Angewandte Chemie DOI: 10.1002/anie.201405990 Natural Products Hot Paper Forazoline A: Marine-Derived Polyketide with Antifungal In Vivo Efficacy** Thomas P. Wyche, Jeff S. Piotrowski, Yanpeng Hou, Doug Braun, Raamesh Deshpande, Sean McIlwain, Irene M. Ong, Chad L. Myers, Ilia A. Guzei, William M. Westler, David R. Andes, and Tim S. Bugni* Abstract: Forazoline A, a novel antifungal polyketide with The high mortality rate[2] combined with the continued rise in in vivo efficacy against Candida albicans, was discovered using fungal resistance to current therapeutics[4] demonstrates the LCMS-based metabolomics to investigate marine-invertebrate- ever-present need for new therapeutics. associated bacteria. Forazoline A had a highly unusual and As part of a drug discovery program to discover novel unprecedented skeleton. Acquisition of 13C–13C gCOSY and antifungal therapeutics, we analyzed a collection of marine- 13C–15N HMQC NMR data provided the direct carbon–carbon invertebrate-associated bacteria using LCMS-based metab- and carbon–nitrogen connectivity, respectively. This approach olomics in conjunction with disc diffusion assays. Metabolo- represents the first example of determining direct 13C–15N mics methodology has shown great promise for streamlining connectivity for a natural product. Using yeast chemical the discovery of novel natural products[5] and overcoming genomics, we propose that forazoline A operated through historic barriers such as the high rate of rediscovery of known a new mechanism of action with a phenotypic outcome of compounds.[6] disrupting membrane integrity. Using metabolomics-based strategies which we previously published,[5] we analyzed LCMS profiles of 34 marine-derived Fungal infections result in over 1.5 million deaths annually bacterial extracts by principal component analysis (PCA) and worldwide and cost $12 billion to treat.[1] Candida spp. are the identified the strain WMMB-499, an Actinomadura sp. most common fungal infections, especially in intensive care cultivated from the ascidian Ecteinascidia turbinata (Herd- units, in solid-organ transplant patients, and in blood- and man, 1880), as a metabolic outlier. After fermentation and marrow-transplant patients.[2] Of the more than one hundred subsequent purification, WMMB-499 was found to produce known Candida spp., Candida albicans is the most common three distinct classes of novel compounds. The first class, cause of fungal-born human disease.[2] C. albicans causes halogenated electrophilic polyketides halomadurones A–D various infections, including candidiasis, which affects about which activate the Nrf2-ARE pathway, was recently de- 400000 people per year, with an astonishingly high mortality scribed.[7] Forazoline A (1) and B (2), described herein, rate between 46 and 75%.[2] represented the second novel class. The third class was While amphotericin B has remained the standard for represented by a potent antibiotic, which is still under study. treatment of severe systemic fungal infections, it suffers from Forazoline A (1) was the lead antifungal agent with a highly low solubility and is associated with dose-limiting toxicity.[3] unusual and unprecedented structure. Forazoline A (1) [*] T. P. Wyche, Dr. Y. Hou, D. Braun, Prof. Dr. T. S. Bugni J.P., I.O., and S.M. are funded by the DOE Great Lakes Bioenergy Pharmaceutical Sciences Division Research Center (DOE BER Office of Science DE-FC02-07ER64494). University of Wisconsin-Madison, Madison, WI 53705 (USA) C.M. and R.D. are supported by grants from the National Institutes E-mail: [email protected] of Health (1R01HG005084-01A1, 1R01M104975-01, Dr. J. S. Piotrowski, S. McIlwain, I. M. Ong R01HG005853), a grant from the National Science Foundation (DBI Great Lakes Bioenergy Research Center 0953881), and by the CIFAR Genetic Networks Program. We would University of Wisconsin-Madison, Madison, WI 53726 (USA) like to thank the Analytical Instrumentation Center at the University of Wisconsin-Madison for the facilities to acquire spectroscopic R. Deshpande, C. L. Myers data. This study made use of the National Magnetic Resonance Department of Computer Science and Engineering, University of Facility at Madison, which is supported by NIH grants P41RR02301 Minnesota-Twin Cities, Minneapolis, MN 55455 (USA) (BRTP/NCRR) and P41M66326 (NIGMS). Additional equipment Dr. I. A. Guzei was purchased with funds from the University of Wisconsin, the Department of Chemistry, University of Wisconsin-Madison NIH (RR02781, RR08438), the NSF (DMB-8415048, OIA-9977486, Madison, WI 53706 (USA) BIR-9214394), and the USDA. We would like to thank D. Demaria for Dr. W. M. Westler assistance with collection and Dr. R. McClain for assistance with Department of Biochemistry, University of Wisconsin-Madison ICP-AES. The yeast deletion collection was kindly provided by Madison, WI 53706 (USA) Charlie Boone. CD data were obtained at the University of Prof. Dr. D. R. Andes Wisconsin-Madison Biophysics Instrumentation Facility, which was School of Medicine, University of Wisconsin-Madison established with support from the University of Wisconsin and Madison, WI 53705 (USA) grants BIR-9512577 (NSF) and S10RR13790 (NIH). [**] We acknowledge financial support from the University of Wisconsin- Supporting information for this article is available on the WWW Madison School of Pharmacy. This work was also funded by the under http://dx.doi.org/10.1002/anie.201405990. NIH, NIGMS Grant R01 GM092009, and in part by R01 GM104192. Angew. Chem. Int. Ed. 2014, 53, 1 – 5 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 These are not the final page numbers! Ü Ü Angewandte. Communications that N43 (d = 140.2 ppm) was attached to C42 (d = 166.5 ppm) and C38 (d = 76.2 ppm), and N37 (d = 302.6 ppm) was attached to C36 (d = 170.3 ppm) and C34 (d = 76.3 ppm). The spectrum also confirmed the presence of two N-dimethyl groups. The downfield shift of N37 and the downfield shift of C36, combined with the 13C–15N correla- tions, confirmed the thiazoline structure. While analysis of the 13C–13C COSY and 13C–15N HMQC data rapidly provided the majority of the planar structure, additional data were necessary to complete structure eluci- dation. The presence of the sulfoxide was indicated by an absorption in the IR spectrum at 1060 cmÀ1. Additionally, demonstrated in vivo efficacy against the fungal pathogen having accounted for all carbon substituents, logically, the Candida albicans and works by a putative novel mechanism. remaining oxygen atom was likely attached to the sulfur atom. Forazoline B (2), a brominated analogue, was also produced Apratoxin, isolated from the marine cyanobacterium Lyng- to aid in structure elucidation. bya majuscula,[10] had a thiazoline ring. And more recently, Analysis of forazoline A (1) by HRMS supported the apratoxin sulfoxide, which contains a thiazoline moiety with [11] 13 molecular formula of C43H69ClN4O10S2. The MS isotopic a sulfoxide, was isolated. The C NMR shift of the distribution suggested the presence of one chlorine atom thiazoline methylene in apratoxin and apratoxin sulfoxide 13 and two sulfur atoms. HRMS of 1 in CD3OD and D2O was d = 37.6 and 58.3 ppm, respectively. The C NMR shift of revealed the presence of two exchangeable protons. Acquis- the thiazoline methylene (C35) in 1 was d = 38.8 ppm, similar ition of a 1H–15N HSQC allowed us to conclude that one of to that of apratoxin and indicating that the sulfoxide was most the exchangeable protons was on an enamine (dH = likely not part of the thiazoline moiety in 1. To support this 14.58 ppm, dN = 70.3 ppm). To determine the location of the hypothesis, low-energy conformers of the two possible other exchangeable proton, 1 was acetylated.[8] Two major regioisomers were determined with Spartan10,[12] and DFT products, mono- and bis(acetyl) analogues were formed, and NMR calculations (Gaussian09[13]) of the low energy con- acquisition of one-dimensional (1D) and two-dimensional formers were analyzed with the DP4 probability method. The (2D) NMR spectra determined that the other exchangeable DP4 method uses a mathematical algorithm to compare proton was an OH at C11. calculated and experimental NMR shifts and determine Extensive 1D and 2D NMR data[8] (see Table S14 in the which structure fits the experimental data better.[14] It Supporting Information) were analyzed to establish the should be noted that these modeling studies were done after majority of the planar structure. However, the structure much of the configuration had been established as outlined elucidation presented several challenges. Therefore, we below. Each of the two possible sulfoxide diastereomers was unambiguously determined the carbon backbone by fermen- also investigated using Spartan10 and Gaussian09.[8] After tation with uniformly labeled 13C glucose and acquisition of comparing the calculated 13C chemical shifts of the two a 13C–13C gCOSY spectra, an approach we previously structures and the corresponding set of diastereomers with demonstrated as useful for determining carbon–carbon con- the observed chemical shifts of 1, the DP4 probability method nectivity.[9] Fermentation of WMMB-499 with 13C-labeled predicted a 100.0% probability that the sulfoxide was glucose yielded 1 with approximately 75% 13C incorporation. attached to C40 and C42. Notably, for the structure with the The 13C–13C gCOSY spectra was acquired in 30 minutes on sulfoxide in the thiazoline moiety, DFT calculations resulted 7.0 mg of 1 and allowed complete assignment of the carbon in a 13C chemical shift of d = 58.6 ppm for C35, which is nearly backbone. identical to the methylene in apratoxin sulfoxide (Figure 1). To help confirm the location of the chlorine atom, the Meanwhile, DFT calculations for 1 predicted a 13C chemical amount of KBr in the fermentation medium ASW-A was shift of d = 43.3 ppm at C35. Thus, the location of the increased from 0.1 glÀ1 to 10 glÀ1 to produce the brominated sulfoxide in 1 was supported by both chemical shifts of analogue 2.