Bioactive Diterpenoids from the Leaves of Callicarpa macrophylla

Article pubs.acs.org/jnp

Bioactive Diterpenoids from the Leaves of Callicarpa macrophylla Jing Xu,†,‡ Yihang Sun,†,‡,¶ Meicheng Wang,†,‡,¶ Quanhui Ren,†,‡ Shen Li,†,‡ Hao Wang,†,‡ Xiaocong Sun,†,‡ Da-Qing Jin,§ Hongwei Sun,⊥ Yasushi Ohizumi,∥ and Yuanqiang Guo*,†,‡ †

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, ‡Tianjin Key Laboratory of Molecular Drug Research, §School of Medicine, and ⊥Computational Centre for Molecular Science, College of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China ∥ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan S Supporting Information *

ABSTRACT: A phytochemical investigation of the leaves of Callicarpa macrophylla led to the isolation of five new diterpenoids (1−5), macrophypenes A−E, and nine known analogues (6−14). The structures of 1−5 were established on the basis of extensive analysis of NMR spectroscopic data, Xray diffraction data, and experimental and calculated electronic circular dichroism spectra. Compound 1 is a spiroditerpenoid with a novel skeleton, and compound 5 is a rare ent-abietane diterpenoid possessing a peroxide bridge. Compounds 1, 5−7, and 11−14 stimulate nerve growth factor mediated neurite outgrowth from PC12 cells.

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RESULTS AND DISCUSSION The EtOAc-soluble part of the MeOH extract of the leaves of C. macrophylla afforded five new (1−5) and nine known (6− 14) diterpenoids.

he genus Callicarpa, belonging to the Verbenaceae plant family, contains about 190 species that are distributed mainly in tropical and subtropical Asia and Oceanica.1 Some Callicarpa species have been used traditionally as folk medicines for the treatment of various medical indications. Components from this genus include terpenoids,2−10 especially diterpenoids, phenylethanoids,11,12 lignans,13 volatile oils,14,15 and flavonoids,16 displaying diverse biological effects, such as cytotoxic,2 anti-inflammatory,3,9 antitubercular,4 and antiplatelet aggregation activities.10 The species Callicarpa macrophylla Vahl. is a shrub distributed mainly in southern mainland China,17 and its leaves or roots have been used as a folk medicine to relieve pain, stop bleeding, and eliminate stasis to subdue swelling.18 Although many bioactive constituents from the genus Callicarpa have been reported, phytochemical and pharmacological studies on its subordinate species C. macrophylla used as a folk medicine are limited.19,20 In our continuous survey on the chemical composition of folk medicines and search for natural bioactive substances,21−23 the chemical constituents of the leaves of C. macrophylla, a traditional folk medicine, have been investigated. This procedure led to the isolation of five new diterpenoids, macrophypenes A−E (1−5), together with nine known analogues (6−14). The structures of these new compounds were established by analysis of their NMR spectroscopic data, X-ray diffraction data, and experimental and calculated electronic circular dichroism (ECD) spectra. Herein, the isolation and structural elucidation of these compounds as well as their ability to stimulate nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells are described. © XXXX American Chemical Society and American Society of Pharmacognosy

Compound 1 was obtained as colorless crystals from MeOH. Its molecular formula, C20H30O4, was deduced from the 13C NMR data and the HRESIMS ion at m/z 333.2070 [M − H]− Received: January 13, 2015

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DOI: 10.1021/acs.jnatprod.5b00018 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 13C NMR Data for Compounds 1−5 (δ in ppm, 100 MHz)a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCH3 a

1 34.4 17.7 37.0 48.0 47.6 23.1 35.6 55.2 211.8 47.7 21.4 30.1 71.5 67.4 30.2 18.8 18.5 17.2 180.2 18.5

2 CH2 CH2 CH2 C CH CH2 CH2 C C C CH2 CH2 C CH CH CH3 CH3 CH3 C CH3

3

39.0 19.8 43.3 72.4 52.2 22.5 127.0 137.5 46.9 36.0 20.3 27.5 41.0 79.6 146.5 113.5 22.1 23.3

CH2 CH2 CH2 C CH CH2 CH C CH C CH2 CH2 C CH CH CH2 CH3 CH3

14.4

CH3

34.5 18.2 36.0 47.0 41.2 25.0 78.2 125.3 141.8 37.7 21.0 35.3 34.9 39.1 145.9 111.4 27.4 16.5 183.3 18.4 56.1

4 CH2 CH2 CH2 C CH CH2 CH C C C CH2 CH2 C CH2 CH CH2 CH3 CH3 C CH3 CH3

34.6 18.3 34.5 38.2 39.8 22.9 79.0 125.2 142.3 37.2 21.0 35.8 34.9 39.4 146.1 111.3 27.3 17.6 71.8 18.4 57.3

5 CH2 CH2 CH2 C CH CH2 CH C C C CH2 CH2 C CH2 CH CH2 CH3 CH3 CH2 CH3 CH3

33.5 17.5 36.6 46.8 37.8 31.4 65.3 146.4 81.6 38.6 24.1 27.1 79.9 131.1 32.2 17.4 17.2 17.7 182.6 18.0

CH2 CH2 CH2 C CH CH2 CH C C C CH2 CH2 C CH CH CH3 CH3 CH3 C CH3

Compound 1 was recorded in pyridine-d5, and the others were recorded in CDCl3.

Table 2. 1H NMR Data for Compounds 1−5 (δ in ppm, J in Hz, 400 MHz)a position 1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11a/α b/β 12a/α b/β 13 14

1

2

1.65 1.98 1.41 1.68 2.00 1.73

m m m m m m

1.10 1.80 1.62 1.42 1.82 1.33

m m m m m m

2.44 1.81 1.55 1.58 1.90

dd (12.2, 1.7) m m m m

1.47 dd (12.2, 4.1) 2.24b 1.92b 5.75 dd (3.6, 2.2)

3 1.52 1.29 1.63 1.59 1.84 1.57

m m m m m m

4 1.51 1.28 1.65 1.53 1.49 1.26

m m m m m m

2.19 dd (12.5, 2.0) 1.66b 1.56b

1.63b 1.81 dt (14.0, 1.5) 1.45b

3.33 d (2.8)

3.28 d (2.3)

1.94 1.92 1.13 1.72

1.94 1.92 1.04 1.70

5 1.59 1.67 1.60 1.54 1.57 1.69

m m m m m m

2.24 dd (11.8, 5.2) 2.07b 1.40b 4.76 t (4.4)

2.15 dd (12.5, 4.9) 1.70 2.07 1.10 1.80

m m m m

1.60 1.38 1.95 1.33

m m m m

3.79 s

3.63 s

15 16

2.84 sept (6.8) 0.97 d (6.8)

17 18 19

0.97 d (6.8) 1.47 s

5.90 5.14 5.10 0.89 1.22

20 OCH3

1.37 s

0.82 s

dd (17.6, 10.9) dd (10.9, 1.2) dd (17.6, 1.2) s s

m m m m

α 2.36 d (16.8) β 1.61b 5.72 dd (17.5, 10.7) 4.98 d (17.5) 4.89 d (10.7) 0.97 s 1.19 s

0.95 s 3.30 s

m m m m

α 2.32 dd (16.8, 1.2) β 1.65b 5.75 dd (17.5, 10.8) 4.99 dd (17.5, 1.4) 4.91 dd (10.8, 1.4) 0.98 s 0.80 s 3.49 d (11.2) 3.10 d (11.2) 0.97 s 3.36 s

2.31 1.64 2.05 1.41

m m m m

6.60 d (1.3) 1.92 sept (6.8) 1.00 d (6.8) 0.99 d (6.8) 1.28 s

1.06 s

a

Compound 1 was recorded in pyridine-d5, and the others were recorded in CDCl3. bSignals were in overlapped regions of the spectra, and the multiplicities could not be discerned.

B



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Figure 1. 1H−1H COSY and key HMBC correlations of compounds 1−3 and 5.

Figure 2. Key NOESY correlations of compounds 1−3 and 5.

(calcd for C20H29O4, 333.2066). The 1H NMR spectrum of 1 exhibited four methyl groups [δH 0.97 (6H, d, J = 6.8 Hz, H3-16 and H3-17), 1.47 (3H, s, H3-18), and 1.37 (3H, s, H3-20)] and one oxygenated methine proton [δH 3.79 (1H, s, H-14)]. The 13 C NMR spectrum of 1 showed 20 carbon resonances (Table 1). From the 1H and 13C NMR spectra, an isopropyl moiety was deduced and defined from the observation of the carbon signals at δC 30.2, 18.8, and 18.5 and the corresponding proton signals (Table 2). Apart from the above three carbon resonances for the isopropyl group, the remaining 17 signals were assigned as two methyls [δC 17.2 (C-18) and 18.5 (C20)], seven methylenes [δC 34.4 (C-1), 17.7 (C-2), 37.0 (C-3), 23.1 (C-6), 35.6 (C-7), 21.4 (C-11), and 30.1 (C-12)], two methines [δC 47.6 (C-5) and 67.4 (C-14)], three quaternary carbons [δC 48.0 (C-4), 55.2 (C-8), and 47.7 (C-10)], two carbonyls [211.8 (C-9) and 180.2 (C-19)], and one oxygenated tertiary carbon [71.5 (C-13)] with the aid of DEPT and HMQC spectra. The 20 carbon resonances indicated the diterpenoid skeleton.24−28 From the HMBC spectrum, the long-range couplings of H3-18 to C-3, C-4, and C-5, H3-20 to C-1, C-5, C-9, and C-10, H2-7 to C-5, C-6, C-8, and C-9, and H-5 to C-3, C-4, C-6, C-7, and C-10 revealed the presence of two fused six-membered rings A and B, which was supported by the cross-peaks in the 1H−1H COSY spectrum (Figure 1). In addition to these two fused six-membered rings, another fivemembered ring C carrying the above-mentioned isopropyl moiety at C-13 was also demonstrated by the HMBC

correlations of H-14 (δH 2.44) with C-8, C-11, C-12, C-13, and C-15 and H-15 with C-12, C-13, and C-14, as well as the 1 H−1H COSY correlations (Figure 1). The linkage of rings B and C via C-8 (δ C 55.2) was demonstrated by the corresponding HMBC correlations shown in Figure 1. By further analyses of the HMQC, HMBC, and 1H−1H COSY spectra, the proton and carbon signals were assigned unambiguously, which resulted in the establishment of the planar structure for compound 1. However, the molecular formula based on this planar structure for compound 1 was not compatible with the HRESIMS data, indicating the presence of another ring according to the index of hydrogen deficiency. The chemical shifts of carbons and HRESIMS data of compound 1 strongly pointed toward a 13,14-epoxy structural moiety. Thus, the planar structure of compound 1 was defined. The relative configuration of compound 1 was established via NOESY data and Chem3D modeling (Figure 2). NOESY correlations of H3-20/H-2β, H3-20/H3-18, H3-20/H-6β, H318/H-6β, H-1α/H-5, H-5/H-3α, H-5/H-7α, H-7β/H-11b, H11b/H-14, H-7β/H-14b, H-11b/H-12b, and H-11a/H-12a, together with Chem3D modeling, implied a conformation for compound 1, as depicted in Figure 2, where the two sixmembered rings A and B were trans-fused and existed in a chair conformation. The five-membered ring C, orthogonal to ring B, had an envelope conformation. According to this arrangement of the three rings and the observed NOESY correlations, the relative configuration of 1 was inferred, in which H-5, C-11, and C



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C-19 were α-oriented, C-14, C-18, and C-20 were β-oriented, and the epoxy group, H-11a, and H-12a, relative to ring C, were on the same side of ring C. Such a relative configuration of 1 was confirmed by a single-crystal X-ray crystallographic analysis using anomalous scattering of Mo Kα radiation, and a drawing of thermal ellipsoid representation, with the atom numbering indicated, is shown in Figure 3. The absolute configuration was

corresponding carbons (Figure 1) revealed the existence of three fused six-membered rings, which was supported by the 1 H−1H COSY correlations shown in Figure 1. Further analyses of the HMQC, HMBC, and 1H−1H COSY data permitted the assignments of all the proton and carbon signals. Thus, the planar structure of 2 was shown to be a 19-norpimarane diterpenoid. The relative configuration of 2 was deduced from the NOESY spectrum and Chem3D modeling. The NOESY correlations observed for H3-20/H3-18, H3-20/H-2β, H3-20/H6β, H3-18/H-6β, H3-20/H-11β, H-11β/H3-17, H3-17/H-14, H14/H-7, H-1α/H-5, H-1α/H-9, H-5/H-3α, H-5/H-9, and H12α/H-9 revealed a conformation for compound 2, as depicted in Figure 2. Based on this conformation, the two six-membered rings A and B were trans-fused; the three methyl groups Me-17, Me-18, and Me-20 were all in β-axial positions; H-9 and the C14 hydroxy group were both in α-axial positions, and the C-4 hydroxy group was in an α-equatorial position. The absolute configuration of 2 was established as in the case of 1. Through conformation searches, geometry optimizations, time-dependent density functional theory (TDDFT) ECD calculations, and the comparison of the calculated and experimental ECD spectra (Supporting Information Figure S13), the absolute configuration of 2 was assigned as 4R, 5R, 9R, 10R, 13R, and 14S, and the compound was named macrophypene B. Compound 3 (macrophypene C) was obtained as a colorless oil. Its molecular formula was deduced as C21H32O3 from the 13 C NMR and HRESIMS (m/z 331.2278 [M − H]−, calcd for C21H31O3, 331.2273) data. The 1H NMR data for 3 (Table 2) exhibited three methyl singlets, three olefinic protons, and one oxymethine proton. Additionally, a methoxy signal was present in the 1H NMR spectrum. The 13C NMR data of 3 (Table 1) showed the methoxy carbon (δC 56.1) and additional 20 resonances, which included three methyls, eight methylenes, three methines, and three quaternary carbons, based on DEPT and HMQC experiments. The above spectroscopic features and the 20 skeletal carbons in the 13C NMR spectrum suggested that compound 3 is a diterpenoid possessing one methoxy group.37−39 By comparison of the chemical shifts of C-1−C-20 of 3 with those of related reported compounds,39 the presence of a pimarane-type diterpenoid skeleton for 3 was deduced. The interpretation of 2D NMR (HMBC and 1H−1H COSY) spectra resulted in the validation of this skeletal type for 3 and the designations of the carbonyl, olefinic, and oxygenated carbon signals at δC 183.3 (C-19), 125.2 (C-8), 141.8 (C-9), 145.9 (C-15), 111.4 (C-16), and 78.2 (C-7). Subsequently, the methoxy group was located at C-7 by the HMBC correlations (Figure 1). By further analyzing the HMQC, HMBC, and 1 H−1H COSY data (Figure 1), all the proton and carbon signals were assigned unambiguously. The planar structure of 3 was therefore characterized. The NOESY spectrum allowed the relative configuration to be assigned, which showed correlations of H3-20/H3-18, H3-20/H-2β, H3-20/H-6β, H3-18/H-6β, H320/H-11β, H-11β/H3-17, H3-17/H-14β, H3-17/H-12β, H-7/ H-6β, H-1α/H-5, H-3α/H-5, and H-12α/H-14α. These NOESY correlations, together with Chem3D modeling, revealed a molecular conformation for compound 3 (Figure 2), where the two six-membered rings A and B were trans-fused with a β-axial orientation for Me-20 and an α-axial orientation for H-5. The methyl groups Me-17 and Me-18 were both βaxially oriented. The C-7 methoxy group was α-axially oriented. Thus, the structure of 3 was elucidated, as depicted in Figure 2.

Figure 3. ORTEP drawing of 1.

determined via experimental and calculated ECD data, a powerful tool to assign the absolute configuration of natural products.29,30 Through systematic conformational search and geometry optimizations,31−33 the ECD spectra were calculated at the CAM-B3LYP/SVP level with the conductor-like polarizable continuum model (CPCM) in acetonitrile. The calculated ECD spectrum of 1a (Supporting Information Figure S7) matched the experimental spectrum closely, which suggested an absolute configuration of 4R, 5R, 8S, 10S, 13S, and 14S. The structure of 1 was therefore elucidated as shown, and the compound was named macrophypene A. Compound 2, a colorless oil, possessed a molecular formula of C19H30O2, as determined from the 13C NMR and HRESIMS (m/z 289.2173 [M − H]−, calcd for C19H29O2, 289.2168) data. From the 1H NMR spectrum of 2, three methyl groups, four olefinic protons, and one oxymethine proton were observed (Table 2). Besides the corresponding four olefinic and two oxygenated carbons, the 13C NMR spectrum exhibited 13 additional resonances (Table 1). The 19 carbon resonances suggested that compound 2 may be a nor-diterpenoid based on the reported related diterpenoids.34−36 With the aid of DEPT and HMQC spectra, the 19 carbons were sorted as two quaternary carbons [δC 36.0 (C-10) and 41.0 (C-13)], five methines [δC 52.2 (C-5), 127.0 (C-7), 46.9 (C-9), 79.6 (C-14), and 146.5 (C-15)], seven methylenes [δC 39.0 (C-1), 19.8 (C2), 43.3 (C-3), 22.5 (C-6), 20.3 (C-11), 27.5 (C-12), and 113.5 (C-16)], three methyls [δC 22.1 (C-17), 23.3 (C-18), and 14.4 (C-20)], one oxygenated tertiary carbon [δC 72.4 (C-4)], and one quaternary olefinic carbon [δC 137.5 (C-8)] (Table 1). In the HMBC spectrum, the cross-peaks of the olefinic H-7 with C-5, C-6, C-8, C-9, and C-14, the oxygenated methine H-14 with C-7, C-8, C-9, C-12, C-13, C-15, and C-17, the methine H-9 with C-1, C-5, C-7, C-8, C-10, C-11, C-12, and C-14, and the methyl protons H3-17, H3-18, and H3-20 with the D



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ring C. Consequently, H-5 and C-11 were β-oriented; C-18 and C-20 were α-oriented with axial orientations, and the C-7 hydroxy group was β-axially oriented, which was supported by the coupling constant (J7,6α/β = 4.4 Hz) between H-7 and H-6α (β). These configurational assignments demonstrated the peroxide bridge and the C-20 methyl group to both be αoriented, which was also supported by comparative analysis of the 13C NMR data of 5 with those published for similar compounds.42,43 TDDFT ECD calculations were performed to elucidate the absolute configuration of 5. By comparison of its experimental and calculated ECD spectra (Supporting Information Figure S31), the absolute configuration of 5 was determined as 4S, 5S, 7S, 9S, 10R, and 13R. The structure of compound 5 (macrophypene E) was therefore elucidated and comprised a rare ent-abietane diterpenoid with a peroxide bridge. The nine known diterpenes were identified by comparison of experimental and literature spectroscopic data as calliphyllin (6), 4 4 14α,18-dihydroxy-7,15-isopimaradiene (7), 4 4 8α,9α,13α,14α-diepoxyabietan-18-oic acid (8),45 7α-hydroxydehydroabietic acid (9),46 7-oxodehydroabietic acid (10),47 abieta-8,11,13,15-tetraen-18-oic acid (11),45 17-acetoxy-16βhydroxy-3-oxo-ent-kaurane (12),48 ent-16α,17-dihydroxykauran-3-one (13),48 and 3-oxoanticopalic acid (14).49 Pharmacological studies of NGF have demonstrated that bioactive substances to promote the neurite outgrowth of nerve cells against neuron degeneration may be potentially useful for the treatment of Alzheimer’s disease.50,51 Compounds 1−14 were thus evaluated for their NGF-potentiating activities as described previously.23 NGF was used as the positive control.23,50 Compounds 1, 5−7, and 11−14 stimulated NGF-mediated neurite outgrowth from PC12 cells. The EC50 values of these active diterpenes to enhance NGF action dosedependently are shown in Table 3. However, compounds 2−4 and 8−10 had no effects on neurite outgrowth from PC12 cells in either the absence or the presence of NGF (20 ng/mL).

The molecular formula of compound 4 was determined as C21H34O2 based on the 13C NMR and HRESIMS (m/z 341.2454 [M + Na]+, calcd for C21H34NaO2, 341.2457) data. Its 1H and 13C NMR data resembled those of compound 3, which implied that 4 is also a pimarane-type diterpenoid. Apart from the characteristic olefinic, methyl, methoxy, and oxygenated methine protons present in compound 3, a pair of oxygenated methylene protons was observed in the 1H NMR spectrum of 4, which was supported by the corresponding carbon resonance at δC 71.8 (C-19) in the DEPT spectrum. These similar spectroscopic features implied that compounds 3 and 4 were C-4 structural isomers. By interpretation of the 2D NMR spectra, the assignments of the proton and carbon signals were accomplished, which substantiated that the hydroxycarbonyl group in 3 was replaced by a hydroxymethyl group in 4. The same relative configuration was inferred for macrophypene D (4) and macrophypene C (3) on the basis of comparison of their NOESY spectra. Compound 4 was therefore characterized and named macrophypene D. It should be noted that the ECD spectra of compounds 3 and 4 were conspicuously devoid of Cotton effects at wavelengths above 200 nm. Thus, only relative configurations could be defined for these compounds. Compound 5 was obtained as an amorphous white powder with the molecular formula C20H30O5, as suggested by the 13C NMR and HRESIMS (m/z 349.2020 [M − H]−, calcd for C20H29O5, 349.2015) data. From the 1H and 13C NMR data (Tables 1 and 2), the same isopropyl moiety as in compound 1 was evident from the corresponding proton and carbon signals. In addition, the same carbonyl group as that in 1 and 3 was also revealed by the 13C NMR spectrum. Apart from the above four signals for the isopropyl moiety and carbonyl group, the remaining 16 resonances in the 13C NMR spectrum were assigned to two methyls, six methylenes, three methines, two quaternary carbons, two oxygenated tertiary carbons, and one disubstituted olefinic carbon based on DEPT and HMQC spectra. These spectroscopic features implied that compound 5 should be a diterpenoid having a carbonyl group and a isopropyl moiety.40 Comparison of the chemical shifts of C-1− C-20 of compound 5 with those of related compounds40 suggested the presence of an abietane-type diterpenoid skeleton for 5. Using the same HMQC, HMBC, and 1H−1H COSY experiments as used for compounds 1−4, the abietane-type skeleton for 5 was confirmed, and the carbonyl, olefinic, and oxygenated carbon signals at δC 182.6, 146.4, 131.1, 65.3, 81.6, and 79.9 were assigned to C-19, C-8, C-14, C-7, C-9, and C-13, respectively. By further analyzing the HMQC, HMBC, and 1 H−1H COSY spectra (Figure 2), the proton and carbon signals were assigned and the planar structure for compound 5 was inferred. However, the molecular formula of this structure for 5 was inconsistent with the HRESIMS data, implying the presence of another ring according to the index of hydrogen deficiency. According to the NMR data of compound 5, a peroxide bridge between C-9 and C-13 was proposed.41 Thus, the planar structure of compound 5 was determined. The relative configuration of compound 5 was elucidated as follows. The NOESY spectrum displayed the correlations of H3-20/H3-18, H3-20/H-2α, H3-20/H-6α, H3-18/H-6α, H-6α/ H-7, H-7/H-14, H-14/H-12b, H-12b/H-11b, H-11b/H-5, H5/H-1β, H-5/H-3β, H-1β/H-3β, and H-11a/H-12a (Figure 2). According to these correlations and the Chem3D modeling, the conformation of compound 5 comprised two trans-fused sixmembered rings A and B, both assuming chair conformations. The peroxide bridge occupied the α-face of the six-membered

Table 3. EC50 Values of Compounds 1, 5−7, and 11−14 Stimulating NGF-Mediated Neurite Outgrowth from PC12 Cells compound

EC50a (μM)

compound

EC50 (μM)

1 5 6 7

>30 10.6 16.0 7.6

11 12 13 14

>30 11.0 19.7 6.0

NGF was used as a positive control (EC50 value, 5.1 × 10−2 μg/mL). Data are presented based on three experiments. a

In conclusion, our present phytochemical investigation on C. macrophylla has led to the isolation and structure elucidation of five new (1−5) and nine known (6−14) diterpenoids. Besides their relative configurations, the absolute configurations of compounds 1, 2, and 5 were determined on the basis of NMR data analysis, X-ray diffraction, and their experimental and calculated ECD spectra. Among these new compounds, compound 1 is a spiroditerpenoid with a novel skeleton and compound 5 is a rare ent-abietane diterpenoid having a peroxide bridge. Compounds 1, 5−7, and 11−14 showed potentiating activities of NGF-mediated neurite outgrowth from PC12 cells, with compound 14 exerting the most significant promotion of NGF-mediated neurite outgrowth from PC12 cells. These bioactive diterpenoids may be useful for E



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23 min, 344.6 mg) and 13 (tR = 21 min, 17.9 mg) were obtained from F7‑2 (80% MeOH in H2O). Macrophypene A (1): colorless crystals (MeOH); mp 234−235 °C; [α]D21 −15 (c 0.1, CH2Cl2); ECD (CH3CN) 192 (Δε −15.03), 219 (Δε +1.13), 303 (Δε +1.93) nm; IR (KBr) νmax 3444, 2935, 2873, 1721, 1453, 1163, 933, 739 cm−1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 333 [M − H]−; HRESIMS m/z 333.2070 [M − H]− (calcd for C20H29O4, 333.2066). X-ray Crystal Data of Macrophypene A (1): C20H30O4, Mr = 334.44, orthorhombic, space group P2(1)2(1)2(1), a = 7.5870 (15) Å, b = 9.6985 (19) Å, c = 25.106 (5) Å, V = 1847.4 (6) Å3, Z = 4, Dcalc = 1.202 g/cm3, crystal dimensions 0.25 × 0.20 × 0.15 mm were used for measurements. The total number of reflections measured was 15 232, of which 3429 were unique and 2825 were observed, I > 2σ(I). Final indices: R1 = 0.0611, wR2 = 0.1745 for observed reflections, and R1 = 0.0753, wR2 = 0.1842 for all reflections. Crystallographic data of this compound have been deposited in the Cambridge Crystallographic Data Centre (CCDC 1406620). Macrophypene B (2): colorless oil; [α]D21 −14 (c 0.1, CH2Cl2); ECD (CH3CN) 193 (Δε +0.66), 213 (Δε −1.46) nm; IR (KBr) νmax 3458, 2926, 2855, 1636, 1364, 1276, 750 cm−1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 289 [M − H]−; HRESIMS m/z 289.2173 [M − H]− (calcd for C19H29O2, 289.2168). Macrophypene C (3): colorless oil; [α]D20 +34 (c 0.1, CH2Cl2); ECD (CH3CN) 190 (Δε +18.46), 221 (Δε +0.33), 228 (Δε +0.44), nm; IR (KBr) νmax 3467, 2925, 2868, 1722, 1694, 1454, 1275, 1187, 749 cm−1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 331 [M − H]−; HRESIMS m/z 331.2278 [M − H]− (calcd for C21H31O3, 331.2273). Macrophypene D (4): colorless oil; [α]D21 +31 (c 0.1, CH2Cl2); ECD (CH3CN) 190 (Δε +16.52), 243 (Δε −0.11) nm; IR (KBr) νmax 3307, 2926, 2857, 1653, 1457, 1266, 1081, 968, 741 cm−1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 341 [M + Na]+; HRESIMS m/z 341.2454 [M + Na]+ (calcd for C21H34NaO2, 341.2457). Macrophypene E (5): amorphous white powder; [α]D20 −27 (c 0.1, CH2Cl2); CD (CH3CN) 198 (Δε −5.16), 226 (Δε +2.06) nm; IR (KBr) νmax 3455, 2935, 2874, 1698, 1651, 1465, 1389, 1270, 1189, 947, 736 cm−1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 349 [M − H]−; HRESIMS m/z 349.2020 [M − H]− (calcd for C20H29O5, 349.2015). Computations. Conformational searches were performed by the MOE software using the MMFF94S force field.31 The single-crystal Xray diffraction data and the obtained conformers were used for geometry reoptimizations at the B3LYP/6-31+G(d,p) level in the Gaussian 09 package.32 The ECD spectra for the optimized conformers were calculated at the CAM-B3LYP/SVP level with a CPCM solvent model in acetonitrile, and the calculated ECD spectra of different conformers were simulated with a half bandwidth of 0.3− 0.4 eV. The ECD curves were extracted by SpecDis 1.62 software.33 The overall ECD curves of all the compounds were weighed by Boltzmann distribution after UV correction. Bioassay for Neurite Outgrowth. PC12 cells were cultured at 37 °C in Dulbecco’s modified Eagle medium supplemented with 5% (v/ v) inactivated fetal bovine serum (FBS), 5% (v/v) inactivated horse serum (HS), and 100 U/mL penicillin/streptomycin under a watersaturated atmosphere of 95% air and 5% CO2. The cells were disassociated by incubation with 1 mM of ethylene glycol/bis(2aminoethyl ether)-N,N,N′,N′-tetraacetic acid in phosphate-buffered saline for 15 min and then seeded in 24-well culture plates (3 × 104 cells/well) coated with poly-L-lysine. After 24 h, the medium was changed to test medium containing various concentrations of NGF (100 ng/mL for positive control, 20 ng/mL for test samples and significant difference control), 1% FBS, 1% HS, and various concentrations of test compounds. After a continuous incubation of 96 h, the neurite outgrowth was assessed under a phase contrast microscope. Neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite-bearing

the development of anti-neurodegenerative agents to combat Alzheimer’s disease and other neurological disorders.51

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting point was measured with an XT-4 microscopic thermometer (Beijing Tech Instrument Co., Ltd., Beijing, People’s Republic of China). Optical rotations were measured in CH2Cl2 using an Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). ECD spectra were obtained on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). IR spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer with KBr disks. One- and twodimensional NMR spectra were recorded on a Bruker AV 400 instrument (Bruker, Switzerland, 400 MHz for 1H and 100 MHz for 13 C) with tetramethylsilane as an internal standard. ESIMS spectra were acquired on a Thermo Finnigan LCQ-Advantage mass spectrometer. HRESIMS spectra were recorded by IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA). HPLC separations were performed on a CXTH system, equipped with a UV3000 detector at 210 nm (Beijing Chuangxintongheng Instruments Co., Ltd., Beijing, People’s Republic of China), and a YMC-pack ODS-AM (20 × 250 mm) column (YMC Co. Ltd., Kyoto, Japan). Silica gel was used for column chromatography (200−300 mesh, Qingdao Haiyang Chemical Group Co., Ltd., Qingdao, People’s Republic of China). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Yuanli Co., Ltd., Tianjin, People’s Republic of China. Biological reagents were from Sigma Chemical Co. The PC12 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, People’s Republic of China). Plant Material. The leaves of C. macrophylla native to the Guangxi province, People’s Republic of China, were purchased in July 2013 from Anguo Materia Medica Market, Hebei Province, People’s Republic of China. The botanical identification was made by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University), and a voucher specimen (no. 20130809) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University. Extraction and Isolation. The air-dried leaves of C. macrophylla (10.0 kg) were extracted with MeOH (3 × 60 L) under reflux. The organic solvent was evaporated to afford a crude extract (1.0 kg). The extract was suspended in H2O (1.2 L) and partitioned with EtOAc (3 × 1.2 L). The EtOAc-soluble portion (360.0 g) was subjected to silica gel column chromatography, using a gradient of acetone in petroleum ether (0−30%), to yield seven fractions (F1−F7) based on thin-layer chromatography analysis. Fraction F5 (66.5 g) was further fractionated by silica gel column chromatography, using a gradient of acetone in petroleum ether (0−11%), to provide six subfractions (F5‑1−F5‑6). These subfractions F5‑2, F5‑3, F5‑4, F5‑5, and F5‑6 were subjected to medium-pressure liquid chromatography (MPLC) over octadecylsilane (ODS), eluting with a step gradient from 70 to 95% MeOH in H2O, to give subfractions F5‑2‑1−F5‑2‑4, F5‑3‑1−F5‑3‑4, F5‑4‑1−F5‑4‑5, F5‑5‑1−F5‑5‑5, and F5‑6‑1−F5‑6‑5. The purification of F5‑3‑1 by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 85% MeOH in H2O) afforded compound 1 (tR = 24 min, 18.4 mg). Compounds 2 (tR = 27 min, 21.0 mg), 8 (tR = 18 min, 19.6 mg), and 9 (tR = 25 min, 18.0 mg) were obtained from F5‑6‑2 (85% MeOH in H2O) by the above HPLC method. Using the same HPLC system, F5‑3‑4 (92% MeOH in H2O) provided compounds 3 (tR = 38 min, 21.2 mg), 4 (tR = 42 min, 15.9 mg), and 6 (tR = 25 min, 13.8 mg); F5‑2‑2 (85% MeOH in H2O) yielded compounds 7 (tR = 30 min, 13.0 mg) and 14 (tR = 18 min, 14.3 mg); F5‑4‑2 (84% MeOH in H2O) afforded compound 10 (tR = 33 min, 12.8 mg); and F5‑5‑2 (77% MeOH in H2O) produced compound 12 (tR = 28 min, 13.6 mg). In addition to fraction F5, another fraction F3 (22.5 g) was further separated by the above MPLC, eluting with a step gradient from 82 to 91% MeOH in H2O, to give seven subfractions (F3‑1−F3‑7). Subfraction F3‑6 was purified by the same HPLC (88% MeOH in H2O) method to give compound 11 (tR = 45 min, 12.2 mg). Using the same protocols as those for fraction F3, F7 (63.5 g) yielded nine subfractions F7‑1−F7‑9, and compounds 5 (tR = F



Article

cells. The ratio of the neurite-bearing cells to total cells (with at least 100 cells examined/viewing area; three viewing areas/well; six wells/ sample) was determined and expressed as a percentage. Each sample was performed in three replicates. The EC50 values were determined on the basis of linear or nonlinear regression analysis of the concentration−response data curves.

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(22) Xu, J.; Guo, Y.; Xie, C.; Li, Y.; Gao, J.; Zhang, T.; Hou, W.; Fang, L.; Gui, L. J. Nat. Prod. 2011, 74, 2224−2230. (23) Xu, J.; Zhang, Q.; Wang, M.; Ren, Q.; Sun, Y.; Jin, D. Q.; Xie, C.; Chen, H.; Ohizumi, Y.; Guo, Y. J. Nat. Prod. 2014, 77, 2182−2189. (24) Whitson, E. L.; Thomas, C. L.; Henrich, C. J.; Sayers, T. J.; McMahon, J. B.; McKee, T. C. J. Nat. Prod. 2010, 73, 2013−2018. (25) Fullas, F.; Hussain, R. A.; Chai, H. B.; Pezzuto, J. M.; Soejarto, D. D.; Kinghorn, A. D. J. Nat. Prod. 1994, 57, 801−807. (26) Burmistrova, O.; Simões, M. F.; Rijo, P.; Quintana, J.; Bermejo, J.; Estévez, F. J. Nat. Prod. 2013, 76, 1413−1423. (27) Pan, L.; Terrazas, C.; Lezama-Davila, C. M.; Rege, N.; Gallucci, J. C.; Satoskar, A. R.; Kinghorn, A. D. Org. Lett. 2012, 14, 2118−2121. (28) Li, J.; Pan, L.; Fletcher, J. N.; Lv, W.; Deng, Y.; Vincent, M. A.; Slack, J. P.; McCluskey, T. S.; Jia, Z.; Cushman, M.; Kinghorn, A. D. J. Nat. Prod. 2014, 77, 1739−1743. (29) Li, X. C.; Ferreira, D.; Ding, Y. Curr. Org. Chem. 2010, 14, 1678−1697. (30) Mazzeo, G.; Santoro, E.; Andolfi, A.; Cimmino, A.; Troselj, P.; Petrovic, A. G.; Superchi, S.; Evidente, A.; Berova, N. J. Nat. Prod. 2013, 76, 588−599. (31) MOE2013.08; Chemical Computing Group Inc., www. chemcomp.com. (32) Frisch, M. J.; et al.; Gaussian 09, revision B. 01; Gaussian, Inc.: Wallingford, CT, 2010. (33) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis, version 1.62; University of Wuerzburg: Wuerzburg, Germany, 2014. (34) Andolfi, A.; Maddau, L.; Basso, S.; Linaldeddu, B. T.; Cimmino, A.; Scanu, B.; Deidda, A.; Tuzi, A.; Evidente, A. J. Nat. Prod. 2014, 77, 2352−2360. (35) Evidente, A.; Venturi, V.; Masi, M.; Degrassi, G.; Cimmino, A.; Maddau, L.; Andolfi, A. J. Nat. Prod. 2011, 74, 2520−2525. (36) Cimmino, A.; Andolfi, A.; Zonno, M. C.; Avolio, F.; Santini, A.; Tuzi, A. J. Nat. Prod. 2013, 76, 1291−1297. (37) Fullas, F.; Soejarto, D. D.; Kinghorn, A. D. Phytochemistry 1992, 31, 2543−2545. (38) Williams, R. B.; Norris, A.; Miller, J. S.; Birkinshaw, C.; Ratovoson, F.; Andriantsiferana, R.; Rasamison, V. E.; Kingston, D. G. I. J. Nat. Prod. 2007, 70, 206−209. (39) Liu, N.; Li, R. J.; Wang, X. N.; Zhu, R. X.; Wang, L.; Lin, Z. M.; Zhao, Y.; Lou, H. X. J. Nat. Prod. 2013, 76, 1647−1653. (40) Yang, L.; Qiao, L.; Ji, C.; Xie, D.; Gong, N. B.; Lu, Y.; Zhang, J.; Dai, J.; Guo, S. J. Nat. Prod. 2013, 76, 216−222. (41) Delgado, G.; Sánchez, E.; Hernández, J.; Chávez, M. I.; Alvarez, L.; Martínez, E. Phytochemistry 1992, 31, 3159−3161. (42) Monaco, P.; Parrilli, M.; Previtera, L. Tetrahedron Lett. 1987, 28, 4609−4610. (43) Barrero, A. F.; Sanchez, J. F.; Alvarez-Manzaneda, E. J.; Munoz Dorado, R. M.; Haidour, A. Phytochemistry 1991, 30, 593−597. (44) Bruno, M.; Savona, G.; Fernandez-Gadea, F.; Rodriguez, B. Phytochemistry 1986, 25, 475−477. (45) Tanaka, R.; Ohtsu, H.; Matsunaga, S. Phytochemistry 1997, 46, 1051−1057. (46) Ohmoto, T.; Saito, M.; Yamaguchi, K. Chem. Pharm. Bull. 1987, 35, 2443−2447. (47) Lee, C. K.; Fang, J. M.; Cheng, Y. Phytochemistry 1994, 35, 983− 986. (48) Jaensch, M.; Jakupovic, J.; Sanchez, H.; Dominguez, X. A. Phytochemistry 1990, 29, 3587−3590. (49) Zinkel, D. F.; Magee, T. V. Phytochemistry 1987, 26, 769−774. (50) Kubo, M.; Ishii, R.; Ishino, Y.; Harada, K.; Matsui, N.; Akagi, M.; Kato, E.; Hosoda, S.; Fukuyama, Y. J. Nat. Prod. 2013, 76, 769−773. (51) Siegel, G. J.; Chauhan, N. B. Brain Res. Rev. 2000, 33, 199−227.

ASSOCIATED CONTENT

S Supporting Information *

NMR, HRESIMS, and ECD spectra of compounds 1−5 and the CIF file of the X-ray data of compound 1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00018.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: 86-22-23502595. E-mail: [email protected]. Author Contributions ¶

Y.S. and M.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Editorial Committee of the Flora of China, Chinese Academy of Sciences. Flora of China, Science Press: Beijing, 1999; Vol. 65 (1), p 25. (2) Jones, W. P.; Lobo-Echeverri, T.; Mi, Q.; Chai, H. B.; Soejarto, D. D.; Cordell, G. A.; Swanson, S. M.; Kinghorn, A. D. J. Nat. Prod. 2007, 70, 372−377. (3) Liu, Y. W.; Cheng, Y. B.; Liaw, C. C.; Chen, C. H.; Guh, J. H.; Hwang, T. L.; Tsai, J. S.; Wang, W. B.; Shen, Y. C. J. Nat. Prod. 2012, 75, 689−693. (4) Chen, J. J.; Wu, H. M.; Peng, C. F.; Chen, I. S.; Chu, S. D. J. Nat. Prod. 2009, 72, 223−228. (5) Xu, J.; Harrison, L. J.; Vittal, J. J.; Xu, Y. J.; Goh, S. H. J. Nat. Prod. 2000, 63, 1062−1065. (6) Singh, A. K.; Agrawal, P. K. Phytochemistry 1994, 37, 587−588. (7) Cantrell, C. L.; Klun, J. A.; Bryson, C. T. J. Agric. Food Chem. 2005, 53, 5948−5953. (8) Liu, H. Y.; He, H. P.; Gao, S.; Chen, C. X.; Shen, Y. M.; Hao, X. J. Helv. Chim. Acta 2006, 89, 1017−1022. (9) Zhang, L.; Dong, L.; Huang, J.; Liu, M.; Li, G.; Zhang, C.; Zhang, K.; Wang, J. Fitoterapia 2013, 89, 218−223. (10) Zhou, Z.; Wei, X.; Fu, H.; Luo, Y. Fitoterapia 2013, 88, 91−95. (11) Koo, K. A.; Sung, S. H.; Park, J. H.; Kim, S. H.; Lee, K. Y.; Choong-Kim, Y. Planta Med. 2005, 71, 778−780. (12) Wu, A. Z.; Zhai, Y. J.; Zhao, Z. X.; Zhang, C. X.; Lin, C. Z.; Zhu, C. C. Fitoterapia 2013, 84, 237−241. (13) Shao, Y.; Hu, L. H.; Sim, K. Y.; Goh, S. H. Helv. Chim. Acta 2006, 89, 64−72. (14) Kim, Y. S.; Shin, D. H. J. Agric. Food Chem. 2004, 52, 781−787. (15) Kobaisy, M.; Tellez, M. R.; Dayan, F. E.; Duke, S. O. Phytochemistry 2002, 61, 37−40. (16) Hu, Y. M.; Shen, Y. M.; Gu, Q. X.; Zuo, G. Y.; Hao, X. J. Chin. Tradit. Herb. Drugs 2001, 32, 1063−1065. (17) Editorial Committee of the Flora of China, Chinese Academy of Sciences. Flora of China; Science Press: Beijing, 1999; Vol. 65 (1), pp 38−39. (18) Nanjing University of Chinese Medicine. Dictionary of Chinese Herb Medicines, 2nd ed.; Shanghai Scientific and Technologic Press: Shanghai, 2006; p 186. (19) Singh, A. K.; Chanotiya, C. S.; Yadav, A.; Kalra, A. Nat. Prod. Commun. 2010, 5, 269−272. (20) Pan, P.; Sun, Q. Shenyang Yaoke Daxue Xuebao 2006, 23, 565− 567. (21) Guo, P.; Li, Y.; Xu, J.; Liu, C.; Ma, Y.; Guo, Y. J. Nat. Prod. 2011, 74, 1575−1583. G


Bioactive Diterpenoids from the Leaves of Callicarpa macrophylla

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