Synthesis and biological evaluation of sparsomycin analogues

Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 1981 Synthesis and biological evaluation of sparsomycin analogues Scherer Preston Sanders Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medicinal and Pharmaceutical Chemistry Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/5601 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass For more information, please contact libcompass@vcu.edu SYNTHES I S AND B I OLOG I CAL EVALUATION OF SPARSOMYC I N ANALOGUE S by S c h e r e r P r e s t on S an d e r s Duk e B S , C o l lege o f W i l l i am and Mary , 1977 Thes i s subm i t t e d i n part i a l f u l f i l lme n t o f t h e requi reme n t s f o r t h e Degree o f Doc t o r o f Ph i l osophy i n t h e Departme n t o f Pharmaceut i c a l Chem i s t ry at t h e Me d i c a l C o l l e ge o f V i r g i n i a , V i r g i n i a Commonwe a l t h Un i ve rs i t y R i chmond , V i r g i n i a August , 19 ii Th i s t h e s i s b y S c h e r e r P r e s t o n Sanders Duke i s a c ce p t e d i n i t s p r e s e n t form as s a t i s f y i n g t h e t he s i s r e q u i reme n t f o r t h e degree o f D o c t o r o f Ph i l os ophy Date : - TJl ':7 · • · · • • • 1- l J • t• I � • Approve d : � • I • • !� r ! .� �J.) (q.ft [v: /) � · · :r u.l'f �l Cf4r Approved : t I g 8-l ,;2/., 1'1.f1 Ch a i rman , MCV Graduat e Counc i l ; Dean , Schoo l o f Bas i c Sciences iii S ch e re r Pres t on Sanders Duke A l l R i gh t s Re served 1981 iv ACKNOWLEDGMENTS I w i s h to t h an k t h e memb e r s o f my graduate commi t te e f o r overs e e i n g t h i s proj e c t an d e s p e c i a l l y my adv i sor , D r Mar v i n R Boot s , f o r h i s g u i dan ce a n d e n courageme n t t h roughout t h e course o f t h i s res earch a n d t h e preparat ion o f t h i s t h es i s I a l s o appre c i at e t h e h e l p f u l adv i ce o f Dr Grah am W i nd r i dge and h i s generous g i f t of t h e t e r t - but y loxyaz i f orma t e used i n t h i s proj e c t I am grat e f u l t o Dr A l Mun s on , Dr P a a l K l y kken , and Ms Bern i e Kauf fmann for t h e i r i nv a l u ab l e g u i dance and ass i s t ance with t h e ph armaco l o g i c a l a s p e c t s o f t h e proj e c t I g r at e fu l l y acknowle dge t h e f i n anc i a l support provi ded b y A H Rob i n s , In c , the A D W i l l i ams Fund , and the De­ partment of Ph armaceu t i ca l Chemi s t ry , MCV / VCU I t h an k Mrs Gay le H y l t on for t y p i n g t h e man usc r i pt A spec i a l t h anks i s e x t ended t o my paren t s , Mr and Mrs Carl W Sanders , and my b ro t h e r , Car l , f o r t h e i r i n ­ e s t i mab l e e n courageme n t a n d i n t e res t i n m y care e r I espec i a l l y apprec i at e t h e w i se adv i ce g iv e n me by my fat h e r Las t ly , I w i s h t o express my s i nc e re grat i t ude t o my husban d , George , f o r h i s p at i en ce , encouragemen t , and un der­ s t an d i n g It i s t o h i m t h i s work i s de d i cat ed v V I TA vi TABLE OF CONTENTS COPYR I GHT PAGE - - - - - - - - - - - - - - - - - - - - - - - - - - - V I TA - - - - ACKNOWLEDGMENTS - - - - - iii iv - - - v TABLE OF CONTENTS - - - - - - - - - - - - - - - - - - - - - - - - - vi L I ST O F TABLE S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - xi L I ST OF F I GURES - - - - - - - - - - - - - - - - - - - - - - - - - - - - - xii i L I ST OF ABBREV I A T I ONS - - - - - - - - - - - - - - - - - - - xiv ABSTRACT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - xv I NTRODUCT10N- - - - - - - - - - - - - - - - - - - - - - - - - - - - I I RESEARCH A I M - - - - - - - - - - - - - - - - - - - - - - - - - 23 I I I D I SCUS S I ON O F SYNTHES I S - - - - - - - - - - 33 I V D I SCUS S I ON OF PHARMACOLOG I CAL RESULTS - - 57 V CONCLUS I ONS - - - - - - - - - - - - - - - - - - - - - - - - 87 V I EXPE R I MENT AL - - - - - - - - - - - - - - - - - 94 A Gen e r a l - - - - - - - - - - - - - - - - - - - 94 B Compoun ds S y n t h e s i z e d - - - - - - - - - - - - - 96 , - D i h ydroxy - 5- h y droxyme t h y l - me t h y l p y r im i d i ne (�) - - - - - - - - 96 ( , -D i h y droxy - - me t h y l - 5-py r im i d i n y l ) carbox a l dehyde (1) - 96 Carb e t hoxyme t h y l t r i ph e n y l phosphon i um Brom i d e ( 4a ) - - - - - - - - - - - - - - - - - - - - - - - 97 Carbet hoxyme t h y l en e t r i p h en y l sphorane (i) - - - - - - - - - - - - - - - - - 97 ( E ) -Me t h y l - ( , - D i h y droxy - - met h y l - 5pyr im i d i n y l ) prope noa t e (�) - 98 I - - - - - - - vi i Page ( E ) - - ( , - D i h ydroxy- 6-m e t h y l- 5-py­ rIm i d i n y l ) propeno i c Acid ( Q ) - - - - - - - - - 99 ( E ) - ( + ) - N - ( 2- H e p t an y l ) - 3- ( , - d i hy ­ droxy=6-me t h y l - - p y r i m i d i n y l ) - propen am i de ( 19 ) - - - - - - - - - - - - - - - - - - - 99 Met h y l - Ami n o- -phen y l propanoate H y ­ droch l or i de ( ) - - - - - - - - - - - - - - - - - - - 100 -Am i no - - phen y l - l - propan o l ( ) - - - - - 101 ( E ) - ( + ) -N- ( 1-H yd roxy- - p he n y l- - propy l ) 3= ( , - dIhy drox y - 6-met h y l - 5- p y r i m i d i n y l ) - propen ami de ( 2 ) - - - - - - - - - - - - - - - - - - - 1 - Am i n o - - ( - b romopheny l ) propano l ( ) 102 ( E ) - ( + ) -N- [ 1-h y droxy- - ( 4- b romopheny l ) 2=prop y l] - - ( , - d i h ydroxy- 6-me t h y l - py r im i d i n y l ) - 2- propenam i de ( 24 ) - - - - - - D i e t h y l -A c e t am i do- - ( -me t h y l b e n ­ z y l ) ma lo na t e ( ) - - - - - - - - - - - - - - - - - - 104 -Am i n o - - ( - t o l y l ) pr op i o n i c A c i d ( ) 105 - ( - Ami no- -h ydroxyprop y l ) t o luene ( ) 106 ( E ) - ( + ) -N- [ - H y d roxy - - ( 4-me t h y l ­ ph e n yl ) - - p ropy l ] - 3- ( , 4- d ih y droxy6 - me t h y l - 5- py r i m i d i ny l ) - -propenamide ( ) - - - - - - - - - - - - - - - - - - - - - - - - - 107 - M e th oxybe n z y l Brom i de ( ) - - - - - - - - 107 E t h y l - A c e t am i - - carbet hoxy- 3- ( methoxyphen y l ) propanoate ( 30 ) - - - - - - - 108 - Ac e t am i -2- carb e t hoxy - - ( -met hoxy­ pheny l ) p ropan o i c Ac i d ( ) - - - - - - - - 109 E t h y l - A c e t am i - - ( 4-methoxypheny l ) propanoat e ( ) - - - - - - - - - - - - - - - 109 - A c e t am i - - ( - me thoxyph e n y l ) prop a n o l ( 3 ) - - - - - - - - - - - - - - - - - - - - - - 110 - Am in o - - ( 4-methoxyphen y l ) propano l Hydroc h l o r i de ( 34 ) - - - - - - - - - - - - - - - - 111 vi i i Page ( E ) - ( + ) - N - � - Hy droxy- - ( - methoxy­ ph enyl ) - 2- propy l]- - ( , 4-d i h ydroxy - met h y l-5 - p y r i m i d i n y l ) - - propenam i de ( ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 ( E ) - ( + ) - N - ( - Pheny l e t hy l ) - - ( , dlhy droxy - - met h y l - - p y r im i di n y l ) - prope nam i de ( 36 ) - - - - - - - - - - - - - - - - 112 ( E ) - ( + ) -N- [ - ( - M e t h y l ph e n y l ) - e t hy l]3= ( , - dlh ydroxy- 6-met h y l - - p y r im i ­ d i n y l ) - - p ropen amide ( ) - - - - - - - 1 ( E ) - ( + ) - N - [ - ( - M e t h o xyphen y l ) - e t h y l ] 3=( , - dlh ydroxy- 6-me t h y l - - p y r i m i ­ d i ny l ) - - propenam i de ( 38 ) - - - - - - - 14 ( E ) - ( + ) - N - [ - ( - Bromophen y l ) - e t h y l ] 3= ( , - dlhy droxy - - me t h y l - 5-pyr imi­ d i ny l ) - - propena mide ( 39 ) - - - - - - - - - - 14 3- ( t e r t - But y l oxyc arbon y l am ino ) pro­ p an o i c A c i d ( ) - - - - - - - - - - - - - - - - - 115 , , - T r i c h l orophen y l - ( t e r t - But y­ loxyc arbon y l am i n o ) propanoa t e ( ) - 116 ( + ) - N - Ben z y l - - ( t ert -but y l ox y carbo­ ny l am ino ) propanamide ( ) - - - - - - - - - 116 - Ami n o - N - benzy l propan ami de Tr i­ f l uo r o acet at e ( ) - - - - - - - - - - - - - - - - 117 ( E ) - ( + ) -N- Ben z y l - - [ - ( , - d i hydroxy 6=me thy l=5- py r im i d i n y l ) - 2- propana­ m i do]propanam i de ( 44 ) - - - - - - - - - - - 1 ( + ) - N - ( - Met h y l benzy l ) - - ( t e r t ­ but yloxyc arbon y l am i n o ) propanam i de ( 5a ) - - - - - - - - - - - - - - - - - - - - - - - - - - - 119 ( � )-( � ) ��_( - M e t h y lbenzy l ) - - [ - ( , d i h y droxy - - me t h y l - - p y r i m i d in y l ) - propenami do]propanami de ( ) - - - - 19 ( + ) - N - ( - M e t hoxybenzy l ) - - ( t e r t ­ but ylox y c arbon y l am i n o ) propanamide ( 47a ) - - - - - - - 121 ( E ) - ( + ) - N - ( - Met hoxyben z y l ) - - [ - ( , 4=d i hydroxy - - me t h y l - - p y r i m i d i n y l ) 121 - propenami do]propanam i d e ( ) ix Page ( + ) -N- ( 4- Bromobenzy l ) - 3- ( t e r t - buty l oxy­ carbon y l am i n o ) propanam i de ( 49a ) - - - - - - - 123 ( E �+ ) -N- ( 4- Bromobe n zy l ) - - [ 3- ( , 4dIhydroxy-6 - me t h y l - 5- py r im i d i n y l -propen am i ] propanam i d e ( 50 ) - - - - - - - - 124 ( + ) - N- ( 3,4 - D i c h l oroben zy l ) - 3- ( t e r t ­ butyloxycarbon y l am i n o ) propa' n am i de ( 1a ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 125 ( E ) - ( + ) - N - ( 3,4- D i c h l orob enzy l ) - 3[ - ( � - d i h y droxy- -met h y l - pyr i m i d in y l ) - -propenam i d o ] pro panam i de ( 52 ) - - - - - - - - - - - - - - - - - - - - - - - - 126 � -Pht h a l y l -DL - aspart i c An hydr i de (�) 127 N - Ben z y l -N - P h t h a l y l -DL- aSparag i n e ( 54 ) -= -� N - Ben z y l oxycarbon y l -DL- aspart i c Ac i d ( 60 ) � N-Benzy l oxycarbony l -DL- aspar t i c Anhydr i d e ( ) - - - � - - - - - - - - - - - - - - - - - - 129 a - Benzy l N- Ben zy l ox y c arbo n y l - DL­ aspar t a t e- ( 62 ) - - - - - - - - - - - - - � - - - - - - - - Be n z y l N-Ben zy l oxycarbon y l - DL­ � aspa r t a t e- ( 62 a ) 131 N - ( - Bromob enzy l ) - 3-benzy l oxycarbo n y l ­ ami n o- , - p y rro l i d i n e d i o n e ( 62b ) - - - - - - 132 - - Ben z y l � - Ben z Y l oxycarbon y l -�4 - ( 4-bromo­ b e n zy l ) -DL- asparag i na t e ( ) - - - - - - 3 C �- ( 4-Bromobenzy l ) - 3-benzy l oxycarbo­ n y l am i n o - - hydroxybut anam i d e ( 64 ) - - - - 133 Pharmaco l o g i c a l Mat e r i a l s and Methods - 135 Exper imen t a l An ima l s - - - - - - - - - - - 135 Preparat i o n o f Med i a - - - - - - - - - - - - a A l pha Mod i f i cat ion o f Eag l e ' s M i n imum E s s en t i a l Med i um - - - b Comp l e t e Du l b e c c o ' s Mod i f i e d E a g l e ' s M i n imum E s s en t i a l Med i um - - - - - - - - - - - - - - - - - - - - 135 135 36 39 t r i fu ge d f o r t e n m i nu t e s at 300 X g ( I n te r n at i o n a l CRU- 5000 c e n t r i f uge , Damon , IEC D iv i s ion , Needham , MA ) and resus - p e n de d i n S p i n n e r ' s me d i a The P 8 t umo r l in e was ma i n t a i n e d i n DBA / m a l e m i c e b y s e r i a l i n t rape r i t o n e a l ( i p ) pass age o f ce l l s every seven t o t e n days To h a r v e s t t h e t umo r c e l l s , t h e DBA / m i c e we re s a c r i f i c e d b y cerv i c a l d i s l o c a t i on The pe r i t on e a l ce l l s were co l l e c t e d asept i c a l l y b y i n j e c t i n g and w i t hdraw i n g - mL o f S p i n n e r ' s me d i a f rom t he pe r i t on e a l cav i t y were spun a t 300 X g f o r t e n m i n u t e s The c e l l s The supe r n a t a n t was decan t e d and the ce l l s were resuspended in - mL of Sp i n n e r ' s me d i a t a i n i n g 10% heat - in ac t i v a t e d f e t a l c a l f s e r um The ce l l s were e n ume r a t e d on a Cou l t e r coun t e r a n d t h e c o n c e n t r at i o n o f ce l l s was adj us t e d t o X ce l l s / mL b y d i l u t i on w i t h Sp i n n e r ' s me d i a b I n v i t ro P c e l l s , court e s y o f Dr Do l ph Adams , Departme n t o f P at h o l o gy , Duke Un i v e r s i t y Hosp i t a l , Durh am , N o r t h Caro l i n a , were ma i n t a i n e d i n c u l t u r e f l asks ( cm T f l asks , Cor n i n g , Corn i n g , New Yor k ) i n CDMEM The ce l l s were passaged tw i ce wee k l y b y add i n g an a l i quot o f X c e l l s t o mL o f CDMEM W h e n n e eded f o r t h e pro t e i n s y n t he s i s s t ud i e s , t he cen t r at i o n of ce l l s was adj u s t e d t o X ce l l s / mL b y d i lu t i o n w i t h CDMEM Prepar a t i o n o f Sub s t rat e , Drug , and I nh i b i t o r S o l u t i o n s a Cont ro l Drugs 140 Puromy c i n d ih y droch l o r i de ( NSC- 5 ) was ob t a i ned f r om S i gma Chem i cal Compan y , St Lou i s , MO Sparsomy c i n ( NSC- 9NB ) and A r a- C ( NSC - 8 , ami n o - B -Q- arab i no furanos y l - ( lH ) - py r im i d i none ) were generou s l y obt a i ned f r om t h e Nat i o a l Can c e r I n s t i t ut e , B e t h esda , MD So lut i on s o f t h e s e drugs at the concen t r a t i on s spec i f i ed in the d i scus s i on of t h e p h armaco l o g i c a l resu l t s were prepared us i n g t h e appropr i at e med i a f o r t h e assay Rad i o i s ot op es b - - l odo - - deoxyur i d i ne ( IUdR ) was ob t a i ned f rom New E n g l an d Nuc l ear , Bos ton , MA , and prepared as a s o l u t ion -5 125 of �Ci IUdR i n 20 �L o f X M f l uorodeoxyur i d i n e 75 ( FUdR , S i gma Chem i c a l Compan y , S t Lou i s , MO ) L_ [ Se _ s e l e n ome t h ion i n e was ob t a i ned f rom Amer sh am Corporat ion , A r l i ng t o n H e i gh t s , I L , and was prepared as a s o l u t ion o f �Ci [ 75 Se l - s e l enome t h ion i ne p e r � L o f t h e i n d i c at e d med i a f o r t h e a s s ay b e i n g per f o rmed c A n a l o gues o f Sparsomy c i n Compoun ds , 2 , , , 36 , 37 , , 39 , 48 , a n d 50 -2 M aqueous s t o c k s o l ut i o n s us i n g d i s were prepared as t i l l e d wat e r D i lut i on s were made us i n g med i a to obt a i n t h e concen t rat i o n s requ i red f o r exper i men t at i o n w a s i n so lub l e i n wat e r an d was prepared a s a 10 a l coho l i c s o l u t i o n u s i n g ab so l u t e e t h an o l Compound -2 M D i l u t i o n s were made w i t h med i a t o decrease the p e r c e n t age of a l coho l pr es ent i n t h e c e l l c u l t ure Compoun ds 44 , 46 , and were not so l ub l e in wat e r o r e t h a n o l at the requ i re d c e n t r a- 141 t i on t h ereby p r e c l u d i n g t h e t e s t i ng o f t he s e t h ree compoun ds E xpe r imen t a l Proc edures f o r t h e B i o l o g i c a l A s s ay s a DNA S y n t h e s i s I n h ib i t ion A s s a y (1) T ime Cou r s e Determ i n a t ion CD- m i ce were s a c r i f i c e d b y cerv i c a l d i s locat i o n The l ower h a l f o f t h e i r b o d i e s was washed w i t h 5% et hano l , t h e femurs were d i s s e c t e d , and a l l t h e t i ssue surroun d i n g t he b o n e w a s remove d Both ends ( i f necessary ) of the fe- mur were cut o f f and t h e marrow was f lushed out i n t o a p l a s t i c c en t r i fuge t ub e u s i n g a 5- gauge n e e d l e f i l l ed w i t h a MEM Th e bone mar row susp e n s i o n was cen t r i f uged at 300 X g f o r ten m i nu t e s The supe r n a t e n t was deca n t e d an d t h e c e l l s were resuspended i n o n e mL o f a MEM The ce l l s were coun t e d on a Cou l t er coun t e r and t h e conce n t rat i on o f c e l l s adj u s t e d t o X 10 ce l l s / mL b y d i lut i on w i t h aMEM A f t e r prepa r i n g t h e c e l l s , 200 � L of t he ce l l suspen s i on was added t o each w e l l o f a m i crot i t e r p l a t e ( M i c r o t e s t I I , Fa l co n P l a s t i c s , Oxnard , CA ) I n add i t i o n t o t h e c e l l s , I U dR at a concen t rat i on o f � C i I UdR/ 20 20 � L of -5 M FUdR was added t o each we l l The i n cu� L o f X 10 b a t i o n t ime s were var i e d so t h a t s i x we l l s o f ce l l s were harve s t e d a t e ac h t ime po i nt : , 30 , , 60 , and 120 m i n utes Fo l l ow i n g i n cub at i on , t h e c e l l s were h a rv e s t e d on a T i t e rt ek c e l l h a r v e s t e r wh i l e f lush i n g t h e we l l s w i t h n o r m a l s a l i n e ( M sod i um c h l o r i de) for comp l e t e remo v a l o f t he ce l l s T h e f i l t er paper d i s ks t a i n g t h e I U d R 142 i n co rpor a t e d i n t o DNA w e r e p l aced i n t ubes and r a d i o a s s ay e d on a gamma count e r ( mo d e l 0 or model 300 , Beckman I n s t rumen t s , Fu l l er t on , CA ) (2) Bone Marrow C e l l s Bone marrow ce l l s a t a c o n c e n t r a t ion of X 10 ce l l s / mLaMEM were ob t a i ne d a s out l i n e d i n s e c t ion V I C a ( ) A f t er p r epar i n g t h e c e l l s , 200 � L o f t h e c e l l suspe n s ion was added t o each we l l o f a m i crot i t er p l at e ( M i cr o t e s t I I , Fal P l a s t i c s , Oxn a rd , CA ) I n add i t i o n t o t he c e l l s , � L o f I UdR at a conce n t rat ion of � C i I UdR/ 20 �L of X -5 M FUdR was added t o each wel l The com- pounds t o be t e s t e d were added t o t h e m i c r o t i t e r p l a t e i n � L a l i qu o t s o f - M s o l u t i o n s f o r DNA s y n t he s i s , 20 � L o f 10 we l l s o f ce l l s -2 A s a negat i v e t r o l M A r a C was added t o s i x The f in a l m i c ro t i t e r p l at e cont a i n e d : we l l s o f b o n e mar row c e l l s ( BMC ) a n d we l l s o f each t e s t compoun d we l l s of AraC + BMC + + BMC + 125 125 I BdR I UdR I UdR we l l s of I UdR o n l y T h e c e l l s were i ncub a t e d w i t h t he drugs o n a rocker p l at f orm for t wo hour s A f t er i ncubat i on , t h e ce l l s were harve s t e d on a T i t e r t e k c e l l h a r v e s t er an d r a d i o a s s a y e d as desc r ib e d in s e c t i on I V C a ( ) ( 3) P 8 Ce l l s The a s s ay des c r i bed i n sec t i o n V I C a ( ) was re­ pe a t e d u s in g P 8 ce l l s h arve s t e d f rom DBA / mice w i t h mat u r e d P 8 t umor s a c co r d i n g t o s e c t ion V I C a aMEM 143 was s ub s t i t u t e d f o r S p i n ne r ' s med i a b Prot e i n S y n t h es i s I n h ib i t i on Assay ( 1) T ime Cour s e Det e rmi n a t ion P 8 ce l l s were h arves t e d f rom DBA / mice w i t h mat ured P388 t umo rs a c co r d i n g t o s e c t i o n V I C a The p repared c e l l s were added t o a mi crot i te r p l at e ( M i c rot e s t I I , F a l ­ c o n P l as t i cs , Oxn ar d , CA ) i n a l i quot s o f 0 � L p e r we l l To e a c h we l l was added � C i L- [ Se ] - s e l enome t h i o n i n e i n � L o f S p i n ne r ' s med ia I n cub a t i o n t imes were var i ed so t h at s ix we l l s of c e l l s we re h arve s t e d at each t ime p o i n t : , , , , , , and hour s Fo l l o w i n g t h e a l lo t e d i n cub at ion pe r io d , t h e ce l l s w e r e harves t e d on a T i t er t ek c e l l harve s t e r a n d r a d i oassayed a s descr ib e d i n s e c t ion IV C a ( ) The p ro cedu r e was r e p e a t e d u s i n g b o n e mar row c e l l s ob­ t a i n e d as d e s c r i b e d in s e c t i o n V I C a ( ) I n cuba t i o n t ime s w e r e var i e d so t hat t hree we l l s o f ce l l s were harv e s t e d at each t ime p o i n t : , , 14 , , and 24 hours (2) Bone Marrow Ce l l s The b o n e marrow c e l l s were ob t a i n e d a s d e s c r i b e d in Sect i o n V I C a ( ) , us i n g S p i n n e r ' s med i a i n s t e ad o f a MEM A f t e r add i n g 0 � L o f t he prepared b o n e mar row c e l l s t o each we l l of a m i c ro t i t e r p l at e ( M ic r o t e s t I I , F a l P l ast i c s , Oxnar d , CA ) , each o f t h e t e s t compoun ds was added t o four d i f f er e n t we l l s i n 20 �L a l i quot s I n add i t ion , four we l l s o f t r o l ce l l s were p repared b y a d d i n g � L of Sp i nner ' s me d i a t o e a c h o f four we l l s t a i n i n g bone marrow ce l l s 144 A s a n e gat i v e c o n t r o l f o r prot e i n s y n t h e s i s , 20 �L o f 10 -2 M puromy c i n was added t o f our we l l s o f bone marrow c e l l s The ce l l s were i n cub a t e d on a rocker p l at f orm f o r hours A f t er t h e a l l o t t e d i n cub a t i o n per i od , � L o f � C i L- [ Se ] - s e l e n ome t h ion i n e / � L o f S p i n n e r ' s m e d i a was added t o each of the we l l s The f i n a l m i crot i t e r p l a t e c on t a i n e d : we l l s BMC + [ Se ] - s e l e nomet h ion i n e we l l s o f each t e s t compound i on i n e we l l s o f puromy c i n + BMC + + BMC + [ 75 Se ] - s e l enome t h- [ S e ] - s e l enome t h i o n i n e we l l s o f [ Se ] _ s e l enome t h i on i n e o n l y T h e m i c r o t i t e r p l a t e was i ncub a t e d on a rocker p l a t form for twen t y - f o u r hour s The c e l l s were h a r v e s t ed on a T i t er - t e k c e l l h a r v e s t e r and r a d i o as s a y e d as des c r ib e d i n s ec t i o n VI C a ( ) ( 3) P388 c e l l s The a s s ay d e s c r i b e d i n s e c t i o n V I C b ( ) was r e p e a t e d us i n g P 8 c e l l s harve s t ed f rom DBA / m i c e w i t h matu r e d P 8 t umor s acco r d i n g t o s e c t i o n V I C a (4) P ce l l s The assay d e s c r i b e d i n s ec t i on V I C b ( ) was re­ p e a t e d u s i n g P c e l l s ob t a i ne d f rom the c u l t u r e s descr i b ­ e d i n s e c t ion I V C b me d i a CDMEM wa s used i n s t ea d o f S p i n n e r ' s 145 c Ce l l Grow t h S t u d i e s ( 1) T i me Course D e t e rm i nat i o n P c e l l s ma i n t a i n e d i n c u l ture V I C b ) we re added t o four d i f f er e n t m i c ro t i t e r p l at es , ( Co s t a r , Be l l co , V i n e ­ l an d , New J e r s e y ) i n a l i q u o t s t a i n in g X P c e l l s pe r we l l Each o f t h e t e s t compounds , i n a l i q u o t s o f 20 � L , was added t o four d i f f e r e n t we l l s on each p l a t e , l e av i n g four we l l s o f ce l l s as t r o l s ( 20 � L o f CDMEM was added The i n cubat i o n t ime s for each p l a t e to t h e s e four we l l s ) were v a r i e d t o i n c l ude , 24 , 3 , a n d 48 hours o f i n cubat i o n F o l l ow i n g i n cubat i on , t h e c e l l s were e n ume r a t e d us i n g a h emacytome t e r v i ewe d under a b i nocu l ar l i ght m i c roscope (2) ED 50 C a l cu l a t i on P ce l l s ma i n t a i ned i n c u l t ure ( V I C b ) were added t o e ach we l l o f a m i c r o t i t e r p l a t e ( Co s t a r , Be l l co , V i n e l an d , New J e r s e y ) i n � L a l i q uo t s t a i n i n g approx ima t e l y X 10 c e l l s / mL Each o f t h e t e s t compounds , i n � L a l i quot s , was added t o four d i f f erent we l l s o f c e l l s , l e a v i n g four we l l s o f c e l l s as c o n t ro l s added t o t h e s e f our we l l s ) ( 20 � L o f CDMEM was A f t er i n cubat i n g t h e m i crot i t er p l a t e f o r twe n t y - four hours , t h e c e l l s wer e enume r a t e d u s i n g a hemacy t ome t e r v iewed under a b i no c u l a r l i ght m i cros cope Var i a t i o n s were made i n t h e conce n t rat ion o f t h e t e s t drugs to det e rm i n e t h e ED 50 Stat i s t ical Anal y s i s The l e ve l o f f i de n c e f o r a l l expe r imen t s was s e t at n in e t y - f i ve percen t A one-way a n a l y s i s o f var i an c e 146 ( ANOVA ) w i t h a Dun n et t ' s t - t e s t was u s e d t o compare a c o n t r o l t o m o r e t han one e x p e r i me n t a l group ( 99 ) L i near r e gr e s s ion a n a l y s i s was used t o de t e rm i n e t h e s lopes of c e l l grow t h curves 147 B I B L I OGRAPHY 148 B I BL I OGRAPHY E v an s , R M , " T h e Chem i s t ry of t h e An t ib iot i cs Used in M e d i c i ne " ; Pe rgamo n Press , 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