Patent Abstract:
compounds and methods related to nir molecular imaging , in - vitro and in - vivo functional imaging , therapy / efficacy monitoring , and cancer and metastatic activity imaging . compounds and methods demonstrated pertain to the field of peripheral benzodiazepine receptor imaging , metabolic imaging , cellular respiration imaging , cellular proliferation imaging as targeted agents that incorporate signaling agents .

Detailed Description:
embodiments of the present invention include nir agents for the pbr based on nir dyes , including lanthanide chelates . additionally , complimentary imaging agents are disclosed using a novel nir sacharide and nir thymidine agent . aspects of the present invention include both being used separately , as well as where the agents are used together as a cocktail whereby both pbr expression and metabolic and or cellular proliferation status could be simultaneously monitored in - vivo . pbr ligands such as pk11195 have been suggested as therapeutic agents . mitochondria localized anti - death proteins of the bcl - 2 family play a central role in inhibiting apoptosis and therefore present therapeutic targets . pbr shares a close physical association with the permeability transition pore complex ( ptpc ) and binding of pk11195 has been shown to cause bcl - 2 resistant generation of oxidative stress . the agents reported here are unique in that they facilitate in - vivo monitoring of therapeutic delivery and efficacy . as stated above , aspects of the present invention include methods of imaging a molecular event . in a sample , the method steps comprising administering to the sample a probe having an affinity for a target . the probe has at least one of a ligand / signaling agent combination , or conjugable form of a ligand / signaling agent combination . one such ligand / signaling agent combination comprises pbr ligands , or conjugable forms thereof . examples of the pbr ligands of the present invention include conjugable forms , or conjugable analogs of the following compounds : for the purposes of the present invention , the term analog encompases isomers , homologs , or other compounds sufficiently resembling the base compound in terms of structure and do not destroy activity . “ conjugable forms ,” “ conjugable compounds ,” and similar terms describe a form of the compound that can readily form a covalent form a covalent bond with a signaling agent such as an ir dye . for exemplary purposes , conjugable forms of pk11195 , above , include at least the following compounds , and / or analogs or derivatives thereof : wherein r is h or alkyl , n is 0 - 10 , and “ halo ” is fluorine , chlorine , bromine , iodine . in other embodiments of the present invention , halo is chlorine . the term “ halo ” or “ halogen ,” as used herein , includes radio isotopes of halogen compounds , such as i 121 and f 19 . additionally , for exemplary purposes , conjugable forms of ro5 - 4864 include the following and / or analogs or derivatives thereof : wherein the variables are defined above . in other embodiments of the present invention , halo is chlorine . additionally , for exemplary purposes , conjugable forms of daa1106 include the following and / or analogs or derivatives thereof : in other embodiments of the present invention , halo is chlorine or fluorine . additionally , for exemplary purposes , conjugable forms of ssr180575 include the following and / or analogs or derivatives thereof : wherein the variables are defined above . in other embodiments of the present invention , halo is chlorine . non - limited examples of pbr ligands and signaling moieties include the following compounds : or an analog thereof , wherein r 1 is a signaling moiety ; “ halo ” is fluorine , chlorine , bromine , iodine ; and n is 0 - 10 . additionally , other aspects of the present invention include nir - sacharide agents suitable for metabolic imaging in similar fashion to 18 fdg . given the ubiquitous clinical use of 18 fdg , 2 - deoxyglucose derivatives have been extensively biologically characterized . see czernin , j . ; phelps , m . e . annu rev med 2002 , 53 , 89 - 112 . these derivatives are useful metabolic imaging agents given the overexpression of glucose transporters and increased hexokinase activity in tumors . see medina , r . a . ; owen , g . i . biol res 2002 , 35 , 9 - 26 . 2 - deoxyglucose imaging agents are incorporated into cells via the glucose transporter and are subsequently phosphorylated by hexokinase . in phosphorylating the probe , the neutral molecule becomes anionic and membrane impermeable . functionalization at the 2 - position prevents further metabolism , and thus the probe is trapped in the cells , with further uptake leading to significant accumulation . see zhang , m . ; zhang , z . h . ; blessington , d . ; li , h . ; busch , t . m . ; madrak , v . ; miles , j . ; chance , b . ; glickson , j . d . ; zheng , g . bioconjugate chem 2003 , 14 , 709 - 714 . additionally , other aspects of the present invention include a nir - thymidine probe for monitoring cellular proliferation , similar in fashion to [ 18 f ] 3 ′- deoxy - 3 ′ fluorothymidine ( flt ). flt has been used clinically and extensively compared to fdg . see halter et al . general thoracic surgery 2004 , 127 , 1093 - 1099 and francis et al . eur j nucl med mol imaging 2003 , 30 , 988 - 994 . in proliferating cells , flt metabolism takes place within the anabolic arm of the dna salvage pathway . tk1 controls entry into the salvage pathway and converts flt to the mono - phosphate species . the agent is further phosphorylated , but can not be incorporated into dna due to its lack of a hydroxyl group at 3 ′. with respect to the signaling agents used in connection with the present invention , embodiments include near infrared signaling agents . also includes are dyes , such as , for example , near - infrared fluorophores / fluorescent dyes . examples include cyanine dyes which have been used to label various biomolecules . see u . s . pat . no . 5 , 268 , 486 , which discloses fluorescent arylsulfonated cyanine dyes having large extinction coefficients and quantum yields for the purpose of detection and quantification of labeled components . additional examples include dyes available from li - cor , such as ir dye 800cw ™, available from li - cor . wherein r is — oh , — co 2 h , — nh 2 , or — ncs and each of x and y , independently , is an integer selected from 1 to about 10 . in preferred embodiments , each of x and y , independently , is an integer between about 2 and 6 . in a second embodiment , the dye is n -( 5 - carboxypentyl ) n ′-( 4 - sulfonatobutyl ) 3 , 3 , 3 ′, 3 ′- tetramethylbenz ( e ) indodicarbocyanine , which has the formula : these two dyes are embodiments because they have commercially available precursors for the linking groups : 6 - bromohexanol , 6 - bromohexanoic acid and 1 , 4 - butane sultone ( all available from aldrich chemical co ., milwaukee , wis .). the linking groups provide adequate distance between the dye and the biomolecule for efficient attachment without imparting excessive hydrophobicity . the resulting labeled biomolecules retain their solubility in water and are well - accepted by enzymes . these dyes , wherein r is — co 2 h or — oh can be synthesized , as set forth in detail in the us &# 39 ; 709 patent , by reacting the appropriate n -( carboxyalkyl )- or n -( hydroxyalkyl )- 1 , 1 , 2 - trimethyl - 1h - benz ( e ) indolinium halide , preferably bromide , with sulfonatobutyl - 1 , 1 , 2 - trimethyl - 1h - benz ( e ) indole at a relative molar ratio of about 0 . 9 : 1 to about 1 : 0 . 9 , preferably 1 : 1 in an organic solvent , such as pyridine , and heated to reflux , followed by the addition of 1 , 3 , 3 - trimethoxypropene in a relative molar ratio of about 1 : 1 to about 3 : 1 to the reaction product and continued reflux . the mixture subsequently is cooled and poured into an organic solvent such as ether . the resulting solid or semi - solid can be purified by chromatography on a silica gel column using a series of methanol / chloroform solvents . as an alternative , two - step , synthesis procedure , also detailed in u . s . &# 39 ; 709 , n - 4 - sulfonatobutyl - 1 , 1 , 2 - trimethyl - 1h - benz ( e ) indole and malonaldehyde bis ( phenyl ) mine )- monohydrochloride in a 1 : 1 molar ratio can be dissolved in acetic anhydride and the mixture heated . the acetic anhydride is removed under high vacuum and the residue washed with an organic solvent such as ether . the residual solid obtained is dried and subsequently mixed with the appropriate n -( carboxyalkyl )- or n -( hydroxyalkyl )- 1 , 1 , 2 - trimethyl - 1h - benz ( e ) indolinium halide in the presence of an organic solvent , such as pyridine . the reaction mixture is heated , then the solvent is removed under vacuum , leaving the crude desired dye compound . the procedure was adapted from the two step procedure set forth in ernst , l . a ., et al ., cytometry 10 : 3 - 10 ( 1989 ). the dyes also can be prepared with an amine or isothiocyanate terminating group . for example , n -( omega .- amino - alkyl )- 1 , 1 , 2 - trimethyl - 1h - benz ( e ) indolenium bromide hydrobromide ( synthesized as in n . narayanan and g . patonay , j . org . chem . 60 : 2391 - 5 ( 1995 )) can be reacted to form dyes of formula 1 wherein r is — nh 2 . salts of these amino dyes can be converted to the corresponding isothiocyanates by treatment at room temperature with thiophosgene in an organic solvent such as chloroform and aqueous sodium carbonate . these dyes have a maximum light absorption which occurs near 680 nm . they thus can be excited efficiently by commercially available laser diodes that are compact , reliable and inexpensive and emit light at this wavelength . suitable commercially available lasers include , for example , toshiba told9225 , told9140 and told9150 , phillips cql806d , blue sky research ps 015 - 00 and nec ndl 3230su . this near infrared / far red wavelength also is advantageous in that the background fluorescence in this region normally is low in biological systems and high sensitivity can be achieved . the hydroxyl , carboxyl and isothiocyanate groups of the dyes provide linking groups for attachment to a wide variety of biologically important molecules , including proteins , peptides , enzyme substrates , hormones , antibodies , antigens , haptens , avidin , streptavidin , carbohydrates , oligosaccharides , polysaccharides , nucleic acids , deoxy nucleic acids , fragments of dna or rna , cells and synthetic combinations of biological fragments such as peptide nucleic acids ( pnas ). in another embodiment of the present invention , the ligands of the present invention may be conjugated to a lissamine dye , such as lissamine rhodamine b sulfonyl chloride . for example , a conjugable form of daa1106 may be conjugated with lissamine rhodamine b sulfonyl chloride to form a compound of the present invention . lissamine dyes are typically inexpensive dyes with attractive spectral properties . for example , examples have a molar extinction coefficient of 88 , 000 cm − 1 m − 1 and good quantum efficient of about 95 %. it absorbs at about 568 nm and emits at about 583 nm ( in methanol ) with a decent stokes shift and thus bright fluorescence . coupling procedures for the pbr ligands and glucosamine proceed via standard methods and will be recognized by those skilled in the art . in general , the nucleophilic n terminus of the targeting moieties are reactive towards activated carbonyls , for example an nhs ( n - hydroxysuccinimide ester ), sulfonyl chlorides , or other electrophile bearing species . solvent of choice for coupling reactions can be dye specific , but include dimethyl sulfoxide ( dmso ), chloroform , and / or phosphate buffered saline ( pbs buffer ). the resulting conjugates , amides , sulfonamides , etc . resist hydrolysis under physiological conditions , and are thus useful for in - vivo and in - vitro application . the following compound is an example of one of the coupled compounds described above : and analogs thereof , wherein n and x are integers from 1 to 10 . as stated above , the compounds of the present invention can be employed as signaling agents in nir imaging . the resulting signal may be used to image a molecular event . non - limiting examples of specific molecular events associated with the present invention include at least one of peripheral benzodiazepine expression , cell proliferation , glucose uptake , epidermal growth factor receptor expression , coronary disease . thus , the resulting signal may be used to diagnose a disease state such as , for example , cancer , neurodegenerative disease , multiple sclerosis , epilepsy , coronary disease , etc . specifically , brain cancer and breast cancer are two cancers that may be diagnosed with the compounds and methods of the present invention . two additional examples are non - hodgkin &# 39 ; s lymphoma and colon cancer . another embodiment of the present invention is a method of measuring glucose uptake . this method comprises , comprises administering to a sample a conjugate , the conjugate comprising a conjugable glucosamine compound and a signaling agent ; and detecting a signal from said conjugate . as in the other methods , the sample is at least one of cells , tissue , cellular tissue , serum or cell extract . an example of a conjugable glucosamine includes the following compound and conjugable analog thereof : the administration step may be in vivo administration or in vitro administration . the in vivo administration step further comprises at least one time course imaging determination , and in other embodiments , the in vivo administration step further comprises at least one bio distribution determination . other embodiments of the present invention include conjugable compounds associated with this glucosamine method , specifically including the following : the following examples are presented purely for exemplary purposes , and as such the material in this section should be considered as embodiments of the present invention and not to be limiting thereof . this example demonstrates the conjugation of a nir dye of the present invention and a conjugable analog or conjugable form of pk11195 for deep tissue imaging . in this example , irdye800cw ( licor ) is coupled to conjugable pk11195 . dye800cw - pk11195 ( scheme 1 )— to a 10 ml round bottom flask , about 196 . 5 μl of a 1 mg / ml conjugable pk11195 solution ( dmso ) is mixed with about 300 μl of an about 1 mg / ml dye800cw ( dmso ). the reaction proceeds under nitrogen flow for about 1 hour at rt . reaction progress is monitored via hplc and esi ms . this example demonstrates an example of the formulation of a nir - glucosamine conjugate of the present invention . dye800cw - glucosamine ( scheme 2 )— to a 10 ml round bottom flask , about 9 . 3 mg sodium methoxide and about 37 mg d - glucosamine hydrochloride are reacted in about 2 ml dmso . the solution is stirred under nitrogen for about 3 hours at rt . next , about 34 , of the resulting solution are mixed with about 150 μl of an about 1 mg / ml dye800cw / dmso solution in a separate 10 ml flask . the mixture is stirred under nitrogen for another 1 . 5 hours at rt . reaction progress was monitored via hplc and esi ms and the reaction yielded 98 % pure conjugate . this example demonstrates the use of compounds of the present invention in esi ( electrospray ionization ) mass spectra . initially , about 20 μl of the reaction solution of example 1 is diluted to about 180 μl using 5 mm ammonium acetate aqueous solution containing about 0 . 05 % acetic acid . the sample is injected the sample immediately into a mariner esi mass spectrometer . some major instrument settings are : spray tip at about 3 . 4 kv , nozzle potential at about 200v , quadrupole temperature at about 150 ° c . and nozzle temperature at about 150 ° c . spectra is collected every 100 seconds . in spectrum for dye800w - glucosamine complex , the expected molecular peak is observed at 1164da . in the spectrum for dye800cw - pk11195 complex , the expected molecular peak is observed at 1365 . 9da . this example shows a synthetic pathway yielding a conjugable ro5 - 4864 of the present invention , and conjugation to an imaging agent , such as lanthanide chelate or nir - dye . a conjugable form of compounds similar to ro5 - 4864 has been previously reported ( see u . s . pat . no . 5 , 901 , 381 ) and a synthetic procedure in scheme 3 will be used to synthesize a conjugable form of ro5 - 4864 . a solution of koh in methanol will be treated with a solution of 4 - chlorophenyl - acetonitrile 1 and 4 - chloronitro - benzene 2 in benzene . the mixture will be stirred for 3 hours and then poured to ammonium chloride solution . compound 3 will then precipitate out . compound 4 will be produced by stirring compound 3 and dimethyl sulfate for 5 hours , followed by being treated with ethanol , water , iron fillings and hydrochloric acid . see vejdelek z , polivka z , protiva m . synthesis of 7 - chloro - 5 -( 4 - chlorophenyl )- 1 - methyl - 1 , 3 - dihydro - 1 , 4 - benzodiazepin - 2 - one . collection of czechoslovak chemical communications 1985 ; 50 : 1064 - 1069 . compound 4 and semicarbazide , after heated to 210 ° c ., will produce compound 5 . compound 7 will be used as a linker to combine compound 5 and lanthanide chelate / dye800cw . compound 7 can be synthesized by the reaction between compound 6 and thionyl chloride . lanthanide chelate ( with carboxylic acid group ) or dye800cw ( a n - hydroxysuccinimide ester ) can then react with compound 7 in basic solution to produce a compound in the form of compound 8 . the chlorine on the signaling part will react with n — h group in compound 5 to produce the final imaging agent ( compound 9 ). the product can be further chelated by adding lanthanide chloride solution ( lncl 3 , eucl 3 etc ) into product solution with ph 6 . 5 . the synthetic pathway for lanthanide chelate has been reported . see griffin j m m , skwierawska a m , manning h c , marx j n , bornhop d j . simple , high yielding synthesis of trifunctional fluorescent lanthanide chelates . tetrahedron letters 2001 ; 42 : 3823 - 3825 . this example shows a scheme for the synthesis of a conjugable form of daa1106 of the present invention , which cnathen be conjugated to an imaging agent . the synthetic pathway for conjugable dam106 is shown in scheme 4 . compound 2 will be obtained by reaction of compound 1 with phenol in dmf . compound 2 will then be reduced by pto2 under hydrogen flow in methanol . compound 3 can react with acetyl chloride in pyridine to produce compound 4 after the reaction refluxes for 2 hours . the hydroxyl group in compound 5 will be substituted by bromide to produce compound 6 . one hydroxyl group in compound 6 will be deprotected in dmf by sodium ethanethiolate to produce compound 7 . compound 4 and 7 will then react in dmf with the presence of sodium hydride . after compound 8 is obtained , the hydroxyl group will be brominated to form compound 9 . conjugable daa1106 ( compound 10 ) is prepared by treatment of compound 9 with hexane - 1 , 6 - diamine . the conjugation position on dam106 is determined according to another conjugation that has been done on daa1106 which did not affect the biological activity of daa1106 . see zhang m r , maeda j , furutsuka k , yoshida y , ogawa m , suhara t , et al . [ f - 18 ] fmdaa1106 and [ f - 18 ] fedaa1106 : two positron - emitter labeled ligands for peripheral benzodiazepine receptor ( pbr ). bioorganic & amp ; medicinal chemistry letters 2003 ; 13 : 201 - 204 . the product should be conjugable to lanthanide chelator in water / dmf / dioxane / tea mixture . the conjugate will be further chelated by adding lanthanide chloride solution ( lncl 3 , eucl 3 etc ) into ph 6 . 5 product solution . this example shows an example of the synthesis , characterization , and preliminary cell study for an embodiment of the present invention , a dye800cw - daa1106 conjugation , as well as the conjugation of the pbr ligand daa1106 to a nir dye , followed by cell uptake . in this example , dye800cw ( 5 mg , 4 . 3 pimp and conjugable daa1106 ( 5 mg , 10 μmol ) is mixed in dmso ( 1 ml ) in a 10 ml round bottom flask . the solution is stirred under argon flow for 10 hours . the reaction scheme is shown in scheme 5 , below . product is purified through neutral alumina column using 0 . 1 m triethyl ammonium acetate in 80 / 20 acetonitrile / water solution . upon preparing dye800cw - daa1106 , absorption and emission spectra ( table 1 ) are obtained at room temperature with a shimadzu 1700 uv - vis spectrophotometer and iss pci spectrofluorometer respectively . the same sample ( 2 μm ) is used for taking both uv and fluorescence spectrum . uv spectrum was scanned from 190 nm to 900 nm with sampling rate of 1 nm . cuvette path length was 1 cm . fluorescence sample was excited at 797 nm . spectrum was collected from 700 nm to 900 nm with scan rate 1 nm / second . slit width was set to 1 . 5 . photo multiplier tube ( pmt ) voltage was at 75 watts . dye800cw - daa1106 has maximum absorption at 779 nm and fluorescence at 801 nm in methanol . regarding cell uptake , c6 glioma cell lines are a widely used cell line in neurobiological research that has high pbr expression . c6 cells were incubated with 10 μm dye800cw - daa1106 in culture media for half hour and then rinsed and re - incubated with saline before imaging . fig1 shows white light and fluorescence pictures of dosed and un - dosed cells . instrument used is nikon epifluorescence microscope equipped with ludl qimaging camera , nikon s fluor 20x / 0 . 75 objective , mercury lamp and icg filter set . picture b shows cell take - up of dye800cw - daa1106 , while un - dosed cell ( picture d ) does not show any significant fluorescence . this example shows the synthesis , characterization and preliminary cell study of a lissamine - daa1106 conjugation . an example of a lissamine dye has a molar extinction coefficient of 88 , 000 cm − 1 m − 1 and good quantum efficient of about 95 %. it absorbs at 568 nm and emits at 583 nm ( in methanol ) with a decent stokes shift and thus bright fluorescence . lissamine rhodamine b sulfonyl chloride ( 4 mg , 6 . 9 μmol ), conjugable dam106 ( 5 mg , 10 pimp and tri - ethylamine ( 10 μl ) was mixed in dichloromethane ( 0 . 8 ml ) in a 10 ml round bottom flask . the solution was stirred under argon flow for 3 hours . the reaction scheme is shown in scheme 6 . product was purified through column chromatography ( silica gel ) using 19 / 1 dichloromethane / methanol solution . upon preparing lissamine - daa1106 , absorption and emission spectra ( table 2 ) was obtained with a shimadzu 1700 uv - vis spectrophotometer and iss pti spectrofluorometer at room temperature . the same sample ( 2 μm ) was used for taking both uv and fluorescence spectrum . uv spectrum was scanned from 190 nm to 900 nm with sampling rate of 1 nm . cuvette path length was 1 cm . fluorescence sample was excited at 561 nm . spectrum was collected from 700 nm to 900 nm with scan rate 1 nm / second . slit width was set to 1 . 5 . photo multiplier tube ( pmt ) voltage was at 75 watts . lissamine - daa1106 has maximum absorption at 561 nm and fluorescence at 579 nm in methanol . c6 cells were incubated with 10 μm lissamine - daa1106 in culture media for half hour and then rinsed and re - incubated with saline before imaging . fig2 shows white light and fluorescence pictures of dosed and un - dosed cells . instrument used was nikon epifluorescence microscope equipped with ludl qimaging camera , nikon s fluor 20x / 0 . 75 objective , mercury lamp and texas red filter set . picture b shows cell take - up of lissamine - daa1106 at perinuclear location . this observation was expected since pbr is a mitochondrial protein . un - dosed cells ( picture d ) exhibited no fluorescence . this example shows an example of a synthetic pathway yielding a conjugable form of a ssr180575 compound of the present invention . starting from m - chloroaniline , which was diazotised and coupled with ethyl α — methylacetoacetate , the azo - ester was converted into ethyl pyruvate m — chlorophenylhydrazone 1 ( the japp - klingeman reaction ). polyphosphoric acid facilitated the conversion to molecule 2 . next , n - methylation with dimethylcarbonate in presence k 2 co 3 yielded the ester 3 and was treated with hydrazine and converted into hydrazide 4 . the ring was closed in the presence of pocl 3 and compound 5 was obtained . n - phenylation with using phi and cui ( as catalyst ) provide compound 6 . mild hydrolysis with dilute koh in etoh yield acid 7 . to conjugated mono - n - boc protected n - methyl - 1 , 6 - hexanediamine to 7 used bop . removal of protecting group with tfa in ch 2 cl 2 is yields 8 . example 9 demonstrates specific , in vivo tumor labeling using a method of the present invention . a nir - pk 11195 deep tissue imaging agent was made as shown in example 1 . tumor bearing smad3 gene knockout mice and control animals were injected with 10 nmoles of the imaging agent and imaged about 14 hours following injection . specific labeling was observed in the abdominal region of tumor animals and clearance in the control animals . this selective uptake is shown in fig3 . a post - imaging autopsy confirmed localization of the imaging agent in the smad3 animal . this example shows nir - pk 11195 imaging in connection with neurodegenerative processes in experimental autoimmune encephalomyelitis ( eae ), the animal model of multiple sclerosis . additionally , this example shows the use of the present invention to monitor the progression of a disease state . a conjugated imaging agent nir - pk 11195 was made in accordance with example 1 . an eae induced and control animals were injected with nir - pk 11195 and imaged . eae animals demonstrate strong fluorescence along the spinal column indicating activated t cell and macrophage response which signal the onset of the demylenation processes characteristic to eae . the eae / treated mouse was treated with a curcumin composition . fig3 shows images associated with this example that confirm insignificant uptake of the imaging agent in the control , but indicate full onset of a disease state in the eae mice . subsequent imaging shows the progression of the disease after a disease state treatment is administered . throughout this application , various publications are referenced . all such publications , specifically including the publications listed below , are incorporated herein by reference in their entirety . 1 . manning h c , goebel , t . s . thompson , r . c ., and bornhop , d . j . a pbr targeted molecular imaging agent for cellular - 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18 : 1676 - 1688 . 43 . manning h c , goebel t , marx j n , bornhop d j . facile , efficient conjugation of a trifunctional lanthanide chelate to a peripheral benzodiazepine receptor ligand . organic letters 2002 ; 4 : 1075 - 1078 . 44 . zhang m , zhang z h , blessington d , li h , busch t m , madrak v , et al . pyropheophorbide 2 - deoxyglucosamide : a new photosensitizer targeting glucose transporters . bioconjugate chemistry 2003 ; 14 : 709 - 714 . it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention . other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein . it is intended that the specification and example be considered as exemplary only , and not intended to limit the scope and spirit of the invention . unless otherwise indicated , all numbers expressing quantities of ingredients , properties such as reaction conditions , and so forth used in the specification and claims are to be understood as being modified in all instances by the term “ about .” accordingly , unless indicated to the contrary , the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be determined by the present invention . notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations , the numerical values set forth in the experimental or example sections are reported as precisely as possible . any numerical value , however , inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements .