WO2023082354A1 - 一种氘代化合物及其制备方法和应用 - Google Patents

一种氘代化合物及其制备方法和应用 Download PDF

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WO2023082354A1
WO2023082354A1 PCT/CN2021/133645 CN2021133645W WO2023082354A1 WO 2023082354 A1 WO2023082354 A1 WO 2023082354A1 CN 2021133645 W CN2021133645 W CN 2021133645W WO 2023082354 A1 WO2023082354 A1 WO 2023082354A1
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compound
solution
metabolite
deuterated
ast
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PCT/CN2021/133645
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French (fr)
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阮美珍
蔡晓宏
段建新
荣唐纳德•T•
李安蓉
孟滕
孙琳
李兵
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深圳艾欣达伟医药科技有限公司
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Priority to US18/003,402 priority Critical patent/US20240140970A1/en
Priority to EP21946249.6A priority patent/EP4206209A4/en
Publication of WO2023082354A1 publication Critical patent/WO2023082354A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/564—Three-membered rings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D15/08—Selective adsorption, e.g. chromatography
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    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
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    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/004—Acyclic, carbocyclic or heterocyclic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur, selenium or tellurium
    • C—CHEMISTRY; METALLURGY
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    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
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    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal

Definitions

  • the invention relates to the technical field of detection, in particular to a deuterated compound and its preparation method and application.
  • DMPK Drug Metabolism and Pharmacokinetics, Chinese means drug metabolism and pharmacokinetics
  • UVD ultraviolet absorption detector
  • FLD fluorescence detector
  • ELSD evaporative light scattering detector
  • MSD mass spectrometer detector
  • AST2660 (aka AST-2660) is a metabolite of AST-3424 (aka OBI-3424, TH-3424) (Meng, F., Li, W.F., Jung, D., Wang, C.C., Qi, T., Shia, C.S., Hsu, R.Y., Hsieh, Y.C., & Duan, J.(2021).
  • a novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity.
  • American journal of cancer research, 11( 7), 3645–3659.) is also the chemical composition of the prodrug AST-3424 to exert its drug effect.
  • the clinical trial registration number of the OBI-3424 drug in the United States is NCT03592264
  • the trial phase is Phase II
  • the indications are castrated prostate cancer and liver cancer
  • the sponsor is Taiwan Haoding Biotechnology Co., Ltd. OBI; the U.S. OBI-
  • the clinical trial registration number of 3424 drug is NCT04315324
  • the trial phase is Phase II
  • the indication is T-ALLT lymphocytic acute leukemia.
  • the trial phase is Phase II, and the indications are various solid tumors.
  • the sponsor is Shenzhen Aixin Dawei Pharmaceutical Technology Co., Ltd.; the clinical trial registration number of the Chinese AST-3424 drug is CTR20201915, and the trial phase is Phase II, and the indication is T -ALL and B-ALL T lymphocytic acute leukemia and B lymphocytic acute leukemia, the sponsor is Shenzhen Aixin Dawei Pharmaceutical Technology Co., Ltd.)
  • the content of AST-3424 used is from 1mg to 100mg.
  • the present invention provides a deuterated compound and its preparation method and application.
  • the deuterated compound I can be used as an internal standard to quantitatively detect the minimum detection limit of metabolite II in biological samples is 0.5ng/ml, reaching Meet the requirements in drug DMPK research:
  • the deuterated compound I provided by the present invention has certain stability, and can be stored for a long time under experimental conditions (-20°C and -70°C without deterioration and stable quality), and is suitable for clinical trials Long-term storage of samples and various operating temperature (laboratory room temperature) requirements in the DMPK laboratory.
  • the present invention actually solves the requirements of the above-mentioned metabolite II under the low detection limit and DMPK research: the content is low, the sample needs to be detected intensively and thus stored for a long time, and meets the actual requirements of laboratory operation, providing a biological Detection system for low-level metabolite II in samples: deuterated internal standard, LC-MS/MS instrument and method, curve fitting quantification and operating procedures.
  • This application uses deuterated compound I for the quantitative detection of metabolite II in biological samples, which can meet the quantitative detection requirements under the condition of low detection limit, and is suitable for DMPK research in clinical trials.
  • the present invention provides deuterated compound I, its structural formula is as shown in formula (I):
  • A is H or D, and at least one of the eight A is D;
  • M is H or alkali metal, alkaline earth metal, ammonium radical.
  • the deuterated compound I is deuterated bis(aziridin-1-yl)phosphinic acid or its salt, and the salt-forming cations are alkali metals such as Na + , K + , or alkaline earth metal ions Ca 2+ , or ammonium radicals ion NH 4 + .
  • M is Na, K, Li or ammonium.
  • the deuterated compound may exist in the form of acid or salt depending on the condition of the biological sample and the reagents added in the post-processing operation.
  • the deuterated compound is an acid or a salt.
  • the corresponding mass-nuclear ratio m/z in the mass spectrum should be distinguishable from non-deuterated compounds.
  • the mass spectrum peak is not a single value, but a mountain peak shape that descends to both sides around the main peak, and the abscissa value m/z of the main peak is the value of the compound, which is set to x; when a hydrogen atom in the compound After (protium) is replaced by deuterium, the abscissa value m/z of its main peak is x+1.
  • the shape of the main peak is a mountain shape, x and x+1 will overlap each other, which may cause the two to be indistinguishable.
  • the calculation method of the molecular weight of the compound (adding the relative atomic masses of all the atoms in the compound), it can be seen that the more atoms there are, the more isotope atoms with different relative atomic masses of the corresponding atoms, the shape of the peak that descends to both sides of the main peak is The more "thick" the corresponding peak overlaps with the main peaks of other compounds.
  • One way to reduce the overlap is to increase the distance between the two main peaks.
  • the deuterated deuterated compound For the deuterated deuterated compound, it is necessary to increase the number of deuterium atoms in the deuterated compound.
  • the present invention combines the specific situation of the deuterated compound I, and through experimental verification and calculation, there are 3 D (deuterium) in the deuterated compound I After atoms, the non-deuterated compound (metabolite II) can be better distinguished from the deuterated deuterated compound I. If there are fewer D (deuterium) atoms, it is necessary to prepare a mass spectrum with higher resolution.
  • the number of D in the deuterated compound I is 4 or 8.
  • deuterated compound I is preferably a compound of formula (I-1) or formula (I-2) structure:
  • the deuterated compound I is selected from compounds of the following structures:
  • the present invention also provides the method for preparing deuterated compound I, it comprises the following steps:
  • Ia compound reacts with phosphorus oxyhalide POX 3 to obtain Ib compound;
  • the I-b compound is hydrolyzed in an aqueous solution by adding a base or not adding a base, and the corresponding deuterated compound I is obtained;
  • I-a compound is deuterated 2-haloethylamine or its inorganic acid salt, sulfate, phosphate, etc., A is H or D, and at least one A is D in the 4 A of I-a compound, in At least one of the eight A's in I-b and I compounds is D;
  • the base in the hydrolysis reaction is selected from: MOH, M is an alkali metal, alkaline earth metal or ammonium; MH, M is an alkali metal; MOR, R is an alkyl group with 1-4 carbons, M is an alkali metal; salt or bicarbonate,
  • X is halogen
  • the first step reaction formula is that Ia compound deuterated 2-halogenated ethylamine or its hydrohalide reacts with phosphorus oxyhalide POX 3 to obtain Ib compound. There can be one or more reactions in this process .
  • Deuterated 2-haloethylamine or 2-haloethylamine inorganic acid salt can be used, depending on the solvent used for the reaction.
  • the reaction is a violent exothermic reaction, dissolve 2-halogenated ethylamine or 2-halogenated ethylamine hydrohalide in a solvent to cool down, and then slowly add phosphorus oxyhalide POX 3 or trihalogenated oxygen Phosphorus POX 3 solution, stirring reaction (dropping speed packaging reaction system temperature at -78 to -20 ° C).
  • the solvent for the reaction is an organic solvent, such as one or a mixture of dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane or cyclohexane.
  • Phosphorus oxyhalide POX 3 includes phosphorus oxybromide POBr 3 and phosphorus oxychloride POCl 3 .
  • Deuterated 2-halogenated ethylamine inorganic acid salts include deuterated 2-halogenated ethylamine hydrohalic acid (hydrochloride, hydrobromide), inorganic oxo acid salt (sulfate, phosphate, etc.) , preferably, the Ia compound is the hydrochloride and hydrobromide of deuterated 2-halogenated ethylamine.
  • the base that adds comprises inorganic base and organic base, and inorganic base selects weaker base, as the hydroxide (calcium hydroxide) of alkaline earth metal, the carbonate of alkali metal, bicarbonate (sodium carbonate, wormwood carbonate, bicarbonate sodium, potassium bicarbonate).
  • inorganic base selects weaker base, as the hydroxide (calcium hydroxide) of alkaline earth metal, the carbonate of alkali metal, bicarbonate (sodium carbonate, wormwood carbonate, bicarbonate sodium, potassium bicarbonate).
  • the organic base is methylamine, ethylamine, propylamine, isopropylamine, N,N-diethylamine, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, N,N- Diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, tetramethylguanidine , pyridine, N-methyldicyclohexylamine or one or more mixtures of dicyclohexylamine.
  • This reaction operation corresponds to dissolving 2-halogenated ethylamine or 2-halogenated ethylamine hydrohalide in a solvent to cool down, and then slowly adding the solution of phosphorus oxyhalide POX 3 or phosphorus oxyhalide POX 3 to cool down , after cooling down, add alkali or alkali solution, and stir the reaction.
  • the organic solvent is one or a mixture of dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane or cyclohexane, and tetrahydrofuran.
  • reaction of compound Ia with phosphorus oxyhalide POX 3 is carried out under certain atmosphere, and described atmosphere is a kind of in air, nitrogen or argon, preferably, described atmosphere is a kind of in nitrogen or argon, more Preferably, the atmosphere is nitrogen.
  • the second step of the reaction is that the I-b compound is hydrolyzed in an aqueous solution with or without adding a base, and the corresponding deuterated compound I is obtained.
  • the hydrolysis reaction must be carried out by adding water, so the reaction is carried out with the participation of water. If only water is added without adding a base, M in the deuterated compound I after the reaction is H, which corresponds to the form of an acid; if a base is added, the corresponding reaction will generate a salt.
  • the base in the hydrolysis reaction is selected from: MOH, M is an alkali metal, alkaline earth metal or ammonium; MH, M is an alkali metal; MOR, R is an alkyl group with 1-4 carbons, M is an alkali metal; salt or bicarbonate. Preferred are NaOH and KOH.
  • the present invention also provides a detection application for the content of the above-mentioned deuterated compound I, that is, using 31 P-NMR method to detect the content of the above-mentioned deuterated compound I, preferably, using 31 P-NMR method to detect deuterium in the solution containing deuterated compound I
  • a detection application for the content of the above-mentioned deuterated compound I that is, using 31 P-NMR method to detect the content of the above-mentioned deuterated compound I, preferably, using 31 P-NMR method to detect deuterium in the solution containing deuterated compound I
  • the content of substituted compound I or use liquid chromatography to detect the content of above-mentioned deuterated compound I, wherein, the liquid chromatograph condition is:
  • a mobile phase is methanol solution of ammonium acetate
  • B mobile phase is acetonitrile
  • Deuterated compounds need to be quantitatively detected after preparation and purification. There are many methods for quantitative detection, which can be directly weighed after purification, or can be directly detected by HPLC.
  • the finished product of deuterated compound I prepared by the present invention is in an aqueous solution, and the general HPLC method and the accurate weighing method are not very fast. Through experimental verification, the detection content of the 31 P-NMR method has the required accuracy, and is fast and convenient. .
  • the present invention also provides a method for detecting the content of the above-mentioned deuterated compound I, which includes:
  • the content of the deuterated compound I was calculated by substituting the known content of the phosphorus-containing compound.
  • the phosphorus-containing compound is preferably a compound containing one phosphorus atom, more preferably hexamethylphosphoric triamide.
  • the deuterated compound I and the phosphorus-containing compound of known content are added to the solvent and the 31 P-NMR spectrum is tested;
  • the deuterated compound I and the known content of the phosphorus-containing compound are added into water for dissolution, and then the 31 P-NMR spectrum is tested together.
  • the phosphorus-containing compound as the internal standard of 31 P-NMR to quantify the deuterated compound I.
  • the number of phosphorus atoms in the phosphorus-containing compound selected is preferably 1, so that the spectrum signal peak of 31 P-NMR is relatively single , which is convenient for quantification.
  • the chemical shift of the 31 P-NMR spectrum signal peak of the phosphorus-containing compound should be separated from the chemical shift of the 31 P-NMR spectrum signal peak of the deuterated compound I to be large enough to distinguish the signal peaks.
  • the number of scans has a certain influence on the 31 P-NMR spectrum signal peak of the phosphorus-containing compound and the 31 P-NMR spectrum signal peak of the deuterated compound I. Through the experiment Verify that the number of scans should not be less than 64 times.
  • the present invention also provides the application of the above-mentioned deuterated compound I as an internal standard in the detection of metabolite II in biological samples.
  • the present invention provides the above-mentioned deuterated compound I as an internal standard in the detection of DNA alkylating agent prodrugs in biological samples.
  • Prodrugs also known as prodrugs, drug precursors, prodrugs, etc., refer to compounds that have pharmacological effects after in vivo transformation.
  • the prodrug itself has no biological activity or very low activity, and becomes an active substance after metabolism in the body. The purpose of this process is to increase the bioavailability of the drug, strengthen the targeting, and reduce the toxicity and side effects of the drug.
  • Prodrugs are currently divided into two categories: carrier-prodrugs and bioprecursors.
  • the DNA alkylating agent prodrug of the present invention refers to a prodrug that releases the DNA alkylating agent, namely metabolite II, after metabolism.
  • the present invention also provides the application of the above-mentioned deuterated compound I as an internal standard in LC-MS/MS analysis and detection of the metabolite content of the DNA alkylating agent prodrug activated by AKR1C3 or the DNA alkylating agent prodrug activated by hypoxia , wherein the structural formula of metabolite II is shown in formula (II):
  • Deuterated compound I is selected from
  • Metabolite II selected from
  • the DNA alkylating agent prodrug of AKR1C3 activation is selected from the compound of following structural formula 1-5:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , R 9 , and R 10 are as described in the claims of patent application PCT/CN2020/089692, publication number WO2020228685A1. details as follows:
  • R 1 is C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z substituted heterocycle, 5-15 membered heteroaryl or Z substituted heteroaryl, 7-15 membered condensed ring Or Z replaces the fused ring;
  • R2 is hydrogen, halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OMS, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, ether with 1-6 carbon atoms or Z-substituted alkoxy with 1-6 carbon atoms, -CONR 6 R 7 , -SO 2NR6R7 , -SO2R6 , -OCOO - R6 , -COOR6 , -NR6COR7 ,
  • R3 is hydrogen, halogen, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OLCMS, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl , C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z Substituted heterocycle, 5-15 membered heteroaryl or Z substituted heteroaryl, C 1 -C 6 alkoxy or Z substituted C 1 -C 6 alkoxy, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCO-R 6 , -OCOO-R 6 , -COOR 6 , -NR 6 COR 7 ,
  • R 4 and R 5 are each independently hydrogen, halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OLCMS, C 1 -C 6 alkyl or Z-substituted alkyl, C 2 -C 6 Alkenyl or Z substituted alkenyl, C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4 -15-membered heterocycle or Z-substituted heterocycle, 5-15-membered heteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z-substituted C 1 -C 6 alkoxy, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCOO-R 6 , -COOR
  • R 6 and R 7 are each independently hydrogen, cyano or isocyano, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 -C 6 alkyne or Z-substituted alkynyl, C 3 -C 8 cycloalkyl or Z-substituted cycloalkyl, C 6 -C 10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 Elementary heteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z substituted C 1 -C 6 alkoxy, or R 6 , R 7 groups form a 5- 7-membered heterocyclic group or Z-substituted 5-7-membered heterocyclic group;
  • R 8 and R 10 are each independently hydrogen, deuterium, aryl or Z substituted aryl, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 - C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, and one of them must be hydrogen or deuterium;
  • R 9 is a substituted C 6 -C 10 aryl group having at least one fluorine atom or a nitro group, a substituted 4-15 membered heterocycle having at least one fluorine atom or a nitro group, a substituted 4-15 membered heterocycle having at least one fluorine atom or a nitro group Substitute 5-15 membered heteroaryl.
  • Z substituent is halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OMS, C 1 -C 3 alkyl or substituted alkyl, C 1 -C 3 alkoxy or substituted alkoxy , C 2 -C 3 alkenyl or substituted alkenyl, C 2 -C 3 alkynyl or substituted alkynyl, C 3 -C 8 cycloalkyl or substituted cycloalkyl, aromatic ring, heterocyclic ring, heteroaryl ring and fused Ring or substituted aromatic rings, heterocyclic rings, heteroaryl rings and condensed rings, the substitution mode is mono-substitution or gem-di-substitution;
  • Substituted C 6 -C 10 aryl, substituted 4-15 membered heterocyclic, substituted 5-15 membered heteroaryl in R 9 are halogen atom, nitro, cyano or isocyano, hydroxyl, amino , C 1 -C 3 alkyl or alkoxy, alkenyl, alkynyl, cycloalkyl or benzene ring, substituted benzene ring, C 1 -C 3 alkoxy or halogen atom substituted alkoxy.
  • the compound of formula (1) (2) is selected from:
  • AST-342 both are prodrugs of AST-2660 (acid form), which will be activated under the action of AKR1C3 enzyme to produce AST-2660 to exert anticancer effect:
  • the compound includes structural formula 1/2 and its salts, esters, solvates and isotopic isomers.
  • R 1 is H, C 1-6 alkyl, C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein said C 1-6 alkyl, C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl and phenyl are optionally substituted by 1, 2 or 3 R a ;
  • Each R is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl;
  • R 2 is H or C 1-6 alkyl
  • R 1 and R 2 are joined together to form a 4-6 membered heterocycloalkyl group together with the N atom to which they are attached, wherein the 4-6 membered heterocycloalkyl group is optionally substituted by 1, 2 or 3 R b ;
  • each R is independently H, F, Cl, Br, I, -CN, -OH, -NH2 , -OCH3 , -OCH2CH3 , -CH3 , or -CH2CH3 ;
  • R 3 is H, F, Cl, Br, I, -OH, -NH 2 , C 1-3 alkoxy or C 1-3 alkyl;
  • T 1 is -(CR c R d ) m -or -(CR c R d ) n -O-;
  • n 1, 2 or 3;
  • n 1 or 2;
  • T2 is N or CH
  • R c and R d are each independently H, F, C 1-3 alkyl or C 1-3 alkoxy;
  • R 4 , R 5 and R 6 are each independently H, F, Cl, Br, I, C 1-3 alkyl or C 1-3 alkoxy;
  • T is N or CH
  • R 7 and R 8 are each independently H, F, Cl, Br or I;
  • R 9 and R 10 are each independently H, F, Cl, Br, I, -CN or
  • the 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.
  • the compound of formula (3) is selected from:
  • AST-3424 both are prodrugs of AST-2660 (acid form), which will be activated under the action of AKR1C3 enzyme to produce AST-2660 to exert anticancer effect:
  • X 10 is O, S, SO or SO 2 ;
  • R 1 and R 2 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • X, Y and Z are each independently hydrogen, CN, halo, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R is hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered hetero Ring, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R 13 and R 14 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle or ether
  • alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, ether groups are substituted or unsubstituted.
  • the compound of formula (5) is selected from:
  • the compound of structural formula 4 is similar to AST-3424T, and is a prodrug of phosphoramidate alkylating agent, which will be activated under the action of AK R1C3 enzyme to produce AST-2660 (acid form) to exert anticancer effect:
  • R 100 , R 101 and R 102 are each independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 form a 5-7 membered heterocyclic ring together with the nitrogen atom to which they are attached;
  • alkyl group and the aryl group are each substituted by 1-3 halo groups or 1-3 C1-C6 alkyl groups;
  • R 1 and R 2 are each independently phenyl or methyl
  • X, Y, and Z are each independently hydrogen or halo
  • R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl.
  • Cx-Cy or " Cxy " before a group refers to the range of the number of carbon atoms present in the group.
  • C1-C6 alkyl refers to an alkyl group having at least 1 and at most 6 carbon atoms.
  • Alkyl means a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, and in some embodiments 1 to 6 carbon atoms.
  • Cy-y alkyl refers to an alkyl group having x to y carbon atoms.
  • the term includes, for example, straight chain and branched chain hydrocarbon groups such as methyl ( CH3- ), ethyl ( CH3CH2- ), n- propyl ( CH3CH2CH2- ), isopropyl group ((CH 3 ) 2 CH-), n-butyl group (CH 3 CH 2 CH 2 CH 2 -), isobutyl group ((CH 3 ) 2 CHCH 2 -), sec-butyl group ((CH 3 )(CH 3 CH 2 )CH-), tert-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 - ) and neopentyl ((CH 3 ) 3 CCH 2 - ).
  • straight chain and branched chain hydrocarbon groups such as methyl ( CH3- ), ethyl ( CH3CH2- ), n- propyl ( CH3CH2CH2- ), isopropyl group ((CH 3 ) 2 CH
  • Aryl means an aromatic group having from 6 to 14 carbon atoms and containing no ring heteroatoms and having a single ring (for example, phenyl) or multiple condensed (fused) rings (for example, naphthyl or anthracenyl). group.
  • aryl is used when the point of attachment is at an aromatic carbon atom or "Ar” is suitable (eg, 5,6,7,8 tetrahydronaphthalen-2-yl is aryl because its point of attachment is at the 2 position of the aromatic phenyl ring).
  • Arylenyl refers to a divalent aryl group having an appropriate hydrogen content.
  • Cycloalkyl means a saturated or partially saturated cyclic group having from 3 to 14 carbon atoms and no ring heteroatoms and having a single ring or multiple rings including fused, bridged and spiro ring systems.
  • cycloalkyl applies when the point of attachment is at a non-aromatic carbon atom (e.g. 5,6,7, 8-tetrahydronaphthalen-5-yl).
  • cycloalkyl includes cycloalkenyl groups.
  • cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl.
  • Cycloalkylene refers to a divalent cycloalkyl group having an appropriate hydrogen content.
  • Halo refers to one or more of fluorine, chlorine, bromine and iodine.
  • Heteroaryl means an aromatic group having 1 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur and includes monocyclic rings such as imidazolyl-2-yl and imidazol-5-yl) and polycyclic systems (eg imidazopyridinyl, benzotriazolyl, benzimidazol-2-yl and benzimidazol-6-yl).
  • monocyclic rings such as imidazolyl-2-yl and imidazol-5-yl
  • polycyclic systems eg imidazopyridinyl, benzotriazolyl, benzimidazol-2-yl and benzimidazol-6-yl.
  • heteroaryl such as 1,2,3,4-tetrahydroquinolin-6-yl and 5,6,7,8-tetrahydroquinolin-3-yl.
  • the nitrogen and/or sulfur ring atoms of the heteroaryl are optionally oxidized to provide an N-oxide (N ⁇ O), sulfinyl or sulfonyl moiety.
  • heteroaryl includes, but is not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuryl, benzothiofuryl, benzothiophenyl, benzoxa Azolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzothienyl, benzimidazolyl, carbazolyl , NH-carbazolyl, carbolinyl, chromanyl (chromanyl), benzopyranyl (chromenyl), cinnolinyl (cinnolinyl), dithiazinyl, furyl, furyl, Imidazolidinyl
  • Heterocyclic or “heterocycle” or “heterocycloalkyl” or “heterocyclyl” means a heteroatom having 1 to 14 carbon atoms and 1 to 6 selected from the group consisting of nitrogen, sulfur or oxygen and include monocyclic and polycyclic ring systems including fused, bridged and spiro ring systems.
  • heterocyclic For polycyclic ring systems having aromatic and/or non-aromatic rings, the terms “heterocyclic”, “heterocyclic” when at least one ring heteroatom is present and the point of attachment is at an atom of the non-aromatic ring , “heterocycloalkyl” or “heterocyclyl” are suitable (such as 1,2,3,4-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-6-yl and decahydroquinolin-6-yl).
  • the heterocyclyl group herein is a 3-15 membered, 4-14 membered, 5-13 membered, 7-12 or 5-7 membered heterocyclic ring.
  • the heterocycle contains 4 heteroatoms. In some other embodiments, the heterocycle contains 3 heteroatoms. In another embodiment, the heterocycle contains up to 2 heteroatoms. In some embodiments, the nitrogen and/or sulfur atoms of the heterocyclyl are optionally oxidized to provide N-oxide, sulfinyl, sulfonyl moieties.
  • Heterocyclic groups include, but are not limited to, tetrahydropyranyl, hexahydropyridinyl, N-methylhexahydropyridin-3-yl, hexahydropyrazinyl, N-methylpyrrolidin-3-yl, 3 -pyrrolidinyl, 2-pyrrolidinon-1-yl, morpholinyl and pyrrolidinyl.
  • a prefix indicating the number of carbon atoms (eg, C3-10) refers to the total number of carbon atoms in the heterocyclyl moiety excluding the number of heteroatoms.
  • Divalent heterocyclic groups will have suitably adjusted hydrogen content.
  • Biaryl refers to a structure in which two aromatic rings are connected through a C-C single bond, such as biphenyl, bipyridine, etc.
  • a group may be substituted with one or more substituents (eg 1 , 2, 3, 4 or 5 substituents).
  • the substituent is selected from the group consisting of chlorine, fluorine, -OCH 3 , methyl, ethyl, isopropyl, cyclopropyl, -CO 2 H and its salts, and C 1 -C 6 alkyl Esters, CONMe 2 , CONHMe, CONH 2 , -SO 2 Me, -SO 2 NH 2 , -SO 2 NMe 2 , -SO 2 NHMe, -NHSO 2 Me, -NHSO 2 CF 3 , -NHSO 2 CH 2 Cl, -NH 2 , -OCF 3 , -CF 3 and -OCHF 2 .
  • the compound of formula (5) is selected from:
  • the compound of structural formula 6 is similar to AST-3424 and is a prodrug of AST-2660, which will be activated under the action of AKR1C3 enzyme to produce AST-2660 to exert anticancer effect:
  • Hypoxia-activated DNA alkylating agent prodrugs are selected from compounds of the following structural formulas 6-12:
  • R 1 , R 2 , R 3 , and Cx are as described in the claims of patent application PCT/CN2020/114519, publication number WO2021120717A1. details as follows:
  • Cx is a 5-10 membered aromatic ring or aromatic heterocyclic ring, aliphatic heterocyclic ring or cycloalkane, which shares two carbon atoms with the nitrobenzene ring to form a condensed ring structure;
  • R 1 is connected to any skeletal atom of the Cx ring, selected from hydrogen, halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, C 1 -C 6 alkyl or Z-substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl base, 4-15 membered heterocycle or Z substituted heterocycle, 5-15 membered heteroaryl or Z substituted heteroaryl, 1-6 carbon atom alkoxy or Z substituted 1-6 carbon atom alkane Oxygen, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCOO-R 6 , -COOR 6 , -NR
  • R 2 and R 3 are each independently hydrogen, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z substituted heterocycle, 5-15 membered heteroaryl or Z substituted Heteroaryl or R 2 , R 3 and the benzylic carbon atom to which it is bonded together form a 3-6 membered ring;
  • the group can replace the hydrogen atom at any position on the carbon atom of the condensed ring, and the number of substitutions is 1;
  • the Z substituent is a halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, C 1 -C 3 alkyl or substituted alkyl, C 1 -C 3 alkoxy or substituted alkoxy, C 2 - C 3 alkenyl or substituted alkenyl, C 2 -C 3 alkynyl or substituted alkynyl, C 3 -C 8 cycloalkyl or substituted cycloalkyl;
  • R 6 and R 7 are each independently hydrogen, C 1 -C 6 alkyl or Z substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl or Z substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or Z substituted C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl or Z substituted C 3 -C 8 cycloalkyl, C 6 -C 10 aryl or Z substituted C 6 -C 10 aryl, 4-15-membered heterocyclic group or Z-substituted 4-15-membered heterocyclic group, 5-15-membered heteroaryl group or Z-substituted 5-15-membered heteroaryl group, or R 6 , R 7 and the atom to which it is bonded together form a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group.
  • the compound of formula (6) is selected from:
  • the definition of R 17 is as described in the claims of patent application PCT/US2016/039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A). details as follows:
  • R 1 is: hydrogen, -N 3 , CN, halo, NR 21 R 22 , -OR 23 , -SO 2 (C1-C6 alkyl), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 Alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or ether;
  • R 21 and R 22 are each independently hydrogen, hydroxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle , 5-15 membered heteroaryl or -SO 2 (C1-C6 alkyl); or R 21 and R 22 form a 4-15 membered heterocyclic ring or 5-15 membered heteroaryl together with the nitrogen atom to which they are bonded;
  • R 23 is hydrogen, C1-C6 alkyl or C6-C10 aryl
  • R 2 and R 3 are independently hydrogen or halo
  • R 4 is hydrogen, halo, C1-C6 alkoxy, C1-C6 alkyl or C6-C10 aryl,
  • R 5 , R 7 , R 9 , R 12 and R 15 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocyclic ring, 5-15 membered heteroaryl group; or R 4 and R 5 form a C5-C6 cycloalkyl ring with intervening carbon atoms between them;
  • R 6 and R 10 are independently hydrogen or halo
  • R 8 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or 5-15 membered heteroaryl;
  • R 11 are each independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl or C6-C10 aryl;
  • R 13 , R 14 , R 16 and R 17 are independently hydrogen, halo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C1-C6 alkoxy;
  • alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, alkoxy and ether groups are optionally substituted.
  • PCT/US2016/039092 Publication No. WO2016210175A1 (corresponding to Chinese Application No. 2016800368985, Publication No. CN108024974A) in PCT/US2016/039092 for specific definitions and meanings, preparation methods, and spectral data.
  • the compounds of formulas (7)-(12) are selected from the compounds specifically disclosed in the above patent applications.
  • the "compound” in the above chemical formulas 1-12 in this specification also includes the compound itself and the solvate, salt, ester or isotope of the compound.
  • the present invention also provides a method for detecting the content of metabolites in biological samples, which comprises the following steps:
  • the preparation of the standard working solution is to prepare a series of standard working solutions containing the internal standard compound of known concentration and the metabolite II of known concentration.
  • the concentration of the internal standard compound in the series of standard working solutions is consistent, and is consistent with the The concentration of the internal standard compound in the solution is the same, and the concentration of the metabolite II in the series of standard working solutions is different;
  • the solution to be tested is prepared from the biological sample with or without treatment
  • Described internal standard compound is deuterated compound I, and its structural formula is as shown in formula (I):
  • A is H or D, and at least 1 of the 8 A is D;
  • M is H or alkali metal, alkaline earth metal, ammonium
  • A is H
  • M is H or alkali metal, alkaline earth metal, ammonium radical.
  • a method for detecting the content of metabolites in a biological sample comprising the steps of:
  • a quantitative internal standard compound is added to the biological sample solution to be tested, and after extraction, it is used as the solution to be tested;
  • A is H or D, and at least 1 of the 8 A is D;
  • M is H or alkali metal, alkaline earth metal, ammonium
  • A is H
  • M is H or alkali metal, alkaline earth metal, ammonium radical.
  • the conditions of the liquid chromatograph in the liquid chromatography-tandem mass spectrometry are:
  • a mobile phase is methanol solution of ammonium acetate
  • B mobile phase is acetonitrile
  • the mass spectrometry conditions are: electrospray ionization source,
  • the metabolite II monitoring transition is: m/z 147.0 ⁇ m/z 62.9,
  • the deuterated compound I monitoring ion pair is: m/z (147.0+the number of deuterated) ⁇ m/z 62.9;
  • the metabolite II monitoring transition is: m/z 149.0 ⁇ m/z 64.9,
  • the deuterated compound I monitoring ion pair is: m/z (149.0+number of deuterated) ⁇ m/z 64.9.
  • acid such as formic acid
  • deuterated compound I internal standard compound
  • deuterated compound I internal standard compound
  • the deuterated compound I is first added to the matrix solution containing the known different concentrations of the metabolite II, and then the extraction operation is performed.
  • the biological sample is a urine sample or a plasma sample.
  • the corresponding matrix solution is the urine of a patient containing additives without administration; when detecting a blood sample, corresponding The matrix solution was plasma from untreated patients containing anticoagulants.
  • Add the deuterated compound and extract the operation process as follows: add the deuterated compound I solution to the solution to be tested and the standard working solution, add methanol and mix evenly, and centrifuge to get the supernatant to get final product;
  • the additive is Na 2 HPO 4 or K 2 HPO 4
  • the anticoagulant is K 2 EDTA or Na 2 EDTA.
  • urine samples For urine samples, they should be stored at -70°C within 24 hours of collection, preferably within 4 hours, and can be stored at -70°C for 32 days; if processed, they should be stored at room temperature or below for 94 hours The sample injection test is completed within.
  • Figure 1 is the 31 P-NMR spectrum of AST-2660-D8-sodium salt.
  • Figure 2 is the 31 P-NMR spectrum of the first AST-2660-D8-sodium salt aqueous solution, with 64, 128, 256, and 512 scans, respectively, see a, b, c, and d, respectively.
  • Figure 3 is the 31 P-NMR spectrum at zero time, according to which AST-2660-D8 sodium salt solution -20°C (a) and AST-2660-D8 sodium salt -78°C (b).
  • Figure 4 is the 31 P-NMR spectrum after 48 hours, according to which it is AST-2660-D8 sodium salt solution -20°C (a figure), AST-2660-D8 sodium salt solution -78°C (b figure).
  • Fig. 5 is the 31 P-NMR spectrum of AST-2660-D8 sodium salt solution after storage at -20°C for 6 months.
  • Fig. 6 is the 31 P-NMR spectrum of AST-2660-D8 sodium salt solution after storage at -78°C for 6 months.
  • Figure 7 is a typical LC-MS/MS spectrum of AST-2660 (a panel) and internal standard AST-2660-D8 (b panel) in the blank human plasma extract.
  • Figure 8 is a typical LC-MS/MS spectrum of AST-2660 (a panel) and internal standard AST-2660-D8 (b panel) in the zero concentration plasma sample extract.
  • Figure 9 is a typical LC-MS/MS spectrum of AST-2660 (a graph) and internal standard AST-2660-D8 (b graph) of standard solution human plasma extract (0.50ng/ml).
  • Figure 10 is a typical LC-MS/MS spectrum of AST-2660 (a graph) and internal standard AST-2660-D8 (b graph) of standard solution human plasma extract (200ng/ml).
  • Figure 11 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 in blank human urine extract.
  • Figure 12 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 in zero concentration urine sample extract.
  • Figure 13 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 of standard working solution human urine extract (0.50ng/ml).
  • Figure 14 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 of standard working solution human urine extract (200ng/ml).
  • the abscissa is time
  • the ordinate is the mass spectrum ion intensity of LC-MS/MS
  • the mass spectrum ion is a selected ion.
  • the AST-3424 drug developed by the applicant is a prodrug of the DNA alkylating agent AST-2660, which is specifically activated by the highly expressed AKR1C3 enzyme in cancer cells and metabolized into AST-2660 to exert its drug effect.
  • DMPK Drug Metabolism and Pharmacokinetics, Chinese means drug metabolism and pharmacokinetics
  • the content of AST-3424 used in clinical practice is relatively low, ranging from 1mg to 100mg.
  • the external standard method or internal standard method corresponding to conventional liquid chromatography cannot meet the requirements of low detection limit. Quantitative testing requirements. Therefore, it is necessary to provide a detection method that can meet the quantitative detection requirements under low detection limit conditions.
  • the internal standard method is a relatively accurate quantitative method in chromatographic analysis.
  • the internal standard method is to add a certain weight of pure substance to a certain amount of the sample mixture to be analyzed, and then perform chromatographic analysis on the sample containing the internal standard substance, and determine The peak area of the internal standard and the component to be tested can be used to calculate the percentage of the component to be tested in the sample.
  • the selection of internal standard is a very important work.
  • the internal standard should be a known compound that can be obtained in pure form so that it can be added to the sample in an accurate, known amount, and it should be essentially the same or as similar as possible to the sample component being analyzed. Consistent chemical and physical properties (such as chemical structure, polarity, volatility and solubility in solvents, etc.), chromatographic behavior and response characteristics, preferably a homologue of the analyte. Of course, under chromatographic conditions, the internal standard must be fully separated from the components in the sample. For the quantitative analysis of the internal standard method, the selection of the internal standard is very important. It must meet the following conditions: 1.
  • the physical and chemical properties of the internal standard substance and the analyzed substance should be similar (such as boiling point, polarity, chemical structure, etc.); 2.
  • the internal standard substance should be a pure substance that does not exist in the sample ; 3. It must be completely soluble in the tested sample (or solvent), and does not react chemically with the tested sample; and can be completely separated from the chromatographic peaks of each component in the sample; 4. Can add internal standard The amount should be close to the measured component; 5.
  • the position of the chromatographic peak should be close to the position of the chromatographic peak of the measured component, or in the middle of several measured component chromatographic peaks. And there is no total overflow, the purpose is to avoid the difference in sensitivity caused by the instability of the instrument; 6. Select the appropriate amount of internal standard to add, so that the matching of the peak areas of the internal standard and the analyte is greater than 75 %, so as to avoid the sensitivity deviation caused by them being in different response value regions.
  • This application has carried out a large number of screenings on similar compounds of AST-2660, and finally obtained deuterated compound I as an internal standard for detecting metabolite II.
  • the LC-MS/MS method established using the deuterated compound I has a lower detection limit: as low as 0.5 ng/ml.
  • the liquid phase-mass spectrometry/mass spectrometry method established by the present application using the deuterated compound I can be used to detect the content of the metabolite AST-2660 in human plasma and human urine. This method meets the requirements of biological sample analysis, and the sample processing method is simple. The method has high sensitivity, precision and accuracy.
  • Figure 1 is the 31 P-NMR spectrum of AST-2660-D8-sodium salt.
  • different intermediates I-b can be synthesized by selecting different deuterated raw material compounds I-a (deuterated 2-bromoethylamine), and then different bases (NaOH, KOH, LiOH or ammonia) to obtain different deuterated bis(aziridin-1-yl)phosphinic acids or salts thereof.
  • Different deuterium can be synthesized by choosing the deuterated positions and deuterated numbers of the first deuterated 2-bromoethylamine hydrobromide and the second deuterated 2-bromoethylamine hydrobromide.
  • Compound I and various salts are possible to be synthesized by choosing the deuterated positions and deuterated numbers of the first deuterated 2-bromoethylamine hydrobromide and the second deuterated 2-bromoethylamine hydrobromide.
  • the quantitative detection method is specifically described below by taking the AST-2660-D8-sodium salt prepared in Example 1 as an example.
  • Example 2 The method for detecting AST-2660-D8-sodium salt
  • HMPA hexamethylphosphoric triamide
  • HMPA HMPA (33.0mg, 0.1840mmol) and dissolve it in water (2.2mL), the total mass is 2612mg, the concentration of HMPA is 7.050 ⁇ 10 -5 (mmol/mg, the nuclear magnetic shift of 31 P-NMR is 29.890ppm)
  • Fig. 2 is the 31 P-NMR spectrum of the first AST-2660-D8-sodium salt solution, scanned 64, 128, 256, and 512 times from top to bottom, and from left to right, respectively.
  • the amount of HMPA is 0.02235mmol
  • the amount of AST-2660-D8-sodium salt can be calculated according to the NMR peak integral area ratio to be 0.005743mmol, and the concentration should be 1.177 ⁇ 10 -5 mmol/mg, that is, 1.177 ⁇ 10 -2 mmol/g, the corresponding mass content is 2.10 mg/g.
  • the phosphorus spectrum is shown in Figure 2, and the peak integral area ratios are 1:0.264, 1:0.268, 1:0.264, and the concentrations of the same samples are respectively 1.209 ⁇ 10 -5 mmol/mg, 1.227 ⁇ 10 -5 mmol/mg, 1.209 ⁇ 10 -5 mmol/mg, namely 2.15mg/g, 2.18mg/g, 2.15mg/g, the average of the four results is 2.1mg /g.
  • the NMR quantitative method of this embodiment is quick and simple, has acceptable accuracy within a certain range, and can replace the HPLC yield analysis method. If it is necessary to further improve the accuracy of quantification, the HPLC method should be used, and the external standard method should be used for quantification, and the absolute content can be accurately quantified through the standard curve.
  • LC-MS/MS method of embodiment 5 and embodiment 7 is applicable to the low content detection of AST-2660-D8, AST-2660, and its corresponding LC liquid phase method (corresponding MS/MS detector is replaced by conventional differential detector, electrospray detector, evaporative light scattering detector) are also applicable to the constant (mg/ml) content detection of AST-2660 and AST-2660-D8.
  • Embodiment 3 Stability study of AST-2660-D8-sodium salt
  • This example is to study the stability of the AST-2660-D8-sodium salt aqueous solution content detection sample solution: use 31 P-NMR detection, compare the 31 P peak of HMPA as an internal standard within 48 hours and AST-2660-D8-sodium The area ratio of the 31 P peak of the salt is used to characterize the content of AST-2660-D8-sodium salt solution to detect whether the sample solution is degraded and the quality is reduced.
  • HMPA 21.8 mg, 0.1217 mmol
  • water 2.20 mL
  • the total mass was 2127 mg
  • the concentration of HMPA was 5.72 ⁇ 10 -5 mmol/mg.
  • AST-2660-D8-sodium salt solution 588mg, about 0.5mL
  • HMPA 293mg, 1.676 ⁇ 10 -2 mmol
  • Table 1 Stability data of AST-2660-D8 sodium salt stored at -20, -78°C for 48 hours
  • the ratio of the 31 P-NMR NMR peak of HMPA to the peak area of AST-2660-D8-sodium salt solution for 8/16/24/36/48 hours was stable at 1:0.43 ⁇ 1:0.47, However, the ratio is stable at 1:0.21 to 1:0.24 under the storage condition of -78°C, so it can be considered that the AST-2660-D8-sodium salt solution is stable at -20 to -78°C for 48 hours.
  • Figure 3 is the 31 P-NMR spectrum at zero time, from left to right are AST-2660-D8 sodium salt solution -20°C, AST-2660-D8 sodium salt solution -78°C.
  • Figure 4 is the 31 P-NMR spectrum after 48 hours, from left to right are AST-2660-D8 sodium salt solution -20°C, AST-2660-D8 sodium salt solution -78°C.
  • the concentration of AST-2660-D8 sodium salt was 1.89 mg/mL at day 0, and 1.37 mg/mL after storage at -20°C for 6 months.
  • the concentration of AST-2660-D8 sodium salt decreased by 27.5% within 6 months of storage.
  • the 31 P-NMR spectrum after 6 months is shown in FIG. 6 .
  • Fig. 5 is the 31 P-NMR spectrum of AST-2660-D8 sodium salt solution after storage at -20°C for 6 months.
  • the concentration of AST-2660-D8 sodium salt was 1.89 mg/mL at day 0, and 1.71 mg/mL after storage at -78°C for 6 months.
  • the concentration of AST-2660-D8 sodium salt decreased by 9.5% within 6 months of storage. Relatively speaking, storage at -78°C is more stable than storage at -20°C, and the degradation changes slower.
  • the 31 P-NMR spectrum after 6 months is shown in FIG. 7 .
  • Fig. 6 is the 31 P-NMR spectrum of AST-2660-D8 sodium salt solution after storage at -78°C for 6 months.
  • Example 4 Establishment and verification of a detection method for AST-2660 in human plasma
  • the deuterated internal standard (IS) AST-2660-D8 (prepared in Example 1) was used to quantitatively detect the metabolite AST-2660 (in the form of sodium salt) in human plasma containing K 2 EDTA anticoagulant .
  • the structural formula of AST-2660 is as follows:
  • AST-2660 refers to its sodium salt
  • AST-2660-D8 also refers to its sodium salt
  • AST-2660 prepared with non-deuterated raw materials with reference to Example 1 standard stock solution (methanol solution with a concentration of 1.0 mg/ml): AST-2660 appropriate amount, dilute with an appropriate amount of methanol and mix to obtain a 1.0 mg/ml solution.
  • Standard working solution SWS
  • blank matrix blank matrix
  • standard curve standard sample solutions with different concentrations were prepared in the following table.
  • AST-2660-D8 stock solution 50 ⁇ g/ml AST-2660-D8 methanol solution: Add appropriate amount of AST-2660-D8 reference substance, add appropriate amount of methanol to dilute and mix well to obtain a 50 ⁇ g/ml solution. Take 20 ⁇ L AST-2660-D8 internal standard stock solution, add 9980 ⁇ L methanol to dilute and mix well to obtain 100 ng/ml internal standard working solution.
  • AST-2660 quality control stock solution (1.0 mg/ml methanol solution): appropriate amount of AST-2660 reference substance, add appropriate amount of methanol to dilute and mix well to obtain a 1.0 mg/ml solution. Take 100 ⁇ L AST-2660QC stock solution and 9990 ⁇ L methanol to obtain 10.0 ⁇ g/ml quality control working solution;
  • Normal mixed human plasma containing anticoagulant K 2 EDTA was used as the diluent, and diluted according to the following table to prepare quality control samples with different concentrations.
  • the accuracy and precision inspection results are shown in Table 7. It can be seen from Table 7 that both the intra-assay and inter-assay precision are less than 8.3%, and the intra-assay and inter-assay accuracies are both within ⁇ 8.7.
  • the analytical criteria for accuracy and precision are: for LLOQ QC samples, the intra-assay and inter-assay accuracy (RE) must be within ⁇ 20.0%, and the intra-assay and inter-assay CV must not be greater than 20.0%.
  • within-assay and between-assay accuracy (RE) must be within ⁇ 15.0%; within-assay and between-assay precision (CV) must be within each
  • the QC concentration level is not greater than 15.0%. Therefore, the intra-assay and inter-assay precision and accuracy of the quality control samples met the requirements.
  • the precision (%CV) of the internal standard AST-2660-D8 was ⁇ 40.0%, meeting the requirements.
  • the CVs of the extraction recoveries of AST-2660 and the internal standard AST-2660-D8 are all less than 15.0%.
  • Analytical criteria for extraction recovery rate For samples spiked after extraction, the CV of the response rate measured at each concentration level must not be greater than 15.0%. Therefore, the extraction recoveries of AST-2660 and internal standard AST-2660-D8 meet the requirements.
  • liquid chromatography-mass spectrometry/mass spectrometry method established in this example for the detection of AST-2660 in human plasma meets the requirements of biological sample analysis, the sample processing method is simple, the method has high sensitivity, precision and accuracy.
  • Example 4 The detection method established in Example 4 was used to detect the content of AST-2660 in the human plasma to be tested.
  • Matrix Normal pooled human plasma with anticoagulant ( K2EDTA )
  • the solution to be tested (the solution to be tested at this time is not added with an internal standard compound): take the blood sample to be tested, and it is ready.
  • Standard working solution (the internal standard compound is not added to the standard working solution at this time): Take an appropriate amount of AST-2660, weigh it accurately, add methanol to dissolve and dilute to make a solution containing about 1.0mg per 1ml, as a standard working reserve solution, take an appropriate amount of standard working stock solution, and add matrix to dilute it to make standard working solutions with different concentrations of about 200ng, 160ng, 100ng, 50ng, 10ng, 2.0ng, 1.0ng, and 0.5ng per 1ml. (Solution stored at -70°C)
  • Quality control sample solution take an appropriate amount of AST-2660, weigh it accurately, add methanol to dissolve and dilute to make a solution containing about 10 ⁇ g per 1ml, as a quality control working stock solution, take an appropriate amount of quality control working stock solution, add matrix Prepare quality control working solutions with different concentrations of about 150ng, 60ng, 15ng, 1.5ng, and 0.5ng per 1ml. (Solution stored at -70°C)
  • the solution to be tested, the standard working solution, and the quality control sample solution were thawed at room temperature and mixed uniformly. Take 100 ⁇ l of the above solution, add 20 ⁇ l internal standard, add 500 ⁇ l methanol, vortex for 3 minutes to mix evenly, and centrifuge at 2143 rcf for 10 Minutes, take 450 ⁇ l of supernatant to dry, add 150 ⁇ l of methanol to redissolve, sonicate, centrifuge the plate at 4°C and 2143rcf for 3 minutes, take 120 ⁇ l of supernatant, that is.
  • step 4 After sample extraction and addition of internal standard, the solution is subjected to step 4 and injected into LC-MS/MS for detection.
  • Step 3 Set the test conditions of chromatography-mass spectrometry
  • Liquid phase conditions use SHARC 1, 3 ⁇ m, 2.1*50mm or equivalent chromatographic column; mobile phase A is methanol solution containing low content of ammonium acetate, mobile phase B is acetonitrile, perform gradient elution as shown in the table below; column temperature 20 °C; injection volume 5 ⁇ l
  • Mass spectrometry conditions electrospray ion source, negative ion scanning mode; spray voltage -4500V; ion source temperature: 500°C; collision energy (CE) is -22eV; declustering voltage (DP) is -55V; entrance voltage (EP) is - 10V; collision cell exit voltage (CXP) is -16V; dwell time is 100ms; all gases use high-purity nitrogen; AST-2660 ion pair: m/z 147.0 ⁇ m/z 62.9; internal standard AST-2660-D8 Transition: m/z 155.0 ⁇ m/z 62.9.
  • the extracted AST-2660 standard working solution, blank sample, zero concentration sample, sample solution to be tested, and quality control sample solution were injected into LC-MS/MS for detection.
  • a quantitative internal standard compound is added to the biological sample solution to be tested, and after extraction, it is used as the test solution;
  • test solution Draw the test solution, inject it into the LC-MS/MS system for detection, measure the ratio of the metabolite II and the deuterated compound I peak area in the test solution solution, and substitute it into the regression equation to obtain the metabolic rate in the test biological sample solution. Content of Product II.
  • the SOP after the above modification is based on the habits of laboratory operators, using containers of the same volume to prepare solutions of the same concentration. This operation only needs to ensure that the volume of the added solution is the same, and the obtained standard working solution is still of equal concentration.
  • Figures 7 to 10 are some typical spectra of the detection process in plasma samples.
  • Figure 7 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 in blank human plasma extract.
  • Figure 8 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 in zero concentration plasma sample extract.
  • Figure 9 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 of standard solution human plasma extract (0.50ng/ml).
  • Figure 10 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 of standard solution human plasma extract (200ng/ml).
  • Table 12 One-month stability study data of AST-2660 in human plasma at -20°C and -70°C
  • human plasma containing AST-2660 contained little AST-2660 during the freeze-thaw process from -70°C to room temperature, while the decrease was more pronounced during the freeze-thaw process from -20°C to room temperature: that is, according to From the data in Table 12, it can be seen that the rapid temperature change during the experimental process of transporting the samples in human plasma stored at -70°C from storage conditions to room temperature will not affect the stability of AST-2660 in human plasma samples.
  • Table 14 Stability study data of AST-2660-D8 solution stored at -70°C
  • Table 16 Bench stability study data of AST-2660 in human plasma at room temperature
  • the human plasma samples containing AST-2660 are not stable at room temperature, and the content of AST-2660 in the human plasma samples has been decreasing with the storage time and prolongation, which suggests that in relevant clinical patients
  • the collected plasma samples should be stored immediately in the above-mentioned low-temperature environment verified by experiments: it is safest to store them at -70°C, and it is also possible to store them at -20°C. In the refrigerator (4 °C to 0 °C) and should try to transfer to the above-mentioned low temperature environment within 4 hours.
  • the whole cold chain logistics full-process temperature monitoring
  • the temperature should be below -20°C or dry ice insulation.
  • Human plasma samples containing AST-2660 should be stored in the above-mentioned experimentally verified low-temperature environment immediately after collection: it is safest to store at -70°C, and it is also acceptable to store at -20°C, and it can also be stored if conditions are limited. In a conventional refrigerator (4°C to 0°C) and should try to transfer to the above-mentioned low temperature environment within 4 hours. During the process of sending samples to the DMPK sample testing center laboratory, the whole cold chain logistics (full monitoring temperature) should be used. The temperature should be below -20°C or dry ice insulation;
  • the sample should be stored at -70°C or lower temperature. It is stable for 28 days under this condition, and it may remain stable for a longer time according to the trend of change;
  • plasma samples should be stored in an environment below -20°C within 4 hours after collection, preferably in an environment of -70°C, and can be stored at -70°C for 28 days; Plasma samples should be refrigerated at 4°C or below after the above-mentioned extraction process, and the sample injection test should be completed within 54 hours at room temperature in the laboratory; the results of the test by observing these storage temperatures and times are reliable, otherwise it will cause serious accidents.
  • the AST-2660 in the sample changes during storage and the results are untrue and unreliable.
  • Example 6 Establishment and verification of the detection method of AST-2660 in human urine
  • the steps to establish the detection method for AST-2660 in human urine in this example are basically the same as in Example 4, the only difference is that the matrix is different (human plasma in Example 4, and human urine in this example) and the sample extraction steps are slightly different . Pooled normal human (non-dosed) urine with additive Na2HPO4-12H2O was used as diluent and base.
  • the ratio of x represents the concentration of metabolite II in the standard working solution, LLOQ (lower limit of quantification): 0.5ng/ml, ULOQ (upper limit of quantitation): 200ng/ml. It can be seen that the linear relationship is good within the linear range of 0.5-200 ng/ml.
  • the accuracy and precision inspection results are shown in Table 17. It can be seen from Table 17 that both the intra-assay and inter-assay precision are less than 15.5%, and the intra-assay and inter-assay accuracies are both within ⁇ 11.4.
  • the analytical criteria for accuracy and precision are: for LLOQ QC samples, the intra-assay and inter-assay accuracy (RE) must be within ⁇ 20.0%, and the intra-assay and inter-assay CV must not be greater than 20.0%.
  • within-assay and between-assay accuracy (RE) must be within ⁇ 15.0%; within-assay and between-assay precision (CV) must be within each
  • the QC concentration level is not greater than 15.0%. Therefore, the intra-assay and inter-assay precision and accuracy of the quality control samples met the requirements.
  • the precision (%CV) of the internal standard AST-2660-D8 was ⁇ 13.4%, meeting the requirements.
  • Table 17 Intra-assay and inter-assay precision and accuracy results for quality control samples
  • liquid chromatography-mass spectrometry method established in this example for the detection of AST-2660 in human urine meets the requirements of biological sample analysis, the sample processing method is simple, the method has high sensitivity, precision and accuracy.
  • Example 6 The detection method established in Example 6 was used to detect the content of AST-2660 in the human urine to be tested.
  • Matrix pooled normal human urine with additive Na2HPO4 ⁇ 12H2O .
  • the solution to be tested (the solution to be tested at this time is not added with an internal standard compound): take the blood sample to be tested, and it is ready.
  • Standard working solution (the internal standard compound is not added to the standard working solution at this time): Take an appropriate amount of AST-2660, weigh it accurately, add methanol to dissolve and dilute to make a solution containing about 1.0mg per 1ml, as a standard working reserve solution, take an appropriate amount of standard working stock solution, and add matrix to dilute it to make standard working solutions with different concentrations of about 200ng, 160ng, 100ng, 50ng, 10ng, 2.0ng, 1.0ng, and 0.5ng per 1ml. (Solution stored at -70°C)
  • Quality control sample solution Take an appropriate amount of AST-2660, weigh it accurately, add methanol to dissolve and dilute to make a solution containing about 10 ⁇ g per 1ml, as a quality control working stock solution, take an appropriate amount of quality control working stock solution, add matrix Prepare quality control working solutions with different concentrations of about 150ng, 60ng, 15ng, 1.5ng, and 0.5ng per 1ml. (Solution stored at -70°C)
  • test solution standard working solution, and quality control sample solution were thawed at room temperature and mixed evenly. Take 100 ⁇ l of the above solution, add 20 ⁇ l internal standard, add 500 ⁇ l methanol, vortex for 3 minutes to mix evenly, and centrifuge at 2143 rcf for 30 Minutes, take 450 ⁇ l supernatant, centrifuge the 96-well plate at 2143 rcf for 10 minutes, take 300 ⁇ l supernatant, that is.
  • step 4 After sample extraction and addition of internal standard, the solution is subjected to step 4 and injected into LC-MS/MS for detection.
  • Step 3 Set the test conditions of chromatography-mass spectrometry
  • Liquid phase conditions use SHARC 1, 3 ⁇ m, 2.1*50mm chromatographic column or a column with equivalent performance; methanol solution containing low content of ammonium acetate, mobile phase B is acetonitrile, and perform gradient elution in the following table; column temperature 20°C; sample injection Volume 5 ⁇ l.
  • Mass spectrometry conditions electrospray ion source, negative ion scanning mode; spray voltage -4500V; ion source temperature: 500°C; collision energy (CE) is -22eV; declustering voltage (DP) is -55V; entrance voltage (EP) is - 10V; collision cell exit voltage (CXP) is -16V; dwell time is 100ms; all gases use high-purity nitrogen; AST-2660 ion pair: m/z 147.0 ⁇ m/z 62.9; internal standard AST-2660-D8 Transition: m/z 155.0 ⁇ m/z 62.9.
  • the extracted AST-2660 standard working solution, blank sample, zero-concentration sample, test solution, and quality control working solution were injected into LC-MS/MS for detection.
  • a quantitative internal standard compound is added to the biological sample solution to be tested, and after extraction, it is used as the test solution;
  • test solution Draw the test solution, inject it into the LC-MS/MS system for detection, measure the ratio of the metabolite II and the deuterated compound I peak area in the test solution solution, and substitute it into the regression equation to obtain the metabolic rate in the test biological sample solution. Content of Product II.
  • the SOP after the above modification is based on the habits of laboratory operators, using containers of the same volume to prepare solutions of the same concentration. This operation only needs to ensure that the volume of the added solution is the same, and the obtained standard working solution is still of equal concentration.
  • Figures 11 to 14 are some typical spectra of the detection process in urine samples.
  • Figure 11 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 in blank human urine extract.
  • Figure 12 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 in the zero concentration urine sample extract.
  • Figure 13 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 of standard working solution human urine extract (0.50ng/ml).
  • Figure 14 is a typical LC-MS/MS spectrum of AST-2660 and internal standard AST-2660-D8 of standard working solution human urine extract (200ng/ml).
  • Table 21 One-month stability study data of AST-2660 in human urine at -20°C and -70°C
  • Table 23 Stability of human urine samples treated at room temperature (AST-2660 content change)
  • Table 24 Bench stability study data of AST-2660 in human urine at room temperature
  • Samples should be stored at -70°C or lower. It is stable for 32 days under these conditions, and may remain stable for a longer period of time according to the trend of change;
  • AST-2660 During the process of taking out the stored samples to room temperature, that is, the samples in human urine stored at -70°C are transported from storage conditions to room temperature for the rapid temperature change during the experiment process, which will not affect the human urine samples
  • the stability of AST-2660 should be placed in a conventional refrigerator or at room temperature after taking it out. It is stable for up to 24 hours at room temperature.
  • the SOP for the operation of urine samples they should be stored at -70°C within 24 hours after collection, preferably within 4 hours, and can be stored at -70°C for 32 days; if processed, they should be stored
  • the sample injection test should be completed within 94 hours at room temperature or below; the test results obtained by adhering to these storage temperatures and times are reliable, otherwise it will lead to untrue and unreliable results due to changes in the storage process of AST-2660 in the sample.

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Abstract

一种氘代化合物及其制备方法和应用，所述氘代化合物I的结构式如式(I)所示，其中，A为H或D，且8个A中至少有1个为D；M为H或碱金属、碱土金属、铵根。提供了氘代化合物I作为内标在检测生物样本中代谢产物II含量中的应用，其中，代谢产物II的结构式如式(II)所示；其中，A为H，M为H或碱金属、碱土金属、铵根。使用氘代化合物I作为内标定量分析生物样本中低含量的代谢产物II的含量，能够满足低浓度情况下的定量检测要求，适合临床试验中DMPK研究。

Description

一种氘代化合物及其制备方法和应用 技术领域
本发明涉及检测技术领域，尤其涉及一种氘代化合物及其制备方法和用途。
背景技术
在人体临床试验中，需要对受试者的生物样本，如血液、尿液、组织等进行药物和药物代谢物的含量检测，通过检测得到的药物和药物代谢物的含量进行药物的DMPK(Drug Metabolism and Pharmacokinetics,中文意思是药物代谢及药代动力学)研究。在新药开发环节，对药物进行DMPK研究是必须的。
常用的方法是液相色谱法，借助HPLC或UHPLC的高超分离能力和紫外吸收检测器(UVD)二极管阵列检测器(PDAD)、荧光检测器(FLD)、蒸发光散射检测器(ELSD)、示差折光检测器、质谱检测器(MSD)的检测性能可以检测几乎所有类型的药物和药物代谢物。
AST2660(又名AST-2660)是AST-3424(又名OBI-3424、TH-3424)的代谢产物(Meng,F.,Li,W.F.,Jung,D.,Wang,C.C.,Qi,T.,Shia,C.S.,Hsu,R.Y.,Hsieh,Y.C.,& Duan,J.(2021).A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity.American journal of cancer research,11(7),3645–3659.)，也是前药AST-3424发挥药效的化学成分。
Figure PCTCN2021133645-appb-000001
AST-3424代谢为AST-2660(图中2660)的化学反应式
在临床试验中(美国OBI-3424药物的临床试验登记号为NCT03592264，试验阶段为II期，适应症为去势前列腺癌和肝癌，申办方为台湾浩鼎生技股份有限公司OBI；美国 OBI-3424药物的临床试验登记号为NCT04315324，试验阶段为II期，适应症为T-ALLT淋巴细胞急性白血病，申办方为美国西南肿瘤协作组SWOG；中国AST-3424药物的临床试验登记号为CTR20191399，试验阶段为II期，适应症为各种实体瘤，申办方为深圳艾欣达伟医药科技有限公司；中国AST-3424药物的临床试验登记号为CTR20201915，试验阶段为II期，适应症为T-ALL和B-ALL T淋巴细胞急性白血病和B淋巴细胞急性白血病，申办方为深圳艾欣达伟医药科技有限公司)使用的AST-3424含量是1mg开始直至100mg。
药物在较小的剂量下，常规的液相色谱法对应的外标法或内标法并不能满足低给药量情况下生物样本中药物代谢物的定量检测要求。因此，需要开发一种检测方法满足低检测限情况下的定量检测要求，且能满足上述药物DMPK研究要求。
发明内容
有鉴于此，本发明提供氘代化合物及其制备方法和应用，经检测并验证氘代化合物I作为内标可用于定量检测生物样本中的代谢产物II的最低检测限为0.5ng/ml，达到了药物DMPK研究中的要求：
作为DMPK分析中的氘代内标，本发明提供的氘代化合物I具有一定的稳定性，在实验条件下(-20℃和-70℃储存不变质，质量稳定)能长期储存，适合临床试验中DMPK实验室中的样本长期储存和各种操作温度(实验室室温)要求。
总体来讲，本发明实际解决了上述代谢产物II在低检测限下、DMPK研究的要求：含量低、样本因为需要集中检测因而储存实际长、符合实验室操作实际的要求，提供了一种生物样本中低含量代谢产物II的检测体系：氘代内标、LC-MS/MS仪器及方法、曲线拟合定量及操作规程。
本申请使用氘代化合物I用于定量检测生物样本中的代谢产物II，能够满足低检测限情况下的定量检测要求，适合于临床试验中DMPK研究。
本发明提供氘代化合物I，其结构式如式(I)所示：
Figure PCTCN2021133645-appb-000002
其中，A为H或D，且8个A中至少有1个为D；
M为H或碱金属、碱土金属、铵根。
氘代化合物I为氘代的二(氮丙啶-1-基)次膦酸或其盐，成盐的阳离子为碱金属如Na +、K +，或碱土金属离子Ca 2+，或铵根离子NH 4 +。优选的，M为Na、K、Li或铵根。
氘代化合物根据生物样本的情况以及后处理操作中添加的试剂不同，可能是以酸、盐的形式而存在，对应的，氘代化合物就是酸或盐。
优选的，氘代化合物I中的8个A中至少有3个为D。作为氘代内标，在质谱中对应的质核比m/z应该能与非氘代化合物区分。实际上，质谱峰并不是一个单一的值，而是一个围绕主峰往两边下降的山峰形状，其主峰的横坐标值m/z即为该化合物的数值,设为x；当化合物中一个氢原子(氕)被氘取代后，其主峰的横坐标值m/z即为x+1。显然，由于主峰的形状是一个山峰形状，x与x+1会相互重叠，有可能导致两者无法区分。根据化合物分子量的计算方式(将化合物中所有原子的相对原子质量相加)可知，原子越多、对应原子具有的不同相对原子质量的同位素原子种类越多，这个主峰的往两边下降的山峰形状就会越“粗壮”，对应的与其他化合物的主峰的重叠部分就越多。为了减少重叠部分，方法之一是增加两个主峰的距离。对于氘代氘代化合物而言，需要增加氘代化合物中氘原子的数目，本发明结合氘代化合物I的具体情况，并通过实验验证和计算，氘代化合物I中有3 个D(氘)原子后，非氘代化合物(代谢产物II)与氘代氘代化合物I能较好区分，如D(氘)原子较少，则需要配制更高分辨能力的质谱。
优选的，氘代化合物I中D的个数为4或者8。
更优选的，氘代化合物I优选为式(I-1)或式(I-2)结构的化合物：
Figure PCTCN2021133645-appb-000003
具体的，氘代化合物I选自以下结构的化合物：
Figure PCTCN2021133645-appb-000004
本发明还提供制备氘代化合物I的方法，其包括以下步骤：
Figure PCTCN2021133645-appb-000005
I-a化合物与三卤氧磷POX 3反应得I-b化合物；
I-b化合物加入碱或不加入碱在水溶液中进行水解反应，对应得氘代化合物I；
其中，I-a化合物为氘代的2-卤代乙胺或其无机酸盐、硫酸盐、磷酸盐等，A为H或D，且在I-a化合物的4个A中至少有一个A为D，在I-b和I化合物的8个A中至少有1个为D；
水解反应中碱选自：MOH，M为碱金属、碱土金属或铵根；MH，M为碱金属；MOR，R为1-4个碳的烷基，M为碱金属；碱金属的碳酸盐或碳酸氢盐，
X为卤素。
第一步反应式是I-a化合物氘代的2-卤代乙胺或其氢卤酸盐与三卤氧磷POX 3反应得I-b化合物，这个过程中发生的反应可以有一个，也可以有多个。
可以使用氘代的2-卤代乙胺或2-卤代乙胺无机酸酸盐，具体根据反应使用的溶剂确定。
由于该反应为剧烈的放热反应，因此是将2-卤代乙胺或2-卤代乙胺氢卤酸盐溶解在溶剂中降温，然后缓慢滴加三卤氧磷POX 3或三卤氧磷POX 3的溶液，搅拌反应(滴加速度包装反应体系温度在-78至-20℃)。反应的溶剂为有机溶剂，如二氯甲烷、氯仿、氯苯、1,2-二氯乙烷、乙酸乙酯、正己烷或环己烷中的一种或几种的混合。
三卤氧磷POX 3包括三溴氧磷POBr 3和三氯氧磷POCl 3。氘代的2-卤代乙胺无机酸盐 包括氘代的2-卤代乙胺的氢卤酸(盐酸盐、氢溴酸盐)、无机含氧酸盐(硫酸盐、磷酸盐等)，优选的，I-a化合物为氘代的2-卤代乙胺的盐酸盐、氢溴酸盐。
第一步反应中会产生酸，因此加入碱进行反应调节。加入的碱包括无机碱和有机碱，无机碱选择较弱的碱，如碱土金属的氢氧化物(氢氧化钙)，碱金属的碳酸盐、碳酸氢盐(碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾)。优选地，所述有机碱为甲胺、乙胺、丙胺、异丙胺、N,N-二乙胺、三乙胺、正丁胺、异丁胺、4-二甲氨基吡啶、N,N-二异丙基乙胺、1,8-二氮杂二环[5.4.0]十一碳-7-烯、N,N,N',N'-四甲基乙二胺、四甲基胍、吡啶、N-甲基二环己胺或二环己胺中的一种或几种的混合。
这样反应操作对应为将2-卤代乙胺或2-卤代乙胺氢卤酸盐溶解在溶剂中降温，然后缓慢滴加三卤氧磷POX 3或三卤氧磷POX 3的溶液再降温，降温后，加入碱或碱溶液，搅拌反应。
优选地，所述有机溶剂为二氯甲烷、氯仿、氯苯、1,2-二氯乙烷、乙酸乙酯、正己烷或环己烷、四氢呋喃中的一种或几种的混合。
化合物I-a与三卤氧磷POX 3的反应在一定的氛围下进行，所述氛围是空气、氮气或氩气中的一种，优选地，所述氛围是氮气或氩气中的一种,更优选地，所述氛围是氮气。
第二步反应是I-b化合物加入碱或不加入碱在水溶液中进行水解反应，对应得氘代化合物I。
水解反应须加入水进行反应，因此反应是在水的参与下进行的。如仅仅加入水而不加入碱，则反应后的氘代化合物I中M为H，对应的是酸的形式；如加入碱则对应反应会生成盐。水解反应中碱选自：MOH，M为碱金属、碱土金属或铵根；MH，M为碱金属；MOR，R为1-4个碳的烷基，M为碱金属；碱金属的碳酸盐或碳酸氢盐。优选为NaOH和KOH。
本发明还提供上述氘代化合物I含量的检测应用，即使用 31P-NMR法检测上述的氘代化合物I的含量，优选的，使用 31P-NMR法检测含有氘代化合物I的溶液中氘代化合物I的含量；或使用液相色谱法检测上述的氘代化合物I的含量，其中，液相色谱仪条件为：
氢受体型固定相色谱柱，
A流动相为醋酸铵的甲醇溶液，B流动相为乙腈，
A、B流动相梯度洗脱，从A流动相15％体积比逐渐升高至90％体积比，然后逐渐减少到15％体积比。
氘代化合物在制备纯化后需要进行定量检测。定量检测的方法有很多，可以在纯化后直接称量，也可以直接使用HPLC法进行检测。
本发明制备的氘代化合物I成品是在水溶液中，一般的HPLC法、精确称量法都不是十分快捷，通过实验验证，使用 31P-NMR法检测含量具有符合要求的准确度，且快捷方便。
本发明还提供一种检测上述氘代化合物I含量的方法，其包括：
检测氘代化合物I和已知含量的含磷化合物的 31P-NMR，得到谱图；
根据 31P-NMR谱图中氘代化合物I和含磷化合物的化学位移特征峰的峰面积比，代入已知含量的含磷化合物的含量计算得到氘代化合物I的含量。
所述含磷化合物优选为含有一个磷原子的化合物，更优选为六甲基磷酰三胺。
优选的，将氘代化合物I和已知含量的含磷化合物加入到溶剂中一并测试 31P-NMR谱图；
优选的，将氘代化合物I和已知含量的含磷化合物加入到水中溶解后一并测试 31P-NMR谱图。
使用含磷的化合物作为 31P-NMR的内标对氘代化合物I进行定量，显然选用的含磷的化合物的磷原子数目最好是1个，这样 31P-NMR的谱图信号峰比较单一，便于进行定量。 另含磷的化合物的 31P-NMR的谱图信号峰的化学位移应当与氘代化合物I的 31P-NMR的谱图信号峰的化学位移间隔足够大，便于将信号峰区分开。
31P-NMR法检测谱图时，扫描的次数对含磷的化合物的 31P-NMR的谱图信号峰、氘代化合物I的 31P-NMR的谱图信号峰有一定的影响，通过实验验证，扫描的次数应不小于64次。
本发明还提供上述的氘代化合物I作为内标在检测生物样本中代谢产物II的应用，优选的，本发明提供上述氘代化合物I作为内标在检测生物样本中DNA烷化剂前药的代谢产物含量的应用，其中代谢产物II的结构式如式(II)所示：
Figure PCTCN2021133645-appb-000006
其中，A为H；M为H或碱金属、碱土金属、铵根。
前药，也称前体药物(prodrug)、药物前体、前驱药物等，是指经过生物体内转化后才具有药理作用的化合物。前体药物本身没有生物活性或活性很低，经过体内代谢后变为有活性的物质，这一过程的目的在于增加药物的生物利用度，加强靶向性，降低药物的毒性和副作用。目前前体药物分为两大类：载体前体药物(carrier-prodrug)和生物前体(bioprecursor)。
本发明的DNA烷化剂前药是指经代谢后释放出DNA烷化剂即代谢产物II的前药。
本发明还提供上述的氘代化合物I作为内标在LC-MS/MS分析检测生物样本中AKR1C3活化的DNA烷化剂前药或乏氧活化的DNA烷化剂前药的代谢产物含量的应用，其中代谢产物II的结构式如式(II)所示：
Figure PCTCN2021133645-appb-000007
其中，A为H；M为H或碱金属、碱土金属、铵根。
氘代化合物I选自
Figure PCTCN2021133645-appb-000008
代谢产物II选自
Figure PCTCN2021133645-appb-000009
AKR1C3活化的DNA烷化剂前药选自以下结构式1-5的化合物：
Figure PCTCN2021133645-appb-000010
其中，R 1、R 2、R 3、R 4、R 5、R 8、R 9、R 10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A1中的权利要求书所记载。具体如下：
R 1是C 6-C 10芳基或Z取代的芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、7-15元的稠环或Z取代稠环；
R 2是氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、1-6个碳原子的醚或Z取代的1-6个碳原子的烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCOO-R 6、-COOR 6、-NR 6COR 7、-OCOR 6、-NR 6SO 2R 7、-NR 6SO 2NR 6R 7或者R 2和与其所键结的R 1基团上的原子一起形成7-15元的稠环或Z取代稠环；
R 3是氢、卤素、氰基或异氰基、羟基、巯基、胺基、OTs、OLCMS、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C 1-C 6烷氧基或Z取代的C 1-C 6烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCO-R 6、-OCOO-R 6、-COOR 6、-NR 6COR 7，-OCOR 6、-NR 6SO 2R 7；
R 4、R 5各自独立地是氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OLCMS、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C 1-C 6烷氧基或Z取代的C 1-C 6烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCOO-R 6、-COOR 6、-NR 6COR 6、-OCOR 6、-NR 6SO 2R 7或者R 4、R 5和与其所键结的苯环上的原子一起形成7-15元的稠环或Z取代稠环；
R 6和R 7各自独立地是氢、氰基或异氰基、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C 1-C 6烷氧基或Z取代的C 1-C 6烷氧基，或者R 6、R 7基团与其所键结的原子一起形成5-7元杂环基或Z取代5-7元杂环基；
R 8、R 10各自独立地为氢、氘、芳基或Z取代芳基、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基且必有一个为氢、氘；
R 9为至少具有一个氟原子或硝基取代的取代C 6-C 10芳基、至少具有一个氟原子或硝基取代的取代4-15元杂环、至少具有一个氟原子或硝基取代的取代5-15元杂芳基。
Z取代基为卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C 1-C 3烷基或取代烷基、C 1-C 3烷氧基或取代烷氧基、C 2-C 3烯基或取代烯基、C 2-C 3炔基或取代炔基、C 3-C 8环烷基或取代环烷基、芳环、杂环、杂芳环和稠环或取代芳环、杂环、杂芳环和稠环，取代的方式为单取代或偕二取代；
R 9中的取代C 6-C 10芳基、取代4-15元杂环、取代5-15元杂芳基的取代基为卤素原子、硝基、氰基或异氰基、羟基、胺基、C 1-C 3烷基或烷氧基、烯基、炔基、环烷基或苯环、取代苯环、C 1-C 3烷氧基或卤原子取代烷氧基。
具体的，式(1)(2)的化合物选自：
Figure PCTCN2021133645-appb-000011
Figure PCTCN2021133645-appb-000012
Figure PCTCN2021133645-appb-000013
Figure PCTCN2021133645-appb-000014
具体定义和含义以及制备方法、波谱数据参见专利申请PCT/CN2020/089692，公开号WO2020228685A1，在此将该申请全文引入。
显然结构式1/2的化合物与AST-342相同，都是AST-2660(酸形式)的前药，其会在AKR1C3酶的作用下活化而产生AST-2660发挥抗癌效果：
Figure PCTCN2021133645-appb-000015
显然该化合物包括结构式1/2及其盐、酯、溶剂合物、同位素异构体。
Figure PCTCN2021133645-appb-000016
其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1中的权利要求书所记载。具体如下：
Rw为
Figure PCTCN2021133645-appb-000017
R 1为H、C 1-6烷基、C 3-6环烷基、4-6元杂环烷基、5-6元杂芳基或苯基，其中所述C 1- 6烷基、C 3-6环烷基、4-6元杂环烷基、5-6元杂芳基和苯基任选被1、2或3个R a所取代；
各R a独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基；
R 2为H或C 1-6烷基；
或者R 1和R 2连接在一起，与其相连的N原子一起形成4-6元杂环烷基，其中所述4-6 元杂环烷基任选被1、2或3个R b所取代；
各R b独立地为H、F、Cl、Br、I、-CN、-OH、-NH 2、-OCH 3、-OCH 2CH 3、-CH 3或-CH 2CH 3；
R 3为H、F、Cl、Br、I、-OH、-NH 2、C 1-3烷氧基或C 1-3烷基；
或者R 2和R 3连接在一起使结构单元
Figure PCTCN2021133645-appb-000018
为
Figure PCTCN2021133645-appb-000019
T 1为-(CR cR d) m-或-(CR cR d) n-O-；
m为1、2或3；
n为1或2；
T 2为N或CH；
R c和R d各自独立地为H、F、C 1-3烷基或C 1-3烷氧基；
R 4、R 5和R 6各自独立地为H、F、Cl、Br、I、C 1-3烷基或C 1-3烷氧基；
T为N或CH；
R 7和R 8各自独立地为H、F、Cl、Br或I；
R 9和R 10各自独立地为H、F、Cl、Br、I、-CN或
所述4-6元杂环烷基和5-6元杂芳基各自包含1、2、3或4个独立选自N、-O-和-S-的杂原子。
具体的，式(3)的化合物选自：
Figure PCTCN2021133645-appb-000020
具体定义和含义以及制备方法、波谱数据参见专利申请PCT/CN2020/120281，公开号WO2021068952A1，在此将该申请全文引入。
显然结构式3的化合物与AST-3424相同，都是AST-2660(酸形式)的前药，其会在AKR1C3酶的作用下活化而产生AST-2660发挥抗癌效果：
Figure PCTCN2021133645-appb-000021
其中，X、Y、Z、R、A以及X 10的定义如专利申请PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)中的权利要求书所记载，T为
Figure PCTCN2021133645-appb-000022
具体如下：
X 10是O、S、SO或SO 2；
A是C 6-C 10芳基、5-15元杂芳基或-N＝CR 1R 2；
R 1及R 2各自独立是氢、C 1-C 6烷基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环、醚、-CONR 13R 14或-NR 13COR 14；
X、Y及Z各自独立是氢、CN、卤基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环、醚、-CONR 13R 14或-NR 13COR 14；
R是氢、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环、醚、-CONR 13R 14或-NR 13COR 14；
R 13及R 14各自独立是氢、C 1-C 6烷基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环或醚
T为
Figure PCTCN2021133645-appb-000023
且
其中这些烷基、烯基、炔基、环烷基、芳基、杂环、杂芳基、醚基经取代或未经取代。
具体的，式(5)的化合物选自：
Figure PCTCN2021133645-appb-000024
Figure PCTCN2021133645-appb-000025
Figure PCTCN2021133645-appb-000026
Figure PCTCN2021133645-appb-000027
Figure PCTCN2021133645-appb-000028
Figure PCTCN2021133645-appb-000029
Figure PCTCN2021133645-appb-000030
Figure PCTCN2021133645-appb-000031
Figure PCTCN2021133645-appb-000032
Figure PCTCN2021133645-appb-000033
及
Figure PCTCN2021133645-appb-000034
具体定义和含义以及制备方法、波谱数据参见专利申请PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)中的权利要求书所记载，，在此将该申请全文引入。
显然结构式4的化合物与AST-3424T类似，是胺基磷酸酯烷化剂的前药，其会在AK R1C3酶的作用下活化而产AST-2660(酸形式)发挥抗癌效果：
Figure PCTCN2021133645-appb-000035
其中：
A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR 1R 2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO 2、–O-(CH 2)-O-、-CO 2H及其盐、-OR 100、-CO 2R 100、-CONR 101R 102、-NR 101R 102、-NR 100SO 2R 100、-SO 2R 100、-SO 2NR 101R 102、C1-C6烷基、C3-C10杂环基；
其中，R 100、R 101及R 102各自独立是氢、C1-C8烷基、C6-C12芳基；或R 101及R 102与其附 接至的氮原子一起形成5-7元杂环；
其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
R 1及R 2各自独立是苯基或甲基；
X、Y及Z各自独立是氢或卤基；
R是氢或C1-C6烷基或卤素取代烷基。
基团前的“Cx-Cy”或“C x-y”是指存在于该基团中的碳原子数目的范围。举例而言，C1-C6烷基是指具有至少1个且最多6个碳原子的烷基。
“烷基”是指具有1至10个碳原子且在一些实施例中具有1至6个碳原子的单价饱和脂肪族烃基。“Cx-y烷基”是指具有x至y个碳原子的烷基。此术语包括(举例而言)直链及具支链烃基，例如甲基(CH 3-)、乙基(CH 3CH 2-)、正丙基(CH 3CH 2CH 2-)、异丙基((CH 3) 2CH-)、正丁基(CH 3CH 2CH 2CH 2-)、异丁基((CH 3) 2CHCH 2-)、仲丁基((CH 3)(CH 3CH 2)CH-)、叔丁基((CH 3) 3C-)、正戊基(CH 3CH 2CH 2CH 2CH 2-)及新戊基((CH 3) 3CCH 2-)。
“芳基”是指具有6至14个碳原子且不含环杂原子且具有单环(例如，苯基)或多个缩合(稠合)环(例如，萘基或蒽基)的芳香族基团。对于包括不具有环杂原子的具有芳香族环及非芳香族环的稠合、桥连及螺环系统的多环系统而言，当附接点位于芳香族碳原子处时，术语“芳基”或“Ar”适用(例如，5,6,7,8四氢萘-2-基是芳基，此乃因其附接点是位于芳香族苯基环的2位处)。“伸芳基”是指具有适当氢含量的二价芳基。
“环烷基”是指具有3至14碳原子且没有环杂原子且具有单环或包括稠合、桥连及螺环系统的多环的饱和或部分饱和环状基团。对于不具有环杂原子的具有芳香族及非芳香族环的多环系统而言，当附接点是位于非芳香族碳原子处时，术语“环烷基”适用(例如5,6,7,8-四氢萘-5-基)。术语“环烷基”包括环烯基。环烷基的实例包括(例如)金刚烷基、环丙基、环丁基、环戊基、环辛基及环己烯基。“伸环烷基”是指具有适当氢含量的二价环烷基。
“卤基”是指氟、氯、溴及碘中的一或多者。
“杂芳基”是指具有1至14个碳原子及1至6个选自由氧、氮及硫组成的群的杂原子的芳香族基团且包括单环(例如咪唑基-2-基及咪唑-5-基)及多环系统(例如咪唑并吡啶基、苯并三唑基、苯并咪唑-2-基及苯并咪唑-6-基)。对于包括具有芳香族及非芳香族环的稠合、桥连及螺环系统的多环系统而言，若存在至少一个环杂原子且附接点是位于芳香族环的原子处，则应用术语“杂芳基”(例如1,2,3,4-四氢喹啉-6-基及5,6,7,8-四氢喹啉-3-基)。在一些实施例中，杂芳基的氮及/或硫环原子视情况经氧化以提供N-氧化物(N→O)、亚磺酰基或磺酰基部分。术语杂芳基包括(但不限于)吖啶基、吖辛因基(azocinyl)、苯并咪唑基、苯并呋喃基、苯并硫代呋喃基、苯并噻吩基(benzothiophenyl)、苯并恶唑基、苯并噻唑基、苯并三唑基、苯并四唑基、苯并异恶唑基、苯并异噻唑基、苯并噻吩基(benzothienyl)、苯并咪唑啉基、咔唑基、NH-咔唑基、咔啉基、苯并二氢吡喃基(chromanyl)、苯并吡喃基(chromenyl)、噌啉基(cinnolinyl)、二噻嗪基、呋喃基、呋呫基、咪唑啶基、咪唑啉基、咪唑并吡啶基、咪唑基、吲唑基、二氢吲哚基(indolenyl)、吲哚啉基、吲嗪基、吲哚基、异苯并呋喃基、异苯并二氢吡喃基(isochromanyl)、异吲唑基、异吲哚啉基、异吲哚基、异喹啉基(isoquinolinyl)、异喹啉基(isoquinolyl)、异噻唑基、异恶唑基、萘啶基、八氢异喹啉基、恶二唑基、恶唑啶基、恶唑基、嘧啶基、啡啶基、啡啉基、吩嗪基、吩噻嗪基、吩恶噻基、吩恶嗪基、酞嗪基、六氢吡嗪基、蝶啶基、嘌呤基、吡喃基、吡嗪基、吡唑啶基、吡唑啉基、吡唑基、嗒嗪基、吡啶并恶唑基、吡啶并咪唑基、吡啶并噻唑基、吡啶基(pyridinyl)、吡啶基(pyridyl)、嘧啶基、吡咯基、喹唑啉基、喹啉基、喹喏啉基、奎宁环基、四氢异喹啉基、四氢喹啉基、四唑基、噻二嗪基、噻二唑基、噻蒽基、噻唑基、噻吩基(thienyl)、噻吩并噻唑基、噻吩并恶唑基、噻吩并咪唑基、噻吩基(thiophenyl)、三嗪基及呫吨基。“伸杂芳基”是指具有适当氢含量的二价杂芳基。
“杂环状”或“杂环”或“杂环烷基”或“杂环基”是指具有1至14个碳原子及1至6个选自由 氮、硫或氧组成的群的杂原子的饱和或部分饱和环状基团且包括单环及包括稠合、桥连及螺环系统的多环系统。对于具有芳香族及/或非芳香族环的多环系统而言，当存在至少一个环杂原子且附接点是位于非芳香族环的原子处时，术语“杂环状”、“杂环”、“杂环烷基”或“杂环基”适用(例如1,2,3,4-四氢喹啉-3-基、5,6,7,8-四氢喹啉-6-基及十氢喹啉-6-基)。在一些实施例中，此处杂环基是3-15元、4-14元、5-13元、7-12或5-7元杂环。在一些其他实施例中，杂环含有4个杂原子。在一些其他实施例中，杂环含有3个杂原子。在另一实施例中，杂环含有最多2个杂原子。在一些实施例中，杂环基的氮及/或硫原子视情况经氧化以提供N-氧化物、亚磺酰基、磺酰基部分。杂环基包括(但不限于)四氢吡喃基、六氢吡啶基、N-甲基六氢吡啶-3-基、六氢吡嗪基、N-甲基吡咯啶-3-基、3-吡咯啶基、2-吡咯啶酮-l-基、吗啉基及吡咯啶基。指示碳原子数的前缀(例如，C3-10)是指杂环基部分中除杂原子数之外的总碳原子数。二价杂环基将具有适当调整的氢含量。
“联芳基”是指两个芳环通过C-C单键相联的结构，如联苯、联吡啶等。
术语“视情况经取代”是指经取代或未经取代的基团。基团可经一或多个取代基(例如1、2、3、4或5个取代基)取代。较佳地，取代基选自由以下组成的群：侧氧基、卤基、-CN、NO 2、-N 2+、-CO 2R 100、-OR 100、-SR 100、-SOR 100、-SO 2R 100、-NR 100SO 2R 100、-NR 101R 102、-CONR 101R 102、-SO 2NR 101R 102、C 1-C 6烷基、C 1-C 6烷氧基、-CR 100＝C(R 100) 2、-CCR 100、C 3-C 10环烷基、C 3-C 10杂环基、C 6-C 12芳基及C 2-C 12杂芳基或诸如–O-(CH 2)-O-、–O-(CH 2) 2-O-及其1-4个甲基经取代的形式等二价取代基，其中R 100、R 101及R 102各自独立是氢或C 1-C 8烷基；C 3-C 12环烷基；C 3-C 10杂环基；C 6-C 12芳基；或C 2-C 12杂芳基；或R 101及R 102与其附接至的氮原子一起形成5-7元杂环；其中烷基、环烷基、杂环基、芳基或杂芳基各自视情况经1-3个卤基、1-3个C 1-C 6烷基、1-3个C 1-C 6卤烷基或1-3个C 1-C 6烷氧基取代。较佳地，取代基选自由以下组成的群：氯、氟、-OCH 3、甲基、乙基、异丙基、环丙基、-CO 2H及其盐及C 1-C 6烷基酯、CONMe 2、CONHMe、CONH 2、-SO 2Me、-SO 2NH 2、-SO 2NMe 2、-SO 2NHMe、-NHSO 2Me、-NHSO 2CF 3、-NHSO 2CH 2Cl、-NH 2、-OCF 3、-CF 3及-OCHF 2。
具体的，式(5)的化合物选自：
Figure PCTCN2021133645-appb-000036
Figure PCTCN2021133645-appb-000037
Figure PCTCN2021133645-appb-000038
Figure PCTCN2021133645-appb-000039
Figure PCTCN2021133645-appb-000040
Figure PCTCN2021133645-appb-000041
Figure PCTCN2021133645-appb-000042
Figure PCTCN2021133645-appb-000043
Figure PCTCN2021133645-appb-000044
Figure PCTCN2021133645-appb-000045
Figure PCTCN2021133645-appb-000046
及
Figure PCTCN2021133645-appb-000047
具体定义和含义以及制备方法、波谱数据参见PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)；PCT/US2020/120281，公开号WO2021068952A1；PCT/CN2020/089692，公开号WO2020228686所公开，在此将这些申请全文引入。
显然结构式6的化合物与AST-3424类似，是AST-2660的前药，其会在AKR1C3酶的作用下活化而产生AST-2660发挥抗癌效果：
Figure PCTCN2021133645-appb-000048
乏氧活化的DNA烷化剂前药选自选自以下结构式6-12的化合物：
Figure PCTCN2021133645-appb-000049
其中，R 1、R 2、R 3、Cx的定义如专利申请PCT/CN2020/114519，公开号WO2021120717A1中的权利要求书所记载。具体如下：
Cx为5-10元的芳环或芳杂环、脂杂环或环烷烃，其与硝基苯环共用两个碳原子形成稠环结构；
R 1连接在Cx环的任意骨架原子上，选自氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、1-6个碳原子的烷氧基或Z取代的1-6个碳原子的烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCOO-R 6、-COOR 6、-NR 6COR 7、-OCOR 6、-NR 6SO 2R 7、-NR 6SO 2NR 6R 7，
R 2、R 3各自独立地是氢、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基或者R 2、R 3和与其所键结的苄位碳原子一起形成3-6元环；
Figure PCTCN2021133645-appb-000050
基团可取代稠环碳原子上任意位置的氢原子，取代的个数为1；
Z取代基为卤素原子、氰基或异氰基、羟基、巯基、胺基、C 1-C 3烷基或取代烷基、C 1-C 3烷氧基或取代烷氧基、C 2-C 3烯基或取代烯基、C 2-C 3炔基或取代炔基、C 3-C 8环烷 基或取代环烷基；
R 6、R 7各自独立地是氢、C 1-C 6烷基或Z取代的C 1-C 6烷基、C 2-C 6烯基或Z取代的C 2-C 6烯基、C 2-C 6炔基或Z取代的C 2-C 6炔基、C 3-C 8环烷基或Z取代的C 3-C 8环烷基、C 6-C 10芳基或Z取代的C 6-C 10芳基、4-15元杂环基或Z取代的4-15元杂环基、5-15元杂芳基或Z取代的5-15元杂芳基，或者R 6、R 7和与其所键结的原子一起形成5-7元杂环基或Z取代的5-7元杂环基。
具体定义和含义以及制备方法、波谱数据参见PCT/CN2020/114519，公开号WO2021120717A1所公开内容，在此将这些申请全文引入。
具体的，式(6)的化合物选自：
Figure PCTCN2021133645-appb-000051
Figure PCTCN2021133645-appb-000052
Figure PCTCN2021133645-appb-000053
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15、R 16、R 17的定义如专利申请PCT/US2016/039092，公开号WO2016210175A1(对应中国申请号2016800368985，公开号CN108024974A)中的权利要求书所记载。具体如下：
R 1为：氢、-N 3、CN、卤基、NR 21R 22、-OR 23、-SO 2(C1-C6烷基)、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基或醚；
R 21和R 22各自独立地为氢、羟基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基或-SO 2(C1-C6烷基)；或R 21和R 22与其所键接的氮原子一起形成4-15元杂环或5-15元杂芳基；
R 23为氢、C1-C6烷基或C6-C10芳基；
R 2和R 3独立地为氢或卤基；
R 4为氢、卤基、C1-C6烷氧基、C1-C6烷基或C6-C10芳基，
R 5、R 7、R 9、R 12和R 15独立地为氢、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基；或R 4和R 5与其之间的介入碳原子一起形成C5-C6环烷基环；
R 6和R 10独立地为氢或卤基；
R 8为氢、C1-C6烷基、C2-C6烯基、C2-C6炔基或5-15元杂芳基；
R 11各自独立地为C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基或C6-C10芳基；
R 13、R 14、R 16和R 17独立地为氢、卤基、C1-C6烷基、C2-C6烯基、C2-C6炔基或C1-C6烷氧基；
其中所述烷基、烯基、炔基、环烷基、芳基、杂环、杂芳基、烷氧基和醚基团任选地经取代。
具体定义和含义以及制备方法、波谱数据参见PCT/US2016/039092，公开号WO2016210175A1(对应中国申请号2016800368985，公开号CN108024974A)所公开内容，在此将这些申请全文引入。
具体的，式(7)-(12)的化合物选自上述专利申请中具体公开的化合物。
显然，本说明书中上述化学式1-12中的“化合物”也当然包括化合物本身以及该化合物的溶剂合物、盐、酯或同位素异构体等。
本发明还提供一种检测生物样本中代谢产物含量的方法，其包括以下步骤：
制备含有已知浓度的内标化合物的待测溶液供LC-MS/MS进样分析；
标准工作溶液的制备，制备含有已知浓度的内标化合物、已知浓度的代谢产物II的一系列标准工作溶液，该一系列标准工作溶液中内标化合物的浓度一致，且与所述待测溶液中的内标化合物浓度相同，该一系列标准工作溶液中代谢产物II的浓度是不同的；
液相色谱-串联质谱法(LC-MS/MS)测定关系函数，吸取配制好的不同浓度的代谢产物II的标准工作溶液，注入LC-MS/MS系统进行检测，得到关系函数y＝f(x)，其中y代表代谢产物II与内标化合物峰面积的比值，x表示标准工作溶液中代谢产物II的浓度；
利用关系函数测定计算未知浓度的待测溶液中代谢产物II的浓度，吸取加入已知含量的内标化合物的待测溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物峰面积的比值y，代入函数y＝f(x)，求得待测溶液中代谢产物II的浓度x，
其中，所述待测溶液由所述生物样本经处理或不处理而制备，
所述内标化合物为氘代化合物I，其结构式如式(I)所示：
Figure PCTCN2021133645-appb-000054
A为H或D，且8个A中至少有1个为D；
M为H或碱金属、碱土金属、铵根；
代谢产物II的结构式如式(II)所示：
Figure PCTCN2021133645-appb-000055
A为H；
M为H或碱金属、碱土金属、铵根。
一种检测生物样本中代谢产物含量的方法，其包括以下步骤：
待测生物样本溶液中加入定量的内标化合物，经提取处理，作为待测溶液；
使用代谢产物II对照品稀释得到一系列不同浓度的代谢产物II的溶液，加入定量的内标化合物，经提取处理，作为标准工作溶液，吸取系列标准工作溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物的峰面积，以代谢产物II与内标化合物的峰面积比值为纵坐标，以代谢产物II浓度为横坐标，绘制标准曲线，计算回归方程；
吸取待测溶液，注入LC-MS/MS系统进行检测，测得待测溶液中代谢产物II与氘代化合物I峰面积的比值，代入回归方程，求得待测溶液中代谢产物II的含量，
其中，内标物为氘代化合物I，结构式如式(I)所示：
Figure PCTCN2021133645-appb-000056
A为H或D，且8个A中至少有1个为D；
M为H或碱金属、碱土金属、铵根；
代谢产物II的结构式如式(II)所示：
Figure PCTCN2021133645-appb-000057
A为H；
M为H或碱金属、碱土金属、铵根。
优选的，液相色谱-串联质谱法中液相色谱仪条件为：
氢受体型固定相色谱柱，
A流动相为醋酸铵的甲醇溶液，B流动相为乙腈，
A、B流动相梯度洗脱，从A流动相15％体积比逐渐升高至90％体积比，然后逐渐减少到15％体积比；
质谱条件为：电喷雾离子源，
负离子扫描方式下
代谢产物II监测离子对为：m/z 147.0→m/z 62.9，
氘代化合物I监测离子对为：m/z(147.0+氘代的数目)→m/z 62.9；
或
正离子扫描方式下
代谢产物II监测离子对为：m/z 149.0→m/z 64.9，
氘代化合物I监测离子对为：m/z(149.0+氘代的数目)→m/z 64.9。
如使用正离子扫描方式，对应的流动相中建议添加酸，比如甲酸。
优选的，制备含有已知含量的氘代化合物I(内标化合物)的待测溶液时，制备含有已知含量的氘代化合物I的待测溶液时，优选先在生物样本溶液中添加氘代化合物I后，再进行提取操作；
对应的，制备标准工作溶液时，在含已知的不同浓度的代谢产物II的基质溶液中先添加氘代化合物I后，再进行提取操作。
优选的，所述生物样本为尿液样本或血浆样本，对应的，在检测尿液样本时，对应的基质溶液为含有添加剂的、没有给药的患者的尿液；在检测血液样本时，对应的基质溶液为含有抗凝剂的、没有给药的患者的血浆。
加入氘代化合物、提取操作流程为:待测溶液、标准工作溶液加入氘代化合物I溶液，加入甲醇混合均匀，离心取上清液即得；
添加剂为Na 2HPO 4或K 2HPO 4，抗凝剂为K 2EDTA或Na 2EDTA。
对于血液样本，应在采集后4小时内储存到-20℃以下环境中，优选为-70℃环境中，-70℃条件下可储存28天；；如经过处理则应在4℃或以下温度下冷藏且在54小时内完成进样检测
对于尿液样本，应在采集后的24小时内，优选4小时内储存在-70℃条件下，-70℃条件下可储存32天；如经过处理则应在室温或以下温度条件下94小时内完成进样检测。
附图说明
图1为AST-2660-D8-钠盐的 31P-NMR谱图。
图2为第一份AST-2660-D8-钠盐水溶液的 31P-NMR谱图，分别为扫描64、128、256、512次，分别参见a图、b图、c图和d图。
图3为零时刻的 31P-NMR谱图，依此为AST-2660-D8钠盐溶液-20℃(a图)、AST-2660-D8钠盐溶液-78℃(b图)。
图4为48小时后的 31P-NMR谱图，依此为AST-2660-D8钠盐溶液-20℃(a图)、AST-2660-D8钠盐溶液-78℃(b图)。
图5为-20℃储存6个月后AST-2660-D8钠盐水溶液的 31P-NMR谱图。
图6为-78℃储存6个月后AST-2660-D8钠盐水溶液的 31P-NMR谱图。
图7为空白人血浆提取物中AST-2660(a图)和内标AST-2660-D8(b图)的典型LC-MS/MS谱图。
图8为零浓度血浆样品提取物中AST-2660(a图)和内标AST-2660-D8(b图)的的典型LC-MS/MS谱图谱图。
图9为标准品溶液人血浆提取物(0.50ng/ml)的AST-2660(a图)和内标AST-2660-D8(b图)的典型LC-MS/MS谱图。
图10为标准品溶液人血浆提取物(200ng/ml)的AST-2660(a图)和内标AST-2660-D8(b图)的典型LC-MS/MS谱图。
图11为空白人尿液提取物中AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图12为零浓度尿液样品提取物中AST-2660和内标AST-2660-D8的的典型LC-MS/MS谱图谱图。
图13为标准工作溶液人尿液提取物(0.50ng/ml)的AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图14为标准工作溶液人尿液提取物(200ng/ml)的AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图8至图14中的坐标图中，横坐标为时间，纵坐标为LC-MS/MS的质谱离子强度，质谱离子为选定的离子。
具体实施方式
需要说明的是，除非另外定义，本说明书一个或多个实施例使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
申请人开发的AST-3424药物是DNA烷化剂AST-2660的前药，其特异性的被癌细胞高表达的AKR1C3酶所激活而代谢为AST-2660发挥药效。如背景技术部分所示，在人体临床试验中，需要对受试者的生物样本，如血液、尿液、组织等进行药物和药物代谢物的含量检测，通过检测得到的药物和药物代谢物的含量进行药物的DMPK(Drug Metabolism and Pharmacokinetics,中文意思是药物代谢及药代动力学)研究。而临床中使用的AST-3424含量较低，是从1mg开始直至100mg，在较小的剂量下，常规的液相色谱法对应的外标法或内标法并不能满足低检测限情况下的定量检测要求。因此，需要提供一种检测方法能够满足低检测限情况下的定量检测要求。
本申请为了解决上述技术问题，申请人尝试采用内标定量对生物样本中低含量的AST-2660进行定量。内标法是色谱分析中一种比较准确的定量方法，内标法是将一定重量的纯物质加到一定量的被分析样品混合物中，然后对含有内标物的样品进行色谱分析，分别测定内标物和待测组分的峰面积，即可求出被测组分在样品中的百分含量。内标物的选择是一项十分重要的工作。理想地说，内标物应当是一个能得到纯样的已知化合物，这样它能以准确、已知的量加到样品中去，它应当和被分析的样品组分有基本相同或尽可能一致的化学物理性质(如化学结构、极性、挥发度及在溶剂中的溶解度等)、色谱行为和响应特征，最好是被分析物质的一个同系物。当然，在色谱条件下，内标物必须能与样品中各组分充分分离。对于内标法定量分析来说，内标物的选择是十分重要的。它必须满足如下的条件：1.内标物与被分析物质的物理化学性质要相似(如沸点、极性、化学结构等)； 2.内标物应是该试样中不存在的纯物质；3.它必须完全溶于被测样品(或溶剂)中，且不与被测样品起化学反应；并与试样中各组分的色谱峰能完全分离；4.能加入内标物的量应接近于被测组分；5.色谱峰的位置应与被测组分的色谱峰的位置相近，或在几个被测组分色谱峰中间。且又不共溢出，目的是为了避免仪器的不稳定性所造成的灵敏度的差异；6.选择合适的内标物加入量，使得内标物和被分析物质二者峰面积的匹配性大于75％，以免由于它们处在不同响应值区域而导致的灵敏度偏差。
本申请对AST-2660的类似化合物进行了大量筛选，最终筛选得到氘代化合物I作为检测代谢产物II的内标。使用氘代化合物I建立的液相-质谱/质谱联用方法具有较低的检测限：低至0.5ng/ml。而且，本申请使用氘代化合物I建立的液相-质谱/质谱联用方法可用于检测人血浆和人尿液中代谢产物AST-2660含量，该方法符合生物样本分析要求，样本处理方法简便，方法灵敏度高，精密度和准确度高。
下面结合具体的实施例对本发明提供的技术方案做进一步的描述。下述实施例仅用于对本发明进行说明，并不会对本发明的保护范围进行限制。
缩略语的一般列表
Figure PCTCN2021133645-appb-000058
Figure PCTCN2021133645-appb-000059
实施例1 AST-2660-D8-钠盐的合成
Figure PCTCN2021133645-appb-000060
氮气保护下,氘代2-溴乙胺的氢溴酸盐(360mg，1.72mmol)，三氯氧磷(132mg，0.86mmol)加入到无水二氯甲烷中(4mL)(先加入氘代2-溴乙胺的氢溴酸盐，后加入三氯氧磷)，降温至-78℃，滴加入三乙胺(348mg，3.44mmol)的二氯甲烷溶液(2mL)，在-78℃下保温30min，自然升温至0℃，然后在0℃下保温4小时，然后快速抽滤掉固体，低温浓缩母液，直接用于下一步。
将上述所得粗品溶于水中(30.8mL)，缓慢分批加入氢氧化钠固体(275mg，6.88mmol)，反应常温搅拌过夜。然后于-20℃保存。 31P-NMR表征如图1所示：作为内标的HMPA的核磁峰29.886ppm，而AST-2660-D8-钠盐核磁峰24.503PPM。
图1为AST-2660-D8-钠盐的 31P-NMR谱图。
显然，在上述操作中，通过选取不同的氘代原料化合物I-a(氘代2-溴乙胺)可以合成不同的中间体I-b，进而在水解反应中添加不同的碱(NaOH、KOH、LiOH或是氨水)得到不同的氘代二(氮丙啶-1-基)次膦酸或其盐。
Figure PCTCN2021133645-appb-000061
Figure PCTCN2021133645-appb-000062
上述是I-a到I-b一个反应完成，实际上也可以采取分(两)步反应并改变投料顺序和反应物的数量，显然如此操作与上述I-a到I-b一个反应完成是等同的，即：
氮气保护下,氘代2-溴乙胺的氢溴酸盐，三氯氧磷加入到无水二氯甲烷中先加入三氯氧磷，后加入第一个氘代2-溴乙胺的氢溴酸盐，两者摩尔比三氯氧磷：氘代2-溴乙胺的氢溴酸盐大于1)，降温至-78℃，滴加入三乙胺的二氯甲烷溶液，在-78℃下保温30min后再加入第二个氘代2-溴乙胺的氢溴酸盐，滴加入三乙胺的二氯甲烷溶液，在-78℃下保温30min后，自然升温至0℃，然后在0℃下保温4小时，然后快速抽滤掉固体，低温浓缩母液，直接用于下一步。
将上述所得粗品溶于水中，缓慢分批加入氢氧化钠固体，反应常温搅拌过夜。然后于-20℃保存，发生的反应如下：
Figure PCTCN2021133645-appb-000063
通过选取不同的第一个氘代2-溴乙胺的氢溴酸盐和第二个氘代2-溴乙胺的氢溴酸盐的 氘代位置、氘代数目可以合成制备不同的氘代化合物I以及不同的盐。
以下以实施例1制备的AST-2660-D8-钠盐为例具体说明定量检测方法。
实施例2 检测AST-2660-D8-钠盐的方法
本实施例以HMPA(六甲基磷酰三胺)作为磷谱内标用于检测实施例1制备的AST-2660-D8-钠盐的含量。
取HMPA(33.0mg，0.1840mmol)溶于水(2.2mL)，总质量为2612mg，HMPA的浓度为7.050×10 -5(mmol/mg， 31P-NMR的核磁位移为29.890ppm)
以HMPA的水溶液为内标测定磷谱，测定AST-2660-D8-钠盐的含量。
1、第一份样品检测
取实施例1制备的AST-2660-D8-钠盐水溶液，约0.5mL(488mg)和已精密称量配制的HMPA水溶液(317mg)，扫描64次测定磷谱(如图2所示)：核磁中对应的HMPA的核磁峰29.874ppm与AST-2660-D8-钠盐核磁峰24.491的峰积分面积比＝1：0.257。
图2为第一份AST-2660-D8-钠盐水溶液的 31P-NMR谱图，从上到下，从左至右分别为扫描64、128、256、512次。
已知HMPA的量为0.02235mmol，则根据核磁峰积分面积比可计算得到AST-2660-D8-钠盐的量应为0.005743mmol，浓度应为1.177×10 -5mmol/mg，即1.177×10 -2mmol/g，对应的质量含量即2.10mg/g。同样算得，扫描次数为128次、256次、512次时磷谱如图2所示，其峰积分面积比依次为1：0.264，1：0.268，1：0.264，则同样的样品的浓度分别为1.209×10 -5mmol/mg、1.227×10 -5mmol/mg、1.209×10 -5mmol/mg，即2.15mg/g、2.18mg/g、2.15mg/g，四份结果平均为2.1mg/g。
本实施例的核磁定量方法比较快捷简单，在一定范围内具有可接受的准确度，可以代替HPLC产量分析方法。如果需要进一步的提高定量的准确度，应使用HPLC方法，选用外标法定量，通过标准曲线来精确定量绝对含量。
实施例5和实施例7的LC-MS/MS方法适用于AST-2660-D8、AST-2660的低含量检测，其对应的LC液相方法(对应的MS/MS检测器替换为常规的示差检测器、电喷雾检测器、蒸发光散射检测器)同样适用于AST-2660，AST-2660-D8的常量(mg/ml)含量检测。
实施例3 AST-2660-D8-钠盐的稳定性研究
本实施例是研究AST-2660-D8-钠盐水溶液含量检测样品溶液的稳定性：使用 31P-NMR检测，比较在48小时内作为内标的HMPA的 31P峰和AST-2660-D8-钠盐的 31P峰的面积比来表征AST-2660-D8-钠盐水溶液含量检测样品溶液是否发生降解而质量减少。
1.分析方法
在NMR上运行样品的 31P-NMR。
HMPA(21.8mg，0.1217mmol)溶于水(2.20mL)，总质量为2127mg，HMPA的浓度为5.72×10 -5mmol/mg。AST-2660-D8-钠盐溶液(588mg，约0.5mL)和HMPA(293mg，1.676×10 -2mmol)溶液混合在一起作为样品在-20℃储存条件下在不同时间进行 31P-NMR分析。并计算核磁中对应的HMPA的核磁峰(29.874ppm左右)与AST-2660-D8-钠盐核磁峰(24.491ppm左右)的峰积分面积比，并且将HMPA的核磁峰峰积分设置为1。
同样的操作，配制另一份浓度的样品进行检测，在-78℃储存条件下在不同时间进行 31P-NMR分析。。
2. -20到-78℃储存48小时稳定性结果
在-20、-78℃储存48小时，并分别在0h、2h、4h、6h、8h、16h、24h、36h、48h分别检测并记录，结果如下表1。
表1：AST-2660-D8钠盐在-20、-78℃储存48小时稳定性数据
Figure PCTCN2021133645-appb-000064
在-20℃储存条件下，8/16/24/36/48小时的HMPA的 31P-NMR核磁峰与AST-2660-D8-钠盐溶液峰面积比稳定在1：0.43～1：0.47，而-78℃储存条件下比值稳定在1：0.21～1：0.24，因此可以认为：AST-2660-D8-钠盐溶液，在-20到-78℃储存48小时是稳定的。
代表性谱图为图3、图4。
图3为零时刻的 31P-NMR谱图，从左至右依此为AST-2660-D8钠盐溶液-20℃、AST-2660-D8钠盐溶液-78℃。
图4为48小时后的 31P-NMR谱图，从左至右依此为AST-2660-D8钠盐溶液-20℃、AST-2660-D8钠盐溶液-78℃。
3.在-20℃下储存6个月的稳定性
使用上述记载的方法测得AST-2660-D8钠盐的浓度在0天时为1.89mg/mL，并且-20℃储存6个月后浓度为1.37mg/mL。AST-2660-D8钠盐在储存6个月内浓度降低了27.5％。
6个月后的 31P-NMR谱图如图6所示。
图5为-20℃储存6个月后AST-2660-D8钠盐水溶液的 31P-NMR谱图。
4.样品在-78℃下储存6个月的稳定性
使用上述记载的方法测得AST-2660-D8钠盐的浓度在0天时为1.89mg/mL，并且-78℃储存6个月后浓度为1.71mg/mL。AST-2660-D8钠盐在储存6个月内浓度降低了9.5％，相对而言，-78℃储存比-20℃储存更稳定，降解变化更慢。
6个月后的 31P-NMR谱图如图7所示。
图6为-78℃储存6个月后AST-2660-D8钠盐水溶液的 31P-NMR谱图。
由上述稳定性实验结果可知，AST-2660-D8钠盐水溶液含量检测样品溶液在-20至-78℃下放置2天是稳定的，：当AST-2660-D8钠盐水溶液分别在-20℃和-78℃储存6个月时，浓度分别降低27.5％和9.5％。
实施例4 人血浆中AST-2660的检测方法的建立及验证
本实施例使用氘代内标(IS)AST-2660-D8(实施例1制备)对含有K 2EDTA抗凝剂的人血浆中的代谢产物AST-2660(以钠盐形式存在)进行定量检测。其中，AST-2660的结构式如下所示：
Figure PCTCN2021133645-appb-000065
AST-2660钠盐
分子式：C 4H 8N 2O 2PNa
分子量：170.08
使用的氘代化合物AST-2660-D8钠盐的结构如下所示：
Figure PCTCN2021133645-appb-000066
AST-2660-D8钠盐
分子式：C 4D 8N 2O 2PNa
分子量：178.13
以下的AST-2660均指其钠盐，AST-2660-D8也是均指其钠盐。
1、LC-MS/MS实验方法。
给出了LC-MS/MS实验方法的相关参数。
表2：色谱分析法仪器参数
Figure PCTCN2021133645-appb-000067
表3：质谱仪MS/MS的实验参数
Figure PCTCN2021133645-appb-000068
表4：质谱仪MS/MS的实验参数可调参数(列出了典型值)
温度： 500℃
离子喷雾电压： -4500V
2、样品制备
2.1标准品工作溶液的制备
AST-2660(参照实施例1选用非氘代原料制备)标准品贮备溶液(浓度1.0mg/ml的甲醇溶液)：AST-2660适量，加甲醇适量稀释并混匀，得到1.0mg/ml溶液。
取100μL AST-2660标准品贮备溶液，加9900μL甲醇稀释并混匀，得到10.0μg/ml的 校准标准品工作溶液。
2.2标准曲线制备
将标准品工作溶液(SWS)和空白基质(稀释剂)放置至室温。使用前对标准品工作溶液进行涡旋，其中，将含有抗凝剂(K 2EDTA)的正常混合人血浆用作稀释剂和空白基质，按下表制备不同浓度的标准曲线标准品样品溶液。
表5：标准品工作溶液(SWS)和空白基质(稀释剂)稀释数据
Figure PCTCN2021133645-appb-000069
2.3内标工作溶液(标准工作溶液)的制备
AST-2660-D8贮备溶液(50μg/ml AST-2660-D8甲醇溶液)：AST-2660-D8对照品适量，加入适量甲醇适量稀释并混匀，得到50μg/ml溶液。取20μL AST-2660-D8内标贮备溶液，加9980μL甲醇稀释并混匀，得到100ng/ml的内标工作溶液。
2.4质控样品的制备
AST-2660质控贮备溶液(1.0mg/ml的甲醇溶液)：AST-2660对照品适量，加入适量甲醇稀释并混匀，得到1.0mg/ml溶液。取100μL AST-2660QC贮备溶液和9990μL甲醇，得到10.0μg/ml的质控工作溶液；
将含有抗凝剂(K 2EDTA)的正常混合人血浆用作稀释剂，按下表进行稀释，制备不同浓度的质控样品。
表6：质控样品的制备
Figure PCTCN2021133645-appb-000070
3、样品的前处理
使用蛋白质沉淀法提取样品的步骤：
1.确认标准曲线样品、质控样品和血浆样品的标签和顺序；
2.解冻后，将所有样品放在多管涡旋仪上涡旋均匀；
3.每份样品取100μl加入96孔板中；
4.除空白样品外，加入20μl AST-2660-D8质控工作溶液氘代内标(即100ng/ml AST-2660-D8甲醇溶液)。在空白样品中加入20μl甲醇；
5.加入500μl甲醇；
6.以中速将孔板涡旋3分钟；
7.在2143rcf(离心力)下离心96孔板10分钟；
8.将450μl上清液转移至一新的96孔板中；
9.用96孔Termovap样品浓缩仪干燥上清液；
10.用150μl甲醇复溶；
11.将平板超声30秒；
12.在4℃和2143rcf(离心力)下离心平板3分钟；
13.将120μl上清液转移至一块新的96孔板中。
4、实验结果
4.1标准曲线
人血浆中AST-2660标准曲线参数：斜率：0.063846，截距：-0.003788，相关系数0.9939，则关系函数y＝f(x)＝0.063846x-0.003788，其中y代表代谢产物II与内标化合物峰面积的比值，x表示标准工作溶液中代谢产物II的浓度，LLOQ(定量下限):0.5ng/ml，ULOQ(定量上限):200ng/ml。由此可以看出，在0.5～200ng/ml的线性范围内，线性关系良好。
4.2质控样品的准确度和精密度考察结果
准确度和精密度定义如下：
准确度表示为相对误差(RE)。
RE＝[(平均值-标称值)/标称值]×100
精密度表示为变异系数(CV)。
CV＝(标准差/平均值)×100。
准确度和精密度考察结果如表7所示。由表7可以看出，批内和批间的精密度均小于8.3％，批内和批间的准确度均在±8.7之间。准确度和精密度的分析标准为：对于LLOQ QC样品，批内和批间准确度(RE)必须在±20.0％范围内，并且批内和批间CV必须不大于20.0％。对于低、几何平均、中、高和稀释QC(如适用)样品，批内和批间准确度(RE)必须在±15.0％范围内；批内和批间精密度(CV)必须在每个QC浓度水平处不大于15.0％。因此，质控样品的批内和批间的精密度和准确度符合要求。内标AST-2660-D8的精密度(％CV)≤40.0％，符合要求。
表7：质控样品的批内和批间的精密度和准确度结果
Figure PCTCN2021133645-appb-000071
4.3 AST-2660和内标AST-2660-D8的提取回收率考察结果
表8：AST-2660和内标AST-2660-D8的提取回收率考察结果
Figure PCTCN2021133645-appb-000072
注：％回收率＝(提取前平均值/提取后平均值)×100
CV＝(SD/平均值)×100
由表8可以看出，AST-2660和内标AST-2660-D8的提取回收率的CV均小于15.0％。提取回收率的分析标准：对于提取后加标样品，在每个浓度水平处所测得的响应率的CV必须不大于15.0％。因此，AST-2660和内标AST-2660-D8的提取回收率符合要求。
4.4基质效应考察结果
表9:：基质效应结果
Figure PCTCN2021133645-appb-000073
根据表9的结果可知，AST-2660基质效应的批间基质准确度和精密度(CV)均小于15％，符合要求。
4.5稳定性
表10：AST-2660工作溶液和AST-2660-D8(内标)工作溶液的稳定性结果
Figure PCTCN2021133645-appb-000074
由表10可以看出，AST-2660工作溶液和内标AST-2660-D8工作溶液的稳定性的准确度在±8.1％范围内，精密度均小于5.5％。稳定性分析标准：贮藏稳定性溶液与新鲜配制的溶液之间的差异必须在±10.0％范围内，并且每组重复实验的CV必须不大于10.0％。因此，在各种储存条件下，AST-2660和AST-2660-D8(内标)工作溶液均稳定性良好，符合要求。
本实施例建立的液相-质谱/质谱联用方法检测人血浆中AST-2660的方法符合生物样本分析要求，样本处理方法简便，方法灵敏度高，精密度和准确度高。
实施例5 实际人血浆中AST-2660含量的检测
使用实施例4建立的检测方法对待测人血浆中的AST-2660含量进行检测。
以下为根据实施例4的方法学和验证后建立的检测标准操作流程SOP：
步骤一、溶液配制
基质：含有抗凝剂(K 2EDTA)的正常混合人血浆
a：内标工作溶液：取AST-2660-D8，加甲醇溶解并稀释制成每1ml中约含100ng的溶液。
b：待测溶液(此时的待测溶液未添加内标化合物)：取待测血液样品，即得。
c：标准工作溶液(此时的标准工作溶液未添加内标化合物)：取AST-2660适量，精密称定，加甲醇溶解并稀释制成每1ml中约含1.0mg的溶液，作为标准工作储备液，取标准工作储备液适量，分别加基质稀释制成每1ml中约含200ng、160ng、100ng、50ng、10ng、2.0ng、1.0ng、0.5ng的不同浓度的标准工作溶液。(溶液贮藏在-70℃)
d：质控样品溶液：取AST-2660适量，精密称定，加甲醇溶解并稀释制成每1ml中约含10μg的溶液，作为质控工作储备液，取质控工作储备液适量，加基质制成每1ml中约含150ng、60ng、15ng、1.5ng、0.5ng的不同浓度的质控工作溶液。(溶液贮藏在-70℃)
步骤二、样品提取
待测溶液、标准工作溶液、质控样品溶液，分别在室温状态下解冻并混合均匀，分别取100μl上述溶液，加入20μl内标，加入500μl甲醇，涡旋3分钟混合均匀，在2143rcf下离心10分钟，取450μl上清液干燥，加150μl甲醇复溶，超声，在4℃和2143rcf下离心平板3分钟，取120μl上清液，即得。
取100μl空白血液，加入20μl甲醇，同法提取，即得空白样品。
取100μl空白血液，加入20μl内标工作溶液，同法提取，即得零浓度样品。
经过样品提取、添加内标后的溶液才进行步骤四进样到LC-MS/MS进行检测。
步骤三、设置色谱-质谱测试条件
使用液相色谱-串联质谱(LC-MS/MS)测定，并进行如下仪器测试条件设置：
液相条件：用SHARC 1，3μm，2.1*50mm或性能相当的色谱柱；流动相A为含有低含量醋酸铵的甲醇溶液，流动相B为乙腈，按下表进行梯度洗脱；柱温20℃；进样体积5μl
表11：实施例5中的液相色谱仪测试时梯度洗脱条件
时间 流速 ％A ％B
初始 0.8 15 85
1.5 0.8 50 50
2.0 0.8 90 10
2.5 0.8 15 85
质谱条件：电喷雾离子源，负离子扫描方式；喷雾电压-4500V；离子源温度：500℃；碰撞能(CE)为-22eV；去簇电压(DP)为-55V；入口电压(EP)为-10V；碰撞室出口电压(CXP)为-16V；驻留时间为100ms；所有气体均使用高纯氮气；AST-2660离子对：m/z 147.0→m/z 62.9；内标AST-2660-D8离子对：m/z 155.0→m/z 62.9。
步骤四、测定
分别取经提取后的不同浓度的AST-2660标准工作溶液、空白样品、零浓度样品、待测样品溶液、质控样品溶液，注入LC-MS/MS进行检测。
接受标准：标准工作溶液测定回收浓度，质控样品溶液的准确度以及空白样品中AST-2660以及内标物峰残留应符合2020年版中国药典4部9012中要求。
吸取配制好的不同浓度的代谢产物II的标准工作溶液，注入LC-MS/MS系统进行检测，得到关系函数y＝f(x)，其中y代表代谢产物II与内标化合物峰面积的比值，x表示标准工作溶液中代谢产物II的浓度；
计算结果：由不同浓度的AST-2660标准工作溶液得到AST-2660与内标峰面积比值的关系函数y＝f(x)，将提取后的供试品溶液测得AST-2660与内标峰面积比值，代入关系函数y＝f(x)，计算待测溶液中代谢产物II的浓度。
利用关系函数测定计算未知浓度的待测溶液中代谢产物II的浓度，吸取加入已知含量的内标化合物的待测溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物峰面积的比值y，代入函数y＝f(x)，求得待测溶液中代谢产物II的浓度x。
作为一种选择，上述的SOP将相关操作步骤先后顺序略作变通，得到以下的操作SOP：
待测生物样本溶液中加入定量的内标化合物，经提取处理，作为供试品溶液；
使用代谢产物II对照品稀释得到一系列不同浓度的代谢产物II的溶液，加入定量的内标化合物，经提取处理，作为标准工作溶液，吸取系列标准工作溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物的峰面积，以代谢产物II与内标化合物的峰面积比值为纵坐标，以代谢产物II浓度为横坐标，绘制标准曲线，计算回归方程；
吸取供试品溶液，注入LC-MS/MS系统进行检测，测得供试品溶液溶液中代谢产物II与氘代化合物I峰面积的比值，代入回归方程，求得待测生物样本溶液中代谢产物II的含量。
上述变通后的SOP根据实验室操作人员习惯，使用相同容积的容器配制相同浓度的溶液，如此操作只需要保证添加的溶液体积相同，所得到的标准工作溶液依然是等浓度的。
图7至图10为血浆样本中检测过程的部分典型谱图。
图7为空白人血浆提取物中AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图8为零浓度血浆样品提取物中AST-2660和内标AST-2660-D8的的典型LC-MS/MS谱图谱图。
图9为标准品溶液人血浆提取物(0.50ng/ml)的AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图10为标准品溶液人血浆提取物(200ng/ml)的AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
使用本实施例提供的人血浆中的AST-2660含量检测的操作规程对部分样品进行检测。结果如下表12-16所示。
表12：人血浆中AST-2660在-20℃和-70℃下的一个月稳定性研究数据
Figure PCTCN2021133645-appb-000075
Figure PCTCN2021133645-appb-000076
很明显，含有AST-2660的人血浆在-70℃条件下储存28天后，含有的AST-2660几乎没有减少，而-20℃条件下减少较为明显，因此应尽量将AST-2660的人血浆样品储存在-70℃条件下，并且在储存28天后没有变化。
表13：人血浆中AST-2660在-20℃和-70℃下的冷冻/融化稳定性研究数据
Figure PCTCN2021133645-appb-000077
Figure PCTCN2021133645-appb-000078
a冻融循环稳定性样品首先在-20℃、-70℃标称温度下冷冻至少24小时，然后在室温下解冻。将样品冷冻至少12小时，以进行后续循环。
很明显，含有AST-2660的人血浆在-70℃到室温的冻融过程中含有的AST-2660几乎没有减少，而-20℃到室温的冻融过程中减少较为明显：也就是说，根据表12的数据可知，将储存在-70℃条件下的人血浆中的样品从储存条件转运到室温进行实验过程的温度急剧变化不会影响到人血浆样本中AST-2660的稳定性。
表14：AST-2660-D8溶液在-70℃下贮藏的稳定性研究数据
Figure PCTCN2021133645-appb-000079
很明显，根据表14的数据可知使用和AST-2660一样的-70℃条件下储存26天后，AST-2660-D8的甲醇溶液具有较强的稳定性。
表15：AST-2660在4℃下的处理后人血浆样品稳定性研究数据
Figure PCTCN2021133645-appb-000080
很明显，根据表15的数据可知AST-2660甲醇溶液在常规的冷冻条件(实验室常用的冰柜)下，54小时储存含量稳定。
表16：室温下人血浆中AST-2660实验台稳定性研究数据
Figure PCTCN2021133645-appb-000081
Figure PCTCN2021133645-appb-000082
很明显，根据表16的数据含有AST-2660的人血浆样本在室温下不稳定，随着存放时间的与延长，人血浆样本中AST-2660的含量一直在减少，这提示在进行相关临床患者人血浆样本采集过程中，采集到的血浆样本应立即储存在上述经过实验验证的低温环境中：储存在-70℃最稳妥，储存在-20℃也可以，如条件所限也也储存在常规的冰箱中(4℃至0℃)并且应尽量在4小时内转移到上述低温环境中。在样本送往DMPK样本检测中心实验室过程中应使用全程冷链物流(全程监控温度)温度选择为-20℃以下或干冰保温。
根据以上实验结果，可知：
(1)含有AST-2660的人血浆样本应在采集后立即储存在上述经过实验验证的低温环境中：储存在-70℃最稳妥，储存在-20℃也可以，如条件所限也也储存在常规的冰箱中(4℃至0℃)并且应尽量在4小时内转移到上述低温环境中。在样本送往DMPK样本检测中心实验室过程中应使用全程冷链物流(全程监控温度)温度选择为-20℃以下或干冰保温；
(2)样本应储存在-70℃或更低温度条件下，在此条件下储存28天是稳定的，而且根据变化趋势，更久的时间也许依然是稳定的；
(3)在取出储存的样本到室温过程中，即将储存在-70℃条件下的人血浆中的样品从储存条件转运到室温进行实验过程的温度急剧变化不会影响到人血浆样本中AST-2660的稳定性，取出后应放置在常规的冰箱中(4℃至0℃)，且常规冰箱中放置时间应不大于4 小时，如放置在室温环境下将导致AST-2660含量降低而不能使用；
(4)AST-2660-D8的甲醇溶液在-70℃条件下储存26天后含量不会发生变化，具有较强的稳定性，而且根据变化趋势，更久的时间也许依然是稳定的。
因此，上述，对于血液样本的操作SOP中，应在采集后4小时内将血浆样本储存到-20℃以下环境中，优选为-70℃环境中，-70℃条件下可储存28天；如血浆样本经过上述提取处理则应在4℃或以下温度下冷藏且在实验室室温环境下、54小时内完成进样检测；遵守这些储存温度和时间所检测的结果是可靠的，否则将导致因样本中AST-2660储存过程中变化而结果不真实、不可靠。
实施例6 人尿液中AST-2660的检测方法的建立及验证
本实施例中人尿液中AST-2660的检测方法的建立步骤与实施例4基本相同，区别仅在于基质不同(实施例4为人血浆，本实施例为人尿液)以及样品提取步骤略有差异。将含有添加剂Na 2HPO 4·12H 2O的混合正常人(不给药)尿液用作稀释剂和基质。
1、样品的前处理
使用蛋白质沉淀法提取样品的步骤：
1.确认标准品、质控样品和血浆样品的标签和顺序；
2.解冻后，将所有样品放在多管涡旋仪上涡旋均匀；
3.每份样品取100μl加入96孔板中；
4.除空白样品外，加入20μl AST-2660质控工作溶液(100ng/ml AST-2660-D8甲醇溶液)。在空白样品中加入20μl甲醇；
5.加入500μl甲醇；
6.以中速将孔板涡旋3分钟；
7.在2143rcf(离心力)下离心96孔板10分钟；
8.将450μl上清液转移至一新的96孔板中；
9.在2143rcf(离心力)下离心96孔板10分钟；
10.将300μl上清液转移至一块新的96孔板中。
2.实验结果
2.1标准曲线
人尿液中AST-2660标准曲线参数：斜率：0.001400，截距：0.000016，相关系数0.9982，关系函数y＝f(x)＝0.001400x+0.000016，其中y代表代谢产物II与内标化合物峰面积的比值，x表示标准工作溶液中代谢产物II的浓度，LLOQ(定量下限):0.5ng/ml，ULOQ(定量上限):200ng/ml。由此可以看出，在0.5～200ng/ml的线性范围内，线性关系良好。
2.2质控样品的准确度和精密度考察结果
准确度和精密度定义如下：
准确度表示为相对误差(RE)。
RE＝[(平均值-标称值)/标称值]×100
精密度表示为变异系数(CV)。
CV＝(标准差/平均值)×100。
准确度和精密度考察结果如表17所示。由表17可以看出，批内和批间的精密度均小于15.5％，批内和批间的准确度均在±11.4之间。准确度和精密度的分析标准为：对于LLOQ QC样品，批内和批间准确度(RE)必须在±20.0％范围内，并且批内和批间CV必须不大于20.0％。对于低、几何平均、中、高和稀释QC(如适用)样品，批内和批间准确度(RE)必须在±15.0％范围内；批内和批间精密度(CV)必须在每个QC浓度水平处不大于15.0％。因此，质控样品的批内和批间的精密度和准确度符合要求。内标AST-2660-D8的精密度(％CV)≤13.4％，符合要求。
表17：质控样品的批内和批间的精密度和准确度结果
Figure PCTCN2021133645-appb-000083
2.3基质效应考察结果
表18：基质效应结果
Figure PCTCN2021133645-appb-000084
根据表18的结果可知，AST-2660基质效应的批间基质准确度和精密度(CV)均小于15％，符合要求。
2.4稳定性
表19：AST-2660工作溶液的稳定性结果
Figure PCTCN2021133645-appb-000085
由表19可以看出，AST-2660工作溶液的稳定性的准确度在±11.8％范围内，精密度均小于11.5％。稳定性分析标准：贮藏稳定性溶液与新鲜配制的溶液之间的差异必须在±10.0％范围内，并且每组重复实验的CV必须不大于15.0％。因此，在各种储存条件下，AST-2660工作溶液稳定性良好，符合要求。
本实施例建立的液相-质谱联用方法检测人尿液中AST-2660的方法符合生物样本分析要求，样本处理方法简便，方法灵敏度高，精密度和准确度高。
实施例7 实际人尿液中AST-2660含量的检测
使用实施例6建立的检测方法对待测人尿液中的AST-2660含量进行检测。
以下为根据实施例6的方法学和验证后建立的检测标准操作流程SOP：
步骤一、溶液配制
基质：含有添加剂Na 2HPO 4·12H 2O的混合正常人尿液。
a：内标工作溶液：取AST-2660-D8，加甲醇溶解并稀释制成每1ml中约含100ng的溶液。
b：待测溶液(此时的待测溶液未添加内标化合物)：取待测血液样品，即得。
c：标准工作溶液(此时的标准工作溶液未添加内标化合物)：取AST-2660适量，精密称定，加甲醇溶解并稀释制成每1ml中约含1.0mg的溶液，作为标准工作储备液，取标准工作储备液适量，分别加基质稀释制成每1ml中约含200ng、160ng、100ng、50ng、10ng、2.0ng、1.0ng、0.5ng的不同浓度的标准工作溶液。(溶液贮藏在-70℃)
d：质控样品溶液：取AST-2660适量，精密称定，加甲醇溶解并稀释制成每1ml中约含10μg的溶液，作为质控工作储备液，取质控工作储备液适量，加基质制成每1ml中约含150ng、60ng、15ng、1.5ng、0.5ng的不同浓度的质控工作溶液。(溶液贮藏在-70℃)
步骤二、样品提取
供试品溶液、标准工作溶液、质控样品溶液，在室温状态下解冻并混合均匀，分别取100μl上述溶液，加入20μl内标，加入500μl甲醇，涡旋3分钟混合均匀，在2143rcf下离心30分钟，取450μl上清液，在2143rcf下离心96孔板10分钟，取300μl上清液，即得。
取100μl空白人尿液，加入20μl甲醇，同法提取，即得空白样品。
取100μl空白人尿液，加入20μl内标工作溶液，同法提取，即得零浓度样品。
经过样品提取、添加内标后的溶液才进行步骤四进样到LC-MS/MS进行检测。
步骤三、设置色谱-质谱测试条件
使用液相色谱-串联质谱(LC-MS/MS)测定，并进行如下仪器测试条件设置：
液相条件：用SHARC 1，3μm，2.1*50mm或性能相当的色谱柱；含有低含量醋酸铵的甲醇溶液，流动相B为乙腈，按下表进行梯度洗脱；柱温20℃；进样体积5μl。
表20：实施例7中的液相色谱仪测试时梯度洗脱条件
时间 流速 ％A ％B
初始 0.8 15 85
1.5 0.8 50 50
2.0 0.8 90 10
2.5 0.8 15 85
质谱条件：电喷雾离子源，负离子扫描方式；喷雾电压-4500V；离子源温度：500℃；碰撞能(CE)为-22eV；去簇电压(DP)为-55V；入口电压(EP)为-10V；碰撞室出口电压(CXP)为-16V；驻留时间为100ms；所有气体均使用高纯氮气；AST-2660离子对：m/z 147.0→m/z 62.9；内标AST-2660-D8离子对：m/z 155.0→m/z 62.9。
步骤四、测定
分别取提取后的不同浓度的AST-2660标准工作溶液、空白样品、零浓度样品、供试品溶液、质控工作溶液，注入LC-MS/MS进行检测。
接受标准：标准工作溶液测定回收浓度，质控工作溶液的准确度以及空白样品中AST-2660以及内标物峰残留应符合2020年版中国药典4部9012中要求。
吸取配制好的不同浓度的代谢产物II的标准工作溶液，注入LC-MS/MS系统进行检测，得到关系函数y＝f(x)，其中y代表代谢产物II与内标化合物峰面积的比值，x表示标准工作溶液中代谢产物II的浓度；
计算结果：由不同浓度的AST-2660标准工作溶液得到AST-2660与内标峰面积比值的关系函数y＝f(x)，将提取后的待测溶液测得AST-2660与内标峰面积比值，代入关系函数y＝f(x)，计算待测溶液中代谢产物II的浓度。
利利用关系函数测定计算未知浓度的待测溶液中代谢产物II的浓度，吸取加入已知含量的内标化合物的待测溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物峰面积的比值y，代入函数y＝f(x)，求得待测溶液中代谢产物II的浓度x。
作为一种选择，上述的SOP将相关操作步骤先后顺序略作变通，得到以下的操作SOP：
待测生物样本溶液中加入定量的内标化合物，经提取处理，作为供试品溶液；
使用代谢产物II对照品稀释得到一系列不同浓度的代谢产物II的溶液，加入定量的内标化合物，经提取处理，作为标准工作溶液，吸取系列标准工作溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物的峰面积，以代谢产物II与内标化合物的峰面积比值为纵坐标，以代谢产物II浓度为横坐标，绘制标准曲线，计算回归方程；
吸取供试品溶液，注入LC-MS/MS系统进行检测，测得供试品溶液溶液中代谢产物II与氘代化合物I峰面积的比值，代入回归方程，求得待测生物样本溶液中代谢产物II的含量。
上述变通后的SOP根据实验室操作人员习惯，使用相同容积的容器配制相同浓度的溶液，如此操作只需要保证添加的溶液体积相同，所得到的标准工作溶液依然是等浓度的。
图11至图14为尿液样本中检测过程的部分典型谱图。
图11为空白人尿液提取物中AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图12为零浓度尿液样品提取物中AST-2660和内标AST-2660-D8的的典型LC-MS/MS谱图谱图。
图13为标准工作溶液人尿液提取物(0.50ng/ml)的AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
图14为标准工作溶液人尿液提取物(200ng/ml)的AST-2660和内标AST-2660-D8的典型LC-MS/MS谱图。
使用本实施例提供的人尿液中的AST-2660含量检测的操作规程对部分样品进行检测。结果如下表21-24所示。
表21：人尿液中AST-2660在-20℃和-70℃下的一个月稳定性研究数据
Figure PCTCN2021133645-appb-000086
Figure PCTCN2021133645-appb-000087
很明显，含有AST-2660的人尿液在-70℃条件下储存32天后，含有的AST-2660几乎没有减少，而-20℃条件下减少了近90％，因此必须将AST-2660的人尿液样品储存在-70℃条件下，在此储存条件下储存32天的样品依然是稳定的。
表22：人尿液中AST-2660在-20℃和-70℃下的冷冻/融化稳定性研究数据
Figure PCTCN2021133645-appb-000088
Figure PCTCN2021133645-appb-000089
a冻融循环稳定性样品首先在-20℃、-70℃标称温度下冷冻至少24小时，然后在室温下解冻。将样品冷冻至少12小时，以进行后续循环。
很明显，含有AST-2660的人尿液在-70℃到室温的冻融过程中含有的AST-2660几乎没有减少，而-20℃到室温的冻融过程减少了近90％：也就是说，根据表22的数据可知，将储存在-70℃条件下的人血浆中的样品从储存条件转运到室温进行实验过程的温度急剧变化不会影响到人血浆样本中AST-2660的稳定性。
表23：在室温下处理人尿液样品稳定性(AST-2660含量变化)
Figure PCTCN2021133645-appb-000090
表24：室温下人尿液中AST-2660实验台稳定性研究数据
Figure PCTCN2021133645-appb-000091
Figure PCTCN2021133645-appb-000092
根据表23可知，在室温下经处理后AST-2660的人尿液样本放置94小时是稳定的。
根据表24可知，在室温下未处理的含AST-2660的人尿液样本放置24小时是稳定的。根据以上实验结果，可知：
(1)含有AST-2660的人尿液样本应在采集后24小时内(最晚)储存在上述经过实验验证的低温环境中：储存在-70℃。在样本送往DMPK样本检测中心实验室过程中应尽量低温(室温或更低)；
(2)样本应储存在-70℃或更低温度条件下，在此条件下储存32天是稳定的，而且根据变化趋势，更久的时间也许依然是稳定的；
(3)经处理后含有AST-2660的人尿液样本可在室温或更低的温度下放置94小时稳定；
(4)在取出储存的样本到室温过程中，即将储存在-70℃条件下的人尿液中的样品从储存条件转运到室温进行实验过程的温度急剧变化不会影响到人尿液样本中AST-2660的稳定性，取出后应放置在常规的冰箱或室温是容许的，放置在室温环境下长达24小时是稳定的。
(5)对于人尿液样本，应在采集后的24小时内，优选4小时内储存在-70℃条件下；如经过处理则应在室温条件下94小时内完成进样检测。
因此，上述，对于尿液样本的操作SOP中，应在采集后的24小时内，优选4小时内储存在-70℃条件下，-70℃条件下可储存32天；；如经过处理则应在室温或以下温度条件下94小时内完成进样检测；遵守这些储存温度和时间所检测的结果是可靠的，否则将导致因样本中AST-2660储存过程中变化而结果不真实、不可靠。

Claims (23)

  1. 氘代化合物I，其结构式如式(I)所示：
    Figure PCTCN2021133645-appb-100001
    其中，A为H或D，且8个A中至少有1个为D；
    M为H或碱金属、碱土金属、铵根。
  2. 根据权利要求1所述的氘代化合物I，其中，8个A中至少有3个为D。
  3. 根据权利要求1所述的氘代化合物I，其中，D的个数为4或者8。
  4. 根据权利要求1所述的氘代化合物I，其中，M为Na、K、Li、铵根。
  5. 根据权利要求1所述的氘代化合物I，其中，氘代化合物I的结构式如式(I-1)或式(I-2)所示：
    Figure PCTCN2021133645-appb-100002
  6. 根据权利要求1所述的氘代化合物I，其中，氘代化合物I选自以下结构的化合物：
    Figure PCTCN2021133645-appb-100003
  7. 一种制备氘代化合物I的方法，其包括以下步骤：
    Figure PCTCN2021133645-appb-100004
    I-a化合物与三卤氧磷POX 3反应得I-b化合物；
    I-b化合物加入碱或不加入碱在水溶液中进行水解反应，对应得化合物I；
    其中，I-a化合物为氘代的2-卤代乙胺或其无机酸盐，A为H或D，且在I-a化合物的4个A中至少有一个A为D，在I-b和I化合物的8个A中至少有1个为D；
    水解反应中碱选自：MOH，M为碱金属、碱土金属或铵根；MH，M为碱金属；MOR，R为1-4个碳的烷基，M为碱金属；碱金属的碳酸盐或碳酸氢盐，
    X为卤素。
  8. 根据权利要求7所述的方法，其中，I-a化合物与三卤氧磷POX 3在有机溶剂中降温， 滴加有机碱溶液反应得I-b化合物；
    优选地，所述降温为降温至-78至-20℃；和/或
    优选地，所述有机溶剂为二氯甲烷、氯仿、氯苯、1,2-二氯乙烷、乙酸乙酯、正己烷或环己烷、四氢呋喃中的一种或几种的混合；和/或
    优选地，所述有机碱为甲胺、乙胺、丙胺、异丙胺、N,N-二乙胺、三乙胺、正丁胺、异丁胺、4-二甲氨基吡啶、N,N-二异丙基乙胺、1,8-二氮杂二环[5.4.0]十一碳-7-烯、N,N,N',N'-四甲基乙二胺、四甲基胍、吡啶、N-甲基二环己胺或二环己胺中的一种或几种的混合；和/或
    I-a化合物与三卤氧磷POX 3的反应在一定的氛围下进行，所述氛围是空气、氮气或氩气中的一种，优选地，所述氛围是氮气或氩气中的一种,更优选地，所述氛围是氮气。
  9. 使用 31P-NMR法检测权利要求1-6中任意一项所述的氘代化合物I的含量，优选的，使用 31P-NMR法检测含有氘代化合物I的溶液中氘代化合物I的含量；或使用液相色谱法检测1-6中任意一项所述的氘代化合物I的含量，其中，液相色谱仪条件为：
    氢受体型固定相色谱柱，
    A流动相为醋酸铵的甲醇溶液，B流动相为乙腈，
    A、B流动相梯度洗脱，从A流动相15％体积比逐渐升高至90％体积比，然后逐渐减少到15％体积比。
  10. 一种检测权利要求1-6中任意一项所述的氘代化合物I含量的方法，其包括：
    检测氘代化合物I和已知含量的含磷化合物的 31P-NMR，得到谱图；
    根据 31P-NMR谱图中氘代化合物I和含磷化合物的化学位移特征峰的峰面积比，代入已知含量的含磷化合物的含量计算得到氘代化合物I的含量。
  11. 根据权利要求10所述的方法，其中，
    所述含磷化合物优选为含有一个磷原子的化合物，更优选为六甲基磷酰三胺；
    将氘代化合物I和已知含量的含磷化合物加入到溶剂中一并测试 31P-NMR谱图，优选的，将氘代化合物I和已知含量的含磷化合物加入到水中溶解后一并测试 31P-NMR谱图。
  12. 权利要求1-6中任一项所述的氘代化合物I作为内标在检测生物样本中代谢产物II的应用，代谢产物II结构式如式(II)所示：
    Figure PCTCN2021133645-appb-100005
    其中，A为H；M为H或碱金属、碱土金属、铵根。
  13. 权利要求1-6中任一项所述的氘代化合物I作为内标在LC-MS/MS分析检测生物样本中AKR1C3活化的DNA烷化剂前药或乏氧活化的DNA烷化剂前药的代谢产物II含量的应用，其中代谢产物II的结构式如式(II)所示：
    Figure PCTCN2021133645-appb-100006
    其中，A为H；M为H或碱金属、碱土金属、铵根。
  14. 根据权利要求13或14所述的应用，其中，
    氘代化合物I选自
    Figure PCTCN2021133645-appb-100007
    代谢产物II选自
    Figure PCTCN2021133645-appb-100008
  15. 根据权利要求14所述的应用，AKR1C3活化的DNA烷化剂前药选自以下结构式1-5的化合物：
    Figure PCTCN2021133645-appb-100009
    其中，R 1、R 2、R 3、R 4、R 5、R 8、R 9、R 10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A1中的权利要求书所记载；
    或
    Figure PCTCN2021133645-appb-100010
    其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1中的权利要求书所记载；
    或
    Figure PCTCN2021133645-appb-100011
    其中，X、Y、Z、R、A以及X 10的定义如专利申请PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)中的权利要求书所记载，T为
    Figure PCTCN2021133645-appb-100012
    或
    Figure PCTCN2021133645-appb-100013
    其中：
    A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR 1R 2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO 2、–O-(CH 2)-O-、-CO 2H及其盐、-OR 100、-CO 2R 100、-CONR 101R 102、-NR 101R 102、-NR 100SO 2R 100、-SO 2R 100、-SO 2NR 101R 102、C1-C6烷基、C3-C10杂环基；
    其中，R 100、R 101及R 102各自独立是氢、C1-C8烷基、C6-C12芳基；或R 101及R 102与其附接至的氮原子一起形成5-7元杂环；
    其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
    R 1及R 2各自独立是苯基或甲基；
    X、Y及Z各自独立是氢或卤基；
    R是氢或C1-C6烷基或卤素取代烷基。
  16. 根据权利要求14所述的应用，乏氧活化的DNA烷化剂前药选自选自以下结构式6-12的化合物：
    Figure PCTCN2021133645-appb-100014
    其中，R 1、R 2、R 3、Cx的定义如专利申请PCT/CN2020/114519，公开号WO2021120717A1中的权利要求书所记载；
    或
    Figure PCTCN2021133645-appb-100015
    Figure PCTCN2021133645-appb-100016
    其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15、R 16、R 17的定义如专利申请PCT/US2016/039092，公开号WO2016210175A1(对应中国申请号2016800368985，公开号CN108024974A)中的权利要求书所记载。
  17. 一种检测生物样本中代谢产物含量的方法，其包括以下步骤：
    制备含有已知浓度的内标化合物的待测溶液供LC-MS/MS进样分析；
    标准工作溶液的制备，制备含有已知浓度的内标化合物、已知浓度的代谢产物II的一系列标准工作溶液，该一系列标准工作溶液中内标化合物的浓度一致，且与所述待测溶液中的内标化合物浓度相同，该一系列标准工作溶液中代谢产物II的浓度是不同的；
    液相色谱-串联质谱法(LC-MS/MS)测定关系函数，吸取配制好的不同浓度的代谢产物II的标准工作溶液，注入LC-MS/MS系统进行检测，得到关系函数y＝f(x)，其中y代表代谢产物II与内标化合物峰面积的比值，x表示标准工作溶液中代谢产物II的浓度；
    利用关系函数测定计算未知浓度的待测溶液中代谢产物II的浓度，吸取加入已知含量的内标化合物的待测溶液，注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物峰面积的比值y，代入函数y＝f(x)，求得待测溶液中代谢产物II的浓度x，
    其中，所述待测溶液由所述生物样本经处理或不处理而制备，
    所述内标化合物为氘代化合物I，其结构式如式(I)所示：
    Figure PCTCN2021133645-appb-100017
    A为H或D，且8个A中至少有1个为D；
    M为H或碱金属、碱土金属、铵根；
    代谢产物II的结构式如式(II)所示：
    Figure PCTCN2021133645-appb-100018
    A为H；
    M为H或碱金属、碱土金属、铵根。
  18. 一种检测生物样本中代谢产物含量的方法，其包括以下步骤：
    待测生物样本溶液中加入定量的内标化合物，经提取处理，作为待测溶液；
    使用代谢产物II对照品稀释得到一系列不同浓度的代谢产物II的标准溶液，加入定量的内标化合物，经提取处理，作为标准工作溶液，吸取系列标准工作溶液，分别注入LC-MS/MS系统进行检测，测得代谢产物II与内标化合物的峰面积，以代谢产物II与内标化合物的峰面积比值为纵坐标，以代谢产物II浓度为横坐标，绘制标准曲线，计算回归方程；
    吸取待测溶液，注入LC-MS/MS系统进行检测，测得待测溶液中代谢产物II与内标化合物峰面积的比值，代入回归方程，求得待测溶液中代谢产物II的含量，
    其中，内标物为氘代化合物I，结构式如式(I)所示：
    Figure PCTCN2021133645-appb-100019
    A为H或D，且8个A中至少有1个为D；
    M为H或碱金属、碱土金属、铵根；
    代谢产物II的结构式如式(II)所示：
    Figure PCTCN2021133645-appb-100020
    A为H；
    M为H或碱金属、碱土金属、铵根。
  19. 根据权利要求17、18所述的方法，其中，
    液相色谱-串联质谱法中液相色谱仪条件为：
    氢受体型固定相色谱柱，
    A流动相为醋酸铵的甲醇溶液，B流动相为乙腈，
    A、B流动相梯度洗脱，从A流动相15％体积比逐渐升高至90％体积比，然后逐渐减少到15％体积比；
    质谱条件为：电喷雾离子源，
    负离子扫描方式下
    代谢产物II监测离子对为：m/z 147.0→m/z 62.9，
    氘代化合物I监测离子对为：m/z(147.0+氘代的数目)→m/z 62.9；
    或
    正离子扫描方式下
    代谢产物II监测离子对为：m/z 149.0→m/z 64.9，
    氘代化合物I监测离子对为：m/z(149.0+氘代的数目)→m/z 64.9；
  20. 根据权利要求17、19、20中任一项所述的方法，其中，
    制备含有已知含量的氘代化合物I的待测溶液时，优选先在生物样本溶液中添加氘代化合物I后，再进行提取操作；
    对应的，制备标准工作溶液时，在含已知的不同浓度的代谢产物II的基质溶液中先添加氘代化合物I后，再进行提取操作。
  21. 根据权利要求18或20所述的方法，其中，所述生物样本为尿液样本或血浆样本，对应的，在检测尿液样本时，对应的基质溶液为含有添加剂的、没有给药的患者的尿液；在检测血液样本时，对应的基质溶液为含有抗凝剂的、没有给药的患者的血浆。
  22. 根据权利要求21所述的方法，其中，
    加入氘代化合物、提取操作流程为:待测溶液、标准工作溶液加入氘代化合物I溶液，加入甲醇混合均匀，离心取上清液即得；
    添加剂为Na 2HPO 4或K 2HPO 4，抗凝剂为K 2EDTA或Na 2EDTA。
  23. 根据权利要求21所述的方法，其中，
    对于血液样本，应在采集后4小时内储存到-20℃以下环境中，优选为-70℃环境中，-70℃条件下可储存28天；如经过处理则应在4℃或以下温度下冷藏且在54小时内完成进样检测；
    对于尿液样本，应在采集后的24小时内，优选4小时内储存在-70℃条件下，-70℃条件下可储存32天；如经过处理则应在室温或以下温度条件下94小时内完成进样检测。
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