Isolation, Characterization, and StructureâActivity Relationship

Article Cite This: J. Nat. Prod. 2018, 81, 998−1006

pubs.acs.org/jnp

Isolation, Characterization, and Structure−Activity Relationship Analysis of Abietane Diterpenoids from Callicarpa bodinieri as Spleen Tyrosine Kinase Inhibitors Jun-Bo Gao,†,‡,§,⊥ Shuang-Jing Yang,†,⊥ Zi-Ru Yan,‡ Xing-Jie Zhang,† De-Bing Pu,† Li-Xia Wang,† Xiao-Li Li,† Rui-Han Zhang,*,† and Wei-Lie Xiao*,†,‡ †

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan Province, School of Chemical Science and Technology, Yunnan University, Kunming, Yunnan 650091, People’s Republic of China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Species belonging to the genus Callicarpa are used traditionally in Chinese medicine for the treatment of inflammation, rheumatism, and pain. Investigation of the leaves and twigs of Callicarpa bodinieri resulted in the isolation of nine new abietane diterpenoids, bodinieric acids A−I (1− 9), along with six known compounds (10−15). The structures of 1−9 were elucidated on the basis of the interpretation of their HRESIMS and NMR data and by ECD calculations. To explore the potential therapeutic target of this plant for immune-mediated disease, the inhibitory activities of the isolates obtained were determined against 13 kinase enzymes. Eight compounds exhibited moderate inhibitory effects on spleen tyrosine kinase (SYK), and the IC50 values of compounds 2 and 6 were 7.2 and 10.7 μM, respectively. In addition, a preliminary structure−activity relationship of this scaffold was analyzed with both molecular docking and a 3D-QSAR pharmacophore model.

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screened for their inhibitory activities against different kinases. Also, both molecular docking and a 3D-QSAR pharmacophore model were employed to develop preliminary structure− activity relationship information on this scaffold for SYK inhibitory activity.

he genus Callicarpa (beautyberry) contains about 190 species of herbaceous plants, which belong to the family Verbenaceae, and 46 species have been found in southern mainland China.1 Callicarpa bodinieri is one of the species in the genus Callicarpa that has been used widely in traditional medicine, for the treatment of inflammation,2 rheumatism, analgesia,3 hematemesis, gastrointestinal bleeding,4 scrofula, and swollen sores.5 The diverse therapeutic applications of plants in the genus Callicarpa may be attributed to their bioactive constituents, including terpenoids, lignans, glycosides,6−9 flavonoids,10 phenolic acids,11−13 and their volatile oils.14,15 The diterpenoids isolated from this genus have been reported to have anti-inflammatory activities in different disease models;16−18 however, their target has not been identified. Spleen tyrosine kinase (SYK), a double SH2-domaincontaining cytoplasmic protein,19 is an important drug target for immune-mediated disorders, such as allergic inflammation, rheumatism, and cancer.20,21 However, the similarity of the binding site in kinases brings the problem of poor target selectivity to most kinase inhibitors. In this report, the isolation of nine new (1−9) and six known (10−15) abietane-type diterpenoids is described from the leaves and twigs of C. bodinieri H. Lév. (Lamiaceae). Based on the indigenous medicinal utilization of this plant, these isolates were then © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A crude extract of the leaves and twigs of C. bodinieri was chromatographed over a silica gel column and eluted with a CHCl3−acetone gradient solvent system (1:0−0:1). The resulting fractions were separated by repeated column chromatography and preparative HPLC to afford nine new abietane-type diterpenoids (1−9, bodinieric acids A−I) and six known compounds, which were identified as 13-hydroxy8,11,13-podocatpatrien-18-oic acid22 (10), 18-oxoferruginol23 (11), 15-hydroxydehydroabietic acid24 (12), dehydroabietic acid25 (13), dehydroabietinol26 (14), and taxodascen C27 (15). Bodinieric acid A (1) was isolated as an amorphous, colorless powder, with [α]22D +121 (c 0.52, MeOH). The sodiated molecular formula, C19H24O4Na, was established from the sodium adduct molecular ion at m/z 339.1568 in the positiveReceived: December 29, 2017 Published: March 26, 2018 998

DOI: 10.1021/acs.jnatprod.7b01082 J. Nat. Prod. 2018, 81, 998−1006

Journal of Natural Products

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Chart 1

and H-6α (Figure 1). The absolute configuration of 1 was elucidated by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra (Figure 2). These two curves both exhibited a negative Cotton effect at 206 nm and a positive Cotton effect at 255 nm. Thus, the structure of 1 was established as (4S,5R,10S)-16-nor-18-hydroxy-15-oxoabieta-8,11,13-triene-19-oic acid. The molecular formulas of bodinieric acids B and C (2 and 3) were established as C20H28O3 (m/z 339.1931 [M + Na]+) and C20H26O3 (m/z 337.1774 [M + Na]+), respectively, based on the observed sodium adduct ions in the positive-ion mode HRESIMS. Both the 1D NMR data (Tables 1 and 2) and ROESY correlations (Figure 1 and Figure S88, Supporting Information) of 2 and 3 were similar to those of 1, except for the additional presence of signals of a methyl group (C-17, δC 24.3) in 2 and an olefinic methylene unit (C-16, δC 111.6) in 3. These proposed structures were verified by the coupling of H15/H-16/H-17 in the 1H−1H COSY spectrum together with the HMBC correlations from H2-16 to C-13 and C-17 and from H3-17 to C-13, C-15, and C-16 (Figures 1 and S88, Supporting Information). Thus, the structure of 2 was defined as 18-hydroxydehydroabietic acid. The absolute structure of 3 was determined as (4S,5R,10S)-18-hydroxyabieta-8,11,13,15tetraen-19-oic acid, and its experimental ECD spectrum was in good agreement with the calculated ECD spectrum (Figure 2). Bodinieric acid D (4) was purified as colorless needles. The sodium-adduct HRESIMS ion ([M + Na]+ at m/z 397.1983), along with the 13C NMR data (Table 2), were consistent with a

ion HRESIMS. The IR spectrum exhibited absorption bands at 3430, 1680, and 1603 cm−1 and indicated the presence of hydroxy, carboxyl, and aryl groups. Its 1H NMR spectrum (Table 1) displayed signals corresponding to two methyl groups at δH 1.18 (s, Me-20) and 2.51 (s, Me-17), an oxygenated methylene δH 3.85 (d, J = 10.3 Hz, Hβ-18) and 3.68 (d, J = 10.2 Hz, Hα-18), a methine at δH 1.86 (dd, J = 12.3, 1.7 Hz, H-5), and three aromatic methines at δH 7.44 (d, J = 8.3 Hz, H-11), 7.71 (d, J = 8.3 Hz, H-12), and 7.65 (s, H-14). The 13C NMR (Table 2) and DEPT spectra of 1 showed the presence of 19 carbon signals ascribable to two methyls, six methylenes (of which one was oxygenated), four methines, five quaternary carbons, a carboxylic acid carbon, and a ketocarbonyl carbon. According to the degrees of unsaturation, the two carbonyl carbons (δC 198.1, 177.6) and an aromatic ring group accounted for six indices of hydrogen deficiency, so the remaining two indices required the presence of two additional rings. In the 1H−1H COSY spectrum of 1, the correlations of H2-1/H2-2/H2-3, H2-6/H2-7, and H-11/H-12 were used to establish the presence of three fragments as shown in Figure 1. Based on the HMBC correlations of H3-20 with C-1, C-5, and C-9; H-5 with C-3, C-7, and C-19; H2-18 with C-3, C-4, and C5; H3-17 with C-12, C-13, and C-15; H-14 with C-7, C-9, and C-12 (Figure 1), the planar structure of compound 1 could be assigned as an abietane-type diterpenoid. The relative configuration was inferred from a ROESY experiment, which showed correlations between the following proton pairs: H3-20 and H-1/3β, H-6β; H-5 and H-1/3α, H-6α, H-18α; H-18 α/β 999



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Table 1. 1H NMR Spectroscopic Data Assignments (600 MHz, in Acetone-d6) for Compounds 1−9 (δ in ppm, J in Hz) position

1

2

3

4

5

6

7

8

9

1α

1.34, oa

1.31, o

1.32, o

1.30, o

2.31, o 1.61, dt (13.2) 2.08, m 1.31, o

2.31, o 1.62, dt (13.7, 3.6) 2.11, o 1.32, o

2.36, o 1.66, m

2.32, o 1.62, dt (13.6, 3.7) 2.11, m 1.23, m

2.35, o 1.86, dd (12.3, 1.7) 2.01, m 2.18, m 2.89, m 7.44, d (8.3) 7.71, d (8.3) 7.65, s

2.31, o 1.79, d (12.4) 1.97, m 2.13, m 2.78, m 7.20, m 6.97, m 6.86, s 2.78, m 1.17, d (6.9)

2.31, m 1.81, d (12.3)

2.28, o 1.73, m

1.30, dt (13.4, 4.0) 2.29, o 1.61, dt (14.8, 3.8) 2.08, m 1.21, td (13.3, 5.0) 2.29, o 1.75, m

1.32, m

2.35, o 1.65, dt (13.7, 3.6) 2.12, m 1.34, o

1.34, dt (13.4, 4.0) 2.32, o 1.63, dt (14.2, 3.7) 2.13, m 1.22 td (13.8, 4.5) 2.32, o 1.79, d (12.2)

1.36, m

1β 2α

1.35, dt (13.4, 4.1) 2.35, o 1.65, m

1.97, m 2.15, m 2.78, m 7.24, m 7.20, m 7.09, s

2.04, m 2.13, m 2.84, m 7.25, d (2.6) 7.25, d (2.6) 7.14, s

2.08, m 2.73, m 7.10, d (8.6) 6.59, d (8.5) 6.46, d (2.7)

5.33, s

2β 3α 3β 5 6α 6β 7 11 12 14 15 16

17

2.51, s

18

3.85, d (10.3) 3.68, d (10.2)

20 AcO− a

1.18, s

1.17, d (6.9) 3.87, d (10.1) 3.64, d (10.1) 1.14, s

2.28, o 1.58, d (13.3) 2.08, m 1.30, o

1.98, m 2.15, m 2.81, m 7.25, m 7.25, m 7.12, s

2.08, m 1.24 td (13.5, 4.4) 2.35, o 1.81, dd (12.2, 1.9) 1.99, m 2.14, m 2.85, m 7.25, m 7.25, m 7.18, d (1.8)

5.32, s

1.47, s

5.36, d (1.5) 5.29, d (1.8)

4.99, s 2.08, s

1.47, s

4.42, s

5.00, m 2.09, s

3.85, d (10.2)

4.42, d (10.5)

4.25, m

4.38, d (10.4)

4.38, d (10.5)

4.41, d (10.4)

4.38, d (10.5)

3.65, d (10.2)

4.14, d (10.4)

4.16, d (10.4)

4.12, d (10.3)

4.14, d (10.5)

4.14, d (10.4)

1.15, s

1.17, s 2.02, s

1.17, s 2.00, s

1.12, s 1.99, s

1.19, s 2.00, s

1.15, s 2.00, s

1.18, s 1.98, s

2.15, m 1.24, m 2.36, o 1.83, dd (12.4, 1.8) 1.98, m 2.15, m 2.92, m 7.45, d (8.4) 7.71, d (8.3) 7.66, d (1.9)

2.32,o 1.78, d (12.1)

2.51, s

1.18, d (6.9)

2.11, m 2.80, m 7.21, d (8.2) 6.98, d (8.3) 6.87, m 2.80, m 1.18, d (6.9)

o = overlapped.

Table 2. 13C NMR Spectroscopic Data Assignments (150 MHz, in Acetone-d6) for Compounds 1−9 (δ in ppm) position

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AcO−

40.1, CH2 20.7, CH2 32.7, CH2 51.0, C 47.3, CH 21.8, CH2 32.6, CH2 135.9, C 155.0, C 39.9, C 127.1, CH 126.7, CH 136.9, C 130.4, CH 198.1, C

40.0, CH2 20.4, CH2 32.4, CH2 50.5, C 47.4, CH 21.6, CH2 32.3, CH2 135.7, C 146.7, C 38.7, C 126.1, CH 124.6, CH 146.2, C 127.5, CH 34.2, CH 24.3, CH3 24.3, CH3 70.2, CH2 177.4, C 23.9, CH3

39.9, CH2 20.3, CH2 32.4, CH2 50.5, C 47.3, CH 21.5, CH2 32.2, CH2 135.8, C 148.7, C 38.9, C 126.1, CH 123.7, CH 143.9, C 126.7, CH 135.8, C 111.6, CH2 21.8, CH3 70.0, CH2 177.6, C 23.8, CH3

39.7, CH2 20.1, CH2 32.8, CH2 48.2, C 48.1, CH 21.8, CH2 32.3, CH2 134.9, C 148.2, C 38.7, C 125.6, CH 123.1, CH 146.5, C 125.8, CH 71.7, C 32.3, CH3 32.3, CH3 71.8, CH2 176.0, C 23.6, CH3 170.8, C



39.9, CH2 20.1, CH2 32.9, CH2 48.3, C 48.2, CH 21.8, CH2 32.2, CH2 136.9, C 140.1, C 38.4, C 127.2, CH 114.1, CH 155.6, C 115.4, CH

39.3, CH2 20.0, CH2 32.7, CH2 48.3, C 47.6, CH 21.5, CH2 32.0, CH2 135.6, C 154.1, C 39.5, C 126.6, CH 126.4, CH 136.3, C 130.0, CH 197.7, C

39.7, CH2 20.1, CH2 32.9, CH2 48.3, C 48.0, CH 21.8, CH2 32.2, CH2 135.5, C 146.4, C 38.8, C 126.1, CH 124.7, CH 146.3, C 127.5, CH 34.2, CH 24.3, CH3 24.3, CH3 71.8, CH2 176.3, C 23.7, CH3 170.8, C

27.0, CH3 70.3, CH2 177.6, C 24.1, CH3

71.8, CH2 176.4, C 23.8, CH3 170.8, C

26.6, CH3 71.7, CH2 176.0, C 23.4, CH3 170.8, C

13 and C-15 suggested the location of a hydroxylated quaternary carbon (C-15, δC 71.7) connected to C-13. In addition, the 1H−1H COSY correlations of H2-1/H2-2/H2-3 and H2-6/H2-7, coupled with the HMBC cross-peaks from H320 to C-1, C-5, and C-9; H2-2 to C-1 and C-3; H-5 to C-18 and

molecular formula of C22H30O5. The absorption bands at 1746 and 1702 cm−1 in the IR spectrum indicated the presence of carboxylic acid and lactone carbonyl groups. The structure of 4 was inferred from extensive 2D NMR experiments (Figure S89, Supporting Information). The cross-peaks from H3-16/17 to C1000



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highly consistent with those of 4 (Figure S89, Supporting Information). Therefore, the structure of 5 was established as 18-acetoxy-17-hydroxyabieta-8,11,13,15-tetraen-19-oic acid. Bodinieric acid F (6) was obtained as an amorphous, colorless powder with a molecular formula of C22H28O4 based on its HRESIMS and 13C NMR data, indicating that it has one less oxygen atom than 5. A side-by-side comparison of the 1D NMR spectroscopic data of 6 with 5 revealed that these two compounds possess similar structural features, with the only difference being due to the absence of a hydroxy group at C-17 in 6. This was confirmed by analysis of the HMBC correlations from H2-16 to C-14 and C-17 and from H3-17 to C-14 and C16 (Figure S89, Supporting Information). Furthermore, comparison of the experimental and calculated ECD curves (Figure 3) indicated a 4R,5R,10S configuration for 6. Thus, compound 6 was elucidated as (4R,5R,10S)-18-acetoxyabieta8,11,13,15-tetraen-19-oic acid. The molecular formula of bodinieric acid G (7) was determined to be C19H24O5 on the basis of its HRESIMS, 13 C NMR, and DEPT data. The similarity of the NMR spectra of 7 (Tables 1 and 2) to those of 4−6 indicated that 7 is a structural analogue of these compounds. The main difference was the absence of an isopropyl group in the abietane diterpenoid skeleton at C-13 and its replacement by a hydroxy group, as evidenced by the chemical shift of C-13 at δC 155.6. This was confirmed by HMBC correlations from H-11 to C-13, H-12 to C-13, and H-14 to C-13 (Figure S89, Supporting Information). The ECD spectrum generated for the configuration of 4R, 5R, 10S showed two positive Cotton effects at 203 and 228 nm, which were in good agreement with the experimental data obtained for 7 (Figure 3). Thus, the structure of 7 was proposed as (4R,5R,10S)-18-acetoxy-13-hydroxy8,11,13-podocarptrien-19-oic acid. By comparison of the HRESIMS and NMR data (Tables 1 and 2) with those of 4−6, bodinieric acid H (8) was seen to be related to 6 structurally and differed from the replacement of a terminal olefinic methylene (C-16) by a carbonyl group. Similarly, the observed spectra of bodinieric acid I (9) were also quite similar to those of 4, differing only in the absence of a hydroxy group at C-15. Data analysis of the HMBC spectrum from H3-17 to C-15 and from H-14 to C-13 and C-15 in 8, and in turn, H3-16/17 to C-17/16 and C-15 and H-15 to C-13 and C-14 in 9, confirmed these proposals and provided complete NMR spectroscopic assignments. The relative configurations of

Figure 1. Key 1H−1H COSY, HMBC, and ROESY correlations of 1 and 2.

C-19; H2-18 to C-3 and C-19; H2-7 to C-5 and C-6; and H-14 to C-7, C-9, C-12, and C-15 suggested that compound 4 possesses the carbon skeleton of an abietane-type diterpenoid (Figure S89, Supporting Information). The additional CH3CO− was bonded with the C-18 hydroxy group to generate an ester, which was confirmed by the correlations from H2-18 to C-OAc. Subsequently, the ROESY correlations of H320 with H-1/3β, H3-AcO and of H-5 with H-1/3α (Figure S89, Supporting Information) revealed that the stereoconfiguration of 4 was completely in accordance with other abietane diterpenoids.28 Consequently, the structure of 4 was established as 18-acetoxy-15-hydroxydehydroabietic acid. Bodinieric acid E (5) was determined to have the molecular formula C22H28O5, based on its HRESIMS and 13C NMR spectroscopic data. The NMR data (Tables 1 and 2) displayed signals that resembled those of 4, except that resonances for two methyls (Me-16, 17, δC 32.3) and an oxygenated quaternary carbon (C-15, δC 71.7) in 4 were replaced by those for a terminal olefin (C-16, δC 110.4) and a hydroxylated methylene (C-17, δC 64.2) in 5. This was supported by HMBC correlations from H2-16 to C-13 and C-17; H2-17 to C-13; and H-14 to C-15. Comparison of the ROESY spectrum indicated that the relative configurations of all chiral centers in 5 were

Figure 2. Experimental and calculated ECD spectra of bodinieric acid A (1) and bodinieric acid C (3). 1001



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Figure 3. Experimental and calculated ECD spectra of bodinieric acid F (6), bodinieric acid G (7), bodinieric acid H (8), and bodinieric acid I (9)

these two compounds were proposed by the analysis of ROESY correlations (Figure S89, Supporting Information) when compared with those of 4−6. In turn, the absolute configurations of 8 and 9 were both established as 4R,5R,10S, based on their ECD similarities with those of 6 and 7 (Figure 3). As a result, compounds 8 and 9 were determined respectively as (4R,5R,10S)-16-nor-18-acetoxy-15-oxoabieta8,11,13-trien-19-oic acid and (4R,5R,10S)-18-acetoxydehydroabietic acid. According to the applications of C. bodinieri in traditional Chinese medicine for the treatment of immune-related diseases, compound 13, identified as dehydroabietic acid with relatively less substituent groups, was screened against 13 kinases at 50 μg/mL (∼166 μM) to evaluate whether this scaffold is able to inhibit any kinases involved in an immune response. As shown in Table 3, compound 13 exhibited inhibition activities (>50%) for seven of the kinases against which it was evaluated at this concentration. The highest inhibition rate was obtained on spleen tyrosine kinase with 81%, followed by Janus kinase 3 (JAK3) with 74.9%, which are two well-known drug targets for treating immune disorders such as rheumatoid arthritis.20 Thus, the inhibitory activities of all the diterpenoids isolated (except for 5, 10, and 15 due to their limited amounts available) were then tested against SYK and JAK3. Eight compounds show moderate to weak inhibitory activity against SYK (IC50 < 100 μM), whereas none of them were active against JAK3 (IC50 > 100 μM). Compound 2 showed the best SYK inhibitory activity with IC50 = 7.2 μM. Five other compounds, 6, 9, 11, 13, and 14, showed IC50 values ranging from 10.7 to 46.4 μM. On analysis of the data in Table 3, it was shown that this scaffold of compounds may show inhibitory selectivity for SYK among the 13 kinases tested.

Table 3. Inhibitory Activities of Dehydroabietic Acid (13) on Different Kinases

FAK BRAF PKCθ SYK PKA ALK JAK3 ZAP70 SRC AKT1 GSK3b PIM1 PDK1

compound 13a inhibition rate with 50 μg/ mL compound (%)

staurosporineb IC50 (nM)

± ± ± ± ± ± ± ± ± ± ± ± ±

11 8.7 5.5 0.86 2.2 7.1 0.12 1.3 2.6 12 24 58 10

67.1 −7.3 0.9 81.0 69.9 17.6 74.9 52.5 61.2 53.6 20.1 10.7 46.1

2.12 6.71 14.8 2.78 3.53 6.04 1.87 6.67 3.67 1.26 3.79 5.39 2.16

a Values are the means ± SD, n = 2. bStaurosporine was used as a positive control.

To better understand the structure−activity relationship of this series of abietane-type diterpenoids, the molecular docking of all compounds was performed on the binding site of SYK (PDB ID: 5TIU).29 As shown in Figure 4 (left), the binding conformation of a cocrystallized inhibitor (7CU) may be reproduced by the protocol used. The binding modes were determined of the two most active diterpenoids obtained (2 and 6, Figure 4), when docked into the binding site of SYK. Polar interactions were formed between the R1/R2 groups of 2 and Arg498, ASN499, and Asp512. However, for compound 6, only hydrogen bonds between the R2 carboxylic acid group and 1002



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Figure 4. Docked model of 2 (B) and 6 (C) in the SYK binding pocket (PDB ID: 5TIU). The cocrystalized inhibitor was docked initially into the binding site (7CU_Auto Dock) and compared with the crystallized conformation (7CU_5TIU) (A). Polar interactions are shown as yellow dashed lines.

FitValue was not perfectly correlated with the IC50 value. Nevertheless, pharmacophore 1 can be used to qualitatively predict the activity of those compounds (5, 10, 15) that were not tested. As a result (dashed box in Figure 6), compound 5 might possess an inhibitory activity against SYK comparable in potency to 11, while compound 10 probably has only weak activity, and compound 15 may be predicted to be inactive. Accordingly, the discovery of this scaffold for the plantderived diterpenoids investigated as SYK inhibitors without affecting JAK3 could provide insight on the development of anti-inflammatory and immunomodulation drugs targeting SYK and to prevent the side effects of unbiased kinase inhibition.

Arg498 were detected, since R1 is a hydrophobic and structurally hindered group. For this diterpenoid scaffold, a πsulfur interaction could also be found between the benzene ring and Met448. The influence of the substituent groups on this scaffold was elucidated further utilizing a 3D-QSAR pharmacophore analysis method. As shown in Figure S90 (Supporting Information), pharmacophore 1 displayed the best correlation between the FitValue and the IC50 value. This optimal model showed three favorable features for SYK inhibitory activity (Figure 5): the

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. ECD spectra were measured on a Chirascan instrument. IR spectra are obtained on a Tenor 27 spectrophotometer with KBr pellets. One-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded on Bruker AV-600 spectrometers with tetramethylsilane as the internal standard. Chemical shifts (δ) were expressed in parts per million with reference to the solvent signals. HREIMS was performed on an Agilent G6230 time-of-flight spectrometer. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph system with a Zorbax SB-C18 (9.4 mm × 250 mm, 5 μm) column at a flow rate of 3 mL/min. Column chromatography (CC) was performed on silica gel (100−200 and 200−300 mesh; Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), LiChroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20 (Pharmacia). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 5% H2SO4 in ethanol, followed by

Figure 5. Mapping of pharmacophore 1 and compound 6. Pharmacophore 1 contains two hydrophobic centers (blue) and two hydrogen-bond acceptors (green).

carboxylic acid group at C-19 as a hydrogen-bond acceptor, and the B-ring and the isopropyl group at C-13 as hydrophobic interaction sites. As shown in Figure S90, pharmacophore 1 was capable of distinguishing the most potent compounds (2, 6, 9, 13, 11) from the inactive compounds (7, 8, 4, 1), whereas for the compounds with only weak activity (IC50, 40−100 μM), the

Figure 6. Activity prediction of compounds 5, 10, and 15 by pharmacophore 1. Compounds 1−15 (compound label in the box) were arranged left to right with the increasing of IC50 value against SYK. The position of compounds 5, 10, and 15 were decided by their FitValue. 1003



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Table 4. Kinase Inhibition Activities of Compounds on SYK and JAK3

a

Compound 14 was not measured for JAK3 inhibition activity. bStaurosporine was used as a positive control.

heating. The purity of obtained isolates were determined by HPLC analysis (Agilent SB-C18, 4.6 mm × 250 mm, 5 μm, 1 mL/min), and all of them were ≥95%. Plant Material. The leaves and twigs of Callicarpa bodinieri were collected in September 2015 at Xishuangbanna, Yunnan Province, People’s Republic of China, and identified by Prof. Yu Chen, Kunming Institute of Botany. A voucher specimen (YNU 20150002) has been deposited in the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan University. Extraction and Isolation. The dried and powdered leaves along with twigs of C. bodinieri (7.8 kg) were extracted three times (20 L, each) with 85% methanol−water at room temperature. The total dried extracts obtained (540 g) were suspended in water, and the resulting aqueous layer was successively partitioned with petroleum ether, ethyl acetate (EtOAc), and n-butyl alcohol. The EtOAc solution was then concentrated under reduced pressure, and the residue (124 g) was chromatographed on a silica gel column (600 g, 100−200 mesh) and eluted stepwise with a CHCl3−acetone gradient system (1:0 → 0:1) to

afford the major fractions A−E. Fraction B was subjected to separation over RP-C18, eluting with MeOH−H2O mixtures (6:4→1:0), to yield 10 fractions. Then, the 85% MeOH sample was applied to silica gel CC eluted with petroleum ether−acetone (8:3) to yield 13 (378.6 mg) and 14 (31.9 mg). Fraction D was subjected to silica gel CC to give three fractions, D1−D3, eluting with CHCl3−acetone (50:1→1:1). D1 was purified on Sephadex LH-20, then followed by passage over an RP-C18 column, to furnish 2 (2.7 mg), 10 (1.2 mg), and 12 (8.3 mg), along with 11 (3.1 mg), which was crystallized from the 65% MeOH− H2O solution. D3 was fractionated by silica gel CC using EtOAc− acetone (9:2) and purified by preparative HPLC to afford 1 (4.5 mg, tR = 18.8 min), 4 (13.8 mg, tR = 9.2 min), 5 (1.3 mg, tR = 6.8 min), 6 (2.4 mg, tR = 11.4 min), and 9 (6.7 mg, tR = 15.3 min). Fraction E was separated employing RP-C18 with a gradient mixture of MeOH−H2O (1:9 → 1:0) to produce four subfractions, E1−E4, and Fr. E2 was further subjected to a silica gel CC, eluting with a CHCl3−MeOH (10:1, 9:2, 8:3, 6:4) gradient system, providing 3 (4.5 mg), 7 (5.1 mg), and 8 (21.6 mg) as well as 15 (1.5 mg). 1004



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Bodinieric acid A (1): amorphous, colorless powder; [α]22D +121 (c 0.52, MeOH); UV (MeOH) λmax (log ε) 210 (4.31), 258 (4.12) nm; IR (KBr) νmax 3430, 2962, 2935, 1680, 1603, 1415, 1383, 1271, 1189, 1064 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion ESIMS m/z 315 [M − H]−; positive-ion HRESIMS [M + Na]+ m/z 339.1568 (calcd for C19H24NaO4, 339.1572). Bodinieric acid B (2): white powder; [α]22D +57.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (4.13), 213 (3.96), 255 (3.04) nm; IR (KBr) νmax 3423, 2960, 2933, 1700, 1498, 1459, 1189, 1025 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion EIMS m/z 339 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 339.1931 (calcd for C20H28NaO3, 339.1934). Bodinieric acid C (3): colorless powder; [α]22D +114 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 211 (4.39), 250 (4.08) nm; IR (KBr) νmax 3390, 2963, 2936, 1700, 1499, 1451, 1376, 1227, 1189, 1071, 1025 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion EIMS m/z 337 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/ z 337.1774 (calcd for C20H26NaO3, 337.1775). Bodinieric acid E (4): colorless needles; [α]22D +89.4 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 201 (4.33), 212 (4.11), 268 (2.99) nm; IR (KBr) νmax 3427, 2970, 2935, 1746, 1702, 1379, 1237, 1039 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 397 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 397.1983 (calcd for C22H30NaO5, 397.1991). Bodinieric acid D (5): amorphous powder; [α]22D +53.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 211 (4.38), 250 (4.07) nm; IR (KBr) νmax 3423, 2958, 1737, 1456, 1381, 1242, 1036 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; positive-ion EIMS m/z 395 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 395.1832 (calcd for C20H26NaO3, 395.1829). Bodinieric acid F (6): amorphous, colorless powder; [α]22D +74.9 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 211 (4.37), 250 (4.05) nm; IR (KBr) νmax 3420, 2963, 2937, 1742, 1454, 1441, 1379, 1235, 1039 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion EIMS m/z 379 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 379.1880 (calcd for C22H28NaO4, 379.1879). Bodinieric acid G (7): amorphous powder; [α]22D +117 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 203 (4.46), 216 (4.06), 280 (3.46) nm; IR (KBr) νmax 3420, 2960, 2937, 1714, 1611, 1500, 1442, 1381, 1243, 1041 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion EIMS m/z 355 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/ z 355.1516 (calcd for C19H24NaO5, 355.1518). Bodinieric acid H (8): white, microcrystalline solid; [α]22D +76.3 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 207 (4.17), 257 (3.95) nm; IR (KBr) νmax 2916, 2938, 1741, 1604, 1363, 1237, 1039 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 381 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 381.1674 (calcd for C21H26NaO5, 381.1678). Bodinieric acid I (9): amorphous, colorless powder; [α]22D +91.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 204 (4.37), 210 (4.20), 254 (3.31) nm; IR (KBr) νmax 3429, 2960, 2933, 2873, 1742, 1498, 1461, 1381, 1237, 1039 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion EIMS m/z 381 [M + Na]+; positive-ion HRESIMS [M + Na]+ m/z 381.2036 (calcd for C22H30NaO4, 381.2035). Kinase Inhibition Assay. The in vitro inhibitory activity of the compounds was tested by ChemPartner Co., Ltd., Shanghai, People’s Republic of China. The mobility shift assay was performed for ALK, PKCθ, PKA, PIM1, AKT1, SRC, SYK, GSK3b, PDK1, JAK3, and ZAP70, and the Latha screen assay was performed for BRAF and FAK. Staurosporine was used as a reference compound. The inhibition rates at single compound concentrations were tested in duplicate. For IC50 estimations, 10 concentrations were measured for each compound, with the starting point of 100 μM and gradient 3-fold dilution. Molecular Docking. Docking study of the diterpenoids was performed with AutoDock 4 using the Lamarckian genetic algorithm.30 The receptor binding site was defined based on the crystal structure of SYK when complexed with a 2-aminopropyl inhibitor (PDB ID: 5TIU),29 which is of similar size to the diterpenoids that were investigated. The docking grid box was centered at the ligand in the crystal structure, with 50 points for each of the x, y, and z directions.

The grid space was 0.375 Å. R498, N499, A451, and D512 were selected as flexible residues during conformational searching. The top 10 docking poses of each ligand were clustered based on the RMSD compared with the starting conformation (cutoff = 2.0 Å). Then, the best docking pose in the largest cluster was selected for analysis. 3D-QSAR Pharmacophore Generation and Analysis. The pharmacophore generation and QSAR study were performed using Discovery Studio 4.0 software (Accelrys, San Diego, CA, USA). The structures of the abietane diterpenoids initially were optimized by ChemBio3D with the MM2 force field. Compounds 2, 3, 7, 8, 12, and 13 were selected as the training set, and the others remained in the test set. The IC50 threshold value of the inactive compounds (>100 μM) was set to 10 000 to emphasize their inactivity. Based on the CHARMM27 force field, maximum numbers of 250 conformations of each compound were generated using the FAST method with an energy threshold of 10 kcal/mol. According to the binding mode (predicted by molecular docking) of the top active compounds, three of the chemical features: HB_ACCEPTOR, HB_DONOR, and HYDROPHOBIC were selected by the Feature Mapping module. Pharmacophore models were obtained using the 3D-QSAR Pharmacophore Generation protocol, and the top nine scoring hypotheses were collected. The test set was then mapped to the pharmacophore models using the Ligand Profiler module for evaluation. The best model was determined by the correlation between the experimental activity and the predicted FitValue. Finally, the SYK inhibitory activity of the three undetermined compounds was predicted by the most ideal pharmacophore model. ECD Calculations. The conformational analysis studies were carried out for compounds 1, 3, and 6−9 using the program BALLOON.31,32 The predominant conformers were then optimized using DFT calculation at the B3LYP/6-311G(d, p) level by Gaussian 09.33 The equilibrium population of each conformer at 298.15 K was calculated from its relative free energies using Boltzmann statistics. The theoretical calculations of ECD were performed using TDDFT at the B3LYP/def2TZVP level in MeOH. For comparisons of the calculated curves and experimental ECD spectra, the program SpecDis was used.34

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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.7b01082. 1 H, 13C NMR, DEPT, HSQC, HMBC, 1H−1H COSY, ROESY, HRESIMS, IR, ECD, and UV spectra of compounds 1−9; key 1H−1H COSY, HMBC, and ROESY correlations of 1−9; evaluation of the pharmacophores; ECD calculation details of compounds 1, 3, and 6−9 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-13808730113. Fax: +86-871-67357014. E-mail: [email protected] (R.-H. Zhang). *Tel: +86-15912172465. Fax: +86-871-67357014. E-mail: [email protected] (W.-L. Xiao). ORCID

Wei-Lie Xiao: 0000-0001-6826-1993 Author Contributions ⊥

J. Gao and S. Yang contributed equally.

Notes

The authors declare no competing financial interest. 1005



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(26) Gonzalez, M. A.; Perez-Guaita, D.; Correa-Royero, J.; Zapata, B.; Agudelo, L.; Mesa-Arango, A.; Betancur-Galvis, L. Eur. J. Med. Chem. 2010, 45, 811−816. (27) Ye, Y.; Wang, Y. P.; Yao, S.; Zhao, J.; Tang, C. P. U. S. Patent 7517542B2, 2009. (28) Bajpai, V. K.; Kang, S. C. J. Biosci. 2010, 35, 533−538. (29) Ellis, J. M.; Altman, M. D.; Cash, B.; Haidle, A. M.; Kubiak, R. L.; Maddess, M. L.; Yan, Y. W.; Northrup, A. B. ACS Med. Chem. Lett. 2016, 7, 1151−1155. (30) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785− 2791. (31) Vainio, M. J.; Johnson, M. S. J. Chem. Inf. Model. 2007, 47, 2462−2474. (32) Puranen, J. S.; Vainio, M. J.; Johnson, M. S. J. Comput. Chem. 2010, 31, 1722−1732. (33) Frisch, M. J. T.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. J. A. Gaussian 09, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2009. (34) Bruhn, T.; Schaumloffel, A.; Hemberger, Y.; Bringmann, G. Chirality 2013, 25, 243−249.

ACKNOWLEDGMENTS This project was supported financially by grants from the Natural Science Foundation of China (81422046 and 21762048), the Yunnan Applicative and Basic Research Program (2015BC002), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R94) and Key Program of Natural Science of Yunnan Province. We are grateful to the high-performance computing centers of Kunming Institute of Botany (CAS) and Yunnan University for providing the calculation resources.

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