Diterpenoids from the aerial parts of Flueggea acicularis and their activity against RANKL-induced osteoclastogenesis

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Diterpenoids from the aerial parts of Flueggea acicularis and their activity against RANKL-induced osteoclastogenesis Dane Huanga,1, Xiangkun Luoa,1, Zhiyong Yina, Jun Xua,b, , Qiong Gua,b, ⁎

a b

⁎

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, People’s Republic of China

ARTICLE INFO

ABSTRACT

Keywords: Flueggea acicularis Clesistanthane diterpenoids Osteoclast Osteoclastogenesis

Compounds 1–11 were isolated from the aerial parts of Flueggea acicularis Croizz Webster, including three new rearranged clesistanthane diterpenoids fluacinoids A-C (1–3) and five new norditerpenoid fluacinoids D–H (4–8). The new compounds were identified from spectroscopic data combined with single crystal X-ray diffraction analysis, modified Mosher’s methods, and ECD data analyses. All the isolated compounds were evaluated for their activities on RANKL-induced osteoclastogenesis in bone marrow monocytes (BMMs). Compound 6 showed the most potent inhibition against osteoclast differentiation (IC50, 0.7 μM) and decreased the expression level of osteoclast-related genes. Moreover, compound 6 prompted the apoptosis of osteoclasts. Compound 6 also suppressed RANKL-induced NF-κB activation. This study reveals that norditerpenoids may be resource for anti-osteoporosis agents.

1. Introduction Osteoporosis is a metabolic bone disease characterized by osteoclast formation while bone resorption leads to a high risk of fractures [1]. Bone resorption and formation modulates bone remodeling. The receptor activator of nuclear factor kappa B ligand (RANKL) contributes to osteoclastogenesis or formation of cells that break down bone tissue. RANKL/RANK interactions activate the NF-κB signal pathway and the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1). These activations induce osteoclast differentiation and result in production of mature multinucleated osteoclasts. However, the efficacy of these medications is not satisfactory [2,3], thus new pharmaceutical options are demanding. Consequently, inhibition of the RANKL/RANK interactions to reduce osteoclastogenesis is a promising therapeutic strategy. Natural germacrane type sesquiterpenoids [4] and ent-rosane diterpenoids [5] have been reported to inhibit RANKL-induced osteoclastogenesis, and in a continuing search for anti-osteoporosis agents, the chemical constituents of Flueggea acicularis Croizz Webster (Euphorbiaceae) were studied. Plants from the Flueggea genus have been reported to produce securinine and norsecurinine alkaloids [6–9] and various biological activities [6,10–13]. There are only a few reports about diterpenoid compounds [14] from this genus, and the chemical composition of F. acicularis has not yet been studied. Here, the isolation

of anti-osteoporosis diterpenoids from extracts of F. acicularis is reported. Eight new 1–8 and three known diterpenoids 9–11 were identified. Compounds 6, 7, and 8 inhibit osteoclastogenesis with IC50 values of 0.7, 2.2, and 4.0 μM, respectively. 2. Results and discussion Compound 1 (fluacinoid A) has a molecular formula C20H24O3 from the negative HR-ESI-MS ion at m/z 311.1657 [M−H]− and the 13C NMR data. The IR absorption maximum at 3423 cm−1 indicated the presence of hydroxy groups. The NMR data (Tables 1 and 2) showed two benzylic methyl groups, an isopropyl group, two aromatic protons, and a vinylic group. This analysis suggests that the structure of compound 1 is similar to phyllanes A and B [15,16], which possess a 4,5seco-rearranged cleistanthane diterpenoid skeleton. HMBC signal from H-4 to C-11/C-2 and from H-2 to C-10/C-11 suggest that C-3 is connected to C-11 (Fig. 1). Analysis of the 2D NMR data suggests compound 1 has the same 2D structure as that of phyllane A [15]. NOESY correlations of H-2/H3-19 indicated that the two hydroxy groups are cofacial (Fig. S12, Supporting Information). The X-ray crystallographic data (Fig. 4) reveals a (2R, 3S) absolute configuration that was confirmed by analysis of the experimental and computational ECD data (Fig. 2).

Corresponding authors at: Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China. E-mail addresses: [email protected] (J. Xu), [email protected] (Q. Gu). 1 D.E. Huang and X.K. Luo contributed equally. ⁎

https://doi.org/10.1016/j.bioorg.2019.103453 Received 5 October 2019; Received in revised form 13 November 2019; Accepted 16 November 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Dane Huang, et al., Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2019.103453

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NMR informations of 4 is similar to those of 3α-hydroxy-12-methoxy13-methyl-ent-podocarp-6,8,11,13-tetraene [14], with an additional vinyl group and the absence of one singlet methyl group. HMBC correlations from H-16 to C-13 and from H-15 to C-12 and C-14 further confirm that 4 contains a vinyl fragment at C-13 (Fig. 1). NOESY cross-peaks of H-3/H-5, H-3/H3-18, and H-3/H-1β show they are β-orientated. The CH3-20 and H-1α are α-orientation elucidated from NOESY signal of CH3-20/H-1α. Compound 4 was determined as 3R by the Mosher ester method (Fig. 3), and this is confirmed by the experimental and calculated ECD spectra (Fig. 2). Compounds 5 and 4 have similar NMR signal (Tables 3 and 4), except for two additional methylenes (δc 19.1, CH2; 30.0, CH2) and the absence of the double bond in 5. HMBC correlations between the proton signal at δH 7.14 (s) and C-7 at δC 30.0 combination with COSY crosspeaks of H-5/H-6/H-7 confirm that compound 4 is as shown. Compounds 5 and 4 have the same relative configuration from the NOESY signals of H-3/H-5, H-3/H-1β, and H-3/H3-18, and H3-20/H-1α. Mosher’s method on 5 reveals its 3R-configuration (Fig. 3) and this is confirmed through the ECD spectrum (Fig. 2). Compound 5 was named fluacinoid E. Compound 6 (fluacinoid F, C20H24O3) is similar to 4, but has an additional α,β-unsaturated ketone in the B ring [δH 6.54 (1H, s); δc 171.1 (C), 125.9 (CH) and 185.0 (C)]. The HMBC cross peaks from the olefinic hydrogen (δH 6.54, s) of the α,β-unsaturated carbonyl group to C-4 (δC 43.5), C-8 (δC 123.5), and C-10 (δC 41.2) reveals that this group locates at C-5, C-6, and C-7. NOESY correlations of H3-20/H3-19/H-1α and H-3/H-1β/H3-18 show the α-orientation of CH3-20 and CH3-19 and the β-orientation of H-3. Compound 6 was defined as 3R (Fig. 3) by the Mosher method as well as the experimental and calculated ECD (Fig. 2). Compound 7 (fluacinoid G), C20H28O3, displays IR absorption bands at 3426 cm−1 (hydroxyl), 1611 cm−1 (aromatic ring), and 1564, 1499 cm−1 (double bonds). As suggested from Tables 3 and 4, compound 7 is very similar to 5 but with a hydroxyl attached to C-2. HMBC correlations from H-2 to C-1, C-3, C-4, and C-10 further confirm this 2D structure. In view of the small observed coupling constant (J2-3 = 3.5 Hz), the 2,3-diol in 7 was defined as the cis-diol. NOESY correlations of H-3/H1β, H-3/H-5, and H-20/H-1α reveal that H-3 and H-5 are co-facial and have the same orientation as CH3-19 and CH3-20. Snatzke’s method [18–21] was used to define the absolute configuration of the 2,3-diol. A negative Cotton effect at λmax 312 nm (Δε = -0.891) was seen in the ECD spectra induced by molybdenum tetraacetate [Mo2(OAc)4] in DMSO (Fig. 5). Consequently, a negative dihedral angle for O-C2-C3-O of the 2,3-diol fragment was determined and compound 7 is assigned as 2R, 3S, which is supported by ECD experiments (Fig. 2). Compound 8 (fluacinoid H) has a molecular formula of C20H26O3, suggesting two hydrogens less than that of 7. Analysis of the NMR data of 8 and 7 suggests a C6]C7 double bond in compound 8, instead of the two aliphatic methylenes (δc 18.8, CH2; 30.0, CH2) in 7. Compound 8 is therefore a dehydrogenated derivative of 7. This is supported by 2D NMR. NOESY cross-peaks of H-3/H-1β, H-3/H-5, and H-20/H-1α suggest 8 has the same relative configuration as that of 7. Snatzke’s method [18–21] defined the configuration of compound 8 as 2R, 3S based on the negative Cotton effect at λmax 302 nm (Δε = −1.525) in the induced ECD spectrum (Fig. 5). Comparing the experimental and calculated ECD further confirms this identification (Fig. 2). Compounds 9–11 are identified as spruceanol (9) [22], phyllanflexoid B (10) [16], and cleistanthol (11) [23] by comparison with published data. Effects of Compounds 1–11 on Osteoclastogenesis. Compounds 1–11 at 10 μM were assayed for their effects on RANKL-induced osteoclasts production in bone marrow mononuclear (BMMs) cells. Parthenolide, as a positive control, inhibits RANKL-induced osteoclast formation with an inhibition rate of 54.6% at 10 μM. Compounds 1–3, which are rearranged clesistanthane-type diterpenoids, show weak

Table 1 1 H NMR Chemical Shifts (δ) of Compounds 1–3 in CDCl3 (400 MHz). No.

1

2

3

1 2 3 4 6 7 11 15 16

3.23, t-like 4.28, m

3.22, d, (2.9) 4.28, m

3.33, m 4.65, m

1.89, sept (6.8) 7.14, d (8.5) 7.84, d (8.5)

1.87, sept (6.9) 7.23, d (8.4) 8.13, d (8.4)

2.50, 7.22, 8.13, 7.36,

3.68, s

3.69, s

2.56, s 1.00, d (6.9) 1.03, d (6.8) 2.38, s 10.25, brs

2.56, 1.04, 0.96, 2.49,

6.99, dd (17.9, 11.5) 5.76, dd (11.5, 2.3) 5.37, dd (17.9, 2.3) 2.36, s 1.04, d (6.8) 1.02, d (6.8) 2.38, s 10.28, brs

17 18 19 20 12-OH

overlap d (8.5) d (8.5) s

s d (7.0) d (6.9) s

Table 2 13 C NMR Chemical Shifts (δ) of Compounds 1–3 in CDCl3 (100 MHz). No.

1

2

3

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

32.2, CH2 70.4, CH 81.9, C 35.6, CH 134.0, C 126.0, CH 124.3, CH 125.5, C 127.7, C 123.4, C 111.6, C 156.1, C 125.2, C 136.7, C 134.6, CH 121.4, CH2 13.9, CH3 16.2, CH3 17.4, CH3 19.8, CH3

32.2, CH2 70.4, CH 82.0, C 35.5, CH 134.7, C 127.2, CH 124.8, CH 127.3, C 127.5, C 123.7, C 114.2, C 155.6, C 120.0, C 132.4, C 80.7, C 86.3, CH 15.2, CH3 17.3, CH3 16.2, CH3 19.9, CH3

34.5, CH2 75.1, CH 217.3, C 37.7, CH 134.8, C 127.9, CH 125.4, CH 128.8, C 131.9, C 128.3, C 107.1, CH 152.8, C 121.0, C 130.0, C 80.7, C 86.4, CH 14.7, CH3 19.5, CH3 17.3, CH3 20.8, CH3

Compound 2 (fluacinoid B), C20H22O3, has a hydroxy group (3410 and 3280 cm−1), an alkynyl group (2097 cm−1), and a phenyl ring (1607, 1591, and 1509 cm−1) in its IR spectrum. In comparison to 1, the NMR data of compound 2 has a characteristic alkynyl group [δC 86.3 (CH), δC 80.7 (C); δH 3.68, (1H, s)] replacing the vinyl group in 1. HMBC correlations between H-15 (δH 3.68) and C-8 (δC 127.3), C-13 (δC 120.0), and C-14 (δC 132.4) also reveal an alkynyl group in 2 in place of the vinyl group in 1. ROESY correlations of H-2/H3-19 suggest that two hydroxy groups are cofacial. The experimental and calculated ECD spectra further confirmed the structure of 2 as 2R, 3S (Fig. 2). Structurally, compound 3 (fluacinoid C, C20H22O3) resembles to 1(7-hydroxy-2,6-dimethyl-1-naphthyl)-4-methyl-3-pentanone[17] but it has an additional OH located at C-2 and an additional alkynyl group at C-14. HMBC correlations from H-1 to C-2, C-3, C-5, and C-9 together with COSY cross-peaks of H1/H2 suggest a 2-OH in 3 (Fig. 1). HMBC signal between H-16 and C-8, C-13, and C-14 reveal an alkynyl moiety at C-14. The 2S configuration was established by the ECD spectra of 3 (Fig. 2). The IR spectrum of compound 4 (fluacinoid D, C20H26O2) shows an OH (3410 cm−1), a C]C (1623 cm−1), and a phenyl (1603, 1556, 1495 cm−1) groups. Tables 3 and 4 reveal compound 4 has one methoxy group (δH 3.85, s), ten sp2 carbons including one vinyl group (δH 6.99, dd, J = 17.8, 11.2 Hz; 5.69, dd, J = 17.8, 1.5 Hz; 5.22, dd, J = 11.2, 1.5 Hz), and three methyl singlets (δH 1.10, 1.03, 1.04). The 2

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Fig. 1. 1H-1H COSY (−) and key HMBC (→) correlations of compounds 1–8.

activity, and compounds 9–11, clesistanthane-type compounds, show no inhibition. Compounds 4–8 are norditerpenoid type compounds. With one hydroxy group, compounds 4 and 5 fail to show inhibition, while compound 6, with an additional α,β-unsaturated ketone, exhibits significant inhibition with IC50 = 0.7 μM (Fig. 6). Compounds 7 and 8, each with two hydroxy groups, also exhibit potent inhibition with IC50 = 2.2 and 4.0 μM, respectively (Figs. S68–S69, Supporting Information). Compounds 6, 7, and 8 at concentrations from 1.5 to

25 μM fail to show cytotoxicity for 72 h in RAW264.7 cells (Fig. S70, Supporting Information). These results suggest that the norditerpenoidtype compounds have a skeleton consistent with RANKL-induced osteoclastogenesis, while the clesistanthane-type or rearranged clesistanthane-type diterpenoids, with their different skeleton, fail to display inhibition. Therefore compound 6, the most potential compound, was further investigated for its effects on RANKL-induced osteoclastogenesis. 3

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Fig. 2. Experimental ECD spectra (200–400 nm) and TD-DFT-calculated ECD spectra for compounds 1–8.

Compound 6 Inhibits Osteoclastogenesis-related Gene. To further evaluate the role of compound 6 in osteoclast differentiation, realtime quantitative PCR (RT-qPCR) experiments were performed. As shown in Fig. 7, compound 6 suppressed the expression of osteoclastic marker genes RANK, CTSK, MMP9, and TRACP in RANKL-induced RAW264.7 cells.

Effects of Compound 6 on the Transcription Factors of NFATc1 and c-Fos in RANKL-induced BMMs. We further examined whether compound 6 changed the expression of NFATc1 and c-Fos stimulated by RANKL, as c-Fos and NFATc1 are key transcription factors for osteoclast differentiation [24]. Bone marrow mononuclear (BMMs) cells were cultured in the absence or presence of 6 for 6 days in differentiation 4

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Table 3 1 H NMR Chemical Shifts (δ) of Compounds 4–8 in CDCl3 (400 MHz). No.

4

1α 1β 2α 2β 3β 4 5β 6α 6β 7α 7β 11 14 15 16

2.20, 1.80, 1.89, 1.82, 3.38,

18 19 20 OMe

5

6

dd (8.8, 2.9) m m m m

2.29, 1.58, 1.83, 1.77, 3.31,

dt (12.9, 3.3) m m m dd (11.0, 5.1)

2.11, t (2.9) 5.90, dd (9.6, 2.7)

1.32, 1.74, 1.88, 2.91, 2.80, 6.73, 7.14, 6.96, 5.68, 5.20, 1.08, 0.90, 1.21, 3.81,

dd m m dd m s s dd dd dd s s s s

6.55, dd (9.6, 3.1) 6.69, 7.18, 6.99, 5.69, 5.22, 1.10, 1.03, 1.04, 3.85,

s s dd (17.8, 11.2) dd (17.8, 1.5) dd (11.2, 1.5) s s s s

(12.2, 2.0)

2.44, 1.69, 2.08, 2.00, 3.45,

dt (13.4, 3.5) td (13.5, 4.2) m m dd (11.5, 4.7)

6.54, s

(16.5, 5.9)

(17.8, 11.2) (17.8, 1.2) (11.2, 1.2)

6.86, 8.24, 6.99, 5.89, 5.33, 1.37, 1.31, 1.55, 3.92,

medium. Results of RT-qPCR suggested that compound 6 treatment significantly reduced the expression of NFATc1 and c-Fos in RANKLstimulated BMMs (Fig. 8). Compound 6 Suppresses RANKL-induced NF-κB Activation. NFκB luciferase reporter gene assay was performed to assay the effect of compound 6 on RANKL-induced signal transduction pathway. Compound 6 significantly inhibits the NF-κB signal pathway in a dosedependent manner (Fig. 9). Compound 6 Induces Apoptotic Cell Death. The effect of compound 6 on mature osteoclast-like (OCL) cells was examined at various concentrations. BMMs stimulated with M-CSF and RANKL to become mature OCL cells were treated with compound 6 for 1 day, and then Trap-stained to visualize the cytoskeleton. As shown in Fig. 10, compound 6 induces apoptosis of mature OCL cells with IC50 = 7.8 μM (Fig. 10).

s s dd (17.8, 11.2) d (17.8, 1.4) d (11.2, 1.3) s s s s

7

8

2.69, dd, (14.4, 2.9) 1.76, dd, (14.4, 3.5) 4.25, m

2.62, dd (14.2, 2.9) 1.98, dd (14.3, 3.4) 4.33, m

3.29, d (3.5)

3.34, d (3.5)

1.40, 1.84, 1.90, 2.82, 2.91, 6.76, 7.13, 6.96, 5.68, 5.19, 1.09, 1.11, 1.46, 3.82,

2.18, d (2.9) 5.94, dd (9.6, 2.8)

d (11.7) m m m dd (16.8, 6.4) s s dd (17.8, 11.2) d (17.8) d (11.2) s s s s

6.57, dd (9.6, 2.9) 6.71, 7.18, 6.98, 5.69, 5.21, 1.11, 1.24, 1.29, 3.86,

s s dd (17.8, 11.2) d (17.8) d (11.2) s s s s

were recorded using a Shimadzu UV-2450 spectrophotometer. CD spectra were obtained on an Applied Photophysics Chirascan spectrometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. The 1H (400 MHz), 13C (100 MHz), and 2D NMR spectra were obtained on a Bruker AM-400 with TMS as an internal reference at 25 °C. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. HRESIMS were acquired on a Shimadzu LCMS-IT-TOF instrument, and the ESIMS data were measured on an Agilent 1200 series LC-MS/MS system. RP-C18 silica gel (Fuji, 40–75 μm), MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (200–300 mesh, Marine Chemical Ltd., Qingdao, People’s Republic of China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography (CC). Analytical and semipreparative HPLC separation were carried out on an LC-20AT Shimadzu liquid chromatography system with an Agilent SB-C18 column connected with an SPD-M20A diode array detector. Semipreparative chiral HPLC separation were carried out on an LC-20AT Shimadzu liquid chromatography system with Phenomenex Lux cellulose-2 chiral-phase column (250 × 10 mm, 5 μm). TLC analysis was carried out on silica gel plates (Marine

3. Experimental section General Experimental Procedures. The Optical rotations were measured on a PerkinElmer 341 automatic polarimeter. UV spectra Table 4 13 C NMR Chemical Shifts (δ) of Compounds 4–8 in CDCl3 (100 MHz). No.

4

5

6

7

8

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

34.4, CH2 27.8, CH2 78.8, CH 38.5, C 50.4, CH 127.1, CH 127.8, CH 126.0, C 148.8, C 38.2, C 105.3, CH 156.6, C 124.3, C 124.7, CH 131.4, CH 114.0, CH2

37.2, CH2 28.1, CH2 78.8, CH 39.2, C 49.9, CH 19.1, CH2 30.0, CH2 127.2, C 150.2, C 38.0, C 107.2, CH 155.2, C 124.6, C 127.0, CH 131.6, CH 113.9, CH2

34.8, CH2 27.4, CH2 76.5, CH 43.5, C 171.1, C 125.9, CH 185.0, C 123.5, C 154.9, C 41.2, C 106.3, CH 160.6, C 126.3, C 124.9, CH 130.5, CH 116.3, CH2

42.8, CH2 71.4, CH 78.4, CH 38.5, C 49.9, CH 18.8, CH2 30.0, CH2 126.7, C 150.8, C 37.5, C 107.5, CH 155.3, C 124.6, C 127.1, CH 131.6, CH 114.0, CH2

40.3, CH2 71.3, CH 78.3, CH 37.9, C 50.3, CH 126.5, CH 128.0, CH 125.6, C 149.2, C 37.7, C 105.5, CH 156.6, C 124.3, C 124.9, CH 131.4, CH 114.1, CH2

28.0, 16.7, 20.3, 55.9,

28.3, 15.6, 24.9, 55.8,

27.6, 22.6, 32.4, 55.8,

29.9, 17.2, 26.8, 55.8,

29.5, 18.1, 22.1, 55.9,

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3

5

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3

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Fig. 3. 1H NMR chemical shift differences of MTPA esters of 4–6.

in H2O (1.5 L) and successively partitioned with CH2Cl2 (4 × 5 L) and n-BuOH (4 × 3 L) to obtain two portions. The CH2Cl2-soluble extract (76.0 g) was separated using a silica gel column with CH2Cl2 − MeOH (100:0, 200:1, 100:1, 50:1, 20:1, 10:1) to yield six fractions (Fr. A-F). Fr. D (8.5 g) was chromatographed over MCI gel CHP-20P, eluted with MeOH-H2O (85:15) and analyzed by silica gel chromatography to afford three sub-fractions (Fr. D1-D3). Fr. D1 was purified on a silica gel column to give 2 (6.7 mg) using CH2Cl2 as eluent. Compounds 1 (10.0 mg, tR = 15 min), 3 (2.6 mg, tR = 25 min), 4 (18.0 mg, tR = 35 min), and 5 (16.3 mg, tR = 38 min) were identified from Fr. D2 through semi-preparative HPLC (SP-HPLC) with CH3CN-H2O (80:20). Fr. E (9.0 g) was analyzed on an RP-18 column using MeOH-H2O (70:30, 80:20 and 90:10) as eluents to produce Fr. E1-E3. Compounds 6 (5.5 mg) and 7 (7.2 mg) were isolated from Fr. E1 using SP-HPLC. Fr. E3 was purified by semi-preparative HPLC, eluted with 70% CH3CNH2O to yield 9 (7.2 mg, tR = 40 min). Fr. F (10.2 g) was further separated by column chromatography to give compounds 10 (4.5 mg), 11 (6.0 mg), and 8 (5.3 mg). Fluacinoid A (1): colorless crystals; [ ]20 D +54° (c 0.5, MeOH); UV (MeOH) λmax (log ε) 240 (4.78), 268 (3.59) nm; ECD (MeOH) λmax (Δε) 204 (-9.78), 236 (+1.99), 337 (+3.84) nm; IR(KBr) νmax 3423, 2921, 2852, 1595, 1542, 1513, 1464, 1379, 1314, 1199, 1077, 998, 925, 870, 816, 724 cm−1; 1H/13C NMR data, see Tables 1 and 2; HRESIMS m/z 311.1657 [M−H]− (calcd for C20H23O3, 311.1653). Fluacinoid B (2): amorphous solid; [ ]20 D +53° (c 0.2, MeOH); UV (MeOH) λmax (log ε) 236 (4.51), 272 (3.19) nm; ECD (MeOH) λmax (Δε) 206 (−3.09), 217 (−2.05), 231 (−3.35), 248 (+0.48), 261 (−0.60), 345 (+2.98) nm; IR(KBr) νmax 3410, 3280, 3060, 2964, 2924, 2097, 1705, 1607, 1591, 1509, 1404, 1320, 1223, 1177, 999, 971, 870, 818, 1 H/13C NMR data, see Tables 1 and 2; HRESIMS m/z 777, 601 cm−1 , 309.1495 [M−H]− (calcd for C20H21O3, 309.1496). Fluacinoid C (3): amorphous solid; [ ]20 D +63° (c 0.8, MeOH); UV (MeOH) λmax (log ε) 234 (4.22) nm; ECD (MeOH) λmax (Δε) 215 (−1.56), 248 (+1.33), 298 (−0.73) nm; IR (KBr) νmax 3408, 3279,

Fig. 4. ORTEP representation of crystal structure of 1.

Chemical Ltd.). Fractions were monitored by TLC and visualized by heating plates sprayed with 5% H2SO4 in EtOH. All solvents were of analytical grade (Guangzhou Chemical Reagents Company Ltd., Guangzhou, People’s Republic of China). Plant Material. The plant materials for twigs and leaves of F. acicularis (11.0 kg) were collected in Lincang District, Yunnan Province, People’s Republic of China and identified by Dr. Chunyan Han. A voucher specimen (XG-2014004) is held at our laboratory. Extraction and Purification. The twigs and leaves of F. acicularis (11.0 kg) were soaked with 95% EtOH (3 × 50 L) at rt for 72 h. Removal of EtOH yielded an extract (820 g), which was then suspended

Fig. 5. IECD spectra of compounds 7 and 8. 6

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Fig. 6. Inhibitory effects of compound 6 on RANKL-induced osteoclast formation. Osteoclastogenesis inhibition ratio and the IC50 curve of compound 6 (left). Representative TRAP-stained images (right) (n = 5 images taken in each well, one image was selected from each well, with triplicate repeated wells of three biological replicates of BMM cells isolated from BMMs from C57BL/6 mice cultured with M-CSF and/or without RANKL and treated with different doses of compound 6 for 6 days. Scale bars, 250 μm.

2963, 2925, 2855, 2096, 1706, 1593, 1509, 1466, 1406, 1261, 1095, 1 1025, 972, 869, 801 cm−1 H/13C NMR data, see Tables 1 and 2; , HRESIMS m/z 311.1638 [M+H]+ (calcd for C20H23O3, 311.1642). Fluacinoid D (4): colorless oil; [ ]20 D +88° (c 0.8, MeOH); UV (MeOH) λmax (log ε) 248 (4.33) nm; ECD (MeOH) λmax (Δε) 203 (−10.34), 244 (+17.39), 277 (−5.48) nm; IR (KBr) νmax 3410, 3082, 2927, 2856, 1711, 1623, 1603, 1556, 1495, 1422, 1369, 1261, 1210, 1132, 1704, 1 1039, 898, 850, 801, 768, 714 cm−1 H/13C NMR data, see Tables 3 , and 4; HRESIMS m/z 299.2005 [M+H]+ (calcd for C20H27O2, 299.2006). Fluacinoid E (5): colorless oil; [ ]20 D −2° (c 0.8, MeOH); UV (MeOH) λmax (log ε) 217 (4.47), 239 (4.09), 253 (4.24), 309 (3.79) nm; ECD (MeOH) λmax (Δε) 208 (+0.42), 217 (+1.94), 233 (−4.35) nm; IR (KBr) νmax 3421, 3083, 2928, 2856, 2656, 1808, 1721, 1624, 1610, 1499, 1463, 1417, 1374, 1322, 1259, 1206, 1132, 1071, 1041, 969, 1 939, 902, 849 cm−1 H/13C NMR data, see Tables 3 and 4; HRESIMS m/ , + z 301.2152 [M+H] (calcd for C20H29O2, 301.2162). Fluacinoid F (6): colorless oil; [ ]20 D +13° (c 1.0, MeOH); UV (MeOH) λmax (log ε) 208 (4.06), 230 (3.94), 253 (4.19), 278 (3.69) nm; ECD (MeOH) λmax (Δε) nm 251 (−4.40), 297 (+1.44), 349 (−0.32) nm; IR (KBr) νmax 3419, 3083, 2926, 2855, 1646, 1594, 1494, 1460, 1304, 1 1260, 1142, 1091, 1039, 884, 802 cm−1 H/13C NMR data, see Tables 3 , and 4; HRESIMS m/z 313.1798 [M+H]+ (calcd for C20H25O3, 313.1798). Fluacinoid G (7): colorless oil; [ ]20 D −3° (c 1.2, MeOH); UV (MeOH) λmax (log ε) 215 (4.04), 238 (3.63), 253 (3.73), 309 (3.30) nm; ECD (MeOH) λmax (Δε) 204 (−1.14), 215 (+0.90) 232 (−1.34) nm; IR (KBr) νmax 3426, 3082, 2961, 2925, 2855, 1709, 1669, 1611, 1564, 1499, 1463, 1415, 1366, 1261, 1200, 1074, 1035, 967, 940, 900, 1 800 cm−1 H/13C NMR data, see Tables 3 and 4; HRESIMS m/z , 317.2099 [M+H]+ (calcd for C20H29O3, 317.2111). Fluacinoid H (8): colorless oil; [ ]20 D +68° (c 0.9, MeOH); UV (MeOH) λmax (log ε) 243 (4.00), 301 (3.22) nm; ECD (MeOH) λmax (Δε) 203 (−3.49), 244 (+6.11), 277 (−1.77) nm; IR (KBr) νmax 3421, 2961,

2925, 2855, 1713, 1604, 1556, 1495, 1462, 1369, 1261, 1207, 1084, 1 H/13C NMR data, see Tables 3 and 4; 1022, 898, 848, 801 cm−1 , HRESIMS m/z 337.1781 [M+Na]+ (calcd for C20H26O3Na, 337.1774). Crystallographic and X-ray Structural Data of 1. Crystallographic data of compound 1 (CCDC 1472727): C20H24O3 (M = 312.17); monoclinic, space group P21 (no. 4), a = 13.56309(19) Å, b = 55.6355(6) Å, c = 14.1821(2) Å, V = 9509.0(3) Å3, Z = 2, T = 100(2) K, μ(Cu Kα) = 0.666 mm−1, Dcalc = 1.2396 g/cm3, 177,738 reflections collected, 37,446 independent reflections (Rint = 0.0764, Rsigma = 0.0469). The final R1 was 0.0503 and wR2 was 0.1275 (all data). Goodness-of-fit on F2 = 1.025. Flack parameter = −0.03 (5). Synthesis of (S)- and (R)-MTPA Esters of Compounds 4–6. (R)MTPA chloride (10 μL) was mixed with the secondary alcohol (2.0 mg each) and 50 μL DMAP (4-dimethylamonipyridine) in chloroform (1.0 mL), and the mixture stirred at room temperature for 10 h. The solvent in crude was removed to give a residue and further chromatographed over a silica gel column (n-hexane-EtOAc, 4:1) to obtain the (S)-MTPA esters, compounds 4a, 5a, and 6a. The (R)-MTPA esters 4b, 5b, and 6b were prepared using the same procedure with (S)-MTPA chloride, each prepared from 2.0 mg of its corresponding alcohol. Selected 1H NMR (CDCl3, 400 MHz) of compound 4a: δH 7.602–7.399 (5H, m, Ph), 7.188 (1H, s, H-14), 6.987 (1H, dd, J = 17.7, 11.2 Hz, H15), 6.673 (1H, s, H-11), 6.558 (1H, dd, J = 9.6, 3.1 Hz, H-7), 5.832 (1H, dd, J = 9.6, 2.7 Hz, H-6), 5.697 (1H, dd, J = 17.7, 1.5 Hz, H-16), 5.225 (1H, dd, J = 11.2, 1.5 Hz, H-16), 4.838 (1H, dd, J = 11.2, 4.2 Hz, H-3), 3.858 (3H, s, OCH3), 3.594 (3H, s, OCH3), 2.249 (1H, m, H-1a), 2.206 (1H, m, H-5), 2.116 (1H, m, H-2a), 2.008 (1H, m, H-2b), 1.925 (1H, m, H-1b), 1.605 (3H, s, CH3, H3-18 or H3-19 or H3-20), 1.304 (3H, s, CH3, H3-18 or H3-19 or H3-20), 0.930 (3H, s, CH3, H3-18 or H3-19 or H3-20). Selected 1H NMR (CDCl3, 400 MHz) of compound 4b: δH 7.580–7.403 (5H, m, Ph), 7.188 (1H, m, H-14), 6.987 (1H, dd, J = 17.8, 11.2 Hz, H-15), 6.670 (1H, m, H-11), 6.562 (1H, dd, J = 9.6, 3.0 Hz, H-7), 5.845 (1H, dd, J = 9.6, 2.6 Hz, H-6), 5.696 (1H, dd, 7

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Fig. 7. Compound 6 suppresses the gene expression of RANK, CTSK, MMP9, and TRACP. RT-qPCR was used to analysis osteoclast marker genes expression in BMMs cultured in M-CSF, or M-CSF and RANKL, or M-CSF, RANKL, with different doses of compound 6 after 6 days’ treatment. Gene expression was normalized to 18S gene. n = 3 per group, p < 0.05 is significant. Fig. 8. Compound 6 reduces the expression of transcription factor of NFATc1 and c-Fos. RT-qPCR was used to analyze the expression of osteoclast marker genes in BMMs cultured in M-CSF, or M-CSF and RANKL, or M-CSF, RANKL, and different doses of compound 6 after 6 days’ treatment. Gene expression was normalized to 18S gene. n = 3 per group, p < 0.05 is significant.

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2.334 (1H, dt, 13.0, 3.2 Hz, H-1a), 2.044 (1H, m, H-2a), 1.955 (1H, m, H-6b), 1.873 (1H, m, H-2b), 1.764 (1H, m, H-6a), 1.689 (1H, m, H-1b), 1.420 (1H, dd, J = 12.1, 2.2 Hz, H-5), 1.254 (3H, s, CH3, H3-18 or H319 or H3-20), 1.237 (3H, s, CH3, H3-18 or H3-19 or H3-20), 0.911 (3H, s, CH3, H3-18 or H3-19 or H3-20). 1H NMR (CDCl3, 400 MHz) of compound 5b: δH 7.605–7.346 (5H, m, Ph), 7.141 (1H, s, H-14), 6.959 (1H, dd, J = 17.8, 11.2 Hz, H-15), 6.698 (1H, s, H-11), 5.682 (1H, dd, J = 17.7, 1.6 Hz, H-16), 5.204 (1H, dd, J = 11.2, 1.6 Hz, H-16), 4.766 (1H, dd, J = 11.7, 4.5 Hz, H-3), 3.807 (3H, s, OCH3), 3.546 (3H, s, OCH3), 2.919 (1H, dd, J = 16.7, 6.6 Hz, H-7a), 2.805 (1H, m, H-7b), 2.305 (1H, dt, J = 13.1, 3.6 Hz, H-1a), 1.977 (1H, m, H-6b), 1.867 (1H, m, H-2a), 1.813 (1H, m, H-6a), 1.737 (1H, m, H-2b), 1.676 (1H, m, H1b), 1.423 (1H, dd, J = 12.2, 2.2 Hz, H-5), 1.209 (3H, s, CH3, H3-18 or H3-19 or H3-20), 0.996 (3H, s, CH3, H3-18 or H3-19 or H3-20), 0.921 (3H, s, CH3, H3-18 or H3-19 or H3-20). 1H NMR (CDCl3, 400 MHz) of 6a: δH 8.240 (1H, s, H-14), 7.629–7.394 (5H, m, Ph), 6.992 (1H, dd, J = 17.6, 11.2 Hz, H-15), 6.843 (1H, s, H-11), 6.525 (1H, s, H-6), 5.895 (1H, dd, J = 17.7, 1.3 Hz, H-16), 5.345 (1H, dd, J = 11.2, 1.3 Hz, H16), 4.852 (1H, dd, J = 11.6, 4.7 Hz, H-3), 3.929 (3H, s, OCH3), 3.598 (3H, s, OCH3), 2.494 (1H, dt, J = 13.5, 3.3 Hz, H-1a), 2.222 (1H, m, H2a), 2.193 (1H, m, H-2b), 1.837 (1H, m, H-1b), 1.576 (3H, s, CH3, H318 or H3-19 or H3-20), 1.307 (3H, s, CH3, H3-18 or H3-19 or H3-20), 1.183 (3H, s, CH3, H3-18 or H3-19 or H3-20). 1H NMR (CDCl3, 400 MHz) of compound 6b: δH 8.238 (1H, s, H-14), 7.592–7.412 (5H, m, Ph), 6.990 (1H, dd, J = 17.7, 11.2 Hz, H-15), 6.835 (1H, s, H-11), 6.535 (1H, s, H-6), 5.892 (1H, dd, J = 17.7, 1.4 Hz, H-16), 5.343 (1H, dd, J = 11.2, 1.4 Hz, H-16), 4.807 (1H, dd, J = 11.6, 4.7 Hz, H-3), 3.924 (3H, s, OCH3), 3.534 (3H, s, OCH3), 2.462 (1H, dt, J = 13.9, 3.5 Hz, H-1a), 2.152 (1H, m, H-2a), 2.059 (1H, m, H-2b), 1.799 (1H, m, H-1b), 1.546 (3H, s, CH3, H3-18 or H3-19 or H3-20), 1.294 (3H, s, CH3, H3-18 or H3-19 or H3-20), 1.250 (3H, s, CH3, H3-18 or H3-19 or H3-20). ECD Computational Calculations of 1–8 and Induced ECD Experiments for Compounds 7–8. The ECD spectra were analyzed using Gauss View (version 5.0) and calculated using Gaussian 09 at the density functional theory (DFT) and time-dependent DFT levels. The Discovery Studio 3.5 (Accelrys Inc., San Diego, CA) was employed to generate conformations of these new compounds. At the B3LYP/6-

Fig. 9. Compound 6 inhibits RANKL-induced activation of NF-κB. NF-κB transfected RAW264.7 cells were treated with different doses of compound 6 and stimulated with RANKL. NF-κB luciferase activity was measured after 6 h of treatment. n = 3 per group, p < 0.05 is significant.

J = 17.8, 1.6 Hz, H-16), 5.224 (1H, dd, J = 11.2, 1.6 Hz, H-16), 4.807 (1H, dd, J = 11.2, 4.3 Hz, H-3), 3.854 (3H, s, OCH3), 3.548 (3H, s, OCH3), 2.235 (1H, m, H-1a), 2.210 (1H, m, H-5), 2.046 (1H, m, H-2a), 1.880 (1H, m, H-2b), 1.857 (1H, m, H-1b), 1.043 (3H, s, CH3, H3-18 or H3-19 or H3-20), 1.039 (3H, s, CH3, H3-18 or H3-19 or H3-20), 1.016 (3H, s, CH3, H3-18 or H3-19 or H3-20). 1H NMR (CDCl3, 400 MHz) of compound 5a: δH 7.617–7.349 (5H, m, Ph), 7.138 (1H, s, H-14), 6.958 (1H, dd, J = 17.8, 11.2 Hz, H-15), 6.705 (1H, s, H-11), 5.682 (1H, dd, J = 17.7, 1.7 Hz, H-16), 5.203 (1H, dd, J = 11.2, 1.6 Hz, H-16), 4.975 (1H, dd, J = 11.8, 4.6 Hz, H-3), 3.811 (3H, s, OCH3), 3.589 (3H, s, OCH3), 2.915 (1H, dd, J = 16.5, 6.5 Hz, H-7a), 2.800 (1H, m, H-7b),

Fig. 10. Compound 6 induces apoptosis of mature osteoclast cell lines. Osteoclast apoptosis ratio and IC50 curve of compound 6 (left). Representative TRAP- stained images (right) (n = 5 images taken in each well, one image was selected from each well, with triplicate repeated wells of three biological replicates) of survival osteoclasts after treatment with different doses of compound 6 for 24 h. Scale bars, 250 μm. 9

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31+G (d, p) level in the gas phase, the minimum geometries were optimized to get preferential conformations. The ECD spectrum were simulated at the B3LYP/6-311+G (2d, p) level in MeOH. The calculated ECD data were generated in the SpecDis software package. Induced ECD Experiments. Compound 7 (1.0 mg) was dissolved in anhydrous DMSO (1 mL) and mixed with [Mo2(OAc)4]. After mixing, the ECD spectrum of the mixture [molar ratio ca. 1:1.2 diol/ Mo2(OAc)4] was measured instantly from 260 nm to 450 nm. The IECD spectrum was subtracted from the inherent ECD spectrum [18]. The sign of the OeCeCeO dihedral angles and the relation between the absolute configurations of the 1, 2-diol unit of compound 7 was assigned following a reported procedure [18–21]. The IECD spectrum of compound 8 was measured using the same procedure. RANKL-induced Osteoclastogenesis Assays. Osteoclastogenesis assays were conducted according to the previously reported method [25]. Bone marrow macrophages (BMMs) were isolated from 6-weekold C57BL/6 mice and then seeded in complete α-MEM supplemented with 20 ng/mL macrophage-colony stimulating factor (M-CSF), purchased from R&D Systems (Minnneapolis, MN, USA) and cultured in 5% CO2 at 37 °C. The differentiation experiments were conducted in triplicate. BMMs were seeded into 96-well plates at a concentration of 1.5 × 104 cells per well. Cells were stimulated with 100 ng/mL RANKL (Proteintech, Rosemont, IL, USA) and 20 ng/mL M-CSF. Compounds and medium were replaced every 2 days. In the sixth day, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and further stained using the TRAP-staining kit (Sigma-Aldrich, 387A-1KT, USA). TRAP-positive multinucleated cells with > 3 nuclei were scored as osteoclasts. Photographs of individual wells were taken using Leica DMi8/DPC7000T (Leica Microsystems, Wetzlar and Mannheim, Germany). NF-κB Luciferase Reporter Gene Assay. RAW264.7 cells stably transfected with a luciferase reporter gene (3κB-Luc-SV40) were used to evaluate the effects of compound 6. RAW264.7 cells stably transfected with a luciferase reporter gene (3κB-Luc-SV40), gifted from Professor Jiake Xu from the School of Pathology and laboratory Medicine, the University of Western Australia, were used to evaluate the effects of compound 6. Cells were cultured in complete α-MEM supplemented and plated in 96-well plates at a density of 1.5 × 105 cells/well and treated with compound 6 with or without RANKL (100 ng/mL) stimulation for 6 h. Cells were harvested, and luciferase activity measured using the Promega Luciferase Assay System according to the manufacturer’s instructions (Progema, Madison, WI, USA). Cell Apoptosis Assay. BMMs cells (1.5 × 104 cells/well) planted in 96 well plates were cultured for 24 h. Complete media with RANKL (100 ng/mL) and M-CSF was replaced every other day. After osteoclast formation was observed, the media were replaced with fresh media together with M-CSF, presence or absence of compounds and incubated for 24 h, then TRAP-stained to determine the amount of live OC cells. Cytotoxicity Assays. The cell survival was tested by 3-(4,5-dimetrylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. BMMs 1.5 × 104 cells/well were seeded into 96-well plate overnight and treated with either compounds or only DMSO for another 72 h. Next, 20 μL of MTT solution (5 mg/mL) was added to each well and the cells were incubated in 37 °C for 4 h. After removing the supernatant and DMSO (120 μL/well) was added for solubilization of formazan. The optical density at 490 nm was measured by a microplate reader (FlexStation 3, Molecular Devices, US). RT–qPCR Analysis. The detail experimental methods were given in the Supporting Information.

4. Notes Authors declare no competing financial interest. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgments This study was supported in part by the National Natural Science Foundation of China (No. 81573310, 81471138) and the Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030). Appendix A. Supplementary material The Supplementary data is given on the ACS Publications website and includes: IR, ESIMS, HRESIMS, and NMR data for 1–8, 1 H NMR for 4a, 4b, 5a, 5b, 6a, 6b, and osteoclastogenesis inhibition data for 7–8. Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioorg.2019.103453. References [1] C. Topini, D. Topini, G. Cerica, F. Nardocci, G. Topini, Clin. Cases Miner. Bone Metab. 11 (2014) 129–131. [2] G.A. Rodan, T.J. Martin, Science 289 (2000) 1508–1514. [3] S. Tanaka, K. Nakamura, N. Takahasi, T. Suda, Immunol. Rev. 208 (2005) 30–49. [4] S. Qin, E. Ang, L. Dai, X. Yang, D. Ye, H. Chen, L. Zhou, M. Yang, D. Teguh, R. Tan, J. Xu, J. Tickner, N.J. Pavlos, J. Mol. Sci. 16 (2015) 26599–26607. [5] S.N. Liu, D. Huang, S.L. Morris-Natschke, H. Ma, Z.H. Liu, N.P. Seeram, J. Xu, K.H. Lee, Q. Gu, Org. Lett. 18 (2016) 6132–6135. [6] L.S. Gan, C.Q. Fan, S.P. Yang, Y. Wu, L.P. Lin, J. Ding, J.M. Yue, Org. Lett. 8 (2006) 2285–2288. [7] B.X. Zhao, Y. Wang, D.M. Zhang, R.W. Jiang, G.C. Wang, J.M. Shi, X.J. Huang, W.M. Chen, C.T. Che, W.C. Ye, Org. Lett. 13 (2011) 3888–3891. [8] B.X. Zhao, Y. Wang, D.M. Zhang, X.J. Huang, L.L. Bai, Y. Yan, J.M. Chen, T.B. Lu, Y.T. Wang, Q.W. Zhang, W.C. Ye, Org. Lett. 14 (2012) 3096–3099. [9] H. Zhang, C.R. Zhang, K.K. Zhu, A.H. Gao, C. Luo, J. Li, J.M. Yue, Org. Lett. 15 (2013) 120–123. [10] M. Chen, L. Hou, Chih Wu Hsueh Pao 27 (1985) 625–629. [11] L.S. Gan, J.M. Yue, Nat. Prod. Commun. 1 (2006) 819–823. [12] E.V. Dehmlow, M. Guntenhoner, T. Van Ree, Phytochemistry 52 (1999) 1715–1716. [13] G.O. Iketubosin, D.W. Mathieson, J. Pharm. Pharmacol. 15 (1963) 810–815. [14] C.H. Chao, J.C. Cheng, D.Y. Shen, T.S. Wu, J. Nat. Prod. 77 (2014) 22–28. [15] T.H. Duong, X.H. Bui, P. Le Pogam, H.H. Nguyen, T.T. Tran, T.A.T. Nguyen, W. Chavasiri, J. Boustie, K.P.P. Nguyen, Tetrahedron 73 (2017) 5634–5638. [16] J.Q. Zhao, J.J. Lv, Y.M. Wang, M. Xu, H.T. Zhu, D. Wang, C.R. Yang, Y.F. Wang, Y.J. Zhang, Tetrahedron Lett. 54 (2013) 4670–14574. [17] W. Yuan, Z. Lu, Y. Liu, C. Meng, K.D. Cheng, P. Zhu, Chem. Pharm. Bull. 53 (2005) 1610–1612. [18] G. Snatzke, U. Wagner, H.P. Wolff, Tetrahedron 37 (1981) 349–361. [19] L. Di Bari, G. Pescitelli, C. Pratelli, D. Pini, P. Salvadori, J. Org. Chem. 66 (2001) 4819–4825. [20] J. Frelek, W.J. Szczepek, Tetrahedron Asymm. 10 (1999) 1507–1520. [21] H. Li, J.J. Zhao, J.L. Chen, L.P. Zhu, D.M. Wang, L. Jiang, D.P. Yang, Z.M. Zhao, Phytochemistry 117 (2015) 400–409. [22] S.P. Gunasekera, G.A. Cordell, N.R. Farnsworth, J. Nat. Prod. 42 (1979) 658–662. [23] E.J. McGarry, K.H. Pegel, L. Philips, E.S. Waight, J. Chem. Soc. (C) 2 (1971) 904–909. [24] H. Takayanagi, S. Kim, T. Koga, H. Nishina, M. Isshiki, H. Yoshida, A. Saiura, M. Isobe, T. Yokochi, J. Inoue, E.F. Wagner, T.W. Mak, T. Kodama, T. Taniguchi, Dev. Cell 3 (2002) 889–901. [25] Q. Liu, H.F. Wu, S.M. Chim, L. Zhou, J.M. Zhao, H.T. Feng, Q.L. Wei, Q. Wang, M.H. Zheng, R.X. Tan, Q. Gu, J. Xu, N. Pavlos, J. Tickner, J.K. Xu, Biochem. Pharmacol. 86 (2013) 1775–1783.

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