Discovery of diverse diterpenoid scaffolds from Euphorbia antiquorum and their activity against RANKL-induced osteoclastogenesis

Bioorganic Chemistry 92 (2019) 103292

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Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg

Discovery of diverse diterpenoid scaffolds from Euphorbia antiquorum and their activity against RANKL-induced osteoclastogenesis

T

Zhi-Yong Yin1, Yan Dai1, Pei Hua, Zhe-Jun Sun, Yan-Fang Cheng, Sheng-Heng Yuan, ⁎ Zi-Yang Chen, Qiong Gu Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China

ARTICLE INFO

ABSTRACT

Keywords: Euphorbia antiquorum Diterpenoids Osteoporosis

Seven new diterpenoids, euphorantones A–D (1, 3, 6, and 10), 8,12,13-epi-3,7,12-O-triacetyl-8-O-(2-methylbutanoyl)-ingol (9), 8,12,13-epi-3,12-O-diacetyl-7-O-benzoyl-8-methoxyingol (11), 2,3-epi-7,12-diacetate-8benzoate-ingol (12), together with eighteen known compounds (2, 4–5, 7–8, and 13–25), were isolated from the aerial parts of Euphorbia antiquorum L.. The structures of new compounds 1, 3, 6, and 9–12 were elucidated by extensive spectroscopic analyses. The absolute configurations of new compounds were assigned using X-ray diffraction, Rh2(OCOCF3)4-induced CD spectrum, and confirmed through comparison of the calculated and experimental 13C NMR and electronic circular dichroism (ECD) data. Compounds 1–25 were evaluated for their inhibition of RANKL-induced osteoclastogenesis. Compound 1 showed the most potent inhibition of RANKLinduced osteoclastogenesis with IC50 value of 0.3 μM. It inhibited NFAT transcript activity and osteoclast related genes TRAcP, CTSK, and NFATc1 expression.

1. Introduction

automatic polarimeter. The UV spectra were recorded using a Shimadzu UV-2450 spectrophotometer. ECD spectra were acquired on an Applied Photophysics Chirascan spectrometer. IR spectra were determined using a Bruker Tensor 37 infrared spectrophotometer. The NMR spectra were tested on BRUKER AV-400-III and BRUKER AV-500-III spectrometers with TMS as an internal reference. HRESIMS were acquired on a Shimadzu LCMS-IT-TOF, 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). Preparative and semi-preparative HPLC separations were carried out on a LC-20AT Shimadzu liquid chromatography system with an YMC ODS-A column (250 × 4.6 mm, 5 μm) connected with an SPD-M20A diode array detector. TLC analysis was carried out on silica gel plates (Marine Chemical Ltd., Qingdao, China). Fractions were monitored by TLC and visualized by heating plates sprayed with 5% H2SO4 in EtOH. BMMs (Bone marrow macrophage cells) were cultured on α-MEM medium (Gibco, China) containing 10% FBS (Gibco, America), 100 IU/mL penicillin, and 100 mg/ mL streptomycin (Hyclone, Shanghai). MTT (Sigama, America) assay was measured on Microplate reader (Thermo, Electroncorporation, China).

Euphorbia, the largest genus of Euphorbiaceae family, is well-known for chemical diverse secondary metabolites [1]. Chemical investigations on this genus plants have led to a variety of diterpenoids including cembrane, ent-abietane, ent-isopimarane, ent-atisane, jatrophane, tigliane, myrsinol, and ingol types. Some of them show multiple biological activities including cytotoxic, anti-inflammatory, and anti-HIV activities [2]. Euphorbia antiquorum L. has been used in the Chinese medicinal system for the treatment of toothache, dropsy, palsy, and amaurosis [3,4]. Some diterpenoids and triterpenoids have been isolated from this plant [3–5]. As part of our continuing study on the structurally unique and bioactive constituents from Euphorbiaceae family plants, here we report the chemical constituents of the aerial parts of E. antiquorum and their activity against RANKL-induced osteoclastogenesis. This results in the isolation of twenty-five diterpenoids including seven new ones (Fig. 1). 2. Materials and methods 2.1. General experimental procedures Optical rotations data were obtained with the Anton Paar MCP 200

Corresponding author. E-mail address: [email protected] (Q. Gu). 1 These authors contributed equally. ⁎

https://doi.org/10.1016/j.bioorg.2019.103292 Received 19 April 2019; Received in revised form 10 September 2019; Accepted 16 September 2019 Available online 18 September 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Bioorganic Chemistry 92 (2019) 103292

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Fig. 1. Structures of compounds 1–25 from the aerial parts of E. antiquorum.

2.2. Plant material

with MeOH as eluents and further purified by semi-preparative HPLC with 60% MeCN–H2O to give compounds 6 (5 mg), 23 (10 mg), and 25 (28 mg).

The aerial parts of E. antiquorum (3.0 kg) were collected from Xishuangbanna in Yunnan Province, People’s Republic of China, on August 21, 2016, and identified by Dr. Chunyan Han from the Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (2016003G) was deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.

2.4. Spectroscopic data Euphorantone A (1): White powder; [α]25 D −47.1° (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (1.50) nm; ECD (MeOH) λmax (Δε) 203 (−3.98), 287 (−0.36); IR (KBr) νmax 3436, 2929, 1704, 1448, 1222, 1018 and 661 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS: m/z 331.2233 [M+Na]+ (calcd for C19H32O3Na, 331.2244). Euphorantone B (3): White powder; [α]25 D +169.9° (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (1.48) nm; ECD (MeOH) λmax (Δε) 234 (−22.4), 275 (+48.67) nm; IR (KBr) νmax 2923, 1747, 1702, 1666, 1091, 1027 and 677 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS: m/z 315.1947 [M+H]+ (calcd for C20H26O3, 315.1955). Euphorantone C (6): White powder; [α]25 D −18.8° (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (0.33), 228 (0.31), 273 (0.03) nm; CD (MeOH) λmax (Δε) 204 (−6.2), 249 (+0.4), 294 (−2.0) nm; IR (KBr) νmax 3361, 2919, 1712, 1656, 1268, 1024 and 798 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS: m/z 441.2634 [M+H]+ (calcd for C27H36O5, 441.2636). 8,12,13-epi-3,7,12-O-triacetyl-8-O-(2-methylbutanoyl)-ingol (9): Colorless syrup; [α]25 D −44.0° (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (0.82) nm; CD (MeOH) λmax (Δε) 231 (+8.2), 268 (+0.6), 303 (+2.1) nm; IR (KBr) νmax 2935, 1737, 1373, 1240 and 1018 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS: m/z 599.2810 [M+Na]+ (calcd for C31H44O10Na, 599.2827). Euphorantone D (10): White solid; [α]25 D +218.5° (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (0.99), 230 (0.71) nm; CD (MeOH) λmax (Δε) 239 (+23.6), 275 (+5.1), 302 (+10.0) nm; IR (KBr) νmax 3390, 2927, 1687, 1454, 1375, 1081 and 661 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS: m/z 403.2090 [M+Na]+ (calcd for C21H32O6Na, 403.2091). 8,12,13-epi-3,12-O-diacetyl-7-O-benzoyl-8-methoxyingol (11): White solid; [α]25 D −144.6° (c 0.3, MeOH); UV (MeOH) λmax (log ε) 204 (1.31), 234 (0.37) nm; CD (MeOH) λmax (Δε) 229 (+12.2), 254 (+1.7), 304 (+6.0) nm; IR (KBr) νmax 2931, 1724, 1542, 1454, 1243, 1018 and

2.3. Extraction and isolation The air-dried and powdered aerial parts of E. antiquorum (3.0 kg) were extracted with 95% EtOH to obtain an extract, which was partitioned with CH2Cl2 (3 × 3 L) and H2O. The CH2Cl2 fraction (80 g) was subjected to silica gel column chromatography (CC) using dichloromethane/methanol (1:0, 200:1, 50:1, 30:1, and 10:1 v/v) to afford fractions A–E. Fraction B (7.8 g) was separated using MCI gel CHP20P eluting with MeOH-H2O (85:15) and further purified by silica gel columns to afford three sub-fractions (B1–B3). Fraction B1 was purified by silica gel columns to give 3 (5 mg), 20 (2 mg), and 22 (3 mg) by using CH2Cl2-MeOH as eluents. Compounds 11 (6 mg), 12 (7 mg), 13 (4 mg), and 14 (22 mg) were isolated from fraction B2 by semi-preparative HPLC with MeCN-H2O (80:20). B3 was purified with semipreparative HPLC eluting with MeCN-H2O (70:30) to obtain compounds 9 (16 mg), 15 (12 mg), 16 (9 mg), 17 (13 mg), and 18 (7 mg). Fraction C (11 g) was submitted to an RP-C18 column eluting with a step gradient of MeOH–H2O from 50% to 100% to afford four subfractions (C1–C4). Fraction C1 was further separated using semi-preparative HPLC with 45% MeOH–H2O as the solvent to produce compounds 7 (7 mg), 8 (6 mg), and 19 (4 mg). Fraction C2 was subjected to semipreparative HPLC eluting with 37% CH3CN–H2O and further purified through semi-preparative HPLC eluting with 55% MeOH–H2O to give compounds 4 (6 mg), 5 (7 mg), and 10 (6 mg). Fraction C3 was chromatographed over a silica gel column with CH2Cl2–MeOH (100:1, 9:1) and further purified by semi-preparative HPLC using 40% MeCN–H2O to afford 21 (14 mg) and 24 (3 mg). Compounds 1 (3 mg) and 2 (2 mg) were obtained by semi-preparative RP-HPLC with 40% aqueous MeOH as the solvent. Fraction C4 was chromatographed over Sephadex LH-20 2

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Table 1 1 H NMR and

13

C NMR Data of Compounds 1, 3 and 6. δC

1a δH (J in Hz)

δC

1

46.2

1.31, m

215.7

2

36.2

4.54, dd (4.5, 10.7)

36.2

3

125.9

5.20, d (10.7)

43.8

4 5

139.1 36.0

6

19.2

7

35.7

8 9

212.7 40.2

10

27.9

11

70.4

12 13

74.6 33.7

14

18.6

15

Position

3a δH (J in Hz)

δC 39.1

2.91, 2.21, 1.89, 1.71,

33.9 57.7

m m dd (4.9, 13.8) overlap

213.1

20.0

1.61, m

39.3

1.46, m 1.18, m

2.51, 2.32, 2.13, 1.55, 3.70,

m m t (3.7) m m

29.1

2.24, 1.27, 1.62, 1.32, 1.14,

m m m m m

16 17

20.4 20.9

0.70, d (6.5) 0.86, d (6.6)

175.4 8.5

1.84, s

74.1 69.1

18 19

14.3 23.3

1.66, s 1.05, s

32.6 22.5

0.97, s 1.11, s

20.5 66.9

15.0

1.30, s

14.4 166.6 138.1 129.8 128.6 133.3

36.9 150.8 44.3

m m t (13.6) t (13.6)

2.81, t (8.8)

56.1

33.0 51.3

1.35, d (3.7)

37.5

30.2 76.4 155.5 115.8

2.60, dd (6.6, 14.3) 1.74, dd (1.3, 4.8) 4.76, dd (6.6, 13.6)

23.4

6.34, s

27.4

117.1

20 OBz

Recorded in CDCl3 (1H NMR 400 MHz,

1.81, 1.57, 2.50, 2.13,

m m td (6.0, 14.4) td (2.7, 6.0)

1.40, m

m m m m ddd (4.1, 12.6, 18.9) d (3.4)

24.3

1.94, 1.39, 2.80, 2.32,

52.6 57.9

2.38, 2.07, 2.00, 1.62, 2.76, 2.17,

a

1.53, m

34.9

6a δH (J in Hz)

32.2 23.4

52.5

2.05, 1.25, 1.83, 1.63, 1.49, 1.86, 0.83, 1.22, 1.14,

d (2.9) d (2.9) m m m m t (6.7) overlap overlap

3.58, 3.44, 1.27, 4.85, 4.22, 1.29,

d d s d d s

(10.9) (10.9) (11.3) (11.3)

7.97, m 7.42, m 7.54, m

13

C NMR 100 MHz).

667 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS: m/z 591.2558 [M+Na]+ (calcd for C32H40O9Na, 591.2565). 2,3-epi-7,12-diacetate-8-benzoate-ingol (12): White solid; [α]25 D −40.5° (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (0.47), 215 (0.20) nm; CD (MeOH) λmax (Δε) 234 (+2.4), 270 (+0.4), 304 (+1.5) nm; IR (KBr) νmax 3743, 2917, 1704, 1542, 1236, 1105 and 1024 cm−1; 1H and 13 C NMR data, see Table 1; HRESIMS: m/z 577.2420 [M+Na]+ (calcd for C31H38O9Na, 577.2408).

number CCDC 1894142. 2.6. Determination of the absolute configuration of the secondary alcohol unit of compound 1 Compound 1 (0.5 mg) was dissolved in anhydrous CH2Cl2 (1 mL) and mixed with [Rh2(OCOCF3)4]. After mixing, the ECD spectrum of the mixture [molar ratio ca. 1:2 secondary alcohol/Rh2(OCOCF3)4] was measured instantly from 270 nm to 450 nm, and until it reached a stable phase (about 30 min) with the time course monitored. The induced ECD (IECD) spectrum was subtracted from the inherent ECD spectrum. The observed sign of the band at around 350 nm in the induced CD spectrum is correlated to the absolute configuration of the secondary alcohol [6].

2.5. X-ray crystallographic analysis of compound 9 The crystal structure and absolute configuration of 9 were determined by single crystal X-ray diffraction analysis. A suitable crystal was selected and tested on an Xtalab Synergy, Rigaku diffractometer with the temperature kept at 99.99(10) K during data collection. The structure was solved and refined using the programs XS and olex2.refine, respectively. The program X-Seed was used as an interface to the SHELX programs, and to prepare figures. Crystal data for compound 9: C31H44O10 (M = 576.30); colourless plate, space group P212121 (no. 0), a = 9.8679(3) Å, b = 13.4497 (3) Å, c = 13.7878 (3) Å, V = 1713.76 (8) Å; Z = 2, T = 99.99(10) K, μ(Cu Kα) = 0.730 mm−1, Dcalc = 1.180 g/cm3, 62,248 reflections collected, 12,897 unique (Rint = 0.0604), which were used in all calculations. The final R1 was 0.0796 and wR2 was 0.2219 (all data). Flack parameter = 0.00(7). Crystallographic data for 9 have been deposited at the Cambridge Crystallographic Data Centre under the reference

2.7. ECD and

13

C NMR computational methods

The ECD and 13C NMR spectra were calculated according to the reported methods [7]. Compounds 1, 3, 6, and 9–12 were calculated via density functional theory (DFT) and time-dependent DFT (TDDFT) using Gaussian 09. The structure at the HF/6-31G level in the gas phase was optimized. Next, the corresponding minimum geometries were further optimized at the B3LYP/6-31+G (d, p) level in the gas phase. The ECD spectra were calculated at the B3LYP/6-31++G (2d, 2p) level in MeOH. The computational ECD data were fitted in the SpecDis 3

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O H

H

H

O

O

O

H HO

H O

H 3

1

OH

HO H

O

6

H O O

O

3

1

O

O

O H

O

O

OH H

O H

O

O O

O

O

O

9

10

9

10 1

1

Fig. 2. Key H– H COSY (

), HMBC (

O

11

), and NOESY (

H

HO

O O

O

O 12

O

O

12 ) correlations of compounds 1, 3, 6, and 9–12.

MEM supplemented with 10% FBS with M-CSF) [8]. 2.9. Assay for cell viability BMMs were seeded into a 96-well plate at 6×103 cells/well. The second day, the cells were incubated with different concentrations of compounds for 72 h. Then, MTT solution (5 mg/mL) was added and incubated with cells for 4 h. The absorbance at 570 nm was assayed with a multi-functional microplate reader.

2.8. Cell cultures Bone marrow macrophage cells (BMMs) were obtained from 4/6week-old C57BL/6J mice to prepare osteoclasts. After isolation, we got marrow cells and they were cultured in complete α-MEM medium (αExpt l ECD of 1

1.0

Calcd ECD of 1

IECD spectrum of 1

ent-Calcd ECD of 1

3

0.5

1

∆ε

∆ε

O

O H

O

11

software package. The 13C NMR chemical shifts were calculated at the mPW1PW91/6-311++G (2df, 2pd) level in chloroform. The computational data were fitted in the GraphPad Prism 5 (GraphPad Inc., La Jolla, CA, USA).

5

O H H

O

O

HO

O O

O

H

HO

6

O

-1

0.0

-3 -5 200

250

300

Wavelength[nm]

350

-0.5

400

300

350

400

450

Wavelength[nm]

Fig. 3. Experimental ECD spectra (200–400 nm) and TD-DFT-calculated ECD spectra for compound 1 (left), The Rh2(OCOCF3)4 induced CD spectrum (270–450 nm) of 1 in CH2Cl2. 4

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2.10. TRAcP staining

2.12. NFAT luciferase assay

BMM cells were planted in 96-well plate, and then cultured with the completed α-MEM medium supplemented M-CSF. Overnight, compounds with varied concentrations (0.03, 0.1, 0.3, 1, 3, 10 μM) and RANKL (50 ng/mL) were added in 96-well plate. The culture medium was changed every two days. After 4/5 days, 4% paraformaldehyde was used to fix cells for 30 min and washed softly with ddH2O for three times. Then cells were stained towards TRAcP (tartrate resistant acid phosphatase) for 1 h at 37 °C.

RAW264.7 cells stably transfected with an NFAT luciferase reporter construct were seeded into a 96-well plate. The second day, cells were treated with 10 μM compound 1 for 1 h and then treated with RANKL (50 ng/mL) for 24 h. Then luciferase activity was detected using the luciferase reporter assay system referring to the manufacturer’s protocol (Promega, Sydney, Australia).

2.11. RNA extraction and analysis

Euphorantones A (1) was obtained as white powder. The HRESIMS displayed a sodiated molecular ion peak at m/z 331.2233 [M+Na]+ (calcd for C19H32O3Na 331.2244) corresponding to a molecular formula of C19H32O3, which indicated four unsaturated degrees. The IR spectrum showed absorption bands at 3436 and 1704 cm−1 indication of hydroxy and carbonyl groups, respectively. The 1H NMR data of 1 revealed that four methyls including one tertiary (δH 1.05, s), one olefinic (δH 1.66, s), and two secondary methyls (δH 0.70, d, J = 6.5 Hz; δH 0.86, d, J = 6.6 Hz). The 13C NMR data of 1 (Table 1) displayed 19 carbon signals comprising four methyls, seven methylenes, three methines (one olefinic), one carbonyl, one quaternary carbon (olefinic), two oxygenated methine carbons, and one oxygenated tertiary carbon. One double bond and the carbonyl group accounted for two unsaturated degrees. Thus, the remaining two degrees of unsaturation defined 1 as a bicyclic molecule. The aforementioned data, in particular the NMR data of 1 (Table 1) exhibited the characteristic resonances for a cembrane-type diterpenoids [9]. Comparing its NMR data with those of quorumolide B (2) [10] demonstrated that the presence of an additional ketone carbonyl carbon signal at δC 212.7 and methylene signal at δC 35.7, and the absence of one carboxylic acid signal and two ole-

3. Results and discussion

After osteoclasts were formed, total RNA was extracted according to the manufacturer’s protocol. For reverse transcription (RT)-PCR, singlestranded cDNA was reverse transcribed from 1 mg total RNA. The cycling parameters for PCR were set as follows: 94 °C for 5 min, followed by 30 cycles of 94 °C (40 s), 60 °C (40 s); 72 °C (40 s), followed by an elongation step of 5 min at 72 °C. The following specific primers (based on the mouse sequences) were used: Target genes

PCR primer sequences

TRAcP-F TRAcP-R CTSK-F CTSK-R NFATC-F NFATC-R β-actin-F β-actin-R

5′-TGTGGCCATCTTTATGCT-3′ 5′-GTCATTTCTTTGGGGCTT-3′ 5′-GGGAGAAAAACCTGAAGC-3′ 5′-ATTCTGGGGACTCAGAGC-3′ 5′-CCCGTCACATTCTGGTCCAT-3′ 5′-CAAGTAACCGTGTAGCTGCACAA-3′ 5′-CCTTCTACAATGAGC-3′ 5′-ACGTCACACTTCATG-3′

60

Exptl ECD of 3

210

ent-Calcd ECD of 3

(ppm)

0

calcd

∆ε

20

-20

180 150 120 90 60

-40 -60 200

y = 1x + 0.001 R2 = 0.9991

240

Calcd ECD of 3

40

30

250

300

350

Wavelength[nm]

0

400

0

30

60

90

120 150 180 210 240 expt

(ppm)

Fig. 4. Experimental ECD spectra (200–400 nm) and TD-DFT-calculated ECD spectra for compound 3 (left), Linear regression analysis of experimental vs calculated 13 C NMR chemical shifts of 3.

Exptl ECD of 6

8

210

ent-Calcd ECD of6

(ppm)

4 0

calcd

∆ε

2 -2

180 150 120 90

-4

60

-6

30

-8 200

250

300

350

Wavelength[nm]

y = 1x - 0.0014 R2 = 0.9975

240

Calcd ECD of 6

6

0

400

0

30

60

90

120 150 180 210 240 expt

(ppm)

Fig. 5. Experimental ECD spectra (200–400 nm) and TD-DFT-calculated ECD spectra for compound 6 (left), Linear regression analysis of experimental vs calculated 13 C NMR chemical shifts of 6. 5

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y = 1.0005x - 0.0703 R2 = 0.9980

240

210 180

(ppm)

180 150 120 90

150 120 90

60

60

30

30

0

0

30

60

90

y = 1x + 0.0012 R2 = 0.9968

240

calcd

calcd

(ppm)

210

0

120 150 180 210 240

0

30

60

90

expt (ppm)

expt

Fig. 6. Linear regression analysis of experimental vs calculated

finic carbon signals in 1. These suggested that the carboxylic acid of quorumolide B (2) was lost and olefinic carbon C-8 was changed to ketone carbonyl in 1. This was further supported by the 1H–1H COSY and HMBC spectra (Fig. 2). Three spin coupling system a (C-1 to C-3), b (C-5 to C-7), and c (C-9 to C-11) were lined out as drawn with bold bonds by 1H–1H correlations. The key HMBC correlations from H2-7 to C-8 (δC 212.7) and C-9 (δC 40.2), and H2-9 to C-7 (δC 35.7) and C-8 (δC 212.7), linked the fragments b and c via the ketone carbonyl carbon (Fig. 2). The relative configuration of 1 was assigned to be identical to that of quorumolide B (2) based on their very similar NMR data and NOESY spectrum. The absolute configuration of C-11 was assigned by the Rh2(OCOCF3)4-induced CD analysis. On the basis of the bulkiness rule Table 2 1 H NMR and Position

13

δC 31.5

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

29.5 76.4 73.5 117.0 139.8 77.0 71.2 25.0 19.4 30.8 70.9 43.1 207.7 71.2 17.1 17.5 29.2 16.4 13.5

1′ 2′ 3′

170.8 21.1 169.8 21.1 170.5 20.6 176.2 41.0 26.8

4′ 5′

11.6 16.4

Ac-12

a b

13

(ppm)

C NMR chemical shifts of 9 (left) and 10 (right).

for secondary alcohols, a positive Cotton effect at around 350 nm (E band) in the Rh2(OCOCF3)4-induced CD spectrum indicated a S-configuration, while negative Cotton effect implied a R-configuration [6]. Thus, a positive Cotton effect at 350 nm in the Rh2(OCOCF3)4-induced CD spectrum of 1 assigned C-11 as 11S configuration. Moreover, the absolute configuration of 1 was further confirmed as 3E, 1R, 2R, 11S, and 12S by comparing the experimental and calculated electron circular dichroism (ECD) spectra (Fig. 3). Compound 3 was obtained as a white powder. Its molecular formula was determined as C20H26O3 based on the [M+H]+ ion peak at m/z 315.1947 (calcd for 315.1955) in the HRESIMS. Further analysis of NMR data revealed that the structure of 3 resembled to that of antiquorine A (5) [11], except for the presence of a carbonyl group at C-1

C NMR Data of Compounds 9–12.

1

Ac-7

120 150 180 210 240

9a δH (J in Hz) 2.77, 1.68, 2.45, 5.32,

δC

dd (9.0, 15.0) m m d (8.5)

31.4

5.57, d (1.7) 5.07, d (2.0) 4.57, dd (2.0, 10.9) 1.29, m 1.05, overlap 4.85, dd (3.9, 11.1) (3.9, 11.0) 2.92, qd (3.9, 7.3) 0.96, 2.09, 1.10, 1.08, 1.05,

d (7.4) s s s d (7.3)

31.5 76.3 72.7 117.7 141.0 73.6 80.8 26.1 18.5 34.3 71.9 43.2 213. 7 76.7 16.3 18.3 29.6 16.3 14.8 56.8

10b δH (J in Hz) 2.70, 1.64, 2.39, 4.36,

m m m d (8.4)

5.85, brs 4.32, brs 2.73, m 1.15, m 0.61, t (10.0) 3.19, m 2.67, m 1.05, 1.99, 1.12, 1.09, 1.24, 3.34,

d (7.5) d (1.2) s s d (7.4) s

2.06, s 2.11, s

p (6.9) m m t (7.7) d (6.7)

Recorded in CDCl3 (1H NMR 400 MHz, Recorded in CDCl3 (1H NMR 500 MHz,

31.6 29.5 77.0 73.6 117.2 139.8 74.7 79.0 27.3 19.5 30.6 71.3 43.2 207.7 71.1 17.1 17.9 29.5 16.7 13.5 56.7 170.8 21.2 170.5 20.6 165.6 133.3 129.7

2.09, s 2.31, 1.63, 1.44, 0.86, 1.09,

δC

128.6 130.1 13

C NMR 100 MHz). C NMR 125 MHz).

13

6

11a δH (J in Hz) 2.78, 1.68, 2.50, 5.16,

dd (9.0, 15.0) d (15.0) m d (8.5)

5.68, s 5.55, brs 3.01, dd (1.8, 10.0) 1.26, m 1.08, overlap 4.92, dd (4.0, 11.0) 2.97, m 0.91 d (7.5) 2.13, s 1.14, s 1.00, s 1.09, d (7.1) 3.36, s 2.13, s

δC 32.0 32.3 76.4 75.9 117.7 139.6 77.4 72.5 25.0 19.6 31.1 70.8 43.4 207.8 73.1 16.2 17.8 29.4 16.4 13.5

8.03, d (7.6)

169.5 21.3 170.5 21.1 165.9 132.0 129.7

7.45, t (7.6) 7.57, t (7.4)

128.6 133.3

2.05, s

12a δH (J in Hz) 2.79, 1.65, 2.43, 4.33,

dd (9.0, 15.0) dd (2.1, 15.0) m d (8.4)

5.74, s 5.41, brs 4.84, dd (1.8, 10.7) 1.40, m 1.21, m 4.93, dd (3.9, 11.0) 2.97, m 1.05, 2.18, 1.15, 0.84, 1.09,

d (7.4) s s s d (7.4)

2.13, s 2.09, s 7.98, d (7.6) 7.44, t (7.6) 7.57, t (7.4)

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Exptl ECD of 9

40

Exptl ECD of 10 Exptl ECD of11

∆ε

20

Exptl ECD of12

0 -20 -40 200

250

300

350

400

Wavelength[nm] Fig. 9. Experimental ECD spectra (200–400 nm) of 9–12.

these were assigned arbitrarily in a β-orientation. The crystal of 9 was obtained in MeOH and then subjected to X-ray diffraction using Cu Kα radiation (Fig. 7). Therefore, the absolute configuration of 9 was determined as 5E, 2S, 3S, 4S, 7R, 8S, 9S, 11R, 12R, 13R, 15R and 2′S, and given a name as 8,12,13-epi-3,7,12-O-triacetyl-8-O-(2-methylbutanoyl)ingol. Compound 10 was obtained as a white powder. Its HRESIMS displayed a molecular ion at m/z 403.2090 [M+Na]+ (calcd for 403.2091), consistent with a molecular formula of C21H32O6. Its NMR data were highly similar to those of 9, and except for the absence of 3Ac, 7-Ac, 8-MeBu, 12-Ac. There is a methoxy group at C-8 based on the HMBC correlations from H-8 to the carbon of methoxyl. The relative configuration of 10 was assigned by analysis of its 1H NMR and NOESY spectra. NMR chemical shift calculations have been used to predict the planar structure and relative configuration of new natural products [7]. In the present study, the 13C NMR chemical shifts of 10 were calculated at the mPW1PW91/6-311++G (2df, 2pd) level. The predicted 13C NMR spectral data for the suggested configuration of 10 were consistent with the experimental data, the correlation coefficient (R2) between experimental and calculated data was 0.9968 (data obtained by linear analysis, Fig. 6). Taken together, these data confirmed the relative configuration of 10. The ECD data of 10 was in agreement with compound 9 (Fig. 9). Thus, the absolute configuration of 10 was determined as 5E, 2S, 3S, 4S, 7R, 8S, 9S, 11R, 12R, 13R, 15R. The molecular formula of 11 was determined as C32H40O9 by HRESIMS. In its NMR spectrum, compound 11 was found to exhibit highly similarities to that of 3,12-O-diacetyl-7-O-benzoyl-8-methoxyingol [14]. Extensive analysis their NMR data suggested that compound 11 is an epimer at C-8, C-12, and C-13 of 3,12-O-diacetyl-7-Obenzoyl-8-methoxyingol [14]. The NOESY correlations of H3-17/H-7, H3-17/H-8, H3-19/H-8, H3-19/H-12, H-12/H-8, and H-13/H-8 indicate that H-7, H-8, H3-19, H-12, and H-13 are β-oriented. Based on the calculated NMR data (Fig. 8), the relative configuration of 11 were determined. The ECD spectrum of 11 was in accordance with

Fig. 7. ORTEP drawing of compound 9.

replacing the hydroxy group in 5. This deduction was verified by the HMBC correlations from H3-20, H-2, and H-3 to C-1 at δC 215.7 (Fig. 2). Biogenetically, compound 3 has the same relative configuration as that of 5. The absolute configuration of compound 3 was assigned as 8Z, 13Z, 5R, 9R, 10R, and 12R, which was confirmed by comparing the experimental and calculated electron circular dichroism (ECD) spectra and 13C NMR (Fig. 4). Compound 6 was obtained as a white powder. Its molecular formula was assigned as C27H36O5 on the basis of the [M+H]+ ion peak at m/z 441.2634 (calcd for 441.2636) in the HRESIMS. Initial analysis of the 1 H and 13C NMR data showed the presence of a benzoyl group. Its NMR data (Table 1) showed highly similarities to those of ent-16α,17-dihydroxyatisan-3-one (7) [11], with the only difference being the presence of a benzoyl group at C-19 in compound 6. This was deduced from the HMBC correlations from H2-19 to C-3, C-4, C-5, and C-18. The relative configuration of 6 was established as identical to that of 7 due to their similar NMR patterns and presumed biosynthetic similarities. The absolute configurations of 6 were determined by the calculated ECD spectra and 13C NMR data (Fig. 5). The structure of 6 was thus assigned as 4R, 5S, 8S, 9R, 10S, 12S and 16S. Compound 9 was obtained as a colorless syrup. Its molecular formula was deduced to be C31H44O10 based on the HRESIMS ion at m/z 599.2810 [M+Na]+ (calcd for C31H44O10Na, 599.2827). Its NMR data (Table 2) show that compound 9 is an epimer of 3,7,12-O-triacetyl-8-O(2-methylbutanoyl)-ingol [13]. The relative configuration of 9 was assigned by analysis of its 1H NMR and NOESY spectra. The coupling constant of J2, 3 = 8.5 Hz indicated the H-2 and H-3 protons in an αorientation, and this was supported by the NOESY correlations of H-1β/ H3-16, H-1α/H-2, H-1α/H-3, H-2/H-3, and H-3/H-5. In addition, the NOESY correlations of H3-17/H-7, H3-17/H-8, H-8/H3-19, H-12/H3-19, and H-8/H-13 revealed that H-7, H-8, H-12, and H-13 are cofacial, and

y = 1x + 0.0033 R2 = 0.9972

240

210

(ppm)

180 150 120 90

180 150 120 90

60

60

30

30

0

0

30

60

90

0

120 150 180 210 240 expt

y = 1x + 0.0014 R2 = 0.9966

240

calcd

calcd

(ppm)

210

(ppm)

0

30

60

90

120 150 180 210 240 expt

Fig. 8. Linear regression analysis of experimental vs calculated 7

13

(ppm)

C NMR chemical shifts of 11 (left) and 12 (right).

Bioorganic Chemistry 92 (2019) 103292

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Fig. 10. Compounds inhibited RANKL-induced osteoclastogenesis. (A) Twenty-five compounds were screened for the activity of anti-osteoclastogenesis by BMMs. BMMs were stimulated with RANKL and cultured with compounds at 10 μM. After five days, the cells were fixed and stained with TRAcP. Negative means cells was treated with RANKL, and no compounds were added. *P<0.05, **P<0.01, ***P<0.001 relative to RANKL-stimulated. MNCs = multinucleated cells. (B) Survival of BMMs in the presence of compounds as assessed by MTS cell viability assay (n = 3). (C) Representative images of RANKL-induced osteoclastogenesis in the presence of 1, 3, 5, 12, 13, 16 and 19 at 10 μM. The images of cells were taken by light microscope at original magnification, ×10 (scale bar, 400 μm). Control group cells was cultured with no RANKL. (D) Quantification of the effect of 1, 3, 5, 12, 13, 16 and 19 treatment by counting TRAcP-positive multinucleated cells (nuclei>3). (n ≥ 3); “+” means RANKL treated. **P<0.01, ***P<0.001 relative to RANKL-stimulated.

Fig. 11. Compound 1 suppresses RANKL-induced gene expression during osteoclastogenesis in BMMs. The cells were treated with 1 (10 μM) and RANKL (50 ng/mL) for 5 days. The expression of maker genes of osteoclast (TRAcP, CTSK, NFATC) were assayed normalized to β-actin and compared to only RANKL-induced group. ***P<0.001 relative to RANKL-stimulated.

8

Bioorganic Chemistry 92 (2019) 103292

Z.-Y. Yin, et al.

certification for the plenty structures from Euphorbia genus plants. It is worthy noted that compound 1 is the first discovery of cembrane-type norditerpenoid, and compound 19 is firstly isolated from nature. Here, we also firstly establish the absolute configurations of ingol-type diterpenoids from E. antiquorum. Their anti-osteoporosis activities toward BMM cells are also described. Compound 1 decreased the expression level of osteoclast maker genes TRAcP, cathepsin K, and NFATC expression, and also showed suppression of the NFAT transcript activity. The present research enriches the structural diversity of E. antiquorum and indicates that diterpenoids may be new chemical agents for the treatment of osteoporosis. Fig. 12. Compound 1 reduces RANKL-stimulated NFAT activity. RAW264.7 cells stably transfected with an NFAT luciferase reporter construct were pretreated with 1 for 1 h, then induced with RANKL for another 24 h. *P < 0.05, **P < 0.01, ***P < 0.001 relative to RANKL-stimulated, 1-untreated controls.

Declaration of Competing Interest

compound 9 (Fig. 9), the absolute configuration of 11 was therefore determined as 5E, 2S, 3S, 4S, 7R, 8S, 9S, 11R, 12R, 13R, 15R and named as 8,12,13-epi-3,12-O-diacetyl-7-O-benzoyl-8-methoxyingol. The molecular formula of compound 12 was established as C31H38O9 based on its HRESIMS. Its NMR data were very similar to those of 2,3-diepiingol 7,12-diacetate 8-benzoate [15,16], indicating that 12 was the epimer of 2,3-diepiingol 7,12-diacetate 8-benzoate. The coupling constant of J2, 3 = 8.4 Hz indicated the H-2 and H-3 protons in an α-orientation. Similarities of NOESY cross-peaks (Fig. 1) between compounds 9 and 12 demonstrate that they have the same relative configuration. Since the calculated 13C NMR data for 5E, 2S, 3S, 4S, 7R, 8S, 9S, 11R, 12R, 13R, 15R-12 is consistent with the experimental data of 12 (Fig. 8), and the ECD spectrum of 12 displayed similar Cotton effects to the ECD curve of compound 9 (Fig. 9), its absolute configuration was defined as 5E, 2S, 3S, 4S, 7R, 8S, 9S, 11R, 12R, 13R, 15R, and named as 2,3-epi-7,12-diacetate-8-benzoate-ingol. In addition, twenty known compounds were identified as quorumolide B (2) [10], (1S, 4S, 5R, 9R, 10S, 12R)-1α,18-dihydroxy-entabieta-8(14),13(15)-dien-12α,l6-olide (4) [10], antiquorine A (5) [10], ent-16α,17-dihydroxyatisan-3-one (7) [17], eurifoloid R (8) [12], triester 3,12-di-O-acetyl-8-O-tigloyl-ingol (13) [12], 3,12-diacetyl-8-benzoylingol (14) [19], euphorantin I (15) [4], 3,7,12-triacetyl-8-benzoylingol (16) [20], 3,7,12-O-triacetyl-8-O-trigloylingol (17) [21], 3,12diacetyl-7-angeloyl-8-methoxyingol (18) [14], 19-hydroxy-(-)-kaur-16en-3-one (19) [22], 14β-hydroxy-3-oxo-ent-kaur-16-ene (20) [23], ent3-α-hydroxy-kaur-16-en-18-ol (21) [24], eurifoloid O (22) [12,18], oryzalexin F(23) [25], ent-2β-hydroxymanool (24) [26], and ingenol (25) [27] with those reported in literature. All the isolated twenty-five compounds were evaluated for their anti-osteoporosis activity in BMM cells. As shown in Fig. 10, compounds 1, 3, 5, 12, 13, 16, and 19 could inhibit the formation and function of osteoclasts without cytotoxicity. The new compound 1 showed the most potent inhibition of BMM cells differention and the expression level of osteoclast maker genes TRAcP, cathepsin K, and NFATC (Fig. 11). Furthermore, compound 1 dose-dependently reduced RANKL-stimulated NFAT activity suggested by NFAT luciferase reporter assay (Fig. 12).

This study was supported in part by the National Natural Science Foundation of China (No. 81573310), the Science and Technology Program of Guangzhou (201604020109), and the Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030).

The authors declare no competing financial interests. Acknowledgements

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4. Conclusion In summary, seven new diterpenoids (1, 3, 6, and 9–12), together with eighteen known diterpenoids (2, 4–5, 7–8, and 13–25), were isolated from the aerial parts of E. antiquorum. The isolated diterpenoids scaffolds are variety including two cembrane (1 and 2), three entabietane (3–5), four ent-atisane (6–8, and 22), ten ingol (9–18), three ent-kaurane (19–21), one ent-isopimarane (23), one labdane (24), and one ingenane type (25) diterpenoids. This research is a good

9