Article
Terpenoids from the Marine-Derived Fungus Aspergillus fumigatus YK-7 Yu Wang 1 , Da-Hong Li 2,3 , Zhan-Lin Li 2,3 , Yan-Jun Sun 4 , Hui-Ming Hua 2,3 , Tao Liu 1, * and Jiao Bai 2,3, * Received: 29 November 2015 ; Accepted: 21 December 2015 ; Published: 28 December 2015 Academic Editor: Isabel C. F. R. Ferreira 1 2
3 4
*
Department of Natural Products Chemistry, School of Pharmacy, China Medical University, Shenyang 110122, China;
[email protected] Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China;
[email protected] (D.-H.L.);
[email protected] (Z.-L.L.);
[email protected] (H.-M.H.) School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450046, China;
[email protected] Correspondence:
[email protected] (T.L.);
[email protected] (J.B.); Tel.: +86-24-3193-9078 (T.L.); +86-24-2398-6465 (J.B.)
Abstract: Two new β-bergamotane sesquiterpenoids, E-β-trans-5,8,11-trihydroxybergamot-9-ene (1) and β-trans-2β,5,15-trihydroxybergamot-10-ene (2), were isolated from the marine-derived fungus Aspergillus fumigatus YK-7, along with three known terpenoids 3–5. Their structures were determined by spectroscopic methods (1D and 2D NMR, HR-ESI-MS). Antiproliferative effects on human leukemic monocyte lymphoma U937 and human prostate cancer PC-3 cell lines were measured in vitro. Compound 4 exhibited potent activity against the U937 cell line with an IC50 value of 4.2 µM. Keywords: marine-derived fungus; Aspergillus fumigatus; terpenoid; cell growth inhibition
1. Introduction Microbial secondary metabolites are an important source of lead compounds for new drug development [1,2]. Aspergillus fumigatus has been found to generate many structurally and biologically diversified metabolites [3]. Among them, fumagillin is a meroterpenoid, and its derivatives have been studied for their potential use in the treatment of microsporidiosis [4] and amebiasis [5], and for their antiangiogenic properties exemplified by the irreversible inhibition of human type 2 methionine aminopeptidase (MetAP2) [6]. In our search for novel antitumor compounds from marine microorganisms, an extract of the fungus Aspergillus fumigatus YK-7, which was isolated from the sea mud of intertidal zone collected from Yingkou, China, exhibited significant activity against the U937 human leukemic monocyte lymphoma cell line (IC50 < 6.25 µg/mL). Previous investigation of this fungus had led to the isolation of fourteen 2,5-diketopiperazines [7]. In the course of our ongoing study on this fungus, two new β-bergamotane sesquiterpenoids 1 and 2 having a rare skeleton among fungal-derived metabolites and three known terpenoids 3–5 (Figure 1) were isolated from its fermentation broth. Compounds 1 and 2 may be the important intermediates in the biosynthesis of fumagillin and its derivatives [8]. Details of the isolation, structure elucidation, and cell growth inhibitory activities of these metabolites against U937 human leukemic monocyte lymphoma and PC-3 human prostate cancer cell lines are described here.
Molecules 2016, 21, 31; doi:10.3390/molecules21010031
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Figure1.1.Structures Structures of Figure of compounds compounds1–5. 1–5.
2. Results and Discussion
2. Results and Discussion
Compound 1 was obtained as a colorless oil. The molecular formula was demonstrated to be
Compound 1 was four obtained asofa unsaturation, colorless oil.based The on molecular formula was demonstrated C15H24O3, indicating degrees HR-ESI-MS (m/z 275.1587 [M + Na]+; calc.to be 13 C15 H275.1623) O , indicating four degrees of unsaturation, based on HR-ESI-MS (m/z 275.1587 [M + Na]+ ; 24 3 in combination with NMR data. The C-NMR spectrum showed 15 carbon signals. Analyses calc. of 275.1623) combination with NMRofdata. The C-NMR showed 15 carbon signals. the 1H-, 13in C-NMR, and HSQC spectra 1 (Table 1) 13 revealed thespectrum presence of three tertiary methyls 1 13 C 10.9; 1.33, δC 29.8; 1.33, δCspectra 30.1), terminal (δH 4.57 and 4.67,the δC 108.0; δC 147.9) andtertiary 1, (δH 0.80,δ Analyses of the H-,δH C-NMR, andδHHSQC of 1 (Table 1) revealed presence of three 2-disubstituted H10.9; 5.66, δ C 125.4; δ H 5.88, δ Cδ140.5) double bonds, an oxymethine (δ H 4.85, δ C 74.5), methyls (δH 0.80, δ(δ δ 1.33, δ 29.8; 1.33, δ 30.1), terminal (δ 4.57 and 4.67, δ C H C H C H C 108.0; and two oxygenated quaternary carbons (δ C 70.9; 76.7). These spectroscopic features together with δC 147.9) and 1, 2-disubstituted (δH 5.66, δC 125.4; δH 5.88, δC 140.5) double bonds, an oxymethine the molecular formula indicated that 1 was a sesquiterpenoid. Since two double bonds accounted for (δH 4.85, δC 74.5), and two oxygenated quaternary carbons (δC 70.9; 76.7). These spectroscopic two of the four degrees of unsaturation, 1 was concluded to be bicyclic. The HMBC spectrum of 1 features together with the molecular formula indicated that 1 was a sesquiterpenoid. Since two double (Figure 2) showed that the exomethylene protons H-15 (δH 4.57 and 4.67) were correlated with C-1 (δC bonds accounted for two of (δ the four degrees of unsaturation, 1 was concluded to be bicyclic. 42.2), C-2 (δC 147.9), and C-3 C 25.3), and H-3 (δH 2.32 and 2.61) and H-7 (δH 1.91) were correlated with The HMBC spectrum of 1 (Figure 2) that the exomethylene H-15 (δH 4.57 the oxygenated carbon C-5 (δC 76.7).showed These results, as well as the COSYprotons correlations (Figure 2) ofand H-1 4.67) were (δ correlated with C-1 (δCand 42.2), 147.9), andand C-32.61) (δC with 25.3), and 2.61) and H 2.33) with H-7 (δ H 1.91 2.47)C-2 and(δofC H-3 (δH 2.32 H-4 (δHH-3 1.79 (δ and 1.98),and indicated H 2.32 H-7 (δ 1.91) were correlated with the oxygenated carbon C-5 (δ 76.7). These results, as well as the presence of a 4-methylene cyclohexanol ring. Specifically, the HMBC correlations of H-1 (δ H 2.33), H C H-7 (δHcorrelations 1.91), and H-4(Figure (δH 1.79)2) with C 52.5), and of CH 3-14 (δH 0.80) with C-1 (δ C 42.2), C-5 (δ C 76.7), the COSY of C-6 H-1(δ(δ 2.33) with H-7 1.91 and 2.47) and of H-3 (δ H H H 2.32 and C-6 (δ C 52.5), led to the assignment of a 6-methylbicyclo[3.1.1]heptane skeleton. Additionally, the and 2.61) with H-4 (δH 1.79 and 1.98), indicated the presence of a 4-methylene cyclohexanol ring. COSY correlations H-8 to H-10, and(δthe2.33), HMBC correlations theH-4 olefinic H-9 C-6 (δH 5.66) Specifically, the HMBCfrom correlations of H-1 H-7 (δH 1.91),ofand (δH proton 1.79) with (δC 52.5), H with the oxygenated carbon C-11 (δC 70.9), and of the olefinic proton H-10 (δH 5.88) with C-12 (δC 29.8) and of CH3 -14 (δH 0.80) with C-1 (δC 42.2), C-5 (δC 76.7), and C-6 (δC 52.5), led to the assignment of and C-13 (δC 30.1) suggested the presence of a 1,4-dihydroxy-4-methylpent-2-enyl side chain in 1. The a 6-methylbicyclo[3.1.1]heptane skeleton. Additionally, the COSY correlations from H-8 to H-10, and linkage of the two moieties was secured by the HMBC correlations of H-8 (δH 4.85) and H-9 (δH 5.66) the HMBC correlations of the olefinic proton (δH 5.66) the oxygenated carbon C-11[9,10]. (δC 70.9), with C-6 (δC 52.5). Therefore, compound 1 wasH-9 established as awith β-5,8,11-trihydroxybergamot-9-ene and of theThe olefinic proton H-10 with C-12 (δC 29.8) C-13 (δCof30.1) suggested the presence H 5.88) double bond was assigned as Eand on the basis a coupling constant 15.7 geometry of the Δ9 (δ of a 1,4-dihydroxy-4-methylpent-2-enyl side chain 1. The linkage of the two moieties was3)secured Hz (J H-9,10). The 6-methyl-endo configuration was in determined by NOESY correlations (Figure of by theH-4β HMBC correlations 4.85) and H-9 (δ 5.66) with C-6 (δ 52.5). Therefore, compound (δH 1.98) with CH3of -14H-8 (δH (δ 0.80), H-7 (δ H 1.91) with H-4α (δ H 1.79), and of H-7 (δ H 2.47) with H-8 H H C 1H-NMR chemical shift data for CH3-14 H 4.85), and as wasa further confirmed by the comparison of[9,10]. 1 was(δestablished β-5,8,11-trihydroxybergamot-9-ene (δH 0.80) with the literature (δH 0.71bond for β-trans-bergamotene; δH 1.23 for β-cis-bergamotene) [11]. The geometry of the values ∆9 double was assigned as E on the basis of a coupling Thus, the structure of 1 was assigned as E-β-trans-5,8,11-trihydroxybergamot-9-ene, although the constant 15.7 Hz (J H´9,10 ). The 6-methyl-endo configuration was determined by NOESY correlations absolute configuration was not defined. (Figure 3) of H-4β (δH 1.98) with CH3 -14 (δH 0.80), H-7 (δH 1.91) with H-4α (δH 1.79), Compound 2 was obtained as colorless needles. The molecular formula C15H26O3 from HR-ESI-MS and of H-7 (δH 2.47) with H-8 (δH 4.85), and was further confirmed by the comparison indicated that it possessed two more hydrogen atoms than (m/z 277.1737 [M + Na]+; calc. 277.1780) 1 of H-NMR chemical fordata CH (δH 1)0.80) with the literature (δH 0.71 compound 1. The 1H-shift and 13data C-NMR of3 -14 2 (Table showed similarity to those ofvalues 1, suggesting the for β-trans-bergamotene; δ 1.23 for β-cis-bergamotene) [11]. Thus, the structure of 1 was assigned presence of another H β-bergamotane skeleton. However, a hydroxymethyl group (δH 3.34 and 3.47; as E-β-trans-5,8,11-trihydroxybergamot-9-ene, absolute was not δC 69.4) linked to the oxygenated carbon C-2although (δC 76.5) inthe 2 replaced theconfiguration exomethylene group in 1,defined. which Compound 2 was obtained as colorless needles. The molecular formula C15 H26 O3 from HR-ESI-MS (m/z 277.1737 [M + Na]+ ; calc. 277.1780) indicated that it possessed two more hydrogen atoms than compound 1. The 1 H- and 13 C-NMR data of 2 (Table 1) showed similarity to those of 1,
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suggesting the presence of another β-bergamotane skeleton. However, a hydroxymethyl group (δH 3.34 and 3.47; δC 69.4) linked to the oxygenated carbon C-2 (δC 76.5) in 2 replaced the exomethylene group in 1, which was confirmed by the HMBC correlations (Figure 2) of H-15 (δH 3.34 and 3.47) with C-1 (δC 39.7), C-2 (δC 76.5), and C-3 (δC 29.4). Moreover, the side chain in 2 was different from Molecules 2016, 21, 31 3 of 6 that in 1, which was established as 4-methylpent-3-enyl by the COSY correlations (Figure 2) from H-8 to H-10, and thebyHMBC correlations H-9 (δ 2.10) (δC(δ131.8), and of the was confirmed the HMBC correlationsof (Figure 2)Hof2.03 H-15and (δH 3.34 andwith 3.47)C-11 with C-1 C 39.7), C-2 olefinic (δproton H-10 (δ 5.15) with C-12 (δ 17.8) and C-13 (δ 25.8). NOESY correlations (Figure 3 C 76.5), and C-3 H (δC 29.4). Moreover, theC side chain in 2 was C different from that in 1, which was Molecules 2016, 21, 31 3 of 6 established as 4-methylpent-3-enyl by the COSY correlations (Figure 2) from H-8 to H-10, and the of H-4β (δH 2.05)/CH3 -14 (δH 1.18), H-4α (δH 1.75)/H-7 (δH 1.47), and H-7 (δH 1.47)/H-15 (δH 3.34 HMBC correlations (δH 2.03 andconfiguration 2.10) with 2) C-11 (δ C 131.8), and the olefinic H-10 was confirmed by of theH-9 HMBC correlations (Figure of H-15 (δthe H 3.34 andof3.47) with C-1 (δ C 39.7), C-2(δH and 3.47) indicated the 6-methyl-endo and α-orientation ofproton the hydroxymethyl 5.15) C-12 (δ C 17.8) and C-13 (δC 25.8). NOESY correlations (Figurefrom 3) of that H-4β (δ H which 2.05)/CH 3-14 (δ Cwith 76.5), and C-3 (δ C 29.4). Moreover, the side chain in 2 was different in 1, was group. Consequently, the structure of 2 was defined as β-trans-2β,5,15-trihydroxybergamot-10-ene. (δHestablished 1.18), H-4α H 1.75)/H-7 (δH 1.47), and H-7correlations (δH 1.47)/H-15 (δH2)3.34 indicated as (δ 4-methylpent-3-enyl by the COSY (Figure fromand H-83.47) to H-10, and thethe The known compounds alismol (3) [12],α-orientation pyripyropene Ehydroxymethyl (4) [13], and helvolic acid (5) [14,15], were HMBC correlations of H-9 (δand H 2.03 2.10) with C-11 (δC 131.8), and of the group. olefinic Consequently, proton H-10 (δHthe 6-methyl-endo configuration theand of the identified by comparison of their spectroscopic data with those reported in the literature. 5.15) with C 17.8) and (δC 25.8). NOESY correlations (Figure 3) ofThe H-4βknown (δH 2.05)/CH 3-14 structure of 2C-12 was(δdefined as C-13 β-trans-2β,5,15-trihydroxybergamot-10-ene. compounds (δH 1.18), H-4α (δH 1.75)/H-7E (δ 1.47),and andhelvolic H-7 (δHacid 1.47)/H-15 (δH were 3.34 and 3.47) indicated the alismol (3) [12], pyripyropene (4)H [13], (5) [14,15], identified by comparison 1 H- and 13the 6-methyl-endo configuration and α-orientation ofcompounds the hydroxymethyl Consequently, the Table 1. C-NMR data 1 and 2group. in CDCl . of their spectroscopic data with those reported in for the literature. 3 structure of 2 was defined as β-trans-2β,5,15-trihydroxybergamot-10-ene. The known compounds alismol (3) [12], pyripyropene (4) 13[13], and helvolic acid (5) [14,15], were identified by comparison Table 1. 1H-Eand 1C-NMR data for compounds 1 and 2 in2CDCl3. of their spectroscopic data with those reported in the literature.
2 δH b (J in Hz) δC a δC a δH b1(J in Hz) a and 13C-NMR Table 1. data for compounds CDCl 3. Position δC1HδH b(J in Hz) δC a 1 andδ2Hinb(J in Hz) 42.2 2.33, d (7.5) 39.7 2.15, m 1 42.2 2.33, d (7.5) 39.7 2.15, m 1 2 147.9 76.5 2 147.9 a Position δH b2.61, (J in Hz) δ76.5 C29.4 δH b(J in Hz)1.86, m 25.3 δC a 2.32, m 1.82, 3 1 31.5 25.3 2.32, 2.61, m m 39.7 29.4 1.82, m (β), m 42.21.79 (α), 2.33, d (7.5) 2.15,1.86, m 2.05 1.98 (β), 30.8 1.75 (α), 4 2 76.7 31.5 30.8 1.75 (α), 2.05 (β), m 147.9 1.79 (α), 1.98 (β), m 76.5 76.2 5 3 52.5 76.7 76.2 25.3 2.32, 2.61, m 29.4 46.9 1.82, 1.86, m 64 52.5 46.9 1.75 (α), 2.05 (β), m 31.5 1.79 1.98 (β), m 30.8 1.91,(α), d (10.0) 1.47, 2.17, m 36.1 36.1 1.91, d (10.0) 5 76.7 76.2 7 36.1 2.47, dd (10.0, 7.5) 36.1 1.47, 2.17, m dd (10.0, 7.5) 6 74.5 52.5 2.47, 46.9 4.85, d (6.1) 34.5 1.45, 1.70, m 1.91, d 8 125.474.5 5.66, 4.85, d (10.0) (6.1) 34.5 1.45, 2.03, 1.70, m dd (15.7, 6.1) 23.2 1.47, 7 36.1 36.1 2.17, m2.10, m 2.47, dd (10.0, 7.5) 9 140.5125.4 5.66, (15.7, 6.1) 23.2 2.03, 2.10, 5.88,dd d (15.7) 124.9 5.15,m t (7.1) 74.5 4.85,dd(15.7) (6.1) 34.5 1.70, m 108 70.9140.5 5.88, 124.9 5.15, t (7.1) 131.8 1.45, 125.4 5.66,1.33, dd (15.7, 6.1) 23.2 m s s 17.8 2.03, 2.10,1.62, 119 29.8 70.9 131.8 140.5 5.88, d s(15.7) 124.9 5.15,1.62, t (7.1) 1.33, 25.8 1.68, s 121030.1 29.8 1.33, s 17.8 s 70.9 131.8 0.80, 17.8 s 131110.9 30.1 1.33,s s 25.8 1.68, 1.18, s 12 29.8 1.33, 17.8 1.62, s d (10.8) 4.57, br. sss 3.34, 14 10.9 0.80, 17.8 1.18, s 69.4 13108.0 30.1 1.33, ss 25.8 1.68, s d (10.8) 4.67, 4.57,br. br. s 3.34, 3.47, d (10.8) 1514 108.0 69.4 10.9 0.80, s 17.8 1.18, s a Recorded at4.67, br. sb Recorded at 300 MHz. 3.47, d (10.8) 75 MHz;
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
4.57, br. s 3.34, d (10.8) a Recorded at 75 MHz; b Recorded 108.0 69.4at 300 MHz. 4.67, br. s 3.47, d (10.8)
15
a
Recorded at 75 MHz; b Recorded at 300 MHz. based on 1H-1H COSY
based on 1HMBC based on H-1H COSY based on HMBC
OH OH
OH OH
1
1
OH
HO
OH
HO
HO HO
OH OH
2
2
1 H-1H2. Key H COSYand andHMBC HMBC correlations of compounds 1 and12.and 2. FigureFigure 2. Key H COSY correlations of compounds 1 1 1
1
Figure 2. Key H- H COSY and HMBC correlations of compounds 1 and 2.
Figure 3. Key NOESY correlations of compounds 1 and 2.
3. Key NOESYcorrelations correlations ofof compounds 1 and12.and 2. FigureFigure 3. Key NOESY compounds
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All compounds were evaluated in vitro for cell growth inhibitory activities against the U937 and PC-3 cell lines (IC50 values are shown in Table 2). Compound 4 exhibited potent selective inhibition against U937 cell line, with the IC50 value of 4.2 µM, and 1, 3, and 5 exhibited weak activities against U937 cell line with IC50 values of 84.9, 61.7, and 57.5 µM, respectively. All the compounds didn’t show antiproliferative effect in PC-3 cell lines. Table 2. Antiproliferative activity (IC50 (µM)) of compounds 1–5 on U937 and PC-3 cells a . Compound
U937 Cells
PC-3 Cells
1 2 3 4 5 Doxorubicin hydrochloride
84.9 ˘ 2.4 >100 67.1 ˘ 1.9 4.2 ˘ 0.3 57.5 ˘ 3.2 0.021 ˘ 0.002
>100 >100 >100 >100 >100 0.73 ˘ 0.04
a
U937 cells were treated for 3 days, and PC-3 cells were treated for 4 days. IC50 value is the concentration that inhibited 50% of cell growth. The data shown are means ˘ S.D. of three independent experiments.
3. Experimental Section 3.1. General Procedures Optical rotations were obtained on a Perkin-Elmer 241MC polarimeter (Perkin-Elmer, Waltham, MA, USA). The IR spectra were recorded on Bruker IFS-55 spectrometer (Bruker Optics, Ettlingen, Germany). NMR spectra were recorded on Bruker ARX-300 or AV-600 NMR spectrometers (Bruker Biospin, Fallanden, Switzerland), with TMS as the internal standard. HR-ESI-MS was performed on a Bruker microTOF-Q mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Chromatographic silica gel (200–300 mesh) was purchased from Qingdao Marine Chemical Factory (Qingdao, China), and ODS (50 µm) was obtained from YMC Co. Ltd. (Kyoto, Japan). The RP-HPLC analysis and semi-preparation were conducted using a Hitachi L2130 series pumping system (Hitachi, Tokyo, Japan) equipped with a Hitachi L2400 UV detector (Hitachi, Tokyo, Japan) and a YMC-PACK ODS-AM column (250 ˆ 10 mm, 5 µm, YMC, Kyoto, Japan). TLC spots were visualized under UV light (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd., Zhengzhou, China) and with 10% H2 SO4 in EtOH followed by heating. 3.2. Fungal Material The fungus, YK-7, was isolated from an intertidal zone sea mud sample collected from Yingkou, China, and identified as Aspergillus fumigatus by its morphological characteristics and ITS sequences [7,16]. A voucher strain was deposited at ´80 ˝ C in School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University (Shenyang, China). 3.3. Extraction and Isolation The fungus was cultivated at 28 ˝ C for 7 days while shaking at 165 rpm in 300 500 mL flasks containing a liquid medium (150 mL per flask) composed of 3 g of yeast extract, 1 g of corn steep liquor, 20 g of mannitol, 10 g of monosodium glutamate, 10 g of glucose, 20 g of maltose, 0.5 g of KH2 PO4 , and 0.3 g of MgSO4 ¨ 7H2 O, per 1000 mL seawater at pH 6.5. The fermented whole broth (45 L) was filtered through a cheesecloth into the supernatant and the mycelia. The supernatant was concentrated under reduced pressure to about 5 L, partitioned with EtOAc (3 ˆ 5 L) at room temperature, and then dried by rotary evaporation to yield a crude extract (21 g), which showed significant growth inhibitory activity against the U937 cell line (IC50 < 6.25 µg/mL). The crude extract was subjected to column chromatography (CC) (SiO2 ; CHCl3 /MeOH gradient) to yield 17 fractions, Fr. 1–17. Fr. 2 (100:1) was purified by repeated CC (SiO2 ; petroleum ether (PE)/acetone 100:15; and ODS; MeOH/H2 O 65:35) to afford 5 (15 mg). Fr. 3 (100:2) was subjected
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to repeated CC (SiO2 ; PE/acetone 100:25; and ODS; MeOH/H2 O 90:10) to afford 3 (21 mg). Fr. 5 (100:5) was fractionated by CC (ODS; MeOH/H2 O) to give seven subfractions, subfrs. 5-1–5-7. Subfr. 5-3 (40:60) was purified by CC (SiO2 ; PE/acetone 2:1) to afford 2 (3 mg). Subfr. 5-4 (50:50) was further subjected to CC (SiO2 ; PE/acetone 4.5:1) to yield 1 (3 mg). The mycelia were extracted with acetone (3 ˆ 3 L) at room temperature and then dried by rotary evaporation. The crude extract (250 g; IC50 < 6.25 µg/mL) was subjected to CC (SiO2 ; CHCl3 /MeOH gradient) to yield 14 fractions, Fr. 1–14. Fr. 2 (100:1) was separated by CC (SiO2 ; PE/acetone gradient) to give six subfractions, subfrs. 2-1–2-6. Subfr. 2-4 (5:1) was further purified by semipreparative HPLC (MeOH/H2 O 85:15; tR = 38 min) to afford 4 (6 mg). E-β-trans-5,8,11-trihydroxybergamot-9-ene (1): colorless oil; rαs22 D –21.6 (c 0.11, MeOH); IR(KBr) νmax 3426, 2920, 2851, 1643, 1460, 1384, 1129, 879 cm´1 ; 1 H- and 13 C-NMR data, see Table 1; HR-ESI-MS m/z 275.1587 [M + Na]+ (Calcd for C15 H24 O3 Na, 275.1623). The IR, NMR, and HR-MS spectra of compound 1 can be found at Supplementary Material (Figures S1–S8). β-trans-2β,5,15-trihydroxybergamot-10-ene (2): colorless needles (MeOH); mp 115–116 ˝ C; rαs22 D –18.5 (c 0.10, MeOH); IR(KBr) νmax 3405, 2921, 2852, 1642, 1452, 1383, 1150, 1052, 954 cm´1 ; 1 H- and 13 C-NMR data, see Table 1; HR-ESI-MS m/z 277.1737 [M + Na]+ (Calcd for C15 H26 O3 Na, 277.1780). The IR, NMR, and HR-MS spectra of compound 2 can be found at Supplementary Material (Figures S9–S16). 3.4. Cell Culture and Growth-Inhibition Assay The growth inhibitory assay was performed as described previously [17,18]. Human leukemic monocyte lymphoma U937 and human prostate cancer PC-3 cell lines (American Type Culture Collection, Rockville, MD, USA) were cultured in RPMI-1640 medium (Gibco, New York, NY, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mmol glutamine, and 10% heat-inactivated fetal bovine serum. The growth-inhibitory ability of these crude extracts and isolated compounds was calculated and expressed as the ratio of the cell number in treated group to that of the untreated group. The concentration that inhibited half of the cell growth, IC50 , was calculated. Doxorubicin hydrochloride (Hua Bo Technology Co. Ltd., Beijing, China) was used as a positive control, and 0.1% DMSO was used as a negative control. Supplementary Materials: The IR, NMR, and HR-MS spectra of compounds 1 and 2 are available online at: http://www.mdpi.com/1420-3049/21/1/31/s1. Acknowledgments: This work was supported by the National Natural Science Foundation of China (Grant No. 81302672). Author Contributions: Y.W. performed the experiments for the isolation, structure elucidation, and cell growth-inhibition assay; Y.W., D.-H.L., Z.-L.L., and Y.-J.S. analyzed the spectroscopic data and elucidated the structure of the new molecules; Y.W. wrote the paper; T.L., J.B., and H.-M.H. supervised the research work and revised the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Sample of the compound 5 is available from the authors. © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
