LC-MS-Guided Isolation of Insulin-Secretion ... - ACS Publications

Article Cite This: J. Nat. Prod. 2018, 81, 2371−2380

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LC-MS-Guided Isolation of Insulin-Secretion-Promoting Monoterpenoid Carbazole Alkaloids from Murraya microphylla Xiao-Li Ma,† Jun Li,‡ Jiao Zheng,‡ Xiao-Pan Gu,‡ Daneel Ferreira,§ Jordan K. Zjawiony,§ Ming-Bo Zhao,† Xiao-Yu Guo,† Peng-Fei Tu,† and Yong Jiang*,†

J. Nat. Prod. 2018.81:2371-2380. Downloaded from pubs.acs.org by UNIV OF GOTHENBURG on 12/01/18. For personal use only.

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State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China § Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *

ABSTRACT: Fifteen new structurally unique monoterpenoid carbazole alkaloids, including two pairs of epimers (1/2 and 3/4), three pairs of enantiomers (6a/6b, 7a/7b, and 8a/8b), and five optically pure analogues (5, 9−12), were obtained from a 95% aqueous EtOH extract of Murraya microphylla by a combination of bioassay- and LC-MS-guided fractionation procedures. Their structures were established based on NMR and HRESIMS data interpretation. The absolute configuration of compound 1 was determined via X-ray crystallographic data analysis and for all compounds by comparison of experimental and calculated ECD data. Compounds 1−5 were assigned as five new thujane−carbazole alkaloids, and compounds 6−12 as 10 new menthene−carbazole alkaloids linked through an ether or carbon−carbon bond. Compounds 1−12 promoted insulin secretion in the HIT-T15 cell line, 1.9−3.1-fold higher than the gliclazide control at 100 μM.

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As part of a search for antidiabetic natural products from medicinal herbs, the CH2Cl2 fraction of the 95% aqueous EtOH extract of M. microphylla was found to promote insulin secretion using a HIT-T15 cell line (2.6-fold more than the control at 100 μg/mL). Bioassay-guided fractionation of the CH2Cl2 fraction afforded a more active subfraction (3.9-fold more than the control at 100 μg/mL), containing a variety of monoterpenoid carbazole alkaloids as revealed by LC-MS analysis. Further neutral loss screening of 136.0 Da (C10H16) and LCMS-guided separation led to the isolation of 15 structurally unique monoterpenoid prenylcarbazole alkaloids. The structures of the isolates were deduced by NMR and HRESIMS data interpretation, and the absolute configurations were established by X-ray crystallographic data analysis (for compound 1) and by comparing the calculated and experimental electronic circular

he prevalence of diabetes mellitus has significantly increased worldwide over the past three decades. In order to search for potential antidiabetic drugs, more and more scientists are shifting their focus to natural products.1,2 Carbazole alkaloids, consisting of two six-membered benzene rings fused on either side of a five-membered nitrogen-containing ring,3 have been found to have antidiabetic properties via multiple underlying mechanisms.4,5 Carbazole alkaloids have been isolated predominantly from four taxonomically related plants of the genera Clausena, Glycosmis, Micromelum, and Murraya (Rutaceae),3,6−8 with Murraya species being the most common source of this type of compound. Murraya microphylla (Merr. et Chun) Swingle is a shrub distributed across the coastal regions of the Hainan Province of the People’s Republic of China. In consideration of a close taxonomic relationship with Murraya koenigii (L.) Spreng,9 M. microphylla is suspected to contain abundant carbazole alkaloids, and several investigations have confirmed their occurrence.10,11 © 2018 American Chemical Society and American Society of Pharmacognosy

Received: May 4, 2018 Published: November 1, 2018 2371

DOI: 10.1021/acs.jnatprod.8b00338 J. Nat. Prod. 2018, 81, 2371−2380

Journal of Natural Products

Article

Chart 1

Figure 1. Key HMBC (arrows) and 1H−1H COSY (bold lines) correlations of 1/2, 5, and 6.

5.72 (1H, d, J = 9.6 Hz), 1.46 (3H, s), and 1.45 (3H, s), and a set of aliphatic protons, including those for an isopropyl group at δΗ 1.60 (1H, q, J = 6.8 Hz), 1.10 (3H, d, J = 6.8 Hz), and 1.05 (3H, d, J = 6.8 Hz) and a methyl singlet at δΗ 1.51 (3H, s). In the 13 C NMR data of 1, 29 carbon resonances, comprising five sp2 quaternary, three oxygenated sp2 tertiary (δC 149.1, 144.4, 143.7), two nitrogenated sp2 tertiary, two sp3 quaternary, an oxygenated sp3 tertiary (δC 76.2), three sp3 methylene, two sp3 methine, four sp2 methine, and seven methyl carbons, were observed. The above data indicated 1 to be a derivative of a pyrano[3,2a]carbazole12 substituted by a 10-carbon moiety. The structure of the carbazole moiety was similar to koenigine9 except for the absence of H-8 in 1, while the 10-carbon substituent was similar to thujane13 except for the lack of H-1″ in 1. The HMBC and 1 H−1H COSY spectra (Figure 1) confirmed the above deductions. The HMBC correlations of H3-7″ with C-8 suggested that the thujane unit is attached to C-8 of the carbazole unit. Therefore, the 2D structure of microphyline A (1) was defined as depicted. In the NOESY spectrum, the correlations of H-2″α/H-6″α and H3-7″/H-2″β (Figure 2) indicated a cis-configuration of the thujane unit,14,15 implying that the methyl and isopropyl groups are cofacial. The calculated ECD curve of (1″S,4″S,5″R)-1 was well-matched with the experimental data [(1″S,4″S,5″R) is labeled as (8″S,13″S,12″R) in the crystal structure] (Figure 3). Therefore, its absolute configuration was defined as (1″S,4″S,5″R)

dichroism (ECD) data (for all compounds). Herein, the isolation, structure characterization, and insulin-secretion-promoting activities of the new alkaloids are reported.

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RESULTS AND DISCUSSION Compounds 1 and 6 were obtained from the active subfraction mentioned above. Based on the mass fragmentation analysis of 1 and 6, the neutral loss of 136.0 Da (C10H16) in the positive-ion MS was found to be a characteristic fragment for the monoterpene unit (Figure S1, Supporting Information). Thus, a screening of the neutral loss of 136.0 Da was adopted for the LC-MS-guided fractionation, which afforded a further 10 analogues (2−5 and 7−12). Microphyline A (1) was isolated as colorless needles (mp 180−182 °C), [α]25 D −87 (c 0.1, MeOH). The protonated molecular ion at m/z 446.2673 [M + H]+ (calcd for C29H36NO3, 446.2695) in the HRESIMS together with the 13C NMR spectroscopic data supported a molecular formula of C29H35NO3, implying 13 degrees of unsaturation. Its UV spectrum exhibited typical absorptions at λmax 230, 300, and 357 nm, indicating the presence of a pyrano[3,2-a]carbazole moiety in 1.12 The 1 H NMR spectrum displayed two proton signals at δΗ 9.31 (1H, br s) and 7.12 (1H, br s), two phenyl singlets at δΗ 7.58 (1H, s) and 7.43 (1H, s), a methoxy group singlet at δΗ 3.94 (3H, s), and a methyl singlet at δΗ 2.29 (3H, s), a set of 2,2dimethyl-2H-pyran moiety signals at δΗ 6.86 (1H, d, J = 9.6 Hz), 2372



Article

Figure 2. Key NOE correlations of 1, 2, 6, and 10.

Microphyline B (3) was obtained as an amorphous powder. The 13C NMR and HRESIMS (m/z 416.2578 [M + H]+, calcd for C28H34NO2, 416.2590) data were used to establish its molecular formula as C28H33NO2, with 30 mass units less than 1. The NMR data of 3 were similar to those of 1, except for the lack of signals due to a C-6 methoxy group. This deduction was supported based on the shielded C-6 signal (δC 110.8; ΔδC −32.9) in 3 compared with 1, as well as the presence of two ortho-coupled phenyl protons [δH 7.59 (1H, d, J = 8.4 Hz) and 6.74 (1H, d, J = 8.4 Hz)]. The similar NOE correlations of 1 and 3 revealed these two compounds have the same relative configurations. The comparable sign and value of the specific rotations of 3 and 1, as well as the ECD spectroscopic analysis (Figure S4, Supporting Information), were used to define the absolute configuration of 3 as (1″S,4″S,5″R). Epimicrophyline B (4) gave an identical molecular formula of C28H33NO2 to 3, as deduced from the HRESIMS (m/z 414.2429 [M − H]−, calcd for C28H32NO2, 414.2433). From the NMR data, 4 could be assigned as an epimer of 3 differing in the C-1 configuration of the thujane unit. The similarity of the ECD Cotton effects (Figure S5, Supporting Information) and NOE correlations to those of 2 suggested that they share the same absolute configuration (1″R,4″S,5″R). Thus, the structure of epimicrophyline B (4) was deduced as shown. Microphyline C (5) was obtained as an amorphous powder, [α]25 D −67 (c 0.1, MeOH). Its deprotonated ion peak at m/z 482.3060 [M − H]− in the negative-ion HRESIMS was used to determine the molecular formula of 5 as C33H41NO2, along with its 13C NMR data. The NMR data of 5 were comparable to those of 3, apart from containing an isopentenyl group [δH 5.13 (1H, t, J = 7.2 Hz), 2.18 (1H, dt, J = 7.2, 8.4 Hz), 1.63 (3H, s), and 1.57 (3H, s); δC 131.9, 125.2, 25.8, 23.5, and 17.6] in 5. This group was located at C-6′ from the HMBC correlations between H2-6′ and C-3′/C-8′ and between H2-7′ and C-2′/C-8′/C-9′ (Figure 1). The relative configurations of C-1″, C-4″, and C-5” were assigned

Figure 3. Experimental and calculated ECD spectra of 1 (in MeOH).

and supported by X-ray crystallographic analysis [Flack parameter of −0.05(6)] (Figure 4). Epimicrophyline A (2) was obtained as an amorphous powder, [α]25 D +34 (c 0.1, MeOH). Its molecular formula (C29H35NO3) was identical with 1, as defined from the HRESIMS (m/z 446.2683 [M + H]+, calcd for C29H36NO3, 446.2695) and 13C NMR data. Slight differences in the 1D NMR data of 2 compared to 1 suggested they could be stereoisomers. The HSQC, HMBC, and 1H−1H COSY spectroscopic analysis facilitated the assembly of the structure of 2, which was similar to 1. The NOE correlations of H3-7″ and H-2″α, H3-7″ and H-6″α, and H-6″β and H3-9″ (Figure 2) indicated a trans-configuration of the thujane unit in 2. The absolute configuration of epimicrophyline A (2) was established as (1″R,4″S,5″R) by ECD spectroscopic analysis (Figure S2, Supporting Information). 2373



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Figure 4. ORTEP drawings of the X-ray structures of 1 and 6.

as the same as those of 1 and 3, as deduced from the NOESY data (Figure S6, Supporting Information). The analysis of the ECD data supported the absolute configuration of 5 as (1″S,4″S,5″R) (Figure S7, Supporting Information). The weak positive Cotton effect (inflection) observed near 300 nm was found to be reminiscent of the 1Lb electronic transition of the (2′R)-chromene moiety.16 Hence, microphyline C (5) was characterized as depicted. Microphyline D (6) was isolated as colorless needles (mp 175−176 °C) in an optically active form, with [α]25 D +51 (c 0.1, MeOH). Its molecular formula was deduced as C29H35NO3, based on the 13C NMR data and the positive-ion HRESIMS (m/z 446.2674 [M + H]+, calcd for C29H36NO3, 446.2695). The NMR spectroscopic data (Table 2) of 6 were similar to those of koenigine,9 except for the presence of a monoterpene unit in 6.17 This unit was defined as 1-p-menthene based on the 1H−1H COSY correlations of H-2″/H2-3″, H2-5″/H2-6″, and H-7″/ H3-8″/H3-9″ and the HMBC correlations from H-7″ to C-3″, C-4″, C-5″, C-8″, and C-9″; from H3-8″ to C-4″, C-7″, and C-9″; and from H3-10″ to C-1″, C-2″, and C-6″ (Figure 1). Moreover, the 1-p-menthene group was deduced to be linked with the koenigine moiety via a (C-4″−O−C-7)-ether bond based on the deshielded C-7 (δC 144.1) and C-4″ (δC 84.3) resonances. X-ray crystallographic data analysis confirmed the 2D structure of 6 (Figure 4), but the crystal structure data suggested 6 to be a scalemic mixture. Subsequent chiral-phase HPLC resolution gave a pair of enantiomers, (−)-6a and (+)-6b, in a 1:3.6 ratio (Figure 5A). These two compounds were found to possess antithetical specific rotations and ECD curves (Figure 5B), and their absolute configurations were determined as (4″S) and (4″R), respectively. Microphyline E (7) was purified as an amorphous powder. It showed a deprotonated molecular ion at m/z 414.2436 [M − H]− in the HRESIMS and indicated a molecular formula of C28H33NO2, with 30 mass units less than 6. The main difference with 6 was the absence of a C-6 methoxy group in 7. This was deduced on the basis of an ABX spin system at δH 6.76 (1H, dd, J = 8.4, 2.0 Hz), 7.01 (1H, d, J = 2.0 Hz), and 7.76 (1H, d, J = 8.4 Hz) in the 1H NMR spectrum and the HMBC correlations of H-5 with C-4a, C-8a, and C-7; H-6 with C-8 and C-4b; and H-8 with C-4b and C-6 (Figure S3, Supporting Information). Compound 7 was also obtained as a scalemic mixture and was separated by chiral-phase HPLC to give the enantiomers (−)-7a and (+)-7b in a 1:3.5 ratio (Figure S8A, Supporting Information). Based on their specific rotations and experimental and calculated ECD data, the absolute configurations of (−)-microphyline E (7a) and (+)-microphyline

E (7b) were established as (4″S) and (4″R) (Figure S8B, Supporting Information), respectively. Microphyline F (8) was isolated as an amorphous powder. A deprotonated molecular ion at m/z 482.3058 [M − H]− in the HRESIMS was used to assign its molecular formula as C33H41NO2. NMR data analysis indicated that 8 has a similar skeleton to 7, except for the presence of a set of resonances for an isopentenyl group [δH 5.13 (1H, t, J = 7.2 Hz), 2.20 (1H, dt, J = 8.4, 7.2 Hz), 1.63 (3H, s), and 1.56 (3H, s); δC 131.9, 125.2, 25.8, 23.5, and 17.6] in 8. The HMBC correlations from H2-6′ to C-3′ and C-8′ and from H2-7′ to C-2′, C-8′, and C-9′ indicated that the isopentenyl group is linked at C-6′. The similarity between their structures of 6, 7, and 8 suggested 8 could also be a scalemic mixture. This was then proven by obtaining a pair of enantiomers of (−)-8a and (+)-8b in a 1:3.4 ratio after chiralphase HPLC resolution (Figure S9A, Supporting Information). (−)-Microphyline F (8a) and (+)-microphyline F (8b) were deduced to have a (4″S)- and (4″R)-configuration, respectively, from their ECD data. Comparable to microphyline C (5), the ECD spectrum of 8b showed a positive Cotton effect at around 300 nm based on the 1Lb electronic transition of the (2′R)chromene moiety (Figure S9B, Supporting Information).16 Accordingly, a (2′S)-configuration was assigned for 8b from the mirror-like ECD spectrum at around 300 nm, as compared with that of 8a. Microphyline G (9) was isolated as an amorphous powder. The HRESIMS showed a deprotonated molecular ion at m/z 444.2536 [M − H]−, suggesting a molecular formula of C29H35NO3. Compound 9 was deduced to have a close structural resemblance with microphyline D (6) based on a comparison of their NMR data. The main difference is that the 1-p-menthene and koenigine moieties are linked via a (C-7−O− C-7″)-ether bond in 9, as deduced by the 1H−1H COSY correlations of H-2″/H2-3″/H3-10″, H-4″/H2-5″, and H2-5″/ H2-6″, as well as the HMBC correlations from H-8″ to C-4′, C-7″, and C-9″ and from H-9″ to C-4″, C-7″, and C-8” (Figure S3, Supporting Information). The absolute configuration of 9 was determined as (4″S) by comparing the experimental and calculated ECD curves (Figure S10, Supporting Information). Microphyline H (10) showed an identical molecular formula of C29H35NO3 to 9, as deduced by the 13C NMR and HRESIMS data. Comparison of the NMR data of 10 (Table 3) with those of 9 indicated the linkage between the koenigine and 1-p-menthene units is via a C-8−C-3″ bond based on the HMBC correlations of H-2″/C-8 (Figure S3, Supporting Information). The NOE correlations of H-3″/H-5″β/H-7″ and H-4″/H-6″α (Figure 2) were consistent with a trans-configured C-3″−C-4″ bond. Comparison of experimental and calculated ECD spectra 2374



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Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of 1−5 in Acetone-d6 (δH in ppm, J in Hz) 1 position 1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 1″ 2″α 2″β 3″α 3″β 4″ 5″ 6″α 6″β 7″ 8″ 9″ 10″ CH3-3 OCH3-6 OH NH

δH (J in Hz)

7.58 s

7.43 s

5.72 d (9.6) 6.86 d (9.6) 1.45 s 1.46 s

2 δC, type 105.2, C 149.1, C 117.6, C 120.5, CH 116.6, C 118.1, C 100.0, CH 143.7, C 144.4, C 118.6, C 133.5, C 135.3, C 76.2, C 129.5, CH 118.8, CH 27.8, CH3 27.9, CH3

δH (J in Hz)

7.57 s

7.46 s

5.72 d (9.6) 6.91 d (9.6) 1.44 s 1.44 s

3 δC, type

δH (J in Hz)

4 δC, type

105.5, C 149.2, C 118.2, C 120.4, CH 7.56 s 116.7, C 118.0, C 100.1, CH 7.59 d (8.4) 143.8, C 6.74 d (8.4) 143.9, C 118.0, C 134.2, C 135.3, C

105.2, C 149.3, C 117.8, C 120.5, CH 117.8, C 118.8, C 117.8, CH 110.8, CH 154.6, C 117.8, C 138.9, C 134.6, C

76.1, C 129.5, CH 118.9, CH 27.8, CH3 27.8, CH3

76.2, C 129.7, CH 118.8, CH 27.8, CH3 27.9, CH3

5.74 d (9.6) 6.87 d (9.6) 1.46 s 1.46 s

δH (J in Hz)

7.55 s

7.60 d (8.4) 6.74 d (8.4)

5.73 d (9.6) 6.90 d (9.6) 1.44 s 1.44 s

5 δC, type 105.4, C 149.3, C 118.2, C 120.4, CH 119.1, C 117.8, C 117.8, CH 111.6, CH 153.9, C 117.7, C 140.5, C 135.4, C 76.1, C 129.6, CH 118.8, CH 27.8, CH3 27.8, CH3

δH (J in Hz)

7.54 s

7.57 d (8.4) 6.72 d (8.4)

5.71 d (9.6) 6.89 d (9.6) 1.43 s 1.75 t (8.4) 2.18 dt (8.4, 7.2) 5.13 t (7.2) 1.63 s 1.57 s

1.54 m 2.71 dd (8.4, 13.2) 1.64 m 1.85 ddd (8.4, 11.2, 11.2) 1.98 dd (4.0, 7.6) 0.75 dd (4.4, 7.6) 0.68 dd (4.0, 4.4) 1.51 s 1.60 q (6.8) 1.10 d (6.8) 1.05 d (6.8) 2.29 s 3.94 s 7.12 br s 9.31 br s

47.7, C 35.5, CH2

24.9, CH2

36.2, C 35.2, CH 15.0, CH2

26.3, CH3 33.7, CH 19.9, CH3 20.6, CH3 16.3, CH3 57.0, CH3

1.37 m 2.73 ddd (4.2, 4.2, 7.8) 1.67 m 1.70 m

2.30 dd (4.0, 8.0) 0.58 dd (4.0, 8.0) 0.43 dd (4.0, 4.8) 1.66 s 1.71 q (6.8) 0.94 d (6.8) 0.85 d (6.8) 2.26 s 3.96 s 7.27 br s 8.84 br s

47.6, C 35.7, CH2

29.8, CH2

36.1, C 34.1, CH 10.3, CH2

25.3, CH3 32.1, CH 20.5, CH3 20.6, CH3 16.2, CH3 57.0, CH3

1.54 m 2.72 dd (8.0, 13.2) 1.64 m 1.85 dd (8.0, 12.0, 12.0) 1.99 dd (4.0, 7.6) 0.77 dd (4.4, 7.6) 0.71 dd (4.4, 4.0) 1.52 s 1.60 q (6.8) 1.11 d (6.8) 1.06 d (6.8) 2.28 s 8.11 br s 9.47 br s

47.6, C 35.7, CH2

24.9, CH2

36.4, C 35.4, CH 15.0, CH2

26.4, CH3 33.8, CH 19.9, CH3 20.6, CH3 16.2, CH3

1.37 m 2.71 ddd (3.2, 3.2, 8.0) 1.67 m 1.70 m

2.30 dd (3.2, 8.0) 0.58 dd (8.0, 4.8) 0.43 dd (4.0, 4.8) 1.65 s 1.71 q (6.8) 0.94 d (6.4) 0.83 d (6.4) 2.26 s 8.16 br s 8.93 br s

47.4, C 35.8, CH2

29.8, CH2

36.1, C 34.3, CH 10.3, CH2

25.5, CH3 32.1, CH 20.5, CH3 20.6, CH3 16.2, CH3

1.55 m 2.71 dd (8.0, 13.2) 1.63 m 1.84 dd (8.0, 13.2) 1.97 dd (4.0, 7.6) 0.76 dd (4.4, 7.6) 0.69 dd (4.0, 4.4) 1.51 s 1.60 q (6.8) 1.11 d (6.8) 1.05 d (6.8) 2.28 s

δC, type 105.0, C 149.3, C 117.6, C 120.6, CH 117.7, C 118.8, C 117.7, CH 110.7, CH 154.6, C 117.6, C 139.8, C 135.5, C 78.6, C 128.9, CH 119.1, CH 26.2, CH3 41.5, CH2 23.5, CH2 125.2, CH 131.9, C 17.6, CH3 25.8, CH3 47.6, C 35.7, CH2

24.9, CH2

36.4, C 35.4, CH 15.0, CH2

26.4, CH3 33.8, CH 19.9, CH3 20.6, CH3 16.2, CH3

8.08 br s 9.45 br s

[M − H]− and the 13C NMR data. By comparing the NMR data of 12 with those of 11, a difference was found in that an isopentenyl group [δH 5.13 (1H, t, J = 7.2 Hz), 2.06 (1H, dt, J = 8.4, 7.2 Hz), 1.63 (3H, s), and 1.56 (3H, s); δC 131.9, 125.2, 25.8, 23.5, and 17.6] occurs in 12. The isopentenyl group was determined to be linked to C-6′ based on the correlations of H-6′ with C-3′/C-8′ and of H-7′ with C-2′/C-8′/C-9′, as observed in the HMBC spectrum (Figure S3, Supporting Information). Compound 12 was assigned the same (3″S,4″S) absolute configuration as 10 and 11 from their similar specific rotations, NOE correlations, and ECD spectra (Figure S13, Supporting Information). In the ECD spectrum, a positive Cotton effect (inflection) near 300 nm permitted the assignment of a (2′R) absolute configuration in 12, as in 8a.16

(Figure S11, Supporting Information) permitted assignment of the absolute configuration of 10 as (3″S,4″S). Microphyline I (11) was purified as an amorphous powder. Its molecular formula was assigned as C28H33NO2 based on the 13C NMR and HRESIMS data. The NMR data of 11 (Table 3) showed a close resemblance to those of 10, indicating their comparable structural scaffolds. The main difference found involved the absence of the C-6 methoxy group in 11, as deduced by 2D NMR spectroscopic data analysis (Figure S3, Supporting Information). The similar NOE correlations and ECD data of 11 (Figure S12, Supporting Information) compared to 10 suggested its absolute configuration as (3″S,4″S). The molecular formula of microphyline J (12) was determined as C33H41NO2 by the HRESIMS ion at m/z 482.3070 2375



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Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data of 6−9 in Acetone-d6 (δH in ppm, J in Hz) 6 position 1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 1″ 2″ 3″α 3″β 4″ 5″α 5″β 6″α 6″β 7″ 8″ 9″ 10″ CH3-3 OCH3-6 NH

δH (J in Hz)

7.62 s

7.51 s

7.04 s

5.74 d (9.6) 6.86 d (9.6) 1.45 s 1.45 s

7 δC, type 104.0, C 149.7, C 117.9, C 121.3, CH 118.0, C 119.4, C 107.8, CH 149.9, C 144.1, C 104.0, CH 135.4, C 136.7, C 76.4, C 129.7, CH 118.7, CH 27.9, CH3 27.9, CH3

δH (J in Hz)

7.62 s

7.76 d (8.4) 6.76 dd (8.4, 2.0) 7.01 d (2.0)

5.75 d (9.6) 6.89 d (9.6) 1.45 s 1.45 s

8 δC, type 105.5, C 149.8, C 117.6, C 121.3, CH 118.1, C 120.1, C 119.5, CH 116.5, CH 154.0, C 106.2, CH 141.7, C 136.5, C 76.4, C 129.8, CH 118.6, CH 27.8, CH3 27.8, CH3

δH (J in Hz)

7.62 s

7.76 d (8.4) 6.76 dd (8.4, 2.0) 7.01 d (2.0)

5.74 d (9.6) 6.92 d (9.6) 1.43 s 1.76 t (8.4) 2.20 dt (8.4, 7.2) 5.13 t (7.2) 1.63 s 1.56 s

5.19 br s 2.20 dd (4.0, 8.6) 2.28 m 1.92 m 2.16 dd (3.6, 12.6) 1.98 m 2.12 m 2.17 q (6.8) 1.13 d (6.8) 1.08 d (6.8) 1.60 br s 2.27 s 3.84 s 9.89 br s

133.9, C 120.0, CH 32.1, CH2 84.3, C 30.0, CH2 29.6, CH2 32.9, CH 17.6, CH3 17.7, CH3 23.1, CH3 16.2, CH3 57.0, CH3

5.22 br s 2.10 dd (4.0, 8.6) 2.19 m 1.96 m 2.24 ddd (3.6, 12.6) 1.79 m 2.12 m 2.20 q (6.8) 1.11 d (6.8) 1.01 d (6.8) 1.66 br s 2.28 s

134.1, C 119.6, CH 31.7, CH2 83.3, C 28.9, CH2 28.7, CH2 33.6, CH 17.7, CH3 17.9, CH3 23.3, CH3 16.2, CH3

10.09 br s

5.23 br s 2.10 dd (4.0, 8.6) 2.19 m 1.96 m 2.24 ddd (3.6, 12.6) 1.79 m 2.12 m 2.20 q (6.8) 1.12 d (6.8) 1.02 d (6.8) 1.66 br s 2.30 s 10.03 br s

9 δC, type 105.3, C 149.9, C 117.6, C 121.4, CH 117.6, C 120.1, C 119.6, CH 116.5, CH 154.1, C 106.2, CH 141.8, C 136.6, C 78.8, C 129.0, CH 119.0, CH 26.2, CH3 41.6, CH2 23.5, CH2 125.2, CH 131.9, C 17.6, CH3 25.8, CH3 134.2, C 119.6, CH 31.8, CH2 83.4, C 29.1, CH2 28.8, CH2 33.7, CH 17.7, CH3 18.0, CH3 23.4, CH3 16.2, CH3

δH (J in Hz)

105.5, C 149.8, C 117.9, C 121.3, CH 118.0, C 119.9, C 108.6, CH 150.1, C 144.3, C 104.1, CH 135.5, C 136.8, C

7.65 s

7.05 s

7.56 s

5.76 d (9.6) 6.88 d (9.6) 1.45 s 1.45 s

5.44 br s 2.04 m 2.20 m 1.90 m 2.02 m 2.10 dd (2.0, 10.0) 1.41 ddd (2.0, 8.0, 10.0) 2.21 m 1.24 s 1.24 s 1.68 br s 2.29 s 3.88 s 9.96 br s

δC, type

76.4, C 129.8, CH 118.7, CH 27.9, CH3 27.9, CH3

134.3, C 121.9, CH 27.9, CH2 45.5, CH 31.9, CH2 25.0, CH2 84.3, C 17.6, CH3 23.3, CH3 24.2, CH3 16.2, CH3 56.9, CH3

Figure 5. Chiral HPLC separation (A) and comparison of the experimental ECD spectra of 6a/6b in MeOH with the calculated ECD spectra of (4″S)-6a and (4″R)-6b (B). 2376



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Table 3. 1H (400 MHz) and 13C (100 MHz) NMR Data of 10−12 in Acetone-d6 (δH in ppm, J in Hz) 10 position 1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2 3′ 4 5′ 6′ 7′ 8′ 9′ 10′ 11′ 1″ 2″ 3″ 4″ 5″α 5″β 6″α 6″β 7″ 8″ 9″ 10″ CH3-3 OCH3-6 OH NH

δH (J in Hz)

7.56 s

7.42 s

5.73 d (9.6) 6.71 d (9.6) 1.44 s 1.44 s

11 δC, type 105.4, C 149.1, C 117.9, C 120.7, CH 116.1, C 118.5, C 100.3, CH 143.5, C 144.1, C 114.2, C 134.9, C 135.8, C 76.3, C 129.8, CH 118.4, CH 27.8, CH3 27.9, CH3

δH (J in Hz)

12 δC, type 105.4, C 149.3, C 118.0, C 120.8, CH 118.2, C 118.2, C 118.2, CH 105.4, CH 153.9, C 113.9, C 141.0, C 135.7, C

7.55 s

7.59 d (8.4) 6.73 d (8.4)

76.3, C 129.9, CH 118.4, CH 27.8, CH3 27.9, CH3

5.74 d (9.6) 6.73 d (9.6) 1.44 s 1.44 s

δH (J in Hz)

105.2, C 149.4, C 117.9, C 120.8, CH 117.7, C 118.7, C 118.2, CH 109.8, CH 154.0, C 119.2, C 140.4, C 135.7, C

7.55 s

7.59 d (8.4) 6.73 d (8.4)

5.73 d (9.6) 6.74 d (9.6) 1.42 s 1.75 t (8.4) 2.06 dt (8.4, 7.2) 5.13 t (7.2) 1.63 s 1.56 s

5.45 br s 4.08 d (8.0) 2.10 m 2.11 dd (5.2, 10.0) 1.46 m 2.38 m 2.15 m 1.62 dq (7.2, 7.2) 0.90 d (7.2) 0.79 d (7.2) 1.80 br s 2.26 s 3.94 s 7.21 br s 8.53 br s

135.5, C 126.9, CH 38.3, CH 43.6, CH 23.4, CH2 31.5, CH2 28.9, CH 16.9, CH3 21.9, CH3 23.6, CH3 16.2, CH3 56.9, CH3

5.47 br s 4.07 d (8.0) 2.10 m 2.11 dd (5.2, 10.0) 1.46 m 2.38 m 2.15 m 1.62 dq (7.2, 7.2) 0.90 d (7.2) 0.79 d (7.2) 1.81 br s 2.25 s 7.99 br s 8.62 br s

135.6, C 127.1, CH 38.0, CH 43.9, CH 23.4, CH2 31.5, CH2 28.8, CH 16.9, CH3 21.9, CH3 23.6, CH3 16.2, CH3

δC, type

5.48 br s 4.07 d (8.0) 2.10 m 2.11 dd (5.2, 10.0) 1.46 m 2.38 m 2.15 m 1.62 dq (7.2, 7.2) 0.90 d (7.2) 0.79 d (7.2) 1.82 br s 2.28 s

78.7, C 129.1, CH 118.7, CH 26.2, CH3 41.5, CH2 23.5, CH2 125.2, CH 131.9, C 17.6, CH3 25.8, CH3 135.7, C 127.1, CH 38.0, CH 43.9, CH 23.4, CH2 31.5, CH2 28.8, CH 16.9, CH3 22.0, CH3 23.6, CH3 16.2, CH3

7.94 br s 8.64 br s

test compounds increased insulin secretion by 1.9−3.1-fold in comparison with the control at a concentration of 40 μM. Gliclazide, as a clinically used insulin secretagogue, was adopted as a positive control in this test.22 Figure 6 indicated that most of these monoterpenoid carbazole alkaloids showed a better insulinotropic activity, even at a lower concentration. In addition, the influence on cell proliferation/viability of the isolates was tested by an MTT method. None of these compounds showed cytotoxicity against HIT-T15 cells at a test concentration of 40 μM.

Consequently, the structure of microphyline J (12) was defined as depicted. Compounds 1−5 are thujane−pyranocarbazole alkaloids, while compounds 6−12 are menthene−pyranocarbazole alkaloids. Putative biosynthesis pathways to compounds 1 and 10 are illustrated in Scheme 1. Both 1 and 10 are possibly derived from koenigine, a common carbazole alkaloid from Murraya species, via an electrophilic substitution reaction with monoterpene carbocation intermediates. Koenigine was deduced to be biosynthesized from 3-methylcarbazole by a sequence of oxidation, prenylation, and cyclization procedures.18,19 The terpinyl and thujyl cations were reported to be produced from geranyl diphosphate (GPP) by a series of isomerization, cyclization, hydride shift, and other rearrangements.20 It is generally acknowledged that enzyme-catalyzed reactions are stereoselective;21 thus, the presence of (1″S)-1/3/5 and (1″R)-2/4 indicates that they could be the nonenzymatic products. Compounds 1−12 were evaluated for their insulin-secretionpromoting effects in HIT-T15 cells. As shown in Figure 6, all the

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an X-4 micro melting point apparatus (Beijing Fukai Tech Instruments Co., Beijing, People’s Republic of China). Optical rotations were recorded on a Rudolph Autopol IV automatic polarimeter (Hackettstown, NJ, USA). UV data were measured on a Shimadzu UV 2450 UV spectrophotometer (Tokyo, Japan). ECD data were acquired on a JASCO J-810 CD spectrophotometer (Tokyo, Japan). IR spectra were recorded on a Thermo/Nicolet Nexus 470 FT-IR 2377



Article

Scheme 1. Putative Biosynthetic Pathways to 1 and 10

specimen (no. MM201507) was deposited at the Modern Research Center for Traditional Chinese Medicine, Peking University. Extraction and Isolation. Air-dried leaves and stems of M. microphylla (16 kg) were powdered and extracted under reflux with 95% aqueous EtOH (2 × 160 L). After evaporation of the organic solvents, the extract (730 g) was suspended in H2O and fractionated with CH2Cl2 and n-BuOH, successively. The CH2Cl2 fraction (410 g) was isolated into eight fractions (A−H) on a silica gel column that was eluted with a stepwise gradient of petroleum ether−acetone (98:2 to 40:60, v/v). Then an LC-MS-guided separation was carried out to search for compounds with the characteristic neutral loss of 136.0 Da. Fraction B (55 g), containing the target monoterpene carbazole alkaloids, was chromatographed on silica gel CC and eluted with petroleum ether−CH2Cl2 (98:2 to 80:20, v/v), and five subfractions (B1−B5) were produced. Subfraction B3 (11.2 g) was separated by Sephadex LH-20 CC and eluted with MeOH−CH2Cl2 (1:1, v/v) to give three fractions (B3a−B3c). Fraction B3b (1.2 g) was subjected to silica gel CC eluting with petroleum ether−acetone (98:2 to 90:10, v/v) and further chromatographed on a silica gel plate GF254 with petroleum ether−acetone (98:2) as developing solvent to give 1 (12.0 mg) and 6 (4.0 mg). Fraction B3b2 (430 mg) was purified by semipreparative HPLC [CH3CN−H2O (85:15), 3.0 mL/min] to yield 8 (5.0 mg, tR 17.8 min) and 11 (4.0 mg, tR 20.1 min). Fraction B5 (10.9 g) was separated subsequently on Sephadex LH-20 CC (MeOH−CH2Cl2, 1:1, v/v) and MCI (MeOH as eluent) gel columns to yield five fractions (B5b1−B5b5). Fraction B5b4 (430 mg) was initially chromatographed on semipreparative HPLC [CH3CN−H2O (75:25), 3.0 mL/min], to give seven subfractions (B5b4H1−B5b4H7). Further purification of fractions B5b4H3 and B5b4H5−B5b4H7 by semipreparative HPLC [CH3CN−H2O (80:20), 3.0 mL/min] afforded 10 (5.0 mg, tR = 17.6 min), 2 (7.0 mg, tR = 19.5 min), 4 (10.0 mg, tR = 28.4 min), 3 (5.0 mg, tR = 30.6 min), 12 (7.0 mg, tR = 31.2 min), 5 (5.0 mg, tR = 35.2 min), 7 (6.0 mg, tR = 44.1 min), and 9 (5.0 mg, tR = 46.6 min), respectively. Microphyline A (1): colorless needles (acetone−H2O, 9:1); mp 180−182 °C; [α]25 D −87 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.47), 300 (4.36), 357 (4.10) nm; IR (KBr) νmax 3421, 2914, 2848, 1025 cm−1; ECD (MeOH) λmax (Δε) 237 (+22.1), 302 (+2.12), 357

Figure 6. Relative insulin secretion levels of HIT-T15 cells after compounds 1−12 (40 μM) and gliclazide (positive control, 100 μM) treatment (**, p < 0.01), compared with the control. spectrometer (Waltham, MA, USA). 1D and 2D NMR spectra were obtained on a Bruker Avance II 400 NMR spectrometer (Billerica, MA, USA), using solvent residual signals as a reference. HRESIMS data were measured on a Waters Xevo G2 QTOF mass spectrometer (Milford, MA, USA). X-ray crystallographic data were acquired with a MicroMax003 Compact Homelab CCD X-ray single-crystal diffractometer (Rigaku Americas Co., Woodlands, TX, USA) with Cu Kα radiation. LC-MS analysis was carried out on a Waters Acquity UPLC H-Class (Waters Co.) coupled with an AB Sciex Qtrap 4500 mass spectrometer (Foster, CA, USA). Silica gel (200−300 mesh) for column chromatography (CC) and precoated silica gel GF254 plates were purchased from Qingdao Marine Chemical Co. Ltd. (Qingdao, People’s Republic of China). A Zorbax Eclipse XDB-C18 column (10 × 250 mm, 5 μm) was used for semipreparative HPLC on an Agilent 1200 series LC instrument (Palo Alto, CA, USA). Plant Material. The dried leaves and stems of Murraya microphylla were harvested in July 2015 from Lingshui County, Hainan Province, People’s Republic of China, and were identified by P.F.T. A voucher 2378



Article

Microphyline I (11): amorphous powder; [α]25 D +5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 239 (4.45), 298 (4.39), 348 (4.13) nm; IR (KBr) νmax 3439, 2924, 1453 cm−1; ECD (MeOH) λmax (Δε) 230 (+10.7), 258 (+3.3), 287 (+2.12) nm; 1H and 13C NMR spectroscopic data, see Table 3; negative-ion HRESIMS m/z 414.2431 [M − H]− (calcd for C28H32NO2, 414.2433). Microphyline J (12): amorphous powder; [α]25 D +14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 245 (4.47), 294 (4.37), 347 (4.12) nm; IR (KBr) νmax 3726, 3345, 2955, 2921, 1734 cm−1; ECD (MeOH) λmax (Δε) 223 (+25.47), 293 (+1.7), 350 (−1.36) nm; 1H and 13C NMR spectroscopic data, see Table 3; negative-ion HRESIMS m/z 482.3070 [M − H]− (calcd for C33H40NO2, 482.3059). X-ray Crystallographic Analysis. Compounds 1 and 6 were crystallized from acetone−H2O (9:1, v/v). The solved methods for their crystal structures were the same as previously reported.23 The detailed crystallographic data of 1 and 6 are listed in Table S1, Supporting Information, and deposited at Cambridge Crystallographic Data Center [Deposition Nos. CCDC 1573194 (1) and CCDC 1573193 (6)]. ECD Calculations. The ECD calculations for compounds 1−12 were the same as previously reported.24,25 Insulin-Secretion-Promoting Assay. HIT-T15 pancreatic islet cells were provided by Prof. Guan Y. F. (Peking University, Beijing, People’s Republic of China). The procedure for the assay of insulin secretion was the same as previously described, and each experiment was performed three times.26 Gliclazide (100 μM) was utilized as a positive control. A Student t-test was conducted for repeated measurements, and p < 0.05 was statistically significant.

(−5.81) nm; 1H and 13C NMR spectroscopic data, see Table 1; positive-ion HRESIMS m/z 446.2673 [M + H]+ (calcd for C29H36NO3, 446.2695). Epimicrophyline A (2): amorphous powder; [α]25 D +34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 232 (4.45), 300 (4.35), 346 (4.09) nm; IR (KBr) νmax 3489, 2956, 2850, 1721 cm−1; ECD (MeOH) λmax (Δε) 246 (−17.3), 300 (−10.6), 369 (+2.03) nm; 1H and 13 C NMR spectroscopic data, see Table 1; positive-ion HRESIMS m/z 446.2683 [M + H]+ (calcd for C29H36NO3, 446.2695). Microphyline B (3): amorphous powder; [α]25 D −62 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (4.48), 295 (4.37), 347 (4.11) nm; IR (KBr) νmax 3478, 2922, 2867, 1019 cm−1; ECD (MeOH) λmax (Δε) 213 (+11.6), 227 (−6.7), 250 (+28.9), 289 (+8.5), 350 (−5.5) nm; 1H and 13 C NMR spectroscopic data, see Table 1; positive-ion HRESIMS m/z 416.2578 [M + H]+ (calcd for C28H34NO2, 416.2590). Epimicrophyline B (4): amorphous powder; [α]25 D +20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (4.49), 297 (4.38), 346 (4.12) nm; IR (KBr) νmax 3492, 2956, 2954, 2852, 1713 cm−1; ECD (MeOH) λmax (Δε) 225 (+12.5), 255 (−22.8), 350 (+6.7) nm; 1H and 13 C NMR spectroscopic data, see Table 1; negative-ion HRESIMS m/z 414.2429 [M − H]− (calcd for C28H32NO2, 414.2433). Microphyline C (5): amorphous powder; [α]25 D −67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 237 (4.46), 298 (4.36), 347 (4.09) nm; IR (KBr) νmax 3474, 2956, 2925, 2868, 1707 cm−1; ECD (MeOH) λmax (Δε) 217 (+10.7), 253 (+60.6), 295 (+11.6), 353 (−6.8) nm; 1H and 13 C NMR spectroscopic data, see Table 1; negative-ion HRESIMS m/z 482.3060 [M − H]− (calcd for C33H40NO2, 482.3059). Microphyline D (6): colorless needles (acetone−H2O, 9:1); mp 175−176 °C; [α]25 D +51 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 239 (4.39), 300 (4.29), 350 (4.02) nm; IR (KBr) νmax 3365, 2970, 2925, 1709, 1491 cm−1; ECD (MeOH) λmax (Δε) 230 (+3.3), 300 (+1.7), 353 (+0.98) nm; 1H and 13C NMR spectroscopic data, see Table 2; positive-ion HRESIMS m/z 446.2674 [M + H]+ (calcd for C29H36NO3, 446.2695). (−)-Microphyline D (6a): [α]25 D −87 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 228 (+3.01), 300 (+0.73) nm. (+)-Microphyline D (6b): [α]D25 +92 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 228 (−2.55), 300 (−0.70) nm. Microphyline E (7): amorphous powder; [α]25 D +26 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (4.39), 298 (4.28), 339 (4.01) nm; IR (KBr) νmax 3420, 2924, 1621 cm−1; ECD (MeOH) λmax (Δε) 212 (−9.87), 230 (+9.87), 250 (+9.77), 290 (+3.76), 347 (+0.60) nm; 1 H and 13C NMR spectroscopic data, see Table 2; negative-ion HRESIMS m/z 414.2436 [M − H]− (calcd for C28H32NO2, 414.2433). (−)-Microphyline E (7a): [α]D25 −80 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 225 (+7.51), 237 (+8.71), 283 (+4.21) nm. (+)-Microphyline E (7b): [α]25 D +80 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 225 (−7.51), 237 (−8.01), 283 (−2.41) nm. Microphyline F (8): amorphous powder; [α]25 D +27 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 237 (4.38), 300 (4.27), 349 (4.00) nm; IR (KBr) νmax 3323, 2922, 1456 cm−1; ECD (MeOH) λmax (Δε) 227 (+5.57), 235 (+5.36), 287 (+0.59), 297 (+0.57) nm; 1H and 13C NMR spectroscopic data, see Table 2; negative-ion HRESIMS m/z 482.3058 [M − H]− (calcd for C33H40NO2, 482.3059). (−)-Microphyline F (8a): [α]25 D −100 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 223 (+4.51), 245 (+3.91), 262 (+2.01) nm. (+)-Microphyline F (8b). [α]25 D +105 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 222 (−3.51), 238 (−4.61), 287 (−1.51) nm. Microphyline G (9): amorphous powder; [α]25 D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 235 (4.37), 300 (4.25), 347 (4.00) nm; IR (KBr) νmax 3363, 2924, 1711 cm−1; ECD (MeOH) λmax (Δε) 225 (−19.7), 287 (+22.6), 350 (−5.7) nm; 1H and 13C NMR spectroscopic data, see Table 2; negative-ion HRESIMS m/z 444.2536 [M − H]− (calcd for C29H34NO3, 444.2539). Microphyline H (10): amorphous powder; [α]25 D +7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 237 (4.37), 300 (4.27), 347 (4.01) nm; IR (KBr) νmax 3442, 2065, 1642 cm−1; ECD (MeOH) λmax (Δε) 227 (−15.7), 252 (+17.6), 297 (+10.2), 337 (−7.6) nm; 1H and 13C NMR spectroscopic data, see Table 3; positive-ion HRESIMS m/z 446.2685 [M + H]+ (calcd for C29H36NO3, 446.2695).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00338.

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Neutral loss scanning and UV spectrum of the extract of M. microphylla; key HMBC and 1 H− 1 H COSY correlations of 3/4, 9, and 10, and NOE correlations of 5 and 9; experimental and calculated ECD spectra of 2−5 and 7−12; chiral-phase HPLC separation of 7 and 8; detailed NMR and HRESIMS spectra of 1−12 (PDF) X-ray crystallographic data for 1 (CIF) X-ray crystallographic data for 6 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-82802719. E-mail: [email protected]. ORCID

Jun Li: 0000-0001-8243-5267 Daneel Ferreira: 0000-0002-9375-7920 Jordan K. Zjawiony: 0000-0001-5242-2799 Peng-Fei Tu: 0000-0003-3553-1840 Yong Jiang: 0000-0002-8450-7786 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by National Natural Science Foundation of China (nos. 81473106 and 81773864) and National Key Technology R&D Programs “New Drug Innovation” of China (no. 2018ZX09711001-008-003).

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REFERENCES

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