The Southern Indian Ocean marine worm Cephalodiscus gilchristi has been found to yield new cephalostatins 7-9. Tube-inhabiting marine animals of the genus Cephalodiscus (one of two divisions in the class Pterobranchia, Hemichordata Phylum) are rarely encountered. Only some 18 species are presently known and confined primarily to Antarctica. One Southern Hemisphere temperate region species Cephalodiscus gilchristi was recorded off the coast of South Africa in 1906 and described in more detail in 1915-17. In 1988 we summarized results from the first chemical study of this genus and isolation of the powerful (P388 EDw 10-7-10-9 pg/mL) cell growth inhibitor cephalostatin 1 (1) from C. gilchristi. Subsequently we described cephalostatins 2-4 and 5 where introduction of an aromatic C'-ring (cf. 2 corresponding to cephalostatin 6) was found to greatly reduce (P388 EDw N pg/mL) the cytostatic activity. We now report that further detailed investigation of C. gilchristi antineoplastic constituents has led to the discovery of three new and remarkable cephalostatins designated 7-9 that exhibit potent growth inhibitory and cytotoxic activity against diverse human solid tumor types in the US. National Cancer Institute's new in vitro, disease-oriented antitumor screen. A 28-g methylene chloride fraction prepared from 166 kg (wet wt) of C. gilchristi was separated (P388 lymphocytic leukemia cell line guided) to provide fractions h (8 mg), 1 (20 mg), and m (33 mg) using a series of steric exclusion and partition chromatographic procedures followed by gradient HPLC, as previously outlined. Continued bioassay-directed separation of fraction m employing a gel (Sephadex LH-20) partition - HPLC - gel permeation sequence afforded (18.7 mg, 1.1 X 10^-6% yield) cephalostatin 7 (3); amorphous powder, dp 315 "C; Rf 0.39 (SiO2) in 90:10:0.8 CH2Cl2/CH3OH/H2O; [(Y]D +106" (c 0.244 in CH3OH); SP-SIMS found 967.5067 [M + K]+ for C51H76N2O11 (calcd 967.6413 and 928.5450 for M+); W (CH3OH) A,, 286 (c 17400) and 310 (shoulder) nm; IR (KBr) 3430, 2960, 2920, 2880, 2860, 1715 (weak, broad), 1650-1615 (broad), 1450,1400,1385,1056,1043, and 950 cm^-1; for the 'H- and 13C-NMR see ref. 12. Analogous separation of fractions h and 1 provided (3.4 mg, 2 X 10^-7% yield and 9.2 mg, 5.5 X recovery, respectively) cephalostatin 8 (4) [powder, dp 313 "C; Rf 0.35 (SiO2) in 90:10:0.8 CH2Cl2/CH3OH/H2O; [(Y]D +110" (c 0.10 in CH3OH); SP-SIMS found 965.5261 [M + K]+ for C55H78N2O10 (calcd 965.6640 and 926.5657 for M+); W (CH3OH) A- 286 (e 20800), 310 (shoulder) nm; IR (KBr) 3400,2970,2920,2880,2860,1715 (weak, broad), 1650-1600 (broad), 1445, 1400, 1385, 1037, and 965 cm^-1] and cephalostatin 9 (5) [powder dp 307 "C; Rf 0.38 (SiO2 90:10:0.8 CH2Cl2/CH3OH/H2O; [(Y]D +105" (c, 0.496 in CH3OH); SP-SIMS found 929.5527 [M + HI+ for C54H78N2O11 (calcd 929.5527 and 928.5450 for M+); W CH3OH A- 286 (e 18600) and 310 (shoulder) nm; IR (KBr) 3400, 2990, 2930,2880,2860,1710 (strong, sharp), 1645-1630 (broad), 1445,1400,1385,1055, and 950 cm^-1]. With the X-ray crystal structure of cephalostatin 1 (1) and the corresponding 'H- and 13C-NMR assignments providing a solid foundation for related structural determinations we were able to assign structures 3-5 respectively to cephalostatins 7-9. While the right-side moiety of pyridizines 7-9 proved identical with that of cephalostatin 1 (l), beyond the left-side C'-ring of each were found substantial differences. Structural elucidation of cephalostatin 7 (3) primarily by high field two-dimensional 'H- and 13C-NMR provides an appropriate illustration. Partial structure A for rings D'-F' was derived from the result of heteronuclear multiple bond correlations (HMBC). Application of comparable H,H-relayed COSY, NOE, and HMBC experiments to cephalostatin 8 resulted in structure 4 as most consistent with the spectral data with only the stereochemistry at (2-22' remaining equivocal. The same approach was also successful with cephalostatin 9 (5) again excepting the stereochemistry at C-22'. The configuration of the C-23' hydroxyl is likely R as shown, and established in cephalostatin 1 (1). Cephalostatins 7-9 (3-5) displayed remarkable potency with TIb0 (molar) values of 10-9-<10-10 against a number of (e.g., non-small cell lung HOP 62, small cell lung DMS-273, renal RXF-393, brain U-251 and SF-295, and leukemia CCRF-CEM, HL-60, and RPM1-8226) cell lines and values of 10-8-10-9 for the breast MCF-7 cell line; the "mean graphs" (see ref 10 for definition and interpretation) showing the patterns of relative cellular sensitivity across the panel of 60 cell lines were remarkably similar, if not essentially indistinguishable, for cephalostatins 1-4 and 7-9. By contrast, cephalostatins 5 and 6 (2) proved to be modestly cytotoxic against only two of these human cell lines (renal SNl2Kl and CNS U-251) with potency reduced to GIM 10-7-10-8 molar. Discovery of cephalostatins 1-4 and 7-9 with potent cytotoxicity against certain human cancer cell lines suggests that the pyridizine rightrside unit is easential for such biological activity. Minor configuration and substitution (including an additional methyl in cephalostatin 8) alterations in the left-side E'- and F'-rings appear to have little influence on cytotoxic activity, but aromatization of the C'-ring with concommitant bonding to the side-chain system (cf. 2) markedly diminishes the potency. Perhaps cephalostatins 5 and 6 (2) represent a biosynthetic misadventure in the long evolutionary history of Cephalodiscus. Equilibrium constanta in THF for dimerization of several cesium enolates of benzylic ketones show a decrease as the ion-pair acidities of the parent ketones increase when steric factors are comparable. Many enolate salts are known to be aggregated in synthetically important ethereal solvents such as THF. With increasing delocalization of the carbanionic charge the enolate ion becomes less effective in solvating the cation compared to solvent dipoles and aggregation is expected to be less important. This principle has been established qualitatively but not quantitatively. Arnett and Moe have recently shown that aggregation numbers of some lithium enolates and related phenolates depend also on steric bulk and the type of acidic group. In the present work we present the first determinations of equilibrium constants for dimerization of some cesium enolates and show that the principle is established quantitatively for several ketones when steric effects are approximately constant. The ketones selected for study are the aryl-substituted acetones, 1,3-diphenyl- (l), 1,3-di(l-naphthyl)- (2), 1,3 di(Cbiphenyly1)- (3), and 1,1,3,3-tetraphenylacetone (4).