Tetrahedron Letters 53 (2012) 6372–6375

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Tetrahedron Letters

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Fusarimine, a novel polyketide isoquinoline alkaloid, from the endophyticfungus Fusarium sp. LN12, isolated from Melia azedarach

Sheng-Xiang Yang a,b,d, Jian Xiao a, Hartmut Laatsch b, Julian J. Holstein c, Birger Dittrich c, Qiang Zhang a,Jin-Ming Gao a,⇑a Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, College of Science, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, Chinab Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germanyc Institute for Inorganic Chemistry, University of Göttingen, Tammannstrasse 4, D-37077 Göttingen, Germanyd College of Science, Zhejiang A&F University, Lin’an, Hangzhou 311300, Zhejiang, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 April 2012Revised 2 September 2012Accepted 7 September 2012Available online 24 September 2012

Keywords:Fungal metaboliteFusarium sp.Isoquinoline alkaloidAzaphilone

0040-4039/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tetlet.2012.09.031

⇑ Corresponding author. Tel.: +86 29 87092335.E-mail address: jinminggao@nwsuaf.edu.cn (J.-M.

Fusarimine (1), a novel polyketide-derived isoquinoline alkaloid with a rare N-ethyl-4-methyl-7-carb-oxyisoquinoline carbon skeleton, was isolated from cultures of Fusarium sp. LN-12, an endophytic fungusisolated from the leaves of Melia azedarach. The structure of 1 was elucidated on the basis of extensivespectroscopic data, especially by 2D NMR; X-ray crystallographic analysis confirmed the structure anddelivered also the absolute configuration.

� 2012 Elsevier Ltd. All rights reserved.

H

O N

CH3

OH

O

OHO

+-1

4a5

8a

910

11

1213

14 1516

346

7 8

R

1

O

HO

HOOC

S

Plant endophytic fungi are proving to be a productive source ofstructurally unique and biologically active natural products.1 As aresult, the study of fungal endophytes is currently considered apromising approach for the discovery of novel pharmacologicallyactive natural products. In continuation of our search for bioactivemetabolites from endophytes of the renowned insecticidal plantMelia azedarach (Meliaceae),2 we isolated from cultures of thefungal strain Fusarium sp. LN-12 a novel azaphilone-type alkaloidmetabolite, fusarimine A (1) (Fig. 1), possessing an unprecedentedskeleton. Fusarimine (1) is the first member of this hexasubstitutedisoquinoline alkaloid type isolated from natural sources. Here, wereport the isolation and structural elucidation of this isoquinoline,as well as its antimicrobial and phytotoxic activities.

The fungus was cultured on a rotary shaker at 28 �C for 5 days in1000 mL flasks each containing the liquid medium (200 mL/flask)composed of CaCl2 0.5 g, KH2PO4 0.1 g, KCl 0.05 g, MgSO4�7H2O0.1 g, glucose 20.0 g, peptone 15.0 g, and 1000 mL H2O. Thefermented broth (20 L) was filtered through a cheese cloth to givesupernatant and mycelia. The former was adsorbed onto polymericresin Amberlite XAD-16. Salts and high molecular materials werewashed out with water, and other adsorbed organic materials wereeluted with MeOH to give a crude extract (6.0 g) after removing thesolvent in vacuum. The residue was successively extracted with

ll rights reserved.

Gao).

petroleum ether and EtOAc. The EtOAc extract (2.9 g) was fraction-ated on a silica gel column using CH2Cl2–MeOH (CH2Cl2, 50:1, 30:1,20:1, 10:1, 1:1) to provide six fractions A–F. Fraction C (1.20 g) wassuccessively chromatographed over Sephadex LH-20 column(CH2Cl2–MeOH 1:1, MeOH). Subfraction C4b containing 1 wassubjected to RP-18 column eluted with MeOH–H2O (70:30,90:10) to afford the crude product, which was further purified byrecrystallization from MeOH to furnish compound 1 (21 mg).

O2

Figure 1. Structures of isoquinochitine A (1) and ascochitine (2).

Table 11H (500 MHz) and 13C (125 MHz) NMR Data of 1 in DMSO-d6

a (d ppm)

Position dH (J in Hz) dC

(DEPT)HMBC (H ? C) COSY

1 9.16 (1H, s) 144.9 (d) 3, 4, 4a, 8, 8a, 1523 148.2 (s)4 127.4 (s)4a 141.7 (s)5 6.50 (1H, s) 94.4 (d) 4, 6, 7, 8a

6 169.4 (s)7 101.3 (s)8 169.5 (s)8a 112.9 (s)9 3.46 (1H, m) 35.0 (d) 3, 4, 11, 12, 13 10, 1210 1.88 (2H, m) 27.5 (t) 3, 11, 12 9, 1111-CH3 0.90 (3H, t, 7.2 Hz) 12.3 (q) 9, 1012-CH3 1.44 (3H, d, 7.2 Hz) 18.0 (q) 3, 9, 1013-CH3 2.50 (3H, s) 15.2 (q) 3, 4, 4a, 914 174.2 (s)15 4.66 (1H, m), 4.72 (1H, m) 59.7 (t) 1, 3, 16 1616 3.79 (2H, m) 59.8 (t) 15 156-OH 14.8 (1H, s) 5, 6, 716-OH 5.16 (1H, s)

a Assigned by COSY, TOCSY, HSQC, NOESY, and HMBC experiments; referenced onDMSO-d6 with dC = 39.5, dH = 2.50.

N

OH

OH

OOC OH

H

H+-

COSY HMBC

a

b

NOESY

Figure 2. Key HMBC and COSY correlations of 1.

Figure 4. ORTEP diagram of 1.

S.-X. Yang et al. / Tetrahedron Letters 53 (2012) 6372–6375 6373

Compound 1 was obtained as colorless needles.3 The molecularformula of 1 was determined to be C17H21NO5 from pseudomolec-ular ion peaks at m/z 342.1307 ([M+Na]+, calculated forC17H21NO5Na 342.1312) and 318.1358 ([M�H]�, calculated forC17H20NO5 318.1347) by HRESIMS, with eight double bond equiv-alents. The IR spectrum exhibited hydroxyl (3277–3441 cm�1) andcarboxyl (1651 cm�1) functionalities. Its UV spectrum withabsorption at kmax 203, 275 and 395 nm was indicative of anisoquinoline skeleton.4

The 1H NMR and 13C NMR data for 1 (Table 1) in DMSO-d6

showed three methyls, three methylenes, three methines, andeight quaternary carbons. Among them, besides one carboxylic car-bonyl carbon at dC 174.2 (C-14), nine aromatic carbons includingtwo methine carbons were observed; two of them were oxygen-bearing (C-6, C-8), and two carbons (C-1, C-3) were connected withnitrogen. Furthermore, one sp3 N-methylene at dC 58.8 (t, C-15),

N

CH3

OH

OH

OOC OH O+-1

m/z 319

-H2Om/z 301

-CO 2m/z 275

-m/z 27

m/z 58m/z 261

Figure 3. Proposed major EI-MS

one sp3 oxymethylene at dC 59.6 (t, C-16), as well as one aromaticmethyl at dC 15.2 (C-13), one secondary methyl at dC 18.0 (C-12),and a primary methyl at dC 12.3 (C-11) were observed. In the 1HNMR spectrum of 1 (Table 1), two isolated aromatic proton singlets[dH 9.12 (s, H-1), 6.46 (s, H-5)], an aromatic methyl [dH 2.47 (H3-13)], one secondary methyl [dH 1.41 (d, J = 7.2 Hz, H3-12)], andone primary methyl [dH 0.87 (t, J = 7.2 Hz, H3-11)] were seen. Thesedata revealed that 1 was an isoquinoline alkaloid with substituentsat carbons 3, 4, 6, 7, and 8.

The gross structure of compound 1 was elucidated by 1H–1HCOSY, NOESY, and HMBC spectra. The HMBC spectrum of 1 showedcorrelations from H-1 to C-3, C-4a, C-8, C-8a, C-15, from H-5 to C-4,C-6, C-7, and C-8a, and from CH3-13 to C-3, C-4, C-4a, and (weakly)C-9. These data along with a NOESY correlation between CH3-13and H-5, H-9 and CH3-12 confirmed that the C-13 methyl waslocated at C-4. Additional HMBC correlations from the 6-OH protonsinglet (dH 14.8) to C-5, C-6, and C-7, together with an intramolec-ular hydrogen bond observed between 6-OH and the carboxylicgroup due to strongly downfield shifted proton signal at dH 14.8supported that the carboxylic moiety was linked to C-7 (dC 101.3)on the isoquinoline scaffold. This evidence suggested that 1 con-tains a 7-carboxy-6,8-dihydroxy-4-methylisoquinoline moiety.

Two further partial structures of 1 were identified by 1H–1HCOSY experiments as (a) –CH(CH3)CH2CH3 and (b) –CH2CH2OHfragments, as depicted in Figure 2. The attachment of the isobutylgroup (fragment a) to C-3 of the isoquinoline skeleton was indi-cated by HMBC correlations of H-12 with C-3 (Fig. 2) and by the al-ready mentioned NOESY correlations of Me-13. Further diagnosticHMBC correlations were observed between the methylene protonsH2-15 and C-1 and C-3. These observations as well as the shifts ofC/H-15/16 indicated the attachment of the 2-hydroxyethyl moiety(fragment b) to a nitrogen atom. The resulting structure implicatedthat the nitrogen atom was quaternary and the carboxylic acid wasionized. The structure of 1 was further confirmed by the EIMS spec-trum (Fig. 3), which gave rise to prominent fragment ion peaks atm/z 301 (M+�H2O), 275 (M+�C2H4O), and 261 (M+�C4H10), in addi-tion to a molecular ion at m/z 319 (M+).

-CH3 m/z 286

-CH2CH2OHm/z 230

5-CO 2 m/z 231

-CH3m/z 216

-COm/z 203

-CHOm/z 174

fragmentation pathway of 1.

O

HO

O NH

OH

HOOC

N

O

OH

OH

HO

HOOCN

OOH

HO

HOOC

O

SCoA

O

O

OO

O

OH

HO

OCO2H

N

OHOH

HO

OOC+_

OH

HO

OCHO

O

HO

OHOOC

O

OH

OHOOC O

HO

OH NH

OH

HOOC

H

1

intermediate

HexaketideAscochitine (2)

NH2CH2COOH

NH2CH2CH2OH (3)

+

Figure 5. Proposed biosynthetic pathway of 1.

6374 S.-X. Yang et al. / Tetrahedron Letters 53 (2012) 6372–6375

The structure of 1 was finally confirmed by single-crystal X-rayanalysis (Fig. 4).5 The conventional enantiomorph distinguishingparameter6 and three more recently developed methods7 wereused to determine the absolute configuration. While the Flackparameter x = �0.3(3) had no sufficient distinguishing power,invarioms reduced both absolute value and standard uncertaintyto �0.10(29). Both Hooft’s y = �0.10(11) and Parsons’ x = �0.08(11) enhanced the precision and allowed the assignment of theabsolute configuration at C-9 as (R). Thus, the structure of 1 wasestablished as 3-((R)-sec-butyl)-7-carboxy-6,8-dihydroxy-2-(2-hydroxyethyl)-4-methylisoquinolinium, which we namedfusarimine.

It should be mentioned that fusarimine (1) showed a negativesign of optical rotation (½a�20

D �87.9 (c 0.17, MeOH), which is thesame as that of ascochitine (2) [lit.,8�86 (c 1.0, CHCl3), ½a�27

D

�65.4 (c 0.1, CHCl3),9 although the configuration of 2 is publishedas (S).10 As details of this determination were not available, itremains an open question if Mishima’s data can be interpreted ina different way.10 Alternatively, (R)-ascochitine (ent-2) might alsooccur naturally, if not an inversion of 2 happened duringbiosynthesis.

Isoquinoline alkaloids, one of the largest groups of natural prod-ucts, are mainly occurring in higher plants,11 where they arebiosynthesized from tyrosine. Only very few isoquinolines areknown from fungi (e.g., Penicillium,4,12 Aspergillus,13 or Chaetomiumspp.),14 from mycobionts of lichens,15 from Streptomycetes,16 andfrom sponges,17 where they are produced biogenetically by polyke-tide pathway accompanied with amination.

In the same way, fusarimine (1) could be formed biogeneticallyfrom ascochitine (2),9 a phytotoxic fungal metabolite derived froma single hexaketide chain in Ascochyta fungi (Fig. 5).18 Although thecompound 2 was not isolated from cultures of the genus Fusarium,compound 1 may be produced by the Michael addition reaction of2 with aminoethanol (3) (Fig. 5). A similar O/N exchange has beenobserved in the formation of pyridones from pyrones (e.g. pestala-mide C from pestalamide B), of lactams from lactones (e.g. thesalfredins from respective phthalides), or of pyrroles from furans(bhimamycin C from bhimamycin A).19 The aminoethanol couldbe formed by the reduction of glycin or of the respective glycin ad-ducts as for salfredin A4.

Fusarimine (1) was evaluated for its antimicrobial activitiesagainst Escherichia coli, Bacillus subtilis, Streptomyces viridochrom-ogenes, Staphylococcus aureus, Candida albicans, and Mucor mieheiin the agar diffusion test at a concentration of 40 lg/paper disk,and for in vitro toxicity toward brine shrimps (Artemia salina) ata concentration of 10 lg/ml, as well as for phytotoxicity towardAmaranthus mangostanus, Raphanus sativus, Lactuca sativa, andMedicago sativa in the Petri dish assay at 100 lg/ml, by usingpreviously reported methods.20 Fusarimine (1) was found to beinactive in these assays.

This is the first report of a quaternary isoquinoline alkaloid,fusarimine (1), in fungi, which was derived from polyketide. Theabsolute configuration of 1 could be tentatively determined as R.This compound represents an unprecedented carbon skeletonand is an internal salt formed between the nitrogen atom andthe carboxylic acid.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Grant No. 21102114), the Program for NewCentury Excellent Talents in University (NCET-05-0852), and. BDand JJH would like to acknowledge funding from the DFG (DI921/3-1). We also thank Dr. H. Frauendorf and R. Machinek, Insti-tute for Organic and Biomolecular Chemistry, University of Göttin-gen, Germany, for the MS and NMR measurements.

Supplementary data

Supplementary data (1D and 2D NMR spectra and X-ray crystaldata of fusarimine (1)) associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.tetlet.2012.09.031.

References and notes

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21. Crystallographic data for compound 1 have been deposited with the CambridgeCrystallographic Data Centre (deposition number CCDC 865287). Copies of thedata can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif,or by emailing data_request@ccdc.cam.ac.uk, or by contacting The CambridgeCrystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +441223 336033.