Characterization and Biological Evaluation of Diterpenoids from

Article pubs.acs.org/jnp

Characterization and Biological Evaluation of Diterpenoids from Casearia graveolens Jing Xu,†,‡ Feifei Ji,†,‡,⊥ Xiaocong Sun,†,‡ Xiangrong Cao,†,‡ Shen Li,†,‡ Yasushi Ohizumi,∥ and Yuanqiang Guo*,†,‡ †

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy and ‡Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People’s Republic of China ⊥ College of Pharmacy, Harbin University of Commerce, Harbin 150076, People’s Republic of China ∥ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan S Supporting Information *

ABSTRACT: Biologically active substances that promote the neurite outgrowth of nerve cells against neuron degeneration may be useful for the treatment of Alzheimer’s disease. In a continuing search for bioactive compounds from plants, an ethyl acetate-soluble extract of the twigs of Casearia graveolens showed moderate stimulatory activity of neurite outgrowth from PC12 cells. Further investigation to obtain bioactive compounds led to the isolation of 10 new clerodane diterpenoids, graveopenes A− J (1−10). Their structures including absolute configurations were elucidated based on analysis of their NMR spectroscopic data and experimental and calculated ECD spectra. Compounds 3−6 and 8 were shown to stimulate NGF-mediated neurite outgrowth from PC12 cells.

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Province of mainland China,26 and preliminary reports have described the presence of several coumarins in this plant.27,28 In the course of an ongoing search for bioactive substances from plants,24,29−31 attention has been given to the occurrence of bioactive compounds having nerve growth factor (NGF)potentiating effects, since active compounds of this type are expected to be potentially useful for the treatment of AD and other neurological disorders.4,32 In a preliminary screening procedure, an ethyl acetate-soluble extract of the twigs of C. graveolens showed moderate stimulatory activity of neurite outgrowth from PC12 cells, which prompted further investigation of the bioactive constituents of this plant. As a result, 10 new compounds, graveopenes A−J (1−10), were isolated from the twigs of C. graveolens. Their structures, including their absolute configurations, were established by analysis of their NMR spectroscopic data and their experimental and calculated ECD spectra. 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.

lzheimer’s disease (AD), the most common neurodegenerative disorder, affects about 2% of the population over 65 years of age and is a major threat to the health of elderly persons.1 Patients with AD usually have characteristic symptoms of progressive impairment in memory, decision making, orientation to physical surroundings, judgment, and language, all of which deteriorate the quality of life severely and cause great concern to themselves and their families.1 In light of the severity of AD, some drugs, such as donepezil, galantamine, and rivastigmine, have been approved for the treatment of AD. Although these drugs are beneficial in improving cognitive and behavioral symptoms, their effectiveness has been questioned since they cannot prevent or delay neurodegeneration.2,3 Therefore, there is an urgent need to develop new agents to treat AD effectively.4,5 One strategy to develop new agents for AD is to search for biologically active substances or lead compounds from plant secondary metabolites, which have been proven to be an effective and practicable method for many new medicinal agents.6 The genus Casearia, a member of the family Flacourtiaceae, contains about 180 species that are distributed widely in South America, Asia, tropical Africa, and northwest Australia.7 Some Casearia species have been used traditionally as folk medicines for the treatment of various medical indications.7 Compounds reported from this genus include mainly terpenoids, phenylethanoids, flavonoids, phenolics, steroids, and volatile oil constituents,7−25 displaying diverse biological effects, such as antifungal, cytotoxic, antimalarial, antimicrobial, and DNAmodifying activities.7 The species Casearia graveolens Dalzell, a nonmedicinal plant, is a tree distributed mainly in Yunnan © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The EtOAc-soluble part of the MeOH extract of the twigs of C. graveolens afforded 10 new diterpenoids (1−10). Compound 1 was obtained as a colorless oil. Its molecular formula was determined as C26H38O6 by HRESIMS, from the Received: July 2, 2015

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DOI: 10.1021/acs.jnatprod.5b00583 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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butyryl moiety was deduced and defined from the observation of the following carbon signals (δC 171.8, 36.1, 18.0, and 13.6) and the corresponding methyl and methylene proton signals (Table 2).29 Apart from the above six carbon signals for the substituent groups, there were an additional 20 resonances displayed in the 13C NMR spectrum, including four olefinic, one ketone carbonyl, and two acetal carbons based on the DEPT and HMQC spectra. These 20 typically skeletal carbons, especially the two acetal carbons and the four olefinic ones forming two terminal double bonds, implied that compound 1 possesses a characteristic clerodane-type diterpene skeleton as shown in Figure 1, based on the comparison of its chemical shifts with those of related compounds.33−36 This characteristic skeleton of a clerodane-type diterpene was confirmed by HMBC and 1H−1H COSY experiments, and the four olefinic, one ketone carbonyl, and two acetal carbon signals at δC 145.0, 140.1, 112.5, 115.3, 212.5, 96.9, and 99.7 were assigned to C13, C-14, C-15, C-16, C-2, C-18, and C-19, respectively. The positions of the two acyloxy groups were deduced from the HMBC spectrum. The HMBC correlation of the carbonyl signal at δC 171.8 with the proton signal at δH 6.50 (H-18) demonstrated that a butyryloxy group is attached at C-18. Similarly, the long-range couplings of the carbonyl carbon signal at δC 169.4 with the proton signal at δH 6.22 (H-19) disclosed the presence of the acetoxy group at C-19. Further

peak at m/z 469.2562 [M + Na]+ (calcd for C26H38NaO6, 469.2566). The 1H NMR spectrum of 1 exhibited signals for five olefinic protons [δH 6.43 (1H, dd, J = 17.6, 10.9 Hz, H-14), 5.23 (1H, d, J = 17.6 Hz, H-15a), 5.04 (1H, d, J = 10.9 Hz, H15b), and 5.04 and 4.94 (each 1H, s, H2-16)] and two oxygenated methine protons [δH 6.50 (1H, d, J = 7.1 Hz, H-18) and 6.22 (1H, s, H-19)]. Additionally, signals for four methyl groups [δH 0.90 (3H, d, J = 6.3 Hz, H3-17), 0.93 (3H, s, H320), 0.93 (3H, t, J = 7.5 Hz, COCH2CH2CH3-18), and 1.92 (3H, s, COCH3-19)] were also observed. The 13C NMR spectrum of 1 showed 26 carbon resonances (Table 1). From the 1H and 13C NMR spectra, one acetyl group was apparent from the methyl singlet (δH 1.92 s) and the corresponding carbons (δC 169.4 and 21.3). In addition to this acetyl group, a

Table 1. 13C NMR Spectroscopic Data for Compounds 1−10 (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 OR-2b

OCH3-6b OR-18b

OR-19b

1

2

3

4

5

6

7

8

9

10

37.8 212.5 37.2 47.8 49.5 34.0 27.6 37.7 37.1 35.4 27.1 23.7 145.0 140.1 112.5 115.3 15.5 96.9 99.7 26.2

28.9 64.2 123.7 145.5 49.3 29.6 27.2 37.2 37.2 33.4 28.2 23.6 145.4 140.3 112.5 115.3 15.8 94.4 99.8 26.1

27.1 66.2 121.2 146.3 53.0 81.9 31.1 36.8 37.4 36.5 27.9 23.8 145.1 140.4 112.3 115.6 15.9 96.0 98.4 25.6 174.0 28.0 9.3

27.2 66.1 120.9 146.9 52.8 82.3 31.1 36.8 37.4 36.4 27.9 23.7 145.3 140.4 112.3 115.4 15.9 104.4 98.0 25.7 174.1 27.9 9.2

29.6 64.3 124.2 147.4 54.1 82.8 31.4 36.9 37.5 35.0 28.6 23.5 146.2 140.2 112.7 113.6 15.9 103.8 105.3 25.7

26.2 66.5 120.4 147.3 49.2 29.4 27.2 37.2 37.2 34.3 28.1 23.6 145.3 140.4 112.2 115.4 15.7 94.3 99.6 26.0 173.1 36.6 18.7 13.6

26.2 66.4 120.4 147.1 49.2 29.4 27.2 37.1 37.1 34.3 28.1 23.6 145.3 140.4 112.2 115.4 15.7 94.4 99.6 26.0 173.1 36.6 18.7 13.6

172.9 36.3 18.3 13.6 170.1 21.4

57.5 172.8 36.4 18.3 13.5 169.8 21.7

57.5 55.6

27.2 66.1 120.9 147.0 52.9 82.3 31.1 36.9 37.4 36.4 27.8 23.8 145.3 140.5 112.2 115.5 15.9 104.4 98.0 25.7 173.3 36.5 18.6 13.7 57.5 55.5

29.3 64.5 124.4 145.9 47.4 29.8 27.4 37.1 37.2 33.2 28.0 23.7 145.6 140.3 112.5 115.3 15.8 103.8 99.8 26.0

171.8 36.1 18.0 13.6 169.4 21.3

27.0 66.2 121.2 146.1 53.0 81.9 31.0 36.4 37.4 36.8 27.8 23.7 145.1 140.4 112.2 115.5 15.9 96.1 98.3 25.5 173.1 36.5 18.7 13.6 57.5 170.2 21.3

56.5

57.8 55.5

170.2 21.4

169.8 21.7

170.2 21.8

170.6 21.6

172.8 36.3 18.2 13.5 170.0 21.3

1 2 3 4 1 2 3 4 1 2

170.2 21.8

54.5

170.0 21.2

a

Assignments of 13C NMR data are based on DEPT, HMQC, and HMBC experiments. bThe number with the superscript indicates the location of the substituent group in the parent skeleton. B



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Table 2. 1H NMR Spectroscopic Data for Compounds 1−5 (δ in ppm, J in Hz, in CDCl3)a position

1 2.45b 2.31b

1α 1β 2 3α 3β 4 6α 6β 7α 7β 8 10 11 12 14 15 16 17 18 19 20 OR-2c

OCH3-6c OR-18c

OR-19c

2 2.04 1.93 4.37 6.91

3

m m br s d (4.3)

2.74 t (16.1) 2.16 dd (16.1, 4.2) 2.35b 1.85 m 1.53 m 1.48 m 1.28 td (13.3, 4.2) 1.54 m 2.48 dd (13.1, 3.0) 1.54 m 1.40 m 2.08 m 6.43 dd (17.6, 10.9) 5.23 d (17.6) 5.04 d (10.9) 5.04 s 4.94 s 0.90 d (6.3) 6.50 d (7.1) 6.22 s 0.93 s

1.72 m 1.46 m 1.50 m 1.39 m 1.68 m 2.14b 1.50 m 1.37 m 2.10 m 6.42 dd (17.6, 10.9) 5.23 d (17.6) 5.03 d (10.9) 5.04 s 4.94 s 0.88 d (6.7) 6.70 s 6.31 s 0.98 s

2.25 t (7.5) 1.60b 0.93 t (7.5) 1.92 s

2.30 1.66 0.94 1.89

2.01 1.87 5.46 5.90

2 3 4 2 3 4 2

t (7.4) sex (7.4) t (7.4) s

m m br s d (3.4)

4 2.10 1.98 5.49 6.01

m m br s d (3.4)

5 2.03 1.93 5.43 5.90

m m br s d (3.4)

3.29 dd (10.8, 3.8)

3.28 dd (11.1, 3.5)

3.28 dd (11.9, 3.8)

1.86 m 1.49 m 1.73 m 2.27b 1.50 m 1.27,m 2.08 m 6.43 dd (17.5, 10.8) 5.21 d(17.5) 5.04 d (10.8) 5.05 s 4.94 s 0.95 d (7.3) 6.68 s 6.44 s 0.93 s 2.40 q (7.6) 1.19 t (7.6)

1.85 1.50 1.72 2.28 1.49 1.25 2.08 6.43 5.19 5.02 5.04 4.95 0.95 5.42 6.42 0.92 2.39 1.17

3.30 2.32 1.64 0.92 1.90

3.30 s 3.41 s

1.85 1.47 1.70 2.27 1.46 1.24 2.05 6.41 5.18 5.01 5.02 4.91 0.92 6.63 6.41 0.90 2.35 1.68 0.97 3.28 2.05

1.87 s

1.86 s

s t (7.3) sex (7.4) t (7.3) s

m m m dd (12.8, 3.2) m m m dd (17.5, 10.8) d (17.5) d (10.8) s s d (6.6) s s s q (7.5) t (7.5)

m m m dd (12.6, 4.6) m m m dd (17.6, 10.9) d (17.6) d (10.9) s s d (6.8) s s s t (7.3) sex (7.3) t (7.3) s 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. cThe number with the superscript indicates the location of the substituent group in the parent skeleton.

to be in an α-equatorial position, and C-19 and the side chain of C-11−C-16 were in an α-axial position. Relative to ring A, H-4 was assigned as β-axially oriented, which was supported by the coupling constant (J4,3α = 16.1 Hz) between H-4 and H-3α. In turn, H-18 and H-19, relative to ring C, were on the same side of ring C and both β-oriented. The relative configuration of 1 was therefore designated as depicted in Figure 2. The absolute configuration of 1 was established via experimental and calculated electronic circular dichroism (ECD) data, a tool to assign the absolute configuration of natural products.37,38 Starting from the conformation of 1 deduced from its NOESY spectrum and Chem3D modeling, conformational searches with the MMFF94 force field by MOE software39 and geometry optimizations by the Gaussian 09 package40 were performed. Then, the ECD spectra41 were calculated at the CAM-B3LYP/ SVP level with the CPCM model in acetonitrile. The ECD spectrum of 1 obtained (Figure 3) matched the experimental results closely, which suggested an absolute configuration of 4R, 5S, 8R, 9R, 10S, 18R, and 19S for compound 1. This absolute configuration of 1 was further substantiated by the octant rule.42 The experimental ECD spectrum of 1 displayed a negative Cotton effect at 291 nm, corresponding to the n−π* transition of the cyclohexanone chromophore, which verified the absolute configuration of 4R, 5S, 8R, 9R, 10S, 18R, and 19S unequivocally, according to the octant rule.42 Molecular orbital

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

analysis of the HMQC, HMBC, and 1H−1H COSY spectra (Figure 1) led to the assignments of all the proton and carbon signals. Thus, the planar structure for 1 could be established. The configuration of 1 was elucidated as follows. NOESY correlations observed for H-1β/H-8, H-8/H-6β, H-1β/H-6β, H-3β/H-4, H-4/H-18, H-18/H-19, H-19/H2-11, H-19/H-7α, H-7α/H2-11, H-7α/H3-17, H-10/H2-11, and H-10/H-3α, together with Chem3D modeling, supported a conformation for compound 1 as shown in Figure 2. In this molecular arrangement of 1, the two six-membered rings A and B were cisfused, with ring A having a twisted boat conformation, ring B a normal chair conformation, and ring C an envelope conformation. Relative to ring B, H-10 and C-17 were found C



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Figure 2. Conformations and key NOESY correlations of compounds 1 and 2.

s, H-2) for compound 2, suggesting the presence of an additional double bond and one more oxygenated carbon. To assign this double bond and the oxygenated carbon and verify the skeleton of 2, HMQC, HMBC, and 1H−1H COSY experiments were carried out. By interpretation of these 2D NMR spectra, this double bond was assigned between C-3 and C-4, the oxygenated carbon was assigned to C-2, and the clerodane diterpene skeleton was thus elucidated.45−48 A total of 26 carbons from the HRESIMS data and the two ester carbonyl carbons at δC 172.9 and 170.1 suggested the occurrence of two acyloxy groups (one acetoxy and one butyryloxy group) in 2, which were confirmed by the 2D NMR data. These acetoxy and butyryloxy groups were demonstrated to be attached at C-19 and C-18, respectively, by the HMBC correlations of H-19 (δH 6.31) and H-18 (δH 6.70) to the corresponding carbonyl carbons. All of the above information allowed the planar structure of compound 2 to be established. The relative configuration of 2 was deduced from the NOESY spectrum and Chem3D modeling (Figure 2). NOESY correlations observed for H-1β/H-8, H-8/H-6β, H-1β/H-6β, H-18/H-19, H-19/H2-11, H-19/H-7α, H-7α/H2-11, H-7α/H317, and H-10/H2-11, together with Chem3D modeling, implied a conformation for compound 2 as depicted in Figure 2. According to this arrangement and these NOESY correlations, two six-membered rings were cis-fused with C-19 and H-10 both in an α-position, and ring A was shown to have a twisted boat conformation with an α-orientation for the C-2 hydroxy group, ring B had a normal chair conformation with an αequatorial orientation for Me-17 and an α-axial orientation for the side chain of C-11−C-16, while ring C presented an envelope conformation with a β-orientation for both H-18 and H-19. The absolute configuration of 2 was established by comparison of the experimental and calculated ECD spectra. On the basis of the relative configuration of 2 deduced from the NOESY spectrum, the same procedures, conformational searches, geometry optimizations, and calculated ECD spectra applied as in the case of 1 were performed to determine the absolute configuration. As shown in Figure 4, the calculated ECD spectrum for (2R,5S,8R,9R,10S,18R,19S)-2 was in good agreement with the experimental data. Thus, compound 2 (graveopene B) was characterized as (2R,5S,8R,9R,10S,18R,19S)-18-butyryloxy-19-acetoxy-18,19-epoxycleroda-3,13(16),14-trien-2-ol. The molecular formula of compound 3 was determined as C30H44O8 based on the HRESIMS (m/z 555.2930 [M + Na]+, calcd for C30H44NaO8, 555.2934). Its 1H and 13C NMR spectra

Figure 3. Calculated ECD spectra of 1 and its enantiomer and the experimental ECD spectrum of 1 in acetonitrile.

(MO) analysis of the predominant conformer of 1 at the CAMB3LYP/SVP level in acetonitrile gave us more information to better understand the ECD spectrum (Figure 3 and Supporting Information Figure S47).43,44 The wave trough at 291 nm in the experimental spectra might be caused by the electronic transitions from MO120 to MO123 involving the n−π* transition in the ketone carbonyl. In addition, the other wave trough at 213 nm may be caused by the electronic transitions from MO121 to MO122 involving the π−π* transition in the conjugated diene. On the basis of the above evidence, the structure of 1 was established as (4R,5S,8R,9R,10S,18R,19S)18-butyryloxy-19-acetoxy-18,19-epoxycleroda-13(16),14-dien2-one, which has been named graveopene A. Compound 2 was also obtained as a colorless oil. Its HRESIMS showed a molecular ion at m/z 469.2562 [M + Na]+, corresponding to the molecular formula C26H38O6. The 1 H NMR spectrum of 2 displayed two similar acetal proton signals to those shown in compound 1 and five olefinic resonances (Table 2) attributable to two terminal double bonds. Corresponding to these diagnostic protons, two acetal carbon resonances and four olefinic signals representative of two terminal double bonds were evident from the 13C NMR spectrum. These spectroscopic features implied that 2 is a clerodane-type diterpene related structurally to 1. Comparison of the 1H NMR spectra of 1 and 2 indicated an additional olefinic proton signal at δH 6.91 (1H, d, J = 4.3 Hz, H-3) and an additional oxygenated methine proton signal at δH 4.37 (1H, br D



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ECD spectra of compounds 2 and 3, the absolute configuration was assigned as 2R, 5S, 6S, 8R, 9R, 10S, 18R, and 19S. Compound 3 (graveopene C) was elucidated therefore as (2R,5S,6S,8R,9R,10S,18R,19S)-2-propionyloxy-6-methoxy-18butyryloxy-19-acetoxy-18,19-epoxycleroda-3,13(16),14-triene. Compound 4 (graveopene D) gave a molecular formula of C27H40O7 based on the HRESIMS (m/z 499.2672 [M + Na]+, calcd for C27H40NaO7, 499.2672). The 1H and 13C NMR spectra suggested this compound to have the same scaffold as compound 3 with four substituent groups (one acetyl, one propionyl, and two methoxy groups) present, as supported by the 2D NMR data. The interpretation of the HMBC spectrum allowed these substituent groups to be assigned, indicating that the C-18 butyryloxy group in 3 is replaced by a methoxy group in compound 4. The relative configuration was deduced from the NOESY spectrum, which was the same as that of 3. On the basis of the same relative configuration and comparison of the ECD spectra of 2−4 (Figure 5), the absolute configuration of 4 was characterized as 2R, 5S, 6S, 8R, 9R, 10S, 18S, and 19S. Compound 4 was thus elucidated as (2R,5S,6S,8R,9R,10S,18S,19S)-2-propionyloxy-6,18-dimethoxy-19-acetoxy-18,19-epoxycleroda-3,13(16),14-triene. Analysis of the 13C and 1H NMR spectra of compound 5 (Tables 1 and 2) revealed that this compound has the same 2,6,18,19-tetrasubstituted-3,13(16),14-triene diterpene skeleton as compounds 3 and 4 and four substituent groups. These four substituents were determined as two acetoxy groups, a butyryloxy group, and a methoxy group, and their locations were determined from the HMBC spectrum. By comparison of the NOESY spectra and the ECD spectra of compounds 2−4, compound 5 (graveopene E) was characterized as (2R,5S,6S,8R,9R,10S,18R,19S)-2-butyryloxy-6-methoxy-18,19diacetoxy-18,19-epoxycleroda-3,13(16),14-triene. The 1H NMR and 13C NMR spectra of compound 6 were very similar to those of 5. The only difference found between these two compounds was that an acetoxy group in 5 is replaced by a methoxy group in 6. This substituted methoxy group in 6 was found to be located at C-18 instead of an acetoxy group in 5 based on the analysis of 2D NMR spectra. Additional butyryloxy, acetoxy, and methoxy groups in 6 were demonstrated to be attached at C-2, C-19, and C-6, respectively, by the HMBC correlations of H-2, H-19, and H6 to the corresponding carbonyl carbons. The structure of 6 (graveopene F) was thus assigned as (2R,5S,6S,8R,9R,10S,18S,19S)-2-butyryloxy-6,18-dimethoxy-19acetoxy-18,19-epoxycleroda-3,13(16),14-triene. Compound 7 was based on the same scaffold as those of compounds 2−6 and contained a methoxy group, an acetoxy group, and a hydroxy group, which were supported by the 2D NMR and HRESIMS data. The methoxy and acetoxy groups were located at C-18 and C-19, respectively, by interpretation of the HMBC spectrum. This compound could be assigned with the same relative configuration as for compounds 2−6; deduced from the NOESY and the ECD spectra, compound 7 (graveopene G) was elucidated as (2R,5S,8R,9R,10S,18S,19S)18-methoxy-19-acetoxy-18,19-epoxycleroda-3,13(16),14-trien2-ol. Compound 8 (graveopene H) was also obtained as a colorless oil. The 1H and 13C NMR spectra of this compound revealed it to be based on the same scaffold as that of 2−7. No acyloxy groups were apparent, but three methoxy moieties could be assigned from the 13C NMR spectrum. These methoxy moieties and one hydroxy group were attached at


closely resembled those of compound 2, which implied that 3 is also a clerodane diterpene. Besides the same acetyl and butyryl groups in these two compounds, an additional propionyl moiety in 3 was deduced and defined from the corresponding 13 C and 1H NMR signals (Tables 1 and 2), consistent with reported diterpenoids with such acyl groups in the literature.29,31 Additionally, one methoxy group was also deduced from a methoxy carbon signal (δC 57.5) and the corresponding proton signals. Apart from these substituent groups, 20 residual carbons were displayed in the 13C NMR spectrum, of which one more oxygenated carbon was apparent when compared to those of compound 2. This oxygenated carbon signal was assigned at C-6 by interpretation of the HMQC and HMBC spectra. Further analysis of the 2D NMR spectra enabled the establishment of the planar structure for 3, which had the same scaffold as that of 2. Compound 3 was found to possess one more methoxy group at C-6 as well as a propionyloxy group at C-2 instead of the hydroxy moiety in compound 2. The same skeleton for compounds 2 and 3 suggested the same skeletal configuration for both substances, which was corroborated by a NOESY experiment. Consequently, by interpretation of the NOESY spectrum, the C-2 propionyloxy, C-6 methoxy, C-18 butyryloxy, and C-19 acetoxy groups were found to be all α-oriented. After defining the relative configuration, an experimental ECD spectrum was recorded, which was almost identical to that of 2 (Figure 5). On the basis of the same relative configuration and the identical

Figure 5. Experimental ECD spectra of compounds 2−8 in acetonitrile. E



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C-6, C-18, C-19, and C-2, respectively, by analysis of the 2D NMR and HRESIMS data. By comparison of its NOESY and ECD spectra with those of compounds 2−7, compound 8 was characterized as (2R,5S,6S,8R,9R,10S,18S,19R)-6,18,19-trimethoxy-18,19-epoxycleroda-3,13(16),14-trien-2-ol. The molecular formula of compound 9 was deduced as C30H44O7 through the presence of an ion peak at m/z 539.2980 [M + Na]+ (calcd for C30H44NaO7, 539.2985) in its HRESIMS. Using the same NMR experiments as for compounds 1−8, the skeleton of 9 was elucidated and one acetoxy and two butyryloxy groups were located, supporting the structure, 2,18-dibutyryloxy-19-acetoxy-18,19-epoxycleroda-3,13(16),14triene.11,12,18 Although compound 9 showed the same relative configuration as 2−8 based on its NOESY spectrum, the ECD spectrum of 9 was found to be different. To obtain further information on its absolute configuration, the TDDFT CD calculations were performed for 9 using the same procedures as utilized for compounds 1 and 2. The calculated ECD spectrum (Figure 6) of 9 was in good agreement with the experimental


nerve cells as candidates for the treatment of AD, clerodane diterpenoids 1−10 were evaluated for their enhancing activities of NGF-induced neurite outgrowth from PC12 cells, according to a previously reported method.49 NGF was used as the positive control.50−52 Compounds 3−6 and 8 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 diterpenoids to stimulate NGF-mediated neurite outgrowth dose-dependently are shown in Table 4. However, compounds 1, 2, 7, 9, and 10 showed no activities on the proportion of neurite-bearing cells in either the absence or presence of NGF (20 ng/mL). In summary, the present bioassay-guided investigation to obtain bioactive compounds led to the isolation and characterization of 10 new (1−10) clerodane diterpenoids from C. graveolens. Besides their relative configurations, the absolute configurations of compounds 1−10 were determined on the basis of NMR data analysis and their experimental and calculated ECD spectra. In the biological screening performed, compounds 3−6 and 8 displayed potentiating activities of NGF-mediated neurite outgrowth from PC12 cells, and, of these, compound 8 exerted the greatest degree of promotion of NGF-mediated neurite outgrowth from PC12 cells. These five bioactive diterpenoids may be useful for the development of antineurodegenerative agents for Alzheimer’s disease and other neurological disorders.4,16


data, implying an absolute configuration of 2R, 5S, 8R, 9R, 10S, 18R, and 19S. Thus, compound 9 (graveopene I) was characterized as (2R,5S,8R,9R,10S,18R,19S)-2,18-dibutyryloxy19-acetoxy-18,19-epoxycleroda-3,13(16),14-triene. Compound 10 (graveopene J), also obtained as a colorless oil, gave a molecular formula of C28H40O7 as determined from the HRESIMS (m/z 511.2667 [M + Na] +, calcd for C28H40NaO7, 511.2672). The 1H and 13C NMR spectra of compound 10 suggested a characteristic 2,18,19-trisubstituted3,13(16),14-triene clerodane scaffold in this molecule.12,18,19 This skeleton was verified by conducting HMQC, HMBC, and 1 H−1H COSY experiments, and the three substituent groups present were defined as one butyryloxy and two acetoxy groups. On the basis of the relative configuration from the NOESY spectrum and the comparison of calculated and experimental ECD spectra (Figure 7), compound 10 was elucidated as (2R,5S,8R,9R,10S,18R,19S)-2-butyryloxy-18,19-diacetoxy18,19-epoxycleroda-3,13(16),14-triene. Biologically active substances from plants play an important role in the research and development of new drugs.7 For AD, it has been proven that agents able to promote the neurite outgrowth of nerve cells against the neuron degeneration may be useful for the treatment of this disease.4,32 In order to screen for bioactive substances to promote the neurite outgrowth of

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EXPERIMENTAL SECTION

General Experimental Procedures. 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. 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 LCQ-Advantage mass spectrometer. HRESIMS were recorded by an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA, USA). HPLC separations were performed on a CXTH system, equipped with a Shodex RI-102 detector (Showa Denko Co., Ltd., Tokyo, Japan) 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. F



Article

Table 3. 1H NMR Spectroscopic Data for Compounds 6−10 (δ in ppm, J in Hz, in CDCl3)a position

6

1α 1β 2 3α 3β 4 6α 6β 7α 7β 8 10 11

1.97 1.87 5.48 6.01

7

m m br s d (3.8)

2.05 1.94 4.36 6.03

3.28 dd (12.2, 3.9)

12 14 15 16 17 18 19 20 OR-2c

OCH3-6c OR-18c

OR-19c

2 3 4 5 2 3 4 2

1.85 1.50 1.70 2.28 1.50 1.27 2.08 6.43 5.18 5.02 5.05 4.95 0.94 5.42 6.42 0.91 2.35 1.69 0.99

m m m dd (12.8, 4.4) m m m dd (17.5, 10.8) d (17.5) d (10.8) s s d (6.8) s s s t (7.4) sex (7.4) t (7.4)

8

m m br s d (4.0)

1.92 1.78 4.33 6.05

1.68 m 1.46 m 1.49 m 1.38 m 1.63 m 2.08b 1.49 m 1.29 m 2.08 m 6.45 dd (17.8, 10.7) 5.23 d (17.8) 5.03 d (10.7) 5.04 s 4.94 s 0.88 d (6.6) 5.20 s 6.38 s 0.96 s

m m br s d (4.0)

3.24 dd (12.2, 3.9) 1.80 1.43 1.70 2.29 1.52 1.27 2.40 6.42 5.23 5.02 5.00 4.98 0.94 5.36 5.00 0.94

m m m dd (13.6, 3.7) m m m; 1.96 m dd (17.6, 10.9) d (17.6) d (10.9) s s d (6.7) s s s

9 2.11 1.92 5.39 5.88

m m br s d (3.3)

10 2.13 1.91 5.40 5.89

m m br s d (4.3)

1.73 m 1.49 m 1.51 m 1.41 m 1.64 m 2.18b 1.41 m 1.23 m 2.09 m 6.44 dd (17.6, 10.5) 5.21 d (17.6) 5.03 d (10.5) 5.05 s 4.94 s 0.88 d (6.5) 6.70 t (1.4) 6.31 s 0.95 s 2.35 t (7.3) 1.69 sex (7.3) 1.00 t (7.3)

1.73 1.48 1.51 1.40 1.63 2.17 1.48 1.23 2.07 6.44 5.21 5.04 5.05 4.94 0.88 6.68 6.31 0.95 2.37 1.71 1.00

m m m m m dd (10.5, 5.8) m m m dd (17.6, 10.9) d (17.6) d (10.9) s s d (6.6) s s s t (7.3) sex (7.3) t (7.3)

3.30 s 3.40 s

3.46 s

3.30 s 3.49 s

2.30 t (7.4) 1.64 sex (7.4) 0.94 t (7.4)

1.91 s

1.87 s

1.87 s

3.17 s

1.89 s

2.08 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. cThe number with the superscript indicates the location of the substituent group in the parent skeleton. based on TLC analysis. Fraction F3 was separated by MPLC over ODS eluting with a step gradient of 60−90% MeOH in H2O to give four subfractions (F3‑1−F3‑4). Subfraction F3‑3 was purified by preparative HPLC (YMC-Pack ODS-AM, 20 × 250 mm, 83% MeOH in H2O) to afford compounds 1 (tR 23 min, 15.9 mg) and 3 (tR 25 min, 14.1 mg). Using the same MPLC separation, fraction F5 yielded subfractions F5‑1−F5‑6, and purification of F5‑5 gave compound 2 (tR 26 min, 13.7 mg) with the above-mentioned HPLC system (82% MeOH in H2O). Compounds 4 (tR 41 min, 20.9 mg), 5 (tR 26 min, 15.0 mg), and 6 (tR 34 min, 11.9 mg) were isolated from subfraction F4‑2 (84% MeOH in H2O), which was obtained from fraction F4 by fractionation using the same MPLC procedure. Using the same protocols for the above fractions and subfractions, fractions F2, F6, and F7 afforded subfractions F2‑1−F2‑7, F6‑1−F6‑6, and F7‑1−F7‑3, respectively. Purification of F6‑3 (85% MeOH in H2O) and F7‑3 (86% MeOH in H2O) with the abovementioned HPLC yielded compounds 7 (tR 36 min, 8.9 mg) and 8 (tR 28 min, 12.7 mg), respectively. Compound 9 (tR 36 min, 8.9 mg) was isolated from F2‑6 (76% MeOH in H2O) and compound 10 (tR 28 min, 12.7 mg) was obtained from F2‑3 (86% MeOH in H2O) with the same HPLC system. Graveopene A (1): colorless oil; [α]15 D −72 (c 1.30, CH2Cl2); ECD (CH3CN) 213 (Δε −1.2), 291 (Δε −2.6); IR (KBr) νmax 2966, 2932, 2877, 1751, 1716, 1596, 1454, 1373, 1221, 1045, 953 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 469 [M + Na]+; HRESIMS m/z 469.2562 [M + Na]+ (calcd for C26H38NaO6, 469.2566).

Table 4. EC50 Values of Compounds 3−6 and 8 Stimulating NGF-Mediated Neurite Outgrowth from PC12 Cells compound

EC50 (μM)a

compound

EC50 (μM)

3 4 5

>45 4.4 28.7

6 8

20.6 3.3

a NGF was used as a positive control (EC50 value, 5.0 × 10−2 μg/mL). Data are presented based on three experiments.

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. graveolens were collected from Xishuangbanna, Yunnan Province, People’s Republic of China, in August 2013. The botanical identification was made by one of us (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. graveolens (16.5 kg) were powdered and extracted with MeOH (3 × 99 L) under reflux. The organic solvent was evaporated to afford a crude extract (850 g). The extract was suspended in H2O (1.0 L) and partitioned with EtOAc (3 × 1.0 L). The EtOAc-soluble portion (255 g) was subjected to silica gel column chromatography, using a gradient of acetone in petroleum ether (1−30%), to give eight fractions (F1−F8) G



Article

Graveopene B (2): colorless oil; [α]15 D −8 (c 0.13, CH2Cl2); ECD (CH3CN) 203 (Δε +3.3), 224 (Δε +0.3); IR (KBr) νmax 3435, 2963, 2931, 2876, 1735, 1457, 1373, 1224, 1056, 955 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 469 [M + Na]+; HRESIMS m/z 469.2562 [M + Na]+ (calcd for C26H38NaO6, 469.2566). Balanspene C (3): colorless oil; [α]15 D −21 (c 0.13, CH2Cl2); ECD (CH3CN) 203 (Δε +16.8), 226 (Δε −1.9); IR (KBr) νmax 2965, 2930, 2879, 1735, 1461, 1371, 1221, 1171, 1064, 947 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 555 [M + Na]+; HRESIMS m/z 555.2930 [M + Na]+ (calcd for C30H44NaO8, 555.2934). Graveopene D (4): colorless oil; [α]15 D −60 (c 0.06, CH2Cl2); ECD (CH3CN) 199 (Δε +13.6), 226 (Δε 3.4); IR (KBr) νmax 2955, 2928, 2857, 1752, 1455, 1368, 1023, 955 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 499 [M + Na]+; HRESIMS m/z 499.2672 [M + Na]+ (calcd for C27H40NaO7, 499.2672). Graveopene E (5): colorless oil; [α]15 D +17 (c 0.09, CH2Cl2); ECD (CH3CN) 202 (Δε +16.3), 226 (Δε −1.7); IR (KBr) νmax 2965, 2932, 2878, 1754, 1731, 1455, 1373, 1227, 1171, 1099, 952 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 541 [M + Na]+; HRESIMS m/z 541.2772 [M + Na]+ (calcd for C29H42NaO8, 541.2777). Graveopene F (6): colorless oil; [α]15 D +21 (c 0.11, CH2Cl2); ECD (CH3CN) 201 (Δε +7.8), 228 (Δε −0.8); IR (KBr) νmax 2962, 2929, 2878, 1730, 1454, 1372, 1273, 1107, 1057, 948 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 513 [M + Na]+; HRESIMS m/z 513.2928 [M + Na]+ (calcd for C28H42NaO7, 513.2828). Graveopene G (7): colorless oil; [α]15 D −40 (c 0.16, CH2Cl2); ECD (CH3CN) 200 (Δε +3.8), 228 (Δε −1.9); IR (KBr) νmax 3445, 2962, 2932, 2878, 1752, 1453, 1373, 1224, 1057, 1005, 946 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 408 [M + NH4]+; HRESIMS m/z 408.2754 [M + NH4]+ (calcd for C23H38NO5, 408.2750). Graveopene H (8): colorless oil; [α]15 D +12 (c 0.17, CH2Cl2); ECD (CH3CN) 201 (Δε +6.3), 228 (Δε −0.8); IR (KBr) νmax 3437, 2931, 2880, 2831, 1728, 1453, 1371, 1195, 1108, 1021, 996, 957 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 415 [M + Na]+; HRESIMS m/z 415.2459 [M + Na]+ (calcd for C23H36NaO5, 415.2460). Graveopene I (9): colorless oil; [α]15 D +9 (c 0.13, CH2Cl2); ECD (CH3CN) 190 (Δε +1.1); IR (KBr) νmax 2964, 2936, 2876,1753, 1731, 1457, 1373, 1220, 1180, 1061, 948 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 539 [M + Na]+; HRESIMS m/z 539.2980 [M + Na]+ (calcd for C30H44NaO7, 539.2985). Graveopene J (10): colorless oil; [α]15 D +1 (c 0.29, CH2Cl2); ECD (CH3CN) 191 (Δε +5.9), 223 (Δε −0.5); IR (KBr) νmax 2964, 2939, 2877, 1754, 1731, 1454, 1373, 1226, 1026, 957 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 511 [M + Na]+; HRESIMS m/z 511.2667 [M + Na]+ (calcd for C28H40NaO7, 511.2672). Computational Studies. Conformational searches were performed by the MOE software using the MMFF94 force field.39 The obtained conformers were used for geometry reoptimizations at the B3LYP/6-31G(d) level in the Gaussian 09 package.40 The ECD spectra for the optimized conformers were calculated at the CAMB3LYP/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 1.6 software.41 The overall ECD curves of all the compounds were weighted by Boltzmann distribution after UV correction. 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 dissociated by incubation with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

(EGTA) in phosphate-buffered saline (PBS) for 15 min and then seeded in 24-well culture plates (3 × 104 cells/well) coated with polyL-lysine. After 24 h, the medium was changed to a test medium containing various concentrations of NGF (Millipore, Billerica, MA, USA; 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.

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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.5b00583. NMR spectra of compounds 1−10, computational details, and some molecular orbitals of 1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax (Y. Guo): 86-22-23502595. E-mail: victgyq@nankai. edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (No. 21372125). REFERENCES

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