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Bioactive Clerodane Diterpenoids from the Twigs of Casearia balansae Jing Xu,†,‡ Qiang Zhang,§ Meicheng Wang,†,‡ Quanhui Ren,†,‡ Yihang Sun,†,‡ Da-Qing Jin,⊥ Chunfeng Xie,†,‡ Hongqiang Chen,†,‡ Yasushi Ohizumi,∥ and Yuanqiang Guo*,†,‡ †

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, ‡Tianjin Key Laboratory of Molecular Drug Research, and ⊥School of Medicine, Nankai University, Tianjin 300071, People’s Republic of China § College of Science, Northwest A&F University, Yangling 712100, People’s Republic of China ∥ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan S Supporting Information *

ABSTRACT: Eight new clerodane diterpenes, balanspenes A−H (1−8), along with two known analogues (9 and 10), were isolated from the twigs of Casearia balansae. The structures of 1−8 were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of compounds 1, 4, and 7 were confirmed by comparing their experimental CD spectra with those calculated by the time-dependent density functional theory method. Compounds 4−7, 9, and 10 were found to possess the property of being able to stimulate NGF-mediated neurite outgrowth from PC12 cells.

T

he genus Casearia, a member of the Flacourtiaceae plant family, contains about 180 species that are distributed widely in tropical Africa, Asia, northwest Australia, and South America.1 Some Casearia species, such as C. sylvestris, C. grewiifolia, and C. esculenta, have been used traditionally as folk medicines for the treatment of various diseases.1 Previous phytochemical investigations on the genus Casearia have revealed that terpenoids, in particular clerodane diterpenoids, are the predominant and representative constituents,2−14 displaying a broad spectrum of biological activities, such as cytotoxic, antimicrobial, antifungal, antimalarial, and DNAmodifying activities.1 The species Casearia balansae Gagnep. is a small tree distributed mainly in southern mainland China,15 and several cytotoxic terpenoids from this plant were reported.16,17 Although many bioactive constituents of the genus Casearia have been reported, phytochemical and pharmacological studies on the plant C. balansae are limited.16,17 In the course of an ongoing search for bioactive diterpenoids from plants,18−20 much attention has been given to the occurrence of compounds having nerve growth factor (NGF)-potentiating effects, since these compounds are expected to be potentially useful for the treatment of Alzheimer’s disease and other neurological disorders.21,22 An ethyl acetate-soluble extract of the twigs of C. balansae showed moderate stimulatory activity of neurite outgrowth from PC12 cells. This extract was fractionated, leading to the isolation of eight new clerodane diterpenoids, balanspenes A−H (1−8), together with two known analogues, caseabalansin F (9) and caseamembrin B (10). The structures of the new compounds © XXXX American Chemical Society and American Society of Pharmacognosy

were elucidated on the basis of 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of compounds 1, 4, and 7 were confirmed by comparing their experimental CD spectra with those calculated by the time-dependent density functional theory (TDDFT) method. Herein, the isolation and structural elucidation of these compounds as well as their ability to stimulate NGF-mediated neurite outgrowth from PC12 cells are described.

■

RESULTS AND DISCUSSION

The ethyl acetate-soluble part of the methanol extract of the twigs of C. balansae was fractionated by column chromatography and purified by HPLC to obtain eight new (1−8) and two known (9 and 10) compounds. The known compounds were identified by comparison of spectroscopic data with those reported in the literature as caseabalansin F (9)16 and caseamembrin B (10).3 Compound 1 was obtained as a colorless oil. Its HRESIMS provided the molecular formula C24H34O6, through the presence of a peak at m/z 436.2695 [M + NH4]+ (calcd for C24H38NO6, 436.2699), which was consistent with the NMR data (Tables 1 and 2). The 1H NMR spectrum for 1 exhibited four methyl groups [δH 0.89 (3H, d, J = 6.0 Hz, H3-17), 0.93 (3H, s, H3-20), 2.02 (3H, s, COCH3-18), and 1.93 (3H, s, COCH3-19)], five olefinic protons [δH 6.43 (1H, dd, J = 17.6, Received: April 21, 2014

A

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above methyl singlets and the corresponding carbon resonances (δC 169.3, 20.9, 169.5, and 21.3). Apart from the above four signals for two acetyl groups, there were additional 20 resonances occurring for the parent skeleton in the 13C NMR spectrum, which were assigned for two methyls [δC 15.5 (C-17) and 26.2 (C-20)], eight methylenes [δC 37.9 (C-1), 37.2 (C-3), 34.1 (C-6), 27.7 (C-7), 27.2 (C-11), 23.8 (C-12), 112.5 (C15), and 115.3 (C-16)], six methines [δC 48.0 (C-4), 37.8 (C8), 35.5 (C-10), 140.1 (C-14), 99.8 (C-18), and 97.2 (C-19)], and four quaternary carbons [δC 212.4 (C-2), 49.5 (C-5), 37.9 (C-9), and 145.0 (C-13)], based on DEPT and HMQC experiments. The above spectroscopic features and the 20 skeletal carbons displayed in the 13C NMR spectrum suggested that compound 1 is a diterpenoid having two acetoxy groups.18,23−29 On comparing the chemical shifts of C-1−C20 of compound 1 with those of related compounds reported in the literature,7−10 the presence of a clerodane-type diterpene skeleton for 1 was evident. To corroborate the above deductions and confirm this skeleton, HMBC and 1H−1H COSY experiments were performed (Figure 1). The following interpretation of HMQC, HMBC, and 1H−1H COSY spectra led to the confirmation of the skeletal type for 1, where the ketone carbonyl, olefinic, and acetal carbon signals at δC 212.4, 145.0, 140.1, 112.5, 115.3, 99.8, and 97.2 were assigned to C-2, C-13, C-14, C-15, C-16, C-18, and C-19, respectively. The

10.8 Hz, H-14), 5.22 (1H, d, J = 17.6 Hz, H-15a), 5.04 (1H, d, J = 10.8 Hz, H-15b), and 5.04 and 4.93 (each 1H, s, H2-16)], and two oxygenated methine protons [δH 6.48 (1H, d, J = 7.2 Hz, H-18) and 6.22 (1H, s, H-19)]. The 13C NMR spectrum of 1 showed 24 carbon resonances. From the 1H and 13C NMR spectra, two acetyl groups were deduced and defined from the

Table 1. 13C NMR Data for Compounds 1−8 (δ in ppm, 100 MHz, in CDCl3)a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OR-2

OCH3-6 OR-18

OR-19

a

1

2

3

4

5

6

7

8

37.9 212.4 37.2 48.0 49.5 34.1 27.7 37.8 37.9 35.5 27.2 23.8 145.0 140.1 112.5 115.3 15.5 99.8 97.2 26.2

35.4 199.1 123.8 165.7 50.4 29.9 27.6 36.6 37.7 39.1 27.0 23.7 145.0 140.1 112.6 115.3 15.6 93.5 99.4 25.1

26.1 66.4 120.4 147.3 49.3 29.3 27.2 37.3 37.2 34.2 28.1 23.6 145.3 140.4 112.0 115.4 15.7 99.5 94.3 26.0 175.8 41.2 27.0 11.6 16.6

27.1 66.3 121.2 146.4 53.1 81.9 31.1 36.9 37.5 36.5 27.9 23.8 145.1 140.5 112.2 115.6 15.9 96.0 98.4 25.5 173.1 36.5 18.7 13.6

25.9 64.7 140.1 149.4 54.9 81.5 32.7 35.9 37.8 39.7 31.7 23.0 146.5 139.0 112.9 116.1 15.6 191.2 202.3 25.7 176.0 40.9 26.9 11.6 16.4 57.1

172.2 36.1 18.2 13.5 169.5 21.2

172.7 36.3 18.2 13.5 169.8 21.2

27.8 66.2 120.9 146.9 53.0 82.2 31.2 37.0 37.4 36.3 27.2 23.8 145.2 140.5 112.1 115.4 15.9 104.4 97.9 25.6 176.2 40.9 27.1 11.7 16.4 57.5 55.2

26.6 71.1 123.0 146.1 52.8 83.2 31.3 36.8 38.1 41.1 27.4 23.7 145.1 140.2 112.4 115.2 15.8 103.8 97.5 25.5 170.5 21.1

169.5 21.3

27.0 66.2 121.3 146.4 53.2 81.9 31.1 37.0 37.4 36.4 27.8 23.8 145.0 140.5 112.1 115.5 15.8 95.9 98.3 25.5 175.8 41.2 27.0 11.6 16.6 57.5 172.7 36.4 18.3 13.5 169.7 21.5

170.0 21.7

169.9 21.6

1 2 3 4 5 1 2 3 4 1 2

169.3 20.9

57.5 172.8 36.4 18.3 13.5 169.7 21.6

57.4 55.3

Assignments of 13C NMR data are based on DEPT, HMQC, and HMBC experiments. B

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Table 2. 1H NMR Data for Compounds 1−4 (δ in ppm, J in Hz, 400 MHz, in CDCl3)a position

1

1α 1β 2 3α 3β 4 6α 6β 7α 7β 8 10 11 12 14 15

16 17 18 19 20 OR-2

OCH3-6 OR-18

OR-19

2 2.48b 2.61b

2.73 t (16.6) 2.17 dd (16.6, 4.0) 2.33b 1.86 m 1.51 m

6.05 s

2.09 1.92 5.37 5.89

1.87 m 1.64 m

1.74 m 1.50 m

1.52 m 1.25 td (12.8, 4.0) 1.53 m 2.46 dd (14.3, 3.2) 1.54 m 1.41 m 2.09 m 6.43 dd (17.6, 10.8) 5.22 d (17.6) 5.04 d (10.8) 5.04 s 4.93 s 0.89 d (6.0) 6.48 d (7.2) 6.22 s 0.93 s

1.52 m 1.24 m

1.41 m 1.51 m

1.74 m 2.60b

1.64 m 2.22 dd (11.1, 5.3) 1.42 m 1.23 m 2.10 m 6.44 dd (17.6, 10.7) 5.18 d (17.6) 5.03 d (10.7) 5.04 s 4.94 s 0.89 d (6.6) 6.70 s 6.30 s 0.94 s 2.44 m 1.64 m 0.97 t (7.4) 1.18 d (6.9)

2.02 s

2.30 1.65 0.94 1.90

1.54 m 1.30 m 2.08 m 6.42 dd (17.6, 10.9) 5.22 d (17.6) 5.04 d (10.9) 5.05 s 4.93 s 0.93 d (7.2) 6.85 d (1.4) 6.39 s 0.94 s

2 3 4 5 2 3 4 2

3

2.31b 2.42b

1.93 s

t (7.3) m t (7.5) s

2.29 1.63 0.93 1.88

m m br s d (4.2)

t (7.3) m t (7.5) s

demonstrated that the other acetoxy group could be located at C-19. By further analyzing the HMQC, HMBC, and 1H−1H COSY spectra (Figure 1), all the proton and carbon signals were assigned unambiguously, which resulted in the establishment of the planar structure for 1. The relative configuration of compound 1 was established based on the NOESY spectrum and Chem3D modeling (Figure 2). NOESY correlations observed for H-1β/H-8, H-8/H-6β, H-

4 2.08 1.91 5.44 5.91

m m br s d (3.0)

3.29 dd (11.8, 4.0) 1.48 m 1.88 m 1.70 m 2.30b 1.49 m 1.24 m 2.09 m 6.43 dd (17.5, 11.2) 5.20 d (17.5) 5.03 d (11.2) 5.05 s 4.94 s 0.94 d (6.7) 6.68 t (1.5) 6.44 s 0.93 s 2.36 t (7.2) 1.71 m 1.00 t (7.4) 3.30 2.30 1.64 0.93 1.87

Figure 2. Key NOESY correlations of compounds 1−3 and 7.

1β/H-6β, H-3β/H-4, H-4/H-18, H-18/H-19, H-19/H2-11, H19/H-7α, H-7α/H2-11, H-7α/H3-17, H-10/H2-11, H-10/H-3α, and H-1α/H3-20, together with Chem3D modeling, suggested a conformation for compound 1 as depicted in Figure 2, where the two six-membered rings A and B are cis-fused and exist in a twist-boat and a chair conformation, respectively. Relative to ring B with a chair conformation, H-10 and C-17 are in an αposition with an equatorial orientation, while C-4 and C-20 are in a β-position with an equatorial orientation, C-19 and C-11 are in an α-position with an axial orientation, and C-1 is in a βposition with an axial orientation. The H-4 proton, relative to ring A, was found to be β-oriented, and H-18 and H-19, relative to ring C, were both β-oriented. Thus, the relative configuration of 1 was assigned as depicted. The absolute figuration of 1 was established from the CD spectrum and the octant rule. Compound 1 has a ketone carbonyl, and its CD spectrum displayed a negative Cotton effect at 293 nm (Δε = −6.01), corresponding to the n−π* transition of the cyclohexanone chromophore. From the (back) octant rule for cyclohexanones30 and the relative configuration of 1, the absolute configuration of 1 was assigned as 4R, 5S, 8R, 9R, 10S, 18R, and 19S, which was confirmed by comparing the experimental CD spectrum with those calculated by the TDDFT method.31 The optimized geometries were obtained by a systematic conformational search with the MMFF94 force field and further optimized at the B3LYP/6-31+G(d,p) level by the Gaussian 09 package.32,33 Then, the ECD spectra were calculated at the CAM-B3LYP/SVP level with the CPCM model in acetonitrile. The calculated ECD spectrum of 1b (Figure 3) matched the experimental results closely. All of the above evidence was used to confirm the structure of 1 as (4R,5S,8R,9R,10S,18R,19S)-

s t (7.3) m t (7.4) s

a

Assignments of 1H NMR data are based on 1H−1H COSY, HMQC, and HMBC experiments. bSignals were in overlapped regions of the spectra, and the multiplicities could not be discerned.

Figure 1. 1H−1H COSY and key HMBC correlations of compound 1.

positions of the two acetoxy groups were determined via HMBC correlations. Thus, the HMBC correlation of the acetal proton H-18 (δH 6.48) with the carbonyl signal at δC 169.5 (CO of the acetoxy) indicated the presence of an acetoxy group at C-18. Similarly, the long-range coupling of the proton signal at δH 6.22 (H-19) with the carbonyl carbon signal at δC 169.3 C

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(5S,8R,9R,10S,18R,19S)-19-acetoxy-18-butanoyloxy-18,19-epoxycleroda-3,13(16),14-trien-2-one. The molecular formula of compound 3 was determined as C31H46O7 based on the HRESIMS (m/z 548.3576 [M + NH4]+, calcd for C31H50NO7, 548.3587). Its 1H and 13C NMR spectra showed close similarities to those of compound 2, which implied that 3 is also a clerodane diterpene. Besides the same acetoxy and butanoyloxy groups in these two compounds, an additional 2-methylbutanoyloxy group in 3 was deduced and defined from the corresponding carbon and proton signals (Tables 1 and 2), based on data reported for other diterpenoids from the genus Casearia in the literature.7−10 Following the careful comparison of the chemical shifts of the skeletal carbons (C-1−C-20), it could be shown that the conjugated ketone carbonyl carbon [δC 199.1 (C-2)] in 2 is replaced by an oxygenated carbon [δC 66.4 (C-2)] in compound 3. To assign the proton and carbon signals unambiguously, HMQC, HMBC, and 1H− 1H COSY experiments were carried out. By interpretation of these 2D NMR spectra, the assignments of the skeletal proton and carbon signals were accomplished, which verified the presence of an oxygenated carbon at δC 66.4 in 3 instead of a carbonyl carbon at δC 199.1 in 2. The locations of the acyloxy groups were determined on the basis of the HMBC spectrum, in which the correlations of the protons at δH 5.37 (H-2), 6.70 (H-18), and 6.30 (H-19) with the carbonyl carbons at δC 175.8, 172.7, and 169.8 demonstrated the 2methylbutanoyloxy, butanoyloxy, and acetoxy groups to be attached at C-2, C-18, and C-19, respectively. Thus, the planar structure for 3 was established. The NOESY correlations of H1β/H-8, H-8/H-6β, H-1β/H-6β, H-18/H-19, H-19/H2-11, H19/H-7α, H-7α/H2-11, H-7α/H3-17, H-10/H2-11, and H-1α/ H3-20 and Chem3D modeling revealed a molecular conformation for compound 3 as depicted in Figure 3. Thus, H-10 and C-17, relative to ring B, were assigned in an α-position with an equatorial orientation, C-4 and C-20 in a β-position with an equatorial orientation, C-19 and C-11 in an α-position with an axial orientation, and C-1 in a β-position with an axial orientation. The H-18 and H-19 protons, relative to ring C, were assigned as both β-oriented, and the H-2 proton, relative to ring A, was assigned as β-oriented. Considering the biosynthetic origin of compound 3, and by comparison with compound 1, the absolute configurations of C-5, C-8, C-9, C10, C-18, and C-19 were inferred as 5S, 8R, 9R, 10S, 18R, and 19S, respectively, and the absolute configuration of C-2, consequently, was determined as 2R. However, the absolute configuration of the chiral carbon of 2-methylbutanoyloxy was not able to be determined, since X-ray diffraction was not used for either compound 3 or one of its derivatives.10,25 Compound 3 (balanspene C) was therefore characterized as (2R,5S,8R,9R,10S,18R,19S)-19-acetoxy-18-butanoyloxy-18,19epoxy-2-(2ξ-methylbutanoyloxy)cleroda-3,13(16),14-triene. The 1H and 13C NMR spectra of compounds 4−6 were found to be similar to each other. Analyses of the 13C and 1H NMR data (Tables 1−3) of the three compounds revealed that compounds 4−6 all have the same clerodane-type diterpene skeleton as in compound 3.7−10 The main differences between compounds 3−6 were found to be in the different substituent groups present in each case. For compound 4, in addition to a methoxy group (δH 3.30 s; δC 57.5), an acetoxy and two butanoyloxy groups were deduced and determined according to its 13C and 1H NMR spectra. Using the same HMBC and NOESY experiments as for compounds 1−3, the positions of these substituent groups and the relative configuration of

Figure 3. Calculated ECD spectra of 1a (4S,5R,8S,9S,10R,18S,19R)and 1b (4R,5S,8R,9R,10S,18R,19S)-isomers and the experimental ECD spectrum of 1 in acetonitrile.

18,19-diacetoxy-18,19-epoxycleroda-13(16),14-dien-2-one, which has been named balanspene A. Compound 2, a colorless oil, gave a molecular formula of C26H36O6 as determined from the HRESIMS (462.2850 [M + NH4]+, calcd for C26H40NO6, 462.2856). From the 1H NMR spectrum of 2, four methyl groups, six olefinic protons, and two oxygenated methine protons (Table 2) were displayed. The 13C NMR spectrum of 2 showed 26 carbon resonances. From the 1 H and 13C NMR spectra, an acetoxy and a butanoyloxy group were deduced and defined from the observation of the following carbon signals (δC 169.5, 21.2, 172.2, 36.1, 18.2, and 13.5) and the corresponding proton signals (Table 2), based on reported diterpenes with acyloxy groups from the genus Casearia.7−10 Apart from the above six resonances for the substituent groups, the remaining 20 carbon resonances observed in the 13C NMR spectrum of compound 2 constituted a clerodane-type diterpene skeleton with an additional double bond at C-3 and C-4 (Table 1).7−10 Furthermore, an HMBC experiment confirmed the presence of a clerodane diterpene scaffold for compound 2, and the ketone carbonyl carbon at δC 199.1 conjugated with the double bond of C-3 and C-4 was assigned to C-2. As in the case of compound 1, the interpretation of the HMBC spectrum allowed the two acyloxy groups to be assigned. The HMBC correlations of the acetal proton signals at δH 6.85 (H-18) and 6.39 (H-19) with the carbonyl carbons at δC 172.2 and 169.5, respectively, demonstrated the butanoyloxy group to be attached at C-18, and the acetoxy group at C-19. Further analysis of the HMQC, HMBC, and 1H−1H COSY spectroscopic data led to the assignments of all the proton and carbon signals. Thus, the planar structure for 2 could be disclosed. The relative configuration of this compound was deduced on the basis of the NOESY spectrum and Chem3D modeling as in the case for compound 1. The NOESY correlations observed for H-1β/H8, H-8/H-6β, H-1β/H-6β, H-18/H-19, H-19/H2-11, H-19/H7α, H-7α/H2-11, H-7α/H3-17, H-10/H2-11, and H-1α/H3-20 supported a molecular arrangement for compound 2 as depicted in Figure 3, which was almost identical to that of 1 except for the additional double bond between C-3 and C-4. Hence, from a biosynthetic standpoint, the absolute configurations of chiral carbons C-5, C-8, C-9, C-10, C-18, and C-19 in 2 should be the same as those in 1. Compound 2 (balanspene B) was therefore elucidated as D

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Table 3. 1H NMR Data for Compounds 5−8 (δ in ppm, J in Hz, 400 MHz, in CDCl3)a position

5

6

1α 1β 2

2.08 m 1.92 m 5.42 m

1.92 m 1.71 m 5.46 m

3

6.00 d (3.0)

6

5.92 dd (4.2, 1.5) 3.33b

7α 7β 8 10

1.48 1.86 1.72 2.35

1.49 1.85 1.71 2.32

11

1.51 m 1.28 m 2.08 m 6.44 dd (17.5, 10.8) 5.16 d (17.5) 5.02 d (10.8) 5.04 s 4.94 s 0.93 d (6.7) 6.68 t (1.6) 6.43 s 0.92 s 2.45 m 1.68 m 0.96 t (7.4) 1.18 d (7.0) 3.30 s 2.29 t (7.3) 1.63 m 0.94 t (7.3) 1.86 s

12 14 15

16 17 18 19 20 OR-2

OCH3-6 OR-18

OR-19

2 3 4 5 2 3 4 2

m m m t (8.7)

3.30b m m m t (8.4)

7

8

2.18 m 1.67 m 5.57 dd (8.5, 7.0) 5.93 s

1.78 m 1.89 m 5.53 m

3.50 dd (12.0, 3.6) 1.50 m 1.88 m 1.77 m 2.34 dd (13.9, 2.5) 1.48 m 1.23 m 2.07 m 6.41 dd (17.6, 10.9) 5.21 d (17.6) 5.03 d (10.9) 5.02 s 4.92 s 0.95 d (6.5) 5.38 s 6.34 s 0.95 s 2.09 s

3.35b

1.49 m 1.32 m 2.08 m 6.42 dd (17.6, 10.8) 5.14 d (17.6) 5.01 d (10.8) 5.04 s 4.94 s 0.95 d (6.8) 5.44 s 6.40 s 0.90 s 2.43 m 1.69 m 0.97 t (7.4) 1.16 d (7.0) 3.29 s 3.39 s

3.33 s 3.38 s

1.86 s

1.84 s

6.85 d (5.0)

1.06 m 1.51 m 1.67 m 2.45 dd (12.9, 5.2) 1.97 m 1.66 m 2.00 m 6.34 dd (17.7, 11.0) 5.20 d (17.7)

Figure 4. Calculated ECD spectra of 4a (2R,5S,6S,8R,9R,10S,18R,19S)- and 4b (2S,5R,6R,8S,9S,10R,18S,19R)-isomers and the experimental ECD spectrum of 4 in acetonitrile.

therefore elucidated as (2R,5S,6S,8R,9R,10S,18R,19S)-19-acetoxy-2,18-dibutanoyloxy-18,19-epoxy-6-methoxycleroda-3,13(16),14-triene, (2R,5S,6S,8R,9R,10S,18R,19S)-19-acetoxy-18butanoyloxy-18,19-epoxy-6-methoxy-2-(2ξmethylbutanoyloxy)cleroda-3,13(16),14-triene, and (2R,5S,6S,8R,9R,10S,18S,19S)-19-acetoxy-18,19-epoxy-6,18-dimethoxy-2-(2ξ-methylbutanoyloxy)cleroda-3,13(16),14-triene, which have been named balanspenes D−F, respectively. Compound 7 gave a molecular formula of C26H38O7 based on the HRESIMS (m/z 485.2509 [M + Na]+, calcd for C26H38NaO7, 485.2515). The 1H and 13C NMR spectra suggested this compound to have the same scaffold as compounds 4−6 and four substituent groups (two acetoxy moieties and two methoxy groups) present, as supported by the 2D NMR data. The HMBC correlations of the protons at δH 5.57 (H-2) and 6.34 (H-19) with the corresponding carbonyl carbons at δC 170.5 and 169.9, respectively, indicated the two acetoxy groups to be attached to C-2 and C-19. In turn, the locations of the two methoxy groups were verified at C-6 and C-18, respectively, from the long-range HMBC couplings of H6 (δH 3.50) and H-18 (δH 5.38) to the corresponding methoxy carbons. The same clerodane skeleton for 7 as those of compounds 4−6 was proposed, as supported by NOESY data and Chem3D modeling. For the orientation of the H-2 proton, the coupling constants (J2,1 = 8.5, 7.0 Hz), the observed NOESY correlation of H-2/H-1α, and the chemical shift of C-2 shifted downfield by about 5 ppm compared to those in compounds 4−6 revealed an α-orientation of H-2 in 7 instead of the β-orientation of H-2 in compounds 4−6. The two methoxy groups at C-6 and C-18 and the acetoxy group at C-19 were determined as being in α-positions by the corresponding NOESY correlations. After defining the relative configuration of 7, TDDFT CD calculations were performed to elucidate the absolute configuration of 2S, 5S, 6S, 8R, 9R, 10S, 18R, and 19S, which was deduced from the same biosynthetic origin of compounds 1−7. By comparison of its experimental and calculated CD spectra (Figure 5), the absolute configuration of 7 was determined as being the same, again taking into account the biosynthetic origin. On the basis of the above evidence, the structure of compound 7 (balanspene G) was established as (2S,5S,6S,8R,9R,10S,18S,19S)-2,19-diacetoxy-18,19-epoxy-6,18dimethoxycleroda-3,13(16),14-triene.

5.03 d (11.0) 5.04 s 4.99 s 0.92 d (6.1) 9.35 s 10.47 s 0.94 s 2.45 m 1.68 m 0.93 t (7.2) 1.16 d (6.9) 3.27 s

a

Assignments of 1H NMR data are based on 1H−1H COSY, HMQC, and HMBC experiments. bSignals were in overlapped regions of the spectra, and the multiplicities could not be discerned.

compound 4 were determined, with the methoxy group attached at C-6 with an α-equatorial orientation, the acetoxy group at C-19 with an α-orientation, and the two butanoyloxy groups at C-2 and C-18 both in α-positions. The absolute configuration of 4 was assigned as 2R, 5S, 6S, 8R, 9R, 10S, 18R, and 19S, by comparison of its experimental CD spectrum (Figure 4) with those calculated by the TDDFT CD method,31 which was also supported from a biosynthetic standpoint. For compound 5, besides the same methoxy group and butanoyloxy group as present in compound 4, a 2-methylbutanoyloxy group was deduced and defined on the basis of its 13C and 1H NMR data, which was located at C-2, replacing the butanoyloxy group in compound 4. Compound 6 was found to possess a methoxy moiety at C-18 instead of the butanoyloxy moiety in 5. The same relative configuration for compounds 4−6 was revealed by the careful comparison of their NOESY spectra. On the basis of the above spectroscopic evidence, biosynthetic considerations, and the absolute configuration of 4 elucidated by the TDDFT CD method, the structures of compounds 4−6 were E

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method previously reported.34 NGF was used as the positive control.35−38 Compounds 4−7, 9, and 10 had no effects on neurite outgrowth from PC12 cells in the absence of NGF, but markedly increased the NGF (20 ng/mL)-induced proportion of neurite-bearing cells. The EC50 values of these active clerodane diterpenes to stimulate NGF-mediated neurite outgrowth dose-dependently are shown in Table 4. However, compounds 1−3 and 8 showed no activities on the proportion of neurite-bearing cells in either the absence or presence of NGF (20 ng/mL). Table 4. EC50 Values of Compounds 4−7, 9, and 10 Stimulating NGF-Mediated Neurite Outgrowth from PC12 Cells compound

EC50 (μg/mL)

compound

EC50 (μg/mL)

4 5 6 7

20.2 12.7 4.0 2.4

9 10 NGFa

2.5 7.5 4.8 × 10−2

Figure 5. Calculated ECD spectra of 7a (2S,5S,6S,8R,9R,10S,18S,19S)and 7b (2R,5R,6R,8S,9S,10R,18R,19R)-isomers and the experimental ECD spectrum of 7 in acetonitrile. a

NGF was used as a positive control. Data are presented based on three experiments.

Compound 8 (balanspene H) was obtained as a colorless oil. It gave the molecular formula C26H38O5 as determined from the HRESIMS (m/z 431.2791 [M + H]+, calcd for C26H39O5, 431.2798). The 1H and 13C NMR spectra revealed the presence of a 2-methylbutanoyloxy and a methoxy group based on the substituent groups in compounds 2−7. Apart from the signals for the substituent groups, there were additional 20 skeletal carbons displayed in the 13C NMR spectrum, which again gave the typical characteristics of a clerodane diterpene like in compounds 1−7.7−10,16 On comparison of the chemical shifts of skeletal carbons of 8 with those of 4−7, the main difference was that two aldehyde carbonyl signals were present in 8 instead of the two acetal carbon signals of C-18 and C-19 in compounds 4−7. From the HMQC and HMBC spectra, the presence of a clerodane-type diterpene skeleton could be corroborated, with aldehyde carbonyl carbons at δC 191.2 and 202.3 and the oxygenated carbons at δC 64.7 and 81.5 attributed to C-18, C-19, C-2, and C-6, respectively. Furthermore, a 2-methylbutanoyloxy group at C-2 and a methoxy group at C-6 were substantiated, respectively, from the long-range couplings of H-2 (δH 5.53) to the carbonyl carbon of the acyloxy group at δC 176.0 and H-6 (δH 3.35) to the methoxy carbon at δC 57.1. The planar structure of 8 was therefore elucidated. The similar clerodane-type diterpene skeleton of 8 compared to those of compounds 1−7 implied the same cis-fusion of the two six-membered rings, and further analysis of the NOESY spectrum led to the elucidation of the relative configuration of 8, where H-2 and H-6 are both in a βposition. On the basis of above evidence, and from biosynthetic considerations, the same conformations of the two sixmembered rings as in compounds 1−7 could be inferred, so compound 8 was characterized as (2R,5S,6S,8R,9R,10S)-6methoxy-2-(2ξ-methylbutanoyloxy)cleroda-3,13(16),14-triene18,19-dial. Studies on the biology of NGF have demonstrated that compounds to promote the neurite outgrowth of nerve cells against neuron degeneration may be useful for the treatment of Alzheimer’s disease.21,22 In order to search for bioactive substances to promote the neurite outgrowth of nerve cells as candidates for the treatment of neurodegenerative diseases, clerodane diterpenes 1−10 isolated from the twigs of C. balansae were evaluated for their enhancing activities of NGFinduced neurite outgrowth from PC12 cells, according to a

In summary, the present phytochemical investigation on C. balansae has led to the isolation and characterization of eight new (1−8) and two known (9 and 10) clerodane diterpenes. Besides their relative configurations, the absolute configurations of compounds 1−8 were determined on the basis of NMR data analysis, and their experimental and calculated ECD spectra, and from the compound biosynthetic origin. This is the first report of the absolute configuration of clerodane diterpenes from the species C. balansae. In the biological screening conducted, compounds 4−7, 9, and 10 exhibited potentiating activities of NGF-mediated neurite outgrowth from PC12 cells, and, of these, compound 7 exerted the greatest promotion of NGF-mediated neurite outgrowth from PC12 cells. These five bioactive diterpenes may be useful for the development of antineurodegenerative agents for Alzheimer’s disease and other neurological disorders.21,22

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). CD spectra were obtained on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). IR spectra were taken on a Bruker Tensor 27 FT-IR spectrometer with KBr disks. 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (Bruker, Switzerland, 400 MHz for 1H and 100 MHz for 13C) with TMS as an internal standard. ESIMS were acquired on a Thermo Finnigan LCQAdvantage mass spectrometer. HRESIMS were recorded by an Agilent 6520 Q-TOF LC/MS. 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 twigs of C. balansae were collected from Xishuangbanna, Yunnan Province, People’s Republic of China, in F

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Balanspene G (7): colorless oil; [α]12 D −22.4 (c 0.42, CH2Cl2); CD (CH3CN) 194 (Δε −31.4) nm; IR (KBr) νmax 2966, 2933, 1739, 1596, 1452, 1373, 1227, 1106, 949, 737 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 485 [M + Na]+; HRESIMS m/z 485.2509 [M + Na]+ (calcd for C26H38NaO7, 485.2515). Balanspene H (8): colorless oil; [α]12 D + 431.7 (c 0.12, CH2Cl2); IR (KBr) νmax 2968, 2930, 1712, 1596, 1276, 1261, 750 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 431 [M + H]+; HRESIMS m/z 431.2791 [M + H]+ (calcd for C26H39O5, 431.2798). Computational Methods. For those compounds for which the absolute configurations were determined, their calculated ECD spectra were obtained using the TDDFT method as follows. A preliminary conformational search was carried out in Conflex 6.7 using the MMFF94 force field.32 Conformers within 6 kcal/mol were saved and further optimized at the B3LYP/6-31+G(d,p) level in the Gaussian 09 package.33 The stable conformers with populations greater than 1% were submitted to ECD calculation by the TDDFT [B3LYP/631+G(d,p)] method with the CPCM model in acetonitrile, and the calculated ECD spectra of different conformers were simulated with a half-bandwidth of 0.3 eV. The final ECD curves were generated according to the Boltzmann distribution of each conformer. Bioassay for Neurite Outgrowth. PC12 cells were cultured at 37 °C in DMEM 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 water-saturated atmosphere of 95% air and 5% CO2. The cells were disassociated by incubation with 1 mM ethylene glycol-bis(2-aminoethyl 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 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.

August 2013. The botanical identification was made by one of the authors (Y.G.), and a voucher specimen (No. 20130808) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University. Extraction and Isolation. The air-dried twigs of C. balansae (8.0 kg) were powdered and extracted with MeOH (3 × 48 L) under reflux. The organic solvent was evaporated to afford a crude extract (510 g). The extract was suspended in H2O (0.8 L) and partitioned with EtOAc (3 × 0.8 L). The EtOAc-soluble portion (135.0 g) was subjected to silica gel column chromatography, using a gradient of acetone in petroleum ether (1−40%), to give seven fractions (F1−F7) based on TLC analysis. Fraction F4 was separated by MPLC over ODS eluting with a step gradient of 70−90% MeOH in H2O to give four subfractions (F4‑1−F4‑4). Subfraction F4‑2 was purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 79% MeOH in H2O) to afford compounds 1 (tR = 27 min, 18.9 mg), 6 (tR = 41 min, 20.9 mg), and 7 (tR = 33 min, 14.9 mg). Compound 4 (tR = 24 min, 13.7 mg) was isolated from subfraction F4‑3 (82% MeOH in H2O), and compound 5 (tR = 26 min, 15.0 mg) was obtained from subfraction F4‑4 (85% MeOH in H2O) using the same HPLC system. Fraction F3, with the same procedures as for F4, provided three subfractions, F3‑1− F3‑3, and compound 2 (tR = 34 min, 11.9 mg) was obtained from F3‑1 (87% MeOH in H2O). Fraction F2 was subjected to the above MPLC procedure to yield three subfractions, F2‑1−F2‑3, and the further purification of F2‑3 with the same HPLC system conditions using 90% MeOH in H2O for elution resulted in the isolation of compound 3 (tR = 27 min, 24.4 mg). Using the same protocols for the above fractions and subfractions, F5 yielded five subfractions, F5‑1−F5‑5. Compound 8 (tR = 36 min, 8.9 mg) was isolated from F5‑5 (82% MeOH in H2O), compound 9 (tR = 28 min, 12.7 mg) was obtained from F5‑2 (73% MeOH in H2O), and the purification of F5‑4 (76% MeOH in H2O) afforded compound 10 (tR = 28 min, 13.2 mg). Balanspene A (1): colorless oil; [α]12 D + 46.7 (c 0.18, CH2Cl2); CD (CH3CN) 217 (Δε −2.62), 236 (Δε +0.48), 293 (Δε −6.01) nm; IR (KBr) νmax 2966, 2928, 1752, 1716, 1596, 1374, 1232, 1046, 953, 749 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 436 [M + NH4]+; HRESIMS m/z 436.2695 [M + NH4]+ (calcd for C24H38NO6, 436.2699). Balanspene B (2): colorless oil; [α]12 D +193.1 (c 0.26, CH2Cl2); IR (KBr) νmax 2966, 2937, 1754, 1677, 1597, 1374, 1213, 1062, 949, 750 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 462 [M + NH4]+; HRESIMS m/z 462.2850 [M + NH4]+ (calcd for C26H40NO6, 462.2856). Balanspene C (3): colorless oil; [α]12 D −35.8 (c 0.33, CH2Cl2); IR (KBr) νmax 2966, 2937, 1753, 1730, 1596, 1375, 1217, 1060, 1010, 932, 750 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 548 [M + NH4]+; HRESIMS m/z 548.3576 [M + NH4]+ (calcd for C31H50NO7, 548.3587). Balanspene D (4): colorless oil; [α]12 D +50.7 (c 0.15, CH2Cl2); CD (CH3CN) 203 (Δε +27.4) nm; IR (KBr) νmax 2966, 2936, 1753, 1731, 1596, 1372, 1276, 1099, 1065, 1007, 750 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 564 [M + NH4]+; HRESIMS m/z 564.3533 [M + NH4]+ (calcd for C31H50NO8, 564.3536). Balanspene E (5): colorless oil; [α]12 D +38.3 (c 0.23, CH2Cl2); IR (KBr) νmax 2966, 2932, 1753, 1730, 1596, 1461, 1373, 1222, 1098, 1065, 947, 749 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 578 [M + NH4]+; HRESIMS m/z 578.3685 [M + NH4]+ (calcd for C32H52NO8, 578.3693). Balanspene F (6): colorless oil; [α]12 D +481.9 (c 0.21, CH2Cl2); IR (KBr) νmax 2965, 2931, 1752, 1728, 1596, 1461, 1373, 1225, 1108, 948, 750 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 522 [M + NH4]+; HRESIMS m/z 522.3424 [M + NH4]+ (calcd for C29H48NO7, 522.3431).

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ASSOCIATED CONTENT

S Supporting Information *

The 1D and 2D NMR spectra of compounds 1−8. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 86-22-23502595. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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