Deuterated compounds and methods for their production and use

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 in particular to deuterated compounds, and methods for their preparation and use. [Background technology]

[0002] In human clinical trials, it is necessary to measure the contents of drugs and drug metabolites in the subjects' biological samples (blood, urine, tissues, etc.), and to conduct drug metabolism and pharmacokinetic (DMPK) studies based on the measured contents of drugs and drug metabolites. 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 taking advantage of the ultra-high separation power of high performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (UHPLC) and the detection capabilities of ultraviolet absorbance 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, C.C., Qi, T., Shia, C.S., Hsu, R.Y., Hsieh, Y.C., & Duan, J (2021)), a novel selective AKR1C3-activating prodrug AST-3424 / OBI-3424 exhibits broad antitumor activity (American Journal of Cancer Research, 11(7), 3645), and is the chemical moiety that exerts the activity of the prodrug AST-3424.

[0005] [ka]

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

[0007] The internal or external standard method used in conventional liquid chromatography cannot meet the requirements for quantitative analysis of drug metabolites in biological samples when the drugs are administered in small amounts. Therefore, it is necessary to develop an assay method that can not only meet the requirements for the lower limit of quantification, but also meet the requirements for DMPK testing of the above drugs. Summary of the Invention [Problem to be solved by the invention]

[0008] To achieve the above object, the present invention provides a deuterated compound, as well as its preparation method and use. It has been tested and confirmed that deuterated compound I can be used as an internal standard for the quantitative analysis of metabolite II in biological samples, with a minimum detection limit of 0.5ng / ml, which meets the requirements of DMPK testing.

[0009] Deuterated Compound I, which serves as a deuterated internal standard in DMPK analysis, has sufficient stability and can be stored for longer periods under experimental conditions (stable quality and characteristics when stored at -20°C and -70°C), and can meet the requirements for long-term storage of samples and various operating temperatures (ambient laboratory temperature) in DMPK laboratories used in clinical trials.

[0010] Generally speaking, the present invention is a system for measuring low content levels of metabolite II in biological samples, including a deuterated internal standard, an LC-MS / MS instrument and method, a curve-fitting algorithm for quantification, and an operation procedure, which actually meets the requirements of DMPK testing of the above metabolite II under the lower limit of quantification (since it exists in small amounts and needs to undergo centralized analysis, the sample must be stored for a longer period of time, and meets the actual requirements of laboratory operation).

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

[0012] The present invention is a deuterated compound I having the structure shown in formula (I). [ka] In the formula, A is 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(aziridin-1-yl)phosphinic acid or a salt thereof, and the salt-forming cation is an alkali metal such as Na+, K+, or an alkaline earth metal ion Ca2+, or an ammonium ion NH4+. Preferably, M is Na, K, Li, or an ammonium radical.

[0014] Depending on the different reagents added in the biological sample and subsequent manipulations, the deuterated compound may be present in an acid or salt form, and correspondingly, 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 a deuterated internal standard, the corresponding mass-to-nucleus ratio m / z in the mass spectrum is distinguishable from non-deuterated compounds. In fact, the mass spectrum peak is not a single value, but a mountain shape that descends on both sides around the main peak, and the abscissa value m / z of the main peak is the value of the compound set as x, and if the hydrogen atom (protium) in the compound is replaced by deuterium, the abscissa value m / z of the main peak is x+1. Since the main peak is a mountain shape, it is obvious that x and x+1 may overlap with each other, resulting in them being indistinguishable. According to the method of calculating the molecular weight of a compound (i.e., the method of summing the relative atomic masses of all atoms in the compound), it can be concluded that the more atoms there are, the more isotopic atomic species with different relative atomic masses of the corresponding atoms, and the more "robust" the shape of the main peak that descends on both sides will be, and the wider the overlap area with the main peak of another compound. One possible approach to narrow the overlap area is to extend the distance between two main peaks. For the deuterated compound, the number of deuterium atoms needs to be increased. Based on the individualized situation of the deuterated compound I, and through experimental verification and calculation, the present invention finds 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 the deuterated compound I has fewer D (deuterium) atoms, it is necessary to provide a mass spectrum with higher resolution.

[0016] Preferably, the number of D in said 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, said deuterated compound I is selected from compounds having the following structure: [ka] [ka]

[0019] The present invention also relates to a method for preparing a deuterated compound I, comprising the steps of: [ka] reacting compound Ia with phosphorus oxyhalide POX3 to obtain compound Ib; hydrolyzing said compound Ib in aqueous solution with or without a base to obtain the corresponding deuterated compound I; Including, The compound Ia is a deuterated 2-halogenated ethylamine or an inorganic acid salt, sulfate, phosphate, etc., where A is H or D, at least one of the four A's of the compound Ia is D, and at least one of the eight A's of the compound Ib and the compound I is D; the base in the hydrolysis reaction is selected from MOH (wherein M is an alkali metal, an alkaline earth metal, or an ammonium radical), MH (wherein M is an alkali metal), and MOR (wherein R is an alkyl group having 1 to 4 carbon atoms and M is an alkali metal, an alkali metal carbonate, or an alkali metal 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 give compound Ib. This reaction process may include one or more reactions.

[0021] The deuterated 2-haloethylamine or 2-haloethylamine inorganic acid salt that can be used is determined based on the solvent used in the reaction.

[0022] Since this reaction is a violently exothermic reaction, 2-haloethylamine or 2-haloethylamine hydrohalide is dissolved in a solvent and cooled. Then, an aqueous solution of phosphorus oxyhalide POX3 or phosphorus oxyhalide is slowly dripped and reacted with stirring (dropped so that the temperature of the reaction system is -78°C to -20°C). The solvent used in the reaction is an organic solvent, such as one or a mixture of two or more of dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, or cyclohexane.

[0023] The phosphorus oxyhalide POX3 includes phosphorus oxybromide POBr3 and phosphorus oxychloride POCl3. The deuterated 2-haloethylamine inorganic acid salt includes deuterated 2-haloethylamine hydrohalide (hydrochloride, hydrobromide), inorganic oxo acid salt (sulfate, phosphate, etc.), and preferably, the compound Ia is a deuterated 2-haloethylamine hydrochloride or hydrobromide.

[0024] When an acid is produced in the first reaction, a base is added to adjust the pH of the reaction. The base added includes inorganic and organic bases. The inorganic base is 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 a mixture of two or more of methylamine, ethylamine, propylamine, isopropylamine, N,N-diethylamine, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, tetramethylguanidine, pyridine, N-methyldicyclohexylamine, or dicyclohexylamine.

[0025] The reaction procedure includes a step of lowering the temperature of 2-haloethylamine or 2-haloethylamine hydrohalide in a solvent, a step of slowly dropping a solution of phosphorus oxyhalide POX3 or phosphorus oxyhalide POX3, further lowering the temperature, and a step of adding a base or a basic solution after lowering the temperature and reacting with stirring.

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

[0027] The reaction of the compound Ia with the phosphorus oxyhalide POX3 is carried out in an atmosphere, the atmosphere being one of air, nitrogen or argon, preferably the atmosphere being one of nitrogen or argon, more preferably the atmosphere being nitrogen.

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

[0029] The hydrolysis reaction must be carried out with the addition of water. Thus, the reaction is carried out with the participation of water. If only water is added without the addition of a base, M in the deuterated compound I after the reaction becomes H, and the compound is present in the corresponding acid form, and if a base is added, a salt is formed in the corresponding reaction. The base in the hydrolysis reaction is selected from MOH (M is an alkali metal, an alkaline earth metal or an ammonium radical), MH (M is an alkali metal), MOR (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] The present invention also relates to a method for measuring the content of deuterated compound I, that is, a method for measuring the content of the deuterated compound I using a 31P-NMR method, preferably a method for measuring the content of the deuterated compound I in a solution containing the deuterated compound I using a 31P-NMR method, or a method for measuring the content of the deuterated compound I using liquid chromatography, comprising: 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. Gradient elution uses mobile phases A and B, where mobile phase A is gradually increased from 15% to 90% by volume, and then mobile phase A is gradually decreased to 15% by volume.

[0031] The deuterated compounds are quantitatively analyzed after preparation and purification. There are various methods for quantitative analysis. The deuterated compounds can be directly weighed after purification or directly analyzed by HPLC.

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

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

[0034] The phosphorus-containing compound is preferably a compound containing one phosphorus atom, and more preferably hexamethylphosphoric acid triamide.

[0035] Preferably, the deuterated compound I and a known content of the phosphorus-containing compound are added to a solvent and examined together for their 31P-NMR spectra.

[0036] Preferably, the deuterated compound I and a known content of the phosphorus-containing compound are added to water and examined together for their 31P-NMR spectra after they are dissolved.

[0037] The deuterated compound I is quantitatively analyzed by using a phosphorus-containing compound as the internal standard of 31P-NMR. It is clear that the number of phosphorus atoms in the selected phosphorus-containing compound is preferably 1, so that the 31P-NMR spectrum has a relatively simple signal peak, which is convenient for quantification. In addition, the chemical shift of the 31P-NMR spectrum signal peak of the phosphorus-containing compound should be sufficiently distant from the chemical shift of the 31P-NMR spectrum signal peak of the deuterated compound I, so that the two signal peaks can be easily distinguished.

[0038] When analyzing the spectrum by 31P-NMR method, the number of scans has a certain effect on the 31P-NMR spectrum signal peaks of phosphorus-containing compounds as well as the 31P-NMR spectrum signal peaks of deuterated compound I. It has been demonstrated that the number of scans needs to be more than 64.

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

[0040] A "predrug" (also known as a prodrug, drug precursor, precursor drug, etc.) refers to a compound that has a pharmacological effect only after being converted in vivo. The prodrug itself is biologically inactive or has low activity, but becomes active after being metabolized in the body. The purpose of this process is to increase the bioavailability and targeting ability of the drug while decreasing its toxicity and side effects. Currently, prodrugs can be classified into two major families: carrier prodrugs and bioprecursors.

[0041] A DNA alkylating agent prodrug of the present invention refers to a prodrug that releases a DNA alkylating agent (ie, Metabolite II) after metabolism.

[0042] The present invention also 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 the metabolite II has a structure shown in formula (II). [ka] where A is H and M is H or an alkali metal, alkaline earth metal, or ammonium radical; The deuterated compound I is [ka] is selected from The metabolite II is [ka] is selected from.

[0043] The AKR1C3 activating DNA alkylating agent prodrug is selected from compounds having the structures shown in Formulas 1-5. [ka] In the formula, the definitions of R1, R2, R3, R4, R5, R8, R9, and R10 are as set forth in the claims of PCT / CN2020 / 089692 (International Publication No. 2020 / 228685).

[0044] Specifically, the groups are defined as follows: R1 is a C6-C10 aryl or Z-substituted aryl, a 4-15 membered heterocycle or Z-substituted heterocycle, a 5-15 membered heteroaryl or Z-substituted heteroaryl, or a 7-15 membered fused ring, or a Z-substituted fused ring; R2 is hydrogen, a halogen atom, cyano or isocyano, hydroxy, sulfhydryl, amino, OT, OMS, 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-C10 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, -CONR6R7, -SO2NR6R7, -SO2R6, -OCOO-R6, -COOR6, -NR6COR7, -OCOR6, -NR6SO2R7 or -NR6SO2NR6R7, or R2 together with the atoms in group R1 to which it is attached to form a 7-15 membered fused ring or Z-substituted fused ring; R3 is hydrogen, halogen, cyano or isocyano, hydroxy, sulfhydryl, amino, OT, 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-C10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR6R7, -SO2NR6R7, -SO2R6, -OCO-R6, -OCOO-R6, -COOR6, -NR6COR7, -OCOR6, or -NR6SO2R7; R4 and R5 are each independently hydrogen, a halogen atom, cyano or isocyano, hydroxy, sulfhydryl, amino, OT, 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-C10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR6R7, -SO2NR6R7, -SO2R6, -OCOO-R6, -COOR6, -NR6COR6, -OCOR6 or -NR6S02R7, or R4 and R5 together with the atoms in the benzene ring to which they are bonded to form a 7-15 membered fused ring or Z-substituted fused ring; R6 and R7 are each 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-C10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, or C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, or R6 and R7 together with the atoms to which they are attached to form a 5-7 membered heterocyclyl or Z-substituted 5-7 membered heterocyclyl; R8 and R10 are each 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 at least one of R8 and R10 must be hydrogen or deuterium; R9 is a substituted C6-C10 aryl substituted with at least one fluorine atom or nitro group, a substituted 4-15 membered heterocycle substituted with at least one fluorine atom or nitro group, or a substituted 5-15 membered heteroaryl substituted with at least one fluorine atom or nitro group; the substituent Z is a halogen atom, cyano or isocyano, hydroxy, sulfhydryl, amino, OT, 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, heterocyclic ring, aromatic heterocyclic ring and fused ring or substituted aromatic ring, heterocyclic ring, or aromatic heterocyclic ring and fused ring, the substitution pattern being mono- or geminal di-substitution; The substituent in R9 in the substituted C6-C10 aryl, substituted 4- to 15-membered heterocycle or substituted 5- to 15-membered heteroaryl is a halogen atom, 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 formula (1) and (2) [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0046] The definitions and meanings of the groups, as well as methods for preparing the compounds and spectral data, are described in PCT / CN2020 / 089692 (WO 2020 / 228685), which is incorporated herein by reference in its entirety.

[0047] It is clear that the compounds of structural formula 1 or 2, like AST-342, are prodrugs (acid forms) of AST-2660, and can be activated by the AKR1C3 enzyme to form AST-2660 and exhibit their anticancer effects.

[0048] [ka] [ka]

[0049] It is understood that the compounds include compounds of formula 1 or 2, as well as salts, esters, solvates, and isotopic isomers thereof.

[0050] [ka] In the formula, the definition of Rw is set forth 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, C1-6 alkyl, C3-6 cycloalkyl, 4- to 6-membered heterocycloalkyl, 5- to 6-membered heteroaryl or phenyl group, wherein the C1-6 alkyl, C3-6 cycloalkyl, 4- to 6-membered heterocycloalkyl, 5- to 6-membered heteroaryl and phenyl group are optionally substituted with 1, 2 or 3 Ra; each Ra is independently H, F, Cl, Br, I, -CN, -OH, a C1-3 alkoxy group or a C1-3 alkyl; R2 is H or C1-6 alkyl; or R and R together with N which combine to form a 4- to 6-membered heterocycloalkyl, said 4- to 6-membered heterocycloalkyl being optionally substituted with 1, 2 or 3 R; each Rb is independently H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3, or -CH2CH3; R3 is H, F, Cl, Br, I, -OH, -NH2, a C1-3 alkoxy group or a C1-3 alkyl; or R2 and R3 are bonded together to form the structural unit [ka] of, [ka] Let us assume that. T1 is -(CRcRd)m- or -(CRcRd)nO-, m is 1, 2 or 3; n is 1 or 2; T2 is N or CH; Rc and Rd are each independently H, F, a C1-3 alkyl or a C1-3 alkoxy group; R4, R5 and R6 are each independently H, F, Cl, Br, I, a C1-3 alkyl or C1-3 alkoxy group; T is N or CH; R7 and R8 are each independently H, F, Cl, Br or I; R9 and R10 are each independently H, F, Cl, Br, I, or -CN; The 4- to 6-membered heterocycloalkyl and the 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: [ka]

[0053] The definitions and meanings of the groups, as well as methods for preparing the compounds and spectral data, are described in PCT / CN2020 / 120281 (WO 2021 / 068952), the entire contents of which are incorporated herein by reference.

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

[0055] [ka] In the formula, the definitions of X, Y, Z, R, A and X10 are as described in the claims of PCT / US2016 / 021581 (corresponding to International Publication No. 2016 / 145092 (Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556))), and T is [ka] It is.

[0056] Specifically, the groups are defined as follows: X10 is O, S, SO or SO2; A is C6-C10 aryl, 5-15 membered heteroaryl, or -N=CR1R2; R1 and R2 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, a 4-15 membered heterocycle, an ether, -CONR13R14, or -NR13COR14; X, Y, and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, a 4-15 membered heterocycle, an ether, -CONR13R14, or -NR13COR14; R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, ether, -CONR13R14, or -NR13COR14; R13 and R14 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl. aryl, C6-C10 aryl, 4-15 membered heterocycle, or ether; T is [ka] wherein the alkyl group, the alkenyl group, the alkynyl group, the cycloalkyl group, the aryl group, The heterocyclic, heteroaryl, and ether groups may be optionally substituted.

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

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

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

[0060] [ka] In the formula, A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl, or -N=CR1R2, wherein the substituents are selected from the group consisting of halogeno, -CN, -NO2, -O-(CH2)-O-, -CO2H and salts thereof, -OR100, -CO2R100, -CONR101R102, -NR101R102, -NR100SO2R100, -SO2R100, -SON2NR101R102, C1-C6 alkyl, and C3-C10 heterocyclyl; In the formula, R100, R101 and R102 are each independently hydrogen, C1-C8 alkyl, or C6-C12 aryl, or R101 and R102 together with the nitrogen atom to which they are bonded form a 5- to 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; R1 and R2 are each independently phenyl or methyl; X, Y and Z are each independently hydrogen or halogen; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl.

[0061] "Cx-Cy" or "Cx-y" before a group refers to the range of numbers of carbon atoms present in the group. For example, C1-C6 alkyl refers to an alkyl group having at least 1 and up to 6 carbon atoms.

[0062] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, in some embodiments 1 to 6 carbon atoms. "Cx-y alkyl" refers to an alkyl group having from x to y carbon atoms. This term includes (by way of example) straight-chain and branched-chain 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 6-14 carbon atoms, no ring heteroatoms, and either a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). For polycyclic systems, including fused, bridged, and spiro ring systems, with aromatic and non-aromatic rings that have no ring heteroatoms, "aryl" or "Ar" applies when the point of attachment is at an aromatic carbon atom (e.g., 5,6,7,8 tetrahydronaphthalen-2-yl is an aryl group where the point of attachment is at the 2-position of the aromatic phenyl ring). "Arylene" refers to a divalent aryl group with the appropriate hydrogen content.

[0064] "Cycloalkyl" refers to saturated or partially saturated cyclic groups having 3 to 14 carbon atoms, no ring heteroatoms, and monocyclic or polycyclic rings, including fused ring systems, bridged ring systems, and spiro ring systems. For polycyclic systems having aromatic and non-aromatic rings with no ring heteroatoms, "cycloalkyl" applies when the point of attachment is at a non-aromatic carbon atom (e.g., 5,6,7,8,-tetrahydronaphthalen-5-yl). "Cycloalkyl" includes cycloalkenyl groups. Cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl. "Cycloalkylene" refers to divalent cycloalkyl groups with the appropriate hydrogen content.

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

[0066] "Heteroaryl" refers to an aromatic group having 1-14 carbon atoms and 1-6 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, including single ring (e.g., imidazolyl-2-yl and imidazol-5-yl) and polycyclic systems (e.g., imidazopyridyl, benzotriazolyl, benzimidazol-2-yl, and benzimidazol-6-yl). For polycyclic systems, including fused, bridged, and spiro ring systems having aromatic and non-aromatic rings, "heteroaryl" applies when at least one ring heteroatom is present and the point of attachment is to an atom of the aromatic ring (e.g., 1,2,3,4-tetrahydroquinolin-6-yl and 5,6,7,8-tetrahydroquinolin-3-yl). In some embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group may be oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties.Examples of heteroaryl include acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzothienyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazopyridyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazo These include, but are not limited to, aryl, oxazolidinyl, oxazolyl, pyrimidinyl, phenanthridinyl, phenanthrinyl, phenanthrinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, triazinyl and xanthenyl. "Heteroarylene" refers to a divalent heteroaryl group having appropriate hydrogen content.

[0067] "Heterocyclic" or "heterocycle" or "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially saturated cyclic group having 1-14 carbon atoms and 1-6 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, including monocyclic and polycyclic systems, including fused, bridged, and spiro ring systems. For multiple ring systems having aromatic and / or non-aromatic rings, "heterocycle", "heterocycle", "heterocycloalkyl" or "heterocyclyl" applies when at least one ring heteroatom is present and the point of attachment is to an atom of the non-aromatic ring (e.g., 1,2,3,4-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-6-yl, and decahydroquinolin-6-yl). In some embodiments, the heterocyclic groups herein are 3-15 membered, 4-14 membered, 5-13 membered, 7-12 membered, or 5-7 membered heterocycles. In some embodiments, the heterocycle contains 4 heteroatoms. In some embodiments, the heterocycle contains 3 heteroatoms. In some embodiments, the heterocycle contains up to 2 heteroatoms. In some embodiments, the nitrogen and / or sulfur atoms of the heterocycle group may be oxidized to provide N-oxide, sulfumyl, or sulfonyl moieties. Heterocyclyl includes, but is not limited to, tetrahydropyranyl, piperidinyl, N-methylpiperidin-3-yl, piperazinyl, N-methylpyrrolidin-3-yl, 3-pyrrolidinyl, 2-pyrrolidon-1-yl, morpholinyl, and pyrrolidinyl. A prefix indicating the number of carbon atoms (e.g., C3-10) refers to the total number of carbon atoms in the portion of the heterocyclyl group excluding the number of heteroatoms. Divalent heterocyclic groups have the hydrogen content appropriately adjusted.

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

[0069] "Optionally substituted" refers to a group that is substituted or unsubstituted. The 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+, -CO2R100, -OR100, -SR100, -SOR100, -SO2R100, -NR100SO2R100, -NR101R102, -CONR101R102, -SON2NR101R102, C1-C6 alkyl, C1-C6 alkoxy, -CR100=C(R100)2, -CCR100, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C6-C12 aryl and C2-C12 heteroaryl, or divalent substituents thereof (e.g., -O-(CH2)-O-, -O-(CH2)2-O-, and and 1-4 methyl substituted groups), R100, R101 and R102 are each independently selected from hydrogen or C1-C8 alkyl, C3-C12 cycloalkyl, C3-C10 heterocyclyl, C6-C12 aryl, or C2-C12 heteroaryl, or R101 and R102 together with the nitrogen atom to which they are bonded form a 5-7 membered heterocycle, and each alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl may be substituted with 1-3 halogen, 1-3 C1-C6 alkyl, 1-3 C1-C6 haloalkyl or 1-3 C1-C6 alkoxy groups. It is preferred that the substituents are selected from the group consisting of chloro, fluoro, -OCH3, methyl, ethyl, iso-propyl, cyclopropyl, -CO2H and their salts and C1-C6 alkyl esters, CONMe2, CONHMe, CONH2, -SO2Me, -SO2NH2, -SON2NMe2, -SON2NHMe, -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] is selected from the group consisting of:

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

[0072] It is clear that the compound of formula 6, like AST-3424, is a prodrug of AST-2660, which can be activated by AKR1C3 enzyme to form AST-2660 and exhibit its anti-cancer effect. [ka]

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

[0074] Specifically, the groups are defined as follows: Cx is a 5- to 10-membered aromatic ring or aromatic heterocycle, alicyclic heterocycle, or cycloalkane that forms a condensed ring structure with a nitrobenzene ring by sharing two carbon atoms; R1 bonded to any skeletal atom of the Cx ring is selected from hydrogen, halogen atoms, cyano or isocyano, hydroxyl, mercapto, amine, OT, 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-C10 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, -CONR6R7, -SONR6R7, -SO2R6, -OCOO-R6, -COOR6, -NR6COR7, -OCOR6, -NR6SO2R7, and -NR6SO2NR6R7; R2 and R3 are each 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-C10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, or R2 and R3, together with the carbon atoms of benzyl which are bonded to form a 3-6 membered ring; [ka] The group can replace a hydrogen atom at any position on the carbon atom of the fused ring, and the number of replacements is one. Z-substituent is a halogen atom, a cyano or isocyano group, a hydroxyl group, a mercapto group, an amino group, a C1-C3 alkyl group or a substituted alkyl group, a C1-C3 alkoxy group or a substituted alkoxy group, a C2-C3 alkenyl group or a substituted alkenyl group, a C2-C3 alkynyl group or a substituted alkynyl group, a C3-C8 cycloalkyl group or a substituted cycloalkyl group; R6 and R7 are each 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, C6-C10 aryl or Z-substituted C6-C10 aryl, 4-15 membered heterocyclyl or Z-substituted 4-15 membered heterocyclyl, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R6 and R7 together with the atoms to which they are bonded to form a 5-7 membered heterocyclyl or Z-substituted 5-7 membered heterocyclyl.

[0075] Specific definitions and meanings of groups, as well as methods for preparing the compounds and spectral data, are described in PCT / CN2020 / 114519 (WO 2021 / 120717), the entirety of which is incorporated herein by reference.

[0076] Specifically, the compound of formula (6) is [ka] [ka] [ka] is selected from the group consisting of In the formula, the definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, and R17 are as described 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 groups are defined as follows: R1 is hydrogen, -N3, CN, halogen, NR21R22, -OR23, -SO2(C1-C6 alkyl), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, or ether; R21 and R22 are each independently hydrogen, hydroxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, or -SO2(C1-C6 alkyl), or R21 and R22, together with the nitrogen atom to which they are attached, form a 4-15 membered heterocycle or a 5-15 membered heteroaryl; R23 is hydrogen, C1-C6 alkyl, or C6-C10 aryl; R2 and R3 are independently hydrogen or halogen; R4 is hydrogen, halogen, C1-C6 alkoxy, C1-C6 alkyl, or C6-C10 aryl; R5, R7, R9, R12 and R15 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, or R4 and R5 together with the carbon atom between them forming a C5-C6 cycloalkyl ring; R6 and R10 are independently hydrogen or halogen; R8 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or 5-15 membered heteroaryl; each R11 is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, or C6-C10 aryl; R13, R14, R16, and R17 are independently hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C6 alkoxy; The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, alkoxy and ether groups may be substituted.

[0078] Specific definitions and meanings of groups, as well as preparation methods and spectral data of the compounds 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 is incorporated herein by reference in its entirety.

[0079] In particular, the compounds of formulae (7) to (12) are selected from the compounds specifically disclosed in the above mentioned patent applications.

[0080] It is clear that the "compound" in the above formulas 1-12 disclosed herein includes the compound itself, as well as solvates, salts, esters, or isotopic isomers thereof.

[0081] The present invention also provides a method for measuring the content of a metabolite in a biological sample, comprising the steps of: preparing a test solution containing a known concentration of an internal standard compound for injection analysis by LC-MS / MS; preparing a series of standard working solutions containing a known concentration of an internal standard compound and a known concentration of Metabolite II, wherein the concentration of the internal standard compound in the series of standard working solutions is consistent and is the same as the concentration of the internal standard compound in the test solution, and the concentration of Metabolite II in the series of standard working solutions is different; Absorbing the prepared standard working solutions of metabolite II with different concentrations, injecting them into a detection LC-MS / MS system to obtain a relationship 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 relationship function by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A step of absorbing the test solution to which the internal standard compound of known concentration has been added, injecting the test solution into the LC-MS / MS system for detection to obtain a ratio y between the peak area of ​​the metabolite II and the peak area of ​​the internal standard compound, and substituting the ratio into a function y=f(x) to obtain the concentration x of the metabolite II in the test solution, and determining and calculating the concentration of the metabolite II in the test solution of unknown concentration using a relational function; Including, The test solution is prepared from a biological sample, whether or not it has been treated; The internal standard compound is a deuterated compound I having the structure shown in formula (I), [ka] In the formula, A is 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 metabolite II has the structure shown in formula (II): [ka] where A is H; M is H or an alkali metal, alkaline earth metal, or ammonium radical.

[0082] The present invention also provides a method for measuring the content of a metabolite in a biological sample, comprising the steps of: adding a quantitative internal standard compound to the test biological sample solution to obtain a test solution after extraction treatment; Dilute the metabolite II standard substance to obtain a series of metabolite II solutions with different concentrations, add the quantitative internal standard compound to obtain a standard working solution after extraction processing, absorb the series of standard working solutions respectively and inject them into a detection LC-MS / MS system to obtain the peak areas of the metabolite II and the internal standard compound, create a standard curve with the ratio of the peak area of ​​the metabolite II to the peak area of ​​the internal standard compound as the vertical axis and the concentration of the metabolite II as the horizontal axis, and calculate a regression equation; absorbing the test solution and injecting it into the LC-MS / MS system for detection to obtain a ratio of the peak area of ​​the metabolite II in the test solution to the peak area of ​​the deuterated compound I, and substituting the ratio into the regression equation to obtain the content of the metabolite II in the test solution; Including, The internal standard is a deuterated compound I having the structure shown in formula (I), [ka] In the formula, A is 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 metabolite II has the structure shown in formula (II): [ka] where A is H; M is H or an alkali metal, alkaline earth metal, or ammonium radical.

[0083] Preferably, the liquid chromatography conditions in the liquid chromatography-tandem mass spectrometry method 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. Gradient elution was performed using mobile phases A and B, with mobile phase A gradually increasing from 15% to 90% by volume, then gradually decreasing mobile phase A to 15% by volume. Mass spectrometry conditions: electrospray ion source In negative ion scanning mode, Monitoring ion pair for metabolite II: m / z 147.0 → m / z 62.9 Monitoring ion pair for deuterated compound I: m / z (147.0 + deuteration number) → m / z 62.9, or In positive ion scanning mode, Monitoring ion pair for metabolite II: m / z 149.0 → m / z 64.9 The monitoring ion pair of deuterated compound I is: m / z (149.0 + deuteration number) → m / z 64.9.

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

[0085] Preferably, in preparing a test solution containing a known concentration of a 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 preparing 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, and correspondingly, if a urine sample is measured, the corresponding matrix solution is untreated patient urine containing an additive, and if a blood sample is measured, the corresponding matrix solution is untreated patient plasma containing an anticoagulant.

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

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

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

[0090] The urine samples should be stored at -70°C within 24 hours, preferably within 4 hours, of collection, and can be stored at -70°C for 32 days, and if processed, injection detection should be completed within 94 hours at room temperature or below. [Brief description of the drawings]

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

[0092] Unless otherwise defined, all technical or scientific terms used in one or more embodiments of the present specification have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0093] The experimental methods in the following examples are conventional methods unless otherwise specified. All medicinal materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0094] AST-3424, developed by the present applicant, is a prodrug of the DNA alkylating agent AST-2660, which is highly expressed in cancer cells and is specifically activated by the AKR1C3 enzyme, which is metabolized to AST-2660, to exert its pharmacological effect. As shown in the background art, in human clinical trials, it is necessary to measure the contents of drugs and drug metabolites in the subjects' biological samples (blood, urine, tissue, etc.), and further conduct drug metabolism and pharmacokinetic (DMPK) tests based on the measured contents of drugs and drug metabolites. However, AST-3424 is used in clinical trials in a relatively small amount of 1 mg to 100 mg. The internal standard method or external standard method used in conventional liquid chromatography cannot meet the requirements for quantitative analysis at the lower limit of quantification. Therefore, it is necessary to develop an assay method that can meet the requirements for the lower limit of quantification.

[0095] In order to solve the above technical problems, the applicant has attempted to quantify low contents of AST-2660 in biological samples using an internal standard quantitative method. The internal standard method is a fairly accurate quantitative method in chromatographic analysis. The internal standard method is a method in which a certain amount of a pure substance is added to a certain amount of a sample mixture to be analyzed, the sample containing the internal standard substance is analyzed by chromatography, the peak area of ​​the internal standard and the measured component are measured, respectively, and the percentage of the measured component in the sample is calculated. The selection of the internal standard is very important. 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. In addition, the internal standard should have chemical and physical properties (such as chemical structure, polarity, volatility and solubility in solvents), chromatographic behavior, and response properties that are substantially the same as or as close as possible to those of the analyte, preferably a homologue of the analyte. Indeed, the internal standard must be sufficiently separated from each component in the sample under chromatographic conditions. The selection of the internal standard is very important for quantitative analysis of the internal standard method. It must satisfy the following requirements: 1. The internal standard has similar physical and chemical properties (such as boiling point, polarity, and chemical structure) to those of the analyte. 2. An internal standard is a pure substance that is not present in the test sample. 3. The internal standard is completely dissolved in the test sample (or solvent) and does not chemically react with the test sample, and 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 component being measured. 5. The position of the chromatographic peak of the internal standard is close to the position of the chromatographic peak of the measured components or is in the middle of the positions of the chromatographic peaks of several measured components without co-overflow. The purpose is to avoid sensitivity differences caused by instrument instabilities. 6. The appropriate amount of internal standard added is selected to ensure that the matching of peak areas 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 the present application, a large number of similar compounds of AST-2660 were screened, and deuterated compound I was finally screened as an internal standard for the detection of metabolite II. The detection limit of the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established using deuterated compound I is as low as 0.5ng / ml. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method established using deuterated compound I according to the present application can be used to measure the content of metabolite AST-2660 in human plasma and urine. This method meets the analytical requirements of biological samples, with simple and convenient sample processing methods, high sensitivity, precision, and accuracy. EXAMPLES

[0097] The technical solutions of the present invention are described below in specific examples. 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 [ka]

[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, then phosphorus oxychloride was added), cooled to -78°C, and triethylamine (348 mg, 3.44 mmol) in dichloromethane solution (2 ml) was added dropwise. The reaction mixture was kept at -78°C for 30 minutes, allowed to warm to 0°C naturally, and then kept at 0°C for 4 hours. The solid was then quickly filtered off with suction. The mother liquor was concentrated at low temperature and used as it was in the next step.

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

[0102] FIG. 1 is a 31P-NMR spectrum of AST-2660-D8-sodium salt.

[0103] It is clear that in the above procedure, by selecting different deuterated raw compound Ia (deuterated 2-bromoethylamine), different intermediate Ib can be synthesized, and then different bases (NaOH, KOH, LiOH or aqueous ammonia) are added in the hydrolysis reaction to obtain different deuterated bis(aziridin-1-yl)phosphinic acids or their salts.

[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, and the feeding order and the amount of reactants can be changed. It is clear that such an operation is equivalent to the above reaction from compound Ia to compound Ib being completed in one step. Under nitrogen protection, deuterated 2-bromoethylamine hydrobromide and phosphorus oxychloride were added to anhydrous dichloromethane (phosphorus oxychloride was added first, and then the first deuterated 2-bromoethylamine hydrobromide was added. At that time, the molar ratio of phosphorus oxychloride to the deuterated 2-bromoethylamine hydrobromide was greater than 1), cooled to -78°C, and triethylamine in dichloromethane solution was added dropwise. The reaction mixture was kept at -78°C for 30 minutes, and then the second deuterated 2-bromoethylamine hydrobromide was added. Triethylamine in dichloromethane solution was added dropwise. The reaction mixture was kept at -78°C for 30 minutes, allowed to warm to 0°C naturally, and then kept at 0°C for 4 hours. The solid was then quickly filtered off with suction. The mother liquor was concentrated at low temperature and used directly in the next step.

[0106] The crude product obtained in the above step was dissolved in water, and solid sodium hydroxide was slowly added in portions. The reaction was stirred at room temperature overnight. 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 hydrobromide salts 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 (hexamethylphosphoric triamide) was used as an internal standard for phosphorus spectrum to measure the content of 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 (mmol / mg, 31P-NMR nuclear magnetic shift was 29.890 ppm).

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

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

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

[0113] Since the amount of HMPA was found to be 0.02235 mmol, the amount of AST-2660-D8-sodium salt was calculated to be 0.005743 mmol from the integrated peak area ratio in the nuclear magnetic spectrum, and the concentration was 1.177×10-5 mmol / mg, i.e., 1.177×10-2 mmol / g, with a corresponding mass content of 2.10 mg / g. Similarly, the phosphorus spectra for 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. Thus, the concentrations of the same sample were 1.209×10-5 mmol / mg, 1.227×10-5 mmol / mg, and 1.209×10-5 mmol / mg, respectively, i.e., 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 quantitative method in this embodiment is relatively fast and simple, has acceptable accuracy within a certain range, and can be replaced by HPLC yield analysis.If the accuracy of quantitative determination needs to be further improved, HPLC method is used.External standard method is used for quantitative determination, and absolute content is accurately determined using standard curve.

[0115] The LC-MS / MS methods of Example 5 and Example 7 are suitable for detecting low contents of AST-2660-D8 and AST-2660, and their corresponding LC liquid-phase methods (where the corresponding MS / MS detectors are replaced by conventional differential detectors, electrospray detectors, and evaporative light scattering detectors) are also suitable for detecting constant contents (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 examined by detecting the content of AST-2660-D8-sodium salt in the aqueous solution. 31P-NMR was used for detection, and the content of AST-2660-D8-sodium salt in the aqueous solution was characterized by comparing the area ratio of the 31P peak of HMPA as an internal standard with the area ratio of the 31P peak of AST-2660-D8-sodium salt within 48 hours to determine whether the mass of the sample solution had decreased due to decomposition.

[0117] 1.Analysis method 31P-NMR of the sample was performed on 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 5.72×10-5 mmol / mg. A solution of AST-2660-D8-sodium salt (588 mg, about 0.5 ml) and a solution of HMPA (293 mg, 1.676×10-2 mmol) were mixed together as a sample for 31P-NMR analysis at different times under storage conditions of -20°C. The ratio of the integrated peak area of ​​the corresponding nuclear magnetic peak of HMPA (about 29.874 ppm) to the integrated peak area of ​​the nuclear magnetic peak of AST-2660-D8-sodium salt (about 24.491 ppm) in the nuclear magnetic spectrum was calculated, and the integrated peak of the nuclear magnetic peak of HMPA was set to 1.

[0118] Using the same procedure, samples of different concentrations were prepared for detection and subjected to 31P-NMR analysis at different times under storage conditions at -78°C.

[0119] 2. Stability results when stored at -20℃~-78℃ 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 storage conditions at -20°C, the ratio of the peak area of ​​the 31P-NMR peak of HMPA to the peak area of ​​the 31P-NMR peak of the AST-2660-D8 sodium salt solution at 8, 16, 24, 36, and 48 hours 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 is considered that the AST-2660-D8 sodium salt solution was stable when stored at -20°C to -78°C for 48 hours.

[0122] Representative spectra are shown in FIGS.

[0123] FIG. 3 is a 31P-NMR spectrum at time 0, showing, from left to right, an AST-2660-D8 sodium salt solution at −20° C. and an AST-2660-D8 sodium salt solution at −78° C.

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

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

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

[0127] FIG. 5 shows the 31P-NMR spectrum of the AST-2660-D8 sodium salt solution after storage at −20° C. for 6 months.

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

[0129] The 31P-NMR spectrum after 6 months is shown in FIG.

[0130] FIG. 6 shows the 31P-NMR spectrum of the AST-2660-D8 sodium salt solution after storage at −78° C. for 6 months.

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

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

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

[0134] 1. LC-MS / MS Experimental Method Relevant parameters of 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 solutions AST-2660 (prepared by selecting non-deuterated raw materials with reference to Example 1) standard stock solution (methanol solution with a concentration of 1.0 mg / ml): Take an appropriate amount of AST-2660, add an appropriate amount of methanol, dilute, and mix thoroughly to obtain a 1.0 mg / ml solution.

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

[0140] 2.2 Preparation of standard curve 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 according to the following table using normal pooled human plasma containing anticoagulant (K2EDTA) as diluent and blank matrix.

[0141] [Table 5]

[0142] 2.3 Preparation of internal standard working solution (standard working solution) AST-2660-D8 stock solution (AST-2660-D8 methanol solution at 50μg / ml): Take an appropriate amount of AST-2660-D8 reference material, add an appropriate amount of methanol, dilute, and mix thoroughly to obtain a 50μg / ml solution. Take 20μl of AST-2660-D8 internal standard stock solution, add 9980μl of methanol, dilute, and mix thoroughly to obtain a 100ng / ml internal standard working solution.

[0143] 2.4 Preparation of quality control samples AST-2660 quality control stock solution (1.0 mg / ml solution in methanol): Take an appropriate amount of AST-2660 reference material, add an appropriate amount of methanol, dilute, and mix thoroughly to obtain a 1.0 mg / ml solution. Take 100 μl of AST-2660 QC stock solution and 9990 μl of methanol 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 according to the table below.

[0145] [Table 6]

[0146] 3. Sample pretreatment The steps for extracting a sample using protein precipitation are as follows. Standard curve samples, quality control samples, and plasma samples are labeled and sequenced. 2. Place all samples on a multi-tube vortexer for uniform vortexing after thawing. 3. Take 100 μl from each sample and add to a 96 well plate. 4. In addition to the blank sample, add 20 μl of AST-2660-D8 quality control working solution deuterated internal standard (ie, 100 ng / ml AST-2660-D8 in methanol) 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. 7. Centrifuge the 96-well plate at 2143 rcf (centrifugal force) for 10 minutes. 8. Transfer 450 μl of the supernatant to a new 96-well plate. 9. Dry the supernatant in a 96-well Termovap sample concentrator. 10. Redissolve in 150 μl of methanol. 11. Sonicate the plate for 30 seconds. 12. Centrifuge the plate at 4°C and 2143rcf (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 of 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 (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 above, the linear relationship was good in the linear range of 0.5 to 200 ng / ml.

[0148] 4.2 Accuracy and precision test results for quality control samples Accuracy and precision are defined as follows: Accuracy is expressed as the relative error (RE). RE = [(average value - nominal value) / nominal value] x 100 Precision represents the coefficient of variation (CV). CV = (standard deviation / mean value) x 100

[0149] The accuracy and precision test results were shown in Table 7. From Table 7, it can be seen that the intra-assay and inter-assay precision were both less than 8.3%, and the intra-assay and inter-assay accuracy were within ±8.7. The analytical criteria for accuracy and precision were as follows: For the LLOQ QC samples, the intra-assay and inter-assay precision (RE) should be within ±20.0%, and the intra-assay and inter-assay CV should not exceed 20.0%. The intra-assay and inter-assay precision (RE) should be within ±15.0% for the low, geometric mean, medium, high, and dilute QC (if applicable) samples, and the intra-assay and inter-assay precision (CV) should 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 less than or equal to 40.0%, meeting the requirements.

[0150] [Table 7]

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

[0152] From Table 8, it can be seen that the CVs of the extraction recoveries of AST-2660 and the internal standard AST-2660-D8 were all less than 15.0%. The analytical criteria for extraction recoveries were as follows: the CVs of the reactions measured at each concentration level for the post-extraction spiked samples must be less than or equal to 15.0%. Therefore, the extraction recoveries of AST-2660 and the internal standard AST-2660-D8 met the requirements.

[0153] 4.4 Test results for matrix effects [Table 9]

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

[0155] 4.5 Stability [Table 10]

[0156] From Table 10, it can be seen that the accuracy of the stability of AST-2660 working solution and 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 storage stable solution and the freshly prepared solution should be within ±10.0%, and the CV of each replicate should not exceed 10.0%. Therefore, both AST-2660 and AST-2660-D8 (internal standard) working solutions had good stability and met the requirements under various storage conditions.

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

[0158] [Example 5] Detection of AST-2660 content in real human plasma The detection method established in Example 4 was used to detect the content of AST-2660 in the test human plasma.

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

[0160] Step 2. Extract the sample The test solution, standard working solution and quality control sample solution were thawed at room temperature and mixed uniformly. 100 μl of each of the above solutions was taken, 20 μl of internal standard and 500 μl of methanol were added, vortexed for 3 minutes to mix uniformly, and then centrifuged at 2143 rcf for 10 minutes. 450 μl of the supernatant was taken, dried, 150 μl of methanol was added for redissolution, and sonicated. The plate was then centrifuged at 4°C for 3 minutes. 200 μl of the supernatant was taken.

[0161] Blank samples were obtained by collecting 100 μl of blank blood, adding 20 μl of methanol, and extracting in the same manner.

[0162] 100 μl of blank blood was collected, to which 20 μl of the internal standard working solution was added, and extracted in the same manner to obtain a zero concentration sample.

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

[0164] Step 3. Setting up chromatography-mass spectrometry test conditions The 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 content of ammonium acetate, and the mobile phase B was acetonitrile. Gradient elution was performed according to the table below, with a column temperature of 20° C. and an injection volume of 5 μl.

[0165] [Table 11]

[0166] Mass spectrometry conditions were as follows: electrospray ion source in negative ion scanning mode, spray potential: -4500 V, ion source temperature: 500 °C, collision energy (CE): -22 eV, declustering potential (DP): -55 V, entrance potential (EP): -10 V, collision chamber exit potential (CXP): -16 V, residence time: 100 ms, high purity nitrogen was 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 collected and injected into LC-MS / MS for detection.

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

[0169] The prepared standard working solutions of metabolite II with different concentrations were absorbed and injected into the LC-MS / MS system for detection, and the relationship function y = f(x) was obtained (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).

[0170] The calculation results were as follows. The relationship function y=f(x) of 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 obtained from the test solution after extraction and substituted into the relationship function y=f(x) to calculate the concentration of metabolite II in the test solution.

[0171] The relationship function was used to measure and calculate the concentration of metabolite II in the test solution with an unknown concentration. The test solution with a known content of the internal standard compound was absorbed and injected into the 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 calculated and substituted into the function y = f(x) to obtain the concentration x of metabolite II in the test solution.

[0172] Optionally, the sequence of the operational steps related to the above SOP may be slightly modified to obtain the SOP for the following operations: A quantitative internal standard compound is added to a test biological sample solution, which is then subjected to an extraction process to obtain a test solution; A series of solutions of metabolite II with different concentrations are obtained by diluting the metabolite II standard substance, and a quantitative internal standard compound is added to obtain standard working solutions after extraction treatment. The series of standard working solutions are absorbed respectively, and then injected into the LC-MS / MS system for detection, and the peak areas of metabolite II and the internal standard compound are obtained. A standard curve is drawn with the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound as the vertical axis and the concentration of metabolite II as the horizontal axis, and the regression equation is calculated. The test solution was absorbed and injected into the LC-MS / MS system for detection, and the ratio of the peak area of ​​metabolite II in the test solution to the peak area of ​​deuterated compound I was calculated and substituted into the regression equation to determine the content of metabolite II in the test biological sample solution.

[0173] Solutions of the same concentration were prepared by the modified standard operating procedure described above, by using containers of the same volume, according to the practice of the laboratory operators, so long as the volumes of solutions added were the same and the resulting standard working solutions remained at the same concentration.

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

[0175] FIG. 7 is a representative LC-MS / MS spectrum of AST-2660 and the internal standard AST-2660-D8 in a blank human plasma extract.

[0176] FIG. 8 is a representative LC-MS / MS spectrum of AST-2660 and the internal standard AST-2660-D8 in a plasma sample extract at 0% concentration.

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

[0178] FIG. 10 shows a representative LC-MS / MS spectrum 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 by the procedure for detecting AST-2660 content in human plasma provided in this example, and the results are shown in Tables 12 to 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 storage at -70° C. for 28 days, but there was a significant decrease at -20° C. Therefore, human plasma samples containing AST-2660 should be stored at -70° C. as much as possible, and under such conditions the samples remained unchanged after storage for 28 days.

[0182] [Table 13]

[0183] As is evident from the table, human plasma containing AST-2660 showed little decrease in AST-2660 content during freeze-thaw treatment from -70°C to room temperature, but there was a significant decrease during freeze-thaw treatment from -20°C to room temperature. In other words, the data in Table 12 show that the rapid temperature change from the storage condition of the human plasma samples stored at -70°C to room temperature for experimental treatment 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, the methanol solution of AST-2660-D8 has strong stability after being stored at -70°C for 26 days under the same conditions as AST-2660.

[0186] [Table 15]

[0187] As can be seen from the data in Table 15, the content of AST-2660 in methanol solution was stable after storage for 54 hours under conventional freezing conditions (freezers commonly used in laboratories).

[0188] [Table 16]

[0189] As is evident from the data in Table 16, human plasma samples containing AST-2660 are not stable at room temperature. The content of AST-2660 in human plasma samples decreases with the prolongation of storage time, suggesting that when collecting human plasma samples in the clinical patients, the collected plasma samples should be immediately stored in the above experimentally confirmed low temperature environment, i.e., storage at -70°C is the safest, storage at -20°C is also acceptable, and when storage conditions are limited, they should be stored in a conventional refrigerator (4-0°C) and transferred to the above low temperature environment within 4 hours. During the transportation of samples to the DMPK sample testing center laboratory, the whole process of cold chain logistics (the temperature should be monitored during the whole process) should be used, where the temperature should be selected to be below -20°C, or dry ice should be used for insulation.

[0190] From the above experimental results, the following can be seen. (1) After collection, human plasma samples containing AST-2660 should be stored immediately under the above experimentally confirmed low temperature environment, i.e., storage at -70°C is the safest, and storage at -20°C is also acceptable. In addition, if storage conditions are limited, they should be stored in a conventional refrigerator (4-0°C) and transferred to the above low temperature environment within 4 hours. During the transportation of samples to the DMPK sample testing center laboratory, the entire process of cold chain logistics (the temperature should be monitored during the entire process) should be used, where the temperature should be selected to be below -20°C, or dry ice should be used for insulation. (2) The samples should be stored at temperatures below -70°C and were stable for 28 days under these conditions, and may be stable for longer periods depending on the change trends. (3) The process of removing the stored samples to room temperature, i.e., the sudden temperature change from the storage conditions of the samples to room temperature for experimental processing in human plasma stored at -70°C, did not affect the stability of AST-2660 in the human plasma samples. After removal, the samples 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 content of AST-2660 will decrease and it will become unusable. (4) The methanol solution of AST-2660-D8 has strong stability, with no change in content even after storage at -70°C for 26 days. Furthermore, it may be stable for a longer period depending on the change tendency.

[0191] Therefore, in the standard operating procedure for the above blood samples, they should be stored in an environment below -20°C, preferably at -70°C, within 4 hours after collection, and can be stored at -70°C for 28 days. When processing blood samples using the above extraction method, they must be refrigerated at a temperature below 4°C 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 will change during storage, and the results will not be correct and unreliable.

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

[0193] 1. Sample pretreatment The steps for extracting a sample using protein precipitation are as follows. 1. Verify the labeling and sequence of standard curve samples, quality control samples, and plasma samples. 2. Place all samples on a multi-tube vortexer for uniform vortexing after thawing. 3. Take 100 μl from each sample and add to a 96 well plate. 4. In addition to the blank sample, add 20 μl of AST-2660-D8 quality control working solution (i.e., 100 ng / ml AST-2660-D8 in methanol) 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. 7. Centrifuge the 96-well plate at 2143 rcf (centrifugal force) for 10 minutes. 8. Transfer 450 μl of the supernatant to a new 96-well plate. 9. Centrifuge the 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 of the standard curve for AST-2660 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 (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 above, the linear relationship was good in the linear range of 0.5 to 200 ng / ml.

[0195] 2.2 Accuracy and precision test results of quality control samples Accuracy and precision are defined as follows: Accuracy is expressed as the relative error (RE). RE = [(average value - nominal value) / nominal value] x 100 Precision represents the coefficient of variation (CV). CV = (standard deviation / mean value) x 100

[0196] The accuracy and precision test results were shown in Table 17. From Table 17, it can be seen that the intra-assay and inter-assay precision were both less than 15.5%, and the intra-assay and inter-assay accuracy were within ±11.4. The analytical criteria for accuracy and precision were as follows: For the LLOQ QC samples, the intra-assay and inter-assay precision (RE) should be within ±20.0%, and the intra-assay and inter-assay CV should not exceed 20.0%. The intra-assay and inter-assay precision (RE) should be within ±15.0% for the low, geometric mean, medium, high, and dilute QC (if applicable) samples, and the intra-assay and inter-assay precision (CV) should 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 less than or equal to 13.4%, meeting the requirements.

[0197] [Table 17]

[0198] 2.3 Test results for matrix effect [Table 18]

[0199] The results in Table 18 show that the inter-assay matrix accuracy and precision (CV) of the matrix effect of AST-2660 were both less than 15%, which met the requirements.

[0200] 2.4 Stability [Table 19]

[0201] From Table 19, it can be seen that the accuracy of the stability of the AST-2660 working solution was within ±11.8%, and the precision was less than 11.5%. The stability analysis criteria were as follows: the difference between the storage stable solution and the freshly prepared solution should be within ±10.0%, and the CV of each replicate should not exceed 15.0%. Therefore, the AST-2660 working solution had good stability and met the requirements under various storage conditions.

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

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

[0204] The standard operating procedure (SOP) for detection established after the methodology and validation according to Example 6 was as follows: Step 1. Preparation of the solution Matrix: Mixed normal human urine with additive Na2HPO4·12H2O a: Internal standard working solution: AST-2660-D8 was collected, dissolved in methanol, and diluted to prepare a solution containing approximately 100 ng per ml. b: Test solution (no internal standard compound was added to the test solution at this point): A test blood sample was obtained. c: Standard working solution (no internal standard compound was added to the standard working solution at this point): 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 ml, which was used as the standard working stock solution. An appropriate amount of the standard working stock solution was taken, added with 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 ml, respectively. (The solutions were stored at -70°C.) d: Quality control sample solution: An appropriate amount of AST-2660 was taken, accurately measured, dissolved in methanol, and diluted to prepare a solution containing approximately 10 μg per ml, which was used as the quality control working stock solution. An appropriate amount of the quality control working stock solution was taken and added with the matrix to prepare quality control working solutions of different concentrations containing approximately 150 ng, 60 ng, 15 ng, 1.5 ng, and 0.5 ng per ml, respectively. (The solutions were stored at -70°C.)

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

[0206] Blank samples were obtained by collecting 100 μl of blank human urine, adding 20 μl of methanol, and extracting in the same manner.

[0207] 100 μl of blank human urine was collected, 20 μl of the internal standard working solution was added, and extracted in the same manner to obtain a zero concentration sample.

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

[0209] Step 3. Setting up chromatography-mass spectrometry test conditions The 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, a methanol solution containing a low content of ammonium acetate was used, mobile phase B was acetonitrile, and gradient elution was performed according to the table below, with a column temperature of 20° C. and an injection volume of 5 μl.

[0210] [Table 20]

[0211] Mass spectrometry conditions were as follows: electrospray ion source in negative ion scanning mode, spray potential: -4500 V, ion source temperature: 500 °C, collision energy (CE): -22 eV, declustering potential (DP): -55 V, entrance potential (EP): -10 V, collision chamber exit potential (CXP): -16 V, residence time: 100 ms, high purity nitrogen was 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 collected and injected into LC-MS / MS for detection.

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

[0214] The prepared standard working solutions of metabolite II with different concentrations were absorbed and injected into the LC-MS / MS system for detection, and the relationship function y = f(x) was obtained (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).

[0215] The calculation results were as follows. The relationship function y=f(x) of 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 obtained from the test solution after extraction and substituted into the relationship function y=f(x) to calculate the concentration of metabolite II in the test solution.

[0216] The relationship function was used to measure and calculate the concentration of metabolite II in the test solution with an unknown concentration. The test solution with a known content of the internal standard compound was absorbed and injected into the 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 calculated and substituted into the function y = f(x) to obtain the concentration x of metabolite II in the test solution.

[0217] Optionally, the sequence of the operational steps related to the above SOP may be slightly modified to obtain the SOP for the following operations: A quantitative internal standard compound is added to a test biological sample solution, which is then subjected to an extraction process to obtain a test solution; A series of solutions of metabolite II with different concentrations are obtained by diluting the metabolite II standard substance, and a quantitative internal standard compound is added to obtain standard working solutions after extraction treatment. The series of standard working solutions are absorbed respectively, and then injected into the LC-MS / MS system for detection, and the peak areas of metabolite II and the internal standard compound are obtained. A standard curve is drawn with the ratio of the peak area of ​​metabolite II to the peak area of ​​the internal standard compound as the vertical axis and the concentration of metabolite II as the horizontal axis, and the regression equation is calculated. The test solution was absorbed and injected into the LC-MS / MS system for detection, and the ratio of the peak area of ​​metabolite II in the test solution to the peak area of ​​deuterated compound I was calculated and substituted into the regression equation to determine the content of metabolite II in the test biological sample solution.

[0218] Solutions of the same concentration were prepared by the modified standard operating procedure described above, by using containers of the same volume, according to the practice of the laboratory operators, so long as the volumes of solutions added were the same and the resulting standard working solutions remained at the same concentration.

[0219] 11 to 14 are some representative spectra of the detection process of a urine sample.

[0220] FIG. 11 is a representative LC-MS / MS spectrum of AST-2660 and the internal standard AST-2660-D8 in a blank human urine extract.

[0221] FIG. 12 is a representative LC-MS / MS spectrum of AST-2660 and the internal standard AST-2660-D8 in a urine sample extract at 0% concentration.

[0222] FIG. 13 is a representative LC-MS / MS spectrum of AST-2660 and the internal standard AST-2660-D8 in a standard sample solution (0.50 ng / ml) of human urinary extract.

[0223] FIG. 14 shows a representative LC-MS / MS spectrum 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 by the procedure for detecting AST-2660 content in human urine provided in this example, and the results are shown in Tables 21 to 24 below.

[0225] [Table 21]

[0226] As can be seen from Table 21, human urine containing AST-2660 showed little loss of AST-2660 content after storage at -70°C for 32 days, but nearly 90% loss at -20°C. Therefore, human urine samples containing AST-2660 should be stored at -70°C, and under such conditions, samples stored for 32 days were still stable.

[0227] [Table 22]

[0228] As can be seen from Table 22, human urine containing AST-2660 showed little decrease in AST-2660 content during freeze-thaw treatment from -70°C to room temperature, but decreased by nearly 90% during freeze-thaw treatment from -20°C to room temperature. In other words, the data in Table 22 show that the rapid temperature change from the storage condition of the human plasma samples stored at -70°C to room temperature for experimental treatment did not affect the stability of AST-2660 in the human plasma samples.

[0229] [Table 23]

[0230] [Table 24]

[0231] As can be seen from Table 23, the processed human urine samples containing AST-2660 were stable for 94 hours at room temperature.

[0232] As is apparent from Table 24, untreated human urine samples containing AST-2660 were stable for 24 hours at room temperature. (1) Human urine samples containing AST-2660 should be stored in the above experimentally confirmed low temperature environment, −70°C, within 24 hours (at the latest) after collection. When sent to the DMPK Sample Testing Center laboratory, they should be kept as cold as possible (below room temperature). (2) The samples should be stored at temperatures below -70°C, where they were stable for 32 days, and may be stable for longer periods depending on the change patterns. (3) Processed human urine samples containing AST-2660 were stable for 94 hours at or below room temperature. (4) The process of removing the stored samples to room temperature, i.e., the sudden temperature change from the storage condition of the 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 samples. After removing the samples, they should be placed in a conventional refrigerator or at room temperature, and were stable at room temperature for up to 24 hours. (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 operating procedure for the above urine sample manipulation, it 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. When processing, 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 results cannot be said to be correct and cannot be trusted because the AST-2660 in the sample changes during storage.

Claims

1. A deuterated compound I having the structure shown in formula (I): [Chemical formula I] In the formula, A is 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.

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

3. 2. The deuterated compound I according to claim 1, wherein the number D is 4 or 8.

4. 2. The deuterated compound I according to claim 1, wherein M is Na, K, Li or an ammonium radical.

5. The deuterated compound I according to claim 1, which is a compound having a structure represented by formula (I-1) or (I-2): [Chemical formula I-1] [Chemical formula I-2]

6. 2. The deuterated compound I of claim 1, selected from compounds having the following structure: [Chemical formula I-3] [Chemical formula I-4-1]

7. 【Chemical I-5】 reacting compound I-a with phosphorus oxyhalide POX3 to obtain compound I-b; hydrolyzing said compound I-b in aqueous solution with or without a base to obtain the corresponding deuterated compound I; Including, The compound I-a is a deuterated 2-halogenated ethylamine or an inorganic acid salt thereof, A is H or D, at least one of the four A's of the compound I-a is D, and at least one of the eight A's of the compound I-b and the compound I is D; the base in the hydrolysis reaction is selected from MOH (wherein M is an alkali metal, an alkaline earth metal, or an ammonium radical), MH (wherein M is an alkali metal), and MOR (wherein R is an alkyl group having 1 to 4 carbon atoms and M is an alkali metal, an alkali metal carbonate, or an alkali metal bicarbonate); A method for preparing a deuterated compound I, wherein X is a halogen.

8. The compound Ia and the phosphorus oxyhalide POX3 are cooled in an organic solvent, and an organic base solution is added dropwise to obtain compound Ib; Preferably, the temperature is lowered to −78 to −20° C., and / or Preferably, the organic solvent is one or a mixture of two or more of dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane or cyclohexane and tetrahydrofuran; and / or Preferably, the organic base is one or a mixture of two or more of methylamine, ethylamine, propylamine, isopropylamine, N,N-diethylamine, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, tetramethylguanidine, pyridine, N-methyldicyclohexylamine or dicyclohexylamine, and / or 8. The process according to claim 7, wherein the reaction of the compound I-a with the phosphorus oxyhalide POX3 is carried out in an atmosphere, the atmosphere being one of air, nitrogen or argon, preferably the atmosphere being one of nitrogen or argon, more preferably the atmosphere being nitrogen.

9. A method for measuring the content of the deuterated compound I according to any one of claims 1 to 6 by using a 31P-NMR method, preferably a method for measuring the content of the deuterated compound I in a solution containing the deuterated compound I by using a 31P-NMR method, or a method for measuring the content of the deuterated compound I according to any one of claims 1 to 6 by 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; Gradient elution involves using mobile phases A and B, gradually increasing mobile phase A from 15% to 90% by volume, and then gradually decreasing mobile phase A to 15% by volume.

10. Detecting the 31P-NMR of the deuterated compound I and a phosphorus-containing compound having a known content to obtain their spectra; and calculating the content of the deuterated compound I by substituting the content of the known content of the phosphorus-containing compound based on a peak area ratio between a chemical shift characteristic peak of the deuterated compound I and a chemical shift characteristic peak of the phosphorus-containing compound in the 31P-NMR spectrum, thereby determining the content of the deuterated compound I.

11. The phosphorus-containing compound is preferably a compound containing one phosphorus atom, more preferably hexamethylphosphoric acid triamide; 11. The method of claim 10, wherein the deuterated compound I and the phosphorus-containing compound with a known content are added to a solvent and examined together for their 31P-NMR spectra, preferably the deuterated compound I and the phosphorus-containing compound with a known content are added to water and examined together for their 31P-NMR spectra after they are dissolved.

12. Use of the deuterated compound I of any one of claims 1 to 6 as an internal standard for detecting metabolite II in a biological sample, said metabolite II having the structure shown in formula (II): 【Chemistry II】 where A is H and M is H or an alkali metal, alkaline earth metal, or ammonium radical.

13. Use of the 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 the metabolite II has the structure shown in formula (II). 【Chemistry II】 where A is H and M is H or an alkali metal, alkaline earth metal, or ammonium radical.

14. The deuterated compound I is [Chemical formula I-3] is selected from The metabolite II is [Chemical formula I-6] The use according to claim 13 or 14, selected from the group consisting of

15. The use according to claim 14, wherein the AKR1C3 activating DNA alkylating agent prodrug is selected from compounds having the structures shown in formulas 1-5. 【Chemistry 1-2】 In the formula, the definitions of R1, R2, R3, R4, R5, R8, R9, and R10 are as described in the claims of PCT / CN2020 / 089692 (WO 2020 / 228685), or 【Chemistry 3】 In the formula, the definition of Rw is as described in the claims of PCT / CN2020 / 120281 (WO 2021 / 068952), or 【Chemistry 4】 In the formula, the definitions of X, Y, Z, R, A and X10 are as described in the claims of PCT / US2016 / 021581 (corresponding to WO 2016 / 145092 (Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556))), and T is [Chemical formula I-7] and or 【Chemistry 5】 wherein A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl, or -N=CR1R2, said substituents being selected from the group consisting of halogeno, -CN, -NO2, -O-(CH2)-O-, -CO2H and salts thereof, -OR100, -CO2R100, -CONR101R102, -NR101R102, -NR100SO2R100, -SO2R100, -SON2NR101R102, C1-C6 alkyl, and C3-C10 heterocyclyl; wherein R100, R101 and R102 are each independently hydrogen, C1-C8 alkyl, or C6-C12 aryl, or R101 and R102 together with the nitrogen atom to which they are attached form a 5- to 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; R1 and R2 are each independently phenyl or methyl; X, Y and Z are each independently hydrogen or halogeno; R is hydrogen or C1-C6 alkyl or halogen substituted alkyl.

16. The use of claim 14, wherein the hypoxia activated DNA alkylating agent prodrug is selected from the group consisting of compounds having the structures shown in formulas 6-12. 【Chemistry 6】 In the formula, the definitions of R1, R2, R3 and Cx are as described in the claims of PCT / CN2020 / 114519 (WO 2021 / 120717), or 【Chemistry 7-12】 In the formula, the definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16 and R17 are as described in the claims of PCT / US2016 / 039092 (WO 2016 / 210175 (corresponding to Chinese Patent Application No. 2016800368985 (Chinese Patent Application Publication No. CN108024974))).

17. preparing a test solution containing a known concentration of an internal standard compound for injection analysis by LC-MS / MS; preparing a series of standard working solutions containing a known concentration of an internal standard compound and a known concentration of Metabolite II, wherein the concentration of the internal standard compound in the series of standard working solutions is consistent and is the same as the concentration of the internal standard compound in the test solution, and the concentration of Metabolite II in the series of standard working solutions is different; Absorbing the prepared standard working solutions of metabolite II with different concentrations, injecting them into a detection LC-MS / MS system to obtain a relationship 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 relationship function by liquid chromatography-tandem mass spectrometry (LC-MS / MS); A step of absorbing the test solution to which a known content of the internal standard compound has been added, injecting the test solution into the LC-MS / MS system for detection to obtain a ratio y between the peak area of ​​the metabolite II and the peak area of ​​the internal standard compound, and substituting the ratio y into a function y=f(x) to obtain the concentration x of the metabolite II in the test solution, using a relational function to determine and calculate the concentration of the metabolite II in the test solution of unknown concentration; Including, The test solution is prepared from a biological sample, whether or not it has been treated; The internal standard compound is a deuterated compound I having the structure shown in formula (I), [Chemical formula I] In the formula, A is 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 metabolite II has the structure shown in formula (II): 【Chemistry II】 wherein A is H; 2. A method for measuring the content of a metabolite in a biological sample, wherein M is H or an alkali metal, an alkaline earth metal, or an ammonium radical.

18. adding a quantitative internal standard compound to the test biological sample solution to obtain a test solution after extraction treatment; Dilute the metabolite II standard material to obtain a series of metabolite II standard solutions with different concentrations, add the quantitative internal standard compound to obtain a standard working solution after extraction processing, absorb the series of standard working solutions respectively and inject them into a detection LC-MS / MS system to obtain the peak areas of the metabolite II and the internal standard compound, create a standard curve with the ratio of the peak area of ​​the metabolite II to the peak area of ​​the internal standard compound as the vertical axis and the concentration of the metabolite II as the horizontal axis, and calculate a regression equation; a step of absorbing the test solution, injecting the test solution into the LC-MS / MS system for detection, determining a ratio of a peak area of ​​the metabolite II in the test solution to a peak area of ​​the internal standard compound, and substituting the ratio into the regression equation to determine the content of the metabolite II in the test solution; Including, The internal standard is a deuterated compound I having the structure shown in formula (I), [Chemical formula I] In the formula, A is 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 metabolite II has the structure shown in formula (II): 【Chemistry II】 wherein A is H; 2. A method for measuring the content of a metabolite in a biological sample, wherein M is H or an alkali metal, an alkaline earth metal, or an ammonium radical.

19. The liquid chromatography conditions in the liquid chromatography-tandem mass spectrometry method 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; Gradient elution was performed using mobile phases A and B, with mobile phase A gradually increasing from 15% to 90% by volume, and then gradually decreasing mobile phase A to 15% by volume. Mass spectrometry conditions: electrospray ion source In negative ion scanning mode, Monitoring ion pair for metabolite II: m / z 147.0 → m / z 62.9 Monitoring ion pair of deuterated compound I: m / z (147.0 + deuteration 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 17 or 18, wherein the monitoring ion pair of deuterated compound I is: m / z (149.0 + deuteration number) → m / z 64.

9.

20. In preparing a test solution containing the deuterated compound I at a known content, the deuterated compound I is preferably added to a biological sample solution, followed by an extraction operation; 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.

21. 21. The method according to claim 18 or 20, wherein the biological sample is a urine sample or a plasma sample, and correspondingly, when the urine sample is measured, the corresponding matrix solution is urine of an untreated patient containing an additive, and when the blood sample is measured, the corresponding matrix solution is plasma of an untreated patient containing an anticoagulant.

22. the operation step of adding and extracting the deuterated compound comprises adding the test solution and the standard working solution to a solution of the deuterated compound I, adding methanol to the solution and mixing uniformly, and centrifuging the solution to obtain a supernatant; 22. The method of claim 21, wherein the additive is Na2HPO4 or K2HPO4 and the anticoagulant is K2EDTA or Na2EDTA.

23. The blood sample should be stored at -20°C or below, preferably -70°C, within 4 hours after collection. It can be stored at -70°C for 28 days. When processing, it should be refrigerated at 4°C or below, and injection detection should be completed within 54 hours. The method of claim 21, wherein the urine sample needs to be stored at -70°C within 24 hours, preferably within 4 hours, of collection, and can be stored at -70°C for 32 days, and if processed, injection detection needs to be completed within 94 hours at room temperature or below.

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