Personal pdf file for Jing Xu, Yong Chun Song, Ye Guo, Ya Ning Mei, Ren Xiang Tan With compliments of Georg Thieme Verlag www.thieme.de Fumigaclavines D–H, New Ergot Alkaloids from Endophytic Aspergillus fumigatus DOI 10.1055/s-0034-1382958 Planta Med 2014; 80: 1131–1137 For personal use only. No commercial use, no depositing in repositories. Publisher and Copyright: © 2014 by Georg Thieme Verlag KG Rüdigerstraße 14 70469 Stuttgart ISSN 0032‑0943 Reprint with the permission by the publisher only Original Papers 1131 Fumigaclavines D–H, New Ergot Alkaloids from Endophytic Aspergillus fumigatus Authors Jing Xu1, 2, Yong Chun Song1, Ye Guo1,YaNingMei3, Ren Xiang Tan1 Affiliations 1 Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, P.R. China 2 Key Laboratory of Protection and Development Utilization of Tropical Crop Germplasm Resources, Ministry of Education, Hainan University, Haikou, P.R. China 3 Department of Clinical Laboratory, the First Affiliated Hospital of Nanjing Medical University, Nanjing, P.R. China Key words Abstract loids featuring a reverse prenyl moiety at C-2, l" Aspergillus fumigates ! with 1–4, 6,and8 bearing additional substitu- l" Trichocomaceae Ergot alkaloids are toxins which are produced ents, e.g., an OH or OAc group at C-9. Compounds l" Cynodon dactylon biotechnologically on an industrial scale. The 2, 4,and6–8 showed a broad spectrum of antimi- l" Poaceae chemical investigation of endophytic Aspergillus crobial activity against a panel of anaerobic mi- l" clavine‑type ergot alkaloids l" antimicrobial activity fumigatus resulted in the isolation of five new er- croorganisms, of which compounds 4 and 6 were got alkaloids named fumigaclavines D–H(2–6), the most active against Veillonella parvula with an along with three known analogues, fumigaclavine MIC = 16 µg/mL compared to that (0.12 µg/mL) of C(1), festuclavine (7), and fumigaclavine A (8). tinidazole, co-assayed as a positive reference. Their structures were unequivocally elucidated by extensive spectroscopic analyses in association Supporting information available online at received March 11, 2014 revised June 27, 2014 with X‑ray single-crystal diffraction. Fumigacla- http://www.thieme-connect.de/products accepted July 9, 2014 vines D–H are interesting clavine-type ergot alka- Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382958 Introduction activity traditionally requires testing of large bat- Published online August 15, ! teries of different microbial strains [8]. During our 2014 Fungal endophytes are receiving increasing atten- ongoing search for chemically new and biologi- Planta Med 2014; 80: tion due to their productivity of biologically po- cally potent metabolites from plant endophytes 1131–1137 © Georg Thieme – Verlag KG Stuttgart · New York · tent and structurally unforeseeable compounds, [9 13], we recently described the isolation and ISSN 0032‑0943 which are of significance in drug discovery [1–3]. identification of several new ergot alkaloids from The genus Aspergillus (Trichocomaceae), which the endophytic fungus A. fumigatus isolated from Correspondence Prof. Dr. Yongchun Song contains about 180 recognized species, has been a healthy stem of Cynodon dactylon (Poaceae) [14, Institute of Functional proven to be a rich source of bioactive metabolites 15]. A continued investigation of the minor con- Biomolecules [4]. Among the species of this genus, Aspergillus stituents obtained after the fermentation of the Electronic reprint for personal use State Key Laboratory of Pharmaceutical Biotechnology fumigatus Fresen. is a common airborne fungus fungus on solid medium afforded five new clav- Nanjing University and the most important Aspergillus species caus- ine-type metabolites named fumigaclavines D–H 22 Hankou Road ing infective lung diseases. This species proved to (2–6), along with three previously known com- Nanjing 210093 P.R. China be particularly productive regarding the accumu- pounds, fumigaclavine C (1), festuclavine (7), and Phone: + 862583593201 lation of diverse natural products and resulted in fumigaclavine A (8). Some clinically relevant an- Fax:+862583593201 the discovery of over 226 potentially bioactive aerobic microorganisms, Peptostreptococcus [email protected] secondary metabolites with 18 of them being er- anaerobius, Bacteroides diatasonis, Veillonella par- Correspondence got alkaloids [5]. The ergot alkaloids are a com- vula, Actinomyces israelii, Bacteroides vulgatus, Prof. Dr. Ren Xiang Tan plex family of indole-derived mycotoxins that in- and Streptococcus anaerobius, were adopted for Institute of Functional Biomolecules teract with several monoamine receptors of the the antimicrobial screening. In the current paper, State Key Laboratory of central nervous system, negatively affecting car- the isolation, structure elucidation, and antimi- Pharmaceutical Biotechnology diovascular, nervous, reproductive, and immune crobial activities of these compounds are re- Nanjing University 22 Hankou Road systems of exposed humans and animals [6,7]. ported herein. Nanjing 210093 The intensive use of antibiotics inevitably selects P.R. China resistant microorganisms, which issue a continu- Phone: + 862583592945 Fax:+862583302728 ing and cyclical need for new antibiotics. A search [email protected] for new chemical structures with antimicrobial Xu J et al. Fumigaclavines D–H, New… Planta Med 2014; 80: 1131–1137 1132 Original Papers Fig. 1 Single-crystal X‑ray structure of 1. (Color figure available online only.) Results and Discussion sic tetracyclic ergoline ring skeleton, except for the absence of an ! N-CH3 signal within 2. This conclusion was supported by the dis- The mycelia and culture medium of the fungus A. fumigatus were cernible HMBC interaction of H-7 with C-5 (δC 55.2), C-9 (δC extracted with ethyl acetate. The resulting ethyl acetate extract 73.2), and C-18 (δC 15.7). In addition, the remarkable upfield was dried and chromatographed on silica gel and Sephadex LH- shifts for C-5 (δC 55.2) and C-7 (δC 47.3) and downfield shifts for 20 followed by reversed-phase chromatography to yield eight al- H-5 (δH 3.17, m) and H-7 [δH 3.19, dd, 13.3, 4,4 Hz, H-7α; δH 2.71, kaloids (1–8). Compounds 2, 4,and6–8 showed a broad spec- br d, 13.3 Hz, H-7β] were observed. The NOESY correlations be- trum of antimicrobial activity against a panel of anaerobic micro- tween H-10 and H-9, H-18, and between H-5 and H-25 were organisms, of which compounds 4 and 6 were the most active found in both compounds 1 and 2 (l" Fig. 2) and corroborated against V. parvula. that fumigaclavines C and D have the same relative configuration. Compound 1 was obtained as a colorless crystal. Its molecular Hence, 2 was finally identified as (5R,8R,9S,10R)-6-demethy- formula was determined as C23H30 N2O2 by HR‑ESI‑MS (m/z lated fumigaclavine C. 367.37 860, calcd. for [M + H]+ 367.23 855). Compound 1 was Fumigaclavine E (3) was found to have the molecular formula 1 13 identified as fumigaclavine C [16] according to its Hand C C21H28 N2O, established by HR‑ESI‑MS (m/z 325.33131, calcd. for NMR spectra along with a set of 2D NMR experiments (1H-1H [M + H]+ 325.22 799), which differed from that of 1 by 43 amu, COSY, NOESY, HSQC and HMBC). Although this compound was suggesting the lack of an acetyl group. Comparison of NMR data discovered as early as 1977, its absolute configuration has not indicated the compound featured the same core structure as 1 ever been reported so far, which somewhat causes confusion and possessed a hydroxyl group at C-9 rather than an acetoxy about the C-8 stereochemistry with both 8S and 8R configura- group as in 9-deacetylfumigaclavine C, previously isolated in our Electronic reprint for personal use tions being assigned in the literature [17]. In this paper, we report laboratory [14]. The position of the substituent was confirmed by herein the absolute configuration of 1 by using low-temperature the significant downfield shift of H-9 (δH 4.52, br s) and COSY cor- (100 K) single-crystal X‑ray diffraction with Cu Kα radiation anal- relations from H-9 to H-8 (δH 2.09, m) and H-10 (δH 3.15, d, ysis (l" Fig. 1), and a (5R,8R,9S,10R) stereochemistry of fumiga- 10.6 Hz). The relative stereochemistry of 3 was the same as 1,as clavine C (1) was allowed to be established for the first time. evidenced from the similar NMR data and NOESY relationships Fumigaclavine D (2), a white amorphous powder, has the molec- (l" Fig. 2). Especially the NOSEY correlations between H-10 and ular formula C22H28 N2O5, established by HR‑ESI‑MS (m/z H-9, H-18 but not with H-5 are of importance as they directly 353.22 165, calcd. for [M + H]+ 353.22 290), implying ten degrees proved the relative configuration of C-10. The similar Cotton ef- of unsaturation. The 1Hand13C NMR data of 2 (l" Tables 1 and 2) fect in comparison with those of 1 thus supposed the assignment indicated that six of the ten units of unsaturation come from five of the same stereochemistry of 3 as (5R,8R,9S,10R)-9-deacetyl- carbon–carbon double bonds and a carbonyl. Therefore, the other fumigaclavine C. four units of unsaturation come from four rings. The 1H NMR data Fumigaclavine F (4) shared the same molecular formula as 3,as of 2 and the information from its 1H-1H COSY spectrum revealed determined by the HR‑ESI‑MS ([M + H]+ m/z 325.32 954, calcd. 1 13 the presence of a three-spin system made of vicinal aromatic hy- for C21H28 N2O, 325.22799) and NMR data. The Hand CNMR drogens (H-12 to H-14) and the partial structures of the dihy- data of 4 (l" Tables 1 and 2) and HMBC correlations, which closely droergine moiety (H-2 to H-10) and ethylene group (H-22 to H- resemble those of 3, showed that these two compounds had the 23).