Deuterated compounds, as well as methods for producing and using the same.

Abstract

The present invention relates to a deuterated compound, and its preparation method and use. The deuterated compound I has the structure shown in formula (I), where A is H or D, at least one of the eight A's is D, and M is H or an alkali metal, an alkaline earth metal, or an ammonium radical. The present invention provides the use of the deuterated compound I as an internal standard for measuring the content of metabolite II in a biological sample, where the metabolite II has the structure shown in formula (II), where A is H, and M is H or an alkali metal, an alkaline earth metal, or an ammonium radical. The present invention uses the deuterated compound I as an internal standard to quantitatively analyze the content of low-content metabolite II in a biological sample, which can not only meet the requirements of the lower limit of quantification, but also meet the requirements of DMPK testing in clinical trials. [Chemical formula I] JPEG2025500718000110.jpg34170 [Case II] JPEG2025500718000111.jpg34170

Description

[Technical Field]

[0001] The present invention relates to the field of assay technology, and more particularly to deuterated compounds, as well as methods for preparing and using them. [Background technology]

[0002] In human clinical trials, it is necessary to measure the content of drugs and drug metabolites in the subjects' biological samples (blood, urine, tissue, etc.), and to conduct drug metabolism and pharmacokinetic (DMPK) studies based on the measured drug and drug metabolite content. Such studies are essential for the development of new drugs.

[0003] Liquid chromatography, the most common method, can detect almost all types of drugs and drug metabolites by utilizing the ultra-high resolution of high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC) and the detection capabilities of ultraviolet absorption detectors (UVD), diode array detectors (PDAD), fluorescence detectors (FLD), evaporative light scattering detectors (ELSD), differential refractometers (DR), or mass spectrometry detectors (MSD).

[0004] AST2660 (also known as AST-2660) is a metabolite of AST-3424 (also known as OBI-3424 or TH-3424) (Meng, F., Li, WF., Jung, D., Wang, CC., Qi, T., Shia, CS., Hsu, RY., Hsieh, YC., & Duan, J (2021)), and the novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad antitumor activity (American Journal of Cancer Research, 11(7), 3645), and is the chemical component that exerts the activity of the prodrug AST-3424.

[0005] [ka]

[0006] Clinical trials are underway (OBI-3424 is being investigated in the US Phase II clinical trial NCT03592264 (sponsored by OBI Pharma, Inc., a Taiwan-based biopharmaceutical company, and is targeting patients with castration-resistant prostate cancer (CRPC) and liver cancer), and in the US Phase II clinical trial NCT04315324 (sponsored by Southwest Oncology Group (SWOG), and is targeting patients with T-cell acute lymphoblastic leukemia (T-ALL)), and AST-3424 is being investigated in the China Phase II clinical trial CTR20191399 (sponsored by Ascentawits Pharmaceuticals, Ltd., and is targeting patients with solid tumors), and in the China Phase II clinical trial CTR20201915 (Ascentawits In a study sponsored by Pharmaceuticals, Ltd. and targeting patients with T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic lymphoma (B-ALL), AST-3424 is used in doses ranging from 1 mg to 100 mg.

[0007] Conventional internal or external standard methods used in liquid chromatography cannot meet the requirements for quantitative analysis of drug metabolites in biological samples when drugs are administered in small amounts. Therefore, there is a need to develop an assay method that not only meets the requirements for the limit of quantification but also satisfies the requirements for DMPK testing of the aforementioned drugs. [Overview of the project] [Problems that the invention aims to solve]

[0008] To achieve the aforementioned objectives, the present invention provides deuterated compounds, as well as methods for their preparation and use. Deuterated compound I has been tested and confirmed to be usable as an internal standard for the quantitative analysis of metabolite II in biological samples with a minimum detection limit of 0.5 ng / ml, meeting the requirements of the DMPK test.

[0009] Deuterated compound I, provided as a deuterated internal standard in DMPK analysis, possesses sufficient stability and can be stored for longer periods under experimental conditions (stable quality and properties are obtained when stored at -20°C and -70°C), meeting the requirements for long-term sample storage and various operating temperatures (laboratory ambient temperature) in DMPK laboratories used in clinical trials.

[0010] Generally speaking, the present invention is a system for measuring low levels of metabolite II in biological samples, comprising a deuterated internal standard, LC-MS / MS instrument and method, a curve-fitting algorithm for quantification, and an operating procedure, which actually meets the requirements of DMPK testing of metabolite II below the limit of quantification (it is present in small amounts, requires concentrated analysis, thus requiring longer sample storage, and thus meets the practical requirements of laboratory operations).

[0011] According to the present invention, deuterated compound I can be used for the quantitative analysis of metabolite II in biological samples, satisfying the requirements for quantitative analysis below the limit of quantification, and is also suitable for DMPK testing in clinical trials. [Means for solving the problem]

[0012] The present invention relates to a deuterated compound I having the structure shown in formula (I). [ka] In the formula, A is either H or D, and at least one of the eight A's is D. M is H, or an alkali metal, alkaline earth metal, or ammonium radical.

[0013] The deuterated compound I is deuterated bis(aziridine-1-yl)phosphinic acid or a salt thereof, and the salt-forming cation is Na + , K + , or alkaline earth metal ions Ca 2+ , or ammonium ion NH4 +These are alkali metals such as Na, K, Li, or ammonium radicals.

[0014] Depending on the biological sample and the different reagents added in subsequent operations, the deuterated compound may exist in the form of an acid or a salt, and accordingly, the deuterated compound is an acid or a salt.

[0015] Preferably, at least three of the eight A's in the deuterated compound I are D's. As an internal deuterated standard, the corresponding mass-to-nuclear ratio m / z in the mass spectrum is distinguishable from the non-deuterated compound. In fact, the mass spectral peak is not a single value but a bell shape that slopes downward on both sides around a main peak, and the x-axis value m / z of the main peak is the value of the compound set as x, and when a hydrogen atom (protium) in the compound is replaced with deuterium, the x-axis value m / z of the main peak is x+1. Since the main peak is a bell shape, it is clear that x and x+1 may overlap and become indistinguishable. According to the method for calculating the molecular weight of a compound (i.e., the method for summing the relative atomic masses of all atoms in the compound), it can be concluded that the more atoms there are, the more isotopic species with different relative atomic masses the corresponding atoms have, the more "robust" the shape of the main peak that slopes downward on both sides becomes, and the wider the overlap area with the main peak of another compound. One possible approach to reduce the overlap area is to increase the distance between the two main peaks. For the deuterated compound, it is necessary to increase the number of deuterium atoms. Based on the individualized context of the deuterated compound I, and through experimental verification and calculations, the present invention has found that when the deuterated compound I contains three D (deuterium) atoms, the non-deuterated compound (metabolite II) can be better distinguished from the deuterated compound I. When there are fewer D (deuterium) atoms in the deuterated compound I, it is necessary to provide a mass spectrum with higher resolution.

[0016] Preferably, the number of D atoms in the deuterated compound I is 4 or 8.

[0017] More preferably, the deuterated compound I is a compound having the structure shown in formula (I-1) or (I-2). [ka] [ka]

[0018] In particular, the deuterated compound I is selected from compounds having the following structure. [ka] [ka]

[0019] Furthermore, the present invention relates to a method for preparing deuterated compound I, [ka] The process involves reacting compound Ia with phosphorus oxyhalogenate POX3 to obtain compound Ib, The process involves hydrolyzing the aforementioned compound Ib in an aqueous solution, with or without the presence of a base, to obtain the corresponding deuterated compound I. Includes, The compound Ia is a deuterated 2-halogenated ethylamine or its inorganic salt, sulfate, phosphate, etc., where A is H or D, at least one of the four A's in compound Ia is D, and at least one of the eight A's in compound Ib and compound I is D. The base in the hydrolysis reaction is selected from MOH (where M is an alkali metal, alkaline earth metal, or ammonium radical), MH (where M is an alkali metal), and MOR (where R is an alkyl group having 1 to 4 carbon atoms, and M is an alkali metal, an alkali metal carbonate, or a bicarbonate). X is a halogen.

[0020] In the first reaction, compound Ia (deuterated 2-haloethylamine or its hydrogen halide) is reacted with phosphorus oxyhalide POX3 to obtain compound Ib. This reaction process may include one or more reactions.

[0021] The available deuterated 2-haloethylamine or 2-haloethylamine inorganic salts are determined based on the solvent used in the reaction.

[0022] Since this reaction is a vigorous exothermic reaction, 2-haloethylamine or 2-haloethylamine hydrohalide is dissolved in a solvent and cooled. Subsequently, phosphorus oxyhalide POX3 or an aqueous solution of phosphorus oxyhalide is slowly added dropwise while stirring (added dropwise so that the temperature of the reaction system is between -78°C and -20°C). The solvent used in the reaction is an organic solvent, such as one or more of dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, or cyclohexane.

[0023] The oxyhalogenated phosphorus POX3 includes oxybromide phosphorus POBr3 and oxychloride phosphorus POCl3. Examples of deuterated 2-haloethylamine inorganic salts include deuterated 2-haloethylamine hydrohalides (hydrochloride, hydrobromide) and inorganic oxo salts (sulfate and phosphate, etc.), and preferably, compound Ia is the hydrochloride or hydrobromide of deuterated 2-haloethylamine.

[0024] When an acid is produced in the first reaction, a base is added to adjust the pH of the reaction. The bases to be added include inorganic bases and organic bases. Inorganic bases are selected from weak bases such as alkaline earth metal hydroxides (calcium hydroxide), alkali metal carbonates, and bicarbonates (sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate). Preferably, the organic base is one or more of the following: methylamine, ethylamine, propylamine, isopropylamine, N,N-diethylamine, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undeca-7-ene, N,N,N',N'-tetramethylethylenediamine, tetramethylguanidine, pyridine, N-methyldicyclohexylamine, or dicyclohexylamine.

[0025] The reaction procedure includes the steps of cooling 2-haloethylamine or 2-haloethylamine hydrohalide in a solvent, then slowly adding a solution of phosphorus oxyhalogenate POX3 or phosphorus oxyhalogenate POX3 dropwise and further cooling, and after cooling, adding a base or basic solution and reacting while stirring.

[0026] Preferably, the organic solvent is a mixture of one or more of the following: dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, or cyclohexane and tetrahydrofuran.

[0027] The reaction between compound Ia and phosphorus oxyhalogenate POX3 is carried out in the atmosphere, which is one of air, nitrogen, or argon, preferably one of nitrogen or argon, and more preferably nitrogen.

[0028] In the second step, compound Ib is hydrolyzed in an aqueous solution, with or without a base, to obtain the corresponding deuterated compound I.

[0029] The hydrolysis reaction must be carried out with the addition of water. Therefore, the reaction is carried out with the involvement of water. When only water is added without the addition of a base, M in the deuterated compound I after the reaction becomes H, and the compound exists in the form of an acid. When a base is added, a salt is formed in the corresponding reaction. The base in the hydrolysis reaction is selected from MOH (where M is an alkali metal, alkaline earth metal, or ammonium radical), MH (where M is an alkali metal), and MOR (where R is an alkyl group having 1 to 4 carbon atoms, and M is an alkali metal, carbonate, or bicarbonate of an alkali metal). Preferably, the base is NaOH or KOH.

[0030] Furthermore, the present invention relates to the use of measuring the content of deuterated compound I, that is, 31 A method for measuring the content of the deuterated compound I using P-NMR, preferably, 31 A method for measuring the content of deuterated compound I in a solution containing deuterated compound I using P-NMR, or a method for measuring the content of said deuterated compound I using liquid chromatography, The liquid chromatography conditions are as follows: Using a hydrogen-accepting stationary-phase chromatography column, Mobile phase A is a methanol solution of ammonium acetate, and mobile phase B is acetonitrile. For gradient elution, mobile phases A and B are used. Mobile phase A is gradually increased from 15% to 90% by volume, and then gradually decreased back down to 15% by volume.

[0031] The deuterated compound is quantitatively analyzed after preparation and purification. Various methods exist for quantitative analysis. The deuterated compound can be directly weighed after purification or analyzed directly by HPLC.

[0032] The final product of the deuterated compound I prepared according to the present invention is present in an aqueous solution and therefore cannot be rapidly analyzed by general HPLC or accurate weighing methods. 31It has been experimentally confirmed that by using the P-NMR method, the content can be measured with the required accuracy in a rapid and simple manner.

[0033] The present invention also provides a method for measuring the content of the deuterated compound I, comprising: detecting the P-NMR of the deuterated compound I and the phosphorus-containing compound with a known content to obtain their spectra; and 31 calculating the content of the deuterated compound I by substituting the content of the phosphorus-containing compound with the known content based on the peak area ratio of the chemical shift characteristic peak of the deuterated compound I and the chemical shift characteristic peak of the phosphorus-containing compound in the P-NMR spectrum. The 31 The phosphorus-containing compound is preferably a compound containing one phosphorus atom, and more preferably hexamethylphosphoric triamide.

[0034] Preferably, the deuterated compound I and the phosphorus-containing compound with a known content are added to a solvent, and their P-NMR spectra are tested together.

[0035] Preferably, the deuterated compound I and the phosphorus-containing compound with a known content are added to water, and after they are dissolved, their P-NMR spectra are tested together. 31 The deuterated compound I is quantitatively analyzed using a phosphorus-containing compound as an internal standard for P-NMR. It is obvious that the number of phosphorus atoms in the selected phosphorus-containing compound is preferably 1 so that the P-NMR spectrum has relatively simple signal peaks, which is convenient for quantification. Furthermore, the chemical shift of the P-NMR spectrum signal peak of the phosphorus-containing compound is such that the two signal peaks can be easily distinguished from the

[0036] chemical shift of the deuterated compound I. 31 The deuterated compound I is quantitatively analyzed using a phosphorus-containing compound as an internal standard for P-NMR. It is obvious that the number of phosphorus atoms in the selected phosphorus-containing compound is preferably 1 so that the P-NMR spectrum has relatively simple signal peaks, which is convenient for quantification. Furthermore, the chemical shift of the P-NMR spectrum signal peak of the phosphorus-containing compound is such that the two signal peaks can be easily distinguished from the

[0037] The deuterated compound I 31 chemical shift of the deuterated compound I. 31 chemical shift of the deuterated compound I. 31 chemical shift of the deuterated compound I. 31The signal peaks in the P-NMR spectrum must be sufficiently far from the chemical shift.

[0038] 31 When analyzing spectra using P-NMR, the number of scans is determined by the phosphorus-containing compound. 31 The P-NMR spectral signal peak also shows deuterated compound I. 31 It also has a certain effect on the P-NMR spectral signal peaks. It was demonstrated that the number of scans needs to exceed 64.

[0039] Furthermore, the present invention relates to the use of deuterated compound I as an internal standard for detecting metabolite II in a biological sample, and more preferably, to the use of deuterated compound I as an internal standard for measuring the content of metabolites of DNA alkylating agent prodrugs in a biological sample, where metabolite II has the structure shown in formula (II). [ka] In the formula, A is H, and M is H, an alkali metal, an alkaline earth metal, or an ammonium radical.

[0040] A "predrug" (also known as a prodrug, drug precursor, or precursor drug) is a compound that possesses pharmacological effects only after being converted in vivo. While the prodrug itself is biologically inactive or low-activity, it becomes activated after metabolism in the body. The purpose of this process is to increase the bioavailability and targeting ability of a drug while reducing its toxicity and side effects. Currently, prodrugs can be classified into two main families: carrier prodrugs and bioprecursors.

[0041] The DNA alkylating agent prodrug of the present invention refers to a prodrug that releases a DNA alkylating agent (i.e., metabolite II) after metabolism.

[0042] Furthermore, the present invention relates to the use of the deuterated compound I as an internal standard for measuring the content of metabolite II of an AKR1C3-activated DNA alkylating agent prodrug or a hypoxia-activated DNA alkylating agent prodrug in a biological sample by LC-MS / MS analysis, wherein metabolite II has the structure shown in formula (II). [ka] In the formula, A is H, and M is H or an alkali metal, alkaline earth metal, or ammonium radical. The aforementioned deuterated compound I is [ka] Selected from, The aforementioned metabolite II is, [ka] Selected from.

[0043] The AKR1C3-activating DNA alkylating agent prodrug is selected from compounds having the structures shown in formulas 1 to 5. [ka] In the formula, R1, R2, R3, R4, R5, R8, R9, and R 10 The definition is as stated in the claims of PCT / CN2020 / 089692 (International Publication No. 2020 / 228685).

[0044] Specifically, the base is defined as follows: R1 is C6-C 10 It is an aryl or Z-substituted aryl, a 4-15 member heterocycle or Z-substituted heterocycle, a 5-15 member heteroaryl or Z-substituted heteroaryl, or a 7-15 member fused ring, or a Z-substituted fused ring. R2 is hydrogen, halogen atom, cyano or isocyan, hydroxy, sulfhydryl, amino, OT sOMS, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, ether having 1-6 carbon atoms, or Z-substituted alkoxy having 1-6 carbon atoms, -CONR 6 R 7 -SO2NR 6 R 7 , -SO2R 6 -OCOO-R 6 ,-COOR 6、 -NR 6 COR 7 , -OCOR 6 , -NR 6 SO2R 7 or -NR 6 SO2NR 6 R 7 , or R with atoms in group R1 that bond to form a 7-15 membered fused ring or a Z-substituted fused ring 2 And, R3 is hydrogen, halogen, cyano or isocyan, hydroxy, sulfhydryl, amino, OT s , OLCMS, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 member heterocycle or Z-substituted heterocycle, 5-15 member heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR 6 R 7 -SO2NR 6 R 7 , -SO2R 6 ,-OCO-R 6 -OCOO-R 6 ,-COOR 6 , -NR 6 COR 7, -OCOR 6 , or -NR 6 SO2R 7 And, R4 and R5 are, independently, hydrogen, halogen atom, cyano or isocyanone, hydroxyl, sulfhydryl, amino, OT s , OLCMS, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 member heterocycle or Z-substituted heterocycle, 5-15 member heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR 6 R 7 -SO2NR 6 R 7 , -SO2R 6 -OCOO-R 6 ,-COOR 6 , -NR 6 COR 6 , -OCOR 6 or -NR 6 SO2R 7 or R4 and R5 are atoms in a benzene ring that are bonded together to form a 7-15 membered fused ring or a Z-substituted fused ring. R 6 and R 7 These are, independently, hydrogen, cyano or isocyano, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 R with atoms that combine with aryl or Z-substituted aryl, 4-15 member heterocycle or Z-substituted heterocycle, 5-15 member heteroaryl or Z-substituted heteroaryl, or C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, or atoms that combine with them to form a 5-7 member heterocyclyl or Z-substituted 5-7 member heterocyclyl 6 and R 7 And, R8 and R 10 Each of these is independently hydrogen, deuterium, aryl or Z-substituted aryl, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, and R8 and R 10 At least one of them must be hydrogen or deuterium. R9 is a substituted C6-C atom substituted with at least one fluorine atom or nitro group. 10 The aryl group is a substituted 4- to 15-membered heterocyclic ring substituted with at least one fluorine atom or nitro group, or a substituted 5- to 15-membered heteroaryl group substituted with at least one fluorine atom or nitro group. The substituent Z can be a halogen atom, cyano or isocyano, hydroxy, sulfhydryl, amino, or OT. s OMS, C1-C3 alkyl or substituted alkyl, C1-C3 alkoxy or substituted alkoxy, C2-C3 alkenyl or substituted alkenyl, C2-C3 alkynyl or substituted alkynyl, C3-C8 cycloalkyl or substituted cycloalkyl, aromatic ring, heterocycle, aromatic heterocycle and fused ring or substituted aromatic ring, heterocycle, or aromatic heterocycle and fused ring, and the substitution pattern is single substitution - or geminal disubstituted, Substitute C6-C 10 In aryl, substituted 4-15 membered heterocyclic, or substituted 5-15 membered heteroaryl rings, the substituents in R9 are halogen atoms, nitro, cyano or isocyano, hydroxy, amino, C1-C3 alkyl or alkoxy, alkenyl, alkynyl, cycloalkyl or benzene ring, substituted benzene ring, C1-C3 alkoxy, or halogen atom-substituted alkoxy.

[0045] In particular, the compounds of formulas (1) and (2) are [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0046] The definitions and meanings of the groups, as well as the preparation methods and spectral data of the compounds, are described in PCT / CN2020 / 089692 (International Publication No. 2020 / 228685), which is incorporated herein by reference in its entirety.

[0047] It is clear that the compound with structural formula 1 or 2, like AST-342, is a prodrug (acid form) of AST-2660, and can be activated by the AKR1C3 enzyme to form AST-2660, thereby exhibiting its anticancer effects.

[0048] [ka] [ka]

[0049] It is clear that the aforementioned compound includes the compound of structural formula 1 or 2, and its salts, esters, solvates, and isotopic isomers.

[0050] [ka] In the formula, the definition of Rw is as described in the claims of PCT / CN2020 / 120281 (International Publication No. 2020 / 2021068952).

[0051] Specifically, it is defined as follows: Rw is [ka] And, R1 is H, C 1-6 Alkyl, C3-6 a cycloalkyl, 4- to 6-membered heterocycloalkyl, 5- to 6-membered heteroaryl or phenyl group, wherein said C 1-6 alkyl, C 3-6 the cycloalkyl, 4- to 6-membered heterocycloalkyl, 5- to 6-membered heteroaryl and phenyl groups may be substituted with 1, 2 or 3 R a groups, each R a is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 an alkoxy group or C 1-3 alkyl, R2 is H or C 1-6 alkyl, or together with N to which they are attached forms a 4- to 6-membered heterocycloalkyl, R1 and R 2 wherein said 4- to 6-membered heterocycloalkyl may be substituted with 1, 2 or 3 R b groups, each R b is independently H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3 or -CH2CH3,<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​R c and R d each independently represents H, F, C 1-3 alkyl or C 1-3 an alkoxy group, R4, R5 and R6 each independently represent H, F, Cl, Br, I, C 1-3 alkyl or C 1-3 an alkoxy group, T is N or CH, R7 and R8 each independently represent H, F, Cl, Br or I, R9 and R 10 each independently represent H, F, Cl, Br, I, -CN, 4- to 6-membered heterocycloalkyl and 5- to 6-membered heteroaryl each independently contain 1, 2, 3 or 4 heteroatoms selected from N, -O- and -S-.

[0052] Specifically, the compound of formula (3) is selected from the group consisting of the following compounds.

Chemical formula

[0053] The definitions and meanings of the groups, as well as the preparation methods and spectral data of the compounds, are described in PCT / CN2020 / 120281 (International Publication No. WO 2021 / 068952), which are incorporated herein by reference in their entirety.

[0054] It is clear that the compound of structural formula 3, like AST-342, is a prodrug (acid form) of AST-2660, can be activated by the AKR1C3 enzyme to form AST-2660, and can exhibit its anti-cancer effect.

Chemical formula

[0055]

Chemical formula

[0056] Specifically, the base is defined as follows: X 10 These are O, S, SO, or SO2. A is C6-C 10 Aryl, 5-15 member heteroaryl, or -N=CR 1 R 2 And, R 1 and R 2 These are, independently, hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, and C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 , or -NR 13 COR 14 And, X, Y, and Z are independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 , or -NR 13 COR 14 And, R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 , or -NR 13 COR 14 And, R 13and R 14 These are, independently, hydrogen, C1-C6 alkyl, and C3-C8 cycloalkyl. Ru, C6-C 10 It is an aryl, 4-15 membered heterocycle, or ether. T is [ka] The formula comprises the alkyl group, alkenyl group, alkynyl group, cycloalkyl group, and aryl group. The groups, heterocyclic groups, heteroaryl groups, and ether groups may be substituted.

[0057] Specifically, the compound of formula (5) is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0058] The specific definitions and meanings of the groups, as well as the methods for preparing the compounds and spectral data, are described in the claims of PCT / US2016 / 021581 (International Publication No. 2016 / 145092) (corresponding to Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556)), which are incorporated herein by reference in their entirety.

[0059] It is clear that the compound of structural formula 4, like AST-3424T, is a prodrug of a phosphoramidate alkylating agent, and that it can be activated by the AKR1C3 enzyme to form AST-2660 (acid form), which can then exhibit its anticancer effect. [ka]

[0060] [ka] In the formula, A is a substitute or non-substitute C6-C 10 Aryl, biaryl or substituted biaryl, 5-15 member heteroaryl, or -N=CR 1 R 2 The substituents are halogeno, -CN, -NO2, -O-(CH2)-O-, -CO2H and their salts, -OR 100 , -CO2R 100 ,-CONR 101 R 102 , -NR 101 R 102 , -NR 100 SO2R 100 , -SO2R 100 -SO2NR 101 R 102 , C1-C6 alkyl, and C3-C 10 Selected from a group consisting of heterocyclines, In the formula, R 100 , R 101 and R 102 Each of these is independently hydrogen, C1-C8 alkyl, or C6-C 12 It is either aryl or R 101 and R102 These, together with the nitrogen atoms bonded to them, form a 5-7 membered heterocycle. The alkyl group and the aryl group are each substituted with 1 to 3 halogen groups or 1 to 3 C1-C6 alkyl groups. R 1 and R 2 These are, independently, phenyl or methyl, X, Y, and Z are each independently either hydrogen or a halogen. R is hydrogen, a C1-C6 alkyl group, or a halogen-substituted alkyl group.

[0061] The "Cx-Cy" or "Cx-y" prefix before a group indicates the range of the number of carbon atoms present in that group. For example, C1-C6 alkyl refers to an alkyl group having at least one to six carbon atoms.

[0062] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, and in some embodiments, 1 to 6 carbon atoms. "Cx-y alkyl" refers to an alkyl group having x to y carbon atoms. This term includes (examples) linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl (CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0063] "Aryl" refers to an aromatic group having 6 to 14 carbon atoms, no ring heteroatoms, and having a monocyclic (e.g., phenyl) or multiple fused (condensed) rings (e.g., naphthyl or anthryl). For polycyclic systems, including fused ring systems, bridging ring systems, and spirocyclic systems, that have aromatic and non-aromatic rings without ring heteroatoms, "aryl" or "Ar" is applied if the bond site is on an aromatic carbon atom (for example, 5,6,7,8-tetrahydronaphthalene-2-yl is an aryl group with the bond site at position 2 of an aromatic phenyl ring). "Arylene" refers to a divalent aryl group with an appropriate hydrogen content.

[0064] "Cycloalkyl" refers to a saturated or partially saturated cyclic group having 3 to 14 carbon atoms, lacking a ring heteroatom, and possessing polycyclic systems including monocyclic or fused ring systems, bridging ring systems, and spirocyclic systems. For polycyclic systems having aromatic and non-aromatic rings without a ring heteroatom, "cycloalkyl" applies when the bond site is located on a non-aromatic carbon atom (e.g., 5,6,7,8,-tetrahydronaphthalene-5-yl). "Cycloalkyl" includes cycloalkenyl groups. Examples of cycloalkyl groups include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl. "Cycloalkylene" refers to a divalent cycloalkyl group having an appropriate hydrogen content.

[0065] "Halogen" refers to one or more of the fluoro, chloro, bromo, and iodine compounds.

[0066] "Heteroaryl" refers to 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 (e.g., imidazolyl-2-yl and imidazole-5-yl) and polycyclic (e.g., imidazopyridyl, benzotriazolyl, benzimidazole-2-yl and benzimidazole-6-yl). For polycyclic systems including fused ring systems, bridging ring systems, and spirocyclic systems having aromatic and non-aromatic rings, at least one ring heteroatom is present and the bond site is on an atom of the aromatic ring (e.g.,1 The terms "heteroaryl" are applied to ,2,3,4-tetrahydroquinoline-6-yl and 5,6,7,8-tetrahydroquinoline-3-yl). In some embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group may be oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. Examples of heteroaryls include acridinyl, azosinyl, benzimidazolyl, benzofuranil, benzothiofuranil, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzothienyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carborinyl, chromanil, chromenil, sinnolinil, dithiadinyl, furanil, furazanil, imidazolidinyl, imidazolinil, imidazopyridyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolidinyl, indolyl, isobenzofuranil, isochromanil, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthilidinyl, octahydroisoquinolinyl, oxadiazo Examples include, but are not limited to, lyl, oxazolidinyl, oxazolyl, pyrimidinyl, phenanthidinyl, phenanthinyl, phenanthinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridadinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, triazinyl, and xanthenyl. "Heteroarylene" refers to a divalent heteroaryl group having an appropriate hydrogen content.

[0067] "Heterocyclic," "heterocyclic," "heterocycloalkyl," or "heterocyclyl" refers to a saturated or partially saturated cyclic group having 1 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, and includes monocyclic and polycyclic systems, including fused, bridging, and spirocyclic systems. For multiple ring systems having aromatic and / or non-aromatic rings, "heterocyclic," "heterocycloalkyl," or "heterocyclyl" applies when at least one ring heteroatom is present and the bond site is on an atom of the non-aromatic ring (e.g., 1,2,3,4-tetrahydroquinoline-3-yl, 5,6,7,8-tetrahydroquinoline-6-yl, and decahydroquinoline-6-yl). In some embodiments, the heterocyclic groups herein are 3- to 15-membered, 4- to 14-membered, 5- to 13-membered, 7- to 12-membered, or 5- to 7-membered heterocyclic groups. There are also embodiments in which the heterocycle contains four heteroatoms. There are also embodiments in which the heterocycle contains three heteroatoms. Furthermore, there are embodiments in which the heterocycle contains up to two heteroatoms. In some embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group may be oxidized to provide N-oxides, sulfumyls, and sulfonyl moieties. Examples of heterocyclyls include, but are not limited to, tetrahydropyranyl, piperidinyl, N-methylpiperidine-3-yl, piperazinyl, N-methylpyrrolidine-3-yl, 3-pyrrolidinyl, 2-pyrrolidone-1-yl, morpholinyl, and pyrrolidinyl. A prefix indicating the number of carbon atoms (e.g., C) 3-10 ) refers to the total number of carbon atoms in a portion of a heterocyclyl group, excluding the number of heteroatoms. Divalent heterocyclic groups have appropriately balanced hydrogen content.

[0068] "Biaryl" refers to a structure in which two aromatic rings are linked by a single CC bond, such as biphenyl and bipyridine.

[0069] "May be substituted" refers to a substituted or unsubstituted group. This group may be substituted with one or more substituents, for example, 1, 2, 3, 4, or 5 substituents. Preferably, the substituents are oxo, halogen, -CN, NO2, -N2+, -CO2R 100 , -OR 100 , -SR 100 -SOR 100 , -SO2R 100 , -NR 100 SO2R 100 , -NR 101 R 102 ,-CONR 101 R 102 -SO2NR 101 R 102 , C1-C6 alkyl, C1-C6 alkoxy, -CR 100 =C(R 100 )2, -CCR 100 , C3-C 10 Cycloalkyl, C3-C 10 Heterocyclyl, C6-C 12 Aryl and C2-C 12 A heteroaryl compound or a divalent substituent thereof (e.g., -O-(CH2)-O-, -O-(CH2)2-O-, and 1-4 methyl substituted compounds) is selected, R 100 , R 101 and R 102 Each of these is independently hydrogen or C1-C8 alkyl, C3-C 12 Cycloalkyl, C3-C 10 Heterocyclyl, C6-C 12 Aryl, or C2-C 12 Heteroaryl, or R 101 and R 102These atoms, together with the nitrogen atoms bonded to them, form a 5-7 membered heterocycle, and each alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl atom may be substituted with 1-3 halogens, 1-3 C1-C6 alkyls, 1-3 C1-C6 haloalkyls, or 1-3 C1-C6 alkoxy groups. Preferably, the substituents are selected from the group consisting of chloro, fluoro, -OCH3, methyl, ethyl, isopropyl, cyclopropyl, -CO2H and their salts, and C1-C6 alkyl esters, CONMe2, CONHMe, CONH2, -SO2Me, -SO2NH2, -SO2NMe2, -SO2NHMe, -NHSO2Me, -NHSO2CF3, -NHSO2CH2Cl, -NH2, -OCF3, -CF3, and -OCHF2.

[0070] Specifically, the compound of formula (5) is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0071] The specific definitions and meanings of the groups, as well as the methods for preparing the compounds and spectral data, are disclosed in PCT / US2016 / 021581 (International Publication No. 2016 / 145092) (corresponding to Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556)), PCT / US2020 / 120281 (International Publication No. 2021 / 068952), and PCT / CN2020 / 089692 (International Publication No. 2020 / 0228686), which are incorporated herein by reference in their entirety.

[0072] It is clear that the compound of structural formula 6, like AST-3424, is a prodrug of AST-2660, and can be activated by the AKR1C3 enzyme to form AST-2660, thereby exhibiting its anticancer effects. [ka]

[0073] The hypoxia-activated DNA alkylating agent prodrug is selected from the group consisting of compounds having the structures shown in formulas 6 to 12. [ka] In the formula, the definitions of R1, R2, R3, and Cx are as set forth in the claims of PCT / CN2020 / 114519 (International Publication No. 2021 / 120717).

[0074] Specifically, the base is defined as follows: Cx is a 5-10 membered aromatic ring or aromatic heterocycle, alicyclic heterocycle, or cycloalkane that shares two carbon atoms with a nitrobenzene ring to form a fused cyclic structure. R1, which is bonded to any skeletal atom of the Cx ring, can be hydrogen, halogen, cyano or isocyan, hydroxyl, mercapto, amine, or OT. s, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, alkoxy having 1-6 carbon atoms or Z-substituted alkoxy having 1-6 carbon atoms, -CONR 6 R 7 -SO2NR 6 R 7 , -SO2R 6 -OCOO-R 6 ,-COOR 6 , -NR 6 COR 7 , -OCOR 6 , -NR 6 SO2R 7 , and -NR 6 SO2NR 6 R 7 Selected from, R2 and R3 are, independently, hydrogen, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 These are aryl or Z-substituted aryls, 4-15 membered heterocycles or Z-substituted heterocycles, 5-15 membered heteroaryls or Z-substituted heteroaryls, or R2 and R3 which are attached to the carbon atoms of benzyl to form a 3-6 membered ring. [ka] The group can substitute a hydrogen atom at any position on the carbon atom of the fused ring, and the number of substitutions is 1. The Z-substituents are halogen atoms, cyano or isocyano groups, hydroxyl groups, mercapto groups, amino groups, C1-C3 alkyl groups or substituted alkyl groups, C1-C3 alkoxy groups or substituted alkoxy groups, C2-C3 alkenyl groups or substituted alkenyl groups, C2-C3 alkynyl groups or substituted alkynyl groups, C3-C8 cycloalkyl groups or substituted cycloalkyl groups. R 6 and R 7 These are, independently, hydrogen, C1-C6 alkyl or Z-substituted C1-C6 alkyl, C2-C6 alkenyl or Z-substituted C2-C6 alkenyl, C2-C6 alkynyl or Z-substituted C2-C6 alkynyl, C3-C8 cycloalkyl or Z-substituted C3-C8 cycloalkyl, and C6-C 10 Aryl or Z-substituted C6-C 10 R with atoms that combine with aryls, 4-15 member heterocyclils or Z-substituted 4-15 member heterocyclils, 5-15 member heteroaryls or Z-substituted 5-15 member heteroaryls, or atoms that combine with them to form 5-7 member heterocyclils or Z-substituted 5-7 member heterocyclils 6 and R 7 That is the case.

[0075] The specific definitions and meanings of the groups, as well as the preparation methods and spectral data of the compounds, are described in PCT / CN2020 / 114519 (International Publication No. 2021 / 120717), which is incorporated herein by reference in its entirety.

[0076] Specifically, the compound of formula (6) [ka] [ka] [ka] Selected from the group consisting of, In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R12 , R 13 , R 14 , R 15 , R 16 , and R 17 The definition is as stated in the claims of PCT / US2016 / 039092 (corresponding to International Publication No. 2016 / 210175 (Chinese Patent Application No. 2016800368985 (Chinese Patent Application Publication No. 108024974))).

[0077] Specifically, the base is defined as follows: R1 is hydrogen, -N3, CN, halogen, NR 21 R 22 , -OR 23 -SO2(C1-C6 alkyl), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 They are aryl, 4-15 membered heterocyclic rings, 5-15 membered heteroaryl rings, or ethers. R 21 and R 22 These are, independently, hydrogen, hydroxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C6-C 10 R is an aryl, a 4-15 membered heterocycle, a 5-15 membered heteroaryl, or -SO2(C1-C6 alkyl), or a nitrogen atom that is bonded to them to form a 4-15 membered heterocycle or a 5-15 membered heteroaryl. 21 and R 22 And, R 23 This is hydrogen, C1-C6 alkyl, or C6-C 10 It is Ariel, R2 and R3 are independently hydrogen or halogen. R4 is hydrogen, halogen, C1-C6 alkoxy, C1-C6 alkyl, or C6-C 10 It is Ariel, R5, R7, R9, R 12 and R 15These are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C6-C 10 These are aryl groups, 4-15 membered heterocyclic groups, 5-15 membered heteroaryl groups, or R4 and R5 groups interposed between them with carbon atoms forming a C5-C6 cycloalkyl ring. R6 and R 10 These are independently hydrogen or halogen, R8 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or 5-15 member heteroaryl. Each R 11 These are independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, or C6-C 10 It is Ariel, R 13 , R 14 , R 16 , and R 17 These are independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C6 alkoxy. The alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aryl group, heterocyclic group, heteroaryl group, alkoxy group, and ether group may be substituted.

[0078] The specific definitions and meanings of the groups, as well as the methods for preparing the compounds and spectral data, are described in PCT / US2016 / 039092 (International Publication No. 2016 / 210175) (corresponding to Chinese Patent Application No. 2016800368985 (Chinese Patent Application Publication No. 108024974)), which are incorporated herein by reference in their entirety.

[0079] In particular, the compounds of formulas (7) to (12) are selected from the compounds specifically disclosed in the aforementioned patent application.

[0080] It is clear that the “compound” in the above chemical formulas 1 to 12 disclosed herein includes the compound itself, as well as its solvates, salts, esters, or isotopic isomers.

[0081] Furthermore, the present invention relates to a method for measuring the content of metabolites in a biological sample, A step of preparing a test solution containing an internal standard compound of known concentration for injection analysis by LC-MS / MS, A step of preparing a series of standard working solutions containing an internal standard compound of known concentration and a metabolite II of known concentration, wherein the concentration of the internal standard compound in the series of standard working solutions is consistent and the same as the concentration of the internal standard compound in the test solution, and the concentrations of the metabolite II in the series of standard working solutions are different, The process involves absorbing standard working solutions of metabolite II at different concentrations, injecting them into a detection LC-MS / MS system to obtain the relation function y=f(x) (where y represents the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, and x represents the concentration of metabolite II in the standard working solution), and determining the relation function by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The process involves absorbing the test solution to which the internal standard compound of known concentration has been added, injecting it into the detection LC-MS / MS system to determine the ratio y of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, substituting this ratio into the function y=f(x) to determine the concentration x of metabolite II in the test solution, and using this relational function to determine and calculate the concentration of metabolite II in the test solution of unknown concentration. Includes, The aforementioned test solution is prepared from a biological sample, whether or not it has been treated. The aforementioned internal standard compound is a deuterated compound I having the structure shown in formula (I), [ka] In the formula, A is either H or D, and at least one of the eight A's is D. M is H or an alkali metal, alkaline earth metal, or ammonium radical. The aforementioned metabolite II has the structure shown in formula (II), [ka] In the equation, A is H, M is H, or an alkali metal, alkaline earth metal, or ammonium radical.

[0082] Furthermore, the present invention is a method for measuring the content of metabolites in a biological sample, The process involves adding a quantitative internal standard compound to the biological sample solution and performing an extraction to obtain the test solution, The process involves diluting a metabolite II standard substance to obtain a series of metabolite II solutions of different concentrations, adding the quantitative internal standard compound and performing an extraction to obtain standard working solutions, absorbing each of the series of standard working solutions and injecting them into a detection LC-MS / MS system to determine the peak areas of the metabolite II and the internal standard compound, creating a standard curve with the ratio of the peak area of ​​the metabolite II to the peak area of ​​the internal standard compound on the vertical axis and the concentration of the metabolite II on the horizontal axis, and calculating a regression equation. The steps include: absorbing the test solution, injecting it into the detection LC-MS / MS system to determine the ratio of the peak area of ​​metabolite II to the peak area of ​​deuterated compound I in the test solution, and substituting this into the regression equation to determine the content of metabolite II in the test solution; Includes, The aforementioned internal standard is a deuterated compound I having the structure shown in formula (I), [ka] In the formula, A is either H or D, and at least one of the eight A's is D. M is H or an alkali metal, alkaline earth metal, or ammonium radical. The aforementioned metabolite II has the structure shown in formula (II), [ka] In the equation, A is H, M is H, or an alkali metal, alkaline earth metal, or ammonium radical.

[0083] Preferably, the conditions for liquid chromatography in the liquid chromatography-tandem mass spectrometry are as follows: Using a hydrogen-accepting stationary-phase chromatography column, Mobile phase A is a methanol solution of ammonium acetate, and mobile phase B is acetonitrile. For gradient elution, mobile phases A and B are used, with mobile phase A gradually increased from 15% to 90% by volume, and then gradually decreased back down to 15% by volume. Mass spectrometry conditions: Electrospray ion source In negative ion scanning mode, Monitoring ion pair of metabolite II: m / z 147.0 → m / z 62.9 Monitoring ion pair of deuterated compound I: m / z (147.0 + deuterated number) → m / z 62.9, or, In positive ion scanning mode, Monitoring ion pair of metabolite II: m / z 149.0 → m / z 64.9 The monitoring ion pair of deuterated compound I is m / z (149.0 + deuterated number) → m / z 64.9.

[0084] When using the positive ion scanning mode, it is recommended to add an acid (such as formic acid) to the corresponding mobile phase.

[0085] Preferably, in the preparation of a test solution containing a known concentration of deuterated compound I (internal standard compound), the deuterated compound I is first added to the biological sample solution, and then an extraction operation is performed. Correspondingly, in the preparation of the standard working solution, the deuterated compound I is first added to a matrix solution containing different known concentrations of metabolite II, and then an extraction operation is performed.

[0086] Preferably, the biological sample is a urine sample or a plasma sample. Correspondingly, when the urine sample is measured, the corresponding matrix solution is urine from a patient who has not received an additive. When the blood sample is measured, the corresponding matrix solution is plasma from a patient who has not received an anticoagulant.

[0087] The operation steps of adding and extracting the deuterated compound are as follows: adding the test solution and the standard working solution to the solution of the deuterated compound I, adding methanol and mixing uniformly, and obtaining the supernatant by centrifugation.

[0088] The additive is Na2HPO4 or K2HPO4, and the anticoagulant is K2EDTA or Na2EDTA.

[0089] The blood sample needs to be stored at -20°C or below, preferably at -70°C within 4 hours after collection. It can be stored at -70°C for 28 days. When processed, it should be refrigerated at 4°C or below and the injection detection should be completed within 54 hours.

[0090] The urine sample needs to be stored at -70°C within 24 hours, preferably within 4 hours after collection. It can be stored at -70°C for 32 days. When processed, the injection detection should be completed within 94 hours at room temperature or below.

Brief Description of the Drawings

[0091] [Figure 1] Figure 1 is the 31P-NMR spectrum of AST-2660-D8-sodium salt. [Figure 2] Figure 2 is the 31P-NMR spectrum of the first part of the aqueous solution of AST-2660-D8-sodium salt scanned 64, 128, 256, and 512 times, respectively, as shown in a, b, c, and d. [Figure 3]Figure 3 shows the 31P-NMR spectra at time 0, for the AST-2660-D8 sodium salt solution at -20°C (a) and the AST-2660-D8 sodium salt solution at -78°C (b). [Figure 4] Figure 4 shows the 31P-NMR spectra after 48 hours, for the AST-2660-D8 sodium salt solution at -20°C (a) and the AST-2660-D8 sodium salt solution at -78°C (b). [Figure 5] Figure 5 shows the 31P-NMR spectrum of the AST-2660-D8 sodium salt solution after storage at -20°C for 6 months. [Figure 6] Figure 6 shows the 31P-NMR spectrum of the AST-2660-D8 sodium salt solution after storage at -78°C for 6 months. [Figure 7] Figure 7 shows representative LC-MS / MS spectra of AST-2660(a) and the internal standard AST-2660-D8(b) in blank human plasma extracts. [Figure 8] Figure 8 shows representative LC-MS / MS spectra of AST-2660(a) and the internal standard AST-2660-D8(b) in plasma sample extracts at zero concentration. [Figure 9] Figure 9 shows representative LC-MS / MS spectra of AST-2660(a) and the internal standard AST-2660-D8(b) in a standard sample solution (0.50 ng / ml) of human plasma extract. [Figure 10] Figure 10 shows representative LC-MS / MS spectra of AST-2660(a) and the internal standard AST-2660-D8(b) in a standard sample solution (200 ng / ml) of human plasma extract. [Figure 11] Figure 11 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in blank human urine extract. [Figure 12] Figure 12 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a urine sample extract at zero concentration. [Figure 13]Figure 13 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a standard working solution (0.50 ng / ml) of human urine extract. [Figure 14] Figure 14 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a standard working solution (200 ng / ml) of human urine extract. In the graphs from Figures 8 to 14, the horizontal axis represents time, and the vertical axis represents the mass spectral ion intensity of the LC-MS / MS, with the mass spectral ions being selected ions. [Modes for carrying out the invention]

[0092] Unless otherwise defined, all technical or scientific terms used in one or more embodiments of this specification have meanings that are generally understood by those skilled in the art to which the invention pertains.

[0093] Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, all pharmaceutical raw materials, reagent materials, etc. used in the following examples are commercially available products.

[0094] AST-3424, developed by the present applicant, is a prodrug of the DNA alkylating agent AST-2660. It is highly expressed in cancer cells and is specifically activated by the AKR1C3 enzyme, which metabolizes AST-2660, thereby exerting its therapeutic effect. As described in the background art, human clinical trials require measuring the content of the drug and its metabolites in subject biological samples (blood, urine, tissue, etc.) and conducting further drug metabolism and pharmacokinetic (DMPK) studies based on the measured drug and metabolite content. However, AST-3424 is used in relatively small amounts, from 1 mg to 100 mg, in clinical trials. Conventional internal or external standard methods used in liquid chromatography cannot meet the requirement of quantitative analysis at the limit of quantification. Therefore, there is a need to develop an assay method that can meet the requirement of the limit of quantification.

[0095] To address the technical challenges described above, the applicant is attempting to quantify the low content of AST-2660 in biological samples using an internal standard quantification method. The internal standard method is a fairly accurate quantification method in chromatographic analysis. The internal standard method involves adding a fixed amount of a pure substance to a fixed amount of analyte mixture, analyzing the sample containing the internal standard by chromatography, measuring the peak area of ​​the internal standard and the component being measured, and calculating the percentage of the component being measured in the sample. The selection of the internal standard is crucial. Ideally, the internal standard should be a known compound that can be obtained in a pure form so that it can be added to the sample in an accurate and known amount. Furthermore, the internal standard should have substantially the same or as close as possible chemical and physical properties (chemical structure, polarity, volatility and solubility in solvent, etc.), chromatographic behavior, and response characteristics as those of the analyte, preferably its homolog. Indeed, the internal standard must be well separated from each component in the sample under chromatographic conditions. The selection of the internal standard is crucial for quantitative analysis using the internal standard method. The following requirements must be met: 1. The internal standard shall have similar physical and chemical properties (such as boiling point, polarity, and chemical structure) to those of the analyte. 2. The internal standard is a pure substance that is not present in the test sample. 3. The internal standard is completely soluble in the test sample (or solvent) and does not chemically react with the test sample. Furthermore, the peak of the internal standard can be completely separated from the peaks of each component in the test sample. 4. The amount of internal standard that can be added is close to the amount of the measured component that can be added. 5. The position of the internal standard's chromatographic peak is close to the position of the chromatographic peak of the measured component, or midway between the positions of several measured component chromatographic peaks without co-overflow. The purpose is to avoid differences in sensitivity caused by instrument instability. 6. The appropriate amount of the internal standard to be added is selected such that the peak area matching between the internal standard and the analyte is greater than 75%, thereby avoiding sensitivity deviation due to their being in different response value regions.

[0096] In this application, a large number of similar compounds of AST-2660 were screened, and the deuterated compound I was finally screened as the internal standard for the detection of metabolite II. The detection limit of the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established using the deuterated compound I is as low as 0.5 ng / ml. Furthermore, using the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established with the deuterated compound I according to this application, the content of metabolite AST-2660 in human plasma and urine can be measured. This method meets the analysis requirements of biological samples in that the sample treatment method is simple and convenient, and it has high sensitivity, precision, and accuracy.

Example

[0097] Hereinafter, specific examples will be described for the technical solutions of the present invention. The following examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0098]

Table A

[0099] [Example 1] Synthesis of AST-2660-D8-sodium salt

Chemical formula

[0100] Deuterated 2-bromoethylamine hydrobromide (360 mg, 1.72 mmol) and phosphorus oxychloride (132 mg, 0.86 mmol) were added to anhydrous dichloromethane (4 ml) under nitrogen protection (deuterated 2-bromoethylamine hydrobromide was added first, followed by phosphorus oxychloride), the temperature was lowered to -78°C, and triethylamine (348 mg, 3.44 mmol) from a dichloromethane solution (2 ml) was added dropwise. The reaction mixture was held at -78°C for 30 minutes, allowed to rise naturally to 0°C, and then held at 0°C for 4 hours. The solid was then rapidly filtered by suction. The mother liquor was concentrated at low temperature and used directly in the next step.

[0101] The crude product obtained in the above process was dissolved in water (30.8 ml), and solid sodium hydroxide (275 mg, 6.88 mmol) was slowly added little by little. The reaction mixture was stirred at room temperature overnight. The mixture was then stored at -20°C. 31 The P-NMR properties are shown in Figure 1. The nuclear magnetic peak of HMPA as an internal standard is 29.886 ppm, and the nuclear magnetic peak of AST-2660-D8-sodium salt is 24.503 ppm.

[0102] Figure 1 shows the AST-2660-D8 sodium salt. 31 This is a P-NMR spectrum.

[0103] It is clear that by selecting different deuterated starting compound Ia (deuterated 2-bromoethylamine) in the above operation, different intermediates Ib can be synthesized, and then different deuterated bis(aziridine-1-yl)phosphinic acids or their salts can be obtained by adding different bases (NaOH, KOH, LiOH, or aqueous ammonia) in the hydrolysis reaction.

[0104] [ka]

[0105] The above reaction from compound Ia to compound Ib was completed in one step. In fact, it can also be completed in separate (two) steps, by changing the order of supply and the amounts of reactants. It is clear that such an operation is equivalent to the operation in which the above reaction from compound Ia to compound Ib was completed in one step. Under nitrogen protection, deuterated 2-bromoethylamine hydrobromide and phosphorus oxychloride were added to anhydrous dichloromethane (phosphorus oxychloride was added first, followed by the first deuterated 2-bromoethylamine hydrobromide, in which the molar ratio of phosphorus oxychloride to the deuterated 2-bromoethylamine hydrobromide was greater than 1), the temperature was reduced to -78°C, and triethylamine in a solution of dichloromethane was added dropwise. After holding the reaction mixture at -78°C for 30 minutes, a second deuterated 2-bromoethylamine hydrobromide was added. Triethylamine in a solution of dichloromethane was added dropwise. The reaction mixture was held at -78°C for 30 minutes, then allowed to rise naturally to 0°C, and finally held at 0°C for 4 hours. The solid was then rapidly filtered by suction. The mother liquor was concentrated at a low temperature and used directly in the next step.

[0106] The crude product obtained in the above process was dissolved in water, and solid sodium hydroxide was slowly added little by little. The reaction mixture was stirred overnight at room temperature. The reaction mixture was then stored at -20°C, and the reaction proceeded as follows. [ka] Different deuterated compounds I and their salts can be prepared by selecting first and second deuterated 2-bromoethylamine hydrobromides having different deuteration positions and deuteration numbers.

[0107] The quantitative detection method will be specifically described below using the AST-2660-D8-sodium salt prepared in Example 1 as an example.

[0108] [Example 2] Detection method for AST-2660-D8 sodium salt In this example, HMPA (hexamethyl phosphate triamide) was used as the internal standard for the phosphorus spectrum to measure the content of the AST-2660-D8-sodium salt prepared in Example 1.

[0109] HMPA (33.0 mg, 0.1840 mmol) was dissolved in water (2.2 ml). The total mass was 2612 mg, and the concentration of HMPA was 7.050 × 10⁻⁶. -5 (mmol / mg, 31 The nuclear magnetic shift in the P-NMR spectrum was 29.890 ppm.

[0110] The phosphorus spectrum was measured using an aqueous HMPA solution as an internal standard, and the content of AST-2660-D8-sodium salt was determined.

[0111] 1. Detection of the first part of the sample Approximately 0.5 ml (488 mg) of the AST-2660-D8-sodium salt aqueous solution prepared in Example 1 and an HMPA aqueous solution (317 mg) were precisely weighed and mixed, and the phosphorus spectrum was measured by scanning 64 times (see Figure 2). In the nuclear magnetic spectrum, the integral peak area ratio of the corresponding nuclear magnetic peak of HMPA (29.874 ppm) and the nuclear magnetic peak of AST-2660-D8-sodium salt (24.491 ppm) is 1:0.257.

[0112] Figure 2 shows the first part of the AST-2660-D8-sodium salt aqueous solution. 31 These are P-NMR spectra, scanned 64, 128, 256, and 512 times from top to bottom and left to right, respectively.

[0113] Since the amount of HMPA was known to be 0.02235 mmol, the amount of AST-2660-D8-sodium salt was calculated from the integral peak area ratio in the nuclear magnetic spectrum to be 0.005743 mmol, and the concentration was 1.177 × 10⁻⁶. -5 mmol / mg, i.e., 1.177 × 10⁻⁶. -2The concentration was mmol / g, and the corresponding mass content was 2.10 mg / g. Similarly, the phosphorus spectra after 128, 256, and 512 scans are shown in Figure 2. The integrated peak area ratios were calculated to be 1:0.264, 1:0.268, and 1:0.264, respectively. Therefore, the concentrations of the same sample were 1.209 × 10⁻¹⁶, respectively. -5 mmol / mg, 1.227 × 10⁻⁶ -5 mmol / mg, and 1.209 × 10⁻⁶ -5 The values ​​were mmol / mg, specifically 2.15 mg / g, 2.18 mg / g, and 2.15 mg / g, with the average of the four results being 2.1 mg / g.

[0114] The nuclear magnetic quantification method in this example is relatively rapid and simple, possesses acceptable accuracy within a certain range, and can be replaced by HPLC yield analysis. If further improvement in quantitative accuracy is required, the HPLC method should be used. An external standard method is used for quantification, and the absolute content is accurately determined using a standard curve.

[0115] The LC-MS / MS methods of Examples 5 and 7 are suitable for detecting low content of AST-2660-D8 and AST-2660, and their corresponding LC liquid-phase methods (where the corresponding MS / MS detector is replaced by a conventional differential detector, electrospray detector, or evaporative light scattering detector) are also suitable for detecting constant content (mg / ml) of AST-2660 and AST-2660-D8.

[0116] [Example 3] Stability test of AST-2660-D8 sodium salt In this example, the stability of the sample solution was investigated by detecting the content of the aqueous solution of AST-2660-D8-sodium salt. 31 Using P-NMR, the content of the aqueous solution of AST-2660-D8-sodium salt was determined to be that of HMPA as an internal standard. 31 Area ratio of P peak and AST-2660-D8 sodium salt 31The P-peak area ratio is characterized by a comparison within 48 hours to determine whether the mass of the sample solution decreased due to decomposition.

[0117] 1.Analysis method Sample 31 P-NMR was performed using an NMR instrument. HMPA (21.8 mg, 0.1217 mmol) was dissolved in water (2.20 ml). The total mass was 2127 mg, and the concentration of HMPA was 5.72 × 10⁻⁶. -5 The concentration was mmol / mg. This was obtained from a solution of AST-2660-D8-sodium salt (588 mg, approximately 0.5 ml) and a solution of HMPA (293 mg, 1.676 × 10⁻⁶ ml). -2 mmol) was stored at -20°C for different periods of time. 31 The samples were mixed together for P-NMR analysis. The ratio of the integrated peak area of ​​the corresponding nuclear magnetic peak of HMPA (approximately 29.874 ppm) in the nuclear magnetic spectrum to the integrated peak area of ​​the nuclear magnetic peak of AST-2660-D8-sodium salt (approximately 24.491 ppm) was calculated, and the integrated peak of the nuclear magnetic peak of HMPA was set to 1.

[0118] Using a similar procedure, samples of different concentrations were prepared for detection and stored at -78°C for different periods of time. 31 P-NMR analysis was performed.

[0119] 2. Stability results when stored at -20°C to -78°C for 48 hours. The samples were stored at -20°C and -78°C for 48 hours, and detected and recorded at 0, 2, 4, 6, 8, 16, 24, 36, and 48 hours, respectively. The results are shown in Table 1 below.

[0120] [Table 1]

[0121] Under -20°C storage conditions, HMPA levels at 8, 16, 24, 36, and 48 hours were... 31 P-NMR peak area and AST-2660-D8-sodium salt solution31 The ratio of peak areas of the P-NMR peaks was stable at 1:0.43 to 1:0.47. Under storage conditions at -78°C, it was stable in the range of 1:0.21 to 1:0.24. Therefore, it can be concluded that the AST-2660-D8 sodium salt solution was stable when stored at -20°C to -78°C for 48 hours.

[0122] Typical spectra are shown in Figures 3 and 4.

[0123] Figure 3 shows the time at 0. 31 These are P-NMR spectra, showing the AST-2660-D8 sodium salt solution at -20°C and the AST-2660-D8 sodium salt solution at -78°C, from left to right.

[0124] Figure 4 shows the results after 48 hours. 31 These are P-NMR spectra, showing the AST-2660-D8 sodium salt solution at -20°C and the AST-2660-D8 sodium salt solution at -78°C, from left to right.

[0125] 3. Stability after storage at -20°C for 6 months The concentration of AST-2660-D8 sodium salt was determined to be 1.89 mg / ml on day 0 and 1.37 mg / ml after storage at -20°C for 6 months using the method described above. The concentration of AST-2660-D8 sodium salt decreased by 27.5% during the 6-month storage period.

[0126] 6 months later 31 The P-NMR spectrum is shown in Figure 6.

[0127] Figure 5 shows the results after storing the AST-2660-D8 sodium salt solution at -20°C for 6 months. 31 This is a P-NMR spectrum.

[0128] 4. Stability of samples stored at -78°C for 6 months. The concentration of AST-2660-D8 sodium salt was measured using the method described above, at 1.89 mg / ml on day 0 and at 1.71 mg / ml after 6 months of storage at -78°C. The concentration of AST-2660-D8 sodium salt decreased by 9.5% during 6 months of storage. In comparison, sample storage at -78°C was more stable and the degradation was slower than storage at -20°C.

[0129] 6 months later 31 The P-NMR spectrum is shown in Figure 7.

[0130] Figure 6 shows the results after storing the AST-2660-D8 sodium salt solution at -78°C for 6 months. 31 This is a P-NMR spectrum.

[0131] From the stability test results above, it can be seen that the sample solution used to detect the content of AST-2660-D8 sodium salt aqueous solution remained stable after being stored at -20°C to -78°C for 2 days. When the AST-2660-D8 sodium salt aqueous solution was stored at -20°C and -78°C for 6 months, its concentration decreased by 27.5% and 9.5%, respectively.

[0132] [Example 4] Establishment and validation of a method for detecting AST-2660 in human plasma. In this example, the metabolite AST-2660 (existing in the form of a sodium salt) containing the K2EDTA anticoagulant was quantitatively detected in human plasma using the deuterated internal standard (IS) AST-2660-D8 (prepared in Example 1). The structural formula of AST-2660 is as follows. [ka]

[0133] The structure of the deuterated compound AST-2660-D8 sodium salt used is shown below. [ka] The following AST-2660s refer to their respective sodium salts, and AST-2660-D8 also refers to its sodium salt.

[0134] 1. LC-MS / MS experimental method The relevant parameters for the LC-MS / MS experimental method are shown.

[0135] [Table 2]

[0136] [Table 3]

[0137] [Table 4]

[0138] 2. Sample preparation 2.1 Preparation of standard working solution AST-2660 (prepared by selecting a non-deuterated raw material as described in Example 1) standard storage solution (methanol solution with a concentration of 1.0 mg / ml): An appropriate amount of AST-2660 was taken, an appropriate amount of methanol was added, diluted, and thoroughly mixed to obtain a 1.0 mg / ml solution.

[0139] 100 μl of AST-2660 standard storage solution was taken, 9900 μl of methanol was added, diluted, and thoroughly mixed to obtain a 10.0 μg / ml calibration standard working solution.

[0140] 2.2 Preparation of the standard curve The standard working solution (SWS) and blank matrix (diluent) were left at room temperature. The standard working solution was vortexed before use, and standard sample solutions of various concentrations for the standard curve were prepared using normal pooled human plasma containing an anticoagulant (K2EDTA) as the diluent and blank matrix, according to the table below.

[0141] [Table 5]

[0142] 2.3 Preparation of internal standard working solution (standard working solution) AST-2660-D8 preservation solution (50 μg / ml AST-2660-D8 methanol solution): An appropriate amount of AST-2660-D8 reference material was taken, an appropriate amount of methanol was added, diluted, and thoroughly mixed to obtain a 50 μg / ml solution. 20 μl of AST-2660-D8 internal standard preservation solution was taken, 9980 μl of methanol was added, diluted, and thoroughly mixed to obtain a 100 ng / ml internal standard working solution.

[0143] 2.4 Preparation of Quality Control Samples AST-2660 quality control storage solution (1.0 mg / ml solution in methanol): An appropriate amount of AST-2660 reference substance was taken, an appropriate amount of methanol was added, diluted, and thoroughly mixed to obtain a 1.0 mg / ml solution. 100 μl of AST-2660 QC storage solution and 9990 μl of methanol were taken to obtain a 10.0 μg / ml quality control working solution.

[0144] Normal pooled human plasma containing an anticoagulant (K2EDTA) was used as a diluent, and quality control samples of each concentration were prepared by diluting it according to the table below.

[0145] [Table 6]

[0146] 3. Sample pretreatment The procedure for extracting a sample using the protein precipitation method is as follows: Labeling and sequencing of standard curve samples, quality control samples, and plasma samples are performed. 2. Place all samples on a multi-tube vortexer to ensure uniform vortexing after thawing. 3. Take 100 μl from each sample and add it to a 96-well plate. 4. Add 20 μl of AST-2660-D8 quality control working solution deuterated internal standard (i.e., 100 ng / ml AST-2660-D8 methanol solution) to the blank sample, and then add 20 μl of methanol to the blank sample. 5. Add 500 μl of methanol. 6. Vortex the plate at medium speed for 3 minutes. Centrifuge the 7.96-well plate at 2143 rcf (centrifugal force) for 10 minutes. 8. Transfer 450 μl of the supernatant to a new 96-well plate. The supernatant is dried in a 9.96-well Ternovap sample concentrator. 10. Redissolve with 150 μl of methanol. 11. Ultrasonize the plate for 30 seconds. 12. Centrifuge the plate at 4°C and 2143 rcf (centrifugal force) for 3 minutes. 13. Transfer 120 μl of the supernatant to a new 96-well plate.

[0147] 4. Experimental Results 4.1 Standard curve The parameters for the AST-2660 standard curve in human plasma were as follows: Slope: 0.063846, Intercept: -0.003788, Correlation coefficient: 0.9939, Relationship function: y=f(x)=0.063846x-0.003788 (where y represents the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, and x represents the concentration of metabolite II in the standard working solution), LLOQ (lower limit of quantification): 0.5 ng / ml, ULOQ (upper limit of quantification): 200 ng / ml. As shown, the linear relationship was good in the linear range of 0.5 to 200 ng / ml.

[0148] 4.2 Accuracy and precision test results of quality control samples Accuracy and precision are defined as follows: Accuracy is expressed as relative error (RE). RE = [(Average value - Nominal value) / Nominal value] × 100 Precision is expressed as the coefficient of variation (CV). CV = (Standard Deviation / Mean) × 100

[0149] The accuracy and precision test results are shown in Table 7. From Table 7, it can be seen that both the intra-assay and inter-assay precision were less than 8.3%, and the intra-assay and inter-assay precision was within ±8.7. The analytical criteria for accuracy and precision were as follows: For LLOQ QC samples, the intra-assay and inter-assay precision (RE) must be within ±20.0%, and the intra-assay and inter-assay CV must not exceed 20.0%. The intra-assay and inter-assay precision (RE) must be within ±15.0% for low, geometric mean, medium, high, and diluted QC (if applicable) samples, and the intra-assay and inter-assay precision (CV) must not exceed 15.0% at each QC concentration level. 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% or less, which met the requirements.

[0150] [Table 7]

[0151] 4.3 Test results of extraction and recovery of AST-2660 and internal standard AST-2660-D8 [Table 8]

[0152] Table 8 shows that the coefficient of variation (CV) of the extraction recovery rates for both AST-2660 and the internal standard AST-2660-D8 was less than 15.0%. The analytical criteria for extraction recovery were as follows: For spiked samples after extraction, the CV of the reaction measured at each concentration level must be 15.0% or less. Therefore, the extraction recovery rates of AST-2660 and the internal standard AST-2660-D8 met the requirement.

[0153] 4.4 Test Results of Matrix Effects [Table 9]

[0154] As shown in the results in Table 9, the inter-assay matrix accuracy and precision (CV) of AST-2660 matrix effects were both less than 15%, which indicates that the requirements were met.

[0155] 4.5 Stability [Table 10]

[0156] Table 10 shows that the accuracy of the stability of the AST-2660 working solution and the internal standard AST-2660-D8 working solution was within ±8.1%, and the precision was less than 5.5%. The stability analysis criteria were as follows: The difference between the stored stable solution and the newly prepared solution must be within ±10.0%, and the CV of each replicated experiment must not exceed 10.0%. Therefore, both the AST-2660 and AST-2660-D8 (internal standard) working solutions exhibited good stability and met the requirements under various storage conditions.

[0157] To detect AST-2660 in human plasma, the liquid chromatography-tandem mass spectrometry method established in this example met the analytical requirements for biological samples. The sample preparation method was simple and convenient, and the method exhibited high sensitivity, high accuracy, and high precision.

[0158] [Example 5] Detection of AST-2660 content in actual human plasma The AST-2660 content in the test human plasma was detected using the detection method established in Example 4.

[0159] The standard operating procedure (SOP) for detection established after the methodology and verification in Example 4 was as follows: Step 1. Preparation of the solution Matrix: Mixed normal human plasma containing an anticoagulant (K2EDTA) a: Internal standard working solution: AST-2660-D8 was taken, methanol was added, and it was diluted to prepare a solution containing approximately 100 ng per 1 ml. b: Test solution (at this point, no internal standard compound has been added to the test solution): Obtained by collecting a test blood sample. c: Standard working solution (at this point, no internal standard compound has been added to the standard working solution): An appropriate amount of AST-2660 was taken, accurately weighed, dissolved in methanol, and diluted to prepare a solution containing approximately 1.0 mg per 1 ml, which was used as the standard working storage solution. An appropriate amount of the standard working storage solution was taken, added together with the matrix, and diluted to prepare standard working solutions of different concentrations containing approximately 200 ng, 160 ng, 100 ng, 50 ng, 10 ng, 2.0 ng, 1.0 ng, and 0.5 ng per 1 ml, respectively. (The solutions were stored at -70°C) d: Quality control sample solution: An appropriate amount of AST-2660 was taken, accurately weighed, dissolved in methanol, and diluted to prepare a solution containing approximately 10 μg per 1 ml, which was used as the quality control work storage solution. An appropriate amount of the quality control work storage solution was taken and added together with the matrix to prepare quality control work solutions of different concentrations containing approximately 150 ng, 60 ng, 15 ng, 1.5 ng, and 0.5 ng per 1 ml, respectively. (The solutions were stored at -70°C.)

[0160] Step 2. Sample Extraction The test solution, standard working solution, and quality control sample solution were thawed at room temperature and mixed uniformly. 100 μl of each solution was taken, 20 μl of internal standard and 500 μl of methanol were added, and the mixture was vortexed for 3 minutes to ensure uniformity. The mixture was then centrifuged at 2143 rcf for 10 minutes. 450 μl of the supernatant was taken, dried, and 150 μl of methanol was added for redissolution. The mixture was then sonicated. The plate was then centrifuged at 4°C for 3 minutes. The supernatant was taken to obtain the final product.

[0161] A blank sample was obtained by collecting 100 μl of blank blood, adding 20 μl of methanol, and extracting it using the same method.

[0162] A 100 μl blank blood sample was collected, 20 μl of internal standard working solution was added, and a zero-concentration sample was obtained by extraction using the same method.

[0163] After sample extraction and addition of internal standards, in step 4, the solution was injected into an LC-MS / MS for detection.

[0164] Step 3. Setting the chromatography-mass spectrometry test conditions. Measurements were performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the following instrument test conditions were set. The liquid phase conditions were as follows: A SHARC1, 3 μm, 2.1 × 50 mm or equivalent chromatographic column was used. The mobile phase was a methanol solution containing a low amount of ammonium acetate, and mobile phase B was acetonitrile. Gradient elution was performed according to the table below, at a column temperature of 20°C and an injection volume of 5 μl.

[0165] [Table 11]

[0166] The mass spectrometry conditions were as follows: Negative ion scanning mode electrospray ion source, spray potential: -4500V, ion source temperature: 500℃, collision energy (CE): -22eV, declustering potential (DP): -55V, inlet potential (EP): -10V, collision chamber outlet potential (CXP): -16V, residence time: 100ms, high-purity nitrogen used for all gases, AST-2660 ion pair: m / z 147.0 → m / z 62.9, internal standard AST-2660-D8 ion pair: m / z 155.0 → m / z 62.9.

[0167] Step 4. Judgment AST-2660 standard working solution, blank sample, zero-concentration sample, test sample solution, and quality control sample solutions with different concentrations after extraction were each collected and injected into LC-MS / MS for detection.

[0168] The criteria for evaluation were as follows: The recovery concentration of the standard working solution, the accuracy of the quality control working solution, the peak residue of AST-2660, and the internal standard in the blank sample all meet the requirements of the Chinese Pharmacopoeia (2020 edition), Volume 4, 9012.

[0169] Standard working solutions of metabolite II at different concentrations were absorbed and injected into an LC-MS / MS system for detection, and the relation function y=f(x) was obtained (wherein y represents the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, and x represents the concentration of metabolite II in the standard working solution).

[0170] The calculation results were as follows: The relational function y=f(x) for the ratio of the peak area of ​​AST-2660 to the peak area of ​​the internal standard was obtained from AST-2660 standard working solutions of different concentrations. The ratio of the peak area of ​​AST-2660 to the peak area of ​​the internal standard was determined from the extracted test solution, and this was substituted into the relational function y=f(x) to calculate the concentration of metabolite II in the test solution.

[0171] The concentration of metabolite II in a test solution of unknown concentration was measured and calculated using a relational function. The test solution, to which an internal standard compound of known content was added, was absorbed and injected into an LC-MS / MS system for detection. The ratio y of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound was determined, and substituted into the function y=f(x) to find the concentration x of metabolite II in the test solution.

[0172] As an option, the standard operating procedure for the following operations was obtained by slightly modifying the order of the related operating steps in the standard operating procedure described above. A quantitative internal standard compound is added to the test biological sample solution, and after extraction, it is used as the test solution. By diluting the metabolite II reference substance, a series of solutions of metabolite II at different concentrations are obtained. A quantitative internal standard compound is added, and after extraction, these are used as standard working solutions. Each of these standard working solutions is absorbed and injected into an LC-MS / MS system for detection. The peak areas of metabolite II and the internal standard compound are determined. A standard curve is drawn with the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound on the vertical axis and the concentration of metabolite II on the horizontal axis, and a regression equation is calculated. The test solution was absorbed and injected into an LC-MS / MS system for detection. The ratio of the peak area of ​​metabolite II to the peak area of ​​deuterated compound I in the test solution was determined, and this ratio was substituted into a regression equation to determine the content of metabolite II in the test biological sample solution.

[0173] Solutions of the same concentration were prepared using the same volume containers, in accordance with the laboratory operator's practice, and following the modified standard operating procedure described above. This procedure is sufficient if the volume of added solution is the same and the resulting standard working solutions remain at the same concentration.

[0174] Figures 7-10 show some representative spectra of the plasma sample detection process.

[0175] Figure 7 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in blank human plasma extracts.

[0176] Figure 8 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a 0% plasma sample extract.

[0177] Figure 9 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a standard sample solution (0.50 ng / ml) of human plasma extract.

[0178] Figure 10 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a standard sample solution (200 ng / ml) of human plasma extract.

[0179] Some of the samples were detected using the detection procedure for AST-2660 content in human plasma provided in this example. The results are shown in Tables 12-16 below.

[0180] [Table 12]

[0181] As is clear from the table, human plasma containing AST-2660 showed little decrease in AST-2660 content after 28 days of storage at -70°C, but a significant decrease occurred at -20°C. Therefore, human plasma samples containing AST-2660 should be stored at -70°C whenever possible, and under these conditions, the samples remained unchanged even after 28 days of storage.

[0182] [Table 13]

[0183] As is clear from the table, the AST-2660 content in human plasma containing AST-2660 showed almost no decrease during the freeze-thaw treatment from -70°C to room temperature, but decreased significantly during the freeze-thaw treatment from -20°C to room temperature. In other words, according to the data in Table 12, the rapid temperature change from the storage conditions of human plasma samples stored at -70°C to room temperature for experimental processing did not affect the stability of AST-2660 in the human plasma samples.

[0184] [Table 14]

[0185] As is evident from the data in Table 14, a methanol solution of AST-2660-D8 exhibits strong stability after being stored at -70°C for 26 days under the same conditions as AST-2660.

[0186] [Table 15]

[0187] As is clear from the data in Table 15, the methanol solution content of AST-2660 remained stable after 54 hours of storage under conventional freezing conditions (a freezer commonly used in laboratories).

[0188] [Table 16]

[0189] As is evident from the data in Table 16, human plasma samples containing AST-2660 are unstable at room temperature. Since the AST-2660 content in human plasma samples decreases with extended storage periods, it is suggested that when collecting human plasma samples from clinical patients, the collected plasma samples should be immediately stored in the experimentally confirmed low-temperature environment described above. Specifically, storage at -70°C is the safest, with -20°C being acceptable. If storage conditions are limited, samples should be stored in a conventional refrigerator (4-0°C) and transferred to the aforementioned low-temperature environment within 4 hours. During transport of samples to the DMPK Sample Testing Center laboratory, the entire cold chain logistics process (with temperature monitoring throughout) should be used, where the temperature should be below -20°C, or dry ice should be used for insulation.

[0190] From the experimental results above, the following can be concluded. (1) Human plasma samples containing AST-2660 should be stored immediately after collection in the experimentally confirmed low-temperature environment described above, i.e., storage at -70°C is the safest, and storage at -20°C is also acceptable. If storage conditions are limited, samples should be stored in a conventional refrigerator (4-0°C) and transferred to the above low-temperature environment within 4 hours. During transport of samples to the DMPK Sample Testing Center laboratory, the entire cold chain logistics process (temperature should be monitored throughout the entire process) should be used, with the temperature selected to be below -20°C, or dry ice should be used for insulation. (2) The sample should be stored at a temperature of -70°C or below, and was stable for 28 days under these conditions. Depending on the trend of change, it may be stable for a longer period. (3) The process of removing the stored sample to room temperature, i.e., the rapid temperature change from the storage conditions of human plasma stored at -70°C to room temperature for experimental processing, did not affect the stability of AST-2660 in the human plasma sample. After removal, it should be stored in a conventional refrigerator (4~0°C), and the storage time in a conventional refrigerator should be within 4 hours. If stored at room temperature, the AST-2660 content will decrease and the sample will become unusable. (4) A methanol solution of AST-2660-D8 shows no change in content even after being stored at -70°C for 26 days, demonstrating strong stability. Furthermore, it may remain stable for longer periods depending on the trend of change.

[0191] Therefore, in the standard operating procedure for blood samples described above, the samples should be stored at -20°C or below, preferably at -70°C, within 4 hours of collection, and can be stored at -70°C for 28 days. When processing blood samples using the extraction method described above, they must be refrigerated at a temperature of 4°C or below, and injection detection must be completed within 54 hours at room temperature in the laboratory. The results detected according to these storage temperatures and times were reliable. Otherwise, the AST-2660 in the sample changes during storage, making the results inaccurate and unreliable.

[0192] [Example 6] Establishment and verification of a method for detecting AST-2660 in human urine. In this example, the process for establishing the detection method for AST-2660 in human urine was basically the same as in Example 4. The only differences between the two were the matrix (human plasma in Example 4, human urine in this example) and a slight difference in the sample extraction process. Mixed normal human (drug-free) urine containing the additive Na2HPO4·12H2 was used as the diluent and matrix.

[0193] 1. Sample pretreatment The procedure for extracting a sample using the protein precipitation method is as follows: 1. Label and sequence the standard curve sample, quality control sample, and plasma sample. 2. Place all samples on a multi-tube vortexer to ensure uniform vortexing after thawing. 3. Take 100 μl from each sample and add it to a 96-well plate. 4. Add 20 μl of AST-2660-D8 quality control solution (i.e., 100 ng / ml AST-2660-D8 methanol solution) to the blank sample, and add 20 μl of methanol to the blank sample. 5. Add 500 μl of methanol. 6. Vortex the plate at medium speed for 3 minutes. Centrifuge the 7.96-well plate at 2143 rcf (centrifugal force) for 10 minutes. 8. Transfer 450 μl of the supernatant to a new 96-well plate. Centrifuge the 9.96-well plate at 2143 rcf (centrifugal force) for 10 minutes. 10. Transfer 300 μl of the supernatant to a new 96-well plate.

[0194] 2. Experimental Results 2.1 Standard curve The parameters for the AST-2660 standard curve in human urine were as follows: Slope: 0.001400, Intercept: 0.000016, Correlation coefficient: 0.9982, Relationship function: y=f(x)=0.001400x+0.000016 (where y represents the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, and x represents the concentration of metabolite II in the standard working solution), LLOQ (lower limit of quantification): 0.5 ng / ml, ULOQ (upper limit of quantification): 200 ng / ml. As shown, the linear relationship was good in the linear range of 0.5 to 200 ng / ml.

[0195] 2.2 Results of accuracy and precision tests of quality control samples Accuracy and precision are defined as follows: Accuracy is expressed as relative error (RE). RE = [(Average value - Nominal value) / Nominal value] × 100 Precision is expressed as the coefficient of variation (CV). CV = (Standard Deviation / Mean) × 100

[0196] The accuracy and precision test results are shown in Table 17. From Table 17, it can be seen that both the intra-assay and inter-assay precision were less than 15.5%, and the intra-assay and inter-assay precision was within ±11.4. The analytical criteria for accuracy and precision were as follows: For LLOQ QC samples, the intra-assay and inter-assay precision (RE) must be within ±20.0%, and the intra-assay and inter-assay CV must not exceed 20.0%. The intra-assay and inter-assay precision (RE) must be within ±15.0% for low, geometric mean, medium, high, and diluted QC (where applicable) samples, and the intra-assay and inter-assay precision (CV) must not exceed 15.0% at each QC concentration level. 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% or less, which met the requirements.

[0197] [Table 17]

[0198] 2.3 Test Results of Matrix Effects [Table 18]

[0199] According to the results in Table 18, the inter-assay matrix accuracy and precision (CV) of AST-2660 matrix effects were both less than 15%, which meets the requirements.

[0200] 2.4 Stability [Table 19]

[0201] Table 19 shows that the accuracy of the AST-2660 working solution's stability was within ±11.8%, and its precision was less than 11.5%. The stability analysis criteria were as follows: The difference between the stored stable solution and the newly prepared solution must be within ±10.0%, and the CV of each replicated experiment must not exceed 15.0%. Therefore, the AST-2660 working solution exhibited good stability and met the requirements under various storage conditions.

[0202] To detect AST-2660 in human urine, the liquid chromatography-tandem mass spectrometry method established in this example met the analytical requirements for biological samples. The sample preparation method was simple and convenient, and the method exhibited high sensitivity, high accuracy, and high precision.

[0203] [Example 7] Detection of AST-2660 content in actual human urine The AST-2660 content in the urine of the test subjects was detected using the detection method established in Example 6.

[0204] The standard operating procedure (SOP) for detection established after the methodology and verification in Example 6 was as follows: Step 1. Preparation of the solution Matrix: Mixed normal human urine containing the additive Na2HPO4·12H2O a: Internal standard working solution: AST-2660-D8 was taken, dissolved in methanol, and diluted to prepare a solution containing approximately 100 ng per 1 ml. b: Test solution (at this point, the internal standard compound has not been added to the test solution): The test blood sample was obtained. c: Standard working solution (at this point, no internal standard compound has been added to the standard working solution): An appropriate amount of AST-2660 was taken, accurately weighed, dissolved in methanol, and diluted to prepare a solution containing approximately 1.0 mg per 1 ml, which was used as the standard working storage solution. An appropriate amount of the standard working storage solution was taken, added together with the matrix, and diluted to prepare standard working solutions of different concentrations containing approximately 200 ng, 160 ng, 100 ng, 50 ng, 10 ng, 2.0 ng, 1.0 ng, and 0.5 ng per 1 ml, respectively. (The solutions were stored at -70°C) d: Quality control sample solution: An appropriate amount of AST-2660 was taken, accurately weighed, dissolved in methanol, and diluted to prepare a solution containing approximately 10 μg per 1 ml, which was used as the quality control work storage solution. An appropriate amount of the quality control work storage solution was taken and added together with the matrix to prepare quality control work solutions of different concentrations containing approximately 150 ng, 60 ng, 15 ng, 1.5 ng, and 0.5 ng per 1 ml, respectively. (The solutions were stored at -70°C.)

[0205] Step 2. Sample Extraction The test solution, standard working solution, and quality control sample solution were thawed at room temperature and mixed uniformly. 100 μl of each solution was taken, 20 μl of internal standard and 500 μl of methanol were added, and the mixture was vortexed for 3 minutes to ensure uniformity. The mixture was then centrifuged at 2143 rcf for 30 minutes. 450 μl of the supernatant was taken and centrifuged in a 96-well plate at 2143 rcf for 10 minutes. 300 μl of the supernatant was then taken to obtain the final product.

[0206] A blank sample was obtained by collecting 100 μl of blank human urine, adding 20 μl of methanol, and extracting it using the same method.

[0207] A 100 μl blank human urine sample was collected, 20 μl of internal standard working solution was added, and extraction was performed using the same method to obtain a zero-concentration sample.

[0208] After sample extraction and addition of internal standards, in step 4, the solution was injected into an LC-MS / MS for detection.

[0209] Step 3. Setting the chromatography-mass spectrometry test conditions. Measurements were performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the following instrument test conditions were set. The liquid phase conditions were as follows: A SHARC1, 3 μm, 2.1 × 50 mm chromatographic column or one with equivalent performance was used, along with a methanol solution containing a low amount of ammonium acetate. Mobile phase B was acetonitrile, and gradient elution was performed according to the table below, at a column temperature of 20°C and an injection volume of 5 μl.

[0210] [Table 20]

[0211] The mass spectrometry conditions were as follows: Negative ion scanning mode electrospray ion source, spray potential: -4500V, ion source temperature: 500℃, collision energy (CE): -22eV, declustering potential (DP): -55V, inlet potential (EP): -10V, collision chamber outlet potential (CXP): -16V, residence time: 100ms, high-purity nitrogen used for all gases, AST-2660 ion pair: m / z 147.0 → m / z 62.9, internal standard AST-2660-D8 ion pair: m / z 155.0 → m / z 62.9.

[0212] Step 4. Judgment AST-2660 standard working solution, blank sample, zero-concentration sample, test solution, and quality control working solutions with different concentrations after extraction were each collected and injected into LC-MS / MS for detection.

[0213] The criteria for acceptance were as follows: The recovery concentration of the standard working solution, the accuracy of the quality control working solution, the peak residue of AST-2660, and the internal standard in the blank sample all meet the requirements of the Chinese Pharmacopoeia (2020 edition), Volume 4, 9012.

[0214] Standard working solutions of metabolite II at different concentrations were absorbed and injected into an LC-MS / MS system for detection, and the relation function y=f(x) was obtained (wherein y represents the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, and x represents the concentration of metabolite II in the standard working solution).

[0215] The calculation results were as follows: The relational function y=f(x) for the ratio of the peak area of ​​AST-2660 to the peak area of ​​the internal standard was obtained from AST-2660 standard working solutions of different concentrations. The ratio of the peak area of ​​AST-2660 to the peak area of ​​the internal standard was determined from the extracted test solution, and this was substituted into the relational function y=f(x) to calculate the concentration of metabolite II in the test solution.

[0216] The concentration of metabolite II in a test solution of unknown concentration was measured and calculated using a relational function. The test solution, to which an internal standard compound of known content was added, was absorbed and injected into an LC-MS / MS system for detection. The ratio y of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound was determined, and substituted into the function y=f(x) to find the concentration x of metabolite II in the test solution.

[0217] As an option, the standard operating procedure for the following operations was obtained by slightly modifying the order of the related operating steps in the standard operating procedure described above. A quantitative internal standard compound is added to the test biological sample solution, and after extraction, it is used as the test solution. By diluting the metabolite II reference substance, a series of solutions of metabolite II at different concentrations are obtained. A quantitative internal standard compound is added, and after extraction, these are used as standard working solutions. Each of these standard working solutions is absorbed and injected into an LC-MS / MS system for detection. The peak areas of metabolite II and the internal standard compound are determined. A standard curve is drawn with the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound on the vertical axis and the concentration of metabolite II on the horizontal axis, and a regression equation is calculated. The test solution was absorbed and injected into an LC-MS / MS system for detection. The ratio of the peak area of ​​metabolite II to the peak area of ​​deuterated compound I in the test solution was determined, and this ratio was substituted into a regression equation to determine the content of metabolite II in the test biological sample solution.

[0218] Solutions of the same concentration were prepared using the same volume containers, in accordance with the laboratory operator's practice, and following the modified standard operating procedure described above. This procedure is sufficient if the volume of added solution is the same and the resulting standard working solutions remain at the same concentration.

[0219] Figures 11-14 show some representative spectra of the urine sample detection process.

[0220] Figure 11 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in blank human urine extract.

[0221] Figure 12 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a 0% urine sample extract.

[0222] Figure 13 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a standard sample solution (0.50 ng / ml) of human urine extract.

[0223] Figure 14 shows representative LC-MS / MS spectra of AST-2660 and the internal standard AST-2660-D8 in a standard sample solution (200 ng / ml) of human urine extract.

[0224] Some of the samples were detected using the human urine AST-2660 content detection procedure provided in this example. The results are shown in Tables 21-24 below.

[0225] [Table 21]

[0226] As is clear from Table 21, human urine containing AST-2660 showed almost no decrease in AST-2660 content after 32 days of storage at -70°C, but decreased by almost 90% at -20°C. Therefore, human urine samples containing AST-2660 should be stored at -70°C, and under these conditions, samples stored for 32 days remained stable.

[0227] [Table 22]

[0228] As is clear from Table 22, the AST-2660 content in human urine containing AST-2660 showed almost no decrease during the freeze-thaw treatment from -70°C to room temperature, but decreased by almost 90% during the freeze-thaw treatment from -20°C to room temperature. In other words, according to the data in Table 22, the rapid temperature change from the storage conditions of human plasma samples stored at -70°C to room temperature for experimental processing did not affect the stability of AST-2660 in human plasma samples.

[0229] [Table 23]

[0230] [Table 24]

[0231] As is clear from Table 23, treated human urine samples containing AST-2660 remained stable at room temperature for 94 hours.

[0232] As is clear from Table 24, untreated human urine samples containing AST-2660 remained stable at room temperature for 24 hours. From these experimental results, the following can be concluded: (1) Human urine samples containing AST-2660 should be stored at -70°C, the experimentally confirmed low-temperature environment described above, within 24 hours of collection (at the latest). When sending samples to the DMPK Sample Testing Center laboratory, they should be kept at the lowest possible temperature (below room temperature). (2) The sample should be stored at a temperature of -70°C or below, and was stable for 32 days under these conditions. Depending on the trend of change, it may be stable for a longer period. (3) Human urine samples treated with AST-2660 remained stable for 94 hours at room temperature or below. (4) The process of removing the stored sample to room temperature, i.e., the rapid temperature change from the storage conditions of human urine samples stored at -70°C to room temperature for experimental processing, did not affect the stability of AST-2660 in the human urine sample. After removal, the sample should be placed in a conventional refrigerator or at room temperature and remained stable for up to 24 hours at room temperature. (5) Human urine samples should be stored at -70°C within 24 hours of collection, preferably within 4 hours, and if processed, injection detection should be completed within 94 hours at room temperature.

[0233] Therefore, in the standard procedure for handling urine samples as described above, the samples should be stored at -70°C within 24 hours, preferably within 4 hours, after collection, and can be stored at -70°C for 32 days. If processing is required, injection detection must be completed within 94 hours at room temperature or below. The results detected according to these storage temperatures and times were reliable. Otherwise, the AST-2660 in the sample changes during storage, making the results inaccurate and unreliable.

Claims

1. Deuterated compound I shown in formula (I). [Chemistry I] In the formula, A is either H or D, and at least one of the eight A's is D. M is H, an alkali metal, an alkaline earth metal, or an ammonium radical.

2. The deuterated compound I according to claim 1, wherein at least three of the eight A's are D.

3. The deuterated compound I according to claim 2, wherein the number of D is 4 or 8.

4. The deuterated compound I according to claim 3, wherein M is Na, K, Li, or an ammonium radical.

5. Deuterated compound I according to claim 1, which is a compound shown in formula (I-1) or (I-2). [Chemistry I-1] [Chemistry I-2]

6. Deuterated compound I according to claim 1, selected from the compounds shown in the following formula. [Chemistry I-3] [Chemistry I-4-1] 【Request Item 7】 【Chemistry I-5】 Compound I-a is converted to phosphorus oxyhalogenate POX ３ A step to obtain compound I-b by reacting with, A step of hydrolyzing compound I-b in an aqueous solution, with or without the presence of a base, to obtain the corresponding deuterated compound I, Includes, Compound I-a is a deuterated 2-halogenated ethylamine or its inorganic salt, A is H or D, at least one of the four A's in compound I-a is D, and at least one of the eight A's in compound I-b and compound I is D. The base in the hydrolysis reaction is selected from MOH (where M is an alkali metal, alkaline earth metal, or ammonium radical), MH (where M is an alkali metal), and MOR (where R is an alkyl group having 1 to 4 carbon atoms, and M is an alkali metal, alkali metal carbonate, or bicarbonate). X is a method for producing deuterated compound I, which is a halogen.

8. The aforementioned compound I-a and the aforementioned oxyhalogenated phosphorus POX ３ The manufacturing method according to claim 7, wherein the mixture is cooled in an organic solvent and an organic base solution is added dropwise to obtain compound I-b.

9. The aforementioned temperature reduction is to lower the temperature to -78 to -20°C, and / or The organic solvent is a mixture of one or more of the following: dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane or cyclohexane and tetrahydrofuran, and / or The organic base is one or more of the following: methylamine, ethylamine, propylamine, isopropylamine, N,N-diethylamine, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undeca-7-ene, N,N,N',N'-tetramethylethylenediamine, tetramethylguanidine, pyridine, N-methyldicyclohexylamine, or dicyclohexylamine, and / or The aforementioned compound I-a and the oxyhalogenated phosphorus POX ３ The manufacturing method according to claim 8, wherein the reaction is carried out in the atmosphere, and the atmosphere is one of air, nitrogen, or argon.

10. ３１ A method for measuring the content of deuterated compound I according to any one of claims 1 to 6 using P-NMR, or a method for measuring the content of deuterated compound I according to any one of claims 1 to 6 using liquid chromatography, The liquid chromatography conditions are, Using a hydrogen-accepting stationary-phase chromatography column, Mobile phase A is a methanol solution of ammonium acetate, and mobile phase B is acetonitrile. The gradient elution method uses mobile phases A and B, gradually increasing the volume ratio of mobile phase A from 15% to 90%, and then gradually decreasing the volume ratio of mobile phase A back to 15%.

11. The deuterated compound I and phosphorus-containing compounds of known content ３１ The process involves detecting P-NMR and obtaining their spectra, The aforementioned ３１ A method for determining the content of deuterated compound I according to any one of claims 1 to 6, comprising the step of calculating the content of deuterated compound I by substituting the content of the phosphorus-containing compound with the known content based on the peak area ratio of the chemical shift characteristic peak of the deuterated compound I in the P-NMR spectrum and the chemical shift characteristic peak of the phosphorus-containing compound.

12. The phosphorus-containing compound is a compound containing one phosphorus atom, The deuterated compound I and the phosphorus-containing compound in known amounts are added to the solvent. ３１ The method according to claim 11, wherein the P-NMR spectrum is tested together.

13. The phosphorus-containing compound is hexamethylphosphate triamide, The deuterated compound I and the phosphorus-containing compound in known quantities are added to water, and after they dissolve, ３１ The method according to claim 12, wherein the P-NMR spectrum is tested together.

14. The use of deuterated compound I according to any one of claims 1 to 6 as an internal standard for detecting metabolite II in a biological sample, wherein metabolite II is the compound shown in formula (II), 【Chemistry II】 The use of deuterated compound I, in which A is H and M is H or an alkali metal, alkaline earth metal, or ammonium radical.

15. The deuterated compound I is [Chemistry I-3] Selected from, The aforementioned metabolite II is [Chemistry I-6] The use according to claim 14, selected from the above.

16. The use of deuterated compound I according to any one of claims 1 to 6 as an internal standard for measuring the content of metabolite II of an AKR1C3-activated DNA alkylating agent prodrug or a hypoxia-activated DNA alkylating agent prodrug in a biological sample by LC-MS / MS analysis, wherein metabolite II is the compound shown in formula (II), 【Chemistry II】 The use of deuterated compound I, in which A is H and M is H or an alkali metal, alkaline earth metal, or ammonium radical.

17. The deuterated compound I is [Chemistry I-3] Selected from, The aforementioned metabolite II is [Chemistry I-6] The use according to claim 16, selected from the above.

18. The use according to claim 16, wherein the AKR1C3 activating DNA alkylating agent prodrug is selected from the compounds shown in formulas 1 to 5. 【Chemistry 1-2】 During the ceremony, R １ is C ６ -C １０ aryl or Z-substituted aryl, a 4- to 15-membered heterocyclic ring or Z-substituted heterocyclic ring, a 5- to 15-membered heteroaryl or Z-substituted heteroaryl, or a 7- to 15-membered fused ring, or Z-substituted fused ring, and R ２ This includes hydrogen, halogen atoms, cyano, hydroxy, sulfhydryl, amino, OTs, mesylate, and 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- to 15-membered heterocycle or Z-substituted heterocycle, 5- to 15-membered heteroaryl or Z-substituted heteroaryl, -CONR ６ R ７ , -SO ２ NR ６ R ７ , -SO ２ R ６ , -OCOO-R ６ , -COOR ６、 -NR ６ COR ７ , -OCOR ６ , -NR ６ SO ２ R ７ or -NR ６ SO ２ NR ６ R ７ or groups R that bond to form a 7-15 membered condensed ring or a Z-substituted condensed ring. １ R is present together with the atoms inside. ２ And, R ３ These are hydrogen, halogen, cyano, hydroxy, sulfhydryl, 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, 4- to 15-membered heterocycle or Z-substituted heterocycle, 5- to 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 , -OCOR ６ , or -NR ６ SO ２ R ７ And, R ４ and R ５ are each independently hydrogen, a halogen atom, cyano, hydroxy, sulfhydryl, 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, a 4- to 15-membered heterocycle or Z-substituted heterocycle, a 5- to 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 ６ , -NR ６ COR ６ , -OCOR ６ or -NR ６ SO ２ R ７ , or R together with atoms in a benzene ring to which they are attached form a 7- to 15-membered fused ring or Z-substituted fused ring ４ and R ５ are, R ６ and R ７ These are, independently, hydrogen, cyano, and 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- to 15-membered heterocycle or Z-substituted heterocycle, 5- to 15-membered heteroaryl or Z-substituted heteroaryl, or C １ -C ６ Alkoxy or Z-substituted C １ -C ６ R is associated with alkoxys, or atoms that, when bonded, form 5-7 membered heterocyclines or Z-substituted 5-7 membered heterocyclines. ６ and R ７ And, R ８ and R １０ These are, independently, hydrogen, deuterium, aryl or Z-substituted aryl, and 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 It is a cycloalkyl or Z-substituted cycloalkyl, and R ８ and R １０ At least one of them must be hydrogen or deuterium. R ９ is a substituted C that is substituted with at least one fluorine atom or nitro group. ６ -C １０ The aryl group is a substituted 4- to 15-membered heterocyclic ring substituted with at least one fluorine atom or nitro group, or a substituted 5- to 15-membered heteroaryl group substituted with at least one fluorine atom or nitro group. Substituent Z can be a halogen atom, cyano, hydroxy, sulfhydryl, amino, OTs, mesylate, or 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, heterocycle, aromatic heterocycle and fused ring, or substituted aromatic ring, heterocycle, or aromatic heterocycle and fused ring, and the substitution pattern is single substitution or disubstituted. Substitution C ６ -C １０ R in aryls, substituted 4- to 15-membered heterocyclic rings, or substituted 5- to 15-membered heteroaryls ９ The substituents inside are halogen atoms, nitro, cyano, hydroxy, amino, and C. １ -C ３ Alkyl or alkoxy, alkenyl, alkynyl, cycloalkyl or benzene ring, substituted benzene ring, C １ -C ３ It is an alkoxy or halogen atom-substituted alkoxy, or 【Transformation 3】 During the ceremony, R ｗ teeth, 【Chemistry 20】 And, R １ H, C １－６ Alkyl, C ３－６ The C is a cycloalkyl, a 4-6 member heterocycloalkyl, a 5-6 member heteroaryl, or phenyl group. １－６ Alkyl, C ３－６ Cycloalkyl, 4-6 member heterocycloalkyl, 5-6 member heteroaryl, and phenyl groups have 1, 2, or 3 R groups. ａ It may also be replaced with Each R ａ These are independently H, F, Cl, Br, I, -CN, -OH, C １－３ Alkoxy group or C １－３ It is alkyl, R ２ is H or C １－６ Alkyl, Alternatively, R is present with N, which combines with them to form a 4-6 member heterocycloalkyl group. １ and R ２ The 4-6 member heterocycloalkyl group has 1, 2, or 3 R ｂ It may also be replaced with Each R ｂ These are independently H, F, Cl, Br, I, -CN, -OH, -NH ２ , -OCH ３ , -OCH ２ CH ３ ien-CH ３ or -CH ２ CH ３ And, R ３ is H, F, Cl, Br, I, -OH, -NH ２ , C １－３ Alkoxy group or C １－３ It is alkyl, Or, R ２ and R ３ These are combined together to form a structural unit. 【Chemistry 21】 of, 【Chemistry 22】 Let's assume that. T １ is, -(CR ｃ R ｄ ) ｍ - or - (CR ｃ R ｄ ) ｎ -O-, m is 1, 2, or 3. n is either 1 or 2, T ２ is N or CH, R ｃ and R ｄ These are H, F, and C, each independent of the others. １－３ Alkyl or C １－３ It is an alkoxy group, R ４ , R ５ and R ６ These are H, F, Cl, Br, I, and C, respectively, independently. １－３ Alkyl or C １－３ It is an alkoxy group, T is N or CH, R ７ and R ８ Each of these is independently H, F, Cl, Br, or I. R ９ and R １０ These are, independently, H, F, Cl, Br, I, and -CN, Each of the 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl compounds independently comprises 1, 2, 3, or 4 heteroatoms selected from N, -O-, and -S-. or 【Chemistry 4】 During the ceremony, X １０ is O, S, SO or SO ２ And, A is C ６ -C １０ Aryl, 5-15 member heteroaryl, or -N=CR １ R ２ And, R １ and R ２ These are, independently, hydrogen and C １ -C ６ Alkyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ Aryl, 4-15 membered heterocycle, -CONR １３ R １４ , or -NR １３ COR １４ And, X, Y, and Z are, independently, hydrogen, CN, halogen, and C. １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ Aryl, 4-15 membered heterocycle, -CONR １３ R １４ , or -NR １３ COR １４ And, R is hydrogen, C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ Aryl, 4-15 membered heterocycle, -CONR １３ R １４ , or -NR １３ COR １４ And, R １３ and R １４ These are, independently, hydrogen and C １ -C ６ Alkyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ It is an aryl or 4- to 15-membered heterocycle, T is, [Chemistry I-7] In the formula, the alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aryl group, heterocyclic group, and heteroaryl group may be substituted. or 【Transformation 5】 In the formula, A is a substituted or unsubstituted C. ６ -C １０ Aryl, biaryl or substituted biaryl, 5- to 15-membered heteroaryl, or -N=CR １ R ２ The substituents are halogeno, -CN, and -NO. ２ , -O-(CH ２ )-O-,-CO ２ H and its salts, -OR １００ , -CO ２ R １００ , -CONR １０１ R １０２ , -NR １０１ R １０２ , -NR １００ SO ２ R １００ , -SO ２ R １００ , -SO ２ NR １０１ R １０２ , C １ -C ６ Alkyl and C ３ -C １０ Selected from a group consisting of heterocyclines, In the formula, R １００ , R １０１ and R １０２ These are, independently, hydrogen and C １ -C ８ Alkyl, or C ６ -C １２ It is either aryl or R １０１ and R １０２ These, together with the nitrogen atoms bonded to them, form a 5-7 membered heterocycle. The alkyl group and the aryl group each consist of 1 to 3 halogen groups or 1 to 3 carbon atoms. １ -C ６ It is replaced with an alkyl group, R １ and R ２ These are, independently, phenyl or methyl, X, Y, and Z are each independently hydrogen or halogen, R is hydrogen or C １ -C ６ It is an alkyl or halogen-substituted alkyl group.

19. The use according to claim 16, wherein the hypoxia-activated DNA alkylating agent prodrug is selected from the group consisting of compounds shown in formulas 6 to 12. 【Transformation 6】 During the ceremony, Cx is a 5-10 membered aromatic ring or aromatic heterocycle, alicyclic heterocycle, or cycloalkane that forms a fused cyclic structure by sharing two carbon atoms with a nitrobenzene ring. R bonded to any skeletal atom of the Cx ring １ These are hydrogen, halogen atoms, cyano, hydroxyl, mercapto, amine, OTs, and 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- to 15-membered heterocycle or Z-substituted heterocycle, 5- to 15-membered heteroaryl or Z-substituted heteroaryl, alkoxy having 1 to 6 carbon atoms or Z-substituted alkoxy having 1 to 6 carbon atoms, -CONR ６ R ７ , -SO ２ NR ６ R ７ , -SO ２ R ６ , -OCOO-R ６ , -COOR ６ , -NR ６ COR ７ , -OCOR ６ , -NR ６ SO ２ R ７ , and -NR ６ SO ２ NR ６ R ７ Selected from, R ２ and R ３ These are, independently, hydrogen and 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 R with the carbon atoms of benzyl, which is an aryl or Z-substituted aryl, a 4- to 15-membered heterocycle or Z-substituted heterocycle, a 5- to 15-membered heteroaryl or Z-substituted heteroaryl, or which are bonded together to form a 3- to 6-membered ring. ２ and R ３ And, 【Chemistry 45】 The group can substitute a hydrogen atom at any position on the carbon atom of the fused ring, and the number of substitutions is one. Z- substituents include halogen atoms, cyano groups, hydroxyl groups, mercapto groups, amino groups, and C １ -C ３ Alkyl or substituted alkyl group, C １ -C ３ Alkoxy group or substituted alkoxy group, C ２ -C ３ Alkenyl group or substituted alkenyl group, C ２ -C ３ Alkynyl or substituted alkynyl, C ３ -C ８ It is a cycloalkyl or substituted cycloalkyl, R ６ and R ７ These are, independently, hydrogen and 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 Alkenil, C ２ -C ６ Alkynyl or Z-substituted C ２ -C ６ Alkinyl, 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 R with an atom that combines with an aryl, a 4-15 member heterocyclil or a Z-substituted 4-15 member heterocyclil, a 5-15 member heteroaryl or a Z-substituted 5-15 member heteroaryl, or with an atom that combines them to form a 5-7 member heterocyclil or a Z-substituted 5-7 member heterocyclil ６ and R ７ and, or, 【Chemistry 7-12】 During the ceremony, R １ is hydrogen, -N ３ CN, halogen, NR ２１ R ２２ , -OR ２３ , -SO ２ (C １ -C ６ Alkyl), C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ They are aryl, 4- to 15-membered heterocyclic rings, or 5- to 15-membered heteroaryl rings. R ２１ and R ２２ These are, independently, hydrogen, hydroxyl, and C. １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ Aryl, 4- to 15-membered heterocyclic rings, 5- to 15-membered heteroaryl rings, or -SO ２ (C １ -C ６ R is present with a nitrogen atom that is alkyl, or when bonded to them to form a 4-15 membered heterocycle or a 5-15 membered heteroaryl. ２１ and R ２２ And, R ２３ is hydrogen, C １ -C ６ Alkyl, or C ６ -C １０ It is Ariel, R ２ and R ３ These are independently hydrogen or halogen, R ４ is hydrogen, halogen, C １ -C ６ Alkoxy, C １ -C ６ Alkyl, or C ６ -C １０ It is Ariel, R ５ , R ７ , R ９ , R １２ and R １５ These are, independently, hydrogen, C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ Aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, or C interposed between them ５ -C ６ R is present together with the carbon atoms that form the cycloalkyl ring. ４ and R ５ And, R ６ and R １０ These are independently hydrogen or halogen, R ８ is hydrogen, C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkynyl, or 5-15 member heteroaryl, Each R １１ Independently, C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, C ３ -C ８ Cycloalkyl, or C ６ -C １０ It is Ariel, R １３ , R １４ , R １６ , and R １７ These are, independently, hydrogen, halogen, and C １ -C ６ Alkyl, C ２ -C ６ Alkenil, C ２ -C ６ Alkinyl, or C １ -C ６ It is an alkoxy, The alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aryl group, heterocyclic group, heteroaryl group, and alkoxy group may be substituted.

20. A step of preparing a test solution containing an internal standard compound of known concentration for injection analysis by LC-MS / MS, A step of preparing a series of standard working solutions containing an internal standard compound of known concentration and a metabolite II of known concentration, wherein the concentration of the internal standard compound in the series of standard working solutions is consistent, the same as the concentration of the internal standard compound in the test solution, and the concentrations of the metabolite II in the series of standard working solutions are different. The process involves absorbing standard working solutions of metabolite II at different concentrations, injecting them into a detection LC-MS / MS system to obtain the relation function y = f(x) (where y represents the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, and x represents the concentration of metabolite II in the standard working solution), and determining the relation function by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The process involves absorbing the test solution to which the internal standard compound in a known amount is added, injecting it into the detection LC-MS / MS system to determine the ratio y of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound, substituting this ratio into the function y = f(x) to determine the concentration x of metabolite II in the test solution, and using this relational function to determine and calculate the concentration of metabolite II in the test solution at an unknown concentration. Includes, The aforementioned test solution is prepared from a biological sample, whether or not it has been treated. The internal standard compound is the deuterated compound I shown in formula (I), [Chemistry I] In the formula, A is either H or D, and at least one of the eight A's is D. M is H or an alkali metal, alkaline earth metal, or ammonium radical. The aforementioned metabolite II is the compound shown in formula (II): 【Chemistry II】 In the equation, A is H, A method for measuring the content of metabolites in a biological sample, where M is H, an alkali metal, an alkaline earth metal, or an ammonium radical.

21. The conditions for liquid chromatography in liquid chromatography-tandem mass spectrometry are: Using a hydrogen-accepting stationary-phase chromatography column, Mobile phase A is a methanol solution of ammonium acetate, and mobile phase B is acetonitrile. For gradient elution, mobile phases A and B are used, with mobile phase A gradually increased from 15% to 90% by volume, and then gradually decreased back down to 15% by volume. Mass spectrometry conditions: Electrospray ion source In negative ion scanning mode, Monitoring ion pair of metabolite II: m / z 147.0 → m / z 62.9 Monitoring ion pair of deuterated compound I: m / z (147.0 + deuterated number) → m / z 62.9, or, In positive ion scanning mode, Monitoring ion pair of metabolite II: m / z 149.0 → m / z 64.9 The method according to claim 20, wherein the monitoring ion pair of deuterated compound I is m / z (149.0 + deuterated number) → m / z 64.

9.

22. The process involves adding a quantitative internal standard compound to the biological sample solution and performing an extraction to obtain the test solution, The process involves diluting a metabolite II standard substance to obtain a series of metabolite II standard solutions of different concentrations, adding the quantitative internal standard compound and performing an extraction to obtain a standard working solution, absorbing each of the series of standard working solutions and injecting them into a detection LC-MS / MS system to determine the peak areas of the metabolite II and the internal standard compound, creating a standard curve with the ratio of the peak area of ​​the metabolite II to the peak area of ​​the internal standard compound on the vertical axis and the concentration of the metabolite II on the horizontal axis, and calculating a regression equation. The steps include: absorbing the test solution, injecting it into the detection LC-MS / MS system to determine the ratio of the peak area of ​​metabolite II in the test solution to the peak area of ​​the internal standard compound, and substituting this into the regression equation to determine the content of metabolite II in the test solution; Includes, The aforementioned internal standard is deuterated compound I shown in formula (I), [Chemistry I] In the formula, A is either H or D, and at least one of the eight A's is D. M is H or an alkali metal, alkaline earth metal, or ammonium radical. The aforementioned metabolite II is the compound shown in formula (II), 【Chemistry II】 In the equation, A is H, A method for measuring the content of metabolites in a biological sample, where M is H, an alkali metal, an alkaline earth metal, or an ammonium radical.

23. The conditions for liquid chromatography in liquid chromatography-tandem mass spectrometry are: Using a hydrogen-accepting stationary-phase chromatography column, Mobile phase A is a methanol solution of ammonium acetate, and mobile phase B is acetonitrile. For gradient elution, mobile phases A and B are used, with mobile phase A gradually increased from 15% to 90% by volume, and then gradually decreased back down to 15% by volume. Mass spectrometry conditions: Electrospray ion source In negative ion scanning mode, Monitoring ion pair of metabolite II: m / z 147.0 → m / z 62.9 Monitoring ion pair of deuterated compound I: m / z (147.0 + deuterated number) → m / z 62.9, or, In positive ion scanning mode, Monitoring ion pair of metabolite II: m / z 149.0 → m / z 64.9 The method according to claim 22, wherein the monitoring ion pair of deuterated compound I is m / z (149.0 + number of deuterated compounds) → m / z 64.

9.

24. In preparing a test solution containing the deuterated compound I in a known amount, first the deuterated compound I is added to the biological sample solution, and then an extraction operation is performed. Accordingly, the method according to claim 20 or 22, wherein in the preparation of the standard working solution, the deuterated compound I is first added to a matrix solution containing metabolite II at known different concentrations, and then an extraction operation is performed.

25. The method according to claim 21 or 23, wherein the biological sample is a urine sample or a plasma sample, and accordingly, when the urine sample is measured, the corresponding matrix solution is urine from an untreated patient containing additives, and when the blood sample is measured, the corresponding matrix solution is plasma from an untreated patient containing anticoagulants.

26. The steps of adding and extracting the deuterated compound include adding the test solution and the standard working solution to the solution of the deuterated compound I, adding methanol and mixing uniformly, and obtaining the supernatant by centrifugation. Here, the additive is Na ２ HPO ４ or K ２ HPO ４ The anticoagulant is K ２ EDTA or Na ２ The method according to claim 25, wherein EDTA.

27. The aforementioned blood sample must be refrigerated at -20°C or below, or 4°C or below if processing is required, and injection detection must be completed within 54 hours. The method according to claim 25, wherein the urine sample must be stored in an environment of -70°C within 24 hours of collection, and if processing is required, injection detection must be completed within 94 hours at room temperature or below.

28. The aforementioned blood sample must be stored at -70°C within 4 hours of collection, and can be stored at -70°C for 28 days. The method according to claim 27, wherein the urine sample must be stored at -70°C within 4 hours of collection and can be stored at -70°C for 32 days.

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