MARINE A N I M A L BIOSYNTHETIC CONSTITUESTS FOR C A N C E R C H E M O T H E R A P Y ' GEORGER . PETTIT,* YOSHIAKIK.\Y.\No,YOUICHIFUJII,CHERRY L. HERALD, M.isuo ISOUE, PETERBiron x , DEYESY GCST,KEIICHI KITAHARA, DESSISL. DOCBEI;and CLAUDE MICHEL JEASAI. SCHMIDT, Cancer Resecircii Imtituie ond D e p o r t m e n t of Chemistry, .4 rizonii Slate l.-nit,ersiiy, T e m p e , A riioiiii 85281
ABSTR.~CT.-A fifteen year investigation of marine animal components a s sources for new and potentially useful cancer chemotherapeutic drugs has led t o our discovery of a number of such valuable substances. T h e especially productive Indian Ocean sea mg each) a series of very potent hare Dolabella azuicidoriiz has yielded (100 kg+-I cell growth inhibitory substances designated dolastatins 1-9. The first member of this n e a series, dolastatin 1, m a y represent the most potent anticancer agent so far uncovered with, e.g., a curative response (33%) using a dose of 11 pg/kg (T/C 240, to T I C 139 a t 1.37 p g i k g ) in the National Cancer Institute's murine B16 melanoma. Structural elucidation of t h e new antineoplastic agents is underway, and recent progress is illustrated with the peptide dolastatin 3 (P388 EDx 2.7 x lO-'pg'mI).
Fifteen years ago we began the first systematic study of marine animals and plants as a vast untapped resource for discovering new drugs necessary to improve human cancer treatment. After some four )-ears of evaluating a large number of extracts from species representing (on a n-orld-n-ide basisj various marine phyla our original expectations were amply confirmed and summarized in 1970 ( 2 ) . Subsequently a number of new cytotoxic and,'or antineoplastic marine animal constituents were isolated and characterized (3). For the past eleven years considerable efforts have been devoted to isolating the very active, albeit trace, constituents of certain exceptionally promising marine animal ext'racts. A preliminary report of success with one such problem follows. The great Roman natural scientist Gaius Plinius Secundus (Pliny the Elderj in his comprehensive study (4) of about G O h.D. first described a most potent Indian Ocean sea hare2 of the genus Dolabella. Extracts from this animal and t,wo related A plysia species from the Mediterranean were well knon-n for their toxic properties during the reign of Kero (4, 5 ) . By 150 X.D. Sicander (5) recognized the possibility of using such extracts for treatment of cert,ain diseases. However, the potential of the Indian Ocean Dolabella n.ith respect to modern medical problems \vas not recognized until vie uncovered evidence for extremely active anticancer constituents in the Indian Ocean Dolabella auricularia3 (3c). We have now completed the isolation and preliminary characterization of an exceptionally promising series of cancer chemotherapeutic agents designated dolastatins 1-9 from D . auricularia. The dolastatins most probably correspond to the potent D . auricularia constituents recognized from ancient to fairly recent ( 7 ) times. Since dolastatin 1 has been shown (by the C.S. Sational Cancer 'The present contribution is part 7 2 in the series hntineopiastic Agents. For part, T i refer t o (1). *The Romans first designated Mollusca of the family Aplysiidae in this fashion due t o a similarity between the ears of a hare and the auriculate tentacles of these gastropods, consult (5). 3The D . U Z L Y Z ' C I ~ ~ Y ~was ~ I probably t h a t first described b y Pliny and the minor variations recorded in subsequent literature as, e.g., D . andersoni, D . crilifornicci, D . eccizldafn, and D . scupida are actuall?- one species, namely D . i i ~ l r i c t i / o r i osee , (6). 482
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Institute) to cause an 8 8 7 life extension (at a dose of 11 pg kg) n-ith the murine P388 lyniphocytic leukemia and found to afford a curative response (337,) with a dose of 11 pg kg (with T C 240, to T C 139 a t 1.3i p g ml) against the murine B16 melanoma. it may repreqent the most active (lonest dose) presently known antineoplastic agent. A methylene chloride solvent partition (3i,j) fraction from an ethanol extract of D. auricularis n a s very carefully qeparated by use of an extensive sequence of chromatographic techniques (guided by bioassay) utilizing LH-20 Sephadex and silica gel column methods. By this general approach some 100 kg of the Jvet sea hare afforded about 1 mg each of nine very potent cell growth inhibitory qubstances deqignated dolastatins 1-9. Each dolastatin n as obtained as an aniorphouq colorleqs solid: D-1. mp 105-110', D-2. mp 115-121'; D-4, mp 102109'; D-5, mp 52-56'; D-6. mp 57-58"; D-i, nip 142-145";D-S, mp i2-88', and D-9. mp 149-152'. Structural determination of the new antineoplastic peptide. is n ell uiidern ay and current progress n ith dolastatin 3 provides an illustration. Result9 of field desorption mass spectrometry and elemental analyses led to tentative molecular formula C?9H&&6S2 (11- 660.2512) for dolastatin 3 (P3S5 EDjo 2 . i I lo-; pg ml); nip 133-13i': [ C Z ] ~ ~-35.5" ~D (c. 0.09, methanol). hcid hydrolysis experiments combined v i t h interpretation of the nmr, 'H nmr (at 400 1IHz). infrared and ultraviolet (fig. 1) spectral measurementq revealed
FIG.1. Ultraviolet spectrum of dolastatin 3.
that dolastatin 3 was a peptide containing leucine, proline, valine, and two thiazole amino acids. Experiments directed at defining the amino acid sequence and remaining structural features are non- in progreqs. Discovery of the thiazole ring system in dolastatin 3 was especially interesting. Such bio*ynthetic products are relatively rare. but usually very active biologically. For example. among the loner plant components bleomycin (1) ( 8 , 9. 10) in combination with vinblastine produce. a significant level of complete remissions (some curvative) in patients with testicular cancer (11, 3h) and thiostrepton (2) (12), a
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broad spectrum antibiotic, also has antineoplastic activity. Recently the Western Pacific sponge Dysitiea herbacea Keller (13) and ascidian Lissocliiium patella (14) have been found to produce, respectively, the thiazoles dysidenin (3) and ulithiacyclamide (4). The latter two marine animal thiazoles may also possess useful biological activity. Isolation of the dolastatins and completion of their structural elucidation should significantly enhance our insight into the possible role of cysteine derived thiazoles (and or dehydro amino acids and thiazolidines) in the development of curative cancer chemotherapeutic drugs. .ICKSOWLEDGMESTS Financial assistance was provided by the S a t i o n a l Cancer Institute (performed pursuant t o contracts SOl-C;\l-12308, 67018 and 97262 with the Division of Cancer Treatment, S C I , S a t i o n a l Institutes of Health, D H R J and grant numbers (2.1-1691941 through 06 awarded by t h e S a t i o n a l Cancer Institure, DHJT, the Fannie E . Rippel Foundation, Nrs. Mary Dell Pritzlaff, the Olin Foundation (Spencer T. and .Inn IT.), l l r . Jack IT. Whiteman, t h e E d n a Rider Whiteman Foundation, and the Phoenix Coca Cola Bottling Co. For other very helpful assistance we wish t o thank D r s . J . D. Douros and Ll. I . Suffness of the S a t i o n a l Cancer Institute, D r s . F. Ferrari, L . W .Knapp and G . Hendler of t h e Smithsonian Institution, D r . H. Bertsch of t h e San Diego S a t u r a l History hluseum, D r s . J . E . Rippel, C . Djerassi, J . R . Cronin, R. Inners and J . A . Hasler, Messrs. T. Krupa. P. -1.Liddell, I). T a k a t a and L . D . 1-anell, A h . C. H. Duplissa, Mrs. 31. 11. Baughnian and Miss L . 11.Lange. T h e authors gratefully acknowledge the S a t i o n a l Science Foundat ion Regional Facility at the University of South Carolina (CH76-18723 j . ReceiFed 6 February 1981 LITER-ITCRE CITED 1. G. R . P e t t i t and D . P . Gieschen, J . Cnrbohydri~tes,.\;.tcleosides, S u c l e o t i d e s , 8, 67 (1981). 2 . G , R . P e t t i t , ,J. F. D a y , J . L. Hartwell and H . B . Wood, .\-nture, 227, 962 (1970). 3. (a; G . R . P e t t i t , R . H: Ode and T. B . Harvey, 111,.L l o y d i n , 36, 204 11973): (b) G . R . P e t t i t , R . B . \-on Dreele, C;. Bollirer, P . 11.T r a d e r and P. Brown, Esfierientiil, 29, 521 119731; I C I G . I?. P e t t i t . R.H . 0 d e . T L . Herald. R . B . 1-onDreele and C:lIichel, J . .-lmer. C h e m . Sd:., 98, 1677 fl9761: i d ) G: R . P e t t i t , C.' L . Herald and D . L. Herald, J . ' P h a r n i . Sci., 65, 1558 (1976,: ( e ) G . R . P e t t i t , C. L . Herald, 11.S.Allen, R . B . \-on Dreele, L. D . T-anell, J . P. K a o and W .Blake, J . a4ti7er. C h e w . .So[., 99, 262 (1977): t f l G . R . P e t t i t , and R . H. Ode, J . Piinvi. Sci.. 66, 757 (1977): ig) G . R . P e t t i t , C . L . Herald, and L. D . T-anell, J . S c t . P r o d . , 42. 407 (1979): ih! G . R . P e t t i t , Biosynthetic Prodicctsfor Cancer Chemotherapy, 1-01. 1, (Plenum Publishing Co S e w York, S.l*.1977): ti) G . R . P e t t i t and G. 11. Cragg. Biosynthetic Prodiicis .To cer C h e v i o t h e r i i p ~ ,1-01. 2 , (Plenum Publishing Corp., e t t i t and R . H . Ode, Biosynthefic P r o d u c t s f o r Ciincer S e w l-ork, S.T.,1978): ij, G . Ciieniotliernpy, Yol. 3 , (Plenum Publishing Corp., S e x - T o r k , S . l - .1979). , Plinl-, Historin -\-ntzlrnlis, Lib. I S . , Lib. S S S I I , cii. GO A.D. !.B.C. Jfenioirs, 1-01, XS11.. on "Typical British l l a r i n e Plants and la, E d . by W . A . Hardman and J . Johnstone, (Liverpool University Press, 1921I . 6. H . Engel, Zool. M e d . Museitni Leiden, 24, (1945). 7. 1969 P h . D . dissertation of 11,Watson, U . of Hawaii, "Some Aspects of t h e Pharmacology, Chemistry and Biology of t h e Llidgut Gland Toxins of Some Hawaiian Sea Hares, esneciallr Doinbelln ~zitriczdnria and .-I .pivsin _ .Pli~nionicn," C n i v e r s i t r hlicrofilms Inc., Ann ;i;bor, Llichigan. 8. H . -Irai, IT. K . Hagmann, H . Suguna, S. 11. Hecht, J . dnrer. Cheni. SOC.,102, 6631 11980). 9. T. Shomura, S. Omoto, K. Ohba, H . Ogino, 11.Kojima and S. Inouye, J . .Antibiotics, 33, 1243 (19803. 10. H . Umezawa, T.Suhara, T . T a k i t a and K . Llaeda, J . .-Intibiotic-s, Ser. .A 19, 210 11966). 11. G. J . B o d , R . Kwong, P. H . Lange, E . E. Fraley and B . J. Kennedy, Cnncer Trentnzent R e b o r t s . 64.S o . 2-3. 331 119801. 12. M.'Bodanszky, J . *$: Scozzie and I . Muraniatsu, J . Antibiotics, 23, 9 (1970). 13. C . Charles, J . C . Braekman? D. Daloze and B . Tursch, Tetrahedron, 36, 2133 (1980) 14. C. Ireland and P. ,J. Scheurer, -1. d n i e r . Chenz. Soc., 102, 5688 (1980).
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