Synthetic studies of roquefortine C: SPECIAL FEATURE Synthesis of isoroquefortine C and a heterocycle David J. Richard, Bruno Schiavi, and Madeleine M. Joullie´ * Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323 Edited by Kyriacos C. Nicolaou, The Scripps Research Institute, La Jolla, CA, and approved April 14, 2004 (received for review April 9, 2004) The syntheses of isoroquefortine C and a related heterocycle were achieved by implementation of both intra- and intermolecular vinyl amidation reactions. These accomplishments represent a signifi- cant advance in the use of these strategies in the generation of complex molecules. etabolites produced by fungi represent one of the largest Mclasses of natural products. Such compounds vary widely in their structural composition and have found application in pharmaceuticals such as antibiotics, immunosuppressants, anti- fungal agents, and growth promoters (1). Other congeners Fig. 1. The roquefortine class of natural products. within this class display biological properties harmful to humans and other animals. These natural products have been classified as mycotoxins and are produced as secondary metabolites by an containing hemins but no potency toward Gram-negative or- CHEMISTRY array of soilborne and airborne fungi (2). Mycotoxins have been ganisms (15). Additional studies led to the finding that roque- isolated as contaminants of a variety of grain products and have fortine C inhibited bacterial RNA synthesis but only modestly been a topic of great interest for scientists concerned with affected DNA and protein production (16). veterinary health and agricultural safety. The lack of consistent toxicological data, along with the In most cases, eradication of the fungal infection responsible ubiquitous nature of P. roqueforti in human and animal food for mycotoxin production is the most reasonable method for products, establishes roquefortine C as a natural product worthy elimination of these compounds from commercial foodstuff. of synthetic investigation. The compound is also interesting from However, an interesting exception to this generalization is found a synthetic chemistry standpoint, because it possesses distinctive in the roquefortine family of natural products (Fig. 1). Roque- structural characteristics. Roquefortine C contains the unusual fortine C (1) was isolated independently by researchers in Japan E-dehydrohistidine moiety, a system that typically undergoes (3, 4) and France (5, 6) from the Penicillium roqueforti Thom facile isomerization under acidic, basic, or photochemical con- strain. This finding was significant due to the fact that this fungus ditions (17–21). This functional group is found in only two is essential for the production of Roquefort and a number of natural products, the other being oxaline (22). The total syn- other blue-veined cheeses. In subsequent communications, ro- theses of these compounds have yet to be accomplished. quefortines have been found as metabolites of additional P. Isoroquefortine C (3), or the 3,12 double-bond isomer of roqueforti strains as well as other Penicillium species isolated roquefortine C (Fig. 2), was obtained by photochemical irradi- from a variety of contaminated food products such as feed grain ation of the natural product (23). The biological activity of (7, 8), wine (9), and beer (10). The presumed biosynthetic isoroquefortine C has yet to be investigated. The goals of the precursor of roquefortine C (1), the dihydro compound roque- current synthetic efforts have been to develop a strategy appli- fortine D (2), has also been isolated from cultures of the P. cable to both roquefortine C and isoroquefortine C. A successful roqueforti fungal species (11). route to either of these compounds would allow for investigation Roquefortine C has received attention because of its neuro- of their interconversion and stability. toxic properties. Wagener and coworkers (12) described para- We previously reported the synthesis of roquefortine D (24) lytic activities in cockerels, and Frayssinet and Frayssinet found as well as the generation of isoroquefortine C using Wittig– ͞ the LD50 in mice to be 15–20 mg kg after i.p. injection (5). Horner olefination to establish the enamide stereochemistry Symptoms included prostration, seizures, and death. Support for (25). This latter method was not applicable to the synthesis of these findings was provided by Ohmomo (13), who used a similar roquefortine C. We now disclose recent efforts toward genera- ͞ mouse assay and reported an LD50 of 20 mg kg. However, tion of the dehydrohistidine system using copper-catalyzed vinyl Arnold et al. (14) were unable to reproduce this activity and amidation chemistry. This method has allowed for the synthesis found the lethal dose to be an order of magnitude larger than this of both isoroquefortine C and a polycyclic heterocycle (26). value. A nonalkaloid natural product also produced by P. Studies on the stability of isoroquefortine C and efforts to roqueforti, PR toxin, was found to possess greater toxicity. accomplish isomerization to the natural product are described A subsequent publication by Ha¨ggblom (8) further compli- also. cated the biological activity profile of roquefortine C. In this Due to the questionable stability of the dehydrohistidine finding, roquefortine C, but not PR toxin or other mycotoxins, moiety in the roquefortine system, palladium- or copper- was isolated from a grain sample that produced paralytic symp- toms in cows. These effects disappeared as soon as the cows were no longer fed moldy grain. Another report investigated the This paper was submitted directly (Track II) to the PNAS office. effects of this metabolite on bacterial growth and showed it to *To whom correspondence should be addressed. E-mail: [email protected]. possess inhibitory properties toward Gram-positive bacteria © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0401407101 PNAS ͉ August 17, 2004 ͉ vol. 101 ͉ no. 33 ͉ 11971–11976 Downloaded by guest on September 25, 2021 6.6 Hz, 1H), 4.18 (dd, J ϭ 9.6, 6.8 Hz, 1H), 5.09 (d, J ϭ 17.3 Hz, 1H), 5.16 (d, J ϭ 10.7 Hz, 1H), 5.39 (s, 1H), 5.47 (broad s, 2H), 6.01 (dd, J ϭ 17.3, 10.7 Hz, 1H), 6.83 (broad s, 1H), 7.00–7.05 (m, 2H), 7.12–7.40 (m, 17H), 7.52 (s, 1H), 8.21 (s, 1H); 13C NMR ␦ (125 MHz, CDCl3) 23.0, 23.1, 39.1, 41.0, 59.1, 62.3, 76.2, 86.8, 114.7, 114.9, 123.5, 125.5, 125.6, 128.2, 128.3, 129.2, 129.7, 133.4, 134.3, 138.4, 141.9, 143.3, 151.8, 169.5, 172.9; IR (neat) 3,331 (w, broad), 3,170 (w, broad), 2,966 (w), 1,690 (s), 1,661 (m), 1,595 (w), 1,472 (m), 1,449 (m), 1,384 (m) cmϪ1; high-resolution MS ͞ ϩ ϩ (electrospray) m z calculated for C41H37N5O2Na (M Na) : Fig. 2. Photochemical isomerization of roquefortine C to isoroquefortine C. ͓␣͔20 ϭϩ 654.284495; found: 654.283253; D 21.5 (c 0.55, CHCl3). Hexahydropyrroloindoleimidazolidinone (22). A solution of trityl- catalyzed vinyl amidation offered great promise (Fig. 3). The protected imidazole 21 (0.020 g, 0.032 mmol) and hydroxyben- transformation proceeds with stereospecificity and under rela- zotriazole (0.0129 g, 0.095 mmol) in trifluoroethanol (0.5 ml) was tively mild conditions (27, 28). A large number of catalyst stirred at room temperature for 48 h. The solution was diluted O systems have been reported for the analogous nitrogen carbon with water and extracted with ethyl acetate (10 ml). The organic bond-forming reactions involving amines or amides with aryl layer was dried over sodium sulfate and concentrated under halides or triflates. In contrast, the use of vinyl systems with reduced pressure. The crude product was purified by column amines (29, 30) or amides (31, 32) has only recently received chromatography (80% acetone͞hexanes) to yield 22 as a white ϭ ͞ attention. Therefore, investigation offered the potential for foam (0.0099 g, 80%). Rf 0.22 (10% methanol methylene 1 ␦ significant extension of this methodology. chloride); H NMR (500 MHz, CDCl3) 1.01 (s, 3H), 1.13 (s, 3H), 2.64 (s, 1H), 2.69 (dd, J ϭ 14.0, 9.6 Hz, 1H), 3.25 (dd, J ϭ Materials and Methods 14.0, 6.4 Hz, 1H), 4.32 (dd, J ϭ 9.6, 6.4 Hz, 1H), 5.11 (dd, J ϭ Experimental data for compounds 5–9, 11–14, 16, 17, 19, 20, and 17.6, 1.1 Hz, 1H), 5.19 (dd, J ϭ 11.0, 1.1 Hz, 1H), 5.47 (s., 1H), 23–26 are available in Supporting Materials and Methods, which 5.52 (broad s, 1H), 6.02 (dd, J ϭ 17.6, 11.0 Hz, 1H), 6.79 (broad is published as supporting information on the PNAS web site. s, 1H), 6.94 (d, J ϭ 7.7 Hz, 1H), 6.99 (s, 1H), 7.09 (dt, J ϭ 7.7, 1.0 Hz, 1H), 7.26 (m, 1H), 7.29 (m, 1H), 7.34 and 7.43 (m, 1H, Trityl-hexahydropyrroloindoleimidazolidinone (21). Vinyl bromide conformers), 7.69 and 7.94 (m, 1H, conformers); 13C NMR (125 ␦ 20 (0.175 g, 0.238 mmol), cuprous iodide (0.0045 g, 0.024 mmol), MHz, CDCl3) 23.0, 23.1, 39.2, 41.0, 58.9, 62.2, 87.6, 114.8, 115.0, and finely powdered potassium carbonate (0.066 g, 0.476 mmol) 116.8, 124.0, 125.8, 125.9, 129.4, 134.1, 134.4, 136.6, 137.2, 143.1, were added to a thick-walled pressure tube, and the vessel was 151.7, 170.4, 171.9; IR (neat) 3,203 (m, broad), 2,956 (m), 2,923 fitted with a rubber septum.