40 | 1H,m |
1.32~1.22 | 1H,m |
INT6 preparation method:
2- ((1S,2S,3R,8S)) -2- (((tert-butyloxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid
2-((1S,2S,3R,8S)-2-(((tert-butoxycarbonyl)amino)methyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid
INT5(8.15g,37.89mmol) is added into a round-bottom flask, potassium carbonate (15.38g,109.8mmol), water (100mL) and ethanol (10mL) are sequentially added, di-tert-butyl dicarbonate (10.9g,49.36mmol) is slowly and dropwise added into a reaction solution, after the addition, the reaction is carried out at room temperature for 3h, the reaction solution is extracted by petroleum ether (100mL multiplied by 2), liquid separation is carried out, 5mol/L hydrochloric acid solution is dropwise added into an aqueous phase under an ice water bath to adjust the pH to be about 3, white solid is separated out, dichloromethane is extracted (100mL multiplied by 3), organic phases are combined and washed by saturated common salt water (150mL multiplied by 1) anhydrous sodium sulfate, and the mixture is dried and concentrated under reduced pressure to obtain a white solid product, namely 2- ((1S,2S,3R,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid INT6(8.5g, 73%).
INT9 preparation method:
the first step is as follows: (1S,6S,8S)) -2- (nitromethyl) tricyclo [4.2.1.03,8]Nonan-2-ols
(1S,6S,8S)-2-(nitromethyl)tricyclo[4.2.1.0 3,8]nonan-2-ol
INT1(72.3g, 0.53mol), nitromethane (1.0L), DBU (80.8g, 0.53mol) were added to the reaction flask and reacted at room temperature for 18 h. Saturated aqueous ammonium chloride (5L) was added, extracted with dichloromethane (2L. times.3), and the organic layers were combined, dried, filtered, and concentrated. Purification by silica gel column chromatography (100: 1-10:1 petroleum ether/ethyl acetate (v/v)) gave INT7(10.5g, 10.1% yield) as a yellow oil.
1H NMR(400MHz,CDCl 3):δ4.73–4.40(m,2H),2.65–2.58(m,1H),2.51–2.44(m,1H),2.34(qd,2H),1.99–1.86(m,1H),1.80–1.44(m,6H),1.30–1.17(m,1H)。
The second step is that: (1S,6S,8S, E) -2- (nitromethylene) tricyclo [4.2.1.03,8]Nonane
(1S,6S,8S,E)-2-(nitromethylene)tricyclo[4.2.1.0 3,8]nonane
INT7(0.50g, 2.54mmol), acetic anhydride (10mL), p-toluenesulfonic acid (0.44g, 2.54mmol) were added to the reaction flask and reacted at room temperature for 2 h. Cooled to 0 ℃, 50ml of saturated aqueous sodium bicarbonate solution was added, stirred for 1h, extracted with dichloromethane (35ml × 3), and the organic layers were combined, dried, filtered, and concentrated. This gave 0.15g of a colorless liquid, which was dissolved in methanol (8mL), cooled to 0 ℃ and reacted at room temperature for 2 hours with the addition of sodium methoxide (0.03g, 0.61 mmol). 20mL of saturated aqueous ammonium chloride solution was added, extraction was performed with dichloromethane (40 mL. times.3), and the organic layers were combined, dried, filtered, and concentrated. Purification by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) gave INT8(0.08g, yield 74.1%) as a yellow oil.
1H NMR(400MHz,CDCl 3):δ6.87(dd,1H),3.94–3.61(m,1H),3.25(ddd,1H),3.01–2.80(m,1H),2.39(td,1H),2.12–1.76(m,4H),1.73–1.51(m,1H),1.48–1.23(m,3H)。
The third step: tert-butyl 2- ((1S,2R,6S,8S) -2- (nitromethylene) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
tert-butyl 2-((1S,2R,6S,8S)-2-(nitromethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
1M lithium bis (trimethylsilyl) amide (290mL, 0.29mol) was added to the reaction flask, nitrogen blanketed, cooled to-60 deg.C, a solution of tert-butyl acetate (33.62g, 0.29mmol) in tetrahydrofuran (150mL) was added dropwise, stirred for 20mins, a solution of INT8(28.82g,0.16mol) in tetrahydrofuran (250mL) was added dropwise, and the reaction was allowed to proceed at-60 deg.C for 2 h. Saturated aqueous ammonium chloride (400mL) was added, extracted with ethyl acetate (200 mL. times.3), and the organic layers were combined, dried, filtered, and concentrated. Purification by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) gave INT9(39.4g, yield 84.8%) as a colorless liquid.
δH(ppm)(400MHz,CD3OD) | Number of hydrogens and cleavage events |
4.87 | 2H,A-B system,J*=7.10Hz |
2.89 | 1H,m |
2.58 | 1H,m |
2.47 | 2H,s |
2.33 | 1H,m |
2.30 | 1H,m |
2.02,1.56 | 2H,m |
1.77,1.55 | 2H,m |
1.67,1.28 | 2H,m |
1.62,1.42 | 2H,m |
1.45 | 3H,s |
The preparation method of the impurity A comprises the following steps:
the first step is as follows: ethyl-2- ((1S,2S,3R,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
ethyl 2-((1S,2S,3R,8S)-2-(((tert-butoxycarbonyl)amino)methyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
2- ((1S,2S,3R,8S)) -2- (((tert-butyloxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]INT6(2.07g,6.46mmol) of nonan-2-yl) acetic acid was added to a round-bottomed flask, DMF (20mL), potassium carbonate (1.36g,9.7mmol) were sequentially added, ethyl iodide (1.57g,9.7mmol) was added dropwise slowly in an ice-water bath, after addition, the reaction was carried out at room temperature for 2 hours, 30mL of water was added to the reaction solution, extraction was carried out with methyl tert-butyl ether (30 mL. times.2), the organic phases were combined and washed with saturated brine (50 mL. times.3), drying was carried out with anhydrous sodium sulfate, and concentration was carried out under reduced pressure to obtain ethyl-2- ((1S,2S,3R,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetate INT 6-A.
1H NMR(400MHz,CDCl 3):δ4.98(br,1H),4.13(q,2H,J=7.2Hz),3.39(d,2H,J=6.4Hz),2.84(dd,2H,J=12.8Hz,5.2Hz),2.39(m,1H),2.37(s,2H),2.27(dd,1H,J=12.4Hz,5.6Hz),2.07(m,1H),1.98(m,1H),1.75-1.67(m,1H),1.59-1.47(m,5H),1.44(s,9H),1.26(t,3H)。
The second step is that: ethyl 2- ((1S,2S,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetate benzenesulfonate
ethyl 2-((1S,2S,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate benzenesulfonate
Ethyl-2- ((1S,2S,6S,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Adding nonan-2-yl) acetate INT6-A (2.2g,6.502mmol) into a round-bottom flask, sequentially adding benzenesulfonic acid (2.05g,13.0mmol) and acetonitrile 22mL, heating to 80 ℃ after the addition is finished, reacting for 0.5h, cooling to room temperature, concentrating under reduced pressure to obtain a white solid, adding 10mL methyl tert-butyl ether, pulping, stirring for 10min, and filtering to obtain a white solid product, namely impurity A.
Ms m/z(ESI):238.2[M+H +]。
δH(ppm)(400MHz,CDCl 3) | Number of hydrogens and cleavage events |
1.10 | 1,m |
1.15 | 3,t |
1.25 | 1,m |
1.51-1.39 | 4,m |
1.61 | 1,m |
1.92 | 1,m |
2.10 | 1,m |
2.23 | 1,dd |
2.41 | 1,m |
2.53 | 2,s |
2.70 | 1,dd |
3.33 | 2,dd |
4.00 | 2,m |
7.39 | 4,m |
7.77-7.86 | 3,m |
9.63 | 1,br |
The preparation method of the impurity B comprises the following steps:
INT4(2.02g, 4.35mmol) was added to a round-bottom flask, water (10mL), dichloromethane (10mL), and sodium hydroxide (0.18g,4.35mmol) were added in this order under an ice-water bath, stirred at this temperature for 30min, separated by a separatory funnel, the organic layer was dried over anhydrous sodium sulfate, benzenesulfonic acid (0.77g, 4.35mmol) was added to the organic layer, and after completion of addition, concentration was carried out at 20 ℃ under reduced pressure to obtain impurity B as a white solid.
Ms m/z(ESI):266.2[M+H +]
δH(ppm)(400MHz,CD3OD) | Number of hydrogens and cleavage events |
1.13 | 1,m |
1.32 | 1,m |
1.38 | 9,s |
1.49-1.39 | 4,m |
1.63 | 1,m |
δH(ppm)(400MHz,CD3OD) | Number of hydrogens and cleavage events |
1.92 | 1,m |
2.13 | 1,m |
2.22 | 1,dd |
2.47 | 2,s |
2.73 | 1,m |
3.31 | 2,m |
7.35 | 3,m |
7.89 | 2,m |
7.94 | 3,br |
The preparation method of the impurity C comprises the following steps:
the first step is as follows: (±) - (1R,5S,7R) -7- (2-bromoethyl) bicyclo [3.2.0] hept-2-en-6-one (1B-2)
(±)-(1R,5S,7R)-7-(2-bromoethyl)bicyclo[3.2.0]hept-2-en-6-one
Cyclopentadiene (1A-2) (26.4g, 0.4mol) and cyclohexane (1200mL) were added to a reaction flask, and triethylamine (24.2g, 0.24mol) was added after nitrogen blanket, the reaction system was heated to reflux, and a cyclohexane (50mL, 25mL/h) solution of 4-bromobutyryl chloride (44.4g, 0.24mol) was added dropwise, and the reaction was refluxed for 4 hours after completion of dropwise addition. After cooling to room temperature, the reaction mixture was suction-filtered, washed with cyclohexane (100mL × 3), the filtrates were combined, washed with saturated ammonium chloride (500mL × 3) and water (500mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 80:1) to give (+ -) - (1R,5S,7R) -7- (2-bromoethyl) bicyclo [3.2.0] hept-2-en-6-one (1B) (12g, yield: 24%) as a pale yellow oil.
1H NMR(400MHz,CDCl 3)δ5.97-5.85(m,1H),5.80-5.70(m,1H),3.91-3.79(m,1H),3.67(dd,J=9.7,5.5Hz,2H),3.47(t,J=6.8Hz,2H),2.68(ddd,J=18.3,15.2,3.9Hz,1H),2.47-2.31(m,1H),2.13(dq,J=21.0,6.5Hz,1H),1.93(ddd,J=21.5,12.2,7.1Hz,1H)。
The second step is that: (±) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] hept [2] ene-6, 2' - [1,3] dioxolane ] (1C-2)
(±)-1R,5S,7R)-7-(2-bromoethyl)spiro[bicyclo[3.2.0]hept[2]ene-6,2'-[1,3]dioxolane]
(+ -) - (1R,5S,7R) -7- (2-bromoethyl) bicyclo [3.2.0] hept-2-en-6-one (1B-2) (37g, 0.173mol), p-toluenesulfonic acid monohydrate (1.6g, 8.6mmol), ethylene glycol (42.9g, 0.692mol) and toluene (320 mL) were added in this order to a reaction flask, heated to reflux, and partitioned for 5 hours. After cooling, the reaction solution was poured into ice water, a saturated sodium bicarbonate solution was added to a pH of about 7, extraction was performed with ethyl acetate (400mL × 3), the organic phases were combined, the organic phase was dried over anhydrous sodium sulfate, filtration was performed, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give (+ -) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] hept [2] ene-6, 2' - [1,3] dioxolane ] (1C-2) as a yellow oil (27.4g, yield: 61%).
1H NMR(400MHz,CDCl 3)δ5.94-5.83(m,1H),5.67-5.56(m,1H),3.95-3.75(m,4H),3.36-3.25(m,2H),3.23-3.12(m,1H),3.02(ddd,J=22.9,15.7,8.0Hz,2H),2.48-2.25(m,2H),1.99-1.78(m,2H)。
The third step: (±) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] heptane-6, 2' - [1,3] dioxolane ] -2-ol (1D-2)
(±)-(1R,5S,7R)-7-(2-bromoethyl)spiro[bicyclo[3.2.0]heptane-6,2'-[1,3]dioxolan]-2-ol
(1'S,6'R)-spiro[[1,3]dioxolane-2,2'-tricyclo[4.2.1.03,8]nonan]-7'-one
Adding (+ -) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] into a reaction bottle]G2]Alkene-6, 2' - [1,3]Dioxolane](1C-2) (27.4g, 0.11mol) and tetrahydrofuran (330mL), dropwise adding borane dimethyl sulfide solution (55mL, 0.55mol) in an ice-water bath, reacting for 2 hours in an ice-water bath, and then sequentially dropwise adding purified water (1.1mol), sodium hydroxide aqueous solution (3mol/L, 360mL) and hydrogen peroxide (containing H)2O 21.1mol) of the solution, and the reaction was carried out for 3 hours while the temperature was raised to room temperature. Extracting with ethyl acetate (500mL × 3), washing the organic phase with saturated sodium bicarbonate solution (500mL × 2) and water (500mL × 2), drying over anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain a pale yellow oily liquid (±) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [ 3.2.0%]Heptane-6, 2' - [1,3]Dioxolane]2-alcohol (1D-2) (30g) was used directly in the next reaction.
The fourth step: (±) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] heptane-6, 2' - [1,3] dioxolane ] -2-one (1E-2)
(±)-(1R,5S,7R)-7-(2-bromoethyl)spiro[bicyclo[3.2.0]heptane-6,2'-[1,3]dioxolan]-2-one
(+ -) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] heptane-6, 2' - [1,3] dioxolane ] -2-ol (1D-2) (30g, 0.11mol) and dichloromethane (500mL) were added to a reaction flask, and dessimutane oxidant (70g, 0.17mol) was added in portions under ice bath to react at room temperature for 2 hours. Dichloromethane (300mL) and aqueous sodium thiosulfate (2M, 500mL) were added to the reaction solution, stirred for 30 minutes, separated, the aqueous phase was extracted with dichloromethane (300mL × 2), the organic phases were combined, washed with sodium hydroxide solution (2mol/L, 500mL × 2) and water (500mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 10:1) to give a pale yellow oily liquid (±) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0] heptane-6, 2' - [1,3] dioxolane ] -2-one (1E-2) (15g, yield: 50%).
1H NMR(400MHz,CDCl 3)δ4.02-3.81(m,4H),3.40(dd,J=10.3,3.8Hz,2H),3.15(td,J=10.3,4.9Hz,2H),2.61(ddd,J=20.6,14.0,8.1Hz,2H),2.27(ddt,J=18.9,9.6,1.8Hz,1H),2.12-2.00(m,1H),1.99-1.70(m,3H)。
The fifth step: (±) - (1'S,3' R,6'R,8' S) -spiro [ [1,3]Dioxolane-2, 2' -tricyclo [4.2.1.03,8]Nonane]-7' -Ketone (1F-2)
(±)-(1'S,3'R,6'R,8'S)-spiro[[1,3]dioxolane-2,2'-tricyclo[4.2.1.0 3,8]nonan]-7'-one
Adding potassium tert-butoxide (0.58g, 5.2mmol) and toluene (40mL) into a reaction bottle, introducing nitrogen for protection, cooling to-15 ℃, and dropwise adding (+/-) - (1R,5S,7R) -7- (2-bromoethyl) spiro [ bicyclo [3.2.0]]Heptane-6, 2' - [1,3]Dioxolane]-toluene solution of 2-ketone (1E-2) (1.1g, 1mmol, 5mL), -1 hour at 15 ℃ and then raising to 0 ℃ and stirring for 1 hour. Adding saturated ammonium chloride solution dropwise under ice bath until pH is about 7, extracting with ethyl acetate (80mL × 3), washing organic phase with water (80mL × 2), drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the residue with silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 8:1) to obtain light yellow oil (±) - (1' S,3' R,6' R,8' S) -spiro [ [1, 3' R,6' R,8' S ] - ]]Dioxolane-2, 2' -tricyclo [4.2.1.03,8]Nonane]-7' -keto (1F-2) (0.4g, yield: 51%).
1H NMR(400MHz,CDCl 3)δ4.04-3.86(m,4H),3.20-3.07(m,1H),2.99-2.86(m,1H),2.53(ddd,J=8.6,5.6,1.7Hz,1H),2.41-2.24(m,2H),2.24-2.01(m,2H),1.95(d,J=13.2Hz,1H),1.61(dddd,J=14.4,7.6,2.6,0.7Hz,1H),1.51-1.38(m,1H)。
And a sixth step: (±) - (1'S,3' R,6'S,8' S) -spiro [ [1,3]Dioxolane-2, 2' -tricyclo [4.2.1.03,8]Nonane](1G-2)
(±)-(1'S,3'R,6'S,8'S)--spiro[[1,3]dioxolane-2,2'-tricyclo[4.2.1.0 3,8]nonane]
Adding (+ -) - (1'S,3' R,6'S,8' S) -spiro [ [1,3] into a reaction flask]Dioxolane-2, 2' -tricyclo [4.2.1.03,8]Nonane]-7' -ketone (1F-2) (9.5g, 49mmol), diethylene glycol (170mL), hydrazine hydrate (18.4g, 294mmol) and potassium hydroxide (16.5g, 294mmol), reacted at 180 ℃ for 3 hours, water evaporated under reduced pressure at 70 ℃, warmed to 220 ℃ and stirred for 2 hours, cooled to room temperature, added water (200mL) to the reaction solution, extracted with methyl tert-butyl ether (300mL × 3), the combined organic phases washed successively with hydrochloric acid (1M, 500mL × 2) and water (500mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the residue purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 60:1) to give a colorless oil (±) - (1' S,3' R,6' S,8' S-spiro [1,3 [ ]]Dioxolane-2, 2' -tricyclo [4.2.1.03,8]Nonane](1G-2) (5.6G), and the obtained residue was used in the next step without purification.
The seventh step: (±) - (1S,6S,8S) -tricyclo [4.2.1.03,8]Nonane-2-one (1H-2)
(±)-(1S,6S,8S)-tricyclo[4.2.1.0 3,8]nonan-2-one
Adding (+ -) - (1'R,3' S,6'R,8' R) -spiro [ [1,3] into a reaction flask]Dioxolane-2, 2' -tricyclo [4.2.1.03,8]Nonane](1G-2) (5.6G, 31mmol), tetrahydrofuran (60mL) as solvent and water (20mL), adding trifluoroacetic acid (7G, 62mmol) dropwise in ice bath, reacting at 45 ℃ for 3 hours, adding saturated sodium bicarbonate solution dropwise in ice water bath until pH is about 7, extracting with ethyl acetate (100 mL. times.3), washing organic phase with water (200 mL. times.2), drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and collecting the residueSeparating and purifying the product by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to obtain white solid (+/-) - (1S,6S,8S) -tricyclo [4.2.1.03,8]Nonan-2-one (1H-2) (3.5g, yield: 83%).
1H NMR(400MHz,CDCl 3)δ3.47-3.33(m,1H),3.19(dd,J=3.3,1.8Hz,1H),2.84-2.69(m,1H),2.47-2.32(m,1H),2.12-1.97(m,1H),1.93(d,J=12.3Hz,1H),1.82-1.69(m,1H),1.56-1.35(m,4H),1.27-1.10(m,1H)。
Eighth step: tert-butyl 2- (tricyclo [4.2.1.0 ]3,8]Nonoalkyl-2-yliden) acetate (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (1I-2)
(±)-tert-butyl 2-tricyclo[4.2.1.0 3,8]nonan-2-ylidene)acetate(1R,3S,6R,8Rand 1S,3R,6S,8S racemate)
Sodium hydride (60%, 0.8g, 33.4mmol) and tetrahydrofuran (80mL) are added into a reaction bottle, the temperature is reduced to 0 ℃, a solution of diethylphosphonoacetic acid tert-butyl ester (7.5g, 33.4mmol) dissolved in tetrahydrofuran (10mL) is added dropwise, reaction is carried out at 0 ℃ for 20 minutes, and then (+/-) - (1R,3S,6R,8R) -tricyclo [4.2.1.0 ] is added dropwise3,8]Nonane-2-one (1H-2) (3.5g, 25.7mmol) in tetrahydrofuran (10mL) was reacted at room temperature for 2 hours. Water (100mL) and ethyl acetate (100mL) were added to the reaction mixture, followed by liquid separation with stirring, the aqueous phase was extracted with ethyl acetate (100 mL. times.2), the ethyl acetate was combined and dried over anhydrous sodium sulfate, filtration was performed, and the filtrate was concentrated under reduced pressure to give crude tert-butyl 2- (tricyclo [4.2.1.0 ] as a pale yellow oil3,8]Nonanyl-2-yliden) acetate (1R,3S,6R,8R and 1S,3R,6S,8S racemate) (1I-2) (5.1g) was used directly in the next step.
The ninth step: tert-butyl 2- (2- (nitromethyl) tricyclo [4.2.1.03,8]Nonanyl-2-yl) acetate (1R,2R,3S,6R,8R and 1S,2S,3R,6S,8S racemate) (INT10)
(±)-tert-butyl 2-(2-(nitromethyl)tricyclo[4.2.1.03,8]nonan-2-yl)acetate(1R,2R,3S,6R,8R and 1S,2S,3R,6S,8S racemate)
Mixing the crude product (+/-) -2- ((1R,3S,6R,8R) -tricyclo [ 4.2.1.0)3,8]Nonan-2-ylidene) acetic acid tert-butyl ester (1I-2) (5g, 24.3mmol), nitromethane (90mL) and 1, 8-diazabicyclo [5.4.0 ]]Undec-7-ene (6.6g, 43.7mmol) was added to the reaction flask in sequence and the temperature was raised to 70 ℃ for reaction for 6 hours. Ethyl acetate (100mL) and 1M hydrochloric acid solution (100mL) were added to the reaction solution, followed by partition with stirring, extraction of the aqueous phase with ethyl acetate (100mL × 2), drying over anhydrous sodium sulfate, filtration, concentration of the filtrate under reduced pressure, and separation and purification of the residue by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give (+ -) -2- ((1R,2R,3S,6R,8R) -2- (nitromethyl) tricyclo [4.2.1.03, 8) as a colorless oily liquid]Nonan-2-yl) acetic acid tert-butyl ester (INT10) (5g, yield: 70%).
The tenth step: (1' S,2' S,6' S,8' S) -spiro [ pyrrolidine-3, 2' -tricyclo [4.2.1.03,8]None]-5-ketones
(1'S,2'S,6'S,8'S)-spiro[pyrrolidine-3,2'-tricyclo[4.2.1.0 3,8]nonan]-5-one
INT10(75g, 280.6mmol) was added to a three-necked flask, ethanol (400mL) and water (200mL) were added in this order, iron powder (78.34g,1403mmol) and ammonium chloride (75.04g,1403mmol) were added with stirring, the mixture was heated to 100 ℃ and stirred for 5 hours, suction filtration was performed, the filtrate was collected, ethanol was concentrated off, the remaining portion was extracted with ethyl acetate (800mL), ethyl acetate was washed with 2M HCl, the organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 30 ℃ to obtain INT11-A (52.0g,271.87mmol) as a white solid.
The eleventh step: (1' S,2' S,6' S,8' S) -spiro [ pyrrolidine-3, 2' -tricyclo [4.2.1.03,8]None]-5-ketones
(1'S,2'S,6'S,8'S)-spiro[pyrrolidine-3,2'-tricyclo[4.2.1.0 3-8]nonan]-5-one
Putting a compound INT11-A (3g,16mmol) in a three-neck flask, adding 12mL of absolute ethanol, heating to 70 ℃, cooling to room temperature while stirring until all solids are dissolved, separating out solids, carrying out suction filtration, washing a filter cake once with 5mL of ethyl acetate, and draining the solvent to obtain a solid impurity C.
Ms m/z(ESI):192.2[M+H+]。
δH(ppm)(400MHz,CD3OD) | Number of hydrogens and cleavage events |
1.24-1.17 | 1,m |
1.47-1.36 | 2,m |
1.53-1.50 | 1,d |
1.64-1.57 | 1,m |
1.75-1.66 | 2,m |
2.01-1.92 | 1,m |
2.21-2.20 | 3,d |
2.36-2.26 | 1,q |
2.48-2.45 | 1,m |
2.97-2.82 | 1,m |
3.31-3.30 | 1,m |
3.48-3.45 | 1,d |
3.56-3.53 | 1,d |
The preparation method of the impurity D comprises the following steps:
the first step is as follows: methyl-2- ((1S,2S,3R,6S,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
Methyl-2-((1S,2S,3R,6S,8S)-2-(((tert-butoxycarbonyl)amino)methyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
2- ((1S,2S,3R,6S,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]INT6(2.10g,6.79mmol) of nonan-2-yl) acetic acid was added to a round-bottomed flask, DMF (20mL), potassium carbonate (1.35g,9.7mmol) were added successively, methyl iodide (1.35g,9.7mmol) was added dropwise slowly in an ice-water bath, after addition, the reaction was carried out at room temperature for 2 hours, 30mL of water was added to the reaction solution, extraction was carried out with methyl tert-butyl ether (30 mL. times.2), the organic phases were combined and washed with saturated brineWashing (50mL multiplied by 3), drying by anhydrous sodium sulfate, and concentrating under reduced pressure to obtain methyl-2- ((1S,2S,3R,6S,8S) -2- (((tert-butyloxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetate INT12-A (1.9g, 95%).
1H NMR(400MHz,CDCl 3):δ4.96(br,1H),3.65(s,3H),3.39(d,2H,J=10.4Hz),2.84(dd,1H,J=12.8Hz,5.2Hz),2.41-2.37(m,3H),2.28(dd,1H,J=12.0Hz,6.0Hz),2.07(m,1H),1.98(m,1H),1.72(m,1H),1.58-1.51(m,4H),1.44(s,9H),1.25-1.18(m,2H)。
The second step is that: methyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetate benzenesulfonate
methyl 2-((1S,2S,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate benzenesulfonate
Reacting methyl-2- ((1S,2S,3R,6S,8S) -2- (((tert-butyloxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Adding nonan-2-yl) acetate INT12-A (1.9g,5.8mmol) into a round-bottom flask, sequentially adding benzenesulfonic acid (1.83g,11.6mmol) and acetonitrile 20mL, heating to 80 ℃, reacting for 0.5h, cooling to room temperature, concentrating under reduced pressure to obtain a viscous white solid product, adding 10mL methyl tert-butyl ether, pulping, stirring for 10min, and filtering to obtain a white solid, namely methyl 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.0 ]3,8]Nonan-2-yl) acetate benzenesulfonate impurity D.
Ms m/z(ESI):224.2[M+H +]。
δH(ppm)(400MHz,CDCl 3) | Number of hydrogens and cleavage events |
1.14-1.10 | 1,m |
1.31-1.26 | 1,m |
1.49-1.38 | 4,m |
1.65 | 1,m |
1.93 | 1,m |
2.12 | 1,m |
2.23 | 1,dd |
2.42 | 1,dd |
2.56 | 2,s |
2.71 | 1,dd |
3.33 | 2,s |
3.52 | 3,s |
7.39 | 4,m |
7.79-7.87 | 4,m |
The preparation method of the impurity E comprises the following steps:
the first step is as follows:
isopropyl-2- ((1S,2S,3R,6S,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
isopropyl-2-((1S,2S,3R,6S,8S)-2-(((tert-butoxycarbonyl)amino)methyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
2- ((1S,2S,3R,6S,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]INT6(2.02g, 6.47mmol) of nonan-2-yl) acetic acid is added to a round-bottom flask, DMF (20mL), potassium carbonate (1.35g,14.5mmol) are sequentially added, isopropyl iodide (1.62g, 9.7mmol) is slowly added dropwise under an ice-water bath, after addition, the reaction is carried out at room temperature for 2h, 30mL of water is added to the reaction solution, methyl tert-butyl ether is used for extraction (30 mL. times.2), the organic phases are combined and washed with saturated brine (50 mL. times.3), anhydrous sodium sulfate is dried, and the mixture is concentrated under reduced pressure to obtain ethyl-2- ((1R,2R,3S,6R,8R) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetate INT13-A (2.2 g).
1H NMR(400MHz,CDCl 3):δ4.99(m,2H),3.39(d,2H,J=5.6Hz),2.85(m,1H),2.39(m,1H),2.32(d,2H,J=3.2Hz),2.27(m,1H),2.07(m,1H),1.94(m,1H),1.72(m,1H),1.59-1.47(m,5H),1.44(s,9H),1.24(d,6H,J=6.0Hz),1.22(m,1H)。
The second step is that:
isopropyl 2- ((1R,2R,3S,6R,8R) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-Yl) acetate benzenesulfonate
isopropyl 2-((1R,2R,3S,6R,8R)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate benzenesulfonate
Isopropyl-2- ((1R,2R,3S,6R,8R) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Adding nonan-2-yl) acetate INT13-A (2.2g,6.26mmol) into a round-bottom flask, sequentially adding benzenesulfonic acid (1.99g,12.52mmol) and acetonitrile 22mL, heating to 80 ℃, reacting for 0.5h, cooling to room temperature, concentrating under reduced pressure to obtain a white solid, adding 10mL methyl tert-butyl ether, pulping, stirring for 5min, and filtering to obtain a white solid product ethyl 2- ((1R,2R,3S,6R,8R) -2- (aminomethyl) tricyclo [4.2.1.0 ]3,8]Non-2-yl) acetate benzenesulfonate impurity E.
Ms m/z(ESI):252.2[M+H +]。
δH(ppm)(400MHz,CDCl 3) | Number of hydrogens and cleavage events |
1.12 | 1,m |
1.15 | 6,d |
1.29 | 1,m |
1.49-1.36 | 4,m |
1.62 | 1,m |
1.92 | 1,m |
2.10 | 1,m |
2.23 | 1,dd |
2.42 | 1,dd |
2.51 | 2,s |
2.71 | 1,dd |
3.33 | 2,m |
4.90 | 1,m |
7.39 | 4,m |
7.86 | 3,m |
9.60 | 1,br |
The preparation method of the impurity F comprises the following steps:
the first step is as follows: ethyl 2- ((1S,3R,6S,8S) -tricyclo [4.2.1.03,8]Nonane-2-methylene) ethyl ester (+/-)
ethyl 2-((1R,3S,6R,8R)-tricyclo[4.2.1.0 3,8]nonan-2-ylidene)acetate(+/-)
Adding sodium hydride (266.55g,6.109mol) into a three-neck flask, adding 9L of dried tetrahydrofuran under the protection of nitrogen, stirring, cooling a reaction system to 0 ℃, dropwise adding dimethoxyphosphonoacetic acid ethyl ester (1369.6g,6.109mmol), keeping the temperature for 15min after the addition is finished, dropwise adding a tetrahydrofuran solution of a raw material INT1(640g,4.699mol), naturally heating to room temperature after the addition is finished, and reacting for 1 h. TLC monitors the reaction is complete, saturated ammonium chloride is added dropwise in ice water bath until the pH of the reaction solution is 7-8, 8L of water is added, and then liquid separation is carried out. The aqueous phase was extracted with ethyl acetate (4000mL), the organic phases combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure and separated by column chromatography (PE: EA ═ 100:1) to give product INT14(590g,2.860 mol).
The second step is that: ethyl 2- ((1R,3S,6R,8R) -2- (nitromethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) ethyl ester (+/-)
ethyl 2-((1R,3S,6R,8R)-2-(nitromethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate(+/-)
Adding INT14(590g,2.860mol) serving as a raw material into a reaction bottle, adding 6L nitromethane, dropwise adding DBU (826.5g,5.429mol) at room temperature, reacting at 80 ℃ for about 9 hours, pouring the reaction liquid into ice water (4L), extracting DCM (4L multiplied by 2), combining organic phases, drying by anhydrous sodium sulfate, concentrating, and then carrying out column chromatography separation (PE: EA is 200:1-30:1) to obtain INT15(507.9g,1.9 mol).
Third step resolution by SFC resolution of INT15(507.9g,1.9mol), the preparation conditions are as follows: the instrument comprises the following steps: waters UPC2 analytical SFC (SFC-H); a chromatographic column: ChiralPak AD,150 × 4.6mm i.d.,3 μm; mobile phase: a for CO2and B for MeOH (0.05% DEA); gradient: b5-40%; flow rate: 2.5 mL/min; the column temperature was 35 ℃. Two optical isomers were obtained after separation: peak 1 (retention time: 3.247 min), where Peak 1 is INT16(250g, 187.1 mmol).
1H NMR(400MHz,CDCl 3)δ4.74(q,2H),2.86(dt,1H),2.75–2.63(m,2H),2.53(ddd,1H),2.31(tt,2H),2.05(dt,1H),1.84–1.73(m,1H),1.68–1.55(m,4H),1.52(dd,1H),1.48–1.40(m,9H),1.31–1.23(m,1H)。
The fourth step: (1'R,2' R,3'S,6' R,8'R) -spiro [ pyrrolidine-3, 2' -tricyclo [4.2.1.03,8]None]-5-ketones
(1'R,2'R,3'S,6'R,8'R)-spiro[pyrrolidine-3,2'-tricyclo[4.2.1.0 3,8]nonan]-5-one
INT16(50g, 187.1mmol) was added to a three-necked flask, ethanol (200mL) and water (100mL) were added in this order, iron powder (41.78g,748.2mmol) and ammonium chloride (40.02g,748.2mmol) were added with stirring, the mixture was heated to 100 ℃ and stirred for 5 hours, suction filtration was performed, the filtrate was collected, ethanol was concentrated off, the remaining portion was extracted with ethyl acetate (600mL), the organic phase was washed with 2M HCl, the organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 30 ℃ to obtain INT17(40.16g) as a white solid. Dissolving the white solid in 200mL of ethyl acetate for recrystallization, heating to 80 ℃ for dissolution, cooling, stirring, crystallizing, and carrying out suction filtration to obtain a filter cake with the wet weight of 30.65g, and pumping the solvent by an oil pump for 2 hours to obtain a white solid INT17(28.30 g).
Ms m/z(ESI):192.2[M+H +]。
1H NMR(400MHz,CDCl 3):δ6.06(br,1H),3.36-3.61(m,2H)2.81(dt,1H),2.45(ddd,1H),2.06-2.33(m,4H),1.89-2.01(m,1H),1.29-1.73(m,6H),1.12-1.23(m,1H)。
The fifth step: 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate
2-((1S,2S,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid Benzenesulfonate
Adding compound INT17(9.4g,49.2mmol) into 120mL of water, stirring, adding benzenesulfonic acid (52.33g,282.87mmol), heating to 120 ℃, stirring for 4 hours, precipitating white solid, monitoring the reaction by LC-MS until the reaction is complete, cooling to room temperature, performing suction filtration, washing the filter cake for 3 times by using ice water 150mL, performing suction filtration to dry the filter cake, pulping the filter cake for three times by using dichloromethane 150mL, and performing suction filtration to obtain impurity F (13.07 g).
Ms m/z(ESI):210.2[M+H+]。
1H NMR(400MHz,CD3OD):δ7.83(ddd,2H),7.34-7.50(m,3H),3.24-3.39(m,2H),2.86(dt,1H),2.42-2.59(m,3H),2.32(dd,1H),1.94-2.19(m,2H),1.38-1.82(m,6H),1.22-1.31(m,1H)。
And a sixth step: 2- ((1R,2R,3S,6R,8R) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate
2-((1S,2S,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid Benzenesulfonate
Adding impurity F (13g,35.42mmol) into a three-neck flask, adding 15mL of methanol and 34mL of water, heating to 85 deg.C, stirring for 25min, cooling to room temperature, stirring overnight, filtering, and mixing the filter cake with ice (MeOH: H)2O is 3:1), and the mixture is washed and dried by pumping. Obtaining solid impurity F.
Ms m/z(ESI):210.2[M+H +]。
The preparation method of the impurity G comprises the following steps:
the first step is as follows: (1S,3R,6S,8S) -2- (nitromethyl) tricyclo [4.2.1.03,8]Nonan-2-ols
(1S,3R,6S,8S)-2-(nitromethyl)tricyclo[4.2.1.0 3,8]nonan-2-ol
INT1(72.3g, 0.53mol), nitromethane (1.0L), DBU (80.8g, 0.53mol) were added to the reaction flask and reacted at room temperature for 18 h. Saturated aqueous ammonium chloride (5L) was added, extracted with dichloromethane (2L. times.3), the organic layers were combined, dried, filtered and concentrated. Purification by silica gel column chromatography (100: 1-10:1 petroleum ether/ethyl acetate (v/v)) gave INT7(10.5g, 10.1% yield) as a yellow oil.
1H NMR(400MHz,CDCl 3):δ4.73–4.40(m,2H),2.65–2.58(m,1H),2.51–2.44(m,1H),2.34(qd,2H),1.99–1.86(m,1H),1.80–1.44(m,6H),1.30–1.17(m,1H)。
The second step is that: (1S,3R,6S,8S) -2- (nitromethylene) tricyclo [4.2.1.03,8]Nonane
(1S,3R,6S,8S)-2-(nitromethylene)tricyclo[4.2.1.0 3,8]nonane
INT7(0.50g, 2.54mmol), acetic anhydride (10mL), p-toluenesulfonic acid (0.44g, 2.54mmol) were added to the reaction flask and reacted at room temperature for 2 h. Cooled to 0 ℃, 50ml of saturated aqueous sodium bicarbonate solution was added, stirred for 1h, extracted with dichloromethane (35ml × 3), and the organic layers were combined, dried, filtered, and concentrated. This gave 0.15g of a colorless liquid which was used directly in the next reaction, dissolved in methanol (8mL), cooled to 0 ℃ and reacted at room temperature for 2h with sodium methoxide (0.03g, 0.61 mmol). 20mL of a saturated aqueous ammonium chloride solution was added, extraction was performed with methylene chloride (40 mL. times.3), and the organic layers were combined, dried, filtered, and concentrated. Purification by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) gave INT8(0.08g, yield 74.1%) as a yellow oil.
1H NMR(400MHz,CDCl 3):δ6.87(dd,1H),3.94–3.61(m,1H),3.25(ddd,1H),3.01–2.80(m,1H),2.39(td,1H),2.12–1.76(m,4H),1.73–1.51(m,1H),1.48–1.23(m,3H)。
The third step: tert-butyl 2- ((1S,2R,3R,6S,8S) -2- (nitromethylene) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
tert-butyl 2-((1S,2R,3R,6S,8S)-2-(nitromethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
1M lithium bis (trimethylsilyl) amide (290mL, 0.29mol) was added to the reaction flask, nitrogen blanketed, cooled to-60 deg.C, a solution of tert-butyl acetate (33.62g, 0.29mmol) in tetrahydrofuran (150mL) was added dropwise, stirred for 20mins, a solution of INT8(28.82g,0.16mol) in tetrahydrofuran (250mL) was added dropwise, and the reaction was allowed to proceed at-60 deg.C for 2 h. Saturated aqueous ammonium chloride (400mL) was added, extraction was performed with ethyl acetate (200 mL. times.3), and the organic layers were combined, dried, filtered, and concentrated. Purification by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) gave INT9(39.4g, yield 84.8%) as a colorless liquid. Chiral resolution (preparation conditions: instrument: Waters UPC2 analytical SFC (SFC-H); chromatographic column: ChiralPak AD, 150X 4.6mm I.D.,3 μm; mobile phase: A for CO2and B for EtOH (0.05% DEA); gradient: b5-40%; flow rate: 2.5 mL/min; the column temperature was 35 ℃. ) Two optical isomers were obtained after separation: peak 1 (retention time: 2.452 min, 9.33g), wherein peak 1 is (+) -2- ((1R,2S,3S,6R,8R) -2- (nitromethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid tert-butyl ester (INT 18).
1H NMR(400MHz,CDCl 3)δ4.74(q,2H),2.86(dt,1H),2.75–2.63(m,2H),2.53(ddd,1H),2.31(tt,2H),2.05(dt,1H),1.84–1.73(m,1H),1.68–1.55(m,4H),1.52(dd,1H),1.48–1.40(m,9H),1.31–1.23(m,1H)。
The fourth step: tert-butyl 2- ((1S,2R,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
tert-butyl 2-((1S,2R,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
To a reaction flask was added (+) -2- ((1S,2R,3R,6S,8S) -2- (nitromethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid tert-butyl ester (INT18, 8.33g,28.1mmol), ethanol (50mL), water (25mL), iron powder (7.87g, 0.14mol), ammonium chloride (7.56g, 0.14mol), refluxing for 7 h. After cooling to room temperature, filtration, washing of the filter cake with ethanol (150ml), addition of 100ml of water, extraction with dichloromethane (100ml × 3), combination of organic layers, drying of the organic layers, filtration, concentration, purification by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1,10:1) gave INT19(7.49g, 99.4% yield) as a pale yellow oil.
1H NMR(400MHz,CDCl 3)δ3.14(d,2H),2.89–2.72(m,3H),2.52–2.42(m,1H),2.35–2.18(m,2H),2.05–1.93(m,1H),1.78–1.68(m,1H),1.64–1.40(m,13H),1.33–1.17(m,2H)。
The fifth step is 2- ((1S,2R,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid
2-((1S,2R,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid
INT19(7.4g, 2.79mmol), dichloromethane (50mL) were added to the reaction flask, trifluoroacetic acid (50mL) was added dropwise in ice bath, reaction was carried out at room temperature for 2 hours after the addition was completed, concentration was carried out under reduced pressure, the residue was dissolved in dichloromethane (100mL), triethylamine was added to the solution until pH was 7-8, and filtration was carried out to obtain INT20(3.9g, yield: 67.0%) as a white solid.
1H NMR(400MHz,CD 3OD):δ3.00(dt,3H),2.72(s,2H),2.49(s,1H),2.32(d,1H),2.16(s,1H),2.03(t,1H),1.76(s,1H),1.67(d,2H),1.58–1.46(m,2H),1.47–1.37(m,1H),1.30–1.20(m,1H)。
And a sixth step: 2- ((1S,2R,3R,6S,8S) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid
2-((1S,2R,3R,6S,8S)-2-(((tert-butoxycarbonyl)amino)methyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid
A50 ml aqueous solution of INT20(4.58g, 21.9mmol), tetrahydrofuran (60ml) and sodium hydroxide (2.02g, 50.4mmol) was added to the reaction flask, cooled to 0 ℃ and reacted at room temperature for 2h by adding a solution of di-tert-butyl dicarbonate (5.25g, 24.1mmol) in 45ml tetrahydrofuran. Sodium hydroxide (1.01g, 25.2mmol) and di-tert-butyl dicarbonate (2.62g, 12.0mmol) were added and the reaction was carried out at room temperature for 1 hour. Using petroleum ether: washing with a mixed solution of ethyl acetate (5:1) (100 ml. times.3), combining the organic layers, extracting 1 time with 100ml of pure water, combining the aqueous layers, adding sodium dihydrogenphosphate to adjust pH to about 4, extracting with ethyl acetate (100 ml. times.3), combining the organic layers, concentrating, adding 80ml of cyclohexane, stirring, heating to reflux, dissolving, cooling to room temperature to precipitate a solid, filtering to obtain a white solid INT21(6.4g, 99%) which is not purified and is directly used in the next step.
The seventh step: 2- ((1S,2R,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate
2-((1S,2R,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid benzenesulfonate
INT21(5.9g, 19.1mmol), water (47ml), benzenesulfonic acid (7.1g, 38.2mmol) was added to the reaction flask, which was heated to 100 ℃ for 2 h. Cooled to room temperature and filtered to give a white solid as impurity G.
δH(ppm)(400MHz,CD3OD) | Number of hydrogens and cleavage events |
7.84~7.82 | 2H,m |
7.44~7.41 | 3H,m |
3.34 | 2,s |
2.89~2.84 | 1H,m |
2.55~2.54 | 2H,d |
2.50~2.46 | 1H,m |
2.36~2.27 | 1H,m |
2.35~2.30 | 1H,m |
2.17~2.13 | 1H,m |
2.07~2.00 | 1H,m |
1.81~1.74 | 1H,m |
1.70~1.62 | 2H,m |
1.61~1.53 | 2H,m |
1.48~1.42 | 1H,m |
1.31~1.25 | 1H,m |
Ms m/z(ESI):210.2[M+H +]。
The preparation method of the impurity H comprises the following steps:
the first step is as follows: tert-butyl-2- ((1R,2S,3S,6R,8R) -2- (nitromethyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
tert-butyl 2-((1R,2S,3S,6R,8R)-2-(nitromethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
INT9 chiral resolution, preparation conditions: the instrument comprises the following steps: waters UPC2 analytical SFC (SFC-H); a chromatographic column: ChiralPak AD,150 × 4.6mm i.d.,3 μm; mobile phase: a for CO2and B for EtOH (0.05% DEA); gradient: b5-40%; flow rate: 2.5 mL/min; the column temperature was 35 ℃. Two optical isomers were obtained after separation: peak 2 (retention time: 3.247 min, 8.68g), where Peak 2 is (-) -2- ((1R, 2)S,3S,6R,8R) -2- (nitromethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid tert-butyl ester (INT 22).
1H NMR(400MHz,CDCl 3)δ4.74(q,2H),2.86(dt,1H),2.75–2.63(m,2H),2.53(ddd,1H),2.31(tt,2H),2.05(dt,1H),1.84–1.73(m,1H),1.68–1.55(m,4H),1.52(dd,1H),1.48–1.40(m,9H),1.31–1.23(m,1H)。
The second step is that: tert-butyl-2- ((1S,2R,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid ester
tert-butyl-2-((1S,2R,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetate
INT22(7.3g,24.7mmol), ethanol (50mL), water (25mL), iron powder (6.9g, 123.6mmol), ammonium chloride (6.6g, 123.6mmol) were added to the reaction flask and the reaction was refluxed for 6 h. After cooling to room temperature, filtration, washing of the filter cake with ethanol (60ml × 2), combining the filtrates, concentration to remove ethanol, addition of 150ml water, extraction with dichloromethane (300ml × 2), combining the organic layers, drying of the organic layers, filtration, concentration to yield INT23(6.56g, 100% yield) as a white solid.
1H NMR(400MHz,CDCl 3)δ3.14(d,2H),2.89–2.72(m,3H),2.52–2.42(m,1H),2.35–2.18(m,2H),2.05–1.93(m,1H),1.78–1.68(m,1H),1.64–1.40(m,13H),1.33–1.17(m,2H)。
The third step: 2- ((1R,2S,3S,6R,8R) -2- (aminomethyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid
2-((1S,2R,3R,6S,8S)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid
INT23(6.56g, 24.7mmol) and dichloromethane (60mL) were added to the reaction flask, trifluoroacetic acid (60mL) was added dropwise, the reaction was allowed to react at room temperature for 3 hours, concentrated under reduced pressure, the residue was dissolved in dichloromethane (150mL), triethylamine was added to the solution pH 7-8, suction filtered, and the filter cake was washed with dichloromethane (20 mL. times.3) to give INT24(4.0g, yield: 77.3%) as a white solid.
1H NMR(400MHz,CD 3OD):δ3.00(dt,3H),2.72(s,2H),2.49(s,1H),2.32(d,1H),2.16(s,1H),2.03(t,1H),1.76(s,1H),1.67(d,2H),1.58–1.46(m,2H),1.47–1.37(m,1H),1.30–1.20(m,1H)。
The fourth step: 2- ((1R,2S,3S,6R,8R) -2- (((tert-butoxycarbonyl) amino) methyl) tricyclo [4.2.1.03,8]Non-2-yl) acetic acid
2-((1R,2S,3S,6R,8R)-2-(((tert-butoxycarbonyl)amino)methyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid
A50 ml aqueous solution of INT24(4.47g, 22.7mmol), tetrahydrofuran (60ml) and sodium hydroxide (2.27g, 56.7mmol) was added to the reaction flask, cooled to 0 ℃ and a solution of di-tert-butyl dicarbonate (9.91g, 45.4mmol) in 45ml tetrahydrofuran was added dropwise and reacted at room temperature for 2 h. Sodium hydroxide (1.1g, 27.5mmol) and di-tert-butyl dicarbonate (2.62g, 12.0mmol) were added and the reaction was carried out at room temperature for 1 hour. Using petroleum ether: the reaction solution was washed with a mixed solution (100 ml. times.3) of ethyl acetate (5:1), the organic layers were combined, extracted 1 time with 100ml of pure water, the aqueous layers were combined, sodium dihydrogenphosphate was added to adjust the pH to about 4, extracted with ethyl acetate (100 ml. times.3), the organic layers were combined, concentrated, added with 80ml of cyclohexane, stirred, heated to reflux, dissolved, cooled to room temperature to precipitate a solid, filtered to obtain INT25(5.5g, 83.3%) as a white solid, which was not purified directly in the next step.
The fifth step: 2- ((1R,2S,3S,6R,8R) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate
2-((1R,2S,3S,6R,8R)-2-(aminomethyl)tricyclo[4.2.1.0 3,8]nonan-2-yl)acetic acid
INT25(5.0g, 16.2mmol), water (40ml), benzenesulfonic acid (6.0g, 32.4mmol) was added to the reaction flask and heated to 100 ℃ for 2 h. Cooled to room temperature and filtered to give a white solid as impurity H.
δH(ppm)(400MHz,CD3OD) | Number of hydrogens and cleavage events |
7.87~7.78 | 2H,m |
7.49~7.36 | 3H,m |
3.19 | 2H |
2.94~2.86 | 1H,m |
2.77 | 2H |
2.47~2.39 | 1H,m |
2.35~2.29 | 1H,m |
2.25~2.18 | 1H,m |
2.08~1.97 | 1H,m |
1,82~1.71 | 1H,m |
1.70~1.63 | 1H,m |
1.65~1.57 | 1H,m |
1.59~1.51 | 2H,m |
1.45~1.35 | 1H,m |
1.32~1.22 | 1H,m |
Claims (27)
- 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate or 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]A method for detecting related substances I in a composition of nonane-2-yl) acetic acid benzene sulfonate, wherein the structural formula of the related substances I is shown in the specificationThe detection is carried out by an HPLC-CAD method, and comprises the following steps:1) preparation of test solution of related substance I: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate or its composition is added with diluent to prepare test solution of related substance I;2) Preparation of control solutions for substance i of interest: adding a diluent into the related substance I test solution obtained in the step 1) to dilute by 100 times to obtain the test solution;3) injecting the test solution of the related substance I obtained in the step 1) and the reference solution of the related substance I obtained in the step 2) into a liquid chromatograph under the chromatographic conditions that:a chromatographic column: octadecylsilane bonded silica gel column (4.6X 250mm, 5 μm);mobile phase: a: 10mM ammonium formate solution, B: acetonitrile;gradient elution procedure: 0-5 min, A90%; 5-25 min, A90% → 40%; 25-29 min, A40% → 20%; 29-35 min, A20%; 35-36 min, A20% → 90%; 36-40 min, A90%;flow rate: 1.0 mL/min;column temperature: 35 ℃;CAD parameters: the atomization temperature is 50 ℃, the acquisition frequency is 5.0Hz, and the optical filter is 5.
- The detection method according to claim 1, wherein the diluent in step 1) or step 2) is a mixed solvent of ammonium formate and acetonitrile, preferably a mixed solvent of 10mM ammonium formate and acetonitrile, more preferably 10mM ammonium formate: acetonitrile volume ratio of 80: 20, or a mixed solvent thereof.
- The detection method according to claim 1 or 2, wherein the step 1) of preparing a test solution of the substance I comprises: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is dissolved and diluted to scale by adding a diluent in a volumetric flask with the volume of 50mg to 10mL to obtain the product;the diluent is a mixed solvent of ammonium formate and acetonitrile, preferably a mixed solvent of 10mM ammonium formate and acetonitrile, more preferably 10mM ammonium formate: acetonitrile volume ratio of 80: 20, or a mixed solvent thereof.
- The test method according to any one of claims 1 to 3, wherein the concentration of the test solution of the substance of interest I obtained in step 1) is 5 mg/mL.
- The test method according to any one of claims 1 to 4, wherein the step 2) of preparing a control solution of substance I comprises: precisely measuring a 1.0-100 mL volumetric flask of the related substance I test solution obtained in the step 1), adding a diluent to dilute to a scale, and shaking up to obtain the test solution;the diluent is preferably a mixed solvent of ammonium formate and acetonitrile, more preferably a mixed solvent of 10mM ammonium formate and acetonitrile, still more preferably 10mM ammonium formate: acetonitrile volume ratio of 80: 20, or a mixed solvent thereof.
- 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate or 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]A method for detecting related substance II in a composition of nonane-2-yl) acetic acid benzene sulfonate, wherein the related substance II comprises impurity A, B, C, D, E:the detection is carried out by adopting LC-MS, and comprises the following steps:1) preparation of control solutions for related substance II: dissolving related substance II reference substance with diluent to obtain related substance II reference substance solution;2) preparation of related substance II test solution: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof to prepare a test solution of a related substance II;3) injecting the related substance II reference substance solution obtained in the step 1) and the related substance II test solution obtained in the step 2) into a liquid chromatograph under the chromatographic conditions that:a chromatographic column: octadecylsilane chemically bonded silica gel column (4.6X 250mm, 3.5 μm);mobile phase: a: 10mM ammonium formate solution, B: acetonitrile;gradient elution procedure: 0-15 min, A70% → 30%; 15-16 min, A30% → 10%; 16-18 min, A10%; 18-20 min, 10% → 70%; 20-25 min, A70%;flow rate: 1.0 mL/min;column temperature: 30 ℃;MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+).
- The assay of claim 6 wherein the step 1) preparation of a reference solution of related substance II comprises: adding a related substance II reference substance into a diluent to dissolve and dilute to obtain a related substance II reference substance solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water volume ratio 5: 1.
- the test method according to claim 6 or 7, wherein the step 1) of preparing a reference solution of related substance II comprises: precisely weighing a reference substance II of a related substance II in a volumetric flask with the volume of 25mg to 10mL, adding a diluent to dissolve and dilute the reference substance II to a scale mark to be used as a reference substance stock solution of the related substance II; precisely measuring related substance II reference substance stock solution 100 μ L to 100mL volumetric flask, adding diluent to dilute to scale, and shaking up to obtain; orRespectively weighing 10mg of reference substances of impurities A, B, C, D and E, precisely weighing, placing in a 10mL measuring flask, dissolving and diluting with a diluent to prepare a solution containing 1mg per 1mL as reference substance stock solution; precisely measuring 25 mu L of each of reference stock solutions of impurities A, D and E, 50 mu L of reference stock solution of impurity C and 200 mu L to 100mL of reference stock solution of impurity B, and diluting with a diluent to obtain a mixed solution containing about 0.25 mu g of impurity A, D and E, about 2 mu g of impurity B and about 0.5 mu g of impurity C per 1mL as a reference solution;the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water volume ratio 5: 1.
- the test method according to any one of claims 6 to 8, wherein the concentration of the reference substance II obtained in step 1) is 2.5. mu.g/mL or 1. mu.g/mL.
- The detection method according to claim 6, wherein the preparation of the test solution of the substance II of interest in the step 2) comprises: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Adding a diluent to dissolve nonane-2-yl) acetic acid benzene sulfonate or a composition thereof and diluting to obtain a related substance II test sample solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water volume ratio 5: 1.
- the test method according to claim 10, wherein the step 2) of preparing a test solution of the substance II comprises: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Adding a diluent into a volumetric flask with 25 mg-25 mL of nonane-2-yl) acetic acid benzene sulfonate or a composition thereof for dissolving, diluting to scale, and shaking up to obtain the nonane-2-yl) acetic acid benzene sulfonate;the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the diluent is acetonitrile: water volume ratio 5: 1.
- the detection method according to any one of claims 6 to 11, wherein the concentration of the test solution of the substance II of interest obtained in the step 2) is 1 mg/mL.
- 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate or 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]A method for detecting a chiral isomer in a composition of nonan-2-yl) acetic acid benzenesulfonate, wherein the chiral isomer comprises impurity F, G, H, the impurity F, G, H is as follows:the detection is carried out by adopting LC-MS, and comprises the following steps:1) preparation of chiral isomer control solution: taking a proper amount of chiral isomer reference substance, dissolving by using a diluent and preparing an isomer reference substance solution;2) preparation of chiral isomer test solution: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is used for preparing a chiral isomer test solution;3) injecting the chiral isomer reference solution obtained in the step 1) and the chiral isomer test solution obtained in the step 2) into a liquid chromatograph, wherein the chromatographic conditions are as follows:a chromatographic column: CROWNPAK CR-I (+) (3X 150mm, 5 μm);mobile phase: a: perchloric acid solution (ph1.3), B: acetonitrile;elution procedure: mobile phase A: mobile phase B80: 20 isocratic elution for 35 minutes;flow rate: 0.2 mL/min;column temperature: 20 ℃;MS parameters: ESI ion source, atomization chamber pressure 35psig, dry gas flow rate 12L/min, dry gas temperature 350 ℃, detection mode SIM (+).
- The assay of claim 13 wherein step 1) chiral isomer control solution preparation comprises: adding a diluent into a chiral isomer reference substance to dissolve and dilute the chiral isomer reference substance to obtain a chiral isomer reference substance solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferred, acetonitrile: water volume ratio 20: 80.
- the assay of claim 14, wherein the step 1) chiral isomer control solution preparation comprises: precisely weighing a chiral isomer reference substance 10 mg-10 mL volumetric flask, adding a diluent to dissolve and dilute to a scale, and taking the reference substance as a reference substance stock solution; precisely measuring a volumetric flask with the reference substance stock solution of 100 mu L to 100mL, adding a diluent to dilute to a scale, and shaking up to obtain the product; orRespectively weighing 10mg of impurity F and impurity G (or impurity H) reference substances, precisely weighing, placing in a 10mL measuring flask, dissolving and diluting with diluent (acetonitrile: water: 1:4) to prepare 1mg solution per 1mL, and using the solution as reference substance stock solution; precisely measuring 100 mu L of reference stock solution of impurities F and G (or H) to 100mL, diluting with diluent to obtain a mixed solution containing 1 mu G of impurities F and 1 mu G of impurities G (or H) in 1mL, and using the mixed solution as a reference solution;the diluent is preferably a mixed solvent of acetonitrile and water; further preferred, acetonitrile: water volume ratio 20: 80.
- the assay of any one of claims 13-15 wherein the chiral isomer control solution obtained in step 1) has a concentration of 1 μ g/mL.
- The detection method according to any one of claims 13 to 16, wherein the step 2) of preparing a chiral isomer test solution comprises: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Adding a diluent into nonane-2-yl) acetic acid benzene sulfonate or a composition thereof to obtain a chiral isomer test solution; the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the molar ratio of acetonitrile: water volume ratio 20: 80.
- the detection method according to claim 17, wherein the step 2) of preparing the chiral isomer test solution comprises: precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]10mg of nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is added into a 10mL volumetric flask, and a diluent is added for dissolving and diluting to a scale to be used as a stock solution of a test sample; precisely measuring a volumetric flask with 1-10 mL of the stock solution of the sample, adding a diluent to a constant volume to a scale, and shaking up to obtain the test solution;the diluent is preferably a mixed solvent of acetonitrile and water; further preferably, the molar ratio of acetonitrile: water volume ratio 20: 80.
- the detection method according to any one of claims 13 to 18, wherein the concentration of the chiral isomer test solution obtained in step 2) is 0.1 mg/mL.
- 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonan-2-yl) acetic acid benzenesulfonate or 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]The method for detecting the content of the nonane-2-yl) acetic acid benzene sulfonate composition by using an HPLC-CAD method comprises the following steps:1) preparing a content test sample solution: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof to prepare a test solution;2) preparation of content reference solution: taking 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8]Preparing a content reference substance solution by using a nonane-2-yl) acetic acid benzene sulfonate reference substance;3) injecting the content test solution obtained in the step 1) and the content reference solution obtained in the step 2) into a chromatograph, wherein the chromatographic conditions are as follows:a chromatographic column: octadecylsilane bonded silica gel column (4.6X 250mm, 5 μm);mobile phase: a: 10mM ammonium formate solution, B: acetonitrile;gradient elution procedure: 0-5 min, A90%; 5-25 min, A90% → 40%; 25-29 min, A40% → 20%; 29-35 min, A20%; 35-36 min, A20% → 90%; 36-40 min, A90%;flow rate: 1.0 mL/min;column temperature: 35 ℃;CAD parameters: the atomization temperature is 50 ℃, the acquisition frequency is 5.0Hz, and the optical filter is 5.
- The assay of claim 20, wherein the step 1) of preparing a test solution comprises: dissolving 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonane-2-yl) acetic acid benzene sulfonate or a composition thereof in a diluent and diluting to obtain a content test sample solution; preferably, the diluent is a mixed solvent of ammonium formate and acetonitrile; further preferably, the ammonium formate concentration is 10 mM; still further preferably, the 10mM ammonium formate: acetonitrile volume ratio of 80: 20.
- the assay of claim 21, wherein the step 1) of preparing a test solution comprises:precisely weighing 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [ 4.2.1.0)3,8]Nonane-2-yl) acetic acid benzene sulfonate or a composition thereof is dissolved and diluted to a scale by adding a diluent in a volumetric flask with the volume of 10mg to 10mL to be used as a stock solution of a test sample; precisely measuring a volumetric flask with the stock solution of the sample of 0.5mL to 10mL, adding a diluent to dilute to a scale, and shaking up to obtain the test solution;preferably, the diluent is a mixed solvent of ammonium formate and acetonitrile; further preferably, the ammonium formate concentration is 10 mM; still further preferably, the 10mM ammonium formate: acetonitrile volume ratio of 80: 20.
- the assay of any one of claims 20-22 wherein the concentration of the assay sample solution obtained in step 1) is 0.05 mg/mL.
- The assay of claim 20, wherein the step 2) of preparing a content control solution comprises: adding a diluent into the content reference substance to dissolve and dilute the content reference substance to obtain a content reference substance solution; preferably, the diluent is a mixed solvent of ammonium formate and acetonitrile; further preferably, the ammonium formate concentration is 10 mM; still further preferably, the 10mM ammonium formate: acetonitrile volume ratio of 80: 20.
- the assay of claim 24, wherein the step 2) content control solution preparation comprises: precisely weighing a volumetric flask with the content reference substance of 10mg to 10mL, adding a diluent to dissolve and dilute the volumetric flask to a scale, and taking the volumetric flask as a stock solution of the content reference substance; precisely measuring a volumetric flask with the content of reference stock solution of 0.5mL to 10mL, adding a diluent to dilute to a scale, and shaking up to obtain the product;the diluent is a mixed solvent of ammonium formate and acetonitrile; further preferably, the ammonium formate concentration is 10 mM; still further preferably, the 10mM ammonium formate: acetonitrile volume ratio of 80: 20.
- the assay of any one of claims 20-25, wherein the content control solution obtained in step 2) has a concentration of 0.05 mg/mL.
- A method of detecting impurities in 2- ((1S,2S,3R,6S,8S) -2- (aminomethyl) tricyclo [4.2.1.03,8] nonan-2-yl) acetic acid benzenesulfonate or a composition thereof, by combining the method of any one of claims 1 to 5, the method of any one of claims 6 to 12 with the method of any one of claims 13 to 19.
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