A deuterated compound, and preparation method and use thereof

A method for preparing a deuterated compound I, comprising the steps of:

• reacting compound I-a with phosphorus oxyhalide POX3 to obtain compound I-b;
• hydrolyzing compound I-b in an aqueous solution with or without a base to correspondingly obtain the deuterated compound I;
• wherein compound I-a is deuterated 2-halogenated ethylamine or an inorganic acid salt thereof;
• A is H or D;
• and at least one of four As of compound I-a is D;
• and at least one of eight As of compound I-b and 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;
• MOR, wherein R is an alkyl group with 1-4 carbon atoms, and M is an alkali metal, carbonate or bicarbonate of alkali metal;
• X is halogen.

A method for measuring the content of the metabolite in a biological sample, comprising the steps of:

• preparing a solution to be tested containing an internal standard compound with known concentration for injection analysis by LC-MS/MS;
• preparation of a standard working solution:
• preparing a series of standard working solutions containing an internal standard compound with known concentration and a metabolite II with known concentration, wherein the concentration of the internal standard compound in the series of standard working solutions is consistent and the same as the concentration of the internal standard compound in the solution to be tested, and the concentration of the metabolite II in the series of standard working solutions is different;
• determining a relationship function by liquid chromatography-tandem mass spectrometry (LC-MS/MS):
• absorbing the prepared standard working solutions of metabolite II with different concentrations, and injecting them into the LC-MS/MS system for detection to obtain the relationship function y=f(x), wherein y represents the ratio of the peak area of the metabolite II to that of the internal standard compound, and x represents the concentration of the metabolite II in the standard working solutions;
• determining and calculating the concentration of the metabolite II in the solution to be tested with unknown concentration by using the relationship function:
• absorbing the solution to be tested added with the internal standard compound with known content, injecting it into the LC-MS/MS system for detection to obtain the ratio y of the peak area of the metabolite II to that of the internal standard compound, substituting it into the function y=f(x) to obtain the concentration x of the metabolite II in the solution to be tested;
• wherein the solution to be tested is prepared from the biological sample with or without treatment,
• wherein the internal standard compound is a deuterated compound I, which has a structure as shown in a Formula (I):
• A is H or D, and at least one of eight As is D;
• M is H or an alkali metal, an alkaline earth metal, or an ammonium radical;
• wherein the metabolite II has a structure as shown in the formula (II):
• A is H;
• M is H or an alkali metal, an alkaline earth metal, or an ammonium radical;
• preferably, the deuterated compound I is selected from
• preferably, the metabolite II is selected from
• preferably, the metabolite II is a metabolite II of an AKR1C3-activated DNA alkylating agent prodrug or a hypoxia-activated DNA alkylating agent prodrug;
• preferably, the conditions for liquid chromatography in liquid chromatography-tandem mass spectrometry are as follows:
• a hydrogen acceptor type stationary phase chromatography column is used;
• mobile phase A is methanol solution of ammonium acetate, and mobile phase B is acetonitrile;
• mobile phases A and B are used for gradient elution:
• gradually increasing from 15% by volume ratio to 90% by volume ratio of mobile phase A, and then gradually decreasing to 15% by volume ratio of mobile phase A;
• the mass spectrometry conditions:
• electrospray ion source;
• in negative ion scanning mode
• the monitoring ion pair of the metabolite II is:
• m/z 147.0→m/z 62.9;
• the monitoring ion pair of the deuterated compound I is:
• m/z (147.0+number of deuteration)→m/z 62.9;
• or
• in positive ion scanning mode
• the monitoring ion pair of the metabolite II is:
• m/z 149.0→m/z 64.9;
• the monitoring ion pair of the deuterated compound I is:
• m/z (149.0+number of deuteration)→m/z 64.9.

A method for measuring the content of the metabolite in a biological sample, comprising the steps of:

• adding a quantitative internal standard compound to a biological sample solution to be tested and taking it as a solution to be tested after extraction treatment;
• diluting a metabolite II reference substance to obtain a series of standard solutions of metabolite II with different concentrations, adding the quantitative internal standard compound and taking it as a standard working solution after extraction treatment, absorbing the series of standard working solutions, respectively injecting them into an LC-MS/MS system for detection to obtain the peak area of the metabolite II and the internal standard compounds, taking the ratio of the peak area of the metabolite II to that of the internal standard compounds as an ordinate and taking the concentration of the metabolite II as an abscissa to draw a standard curve and calculate a regression equation;
• absorbing the solution to be tested, injecting it into the LC-MS/MS system for detection to obtain the ratio of the peak area of the metabolite II to that of the internal standard compound in the solution to be tested, substituting it into the regression equation to obtain the content of the metabolite II in the solution to be tested;
• wherein the internal standard is a deuterated compound I, which has a structure as shown in a Formula (I):
• A is H or D, and at least one of eight As is D;
• M is H or an alkali metal, an alkaline earth metal, or an ammonium radical;
• wherein the metabolite II has a structure as shown in the formula (II):
• A is H;
• M is H or an alkali metal, an alkaline earth metal, or an ammonium radical;
• preferably, the deuterated compound I is selected from
• preferably, the metabolite II is selected from
• preferably, the metabolite II is a metabolite II of an AKR1C3-activated DNA alkylating agent prodrug or a hypoxia-activated DNA alkylating agent prodrug;
• preferably the conditions for liquid chromatography in liquid chromatography-tandem mass spectrometry are as follows:
• a hydrogen acceptor type stationary phase chromatography column is used;
• mobile phase A is methanol solution of ammonium acetate, and mobile phase B is acetonitrile;
• mobile phases A and B are used for gradient elution:
• gradually increasing from 15% by volume ratio to 90% by volume ratio of mobile phase A, and then gradually decreasing to 15% by volume ratio of mobile phase A;
• the mass spectrometry conditions:
• electrospray ion source;
• in negative ion scanning mode
• the monitoring ion pair of the metabolite II is:
• m/z 147.0→m/z 62.9;
• the monitoring ion pair of the deuterated compound I is:
• m/z (147.0+number of deuteration)→m/z 62.9;
• or
• in positive ion scanning mode the monitoring ion pair of the metabolite II is:
• m/z 149.0→m/z 64.9;
• the monitoring ion pair of the deuterated compound I is:
• m/z (149.0+number of deuteration)→m/z 64.9.