Kurt F. Sundermann

Semisynthesis and Cytotoxicity of Hypothemycin Analogues

This subset of the kinome accounts for less than 10% of all identified kinases but includes several targets implicated in aberrant cellular proliferation such as ERK, MEK, FLT, and PDGFR. In cell culture, hypothemycin displays potent cytotoxicity against cancer lines that are dependent on certain activating kinase mutations, particularly the BRAF V600E mutation found in approximately half of all melanomas. Additionally, hypothemycin demonstrates significant tumor growth inhibition in at least three separate murine xenograft models. Given these promising in vitro and in vivo results, hypothemycin is an attractive lead compound in the search for new targeted anticancer therapeutics. The goal of the research described herein involves the evaluation of the hypothemycin cytotoxicity structure–activity relationship (SAR) to ultimately facilitate the identification of potent analogues with optimized pharmacological properties, such as increased aqueous solubility and improved bioavailability. A number of RALs have previously been identified as kinase inhibitors; however, very little is known about the structure–cytotoxicity relationship for these compounds. Early SAR studies indicate that hypothemycin and related RALs require a C6’–C8’ (Z)-enone for both potency and selectivity of kinase inhibition. The basis for this assertion is the finding that L-783,277 (2) is approximately 400-fold more potent in MEK inhibition assays than its semisynthetic dihydro derivative (3). Furthermore, olefin geometry at C7’–C8’ is of critical importance as RALs with (E)-enones, such as 4, are substantially less potent kinase inhibitors than their (Z)-enone analogues. Structural diversity is better tolerated at C1’–C2’, as RALs containing either a trans-epoxide (1), an (E)-olefin, or a fully saturated C1’–C2’ hydrocarbon (2) inhibit kinases with IC50 values in the low nanomolar range. To identify the structural requirements for cytotoxicity, we focused on semisynthetic modifications of two RALs that are readily available through fermentation, hypothemycin (1) and 4-O-demethylhypothemycin (5). Hypothemycin (1) was subjected to a variety of reaction conditions targeting the manipulation of its C4’,C5’ diol (Scheme 1). Methylation, using excess (trimethylsilyl)diazomethane and tetrafluoroboric acid, provided the three O-methyl analogues 6, 7, and 8. The C4’ hydroxyl group appeared more reactive than its C5’ counterpart based on relative yields of the monomethyl products 6 and 7 (3:1 respectively). Hypothemycin was also treated with 4-toluenesulfonic acid (pTsOH) in dimethoxymethane in an attempt to tether the diol as a cyclic methylidene acetal, but this reaction failed to generate the desired product. Instead acyclic methoxymethyl (MOM) ethers 9, 10, and 11 were isolated. Again, the C4’ hydroxyl appeared more reactive than the C5’ hydroxyl as the mono-MOM ethers 9 and 10 were recovered in 4:1 relative yield. Although cyclic acetal formation was unsuccessful, the C4’,C5’ diol could be tethered as a cyclic carbonate. Treatment of hypothemycin (1) with triphosgene and pyridine did generate a C4’,C5’ carbonate. This reaction, unfortunately, also resulted in undesired isomerization of the C7’–C8’ olefin to provide 12 (Scheme 1). Efforts to prevent olefin isomerization to the (E)-configuration included the use of alternate bases (NaH or di-tert-butyl pyridine) and various carbonylating reagents (phosgene or carbonyl diimidazole), but each failed to generate the desired (Z)-C7’–C8’ isomer of 12. The C4’,C5’ diol of hypothemycin was also reactive toward isocyanates (Scheme 1). The C4’ monocarbamate 13 and dicarbamate 15 were recovered upon treatment of 1 with trichloroacetyl isocyanate followed by hydrolysis. To selectively produce sufficient quantities for characterization and biological evaluation of the C5’ monocarbamate 14, a three-step procedure was employed. Selective 4’-OH silyl protection was followed by carbamoylation of the 5’-OH. Acidic desilylation with concomitant hydrolysis of the intermediate trichloroacetyl group yielded the desired monocarbamate 14. Finally, hypothemycin was selectively activated with trifluoromethanesulfonic anhydride (Tf2O) then treated with 1,8diazabicycloACHTUNGTRENNUNG[5.4.0]undec-7-ene (DBU) to provide the C4’–C5’ [a] Dr. B. R. Hearn, Dr. K. Sundermann, J. Cannoy, Dr. D. V. Santi Department of Chemistry Kosan Biosciences, Inc. 3832 Bay Center Place, Hayward, California 94545 (USA) Fax: (+1)510-732-8401 E-mail : hearn@kosan.com

Fragment Assembly: An Alternative Approach to Generating Complex Polyketides

Abstract A new approach to the preparation of complex polyketide natural products has been outlined in which the products of biosynthesis are used as starting materials for chemical synthesis of difficult‐to‐obtain natural products.