Mycoleptodiscins A and B, Cytotoxic Alkaloids from the EndophyticFungus Mycoleptodiscus sp. F0194Humberto E. Ortega,† Paul R. Graupner,‡ Yumi Asai,§ Karen TenDyke,⊥ Dayong Qiu,⊥

Young Yongchun Shen,⊥ Nivia Rios,∥ A. Elizabeth Arnold,Δ Phyllis D. Coley,O,# Thomas A. Kursar,O,#

William H. Gerwick,□ and Luis Cubilla-Rios*,†,#

†Laboratory of Tropical Bioorganic Chemistry, Faculty of Natural, Exact Science and Technology, University of Panama, Panama‡Natural Products Discovery, Dow AgroSciences, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States§Eisai Co., Ltd., Tsukuba, Ibaraki 300-2635, Japan⊥Eisai Inc., 4 Corporate Drive, Andover, Massachusetts 01810, United States∥Department of Microbiology, University of Panama, PanamaΔSchool of Plant Sciences, The University of Arizona, Tucson, Arizona, United StatesODepartment of Biology, University of Utah, Salt Lake City, Utah, United States#Smithsonian Tropical Research Institute, Panama City, Panama□Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093, United States

*S Supporting Information

ABSTRACT: Two novel reddish-orange alkaloids, mycolep-todiscin A (1) and mycoleptodiscin B (2), were isolated fromliquid cultures of the endophytic fungus Mycoleptodiscus sp.that had been isolated from Desmotes incomparabilis inPanama. Elucidation of their structures was accomplishedusing 1D and 2D NMR spectroscopy in combination with IRspectroscopic and MS data. These compounds are indole-terpenes with a new skeleton uncommon in nature.Mycoleptodiscin B (2) was active in inhibiting the growth ofcancer cell lines with IC50 values in the range 0.60−0.78 μM.

The International Cooperative Biodiversity Group Programin Panama (ICBG-Panama) has been investigating

cytotoxic, antiparasitic, and antimicrobial agents from variousnatural sources such as marine organisms and endophyticfungi.1

Tropical endophytic fungi have been demonstrated to be richand reliable sources of biologically active and chemically novelcompounds.2 Hence, examination of the unique constituents ofendophytic fungi from endemic plants collected in Panama’sprotected areas is one of our major thrusts.Two hundred and thirty-seven endophytic fungi were

isolated from the endemic plant Desmotes incomparabilis(Rutaceae), collected in Coiba National Park, Veraguas,Panama, by Alicia Ibanez (STRI, Panama City). Because itshowed high biological activity in prescreening assays, thefungus Mycoleptodiscus sp. F0194 was chosen for additionalstudy. In this paper we describe the isolation and structuralelucidation of two novel reddish-orange alkaloids, which wehave named mycoleptodiscin A (1) and mycoleptodiscin B (2).Compound 1 was obtained as a reddish-orange solid. The

molecular formula C23H29O2N was determined by HRESIMS(m/z 352.2277 [M + H]+). The 1H NMR spectrum showedfour methyl singlets [δH 0.89 (H-20), 0.91 (H-19), 1.09 (H-21)

and 1.28 (H-18)] and a number of multiplets between 2.2 and1.3 ppm. The DEPT135 spectrum showed six aliphaticmethylenes [δC 18.8 (C-3), 19.6 (C-11), 19.6 (C-7), 38.3(C-12), 41.3 (C-6), and 42.9 (C-8)] and two methines [δC 57.4(C-10) and 57.9 (C-4)]. These provided evidence of theterpenoid moiety of 1.When the 1H NMR spectrum of 1 was obtained in

chloroform, a signal was observed at 9.4 ppm, consistent withthe proton of a secondary amine. HMBC correlations from amethine singlet at δH 7.00 (H-2) to δC 125.0 (C-2a), 131.7 (C-

Received: November 13, 2012Published: April 5, 2013

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17a), and 126.0 (C-17) and from a methine singlet at δH 5.68(H-14) to δC 126.0 (C-17), 167.6 (C-15), 187.2 (C-16), and131.7 (C-17a), combined with a λmax at 400 nm, providedevidence that compound 1 contained an indoloquinonemoiety.3 The quinone assignment was confirmed fromcomparisons of chemical shift data with the literature.3−7 The15N HMBC experiment showed a correlation between 1Hsignals at δH 7.00 (H-2) and a nitrogen at δN 171.4 (referenceCH3NO2 = 0 ppm), consistent with a nitrogen of a pyrrole/indole ring. Connections between the terpenoid moiety and theindoloquinone ring were established by HMBC correlationsfrom the 1H double doublets at δH 2.54 and 2.48 (H-3) to δC131.7 (C-17a), 125.0 (C-2a), and 128.9 (C-2) and from the 1Hsinglet δH 5.68 (H-14) to δC 39.7 (C-13) and 22.3 (C-18). Thekey correlations are shown in Figure 1. NOESY correlations

from H-3 to H-2 and from H-12 to H-14 indicated theorientation of the indoloquinone ring relative to the terpenoidsection; additional NOESY correlations established the relativestereochemistry of compound 1 (Figure 2).

Compound 2 was also obtained as a reddish-orange solid.The molecular formula C23H29O3N was determined byHRESIMS (m/z 368.2220 [M + H]+), 16 units higher thancompound 1, which suggested the addition of one oxygenatom. The location of this oxygen was indicated by thepresence of a new quaternary carbon resonance at δC 82.5 (C-4) in 2 along with an infrared absorption band at 3504 cm−1 fora hydroxy functionality. Mutually coupled doublets at δH 3.01and 2.68 for a methylene (CH2-3, δC 25.8) were noted for theirlack of vicinal coupling. The DEPT135 showed only onebridgehead methine at δC 47.3 (C-10). The 15N HMBCexperiment showed a correlation between the proton at δH 7.03(H-2) and a nitrogen at δN 152 (reference NH3 = 0 ppm),again consistent with the nitrogen of a pyrrole/indole ring.3−7

The additional oxygen atom in 2 considerably affected the

chemical shifts of the carbons in positions C-3, C-13, C-12, C-5, C-10, and C-6.Compound 2 was active in a cell growth inhibition assay

against all four cancer cell lines tested, with IC50 values rangingfrom 0.60 to 0.78 μM (Table 2). To evaluate the effect of

compound 2 on nonproliferating normal cells, an in vitrocytotoxicity assay was developed to distinguish between trueantiproliferative activity and general cellular cytotoxicity,unrelated to proliferation.8 The IC50 of compound 2 was 0.41μM against the quiescent IMR-90 cells. Thus, compound 2does not have an in vitro therapeutic window betweenproliferating and quiescent cells, indicating that the cell growthinhibitory effects observed against human cancer cells resultfrom indiscriminant proliferation-independent cytotoxicity,rather than from inhibited proliferative processes.Further experiments culturing Mycoleptodiscus sp. F0194 in

PDA resulted in the isolation and characterization of several

Figure 1. Selected COSY and HMBC correlations for mycolepto-discins A (1) and B (2).

Figure 2. Selected NOESY correlations for mycoleptodiscins A (1)and B (2).

Table 1. NMR Spectroscopic Data of Mycoleptodiscins A(1) and B (2) (CD3OD;

1H, 600 MHz; 13C, 150 MHz)

mycoleptodiscin A (1) mycoleptodiscin B (2)

position δC δH (J, Hz) δC δH (J, Hz)

2 128.9, CH 7.00, s 130.1, CH 7.03, s2a 125.0, qC 121.6, qC3 18.8, CH2 2.54, dd (16.2; 3.7) 25.8, CH2 3.01, d (16.6)

2.48, dd (16.0;12.1)

2.68, d (16.6)

4 57.9, CH 1.53, m 82.5, qC5 39.7, qC 44.5, qC6 41.3, CH2 0.95, m 33.3, CH2 1.59, m

1.82, m 1.42, m7 19.6, CH2 1.46, m 19.4, CH2 1.50, m

1.68, m8 42.9, CH2 1.18, m 42.7, CH2 1.38, m

1.40, m 1.19, m9 33.9, qC 34.2, qC10 57.4, CH 0.95, m 47.3, CH 1.76, m11 19.6, CH2 1.75, m 19.3, CH2 1.72, m

1.60, m 1.61, m12 38.3, CH2 2.13, m 32.6, CH2 1.97, m

1.54, m 1.84, m13 39.7, qC 45.4, qC13a 166.1, qC 166.2, qC14 115.6, CH 5.68, s 116.4, CH 5.68, s15 167.6, qC 167.7, qC16 187.2, qC 187.1, qC17 126.0, qC 126.0, qC17a 131.7, qC 132.6, qC18 22.3, CH3 1.28, s 25.5, CH3 1.36, s19 34.2, CH3 0.91, s 34.1, CH3 0.93, s20 22.1, CH3 0.89, s 22.1, CH3 0.91, s21 17.1, CH3 1.09, s 18.6, CH3 1.21, s

Table 2. Cancer Cell Growth Inhibition Assay and IMR-90Cytotoxicity Assay IC50’s (μM) for Compound 2

IC50 (μM)

compound H460 A2058 H522-T1 PC-3 IMR-90

2 0.660 0.780 0.630 0.600 0.41vinblastine 0.002 0.004 0.009 0.005carbonyl cyanide 5.00

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well-known antibacterial and cytotoxic compounds, such asverticillin B, melinacidin IV, and verticillin C.9−12 These lattermetabolites are all epipolythiodioxopiperazines (ETPs), toxicsecondary metabolites that are known to be made only byfungi.13 They are characterized by the presence of an internaldisulfide bridge and an indole ring.This intriguing chemical diversity of metabolites produced by

Mycoleptodiscus sp. F0194 suggests the possibility that addi-tional diverse and interesting bioactive secondary metabolitesmight be obtained by using various other fermentation mediaand conditions.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Optical rotations were

measured with a Rudolf Research Analytical Autopol III 6971automatic polarimeter (for compound 1) and a Jasco P-2000polarimeter (for compound 2), UV spectra on a Waters 996 PDAdetector, and IR spectra using a Nicolet IR-100 FT-IR spectropho-tometer using KBr plates. NMR spectra including 1D and 2Dexperiments were recorded in CD3OD on an Avance 600 MHz(Bruker), 5 mm-TCIl-CP, 5 mm-dual-CP. The LCMS and HRMSwere acquired on an Orbitrap (ThermoFisherScientific, USA) and anAgilent 6230 HRESITOFMS. HPLC was carried out on a Waters LCsystem, including a 1515 pump, a 2487 dual detector, and a YMC-PackSIL (150 × 10 mm) NP-HPLC column. All solvents were HPLCgrade.Isolation and Identification of Fungal Strain. The fungal strain

was isolated on malt extract agar from a healthy, mature leaf of D.incomparabilis collected in Coiba National Park, Veraguas, Panama, inSeptember 2004. The strain was characterized as Mycoleptodiscus sp.based on phylogenetic analyses of the DNA sequence of the nuclearribosomal internal transcribed spacer region and the first 600 bp of thenuclear ribosomal large subunit. Sequence data were obtainedfollowing U’Ren et al.14 Taxon sampling was based on comparisonsof the edited consensus sequence with the NCBI GenBank databaseusing BLASTn.14 Analyses were conducted on 100 top hits usingmaximum likelihood.15 The strain was reconstructed as part of a cladethat is sister to Mycoleptodiscus terrestris and comprised a group ofuncultured fungi, endophytes from other environments, andMycoleptodiscus sp. (data not shown). On the basis of support forthis placement we designated the strain asMycoleptodiscus sp. F0194. Avoucher specimen was deposited in sterile water at the SmithsonianTropical Research Institute, Panama (accession F0194).Culture Preparation. A starter culture was prepared by aseptically

transferring a single agar plug (1 cm2) of Mycoleptodiscus sp. F0194,which had been growing for 1 week on potato dextrose agar (PDA;BD Difco), to a flask containing 25 mL of potato dextrose broth(Sigma). Following incubation for 9 days on an orbital shaker (125rpm; 31 °C) the entire culture was transferred to a flask (1 L)containing 250 mL of PDB, which was incubated for a further 9 dayson an orbital shaker (125 rpm; 31 °C). The culture was then dividedbetween nine flasks (1 L), each containing 250 mL of malt extractbroth (BD Difco; 2%), and incubated for a further 9 days (125 rpm at31 °C).16

Cytotoxicity Assay. Cytotoxic effects of compound 2 andvinblastine were evaluated in four human cell lines. The cells werecultured in 96-well plates and grown in the absence or continuouspresence of test compounds for 96 h. Cell growth was assessed usingthe CellTiter-Glo luminescent cell viability assay (Promega).Luminescence was read on the EnVision 2102 MultiLabel Reader(Perkin-Elmer). IC50 values were determined as the concentration of acompound at which cell growth was inhibited by 50% compared tountreated cell populations.17

IMR-90 Cytotoxicity Assay. IMR-90 human fibroblasts, obtainedfrom American Type Culture Collection, were grown for 4 days toconfluency in MEM containing 10% fetal bovine serum andsupplemented with L-glutamine and penicillin/streptomycin. Afterwashing, the medium was replaced with complete MEM containing

0.1% fetal bovine serum, and cells were cultured for an additional 3days under these low-serum conditions to achieve completequiescence. Test compounds were then added followed by incubationfor 72 h at 37 °C. Cell viability was assessed by measurement ofcellular ATP levels using CellTiter-Glo.17

Isolation of Mycoleptodiscins A (1) and B (2). After incubation,the contents of the nine flasks were combined and filtered. Thecombined culture filtrate (2.25 L) was extracted exhaustively withEtOAc. The organic phase was concentrated under vacuum to give adark brown solid (94 mg). This extract did not show biologicalactivity. The mycelium of the nine flasks was extracted exhaustivelywith EtOAc. The organic phase was concentrated under vacuum andresulted in a dark green solid (229 mg). This extract showed activityagainst the cancer cell line MCF-7 (IC50 = 6.65 μg/mL). The crudemycelium was fractionated by NP-HPLC (YMC-Pack SIL 150 × 10mm NP-HPLC column, isocratic flow of 100% CHCl3 over 15 minand then a gradient to 100% EtOAc until 20 min, 254 nm, 1 mL/min)to yield seven major fractions (F1−F7). Fraction F6 was reinjected inthe same NP-HPLC (gradient of 100% CHCl3 to 100% EtOAc in 40min, 254 nm, 1 mL/min) to yield compounds 1 (1 mg) and 2 (13mg).

Mycoleptodiscin A (1): reddish-orange solid; [α]20D = −140 (c0.005, MeOH); UV/vis (MeOH) λmax 400 nm; IR (KBr) νmax 2928,2848, 1739, 1457, 755 cm−1; 1H NMR (600 MHz, CD3OD) and

13CNMR (150 MHz, CD3OD) see Table 1; HRESIMS m/z 352.2277([M + H]+ calcd for C23H30O2N, 352.2271).

Mycoleptodiscin B (2): reddish-orange solid; [α]25D = −174 (c0.600, MeOH); UV/vis (MeOH) λmax 398 nm; IR (KBr) νmax 3504,2924, 2854, 1642, 1459, 1378, 1263, 1031, 804 cm−1; 1H NMR (600MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) see Table 1;HRESITOFMS m/z 368.2220 ([M + H]+ calcd for C23H30O3N,368.2220).

■ ASSOCIATED CONTENT*S Supporting Information1D and 2D NMR spectra for compounds 1 and 2. Thisinformation is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel: (507) 6676-5824. Fax: (507) 264-4450. E-mail: lucr@ancon.up.ac.pa.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by a U.S. NIH grant for theInternational Cooperative Biodiversity Groups Program(ICBG-Panama; 2 U01TW006634-06) and the College ofAgriculture and Life Sciences at the University of Arizona. Weexpress our thanks to Dr. C. Spadafora for conducting theMCF-7 bioassay, M. Gunatilaka for assisting in classifying theisolate, Dr. E. Suh of Eisai Inc. for his encouragement andsupport, and the personnel of Panama’s Autoridad Nacional delAmbiente for facilitating this research.

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