Patent Application: US-68424903-A

Abstract:
method of automated metabolic stability analysis of library of compounds by mass spectrometry using stable isotope labeled internal standards is provided . said internal standards are prepared in situ by reaction of an authentic sample of said library compounds with a stable isotope labeled reagent . in an automated fashion , cytochrome p450 enzyme systems are added to said library compounds ; said reactions are terminated after certain periods of time by addition of an organic solvent containing equal amounts of said labeled internal standards ; said terminated enzyme reactions are treated with a non - labeled version of said stable isotope labeled reagent to convert remaining library compounds to compounds of identical structures , except the labeled atoms , as those of said labeled internal standards ; said conversion reactions are extracted and the extracts are analyzed by isotope dilution mass spectrometry to determine the percentage of library compounds remaining after enzyme reactions .

Description:
the method of the subject invention provides an automated metabolic stability assay and a quantitative analysis of library compounds after reactions with cytochrome p450 enzyme systems using isotope dilution mass spectrometric analysis . chronologically , depending on the type of functional group present in said library compounds , the invention provides methods of synthesis of stable isotope labeled internal standards of said library compounds using only one type of labeled chemical reagent , methods of extraction of said internal standards , methods of synthesis of a non - labeled version of said internal standards using the non - labeled version of said labeled chemical reagent , methods of extraction of said non - labeled version of said internal standards for use in setting up parameters for mass spectrometric analysis , methods of reactions of enzymes of the cytochrome p450 enzyme systems with library compounds , methods of termination of enzyme reactions and addition of said extracts of said stable isotope labeled internal standards to said terminated enzyme reactions , methods of conversion of remaining library compounds in said terminated enzyme reactions to compounds of identical structure as that of said internal standards using said non - labeled version of said labeled chemical reagent , methods of extraction said internal standards and said converted compounds from said reaction medium , methods of determination of molecular ion and product ion ( also called daughter ion ) of said internal standards and said converted compounds in said chemical synthesis extracts , methods of selection of ion ratios of said molecular ions and said daughter ions and use of said ion ratios in mrm mode ( multiple reaction monitoring mode ), methods of mass spectrometric analysis of said extracts of enzyme reactions in mrm mode , methods of using mass spectrometric data of said selected ion ratios to determine percent remaining of said library compounds from said enzyme reactions . specifically , the method of the subject invention provides methods of synthesizing stable isotope labeled amide , carbamate , urea , and thiourea internal standards of primary and secondary amine compounds by reacting said compounds with a stable isotope labeled chemical reagents selected from a group consisting of labeled acid anhydrides or labeled acid chlorides , labeled chloroformates , labeled isocyanates , and labeled thioisocyanates . the invention provides methods of converting said amine compounds in said terminated enzyme reactions into compounds of identical structure as that of said labeled internal standards by adding to said terminated reactions a non - labeled version of said chemical reagents selected from a group consisting of non - labeled acid anhydrides or non - labeled acid chlorides , non - labeled chloroformates , non - labeled isocyanates , and non - labeled thioisocyanates . specifically , the method of the subject invention provides methods of synthesizing stable isotope labeled ester and carbamate internal standards of phenolic or alcoholic compounds by reacting said compounds with a stable isotope labeled chemical reagents selected from a group consisting of labeled acid anhydrides or labeled acid chlorides , and labeled isocyanates . the invention provides methods of converting said phenolic or alcoholic compounds in said terminated enzyme reactions into compounds of identical structure as that of said labeled internal standards by adding to said terminated reactions a non - labeled version of said chemical reagents selected from a group consisting of non - labeled acid anhydrides or non - labeled acid chlorides , and non - labeled isocyanates . specifically , the method of the subject invention provides methods of synthesizing stable isotope labeled oxime and hydrazone internal standards of aldehyde or ketone compounds by reacting said compounds with a stable isotope labeled chemical reagents selected from a group consisting of labeled alkoxylamines , and labeled alkylhydrazines . the invention provides methods of converting said aldehyde and ketone compounds in said terminated enzyme reactions into compounds of identical structure as that of said labeled internal standards by adding to said terminated reactions a non - labeled version - of said chemical reagents selected from a group consisting of non - labeled alkoxylamines and non - labeled alkylhydrazines . specifically , the method of the subject invention provides methods of synthesizing stable isotope labeled ester internal standards of carboxylic acid compounds by reacting said compounds with either a stable isotope labeled alcohol or a labeled alkyl halide . the invention provides methods of converting said carboxylic acid compounds in said terminated enzyme reactions into compounds of identical structure as that of said labeled internal standards by adding to said terminated reactions a non - labeled version of said labeled alcohol or said labeled alkyl halide . specifically , said cytochrome p450 enzyme systems in said metabolic stability assay can be cryopreserved or fresh human and animal hepatocytes , microsomes , s9 fractions or solutions containing individual cytochrome p450 isoenzymes . specifically , said enzyme reactions include reaction at time zero wherein reaction is terminated immediately after enzyme addition . said ms data of said time zero reaction are used as original concentration of library compounds . specifically , said enzyme reactions include reactions at different time intervals wherein each reaction is terminated at specific time after enzyme addition . said ms data of said time reactions are used to calculate the percentage of remaining of library compounds at said time intervals . specifically , the method of the subject invention provides methods of extraction of reactions by solid liquid extraction , liquid liquid extraction , and solid phase extraction . a library of 4 amines was analyzed for their metabolic stability using human microsomal proteins as the cytochrome p450 enzyme system . aliquots of each amine were automatically placed in test tubes which were used for synthesis of its internal standard , synthesis of reference standards , and reactions with cytochrome p450 enzyme system . the internal standard of each amine is the acetamide - d3 compound formed by reaction of amines with acetic anhydride - d6 . at the same time an aliquot of each amine was treated with acetic anhydride to form acetamide reference standard . deuterated acetamide internal standards and acetamides were then separated by an automated extraction procedure . the reactions of amines with human microsomal protein were carried out in test tubes in 37 ° c . bath and was terminated by aspirating equal aliquots of the reaction solutions into test tubes containing equal volume of acetonitrile as terminating reagent . reactions were aspirated at 0 , 30 , 60 , 90 , and 120 minutes . after all reactions were stopped , equal volumes of extracted acetamides - d3 were added to respective reactions as internal standards . aqueous sodium bicarbonate and acetic anhydride were then added to convert remained amine in each reaction to the acetamide while leaving its acetamide - d3 internal standard unchanged . the tubes were mixed gently for 10 minutes . both acetate amide and its acetate amide - d3 were separated from the reaction by an automatic extraction procedure . solutions of extracts containing acetamide - d3 , acetamide , and both from reactions with cytochrome p450 enzyme sytem were analyzed by electrospray mass spectrometry . molecular ion and its product ion ( daughter ion ) of all acetamides and acetamides - d3 were determined in an automated fashion using an autosampler , a mass spectrometer and its analysis software . after all interested ions were determined , analysis of reaction extracts in mrm mode followed . the ion ratios of acetate amide to acetate amide - d3 were calculated and plotted against time . via a standard liquid handler , aliquots of 4 amines ( amine 1 , 2 , 3 , and 4 ) were placed in 3 rows of 4 test tubes at volume of 0 . 005 ml and concentration of 1 mg / ml in methanol . row 1 was for synthesis of internal standards , row 2 for synthesis of reference standards , and row 3 for reactions with human microsomal protein . row 1 test tubes were treated with 0 . 1 ml of 10 % v / v acetic anhydride - d6 in ethyl acetate while row 2 test tubes were treated with 0 . 1 ml of 10 % v / v acetic anhydride in ethyl acetate . next , aliquots of 0 . 1 ml 1m sodium bicarbonate were added to both rows 1 and 2 . tubes were mixed and allowed to stand for 10 minutes . then , reactions were aspirated into filters containing hydromatrix ® powder and aliquots of 0 . 900 ml ethyl acetate were added to filters . filtrates contaning acetate amide - d3 were collected in new row 4 test tubes while filtrates contaning acetate amide were collected in new row 5 test tubes . each of these filtrate solutions were assumed to be at the concentration of 0 . 005 mg / ml in ethyl acetate . row 3 test tubes containing 0 . 005 ml of amine at 1 mg / ml in methanol were treated with 0 . 345 ml of 0 . 1m phosphate buffer ph 7 and 0 . 025 ml of 20 mg / ml human microsomal protein . the test tubes were incubated at 37 ° c . and were added 0 . 125 ml of buffer containing nadp ( 1 . 7 mg / ml ), glucose - 6 - phosphate ( 7 . 8 mg / ml ), glucose - 6 - phosphate dehydrogenase ( 1 . 5 units / ml ), and sodium bicarbonate ( 20 mg / ml ). immediately at time 0 , 0 . 100 ml of row 3 reactions were aspirated to row 6 test tubes containing 0 . 100 ml acetonitrile each . at time 30 minutes , 0 . 100 ml of row 3 reactions were aspirated to row 7 test tubes containing 0 . 100 ml acetonitrile each . at time 60 minutes , 0 . 100 ml of row 3 reactions were aspirated to row 8 test tubes containing 0 . 100 ml acetonitrile each . at time 90 minutes , 0 . 100 ml of row 3 reactions were aspirated to row 9 test tubes containing 0 . 100 ml acetonitrile each . at time 1200 minutes , 0 . 100 ml of row 3 reactions were aspirated to row 10 test tubes containing 0 . 100 ml acetonitrile each . rows 6 , 7 , 8 , 9 , and 10 test tubes contained microsome reactions that was terminated by 0 . 100 ml acetonitrile . an aliquot of 0 . 050 ml solution of acetate amide - d3 in row 4 test tubes was aspirated to each of rows 6 , 7 , 8 , 9 , and 10 test tubes . next , an aliquot of 0 . 1 ml of 10 % v / v acetic anhydride in ethyl acetate and an aliquot of 0 . 1 ml 1m sodium bicarbonate were added to rows 6 , 7 , 8 , 9 , and 10 test tubes . the test tubes were gently shaked for 10 minutes and were aspirated into filters containing hydromatrix ® powder that was sitting above test tubes of rows 11 , 12 , 13 , 14 , and 15 . aliquots of 1 ml ethyl acetate were aspirated into filters and filtrates were collected in rows 11 , 12 , 13 , 14 , and 15 test tubes . aliquots of 0 . 050 ml of rows 4 , 5 , 11 , 12 , 13 , 14 , and 15 test tubes were placed in autosampler vials for mass spectrometric analysis . total of 28 vials ( 1 to 28 ) were programmed for electrospray ms analysis using q1 scan mode and product ion scan mode for reference standards and internal standards ( vial 1 to 8 ), and using mrm mode for microsome reactions ( vials 9 to 28 ). mrm mode of analysis or tandem ms analysis of 4 amines were set up as follows : amine 1 : acetamide 178 . 1 → 91 . 0 ; acetamide - d3 : 181 . 2 → 91 . 0 amine 2 : acetamide 192 . 2 → 91 . 0 ; acetamide - d3 195 . 2 → 91 . 0 amine 3 : acetamide 236 . 2 → 105 . 0 ; acetamide - d3 : 239 . 3 → 105 . 0 amine 2 : acetamide 250 . 0 → 105 . 0 ; acetamide - d3 : 253 . 2 → 105 . 0 ion ratios of acetamide to acetamide - d3 for amine 1 to 4 are as follows : 0 min 30 min 60 min 90 min 120 min amine 1 0 . 45 0 . 31 0 . 33 0 . 28 0 . 27 amine 2 0 . 35 0 . 22 0 . 21 0 . 23 0 . 19 amine 3 0 . 36 0 . 20 0 . 22 0 . 22 0 . 21 amine 4 0 . 92 0 . 60 0 . 69 0 . 80 0 . 72 using the amount of amine at time t = 0 as 100 % , the % of remained amine with time is tabulated as follows : t = 0 min 30 min 60 min 90 min 120 min amine 1 100 % 69 % 73 % 62 % 60 % amine 2 100 % 63 % 60 % 65 % 54 % amine 3 100 % 56 % 61 % 61 % 58 % amine 4 100 % 65 % 75 % 87 % 78 % us patent documents 5 , 559 , 038 sep . 24 , 1996 j . fred kolhouse 6 , 358 , 996 mar . 19 , 2002 michael s . alexander arun k . ghosh et al , “ stereoselective reduction of alpha - hydroxy oxime ethers : a convenient route to cis - 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