DECEMBER
1957
1575
MARINE PRODUCTS. XLIII
Anal. Calcd. for CaHa4: C, 28.9;H, 4.8;N, 66.0. Found:
C, 28.6;H, 4.8;N, 66.5. (c) When Ir was analogously refluxed with either of the above 2 cyanamides in chloroform it was recovered in 92 and 70% yields, respectively, and formed dicyandiamide in %yo yield in the latter experiment. (d) Finally, when Id was refluxed with either dialkyl cyanamide in ethanolic solution for 6 hr. it was recovered in 7&80% yields. (3) With substituted aminoguanidines. ( a ) When aminoguanidine nitrate was refluxed, without further solvent, in either a small or large excess of dimethyl cyanamide for 3 hr. it was recovered in 90% yield. However, when aminoguanidine, as its free base, was refluxed with an equimolar proportion of diethyl cyanamide for 3 hr., fumes of ammonia were readily detectable from the reaction liquor and a white solid, m.p. 88", was isolated, total yield 50% (on the basis of a provisional molecular weight of 140). This has not been identified as yet. Anal. Calcd. for CaHI2N4:C, 51.4;H, 8.6;N, 40.0.Found: C, 51.5;H, 8,3;N, 39.7. The aminoguanidine free base incidentally was used in the form of its aqueous solution and this was obtained by titration of aminoguanidine sulfate in water with a barium
[CONTRIBUTION No. 1454 FROM
solution. ( b ) When triaminoguanidine nitrate was refluxed with an excess of dimethyl cyanamide in ethanol or without further solvent, for 3-hr. periods, it was recovered in 95 and 92% yields, respectively. (c) When p-methoxybenzylidene aminoguanidine (Va) was refluxed with dimethyl cyanamide either in ethanol or in chloroform solution for 3 hr. it was recovered in 95 and 92% yields, respectively. However, when Va was refluxed in dimethyl cyanamide, without further solvent and under anhydrous conditions, again for 3 hr., it yielded 30% unchanged Va and ca. 17% of a cream-colored substance, m.p. 24&250", which proved to be p-methoxybenzylidene l-amino4,4-dimethyl-2-guanylguanidine (Vb). Anal. Calcd. for C,2HulN60: C, 55.0; H, 6.9; N, 32.1. Found: C, 54.7;H,7.0; N, 32.2.
Acknowledgment. The author is indebted to C. L. McCarthy, M.S., and particularly to Dr. M. F. Cashman, for experimental assistance with portions of this work. CORK,IRELAND Los ANGELES 24, CALIF.
THE STERLING CHEMISTRY LABORATORY, AND FROM THE LABORATORY, YALE UNIVERSITY]
BINGHAM OCEANOGRAPHIC
Contributions to the Study of Marine Products. XLIII. The Nucleosides of Sponges. V. The Synthesis of Spongosine' WERNER B E R G M A "
AND
MARTIN F. STEMPIEN, JR.
Received June 9,1967 Spongosine has been synthesized by two m e r e n t methods and shown to be 9-~-~-ribofuranosyl-2-methoxyadenine.
communication deals with two syntheses of spongosine which establish beyond doubt the point and configuration of the junction between the purine and ribose moieties. In the first synthesis 2-methoxyadenine, prepared by a modification of the (1)This investigation was supported by a research grant methcid previously reported,2 was converted to its (G-3789)from the National Institutes of Health, Public chloromercuri salt4 which was treated with 2,3,5Health Service. (2) W. Bergmann and D. C. Burke, J . Org. Chem., 21, tri-0-acyl-D-ribosyl chloride. In this reaction the 226 (1956). triacetyl derivative6afforded a mixture of products (3)It was pointed out in the previous publication2 that difficult to separate. The tribenzoate, however, spongosine not only appears to be the first methoxypurine to be found in nature, but also one of the first 0-methyl which has recently been used with conspicuous succompounds to be isolated from animal tissues. It should be cess by Kissman, Pidacks, and Baker,6 reacted mentioned in this connection that prior to this observation smoothly to give a product which after Odebenthe occurrence of some phenolic methylethers had been zoylation with catalytic amounts of sodium methnoted in animals, such as methanethol in the sponge, Sphecio- oxide afforded the glycoside in a 30% yield. The spongia vesparia [W. Bergmann and W. J. McAleer, J . Am. Chem. Soc., 73, 4969 (1951)],and of quinol monomethyl- identity of the reaction product with spongosine was ether, chavicol, p-methoxyacetophenone, and 5-methoxy- shown by a comparison of the melting points, rotasalicylic acid in the scent gland of the beaver [E. Lederer, tions, and the chromatographic, electrophoretic, Spongosine, one of the unusual nucleosides obtainable from the Caribbean sponge, Cryptotethiu crypta, has recently been shown2to be the ribofuranThe present oside of 2-metho~y-6-aminopurine.~
J . Chem. SOC.,2120 (1949)l. Ferulic acid, m-methoxy-
benzoic acid, and vanillic acid have been isolated from the urine of horses [E. Lederer and J. Polonski, Biochim. et Biophys. Acta, 2, 431 (1948)l and men [M. D. Armstrong, K. N. F. Shaw, and P. E. Wall, J . BioZ. Chem., 218, 293 (1956)l. These compounds may well have been derived from methyl ethers in dietary plant material. More recent observations, however, show that 0-methylations may occur within the animal, cf. N. F. MacClagan and J. H. Wilkinson, Biochem. J., 56, 2111 (1954);J. M. Price and L. W. Dodge, J . BioZ. Chem., 223, 699 (1956);S. Kraychy
and T. F. Gallagher, J . Am. Chem. Soc., 79, 754 (1957); and F. DeEds, A. N. Booth, and F. T. Jones, J . Biol. Chem., 225,615 (1957). (4)J. Davoll and B. A. Lowy, J . Am. Chem. Soc., 73, 1650 (1951). (5) J. Davoll, B. Lythgoe, and A. R. Todd, J . Chem. Soc., 967 (1948). (6)H. M. Kissman, C. Pidacks, and B. R. Bnkcr, J . Am. Chem. Soc., 77, 18 (1955).
1576
BERGMANN AND STEMPIEN
and spectrophotometric behavior of the synthetic and natural material. While this synthesis strongly indicates that the configuration of the glucosidic link in spongosine is 9-p as in I, it cannot be accepted as the final proof. It is true that in the great majority of the naturally occurring purine nucleosides the carbohydrate moiety is attached to the 9-position of the purine ring and in a 8-glycosidic linkage. It is also known that chloride used in the 2,3,5-tri-O-benzoyl-~-ribosyl the present synthesis has so far always afforded the P-ribosides under similar conditions.6Since none of these methods, however, has previously been applied to 0-methylpurines, there remained some doubt concerning the validity of such analogies. The second synthesis was based on the assumption that spongosine is the 2-0-methylderivative of crotonoside, a compound of known onf figuration,^ and that it might be obtainable from it by selective methylation. Although such methylations are not too well known, some promise of success was indicated by the fact that the silver salt of 2-anilino-6oxypyrimidine reacts with methyl iodide to give 2anilin0-6-rnethoxypyimidine.~Treatment of the silver salt of crotonoside with methyl iodide under similar conditions gave a mixture of products which was separated by chromatography on paper into four fractions. Of these, the major product proved to be unreacted crotonoside. One of the other fractions was found to be chromatographically indistinguishable from spongosine in three different solvent systems. Its ultraviolet spectra in both acidic and aqueous solutions were of the same uniqueness as t,hose of spongosine.2The occurrence of spongosine among the methylation products of crotonoside therefore furnishes additional evidence that its structure is that of a 9-p-D-ribofuranosyl2-methoxyadenine (I). NHz
HO OH I EXPERIMENTAL
B-Methoxyadenine.2 A mixture of 3 g. of 2-chloroadenine, a solution of 6 g. of sodium in 120 ml. of absolute methanol, and an additional 80 ml. of absolute methanol was heated in a stainless steel bomb at 150" for 5 hr. After this time, chromatography of B small amount of the reaction mixture dissolved in water, showed the presence of 2-chloroadenine, adenine: and 2-methoxyadenine. Attempts to separate this mixture by the ion-exchange chromatography recommended ( 7 ) 3 . Davoli, J-. Am. Cimn SOC.,7.3, 3174 (1951). (8) T. B. Johnson and F. Vir. Heyl, Am. Chem. 241 (1907).
:,
36,
VOL.
22
by Bergmann and Burke' failed because the purine mixture began to crystallize on the resin as the pH of the eluent dropped to 9.5. The material was recovered by extracting the mixture of resin and purinea with 3 portions of 1 1 each of hot, 10% formic acid. Cooling of the combined extracts afforded a solid, shown chromatographically to consist of Zchloro and Zmethoxyadenine. When this mixture waa dissolved in 300 ml. of aqueous methanol (1:1 ) only 2-methoxyadenine separated upon cooling to room temperature. The pure product wm obtained in a 50% yield. Spcmgosine. C h l o r ~ r c u ~ - ~ - m e t o x ~ a ~To i n ea .suspension of 1.0 g. of 2-methoxyadenine in 2 ml. of water wi19 added 6.5 ml. of N sodium hydroxide, and the solution warmed gently with shaking to dissolve the purine. To this solution, 1.7 g. of mercuric chloride dissolved in 20 ml. of warm ethanol waa added. A heavy, white precipitate formed which was collected by filtration. The precipitate was washed with water and dried in vacuo a t room temperature over sodium hydroxide. d,3,5-Tri-0-benzoyl-~-m'bofuran~sy~One-half g. of 1-0-acetyl-2,3,5tri-O-benzoyl-&~ribose of m.p. 128-129", prepared according to the directions of Kissman e l al,' was dried in vacuo for 5 hr. at 69" and dissolved in a saturated solution (0') of anhydrous hydrogen chloride in 50 ml. of anhydrous ether. The solution was kept for 4 days a t -14" in a flask covered with a polyethylene seal. After this time, the ether and excess hydrogen chloride were removed at reduced pressure, and the residue, a yellowish sirup, dissolved in 20 ml. of anhydrous benzene. The benzene was remeved at reduced pressure, and the process repeated twice to insure complete removal of hydrogen chloride. The crude 2,3,5t~-0-bemoy~-~-ribofuranosyl chloride was used without further purification. The chloromercuric salt of Zmethoxyadenine (0.8 g.) was suspended in 55 ml. of xylene, and about 5 ml. of the solvent was distilled to insure removal of traces of water. To the remaining suspension was added the ribosyl chloride dissolved in a small amount of xylene. The mixture was refluxed with continuous stirring for 4 hr., after which the xylene was removed at reduced pressure and the residue extracted with hot chloroform. The chloroform extracts were washed twice with 35 ml. portions of 30% potassium iodide solution, twice with water, decolorized with Norit and dried over anhydrous sodium sulfate. The solvent was then removed at reduced pressure, and the residue, a dark brown oil (1.49 g.), dissolved in 50 ml. of anhydrous methanol. One xnl. of a N solution of sodium methoxide in methanol was added and the mixture refluxed for 30 min. A second 1 ml. of sodium methoxide solution was added, and the reflux continued until the pH of the solution was greater than 8, indicative of complete debenzoylation (20 min.). The solution was taken down t o dryness in vacuo, and the semi-solid residue triturated with ether to remove methylbemoate. The remaining crude spongosine (0.6 g.) was dissolved in hot water, the solution made slightly basic with sodium hydroxide, and decanted from a brown, oily residue The extract was then made acidic with hydrochloric acid, and all the liquid was removed by freeze-drying. The residual, brown solid was extracted with chloroform, and the extract evaporated to dryness. The white residue of 50 mg. (31% yield) consisted of spongosine, which wzs recrystallized from hot water; needles cJf m.p. 191-19P.5°; [CY]D -43.5' (9.2 mg. in 2 ml. of 8% sodium hydroxide); reporteds: m . p 192-193'; [orla --K?6". A mixed melting point with authentic spongosine showed no depression Infrared spectra of both spthctic. and natural spongosine in a potassium bromide disk showed the same bands. A R ~Calcd. . for CIIHI~NSOS: C, 44.4; R, 5.08; K $23.56; OCAS, 10.44. Found: C, 44.5: H, 5.14; h', 23.31' OCH?, 10.36 A 2.9 mg sample of syi:thetic spoogosinr was dissolver (9) W Bergmann and R.3. Feeney, J . Org.. Chem., 16, 98L (1951).
DECEMBER
1957
in 0.1N sodium hydroxide solution and made up to 50 ml. with each of the following solutions: O.1N hydrochloric acid, pH 7.2 phosphate buffer, and 0.1N sodium hydroxide. The ultraviolet spectra and pH values of these solutions are shown in Table I. TABLE I ULTRAVIOLET SPECTRA OF SYNTHETIC SPONGOSINE PH Max. (mp) Min. (mp)
1577
THYROXINE ANALOGS
1.35 251, 275 (252, 275)'O 257, 228 (258, 229)
7.2 12.2 268 (268) 268 (268) 232 (233) 233 (232)
in 1ml. of water and 1.8 ml. of 0.1N sodium hydroxide solution. The mixture was warmed until the crotonoside had dissolved, and an excess of silver nitrate solution was added with stirring. The precipitate was collected by centrifugation, washed three times with water and twice with methanol, and finally dried in vacuo over anhydrous potassium hydroxide. It was then ground to a fine powder and suspended in 5 ml. of absolute methanol; 2 ml. of methyl iodide was added and the mixture stirred a t room temperature for 2 hr. The precipitate of silver iodide was separated by centrifugation, and the liquid taken to dryness at reduced pressure. The residue was dissolved in a few drops of
TABLE I1 PAPER CHROMATOGRAPHY OF METHYLATED CROTONOSIDE Solvent System
BuOH-NH~-H~O'*
BuOH-EtOH-H20'J
BuOH-H20'* ~~
Solvent front Spongosine Crotonoside Reaction mixture
36.0cm. 13.Ocm. 0.9cm. 0.7cm. 2.4cm. 4.4cm. 13.3cm.
RF 0.36 0.025 0.019 0.067 0.122 0.37
TABLE I11
37.9cm. 12.5cm.
RF 0.33
39.6 cm. 12.9cm.
Rp 0.33
2.2cm. 4.2cm. 12.7cm.
0.057 0.111 0.34
0.4cm. 2.3cm. 4.4cm. 13.9cm.
0.010 0.058 0.111 0.35
hot water and chromatographed on paper in three solvent
ULTRAVIOLETSPECTRAOF METHYLATEDCROTONOSIDEsystems; see Table 11. ( SPONGOSINE)
0.1N NaOH
0. I N HC1
Max. 267 mp (268)IO Min. 232 mp (233) 280/260 0.645 (0.65) 250/260 0.77 (0.85)
Max. 274, 248 mu (275, 251)'O Min. 257, 228 mp (257, 228) 280/260 1.45 ( 1.41) 250/260 1.02 ( 1.0)
fipongosine from crotonoside. A 50-mg. sample of crystalline crotonoside prepared from croton seed" was suspended (10) The bracketed figures are those reported for natural spongosine.2 (11) E. Cherbuliez and K. Bernhard, HeZv. Chim. Acta, 15, 464 (1932).
Two chromatograms were run in the BuOH-NHtHzO solvent system and the spots with RF values corresponding to spongosine were eluted with 0.1N hydrochloric acid and 0.1N sodium hydroxide solution. The principal features of the ultraviolet absorption spectra of these solutions are shown in Table 111. NEWHAVEN, (12) W. S. Macnutt, Biochem. J., 50, 384 (1952). (13) E. Chargaff, E. Vischer, R. Doniger, R. Green, and F. Misani, J. Biol. Chem., 177, 405 (1949). (14) 1-Butanol saturated with water a t 23".
[CONTRIBUTION FROM TFIE WARNER-CHILCOTT LABORATORIES]
Thyroxine Analogs ROBERT I. MELTZER, DAVID M. LUSTGARTEN,
AND
ALEX FISCHMAN
Received December 86, 1966 3,5-Diiodo-4(4'-hydroxyphenoxy)bensoicl 3,5-diiodo-4-(4'-hydroxyphenoxy)phenylacetic, droxyphenoxy)phenyl]propionic acids were prepared by improved procedures.
Reports on the activity of 3,5-diiodo-4-(4'-hy-
and @-[3,5-diiodo-4(4'-hy-
diiodo-4-(4'-hydroxyphenoxy)benzoic
acid
(Va),
droxy-3'-iodophenoxy)phenyl acetic acid as a thy- 3,5 - diiodo - 4 - (4' - hydroxyphenoxy)phenylacetic roxine-like material in rats' in vitro2 and clinically3 acid (Vb), and ~-[3,5-diiodo-4-(4'-hydroxyphenresulted i n these laboratories in a renewed and en- oxy)phenyl] propionic acid (Vc). larged interest in this type of compound. For the Of these compounds, the benzoic4(Va) and phen-
preparation of such compounds we required 3,5- ylacetic6 (Vb) acid analogs had been previously prepared via a comparatively cumbersome proce(1) R. Pitt-Rivers, Lancet, 11, 234 (1953).
(2) 0. Thibault and R. Pitt-Rivers, Lancet, I, 285 (1955). (3) J. Lerman and R. Pitt-Rivers, J . Clin. Endocrinol., 15, 653 (1955).
(4) C. R. Harington and G. Barger, Biochem. J., 21, 169 (19271. (5)'C. R. Harington and R. Pitt-Rivers, Biochem. J., S O , 438 (1952).
.-
