Bisabolane Sesquiterpenoids from the Plant Endophytic Fungus

Mar 11, 2015 - Brasilamides E–J (1–6), the bisabolane sesquiterpenoids with the 3-cyclohexylfuran (1 and 2) and 3-cyclohexylfuranone (3–6) skele...
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Bisabolane Sesquiterpenoids from the Plant Endophytic Fungus Paraconiothyrium brasiliense Ling Liu,†,∥ Xiaoyan Chen,‡,∥ Dong Li,§ Yang Zhang,§ Li Li,⊥ Liangdong Guo,† Ya Cao,*,‡ and Yongsheng Che*,§ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, People’s Republic of China § State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China ⊥ Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Brasilamides E−J (1−6), the bisabolane sesquiterpenoids with the 3-cyclohexylfuran (1 and 2) and 3cyclohexylfuranone (3−6) skeletons, were isolated from the scale-up fermentation cultures of the plant endophytic fungus Paraconiothynium brasiliense Verkley. Although brasilamide E (1) is a known metabolite, its structure elucidation has yet to be described. The structures of 1−6 were elucidated primarily by NMR experiments. Compounds 3−6 were found to be racemic, and 3 was further separated into enantiomers 3a and 3b on a chiral HPLC column. The absolute configurations of 3a and 3b were assigned by electronic circular dichroism calculations. Compound 1 selectively inhibited the proliferation of the breast (MCF-7) and gastric (MGC) cancer cell lines, with IC50 values of 8.4 and 14.7 μM, respectively. Initial mechanistic investigation revealed that compound 1 inhibited the expression of a key energy metabolic enzyme, hexokinase II (HK2), in MCF-7 cells, which resulted in dysfunction of glucose metabolism and ATP depletion and eventually inhibited the proliferation of the breast cancer cells.

T

Pestalotiopsis.10 Terpenoids are the largest group of natural products, showing diverse structural features and interesting biological effects.11 As a group of the sesquiterpene type of natural products, the bisabolane sesquiterpenoids have been isolated from various sources.12 Fungus-derived bisabolane metabolites include three matrix metalloproteinase-3 inhibitors isolated from Penicillium sp.13 and two antibiotic and nematicidal agents isolated from the basidiomycete Cheimonophyllum candidissium.14 Plant metabolites of this class include the altaicalarins, cytotoxic agents isolated from Ligularia altaica;15 ashitabaol A, an antioxidative agent from Angelica keiskei;16 and 3,6-epidioxy-1,10-bisaboladiene, a cytotoxic compound from Cacalia delphiniifolia.17 Although the bisabolane sesquiterpenes represent a large family of bioactive terpenoids,12 naturally occurring bisabolanes with the 3cyclohexylfuran and 3-cyclohexylfuranone skeletons are rare.

he prevalence of cancer is increasing at an alarming rate globally. Recent studies indicated that inhibition of tumor cell glycolysis is a novel therapeutic approach in cancer treatment.1 Cancer cells rely more on glycolysis than glucose oxidative phosphorylation in energy production, which is known as the Warburg effect.2 Increasingly being recognized as a hallmark of cancer,3 the Warburg effect not only supports rapid growth of cancer cells, but also makes them less dependent on oxygen availability. Known to phosphorylate glucose to glucose-6-phosphate, hexokinase II (HK2) is overexpressd in cancer cells, and its expression appears to be crucial for the Warburg effect.4−6 Agents targeting tumor cell glycolysis, such as inhibitors of HK2, showed promising anticancer efficacy.5,7 Natural products or their synthetic derivatives account for over 50% of all anticancer drugs used in clinics.8 A notable example is paclitaxel, a diterpenoid that was initially isolated from the Pacific yew tree Taxus brevifolia9 and later found from several species of plant endophytic fungi in the genus © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 29, 2014

A

DOI: 10.1021/np5009569 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C15H19NO2 (seven degrees of unsaturation). Analysis of its 1H and 13C NMR data (Table 1) revealed the presence of two exchangeable protons (δH 7.48 and 7.03, respectively), one methyl group, four methylene units, one methine, eight olefinic/aromatic carbons (four of which are protonated), and one carboxylic carbon (δC 170.0). The 1H−1H COSY NMR data showed the isolated spin-system of C-1−C-6 (excluding C3). HMBC correlations from the methylene protons H2-2/H2-4 to C-3 and C-15 and from the olefinic protons H2-15 to C-2, C3, and C-4 revealed the connections of C-3 to C-2, C-4, and C15, completing the cyclohexane ring with an exocyclic olefin located at C-3. Correlations from H-1b/H-5b and H-6 to C-7, from H-8 to C-6, C-7, C-9, and C-14, and from H-14 to C-7 and C-8 indicated that C-6, C-8, and C-14 were all attached to C-7. A key correlation from H-14 to C-9 established the furan moiety. The HMBC cross-peaks from H3-13 to C-10, C-11, and C-12 and from H-10 to C-8 and C-9 revealed the connections of the C-10/C-11 olefin to C-9, C-12, and C-13, respectively. The remaining two exchangeable protons were assigned by default as the two amide protons attached to the C-12 carboxylic carbon. On the basis of these data, the gross structure of 1 was established. The C-10/C-11 olefin was assigned the E-geometry based on a NOESY correlation of H-8 with H3-13. The elemental composition of brasilamide F (2) was determined to be C15H21NO4 (six degrees of unsaturation) by HRESIMS, 34 mass units higher than 1. The 1H and 13C NMR spectra of 2 (Table 1) displayed signals for structural features similar to those found in 1, except that the C-3/C-15 olefin (δH 4.64; δC 107.7 and 148.1, respectively) was replaced by two exchangeable protons (δH 4.31 and 4.03), one methylene (δH 3.30; δC 66.3), and one sp3 quaternary carbon (δC 70.5), which was confirmed by HMBC correlations from H2-15 to C-2, C-3, and C-4, from OH-3 to C-2/C-4, C-3, and C-15, and from OH-15 to C-3 and C-15. The relative configuration of 2 was assigned by analysis of the 1H−1H coupling constants and NOESY correlations (Figure 1). The large trans-diaxial-type coupling constant of 13 Hz observed

Precedents include bilobanone from the heartwood of Ginkgo biloba L.18 and bilobanol from the roots of Ginkgo biloba.19 The only known fungal metabolite with the 3-cyclohexylfuranone skeleton was niduloic acid (7),20 a cytotoxic and antibiotic agent isolated from the basidiomycete Nidula candida. Our prior chemical studies of the plant endophytic fungi have afforded a variety of new bioactive natural products.21 During the process, a strain of Paraconiothyrium brasiliense Verkley (M3-3341) isolated from the branches of Acer truncatum Bunge on Dongling Mountain, Beijing, P. R. China, was investigated, leading to the discovery of four new anti-HIV-1 bergamotane sesquiterpenoids.22 Since the crude extract also showed cytotoxic effects and its HPLC chromatogram revealed the presence of minor components that could not be identified, the fungus was fermented in a larger scale on rice. Fractionation of the resulting EtOAc extract led to the isolation of brasilamides E−J (1−6), six bisabolane sesquiterpenes with 3-cyclohexylfuran (1 and 2) and 3-cyclohexylfuranone (3−6) skeletons, respectively. Compounds 2−6 are new metabolites, whereas 1 is known despite its structure elucidation being undisclosed.23 Details of the isolation, structure elucidation, and biological activity of these metabolites are reported herein.

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RESULTS AND DISCUSSION Brasilamide E (1) was isolated as the major component of the crude extract. Compound 1 gave a pseudomolecular ion [M + Na]+ peak by HRESIMS, indicating a molecular formula of Table 1. NMR Data for 1 and 2 (DMSO-d6) 1

a

position

δC , mult.

1a/5a 1b/5b 2a/4a 2b/4b 3 6 7 8 9 10 11 12 13 14 15 OH-3 NH2-12 OH-15

33.9, CH2

a

34.1, CH2 148.1, 33.4, 132.2, 113.5, 151.5, 121.1, 128.5, 170.0, 14.4, 139.2, 107.7,

qC CH qC CH qC CH qC qC CH3 CH CH2

2

δH (J in Hz) b

1.36, 1.97, 2.11, 2.30,

qd (13, 3.6) dt (13, 3.6) td (13, 3.6) dt (13, 3.6)

HMBC 2, 2, 1, 1,

4, 3, 3, 3,

a

6 4, 6, 7 5, 6, 15 5, 6, 15

2.60, tt (13, 3.6)

1, 2, 4, 5, 7, 14

6.65, s

6, 7, 9, 10, 14

7.03, s

8, 9, 11, 12, 13

2.07, s 7.56, s 4.64, s

10, 11, 12 7, 8, 9 2, 3, 4

δC , mult. c

28.7, CH2 33.1, CH2 70.5, 32.4, 131.8, 113.7, 151.4, 121.1, 128.4, 169.9, 14.4, 139.4, 66.3,

qC CH qC CH qC CH qC qC CH3 CH CH2

7.48, br s; 7.03, br s

δHd 1.42, 1.82, 1.31, 1.70,

HMBCc

(J in Hz)

qd (13, 3.6) dt (13, 3.6) td (13, 3.6) dt (13, 3.6)

2, 2, 1, 1,

3, 3, 3, 3,

4, 4, 5, 5,

6, 6, 6, 6,

7 7 15 15

2.49, tt (13, 3.6)

1, 2, 4, 5, 7, 8, 14

6.60, s

6, 7, 9, 10, 14

6.98, s

8, 9, 11, 12, 13

2.06, 7.50, 3.30, 4.03, 7.48, 4.31,

10, 11, 12 7, 8, 9 2, 3, 4 2, 3, 4, 15

s s d (6.0) s br s; 7.03, br s t (6.0)

3, 15

Recorded at 150 MHz. bRecorded at 600 MHz. cRecorded at 125 MHz. dRecorded at 500 MHz. B

DOI: 10.1021/np5009569 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C10−C-11−C-13 subunits of structure 3. Interpretation of the HMBC data of 3 (Table 2) established the same cyclohexyl moiety with an exocyclic olefin located at C-3 as that found in 1. HMBC correlations from H-1a/H-5a to C-7, from H-6 to C7, C-8, and C-14, and from H-8 to C-6, C-7, and C-14 indicated that C-6, C-8, and C-14 were all attached to C-7. In turn, correlations from H-8 to C-9 and C-10, from H-10 to C-8, C-9, C-12, and C-13, and from H3-13 to C-10, C-11, and C-12 indicated that the C-9/C-10 olefin is conjugated to the C-7/C8 double bond, and C-11 is connected to both C-13 and the carboxylic carbon C-12. The remaining two exchangeable protons were assigned as the C-12 amide protons by default. Considering the 13C NMR chemical shifts of C-9 (δC 149.1) and C-14 (δC 170.0), as well as the bicyclic nature of 3, these two carbons have to be connected to the remaining oxygen atom to form an α,β-unsaturated lactone moiety, which was partially supported by an IR absorption at 1751 cm−1. On the basis of these data, the gross structure of 3 was established as shown. The C-9/C-10 olefin was assigned the Z-geometry based on a NOESY correlation of H-8 with H-10. Despite the presence of a stereogenic center at C-11, the measured optical rotation value of 3 was zero, and no Cotton effect was observed in its CD spectrum, suggesting that 3 is a racemate. A portion of 3 (4.0 mg) was subjected to HPLC separation on a chiral column to afford two enantiomers (Figure S13), (+)-brasilamide G (3a; 1.6 mg) and (−)-brasilamide G (3b; 1.6 mg), with the measured specific optical rotation values of +26.8 and −25.6, respectively (c 0.16, MeOH). The absolute configurations of 3a and 3b were deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-

Figure 1. Key NOESY correlations for brasilamides F (2) and I (5).

between H-6 and H-1a/H-5a indicated that these protons are axially oriented, establishing the furan moiety had adopted an equatorial orientation with respect to the corresponding cyclohexyl ring. NOESY correlations of H-1a/H-5a with H215 indicated that these protons are all on the same face of the ring system with OH-3 equatorially orientated. A correlation of H-8 with H3-13 was used to assign the E-geometry of the C-10/ C-11 olefin. Therefore, the relative configuration of brasilamide F was proposed as shown in 2. The molecular formula of brasilamide G (3) was determined to be C15H19NO3 (seven degrees of unsaturation) on the basis of its HRESIMS data. Analysis of its 1H and 13C NMR data (Table 2) revealed two exchangeable protons (δH 6.88 and 6.24, respectively), one methyl group, four methylene units, two methines, six olefinic/aromatic carbons (three of which are protonated), and two carboxylic carbons (δC 170.0 and 174.9, respectively). These data accounted for all the 1H and 13C NMR resonances, suggesting that 3 was a bicyclic compound. The 1H−1H COSY NMR data showed two isolated spinsystems corresponding to the C-1−C-6 (excluding C-3) and Table 2. NMR Data for 3−6 (Acetone-d6) 3 position

δC,a mult.

1a/5a

33.2, CH2

1b/5b 2a/4a

34.8, CH2

2b/4b 3 6

148.9, qC 35.3, CH

7 8 9 10 11

138.5, 137.2, 149.1, 114.8, 38.7,

qC CH qC CH CH

12 13 14 15 16 17 OH-3 NH2-12

174.9, 18.8, 170.0, 108.1,

qC CH3 qC CH2

δHb (J in Hz)

4 HMBCa

δC,a mult.

1.39, qd (13, 3.0) 2.04, dt (13, 3.0) 2.16, td (13, 3.0) 2.36, dt (13, 3.0)

2, 3, 4, 6, 7

33.2, CH2

2.56, tt (13, 3.0)

1, 2, 4, 5, 7, 8, 14

7.31, s

6, 7, 9, 10, 14

5.42, d (10) 3.64, dq (10, 7.0)

8, 9, 12, 13 9, 10, 12, 13

1.26, d (7.0)

10, 11, 12

4.64, s

2, 3, 4

6.88, br s; 6.24, br s

2, 3, 4, 6 1, 3, 5, 6, 15

34.8, CH2

1, 3, 5, 6, 15 148.9, qC 35.4, CH 139.8, 133.1, 149.9, 114.6, 39.0,

qC CH qC CH CH

175.1, 19.2, 170.0, 108.1,

qC CH3 qC CH2

5 δHb (J in Hz)

1.40, qd (13, 3.0) 2.05, dt (13, 3.0) 2.15, td (13, 3.0) 2.36, dt (13, 3.0) 2.58, tt (13, 3.0) 7.68, s 5.67, d (11) 3.54, dq (11, 7.0) 1.28, d (7.0) 4.64, s

δC,a mult. 27.9, CH2

34.4, CH2

71.5, qC 34.7, CH 138.3, 137.5, 149.0, 114.8, 38.7,

qC CH qC CH CH

175.0, 18.8, 170.0, 67.2,

qC CH3 qC CH2

6.80, br s; 6.26, br s

OH-15 a

6 δHb (J in Hz)

1.53, qd (13, 3.0) 1.85, dt (13, 3.0) 1.43, td (13, 3.0) 1.90, dt (13, 3.0) 2.47, tt (13, 3.0) 7.31, s 5.37, d (10) 3.62, dq (10, 7.0) 1.26, d (7.0) 3.49, br s

3.33, s 6.83, br s; 6.19, br s 3.49, br s

δC,c mult. 27.7, CH2

34.6, CH2

70.2, qC 34.3, CH 138.0, 137.6, 149.0, 114.8, 38.7,

qC CH qC CH CH

174.0, 18.8, 170.1, 68.9, 171.1, 20.7,

qC CH3 qC CH2 qC CH3

δHd (J in Hz) 1.55, qd (13, 3.0) 1.93, dt (13, 3.0) 1.55, td (13, 3.0) 1.85, dt (13, 3.0) 2.51, tt (13, 3.0) 7.38, s 5.42, d (10) 3.63, dq (10, 6.6) 1.26, d (6.6) 4.08, s 2.01, s 3.73, s 6.87, br s; 6.23, br s

Recorded at 125 MHz. bRecorded at 500 MHz. cRecorded at 150 MHz. dRecorded at 600 MHz. C

DOI: 10.1021/np5009569 J. Nat. Prod. XXXX, XXX, XXX−XXX

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dependent density functional theory (TDDFT)24 for enantiomers (11R)-3 (3c) and (11S)-3 (3d). The MMFF94 conformational search followed by reoptimaization using TDDFT at the B3LYP/6-31G(d) basis set level afforded 30 lowest energy conformers for 3c (Figure S14). The overall calculated ECD spectrum of 3c was then generated by Boltzmann weighting of their lowest energy conformers by their relative free energies. The absolute configurations of 3a and 3b were then extrapolated by comparison of the experimental and calculated ECD spectra of 3c and 3d (Figure 2). The experimental CD spectrum of 3a was nearly identical to

C-10 olefin. The absence of an observable optical rotation indicated that 4 is also racemic. Subsequent HPLC separation of 4 using a chiral column was unsuccessful (Figure S15). Brasilamide I (5) was assigned the molecular formula C15H21NO5 (six degrees of unsaturation) by analysis of its HRESIMS, which is 34 mass units higher than 3. Interpretation of the 1H and 13C NMR (Table 2) as well as 2D NMR data of 5 established the same 1-(hydroxymethyl)cyclohexanol and 2((1Z)-(5-oxofuran-2(5H)-ylidene)methyl)propanamide subunits as found in 2 and 3, respectively. HMBC correlations from the methylene protons H2-1/H2-5 to C-7 led to the connection of the two moieties in 5 via the same C-6−C-7 linkage as in 2 and 3. Therefore, the gross structure of 5 was established. The relative configuration of the 1(hydroxymethyl)cyclohexanol unit in 5 was deduced to be the same as in 2 by comparison of the 1H−1H coupling constants and NOESY correlations (Figure 1). Again, a NOESY correlation of H-8 with H-10 was used to assign the Zgeometry for the C-9/C-10 olefin. Similarly, 5 was also deduced to be racemic, and subsequent HPLC separation of 5 using a chiral column was also unsuccessful (Figure S16) The elemental composition of brasilamide J (6) was established as C17H23NO6 (seven degrees of unsaturation) by HRESIMS. Analysis of the NMR data of 6 (Table 2) revealed structural features similar to those of 5, except that the oxymethene proton signals (H2-15) were shifted downfield (δH 3.49 in 5; δH 4.08 in 6). In addition, the exchangeable proton located at C-15 (δH 3.49) was replaced by an acetyl unit (δH 2.01; δC 20.7 and 171.1, respectively), which was confirmed by an HMBC correlation from H2-15 to the carboxylic carbon (δC 171.1) of the acetyl group. Therefore, the structure of 6 was determined and its relative configuration was deduced by analogy to 5. The absence of an observable optical rotation indicated that 6 is also racemic. Subsequent HPLC separation of 6 using a chiral column was unsuccessful (Figure S17). Brasilamides E−J (1−6) were tested for inhibitory effects against a panel of human tumor cell lines (Table 3), using HaCaT (human keratinocyte) and NIH-3T3 (mouse embryo fibroblast) cells as the negative controls. In addition to its cytotoxic effect against the MCF-7 cells,23 compound 1 also inhibited the proliferation of MGC cells, with an IC50 value of 14.7 μM (the positive control paclitaxel showing an IC50 value of 6.4 nM). In addition, compound 1 did not show detectable cytotoxicity against the negative controls HaCaT and NIH-3T3 cells at 50 μM, while paclitaxel showed IC50 values of 24 nM and 4.2 μM, respectively, indicating that 1 is a metabolite showing selective cytotoxicity. However, compounds 2, 3, 3a, 3b, and 4−6 did not show detectable inhibitory effects on the cell lines tested at 50 μM. To explore the possible mechanism of action through which compound 1 inhibits the proliferation of the selected tumor cell lines, MCF-7 cells were treated with 20 μM 1 for 12 h, and then

Figure 2. Experimental CD spectra of 3a and 3b in MeOH and the calculated ECD spectra of (11R)-3 (3c) and (11S)-3 (3d).

the calculated ECD curve of (11S)-3 (3d), both showing a positive Cotton effect (CE) in the range of 270−290 nm and a negative CE in the range 210−230 nm (Figure 2). Therefore, the absolute configuration of 3a was deduced to be 11S. Similarly, the experimental CD spectrum of 3b was nearly identical to the calculated ECD curve of 3c (Figure 2), suggesting the 11R absolute configuration. Brasilamide H (4) was determined to have the same molecular formula of C15H19NO3 as 3 by HRESIMS. Interpretation of the 1D and 2D NMR (Table 2) data of 4 established the same gross structure as 3, indicating their isomeric relationship. The C-9/C-10 olefin was assigned the Egeometry on the basis of a NOESY correlation of H-8 with H11. These data indicated that 4 is a trans isomer of 3 at the C-9/ Table 3. Cytotoxicity of Compound 1a

IC50 (μM) b

compound

A549

1 paclitaxel

>50 3.0 × 10−2

CNE1-LMP1

c

>50 4.2 × 10−3

A375

d

>50 8.9 × 10−3

e

MCF-7

MGCf

EC109g

PANC-1h

Hep3B-2i

HaCaTj

NIH3T3k

8.4 1.4 × 10−2

14.7 6.4 × 10−3

>50 0.14

>50 1.1 × 10−3

>50 1.6 × 10−5

>50 0.024

>50 4.2

a Compounds 2−6 were inactive at 50 μM. bHuman lung adenocarcinoma cells. cStable oncoprotein LMP1 integrated nasopharyngeal carcinoma cells. dHuman malignant melanoma cells. eHuman breast cancer cells. fHuman gastric cancer cells. gHuman esophageal cancer cells. hHuman pancreatic carcinoma cells. iHuman hepatoma carcinoma cells. jHuman keratinocyte cells. kMouse embryo fibroblast.

D

DOI: 10.1021/np5009569 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Compound 1 decreased the glucose uptake and lactate secretion rates, blocked ATP generation, and reduced the HK2 level in MCF-7 cells. (A and B) Cells were treated with 20 μM 1 for 12 h, and the glucose uptake rate (A) and lactate secretion rate (B) were assayed with LY294002 as the positive control. (C) Cells were treated with 20 μM 1 for 2, 4, 6, 8, 12, and 24 h, and the ATP contents were measured and expressed as a percentage of the control group. (D) MCF-7 cells were treated with 1 for 1, 3, 6, 12, 24, and 48 h. Cell lysates were subjected to SDSPAGE and Western blotting analysis with α-tublin as the internal control. Each value was expressed as the mean ± SD (**p < 0.01 and ***p < 0.001 as compared to the blank groups).

a 5,6-dihydroxy-2-methylcyclohex-2-enone unit and by having a 2-methylbut-3-enamide subunit in place of 2-methylpropane and methoxy at C-9. Compound 4 is a trans isomer of 3 at the C-9/C-10 olefin, and 5 is an oxidative product of 3, whereas 6 is an acetylation product of 5. Brasilamide E (1) inhibited the expression of the key energy metabolic enzyme HK2 in MCF-7 cells, which resulted in dysfunction of glucose metabolism and ATP depletion, and eventually inhibited the proliferation of the breast cancer cells. The discovery of these metabolites further demonstrated that the plant endophytic fungi are a valuable source for new bioactive metabolites, especially for antitumor natural products that could be used for further investigations.

the glucose and lactate concentration in the medium was examined. The average glucose uptake rate of MCF-7 cells decreased from 5.6 nM to 4.2 nM per hour per 105 cells (Figure 3A), whereas the average lactate secretion rate decreased by 0.5 nM per hour per 105 cells (Figure 3B). However, 1 did not decrease the glucose uptake or lactate secretion rate in normal immortalized HaCaT cells when tested at 20 μM. These results suggested that 1 specifically impaired glucose metabolism of tumor cells, which may provide an approach by which to kill the tumor cells without affecting the normal ones. In addition, 1 also blocked ATP (adenosine triphosphate) generation and significantly reduced the content of ATP in MCF-7 cells treated with 20 μM 1 for 12 and 24 h when compared to the untreated cells (Figure 3C). Therefore, 1 showed an antiproliferative effect on MCF-7 cells partially via the inhibition of glucose consumption, lactate secretion, and ATP generation. In rapidly growing tumor cells, HK2 has been reported to be upregulated by PI3-K/Akt signaling to facilitate glycolysis. To test the hypothesis that HK2 in tumor cells is involved in glucose metabolism regulated by 1, the level of HK2 in MCF-7 cells was also analyzed when treated with 1. Our data indicated that 1 downregulates the level of HK2 in the treated MCF-7 cells in a time-dependent manner (Figure 3D), suggesting that 1 selectively inhibited glycolysis in tumor cells by suppressing the expression of HK2. Brasilamides E (1) and F (2) are bisabolane sesquiterpenoids with the 3-cyclohexylfuran skeleton. Compound 1 is structurally related to niduloic acid (7),20 but differs by having methylene protons at C-2, an exocyclic olefin located at C-3, and an amide at C-12 instead of an olefinic proton, a methyl group, and a carboxylic acid unit, respectively. 2 is an oxidative product of 1. Brasilamides G−J (3−6) are new members of the bisabolane sesquiterpenoids with a 3-cyclohexylfuranone skeleton. Compound 3 is structurally related to bilobanol,19 but differs by having a methylenecyclohexane moiety instead of

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Analytical automatic polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and 13C NMR data were acquired with Varian Mercury-500 and NMR system-600 spectrometers using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1; DMSO-d6: δH 2.49/δC 39.5) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100−1000 at 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA) equipped with a variable-wavelength UV detector. Fungal Material. The fungus P. brasiliense Verkley has been previously described.22 The strain was cultured on slants of potato E

DOI: 10.1021/np5009569 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C-1, 5, 7; H-8 → C-6, 7, 9, 14; H-10 → C-8, 9, 12, 13; H-11 → C-9, 10, 12, 13; H3-13 → C-10, 11, 12; H2-15 → C-2, 3, 4; NOESY correlations (acetone-d6, 500 MHz) H-8 ↔ H-11; H-11 ↔ H-8; HRESIMS m/z 284.1255 (calcd for C15H19NO3Na, 284.1257). Brasilamide I (5): colorless oil; [α]25D +0.42 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 276 (4.12) nm; IR (neat) νmax 3352 (br), 2935, 2866, 1751, 1671, 1571, 1452, 1207, 1047 cm−1; 1H and 13C NMR data see Table 2; HMBC data (acetone-d6, 500 MHz) H2-1 → C-2, 7; H2-2 → C-1, 3, 6; H2-4 → C-3, 5, 6; H2-5 → C-4, 7; H-8 → C-7, 9, 14; H-10 → C-8, 9, 13; H-11 → C-12, 13; H3-13 → C-10, 11, 12; H2-15 → C-2, 3, 4; NOESY correlations (acetone-d6, 500 MHz) H-1a/H-5a ↔ H2-15; H-8 ↔ H-10; H-10 ↔ H-8; H2-15 ↔ H-1a/H-5a; HRESIMS m/z 318.1312 (calcd for C15H21NO5Na, 318.1311). Brasilamide J (6): colorless oil; [α]25D +1.2 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 276 (4.25) nm; IR (neat) νmax 3354 (br), 2938, 2868, 1737, 1674, 1612, 1454, 1239, 1046 cm−1; 1H and 13C NMR data see Table 2; HMBC data (acetone-d6, 600 MHz) H-1a, 5a → C-2, 3, 4, 6; H-1b, 5b → C-2, 3, 6, 4, 7; H-2a, 4a → C-3, 6; H-2b, 4b → C6; H-8 → C-7, 9, 14; H-10 → C-8, 9; H-11 → C-9, 12; H3-13 → C-10, 11, 12; H2-15 → C-2, 3, 4, 16; H3-17 → C-16; NOESY correlations (acetone-d6, 500 MHz) H-1a/H-5a ↔ H2-15; H-8 ↔ H-10; H-10 ↔ H-8; H2-15 ↔ H-1a/H-5a; HRESIMS m/z 360.1417 (calcd for C17H23NO6Na, 360.1418). Chiral HPLC Analysis of 4−6. Compounds 4−6 were analyzed using a Kromasil CelluCoat RP column (250 × 4.6 mm; 1 mL/min). On the HPLC chromatograms, compound 4 was observed as a single peak at 16.25 and 6.88 min (Figure S15), respectively, when using the two different conditions (30% CH3CN in H2O for 20 min and 40% CH3CN in H2O for 20 min). Compound 5 was also observed as a single peak at 6.50 and 3.90 min (Figure S16), respectively, when using the two different conditions (10% CH3CN in H2O for 20 min and 20% CH3CN in H2O for 20 min, respectively). Compound 6 was analyzed using the same conditions with 5, and only partial resolution of 6 was achieved (Figure S17). Computational Details. Systematic conformational analyses for 3a and 3b were performed via the Molecular Operating Environment (MOE) ver. 2009.10 (Chemical Computing Group, Canada) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational analyses were further optimized using TDDFT at the B3LYP/6-31G(d) basis set level. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 30 lowest electronic transitions were calculated, and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.3 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correlation. All quantum computations were performed using the Gaussian03 package25 on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. Cell Survival Assay. Cell viability was determined by MTS assay using the Celltiter 96 Aqueous One Solution cell proliferation assay kit according to the manufacturer’s instructions (Promega Corporation, Madison, WI, USA). Briefly, cells were seeded at a density of 5 × 103 cells per well in 96-well plates for 24 h, before exposed to serial dilutions of the test compounds (5, 10, 15, 20, and 50 μM) in medium supplemented with 10% fetal bovine serum and allowed to grow for the indicated duration. The control cells were treated with the same volumes of DMSO. The cells were incubated with 100 μL of medium containing 20 μL of MTS at 37 °C for 1 h, and absorbance was measured at 490 nm using a Multilabel counter (Bio-Tek Elx-800). Results were expressed as a percentage of the control, and halfmaximal inhibitory concentration (IC50) values were calculated. Glucose and Lactate Assays. Cells were seeded in 12-well plates. After 24 h, culture medium was replaced with or without 20 μM of test

dextrose agar at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions, 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5. After sterilization, three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Spore inoculum was prepared by suspending the seed culture in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/mL. Fermentation was carried out in 12 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days. Extraction and Isolation. The fermented material was extracted repeatedly with EtOAc (4 × 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (9.0 g), which was fractionated by silica gel VLC using petroleum ether− EtOAc gradient elution. The fraction (100 mg) eluted with 72% EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with 1:1 CH2Cl2−MeOH. The resulting subfractions were combined and further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 30% MeOH in H2O for 2 min, followed by 30−60% over 30 min; 2 mL/min) to afford 6 (1.8 mg, tR 24.22 min). The fraction (120 mg) eluted with 32% EtOAc was separated by Sephadex LH-20 CC eluting with 1:1 CH2Cl2−MeOH, and the resulting subfractions were purified by RP HPLC to afford 1 (35.3 mg, tR 19.52 min; 72% MeOH in H2O for 2 min, followed by 72−85% over 30 min; 2 mL/min), 3 (9.8 mg, tR 36.16 min; 30% MeOH in H2O for 2 min, followed by 30−50% over 40 min; 2 mL/min), and 4 (2.1 mg, tR 33.43 min; the same gradient as in purification of 3). The fraction (190 mg) eluted with 80% EtOAc was separated by Sephadex LH-20 CC eluting with 1:1 CH2Cl2− MeOH. The subfractions were combined and further purified by RP HPLC (25% MeOH in H2O for 2 min, followed by 25−47% over 35 min; 2 mL/min) to afford 2 (1.5 mg, tR 26.90 min) and 5 (1.6 mg, tR 17.52 min). A portion of compound 3 (4.0 mg) was subjected to a chiral HPLC column (Kromasil CelluCoat RP column; 4.6 × 250 mm; 30% CH3CN in H2O for 30 min; 1 mL/min) to afford two enantiomers, (+)-brasilamide G (3a, 1.6 mg; tR 16.51 min) and (−)-brasilamide G (3b, 1.6 mg; tR 18.62 min). Brasilamide E (1): white powder; mp 182−185 °C; [α]25D +0.25 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 298 (4.20) nm; IR (neat) νmax 3392, 3173, 2934, 2859, 1760, 1653, 1587, 1378, 1120, 1001 cm−1; 1H, 13 C NMR and HMBC data see Table 1; NOESY correlations (DMSOd6, 500 MHz) H-8 ↔ H3-13; H3-13 ↔ H-8; HRESIMS m/z 268.1307 (calcd for C15H19NO2Na, 268.1308). Brasilamide F (2): pale yellow oil; [α]25D −0.13 (c 0.75, MeOH); UV (MeOH) λmax (log ε) 274 (4.30) nm; IR (neat) νmax 3292 (br), 3175, 2931, 2859, 1761, 1654, 1590, 1381, 1187, 1002 cm−1; 1H, 13C NMR and HMBC data see Table 1; NOESY correlations (DMSO-d6, 500 MHz) H-1a/H-5a ↔ H2-15; H-8 ↔ H3-13; H3-13 ↔ H-8; H2-15 ↔ H-1a/H-5a; HRESIMS m/z 280.1540 (calcd for C15H22NO4, 280.1543). Brasilamide G (3): colorless oil; [α]25D 0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 281 (4.35) nm; IR (neat) νmax 3369, 3188, 2931, 2859, 1751, 1648, 1448, 1285, 1049 cm−1; 1H, 13C NMR and HMBC data see Table 2; NOESY correlations (acetone-d6, 500 MHz) H-8 ↔ H-10; H-10 ↔ H-8; HRESIMS m/z 284.1253 (calcd for C15H19NO3Na, 284.1257). (+)-Brasilamide G (3a): [α]25D +26.8 (c 0.16, MeOH); CD (c 3.0 × 10−3 M, MeOH) λmax (Δε) 226 (−12.58), 278 (+9.15) nm. (−)-Brasilamide G (3b): [α]25D −25.6 (c 0.16, MeOH); CD (c 3.0 × 10−3 M, MeOH) λmax (Δε) 226 (+10.3), 278 (−8.98) nm. Brasilamide H (4): colorless oil; [α]25D 0 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 274 (3.84) nm; IR (neat) νmax 3370, 3187, 2932, 2860, 1750, 1650, 1449, 1287, 1049 cm−1; 1H and 13C NMR data see Table 2; HMBC data (acetone-d6, 500 MHz) H2-1 → C-2, 3, 6, 7; H22 → C-1, 3, 6, 15; H2-4 → C-3, 5, 6, 15; H2-5 → C-3, 4, 6, 7; H-6 → F

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compound in fresh medium containing 10% fetal bovine serum. After 12 h, the supernatants were collected and the levels of glucose and lactate were measured using an automatic biochemistry analyzer (AU680, Beckman Coulter International, Brea, CA, USA) at the Clinical Biochemical Laboratory of Xiangya Hospital (Changsha, China). In the study, LY294002, a PI3K inhibitor, was used as the positive control, which preferentially inhibited glucose transport in human breast cancer cells.26 The cell numbers of the same treatment were simultaneously measured by MTS assay. Glucose uptake rate for each sample was calculated using the formula glucose uptake rate (nmol/h/105cells) = (glucose in blank medium − glucose in the medium of the treatment group)/12 h/cells, while lactate secretion rate for each sample was calculated using the formula lactate secretion rate (nmol/h/105 cells) = (lactate in the medium of the treatment group − lactate in blank medium)/12 h/ cells. ATP Measurement. The ATP content in cells exposed to 20 μM compound 1 was measured by an assay kit from PerkinElmer (Boston, MA, USA) as described in the manufacturer’s protocol, calculated from a standard curve derived from known concentrations of ATP, and expressed as a percentage of the control group. Western Blotting. The cells were lysed with cold lysis buffer and centrifuged at 12000g for 10 min. Loading buffer was mixed with the cell lysates and boiled at 95 °C for 5 min. Proteins (50 μg) were loaded into 10% SDS polyacrylamide gel for electrophoresis and then transferred onto nitrocellulose membranes. The membranes were blocked and incubated with primary antibodies at 4 °C overnight. After washing, the membranes were incubated in horseradish peroxidase-conjugated antibodies at room temperature for 1 h. Enhanced Western Lightening Plus-ECL kit (Pierce, Rockford, IL, USA) was used to incubate the membranes, and Western blotting images were taken and analyzed (α-tublin was used as the internal control). Antibodies against HK2 were purchased from Cell Signaling (Beverly, MA, USA). Antibody against α-tublin and goat anti-rabbit and anti-mouse IgG-HRPs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Statistical Analysis. The values are presented as mean ± SD. Statistical analyses were performed using Student’s t-test (mean *p < 0.05, **p < 0.01, and ***p < 0.001).

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of 1−6, HPLC chromatogram of 3−6, and CD calculations for 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (Y. Cao): [email protected]. *Tel/Fax (Y. Che): +86 10 66932679. E-mail: [email protected]. Author Contributions ∥

L. Liu and X. Chen contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (81273395) and the National Program of Drug Research and Development (2012ZX09301-003).

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REFERENCES

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