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Competitive antagonists of thyroxine and structurally related compounds

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Competitive antagonists of thyroxine and structurally related compounds

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Content COMPETITIVE ANTAGONISTS OF THYROXINE AND STRUCTURALLY RELATED COMPOUNDS A T h esis P re s e n te d t o th e F a c u lty of th e Departm ent of B io ch em istry and N u t r i t i o n U n iv e rs ity of So uthern C a l i f o r n i a In P a r t i a l F u l f i ll m e n t of th e R equirem ents f o r th e Degree of D octor of Philosophy by E a rl F rie d en June 1949 U M I Num ber: DP21540 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. dissertation Publishing UMI DP21540 Published by ProQ uest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQ uest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United S tates Code ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 • f t D . B i o ' i f F a r 4 ! ? This dissertation, written by 1 . E .^J„...Frieden „ ........... under the guidance of A .is... Faculty Committee . f on Studies, and approved by all its members, has Ch^ c j 0 been presented to and accepted by the Council on Graduate Study and Research, in partial ful- > fillment of requirements for the degree of D O C T O R OF P H I L O S O P H Y 2. < s > /J > ¥ ? C om m ittee on Studies Chairman ACKNOWLEDGEMENTS I t i s w ith s in c e r e p le a s u r e t h a t I acknowledge th e in v a lu a b le aid and guidance of Dr* R ich ard J* W in z le r. I wish to e x p re s s my g r a t i tu d e t o Mrs,. Joy F o r r e s t e r and Miss E li z a b e th B* T ukich f o r t h e i r generous t e c h n ic a l a s s i s t a n c e . M y thanks- t o ELi L i l l y and Co* and to th e Committee on R e se arch in E ndocrinology of th e N a tio n a l R e se a rc h Council f o r t h e i r support of t h i s work, as w ell as to th e Hancock Foundation f o r th e f a c i l i t i e s t h a t were made a v a i l a b l e . F i n a l l y , I w ish to e x p re s s my a p p r e c ia tio n t o my w ife , E s th e r , f o r her u n lim ite d p a tie n c e d u r in g my g rad u a te c a r e e r . I TABLE OF CONTENTS CHAPTER PAGE I* INTRODUCTION ,'kk....................... 1 I I . HISTORY AND REVIEW OF THE LITERATURE.................. 2 S t r u c t u r a l re q u ire m e n ts f o r t h y r o x in e - li k e a c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 C om petitive i n h i b i t i o n of th y ro x in e 3 I I I . EXPERIMENTAL METHODS ............................ 6 S y n th e s is of p o t e n t i a l a n ta g o n is ts and com pounds s t r u c t u r a l l y r e l a t e d to t h y r o x in e . 6 B io lo g ic a l assay f o r t h y r o x i n e - l i k e a c t i v i t y and th y ro x in e antagonism 14 A c c e le r a tio n of th e m etam orphosis of Amphibia • 14 G o ite r p re v e n tio n method .............. 20 IV . KINETIC TREATMENT OF THYROXINE-LIKE ACTIVITY AND THYROXINE ANTAGONISM .............. 23 E nzym e-substr ate k i n e t i c s . ! . . . . .............. 25 C om petitive i n h i b i t i o n ............................. 28 N on-com petitive i n h i b i t i o n .............. 29 U ncom petitive i n h i b i t i o n ............ 30 V. RESULTS .............................. 31 C om petitive i n h i b i t i o n of th y ro x in e 31 C om petitive i n h i b i t i o n of th y ro x in e by e t h e r s of 3 ,5 -d iio d o -4 -h y d ro x y b e n z o ic a c i d . . . . . . 40 S p e c i f i c i t y of th y ro x in e i n h i b i t i o n . . . . . . . 45 C o m petitiv e antagonism of t h y r o x i n e - l i k e a c tiv e compounds 46 I I CHAPTER * PAGE Antagonism t o th y ro x in e using th e g o i t e r p re v e n tio n method • • • • » * * • • • * • • « . . • • • • . • • 52 F u r th e r o b s e r v a tio n s on th e t h y r o x in e - li k e a c t i v i t y of compounds s t r u c t u r a l l y r e l a t e d t O ; t hyr ox ine •« *»•**•***•***•**•****--•*•'** 55 The e f f e c t of g o itr o g e n s on the t h y r o x i n e - l i k e a c tio n of 3 ,5 - d i io d o - L - ty r o s in e in Amphibia * . . • • • * • • • . » » • • « • • • • • • • • • * • • * • * • 57 VI* DISCUSSION OF RESULTS ; A HYPOTHESIS FOR THYROX INE-LIKE ACTIVITY AND ANTAGONISM in AMPHIBIA** 60 V I I . SUMMARY ........... 74 BIBLIOGRAPHY *..............***.*.**.*.*-*** * * ..* * .* * .* ..* * * * * * .,* . 76 I l l LIST OF TABLES TABLE PAGE I . C om petitive I n h i b i t i o n of DL.-Thyr oxine by 0 - B e n z y l- N - a c e ty l- 3 ,5 - d iio d o - L - ty r o s in e . . . . . 32 I I . C om petitiv e I n h i b i t i o n of DL-Thyroxine by 0 -B e n z y l- 3 ,5 - d iio d o - D L - ty r o s in e . . . . . 35 I I I * Comparison of K j 's f o r C o m petitiv e I n h i b i t i o n of DL-Thyroxine f o r V arying C o n c e n tra tio n s of 0 -B e n z y l.-3 ,5 -d iio d o -D L -ty ro s in e . . . . . . . . 36 IV. Type of i n h i b i t i o n of Thyroxine by O -B enzyl- 3 ,5 - d iio d o - D L - ty ro s in e . . . . . . . . . . . . . . . . . . . . 38 V* C om petitive I n h i b i t i o n of DL-Thyroxine by 4— B e n zy lo x y -3 ,5 -d iio d o b e n z o ic acid . . . . . . . . . . 43 VI* C om petitive I n h i b i t i o n by 3 ,5 - D i i o d o - 4 - a n i s i c acid . 4 4 V II* The E f f e c t of Thyroxine I n h i b i t o r s on th e Thy r o x i n e - l i k e A c t iv i t y of 3 ,5 - D iio d o - 4 - ( 3 * , 5 ' - d iio d o -4 '-h y d ro x y p h e n o x y )-b e n z o ic acid . . . . 48 V III* C om petitive I n h i b i t i o n of th e T hyroxine— l i k e A c tiv ity of 3 ,5 - D iio d o - 4 -( 3 ' , 5 ' - d i i o d o - 4 ' — hydroxyphenoxy)-benzoic acid . . . . . . . . . . . . . . 50 IX. Absence of an E f f e c t of Thyroxine A n ta g o n ists on th e T h y ro x in e -lik e A c t iv i t y of 3 ,5 -D iio d o - 4_ ( 4 h y d ro x y p h e n o x y )-a n ilin e ............ 53 X. Antagonism t o DL-Thyroxine usin g th e G o ite r P re v e n tio n Method ............. . . . . . . . . . . . . . . . . . . 54 X I. F u r th e r O b s e rv a tio n s of th e T hyroxine-L ike Ac t i v i t y of Compounds S t r u c t u r a l l y R e la te d to Thyroxine .............. 56 X I I . The E f f e c t of G o itro g e n ic Agents on the Thyrox- i n e - l i k e Action of 3 , 5 - d i io d o - L - ty r o s in e •• 58 X I I I . Summary of I n h i b i t i o n of T hyroxine^L ike A ctive Compounds in Amphibia 61 IV LIST OF FIGURES FIGURE PAGE 1 . S y n th e s is of Thyroxine A n ta g o n ists . . . . . . . . . . . . . . . . 7 2* The E f f e c t of DL-Thyroxine on Amphibian M etamorph.. 19 3*. The E f f e c t of DL-Thyroxine on the Thyroid Weight of th e T h io u r a c il Fed Rat • 28 4.. A. The E f f e c t of In c r e a s in g C o n c e n tra tio n s of 0 - Benssyl-N— a c e ty l - 3 ,5 - d i i o d o - L - t y r o s i n e on th e Re sponse of Tadpoles t o DL-Thyroxine c o n c e n tra tio n * 33 B* The R e c ip ro c a l of th e Observed E f f e c t as Meas ured by th e p er c en t D ecrease in th e L ength of T adp oles p l o tt e d as a F u n ctio n of th e R a tio of th e Molar C o n c e n tra tio n of I n h i b i t o r t o DL- T hyroxine . . . . . . . . . . . . . . . . . 33 5* A. The E f f e c t of I n c r e a s in g C o n c e n tra tio n s of 0 - B e n z y l-3 , 5-diiodo-D L -t$?rosine on th e Response of T adpoles to DL-Thyroxine . . . . . . . . . . . 39 B* The R e c ip r o e a l( x 1Q0) of the Observed E f f e c t as Measured by th e p e r cen t D ecrease in th e L ength of T adpoles p l o t t e d as a F u n c tio n of the R e c ip ro c a l of th e Molar C o n c e n tra tio n of DL-Thyroxine f o r V a rio u s L ev els of O -B e n z y l- 3 ,5 - d iio d o - D L -ty r o s in e . 39 6* A* The E f f e c t of I n c re a s in g C o n c e n tra tio n s of 4- B e n z y lo x y -3 ,5 -d iio d o b e n z o ic acid on the Response of T adpoles to DL-Thyroxine • 43 B. The R e c ip r o c a l( x 1.00) of th e Observed E f f e c t as Measured by th e per c en t D ecrease in the Lenght of T adpoles p l o t t e d as a F u n c tio n of th e R a tio of th e Molar C o n c e n tra tio n of i n h i b i t o r to D L-Thyroxine. 43 7 . A. The E f f e c t of In c re a s in g C o n c e n tra tio n s of O-Ben z y l- 3 ,5 - d iio d o - D L - ty r o s in e on th e Response of Tad p o le s t o 3 ,5 - D iio d o - 4 -( 3 1, '-d iio d o -4 '-h y d ro x y p h e n - o x y ^ b e n z o ic a c i d . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 B* The R e c ip r o c a l(x 100) of th e Observed E f f e c t as Measured by the per cent D ecrease in the L ength of T adpoles p l o tt e d as a F u n c tio n of th e R a tio of th e Molar C o n c e n tra tio n s of 0 - B e n z y l- 3 ,5 - d iio d o ty r o s in e t o th e C arbo xylic Acid Analog of Thyroxine . . . . . . 51 8 . T e n ta tiv e Scheme f o r Thyroxine A ction, Antagonism . 67 9 . G ra p h ic al R e p r e s e n ta tio n of P o s s ib le R e la ti o n s h ip s between DL-Thyroxine and 3 ,5 - D iio d o - 4 - ( 3 * , 5 '- d i i o - d o - 4 ' -hyd ro xyp h en o x y )-b en zo ic acid • .* • * .» • . • ., ... 70 CHAPTER I INTRODUCTION Statem ent of th e problem * A l a r g e number of compounds s t r u c t u r a l l y r e l a t e d t o v a r io u s e s s e n t i a l m e ta b o lite s have been shown t o e x e r t b i o l o g i c a l e f f e c t s a n t a g o n is ti c to th e n a t u r a l l y o c c u r rin g m e ta b o lite * The e f f e c t s of such i n h i b i t ors- have been u s e fu l in r e v e a l i n g th e r o l e of v ita m in s , h o r mones and o th e r growth f a c t o r s as well', as th e in te rm e d ia ry s te p s of c e r t a i n key b io sy n th eses* I t was b e lie v e d t h a t th e a p p lic a tio n of t h i s approach t o compounds r e l a t e d to th y ro x ine might be of v a lu e in e l u c i d a t i n g th e mechanism of a c tio n of th e th y r o id hormone, sin c e a n ta g o n is ts t o th y ro x in e might be exp ected t o i n h i b i t th e p e r i p h e r a l a c tio n of the t h y r o id hormone. In fo rm a tio n on th e mechanism o f a c tio n of a hormone of th e p h y s io lo g ic a l im portance of th y ro x in e may be sought from many v iew p o in ts* In t h i s d i s s e r t a t i o n , th e r o l e of th e v a r i ous p-arts: of the th y ro x in e molecule or i t s a c tiv e analogs was brou ght under p a r t i c u l a r s c ru tin y * The s p e c i f i c i t y of th e i n h i b i t i o n of th y ro x in e and s t r u c t u r a l l y r e l a t e d a c tiv e compounds was a ls o examined* CHAPTER I I HISTORY AND'REVIEW OF THE LITERATURE S„. re q u ire m e n ts - f o r th v r o x in e -1 ike a c t i v i t y ♦ A survey of compounds s t r u c t u r a l l y r e l a t e d t o th y ro x in e and th e a v a ila b le d a t a on t h e i r r e l a t i v e th y r o x i n e - l ik e a c t i v i t y i n Amphibia and Mammalia was p r e s e n te d by F rie d e n (1 9 4 7 ). I t was found p o s s ib le t o c l a s s i f y f o r t y - f o u r such compounds as d e r i v a t i v e s of t y r o s i n e in a d d itio n , t o 21 m is c e lla n e o u s compounds of im portance i n th y r o id b io c h e m is tr y . Since t h a t tim e s e v e r a l a d d itio n a l r e p o r t s on th e s y n th e s is and re la c t i v e t h y r o x i n e - l i k e a c t i v i t y of compounds r e l a t e d t o th y ro x ine have been p u b lis h e d (H a rin g to n , 1948; LeBlond and Grad, 1.948;: P i t t R i v e r s , 1948; P i t t R iv e r s , Lerman, 1948; Saul and T r ik o ju s , 1948; Sm ith, 1 9 4 8 ). These p u b l i c a t i o n s have r e i n f o r e e d th e c o n c lu sio n s of F r ie d e n and W inzler(1948a) t h a t th e m ajor s t r u c t u r a l p r e - r e q u i s i t e s f o r a p p re c ia b le th y ro x in e — Like a c t i v i t y , p a r t i c u l a r i l y in Amphibia, could be r e p r e s e n te d by th e fo rm u la g iv e n below . Included in t h i s ( ho) < £ > o - 0 - y fo rm u la are th e fo llo w in g e s s e n t i a l c h a r a c t e r i s t i c s : . 1 . An o rth o d ih a lo g e n o p h e n o lfc d ip h e n y l e th e r c o n f ig u r a t i o n . 2 . A hydroxy group e i t h e r o rth o or p a r a t o th e e th e r oxygen. 3 . A sid e c h a in , Y, which must in c lu d e a f u n c t i o n a l group such as a carb o x y l or amino group or b o t h . From th e r e c e n t work of H a rin g to n (1 9 4 8 ), i t appears t h a t th e t h i o e th e r co rre sp o n d in g t o th y ro x in e p o s s e s s e s ap p r e c ia b le , t h y r o x i n e - l i k e a c t i v i t y . Maximum t h y r o x i n e - l i k e a c t i v i t y i s obtained i f th e s id e c h a in c o n s i s t s of an a la nine group, th e hydroxyl group i s p a r a to th e e th e r lin k a g e , and th e r e are fo u r io d in e atoms in th e 3 ,5 and 3 ' , 5 ' p o s i t i o n s ! These c o n c lu sio n s have in c o r p o r a te d the e a r l i e r r e views and f in d in g s of H a rin g to n (1 9 3 3 ,1939)', Loeser and T rik o - j u s ( I 9 3 9 ) , Niemann, e t al*(L 941), and o t h e r s . C oinpetitive i n h i b i t i o n of T h y ro x in e. For the p u r p o ses of t h i s d i s s e r t a t i o n , a c o m p e titiv e i n h i b i t o r may be d e fin e d as a b i o l o g i c a l a n ta g o n is t, th e e f f e c t s of which may be overcome by th e a d d itio n of a s t r u c t u r a l l y r e l a t e d normal m e ta b o lite . I t i s p o s s ib le t h a t such a n ta g o n is ts act by com p e tin g w ith th e normal m e ta b o lite f o r a c tiv e s i t e s on c e r t a i n enzymes. For the unequivocal d e m o n s tra tio n of a c o m p e titiv e type of i n h i b i t i o n , th e d a t a o b tain ed w ith i t must f i t c e r t a i n q u a n t i t a t i v e and k i n e t i c c o n d itio n s which w i l l be d i s cussed in d e t a i l l a t e r . R ecent rev ie w s of th e b ro a d e r as p e c ts of c o m p e titiv e i n h i b i t i o n have been p u b lis h e d by M cll- w ain(1944), R o b lin (1 9 4 6 ) , W elch(1945), and W oolley(1947). Although th e r e have been a number of r e p o r t s in th e l i t e r a t u r e of s o - c a l l e d a n t i - t h y r o i d su b s ta n c e s , such i n f o r m ation i s d i f f i c u l t t o e v a lu a te , s in c e u s u a lly no d i s t i n c t i o n was made between g o i tr o g e n ic a c tio n , competL&tlfcve antagonism, or o th e r ty p e s of i n h i b i t i o n * Much of th e e a r l y in fo rm a tio n on t h i s s u b je c t has been summarized by Zondek and Sulm an(1942)• A n ti- th y r o id a c t i v i t y h as been observed f o r such d iv e r s e m ate r i a l s as blood e x t r a c t s , c h o l e s t e r o l , a c e t o n i t r i l e , vitam inA , t h i o u r a e i l , th io u r e a , s u lf a n ila m id e , e tc * However, i t i s not l i k e l y t h a t th e s e compounds w i l l prove t o be c o m p e titiv e an t a g o n i s t s of th y ro x in e because of t h e i r la c k of any s t r u c t u r al. s i m i l a r i t y t o th yroxin e* There have been a few i s o l a t e d r e p o r t s on th e th y ro x ine antagonism by su b s ta n c e s more c lo s e ly r e l a t e d in s t r u c t u r e t o the hormone# L itz k a ( 1.936) and K r a f t (1936) observed a n ta g onism between th y ro x in e and v a r io u s f l u o r in e compounds such as 2 -flu o ro b e n z o ic a c id , 3 - f l u o r o t y r o s i n e , 3 ,5 - d iflu o ro - D L - t y r o s in e on amphibian metamorphosis* However, none of th e s im ila r f l u o r o d e r i v a t i v e s t e s t e d f o r t h e i r e f f e c t on mamma l i a n m etabolism by Boyer(1941) showed any a n ti- th y r o x in e ac t i o n . Raab(1933) n oted some i n d i c a t i o n of th e r e d u c tio n of th e b a s a l m etab o lic r a t e of anim als g iv en D L -th y ro n in e . Di— io d o ty ro s in e has been r e p o r te d by Edwards and S h a rp le ss(1 9 4 4 ) t o d e p re ss a p p re c ia b ly th e e le v a te d m etab o lic r a t e produced by p re v io u s i n j e c t i o n of th y r o x in e . In a r e c e n t survey of th e f i e l d of c o m p e titiv e i n h i b i t i o n , W oolley(1944) summarized th e s t r u c t u r a l m o d ific a tio n s of m e ta b o lite s g e n e r a lly used to produce i n h i b i t o r y compounds as fo llo w s : 1. Exchange of a carboxyl group by a s u l f o n i c acid or a sulfonam ide group or to a carbonyl a l i p h a t i c chain gr oup. 2:. Replacem ent of one or more atoms in th e r i n g systems of c y c l i c m e ta b o lite s such as s u b s t i t u t i n g a n i tr o g e n f o r a carbon or a s u l f u r f o r an e th y le n e group. 3 . M isc e lla n e o u s s t r u c t u r a l changes such as sid e chain a l t e r a t i o n , e t c . < > However, t o produce a n ta g o n is ts of th y r o x in e , W oolley(1946) employed none of th e Usual type of s t r u c t u r a l changes l i s t e d above. In s te a d he p rep a red s e v e ra l e th e r s of N - a c e ty l - 3 ,5 - d i io d o - L - ty r o s in e and found t h a t s e v e r a l of th e s e new com pounds i n t e r f e r e d q u a l i t a t i v e l y w ith v a rio u s e f f e c t s of th y ro x in e on Amphibia. The p - n i t r o p h e n y l e t h y l , b e n z y l, p - n i t r o - b e n zy l, and b u ty l e t h e r s were a n ta g o n is ts in th e o rd er l i s t e d . S e v e ra l of th e compounds r e v e a le d t r a c e s of t h y r o x i n e - l i k e a c t i v i t y . Though Woolley r e p o r te d no d a t a which would have i d e n t i f i e d th e ty pe of antagonism observed, i t s c o m p e titiv e n a tu r e was s tr o n g ly susp ected because of th e c lo s e s t r u c t u r a l r e l a t i o n s h i p betw een W oolley’ s compounds and t h y r o x in e . Some of W o o lley 's o b s e r v a tio n s were confirm ed and extended by Wina- l e r and F ried en (1 9 4 8 b ) and by W illia m s, e t a l . ( I 9 4 8 ) . CHAPTER I I I EXPERIMENTAL METHODS S y n th e s is of p o t e n t i al ant a g o n is ts and compounds s t r u c t u r a l l y r e l a t e d t o t h y r o x i n e » The th r e e major ty p e s of i n h i b i t o r s employed in t h i s stu d y are l i s t e d below; The sequence of r e a c t i o n s n e c e s s a ry f o r t h e i r p r e p a r a tio n i s in d ic a te d in F ig u re 1. The s p e c i a l convenience of syn-. te c h n iq u e f o r d e te rm in in g th e e f f e c t s of R on i n h i b i t o r s tr e n g th , e li m in a ti n g th e n e c e s s i t y of many lo n g er preparac t i o n s in v o lv in g types' I I and I I I * The p r e p a r a t i o n of numerous compounds having a p p re c ia b le t h y r o x i n e - l i k e a c t i v i t y has been d e s c r ib e d p r e v io u s ly by F rie d e n and W inzler(1948a)* Other compounds were r e a d i l y a- v a i l a b l e from i n te r m e d ia te s in th e c l a s s i c a l s y n th e s is of th y ro x in e by H a rin g to n and B a rg e r(1.927)• R o f e c o o H i ( i ) r o - O c h * c h c o o h I M UCOCH ( I I ) r o ^ O ch^ hcooh c m ) Q , t h s i s of compounds of ty p e I a ffo rd e d a sim ple sc ree n in g FIGURE 1 SYNTHESIS OF THYROXINE ANTAGONISTS SCTIIODOTYROSINE DERIVATIVES: H o f c c C M C O O H A C d fe-C H .C H C O O H »h 1 NHj N‘ BH ^ NNAc 10 rokSKHjCHCOOH *■-> RofecdiCHCOOH -MS) -*4 Rofe-C^CHCOOH NHAc " * Un tIKAc ' NHj. a5-0l©DO-4 - HYDROXY 8EM201C ACID DERIVATIVES: K o -O -C O O H - T S ! - ) H ( ^ > C O O H K O ^ > C 0 0 K ^ - ^ R o f c > C O O « R= CH3- , O - C H f , O ^ - O - C H j C H j - , C H j- C H - ch3 Q-Be nzv1-N - ac e t y 1 - 3 .5 -d i i od o-L-1y r o s ine (IV ) . T h is compound was p rep a red as d e sc rib e d by Woolley (1946) ex cep t t h a t a b e t t e r c o n d e n sa tio n w ith a 40 p e r cen t y i e l d was o b ta in e d when e x a c tly two. e q u iv a le n ts of NaQH and a 10 p e r cent e x cess of benzyl c h lo r id e were u sed . The m e lt ing p o in t^ o f IV o b ta in e d i n . t h i s manner was 87-93^, s e v e r a l d e g re e s h ig h e r th an t h a t r e p o r te d by W oolley(1946) . The p ro d u ct gave a n e g a tiv e n in h y d r in t e s t and a n e g a tiv e K endall r e a c t i o n fo r o r th o d iio d o p h e n o ls (K e n d a ll,1 9 2 9 ). Q -B en zy l-5 ,5-diio d< teB L -ty ro sine (V ). Ten g . ofIV w.as re lu x e d o v e rn ig h t in one l i t e r of IN NaOH. The s o l u t i o n gave a s tr o n g ninhydrjtn r e a c t i o n at th e end of th e r e f l u x i n g p e rio d # A d ju stin g th e pH of th e so lu t i o n t o 5*0 p r e c i p i t a t e d a s o lid w ith a r e l a t i v e l y low m e lt ing p o i n t . T his s o l i d was e x tr a c te d w ith s e v e r a l 1 - l i t e r p o r t io n s of hot G.2N HC1 and f i l t e r e d while hot# On c o o lin g , th e h y d ro c h lo rid e of V w.as o b tain ed in c r y s t a l l i n e form . N e u t r a l i z a t i o n of th e mother liq u o r gave a d d itio n a l impure V# The h y d ro c h lo rid e w.as d isso lv e d in d i l u t e a l k a l i and th e s o lu t i o n was a d ju ste d to pH 510, whereupon c r y s t a l l i n e V w;as ob t a i n e d in 30 p e r cen t y i e l d , m .p . w ith d eco m positio n 2 0 3 -5 °. 1 All. m e ltin g p o in ts are c o rre c te d and given in d e g re e s c e n t i grade# The pro d u ct gave a p o s i t i v e n in h y d rin , b u t a n e g a tiv e K endall r e a c t i o n . The above re c r y s t a l l i z a t i o n p r o c e s s was re p e a te d on a sample used f o r elem entary a n a l y s i s . CisHigOgNIg. C a l c . , C 3 6 .7 , H 2 . 9 i found, C 3 6 .9 , H 3 . 0 . 4 -B e n zv lo x v -3 .5 -d iio d o b e n z o ic acid ( V I ) . 3 ,5 -D iio d o -4 -h y d ro x y b e n zo ie a c id , m.p. 2 7 5 -7 °, was p r e p a re d in 50 p e r c e n t of the t h e o r e t i c a l y i e l d from 4-hydroxy— b e n zo ic acid (Eastman Kodak, m.p. 214.5-215.5°), u sin g IC1 as d e s c r ib e d by W o o lle tt and Jo h n so n (1.943). Ten g . of th e io — d in a te d phenol were d is s o lv e d in 61mL. of UN NaOH, h e a te d on a b o i l i n g w ater b a th and t r e a t e d w ith 11.9 g . of benzyl c h lo r id e added dropw ise* A fter a t o t a l ' h e a tin g p e rio d of one hour, th e hot s u p e r n a ta n t was d e ca n te d from a t a r r y b y -p ro d uct.. A heavy w hite p r e c i p i t a t e of* th e sodium s a l t of VI was formed on c o o li n g . 4 -B e n z y lo x y -3 ,5 -d iio d o b e n z o ie acid was g e n e ra te d by a c i d if y i n g a su sp en sio n of the i s o l a t e d s a l t . Two r e c r y s t a l l i z a t i o n s from w ater-m eth an o l m ix tu re s gave a 2Q > p e r c e n t y i e l d of V I, m .p. 22.7-8°. The p ro d u c t gave a n e g a tiv e K endall t e s t . Ci4Hi 00 3l 2 . C a lc ., C 35*0, I 52.9;; found, C 3 5 .2 , I 5 2 .9 . An a lt e r n a t e r o u te f o r the s y n t h e s i s of VI c o n s is te d of th e p r e p a r a t i o n of th e d ip o ta ss iu m s;alt of 3 ,5 - d i i o d o - 4 - hydroxybenzoic acid by th e e v a p o ra tio n t o d ry n e s s of a s o lu t i o n of th e l a t t e r compound and two e q u iv a le n ts of KgCOg. The r e s u l t i n g s o lid i s d is s o lv e d in a minimum amount of 50 j p er c en t e th a n o l and^ a f t e r th e a d d itio n of fo u r e q u iv a le n t s of f r e s h b enzyl c h lo r id e , th e s o l u t i o n i s re flu x e d f o r at l e a s t one hour or u n t i l th e K endall t e s t f o r o rth o d iio d o p h e n o ls Was: n e g a t iv e . The r e a c t i o n m ixture was d i l u t e d w ith s e v e r a l v o l£ umes of w a te r , and th e c r y s t a l l i n e s o l i d which appeared on c o o lin g was c o l l e c t e d . S e v e ra l r e c r y s t a l l i z a t i o n s from an a lc o h o l-w a te r m ixture y i e l d e d a so lid s of a maximum m e ltin g p o in t of 120-2°* T his was presumed to be th e benzyl e s t e r of VI, sin c e upon f e f l u x i n g f o r th r e e hours in 50 p er cent e th a n o l made IN in NaOH and su bsequ ent a c i d i f i c a t i o n , a 60 p e r cen t y i e l d of V I, m .p. 226-7° was o b ta in e d . 5 .5 - D i i o d o - 4 - ( 4 l- n itro p h e n v le th o x v ) - b e n z o ic acid ( V I I ) . Ten g . of 3 , 5 -d iio d o -4 -h y d ro x y b e n z o ic acid was d i s solved in 85 ml. of 0.7N m eth an o lic NaOH and t r e a t e d w ith 6 .1 g.. of 4 - n i tr o p h e n y le t h y l brom ide, m .p. 6 4 ° , p rep ared as d e s c rib e d by W oolley(19 4 6 ). The m ixture was r e f lu x e d f o r two h ours and th e n c o n c e n tra te d t o d ry n e s s under reduced p r e s s u r e . The r e s id u e was w ell washed w ith warm w ater t o remove th e r e l a t i v e l y s o lu b le sodium 3 ,5 -d iio d o -4 -h y d ro x y b e n z o a te and an orange c o lo re d im p u rity . The rem ain in g w hite s o lid was f u r t h e r washed w ith 200 ml. dry e t h y l e th e r t o remove any 4 - n itr o p h e n y le th y l a lco h o l or bromide.. T his re s id u e was s u s pended in 100 ml, w a te r, th e pH ad ju ste d t o 3 .0 4 0 .5 w ith HC1, and th e m ixture h e a te d f o r about two h o u rs ." The r e s u l t ing s o l i d was c o ll e c t e d and th o ro u g h ly washed w ith w a te r . 11 A fte r two r e c r y s t a l l i z a t i o n s from an e th a n o l- w a te r m ix tu re, a 20 p e r cent y i e l d of V II, m .p. 219-20®, was o b ta in e d . I t gave a n e g a tiv e K e n d all r e a c t i o n . CI5 h11°5n i 2* C a l c *> 0 N 2.6;: found, C 3 3 .4 , N 2 .5 . 5 .5 - D ii o d o - 4 - a n i s i c acid ( V I I I ) . 3 ,5 - D i i o d o - 4 - a n i s i c acid was p rep a red as d e s c rib e d by Wheeler and L id d le ( 193IG>) ► The m e ltin g p o in t of .V III, o b tain ed in 45 p e r cent y i e l d , was found t o be 257-8° compared to th e r e p o r te d m eltin g p o in t of 2 5 5 -6 ° . 4-B enzyloxybenzoic acid ( I X ) . T his e th e r was s y n th e s iz e d u sin g e s s e n t i a l l y th e same p roced ure d e s c rib e d f o r th e p r e p a r a t i o n of th e c o rre sp o n d in g 3 ^ 5 -d iio d o d e r i v a t i v e , V I. A 30- p a r c en t y i e l d of crude p ro d u c t , m.p. 185— 7 °, which was s e v e r a l d e g re e s below the r e p o rte d v a lu e of 188-90°, was o b ta in e d . 3 .5 -D iio d o -4 — (4*-m e th o x y p h en o x y )-a n ilin e (X ). The p r e p a r a t i o n of X from 3 ,5 - d iio d o - 4 - ( 4 '- m e th o x y - ph e n o x y )-n itro b en z en e u sin g stan n o u s c h lo r id e in g l a c i a l ace t i c acid has been d e s c rib e d by H aring ton and B a r g e r (1927);. A more s a t i s f a c t o r y p ro ced u re was adapted from t h a t used by Block and g'owell (1942) in t h e i r s y n th e s is ; of 3 * , 5 ‘- d i i o d o t h y - ro p ^ n e . Ten g . of t h e ’n i t r o compound were suspended in 200 ml. of a 50 per cen t e th a n o l s o l u t io n c o n ta in in g 10 m l. .g l a c i a l a c e tic a c i d . Five g . each of 30 mesh i r o n f i l i n g s and ir o n psowder were added, and th e m ixture r e f lu x e d and s t i r r e d w ith 12' a mercury s e a l s t i r r e r f o r at l e a s t 3 h o u r s . The alcohol was removed by d i s t i l l a t i o n and the re s id u e co o le d . The s o lid so o b tain ed was r e p e a te d ly e x t r a c t e d w ith a t o t a l of 300 ml. of b o i l i n g benzene. C o n c e n tra tio n of th e benzene s o l u t i o n to about 150 ml* and th e a d d itio n of 80 ml. of pe troleum e th e r gave, upon s c r a tc h in g , 75-80 p e r eent y i e l d of X, m.p-* 1 2 1 -2 °. S im ila r y i e l d s were o b tain ed w ith 100 g . l o t s . A scampie of th e h y d ro c h lo rid e of X p rep ared as above gave no d e p re s s io n in m e ltin g p o in t when mixed w ith an e q u al q u a n ti t y of th e amine h y d ro c h lo rid e p re p a re d by th e method of H arin gto n and B a rg e r(1927),. 3 . 5-Di i od o-4-( 4 1 -h v d r oxvphenoxv) ani 1 ine ( XI) > ■ Five g . of X : were r e f l u x e d f o r two h o u rs in 40- ml. g l a c i a l a c e tic acid and 50 ml’ ., of 42 p e r cent HBr. A fter b ein g d i l u t e d w ith one volume of w a ter and co o lin g o v e rn ig h t, a small, amount of a brow nish im p u rity was removed by f i l t r a > - t i o n . F u r th e r d i l u t i o n of th e f i l t r a t e w ith one a d d it i o n a l volume of w ater gave a 60. p e r c e n t y i e l d of whatKw*as: assumed t o be th e hydrobromide of XI.. Pure XI, m.p;. 2 2 1 .5 - 3 .5 ° , was o b tain ed by c r y s t a l l i z a t i o n of its ; n e u t r a l i z e d s a l t from 95 p e r cen t e th a n o l. For a n a ly s is , a sample was r e c r y s t a l l i z e d s e v e r a l tim es from aqueous e t h a n o l . ^lS^gOgNIg* C a lc .,. C 3 1 .8 , N 3*lj, found, C 32*0, N 3 .0 . 3 .,5 - P iio d o - 4 - ( 5 f .5 l-d ib ro m o -4 t-hydrox yph en oxy )-benzoic acid (X II)'. 3 ,5 -D iio d o -4 -(4 '-h y d ro x y p } h en o x y )-b en zo ic acid p r e p a r e d ’ as d e s c rib e d by H arington and B a rg e r(192.7) w;as brom inated e s - 13 a e n t i a l l y according t o th e method of S c h u e g ra f(1939). Bromine, 1 .3 g . , was added t o 5*0 g* of th e d iio d o acid suspended in 17 ml. of a c e tic a c id . A 75 p e r cen t y i e l d of w h ite c r y s t a l l i n e XII,. m.p. 3 3 7 -8 .5 °C, was o b ta in e d . 3 .5 - D i i o d o - 4 - ( 3 1.5 , - d ic h l o r o - 4 ', -hydroxyphenoxv)-ben2oic acid x i i i h ; The above compound, X I I I , m.p. 3 7 3 -4 .5 ° , was obtained in approxim ately 50 p er cen t y i e l d u sing th e p ro ced u re d e s c rib e d above f o r th e co rre sp o n d in g dibromo compound. N -A ce ty l-D L -th y ro x in e , S jS -d iio d o -l-C S * ,5 , - d i i o d o - 4 ‘- hydroxyphenoxy)-benzoic a c id , N -acety 1 - 3 , 5 - d i i o d o - L - t y r o s i n e , and th e g ly c in e homolog of th y ro x in e were o b tain ed as r e p o r t ed p re v io u s ly by F rie d e n and W inzler(1948 a ) . Ss-Thiouracil and 3 -m ercaptoim id azo le were k in d ly c o n tr ib u te d by E l i L i l l y and Co., I n d i a n a p o lis , In d ia n a . 3 ,5 - D i f l.upro-4-m ethoxyphenol and 3 , 5 - d i c h l o r o - 4 - a n i s i c acid were o b ta in e d from D r. J . F. Mead, Atomic Energy P r o j e c t , U n iv e rs ity of C a l i f o r n i a at Los A ngeles. DL-Thyroxine was p ro v id ed by D r. K. W. Thompson of Roche-Organon, I n c . , N utley 10, New J e r s e y . A ll o th e r com pounds used were th e b e st a v a ila b le grade o b ta in a b le from th e Eastman Kodak Co. O B io lo g ic a l assay f o r t h y r o x i n e - l i k e , a c t i v i t y and th y ro x in e ant agon ism* The a p p li c a t io n of th e concept of compet i t i v e i n h i b i t i o n to th y ro x in e was p a r t i c u l a r i l y d i f f i c u l t be cause no c o n v en ien t _in v i t r o method f o r m easuring th y ro x in e a c t i v i t y was a v a i l a b l e . Nor was any m ic r o b io lo g ic a l system known t o be s e n s i t i v e t o th y r o x in e . Two very s e n s i t i v e in v iv o te c h n iq u e s , th e a c c e l e r a ti o n of amphibian m etam orphosis and th e g o i t e r p re v e n tio n method, were employed t o determ ine t h y r o x in e - li k e a c t i v i t y in th e r e s e a r c h re p o rte d in t h i s d i s s e r t a t i o n . The a c c e l e r a ti o n of amphibian m etam orphosis h as . proved t o be e s p e c i a l l y u s e fu l in stu d y in g th e c o m p e titiv e an tagonism of th y ro x in e and s t r u c t u r a l l y r e l a t e d compounds. A c c e le r a tio n of th e m etam orphosis of Amphibia. The a b i l i t y of t h y r o a c tiv e su b s ta n c e s t o h a s te n amphibian meta m orphosis was f i r s t r e p o r t e d by G u d ern atsch in 1912. Guder- n a ts c h observed t h a t ta d p o l e s of a v a r i e t y of s p e c ie s , when fed w ith o rd in a ry meat, u s u a lly showed d i s t i n c t hind l e g s in 53 d ay s, f o r l e g s in 73 days a f t e r th e b e g in n in g 'o f th e f e e d in g . T ad p o les, fed on t h y r o id , however, grew hind le g s in 9 days and f o r e l e g s in 11 d a y s and completed t h e i r m etam orphosis in 18-20 days a f t e r th e b e g in n in g of th e th y r o id f e e d in g . B e sid e s th e e ru p tio n of t h e lim bs, th e d e c re a se in le n g th , and th e com pletion of m etam orphosis, o th e r c h a r a c t e r i s t i c s of d i f f e r e n t i a t i o n in c lu d in g d im in u tio n of gut le n g th , weight changes, e t c . have been used by o th e r w orkers as e n d p o in ts 15 in th e q u a n t i t a t i v e e v a lu a tio n of th e r e l a t i v e r a t e of m eta morphosis* Our knowledge of t h i s phenomenon h as been extended by many i n v e s t i g a t o r s in c lu d in g A bderhalden, Geddum, Romeis, Sw ingle, Wokes, and o th e rs* The s p e c i f i c a p p li c a t io n of t h i s method t o th e d e t e r m in a tio n of the r e l a t i v e t h y r o x in e - li k e a c t i v i t y of compounds s t r u c t u r a l l y r e l a t e d t o th y ro x in e h as been co n sid ered in de t a i l by F rie d e n (L 9 4 7 )• I t w.as concluded t h a t th y r o x in e — l i k e a c t i v i t y of su b s ta n c e s in Amphibia can be c o r r e l a t e d w ith t h e i r mammalian a c t i v i t y if. i t be assumed t h a t a t e s t sub sta n c e must d em o n strate at l e a s t 0*1 p e r cent of th e a c t i v i t y of D L -thyroxine on amphibian m etam orphosis before i t can be expected to., have any t h y r o x i n e - l i k e a c t i v i t y in Mammalia. T h is s t i p u l a t i o n le a d s t o a good agreement between a v a ila b le d a t a on amphibian re sp o n se and mammalian m etab o lic e f f e c t s f o r a Large number of th y ro x in e -L ik e compounds. The use of th e ta d p o le te c h n iq u e t o study th e compe t i t i v e i n h i b i t i o n of th y ro x in e and r e l a t e d su b s ta n c e s re q u ir e d the developm ent of s u i t a b l e e x p e rim e n ta l c o n d itio n s which would e l i c i t c o n s i s t e n t re s p o n s e s t o D L -thyroxine and i t s ac t i v e a n a lo g s . The method o u tlin e d below w as’ adapted from t h a t of Gaddum(19S8) : T ed p o les, Bufo s p », were c l a s s i f i e d according to le n g th by m easuring them on a wet o i l c l o t h w ith a m illim e te r r u l e Or s u i t a b l e c a l ip e r s * Larvae in th e range of 24 / 3 mm. le n g th .were in tro d u c ed in groups of f i v e i n to d u p l ic a t e d i s h e s c o n ta in in g 2Q0: ml* of 16 tajr w ater s o l u t io n of the t e s t su b sta n c es* The pH of a ll s o l u t i o n s was a d ju ste d t o 8*0 / 0*5. The d is h e s were p la c e d in an in c u b a to r s e t at 31 / I C. When th e ta d p o le s exposed t o 0*50 microgram p e r ml. had sh o rte n ed to about 50/ per c e n t of t h e i r o r i g in a l l'e n g th (u s u a lly about 60-9© hours f o r thyroxine)", th e animals of a l l groups were measured again and th e ex p erim en t te rm in a te d * No change was n o te d i n u n tre a te d c o n tr o l s d u rin g th e s e in c u b a tio n p e r i o d s . The q u a n t i t a t i v e resp o n se of Amphibia t o th y ro x in e i s d ep en dent upon a number of f a c t o r s which are co n sid ere d in th e fo llo w in g d i s c u s s i o n on th e r a t i o n a l e f o r th e -p ro c e d u re employed: 1* The endpoint of d im in u tio n in body le n g th was p a r t i c u l a r l y co n v en ien t and a p p a re n tly adequate f o r th e la r g e number of t e s t s d e s c r ib e d in t h i s work. Changes in body le n g th v a r ie d from z e ro t o 20 mm.• w ith measurements b eing made t o th e n e a r e s t 0*5 mm*, g iv in g an average ex p erim e n tal e r r o r of about 5 p e r cent*. 2* The in c u b a tio n tem p era tu re of 31 / l.°C. was chosen p r im a r ily because t h i s tem p era tu re reduced t o 60-90/ h ours th e time r e q u ir e d t o produce a s i g n i f i c a n t d e c re a s e in l e n g t h of ta d p o le s exposed t o as low as ©*10 microgram of D L -thyroxine p e r ml. Seven t o t e n days were re q u ir e d f o r s i m i l a r re sp o n se s when the t e s t s were conducted at room te m p e ra tu re , 25 / 3°C. T em peratures g r e a t e r th a n 32°C* were f r e q u e n tly l e t h a l to the ta d p o l e . V a r ia t io n of th e te m p e ra tu re w ith in th e in c u b a to r i t s e l f was minimized by c i r c u l a t i n g th e in s id e a i r w ith a sm all high speed f a n . D ishes were r o t a t e d w ith in the in c u b a to r at 2:4 hour i n t e r v a l s in order t o e q u a liz e any rem aining tern- 17 p e r a tu r e g r a d ie n t w ith in th e in c u b a to r* 3* Bufo la rv a e appeared t o su rv iv e b e s t in an in c u b a tin g f l u i d of pH 8*0 / 0*5* The use of t h i s s l i g h t l y a lk a l i n e pH a lso enhanced th e s o l u b i l i t y of many of th e t e s t sub s t a n c e s , some of which e x h ib it a r e l a t i v e l y low s o l u b i l i t y in w ater* 4 . The ta d p o le s were n o t fed d u rin g th e course of th e experim ent t o e lim in a te p o s s ib le v a r i a b l e food i n t a k e , t o ob v i a t e th e n e c e s s i t y of changing the in c u b a tin g s o l u t i o n , and t o reduce th e amount of t e s t su b s ta n c e re q u ire d f o r a g iven s e r i e s of e x p e rim e n ts. I f th e anim als were fe d , th e e f f e c t s of th e food and th e e x c r e t a were u s u a lly to x ic in 48 h o u rs. 5 . In g e n e r a l, la r v a e were se g re g a te d in th e l a b o r a to r y acc o rd in g t o source and can be re g a rd e d as b e in g in th e same n u t r i t i o n a l s t a t e when so s e g r e g a te d . The resp o n se of Amphibia t o m a t e r i a ls having t h y r o x i n e - l i k e a c t i v i t y i s v e ry s e n s i t i v e t o th e l a r v a e ' s developm ental stage* S ince th e t o t a l body le n g th of an amphibian la rv a e i s a r e l i a b l e index of developm ent, ta d p o le s of th e same le n g th or pooled groups of l e n g th s varying: no more th a n 3 mm. and o th erw ise s i m i l a r i l y t r e a t e d were used t o reduce int®.a— ex perim en t v a r i a t i o n s * C o lle c te d d a t a on s e v e r a l independent e x p erim en ts are p re s e n te d in F igure Z-. In t h i s p a r t i c u l a r f i g u r e , th e r e c i p r o c a l of th e f r a c t i o n a l d e c re a se in l e n g t h of th e ta d p o le 18 from i t s o r i g i n a l le n g th at a c o n sta n t tim e , 7 0 / 5 h o u rs , i s p l o t t e d a g a in s t the r e c i p r o c a l of th e th y ro x in e c o n c e n tra t i o n e x p ressed in micrograras p e r ml. In a l l e x p erim en ts w ith t ta d p o le s , i t was found most co nvenien t t o ta k e the v e l o c i t y of th e b i o l o g i c a l r e a c t i o n as th e e x te n t of th e t o t a l e f f e c t o b ta in e d at a c o n s ta n t tim e . In F ig u re 2, v r e p r e s e n t s th e v e l o c i t y of th e m etam orphosis r e a c t i o n as in d ic a te d by th e f r a c t i o n a l d e c re a se in l e n g t h of the ta d p o l e s at the in d ic a te d c o n sta n t tim e , 7 0 / 5 h o u rs . Th&s | i s i d e n t i c a l in number v and meaning w ith th e r a t i o of 10Q over E where E i s th e per cen t d e c re a se in le n g t h of th e ta d p o le s at a c o n s ta n t tim e . The j u s t i f i c a t i o n f o r th e use of th e s e p a r t i c u l a r u n its f o r th e o rd in a te and th e a b s c i s s a w ill, be p r e s e n te d in some d e t a i l l a t e r . T his ty p e of p l o t i s employed to show th e d i r e c t r e l a t i o n s h i p betw een r e a c t i o n v e l o c i t y and s u b s t r a t e c o n c e n tr a tio n at c o n s ta n t enzyme c o n c e n tra tio n .. The d a t a in F ig u re 2. in d ic a t e some, v a r i a t i o n between independent e x p e r i m ents, which might be due t o d i f f e r e n c e s in in c u b a tio n tim e . However, in d iv id u a l e x p erim e n ts show an adequate d i r e c t p ro p o r t i o n a l i t y betw een 1/v and 1 / s to p erm it th e use of t h i s method t o t e s t antagonism of a c tiv e compounds by t r e a t i n g th e ta d p o le w ith the i n h i b i t o r or th e a c tiv e compound to g e t h e r or s e p a r a t e l y . The d e p a r tu r e of th e curve from th e o ry at h ig h th y ro x in e l e v e l s i s not an in f r e q u e n t occu rren ce in graphs of the type of F ig u re 2:* < s FIGURE 2; THE EFFECT OF DL.-THYROXINE ON AMPHIBIAN METAMORPHOSIS E F F E C T OF D L -T H Y R flX II/f ON AMPHIBIAN M E T /m jH O SIS 4 .0 H X 0 a 3 d 1 _ + V ~ o £ 0 4 - A & V ~ • a ft v it * m o o n y 0 X * 1.0 SEPARATE EXPTS. INDICATED BY X ,0 ,0 , *,4-. INCUBATION Tjfff = 70 ± 5 H B U R S 0 1.0 2.0 3 . 0 A-.O i fw " % L . w G o ite r P re v e n tio n Method, The g o i t e r p r e v e n tio n method f o r th e b i o l o g i c a l d e te r m in a tio n of D L-thyroxine and th y ro x in e - l i k e a c t i v i t y h as been employed by Dempsey and Astwood(1943), R einek e, M ixner, and T u rn e r(194,$)', F rie d e n and W inzler(1948a, 1948c), and o th ers* Groups of f i v e or six alb in o r a t s of th e U n iv e rs ity of Southern C a l i f o r n i a s t r a i n , w eighing between 100-200 g . , were used in a ll of th e e x p e rim e n ts. Over th e 15 day assay p e rio d th e e x p erim e n tal anim als were fed _ad lib itu m w ith 0.3-0*5 p er cent 2 - t h i o u r a c i l in c o r p o r a te d i n to t h e i r ^ s t o c k diet*. D aily * f » ’ i n t r a p e r i t o n e a l i n j e c t i o n s of th e t e s t "co'njpounds were given f o r 14 days, th e i n j e c t i o n volume b e in g in the range of 0 .2 0 - 1.00, m l. p e r 100 g. of body w e ig h t. The t e s t anim als were weighed and s a c r i f i c e d on th e f i f t e e n t h d ay. The th y ro id ■ v g lan d s were th en removed and immediately- weighed t o th e n e a r e s t 0 .1 mg. on a t o r s i o n b a la n c e . The room tem p era tu re was in th e range of 24 / 2°C. DL-Thyroxine c o n t r o l s of the same sex were used in each s e p a r a te experim ent* A t y p i c a l sta n d a rd curve showing th e d i r e c t r e l a t i o n s h i p betw een DL-thyroxine dose and th y ro id w eight resp o n se i s shown in F ig u re 3. When th y ro x in e antagonism was s tu d ie d , th e p o t e n t i a l a n ta g o n is t was also i n j e c t e d i n t r a p e r it one a lly d a i l y , u s u a lly 60 m inutes fo llo w in g th e i n j e c t i o n of th e th y r o x in e s o l u t i o n . No. f i n a l d e c i s i o n can be made at t h i s tim e re g a rd in g 21 th e v a l i d i t y of th e assum ption t h a t a l l t h y r o x i n e - l i k e a c tiv e compounds p a r t i c i p a t e d i r e c t l y or i n d i r e c t l y in th e t h y r o i d - p i t u i t a r y a x is . F rie d en and W inzler (1948 a) d id f i n d q u a lita c t i v e agreement between th o se compounds e x e r tin g a c t i v i t y on Amphibia and th o se showing g o i t e r p re v e n tio n a c t i v i t y . Some evidence was also a v a ila b le t o show q u a l i t a t i v e agreement b e tween in c r e a s e in b a sa l m e ta b o lic r a t e of r a t s and th y r o x in e l i k e a c t i v i t y as measured by th e g o i te r p r e v e n tio n method. I t a lso cannot be assumed _a p r i o r i t h a t a compound which p r e v e n ts th e a b i l i t y of th y ro x in e t o overcome t h i o u r a c i l g o i t e r induced by s tim u la tio n of th e p i t u i t a r y gland i s a ls o n e e e s - Siarily a n ta g o n iz in g th e t h y r o x i n e - l i k e in flu e n c e on p e r i p h e r a l t i s s u e . FIGURE 3 THE EFFECT OF DL-THYROXINE ON THE THYROID WEIGHT OF THE THIOURACIL FED RAT % O 30 N O R M A L f — 01-THYROXINE DOSE (y/msm/< l*Y ) CHAPTER IV KINETIC TREATMENT OF THYROXINE-LIKE ACTIVITY AND THYROXINE ANTAGONISM I t i s now w ell accepted t h a t th e pow erful p h y s i o lo g ic a l e f f e c t s of low m olecular w eight t r a c e s u b s ta n c e s such as v i t a mins, hormones, e t c . , can most l i k e l y be accounted f o r by as suming t h e i r d i r e c t p a r t i c i p a t i o n in some enzyme system or sy stem s. The io d in e c o n te n t of normal t i s s u e s a p a rt from the th y ro id g lan d i s s t r i c t l y at th e tr a c e l e v e l of c o n c e n tr a ti o n . The hormonal or p r o t e i n bound io d in e of th e c i r c u l a t o r y s y s - -8 tem i s l e s s th a n 10 M *. The th y r o id hormone of muscle t i s s u e i s p ro bably l e s s th a n 10 M., The normal d a il y s e c r e t i o n of th e th y r o id gland of th e adult r a t , k e p t at 85°C ., ha3 been e s tim a te d to be 8-3 micrograms of L -th y ro x in e p e r 100 g . of body w e ig h t. Thus i t can be proposed w ith some re a s o n t h a t th y ro x in e and t h y r o x i n e - l i k e compounds act by s e r v in g as th e coenzyme or p r o s t h e t i c group of some enzyme or fam ily of en zymes concerned in th e norm al economy of th e animal c e l l , even though no evidence f o r such a d i r e c t p a r t i c i p a t i o n can y e t be m a rs h a lle d . K in e tic tr e a tm e n t of r e a c t i o n s in which enzymes p a r t i c ip a te does not respond t o c l a s s i c a l chem ical r e a c t i o n r a t e th e o ry beeause of th e unusual n a tu r e and th e l i m i t i n g c h a r a c t e r i s t i c s of the p r o t e i n enzyme. For example, th e k i n e t i c s of an en zy m e-affected r e a c t i o n can l i e anywhere from z e ro to 24 f i r s t ord er w ith r e s p e c t to a r e a c t a n t or s u b s t r a t e which un d ergoes monomolecular a tta c k by th e enzyme. The se a rc h f o r an adequate t h e o r e t i c a l e x p la n a tio n of th e mechanism of en- c zyrne a c tio n le d t o th e v i r t u a l l y identical p ro p o s a ls of Mich— a e l i s and Menten(1913) and Van Slyke and C u l le n ( 19 14). T h e ir t h e o r i e s have affo rd e d a p l.au sib le e x p la n a tio n f o r the in flu e n c e of th e c o n c e n tr a tio n s of s u b s tra te ,, enzyme, and r e a c t i o n p ro d u c ts on r e a c t i o n r a t e . S u b se q u e n tly , i t was fonnd p o s s ib le to t r e a t the e f f e c t on r e a c t i o n r a t e of th e coenzyme c o n c e n tr a t i o n in a manner s u b s t a n t i a l l y i d e n t i c a l w ith t h a t employed' f o r th e in flu e n c e of s u b s t r a t e c o n c e n tr a tio n The prim ary o b je c tiv e in employing any k i n e t i c t r e a t ment t o th e d a t a o b ta in e d w ith th y ro x in e and i t s a n ta g o n is ts was t o i d e n t i f y th e ty p e of i n h i b i t i o n o b se rv e d . In th e b i o l o g i c a l system s which are used t o determ ine th y r o x in e - I ik e a c t iv i t y ,, th e r e a c t i o n s in v o lv in g th y ro x in e and r e l a t e d ac t i v e compounds may be reg a rd ed as r a t e lim itin g * The p a r t i c i p a t i o n of th y ro x in e in a key r e a c t i o n r e s u l t s in su c ceed in g re sp o n se s t h a t i n i t i a t e a s e r i e s of m e ta b o lic e v e n ts t h a t are reco g n ized as t h y r o x i n e - l i k e a c t i v i t y * Thus th e o v e r a l l r e a c t i o n t h a t was observed at a con st a n t-tim e was a f u n c tio n of th e amo)unt of th y ro x in e t h a t was a v a ila b le t o an enzyme system or systems*. I t should be em phasized, however, t h a t the a p p li c a t io n of the m ath em atical r e l a t i o n s d e riv e d f o r th e sim ple system in v o lv in g s u b s tr a te ,, enzyme, and, p e rh a p s,' coenzyme t o th e complex system of th e in v iv o re s p o n se of Amphibia to th y ro x in e i s an o v e r s i m p l i f i c a t i o n of q u e s tio n a b le v a l i d i t y . Many o b je c tio n s can be r a i s e d t o t h i s t r e a t m e n t . For exam p l e , th e a n ta g o n is ts might have made o th e r r e a c t i o n s , im p o rt ant t o m etam orphosis, r a t e l i m i t i n g in s te a d of th e t h y r o x i n e - a ff e c te d rea ctio n * . S ince p o r tio n s of th e a n ta g o n is t m o lecules / are a c tiv e by th e m se lv e s, i t i s p o s s ib l e t h a t much more com p lex k i n e t i c s are n e c e ssa ry t o e x p la in th e f i n d in g s r e p o r te d in t h i s d i s s e r t a t i o n * F urtherm ore t h i s type of k i n e t i c t r e i a t - ment a p p lie s only t o r e v e r s i b l e i n h i b i t i o n s , re g a rd le s s - of t h e i r c o m p lex ity . The p o s s i b i l i t y t h a t th e s e i n h i b i t o r s a t ta c k th e t h y r o x in e - c o n ta in in g enzyme i n an i r r e v e r s i b l e way has not been e lim in a te d . On th e o th e r hand, k i n e t i c a n a l y s i s of t h i s type has been of c o n s id e r a b le v a lu e in th e study of th e r o l e of i n h i b i t o r s in th e r e l a t i v e l y complex system of m ic ro b ia l gro w th . I t h a s a ls o y i e l d e d what appear t o be rea so n a b le r e s u l t s in the a n a ly s is of th y ro x in e a n ta g o n is ts r e p o r te d h e r e . Enzyme— s u b s t r a t e k i n e t i c s . M ic h ae lis and Menten(19;i3.) assumed (1) t h a t th e enzyme and s u b s t r a t e r e a c t r a p i d l y and r e v e r s i b l y t o form an e n z y m e -su b stra te complex and (S) t h a t th e d eco m p o sitio n of t h i s complex i s r e l a t i v e l y i r r e v e r s i b l e and r a t e d e te r m in in g . These p o s t u l a t e s may be fo rm u la te d as; 26 / 1 2 , E / S^ _ * E S ----} E / P where E i s th e f r e e enzyme, S i s the s u b s t r a t e , and P and ES are th e p roduct and th e enzyme— s u b s t r a t e complex, r e s p e c t i v e l y . According t o th e r a t e law ▼ = _ d (S ) - d(P) - k (ESI (3) d t ~ d t. I I f th e e q u ilib riu m (1) obeys th e law of mass a c tio n , th e n C4) where Kffl i s th e M ic h a e lis c o n s ta n t, r e p r e s e n t i n g th e d i s s o c i a t i o n c o n sta n t of th e e n zy rae rsu b stra te complex. I f we l e t e r e p r e s e n t th e t o t a l enzyme c o n c e n tr a tio n th e n e = (E) / - (ES) (5) S o lv in g fo r (ES) in e q u a tio n (4 ) th e r e i s o b tain ed (ES) = e '( S > Km / (3) (6) S u b s t i t u t i n g f o r (ES) i n e q u a tio n ( 3 ) , •v = * « .(s> ' ' m Km / - m k x e may be tak e n as equal t o th e maximum p o s s ib le v e l o c i t y , Vm, when v i r t u a l l y a l l of th e enzyme i s bound up in th e form of ES, so t h a t v - Vm (S) K ^ T T s ) (8 ) which i s the c l a s s i c a l form of th e w ell known M ichael-is- Menten e q u a tio n . E q u a tio n (8) i s handled most c o n v e n ie n tly in i t s r e c i p r o c a l form(Linewe aver and B u r k ,1934), where T h is l a t t e r e q u a tio n p e r m its th e ready e v a lu a tio n of Km by p l o t t i n g l / v a g a in s t l/(S '.)r d e te rm in in g Vm from th e i n t e r c e p t , and c a l c u l a t i n g Km from th e p ro d u c t of the slo p e and Ym F ig u re 2 i s such a p l o t f o r s e v e ra l e x p erim e n ts in v o lv in g v a ry in g th y ro x in e c o n c e n tr a tio n in th e absenee of an antago n i s t . The v e l o c i t y i s e x p re sse d in term s of th e f r a c t i o n a l d e c re a se in l e n g t h of th e ta d p o le at a c o n sta n t tim e* Vm i s found t o be 0*71 and th e Km f o r th y ro x in e i s ap pro xim ately 5 x 10~7M. The p re se n c e of an i n h i b i t o r which combines r e v e r s i b l y w ith th e f r e e enzyme a n d /o r th e e n z y m e -su b stra te complex i n t r duces s e v e r a l new e q u i l i b r i a as fo llo w s;; i m E / 1 *---------------- V E I (inactive)*;; Kt — (E) ( I ) (1 1 ) (E l) , (1 8 ) ES / I ^ * ESI ( i n a c t i v e ) ; : IC,„Tr (ES) ( I ) (13) (E SI)- - - where I i s th e 1 i n h i b i t or or c o m p e tito r, E l th e e n z y m e -in h ib i- t t o r complex, and ESI th e en zy m e-su bstr a t e - i n h i b i t o r complex* Combining th e s e e q u i l i b r i a w ith t h a t tin v o lv in g th e s u b s t r a t e e = (E ) / (ES); / (E l) / (E S I) (14) and e l i m in a ti n g E, ES, E l, and ESI by a p p ro p ria te s u b s t i t u t i o n and c a n c e l l a t i o n s i m i l a r t o t h a t employed f o r e q u a tio n ( 8 > 's d e r i v a t i o n * t h e r e i s f i n a l l y o b tain ed e q u a tio n (15) which w.as p u b lis h e d r e c e n t l y by Harmon and Nieraann(1949) and 28 d e riv e d in d e p e n d e n tly by W erkheiser and F r ie d e n ( 1 9 4 8 ) • T his l : ( I A S i 2 ) i / (1 A l i y ( K l / V m) 4 ' (15) . • k e s i , v m . .K j, . e q u a tio n combines m a th e m a tic a lly th e th r e e most commonly en c o u n te red ty p e s of antagonism which are d is c u s s e d in d e t a i l below . I t must be emphasized t h a t only r e v e r s i b l e ty p e s of i n h i b i t i o n are c o n sid ere d in t h i s treatm ent*. To d e te r m in e th e ty p e of i n h i b i t i o n , l / v i s p l o t t e d a g a in st l / S f o r v a r i ous i n h i b i t o r c o n c e n tr a tio n s w ith th e r e s u l t i n g s t r a i g h t l i n e s b e in g in f lu e n c e d in slo p e a n d /o r o rd in a te i n te r c e p t * O bvious ly when (I): i s z e ro , e q u a tio n (15) red u e es t o ( 9 ) , th e r e c i p r o c a l form of th e c l a s s i c a l M ichaelis-M enten eq uatio n * C o m petitive i n h i b i t i o n . In c o m p e titiv e i n h i b i t i o n , s u b s t r a t e and i n h i b i t o r are v y in g f o r the same r e a c t i v e s i t e s on th e enzyme. There i s , th e r e f o r e ,, no tendency t o form ESI, i , or in o th er words KEg j i s i n f i n i t e . E quation (15) th e n be comes e q u a tio n (16): which was f i r s t p u b lish ed by Lineweaver 1 - 1 / (1 / 1 2 L > (KnAm) 1 v * * Vm Kr (S) (16) and B urk(1 9 3 4 ). The e f f e c t of t h i s type of i n h i b i t o r i s t o modify th e slo p e of a l / v versub> l / ( S ) p l o t by th e f a c t o r (1 / ( D / K j ) w ith no a l t e r a t i o n of i n t e r c e p t . When ( I ) /K i Si v ery la rg e w ith r e s p e c t t o one, (16). reduces to 1 - 1 / Km * ( I, v “ Vra Kj Vm I T which i s th e f a m i l i a r " s h o rt" form of the c o m p e titiv e i n h i b i - 29 t i o n e q u a tio n and p e rm its th e com bination of a l l th e d a t a on one l i n e by p l o t t i n g l / v a g a in s t (I)/(S > . g iv in g a-c o n v in cin g d e m o n stra tio n of th e c o m p e titiv e ty p e of antagonism . Equa t i o n (17),' j u s t i f i e s th e use of m olar i n h i b i t o r i n d ic e s , e . g . , th e ( I ) / ( S ) r a t i o r e q u ir e d f o r 50 p e r c en t r e d u c t io n of th e r a t e caused by (S);. Apopular example of th e c o m p e titiv e type of i n h i b i t i o n i s th e r iv a l r y .b e t w e e n p-am inobenzoic acid and s u lf a n ila m id e in th e m etabolism of c e r t a i n m icroorganism s. Other exam ples of c o m p e titiv e i n h i b i t i o n are g iv en by Linew eaver and B u rk (1 9 3 4 ). N on-com petitive i n h i b i t io n . In ease th e a n ta g o n is t in a c t i v a t e s by combining w ith r e g io n s of th e enzyme remote from • th e s i t e of s u b s t r a t e com bination, n o n -c o m p e titiv e i n h i b i t i o n o c c u rs . Since th e enzym e-substr ate i s l i k e l y t o show the same a t t r a c t i o n f o r th e a n ta g o n is t as th e f r e e enzyme, Kggi m ecjf be sbt e q u al to Kj so t h a t (15); r e s o l v e s t o 1 - 1 ( i / H I ); ( l / Km ):. (18) v - v m . . K j . T s ) . A l / v v e rs u s l/( S ) . p l o t y i e l d s a fam ily of l i n e s of v a r i a b l e i n t e r c e p t and v a r i a b le s lo p e , b oth m odified by a f a c t o r of (1 / ( I ) ; / K j ) » The f r e q u e n t ly observed m etal io n i n h i b i t i o n of enzymes c o n ta in in g e s s e n t i a l s u lf h y d r y l groups i s an example of non c o m p e titiv e i n h i b i t l o n • Other i n s ta n c e s are, given by L in e weaver and B urk (1 9 3 4 ). 3 a Uncom petitive i n h i b i t i o n . U ncom petitive i n h i b i t i o n d e s c r i b e s th e system in which the i n h i b i t o r has no a f f i n i t y f o r th e f r e e enzyme, i . e . , Kj i s z e r o , b u t does combine w ith th e e n z y m e -su b stra te complex, a p p a re n tly p re v e n tin g th e decom po sitio n of ES t o y i e l d P and E*. The r e s u l t i n g e q u a tio n (1.9): shows t h a t t h i s type of i n h i b i t i o n a f f e c t s 1 ~ 2 U / ( I ) ) / Km • 2 (19) v ” Vm KEs i Vm (S) only th e i n t e r c e p t of a l / v v e r s u s l / ( S ) p l o t , in c r e a s in g i t by a f a c t o r of (1 / ( I ) ) . Cases of u n c o m p etitiv e i n h i - k e s i b i t i o n are d e s c r ib e d by E b e rs o le , G u tte n ta g , and W ilso n (1 9 4 4 )• 31 CHAPTER V RESULTS C o m petitiv e i n h i b i t i o n of t h y r o x in e . The compound, Q— b en zy l— N— a c e ty 1 - 3 ,5 - d i i o d o t y r o s i n e , among o th e r d e r i v a t i v e s of N - a c e t y l - 3 ,5 - d i i o d o - L - t y r o s i n e , was f i r s t r e p o rte d by W oolley(1946) t o p r o t e c t Amphibia a g a in s t th e t o x i c i t y of r e l a t i v e l y h ig h l e v e l s of th y r o x in e . W inzler and F rie d en (1.948b) and W illiam s, e t a l .,( 1 9 4 8 ) a ls o found t h a t t h i s com pound antagonized th e e f f e c t s of th y r o x in e in in d u cin g th e m etam orphosis of t a d p o l e s . When th e compound was t e s t e d as a c o m p e titiv e a n ta g o n is t of DL— th y ro x in e in the ta d p o le sys tem d e sc rib e d e a r l i e r , i t was found t o have a f a i r l y c o n s is t e n t m olar i n h i b i t o r index of about 550. Table I summarizes one of many ex p erim e n ts in which th e c o m p e titiv e n a tu re of th e i n h i b i t i o n of th y ro x in e by O - b e n z y l- N - a c e ty l- 3 ,5 - d iio d o - L - ty r o s in e on amphibian m etam orphosis i s c l e a r l y i n d i c a t e d . T y p ic al d a t a are p l o t t e d in F ig u re 4 showing t h a t th y ro x in e becomes l e s s e f f e c t i v e at h ig h e r a n ta g o n is t c o n c e n tr a tio n s and t h a t the i n h i b i t o r becomes l e s s a c tiv e at h ig h e r th y ro x in e l e v e l s . The r e c i p r o c a l of th e th y ro x in e e f f e c t i s seen to be an approxim ate f u n c t i o n of th e r a t i o of i n h i b i t o r t o th y ro x in e c o n c e n tr a tio n s and i s r e l a t i v e l y independ e n t of th e a b so lu te c o n c e n tr a tio n s of th e two s u b s ta n c e s . I t had been observed previously(G addura,1929-30; 3 2 TABLE I COMPETITIVE INHIBITION OF DL-THYROXINE BY O-BENZYL-N— ACETYL— 3 ,5 -DIIOD O-L-TYROSINE 0 -Benzyl-N — a c e ty 1 - 3 ,5 - Per c e n t d e c re a s e in p o le s a f t e r 72 h ou rs th e le n g th in c u b a tio n of t ad- d iio d o -L - ty r o s in e m icrogr ams per ml. DL-Thyroxine micrograms per ml. 0 .2 5 0 .5 0 0 .7 5 1.00 1.25 0 26 47 60 60 60 6 2 .5 19 45 55 56 55 125 13 37 45 47 50 250 11 16 25 35 45 375 6 13 20 30 35 500 12 19 25 29 750 - 8 17 17 25 Molar i n h i b i t o r index a 688 550 412 515 550 a The m olar i n h i b i t o r index i s d e fin e d as th e m olar r a t i o of i n h i b i t o r to th y ro x in e necessjary to reduce th e th y r o x in e e f f e c t by 50 p e r cent* No e f f e c t on th e le n g th of t a d p o l e s was observed f o r groups t r e a t e d w ith i n h i b i t o r alone* P E R C EN T DECREASE I N LENGTH 33 FIGURE 4 A., THE EFFECT OF INCREASING CONCENTRATIONS OF Q-BENZYL-N- ACETYL— 3 ,5-DIIODO-L— TYROSINE ON THE RESPONSE OF TADPOLES TO DL-THYROXINE CONCENTRATION. B. THE RECIPROCAL OF THE OBSERVED EFFECT AS MEASURED BY THE PER CENT DECREASE IN THE LENGTH OF TADPOLES PLOTTED AS A FUNCTION OF THE RATIO OF THE MOLAR CONCENTRATION OF INHIBITOR TO DL-THYROXINE. 6 f t - 4 5 - 30 1 5 / N O IN H IBITO R • ^ — I25^ml. • / 2 5 0 % l . TSOfk. 0.40 0.80 1.20 THYROXINE % | | _ . THYROXINE: X-0.50*/ml. ••0.75yriL. a-|.00YS/ml. o-USVml. INHIBIT0R= 1.60 I O-CHjO -jO -C H ^H C O O H m CO I I C H 3 I 350 7 0 0 , 1050 IN H IB IT0 R / % R t ) X l N E 1 4 0 0 FIGURE 4A FIGURE 4B 34 F rie d e n and W in zler,19 48a) t h a t a c e t y l a t i n g th e amino groupc of th e sid e c h ain of th y ro x in e re d u c e s th e t h y r o x i n e - l i k e a c t i v i t y of th e r e s u l t i n g N -a ce ty l-D L -th y ro x in e t o a p p ro x i m ately 3 p e r c en t of th e a c t i v i t y of D L -th y ro x in e * A s im i l a r r e l a t i o n s h i p was observed f o r th e r e l a t i v e t h y r o x i n e - l i k e a c t i v i t i e s in Amphibia of N - a c e t y l - 3 ,5 - d i i o d o - L - t y r o s i n e and 3 ,5 - d i i o d o — L - t y r o s i n e . A ccordin gly, th e p o s s i b i l i t y t h a t the p re se n c e of an N -a c e ty l group m ight reduce th e e f f e c t i v e n e s s of an a n ta g o n is t as w e ll as t h a t of a t h y r o x i n e - l i k e a c tiv e compound was in v e s tig a te d * . The d a t a p r e s e n te d in T able I I c l e a r l y e s t a b l i s h e s the a n t i — th y ro x in e a c t i v i t y of 0 - b e n z y l- 3 ,5 - d iio d o - D L - ty r o s in e in Amphibia, D e sp ite the f a c t t h a t the racem ic compound was used, t h i s su b sta n c e was f i f t e e n tim es as e f f e c t i v e a th y r o x in e i n h i b i t o r as i t s unracem ized N -a c e ty l d e r i v a t i v e t w ith a molar i n h i b i t o r index of about 37* The v a lu e of th e m olar i n h i b i t o r index at the h i g h e s t i n h i b i t o r c o n c e n tr a tio n s i s used in t h i s and subsequent d i s c u s s i o n s because t h i s v a lu e ^ i s l e a s t a ffe c te d by u n s a t u r a t io n of th e system w ith r e s p e c t t o i n h i b i t o r or th y ro x in e * 0 -B e n z y l-3 ,5 - d iio d o -D L - ty ro s in e has been p a r t i c u l a r i l y u s e fu l f o r subsequent s t u d i e s of th e s p e c i f i c i t y of th e i n h i b i t i o n of t h y r o x i n e - l i k e a c tiv e compounds* I t i s r e l a t i v e l y non t o x i c , and th e only l i m i t a t i o n on i t s t e s t i n g i s the f a c t t h a t at a pH of 8 .0 i t i s s o lu b le only t o th e e x te n t of 250 m icrogram s 35 Tj SBLE II COMPETITIVE INHIBITION OF DL-THYR O X INE BY Q-BENZYL— 3, 5 -DIIODO— DL— TYROSINE 0.-B e n zy l- 3 , 5 - d iio d o -D L - t y r o s i n e P er c e n t d e c re a se in th e le n g th of t a d p o le s a f t e r 63 h ou rs in c u b a tio n micrograras p er ml* DL.— T hyroxine, m icrogram s p er ml* 0.2 5 0 .5 0 1 .0 0 2.00 0 28 46* 51* 60* 1*25 28 44* 48* 56* 2: *50. 24 44* 46* 56* 5 .0 0 20 40 44* 50* 12*5 15 25 42* 44* 25 *0 1.0 16 2.8 34 50*0 9 9 15 28 125 4 6 13 24 250 3 3 9 12 Mol. i n h i b i t o r index 74 45 45 37 -5 T h is i n d i c a t e s 80-100 p e r cen t d e a th s o c c u rrin g s e v e ra l h o u rs p r i o r t o o b s e r v a tio n time., The p r o t e c ti v e a c tio n of t h i s i n h i b i t o r a g a in s t h ig h e r c o n c e n tr a tio n s of D L -thyroxine i s th u s c l e a r l y e s t a b l i s h e d . 36 p e r ml. Even so, i t i s p o s s ib le t o e lim in a te th e norm ally pow erful e f f e c t of 2*0 micrograms p e r ml* of D L fthyroxine at th e maximum i n h i b i t o r c o n c e n tr a ti o n . In L onger-term e x p e rim e n ts, when th e a b i l i t y of t h i s a n ta g o n is t t o oppose endogenous th y r o id hormone was being te s te d ,, s i g n i f i c a n t t h y r o x i n e - l i k e a c t i v i t y was observed f o r t h i s compound at 5*0 micrograms p e r ml*, p e rh a p s because of cleavage t o d iio d o t y r o s i n e by phenyl e th e r a s e s * However t h i s .t h y r o x i n e - l i k e a c t i v i t y did n o t i n t e r f e r e w ith the t e s t i n g of t h i s su b sta n c e at h ig h e r l e v e l s i n th e s h o r t term e x p erim e n ts employed t o d em o n stra te c o m p e titiv e in h ib i t io n * The k i n e t i c s of th e i n h i b i t i o n of th y ro x in e by t h i s com pound was, t h e r e f o r e s u b je c te d t o e x te n s iv e a n a l y s i s . F i r s t of a l l the r e l a t i v e co n stan cy of c a l c u l a t i o n s of Kj u sing th e com p e t i t i v e i n h i b i t i o n e q u a tio n (16) w,as t e s t e d u sin g the d a t a ob ta in e d w ith th e v a rio u s i n h i b i t o r c o n c e n tr a tio n s employed in Table I I w ith th e r e s u l t s com piled in Table I I I . These r e s u l t s TABLE I I I COMPARISON OF M s s FOR COMPETITIVE INHIBITION OF THYROXINE FOR VARYING CONCENTRATIONS OF Q-BENZYL-3 ,5 -DIIODOTYROSINE (Km f o r D L -thyroxine - 5 .0 x 10 M. Vm " 0 .7 1 ) ■ . Mol.aritv of i n h i b i t o r Kt irn moles p er l i t e r — 9 ,6 "x 10*6 i “ 1.59 X 10 x s i : ! ^ i s - * x *7 4 x i o - 5 4 7 .8 x " 1*63 " 9 5.6 '• 1.15 2 3 9 " 478 " 1#I 9 Average Kj i»'*T»75 Q.27 x 3L Q ” M * 37 s u g g e st an adequate co n stancy of Ki f o r th e e x p e rim e n ta l method employed w ith an average v a lu e of 1 .7 x 10“5M and an. adherence of th e d a t a of T able I I to e q u a tio n ( 1 6 ) . The d a t a in Table I I I a ls o in d ic a te th e i n a p p l i c a b i l i t y of th e " S h o rt" ,fo rm of th e c o m p e titiv e i n h i b i t i o n e q u a tio n (1.7), sin c e th e v a lu e of ( I ) / K j i s not much g r e a t e r th an one f o r th e whole range of a n ta g o n is t c o n c e n tra tio n s * The o b s e r v a tio n of somewhat g ra d u a lly d e c r e a s in g molar i n h i b i t o r in d ic e s w ith i n c r e a s in g th y ro x in e c o n c e n tr a tio n s shown in Table I I i s r a t i o n a l i z e d in Table IV. Molar i n h i b i t o r i n d ic e s , whose n u m erical v a lu e may be re g a rd e d as th e number of m olecules of a n ta g o n is t n e c e s s a ry t o d is p la c e one m olecule of DL-thyr oxine from an enzyme system , were c a l c u l a t e d f o r th e th r e e r e v e r s i b l e ty p e s of i n h i b i t i o n assuming re a so n a b le v a lu e s f o r Km, Vm, Kj, and th y ro x in e and i n h i b i t o r c o n c e n tr a t i o n s . The observed d a t a f i t s th e c a l c u l a t e d v a lu e s f o r com p e t i t i v e i n h i b i t i o n c o n s id e ra b ly b e t t e r th a n f o r th e o th e r two ty p e s of i n h i b i t i o n . F u rth e r v e r i f i c a t i o n of th e c o m p e titiv e n a tu r e of t h i s . antagonism appears^ in F ig u re 5, A and B. A d i r e c t p l o t of th e p er c en t d e c re a s e in le n g t h a g a in s t th y ro x in e c o n c e n tr a t i o n g iv e s w ith i n c r e a s in g i n h i b i t o r a s e r i e s of c u rv e s w ith p r o g r e s s i v e l y d e c r e a s in g e f f e c t at th e same le v e l of th y r o x ine as in F ig u re 5A. A g rap h of th e r a t i o of 100 over E, where E i s th e p e r c en t d e c re a s e in le n g th at c o n ta n t tim e, TiffiLE IV T Y P E OF . OF THYROXINE RY L N CHCOOH H H i CALCULATED M OLAR THYROXINE MOLARITY * I01 INHIBITOR INDEX F O R : S.O IDO 2 5 .0 5 0 . 0 NON-COMPETITIVE ! : 1 . IDO 5 0 2 0 10 UN-COMPETITIVE * H O 6 0 £4- II C O M PETITIV E SO 7 0 5 0 4 5 OBSERVED INDEX n j 4 5 4 5 3 7 i /■ t ^ i r t I k n crH V R oitj= 5.0*10 m CALCULATED FROM: K i J l + H J t s ] ! W o , ^ . 0 * lO ^ tl INHIBITOR = 5 ‘ lff‘ -.0 0 5 M Mn-TI^ ^613 ^126 FIGURE 5 A. THE EFFECT OF INCREASING CONCENTRATIONS OF Q-BENZYL- 3 ,5 -DIIODO-DL— TYROSINE ON THE RESPONSE OF TADPOLES TO DL-THYR OXINE B* THE RECIPROCAL ( x 10,0) OF THE OBSERVED EFFECT AS MEASURED BY THE PER CENT DECREASE IN THE LENGTH OF TAD POLES PLOTTED AS A FUNCTION OF THE RECIPROCAL OF THE MOLAR CONCENTRATION OF DL-THYROXINE FOR VARIOUS LEVELS OF G— BENZYL-3,5 -DI IODO-DL.-TYROS INE ^ o p c \ g c o ° H = n o IN H IB IT O R INHIBITOR FIGURE 5 A FIGURE 5B 40 y i e l d s a f a m i ly 'o f l i n e s w ith a common i n t e r c e p t and slo p e i n c r e a s in g w ith i n c r e a s in g amounts of a n ta g o n is t, th u s b e in g c h a r a c t e r i s t i c of c o m p e titiv e i n h i b i t i o n . th e a p p li c a t io n of th e above c r i t e r i a p re s e n te d in d e t a i l f o r the d e te r m in a tio n of th e n a tu re of th e i n h i b i t o r a c tio n of 0 - b e n z y l - 3 ,5 - d i iodo -D H -ty ro sin e t o th e o th e r exam p l e s of i n h i b i t i o n d is c u s s e d in t h i s d i s s e r t a t i o n has le a d to th e c o n c lu s io n t h a t th e antagonism s observed are p r i m a r i ly c o m p e titiv e in n a t u r e . The c o m p e titiv e type of i n h i b i t i o n i s a lso sug gested by th e r e l a t i v e constancy of th e molar in h i b i t o r i n d ic e s in T ab les I,, I I , V, VI, V I I , and V III* C o m petitiv e i n h i b i t i o n of th y ro x in e by e t h e r s of 3 , 5 - d iio d o -4 -h y d ro x y b e n z o ic a c id . V a rio u s e t h e r s of 3 ,5 - d i i o d o - 4-hydroxybenzoic acid were o r i g i n a l l y d e v ised to se rv e as i n h i b i t o r s f o r t h y r o x i n e - l i k e compounds in which th e a la n in e sid e c h ain of th y ro x in e i s re p la c e d by a carboxyl g ro u p . be T his s e r i e s , however, was found t o ^ e f f e c t i v e a g a in s t th y ro x in e * As in d ic a te d su b se q u e n tly , th e p a re n t compound, 3 , 5 - d i - i odo-4-hyd ro xyb en zo ic a c id , re v e a le d no antagonism t o th y ro x ine at mole r a t i o s of 10Q0, a lth o u g h i t d id show some th y ro x - i n e - l i k e a c t i v i t y at h ig h e r c o n c e n tr a ti o n s . To p e rm it a com parison w ith th e N - a c e ty l - 3 ,5 - d i io d o - L — t y r o s in e e t h e r s d e s c rib e d by W oolley(1946), th e benzyl and p- n itr o p h e n y le t h y l e th e r s of 3 , 5 - d i i o d o-4-hydrox ybenzo ic acid 41 were p re p a r e d . To observe f u r t h e r th e minimum s t r u c t u r a l r e q u irem en ts f o r an i n h i b i t o r , th e methyl e t h e r was a lso s tu d i e d . I t was n o t p o s s ib l e t o t e s t th e s e compounds at l e v e ls h ig h e r th an th o se rep .a rted because of t h e i r t o x i c i t y t o t a d poles, at such c o n c e n tr a ti o n s . The d a t a p r e s e n te d in Table V d e m o n stra te t h a t 3 , 5 - d i - io d o -4 -b en z y lo x y b e n zo ic acid i s a p o te n t c o m p e titiv e i n h i b i t o r of th e a c t i v i t y of th y ro x in e in Amphibia. Molar r a t i o s of t h i s compound t o th y r o x in e of about 8 reduced the a c t i v i t y of th y r o x in e by 50, p er c e n t . In F igu re 6 , th e s e t y p i c a l d a t a are p l o t t e d t o show g r a p h i c a l l y th e approxim ate dependency of th e e f f e c t of th y ro x in e on th e r a t i o of a n ta g o n is t to a c tiv e compound. Kj c a l c u l a t i o n s average about 2 x 10/ M. T his su b sta n c e i s , t h e r e f o r e , th e most e f f e c t i v e c o m p e titiv e i n h i b i t o r re p o r te d in t h i s d i s s e r t a t i o n . In Table VI, 3 , 5 - d i i o d o - 4 - a n i s i c acid i s shown t o be a le s s, e f f e c t i v e c o m p e titiv e i n h i b i t o r of th y ro x in e th a n the benzyl e th e r w ith an i n h i b i t o r index of about 1 5 0 . The p - n i - tr o p h e n y le th y l e t h e r , though o r i g i n a l l y r e p o r te d by Woolley (1.946)! a s the most e f f e c tiv e , modif i c a t i o n of N - a c e t y l - 3 , 5 - d i - io d o - L - ty r o s in e f o r i n h i b i t i o n , a ls o re v e a le d c o n s id e ra b ly le s s , i n h i b i t i o n th a n 4 .-b en z y lo x y -3 ,5 -d iio d obenzoic acid w ith an i n h i b i t o r index of around 75*. S tu d ie s w ith t h i s l a t t e r compound were lim ite d b ecause of i t s very h igh t o x i c i t y and r e l a t i v e l y poor i n h i b i t o r p r o p e r t i e s . 42 TABLE V COMPETITIVE INHIBITION OF DL— THYR OXINE BY 4-BENZYL0XY-3 ,5-D I IODOBENZ 0 IC AC ID Per c e n t d e c re a s e in le n g th of t a d p o le s 4^-Benzyloxy- a f t e r 69 hours-: of in c u b a tio n 3 , 5-d i i od o— ben zo ic acid _____________________________ ____________ micrograms DL-Thyroxine, m icrogram s p er ml* per ml* O.Q- 0*15 0*30 0 .5 0 0*90- 1*80 o 00 w 30 45 48 55 0 .5 0, a 17 23 35 40 46 1.00 a 8 19 30 32 43 2.50 0, 4 8 20 25 40 5 .00 Q 2 4 L8 20 37 7.50- o 2 4 10 1.2 33 10.0.0 o . _ 10 25 Molar i n h i b i t o r indeae: 9i 8 6|? 5 8 43 FIGURE 6 A* THE EFFECT OF INCREASING CONCENTRATIONS OF 4-BENZYLOXY**- 3,5 — DIIODOBENZOIC ACID ON THE RESPONSE OF TADPOLES TO DL- THYROXINE * B.. THE RECIPROCAL (x 100) OF THE OBSERVED EFFECT AS MEAS URED BY THE PER CENT DECREASE IN THE LENGTH OF TADPOLES PLOTTED AS A FUNCTION OF THE RATIO OF THE MOLAR CONCENTRA TION OF 4-BENZYL0XY-3, 5-DIIODOBENZOIC ACID TO DL-THYROXINE. 1 0 1 c_b „ NO INHIBITOR 7.5 100 E 5.0 T H Y R O X I N E : x-O.50^m l. O .^ /k . •- uoyML. I N H I B I T O R - ^C^O-JO-COOH 030 1.0 0 1.50 " 2.00 t h y r o x i n e IN H IB ITO K /^TH Y R O X IN E 1 2 18 24 - FIGURE 6 A FIGURE 6B 44 TABLE VI COMPETITIVE INHIBITION OF DL-Thyroxine BY 3 , 5-DII0D0-4-ANISIC ACID 3 ,5 -D iio d o -4 a n is ic acid P er cen t d e c re a s e in 6 9 h o u rs in c u b a tio n le n g th a f t e r micrograms per m i l l i l i t e r DL-Thyroxine, 0 0*25 m icrogram s p e r ml* 0*50 0*75 1*25 Q Q j 33 47 48 52 25*50 O 32; 47 47 46 5*QO 0 2B 47 47 50 12*5 0 23 37 39 48 25*0 0 10, 31 35 42 37 *5 0 8 26 34 40 Molar i n h i b i t o r index 153 153 >96 >58 45 The s i g n i f ic a n c e of th e io d in e s in th e 3 and 5 p o s i t i o n s was. s tu d ie d by t e s t i n g th e a n ta g o n is ti c p r o p e r t i e s of 4 - a n i s i c acid and 4-b en zy lo x y b en zo ic a c id . 4 -A n isie acid could be t e s t ed s a t i s f a c t o r i l y up to mole r a t i o s w ith no i n h i b i t i o n ob s e r v a b l e . The b enzyl e t h e r showed some antagonism at mole r a t i o s in th e neighborhood of 350, alth o u g h i t was im p o ssib le to o b ta in c l e a r - c u t d a t a becau se of th e a p p re c ia b le t o x i c i t y of t h i s compound. In any c a s e , t h i s r e p r e s e n te d a marked d e c re a s e from th e i n h i b i t o r s t r e n g t h of th e c o rre sp o n d in g io d in - ated analog which had an i n h i b i t o r index of only 8 . S p e c i f i c i t y of th y ro x in e i n h i b i t i o n . The s p e c i f i c i t y of th e i n h i b i t i o n of th y ro x in e by th e type of c o m p e titiv e a n ta g o n is ts d e s c rib e d in p re v io u s p a ra g ra p h s was t e s t e d by a d m in is te rin g s im u lta n e o u s ly w ith D L -thyroxine a l a r g e number of compounds in c lu d in g s t r u c t u r a l l y r e l a t e d , g o i tr o g e n ic , and p o ly f u n c tio n a l s u b s ta n c e s . DL-Thyroxine was used at a con c e n t r a t i o n of 0 .5 0 micrograms p e r m l., which when g iv e n alone, r e s u lte d , in a d e c re a se in le n g th of 4 5 5 per cen t in 70 * - L. • _ 1 . \ " * 7 ' ’ 1 0 U h o u rs in c u b a tio n at 31 _£ 1°C. A compound was c o n sid e re d t o be w ith o u t i n h i b i t o r y e f f e c t when th e resp o n se to th y ro x in e was n o t a lt e r e d by more th a n 10 p e r cent d e c r e a s e u in l e n g t h . The maximum dosage of a susp ected a n ta g o n is t was d e term in ed by i t s t o x i c i t y , by i t s w ater s o l u b i l i t y at pH 3*0 / Q.5 and f i n a l l y by a l e v e l j u s t below a c t i v i t y in case th e compound 46 i t s e l f e x e rte d t h y r o x i n e - l i k e a c t i v i t y , e . g . , N -acety l-D L - th y r o x in e . A h ig h s p e c i f i c i t y of th e p re v io u s c o m p e titiv e a n ta g o n is ts f o r checking th y ro x in e was in d ic a t e d by th e f a c t t h a t none of th e fo llo w in g compounds blocked th e e f f e c t of th y ro x in e when t e s t e d at the. maximum p o s s ib le mole r a t i o s l i s t e d below; N— acetyl-D L — th y r o x in e , 10; 3 , 5 - d i i o d o - 4 - ( 4 '- hydroxyphenoxy)-benzoic acid, 15; 3 ,5 -d iio d o -4 -h y d ro x y b e n - z.oic acid, 10QO; 3 ,5 -d iflu o ro -4 -m e th o x y p h e n o l, 440 ; 3 , 5 - d i - c h l o r o - 4 - a n i s i c acid , 350; 2 - a m in o -3 ,5 -d iio d o b e n z o ic acid, 10.0; L - ty r o s in e , 2000; N - a c e t y l - 3 ,5 - d i i o d o - L - t y r o s i n e , 1600; 4 -flu o ro b e n z o ic a c id , 5000; 4 - a n i s i c a c id , 500; 3-hydroxyben- z o ic acid , 280; s a l i c y l i c acid, 1700; 4-am inobenzoic acid , 2830; 4 -io d o b e n z o ic acid , 1560; 4 - a n i s i d i n e , 3150; s u l f a n i l i c a c id , 1350; s u lf a n ila m id e , 2250; ben zo ic a c id , 6300; benzyl a lc o h o l, 1800; 2 - t h i o u r a c i l , 2400; 2 -m e rc a p to im id a z o le , 1550; a sc o rb ic acid , 1770; sodium io d id e , 5000; m alonic a c id , 750; a c e t o n i t r i l e , 350. C o m petitiv e ant agoni sm of t h y r o x i n e - l i k e a c tiv e compounds* The t e s t system in v o lv in g Amphibia o ff e r e d th e o p p o rtu n ity to vary a c tiv e component as w ell as a n ta g o n is t* The r e c e n t sug g e s t io n by F rie d e n and W inzler(L948a) r e g a r d in g th e n o n -sp e c i f i c n a tu r e of th e s id e ch ain f u n c t i o n a l group as a s t r u c t u r a l re q u ire m e n t f o r t h y r o x i n e - l i k e a c t i v i t y i n Amphibia made a v a i l able a number of compounds of u se fu l and a p p re c ia b le t h y r o x in e l i k e a c t i v i t y . 47 P re lim in a ry s tu d i e s w ith th e g ly c in e horaolog of th y ro x in e r e v e a le d a resp o n se to i n h i b i t i o n by th e b e n z y l e t h e r s of N - a c e ty l - 3 ,5 - d i io d o - L - t y r o s i n e and 3 ,5 - d iio d o - 4 - h y d r o x y - ben zo ic acid v i r t u a l l y i d e n t i c a l w ith th y r o x in e . Since t h i s ' compound e x h i b i t s o n e -th ir d to o n e -h a lf th e a c t i v i t y of th y — r o x in e ( F rie d e n and ? /in z le r ,1 9 4 8 a ) , th e g ly c in e sid e chain homolog could be t e s t e d at m olar r a t i o s v ery s i m il a r to th o s e employed w ith t h y r o x in e . The i n v e s t i g a t i o n of th e e f f e c t of th y ro x in e c o m p eti t i v e a n ta g o n is ts on th e t h y r o x i n e - l i k e a c t i v i t y of 3 ,5 - d i i o d o - 4— ( 3 * , 5 , - d i i o d o - 4 , — hydroxyphenoxy)— b e n zo ic a c id , a compound p p o s s e s s in g 10 p e r c e n t th e a c t i v i t y of D L -th y ro x in e, Km of 4,8 x 10 M assuming a Vm of 0 ,7 1 , proved t o be of c o n s id e r a b le i n t e r e s t * I t i s seen in Table V II t h a t no i n h i b i t i o n of th e a c t i v i t y of t h i s c a r b o x y lic acid analog of th y ro x in e could be observed w ith any of th e two i n h i b i t o r s t e s t e d at concentrac t i o n s which were able t o red u ce a p p re c ia b ly th e e f f e c t of a c o rre sp o n d in g a c t i v i t y le v e l, of D L -th y ro x in e , However, when a n t i — th y ro x in e compounds were t e s t e d f o r antagonism a g a in s t th e c a r b o x y lic acid analog of th y ro x in e at mol ar r a t i o s c o r re sp o n d in g t o th o se e f f e c t i v e a g a in s t th y ro x in e , a d e f i n i t e r e d u c t io n i n a c t i v i t y was n o te d . Inasmuch as t h i s a c tiv e compound r e q u i r e s a te n f o ld i n c r e a s e in m o la r ity t o produce th e same a c t i v i t y as D L -th y ro x in e, approxim ately t e n fo ld i n c r e a s e s of i n h i b i t o r were r e q u ir e d t o achieve s i m il a r TABLE V II 48 EFFECT QF THYROXINE INHIBITORS ON THE THYROXINE-LIKE ACTIVITY OF 3 ,5 — DIIODO— 4 - ( 3 ' , 5 '-DIIODO— 4 ' -HYDROXYPHENOXY)-BENZOIC ACID ‘ Expjt « No.. I n h i b i t o r micrograms p e r ml. ho| © - « | © - cboh * * M o>l ar V /m l* r a t i o a Per c en t d e c re a se ' in l e n g t h 1 3 5 Q-Benzyl-N— a c e t y l - 3 ,5 — d i i od o— L - ty r o s in e 2.0 a 30 250 2 .0 173 32 0 4 .0 0 48 250 4 .0 86 48 6 Q 2.5 Q 3Q 25Q 2 .5 138 28 10 O 2.5 0 33 750 2.5 413 22 0 6*0 0 45 400 6*0 93 44 600 6 .0 138 43 10 4-Benzyl o x y -3 , 5-d i iodo- ben zo ic acid 2.5 0 33 2*50 2.5 1.5 33 3*75 2*5 2.2 31 5*0 2.5 3*0 25 7*5 2.5 4.5 17 10*0 2.5 6 .0 12 Q . 3.75 a 48 2*50 3*75 1.0 44 3*75 3.75 1.5 39 5 .0 3.75 2 .0 31 7*5 3.75 3 .0 25 0 5 .0 a 52 2*5 5 .0 0 .7 5 48 3.75 5 .0 1.2 46 5 .0 5 .0 1*5 39 0 7.5 Q 56 2 .5 7.5 0 .5 52 3*75 7.5 0.75 46 5*0. 7.5 1.0 42 7.5 7.5 1.5 39 % o la r r a t i o of i n h i b i t o r t o t h y r o x i n e - l i k e a c tiv e compound* bIn c u b a tio n p e rio d was 70_£ 5 hours in a l l r e p o r te d e x p e rim e n ts 49 molar r a t i o n of i n h i b i t o r to th y r o a c ti v e compound. Such c o n c e n tr a tio n s of 0— b e n z y l - N - a e e t y l - 3 ,5 - d ii o d o - L - t y r o s i n e t h a t would.be r e q u ir e d f.or 5Q p e r .c e n t i n h i b i t i o n were t o x i c . At 2*5 microg'rams p e r ml. of th e c a rb o x y lic acid an alo g , 50 p er c e n t i n h i b i t i o n was observed w ith a r a t i o of f i v e m ole- • c u le s of 4 -b e n z y lo x y -3 ,5 -d iio d o b e n z o ic acid to one of th e analo g . Again, an e n t i r e l y adequate study of h ig h e r m olar r a t i o s was p re c lu d e d because of t o x i c i t y . A more com plete study of th e e f f e c t of a n ti- th y r o x in e compounds on th e t h y r o x i n e - l i k e a c t i v i t y of t h i s b e n zo ic acid d e r i v a t i v e was p o s s ib le w ith 0 - b e n z y l- 3 ,5 - d ii o d o - D L - ty r o s in e , the d a t a b e in g p re s e n te d in Table V I I I . The d a t a i n d i c a t e an i n h i b i t o r index of about 36 which i s s u b s t a n t i a l l y th e same as th e m olar i n h i b i t o r index observed f o r t h i s compound w ith r e s p e c t t o th y ro x in e ., The c o m p e titiv e n a tu re of t h i s r i v a l r y betw een 0 - b e n z y l - 3 ,5 - d i i o d o — DL— ty r o s in e and 3 , 5 - d i i o d o - 4 — ( 3 ' , 5 l- d i i o d o - 4 , -h ydrox yp h en o x y )-b en zo ic acid i s also su g g e ste d from F ig u re T* The p o te n t t h y r o x i n e - l i k e a c t i v i t y of 3 , 5 - d i i o d o - 4 - ( 4 '- h y drox yph en oxy )-anil.in e, a compound w ith a b a s ic sid e chain group, p e rm itte d f u r t h e r stu d y of th e s p e c i f i c i t y of th e i n h i b i t i o n of t h y r o x in e — I.ike compounds. Though th e t e t r a i o d o cnmposund would have been more d i r e c t l y com parable, th e d iid o d o compound h a s been observed to possess; about one— t h i r d th e ac t i v i t y of DL— th y ro x in e in A m phibia(Frieden and W in z le r,1 9 4 8 a) • TABLE V III COMPETITIVE INHIBITION OF THE THYROXINE-LIKE ACTIVITY OF 3*5— DIIODO-4— ( 3 ' , 5 • -D II0 D 0 -4 ' -HYDROXYPHENOXY) — BENZOIC ACID BY Q— BENZYL-3 ,5-DIIODO-DL— TYROS INE Her c e n t d e c re a se in th e le n g th of ta d p o l e s a f t e r 72 h o u rs in c u b a tio n 3 * 5— Di i od o-4— ( 3 ' * 5 ' -d d i od o - 4 ' -h y d r oxy ph en o x y )-b en zo ic acid m icrogram s p e r ml* 0*0 2*50 5 .0 0 7 .5 0 10.00 o O 29 46 50 52 10.0 O 25 42 49 52 25 .0 O 21 38 42 48 50 .0 O 18 34 40 44 lQOiO O 13 27 33 38 200.0 0 11 17 20 29 Molar i n h i b i t o r index 50 43 32 36 0-B enzy 1 -3 * 5 - d iiodo -D L - t y r o s i n e micrograms p e r ml. 51 FIGURE 7 A. THE EFFECT OF INCREASING CONCENTRATIONS OF G-BENZYL-3,5- DIIODO-DL-TYROSINE ON THE RESPONSE OF TADPOLES TO 3 ,5 -DIIODO- 4— ( 3 ' , 5 *-DIIODO-4•-HYDROXYPHENOXY) -BENZ 0 IC AC ID. B» THE RECIPROCAL (x IOO)v OF THE OBSERVED EFFECT AS MEASURED BY THE PER CENT DECREASE IN THE LENGTH OF TADPOLES PLOTTED AS A FUNCTION OF THE RATIO OF THE MOLAR CONCENTRATIONS OF G-BEN- ZYL-3,5-DIIODO— DL-TYROSINE TO 3 ,5-D IIO D O -4-(3',5'-D IIO D O -4*- HYDROXYPHENOXY) -BENZ0 IC ACID. 60 C J D £ 30 C-J 1 5 U-l d - MO IN H IB IT O R - JS'Vhl.Z- • looyrtL. 2.50 5.00 7.50 H fl^-o)^C 00H .yML *• .• 10.0 4.5 100 E 3 1.5 ^ - D I I O D O - t ^ S ’ D l l O D O - f H Y D R O X Y P H E N O y ^ E N Z O IC 4 C ID * . x- 5.00% l . * ~ 7 . SOV ml. •-10.00%. I N H IB IT O R 3 < o > - c h 2 o ^ ^ch 2 c h cd o h n h 2 15 30 45 60 I N H I B I T 0 R / | t f 0 - j p - o - | s > - C 0 Q H FIGURE 7 A FIGURE 7B 52 T h e re fo re , i t was p o s s ib l e t o t e s t the i n h i b i t i o n of th e ac t i v i t y of t h i s a n ilin e d e r i v a t i v e at m olar r a t i o s q u i te com p a ra b le to th o se used w ith thyroxine*. The d a t a p r e s e n te d in Table IX show t h a t at molar r a t i o s of O -benzyl-N— a c e ty l - 3 ,5 - d ii o d o - L — ty r o s in e t o 3 ,5 - d i io d o — 4 -(4 * -h y droxyphenoxy) - a n ilin e as h ig h as 5QQ, of 3 ,5 - d i i o d o - 4-benzyloxybenzoic acid up t o 7 and of 0 -.b en zy l-3 ,5 -d iio d o -D L — t y r o s i n e as g r e a t as 170-, no i n h i b i t i o n of the th y r o x in e — l i k e a c t i v i t y of th e a n il i n e d e r i v a t i v e was observed,, d e s p i t e ‘th e a b i l i t y of all', th e s e a n ta g o n is t r a t i o s t o reduce th e e f f e c t of DL— th y ro x in e v ery d r a s t i c a l l y . I t i s p a r t i c u l a r i l y s i g n i f i c a n t to n o te t h a t no i n h i b i t i o n was observed f o r a mole r a t i o of 0 -b e n z y l- 3 ,5 - d iio d o - D L - ty r o s in e f u l l y fo u r tim es as g r e a t as t h a t re q u ir e d t o produce a 5Gt p e r c e n t r e d u c tio n of th e e f f e c t of D L -th y ro x in e . Ant agonism t o th y ro x in e usin g th e g o i t e r p re v e n tio n method» ■ P re lim in a ry t e s t s on th e b lo c k in g of th e g o i t e r p re v e n tio n a c tio n of D L -thyroxine in r a t s by p o t e n t i a l th y ro x in e a n ta g o n is ts are summarized in Table X* In ex perim en ts 2 , 3 , 4 ; 6 , 7 , 8 ; and 1 1 ,1 2 ,1 3 , i t was ob served t h a t th e average t h y r o id w eig hts of r a t s g iv en th e i n d i c a t e d d o ses of Q - b e n z y l- N - a c e ty l- 3 ,5 - d iio d o - L - ty r o s in e and D L -thyroxine are g r e a t e r in every case th a n th o se g iven the same dose of th y ro x in e a lo n e . The d i f f e r e n c e s appeared t o be s i g n i f i e s ^ f o r experim en ts 6 , 7, and 8, where th e e f f e c t of A 53 TABLE IX ABSENCE OF AN EFFECT OF THYROXINE ANTAGONISTS ON THE THYROXINE^ LIKE ACTIVITY OF '.3;?5-DI IO D O ^ -(4.'-HYDROXYPHENOXY) -ANILINE A n ta g o n ist E x p t. m icrogram s No* o er ml*. 3 ,5 - D i i o d o - 4 - ( 4 ' hydroxyphen— oxy)— a n il i n e m ic ro sr ams/ml* mm Molar r a tio ® P er c e n t d e c re a s e in l e n g t h 0 IS- Q-B e n z y l- N - a c e ty l- 3 ,5 - d i io d o t y r o s i n e 1.0 o. 55 250 1..0 200: 55 500 1.0 400 55 0 2 .0 0 56 250; 2 .0 1.00 54 500, 2;.0, 200 56 4-Benzy1 oxy-3, 5 - d iio d o b e n z o ic acid 1 .0 O 55 5*0 1.0, 4*5 56 7*5 1 . 0 6*3 54 O 2*0 Q . 56 5.0 2*0 2 .3 54 7.5 2*0 3 .4 52 19 0 - B e n z y l- 3 ,5 - d iio d o — DL— t y r o s in e 1 .0 0 45 10*0. 1 .0 8.6 42 25 .0 1.0- 22; 44 50 *0 1 .0 43 43 100 1 .0 86 45 200 1*0 172 43 0 2 .0 o. 52 10 2 .0 4 .3 45 2.5 2 .0 11 45 50 2*0 22 42 100 2*0 43 . 51 200 2.0- 86 - * ■ 45 ' ; 0 3*0 Q * ' 52 ‘ 10 3*0. 3 54 25 3 *Q 7 .3 45 50 3*0 15 45 100 3*0 29 48 200-. 3.0, 58 48 % o l a r r a t i o of i n h i b i t o r to t h y r o x i n e - l i k e a c tiv e compound* ^ In c u b a tio n tim e in th e s e ta d p o le ex perim en ts was 80 / 10 hours* TABLE X 54 ANTAGONISM TO DL-THYROXINE USING THE GOITER PREVENTION METHOD a Ex- Ser No. p t . ies. of of No. No. R a ts R ats Aver, DL- Weight th y r o x . Ant ag o n i s t Aver age t h y r o id 3 /lO O g . mgs/lOOg. w eight DL-thy P er cen t ro x in e of DL-thy e q u i- ro x in e l b 2 7 233 g . 0 0 9.5 1 .3 ° 0 2, 2: 6 147 0. a 33.0- 2.6 0 0 3 2 6 135 6 .0 19.3 4.1 6 .0 10Q£ 4 2. 5 141 6 .0 A 6 .0 24.4 3 .0 4.0 67 5 2 5 111 0 A 6 .0 35 .0 4.5 «#. - 6 C 12; 173 0 0 28.1 4.8 «»• mm- 7 C 6 196 2.5 0 10.9 1.3 2.5 100 8 c 6 185 2.5 A 2.5 19.4 3 .4 1.3 52 9 c 6 207 2.5 B 1 .0 13.3 3.6 2 .2 88 10 c 5 166 0 B 1 .0 18.4 1.8 1.5 60 11 F 6 215 0 0. 22.5 1.9 m m mm- 12 F 7 219 2.5 0 . 9.0 2.9 2.5 100 13 F 6 182 2.5 A 5 .0 10.9 2 .4 2 .2 88 14 F 6 159 0 A 5 .0 29.1 6 .9 — . _ 15 F 6 223 2 .5 C 0 .1 7 .0 2.1 2.7 108 16 F 5 254 0 C 0 .1 19.8 3.1 0 .5 20 17 E 5 207 0 0 25.5 7 .3 0 0 18 E 12! 200 2.5 0 19.0 3*8 2 .5 100 19 E 6 174. 2.5 D 1.0, 15 .4 4.3 3 .0 120 20 E 6 198 0 D 1 .0 29.6 6 .0 0 .8 32 21 G 6 226 0 0 24.5 3.6 0 0 22. G 5 215 1.25 0 18.6 2.6 - - 23 G 6 214 2.5 0 12. .6 2.3 2.5 100 24 G 6 207 2.5 e i . a 21.1 1.8 0 .5 20 25 i G 6 210 2.5 F Q-.6 16.8 1.7 1.3 52 S e r i e s 2, C, F, and E were fe m a le s; Grw.as comprised of males* aAll. anim als fed Q .3 $ -0 .5 $ 2— t h i o u r a c i l d i e t ex cept as noted* ^Comparison group m aintain ed on normal sto c k d i e t . .Average d e v i a t i o n from th e mean. “ A n ta g o n ists d e sig n a te d as. follows:.- A— Q - B e n z y l- N - a c e ty l- 3 ,5 - d iio d o - L - ty r o s ir ie . B- 3 ,,5 -D iio d o -4 -(3 ' ,5 , -d iio d o -4 '-h y d ro x y p h e n o x y ) -b e n z o ic C- 3 ,5 - D i i o d o - L - t y r o s i n e • a c id . D- Sodium io d id e . E~ 0 -B e n z y l-3 ,5 -d iio d o -D L -ty ro s in e . F— 4-Benzy 1 oxy -3 |5 G d iio d o b en zo ic a c id . 55 D L -thyroxine i s reduced to 52 per cen t of i t s o r i g i n a l a c t i v i t y but t o be of d o u b tfu l s i g n i f ic a n c e f o r the o th e r experim ents* A comparison of experim ents- 2 and 5 and, e x p erim e n ts 11 and 14 su g g e sts t h a t th e a d m in is tr a tio n of t h i s a n ta g o n is t to the t h i o u r a c i l fed r a t , in c re a s e d th e average th y r o id weight of such anim als, but t h i s augm entation i s of d o u b tfu l s i g n i f i c a n c e in view of th e o v e rla p p in g d e v i a t i o n s . In s e r i e s G, p r e lim in a r y evidence f o r a s i g n i f i c a n t b lo ck in g of th y ro x in e a c tio n by 0 -b e n z y l- 3 ,5 - d iio d o - D L - ty r o s in e and 4 -b e n z y lo x y -3 ,5 -d iio d o b e n z o ic acid which reduced th e th y ro x in e e f f e c t by 80 and 48 p e r cen t r e s p e c t i v e l y . No i n h i b i t i o n by the i n d ic a te d doses of sodium io d id e and 3 ,5 - d i io d o - L - ty r o s in e was found in S e r ie s Fand E* P o s s ib le antagonism by th e c a rb o x y lic acid analog i s i n d ic a t e d in s e r i e s C. Though t h i s compound i s a c tiv e when a d m in iste re d alone, when given w ith a p o te n t dose of D L -thyrox ine, i t reduced the e f f e c t of th y ro x in e t o a sm all e x te n t and th e e x p ected a d d itiv e resp o n se to b o th s u b s ta n c e s by an a p p re c ia b le f r a c t i o n * F u rth e r o b s e r v a tio n s on the t h y r o x in e - li k e a c t i v i t y ,of - compounds s t r u c t u r a l l y r e l ated t o th y ro x in e * Some a d d itio n a l o b s e rv a tio n s on th e t h y r o x i n e - l i k e a c t i v i t y of s e v e r a l com pounds r e l a t e d to D L -thyroxine are p r e s e n te d in Table XI* In experim ents 1-12 , th e e f f e c t of halogen s u b s t i t u e n t s in th e 3 ' , 5 ' p o s i t i o n s of 3 , 5 - d i i o d o - 4 - ( 4 , -hydroxyphenoxy)-ben- z o ic acid i n d i c a t e s t h a t th e o rd er of a c t i v i t y i s io d in e g r e a te r th a n bromine g r e a t e r th an c h lo rin e g r e a t e r th an 56 TABLE XI FURTHER OBSERVATIONS ON THE THYROXINES-LIKE ACTIVITY OF COMPOUNDS STRUCTURALLY RELATED TO THYROXINE Expt No. . S e r i e s T est No • Subst ance m icro grams p e r ml. P er cen t d e cre as e ^ in length"* Per c e n t of D L -thyroxine a c t i v i t y 1 1 3,.5-d iio d o -4 — (4.1 - 0-.5 0 2 5 hyd r oxyphen oxy) - benzoic acid 2*5 25 1.0 3 L 3 *, 5 ’ -^ d ich lo ro - m 0 .5 14 2 4 2 in I f tr 1.2,5 29 2 5 2: it in 2.5 51 6 1 3' ,5 '-dibromo; Q-.5 12; 10 7 2 I I tit 1 .0 12 1.0 8 2 it n 2.5 29 10 9 2 u III 3.75 41 12 1 . 0 2 1 1 I I T 5 .0 53 10 1.1 3 3 ,5 - d i iodo-4~hy— 50 16 Q.2 12 4 droxybenzoic acid 250 30 0 .0 5 1.3 5 in 5QQ 39 Q..05 14 5 ii 1250 55 0 .0 5 15 21. 3 , 5-d i i od o - 4 - ( 4' — methoxyphenoxy}- n itro b e n z e n e 5 .0 53 10 * x 'ln c u b a tio n tim e i n th e s e ta d p o le experim ents v a r i e d from 70-110 h o urs at 3 1 10C. 57 hydrogen(lQ -15#, 10#, 2#, 1# th y ro x in e a c t i v i t i e s ) . T h is o r d e r of r e l a t i v e a c t i v i t y was in agreement w ith p re v io u s r e p o r t s by Abderhalden and W ertheim er(1929) and LeBlond and Grad(194B) on th e a c t i v i t y of v a rio u s h alo g en d e r i v a t i v e s in th e th y ro n in e s e r i e s . The observed ord er of a c t i v i t y also c o r r e s p o n d s 't o th e o rd e r of th e e f f e c t on th e acid s t r e n g t h of th e f r e e phenol group by o r th o d ih a lo g e n s . D e f in ite t h y r o x in e - li k e a c t i v i t y has been observed f o r very h ig h c o n c e n tr a tio n s of 3 ,5 - d i iodo-4-hydroxybenZtoic a c id . The a c t i v i t y of t h i s su b stan ce could be due to i t s i n v i t r o ' or _in v iv o co n v ersio n t o th e c a rb o x y lic acid analog of th y ro x in e in a manner s i m i l a r to th e dem onstrated t r a n s fo rm a tio n of 3 ,5 - d i io d o t y r o s i n e to t h y r o x i n e . The t h y r o x i n e - l i k e a c t i v i t y of 3 , 5 - d i i o d o - 4 - ( 4 '- m e t h - oxypheno xy)-n itrob en zene shown in experim ent 15 s u g g e s ts s t i l l f u r t h e r th e la c k of s p e c i f i c i t y of th e sid e ch ain req u ire m e n t f o r t h y r o x i n e - l i k e a c t i v i t y in Amphibia. The e f f e c t of g o itr o g e n s on th e t h y r o x i n e - l i k e a c tio n of 3 ,5 - d iio d o - L - ty r o s i n e in Amphibia. . The d a t a compiled in Table XII i n d i c a t e t h a t th e t h y r o x in e - li k e a c tio n of 3 , 5 - d i - io d o -L - ty r o s in e in Amphibia i s at l e a s t p a r t i a l l y blocked by th e sim u ltan eo u s a d m in is tr a tio n of adequate amounts of 2 - t h id — u r a c i l or 2-rae rca p to im id az o le , two w ell known g o i tr o g e n ic a g e n ts . In ex p erim e n ts 7 -1 1, th e e f f e c t of t h i o u r a c i l in 58 TABLE XII THE EFFECT OF GOITROGENIC AGENTS ON THE THYROXINE— - LIKE ACTION OF 3;5-DIIODO-L-TYROSINE Expt No* * S e r i e s Na. 3 ,5 - D iio d o - L - ty r o s in e micrograms p e r ml. Per Cent t h i o u r aci Per c en t d e c re a s e * 1 in le n g th 1 2 5 .0 0 9% 2 2 1.5.0 0 25 3 2 25.0 0 45 4 2: 50*0 0. 64 5 3 25.0 O 33 6 3 50 .0 0 40 7 3 100 Q . 45 8 3 100 G.QQOl# 42 9 . 3 100 0 *0010 32 10 3 100 0 .010 25 11 3 100 0 .020 13 12 3 5 0 .Q( aged) a 55 13 3. 5Q*0( aged) Q .020, 56 14 19 25.0 0 39 15 19 25 .0 0 .020 19 16 1.9 75 .0 O 55 L7 19 7 5 .0 0 .0 2 0 10- 18 19 1.25 O 60 19 19 125 0 .020, 8 2-mercap,toimid azole 20. 2 a 1.00 O 56 21 20 1.0Q 0.0 0 5 ^ 5 22 20 100. Q.010# 4 ^ In c u b a tio n tim e in th e s e ta d p o le e x p erim e n ts v a rie d from 80.-1.20 hours at 3 1 ^ 1°C. 59 d im in is h in g the e f f e c t of d iio d o t y r o s i n e i s seen to e n la rg e w ith i n c r e a s in g t h i o u r a c i l c o n c e n tr a ti o n . An aged d i io d o t y r o s i n e s o lu tio n ( a llo w e d to sta n d at room te m p e ra tu re f o r 10 months) was, n o t s u s c e p t i b l e t o t h i s e f f e c t of t h i o u r a c i l as i n d ic a t e d in exp erim en ts 12 and 13. This f i n d i n g can be accounted f o r by assuming some in v i t r o c o n v e rsio n of th e d i i o d o t y r o s i n e t o th y ro x in e d u rin g a g in g . I t cannot y e t be a s c e r ta in e d whether or n o t th e th y - r o x i n e - l i k e a c t i v i t y of f r e s h l y p re p a re d 3 , 5 - d iio d o - L - ty r o s in e i s due t o i t s co n v ersio n t o th y ro x in e e i t h e r in th e in c u b a tio n medium or in th e organism . I t i s w ell known t h a t t h i o u r a c i l e x e r t s no e f f e c t on the a b i l i t y of th y ro x in e t o a c c e le r a te amphibian m etam orphosis. While i t i s w ell accepted t h a t t h i o u r a c i l p r e v e n ts th e in c o r p o r a ti o n of in o rg a n ic io d in e i n t o th e o rg an ic io d in e f r a c t i o n of th e th y r o id g la n d , l i t t l e i s known r e g a r d in g th e e f f e c t of t h i s drug on th e c o n v e rsio n of d i io d o t y r o s i n e to th y r o x in e . These o b s e rv a tio n s i n d i c a t e the p o s s i b i l i t y t h a t th e g o itr o g e n s may a f f e c t t h i s r e a c t i o n as w ell as the fo rm a tio n of o rg an ic io d in e compounds. The d e c i s i o n as t o w hether 3 ,5 - d i io d o - L - ty r o s in e a c t s ' in d e p e n d e n tly or by v i r t u e of i t s co n v ersio n to th y ro x in e may be f a c i l i t a t e d by th e use of th e c o m p e titiv e a n ta g o n is ts of th y ro x in e d e s c rib e d p r e v i o u s l y . Molar i n h i b i t o r l e v e l s s i d e n t i c a l w ith th o se ob tain ed w ith pure th y ro x in e should be found i f th e l a t t e r p o s s i b i l i t y i s th e c a s e . 60 CHAPTER VI DISCUSSION OF RESULTS A HYPOTHESIS FOR THYROXINE-LIKE ACTIVITY AND ANTAGONISM IN AMPHIBIA The a v a i la b l e d a t a from e x p erim e n ts in v o lv in g a ll p a i r s of t h y r o x i n e - l i k e a c tiv e compounds and p o t e n t i a l an ta g o n i s t s in Amphibia i s summarized in T able X III* Any h y p o th e s i s to e x p la in th e s e d a t a should account f o r the f in d in g s ite m iz ed below . I t i s apparent t h a t i n s u f f i c i e n t d a t a are a v a ila b le to extend th e s e o b s e r v a tio n s and f u r t h e r d e d u c tio n s t o th e r a t or h ig h e r a n im als. I . Numerous m o d if ic a tio n s of th e sid e c h ain of th y r o x - i n e - l i k e compounds w ith the 3 ,5 - d i i o d o - 4 - < 4 , -hydroxyphenoxy) - p h e n y l- or 3 ,5 - d i io d o - 4 — (3 ' ,5 ' - d iio d o -4 '-h y d ro x y p h e n o x y )— phenyl, n u c le u s r e s u l t e d only in a quant i t a tiv e v a r i a t i o n in t h y r o x i n e - l i k e a c t i v i t y . The r e l a t i v e o rd er of d e c r e a s in g a c t i v i t y of sid e c h ain s u b s t i t u t i o n s i s al anine ^ g ly c in e y ©arboxyl ) N - a c e t y l a l a n i n e . 3 , 5 - D i i o d o - 4 - ( 4 ' -hydroxyphenoxy)- a n il i n e e x h ib ite d a c t i v i t y in th e same range as t h a t of the g ly c in e homolog of th y ro x in e m entioned above. 3 ,5 - D iio d o - 4 — (4-'-m ethoxyphenoxy)-nitrobenzene showed a c t i v i t y comparable to th e c a r b o x y lic acid analog of t h y r o x i n e . 61 TABLE X III n u m m ary of Inhibition of Thyroxine-Like Active Compounds in A m ph ibia Thyroactive compound DL-Thyroxine Hofe-ofcKH2C H C O O H 1 n h 2 Glycine homologue of th y roxine Carboxylic acid analogue of thvroxine if i • H O -^O ^C O O H if • ■ 3.5 - Diiodo - 4 - (4' - h y droxy phenoxy;-aniline ho- ^ o^ > - nh2 Inhibitor* { /' rW Comment A B D E B 1 3 C A B C 37 550 i o 150 650 10 36 400 see see see : Competitive inhibition, Table II, Fig. JT | Competitive inhibition, I Table ; Competitive inhibition, I Table my FI0 .6 : Competitive inhibition fiojbttY! Probably competitive inhibi tion Probably competitive inhibi tion Competitive inhibition, see Table HT VIII f i g T Competitive inhibition toxic ity prevented testing at high er ratios T a b i c VII Competitive inhibition »» M O I N H I B I T I O N A T 1 1 2 u a •I (I It * Inhibitors are designated A. 0-bonz.yl-3.5-diiodo-ni.-tyrosine; B, O-bonzyl-X- a c e t vi-3.5-diiodo- 1.-tyrosine; C , -l-benzyloxy-3.5-diiodobenzoic acid; D, 3.5-diiodo- 4- fp-nit ropheri vlct hoxv <-benzoic acid ; K, 3 .5-diiodo-4-anisic acid. 62 I I . V a r ia t io n of R in r o -j<S>-Y from 4- hydroxyphepyl or 3 ,5 -d iio d o -4 -h y d ro x y p h e n y l to sim ple groups such as b e n z y l, m ethyl, e t c . produced in a l l c a se s a com peti t i v e a n ta g o n is t of t h y r o x i n e - l i k e a c tiv e compounds r e g a r d l e s s of sid e c h ain but only so long as th e sid e c h ain r e t a i n s an a c id ic group.. In th e th re e v a r i a t i o n s of R e t h e r s of 3 , 5 - d i - io d o-4-hyd ro xyb en zoic acid s tu d ie d th u s f a r , th e r e l a t i v e or d e r of d e c r e a s in g i n h i b i t o r y a c t i v i t y i s benzyl ^ p - n i t r o — phenyl'ethyl ^ m eth y l. I I I . No antagonism of a t h y r o a c ti v e compound w ith an amino group s id e chain co uld be achieved at v ery high m olar r a t i o s of i n h i b i t o r s w ith a c id ic sid e c h a in s . IV. The absence of the 3,5 io d in e s from even th e most e f f e c t i v e of th e c o m p e titiv e i n h i b i t o r s le d t o a p p re c ia b le i f n o t complete l o s s of a n ta g o n is t a c t i v i t y . V . S tu d ie s of th e m olar i n h i b i t o r in d ic e s of th e v a r i ous c o m p e titiv e a n ta g o n is ts a g a in s t D L -th y ro x in e , i t s c a rb o x y lic acid analog, and i t s g ly c in e homolog r e v e a le d s u b s t a n t i a l agree ment of the r e s p e c t i v e molar r a t i o of i n h i b i t o r t o t h y r o x i n e - l i k e compound r e q u ir e d f o r 50 p er cent r e d u c t io n in t h y r o x i n e - l i k e e f f e c t f o r a g iv en i n h i b i t o r . I t has been sugg ested t h a t th y ro x in e and a c tiv e r e l a t e d compounds act by v i r t u e of s e r v in g as a coenzyme or a p r o s t h e t i c group in an enzyme or fam ily of enzymes and/o r as a s u b s t r a t e in some im p o rta n t e n zy m e -a ffe c te d r e a c t i o n . 63 I f t h i s be assumed, we may th e n attem pt t o extend our i n t e r p r e t a t i o n of th e above f a c t s and c o n s t r u c t a s p e c u l a ti v e hy p o t h e s i s t o e x p la in th y r o x in e a c tio n in Amphibia, p a r t i c u l a r i l y from th e s ta n d p o in t of i t s e s s e n t i a l m o lecu lar c o n s t i t u t i o n . I t should be emphasized t h a t th e su b seq u e n t d i s c u s s i o n r e p r e s e n t s a d i s t i n c t d e p a r tu r e from th e p re v io u s s t r i c t l y f a c t u a l o b s e r v a tio n s and i s , t h e r e f o r e , s u b je c t t o a ll th e many h a z a rd s of th e c o n j e c t u r a l approach* A* I t may f i r s t be proposed t h a t t h y r o x i n e - l i k e a c tiv e compounds a s s o c ia te w ith an enzyme by means of th e g ro u p in g j - 0 - 3 Q - Y » where Y can be almost any f u n c t i o n a l g ro u p . T h is s u g g e s tio n accoun ts f o r th e la c k of s p e c i f i c i t y of th e s id e c h ain req u ire m e n t f o r b o th t h y r o x i n e - l i k e a c tiv e and a n ta g o n is ti c s u b s ta n c e s as l i s t e d in the o b s e r v a tio n s I , I I , and IV on pages 60 and 6 2 . The r e l a t i v e o rd er of a c t i v i t y of th e a c id ic sid e c h ain s e rie s ; might b e a r some r e l a t i o n to th e r e l a t i v e s t r e n g t h of th e acid group w ith th e e x c e p tio n of N -a c e ty l-D L -th y ro x in e and D -th y ro x in e . As r e p o r te d e a r l i e r by F rie d e n and W inzler(19 48a}, a o e t y la t io n of th e amino group of e i t h e r th y ro x in e or d i io d o t y r o s i n e m arkedly red u c es t h e i r t h y r o x in e - li k e a c t i v i t y in Amphibia. The a c e ty la te d compounds are e i t h e r i n e f f i c i e n t l y used as such or are only p o o rly h y d ro ly zed t o th e f r e e amino a c id s . P erhaps th e s t e r i c h in d ran c e c o n tr ib u te d by th e a c e ty l group p la y s a r o l e in d e c r e a s i n g th e a v a i l a b i l i t y of th e i n t a c t a c e ty l compound. 64 The im portance of th e s t e r i c f a c t o r i s su g g e ste d by th e r e l a t i v e l y poor t h y r o x i n e - l i k e a c t i v i t y of D - t h y r o x i n e ( P i t t R iv e r s and Lerm an,1948)» A p p aren tly , th e th y ro x in e -c o m b in in g enzyme p r o t e i n can accomodate, at l e a s t in an a c tiv e form, a com pound w ith a marked s t r u c t u r a l m o d if ic a tio n of th e sid e c h a in b e t t e r th an th e enantiom orph of th e n a t u r a l l y o c c u r rin g h o r mone* In any c a s e , i t h a s been p o s s ib l e t o modify th e apparent enzyme^-combining a b i l i t y , and th u s th e p o ten cy , of b o th an a c tiv e and an a n t a g o n is ti c compound in th e same q u a n t i t a t i v e manner by N -a c e ty l at ion* Another s t r u c t u r a l p r e - r e q u i s i t e f o r th e p o s tu la te d combining group of th e s e s u b s ta n c e s i s th e p resen ce of th e 3,5 io d in e s or some h alo g en t o s a t i s f y o b s e rv a tio n IV* The g r e a t l o s s of i n h i b i t o r a c t i v i t y of 4 -b e n z y lo x y -3 ,5- d i i o d o - benzoic acid on th e removal of i t s io d in e s can b e s t be i n t e r p r e t e d as a r e d u c t io n in th e enzyme combining a b i l i t y of 4 -b en zyloxy benzoic acid* T h is argument h o ld s fo r th e th y ro x - i n e - l i k e a c tiv e compound, 3 ,5 - d i io d o t h y r o n i n e , which i s ap p r e c ia b ly a c tiv e b o th in Amphibia and Mammalia, w hile th y ro n ine i s com pletely in ac tiv e (G ad d u m ,1928,1929-30)• The only in fo rm a tio n a v a ila b le on th e t h y r o x i n e - l i k e a c t i v i t y of 3 ' , 5 '- d i i o d o th y r o n i n e i n d i c a t e s t h a t i t p o ss e s se d l e s s th an one- f o u r t h th e a c t i v i t y of D L -thy ro xin e(B lock and P o w e ll,1942)• W hether th e 3 ,5 - d ih a lo g e n s are e s s e n t i a l f o r t h e i r g e o m e tric a l in flu e n c e or w hether th e y c o n tr ib u te in an im p o rtan t way to 6 5 th e a c tu a l combining s t r e n g t h of compounds by v i r t u e of t h e i r in d u c tiv e a c tio n cannot be s t a t e d at p re s e n t* The r e l a t i v e im portance of geometry might be more a c c u ra te ly a s s e s s e d by s t u d i e s of a c t i v i t y or antagonism of compounds w ith th e combining group * . HjC B*. The a c tiv e lo c u s of m o lecu les w ith t h y r o x i n e - l i k e a c t i v i t y i s th e hydroxyphenyl e th e r group , • T his f i t s in w ith o b s e r v a t i o n I I in w hich m o d ific a tio n of R of f t o - 3 g > - Y has le d in every case t o a c o m p e titiv e i n h i b i t o r of th y ro x in e * The p re s e n c e of th e 3 ' , 5 ' io d in e s r e s u l t s in c o n s id e r a b le enhancement of th y ro x in e — l i k e a c t i v i t y . Niemann e t a l* (1942) lik e w is e have su g g e ste d a f u n c t i o n a l r e g io n of t h y r o x i n e - l i k e a c tiv e m o lecu les by th e fo rm a tio n of a q u in o id reso n an ce form in v o lv in g th e p h e n o lic hydroxyl group and th e e t h e r oxygen* In a s e r i e s of i n h i b i t o r s of the s t r u c t u r e RO^^-COOH v a r i a t i o n s of R have le d t o th e fo llo w in g o rd er of th e e f f e c t of R on i n h i b i t o r s tr e n g th :, b en zy l ^ p - n i tr o p h e n y le t h y l ^ methyl* I t i s n o t ex p ec te d t h a t th e s e v a r i a t i o n s in R w i l l a f f e c t th e pK of th e carb o x y l group in an a p p re c ia b ly d i f f e r e n t fa s h io n * The only o th e r obvious d i f f e r e n c e in th e p r o p e r t i e s of t h i s s e r i e s of e t h e r s i s th e d i r e c t r e l a t i o n sh ip between r e l a t i v e i n h i b i t o r s t r e n g t h , i*.e*, enzyme com b in in g a b i l i t y , and in c r e a s in g s t e r i c h in d ra n c e about th e e t h e r 6 6 oxygen which i s most c e r t a i n l y i n the o rd er of b en zy l g r e a t e r th a n p - n i t r o p h e n y le t h y l g r e a t e r th a n m eth y l. C* O b s e rv a tio n I I I , page 62, can be e x p la in e d most sim ply by assuming t h a t th e r e are two fu n d a m e n ta lly d i f f e r e n t com bining re g io n s in th e enzyme system or system s in which th y ro x in e a s s o c i a t e s . These two groups may d i f f e r in a t t r a c t i n g combining groups of d i f f e r e n t p o t e n t i a l p o l a r i t y . As i n d ic a te d in th e summary Table X I I I , th r e e t h y r o x i n e - l i k e a c tiv e compounds w ith p red o m in an tly n e g a t iv e l y charged s id e c h ain s at pHs 6*5- 8 .0 were a l l i n h i b i t e d by s e v e r a l c o m p e titiv e a n ta g o n is ts con t a i n i n g n e g a t iv e l y charged sid e c h a in s at a p p a re n tly s i m il a r m olar r a t i o s . But no b lo c k in g of th e t h y r o x i n e - l i k e a c t i v i t y of 3 ,5 - d i io d o - 4 - ( 4 '- h y d r o x y p h e n o x y ) - a n i li n e was observed w ith any of th e s e a n ta g o n is ts at v ery h ig h m olar r a t i o s , e q u a l li n g or e x ceed in g th o se r a t i o s f o r which a 50 p e r c en t r e d u c t io n in t h y r o x i n e - l i k e a c t i v i t y was e x p e c te d . In F ig u re 8, t h y r o x in e l i k e a c tiv e compounds w ith a c id ic s id e c h a in s are p i c t u r e d as b e in g a t t r a c t e d t o enzyme a c c e p to r r e g io n s w ith b a s i c or p o s i t i v e l y charged g ro u p s. I n h b i t o r s w ith a c i d ic combining groups w i l l be a t t r a c t e d to the same lo c u s . A ctive compounds w ith a b a s i c combining group such as th e above mentioned a n i l i n e d e r i v a t i v e are d e p ic te d as b e in g a t t r a c t e d to a c c e p to r groups of a c id ic n a tu re or p o s s i b l y n e g a t iv e l y c h a rg e d . I n h i b i t o r s w ith acid sid e c h a in s w i l l n ot be o r ie n te d to a s i m i l a r re g io n because of t h e i r l ik e c h a rg e s or l i k e n a tu re of groups and FIGURE 8 T E N T A T IV E SCHEME FOR THYROXINE ACTION AND INHIBITION R o f c > C O ( f I A M 1 * ' p f f l , H0E>-0-OtH,CHC00 H O ^ - O - j ^ H O O + THYROXINE ACCEPTING- v A P O - E N I Y f l t SYSTEM R 0 - p W H 3 | ----------> H K ^ O “f E > N j 6 8 th u s w i l l not compete f o r a c id ic or n e g a t iv e l y charged ac c e p to r g ro u p s. Not p o rtra y e d in F igure 8 i s the p o s s i b i l i t y t h a t th e uncharged a n ilin e compound, because of i t s in te n s e hydrophobic c h a r a c t e r , can p e n e tr a te i n to a re a s of c e l l s which are e f f e c t i v e l y b a rr e d t o the h y d r o p h i li c a la n in e , g ly c in e , and c arb o x y l ate s id e c h a in s . T h is l a t t e r argument might a lso be used t o account f o r the unexp ectedly h ig h o rd er of t h y r o x i n e - l i k e a c t i v i t y of th e compound, 3 ,5 - d i i o d o - 4 - ( 4 h y d ro x y p h e n o x y )-a n ilin e , which la c k s th e 3 ',5 * i o d in e s . D* O b serv ation V i s p erhap s -the most d i f f i c u l t f a c t to r e c o n c ile w ith th e fo re g o in g p o s t u l a t e s . Assuming the same Vm as o b tain ed w ith D L -th y ro x in e , c a l c u l a t i o n s of ap p a re n t Km v a lu e s f o r the c a rb o x y lic acid analog of th y ro x in e (la b e le d C) averaged 4.8 x 1 0"®M, some t e n tim e s th e Km fo r D L -th y ro x in e . O r d i n a r i l y , t h i s Kc valu e(K c i s used f o r the Km of th e c a rb o x y lic acid analog to d i s t i n g u i s h i t from the Kffl v a lu e f o r D L -thyroxine) would be i n t e r p r e t e d as meaning t h a t C combines w ith th e proposed t h y r o x in e - a c c e p tin g p r o t e i n only o n e -te n th as w e ll as th y r o x in e , f o r t h i s compound d i f f e r s from th y ro x in e only in th e n a tu r e of i t s combining g ro u p . I f t h i s were the c a se , th e n th e i n h i b i t o r in d ic e s w ith r e s p e c t to C should be only o n e - te n th of th o se observed w ith r e s p e c t to DL— th y ro x iiie , sin c e th e presen ce of a d i f f e r e n t a c tiv e com pound should in no w f a l t e r the Kj v a lu e f o r a p a r t i c u l a r i n h i b i t o r . I n s te a d , e s s e n t i a l l y i d e n t i c a l molar i n h i b i t o r in d ic e s were observed* I t would also be exp ected t h a t 69 Q -b e n z y l-3 ,5 -d iio d o -D L — t y r i s i n e would show t e n tim e s th e i n h i b i t o r a c t i v i t y of 4 -b e n z y lo x y -3 ,5 -d iio d o b e n z o ic a c id . Yet th e observed i n d ic e s were 37 and 8, r e s p e c t i v e l y * Since th e D- form p ro b a b ly has v e ry l i t t l e a c t i v i t y , i f G - b e n z y l - 3 ,5 - d ii o d o - L - ty r o s in e were a v a i l a b l e , i t would have shown an i n h i b i t o r index of about 18*. The th r e e mast l i k e l y s e t s of r e l a t i o n s h i p s t h a t m ight e x i s t between Km an Vra f o r D L -thyroxine and Kc and Vc f o r th e c a r b o x y lic acid analog are as follow s*: Case I f Kc = Kmi Vc r 1 /1 0 Vm Case I I K c = Km; Vc = Vra A s/ Case I I I : ; Kc = 10 Kra;. Vc r 1/10. Vra These c a se s are r e p r e s e n te d g r a p h i c a l l y i n th e two p l o t s of F ig u re 9*. In F ig u re 9A, t h e o r e t i c a l l i n e s f o r th y ro x in e and f o r C acco rd in g t o th e above t h r e e c a se s are p re s e n te d f o r a l / v v e rs u s l / s p l o t . F ig u re 9B c o n s i s t s of id e a l curves f i t t i n g th y ro x in e and th e in d ic a te d c a s e s f o r C* Kra and Kc v a lu e s , th e s u b s t r a t e c o n c e n tr a tio n at o n e -h a lf maximum v e lo c i t y , are a ls o i n d i c a t e d . Case I acco u n ts f o r a r e s p e c t a b l e f r a c t i o n of th e ob s e r v a t i o n s r e p o r te d h e re on th y ro x in e — l i k e a c t i v i t y and th y ro x in e antagonism . I f Kra — Kc , i.~ e*,, C has the same te n d e n cy to combine w ith th e t h y r o x in e - a c c e p t in g p r o t e i n , th e n each such c om b ination w ith C must be only o n e - te n th as a c tiv e as w ith D L -th y ro x in e . T his might be r a t i o n a l i z e d on th e b a s i s G R A P H I C A L R tP R E S E f T A T I O N O f fO S S IB J: R E L A T I O I I S H I P S B E T W E E N D L - T H Y R O X I N B A N D THYROXINE IxJ SUBSTRATE CONCENTRATION of d i f f e r e n c e s in th e s t e r i c r e l a t i o n s of th e f u n c t i o n a l p a r t of th e two m o le c u le s. Also, i t must be assumed t h a t t h e r e e x i s t s a l a r g e e x ce ss of enzyme a cc ep to r groups w ith r e s p e c t t o th y r o x in e . The e q u a l i t y of Km and Kc and th e r e l a t i v e a c t i v i t i e s of th e two compounds means t h a t te n tim e s as many co m bin ation s in v o lv in g th y r il .i c acid must occur b e fo re i t w ill y i e l d a response e q u iv a le n t to th y ro x in e f o r th e same in c u b a tio n p e r i o d . T h e re fo re , t e n tim es as many i n h i b i t o r m o lecu les w i l l be r e q u i r e d t o b lo c k C 's a c t i v i t y . T his would account f o r a molar i n h i b i t o r index i d e n t i c a l w ith D L-thyroxine., However, i n h i b i t o r s w i l l n o t vary in t h e i r a b i l i t y t o produce an i n a c t i v e e n z y m e -in h ib ito r complex, sin c e once such a com bination h as o c cu rred th e r e i s th en no f u n c t i o n a l end of th e m o le cu le . On t h i s b a s i s , i t would be expected t h a t an e th e r of d iio d o t y r o s i n e should have th e same i n h i b i t o r index as th e co rre sp o n d in g e th e r of 3 ,5 - d i io d o b 4 - hydroxybenzoic acid.- The observed in d ic e s are in th e ratio* of 2 £ l, which i s much c lo s e r to 1:1 th a n th e r a t i o of 1:10 as ex pected from Cases I I and I I I * Case I f a i l s t o e x p la in th e f a c t t h a t v i r t u a l l y i d e n t i c a l Vms were c a l c u l a t e d f o r DL- th y ro x in e and C. Case I I , w hile not accounting f o r s e v e ra l of th e form er f a c t s , does p r e d i c t th e observed Vc * Curve I I , F ig u re 9A, f i t s th e d a t a o b tain ed w ith C.- Case I I I appears t o be the l e a s t l i k e l y of a l l . The app arent i d e n t i t y between th e o bserved Vm and Vc might a r i s e from i n a b i l i t y t o o b ta in re s p o n s e s g r e a t e r th a n Vc , in d ic a t e d by th e l i n e L in F ig u re 9B, b ecause of o th e r l i m i t i n g f a c t o r s . Thus th e c a l c u l a t e d Vm f o r D L -th yro xine would be only an apparent Vra» . T his p o s s i b i l i t y s tr e n g th e n s Case I at th e expense of Case I I . However, no> f i n a l d e c i s i o n as t o which Case h o ld s can be made on th e b a s i s of th e a v a i l able e v id e n c e . I t i s c l e a r from th e fo r e g o in g c o n s i d e r a t i o n s and th e summarized d a t a of T able X I I I t h a t th e p o s s i b i l i t y of th e v a r i o u s t h y r o x i n e - l i k e a c tiv e compounds e x e r t in g t h e i r a c t i v i t y by t h e i r c o n v e rsio n t o th y ro x in e i s v i r t u a l l y p r e c lu d e d , e x c e p tin g , of c o u rs e , d i io d o t y r o s i n e or o th e r s i n g l e r i n g compounds. I f such compounds were tran sfo rm e d t o th y r o x in e , m olar r a t i o s n e c e s s a r y t o reduce th e t h y r o x i n e - l i k e e f f e c t would be lower in th e case of th e l e s s a c tiv e compounds. T h is i s in com plete d isa g re e m e n t w ith th e d a t a r e p o r te d in T able X III* In f a c t no i n h i b i t i o n of 3 ,5 - d i i o d o - 4 — ( 4 ' -hy&fcoxy- p h e n o x y )- a n ilin e could be achieved at v e ry h igh m olar r a t i o s . T h is h y p o th e s is f o r t h y r o x i n e - l i k e a c t i v i t y and t h y ro x in e antagonism in Amphibia r e a d i l y le n d s i t s e l f to t e s t i n g as in d ic a t e d in s e v e r a l of th e many p o s s i b l e e x p e rim e n ts which are o u tlin e d below: ty pe 1.* I t should be p o s s ib le t o p re p a re i n h i b i t o r s of th e w hich should c o m p e titiv e ly i n h i b i t the 73 t h y r o x i n e - l i k e a c t i v i t y of compounds w ith b a s ic sid e c h a in s , b ut t h i s type of i n h i b i t o r would not be ex pected t o a f f e c t compounds w ith a c id ic s id e c h a in s . A w ater s o l u b i l i z i n g group might have t o be in c lu d e d in R, or th e s id e c h a in might be ex tended t o th e more h y d r o p h ilic e th y l amine group*. 2*. O b s e rv a tio n s of th e e f f e c t of sim u lta n e o u s admin i s t r a t i o n of D L -thyroxine and th e c a rb o x y lic a cid analog in ejquimolar q u a n t i t i e s should produce approxim ately 40-50 per cen t r e d u c tio n in th e t o t a l t h y r o x i n e - l i k e e f f e c t , sin c e th e analog w i l l compete w ith th y ro x in e t o form l e s s a c tiv e e n z y m e -su b stra te co m b in atio n s. No such e f f e c t shou ld be ob se rv ed upon the sim u lta n e o u s g iv in g of th y ro x in e and a b a s ic sid e ch ain compound such as 3 ,5 - d iio d o - 4 - ( 4 '- h y d r o x y p h e n o x y ) - a n il i n e ,, sin c e i t i s b e lie v e d t h a t th e y combine w ith d i f f e r e n t s i t e s on th e enzyme system s. In f a c t , in th e l a t t e r case th e e f f e c t of two such compounds should be ad ditive*. Obviously many p a i r s of a c tiv e compounds could be s tu d ie d in t h i s way. 3* A compound such as N - a c e ty lth y r o x in e should be in h i b it e d by th e same a b so lu te amounts of i n h i b i t o r as i s th y ro x in e or about 1/35 th e i n h i b i t o r index observed w ith t h y r o x in e . 4*. The e f f e c t of s t e r i c h in d ra n c e about th e e th e r oxy gen on i n h i b i t o r s t r e n g t h can be f u r t h e r s tu d ie d by th e p re p a r a t i o n and a c t i v i t y s t u d i e s of th e t - b u t y l , i - p r o p y l, or o th e r h in d ere d e t h e r s of 3 , 5 - d i io d o -4 -h y d roxybenzoic a c id , 3 , 5 - d i i o d o - D L -ty ro s in e , e t c . CHAPTER VII SUM M ARY S e v e ra l d e r i v a t i v e s of 3 , 5 - d i i o d o t y r o s i n e and of 3 ,5 - d i~ iod o -4 -h y d ro x y b en zo ic acid have been p re p a re d and observed to i n h i b i t c o m p e titiv e ly th e e f f e c t s of D L -thyroxine on amphibian m etam orphosis. In p a r t i c u l a r , th e r e s p e c t i v e benzyl e t h e r s of 3 .5 -d iio d o -4 -h y d ro x y b e n z o ic acid and of 3 , 5 - d iio d o - D L - ty r o s in e were found t o reduce th e e f f e c t of D L -thyroxine by o n e -h a lf when th e m olar r a t i o of a n ta g o n is t to th y ro x in e was 8 and 37 r e s p e c t i v e l y . The s p e c i f i c i t y of t h i s th y ro x in e i n h i b i t i o n was d e m o n stra te d by th e i n a b i l i t y of many o th e r s u b s ta n c e s to produce any i n h i b i t i o n of D L -thy ro xine at much h ig h e r molar r at i o s . The v a r io u s b en zy l e th e r s a ls o proved to be c o m p e titiv e i n h i b i t o r s of th e t h y r o x i n e - l i k e a c tiv e compounds, th e g ly c in e homolog of th y ro x in e and S j S - d i i o d o ^ - C S 1,5 ' - d i i o d o - 4 ’-h y d ro x y - ph en ox y )-b en zo ic a c id . T h is i n h i b i t i o n was achieved at molar r a t i o s s i m i l a r t o th o se o b ta in e d w ith D L -th y ro x in e . However, no i n h i b i t i o n was observed f o r th e t h y r o x i n e - l i k e a c t i v i t y of 3 . 5 - d iio d o - 4 - ( 4 '- h y d r o x y p h e n o x y ) - a n ilin e at very h ig h molar r a t i o s . The f a c t t h a t c o n c e n tr a tio n s of i n h i b i t o r s which r e duced th e e f f e c t of th y r o x in e a p p re c ia b ly d id not g r e a t l y a l t e r th e a c t i v i t y of th e s e t h y r o x i n e - l i k e compounds i n d i c a t e d t h a t th ey were n o t c o n v e rte d t o th y ro x in e t o e x e r t a c t i v i t y . 75 P re lim in a ry evidence of antagonism of th e e f f e c t of th y ro x in e by s e v e r a l of th e s e i n h i b i t o r s has been o b ta in e d using th e g o i t e r p re v e n tio n method in r a t s . Some new exam ples of t h y r o x i n e - l i k e a c tiv e compounds were g iv en and th e a c tio n of g o i tr o g e n ic agents in re d u c in g th e t h y r o x i n e - l i k e a c t i v i t y of 3 ,5 - d i i o d o - L - t y r o s i n e in Amphibia was noted* These and p re v io u s f i n d i n g s were i n t e g r a t e d i n to a s p e c u la tiv e h y p o th e s is f o r t h y r o x i n e - l i k e a c t i v i t y and th y ro x in e antagonism in Amphibia. Assuming t h a t t h y r o x i n e - l i k e a c tiv e compounds act by v i r t u e of s e r v in g as a coenzyme, p r o s t h e t i c grou p, or. key s u b s t r a t e f o r some enzyme system or s y s tem s, i t h as been su g g ested t h a t th e amino acid sid e chain of th y ro x in e s e rv e s as anenzyme group w ith the 3 ,5 io d in e s i n volved in some as y e t unknown way. The d iio d o h y d ro x y p h en y l e th e r group was proposed as th e a c tiv e or f u n c tio n a l lo c u s of th y r o x i n e - l i k e a c tiv e m o le c u le s. The f a i l u r e of a c id ic sid e chain i n h i b i t o r s to antagonize th e a c t i v i t y of a b a s ic sid e ch ain activ e compound was i n t e r p r e t e d as i n d i c a t i n g the p re se n c e of at l e a s t two d i f f e r e n t combining s i t e s on th e t h y r o x i n e - a c c e p t ing enzyme system* Many o th e r a s p e c ts of t h i s h y p o th e s is have been c o n sid e re d and s e v e ra l f u t u r e t e s t system s have been o u t l i n e . BIBLIOGRAPHY A bderhalden, E ., and E* W ertheim er, Z t s c h r . f u r d . G es. E x p er. Med., 6_8, 563, 1929. B lo ck , J r . , P . , and G. P ow ell, J . Am. Chem. S o c ., 6 4 , 10,70, 1942. Boyer, P . D ., Evfcans, R. J . , and P. H*. P h i l l i p s , J . Pharm. E x p t l • T herap. 73, 176-183, 1941. Dempsey, E. W., and E. B. Astwood, E n d o crin o lo g y , 37, 509, 1943. E b e rs o le , E. R ., G u tte n ta g , C ., and P . W. W ilson, Arch. B iochem ., 3, 399-418, 1944. Edwards, S . A*, and G. R . S h a r p le s s , P a p e rs Mich. Acad. S c i . Arts, and L e t t e r s , 30,, 6 5 3 -9 , 1944. F rie d e n , E ., M a s t e r 's T h e s is , Univ. of South ern C a l . , Aug., 1947. F r ie d e n , E*, and R. J . W inzler, J . B i o l . Chem., 176, 155, 1948a. F rie d e n , E ., and R. J . W inaler, J . Am. Chem. S o c ., _70, 3511, 1948b. F rie d e n , E . , and R . J . W in zler, E n d o crin o lo g y , _43, 40-47, 1948b. Gaddum, J . H ., J * P h y s i o l . , 64 , 246 -254, 1928. Gaddura, J . H ., J * P h y s i o l . , 6 8 , 383-405, 1929-30. Green, D. E*, Advances in Enzvmology, V o l. I , p . 191, 1941. G udernatsch, J . F . , Arch. E n t. Organ. 35, 457-483, 1912. H a rin g to n , C. R ., and G. B a rg er, Biochem . J . , 21, 169-183, 1927. H a rin g to n , C.. R . 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Creator Frieden, Earl (author)

Core Title Competitive antagonists of thyroxine and structurally related compounds

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

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Advisor Winzler, Richard J. (committee chair), Deuel, H.J. (committee member), Kharasch, Norman (committee member), Kloetzel, M.C. (committee member), Marx, Walter (committee member), Visser, Donald (committee member)

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