Evodialones A and B: Polyprenylated Acylcyclopentanone Racemates

A putative biosynthetic pathway of 1 and 2 starting from the monocyclic .... Supporting Information) with relative Gibbs free energies less than 2 kca...
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Evodialones A and B: Polyprenylated Acylcyclopentanone Racemates with a 3‑Ethyl-1,1-diisopentyl-4-methylcyclopentane Skeleton from Evodia lepta Ya-Qi Tang, Yi-Qiu Li, Yi-Bo Xie, Jun-Sheng Zhang, Wei Li, Lan-Lan Lou, Ge Zhang, and Sheng Yin* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China

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S Supporting Information *

ABSTRACT: Two polyprenylated acylcyclopentanone racemates, evodialones A (1) and B (2), featuring a 3-ethyl-1,1diisopentyl-4-methylcyclopentane skeleton, were isolated from an extract of the aerial parts of Evodia lepta. Evodialone A (1) was resolved by chiral-phase HPLC to afford a pair of enantiomers, (+)- and (−)-evodialones A (1b/1a), while evodialone B (2) could not be resolved. Their structures were elucidated by spectroscopic analysis and a combination of computational techniques including gauge-independent atomic orbital calculation of 1D NMR data and experimental and TDDFT-calculated ECD spectra. A putative biosynthetic pathway of 1 and 2 starting from the monocyclic polyprenylated acylphloroglucinols is proposed. All the isolates were screened for the antimicrobial activity in vitro, and 1a and 1b showed moderate inhibitory activities against several pathogenic fungi with MICs values of 17.1−68.3 μM. findings suggest that the exploration of the chemistry and potential bioactivities of PACs may be an intriguing topic. Evodia lepta (Spreng.) Merr. (Rutaceae) is a timber tree distributed in most of the Southeast Asia countries.13 Its roots and leaves have been widely used by local people for the treatment of arthritis, fever, and epidemic influenza.14 Previous chemical investigation of this plant revealed the presences of quinoline-type alkaloids, flavonoids, chromenes, and coumarins, some of which showed inhibitory activity toward acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).15−17 In our continuing efforts to discover structurally intriguing and biologically significant metabolites from medicinal plants,18−20 two unusual polyprenylated acylcyclopentanone racemates, evodialones A (1) and B (2), were isolated from an extract of the aerial parts of E. lepta. Compounds 1 and 2 feature a new 3-ethyl-1,1-diisopentyl-4-methylcyclopentane skeleton, and the further chiral-phase separation of 1 led to a pair of enantiomers, 1a and 1b. The antimicrobial activities of these isolates were evaluated against a panel of bacteria and fungi, and 1a and 1b showed moderate activities against several fungi strains. Herein, the isolation, structural elucidation, putative biosynthetic formation, and antimicrobial activity of 1a, 1b, and 2 are discussed.

P

olyprenylated acylcyclopentanones (PACs) are a group of rare natural products predominantly occurring in Humulus lupulus L. (commonly named hops) (Cannabaceae).1−3 Biosynthetically, they are derived from monocyclic polyprenylated acylphloroglucinols (MPAPs) containing diprenylated αacid and triprenylated β-acid moieties, via an acyloin-type ring contraction.4−6 The structural diversity of PACs mainly arises from their alkanoyl side chains, with different locations and lengths. Although PACs are known as bitter acids in the hop brewing industry, which contribute to the bitter taste and hoppy aroma of beer,2,7 the reported PAC structures are mainly based on analysis of the LC-MS data of the crude hop extract or on chemical transformation of the coexisting MPAPs.2,3 So far, only a few natural PACs, such as isohumulone, isocohumulone, isoadhumulone, cohulupone, and melicolones A and B, were fully characterized.7−9 Recently, PACs have attracted considerable interest due to their broad bioactivities and diverse structures.9−12 For example, isohumulone and isocohumulone were reported to prevent inflammation and cognitive decline in a mouse model of Alzheimer’s disease.11 The mixture of isohumulones could inhibit oxidative damage and alleviate metabolic syndrome both in vitro and in vivo.10,12 Melicolones A and B, with an unusual 9-oxatricyclo[3.2.1.13,8]nonane core, exhibited potent cell-protecting activities against high-glucoseinduced oxidative stress in human vein endothelial cells.9 These © 2018 American Chemical Society and American Society of Pharmacognosy

Received: November 24, 2017 Published: May 30, 2018 1483

DOI: 10.1021/acs.jnatprod.7b00993 J. Nat. Prod. 2018, 81, 1483−1487

Journal of Natural Products

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122.1 and 171.3), five methyls, two sp3 methylenes, an oxymethine carbon (δC 67.5), two oxygenated sp3 tertiary carbons, and an sp3 quaternary carbon. As five of the seven indices of hydrogen deficiency were accounted for by two ketocarbonyls, an ester carbonyl, and two double bonds, the remaining two indices of hydrogen deficiency required that 1 was bicyclic. The 2D structure of 1 was established by analysis of its 2D NMR data. The HMBC correlations from the two allylic methyls (Me-14 and Me-15) to C-12 and C-13 together with the 1H−1H COSY correlations between the olefinic proton (H-12) and the methylene protons (H2-11) showed the presence of an isopentenyl side chain (Figure 1). Similarly, the

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RESULTS AND DISCUSSION The aerial parts of E. lepta were extracted with EtOH/H2O (95:5) at room temperature (rt), and the residue was suspended in H2O and successively partitioned with petroleum ether (PE), EtOAc, and n-BuOH. Subsequent separations of the EtOAc extract using a series of column chromatographies afforded evodialones A (1) and B (2). Evodialone A (1) was isolated as a colorless oil. Its molecular formula C19H26O7 was determined by HRESIMS at m/z 389.1573 [M + Na]+ (calcd 389.1571). The IR spectrum showed absorption bands for hydroxy (3462 cm−1) and carbonyl (1766 and 1742 cm−1) functionalities. The 1H NMR data (Table 1) displayed signals for five methyls [δH 1.26, 1.47, Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data for Evodialones A (1) and B (2) in DMSO-d6 (δ in ppm) 1 no. 1 2 3 4 5 6

7 8 9 10 11

12 13 14 15 16 17 18 19 2-OH 7-OH

δH (J in Hz)

α 1.82, dd (14.4, 3.7) β 1.97, dd (14.4, 4.1) 3.59, q (4.1) 1.47, s 1.26, s a 2.92, dd (14.3, 7.4) b 2.64, dd (14.3, 7.4) 5.07, t (7.4) 1.54, s 1.65, s 2.33, s 3.54, s 6.32, s 5.48, d (4.8)

Figure 1. Selected HMBC (→) and 1H−1H COSY (bold lines) correlations of 1.

2 δC, type 206.4, C 81.4, C 122.1, C 171.3, C 49.7, C 29.0, CH2

67.5, 87.1, 24.0, 25.4, 36.3,

CH C CH3 CH3 CH2

117.9, CH 134.8, C 17.9, CH3 25.8, CH3 192.4, C 30.2, CH3 170.2, C 52.3, CH3

δH (J in Hz)

α 1.41, dd (13.0, 11.3) β 1.97, dd (13.0, 4.3) 3.83, dt (11.3, 5.7) 1.52, s 1.16, s a 2.63, dd (14.6, 8.4) b 2.45, dd (14.6, 6.6) 5.02, t (7.5) 1.58, s 1.68, s 2.32, s 3.56, s 6.42, s 5.42, d (5.3)

δC, type 206.3, C 81.9, C 122.5, C 169.3, C 52.7, C 30.4, CH2

66.5, 86.8, 26.9, 18.7, 34.3,

HMBC correlations from Me-9 and Me-10 to two oxygenated carbons (C-8 and C-7) together with the 1H−1H COSY correlations between an oxymethine (H-7) and the methylene protons (H2-6) showed the presence of a functionalized isopentyl side chain. These two C5 side chains were attached to a quaternary carbon (C-5) by HMBC correlations from H12, H2-11, H-7, and H2-6 to C-5 and from H2-11 to C-6. Furthermore, a ketocarbonyl (C-1) and an sp2 carbon (C-4) were connected to C-5 by HMBC correlations from H2-6 and H2-11 to C-1, C-4, and C-5. The ketocarbonyl was further linked to the other sp2 carbon (C-3) via a tertiary carbon (C-2) bearing a hydroxy and a methoxycarbonyl group, as indicated by HMBC correlations of 2-OH/C-1, C-2, C-3, and C-18. An acetyl group was attached to C-3 by the HMBC correlation of H3-17/C-3. The “unassigned” carbons C-8 and C-4 required an oxygen bridge between these two carbons to form a pyran ring, which was supported by the remaining index of hydrogen deficiency and the deshielded C-4 signal (δC 171.3). Thus, the gross structure of evodialone A was established to possess a 3ethyl-1,1-diisopentyl-4-methylcyclopentane skeleton, containing an unusual cyclopenta[b]pyran core. The relative configuration of 1 was determined by a NOESY experiment, in which the correlations of H2-11/7-OH and 2OH suggested that the isopentenyl group, 7-OH, and 2-OH were cofacial. This was supported by gauge-independent atomic orbital (GIAO) calculation of its 1H and 13C NMR chemical shifts (Tables S3 and S4, Supporting Information). The experimental and calculated data were compared by the improved probability DP4+ method (Figure S2, Supporting Information), which showed a DP4+ probability score at 99.47%. The small specific rotation value of 1 suggested that it was a scalemic mixture. Subsequent chiral-phase separation afforded the anticipated enantiomers 1a and 1b, which showed opposite specific rotation values and electronic circular dichrosim (ECD) curves. The determinations of the absolute configurations of 1a

CH C CH3 CH3 CH2

117.0, CH 135.7, C 17.8, CH3 25.8, CH3 192.5, C 30.2, CH3 169.9, C 52.4, CH3

1.54, 1.65, and 2.33 (each 3H, s)], a methoxy [δH 3.54 (3H, s)], an olefinic proton [δH 5.07 (1H, t, J = 7.4 Hz)], an oxymethine proton [δH 3.59 (1H, q, J = 4.1 Hz)], and four aliphatic protons [δH 1.82 (1H, dd, J = 14.4, 3.7 Hz), 1.97 (1H, dd, J = 14.4, 4.1 Hz), 2.92 (1H, dd, J = 14.3, 7.4 Hz), and 2.64 (1H, dd, J = 14.3, 7.4 Hz)]. The 13C NMR spectrum, associated with DEPT experiments, resolved 19 carbon resonances attributable to two ketocarbonyl groups (δC 192.4 and 206.4), a methyl ester group (δC 170.2 and 52.3), two double bonds (δC 117.9 and 134.8; δC 1484

DOI: 10.1021/acs.jnatprod.7b00993 J. Nat. Prod. 2018, 81, 1483−1487

Journal of Natural Products

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and 1b were a challenge, as modification of its 7-OH either by Mosher’s method21 or by the Rh2(OCOCF3)4 chelating method22 failed. Thus, ECD calculations were employed to resolve the absolute configurations of 1a and 1b. Conformational analysis of the SSS-1 isomer was carried out. All the conformers were reoptimized using density functional theory (DFT) at the B3LYP/6-31G(d) level under vacuum using the Gaussian 09 program, and four conformers (C1−C4, Figure S3, Supporting Information) with relative Gibbs free energies less than 2 kcal/mol were selected for subsequent calculations (Table S5, Supporting Information). The stability of these conformers was further confirmed by calculation of the B3LYP/6-31G(d) harmonic vibrational frequencies. Their energies, rotational strengths, and oscillator strengths of the first 60 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311++G(2d,2p) level in a vacuum.23 The ECD spectra were simulated by the overlapping Gaussian function (σ = 0.6 eV),24 and velocity rotatory strengths of the first 12 excited states were adopted (Table S6, Supporting Information). The ECD spectrum was generated by averaging the simulated spectra of the lowest energy conformers according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). The simulated ECD spectrum of RRR-1 was obtained by direct inversion of SSS-1. In the 180−450 nm region (Figure 2), the experimental ECD curves for 1a and 1b showed three main Cotton effects,

Figure 3. Newman projections along the C-7/C-8 and C-7/C-6 bonds for 1 and 2 (red arrow represents a γ-gauche effect).

tentatively assigned as the (2S*, 5S*, 7R*) isomer of 1 and was named evodialone B. Based on their new 3-ethyl-1,1-diisopentyl-4-methylcyclopentane skeletons, a putative biosynthetic pathway toward the formation of evodialones A (1) and B (2) was proposed (Scheme 1). The well-known MPAPs, β-acids, were considered as the precursors. Oxidation of the β-acid would generate the free radical intermediate i, which would be susceptible to a series of intramolecular carbon radical rearrangements to produce the ring contraction intermediate iv.6 Intermediate iv is epoxidized to v, followed by the acidic epoxy ring-opening to generate intermediate vi. The Michael addition26 of H2O to vi, followed by the loss of H+, would generate the pyran ring containing intermediate vii, which on further oxidation and methylation would afford 1 and 2. As the literature suggested that some PACs could inhibit the growth of beer-spoilage bacterium and play an important role in beer preservation,12 compounds 1a, 1b, and 2 were tested for antimicrobial activity against 13 microorganisms in vitro, including fungi and Gram-positive and Gram-negative bacteria (for the details on antimicrobial testing, see Supporting Information). The results showed that all the compounds were inactive to bacteria (MICs > 200 μM, Table S7, Supporting Information), while 1a and 1b inhibited fungi strains Candida parapsilosis ATCC 22019, C. glabrata ATCC 2001, C. guilliermondii ATCC 6260, and C. lusitaniae ATCC 34449 with MICs of 17.1−68.3 μM (Table 2). PACs are a group of rare natural products mainly found in hops. In the current study, two unusual PAC scalemic mixtures, evodialones A (1) and B (2), featuring a new 3-ethyl-1,1diisopentyl-4-methylcyclopentane skeleton, were isolated from another plant source (E. lepta). Chiral-phase resolution of 1 led to a pair of enantiomers, 1a and 1b, while evodialone B (2) was nonresolvable. The antimicrobial activities of 1a, 1b, and 2 were evaluated against a series of bacterial and fungal strains in vitro, and 1a and 1b showed moderate activities against some of the pathogenic fungi with MICs values ranging from 17.1 to 68.3 μM. This study not only expands the structural diversity of the PAC categories but also provides scientific evidence for explaining the antifungal efficacy of this plant in folk medicine.

Figure 2. Experimental ECD curves of 1a (solid black line) and 1b (solid red line) and B3LYP/6-311++G(2d,2p)//B3LYP/6-31G(d)calculated ECD spectra of SSS-1 (dashed black line) and RRR-1 (dashed red line).

which matched well with the computed curves for SSS-1 and RRR-1, respectively. Therefore, qualitative analysis of the calculated and experimental ECD spectra allowed the assignments of the absolute configuration of 1a as (2S, 5S, 7S) and 1b as (2R, 5R, 7R). Evodialone B (2), a colorless oil, had the same molecular formula, C19H26O7, as that of 1, indicating that it was an isomer of 1. The NMR data of 2 were similar to those of 1, except for the deshielded C-5 signal (δC 52.7 in 2; δC 49.7 in 1) and the shielded C-10 signal (δC 18.7 in 2, δC 25.4 in 1). This implied that the relative configuration of C-7 in 2 was changed, leading to different γ-gauche effects25 on C-5 and C-10 as compared with those of 1 (Figure 3). This was supported by NOE correlations of H-7/H2-11 and 2-OH. The flat ECD spectrum and small specific rotation value revealed that 2 was also a scalemic mixture. However, chiral-phase resolution using different conditions failed. Thus, the structure of 2 was

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EXPERIMENTAL SECTION

General Experimental Procedures. The instruments and materials for the isolation of the compounds from the plant and for the spectroscopic measurements of the purified compounds are presented in the Supporting Information. Plant Material. The aerial parts of E. lepta employed in this work were obtained from Yunnan Province, P. R. China, in July 2016, and were identified by You-Kai Xu, Xishuangbanna Tropical Botanical 1485

DOI: 10.1021/acs.jnatprod.7b00993 J. Nat. Prod. 2018, 81, 1483−1487

Journal of Natural Products

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Scheme 1. Putative Biosynthetic Pathway toward the Formation of 1 and 2

Table 2. Minimal Inhibitory Concentrations (MICs, μM) of 1a, 1b, and 2 against Pathogenic Fungi

Antibacterial Testing. The microbial strains used in the tests were obtained from ATCC. These strains included four Gram-positive bacteria strains (Staphylococcus epidermidis ATCC 12228, S. aureus ATCC 25923, Bacillus subtilis ATCC 21332, and Enterococcus faecalis ATCC 29212), three Gram-negtive bacteria strains (Escherichia coli ATCC 25922, Klebsiellar pneumoniae ATCC 700603, and Enterobacter cloacae ATCC 700323), and six fungi strains (Candida glabrata ATCC 2001, C. parapsilosis ATCC 22019, C. guilliermondii ATCC 6260, C. lusitaniae ATCC 34449, C. krusei ATCC 6258, and C. albicans ATCC 60193). The protocols for the microorganism cultures and compound assay were similar to those described previously.27 More details about the experimental procedures are provided in the Supporting Information.

compound strain C. C. C. C. C. C. a

parapsilosis ATCC 22019 glabrata ATCC 2001 guilliermondii ATCC 6260 lusitaniae ATCC 34449 kruseii ATCC 6258 albicans ATCC 60193

1a

1b

2

amphotericin Ba

17.1 34.2 34.2 68.3 136.6 136.6

17.1 34.2 68.3 68.3 136.6 136.6

>200 >200 >200 >200 >200 >200