Cephalostatins isolation

Ritterella tokioka Kott, a tunicate of the family Polyclinidae, collected from the Izu Peninsula 100 km southwest of Tokyo, on extraction and partitioning between a number of solvent systems followed by repeated chromatography, afforded another series of bis-steroidal pyrazine alkaloids, ritterazines A-M (26-38). Structures of these alkaloids were found to be related to the cephalostatins isolated from the hemichordate Cephalo-discus gilchristii. 

Pettit et al., have proposed a biogenetic pathway for the dimeric pyrazine-containing steroidal alkaloids, the cephalostatins isolated from the marine worm Cephalodiscus gilchristi and the tunicate Ritterella tokioka. These alkaloids contain at least 13 fused rings, which constitutes the largest such system known in marine animals. 

In the last two years, Pettit s group has reported the structure of eight more cephalostatins isolated from a 450 kg collection of C. gilchristi made in 1990. Cephalostatins 10 and 116 (Figure 4) are the C-l and C-l methoxy derivatives of cephalostatin 2 respectively. Cephalostatin 10 thus represents the first member with a structural change in the right half, which has remained constant in cephalostatins 1 through 9. In their cytotoxic effects, both these cephalostatins are of similar activity to cephalostatin 1. 

In general, the cephalostatins isolated recently are in relatively minor abundance compared to cephalostatin 1 (for example, only 3.8 mg, 8xl0 7 % yield, of cephalostatin 17 was obtained), and of lower activity except for cephalostatins 16 and 17. The Pettit group has also detected10 other new cephalostatins in very small quantities (approximate yield of 10 8 %) with promising activity against brain cancer xenografts. 

Together with cephalostatins, isolated from the marine worm Cephalodiscus gilchristi, they make up a imique family of 45 trisdecacyclic bis-steroidal pyrazines (Figures 1 and 2) that display extremely potent cytotoxicity against human tumors with unique cell selectivity and apoptotic response [Reviewed in 109, 110],... 

Cephalostains 1-6 (507-512), powerful cell growth inhibitory substances against the PS cell line, were isolated from the marine worm Cephalodiscus gilchristi collected in the Indian Ocean 406-408). The structure of cephalostatin 1 (507) was determined by X-ray analysis. Cephalostatins apparently result from a biosynthetic condensation of 2-amino-3-oxosteroid units to yield dimeric steroidal molecules connected by a pyrazine ring. 

The synthesis of the potent in vitro active compound, cephalostatin 1, isolated from a rare marine worm, Cephalodiscus gilchristi, has recently been reported, and scale-up... 

Pettit et al. (24), working on the marine worm Cephalodiscus gilchristi (order Cephalodisicida, phyla Hemichordata, class Pterobranchia) collected from the Indian Ocean off Southeast Africa, have isolated a number of bioactive bis-steroidal pyrazine alkaloids, the cephalostatins 1-15 (11-25). 

A sequence (using P-388 guided bioassay) of steric exclusion and partition-type chromatographic separations of the active butanolic fraction by reversed-phase semipreparative HPLC led to the isolation of cephalo-statin 12 (22), CaHyeNzOjz, [a]D 157° (CH3OH), (M + H)+ m/z 945.5444, and cephalostatin 13 (23), [a]D 108° (CH3OH), (M + H)+... 

Four years after the isolation of cephalostatin A, the first synthetic studies in this area were reported. Fuchs etal. (38) reported the conversion of steroidal a-azidoketones 48 to the C-2 symmetrical nonacyclic and trisdecacyclic pyrazines 49 through catalytic reduction (Scheme 1). In the process they also observed an unusual azide-mediated formation of an unsymmetrical heterobenzyl azide (Scheme 2). Use of excess azide served as a base to generate the a-aminoenone 50. Dimerization of 50, followed by SN2 reaction with the hydrazoic acid coproduct yielded the unsymmetrical azidopyra-zine 51. 

The bis-steroidal alkaloids cephalostatins 1-15 (11-25) (24-30) and rit-terazines A-M (26-38) (31-33) isolated from the marine worm Cephalodis-cus gilchristi and the tunicate Ritterella tokioka, respectively, also exhibit powerful cell growth inhibitory activity against a number of cell lines. 

The biosynthesis of these compounds seems to occur in two phases (1) coupling of two steroids via a pyrazine linker, and (2) relatively unselective oxidation at various positions. Some of these compounds are related to others by simple processes the hydration of cephalostatin 1 to its hemiketal form cephalostatin 9 the dehydration of cephalostatin 2 to an enone, which in turn may be an intermediate to cephalostatin 6 the skeletal rearrangement of ritterazine B to ritterazine A, which may be acid catalyzed and the pairs of ritterazines epimeric at C-22. One can speculate whether such reactions are non-enzymatic transformations occurring in the organism or even during the isolation procedure. 

Subsequent to the isolation of the cephalostatins, there was renewed interest in such steroid dimers. An improved procedure for the preparation of 4 was reported16 by the Fuchs group (Scheme 5), in which the intermediate a-amino ketone was produced in higher yield by the sequence of bromination with phenyltrimethylammonium bromide, displacement by azide to give a-azido ketone 5, and hydrogenation (an identical route was independently developed17 in the Heathcock group). 

The most significant group of bioactive metabolites to be isolated from southern African marine worms have undoubtedly been the powerful cell growth inhibitors, cephalostatins 1-17 (1-17), isolated by Pettit et al. from... 

Comparative evaluation of 1 and cephalostatins 14 and 15 (14, 15) in the NCI in vitro primary screen again revealed a reduced average GI50 cytotoxicity (100 nM and 68 nM respectively) for 14 and 15 compared to 1 [23]. The final pair of cephalostatins (16 and 17) isolated from C. gilchristi exhibited the same cytotoxic profile as the majority of the other cephalostatins with the panel averaged GI50 values of 16 (InM) and 17 (4nM) comparable to that of 1 (InM) [24],... 

In 1974, extracts of the tiny ( 5 mm) marine tube worm Cephalodiscus gilchristi collected off South Africa were found to be active in the NCI s primary assay, the murine lymphocytic leukemia P388. Fifteen years of relentless research by Pettit s group culminated in the isolation of 139 mg of the major bioactive component, cephalostatin 1, from 166 kg wet weight of the tube worm, and its structural elucidation. [2] This phase was summarized [3] as follows Interest in such a powerfully antileukemic agent as cephalostatin 1. .. has prompted Americans to dive extensively at a depth of 20 meters to collect... 

The 45 members of the cephalostatin/ritterazine family isolated to date, together with a growing number of analogues and related monosteroidal antineoplastics provide the basis for elueidating some structure -activity relationships (SAR) of these potent cytotoxins and for diseovering the minimum pharmacophore required to maintain potent cancer cell growth inhibitory behavior. 

Subsequently, several other cephalostatins were isolated from the same species, Cephalodiscus gilchristi, the two most recent being cephalostatins 18 and 19, characterized in 1998. All these derivatives have major structural similarities since all cephalostatins currently known are derived mainly from cephalostatin 1, the major component in mixtures, by modification of the west moiety, or from cephalostatin 2 by modification of the east moiety (Figures 27.2 and 27.3). Four publications deal with the structures, antitumor activities, and synthesis of cephalostatins (Ganesan, 1996 Li and Dias, 1997 Dirsch and Vollmar, 2005 Lopez-Anton et al., 2006 Moser, 2008 Rudy et al., 2008). 

Moser, B. (2008) Review of cytotoxic cephalostatins and ritterazines isolation and synthesis./. Nat. Prod., 71, 487—491. 

Pettit, G.R., Ichihara, Y, Xu, J., Boyd, M.R., and Williams, M.D. (1994a) Isolation and structure of the symmetrical disteroidal alkaloids cephalostatin 12 and cephalostatin 13. Bioorg. Med. Chem. Lett., 4,1507-1512. 

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