N,N' DIPHENETHYLUREA, A METABOLITE FROM T H E MARINE ASCIDIA4N D I D E M N U M TERiVA T AA'UM CHRISM. IREL.4h.D* and AUGUSTINE R. DURSO, JR. Section of Medicinal Chemistry and Pharmacognosy, School of Pharmacy, Cniversity of Connecticut, S t o r r s , Connecticut 06268
P a u J. ~ SCHEUER Department of Chemistry, Cnialersity of H a w a i i at M a n o a , Honolulu, H a w a i i 96822
The didemnid ascidians represent a select group of marine chordates which harbor procaryotic algal symbionts (1). Although it has been well documented that unicellular algae occur as endosymbionts in a \vide variety of marine organisms (2), their impact on the secondary metabolism of their host organisms remains illdefined. This is particularly true in cases where the symbiont is a procaryotic alga. As part of our investigation into the molecular basis of marine symbiosis (3), we have undertaken a systematic study of the secondary metabolites of didemnid ascidians from the Western Caroline Islands. We have previously reported the isolation of cyclic peptides, e.g., ulicyclamide (1) from Lissoclinum
patella (4), which suggested that these symbiotic pairs are capable of de novo amino acid synthesis. This is particularly interesting in view of earlier reports that the nitrogen-fixing procaryotes Lyngbya majuscula ( 5 ) and Microcystis aeroginosa (6) produce cyclic peptides. We now report the isolation of S,S'diphenethylurea (2), apparently a product of phenylalanine metabolism, from the didemnid Didemnum ternatanum. This provides further evidence for the existence of amino acid metabolism by such symbiotic pairs. Typically, chromatography of an organic extract of the frozen ascidian yielded crystalline 2, mp 135" (lit. 138-141') (7), as the only secondary metabolite (28% of organic oil). The structure of 2 was confirmed by
H
H
2 c.l
1 H
360
MAY-Jl-X
19811
I R E L A S D , DURSO .4SD S C H E C E R :
synthesis from phenethylamine and phosgene. This represents the first isolation of 2 from a marine organism. Interestingly, its first isolation from a natural source was from a Streptomyces sp. ( i ) . The compound was found t o be a weak depressant lacking acute toxicity.
S,S' DIPHENETHTLUREA
361
ISOL~TIOV OF , ~ , ~ - ' - D I P H E ~ E T B T L T R E ~ . Column chromatography of t h e organic soluble material (2.7 g) first on Sephadex LH-20 (methylene chloride/methanol, 3:1), then on silica gel (ethyl acetate) gave S,S'diphenethylurea (0.76 g), colorless crystals from ethanol, m p 135"; i r (CH2C12) 3450, 3380, 3040, 2940, 2880, 1670, 1650 cm-l; l H nmr iCDC1,) 6 2.70 i t . 2H. J=7 H z ) . 3.30
EXPEKI;\IESTALi COLLECTION 4 h D E X T R i C T I O S OF D z d e m n u m ternatanurn .-Colonies of D . ternatanurn were collected b y snorkeling (-2 t o -4 m ) a t Urukthapal island (7%' S , 120"27', 30" E) in t h e Western Carolines. T h e material was frozen immediately and maintained frozen until eutraction. Three different extraction procedures were employed: Frozen tunicate (455 g) was homogenized in methanol (1liter) in a Waring blender. T h e resulting suspension was filtered through Whatman ~1 paper. T h e solvent was evaporated t o give a water\- residue which was partitioned betn-een m-ater and methylene chloride. T h e methylene chloride layer was evaporated t o give t h e organic soluble material (3.7 g ) . Frozen tunicate (100 g) v a s homogenized in methylene chloride (0.5 liter) in a Waring blender. T h e suspension was filtered through R h a t m a n =1 P S phase separating paper, and t h e methylene chloride was evaporated t o give the organic soluble material (0.82 g ) . Freeze-dried tunicate (3g) was homogenized in methylene chloride (0.25 liter) and treated according t o method 2 t o give t h e organic soluble material (0.064 g ) . Tlc inspection of the extracts indicated t h a t all m-ere similar in composition. 'The ir spectra were recorded on a Beckman 620 MX spectrophotometer. T h e uv spectra were recorded on a C a r y 15 spectrophotometer. Electron impact mass spectra --ere recorded on an AEI-AIS-902 mass spectrometer. 'H nmr spectra were recorded on a Perkin Elmer R-24B GO MHz spectrometer and 13C nmr spectra on a Bruker WP-60 MHz Fourier transform spectrometer. Melting points were measured on a Thomas Hoover apparatus and are uncorrected.
SI-STHESISOF ~ V , A V ' - ~ ~ ~ ~ ~ Phenethylamine (Eastman, 9.5 g, 10 ml) was dissolved in methylene chloride (100 ml) in a 500 ml 2-necked round-bottom flask equipped with reflux condenser and addition funnel. T h e addition funnel was charged with phosgene (12'5 solution in benzene, 10 ml) and methylene chloride (50 m l ) . T h e contents mTere added dropwise t o t h e stirred phenethylamine solution over a 2 h period. F a s t e r addition results in formation of significant amounts of monoacylated product. After addition was completed, stirring was continued for 24 h . T h e reaction, when washed with water (3 X 100 ml), dried over magnesium sulfate and evaporated gave crude product. Recrystallization from ethanol gave white crystals (2.35 g ) , m p 135", identical in every respect (ir, nmr, ms) t o the natural product. ACKXOWLEDGMESTS We thank A h . Marvin Thompson for mass spectra, Mr. Richard Kriwacki for 13C spectra, and t h e Petroleum Research Fund of t h e -4merican Chemical Society for financial support ~ ( 1 2 7 3 0G 4 ) . T h e field collection was supported b y S I H Postdoctoral Fellowship (Ghl 06256) t o C I . Receit'ed 24 S o r e m b e r 1980 LITERATCRE CITED 1. R . A. Lewin and S . Withers, r a t u r e (London),256, 735 (1975). 2 . W.E. 1-ernberg, "Srmbiosis in t h e Sea." University of-' S o i t h Carolina Press. 1974. 3. C . Ireland and P. J. Scheuer, Science, 20.5. 205, 922-23 11979). (1979). 4. C C.. - ?Ireland r e l a i d and P . J. Scheuer, J . A m . C h e m . SOC SOC., , 102, 5688-91 (1980) (1980). 5. R . E E. Moore, BioSczence, 27, 797-802 (1977). 6. P . R . Gorham, Can. I-ef. J . , 1 , 2 3 5 11960). I . T.Iwai,:4. Hirano, J. Am-aya, 8.Matsuo and S. Omura, J . Anfzbiotics, 31, 375-6 (1978).
-
