Cyclopiamines C and D: Epoxide Spiroindolinone Alkaloids from

Feb 28, 2018 - Cyclopiamines C (1) and D (2) were isolated from the extract of Penicillium sp. CML 3020, a fungus sourced from an Atlantic Forest soil...
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Cyclopiamines C and D: Epoxide Spiroindolinone Alkaloids from Penicillium sp. CML 3020 Sara Kildgaard,†,# Lívia S. de Medeiros,‡,# Emma Phillips,§ Charlotte H. Gotfredsen,⊥ Jens C. Frisvad,† Kristian F. Nielsen,† Lucas M. Abreu,∥ and Thomas O. Larsen*,† †

Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark ‡ Departamento de Química, Universidade Federal de São Paulo (UNIFESP), Rua São Nicolau, 210, CEP 09913-030, Diadema-SP, Brazil § German Cancer Research Center, Brain Tumor Translational Targets, Im Neuenheimer Feld 580, Heidelberg D-69120, Germany ⊥ Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, DK-2800 Kgs. Lyngby, Denmark ∥ Departamento de Fitopatologia, Universidade Federal de Viçosa (UFV), Avenida P.H. Rolfs, s/n.° CEP 36570-000, Viçosa-MG, Brazil S Supporting Information *

ABSTRACT: Cyclopiamines C (1) and D (2) were isolated from the extract of Penicillium sp. CML 3020, a fungus sourced from an Atlantic Forest soil sample. Their structures and relative configuration were determined by 1D and 2D NMR, HRMS, and UV/vis data analysis. Cyclopiamines C and D belong to a small subset of rare spiroindolinone compounds containing an alkyl nitro group and a 4,5-dihydro-1Hpyrrolo[3,2,1-ij]quinoline-2,6-dione ring system. NMR and MS/HRMS data confirmed the presence of an epoxide unit (C-17−O−C-18) and a hydroxy group at C-5, not observed for their known congeners. Cytotoxic and antimicrobial activities were evaluated.

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citrinum4,6 were suggested as the possible biosynthetic precursors of cyclopiamines A (5) and B (6), discovered in 1979 by Steyn et al. from Penicillium cyclopium3 (later reidentified as P. griseof ulvum).7,8 The discovery of the coproduction of citrinalin C (7) (exhibits the bicyclo diazaoctane ring system) and 17-hydroxycitrinalin B (8) (displays the loss of one diketopiperazine carbonyl group) by P. citrinum F53 was the first to corroborate the hypothesis that citrinalin- and cyclopiamine-like compounds are likely degradation products from bicyclo[2.2.2]diazaoctane-containing metabolites. Cyclopiamines C and D represent to date the only known natural products to contain a 4,5-dihydro-1H-pyrrolo[3,2,1ij]quinoline-2,6-dione ring system, together with the cyclopiamines (A and B),1 cycloexpansamine B (9),9 and 6hydroxycyclopiamine B (10).10 This paper describes the characterization of cyclopiamines C (1) and D (2), their hypothesized biosynthetic pathway by Penicillium sp. CML 3020 based on the extra structural features, and evaluation of their cytotoxic and antimicrobial activities.

any prenylated indole alkaloids have been isolated from Penicillium and Aspergillus fungal species containing a bicyclo[2.2.2]diazaoctane ring system as depicted by the wellknown structures of notoamides, brevianamides, stephacidins, and macfortines.1,2 Other prenylated indole biomolecules include cyclopiamines,3 citrinalins,4 and citrinadins,5 which contain a spirooxindole core. Due to their unusual lack of the bicyclo[2.2.2]diazaoctane framework, these compounds are becoming increasingly interesting to synthetic organic chemists.6 Cyclopiamines and citrinalins represent rare examples of fungal secondary metabolism, as they possess an alkyl nitro group.6 Two new prenylated spirooxindole alkaloids, cyclopiamines C (1) and D (2), were isolated from an unidentified strain coded as Penicillium sp. strain CML 3020, a soilborne species isolated in 2009 from the Atlantic Forest of Brazil. Unlike their known congeners, NMR and MS/HRMS fragmentation data of 1 and 2 suggested the presence of an epoxide unit connected to the proline-derived pyrrolidine moiety and a hydroxy group at C-5. Recently, the total synthesis and isolation of cyclopiamine and citrinalin congeners have been achieved by Mercado-Marin et al.6 to assist the unambiguous confirmation of the structures’ absolute configuration and their proposed biogenesis. The citrinalins A (3) and B (4) isolated in 2010 by Pimenta et al. from Penicillium © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 27, 2017

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DOI: 10.1021/acs.jnatprod.7b00825 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

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protons at δH 3.25 (H-14), δH 4.52 (H-16), δH 4.15 (H-17), and δH 4.09 (H-18). The aromatic protons H-9 and H-10 were assigned to a 1,2,3,4-tetrasubstituted benzene ring (ring C), based on 13C HMBC correlations from both protons to the aromatic nonprotonated carbons at δC 106.8 (C-7) and δC 160.5 (C-8), together with individual HMBC correlations from H-9 to the quaternary aromatic carbon at δC 121.2 (C-11) and H-10 to the nonprotonated aromatic carbon at δC 148.8 (C-12) (Figure 1). The singlet methoxy group at δH 3.92 (CH3-28) was connected by HMBC correlations to C-8, and the connection was confirmed by a strong NOE correlation in the NOESY spectrum of H3-28 with H-9. The fusion of rings C and B through the nonprotonated aromatic carbons C-7 and C12 was supported by the long-range HMBC correlation from H-9 to the carbonyl carbon at δC 193.1 (C-6) and the correlation from the singlet proton at δH 4.33 (H-5) to C-6. H5 was also correlated to the heteroatom-substituted carbon at δC 62.6 (C-4) and the two methyl carbons assigned to δC 17.3 (C-24) and δC 24.0 (C-25). The H3-24 and H3-25 protons were linked through HMBC correlations to C-4 and to the oxycarbon at δC 79.3 (C-5). HMBC correlations were seen from the aromatic proton H-10 to the quaternary spiro-carbon assigned to δC 61.8 (C-1) and also from the diastereotopic protons H2-23 to C-1, C-11, and the amide carbonyl carbon at δC 183.4 (C-2). The methyl protons H3-26 and H3-27 and the proton at H-14 also correlated through HMBC to C-1. These correlations defined the spirooxindole A,B,C ring system of 1. The diastereotopic protons of H2-23 were furthermore correlated to the nonprotonated carbons at δC 48.4 (C-13) and δC 94.4 (C-22), the methylene carbon at δC 64.3 (C-21), and the methine carbon at δC 49.3 (C-14). Additional correlations included the protons H3-26 and H3-27 to C-13 and C-14, and H-14 to C-13, C-21, and C-22. These HMBC correlations assisted in the assembly of the cyclopentane ring D containing the spirocarbon C-1 and its connection to CH2-21.

RESULTS AND DISCUSSION The chemical structures of 1 and 2 were established by spectroscopic data analysis of 1D and 2D NMR spectra and supported by the data obtained from UV/vis spectroscopy, HRMS, and MS/HRMS fragmentation data (see Supporting Information). Compounds 1 and 2 appeared in the base peak chromatogram of the extract as the two most polar peaks eluting at 5.2 and 5.7 min (Figure S1, Supporting Information) with the molecular formulas C26H31N3O7 and C25H29N3O7 obtained from HRESITOFMS. Noteworthy for these two compounds was the characteristic loss of HNO2 exhibited in their MS/HRMS spectra (Δ 47.0006 between fragment ion and [M + H]+, calcd 47.0007), suggesting the presence of a NO2 group as part of the molecular structures. The UV spectra for 1 and 2 showed similar profiles with absorption maxima for 1 at 206, 232, 266, 281sh, and 357 nm, indicating a conjugated chromophore. The structure of 1 was elucidated using 1H, 13C, DQF-COSY, HSQC, and HMBC spectra (Table 1 and Figure 1). The 1H NMR spectrum of 1 revealed the presence of seven methines (five aliphatic and two aromatic), four methylenes, and five methyl groups. The 13C NMR spectrum identified two carbonyl groups and eight nonprotonated carbons (including four aromatic). Six out of the 13 unsaturations inferred from the molecular formula are accounted for by these moieties and the presence of a nitro group, indicating that compound 1 contains seven rings. The DQF-COSY spectrum of 1 defined two spin systems, besides the four singlet methyl signals at δH 1.21 (CH3-24), δH 1.87 (CH3-25), δH 0.76 (CH3-26), and δH 1.10 (CH3-27) and the two diastereotopic methylenes at δH 3.75/4.26 (CH2-21) and δH 2.47/3.38 (CH2-23) (Figure 1). One spin system included two aromatic protons at δH 6.77 (H9) and δH 7.62 (H-10) displaying an ortho coupling (J9,10 = 8.4 Hz). The second spin system included two methylenes at δH 2.50 (CH2-15) and δH 3.80/3.84 (CH2-19) and four aliphatic B

DOI: 10.1021/acs.jnatprod.7b00825 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data (400 MHz 1H; 100 MHz 13C) of 1 and 2 in CD3OD 1 position

δC/δN,a type

1 2 3N 4 5 6 7 8 9 10 11 12 13 14 15a 15b 16 17 18 19a 19b 20 N 21a 21b 22 22-N 23a 23b 24 25 26 27 28

61.8, C 183.4, C 148.7 (δN) 62.6, C 79.3, CH 193.1, C 106.8, C 160.5, C 105.8, CH 134.5, CH 121.2, C 148.8, C 48.4, C 49.3, CH 21.4, CH2 62.0, 60.0, 53.4, 57.3,

CH CH CH CH2

51.6 (δN) 64.3, CH2 94.4, C −7.2 (δN) 42.7, CH2 17.3, 24.0, 20.3, 24.8, 56.7,

CH3 CH3 CH3 CH3 CH3

2

δH mult. (J in Hz)

δC, type

HMBC

δH mult. (J in Hz)

HMBC

62.4, C 183.9, C

4.33, s

4, 6, 24, 25

6.77, d (8.4) 7.62, d (8.4)

6, 7, 8, 11, 12 1, 7, 8, 9, 12

3.25, 2.51, 2.49, 4.52, 4.15, 4.09, 3.84, 3.80,

1,13, 15, 21, 22, 26, 27 14, 16, 17, 22 14, 16, 17, 22 14, 15, 21 16 19 21 16, 17, 18

dd (11.6, 6.9) m m m d (3.1) d (3.1) d (13.5) d (13.5)

4.26, d (13.5) 3.75, d (13.5)

14, 16, 22 14, 19, 22, 23

63.0, C 78.9, CH 198.0, C 104.3, C 160.4, C 110.2, CH 135.6, CH 119.3, C 147.6, C 48.3, C 49.4, CH 21.4, CH2 61.9, 60.0, 53.4, 57.3,

CH CH CH CH2

64.3, CH2

4.43, s

4, 6, 24, 25

6.58, d (8.4) 7.54, d (8.4)

7, 8, 11 1, 8, 12

3.23, 2.49, 2.47, 4.51, 4.14, 4.09, 3.84, 3.79,

13, 15, 26 14, 16, 17, 22 14, 16, 17, 22

m m m m d (3.1) d (3.1) d (13.5) d (13.5)

16 19 16, 17, 18, 21 16, 17, 18, 21

4.26, d (13.5) 3.74, d (13.5)

14, 16, 22 19, 23

3.37, 2.45, 1.25, 1.89, 0.75, 1.10,

1, 1, 4, 4, 1, 1,

94.4, C 3.38, 2.47, 1.21, 1.87, 0.76, 1.10, 3.92,

d (16.4) d (16.4) s s s s s

1, 1, 4, 4, 1, 1, 8

2, 11, 14, 22 2, 11, 13, 21 5, 25 5, 6, 24 13, 14, 27 13, 14, 26

42.7, CH2 17.4, 24.0, 20.3, 24.9,

CH3 CH3 CH3 CH3

d (16.4) d (16.4) s s s s

2, 11, 14, 22 2, 11, 13, 21 5, 25 5, 6, 24 14, 27 14, 26

a

The 15N HMBC data were obtained at 800 MHz in CD3OD. The reported 15N chemical shifts are referenced through the solvent lock (2H) signal according to IUPAC recommended secondary referencing method with δ NH3 (0 ppm).

connecting the decahydro-1H-cyclopenta[f ]indolizine D,E,F ring system. The presence of an epoxide oxygen between CH17 and CH-18 (ring G) was suggested by their chemical shifts and the need to fulfill the unsaturation index. Further, a strong COSY correlation (J = 3.1 Hz) between H-17 and H-18 supported a 3J cis coupling, similar to what has been reported for dibohemamine A and related analogues.11 These results allowed the complete assembly of the D,E,F,G ring system connected through the spiro-carbon C-1 to the spirooxindole A,B,C ring system for compound 1. Analysis of the 15N HMBC spectrum (Figure 1) showed 3J-bond correlations from H3-24, H3-25, and H-5 and a 4J-bond correlation from H-10 to the amide nitrogen δN 148.7 (N-3). 3J-bond correlations were also seen from the protons H2-15, H-17, and H-18 to the tertiary amine nitrogen δN 51.5 (N-20). Attachment of the nitro group to the nonprotonated carbon C-22 was based on 3J-bond correlations from the protons H-14, H2-21, and H2-23 to the nitrogen δN −7.3 (N-22). The relative configuration of 1 was based on observed coupling constants and NOEs in the NOESY spectra. Correlations of H-10 with H-23a and H3-26 and of H-23a with H-21a and H3-26 placed these protons on the same face of the molecule, while correlations of H-14 with H3-27, H-23b,

Figure 1. Structures with numbering and 13C HMBC and COSY (left) and 15N HMBC (right) correlations for cyclopiamine C (1). All HMBC correlations are from protons to carbon or nitrogen atoms (1H−13C /1H−15N).

The presence of a central nitrogen atom linked to the heteroatom-substituted carbons at δC 57.3 (C-19), δC 62.0 (C-16), and C-21 was supported by the HMBC correlations from H-19 to C-16 and C-21 and from H-21 to C-16 and C-19, C

DOI: 10.1021/acs.jnatprod.7b00825 J. Nat. Prod. XXXX, XXX, XXX−XXX

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respectively (Figures S4 and S5). The likely occurrence of a retro Diels−Alder mechanism from these ions to form the fragment ion [C4H6NO]+ could explain the detection of m/z 84.0448 (−1.2 ppm deviation) observed in the MS/HRMS spectra for compounds 1 and 2. Similarly, the fragment ion [C4H8N]+ of m/z 70 (MS data obtained in low resolution) reported for cyclopiamine B was assigned to the pyrrolidine moiety.3 Looking at the biosynthetic origin of cyclopiamines C and D, the presence of an epoxide unit (C-17−O−C-18) and the hydroxy group at C-5 are structural features not observed for any of their known congeners. This prompted us to hypothesize the biosynthetic pathway by Penicillium sp. CML 3020 for the extra biosynthetic steps displayed for suggested intermediates 11 to 14 toward cyclopiamine C (1) (Figure 4). The hydroxy group at C-5 may originate from oxidations at this position for a suggested precursor (11) similar to the structure of cyclopiamine A as displayed in Figure 4 to form 12 followed by O-methylation at C-8. The epoxy pyrrolidine moiety could be hypothesized to arise from late stage oxygenation at C-17 or from 3-hydroxyproline as an alternative starter unit to proline, similar to what was suggested during labeling experiments for 17-hydroxycitrinalin B (8),6 which is the only other known congener to display an oxygenated site (C-17) attached to the proline-derived moiety. A putative dehydration may be anticipated followed by epoxidation of C17/C-18, likely favored by an enzymatic reaction, to produce the epoxy pyrrolidine moiety seen for cyclopiamine C. This has been observed for other fungal metabolites such as fusarin C from Fusarium moniliforme.12 MS/HRMS data analysis of the Penicillium sp. CML 3020 extract led to the detection of several compounds possibly corresponding to the putative biosynthetic intermediates proposed here to be involved in these extra steps (Figure 4). The hypothesis was corroborated by the occurrence of the diagnostic loss of HNO2 observed in the MS/HRMS spectra of each compound (Figure S24). Additionally, the detection of fragment ions that might correspond to the related pyrrolidine moieties, i.e., m/z 86.0594 and m/z 80.0486, was observed in accordance with 11, 12, 13, and 14. A trace amount of what possibly corresponds to the known congener cyclopiamine A or B was also observed in the extract corroborated by the HNO2 loss and a fragment corresponding to the pyrrolidine moiety, i.e., m/z 70.0653 (Figure S25). We suggest cyclopiamine D to be formed either by a similar biosynthetic route through putative dehydration and epoxidation from (12) or by O-demethylation. Cyclopiamines C (1) and D (2) were subjected to cytotoxicity, antibacterial, and antifungal bioassays. Both compounds were inactive (IC50 > 180 μM) in the tested cytotoxicity assays against NCH421k (glioblastoma), SW480 (colorectal adenocarcinoma), MEC-1 (B-chronic lymphocytic leukemia), and MCF7, EVSA-T, and MDA-MB-231 (breast

and H-21b and of H-23b with H-21b placed these protons on the opposite face of the molecule. A chair conformation of the central six-membered ring E was assumed, with H-14 and the nitro group in axial positions and a trans relationship between H-14 and H-16 (Figure 2) similar to that of citrinalin A.4 This

Figure 2. Selected observed NOE correlations for cyclopiamine C (1).

was supported by the fact that H-14 was observed as a double doublet in the 1H NMR spectrum with coupling constants J14,15 = 11.6, 6.9 Hz, and NOE correlations were observed of H-15b with H-14 and of H-15a with H-16. The suggested trans fusion of rings D and E is in contrast to the cis fusion (H-14 in equatorial position) seen for the rings in the structure of cyclopiamine B (6), for which the absolute configuration was determined by X-ray diffraction analysis.3 Furthermore, NOE correlations were observed of H-17 with H-16 and of H-19a with H-16, H-18, and H-21a, suggesting the cis protons H-17 and H-18 to be on the same side as H-16 and H-19a. NOEs of H-5 with H3-25 and of H3-25 with H3-27 suggest that these protons are on the same face of the molecule. The relative configuration of compound 1 is in agreement with that of cyclopiamine A (5)3 and citrinalin A (3), with the absolute configuration reported by X-ray diffraction analysis of the latter.4 The HRMS data showed a 14.016 Da mass difference between compounds 2 ([M + H]+ m/z 484.2085) and 1 ([M + H]+ m/z 498.2244), indicating the distinction of a CH2 group between the structures. This could likely correspond to the incorporation of either a hydroxy or methoxy group for each analogue, respectively. This was confirmed by highly similar 1H and 13C NMR spectra of both compounds (Table 1) and the fact that the singlet methyl signal corresponding to δH 3.92 (CH3-28) in 1 is missing from the 1H NMR spectrum of 2. The NMR chemical shifts of 2 were assigned completely using the DQF-COSY, HSQC, and HMBC spectra, as indicated in Table 1. The same relative configuration of 1 and 2 was suggested by highly similar chemical shifts and NOESY spectra. Reanalysis of the MS/HRMS data assisted the confirmation of the epoxy pyrrolidine moiety for compounds 1 and 2. First the proposed fragmentation mechanism displayed in Figure 3 supports the loss of HNO2 from each precursor [M + H]+, leading to the detected fragments m/z 451.2235 (base peak) and m/z 437.2078 (base peak) for compounds 1 and 2,

Figure 3. Proposed fragmentation mechanism explaining the detected diagnostic product ions for compounds 1 and 2. D

DOI: 10.1021/acs.jnatprod.7b00825 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 4. Proposed biosynthesis toward cyclopiamine C (1). while for metabolite extraction and purification the applied solvents were HPLC grade (both from Sigma-Aldrich) and the Milli-Q H2O was purified using a Milli-Q system (Millipore). Fungal Strain and Identification. Strain Penicillium sp. CML 3020 was isolated in September 2009, from a soil sample collected in a preserved Atlantic Forest site located in the municipality of São Miguel Arcanjo, São Paulo State, Brazil. It is preserved in the Coleçaõ Micológica de Lavras (CML), Departamento de Fitopatologia, Universidade Federal de Lavras, Brazil, accession number CML 3020. A replica of the strain is preserved in the IBT culture collection, Department of Biotechnology and Biomedicine, Technical University of Denmark, accession number IBT 33750. Colony aspects on standard culture media13 and observed micromorphological features of Penicillium sp. CML 3020 showed some similarities with P. brasilianum. A partial DNA sequence of the second largest subunit of RNA polymerase II (RPB2) of Penicillium sp. CML 3020 (GenBank accession number RPB2: KY420180) was compared with DNA data from reference strains of most accepted Penicillium species.13 Subsequent maximum likelihood phylogenetic analysis conducted on MEGA software14 showed that Penicillium sp. CML 3020 is closely related to, yet distinct from, P. herquei and P. malachiteum (Figure S27). Chemical Analysis of Microextracts. The microextracts of the soilborne Penicillium sp. CML 3020 were three-point inoculated on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) plates and cultivated for 7 and 14 days at 25 °C in the dark. The YES and CYA plates were prepared as described by Frisvad.15 Five plugs (6 mm diameter) were extracted using 800 μL of 1:5 v/v MeOH−EtOAc with 1% formic acid. Vials were placed in an ultrasonic bath for 60 min, and extracts were transferred to a new vial, evaporated to dryness, and redissolved in 500 μL of MeOH for further ultrasonication over 20 min. After 5 min of centrifugation at 13000g, the clear extracts were transferred to new vials and submitted for UHPLC-DAD-HRMS analysis using the Agilent QTOF system. Fermentation, Extraction, and Isolation. Penicillium sp. CML 3020 was cultivated on 200 YES plates for 14 days at 25 °C in the dark. The plate contents were transferred to an Erlenmeyer flask (5 L), and an extraction was performed twice, using 2 L of EtOAc with 1% formic acid. The solvent mixture was filtered and evaporated, which afforded the EtOAc extract (6.3 g). Carbohydrates and fatty acids were removed by liquid−liquid extraction with Milli-Q H2O−MeOH and heptane, respectively. The hydroalcoholic extract (4.5 g) was subjected to the flash chromatography system fitted to a C18 Snap Cartridge (50 g, 66 mL) using a stepwise gradient elution of Milli-Q H2O−MeOH (85:15 → 0:100, v/v) with a flow rate of 40 mL/min over 25 min to afford 11 fractions (Fr.1−Fr.11). Fr.6 (0.080 g) was fractionated on the flash chromatography system using a C18 Snap Cartridge (25 g, 33 mL) with a stepwise gradient elution of Milli-Q H2O−MeCN (90:10 → 10:90, v/v) with a flow rate of 20 mL/min over 25 min to afford six fractions (Fr.12−Fr.17). Fr.14 (0.025 g) was purified by semi-

carcinoma) cell lines. Furthermore, 1 and 2 were inactive at tested concentrations in antibacterial assays against Gramnegative Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, and Pseudomonas aeruginosa and Gram-positive methicillin-resistant Staphylococcus aureus (MICs above 64 μg/ mL). No antifungal effects were detected at the tested concentrations in bioassays against Aspergillus f umigatus and Candida albicans (MICs above 64 μg/mL).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. UV data were recorded on a Shimadzu MultiSpec-1501 UV−vis spectrophotometer. IR data were obtained using a Shimadzu IRPrestige-21 FT-IR spectrophotometer. The NMR spectra, including 1D and 2D experiments, were performed using a 400 MHz (100 MHz for 13C) Bruker Ascend NMR spectrometer with a 5 mm Prodigy Cryoprobe, with the exception of the 1H,15N HMBC experiment, which was run on a 800 MHz Bruker Avance equipped with a 5 mm TCI cryoprobe. NMR experiments were all run using standard pulse sequences. The UHPLC-DADHRMS data were recorded on an Agilent Infinity 1290 UHPLC system equipped with a DAD. Separation of 1 μL samples were performed applying an Agilent Poroshell 120 phenyl-hexyl column (2.1 × 150 mm, 2.7 μm) at 60 °C, using a linear gradient 10% MeCN in Milli-Q H2O buffered with 20 mM formic acid increased to 100% in 15 min, staying there for 2 min, returned to 10% in 0.1 min, and kept there for 3 min before the following run, at a flow rate of 0.35 mL/ min. MS detection was obtained on an Agilent 6545 QTOF MS equipped with an Agilent Dual Jet Stream electrospray ion source with the drying gas temperature of 160 °C, gas flow of 13 L/min, sheath gas temperature of 300 °C, and flow of 16 L/min. Capillary voltage was set to 4000 V, and nozzle voltage to 500 V, in the positive ion mode. Mass spectra were recorded in the range m/z 85−1700, with an acquisition rate of 10 spectra/s. Automated data-dependent acquisition MS/ HRMS analysis was performed for ions detected in the full scan above 50 000, applying fixed CID energies of 10, 20, and 40 eV with a maximum of three selected precursor ions per cycle. Lock mass solution in 95% MeCN was infused in the second sprayer using an extra LC pump at a flow of 10−50 μL/min; the solution contained 1 μM tributylamine (Sigma-Aldrich) and 10 μM hexakis(2,2,3,3tetrafluoropropoxy)phosphazene (Apollo Scientific Ltd.), corresponding to m/z 186.2216 and 922.0098, respectively, as lock masses. Flash chromatography was carried out on a Biotage Isolera One system, using a C18 Snap Cartridge (50 g, 66 mL). Semipreparative HPLC was performed on a Waters 600 system attached to a Waters 996 DAD fitted with a Phenomenex Luna C18 column (250 × 10 mm, 5 μm). For HRMS analysis, LCMS grade chemicals and solvents were used, E

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preparative HPLC using a Luna PFP column (250 × 10 mm, 5 μm, Phenomenex) with a flow rate of 5 mL/min and a gradient elution of Milli-Q H2O−MeCN (80:20 → 27:73, v/v) over 9 min, (27:73 → 0:100, v/v) to 10 min and held at 100% MeCN to 15 min Milli-Q H2O buffered with 50 ppm trifluoroacetic acid. This yielded 8.4 mg of cyclopiamine C (1) and 6.2 mg of cyclopiamine D (2). Cyclopiamine C (1): pale yellow solid; [α]20D +6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 206 (4.51), 232 (4.17), 266 (4.03), 281 (3.97), 357 (3.73) nm; 1H, 13C, and 15N NMR, Table 1; HRMS m/z 498.2244 [M + H]+ (calcd for C26H32N3O7, 498.2240, Δ 0.8 ppm). Cyclopiamine D (2): pale yellow solid; [α]20D +2 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 206 (4.47), 232 (4.13), 266 (3.94), 281 (3.93), 362 (3.67) nm; IR (KBr) νmax 3421 (br), 1687, 1620, 1548, 1371, 1203; 1H and 13C NMR, Table 1; HRMS m/z 484.2085 [M + H]+ (calcd for C25H30N3O7, 484.2083, Δ 0.4 ppm).

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(5) Mugishima, T.; Tsuda, M.; Kasai, Y.; Ishiyama, H.; Fukushi, E.; Kawabata, J.; Watanabe, M.; Akao, K.; Kobayashi, J. J. Org. Chem. 2005, 70, 9430−9435. (6) Mercado-Marin, E. V.; Garcia-Reynaga, P.; Romminger, S.; Pimenta, E. F.; Romney, D. K.; Lodewyk, M. W.; Williams, D. E.; Andersen, R. J.; Miller, S. J.; Tantillo, D. J.; Berlinck, R. G. S.; Sarpong, R. Nature 2014, 509, 318−324. (7) Frisvad, J. C. Arch. Environ. Contam. Toxicol. 1989, 18, 452−467. (8) Frisvad, J. C.; Samson, R. A. Stud. Mycol. 2004, 49, 1−173. (9) Lee, C.; Sohn, J. H.; Jang, J.-H.; Ahn, J. S.; Oh, H.; Baltrusaitis, J.; Hwang, I. H.; Gloer, J. B. J. Antibiot. 2015, 68, 1−4. (10) Kawahara, T.; Nagai, A.; Takagi, M.; Shin-ya, K. J. Antibiot. 2012, 65, 535−538. (11) Fu, P.; Legako, A.; La, S.; MacMillan, J. B. Chem. - Eur. J. 2016, 22, 3491−3495. (12) Song, Z.; Cox, R. J.; Lazarus, C. M.; Simpson, T. J. ChemBioChem 2004, 5, 1196−1203. (13) Visagie, C. M.; Houbraken, J.; Frisvad, J. C.; Hong, S. B.; Klaassen, C. H. W.; Perrone, G.; Varga, J.; Yaguchi, T.; Samson, R. A. Stud. Mycol. 2014, 78, 343−371. (14) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. Mol. Biol. Evol. 2013, 30, 2725−2729. (15) Frisvad, J. C. In Fungal Secondary Metabolism−Methods and Protocols; Keller, N. P.; Turner, G., Eds.; Humana Press: New York, 2012; Vol. 944, Chapter 3, pp 47−58.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00825. HRESITOFMS, MS/HRMS, and UV data together with 1D and 2D NMR spectra of 1 and 2; maximum likelihood phylogenetic analysis from RPB2 DNA sequences of Penicillium sp. CML 3020; experimental procedures for cytotoxicity and antimicrobial assays of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +45 45252632. Fax: +45 45884922. E-mail: [email protected]. dk. ORCID

Sara Kildgaard: 0000-0003-3761-0382 Thomas O. Larsen: 0000-0002-3362-5707 Author Contributions #

S. Kildgaard and L. S. de Medeiros contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank CNPq Conselho Nacional de Desenvolví mento Cientifico e Tecnológico for the scholarships to L.S.M. (202183/2014-9/PDE-CNPq) and L.M.A. (249511/2013-4/ PDE-CNPq). The authors graciously acknowledge Fundación Medina, Granada (Spain) for bioassay screening of pure compounds and the NMR Center•DTU together with the Villum Foundation for 800 MHz NMR time. Agilent Technologies is acknowledged for the Thought Leader Donation of the 6545 UHPLC-QTOF.

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REFERENCES

(1) Finefield, J. M.; Frisvad, J. C.; Sherman, D. H.; Williams, R. M. J. Nat. Prod. 2012, 75, 812−833. (2) Yang, B.; Dong, J.; Lin, X.; Zhou, X.; Zhang, Y.; Liu, Y. Tetrahedron 2014, 70, 3859−3863. (3) Bond, R. F.; Boeyens, J. C. A.; Holzapfel, C. W.; Steyn, P. S. J. Chem. Soc., Perkin Trans. 1 1979, 1, 1751−1761. (4) Pimenta, E. F.; Vita-Marques, A. M.; Tininis, A.; Seleghim, M. H. R.; Sette, L. D.; Veloso, K.; Ferreira, A. G.; Williams, D. E.; Patrick, B. O.; Dalisay, D. S.; Andersen, R. J.; Berlinck, R. G. S. J. Nat. Prod. 2010, 73, 1821−1832. F

DOI: 10.1021/acs.jnatprod.7b00825 J. Nat. Prod. XXXX, XXX, XXX−XXX