Note pubs.acs.org/jnp

Metabolites of Antroquinonol Found in Rat Urine Following Oral Administration Chien-Kuang Chen,† Jaw-Jou Kang,‡ Wu-Che Wen,§ Hui-Fen Chiang,† and Shoei-Sheng Lee*,† †

School of Pharmacy, College of Medicine, National Taiwan University, Taipei 10050, Taiwan, Republic of China Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan, Republic of China § Golden Biotechnology Corporation, New Taipei City 251, Taiwan, Republic of China ‡

S Supporting Information *

ABSTRACT: Four metabolites (1−4) of antroquinonol from rat urine, collected within 24 h after oral administration of antroquinonol, were characterized by HPLC−SPE−NMR. Compounds 1−4 were further isolated by semipreparative HPLC for structure confirmation. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic analyses and HRESIMS data.

T

he cytotoxic agent antroquinonol, which contains a ubiquinone moiety,1 was isolated from a solid-state fermentation of Antrodia cinnamomea Chang & Chou (Polyporaceae), also known as Antrodia camphoratus, and GDAC, an antroquinonol-containing nutraceutical, used as a hepatoprotective agent,2 has been on the Taiwan market for several years. Further development of antroquinonol for the prevention or treatment of cancers3,4 is pending. The metabolic profile of antroquinonol has not been reported. During drug development it is important to clarify the potential toxicity and pharmacological effects of the drug metabolites. The aim of the present study is to identify the metabolites of antroquinonol in rat urine, collected 24 h after oral administration (200 mg/kg). The urines of the antroquinonol-treated rats were fractionated via liquid−liquid partitioning and on an Amberlite XAD-2 column to give two metabolite-rich fractions, UT-M1 and UTM2. The HPLC profile of UT-M2-2, a subfraction obtained from fractionating UT-M2 on a Sephadex LH-20 column, is shown in Figure 1. Each resolvable HPLC peak was trapped by a resin GP (general purpose) cartridge. Thirteen peaks were collected and were characterized by a high performance liquid chromatography−solid phase extraction−tube transfer−nuclear magnetic resonance (HPLC−SPE−TT−NMR) hyphenated technique.5−7 The online 1H NMR spectra of peaks 1−6 indicated they were not metabolites of antroquinonol. Peaks 7− 13, however, showed characteristic 1H NMR spectra for the ubiquinone moiety (Figure S1, Supporting Information (SI)), e.g., δ 1.14 (d, Me-6′), 1.67 (m, H-1′), 4.36 (d, H-2′), 2.47 (dq, H-6′), 3.57 (s, MeO-4′), and 4.04 (s, MeO-3′) in 1 (Table 1), suggesting them to be metabolites of antroquinonol. The four major compounds (1−4, peaks 10−13) were further isolated from fraction UT-M1 via semipreparative RP-18 HPLC for structure confirmation. Additional material is needed prior to characterizing the three minor metabolites (peaks 7−9). Compound 1 (peak 13, Figure 1) had the molecular formula C16H24O6 as deduced from its HRESIMS−. The 1H NMR © 2014 American Chemical Society and American Society of Pharmacognosy

spectrum of 1 (Table 1 and Figure S2, SI) (CD3OD) exhibited signals for one olefinic proton (δ 5.29, t, J = 7.1 Hz, H-5), one methyl singlet (δ 1.68, Me-4), and three methylene groups (δ 2.24, br t, H2-6; δ 2.32, t, H2-3; δ 2.42, t, H2-2) in addition to those for the ubiquinone moiety as indicated above. These data suggested that 1 possessed a ubiquinone skeleton, but its side chain was two olefinic protons, two methylenes and three methyls less than that of the parent antroquinonol.1 This suggestion was supported by the 13C NMR data (Table 1 and Figure S3, SI), showing the carbon signals for the ubiquinone skeleton (C-1′−6′) and a carboxylic group (δ 177.3, s, C-1), the latter being correlated to two methylenes’ protons (H2-3, δ 2.32; H2-2, δ 2.42) in the HMBC spectrum (Table 1 and Figure S7, SI). Analysis of the COSY spectrum of 1 (Figure S4, SI) indicated the following vicinal couplings, H-2′/H-6′ ↔ H-1′ ↔ H2-6 ↔ H-5 (the olefinic proton), and H2-3 ↔ H2-2, and an allylic coupling, H-5 ↔ Me-4. These data together established the structure of 1 as (E)-6-((1R,2R,6R)-2-hydroxy-3,4dimethoxy-6-methyl-5-oxocyclohex-3-enyl)-4-methylhex-4enoic acid. This structure was also confirmed by the NOESY spectrum (Figure S5, SI), which showed key NOE correlations Received: August 30, 2013 Published: March 4, 2014 1061

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Figure 1. HPLC profile of fraction UT-M2-2 for HPLC−SPE−NMR, monitored at 270 nm. For exact conditions see the Experimental Section.

Table 1. 1H and 13C NMR Data of 1−4 and HMBC Data of 1 and 2 (Methanol-d4)a 1 position 1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ Me-4 MeO-3′ MeO-4′ Me-6′ a1

δC 177.3, C 33.8, CH2 35.8, CH 137.2, C 123.4, CH 28.1, CH2 45.6, CH 66.7, CH 164.9, C 137.2, C 199.4, C 41.4, CH 16.2, CH3 58.5, CH3 60.7, CH3 12.8, CH3

δH mult (J/Hz)

2 HMBC (C#)

2.42 t (7.4) 2.32 t (7.4)

1, 3, 4

5.29 t (7.1) 2.24 br t (7.4) 1.67 m

3, Me-4 4, 5, 1′, 2′, 6′

4.36 d (3.1)

1′, 3′, 4′, 6′

2.47 dq (11.4, 6.9) 1.68 s

1′, 5′, Me-6′

4.04 s

3′

3.57 s

4′

1.14 d (6.9)

1′, 5′, 6′

1, 2, 4, 5, Me-4

3, 4, 5

δC 176.5, C 34.5, CH2 118.4, CH 139.5, C 37.2, CH2 27.1, CH2 44.0, CH 66.2, CH 164.6, C 137.3, C 199.5, C 41.5, CH 16.0, CH3 58.5, CH3 60.7, CH3 12.7, CH3

3

δH mult (J/Hz)

HMBC (C#)

3.02 br d (5.5)

1, 3, 4

5.39 br t (6.9)

1, 2, 5, Me-4

2.25 dd (8.7, 13.8) 2.09 dd (8.1, 13.8) 1.68 m, 1.64 m

3, 4, 6, 1′ 3, 4, 6, 1′, Me-4 5, 1′, 2′

1.64 m 4.47 d (2.4)

6, 1′, 3′, 4′, 6′

2.44 dq (10.6, 6.9)

6, 1′, 5′, Me-6′

1.66 s

1, 3, 4, 5

4.06 s

3′

3.57 s

4′

1.12 d (6.9)

1′, 5′, 6′

δC

δH mult (J/Hz)

176.3, C 34.4, CH2 118.2, CH 139.5, C 38.0, CH2 25.8, CH2 45.3, CH 70.0, CH n.o.b 134.1, C n.o.b n.o.b

3.01 d (7.1) 5.35 br t (7.1)

2.14 dt (14.4, 7.2) 2.09 dt (14.4, 7.2) 1.75 dq (14.4, 7.2) 1.38 m 1.85 m 4.36 d (4.3)

2.55 dq (6.2, 6.9)

4 δC 177.3, C 33.9, CH2 35.9, CH2 137.5, C 123.9, CH 26.3, CH2 46.8, CH 70.5, CH n.o.b 133.9, C n.o.b n.o.b

δH mult (J/Hz) 2.40 t (7.6) 2.31 t (7.6)

5.19 t (7.0) 2.25 dt (14.2, 6.4) 1.98 m 1.91 m 4.36 d (4.2)

2.53 dq (4.5, 7.2)

16.1, CH3

1.64 s

16.2, CH3

1.60 s

60.2, CH3 n.o.b

3.57 s

60.3, CH3 n.o.b

3.58 s

1.26 d (6.9)

1.27 d (7.2)

H NMR and HMBC, 600 MHz; 13C NMR, 150 MHz; bSignals were not observed.

spectrum of 2 (Figure S10, SI) indicated the following vicinal couplings: H-6′ (δ 2.44, dq)/H-2′ (δ 4.47, d) ↔ H-1′ (δ 1.64, m) ↔ H2-6 (δ 1.68/1.64, m) ↔ H2-5 (δ 2.25/2.09) and H-3 (δ 5.39, br t) ↔ H2-2 (δ 3.02, br d) and an allylic coupling, H-3 ↔ Me-4 (δ 1.66). These data together established the structure of 2 as (E)-6-((1R,2R,6R)-2-hydroxy-3,4-dimethoxy-6-methyl-5oxocyclohex-3-enyl)-4-methylhex-3-enoic acid. This structure was confirmed by the HMBC spectrum (Table 1 and Figure

of Me-4 to H2-2, H2-3, and H2-6 as listed in the Experimental Section. Compound 2 (peak 12, Figure 1) had the same molecular formula as 1, deduced from its HRESIMS−. The 1H NMR spectrum of 2 (Table 1 and Figure S8, SI) (CD3OD) was similar to that of 1, except for the signals for the side-chain protons. Its 13C NMR spectrum also showed the signals for a ubiquinone skeleton (Table 1, Figure S9, SI). The COSY 1062

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preparative HPLC column (LiChrospher RP-18e, 5 μm, 250 mm × 25 mm, Merck) were used for the isolation of metabolites. Off-line NMR spectra were measured on a Bruker AVIII-600 NMR spectrometer, the latter equipped with an SEI 13C−1H probehead for 2D spectra (methanol-d4, δH 3.30 and δC 49.0 ppm). Mass spectra were recorded on an Esquire 2000 (ESIMS) and a micrOTOF orthogonal ESI-TOF mass spectrometer (Bruker Daltonik) (HRESIMS). Materials and Sample Preparation. Antroquinonol (99.7% purity as determined by HPLC) was supplied by Golden Biotechnology Corp., New Taipei City, Taiwan. Antroquinonol was vortexed with 10% aqueous acacia solution to form a suspension with a concentration of 40 mg/mL. Laboratory Animals. Six male Wistar rats, two as the control group (273 and 257 g) and four as the experimental group (282, 285, 302, and 304 g), were obtained from the Laboratory Animal Center of National Taiwan University (NTU). The animal experiment was conducted according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC), NTU (Approval no. 20080191 to Prof. Kang, J. J.). Animal Dosing and Sample Collection. Each rat was kept in an individual metabolic cage for 24 h before dosing. Normal food and water were available all the time. Four experimental rats were given antroquinonol, dosing being at 200 mg/kg, and two control rats were given vehicle via gavage. The total amounts of antroquinonol administered were about 234 mg. The urine and feces were collected separately for 24 h. Following the initial experiment as described above, a second experiment using 205 mg of antroquinonol was conducted and the urine and feces collected separately for 24 h after oral administration. Pretreatment and Fractionation of Urine Samples. The urines collected were 170 and 73 mL, respectively, for the treated and control groups in the first experiment. The latter was passed through an XADII column (350 g), washed with H2O (200 mL), MeOH−H2O (1:1) (200 mL), and MeOH (400 mL) to give the corresponding fractions, UC-H (1.68 g), UC-M1 (146 mg), and UC-M2 (109 mg) after lyophilization or condensation under reduced pressure. The urine of the treated group (170 mL) was partitioned against CH2Cl2 (100 mL × 3) to give the CH2Cl2-soluble fraction (15 mg) (UT-D). The aqueous fraction was passed through an XAD-II column and was treated in a similar manner to the control group as described above to give fractions UT-H (3.68 g), UT-M1 (242 mg), and UT-M2 (174 mg). UT-M2 (138 mg) was fractionated on a Sephadex LH-20 column (230 mL, MeOH−H2O 4:1) to give a subfraction UT-M2-2 (66 mg), showing spots with similar UV absorption to antroquinonol in TLC analysis. HPLC−SPE−NMR Analysis of UT-M2-2. UT-M2-2 was analyzed on an Agilent 1100 HPLC equipped with an analytical RP-18 column under the following conditions: delivery system, MeCN (A)−0.1% TF (aq) (B), 10% A/B to 35% A/B in 37 min, and 5 min to 95% A/B, both via linear gradient, and 95% A/B for 18 min; amount injected, 1 mg/20 μL (MeOH) × 3; flow rate, 0.6 mL/min; monitored at 270 and 210 nm. A makeup flow of pure water with a flow rate of 1.2 mL/min was added to the postcolumn eluent, and the mixed sample volume of each peak was passed through a resin GP (general purpose) cartridge in a Prospekt 2 automated solid-phase extraction unit. Each compound-loaded cartridge after a drying process by flushing with dry nitrogen for 30 min was transferred by CD3OD into a 2 mm NMR tube via Gilson Liquid Handler 215 (Gilson, Inc., Middleton, WI) and the 1H NMR spectra were measured using the reported procedure.10 Separation of 1 and 2 from UT-M1. UT-M1 (205 mg) was fractionated on a Sephadex LH-20 column (230 mL, MeOH−H2O 4:1) to give five fractions. Fraction 2 (18.5 mg/50 μL × 4, MeOH− H2O 1:3) was further separated by a semipreparative HPLC column delivered by MeCN−0.1% HOAc (aq) (1:3) with a flow rate of 3.0 mL/min and detection at 270 nm to give 2 (1.6 mg) (tR 22.5 min) and 1 (5.5 mg) (tR 25.3 min). Separation of 1−4 from Another Batch of UT-M1. Another batch of UT-M1 (151 mg) was obtained from the second experiment using the same workup procedure as described above for the first experiment. Under a similar semipreparative HPLC conditions as

S13, SI), showing the correlation of H2-2 (δ 3.02) and the olefinic proton (δ 5.39, H-3) to the carboxylic carbon (δ 176.5, C-1), and by the NOESY data (Figure S11, SI), showing key NOE correlations of H-3 to H2-2 and H2-5 as listed in the Experimental Section. The NOESY spectrum also confirmed the assignment of MeO-3′ at δ 4.06 by its correlation with H-2′ (δ 4.47) (Figure S11, SI), thereby designated MeO-4′ at δ 3.57 by elimination. Compounds 3 and 4 (peaks 11 and 10, Figure 1) had identical molecular formula, C15H22O6, as deduced from their HRESIMS+, a CH2 residue less than 1 or 2. The 1H NMR spectra of 3 (Figure S14, SI) and 2 (Figure S8, SI) were similar except that of 3 lacked a MeO-3′ singlet (δ 4.06 in 3) (Table 1). Thus 3 was likely a 3′-O-demethylated analogue of 2. However, the 1H NMR data for H-2′ (J = 4.3 Hz vs 2.4 Hz), H6′ [δ 2.55, dq (6.2, 6.9 Hz) vs δ 2.44, dq (10.6, 6.9 Hz)], and Me-6′ (δ 1.26 vs δ 1.12) were different from those of 2. Accordingly, the structure of 3 was designated as the tautomer of 3′-O-demethylated 2. The 13C NMR data of 3, assigned by comparison with those of 2 (Table 1), supported this suggestion by showing a large chemical shift difference for C2′ (δ 70.0 vs 66.2) and C-4′ (δ 134.1 vs 137.3) relative to the corresponding signals in 2. Therefore, compound 3 was established as (E)-6-((1R,2R,6R)-2,5-dihydroxy-4-methoxy-6methyl-3-oxocyclohex-4-enyl)-4-methylhex-3-enoic acid. The 1 H NMR spectrum of 4 (Figure S16, SI) was similar to that of 1 (Figure S2, SI) except for the absence of the MeO-3′ singlet (δ 4.04 in 1) (Table 1). On the basis of this spectroscopic data, 4 was likely a 3′-O-demethylated analogue of 1. Nevertheless the 1H NMR data for H-2′ (J = 4.2 Hz vs 3.2 Hz), H-6′ [δ 2.53, dq (6.2, 6.9 Hz) vs δ 2.47, dq (10.6, 6.9 Hz)], and Me-6′ (δ 1.27 vs δ 1.12) were different from those of 1. Thus the structure of 4 was designated as the tautomer of 3′O-demethylated 1. While comparing the 13C NMR data of 2 and 4, the latter being assigned by comparison with those of 1 (Table 1), large chemical shift differences for C-2′ (δ 70.5 vs 66.7) and C-4′ (δ 133.9 vs 137.2) were observed, supporting the suggested structure for 4, i.e., (E)-6-((1R,2R,6R)-2,5dihydroxy-4-methoxy-6-methyl-3-oxocyclohex-4-enyl)-4-methylhex-4-enoic acid. Some carbon signals of 3 and 4 were not observed due to limited amount of materials. Compounds 1−4 are apparently the catabolic products of antroquinonol obtained via a metabolic pathway similar to the cleavage of cholesterol side chain.8,9 The biological activity of these metabolites was not determined.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured on a JASCO DIP-370 polarimeter. UV spectra were measured in MeOH on a U-2001 Hitachi double-beam spectrophotometer. The ECD spectra were recorded on a J-710 spectropolarimeter. HPLC-DAD (diode array detector)-SPE-NMR (600 MHz) carried out using an Agilent 1100 liquid chromatograph (Waldbronn, Germany), equipped with a DAD (Bruker, Rheinstetten, Germany), a Knauer K120 HPLC pump (makeup pump), followed by a Prospekt2 automated solid-phase extraction unit (Spark Holland, Emmen, Holland), containing 192 HySphere resin GP cartridges (10 mm × 2 mm, 10−12 μm), a nitrogen separator, and a Bruker AVIII-600 NMR spectrometer equipped with a cryoprobe. An analytical HPLC column (Prodigy ODS3 100A, 250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, California, USA) was used in metabolites profiling, HPLC-SPE-NMR, and HPLC-MS. A semipreparative HPLC column (Prodigy ODS3 100A, 250 mm × 10 mm, 5 μm) and a 1063

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(8) Sih, C. J.; Wang, K. C.; Tai, H. H. J. Am. Chem. Soc. 1967, 78, 1956−1957. (9) Sih, C. J.; Tai, H. H.; Tsong, Y. Y. J. Am. Chem. Soc. 1967, 78, 1957−1958. (10) Lee, S. S.; Lai, Y. C.; Chen, C. K.; Tseng, L. H.; Wang, C. Y. J. Nat. Prod. 2007, 70, 637−642.

described above for separation of 1 and 2 except that 0.1% HOAc (aq) in the eluent was replaced by 0.1% TFA (aq), UT-M1 (151 mg) yielded 4 (0.3 mg, tR 12.3 min), 3 (0.2 mg, tR 13.4 min), 2 (2.6 mg, tR 22.3 min), and 1 (10.1 mg, tR 24.9 min). (E)-6-((1R,2R,6R)-2-Hydroxy-3,4-dimethoxy-6-methyl-5-oxocyclohex-3-enyl)-4-methylhex-4-enoic Acid (1). [α]24D +67 (c 0.69, MeOH); UV (MeCN−0.1%TF (aq) 1:3) λmax 269 nm; CD (c 3.15 × 10−4 M, MeOH) [θ]363 0°, [θ]324 +1 440°, [θ]297 −470°, [θ]263 +5 670°, [θ]230 0°, [θ]215 −2 790°; 1H and 13C NMR, and HMBC, see Table 1; NOESY data H-3 ↔ Me-4 ↔ H-2 ↔ H-3 ↔ H-5 ↔ H-6 ↔ H-1′ ↔ H-2′ ↔ MeO-3′, H-5 ↔ Me-4 ↔ H-6 ↔ Me-6′ ↔ H-1′ and H-6′, H-6 ↔ H-2′; ESI/MS− m/z 311 [M − H]−; HRESIMS− m/z 311.1502 (calcd for C16H24O6−H, 311.1500). (E)-6-((1R,2R,6R)-2-Hydroxy-3,4-dimethoxy-6-methyl-5-oxocyclohex-3-enyl)-4-methylhex-3-enoic Acid (2). [α]24D +50 (c 0.2, MeOH); UV (MeCN−0.1%TFA (aq) 1:3) λmax 269 nm; CD (c 3.15 × 10−4 M, MeOH) [θ]360 0°, [θ]322 +1 430°, [θ]300 −240°, [θ]259 +4 620°, [θ]232 0°, [θ]216 −2 210°; 1H and 13C NMR, and HMBC, see Table 1; NOESY data H-3 ↔ H-2 ↔ Me-4 ↔ H-5 ↔ H-6 ↔ H-1′ ↔ H-2′ ↔ MeO-3′, H-3 ↔ H-5 ↔ H-2′ ↔ H-6 ↔ Me-6′ ↔ H-1′ and H-6′; ESI/MS+ m/z 335 [M + Na]+; ESI/MS− m/z 311 [M − H]−; HRESIMS− m/z 311.1497 (calcd for C16H24O6−H, 311.1500). (E)-6-((1R,2R,6R)-2,5-Dihydroxy-4-methoxy-6-methyl-3-oxocyclohex-4-enyl)-4-methylhex-3-enoic Acid (3). UV (MeCN−0.1%TFA (aq) 1:3) λmax 269 nm from HPLC-DAD; 1H and 13C NMR, see Table 1; HRESIMS− m/z 297.1336 (calcd for C15H22O6−H, 297.1344). (E)-6-((1R,2R,6R)-2,5-Dihydroxy-4-methoxy-6-methyl-3-oxocyclohex-4-enyl)-4-methylhex-4-enoic Acid (4). UV (MeCN−0.1%TFA (aq) 1:3) λmax 269 nm from HPLC-DAD; 1H and 13C NMR, see Table 1; HRESIMS− m/z 297.1345 (calcd for C15H22O6−H, 297.1344).

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ASSOCIATED CONTENT

S Supporting Information *

1D (1H and 13C) and 2D NMR (COSY, NOESY, HSQC, and HMBC) spectra of 1 and 2 and 1D NMR spectra (1H and 13C) for 3 and 4). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/Fax: +886 2 23916127. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by National Science Council, Republic of China, under grants NSC 97-2323-B-002-001 and NSC 100-2325-B-002-021.

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REFERENCES

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