Porphyrin metabolism. IV. Molecular structure of acetamide derivatives affecting porphyrin metabolism

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 66, 289-300 (1957)

Porphyrin M e t a b o l i s m . IV. Molecular Structure of Acetamide Derivatives Affecting Porphyrin M e t a b o l i s m I Ellen L. Talman, Robert F. Labbe and Robert A. Aldrich2 From the Departments of Biochemistry and Pediatrics, University of Oregon Medical School, Portland, Oregon R e c e i v e d March

27, 1956

INTRODUCTION

Although the induction of hepatic porphyria in experimental animals by treatment with allylisopropylacetylcarbamide (Sedormid) (1-3) is a well-established phenomenon, the mechanism of action of the drug is still obscure. Elucidation of this mechanism would be materially aided by a precise knowledge of the molecular structure e]iciting the response. Furthermore, such knowledge might permit selection of a compound equally well-suited to studies of porphyrin metabolism but presenting none of the technical difficulties associated with the use of Sedormid (hypnosis and extreme insolubility). A solution to this problem was sought by examining a number of selected compounds related to Sedormid for porphyria-producing properties, using the chick embryo as the test animal. While this work was in progress, several reports of similar studies, using rabbits, appeared in the literature (4-8). However, the close control of experimental conditions afforded by the chiek embryo allowed more definitive comparison of the potencies of the various compounds. Also, several of the compounds made available to this group had not been mentioned in the other papers. These studies have confirmed Goldberg's observation (4, 6, 7) that a soluble, nonhypnotic i Supported by a special research grant of the Eli Lilly Company; the Helen Hay Whitney Foundation; American Medical Association Council on Pharmacy and Chemistry, William Volker Grant No. 7; National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service (Grant No. A-752). Presented in part at the meeting of the Federation of American Societies for Experimental Biology at San Francisco, April 1955: Talman, E. L., Labbe, R. F., and Aldrich, R. A., Federation Proc. 14, 390 (1955). With the technical assistance of Margaret C. Schropp. 289

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E. L. TALMAN, R. F. LA.BBE, AND R. A. ALDRICH

Sedormid d e r i v a t i v e (allylisopropylacetamide) exerts a p o t e n t p o r p h y r i a p r o d u c i n g action, a n d have led to conclusions regarding the molecular s t r u c t u r e capable of i n d u c i n g h e p a t i c p o r p h y r i a which are similar i n m o s t respects to those a d v a n c e d b y G o l d b e r g (7). P o i n t s of d i s a g r e e m e n t are t h o r o u g h l y discussed below. EXPERIMENTAL

EmbryonatedEggs Pure strain white Leghorn eggs (large), always obtained from the same hatchery after a week's incubation, were maintained in an incubator at 37.5°C. and 55-60~ relative humidity for the duration of the experiment. Eggs were turned at least once daily when candled for viability. Dead embryos were discarded.

Preparation of Injectable Materials Suspensions of the compounds in isotonic glycerol were prepared and sterilized as previously described (3). Concentrations of the suspensions were adjusted so that the dose given was contained in 0.5 ml.

Procedure Drugs were injected into the yolk sacs of 8-day embryonated eggs (wt. range 50-59 g./egg. Av. 53.5 g.) in doses equimolar with 10 rag. of Sedormid. A control group included in each experiment received 0.5 ml. of sterile suspension medium via the same route. At intervals of 1, 2, 4, and 6 days after treatment, six embryos from each group were sacrificed, and their allantoic fluids were collected for analysis as previously described (3). Quantitative determinations of coproporphyrin (9) and uroporphyrin (10) ("uroporphyrin" is used to denote ether-insoluble porphyrin) were carried out on the pooled allantoic fluids from each group of eggs, and the amounts of each porphyrin contained in the allantoic fluid of a single egg were calculated (3). Since the maximal effect of Sedormid is attained 6 days after treatment (3), the final evaluation of relative porphyria-producing potencies is based upon data obtained at that time and was arrived at as follows : (a) The total amounts of eoproporphyrin and uroporphyrin present in the allantoic fluid of a single egg were calculated. (b) These quantities were corrected by subtracting the quantities of each porphyrin found in the control series of that experiment. (c) The figures thus obtained for Sedormid-treated embryos were assigned base values of 100%, and the percentage activities of the other drugs in comparison with Sedormid were computed. (d) The average of these two percentage values for uroporphyrin and coproporphyrin was used for the final evaluation of relative porphyria-producing potency.

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I~ESULTS AND DISCUSSION

The data included in Tables I and II, 3, 4 demonstrate that the porphyrins, like other embryonic excretory products, accumulate in the allantoic fluid during embryonic development. Thus, the amounts of coproporphyrin and uroporphyrin calculated to be present in the excreta of a single egg increase progressively except at three points, and there the decreases are insignificant. Effects of the potent porphyria-producing compounds are apparent 24 hr. after treatment, becoming increasingly obvious with the passage of time. At the end of 24 hr., careful inspection in ultraviolet light of the allantoic fluids from eggs receiving these compounds usually reveals the red fluorescence characteristically emitted by porphyrins exposed to light of these wavelengths. This fluorescence is easily visible at the end of 48 hr. and is very intense in samples collected after 6 days.

Role of the Carbamide Moiety These studies show that the carbamide portion of the Sedormid molecule can be altered considerably without destroying the porphyriaproducing property (Table I; Fig. 1). Thus, removal of the terminal 0

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--C--NH~ group of Sedormid, giving allylisopropylacetamide (AIA), results in a 49 % increase in activity. The presence of a methyl substit3 AllylisopropyIacetic acid and 2-hydroxypropylisopropylacetylcarbamide were prepared in this laboratory, the former by treating allylisopropylacetamide ~dth nitrous acid [J. Am. Chem. Soc. 54, 3438 (1932)] and the latter by reacting allylisopropylacetylcarbamide with sulfuric acid with subsequent hydrolysis in the cold. We are indebted to Dr. A1 Steyermark of Hoffmann-LaRoche, Inc., Nutley, New Jersey, for the following analyses of these compounds: Calculated Found Allyiisopropylacetic acid (CSH1402) Carbon 67.57 65.82 Hydrogen 9.92 9.70 2-Hydroxypropylisopropylacetylcarbamide (CgHlsO~N2) Carbon 53.44 53.54 Hydrogen 8.97 8.88 4 Seconal and Valmid supplied through the courtesy of Eli Lilly and Company, Indianapolis, Indiana; Dial by Ciba Pharmaceutical Products, Summit, New Jersey; a-allylmalonamMe by Parke, Davis and Co., Detroit, Michigan; Phenurone by Abbott Laboratories, North Chicago, Illinois; all other compounds supplied by Hoffmann-LaRoche, Inc., Nutley, New Jersey.

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uent on the nonterminal nitrogen atom (N-Mlylisopropylacetyl-Nmethylearbamide) fails to affect porphyrin output; but methylation of both nitrogen atoms (allylisopropylaeetyl-N,N'-dimethylearbamide) reduces activity to 73 % of Sedormid. Since AIA is soluble, in contrast to the extremely insoluble Sedormid, the greater porphyria-producing capacity of AIA might simply reflect a more efficient absorption of the drug. However, hr-allylisopropylaeetyl-N-methylearbamide is insoluble, yet its effects upon porphyrin excretion compare favorably with those of AIA during the first 2 days of the test period (Table I) even though it exhibited the same activity as Sedormid at the end of 6 days. On the other hand, allylisopropylaeetyl-N,Nr-dimethylearbamide is soluble but gave somewhat lower porphyrin excretions than either AIA or Sedormid throughout the experimental period with a final activity of 73 % of Sedormid. Therefore, it appears that the greater potency of AIA

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E. L. TALMAN, R. F. LABBE, AND R. A. ALDRICH

is probably a reflection of molecular structure, not simply a matter of solubility. Furthermore, it may be that AIA is the true porphyria-produeing agent when Sedormid is given, since monoureides are very readily hydr~lyzed to the corresponding acid amide in alkaline media. When the carbamide group is incorporated into a barbituric acid ring in allylisopropylbarbituric acid (Ahirate), porphyria-producing activity is reduced to 40%. A close relative of Alurate, allyl-(1-methylbutyl)barbituric acid (Seconal) displays an activity of 88 %. However, the barbituric acid derivative bearing two allyl groups (Dial) is nearly devoid of activity. More drastic alterations in the Sedormid molecule destroy porphyriaproducing activity almost completely. Thus, allylisopropylacetic acid exhibits no activity. Isonicotinyl hydrazide (Isoniazid) is not strikingly similar to Sedormid in structure, but the observation that Isoniazidresistant tubercle bacilli are catalase-defieient (11), and Fisher's (12) suggestion that Isoniazid may affect the porphyrin metabolism of mycobacteria, coupled with the greatly reduced Hver catalase activity observed in experimental porphyria (1, 3, 13), made the investigation of Isoniazid advisable. Since none of the eggs receiving Isoniazid survived to the sixth day, final evaluation had to be based upon data obtained on the fourth day. These data show Isoniazid to affect porphyrin excretion only slightly. 1-Aeetyl-3-benzohydrylcarbamide and 1-ethinylcyelohexylcarbamate (Valmid) show activities of 12 and 19 % of Sedormid, respectively. Closer inspection of the figures for these two compounds, however, reveals that their effects on porphyrin excretion were negligible for the first 2 days after treatment. This delayed increase in porphyrin excretion coupled with the relatively slight effect on uroporphyrin output, suggests that the activity observed might reflect some other type of liver damage (14), not a primary porphyria-producing action, a-Allylmalonamide is totally inactive. Role of Substituents on the A cetyl Group

Findings with the barbiturie acid derivatives suggest that the types of substituents on the acetyl group may play important roles in the production of porphyria by drugs. It is apparent that this is actually the case (Table II; Fig. 2), When substitutents are absent (acetylcarbamide), porphyria-producing activity is also absent. Substitution of a n-propyl group for the isopropyl of Sedormid (allyl-n-propylacetylcarbamide) reduces activity to 29 %. In the ease of 2-hydroxypropyliso-

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propylaeetylcarbamide, which may be the metabolic product of Sedormid (15), activity is reduced to 8%. When both substituents are allyl groups (diallylacetylcarbamide), activity is only 6 %. Di-n-propylacetylcarbamide and diethylacetylcarbamide display activities of 2 and 4%, respectively, and allylvinylacetylcarbamide displays only 1% activity. Phenylethylacetylcarbamide is also only weakly active (9%), and phenylacetylcarbamide (Phenurone) is inactive. Compounds exhibiting less than 10 % activity cannot be considered to produce serious alterations in porphyrin metabolism. A fairly precise formulation of the molecular structure required to produce porphyria may be derived from these data. This structure is illustrated below: CH2~CtI--CH2 tI3C

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PORPttYRIN METABOLISM.

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297

1. It must be a dialkylsubstituted acetamide or acetamide derivative. 2. One substituent must be an allyl group. 3. The other substituent must contain at least three carbon atoms, preferably in a branched chain. While Goldberg has concluded (7), on the basis of his studies with rabbits, that the structure affecting porphyrin metabolism is a substituted acetamide or acetamide derivative and that one of the acetyl substituents must be an allyl group, there is some disagreement between his work and the work reported here concerning the nature of the second substituent. In the work described here, diallylbarbiturie acid (Dial) failed to affect the porphyrin output of chick embryos markedly, although Goldberg (5-7) and Stieh (8) report that this compound influences the porphyrin excretion of rabbits to a considerable degree. Our observation that diallylacetylcarbamide also has little effect upon the porphyrin outputs of chick embryos supports the findings with Dial in this species. Furthermore, comparable results have been obtained in each of three experiments using Dial and in two experiments using diallylaeetylcarbamide. The syndrome produced by Sedormid and AIA is so similar in all species studied (rat, rabbit, and chick embryo) that it seems unlikely that this discrepancy arises from a species difference, although this possibility cannot definitely be excluded. Since increased porphyrin excretion is seen in many forms of liver disease (14), great caution must be exercised in interpreting elevated porphyrin outputs following prolonged administration of hepatotoxie agents as indicating porphyria. The deleterious action of certain allyl compounds is clearly shown by the work of Jurgens (16), who found that diallylaeetylcarbamide produced severe liver damage in rabbits, and of Popper (17, 18) who made similar observations in other experimental animals treated with ally1 formate. It is noteworthy that, among Goldberg's Dial-treated rabbits, only three out of eight excreted porphobilinogen plus large amounts of uroporphyrin in the urine, and these three rabbits received the drug, often intramuscularly, for periods ranging from 15 to 35 days. Since the action of the potent porphyria-producing drugs is clearly evident within 2 or 3 days, even when given by mouth, the increased excretion of porphyrins occurring after such prolonged parenteral administration may arise from some hepatic disturbance other than porphyria. The experimental conditions employed by Goldberg were quite variable (differing periods of treatment, routes of administration, dosages per unit body weight, and reuse of the same rabbits after previous exposure to other drugs) making a strict comparison of his findings with those

298

E. L. TALMAN~ R. F. LABBE, AND R. A. ALDRICH

reported here difficult. However, a few general observations can be made. Although Goldberg rates AIA and Sedormid as equal in porphyria-producing capacity (6, 7), his data (7) suggest the possibility of a greater potency for AIA. His report of slight activity for allylisopropylacetie acid based upon increases in eoproporphyrin excretion of more than 5 ~g./day in three of five rabbits (actual increases of 9, 10, and 16 ~g./day) hardly seems justified in view of the normal daily excretions ranging from 1.8 to 19.7 ug. reported in his earlier paper (5). Thus, with the exception of the role of a second ally] substituent on the acetamide derivative, the data obtained regarding the molecular structure capable of inducing porphyria differ from those of Goldberg only in degree. Although the molecular configuration eliciting porphyria has been quite accurately defined, the mechanism of action of these compounds remains uncertain. Stieh (8) has suggested that experimental porphyria may be attributed to a competitive inhibition of succinyl-coenzyme A (CoA) catabolism to succinic acid. There are, however, several clues pointing to a possible implication of vitamin BI~. Thus, embryos developing in Sedormid-treated eggs resemble those developing in eggs from B12-deficient hens (3, 19) in many ways, and a poultry expert 5 consulted in this matter remarked that the symptoms exhibited by chicks hatched from Sedormid-treated eggs resembled those of B~2 deficiency more closely than those of any other nutritional deficiency syndrome, although signs of other nutritional deficiencies were detectable. Furthermore, vitamin B~, influences both purine and porphyrin metabolism (20, 21), and purine as well as porphyrin metabolism has been shown to be altered in experimental porphyria (3, 22, 23). Since it is now known that the vitamin B~2 molecule bears three aeetamide and three propionamide residues (24, 25), such effects of certain acetamide derivatives could conceivably arise from some interference with the function of this vitamin. SUMMARY

Twenty compounds related to Sedormid have been tested for porphyria-producing activity, using the chick embryo as the experimental animal. The molecular structure eliciting this response has been identified as a dialkyl-substituted acetamide or acetamide derivative where one substituent is an allyl group and the other contains at least three 5 Arscott, G. H., Department of Poultry Husbandry, Oregon State College, Corvallis, Oregon. Personal communication.

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METABOLISM.

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carbon atoms, preferably in a branched chain. The possibility that these compounds may interfere with the function of vitamin B12 is considered. ADDENDUM At the time this paper was submitted for publication, the American edition of the Ciba Foundation Symposium on Porphyrin Biosynthesis and Metabolism had not yet appeared. As a result of this circumstance, discussion of the work of Stich and Decker (26) was not so complete as the authors would have liked. The former's findings and conclusions are in accord with those of Goldberg, however, and thus do not alter significantly the structure proposed for porphyria-producing activity. It is noteworthy that Stich and Decker found the presence of a bromine atom on the E-carbon of the allyl g r o u p (e.g., B-bromallylisopropylbarbituric acid) to destroy the porphyria-producing property, thus emphasizing the importance of the free allyl group. Another relevant item (27) appearing in the discussion of Stich and Decker's paper deals with the possible conversion of AIA itself to porphobilinogen and thence to porphyrins. Falk and Dresel investigated this possibility by administering C14-1abeled AIA to rabbits and isolating the porphobilinogen subsequently excreted. This porphobilinogen was devoid of radioactivity, thus excluding conversion of the drug to porphobilinogen as a mechanism of action. I~EFERENCES 1. SCHMID, R., AND SCHWARTZ,S., Prec. See. Exptl. Biol. Med. 81,685 (1952). 2. CASE, J. D., ALDRICH, R. A., AND NEVE, n . A., Prec. Soc. Exptl. Biol. Med. 83, 566 (1953). 3. TALMAN,E. L., CASE, J. D., NEVE, R. A., LABBE, R. F., ANDALDRICH,R. A., J. Biol. Chem. 212, 663 (1955). 4. GOLDBERG, A., Biochem. J. 57, ii (1954). 5. GOLDBERG,A., Biochem. J. 57, 55 (1954). 6. GOLDBERG, A., Biochem. Soc. Symposia (Cambridge, Engl.) 12, 27 (1954). 7. GOLDBERG,A., ANDRIMINGTON,C., Prec. Roy. Soc. (London) B 143,257 (1955). 8. STIcH, W., "The Biosynthesis of Porphyrins and Porphyrin Metabolism," Abstracts, p. 18. Ciba Foundation Symposium, London, 1955. 9. SCHW/~RTZ,S., ZI~V~, L., AND WATSON, C. J., J. Lab. Clin. Med. 37,843 (1951). 10. SCHWARTZ, S., KEPRIOS, M., AND SCHI~/IID,R., Prec. Soc. Exptl. Biol. Med. 79, 463 (1952). 11. MIDDLEBROOK, G., Am. Rev. Tuberc. 69, 471 (1954). 12. FISHER, M. W., Am. Rev. Tuberc. 69, 469 (1954). 13. SC~MID, R., FIGEN, J. F., AND SCHWARTZ,S., J. Biol. Chem. 217, 263 (1955). 14. WATSON, C. J., AND LARSON, ]~. A., Physiol. Revs. 27, 478 (1947). 15. MArNERT, E. W., Federation Prec. 11,625 (1952). 16. JURGENS, R., Arch. exptl. Pathol. Pharmakol. 212, 440 (1951). 17. PoPPy.R, H., Virchow's Arch. Pathol. Anat. u. Physiol. 298, 574 (1936). 18. POPPER, H., Z. klin. Med. 131, 161 (1937). 19. FEROUSON, T. M., AND COUCH, J. •., J. Nutrition 54, 333 (1954). 20. ABBOTT, L. D., JR., AND DODSON, M. J., J. Biol. Chem. 211,845 (1954).

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21. WILLIAMS,J. N., JR., Am. J. Clin. Nutrition 3, 20 (1955). 22. LABBE, R. F., TALMAN,E. L., AND ALDRICH, R. A., Biochim. et Biophys. Acta 15, 590 (1954). 23. LABBE, 1~. F., TALMAN, E. L., AND ALDRICH, R. A., Federation Proc. 14, 241 (1955). 24. HODGKIN,D. C., PICKWORTH,J., t~OBERTSON,J. ~I., TRUEBLOOD, •. •., PROSEN, •. J., AND WHITE, J. G., Nature 176,325 (1955). 25. BONNETT, R., CANNON, J. R., JOHNSON, A. W., SUTHERLAND,I., AND TODD, A. R., Nature 176,328 (1955). 26. STICH,W., AND DECKER, P., "The Biosynthesis of Porphyrins and Porphyrin Metabolism," p. 254. Ciba Foundation Symposium, London, 1955. 27. FALK, J. E., AND DRES]~L, E. I. B., "The Biosynthesis of Porphyrins and Porphyrin Metabolism," p. 261. Ciba Foundation Symposium, London, 1955.