COMPETITIVE ANTAGONISTS OF THYROXINE AND STRUCTURALLY RELATED COMPOUNDS*

BY EARL FRIEDENt AND RICHARD J. WINZLER

(From the Department of Biochemistry and Nutrition, University of Southern California School of Medicine, Los Angeles)

(Received for publication, October 6, 1943)

Woolley (1) has prepared a number of ethers of N-acetyl-3,5-diiodo- tyrosine and found them to interfere with various effects of thyroxine on amphibian larvae. Some of Woolley’s observations were confirmed and extended by Winder and Frieden (2) and by Williams et al. (3).

It is the purpose of this report to investigate the competitive nature of several thyroxine inhibitors and to determine the structural requirements for and the specificity of the inhibition of thyroxine and its active analogues,

Materials

0-Benzyl-3,6-diiodo-DL-tyrosine (I)-10 gm. of 0-benzyl-N-acetyl-3,5- diiodo-L-tyrosine, m.p. 87-90’: prepared as described by Woolley (l), were refluxed overnight in 1 liter of 1 N NaOH. The solid which precipi- tated at pH 5 was extracted with hot 0.2 N HCl, which when cooled yielded the crystalline hydrochloride of I. Free I, m.p. 203-205” with decom- position, was obtained in 30 per cent yield by dissolving its hydrochlo- ride in dilute alkali and adjusting the pH to 5. The product gave a posi- tive ninhydrin, but a negative Kendall, test for orthodiiodophenols. The above recrystallization process was repeated on a sample used for ele- mentary analysis.

CIJIBO~NII. Calculated, C 36.7, H 2.9; found, C 36.9, H 3.0

4-Benzyloxy-3,6-diiodobenzoic Acid (II)-3,5-Diiodo-4-hydroxybenzoic acid, m.p. 275277”, was prepared in 50 per cent of the theoretical yield from 4-hydroxybenzoic acid (Eastman Kodak, m.p. 214.5-215.5”) (4). 10 gm. of the iodinated phenol were dissolved in 60 ml. of 1 N NaOH, heated on a boiling water bath, and treated dropwise with 11.9 gm. of

* We wish to express our appreciation to Eli Lilly and Company and to the Com- mittee on Research in Endocrinology of the National Research Council for their support of this work, as well as to the Hancock Foundation for the facilities that were made available. Contribution No. 190 from the Department of Biochemistry and Nutrition, University of Southern California.

t Some of the data were taken from a thesis presented by Earl Frieden to the Grad- uate School of the University of Southern California in partial fulfilment for the re- quirements for the degree of Doctor of Philosophy.

* All melting points are corrected. 423

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424 ANTAGONISTS OF THYROXINE

benzyl chloride. A heavy white precipitate of the sodium salt of II was formed on complete cooling, after the removal of a tarry by-product on preliminary cooling. II was generated by acidifying a suspension of the isolated salt. Two recrystallizations from a water-methanol mixture re- sulted in a 20 per cent yield2 of II, m.p. 227-228”, which gave a negative Kendall test.

CMHK,O&. Calculated, C 35.0, I 52.9; found, C 35.2, I 52.9

3,5-Diiodo+(p-nitrophenylethoxy)-benzoic Acid-10 gm. of 3,5-diiodo- 4-hydroxybenzoic acid were dissolved in 85 ml. of 0.7 N methanolic NaOH and treated with 6.1 gm. of p-nitrophenylethyl bromide, m.p. 64”, pre- pared as described by Woolley (1). The mixture was refluxed for 2 hours and concentrated to dryness under a vacuum, and the residue was thor- oughly washed with 200 ml. each of warm water and ethyl ether. The washed residue was suspended in 100 ml. of water, the pH adjusted to 2.5 f 0.5 with HCl, and the mixture heated for 2 hours. The resulting washed solid was twice recrystallized from an ethanol-water mixture, giv- ing a 20 per cent yield of the ether, m.p. 219-220”, which gave a nega- tive Kendall test.

G&IO&L. Calculated, C 33.4, N 2.6; found, C 33.4, N 2.5

3,5-Diiodo-4-(Q’-methoxyphenoxy)-aniline (III)-The preparation of III from 3,5-diiodo-4-(4’-methoxyphenoxy)-nitrobenzene was accomplished by adapting a procedure of Block and Powell (5) instead of the earlier method of Harington and Barger (6). 10 gm. of the nitro compound were sus- pended in 200 ml. of a 50 per cent ethanol solution containing 10 ml. of glacial acetic acid. 5 gm. each of 30 mesh iron filings and iron powder were added, and the mixture refluxed for 3 hours. The alcohol was re- moved by distillation, and the residue was cooled and repeatedly extracted with a total of 300 ml. of boiling benzene. Concentration of the benzene solution to about 150 ml. and the addition of 80 ml. of petroleum ether gave, with scratching, 75 to 80 per cent yield of III, m.p. 121-122’.

3,5-Diiodo-d-(4’-hydroxyphenoxy)-aniline (IV)--5 gm. of III were re- fluxed for 2 hours in 40 ml. of glacial acetic acid and 50 ml. of 42 per cent HBr. After diluting with 1 volume of water and cooling overnight, a brownish impurity was removed by filtration. Further dilution of the filtrate with 1 additional volume of water gave a 60 per cent yield of the

z A 60 per cent yield of II has been obtained subsequently by treating the di- potassium salt of 3,5-diiodo-4-hydroxybenzoic acid with 6 equivalents of benzyl chloride in hot 50 per cent ethanol and hydrolyzing the resulting benzyl ester of II with dilute NaOH.

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E. FRIEDEN AND R. J. WINZLER 425

hydrobromide of IV. Pure IV, m.p. 221.5-223.5’, was obtained by crys- tallization of its neutralized salt from 95 per cent ethanol.

C~zHeozNIz. Calculated, C 31.8, N 3.1; found, C 32.0, N 3.0

3,5-Diiodo-4-anisic acid was prepared as described by Wheeler and Liddle (7), m.p. 257-258”, compared to the reported melting point of 255-256”. 4-Benzyloxybenzoic acid was synthesized by essentially the same procedure described for the corresponding 3,5-diiodo derivative. A 30 per cent yield of crude product melting several degrees below the re- ported value of 188-190” was obtained.

N-Acetyl-m-thyroxine, 3,5-diiodo-4-(4’-hydroxyphenoxy)-benzoic acid, 3,5-diiodo-4-(3’) 5’-diiodo-4’-hydroxyphenoxy)-benzoic acid, N-acetyl-3,5- diiodo-n-tyrosine, and the glycine homologue of thyroxine were obtained as described in a previous report (8). 2-Thiouracil and 2-mercaptoimida- zole were kindly contributed by Eli Lilly and Company, Indianapolis, Indiana. 3,5-Difluoro-4-methoxyphenol and 3,5-dichloro-4-anisic acid were obtained through the generosity of Dr. J. F. Mead, Atomic Energy Project, University of California at Los Angeles. r&-Thyroxine was pro- vided by Dr. K. W. Thompson of Roche-Organon, Inc., Nutley 10, New Jersey. All the other compounds tested were the best available grade obtainable from the Eastman Kodak Company.

Methods

The effect of thyroxine and compounds possessing thyroxine-like activity on amphibian metamorphosis afforded a convenient system for the study of compounds for thyroxine antagonism. The technique employed in these studies was similar to that described in previous work on the thy- roxine-like activity of compounds structurally related to thyroxine (8). Duplicate dishes containing a solution of the active compound and the inhibitor were brought to a final pH of 8.0 f 0.5 prior to introducing the tadpoles. In so far as possible, tadpoles of the same history, of the same relative nutritional state, and of the same length or pooled groups of length varying no more than 2 mm. were employed.

Results

Competitive Inhibition of Thyroxine--The results shown in Tables I to III summarize several of many experiments and indicate a competitive antagonism between thyroxine and a number of related compounds.

In Table I the action of thyroxine in inducing the metamorphosis of tad- poles is observed to be inhibited by 0-benzyl-N-acetyl-3,5-diiodo-n-tyro- sine, a compound reported by Woolley (1) to antagonize the toxicity of

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426 ANTAQONISTS OF THYROXINE

thyroxine to tadpoles. The thyroxine effect is reduced by 50 per cent when the molar ratio of inhibitor to thyroxine is about 550, regardless of their absolute concentrations.

Table II shows that 0-benzyl-3,5-diiodo-nn-tyrosine is some 5 times more effective a thyroxine antagonist than its N-acetyl derivative, the molar ratio of inhibitor to thyroxine necessary to reduce the thyroxine

TABLE I Competitive Inhibition of Thyroxine by 0-Benzyl-N-acetyl-b,b-diiodo-L-tyrosine

O-Benzyl-N-acetyl-3, Sdiiodo& tyrosine

y per mc. 0

62.5 125 250 375 500 750

(Z/T)600/o weight7 . . . . . . . . . (Z/T)60% molar1 . . . . . . . . . .

- Per cent decrease in length after 72 hrs. incubation

26 19 13 11 6

500 688

m-Thyroxine per ml.*

0.50 y --

47 45 37 16 13 12 8

400 550

0.75 y 1.00 7

~__ 1.25 y

--

60 60 60 55 56 55 45 47 50 25 35 43 20 30 35 19 25 29 17 17 25

300 375 400 412 515 5508

* No detectable difference in length from untreated controls was observed during the reported incubation period at all the indicated concentrations of theinhibitor in the absence of thyroxine.

t Ratio by weight of inhibitor to thyroxine required for reduction of thyroxine effect by 50 per cent.

1 Molar ratio of inhibitor to thyroxine required for reduction of thyroxine effect by 50 per cent.

Q This value of (I/T) at highest inhibitor concentration is used in subsequent dis- cussions because it is least affected by unsaturation of the system with respect to inhibitor or thyroxine.

effect by one-half being about 37. This is in accord with previous obser- vations (8, 9) that N acetylation reduced thyroxine-like activity.

The observation of gradually decreasing ratios with increasing thyroxine concentrations shown in Table II is in agreement with calculations made from the competitive inhibition equation of Lineweaver and Burk (10) for the relatively non-saturating ranges of concentrations of inhibitor and thyroxine used in the experiments reported in this paper. Further verifi- cation of the competitive nature of this type of antagonism appears in Fig. 1, A and B. A direct plot of per cent decrease in length against thy- roxine concentration gives with increasing inhibitor a series of curves with a progressively decreasing effect at the same level of thyroxine (Fig. 1, A).

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E. FRZEDEN AND E. J. WINZLER

TABLE II

Competitive Inhibition of Thyroxine by 0-Benzyl-S,kliiodo-DL-tyrosine

Per cent decrease in length after 63 brs. incubation ___--.----.---

0.25 y

7Wd.

0 28

1.25 28 2.56 24 5.09 20

12.5 15 25.0 10 50.0 7

125 4 250 3

---- ---

46* 44* 44* 40 25 16 9 6 3

___- _j-

51* 60* 48* 56’ 46* 56* 44* j 50: 42* 44* 28 34 15 28 13 24

-91 l2 (1/2’)60% weight.. . . . . . . . 50 1 30 1 30 (I/T)BvO molar.. . . . . . . . . . . 74 I45 I45 ; :

See corresponding foot-notes to Table I. * Indicates 89 to 100 per cent died several hours prior to observation time. The

protective action of this inhibitor against higher concentrations of thyroxine is thus clearly established.

TABLE III Competitive Inhibition of Thyroxine by .J-Benzyloxy-d,B-diiodobenzoic Acid

i Per cent decrease in length after 69 hrs. incubation

- 4BeDzyloxy-3, S-diiodobenzoic acid 1 Dx.-Thyroxine per ml.

~~__ 0.15 7 0.30 7 / 0.50 7

.-- 7Wd.

0 19 30 45 0.50 17 23 35 1.00 8 19 30 2.50 4 8 20 5.09 2 4 18 7.50 2 4 10

10.0 * * * .--____-.

(I/T)ro% weight . . . . . . . . . . 6 5 4 (I/T)s~Omolar............ 9.5 8 6.5

See the corresponding foot-notes to Table I. * Indicates toxicity of inhibitor dose.

0.90 7

48

40

32 25 20 12 10

3 5

--- 1.80 7

55

46

43

40

37 33 25

5 8

A graph of 100/E against the reciprocal of the molar concentration of m-thyroxine as shown in Fig. 1, B, where E is the per cent decrease in

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428 ANTAGONISTS OF THYROXINE

length at constant time, yields a family of lines with a common intercept and slope increasing with increasing amounts of inhibitor, characteristic of competitive inhibition (10). Non-competitive inhibition would give lines of variable intercept as well as variable slope.

The recent observation (8) that the carboxylic acid analogue3 of thyroxine has a thyroxine-like activity of about 10 per cent of that of thyroxine suggested the investigation of ethers of 3,5-diiodo-4-hydroxybenzoic acid. The benzyl ether proved to be the most potent competitive inhibitor of all, the data in Table III indicating that molar ratios of inhibitor to thyroxine

00

THYROXINE yflL.

e I6

10*x1 / THYROXINE

FIG. 1, A FIG. 1, B FIG. 1, A. The effect of increasing concentrations of 0-benzyl-3,5-diiodo-nn-

tyrosine on the response of tadpoles to nn-thyroxine concentration. FIG. 1, B. The reciprocal of the observed effect as measured by the per cent de-

crease in the length of tadpoles plotted as a function of the reciprocal of the molar concentration of nn-thyroxine for various levels of 0-benzyl-3,5-diiodo-nn-tyrosine.

of 8 produced a 50 per cent reduction in thyroxine effect. The toxicity of this compound prevented its use at concentrations above those listed. The methyl and p-nitrophenylethyl ethers of 3,5-diiodo-4-hydroxybenzoic acid required molar ratios of 150 and 75, respectively, to produce the same antagonism. The importance of the 3 ,biodines was indicated by the failure of 4-anisic acid and 4-benzyloxybenzoic acid to inhibit appreciably at molar ratios of 500 and 350, respectively.

Inhibition of Compounds with Thyroxine-Like Activity-The inhibition by thyroxine antagonists of the thyroxine-like activity of compounds

* This designation has been preferred to the “benzoic acid analogue” nomenclature used to denote the same compound in a previous publication (8).

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E. FRIEDEN AND R. J. WINZLER 429

with altered acid side chains was also observed. The action of the car- boxylic acid analogue of thyroxine, 3,5-diiodo-4-(3’) 5’-diiodo-4’-hydroxy- phenoxy)-benzoic acid, was competitively reduced by thyroxine an- tagonists at molar ratios similar to those for thyroxine. This is illustrated in Table IV which shows that the antagonism by 0-benzyl-3,5-diiodo-nn- tyrosine of the thyroxine-like activity of the carboxylic acid analogue re- quires a molar ratio of 36 to diminish the thyroxine effect by 50 per cent. Ratios of 5 and more than 400, respectively, were required for a similar effect on the activity of this compound by 4-benzyloxy-3,5-diiodobenzoic acid and 0-benzyl-N-acetyl-3,5-diiodo-n-tyrosine.

TABLE IV Competitive Inhibition of Thyroxine-Like Activity of S,b-DiiodoJ-(3’,6’-diiodo-4’-

hydroxyphenoxy)-benzoic Acid by 0-Benzyl-S,b-diiodo-Dr.-tyrosine

i

O-Ben&3, S-diiodo-m-tyrosine 3, S-Diiodo-4-(3’, S’-diiodo-4’-hydroxyphenoxy)-benzoic acid per ml.

y per ml.

0 10.0 25.0 50.0

100.0 200.0

(I/T)so% weight .......... (I/T)60y0 molar ...........

Per cent decrease in length after 72 hrs. incubation

:/

T

0-Y

0 0 0 0 0 0

--

2.5 y

29 25 21 18 13 11

-- 35 50

5.0 7

46 42 38 34 27 17

30 43

7.3r 1 lO.Oy

50 52 49 52 42 48 40 44 33 38 20 29

__- 22 25 32 36

-__ See the corresponding foot-notes to Table I.

No inhibition of the thyroxine-like activity of 3,5-diiodo-4-(4’-hydroxy- phenoxy)-aniline was observed with any of the above thyroxine antago- nists even at molar ratios greater than those which reduced the activity of thyroxine to less than 50 per cent as indicated in sample data reported in Table V.

Specijicity of Thyroxine Inhibition--The specificity of the inhibition of thyroxine by the type of competitive inhibitors described in previous para- graphs was tested by administering simultaneously with m-thyroxine a number of compounds, including structurally related, goitrogenic, and polyfunctional substances. The thyroxine was used at a concentration of 0.50 y per ml. which, when given alone, resulted in a decrease in length of 45 f 5 per cent in 3 days incubation at 31” f lo. A compound was con- cIuded to be without inhibitory effect when the thyroxine response was

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430 ANTAGONISTS OF THYROXINE

not altered by more than a 10 per cent decrease in length. The maximum concentration of potential inhibitor was determined by its toxicity, by its water solubility at pH 8.0 f 0.5, and finally by a level just below activity in case the compound itself exerted thyroxine-like activity (e.g., N-acetyl thyroxine, etc.). A high specificity for thyroxine antagonism was indi- cated by the fact that none of the following compounds inhibited the effect of thyroxine when tested at the maximum possible molecular ratios listed:

Absence of Effect of Thgrozine Inhibitors on Thyroxine-Like Activity of _ - S,6:Diiodo-~-(4’-hydrozyphenozy)-aniline

Exe?

12

19

Inhibitor

0 - Benzyl - iV - acetyl- 3,5 - diiodotyrosine

4 - Benzyloxy - 3,5 - di- iodobenzoic acid

0 - Benzyl - 3,5 - di- iodotyrosine

y jer ml. -rWd.

0 1.0 250 1.0 500 1.0

0 1.0 5.0 1.0 7.5 1.0 0 1.0

10 1.0 25 1.0 50 1.0

100 1.0 200 1.0

0 2.0 10 2.0 25 2.0 50 2.0

100 2.0 200 2.0

,5-Diiodo-4-(4’ hydroxyphen- my)-aniline

0 200 400

0 4.5 6.8 0 8.6

22 43 86

172 0 4.3

11 22 43 86

55 55 55 55 56 54 45 42 45 45 45 45 50 45 45 42 52 45

~~ * Molar ratio of inhibitor to thyroxine-like, active compound. t The incubation time in these experiments was 80 i 5 hours.

N-acetyl-m-thyroxine, 10; 3,5-diiodo-4-(4’-hydroxyphenoxy)-benzoic acid, 15; 3,5-diiodo-4-hydroxybenzoic acid, 1000; 3,5-difluoro-4-methoxyphenol, 440; 3,5-dichloro-4-anisic acid, 350; 2-amino-3,5-diiodobenzoic acid, 100; L-tyrosine, 2000; N-acetyl-3,5-diiodo-L-tyrosine, 1600; 4-fluorobenzoic acid, 5000; 4-anisic acid, 500; 3-hydroxybenzoic acid, 280; salicylic acid, 1700; 4-aminobenzoic acid, 2830; 4-iodobenzoic acid, 1560; 4-anisidine, 3150; sul- fanilic acid, 1350; sulfanilamide, 2250; benzoic acid, 6300; benzyl alcohol, 1800; 2-thiouracil, 2400; 2-mercaptoimidazole, 1550; ascorbic acid, 1770; sodium iodide, 5000; malonic acid, 750; acetonitrile, 350.

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E. FRIEDEN AND It. J. WINZLER 431

DISCUSSION

Table VI summarizes the experiments involving all pairs of thyroid- active and antagonistic compounds. Considerable modification of the structure of thyroxine has resulted in the production of potent competitive antagonists.

TABLE VI Summary of Inhibition of Thyroxine-Like Active Compounds in Amphibia

Thyroxine-like compound

nn-Thyroxine

‘I

“

“

Glycine homologue of thy- roxine

‘I “

I‘

Carboxylic acid analogue of thyroxine

‘I “

“ ‘I

3,5 - Diiodo - 4 - (4’ - hy- droxyphenoxy)-aniline

“

A 37 Competitive inhibition, see Table II, Fig. 1

B 550 Competitive inhibition, see Table I

C 8 Competitive inhibition, see Table III

D 75 Competitive inhibition E 150 “ I‘

B 656 Probably competitive inhibi- tion

C 10 Probably competitive inhibi- tion

A 36 Competitive inhibition, see Table IV

B 400 Competitive inhibition, toxic- ity of B prevented testing at higher ratios

C 5 Competitive inhibition A No inhibition at 172, see Table V

B “ “ “ 466 (1 6‘ “ “ c I‘ “ I‘ , ‘( “ “

* Inhibitors are designated A, 0-benzyl-3,5-diiodo-nn-tyrosine; B, O-benzyl-N- acetyl-3,5-diiodo-L-tyrosine; C, 4-benzyloxy-3,5-diiodobenzoic acid; D, 3,5-diiodo- 4-(p-nitrophenylethoxy)-benzoic acid; E, 3,5-diiodo-4-anisic acid.

i The value of (Z/T) at highest inhibitor concentration is used in this column, since this value is least affected by unsaturation of the system with respect to inhibi- tor or thyroxine.

Inhibitor* U/T),o% t Comment

A most interesting finding is that these antagonists appear to have the same lack of specificity with regard to the side chain that has been pre- viously described for thyroxine-like activity (8). Modification of the other end of the molecule, however, has very pronounced effects on both thyrox- ine-like activity and antagonism. If it is assumed that thyroxine acts as a coenzyme or a prosthetic group in an enzyme or family of enzymes, these facts may suggest that the thyroxine molecule associates with an enzyme

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432 ANTAGONISTS OF THYROXINE

by means of its side chain and exerts its function by means of the ortho- diiodohydroxyphenyl ether structure at the opposite end of the molecule. Niemann et al. (11, 12) likewise have suggested an active locus of the mole- cule by the formation of a quinoid resonance form involving the phenolic hydroxyl group and the ether oxygen. The observation that several potent antagonists of thyroxine and its acid analogues have no influence on the thyroxine-like action of 3,5-diiodo-4-(4’-hydroxyphenoxy)-aniline can most readily be interpreted as an indication that this thyroid-active substance combines with different groups, perhaps of opposite polarity at physio- logical pH values, in the postulated enzyme system than do the thy- roxine-like active compounds with acidic side chains.

In earlier work (8) it was not possible to determine whether or not cer- tain thyroid-active compounds were independently active or were con- verted to thyroxine. If such compounds were converted to thyroxine, molar ratios necessary to reduce the thyroxine-like effect to one-half would be lower in the case of the less active compound. Table VI indicates the similarity of such molar ratios for thyroxine as compared to the carboxylic acid analogue. No influence on the thyroxine-like activity of 3,5-diiodo- 4-(4’-hydroxyphenoxy)-aniline was observed for even larger molar ratios. It is, therefore, suggested that these two compounds are not converted to thyroxine to exert their action on Amphibia.

SUMMARY

Several derivatives of 3,5-diiodotyrosine and of 3,5-diiodo-4-hydroxy- benzoic acid have been prepared and observed to inhibit competitively the effects of thyroxine on amphibian metamorphosis. The respective benzyl ethers of 3,5-diiodo-4-hydroxybenzoic acid and of 3,5-diiodotyrosine were found to reduce the effect of thyroxine by 50 per cent when the molar ratio of the inhibitor to thyroxine was 8 and 37 respectively. The specificity of this inhibition was demonstrated by the inability of many other sub- stances to produce any inhibition at much higher molar ratios.

The benzyl ethers of 3,5-diiodotyrosine and of 3,5-diiodo-4-hydroxy- benzoic acid also proved to be competitive inhibitors of the thyroxine-like active compound, 3,5-diiodo-4-(3’,5’-diiodo-4’-hydroxyphenoxy)-benzoic acid. This inhibition was achieved at molar ratios similar to those ob- tained with thyroxine. However, these inhibitors had no effect upon the thyroxine-like activity of 3,5-diiodo-4-(4’-hydroxyphenoxy)-aniline at very high molar ratios. The fact that concentrations of inhibitors which re- duced the effect of thyroxine appreciably did not alter the activity of these thyroxine-like compounds indicates that they are not converted to thyrox- ine for activity.

Assuming that thyroxine-like active compounds act by virtue of serving

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E. FRIEDEN AND R. J. WINZLER 433

as a prosthetic group or coenzyme for some enzyme system or systems, this and other recent evidence suggests that the side chain of thyroxine serves as an enzyme-associating locus, while the orthodiiodohydroxyphenyl ether group acts as an “active” or “functional” locus of the molecule.

We are greatly indebted to Joy Levin Forrester for assistance in per- forming the syntheses described, and to Elizabeth V. Tukich for assist- ance in the biological tests.

BIBLIOGRAPHY

1. Woolley, D. W., J. Biol. Chem., 164.11 (1946). 2. Winzler, R. J., and Frieden, E., Federation Proc., 7,200 (1948). 3. Williams, R. H., Tagnon, R. F., Jaffe, H., Towery, B. T., and Rogers, W. F., Jr.,

30th meeting of the Association for the Study of Internal Secretions, Chicago, 32 (1948).

4. Woollett, G. H., and Johnson, W. W., Org. Syntheses, coll.2,343 (1943). 5. Block, P., Jr., and Powell, G., J. Am. Chem. Sot., 64,107O (1942). 6. Harington, C. R., and Barger, G., Biochem. J.,21,169 (1927). 7. Wheeler, H. L., and Liddle, L. M., Am. Chem. .I., 42,441 (1910). 8. Frieden, E., and Wineler, R. J., J. Biol. Chem., 176,155 (1948). 9. Gaddum, J. H., J. Physiol., 66,383 (192930).

10. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66,658 (1934). 11. Niemann, C., and Redemann, C. E., J. Am. Chem. Sot., 63,1549 (1941). 12. Niemann, C., and Mead, J. F., J. Am. Chem. SOL, 63,2685 (1941).

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Earl Frieden and Richard J. WinzlerRELATED COMPOUNDS

THYROXINE AND STRUCTURALLY COMPETITIVE ANTAGONISTS OF

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