Monascustin, an Unusual γ-Lactam from Red Yeast Rice - Journal of

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Monascustin, an Unusual γ‑Lactam from Red Yeast Rice Wendi Wei,†,⊥ Sheng Lin,‡,⊥ Minghua Chen,‡ Tianxi Liu,§ Ali Wang,† Jinjie Li,† Qinglan Guo,‡ and Xiaoya Shang*,† †

Beiijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing 100191, People’s Republic of China ‡ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China § Lanzhou University First Affiliated Hospital, Gansu 730000, People’s Republic of China S Supporting Information *

ABSTRACT: Monascustin (1), an unusual γ-lactam, was isolated from an ethanol extract of the Monascus purpureus fermented rice. Its structure including the absolute configuration was determined by spectroscopic data analysis and confirmed by X-ray crystallography. A plausible biosynthetic pathway is discussed on the basis of amino acid derivatization. Compound 1 showed inhibitory activity against histone deacetylase 1.

R

ed yeast rice, otherwise known as red koji or hongqu, is prepared by fermenting Monascus purpureus on steamed rice. It has been extensively used as a folk medicine to decrease blood pressure and lower plasma cholesterol levels and blood sugar in East Asia.1,2 Chemical investigations of red yeast rice have revealed the presence of yellow, orange, and red pigments, monacolin analogues, γ-aminobutyric acid, citrinin, and dimerumic acid.3−6 The discovery of monacolin analogues has drawn much attention to their chemical synthesis and biosynthesis due to their inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), an enzyme involved in the synthesis of cholesterol.7−16 Lovastatin, also known as mevinolin or monacolin K, has been approved for use as a cholesterol-lowering drug for the treatment of familial hypercholesterolemia and heterozygous familial hypercholesterolemia.17−21 As part of a program to assess the chemical diversity of Chinese traditional medicines and study their biological effects, especially focusing on minor constituents, we investigated M. purpureus fermented rice. In a previous study, three azaphilones, three monacolin analogues, and four steroids were characterized in several fractions obtained from an EtOH extract. Among these compounds, three monacolin analogues and four steroids showed selective cytotoxic activity against the A2780 ovarian cancer cell line.22−26 Further bioassay-guided isolation of a remaining fraction with inhibitory activity against TNF-α secretion in mouse peritoneal macrophages led to the isolation of a minor metabolite, monascustin (1) (Figure 1). Its absolute configuration was established using a combination of NMR spectroscopy, CD data analysis, and X-ray crystallography. Herein, we report details of the isolation, structure elucidation, postulated biogenetic pathway, and bioactivity of this compound. Monascustin (1) was obtained as white powder with [α]20D +66.0 (c 0.55, MeOH), and its molecular formula C10H18N2O3, requiring three degrees of unsaturation, was deduced from HRESIMS. This molecular formula was corroborated by NMR © 2016 American Chemical Society and American Society of Pharmacognosy

Figure 1. Structure of monascustin (1).

spectroscopic data (Table 1). The 1H NMR spectrum of 1 in DMSO-d6 showed resonances attributable to one isolated Table 1. NMR Spectroscopic Data (500 MHz, DMSO-d6) for Monascustin (1)a position

δC

1 2a 2b 3 4 5a 5b 6 7 8 9 10 OH-3 NH2 NH

174.6 45.0 77.8 72.1 43.0 24.0 22.5 24.8 173.0 21.6

δH 1.95 d (16.5) 2.43 d (16.5)

1.22 1.85 1.55 0.82 0.87

dd (4.3, 13.5) dd (8.3, 13.5) m d (6.5) d (6.5)

1.27 s 4.90 s 6.60, 7.09 (each 1H, brs) 7.98 s

a

The assignments were based on DEPT, 1H−1H gCOSY, gHSQC, and gHMBC experiments. Received: June 3, 2016 Published: December 27, 2016 201

DOI: 10.1021/acs.jnatprod.6b00493 J. Nat. Prod. 2017, 80, 201−204

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strong positive Cotton effect at λmax = 225 nm (Δε = 4.90) (Figure 3). However, due to the fact that no model compounds

aliphatic methylene at δH 1.95, 2.43 (each 1H, d, J = 16.5 Hz, H-2a and H-2b), an isobutyl group at δH 0.82, 0.87 (each 3H, d, J = 6.5 Hz, H3-7 and H3-8), 1.55 (1H, m, H-6), 1.22, and 1.85 (each 1H, dd, J = 13.5, 4.3 Hz, H-5a; J = 13.5, 8.3 Hz, H-5b), and a tertiary methyl group at δH 1.27 (H3-10). The spectrum also illustrated (in D2O) four exchangeable protons, including one that corresponded to a hydroxy group at δH 4.90 (s, OH-3) and three at lower field, assigned to NH and NH2 at δH 7.98 7.09, and 6.60 (each 1H, s), respectively. In addition to protonated carbon signals corresponding to the above protons, the 13C NMR and DEPT data for 1 showed four carbons that were identified as two carbonyl carbons and two oxygen and/or nitrogen-bearing carbons at δC 72.1 and 77.8. IR absorption bands were observed at 3565−2959 and 1677 cm−1, which when combined with the carbonyl carbons at δC 174.6 and 173.0 indicated the presence of two amide functional groups. The structure of 1 was finally deduced from a comprehensive 2D NMR spectroscopic analysis. The proton and hydrogenbearing carbon resonances in the NMR data of 1 were assigned unambiguously by 1H−1H gCOSY and HSQC spectroscopic data interpretation. In the 1H−1H gCOSY spectrum of 1, the homonuclear coupling correlations of H3-7(H3-8)/H-6/H2-5 confirmed the presence of an isobutyl group (Figure 2, thick

Figure 3. Experimental ECD spectra of 1 (black) and the calculated ECD spectra of (2R,3S)-1 (red) and (2S,3R)-1 (blue).

were found for reference, the absolute configuration of 1 could not be resolved directly by analysis of its CD data. To resolve this, the calculation of electronic circular dichroism (ECD) using time-dependent density functional theory (TDDFT), which has greatly enhanced the value of ECD in determining absolute configuration in recent years,27,28 was applied in combination with the experimental CD data. The calculated ECD data of 1 and its enantiomer in both the gas phase and CH3CN are shown in Figure 3. The calculated ECD of 1 matches very well with the experimental CD, while the calculated ECD of its enantiomer is opposite the experimental value, allowing the assignment of the 2R,3S configuration of 1. Confirmation of the absolute configuration of 1 was achieved by X-ray crystallography (Figure 4). A light atom absolute configuration determination was carried out by the anomalous dispersion protocol with more effective Cu Kα radiation as opposed to the traditional Mo Kα radiation. The absolute

Figure 2. Main 1H−1H COSY (black thick lines) and selected HMBC correlations (red arrows, from 1H to 13C) of monascustin (1).

lines). The HMBC data of 1 showed two- and three-bond correlations from H3-10 to C-2, C-3, and C-4; from H2-5 to C3, C-4, C-6, C-7, and C-9; from H2-2 to C-1, C-3, C-4, and C10; and from OH-3 to C-2, C-3, C-4, and C-10 (Figure 2 and Figures S13−S21, Supporting Information). These correlations, in combination with the chemical shifts of the proton and carbon resonances, indicated the presence of a 4-substituted 4formamide-3-hydroxy-3,6-dimethylheptamide moiety in 1. In order to satisfy the 3 degrees of unsaturation indicated by the molecular formula, it was apparent that one of the nitrogens was involved in a ring closure to form a lactam or a piperidine2,6-dione moiety. Since the two amine protons at δH 7.09 and 6.60 showed no HMBC correlations to C-1, C-4, and C-9, key HMBC correlations from another amine proton at δH 7.98 to C-1, C-2, C-3, and C-4, together with the corresponding chemical shifts, clearly served to construct the γ-lactam in 1, leaving the formamide to be located at C-4. On the basis of the foregoing data, the gross structure of 1 was established as shown. The relative configuration of 1 was deduced from analysis of ROESY data. ROESY correlations form H-2a to H-5a and to the methyl protons H3-10 implied that these protons were in a cofacial position (Figure S18, Supporting Information). To determine its absolute configuration, the CD spectrum of 1 was measured (in CH3CN), which was dominated by a

Figure 4. Final X-ray drawing for monascustin (1) depicting its absolute configuration. 202

DOI: 10.1021/acs.jnatprod.6b00493 J. Nat. Prod. 2017, 80, 201−204

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Scheme 1. Plausible Biosynthetic Pathway of Monascustin (1)

in methanol. Upon cooling, the white precipitate was filtered to afford 1 (150 mg). Monascustin (1): colorless crystal (DMF and MeOH), [α]20D +66.0 nm (log ε) 203 (4.753); CD (MeCN) (c 0.20, MeOH); UV λMeOH max 225 (Δε 4.90) nm; IR νmax 3565, 3491, 3356, 3177, 2959, 2872, 1677, 1460, 1433, 1385, 1288, 1259, 1214, 1169, 1150, 1127, 1102, 1024, 939, 913, 866, 780, 702 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 1; (+)-ESIMS m/z 215 [M + H]+; (−)ESIMS m/z 213 [M − H]−; (+)-HRESIMS m/z (−)-HRESIMS m/z 213.1243 [M − H]− (calcd for C10H17N2O3, 213.1244). X-ray Analysis of 1. Crystals of compound 1 were grown via the slow evaporation of diffusion protocol using an N,N-dimethylformamide (DMF), and MeOH solution. A colorless block of appropriate dimensions was mounted on a sealed tube. The room-temperature (293 ± 2 K), single-crystal X-ray experiments were performed on a Rigaku MicroMax 002+ diffractometer equipped with ConfocalLaser monochromatized Cu Kα radiation by using the ω and κ scan technique to a maximum 2θ value of 146.28°. The structure was established by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXS-97). Seventeen non-hydrogen atoms were refined anisotropically using the least-squares method, and all the hydrogen atoms were positioned by geometrical calculations and difference Fourier overlapping calculation. The reliable factor is R1 = 0.0370, wR2 = 0.0982 (w = 1/σ|F2|), S = 1.114. The absolute configuration was assigned on the basis of the Flack parameter of −0.1(2). Crystal data of 1: colorless, columnar; crystal size 0.29 × 0.35 × 0.57 mm; molecular formula C10H18N2O3·CH3OH; fw 246.31; orthorhombic system; space group P212121; unit cell dimensions a = 7.403(3) Å, b = 8.932(5) Å, c = 20.101(8) Å, V = 1329.2(11) Å3; Z = 4; dcalc = 1.231 g/cm3; reflections collected 676 and independent reflections 2442. Crystallographic data of 1 have been deposited at the Cambridge Crystallographic Data Centre (deposition no. 1445768). Copies of these data can be obtained free of charge via www.ccdc.cam. ac.uk/deposit or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK [fax: (+44) 1223-336-033; or e-mail: [email protected]]. TNF-α Secretive Inhibition Assay. Compound 1 was evaluated for inhibitory activity against TNF-α secretion in mouse peritoneal macrophages, according to a previously described protocol.30 Cytotoxicity Assay. Human colon cancer (HCT-8), human hepatoma (Bel7402), human stomach cancer (BGC-823), human lung adenocarcinoma (A549), and human ovarian cancer (A2780) cell lines were obtained from ATCC. All the cells were seeded in 96-well microtiter plates at 1200 cells/well and were treated 24 h later with various concentrations of compound 1. After 24 h of incubation, MTT was added to all wells. Plates were incubated for another 24 h, and cell viability was measured by observing absorbance at 570 nm on an MK 3 Wellscan (Labsystem Drogon). IC50 values were calculated using Microsoft Excel software. Paclitaxel was used as a positive control. HDAC1 Inhibition Screen of 1. HDAC1 (Cisbio Bioassays No. NP_004955.2) inhibitory activity was measured using 2 ng/μL HDAC1 and 0.2 μmol/L H3(1−21)K9 incubated for 60 min at 37 °C with a range of concentrations of compound 1. Reaction was stopped by addition of 5 μL of SA-XL665 and 2 ng of H3K9me0 antibody. Then, fluorescence signal was measured on a fluorescence microplate reader with an emission wavelength of 665 nm. All dilutions were prepared in a buffer containing 50 mM Tris-HCl, pH 8.0; 137 mM NaCl; 2.7 mM KCl; 1 mM MgCl2; and 0.01% Tween20.

configuration was assigned on the basis of the absolute structure parameter, which refined to a value of −0.1(2). Therefore, the structure and absolute stereochemistry of compound 1 was fully determined, and the name monascustin was assigned. The plausible biosynthetic pathway for monascustin (1) is postulated (Scheme 1). Compound 1 may be produced by coupling of one molecule of L-Leu and two molecules of acetylSCoA, followed by the intramolecular nucleophilic addition. Monascustin (1) was initially tested for inhibitory activity against TNF-α secretion in mouse peritoneal macrophages and for cytotoxicity against several human cancer cell lines,29 but no activity was observed at the highest test concentrations (50 μM against TNF-α secretion and 10 μM for cytotoxicity). In addition, monascustin (1) was evaluated for inhibitory activity against histone deacetylase 1 (HDAC1). Monascustin (1) was only weakly active, with an IC50 of 3.7 μM, compared to the positive control suberoylanilide hydroxamic acid, with an IC50 of 65.2 nM.

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EXPERIMENTAL SECTION

General Experimental Procedures. CD spectra and UV data were determined on a JASCO J-810 circular dichroism spectrometer. IR spectra were recorded as microscope transmission on a Nicolet 5700 FT-IR spectrophotometer. 1D and 2D NMR spectra were acquired in DMSO-d6 with tetramethylsilane as internal standard on Varian 500 MHz spectrometers. Mass spectra were recorded on a JEOL JMS AX-500 spectrometer. Column chromatography was performed with silica gel (160−200 mesh, Qingdao Marine Chemical Inc. China), RP-18 reverse-phase silica gel (43−60 μm), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala Sweden). MPLC separation was performed with Buchi (Buchi Companion). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 8% H2SO4 in 95% EtOH followed by heating. Fungal Material. Monascus purpureus was prepared from cooked paddy rice inoculated with M. purpureus B0708, purchased from the Beijing Dawn Aerospace Biotech Company. The sample was deposited at Beijing Union University, Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing 100191, China. Extraction and Isolation. The dried M. purpureus fermented rice powder (4.5 kg) was extracted with 50 L of 95% EtOH, 80% EtOH, and 60% EtOH, respectively, at room temperature (×3, each for 3 h). After removal of the solvent under reduced pressure, the residue (850.0 g) was suspended in 3 L of H2O and then partitioned sequentially with petroleum ether (three times with 5 L each) and EtOAc (four times with 5 L each) to yield petroleum ether (38.0 g), EtOAc (223.5 g), and H2O (588.5 g) fractions, respectively. The H2O-soluble portion (588.5 g) was fractionated via macroporous adsorption resin column chromatography by eluting with 10% EtOH, 30% EtOH, and 60% EtOH, respectively. The 30% EtOHsoluble portion (123.0 g) was separated into eight (E1−E8) subfractions on preparative reversed-phase MPLC (MeOH−H2O, 5:95 to 100:0). Fraction E3 (38.0 g) was fractionated via silica gel column chromatography by eluting with a gradient of increasing methanol (0−100%) in chloroform to give nine fractions (I−IX) on the basis of TLC analyses. Fraction E3-2 was then subjected to Sephadex LH-20 (CHCl3−MeOH, 2.5:1) to provide three subfractions (E3-2-1−E3-2-4). Fraction E3-2-3 was heated and dissolved 203

DOI: 10.1021/acs.jnatprod.6b00493 J. Nat. Prod. 2017, 80, 201−204

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Suberoylanilide hydroxamic acid was used as positive control with an IC50 of 65.2 nM.

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(16) Barriuso, J.; Nguyen, D. T.; Li, J. W. H.; Roberts, J. N.; MacNevin, G.; Chaytor, J. L.; Marcus, S. L.; Vederas, J. C.; Ro, D. K. J. Am. Chem. Soc. 2011, 133, 8078−8081. (17) Barkas, F.; Elisaf, M.; Milionis, H. Atherosclerosis 2015, 243, 60− 64. (18) Clauss, S. B.; Holmes, K. W.; Hopkins, P.; Stein, E.; Cho, M.; Tate, A.; Johnson-Levonas, A. O.; Kwiterovich, P. O. Pediatrics 2005, 116, 682−688. (19) Pappu, A. S.; Bacon, S. P.; Illingworth, D. R. J. Lab. Clin. Med. 2003, 141, 250−256. (20) Pappu, A. S.; Illingworth, D. R. Atherosclerosis 2002, 165, 137− 144. (21) Kwiterovich, P. O. J. Nut. Metab. Cardiovasc. Dis. 2001, 11, 30− 34. (22) Li, J. J.; Shang, X. Y.; Li, L. L.; Liu, M. T.; Zheng, J. Q.; Jin, Z. L. Molecules 2010, 15, 1958−1966. (23) Liu, M. T.; Li, J. J.; Shang, X. Y.; Li, S.; Li, L. L.; Luan, N.; Jin, Z. L. Magn. Reson. Chem. 2011, 49, 129−131. (24) Shang, X. Y.; Li, J. J.; Liu, M. T.; Li, S.; Liu, Y.; Wang, Y. F.; Huang, X.; Jin, Z. L. Steroids 2011, 76, 1185−1189. (25) Liu, M. T.; Luan, N.; Li, J. J.; Huang, X.; Wang, Y. F.; Wang, A. L.; Shang, X. Y. Magn. Reson. Chem. 2012, 50, 709−712. (26) Liu, M. T.; Wang, A. L.; Sun, Z.; Li, J. J.; Wu, X. L.; Liu, Y. X.; Shang, X. Y. J. Asian Nat. Prod. Res. 2013, 15, 600−609. (27) Diedrich, C.; Grimme, S. J. Phys. Chem. A 2003, 107, 2524− 2539. (28) Chen, M. H.; Lin, S.; Li, L.; Zhu, C. G.; Wang, X. L.; Wang, Y. N.; Jiang, B. Y.; Wang, S. J.; Li, Y. H.; Jiang, J. D.; Shi, J. G. Org. Lett. 2012, 14, 5668−5671. (29) Yuan, Z. Z.; Duan, H. M.; Xu, Y. Y.; Wang, A. L.; Gan, L. S.; Li, J. J.; Liu, M. T.; Shang, X. Y. Phytochem. Lett. 2014, 8, 116−120. (30) Lin, S.; Wang, S. J.; Liu, M. T.; G, M. L.; Li, S.; Yang, Y. C.; Wang, Y. H.; He, W. Y.; Shi, J. G. J. Nat. Prod. 2007, 70, 817−823.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00493. CCDC 1445768 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures. Additional figures (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-62004533. E-mail: [email protected] (X.Y. Shang). ORCID

Xiaoya Shang: 0000-0003-2193-1712 Author Contributions ⊥

W. Wei and S. Lin have contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Sciences Foundation of China (NNSFC; Grant No. 31171669), Beijing Natural Sciences Foundation (Grant No. 7142028), and Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University (Grant No. Zk60201601) is acknowledged.

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REFERENCES

(1) Ma, J.; Li, Y.; Ye, Q.; Li, J.; Hua, Y.; Ju, D.; Zhang, D.; Cooper, R.; Chang, M. J. Agric. Food Chem. 2000, 48, 5220−5225. (2) Akihisa, T.; Tokuda, H.; Yasukawa, K.; Ukiya, M.; Kiyota, A.; Sakamoto, N.; Suzuki, T.; Tanabe, N.; Nishino, H. J. Agric. Food Chem. 2005, 53, 562−565. (3) Wild, D.; Toth, G.; Humpf, H. U. J. Agric. Food Chem. 2002, 50, 3999−4002. (4) Antonio, B. Chin. J. Integr. Med. 2005, 11, 309−313. (5) Liu, B. H.; Wu, T. S.; Su, M. C.; Chung, C. P.; Yu, F. Y. J. J. Agric. Food Chem. 2005, 53, 170−175. (6) Su, N. W.; Lin, Y. L.; Lee, M. H.; Ho, C. Y. J. Agric. Food Chem. 2005, 53, 1949−1954. (7) Alberts, A. W.; Chen, J.; Kuron, G.; Hunt, V.; Huff, J.; Hoffman, F.; Rothrock, J.; Lopez, M.; Joshua, H.; Harries, E.; Patchett, A.; Momaghan, R.; Currie, S.; Stapley, E.; Albers-Schonberg, G.; Hensens, O.; Hirshfield, J.; Hoogsteen, K.; Liesch, J.; Springer, J. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 3957−3961. (8) Endo, A. J. Antibiot. 1979, 32, 852−854. (9) Endo, A.; Hasumi, K.; Negishi, Y. J. Antibiot. 1985, 38, 420−422. (10) Burr, D. A.; Chen, X. B.; Vederas, J. C. Org. Lett. 2007, 9, 161− 164. (11) Witter, D. J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613−2623. (12) Himara, M.; Uei, M. J. Am. Chem. Soc. 1982, 104, 4251−4253. (13) Wovkulich, P. M.; Tang, P. C.; Chadha, N. K.; Batcho, A. D.; Barrish, J. C.; Uskoković, M. R. J. Am. Chem. Soc. 1989, 111, 2596− 2599. (14) Auclair, K.; Sutherland, A.; Kennedy, J.; Witter, D. J.; Van den Heever, J. P. J. Am. Chem. Soc. 2000, 122, 11519−11520. (15) Xie, X. K.; Meehan, M. J.; Xu, W.; Dorrestein, P. C.; Tang, Y. J. Am. Chem. Soc. 2009, 131, 8388−8389. 204

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