Anisomycin

Two naturally occurring 2-aralkyl pyrrolidines are included in this part The first one is the antibiotic anisomycin, which has various important therapeutic values. The second one is preussin, which exhibits antifungal activity. 

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Anisomycin (Sigma) Stock solution of 10 mM in DMSO stored at —20°. 

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

The pyrrolidine derivative 314, a skeletal analog of the antitumor antibiotic anisomycin, was synthesized from the acetal derivative 16b. The 5-OH group of 16b was tosylated and then substituted with sodium azide. Reduction (sodium borohydride) of the lactone group afforded an open-chain derivative, which was selectively protected to give 313. Hydrogenation of the azide function, followed by p-toluenesulfonylation, led to 314 by an intramolecular nucleophilic displacement (284). 

The reactions depicted in Scheme 39 were already conducted in view of a potential use in the synthesis of pyrrolidinols and piperidinols. The structural feature of a 2-arylmethyl-3-hydroxysubstitution is not only found in preussin but also in anisomycin (152) [87] or in the piperidine alkaloid FR 901483 (153) [88] (Fig. 5). 

We have extended our work on a new synthesis of the antiprotozoal antibiotic anisomycin to the necine bases of the pyrrolizidine alkaloids, in particular retronecine and crotanecine. The key intermediate, (2R,3S,4R)-2-(alkoxy-carbonylmethyl)-3,4—isopropylidenedioxypyrrolidine, has been prepared by three distinct routes from D-ribose and g-erythrose, using reactions of high stereoselectivity. 

A new approach to anisomycin from D-erythrose using Wittig methodology is outlined. 

We were first attracted to chiral pyrrolidines by the possibility of applying methods used in C-nucleoside synthesis (1) to the synthesis of the antiprotozoal antibiotic anisomycin (p from D-ribose (2). 

Scheme 2 illustrates a synthesis of the benzyloxycarbonyl derivative (H) of the ester (9a) using chemistry analogous to that for anisomycin (Scheme 1). In the formation of the D-allo-triol (.12),...

The triflate of (39) has been converted into the pyrrolidine (4 directly by treatment with ammonia. The 6-configuration, inappropriate for a synthesis of anisomycin, was expected, assuming that displacement of the triflate group is the first stage in the reaction. We have so far been unable to rearrange the 6-isomer (45) into the required a-series. This work is being continued. 

Alice et al studied the turnover kinetics of Listeria OTonocytogenex-secreted p60 protein (a murein hydrolase) by host cell cytosolic proteasomes. J774 cells, seeded in flasks and incubated overnight in culture medium, were infected with log-phase cultures of E. monocytogenes for 30 min, washed, and incubated in culture medium for 3 h, with gentamicin (50 tg/ml) added after the first 30 min to inhibit extracellular bacterial growth. Cells then were washed and placed in methionine-free medium with spectinomycin, gentamicin, the eukaryotic protein synthesis inhibitors [cycloheximide (50 tg/mL) and anisomycin (30 tg/ml),] and 25 dVI calpain inhibitor I. After 30 min, [ S]methionine was added, and the cells were pulse-labeled for periods of 20 to 60 min. Cells... 

The (—)-anisomycin work is presented in Scheme 35. Its key step centered around the formation of a pyrrolidine ring that possessed all three of the asymmetric centers present in the target this was done by nucleophilic displacement of a 3-tosyloxy function in an appropriately functionalized 6-amino-6-deoxy-p-i.-talose derivative, whose 1,2-diol was later released and oxidatively cleaved with sodium periodate. Grignard coupling, O-acetylation, and catalytic hydrogenation then furnished the desired natural-product target. 

SCHEME 35. Richardson s total synthesis of (—)-anisomycin with Ram Bhatt, John Moffatt, and Julian Verheyden (and others) at Syntex in 1975. 

Another impressive example is the synthesis of ( + )-anisomycin, the unnatural enantiomer, in 88-90% ee and 22% overall yield, the key step being an asymmetric alkylation of 2,5-dihydro-1//-pyrrole44. ... 

J. G. Moffat Chiral synthesis of the antibiotics anisomycin and pentenomycin A. S. Perlin Aspects of the chemistry of glycosides... 

Frey U, Krug M, Reymann KG, Matthies H (1988) Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro. Brain Res 452 57-65... 

Cano, E., Hazzalin, C.A., and Mahadevan, L.C. 1994. Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol Cell Biol 14 7352-7362. 

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