Patent Application: US-94653310-A

Abstract:
the present patent application introduces methods for generating mixture compound libraries from a drug lead . the mixture compound libraries are then screened for the discovery of modified drug lead compounds which possess desired improved drug properties . the process utilizes a non - selective reaction to modify the drug lead compound structure . compared to existing methods of modifying a drug lead compound , this new method can modify more structural positions of a drug lead compound . as a consequence , there will be greater probability of finding a product with improved drug properties .

Description:
in one embodiment of the present method ( whole structure modification ), a drug lead molecule structure may be modified directly by a non - selective reaction ( fig3 ). this embodiment is suitable for those drug lead structures which are stable under the reaction conditions chosen for the non - selective modification of the drug lead . in another embodiment of the present method ( substructure modification ), one or more substructure ( s ) of a drug lead may be modified , separately , by non - selective reactions . the mixture products of each of the substructure modifications may then be jointed together through appropriate coupling reactions , to form the whole of the modified drug lead structure ( fig4 ). the degree of modification will depend on the degree of structural modification of each of the substructures . in another embodiment of the present method ( combination of selective and non - selective modifications ), the mixture compound library may be generated by a combination of the non - selective modification methods described in this method and the traditional methods of modification using selective organic reactions . in some embodiments ( combination of drug leads ), the mixture compound library may include modifications for more than one drug lead . thus , for example , several analog compounds of a drug lead can be put together and this mixture can undergo a non - selective chemical reaction to form a new mixture of compounds for the generation of mixture compound library . in some embodiments ( mixture libraries ), the method of generating the mixture compound library can include , but not limited to : directly using the non - selective modification reaction product mixture of the drug lead , or separating the product mixture into fractions and taking one fraction , or mixing selected fractions together , or mixing one or more fractions with compounds or mixtures of other origins . in some embodiments ( treated libraries ), the mixture compound library is treated with a generic protein ( such as bovine serum albumin ) to remove covalent binders of protein from the mixture compound library and recover the remaining compounds by organic solvent extraction or other extraction methods . in some embodiments , the modified drug lead compound library may be formed by non - selective reactions include , but not limited to : fluorination , fenton reaction , photochemical reaction , radiation reaction , electrochemical reaction , oxidation reaction , free radical reaction , microwave assisted reaction . in some embodiments , the drug properties may include , but are not limited to : affinity toward a biological target , functional activity in a biological assay , metabolic stability , pampa permeability , plasma protein binding , blood - brain barrier ( bbb ), solubility in a drug formulation . in an embodiment of the present method ( structure id first ), the structure of a component of the mixture compound library showing improved drug properties may be determined by lc - ms / ms and / or lc - nmr . this compound may then be synthesized by standard organic synthesis technologies by those skilled in the art of organic synthesis and the improved drug properties can be confirmed by appropriate drug property tests of this synthesized compound . in another embodiment of the present method ( separation first ), sufficient amount of a component of the mixture compound library showing improved drug properties may be separated and purified by hplc or lc - ms . this purified compound may then be evaluated by appropriate drug property tests to confirm the improved drug properties . upon confirmation of improved drug properties , this purified compound may then be structure determined by nmr and / or ms / ms and be synthesized by standard organic synthesis technologies by those skilled in the art of organic synthesis for further development . the following examples are offered to illustrate , but not to limit the claimed method . one of ordinary skill in the art will recognize a variety of non - critical parameters that may be altered without departing from the scope of the claimed method . non - selective modification of a drug lead by fluorination . 300 mg donepezil was dissolved in 200 ml ch2cl2 at − 78 ° c . cooled by dry - ice / acetone bath . a mixture of f2 and n2 gas containing 10 % f2 / 80 % n2 was passed through the reaction vessel continuously at a flow rate of 2 l / min . at time points of 15 , 30 , 45 , and 60 min , one quarter of the content of the reaction was taken out and the solvent was removed under vacuum . dried reaction products were dissolved in 3 ml acetonitrile and the solution was analyzed by lc - ms ( agilent 1200 , agilent eclips 150 × 4 . 6 mm column , gradient elution from 10 % acetonitrile / 90 % 0 . 1 % formic acid in dd - h2o to 90 % acetonitrile / 10 % 0 . 1 % formic acid in dd - h2o , 1 ml / min total flow rate , waters lct tofms in positive ion mode ). lc - ms analysis results of selected products for 30 min time point are shown in fig5 . preparation of mixture compound library for testing of drug properties . reaction mixture of donepezil fluorination was separated on an agilent zorbax c8 250 × 4 . 6 mm column , gradient elution from 30 % meoh / 70 % 0 . 1 % formic acid in dd - h2o to 90 % meoh / 10 % 0 . 1 % formic acid in dd - h2o , 1 ml / min total flow rate ). each fraction was analyzed by lct mass spectrometer ( fig6 ). fractions 7 - 13 contained mostly the unreacted donepezil and were not included in the formation of library . fractions 14 - 16 were mixed to form the mixture compound library ( donepezil fluorination mixture library 1 ) for the subsequent testing of drug properties . affinity testing of compound mixture library . donepezil fluorination mixture library 1 was mixed with a buffered solution containing 10 um ache enzyme ( sigma catalog no c3389 ) at ph 7 . 4 ( 50 mm tris hcl ). the mixture was filtered through a microcon filter with molecular weight cutoff ( mwco ) of 10 kda . new buffer was added to the top of the filter and the solution was filtered through the microcon filter . this process was repeated three times and a portion of the top layer of each filtration was removed and treated with organic solvent acetonitrile to denature the enzyme and to extract the donepezil fluorination mixture library compounds that bound to the enzyme . compound concentration in the top layer was measured by lc - ms analysis ( agilent 1200 , agilent eclips 150 × 4 . 6 mm , gradient elution from 10 % acetonitrile / 90 % 0 . 1 % formic acid in dd - h2o to 90 % acetonitrile / 10 % 0 . 1 % formic acid in dd - h2o , 1 ml / min total flow rate , waters lct tofms in positive ion mode ), and the relative affinity determined based on the concentration change after each round of filtering through microcon filter . a sample containing the ache enzyme but without donepezil fluorination mixture library 1 was treated the same way as the sample with donepezil fluorination mixture library 1 to serve as a compound - negative control . the components with larger decrease in concentration in the sample with donepezil fluorination mixture library 1 after each filtering indicate lower affinity and vice versa . ( fig7 ). the affinity testing results for selected compounds are listed below : compound ( m / z , rt ), affinity toward ache relative to donepezil : liver microsome stability testing of compound mixture library . donepezil fluorination mixture library 1 was mixed with a buffered solution containing human liver microsome ( invitrogen cat . no . hmmc - pl , 1 mg / ml ), 1 mm nadph in 50 mm kpo4 ph7 . 4 for 2 hours at 37 ° c . and then was treated with 4 volumes of organic solvent acetonitrile to stop the metabolism reaction and to extract the donepezil fluorination mixture library 1 compounds . control samples were also done where the donepezil library 1 was replaced by pure donepezil and by reference compound terfenedine each at 1 um . the concentration of compounds before and after the microsome incubation was measured by lc - ms analyses ( agilent 1200 , agilent eclips 150 × 4 . 6 mm , gradient elution from 10 % acetonitrile / 90 % 0 . 1 % formic acid in dd - h2o to 90 % acetonitrile / 10 % 0 . 1 % formic acid in dd - h2o , 1 ml / min total flow rate , waters lct tofms in positive ion mode ), and the relative metabolic stability determined based on the concentration change after incubation . the components with larger decrease in concentration after microsome incubation indicate lower metabolic stability and vice versa . ( fig8 ). the metabolic stability testing results for selected compounds are listed below : compound ( m / z , rt ), metabolic stability relative to donepezil : enzymatic activity assay . donepezil fluorination mixture library 1 is separated by an hplc device ( agilent 1200 , agilent eclips 150 × 4 . 6 mm , gradient elution from 10 % meoh / 90 % 10 mm nh4oac ( ph 7 ) to 40 % meoh / 60 % 10 mm nh4oac ( ph 7 ), 1 ml / min total flow rate . the eluent of the hplc is continuously passed through a buffered flow reactor cell containing ache enzyme with a substrate acetylcholine at ph 7 and analyzed by mass spectrometer ( waters lct - tof in positive ion mode ). the inhibition effect and the identity of the components are monitored by the intensity change of the substrate acetylcholine , the formation of enzymatic reaction product choline and the corresponding mass of the mixture component eluting out at the time . relative inhibition of ache activity of components of the donepezil fluorination mixture library 1 vs . donepezil itself is determined . purification of compound showing improved drug properties . compound don_f — 15 having a molecular weight consistent with m + o + 2f and don_f — 18 having a molecular weight consistent with m + o + 3f were purified first using a preparative hplc ( shimadzu 10advp , supelco discovery rp amide c16 , meoh / 0 . 1 % formic acid - ddh2o gradient ) and then using another preparative hplc column ( thermo pfp gold , 4 . 6 × 250 mm , 80 % meoh / 0 . 1fa / 20 % h2o ) with mass spectrometry and uv detection ( fig9 ). lc - ms / ms ( thermo finnigan lcq orbitrap ) analysis identified the structure of the compound . ( fig1 ). the fragment ions observed from lc - ms / ms and ms3 analysis of purified don_f — 15 are listed below : confirmation of compound showing improved drug properties . the purified compounds don_f — 15 and don_f — 18 were tested individually for affinity , metabolic stability and activity . the procedures were the same as example 3 - 5 except a single purified compound is used in example 7 while a mixture of compounds is used in examples 3 - 5 . the results confirm that don_f — 15 and don_f — 18 , after purification into single pure compounds , show higher affinity toward ache enzyme , greater metabolic stability in human microsomal incubation , and higher activity toward ache enzyme compared to donepezil itself . additionally , ache enzymatic activity assay was conducted using ellman method ( ellman , g . l ., et al , biochem . pharmacol ., 7 , 88 - 95 , 1961 , colorimetric method ; color indicator , dtnb , measuring absorption at 405 nm ; ache enzyme , 10 mu / ml ; substrate acetylthiocholine iodide , 75 mm ; 100 mm phosphate buffer , ph 7 . 4 ) fig1 shows results of ache enzymatic activity assay of donepezil and purified compound don_f — 15 from donepezil fluorination mixture library 1 using the ellman colormetric method . substructure embodiment . compound 1 ( substructure - 1 of gleevec ) is dissolved in ch 2 cl 2 at − 78 ° c . cooled by dry - ice / acetone . a mixture of f2 and n2 gas containing 10 % f2 is passed through the reaction vessel continuously . at time points of 15 , 30 , 45 , and 60 min , one quarter of the content of the reaction is taken out and the solvent removed under vacuum . dried reaction products are dissolved in acetonitrile and the content analyzed by lc - ms ( agilent 1200 , agilent eclips 150 × 4 . 6 mm , gradient elution from 10 % acetonitrile / 90 % 0 . 1 % formic acid in dd - h2o to 90 % acetonitrile / 10 % 0 . 1 % formic acid in dd - h2o , 1 ml / min total flow rate , waters lct tofms in positive ion mode ). the expected products is a mixture and are shown in fig1 a , step 1 . this product mixture is then reacted with compound 2 ( substructure - 2 of gleevec ) under the catalysis of dcc to produce a mixture of modified gleevec as expected products showing in the second step of fig1 a . compound 2 ( substructure - 2 of gleevec ) is dissolved in ch2cl2 at − 78 ° c . cooled by dry - ice / acetone . a mixture of f2 and n2 gas containing 10 % f2 is passed through the reaction vessel continuously . at time points of 15 , 30 , 45 , and 60 min , one quarter of the content of the reaction is taken out and the solvent removed under vacuum . dried reaction products are dissolved in acetonitrile and the content analyzed by lc - ms ( agilent 1200 , agilent eclips 150 × 4 . 6 mm , gradient elution from 10 % acetonitrile / 90 % 0 . 1 % formic acid in dd - h2o to 90 % acetonitrile / 10 % 0 . 1 % formic acid in dd - h2o , 1 ml / min total flow rate , waters lct tofms in positive ion mode ). the expected products is a mixture and are shown in fig6 b , step 1 . this product mixture is then reacted with compound 1 catalyzed by dcc to produce a mixture of modified gleevec as expected products showing in the second step of fig1 b . the product mixture from example 8 and 9 is mixed with a buffered solution containing bcr - abl kinase enzyme at ph 7 . the mixture is filtered through a microcon filter with molecular weight cutoff ( mwco ) of 10 kda . new buffer is added to the top of the filter and the solution is filtered through the microcon filter . this process is repeated three times and a portion of the top layer of each filtration is removed and treated with organic solvent acetonitrile to denature the enzyme and to extract the ibuprofen and its fluorine modification products . compound concentration in the top layer is measured by lc - ms analysis and the relative affinity determined based on the concentration change after each round of filtering through microcon filter . the components with larger decrease in concentration after each filtering indicate lower affinity and vice versa . the product mixture from example 8 and 9 is separated by an hplc device . the eluent of the hplc is continuously passed through a buffered flow reactor cell containing bcr - abl kinase enzyme with a substrate peptide at ph 7 and analyzed by lc - ms . the inhibition effect and the identity of the components are monitored by the intensity change of the substrate peptide , the formation of enzymatic reaction product phosphopeptide and the corresponding mass of the mixture component eluting out at the time . the product mixture from example 9 is mixed with a buffered solution containing human liver microsome preparation and cofactors for metabolism . the mixture is incubated for 30 min at 37 ° c . and then is treated with organic solvent acetonitrile to stop the metabolism reaction and to extract the ibuprofen and its fluorine modification products . the concentration of compounds before and after the microsome incubation is measured by lc - ms , 1h - nmr and 19f - nmr analyses and the relative metabolic stability determined based on the concentration change after incubation . the components with larger decrease in concentration after microsome incubation indicate lower metabolic stability and vice versa . the data from experiments 10 - 12 are examined and the components with improved properties judging by the affinity / activity toward bcr - abl kinase enzymes or metabolic stability in human microsome incubation are identified . these components are purified using a preparative hplc with mass spectrometry and uv detection to obtain 0 . 5 - 1 mg for each compound of interest . nmr and / or ms / ms analyses confirm the structure of the purified compounds . the purified compounds are tested individually for affinity and activity toward bcr - abl kinase enzymes and for metabolic stability with human liver microsome preparation . the procedures are the same as examples 10 - 12 except a single purified compound is used in example 15 while a mixture of compounds is used in examples 10 - 12 . the results confirm that certain modified drug lead compounds , after purification into single pure compounds , show higher affinity and activity toward bcr - abl kinase enzymes or have better metabolic stability than the original drug lead compound . donepezil fluorination mixture library 1 is treated with 1 mg / ml concentration bovine serum albumin for 30 min in 10 mm nh4oac ( ph 7 ) to remove compounds that can covalently modify a protein , and then extracted with an organic solvent ( acetonitrile ) before using the mixture library for testing of drug properties . exemplary ms or lc - ms protocol . samples are analyzed by mass spectrometry alone or by liquid chromatography coupled to mass spectrometry , or other analytical techniques such as nmr , for the quantity and identity of components of the mixture compound libraries described in this method . mass spectrometry : analysis may be performed on , e . g ., time - of - flight mass spectrometers lct ( waters corporation , milford , mass ., usa ) using an z - spray ( electrospray ) ionization source . the electrospray voltage is generally maintained in the range of about 3 . 5 - 4 . 0 kv . ion optics settings are optimized on the day of the analysis to provide the maximum efficiency of ion to the detector . the effective mass range is generally from m / z 100 to m / z 1000 at a rate of about 1 s / scan . liquid chromatography : for example , samples can be introduced through an agilent 1200 ( agilent technologies , santa clara , calif ., usa ) chromatography operating in the gradient mode at a flow rate of 1 ml / min . an eclips c18 base - deactivated column ( 4 . 6 mm × 15 cm ) from agilent is used for sample separation . the mobile phase gradient is h2o + acn 90 / 10 ( v / v ) containing 0 . 1 % formic acid to a h2o + acn 10 / 90 ( v / v ) containing 0 . 1 % formic acid in 10 minutes . samples are introduced through an autosampler as part of the agilent 1200 . the sample injection volumes are generally 1 - 20 μl . it is apparent from the above results and discussion that a novel method of generating mixture compound library and screening the library for the purpose of discovering compounds with improved drug properties are provided by the subject method . these methods can find wide application in drug discovery and development . also provided are novel methods of non - selective modification of a drug lead structure . accordingly , the subject method provides for a significant contribution to the field . all publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference . the citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present method is not entitled to antedate such publication by virtue of prior methods . although embodiments of the method are shown and described therein , it should be understood that various changes and modifications to the presently preferred embodiments will be apparent to those skilled in the art . such changes and modifications may be made without departing from the spirit and scope of the method and without diminishing its attendant advantages . it is , therefore , intended that such changes and modifications be covered by the appended claims . nikolai f . sepetov , et al . u . s . pat . no . 6 , 799 , 120 — nonredundant split / pool synthesis of combinatorial libraries , us patent issued on sep . 28 , 2004 . nogrady , t . and weaver , d . f ., medicinal chemistry : a molecular and biochemical approach , oxford university press , 2005 corey , e . j . and cheng , x .- m ., the logic of chemical synthesis , wiley , new york , 1989 . lam , k . s ., et al . “ the ‘ one - 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