Fig. 1 Structures of compounds 1–4.

Table 1 NMR data for compounds 1 and 2 (500MHz CDCl3).

Position 1 2

δC δH (J in Hz) δC δH (J in Hz)

1 164.1 167.0

2 88.4 5.49, s 89.2 5.55, s

3 170.8 171.0

4 112.3 114.0

5 162.3 159.1

6 134.6 132.6

7 132.1 6.06 (q, 7.1) 132.1 6.06 (q, 6.9)

8 13.7 1.88 (d, 7.1) 14.9 1.67 (d, 6.9)

9 56.4 3.87, s 56.4 3.90, s

10 55.2 4.43, s 55.0 4.34, s

11 57.3 4.41, s 65.0 4.30, s

277Letters

Chaetoglocins A–D, Four NewMetabolites from the EndophyticFungus Chaetomium globosum

Hui Ming Ge, Qiang Zhang, Su Hai Xu, Zhi Kai Guo,Yong Chun Song, Wu Yang Huang, Ren Xiang TanInstitute of Functional Biomolecules, State Key Laboratory of Pharma-ceutical Biotechnology, School of Life Sciences, Nanjing University,Nanjing, P.R. China

Abstract!

Chaetoglocins A–D (1–4), four new secondary metabolites, wereisolated from the solid-fermentation culture of Chaetomiumglobosum (strain no. IFB-E036), an endophytic fungus residing in-side the root of Cynodon dactylon. The structures of these com-pounds were elucidated on the basis of detailed spectroscopicevidence and by comparing spectroscopic data with those in theliterature. Compounds 1 and 2 displayed antimicrobial activityagainst the gram-positive bacteria with minimum inhibitoryconcentrations (MIC) between 8 and 32 µg/mL.

Key wordsChaetomium globosum · Chaetomiaceae · secondary metabo-lites · endophyte · antibiotic

Endophytic fungi, ubiquitous in all naturally growing plants, havebeen demonstrated to be actively involved in the host evolution.Some of the endophytes have been ascertained to be symbiotic orat least nonpathogenic to the host, in addition to the gene trans-formation among host and endophytes [1]. This is why some en-dophytes can produce arrays of functional molecules that may beof ecological and/or pharmacological significance [2–4]. The ge-nus Chaetomium, a large genus of saprobic ascomycetes, com-prises approximately 90 dematiaceous filamentous fungal spe-cies [5]. As a typical Chaetomium species, C. globosum is wellknown to produce chaetoglobosin toxins as important tool mole-cules in cellular andmolecular biology owing to the specific bind-ing to the actin filaments without affecting mitosis [6]. Previ-ously, we have shown that some endophytic Chaetomium speciesare a rich source of novel chaetoglobosin and azaphilone alka-loids with potent bioactivities [7–9]. During our ongoing searchfor new bioactive metabolites from Chaetomium species, a C. glo-bosum strain (no. IFB-E036) was isolated from an apparentlyadaptive plant, Cynodon dactylon (Poaceae), which grows domi-nantly in the coastal beach with high salinity in the suburb ofYancheng City, China. After cultivation of the fungus over solidsubstrate, the EtOAc extract of the resultant fermentation mix-ture showed pronounced antimicrobial activity against gram-positive bacteria. The subsequent fractionation procedure led tothe isolation of four new secondary metabolites, 1–4. We herebywish to present the isolation, structure elucidation, and bioactiv-ities of these metabolites.Chaetoglocin A (1), a white gum, had the molecular formulaC11H14O5 (5 degrees of unsaturation retained) evidenced fromthe quasimolecular ion at m/z 249.0731 ([M + Na]+) exhibited inits high resolution electrospraymass spectrometry (HRESIMS). Incompliance with the formula, the presence of hydroxyl and ester

carbonyl groups in the molecule could be proposed from the IRabsorption bands of 1 at 3396 and 1699 cm−1. In the 13C NMRspectrum of 1, a total of eleven resonance lines could be readilyedited by the DEPT pulse sequences as a carbonyl, four sp2 qua-ternary, two sp2 methine, two sp3 methylene, and two methylcarbons. This was further confirmed by a set of 2DNMR experi-ments that allowed the unequivocal assignment of all 1H and 13CNMR signals (l" Table 1). In the HMBC spectrum of 1, the presenceof an isopentenyl moiety was evidenced from the olefinic protonquartet (J = 7.1 Hz) at δH‑7 6.06 correlating with C-5 (δ 162.3) andC-11 (δ 57.3), and the hydroxymethyl proton at δH‑11 4.41 havingcross-peak with C-5 (l" Fig. 2). Furthermore, the co-occurrence of3-methoxyl and 4-hydroxymethyl groups was indicated by atwo-proton singlet at δH‑10 4.43 showing HMBC correlations withC-3 (δ 170.8) and C-5 (δ 162.3), the former having an additionalcross-peak with a three-proton singlet at δH 3.87. The chemicalshift of C-1 (δ 164.1) and the unsaturation index could only be ex-plained by editing into a δ-lactone ring. This hypothesis was rein-forced by the HMBC correlation of the olefinic singlet at H-2 (δ5.49) with C-1 (δ 164.1), C-3 (δ 170.8), and C-4 (δ 112.3), whichcould be well rationalized by the planar structure of 1 (l" Fig. 1).Chaetoglocin B (2) was also obtained as a white gum, with itsHRESIMS data identical with that of 1, indicating that it pos-

Ge HM et al. Chaetoglocins A–D, Four… Planta Med 2011; 77: 277–280

Fig. 2 Key HMBC correlations of compounds 1–4.

Table 2 NMR data for compounds 3 and 4 (CDCl3).

Position 3a 4b

δC δH (J in Hz) δC δH (J in Hz)

1-NH 5.83, br s 6.24, br s

2 166.2 167.2

3 96.9 97.1

4 172.0 197.5

4-OH 11.05, br s

5 41.6 2.24, m 46.4 2.38, m

6-Ha 65.1 4.28, dd(11.2, 3.3)

65.8 4.38, dd,(11.2,4.2)

6-Hb 65.1 4.16, dd(11.2, 2.7)

65.8 4.10, dd,(11.2,7.0)

7 200.8 174.6

7-OH 11.83, br s

8 31.4 2.53, s 25.3 2.54, s

9-Ha 23.1 1.80, m 20.3 1.86, m

9-Hb 23.1 1.80, m 20.3 1.55, m

10 11.6 1.08, t (7.5) 11.6 1.00, t (7.2)

a Recorded at 300MHz. b Recorded at 600MHz.

278 Letters

sessed a molecular formula of C11H14O5 as well. Moreover, the 1Hand 13C NMR spectra of 2 were very similar to those of 1 exceptfor the C-11 signal shifting downfield to δ 65.0 compared to that(δ 57.3) of 1 (l" Table 1). This observation suggested that com-pound 2 could be a geometric isomer of chaetoglocin A (1). Thiswas confirmed by a set of 2DNMR experiments that led to the ex-act assignment of all 1H and 13C NMR signals of 2 (l" Table 1).To understand the geometric isomerism between the two fungalmetabolites, the 1H and 13C NMR spectra of chaetoglocins A (1)and B (2) were rationalized cautiously after the unambiguous al-location (l" Table 1). Since the most striking NMR spectral dis-crepancy lay in the H-8 and C-11 chemical shifts, the two com-pounds were most probably isomeric in the geometry of the6,7-double bond. We therefore scrutinized the 1H and 13C NMRresonances due to the 2-substituted (Z/E)-but-2-en-1-ol moietyof organic compounds described elsewhere [10–17], concludingthat chaetoglocins A (1) and B (2) possessed 6,7-(Z and E)-doublebonds, respectively. In case of 1, the upfield shifted C-11 (δC 57.3)and downfield moved H-8 (δH 1.88) signals required the syn-lo-cation of the 8-methyl and 11-hydroxymethyl groups, whose car-bon and hydrogen nuclei were shielded and deshielded, respec-tively, presumably via the steric interaction similar to the γ-gauche effect [18]. And in case of 2, the magnitude of the C-11(δC 65.0) and H-8 (δH 1.67) signals could only be explained by as-suming that the 11-hydroxymethyl was vicinal to a proton.Chaetoglocin C (3) was obtained as a white amorphous powder.The HRESIMS analysis of 3 provided the molecular formula ofC9H13NO3 requiring four degrees of unsaturation. The 1H and 13CNMR spectral data (l" Table 2) could be ascribed to a methyl, anethyl, a methylene, a methine, two carbonyl, and a tetrasubsti-tuted ethylene unit in addition to two exchangeable protons.The remaining one degree of unsaturation indicated that themolecule possessed a ring. The COSY spectrum revealed a spinsystem composed of H-10/H-9/H-5/H-6, indicative of a 2-substi-tuted n-butyl group. The two sp2 carbon signals at δC 96.9 (C-3)and 172.0 (C-4) showed HMBC correlations (l" Fig. 2) with H-5(δH 2.24), H-6 (δH 4.28 and 4.16), and H-9 (δH 1.80), respectively,suggesting that the double bond was connected at C-5. Furthercorrelations of the methyl proton at δH‑8 2.53 with a ketone car-

Ge HM et al. Chaetoglocins A–D, Four… Planta Med 2011; 77: 277–280

bonyl (δC‑7 200.8) and C-3 enabled the acetyl group anchored atC-3. Correlations were also observed from H-6 to C-2 (δC 166.2)and H-1 to C-3, suggesting the form of a δ-lactam ring. Finally,the down- (C-4) and up- (C-3) field signals for the quaternary car-bons, together with HMBC correlations of OH-4 with C-3 and C-5,revealed that the hydroxyl group was linked at C-4. Thus, thestructure of 4 was depicted as l" Fig. 1.Chaetoglocin D (4) also had the same molecular formulaC9H13NO3 with 3 on the basis of HRESIMS. The 1H and 13C NMRdata (l" Table 2) of 4were almost identical to those of 3. However,the ketone carbonyl carbon (δC‑4 197.5) showed HMBC correla-tions with H-6 (δH-6 4.38, 4.10) and H-9 (δH-9 1.86, 1.55), whilethe enol carbon (δC‑7 174.6) correlated with the methyl proton(δH‑8 1.00). This observation suggested that 4 was the enol-ketoisomer of 3.Compounds 1–4 were subjected to in vitro antibacterial activitytests against four gram-positive and two gram-negative bacteria,including Bacillus subtilis CICC10285, Streptococcus pyogenesATCC19615, Mirococcus luteus CMCC(B) 28001, Mycobacteriumsmegmatis CGMCC1.562, Escherichia coli ATCC35218, and Pseudo-monas aeruginosa CICC10351. Only compounds 1 and 2 exhibitedmoderate antibacterial bioactivities against four gram-positivebacteria with MIC values from 8 to 32 µg/mL (l" Table 3).

Materials and Methods!

General experimental proceduresHPLC were performed by using a Waters ODS 5 µm column(250 × 10mm), a Hitachi pump L-7100, and a UV detector L-7400. IR spectra were taken on a Nexus 870 FT‑IR spectrometer.UV spectra were recorded on a Hitachi U-3000 UV‑Vis spectrom-eter. Melting points were measured on a XT-4 melting point ap-paratus andwere uncorrected. All mass spectrawere acquired onan Agilent 6210 LC/TOFMS spectrometer, and NMR data were ac-quired on a Bruker DRX500 or DPX300 NMR or Varian Inova-600spectrometer. Silica gel (200–300 mesh) for column chromatog-raphy and GF254 (10–20mm) for TLC were produced by QingdaoMarine Chemical Factory. The ODS silica gel (50 µm) was from

Table 3 Antimicrobial activities of compounds 1–4.

Compounds MIC for test microorganisms (µg/mL)

Bacillus subtilis Streptococcus

pyogenes

Mirococcus luteus Mycobacterium

smegmatis

Escherichia coli Pseudomonas

aeruginosa

1 32 16 8 16 > 32 > 32

2 32 16 16 16 > 32 > 32

3 > 32 > 32 > 32 > 32 > 32 > 32

4 > 32 > 32 > 32 > 32 > 32 > 32

Tetracyclinea 0.12 2 0.25 0.5 1 32

Kanamycina 0.5 16 2 2 16 > 32

a Used as positive controls.

279Letters

YMC. Tetracycline (98%) and kanamycin (99%), the positive con-trols used in the bioassay, were purchased from Sigma-AldrichCo.

Fungal materialThe title fungus was isolated from the fresh and healthy root ofCynodon dactylon collected in July 2001 from the Coastal beachof Yancheng in the east of China and identified by Dr. Y.C. Songas Chaetomium globosum by comparing its morphological charac-ters.

Fermentation, extraction, and isolationFor chemical investigations, the fungal strain was statically culti-vated in solid medium as reported [19]. The dried products ofsolid state fermentation (1.25 kg) were powdered and extractedwith methyl hydrate (10 L) at room temperature for 2 days. Thefiltered solution was evaporated in vacuo to give a crude extract(11.2 g), which was subjected to column chromatography(3 × 80 cm) on silica gel (224 g, 200–300 mesh) eluted with a gra-dient of CHCl3 :MeOH (v/v 100:0, 100:1, 100:2, 100:4, 100:8,100:20, 0 :100, each 1.5 L) to give 6 fractions (Fr.1, 2.5 g; Fr.2,1.7 g; Fr.3, 1.8 g; Fr.4, 2.1 g; Fr.5, 1.4 g; Fr.6, 1.1 g) based on TLCmonitoring. Fraction Fr.3 was isolated by Sephadex LH-20 col-umn (2 × 50 cm) chromatography eluting with MeOH/CHCl3 (v/v1:1, 300mL) to give three subfractions (Fr.1-1 ~ Fr.1-3). Fr.1-2(147mg) was further separated with HPLC (MeOH/H2O, v/v33:67; tR of 1: 15.26, 2: 16.78min) to give compounds 1(25.4mg) and 2 (3.3mg). Fr.4 was separated into five parts, Fr.4-1~Fr.4–5, eluted with H2O/MeOH (v/v 6:4, 5 :5, 4 :6, 3 :7, 0 :10,each 500mL) on a reversed phased ODS column (2.0 × 50 cm).Compounds 3 and 4 were obtained from Fr.4-3 by HPLC(MeOH‑H2O, v/v 60:40) to give 3, 3.7mg (tR: 21.9), and 4,5.3mg (tR: 30.82min).

BioassayThe purity of the tested compounds was determined to be over95% by using the HPLC‑DAD method. The minimum inhibitoryconcentration values of the compounds for each test organismwere determined by a modification of the liquid dilution methodperformed in 96-well micro-trays [20].Chaetoglocin A (1): white wax; mp: 139–141°C; UV (MeOH) λmax

(log ε) 205 (4.8), 291 (3.4); IR(KBr) νmax 3396, 2947, 1699, 1557,1453, 1405, 1244, 1024 cm−1; 1H and 13C-NMR data, seel" Table 1;HRESIMS m/z 227.0908 ([M + H]+, calcd. for C11H15O5, 227.0914),249.0731 ([M + Na]+, calcd. for C11H14O5Na, 249.0733).Chaetoglocin B (2): white wax; mp: 139–141°C; UV (MeOH) λmax

(log ε) 207 (4.5), 284 (3.7); IR(KBr) νmax 3420, 2961, 2923, 1693,

1563, 1408, 1255, 1021 cm−1; 1H‑NMR and 13C‑NMR data, seel" Table 1; HRESIMS m/z 227.0915 ([M + H]+, calcd. for C11H15O5,227.0914), 249.0735 ([M + Na]+, calcd. for C11H14O5Na,249.0733).Chaetoglocin C (3): white amorphous powder; mp: 108–110°C;UV (MeOH) λmax (log ε) 279 (4.01), 235(3.98); IR(KBr) νmax

3306, 2970, 2880, 1678, 1614, 1484, 1396, 1291, 1129 cm−1; 1H-NMR and 13C-NMR data, see l" Table 2; HRESIMS m/z 184.0960([M + H]+, calcd. for C9H14NO3, 184.0968), 206.0783 ([M + Na]+,calcd. for C9H13NO3Na, 206.0788).Chaetoglocin D (4): white amorphous powder; mp: 109–112°C;UV (MeOH) λmax (log ε) 282 (4.09), 235 (3.98); IR(KBr) νmax

3253, 2977, 2885, 1683, 1603, 1461, 1375, 1315 cm−1; 1H-NMRand 13C-NMR data, see l" Table 2; HRESIMS m/z 184.0967 ([M +H]+, calcd. for C9H14NO3, 184.0968), 206.0787 ([M + Na]+, calcd.for C9H13NO3Na, 206.0788).

Acknowledgements!

This work was co-supported by grants for TRX from NSFC(30821006) and for GHM from NSFC (20802035), JSNSF(BK2008270), and MOE (200802841022).

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received May 19, 2010revised July 27, 2010accepted August 5, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1250292Published online September 2, 2010Planta Med 2011; 77: 277–280© Georg Thieme Verlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Dr. R. X. TanState Key Laboratory of Pharmaceutical BiotechnologyInstitute of Functional BiomoleculesNanjing UniversityNanjing 210093P.R. ChinaPhone: + 862583592945Fax: + 862583302728rxtan@nju.edu.cn