Cytotoxic and Antibacterial Quinone Sesquiterpenes from a

Article pubs.acs.org/jnp

Cytotoxic and Antibacterial Quinone Sesquiterpenes from a Myrothecium Fungus Ying Fu,†,‡ Ping Wu,† Jinghua Xue,† and Xiaoyi Wei*,† †

Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, People’s Republic of China ‡ University of Chinese Academy of Sciences, Yuquanlu 19A, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Six new quinone sesquiterpenes, myrothecols A−F (1−6), and hymenopsin B (7) were isolated from cultures of Myrothecium sp. SC0265 by bioassay-guided fractionation. Their structures were elucidated on the basis of 1D and 2D NMR and MS data. The absolute configurations of the new compounds were assigned by CD/TDDFT calculations, and that of and hymenopsin B was confirmed by X-ray diffraction analysis. Compounds 1−5 and hymenopsin demonstrated cytotoxic activity against human carcinoma A549, HeLa, and HepG2 cells. Compounds 1−5 also exhibited antibacterial activity against Staphylococcus aureus and Bacillus cereus.

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RESULTS AND DISCUSSION The producing fungus, Myrothecium sp. SC0265, was isolated from a forest leaf litter sample collected in the Dinghu Mountain Biosphere Reserve, Guangdong, China. The fungus was cultured by solid-substrated fermentation on wheat grains. The EtOH extract of the fermentation mixture was fractionated by successive extraction with petroleum ether, EtOAc, and nBuOH. The EtOAc-soluble fraction, displaying antibacterial activity against S. aureus, was subjected to repeated column chromatography (CC) over silica gel, ODS, and Sephadex LH20 followed by preparative HPLC to yield compounds 1−7. The molecular formula of myrothecol A (1) was determined to be C22H31ClO5 (seven unsaturations) on the basis of HRESIMS and NMR data. The 1H NMR spectrum (Table 1) indicated the presence of two oxygenated methines [δ 4.93, 3.78 (each 1H, d, J = 1.6 Hz, H-5′ and H-6′)], two oxygenated methylenes [δ 4.60, 4.30 (each 1H, d, J = 15.6 Hz, H2-7′); δ 3.00, 3.37 (each 1H, d, J = 11.2 Hz, H2-13)], two methyls [δ 0.78, 0.75 (each 3H, s, H3-15 and H3-14)], and two olefinic protons [δ 4.81, 4.60 (each 1H, br s, H2-12)]. In the 13C NMR spectrum (Table 1), resonances were observed for a carbonyl group [δ 188.9 (C-2′)], four olefinic carbons [(δ 153.1 (C-4′), 150.6 (C-8), 128.2 (C-3′), 107.7 (C-12)], and five oxygenated sp3 carbons [(δ 72.1 (C-12), 64.5 (C-5′), 62.5 (C-6′), 61.5 (C-

uinone sesquiterpenes are a class of secondary metabolites that combine a bicyclic drimane sesquiterpene unit with a quinone or quinol moiety.1 These compounds have provoked much interest because of their abundant structural variants and attractive biological activities, including anti-HIV, antituberculosis, protein kinase inhibition, and immunosuppression.1 Most of these were isolated from sponges, brown aglae, and fungi.1 Macrophorins, representatives of this class of metabolite, demonstrated potent antisepsis, antifungal, and immunomodulatory activity, cytoxicity against tumor cells, and 11β-hydroxysteroid dehydrogenase inhibition.2−5 In contrast to macrophorins, which have a tricyclic carbon skeleton, hymenopsins are tetracarbocyclic quinone sesquiterpenes and were reported to have weak antibacterial activity.6 During our search for bioactive natural products produced by filamentous fungi collected in South China, the methanol extract from a mycelial solid culture of Myrothecium sp. SC0265 was found to show antibacterial activity against Staphylococcus aureus. We therefore investigated the secondary metabolites of this fungus and isolated six new quinone sesquiterpenes, myrothecols A−F (1−6), along with hymenopsin B (7). Herein, we report the isolation, structure elucidation, and antibacterial and tumor cell growth inhibitory activities of these compounds. © 2014 American Chemical Society and American Society of Pharmacognosy

Received: February 15, 2014 Published: August 4, 2014 1791

dx.doi.org/10.1021/np500142g | J. Nat. Prod. 2014, 77, 1791−1799

Journal of Natural Products

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exocyclic double bond corresponding to the olefinic proton signals in the 1H NMR spectrum. The other two olefinic carbons (C-3′ and C-4′) constituted a tetrasubstituted olefin unit. The remaining three degrees of unsaturation revealed the presence of three rings. Analysis of the 1H−1H COSY and HMBC spectra (Figure 1) revealed that 1 shared the same

1′), 61.2 (C-7′)]. According to the HSQC spectrum, 28 hydrogens were bound to carbon atoms, illustrating the existence of three hydroxy groups. The availability of only one oxygen atom and the remaining two oxygenated carbons (C-1′ and C-6′) suggested the presence of an epoxide unit. The carbon signals at δ 150.6 (C-8) and 107.7 (C-12) indicated an

Figure 1. 1H−1H COSY (bold lines) and key HMBC correlations (arrows) of 1.

drimane sesquiterpene-coupled epoxycyclohexenone skeleton as the macrophorins.2−5 The differences between 1 and the

Table 1. NMR Spectroscopic Data (600 MHz, CD3OD) for Compounds 1−3 1 position

δC, type

1

39.8, CH2

2

19.9, CH2

3

36.7, CH2

4 5 6

39.2, C 49.5, CH 25.3, CH2

7

38.9, CH2

8 9 10 11

150.6, 52.9, 40.7, 22.2,

12

107.7, CH2

13

72.1, CH2

14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′

18.2, 15.7, 61.5, 188.9, 128.2, 153.1, 64.5, 62.5, 61.2,

C CH C CH2

CH3 CH3 C C C C CH CH CH2

2

δH, mult. (J in Hz) α 1.21 td (13.3, 3.6) β 1.82 br d (13.3) α 1.60 m β 1.68 m α 1.55 overlapped β 1.26 br d (13.0) 1.55 overlapped α 1.68 m β 1.33 qd (13.1, 4.3) α 2.06 td (13.1, 5.2) β 2.35 br d (13.1) 1.78 d (10.8) α 2.37 br d (14.7) β 2.09 dd (14.7, 10.8) a 4.81 br s b 4.60 br s 3.37 d (11.2) 3.00 d (11.2) 0.75 s 0.78 s

4.93 3.78 4.59 4.39

d d d d

(1.6) (1.6) (15.6) (15.6)

δC, type 39.9, CH2 19.9, CH2 36.6, CH2 39.2, C 49.3, CH 25.0, CH2 38.9, CH2 150.6, 50.6, 40.4, 21.2,

C CH C CH2

108.7, CH2 72.1, CH2 18.3, 15.9, 60.5, 197.1, 121.5, 160.4, 64.6, 63.5, 62.8,

CH3 CH3 C C CH C CH CH CH2

3

δH, mult. (J in Hz) α 1.02 td (13.3, 3.6) β 1.76 br d (13.3) α 1.56 m β 1.64 m α 1.53 overlapped β 1.24 br d (13.3) 1.53 overlapped α 1.67 m β 1.34 qd (13.1, 4.0) α 2.09 td (13.1, 4.8) β 2.39 ddd (12.9, 3.8, 2.3) 1.60 d (10.4) α 2.21 dd (16.3, 1.7) β 2.15 dd (16.3, 10.4) a 4.83 br s b 4.50 br s 3.37 d (11.0) 3.00 d (11.0) 0.75 s 0.80 s

6.05 t (1.5) 4.40 3.50 4.40 4.16 1792

br s d (1.6) dd (17.5, 1.5) dd (17.5, 1.5)

δC, type 39.8, CH2 19.9, CH2 36.7, CH2 39.2, C 49.5, CH 25.3, CH2 39.0, CH2 150.7, 52.9, 40.6, 21.5,

C CH C CH2

107.6, CH2 72.0, CH2 18.3, 15.7, 60.6, 195.8, 121.5, 160.4, 64.3, 63.4, 62.9,


δH, mult. (J in Hz) α 1.19 td (12.9, 3.6) β 1.81 br d (12.9) α 1.58 m β 1.67 overlapped α 1.55 m β 1.24 br d (13.0) 1.53 dd (12.3, 2.2) α 1.67 overlapped β 1.32 qd (13.0, 4.0) α 2.06 td (13.1, 4.7) β 2.35 ddd (12.7, 3.6, 2.2) 1.77 d (10.8) α 2.32 br d (14.3) β 2.03 dd (14.3, 10.8) a 4.79 br s b 4.61 br s 3.37 d (11.1) 2.99 d (11.1) 0.74 s 0.78 s

6.03 t (1.4) 4.42 3.67 4.41 4.16

br s d (1.3) dd (17.3, 1.4) dd (17.3, 1.4)



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1a1 (ΔG = 0.14 kcal/mol in MeOH solution) (Figure 2), the angle was about 69°, while in pair 1b (accounting for about 47% equilibrium population of 1 in MeOH), represented by the global minimum 1b1 (Figure 2), this angle was about −141°. The dihedral angle from C-8 to C-1′ was similar between the two pairs (ca. +81° or +75°). The NOE correlations observed between H-11β/H-12b, H-7β/H-12a, and H-1β/H-11α in the NOESY spectrum (Figure 2) were in agreement with all conformers of both pairs, whereas the NOEs of H-6′ with H11β and H-12b were characteristic of conformers in pair 1a, and those of H-6′ with H-9 and H-11α were indicative of conformers in pair 1b. It was also apparent that H-9 was in an anti relationship with H-11β and a gauche relationship with H11α in conformers of both pairs, which was consistent with the measured 1H NMR coupling constants (J9,11β = 10.8 and J9,11α ≈ 0 Hz). On the basis of the above spectroscopic analysis in combination with theoretical calculations, the relative configuration of 1 was assigned as shown. In order to assign the absolute configuration of 1, we carried out CD/TDDFT calculations on all low-energy conformers using the functional CAM-B3LYP and the basis set SVP. The simulated CD spectra of individual conformers (see Supporting Information) and the Botzmann-weighted spectra in both the gas phase and MeOH solution (Figure 3) exhibited positive

macrophorins were that 1 possessed an additional hydroxy group and a chlorine atom. The presence of HMBC correlations from H2-13 to C-3, C-4, C-5, and C-14 indicated the additional hydroxy group was located at C-13.6 The chlorine atom was deduced to be attached to C-3′ because it was the only possibility, and the shift of C-3′ (δ 128.2 ppm) was in agreement with those reported for other relevant αchlorocyclohexenone derivatives.7 The assignment of the configuration of the epoxycyclohexenone ring in 1 was fairly challenging because of its flattened ring conformation on account of the presence of the epoxide and the double bond. As a result, the NOESY correlations among protons of the ring were meaningless to distinguish the relationship (cis or trans) between H-5′ (δ 4.93) and H-6′ (δ 3.78). It has been demonstrated that the J value between the vicinal epoxymethine and hydroxymethine protons in the cyclohexenone rings differs slightly in cis and trans relationships of the two protons.8 A trans relationship between 1′,6′-epoxy and 5′-OH in 1 was confirmed on the basis of the fact that the J5′,6′ value was 1.6 Hz, which was consistent with that of jesterone (trans),9 but quite different from those of macrophorins (cis)2 and 7-deacetoxyyanuthone A (cis).10 The relative configuration of the sesquiterpene unit was determined to be the same as those of 2′,3′-epoxy-13-hydroxy-4′-oxomacrophorin A6 and phlomistetraol A11 by analysis of the NOESY (Figure 2) and 1D NMR data. However, the sesquiterpene unit and the

Figure 3. Comparison between calculated and measured CD spectra of 1.

Cotton effects (CEs) around 320 and 260 nm and a negative CE around 220 nm, all consistent with the experimental spectrum (Figure 3). This result indicated 1′R,5′S,6′R configurations in the quinone portion and permitted the assignment of 4R,5R,9S,10R configurations to the sesquiterpene moiety on the basis of the relative configuration. This stereochemistry was supported by the co-occurrence of 1 with the known compound hymenopsin B (7);6 the stereochemistry of the latter was confirmed by X-ray diffraction analysis in the present study (see below). Myrothecols B (2) and C (3) had the same formula, C22H32O5, as determined from HRESIMS data. The 1H and 13 C NMR spectra (Table 1) revealed that their structures were similar to that of compound 1, except that the chlorosubstituted olefinic quaternary carbon (C-3′) in 1 was replaced by an olefinic methine in both 2 [δH 6.05 (1H, t, J = 1.5 Hz, H3′); δC 121.5 (C-3′)] and 3 [δH 6.03 (1H, t, J = 1.4 Hz, H-3′); δC 121.5 (C-3′)]. Analysis of the 1H−1H COSY, HSQC, and HMBC spectra supported the assignments of their 1D NMR data (Table 1) and showed that the data for 2 and 3 were almost identical. The only differences found were in the proton

Figure 2. Low-energy conformers and key NOE correlations (curves) of 1. Dashed curves show different correlations between the two conformers.

epoxycyclohexenone moiety in 1 were connected by two single bonds (C-9−C-11 and C-11−C-1′), and the compound should exist as an equilibrium of multiple rotamers of these two bonds. Straightforward analysis of the NOESY spectrum was unsuitable to determine the configurational relationship between the two parts. In order to clarify the dominant lowenergy conformers, we carried out a conformational analysis on this compound by theoretical computations. MMFF conformational search followed by geometry optimization using the DFT method at the B3LYP/6-31G(d,p) level afforded four distinctive low-energy minima (see Supporting Information), which could be grouped into two different pairs (1a and 1b) according to their dihedral angle from C-9 to C-2′. In pair 1a, which accounted for about 43% equilibrium population of the compound in MeOH solution according to the Boltzmann statistics, represented by the second lowest energy minimum 1793



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Figure 4. Comparison between calculated and measured CD spectra of 2 and 3.

Table 2. NMR Spectroscopic Data (600 MHz) for Compounds 4−6 4 in C5D5N position

δC, type

1

38.8, CH2

2

19.0, CH2

3

36.2, CH2

4 5 6

37.4, C 50.0, CH 24.5, CH2

7

37.8, CH2

8 9 10 11

149.0, 49.8, 39.8, 21.1,

C CH C CH2

12

108.9, CH2

13

73.4, CH2

14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′

17.8, 15.5, 60.3, 196.1, 120.9, 162.0, 64.4, 63.8, 62.7,


Ac(CH3) Ac(CO)

21.1, CH3 171.2, C

δH, mult. (J in Hz) α 0.99 td (12.9, 3.9) β 1.67 dt (12.8, 3.0) α 1.41 m β 1.46 m α 1.31−1.34 m β 1.31−1.34 m 1.40 br d (12.3) α 1.54 br d (12.9) β 1.24 qd (12.9, 4.1) α 1.99 td (13.1, 5.4) β 2.32 ddd (13.1, 3.6, 2.2) 1.76 d (10.0) α 2.49 dd (16.2, 2.0) β 2.44 dd (16.2,10.0) a 4.87 br s b 4.67 br s 3.93 d (10.7) 3.77 d (10.7) 0.74 s 0.68 s

6.83 t (1.9) 5.05 3.93 5.05 4.67 2.04

br s d (1.4) dd (17.5, 1.8) dd (17.5, 1.8) s

5 in C5D5N δC, type

19.1, CH2 36.4, CH2 37.6, C 50.2, CH2 24.8, CH2

39.6, CH2 20.0, CH2 36.8, CH2

1.38 dd (12.7, 2.6) α 1.52 br d (12.9) β 1.24 qd (12.9, 4.2) α 1.95 td (13.1, 5.0) β 2.30 ddd (13.1, 3.6, 2.2)

38.3, CH2 C CH C CH2

1.94 br d (10.0) α 2.41 br d (14.7) β 2.34 dd (14.7, 10.8) a 4.95 br s b 4.92 br s 3.92 d (10.9) 3.76 d (10.9) 0.75 s 0.69 s

108.0, CH2 73.5, CH2 17.8, 15.4, 60.1, 195.0, 120.9, 162.0, 64.1, 63.6, 72.7,

δC, type

α 1.13 td (12.9, 4.5) β 1.69 dt (12.8, 3.0) α 1.44−1.54 m β 1.44−1.54 m α 1.31−1.36 m β 1.31−1.36 m

38.8, CH2

149.4, 52.1, 40.0, 21.3,

6 in CD3OD

δH, mult. (J in Hz)


6.78 t (1.9) 5.02 4.13 5.04 5.04 2.03

21.1, CH3 171.2, C

br s d (1.2) dd (17.4, 1.5) dd (17.4, 1.5) s

39.4, C 49.7, CH 25.6, CH2 39.2, CH2 152.0, 56.3, 41.6, 23.2,

C CH C CH2

107.0, CH2 72.2, CH2 18.3, 15.3, 137.2, 144.5, 196.0, 80.5, 80.1, 72.7, 64.5,

CH3 CH3 C C C C CH CH CH2

δH, mult. (J in Hz) α 1.34 overlapped β 1.83 br d (12.9) α 1.53 m β 1.63 m α 1.52 m β 1.24 br d (13.3) 1.59 dd (12.5, 2.6) α 1.68 br d (12.8) β 1.34 overlapped α 2.08 td (13.0, 5.0) β 2.36 ddd (12.8, 4.2, 2.4) 2.45 dd (8.5, 4.0) α 2.70 dd (13.9, 4.0) β 2.55 dd (13.8, 8.5) a 4.81 br s b 4.81 br s 3.36 d (11.1) 2.99 d (11.1) 0.75 s 0.83 s

3.69 4.41 3.82 3.61

d d d d

(7.8) (7.8) (11.5) (11.5)

This was confirmed by their CD spectra (Figure 4), of which one was almost a mirror of the other. Further, the CD spectrum of 3 was found to be very similar to that of 1, while that of 2 was close to the mirror image of that of 1, suggesting that 3 had a stereochemistry identical with that of 1, while 2 had a cyclohexenone ring enantiomeric to that of 1. The stereochemistry of 2 and 3 was finally confirmed by using the CD/ TDDFT approach. The simulated CD spectra for 2 and 3 in the gas phase and MeOH solution are shown in Figure 4, which

and/or carbon chemical shifts of the C-9 methine, C-11 methylene, C-12 olefinic methylene, and C-2′ ketone carbonyl (Table 1). Analysis of the NOESY spectra confirmed that the relative configurations of the sesquiterpene moieties in 2 and 3 were the same as that of 1. The trans relationship between the 1′,6′-epoxy and the 5′-OH in both 2 and 3 was also supported by the J5′,6′ values, which were 1.6 Hz in 2 and 1.3 Hz in 3. These findings showed the structural difference between 2 and 3 was the absolute configurations in their cyclohexenone rings. 1794



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were generated by Boltzmann-weighting from individual spectra of low-energy conformers of the truncated structures (the fragment from C-2 to C-4 was replaced by H atoms). It can be seen that the calculated CD spectra provided excellent fits with the experimental spectra. Therefore, the absolute configurations of the epoxycyclohexenone moieties were determined to be 1′S,5′R,6′S in 2 and 1′R,5′S,6′R in 3. The HRESIMS and 1D NMR data indicated the molecular formulas of myrothecols D (4) and E (5) were the same, determined to be C24H34O6. The 1H and 13C NMR spectra (Table 2) of 4 and 5 were similar to those of 2 and 3 except for the presence of an acetyl group in 4 [δH 2.04 (3H, s, CH3); δC 21.1 (CH3), 171.2 (CO)] and 5 [δH 2.03 (3H, s, CH3); δC 21.1 (CH3), 171.2 (CO)]. The acetyl group in 4 and 5 was readily assigned to be attached at C-13 via an ester linkage on the basis of downfield shifts of H2-13 and C-13 relative to those of 2 and 3 in the NMR spectra as well as the presence of a longrange correlation from H2-13 to the carbonyl carbon (δC 171.2) in their HMBC spectra. The J5′,6′ values of 1.4 Hz in 4 and 1.2 Hz in 5 confirmed the trans relationship between the 1′,6′epoxy and 5′-OH for both compounds. Their CD spectra (Figure 5) were mirror images, of which the spectrum of 4 was

presence of a strong NOE correlation between H-6′ and one (δ 3.61) of H2-7′ in the NOESY spectrum indicated that H-6′ and 4′-CH2OH are oriented on the same side, while H-5′ is on the opposite side. In addition, the NOE interactions of H-6′ with both H2-12 and H-9 were observed in the NOESY spectrum. This observation in association with conformational profiles of the low-energy conformers of 1−3 suggested a 4′S,5′R,6′S configuration for the cyclohexenone unit. Further analysis was performed by DFT and TDDFT calculations using the procedure as described for compound 1. Conformational analysis using the truncated structure yielded seven low-energy minima within relative free energies of 2.0 kcal/mol in MeOH solution (see Supporting Information). The conformational profiles and equilibrium populations of the low-energy minima were in agreement with the above observed NOE correlations. Finally, CD/TDDFT calculations were conducted using all the low-energy conformers. The predicted CD spectra in the gas phase and MeOH solution, generated from individual spectra of the low-energy conformers by Botzmann-weighting, were in excellent agreement with the experimental spectrum of 6 (Figure 6). Therefore, the supposed absolute configuration of the cyclohexanone moiety in 6 was confirmed.

Figure 5. Comparison among measured CD spectra of 2−5.

Figure 6. Comparison between calculated and measured CD spectra of 6.

similar to that of 2 and the spectrum of 5 was consistent with that of 3 (Figure 5), indicating that the absolute configurations of the epoxycyclohexenone moieties in 4 and 5 were the same as those in 2 and 3, respectively. Therefore, the structures of 4 and 5 were determined as depicted. Myrothecol F (6) was determined to have a molecular formula of C22H34O7 (six unsaturations) from the HRESIMS data. The 1H and 13C NMR spectra (Table 2) showed that this compound was also composed of a 13-hydroxy-8(12)-drimene moiety and a hydroxymethylcyclohexenone unit. Analysis of the 1D NMR spectra in combination with 1H−1H COSY and HSQC revealed that the cyclohexenone unit in 6 consisted of a ketone carbonyl [δC 196.0 (C-3′)], a tetrasubstituted olefin [δC 144.5 (C-2′), 137.2 (C-1′)], two vicinal coupled hydroxylated methines [δH 3.69 and 4.41 (each 1H, d, J = 7.8 Hz, H-5′ and H-6′); δC 80.1 (C-5′), 72.7 (C-6′)], an oxygenated quaternary carbon [δC 80.5 (C-4′)], and a hydroxymethyl [δH 3.82, 3.61 (each 1H, d, J = 11.5 Hz, H2-7′); δC 64.5 (C-7′)]. In the HMBC spectrum, long-range correlations were observed from H-9 to C-1′, from H2-11 to C-1′, C-2′, and C-6′, and from H27′ to C-3′, C-4′, and C-5′, indicating a 2′,4′,5′,6′-tetrahydroxy4′-hydroxymethyl-3′-oxocyclohexenyl unit. The J5′,6′ value was observed to be 7.8 Hz, consistent with a trans relationship and axial positions of H-5′ and H-6′.10 This in combination with the

Compound 7 was obtained as the most abundant metabolite from this fungus. Its molecular formula, C22H30O6, was determined on the basis of HRESIMS data. Analysis of the 1D (see Experimental Section) and 2D NMR spectra (COSY, HSQC, HMBC, and NOESY) in CD3OD established a structure identical with that of hymenopsin B, a tetracarbocyclic quinone sesquiterpene recently reported from a Hymenopsis fungus.6 However, when attempting to get NMR data in the same solvent as that reported, we found that 7 was almost insoluble in CHCl3, although hymenopsin B should be soluble in this solvent, as its NMR spectra were recorded in CDCl3. This difference led us to consider that 7 might have had a stereochemistry different from that of hymenopsin B. After several efforts failed to ascertain the stereochemistry, 7 was subjected to acetylation using anhydrous pyridine and acetic anhydride and afforded a tri-O-acetyl derivative (7a) on the basis of its 1H and 13C NMR data (see Experimental Section). A single crystal of 7a was obtained by recrystallization in chloroform and subjected to X-ray diffraction analysis. As a result (Figure 7), 7a was unambiguously determined to be triO-acetylhymenopsin B and the structure of 7 was clarified to be the same as reported for hymenopsin B (the difference in solubility in CHCl3 found in our experiments and that reported in the literature could not be explained).6 The absolute 1795



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Figure 7. X-ray structure of 7a.

Table 3. Cytotoxicity and Antibacterial Activity of 1−7 tumor cell growth inhibition (IC50, μM)a

a

antibacterial activity (MIC, μg/mL)

compound

A549

HeLa

HepG2

S. aureus

B. cereus

1 2 3 4 5 6 7 ADM kanamycin ceftazidime

8.0 ± 0.7 39.8 ± 3.5 41.5 ± 6.6 29.4 ± 1.8 18.0 ± 1.8 >50 1.3 ± 0.2 0.69 ± 0.06

7.9 ± 0.9 29.3 ± 2.0 35.9 ± 1.1 19.2 ± 1.5 19.6 ± 3.8 >50 4.5 ± 0.2 0.47 ± 0.05

15.2 ± 1.9 48.5 ± 4.9 34.2 ± 3.6 37.8 ± 1.8 27.8 ± 3.5 >50 3.2 ± 0.3 1.22 ± 0.02

12.5 50.0 50.0 100.0 50.0 >100.0 >100.0

25.0 100.0 100.0 >100.0 >100.0 >100.0 >100.0 6.25

12.5

Values represent means ± SD based on three individual experiments.

configuration of the sesquiterpene moiety in 7 was consistent with those assigned for 1−6 by DFT and TDDFT calculations, which provided further support to the stereochemistry of these new compounds. The antibacterial activity of compounds 1−7 was evaluated against two Gram-positive (S. aureus and Bacillus cereus) and three Gram-negative bacteria (Escherichia coli, Salmonella typhimurium, and Shigella dysenteriae) using the macroplate Alamar Blue assay.12 Compound 1 showed activity against S. aureus (MIC = 12.5 μg/mL) and B. cereus (MIC = 25.0 μg/ mL). For other compounds, only weak activity was observed for 2−5 against S. aureus and 2 and 3 against B. cereus (Table 3). All the quinone sesquiterpenes were inactive against the three Gram-negative strains. Compounds 1−7 were also evaluated for growth inhibitory activity against human carcinoma A549, HeLa, and HepG2 cells by the MTT method.13 Their IC50 values are presented in Table 3. Compound 7 demonstrated inhibitory activity against the three cell lines with IC50 values of 1.3−4.5 μM, and 2−5 showed activity with values ranging from 7.9 to 48.5 μM. Only 6 was found to be inactive (IC50 > 50 μM) against all the test cell lines. On the basis of the fact that the tumor cytotoxicity of the tricyclic quinone sesquiterpenes 1−5 was stronger than their antibacterial activity, it could be speculated that the antibacterial activity of these compounds is due to their general cytotoxicity instead of the interference with a prokaryotic target. However, the tetracarbocycle 7 demonstrated strong cytotoxicity to the tumor cells, but showed no inhibition against the tested bacterial strains, illustrating that 7 may have specificity on eukaryotes.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Perkin-Elmer 343 spectropolarimeter. UV measurements were performed with a Perkin EImer Lambda 650 UV/vis spectrometer. CD data were collected by a Jasco J-810 CD spectrometer (JASCO, Inc., Japan). 1H NMR (600 MHz), 13C NMR (125 MHz), and 2D NMR spectra were recorded on a Bruker AV-600 instrument with residual solvent peaks as references. HRESIMS data were obtained on a Bruker Bio TOF IIIQ mass spectrometer in positive-ion mode. Preparative HPLC was performed with an HPLC system equipped with a Shimadzu LC-6AD pump and a Shimadzu RID-10A refractive index detector using a YMC-pack ODS-A C 18 column (5 μm, 250 × 20 mm). For column chromatography, silica gel 60 (100−200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), YMC ODS (75 μm), and Sephadex LH-20 were used. Preparative TLC was performed using HSGF254 silica gel plates (0.2 mm, Yantai Jiangyou Silica Gel Development Co. Ltd., Yantai, China). The antibacterial and cytotoxic assays were determined with a Genois microplate reader (Tecan Group, Männedorf, Zürich, Switzerland). Producing Fungus and Fermentation. The producing fungus, Myrothecium sp. SC0265, was isolated from a forest leaf litter sample collected at the district of rare and endangered tree species in the Dinghu Mountain Biosphere Reserve, Guangdong, China, in March 2003. It was authenticated on the basis of its morphological characteristics and ITS DNA sequence data (GenBank accession number KM086710) by Prof. Tai-hui Li, Guangdong Institute of Microbiology, Guangzhou, China. Fermentation of the fungus was performed as previously described.14 Briefly, the mycelia grown on PDA plates were prepared to inoculate fifteen 500 mL Erlenmeyer flasks containing 100 mL of YMG medium (glucose 0.4%, malt extract 1.0%, yeast extract 0.4%, pH 5.5). The flasks were incubated on a rotary shaker (LRH-250-Z oscillatory incubator, Shaoguan Taihong Medical Apparatus and Instruments, Co., Ltd., Shaoguan, China) for 5 days in the dark at 28 °C with shaking at 150 rpm. The seed culture (5 mL per flask) was transferred into two hundred 500 mL Erlenmeyer 1796



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flasks containing 100 mL of YMG medium and 70 g of wheat grains, and the flasks were incubated for 12 days in the dark at 28 °C. Extraction and Isolation. The mycelia culture was extracted with 95% EtOH, and the resultant extract was sequentially partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc-soluble extract (28 g) was separated by silica gel CC and eluted with CHCl3−MeOH mixtures of increasing polarity (98:2−70:30) to afford eight primary fractions (F1−F8). Fractions F3−6, all obtained on elution with CHCl3−MeOH (95:5), were then subjected to ODS CC using decreasing polar aqueous MeOH (40−90%) to obtain seven fractions (F3-a−F3-g) from F3 and five fractions (F4-a−F4-f) from F4. Fraction F3-f, obtained from elution with 70% MeOH, was further separated by preparative HPLC using 65% MeOH to obtain 4 (22.8 mg, tR = 84.0 min) and 5 (40 mg, tR = 105.5 min). Fraction F4-a, afforded by elution with 60% MeOH, was washed with MeOH to give 7 (253 mg). Fraction F4-d, obtained from the 70% MeOH elution, was separated by Sephadex LH-20 CC using MeOH to yield 1 (112.4 mg). The primary fraction F5 was further subjected to silica gel CC using progressive gradient elution (CHCl3−MeOH, 99:1−98:2) to give eight fractions (F-5a−F-5h). Fraction F5-g, obtained from elution with CHCl3−MeOH (98:2), was separated by ODS CC using aqueous MeOH (60−90%). The 60% MeOH subfraction was purified by preparative HPLC using 50% MeOH to afford 2 (35 mg, tR = 43.5 min) and 3 (53 mg, tR = 53.4 min). The primary fraction F7, obtained on elution with CHCl3−MeOH (9:1), was separated by silica gel CC using a CHCl3−MeOH system of increasing polarity (97:3−80:20) to give nine fractions (F7-a−F7-i). Fraction F7-f was further separated by ODS CC eluted with 60% MeOH followed by preparative TLC using a CHCl3−MeOH−H2O system (15:3:1) to yield 6 (2.6 mg, Rf = 0.34). To retain bioactivity, extracts and primary fractions were tested for antibacterial activity against S. aureus by the agar diffusion method with paper disks.15 The test dosage was 500 μg per paper disk, and the activity was assessed by the sizes of the inhibition zones. Myrothecol A (1): yellow, viscous oil; [α]20D +89 (c 0.37, MeOH); UV (MeOH) λmax (log ε) 201 (3.9), 232 (3.9), 257 (3.6); CD (MeOH) Δε 229 (−6.5), 260 (+6.8), 332 (+1.5); 1H and 13C NMR data, see Table 1; HRESIMS m/z 411.1935 [M + H]+ (calcd for C22H32ClO5, m/z 411.1933); m/z 433.1756 [M + Na]+ (calcd for C22H31ClNaO5, m/z 433.1752); m/z 843.3617 [2 M + Na]+ (calcd for C44H62Cl2NaO10, m/z 843.3612); obsd isotopic peak (3:1). Myrothecol B (2): colorless oil; [α]20D −76 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 203 (4.2), 220 (4.0), 250 (3.7); CD (MeOH) Δε 220 (+8.3), 249 (−5.5), 334 (−3.8); 1H and 13C NMR data, see Table 1; HRESIMS m/z 377.2320 [M + H]+ (calcd for C22H33O5, m/z 377.2323); m/z 399.2145 [M + Na]+ (calcd for C22H32NaO5, m/z 399.2142). Myrothecol C (3): yellow oil; [α]20D +103 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 203 (4.1), 220 (3.9), 250 (3.6); CD (MeOH) Δε 220 (−9.0), 248 (+5.8), 334 (+2.7); 1H and 13C NMR data, see Table 1; HRESIMS m/z 377.2313 [M + H]+ (calcd for C22H33O5, m/z 377.2323); m/z 399.2138 [M + Na]+ (calcd for C22H32NaO5, m/z 399.2142). Myrothecol D (4): yellow oil; [α]20D +29 (c 0.47, MeOH); UV (MeOH) λmax (log ε) 202 (4.0), 220 (3.8), 253 (3.6); CD (MeOH) Δε 220 (+7.0), 248 (−4.2), 332 (−2.5); 1H and 13C NMR data, see Table 2; HRESIMS m/z 419.2422 [M + H]+ (calcd for C24H35O6, m/z 419.2428); m/z 441.2250 [M + Na]+ (calcd for C24H34NaO6, m/z 441.2248). Myrothecol E (5): yellow oil; [α]20D +7 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 202 (3.9), 220 (3.6), 250 (3.4); CD (MeOH) Δε 229 (−2.3), 260 (+7.7), 332 (+2.3); 1H and 13C NMR data, see Table 2; HRESIMS m/z 419.2411 [M + H]+ (calcd for C24H35O6, m/z 419.2428); m/z 441.2251 [M + Na]+ (calcd for C24H34NaO6, m/z 441.2248). Myrothecol F (6): yellow oil; [α]20D +34 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 278 (3.6); CD (MeOH) Δε 215 (+0.4), 275 (+2.1); 1H and 13C NMR data, see Table 2; HRESIMS m/z 411.2392 [M + H]+ (calcd for C22H35O7, m/z 411.2377); m/z 433.2193 [M + Na]+ (calcd for C22H34NaO7, m/z 433.2197).

Hymenopsin B (7): white powder; [α]20D +8 (c 0.38, MeOH); UV (MeOH) λmax (log ε) 206 (4.0), 229(3.5); CD (MeOH) Δε 237 (−1.7), 321 (−3.6); 1H NMR (600 MHz, C5D5N) δ 5.51 (1H, br s, H-7), 4.35, 4.29 (each 1H, d, J = 13.5 Hz, H2-7′), 4.26 (1H, s, H-5′), 3.74 (1H, s, H-2′), 3.63, 3.33 (each 1H, d, J = 10.7 Hz, H2-13), 3.15 (1H, br d, J = 14.5 Hz, H-12β), 2.88 (1H, d, J = 10.7 Hz, H-11α), 2.82 (1H, br d, J = 14.5 Hz, H-12β), 2.38 (1H, br d, J = 12.8 Hz, H-9), 2.05 (1H, m, H-6α), 1.95 (1H, m, H-6β), 1.93 (1H, dd, J = 11.3, 2.4 Hz, H5), 1.81 (1H, td, J = 13.3, 3.4 Hz, H-3α), 1.64 (1H, br d, J = 12.4 Hz, H-1β), 1.55 (1H, m, H-2β), 1.45 (1H, m, H-2α), 1.45 (1H, br d, J = 13.5 Hz, H-3β), 1.27 (1H, d, J = 10.7 Hz, H-11α), 1.01 (3H, s, H3-15), 0.96 (1H, td, J = 13.3, 3.4 Hz, H-1α), 0.90 (3H, s, H3-14); 13C NMR (150 MHz, C5D5N) δ 202.5 (C, C-3′), 133.3 (C, C-8), 124.9 (CH, C7), 73.0 (C, C-1′), 71.6 (CH2, C-13), 68.4 (CH, C-5′), 67.9 (C, C-6′), 63.6 (CH, C-2′), 63.4 (C, C-4′), 58.4 (CH2, C-7′), 50.2 (CH, C-9), 43.7 (CH, C-5), 43.6 (CH2, C-12), 39.8 (CH2, C-1), 38.3 (C, C-4), 36.5 (CH2, C-3), 35.7 (C, C-10), 29.2 (CH2, C-11), 24.1 (CH2, C-6), 18.9 (CH2, C-2), 18.9 (CH3, C-14), 16.0 (CH3, C-15); HRESIMS m/ z 391.2116 [M + H]+ (calcd for C22H31O6, m/z 391.2115); m/z 413.1936 [M + Na]+ (calcd for C22H30NaO6, m/z 413.1935). Acetylation of Hymenopsin B (7). A sample of 7 (10 mg) was reacted with anhydrous pyridine (0.5 mL) and acetic anhydride (1.5 mL) with stirring at room tempreture for 60 h, then evaporated to afford the reaction mixture, which was purified by silica gel CC eluting with petroleum ether−acetone (8:1) followed by recrystallization in CHCl3 to yield tri-O-acetylhymenopsin B (7a, 6 mg): colorless crystal; mp 158−160 °C; 1H NMR (600 MHz, CD3OD) δ 5.64 (1H, br s, H7), 4.50, 4.21 (each 1H, d, J = 12.5 Hz, H2-7′), 3.87, 3.69 (each 1H, d, J = 11.0 Hz, H2-13), 3.58 (1H, s, H-5′), 3.46 (1H, s, H-2′), 2.86, 2.75 (each 1H, d, J = 14.8 Hz, H2-12), 2.22 (1H, m, H-9), 2.00−2.07 (2H, overlapped, H-6α, H-11β), 2.05, 2.04, 2.02 (each 3H, s, COCH3 × 3), 1.98 (1H, d, J = 13.6 Hz, H-6β), 1.72 (1H, br d, J = 13.2 Hz, H-1β), 1.60 (1H, br d, J = 13.1 Hz, H-3α), 1.54 (1H, J = 11.3, 5.3 Hz, H-5), 1.50 (1H, m, H-2β), 1.41 (1H, m, H-2α), 1.30 (1H, m, H-3β), 1.22 (1H, d, J = 13.7, 5.8 Hz, H-11α), 1.02 (1H, td, J = 13.3, 3.4 Hz, H-1α), 0.99 (3H, s, H3-15), 0.96 (3H, s, H3-14); 13C NMR (150 MHz, CD3OD) δ 198.6 (C, C-3′), 173.0, 172.2, 171.1 (each C, COCH3 × 3), 131.9 (C, C-8), 126.0 (CH, C-7), 76.0 (C, C-1′), 73.8 (CH2, C13), 68.8 (CH, C-5′), 65.1 (C, C-6′), 63.8 (CH, C-2′), 61.1 (C, C-4′), 60.9 (CH2, C-7′), 50.2 (CH, C-9), 45.4 (CH2, C-12), 41.9 (CH, C-5), 39.9 (CH2, C-1), 37.4 (C, C-4), 37.0 (CH2, C-3), 36.2 (C, C-10), 28.8 (CH2, C-11), 24.7 (CH2, C-6), 21.0, 20.8, 20.5 (each CH3, COCH3 × 3), 18.9 (CH2, C-2), 18.2 (CH3, C-14), 15.5 (CH3, C-15); HRESIMS m/z 517.2429 [M + H]+ (calcd for C28H37O9, m/z 517.2432); m/z 539.2249 [M + Na]+ (calcd for C28H36NaO9 m/z 539.2252). X-ray Crystallographic Analysis of Tri-O-acetylhymenopsin B (7a). C28H36O9, M = 516.57, colorless plate, 0.42 × 0.37 × 0.24 mm3, monoclinic, space group: P 1 21 1, a = 8.99959(15) Å, b = 9.16301(14) Å, c = 15.8136(3) Å, α = γ = 90°, β = 100.2226(17)°, V = 1283.34(4) Å3, Z = 2, Dc = 1.337 g/cm3, F000 = 552. Cu Kα radiation, λ = 1.5418 Å, T = 150(2) K, theta range for data collection: 2.84° to 66.80°; 9971 reflections collected, 4481 unique (Rint = 0.0197), completeness to theta = 66.80° (99.6%), max. and min. transmission: 0.8271 and 0.7239. The structure was refined by full-matrix leastsquares on F2. Final GooF = 1.057, final R indices [I > 2σ(I)]: R1 = 0.0299, wR2 = 0.0763, R indices (all data): R1 = 0.0320, wR2 = 0.0785; absolute structure parameter: Hooft = 0.04(12); largest diff. peak and hole: 0.236 and −0.144 e Å−3. Crystallographic data for the structure of 7a have been deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC 983619. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). Antibacterial Activity Evaluation. Two Gram-positive bacterial strains, S. aureus (ATCC 6538) and B. cereus (CMCC 63302), and three Gram-negative strains, E. coli (ATCC 8739), S. typhimurium (CMCC 50115), and S. dysenteriae (CMCC 51252), were used for the antibacterial activity evaluation. These strains were all obtained from the Microbial Culture Collection Center of Guangdong Institute of Microbiology (Guangzhou, China). Cultures of each strain were 1797



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maintained in 20% aqueous glycerol and stored at −80 °C. Alamar Blue was obtained from Invitrogen Corporation, Carlsbad, America. The activity was evaluated by the microplate Alamar Blue assay.12 Each test strain was incubated in 25 mL of MHB (Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., Guangzhou, China) on a rotary shaker at 150 rpm at 37 °C for 12 h. The count of bacterial suspension of each strain was adjusted to 1 × 105 CFU/mL with MHB. Test compounds were diluted with the medium (DMSO) to give 2-fold gradient concentrations. One hundred microliters of bacterial suspension of each strain (1 × 105 cfu/mL), which contained Alamar Blue (8%, v/v) and the compound solution (4%, v/v), was added into a 96-well microtiter plate in triplicate and incubated at 37 °C in the dark for 7 h, when the color switching from blue to red was stable enough. The final concentrations of each compound in the wells were 100, 50, 25, 12.5, 6.25, 3.125, and 1.562 μg/mL. Negative control wells, which contained DMSO instead of the test compound, and blank control wells, which contained Alamar Blue but without bacteria, were set in the plate, as well. Ceftazidime (in DMSO) was used as a positive control against S. aureus and E. coli, and kanamycin (in sterile water) was used as a positive control against B. cereus, S. typhimurium, and S. dysenteriae. The final concentration of the well that was closest to the red one and remained blue was referred to as the minimal inhibitory concentration (MIC). Cytotoxicity Assay. Human lung adenocarcinoma A549, human hepatoma HepG2, and human cervical carcinoma HeLa cell lines were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C throughout the assay. Cell viability was estimated by the MTT colorimetric assay as previously described.13 The test compounds in DMSO (10 mg/mL) were serially diluted with culture medium. Test cells (100 μL, 5 × 104 cells/ mL) were seeded into a 96-well microplate and incubated about 24 h for cell implantation; then the supernatant was removed, and 100 μL of fresh medium and 100 μL of medium containing a test compound or vehicle control (DMSO) were added. The blank control contained 200 μL of medium without cells. The final concentrations of each compound in the wells were 50, 10, 2, 0.4, 0.08, and 0.016 μg/mL, and the experiments for each concentration were performed in quadruplicate. The conventional antitumor drug adriamycin (ADM) was used as a positive control. Cells were further incubated for 72 h and then treated with MTT (20 μL, 5 mg/mL in DMSO) and shaken for 15 min. After another 4 h of incubation, the supernatant per well was removed and 150 μL of DMSO was added to dissolve the blue formazan crystals. The optical density (OD) of each well was measured on a Genois microplate reader at a wavelength of 570 nm. The inhibitory rate of cell growth was calculated according to the following formula: Inhibition rate (%) = {1 − (ODtreated − ODcontrol)/ (ODcontrol − ODblank)} × 100%. IC50 values were determined by nonlinear regression analysis of logistic dose−response curves (SPSS 16.0 statistic software). Computational Methods. Molecular Merck force field (MMFF) calculations and DFT and TDDFT calculations were performed with the Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and the Gaussian09 program package,16 respectively, using default grids and convergence criteria. MMFF conformational searches were performed using the Monte Carlo search method. The low-energy conformers within a 10 kcal/mol energy window were optimized using the DFT method at the B3LYP/6-31G(d,p) level. Frequency calculations were run at the same level to estimate free energies at 298.15 K. Solvent effects were taken into account by using the polarizable continuum model. The TDDFT calculations were performed using the long-range corrected hybrid CAM-B3LYP and the hybrid B3LYP functionals and Ahlrichs’ basis sets SVP (split valence plus polarization)17 and TZVP (triple-ζ valence plus polarization).18 The number of excited states per each molecule was 20−30. In a preliminary set of calculations with compound 1, the truncated structure (the fragment from C-2 to C-4, including the C-14 methyl and C-13 hydroxymethyl groups attached at C-4, was removed from the intact structure and an H atom was added to C-1 and C-5)

provided similar relative energies and simulated ECD spectra to the intact structure, and the SVP basis set gave consistent results with the TZVP one; therefore, the smaller SVP basis set and the truncated structure were used in all the subsequent calculations in order to save computation time. The functionals CAM-B3LYP and B3LYP were evaluated in each compound using the conformer of the absolute minimum. Because of the better performance in the evaluation, the former was used in TDDFT calculations of compounds 1−3 and the latter was used in those of compound 6. CD spectra were generated by the program SpecDis19 using a Gaussian band shape with a 0.30 eV (1−3) and 0.45 eV (6) exponential half-width from dipole-length dipolar and rotational strengths; the difference in dipole-velocity values was negligible (

Cytotoxic and Antibacterial Quinone Sesquiterpenes from a

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