Assignment of the stereostructures of sesquiterpenoids from the roots of Daphne genkwa via quantum chemical calculations

Journal Pre-proof Assignment of the stereostructures of sesquiterpenoids from the roots of Daphne genkwa via quantum chemical calculations

Jie Wang, Qiang Ren, Yang-Yang Zhang, Rui Guo, Bin Lin, XiaoXiao Huang, Shao-Jiang Song PII:

S0367-326X(19)31520-5

DOI:

https://doi.org/10.1016/j.fitote.2019.104352

Reference:

FITOTE 104352

To appear in:

Fitoterapia

Received date:

25 July 2019

Revised date:

27 August 2019

Accepted date:

29 August 2019

Please cite this article as: J. Wang, Q. Ren, Y.-Y. Zhang, et al., Assignment of the stereostructures of sesquiterpenoids from the roots of Daphne genkwa via quantum chemical calculations, Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2019.104352

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© 2018 Published by Elsevier.

Journal Pre-proof

Assignment of the Stereostructures of Sesquiterpenoids from the Roots of Daphne genkwa via Quantum Chemical Calculations Jie Wanga, Qiang Rena, Yang-Yang Zhanga, Rui Guoa, Bin Linb, Xiao-Xiao Huanga* , Shao-Jiang Songa* Key Laboratory of Computational Chemistry Based Natural Antitumor Drug

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Research & Development, School of Traditional Chinese Materia Medica, Shenyang

School of Pharmaceutical Engineering, Shenyang Pharmaceutical University.

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b

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Pharmaceutical University, Shenyang 110016, People′s Republic of China

*Corresponding author.

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Shenyang 110016, People’s Republic of China

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Song).

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E- mail addresses: [email protected] (X.-X. Huang), [email protected] (S.-J.

Journal Pre-proof ABSTRACT: Five new guaiane-type sesquiterpenoids were obtained from the roots of Daphne genkwa. Their gross structures were established by extensive spectroscopic analyses. Attempts on the assignment of the relative configurations were unsuccessful when based on the NOESY correlations. Therefore, NMR chemical shift calculations based on the gauge independent atomic orbital (GIAO) method in combination with the statistical method DP4+ were employed to establish their relative configurations.

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Furthermore, the absolute configurations were determined by comparing the experimental and calculated electronic circular dichroism (ECD) using time-

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dependent density functional theory (TDDFT). The isolated compounds were

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HepG2 and Hep3B cell lines.

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screened for their cytotoxicity in vitro against two human hepatocellular carcinoma,

Keywords: Daphne genkwa; Sesquiterpenoids; NMR calculations; DP4+; Calculated ECD

Journal Pre-proof 1. Introduction Daphne genkwa Siebold et Zucc. (Thymelaeaceae), is a well-known medicinal plant widely distributed in the China and Korea [1-3]. Its flower buds and roots have been widely used in traditional Chinese medicine for anticancer, antitussive, diuretic, expectorant and others purposes [4-6]. Previous phytochemical investigation of this plant revealed the presence of diterpenoids, flavonoids, lignans, and coumarins [7,8]. However, the previous research on D. genkwa mainly focused on its flower buds [9,10]

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and its roots were largely overlooked [11,12].

In our previous work, the chemical investigation on the roots of D. genkwa resulted

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in the identification of some guaiane-type sesquiterpenoids [13]. As part of our

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continuing efforts to discover novel and structurally diverse natural products, five new

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guaiane-type sesquiterpenoids, daphneguaine A-D (1-4), and (1S, 7R, 10S, 11R)-3oxoguai-4-ene-11,12-diol (5) were isolated from the EtOAc extract of the roots of D.

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genkwa. Their structures were determined on the basis of the comprehensive

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spectroscopic analyses. NMR chemical shift calculations based on gauge independent

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atomic orbital (GIAO) method have emerged as useful tools for full elucidation of the relative configurations of new natural products [14,15]. When attempts on the assignment of the relative configurations were unsuccessful based on the NOESY experiment, quantum chemical calculations of 1 H and

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C NMR chemical shifts

calculations supported by DP4+ probability were performed to help determine the relative configurations. Moreover, by comparing the experimental and calculated electronic circular dichroism (ECD) spectra, their absolute configurations were determined. All compounds were tested for their cytotoxicity in vitro using two human hepatocellular carcinoma cell lines (Hep3B and HepG2). 2. Experimental section

Journal Pre-proof 2.1. General experimental procedures UV-Vis spectra were measured on a Shimadzu UV-1700 spectrometer (Shimadzu, Kyoto, Japan). Optical rotations were determined on a JASCO DIP-370 digital polarimeter. ECD spectra were acquired using a Bio-Logic MOS450 spectrometer. NMR spectra were performed on a Bruker AVANCE HD 600 NMR spectrometer (Switzerland). HRESIMS data were obtained on a Bruker Micro Q-TOF spectrometer. Column chromatography was performed with silica ge l (100–200 mesh, 200–300

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mesh, Qingdao, China), HP-20 macroporous resin (75–150 μm, Tokyo, Japan), and octadecyl silica gel (ODS, 60–80 μm, Germany). Semi-preparative HPLC separation

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was achieved with Shimadzu LC-10AR series instrument with SPD-20A UV-vis

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wavelength detector (Shimadzu, Kyoto, Japan) and YMC C18 column (250 mm × 10

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mm, 5 μm). TLC was conducted on precoated silica gel GF254 on glass plates

2.2. Plant material

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unfolding in TLC expansion cylinder (Qingdao).

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The dried roots of D. genkwa were collected from Xingyi City, Guizhou Province,

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People’s Republic of China, in July 2017. They were identified by Prof. Jin-Cai Lu (Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen (No. 20170721) has been deposited in the Herbarium of Shenyang Pharmaceutical University. 2.3. Extraction and isolation The dried roots of D. genkwa (50 kg) were cut and soaked in 70% EtOH (3 × 50 L) under reflux for three times (3 h, each). The crude extract was filtered and concentrated under reduced pressure. Then, the extract was suspended in H2 O and partitioned sequentially using petroleum ether, ethyl acetate (EtOAc) and n-butanol. The EtOAc extract (1000 g) was chromatographed on silica gel under reduced

Journal Pre-proof pressure, eluting with a step gradient of CH2 Cl2 /MeOH (100:1 → 1:1) to afford four fractions (Fr. 1 ~ 4). Fraction 2 (140 g) was subjected to HP20 macroporous resin with EtOH-H2 O (30%, 60% and 90%) to give four fractions (Fr. A ~ Fr. D). Fr. B (30 g) and Fr. C (70 g) were further purified by ODS with an increasing gradient of EtOH/H2 O (2:10 → 9:1) to afford Fr. B1 ~ Fr. B2 and Fr. C1 ~ Fr. C3, respectively. Fr. B1 (10.5 g) and Fr. B2 (11.0 g) were further subjected to silica gel column chromatography with the mobile phase CH2 Cl2 /MeOH (50:1 → 1:1) to yield fractions

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Fr. B1.1 ~ Fr. B1.10 and Fr. B2.1 ~ Fr. B2.12, respectively. Fr. C1 (15.5 g) and Fr. C2 (12.1 g) were subjected to further silica gel column chromatography and eluted with

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CH2 Cl2 / MeOH (50:1 → 1:1) separately yield fractions Fr. C1.1 ~ Fr. C1.13 and Fr.

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C2.1 ~ Fr. C2.11, respectively. Then Fr. B1.2 was separated over preparative and

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semi-preparative RP-HPLC (CH3 CN-H2 O, 25:75, v/v, 2.5 mL/min) over a YMC C18 column to afford compound 1 (9.8 mg, t R = 80 min) (TLC, CH2 Cl2 -MeOH, 35:1, v/v,

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Rf = 0.4) and compound 2 (3.0 mg, t R = 70 min) (TLC, CH2 Cl2 -MeOH, 30:1, v/v, Rf =

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0.5). Compound 5 (13.0 mg, t R = 57 min) was yielded from Fr. B2.3 eluted with

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CH3 CN-H2 O (23:77 v/v, 2.5 mL/min) (TLC, CH2 Cl2 -MeOH, 30:1, v/v, Rf = 0.6). From similar separating procedures, compound 3 (3.0 mg, t R = 63 min) was subjected to fraction Fr. C1.5 (CH3 CN-H2O, 40:60, v/v, 2.5 mL/min) (TLC, CH2 Cl2 -MeOH, 30:1, v/v, Rf = 0.3). Purification of subfraction Fr. C2.3 yielded 4 (9.8 mg, t R = 59 min) by eluting with CH3 CN-H2 O (45:55, v/v, 2.5 mL/min) (TLC, CH2 Cl2 -MeOH, 35:1, v/v, Rf = 0.35). 2.3.1. daphneguaine A (1) Yellow oil (methanol); [α] 20 +50.0 (c 0.20, MeOH), UV (MeOH) λmax (log ε) 239 D (0.75) nm; ECD (MeOH) λmax (Δε) 216 (+27.2), 245 (–49.6), 323 (+11.1) nm; 1 H and 13

C NMR (600 and 150 MHz, CDCl3 ), shown in Tables 1 and 2; positive- ion

Journal Pre-proof HRESIMS m/z 273.1467 [M + Na]+ (calcd for C15 H22 O 3 Na, 273.1461). 2.3.2. daphneguaine B (2) Yellow oil (methanol); [α] 20 +69.0 (c 0.10, MeOH), UV (MeOH) λmax (log ε) 241 D (1.41) nm; ECD (MeOH) λmax (Δε) 215 (+28.2), 245 (–50.3), 323 (+8.0) nm; 1 H and 13

C NMR (600 and 150 MHz, CDCl3 ), shown in Tables 1 and 2; positive- ion

HRESIMS m/z 273.1462 [M + Na]+ (calcd for C15 H22 O 3 Na, 273.1461).

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2.3.3. daphneguaine C (3)

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Yellow oil (methanol); [α] 20 –1.7 (c 0.10, MeOH), UV (MeOH) λmax (log ε) 230 D

242 (–9.1), 306 (–10.1) nm; 1 H and

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(0.36), 303 (0.20) nm; ECD (MeOH) λmax (Δε) 201 (–27.2), 245 (–16.6), 223 (+10.4), C NMR (600 and 150 MHz, CDCl3 ), shown in

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Tables 1 and 2; positive- ion HRESIMS m/z 271.1311 [M + Na]+ (calcd for

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C15 H20 O 3Na, 271.1305). 2.3.4. daphneguaine D (4)

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Yellow oil (methanol); [α] 20 +20.0 (c 0.20, MeOH), UV (MeOH) λmax (log ε) 238 D

H and

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C NMR (600 and 150 MHz, CDCl3 ), shown in Tables 1 and 2; positive- ion

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1

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(1.09), 303 (0.28) nm; ECD (MeOH) λmax (Δε) 204 (–8.0), 229 (+23.6), 311 (–5.9) nm;

HRESIMS m/z 251.1650 [M + H]+ (calcd for C15 H23 O 3, 251.1642). 2.3.5. (1S, 7R, 10S, 11R)-3-oxoguai-4-ene-11,12-diol (5) Yellow oil (methanol); [α] 20 +2.4 (c 0.10, MeOH), UV (MeOH) λmax (log ε) 243 D (0.35) nm; ECD (MeOH) λmax (Δε) 201 (–8.0), 245 (+20.3), 311 (–2.8) nm; 1 H and 13

C NMR (600 and 150 MHz, CDCl3 ), shown in Tables 1 and 2; positive- ion

HRESIMS m/z 253.1809 [M + H]+ (calcd for C15 H25 O 3, 253.1798). 2.4. NMR calculations The gauge independent atomic orbital (GIAO) shielding constants of all conformers were calculated at the mPW1PW91/6-311+G(d,p) level after geometry optimization at

Journal Pre-proof the B3LYP/6-31G(d) level using CPCM model in CDCl3 solvent with Gaussian 09 software (all conformers: compound 1a, 74, 1b, 96, 1c, 70, 1d, 80; compound 2a, 33, 2b, 21, 2c, 29, 2d, 23; compound 3a, 39, 3b, 62; compound 4a, 75, 4b, 47; compound 5a, 37, 5b, 36) [16]. Boltzmann-weighted averages of the chemical shifts were calculated to scale them against the experimental values. The experimental and calculated data were analyzed by the improved probability DP4+ method for isomeric compounds [17].

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2.5. ECD calculations

The absolute configurations of compounds 1-5 were determined by using TDDFT

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calculations as follows [18]. Conformational search of all the possible conformers of

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compounds 1-5 (16 conformers for compound 1, 15 conformers for compound 2, 18

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conformers for compound 3, 19 conformers for compound 4, and 18 conformers for compound 5) were performed by Spartan14 software package with MMFF94S force

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fields [19]. Then, all of the conformations were optimized at the B3LYP/6-31G(d)

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level with the Gaussian 09 software. ECD calculations were performed on the

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optimized conformers by the TDDFT method at B3LYP/6-31G(d, p) level with the PCM model in MeOH solution. The spectra were produced by SpecDis 1.51 via Boltzmann averaging [20]. 2.6. Cell culture

Human hepatocellular carcinoma Hep3B and HepG2 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, USA). The cells were cultured in DMEM medium (HyClone, Utah, USA) contained 10% foetal bovine serum (FBS), 10 μg/mL streptomycin and 100 U/mL penicillin (HyClone, Utah, USA). The cells were cultured at 37 °C with 5% CO 2 in humid atmosphere. Logarithmically growing cells were used for the experiments.

Journal Pre-proof 2.7. Cytotoxicity Assay The cytotoxic activities were detected by MTT assay. The cells were made into a single-cell suspension by DMEM containing 10% fetal bovine serum and then seeded into 96-well plates at a cell density of 5000–8000 cells per well. After incubation for 12 h at 37 °C with 5% CO 2 , the cells in the 96-well plates were treated with the test compounds at 50 μM concentration and Sorafenib (positive control, solarbio, Beijing, People’s Republic of China, 6–18 μM), and the samples were incubated for 48 h.

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Then, 20 μL of MTT reagent (0.5 mg/mL) was added to each well, and incubation was conducted for 4 h. After removal of the medium, 150 μL of DMSO was added to

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solubilize the residuum. All tests were conducted at least in triplicate. Finally, the

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absorbances were measured at 492 nm using a microplate reader (Thermo Scientific

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Multiskan MK3, Shanghai, China). The cell growth inhibitory ratio was calculated as follows:

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3. Results and discussion

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Inhibition ratio = (A492, control – A492, sample) / (A492, control – A492, blank ) × 100

Compound 1 was isolated as yellow oil. Its molecular formula was C15 H22 O3 with 5

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degrees of unsaturation, as deduced from its HRESIMS (m/z 273.1467 [M + Na]+, calcd for C15 H22 O3Na, 273.1461). The 1 H NMR data (Table 1) exhibited resonances characteristic for a terminal double bond at δH 4.95 (1H, s, H-12a), 5.06 (1H, s, H12b), a oxygenated methylene at δH 4.15 (2H, s, H-13), and two methyl protons at δH 0.77 (3H, d, J = 7.1 Hz, H-14) and 1.66 (3H, s, H-15), four methylenes at δH 1.55 (2H, m, H-8), 1.73 (1H, m, H-9a), 2.18 (1H, m, H-9b), 2.45 (1H, d, J = 18.2 Hz, H-2a), 2.51 (1H, dd, J = 11.8, 19.2 Hz, H-6a), 2.63 (1H, d, J = 18.2 Hz, H-2b), and 2.82 (1H, d, J = 19.2 Hz, H-6b), two methines at δH 2.28 (1H, m, H-10) and 2.89 (1H, m, H-7). The

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C NMR (Table 2) and HSQC data displayed the occurrence of 15 carbon

resonances, including a ketone carbonyl at δC 205.1 (C-3), four olefinic carbons at δC

Journal Pre-proof 108.8 (C-12), 139.0 (C-4), 154.8 (C-11) and 170.8 (C-5), two oxygenated carbons at δC 65.5 (C-13) and 83.1 (C-1), and eight aliphatic carbon signals. The HMBC correlations (Fig. 2) of H-2/C-3 and H-15/C-3 indicated that a ketone carbonyl (δC 205.2) was linked to C-3. Besides, HMBC correlations of H3 -14 (δH 0.77) to C-1, C-8, C-10 indicated that a methyl group was linked to the olefinic carbon. The methyl protons at H3 -15 (δH 1.66) correlated with C-3, C-4, C-5. The cross-peaks from H-2/C1 and H3 -14/C-1 and H-13/C-7, C-11, C-12 corroborated the presence of two hydroxyl

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groups at C-1 and C-13 positions, respectively. Accordingly, these characteristic signals implied that 1 was a guaiane-type sesquiterpenoid, as shown in Fig. 1.

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The relative configuration of 1 was not able to be determined by NOESY

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experiment due to the long distance between the methyl at C-14 and the isopropenyl 13

C NMR chemical shifts of the four

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side chain. Thus, NMR calculations of 1 H and

possible conformers (1R*, 7R*, 10S*-1a, 1S*, 7R*, 10S*-1b, 1R*, 7S*, 10S*-1c, and

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1S*, 7S*, 10S*-1d) were subjected to the GIAO method with the Gaussian 09

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software [21] at mPW1PW91/6-311+G(d,p) level utilizing the CPCM model in

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CDCl3 solvent. The predicted

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C NMR spectral data for the suggested configuration

of 1R*, 7R*, 10S*-1a were consistent with the experimental data, and the linear correlation coefficient (R2 ) between experimental and calculated data was 0.9984 (Fig. 3). Furthermore, the experimental and calculated 1 H and

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C data were compared by

the improved probability DP4+ method, which showed 1R*, 7R*, 10S*-1a with a DP4+ probability at 97.34% (Fig. 3). To prove the above assignments and determine the absolute configuration of 1, ECD calculations for respective (1R, 7R, 10S)-1 and (1S, 7S, 10R)-1 were performed using TDDFT/ECD method at the B3LYP/6-31G(d, p) level with PCM model in MeOH solution (Fig. 4). The experimental ECD spectrum

Journal Pre-proof of 1 was in good agreement with the calculated ECD spectrum for (1R, 7R, 10S)-1. Hence, the absolute configuration of 1 was assigned as 1R, 7R, 10S. The molecular formula of 2 was deduced as C15 H22O3 based on its HRESIMS (m/z 273.1462 [M + Na]+, calcd for C15 H22 O3 Na, 273.1461), implying 5 degrees of unsaturation. The 1 H and

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C NMR spectroscopic features of 2 (Tables 1 and 2) were

resembled to those of 1, except for the additional methyl group at C-10 and hydroxymethyl group at C-13. Based on the 1 H- 1 H COSY and HMBC correlations

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(Fig. 2), the gross structure of 2 was determined as shown in Fig. 1. For the same reason as mentioned for 1, the relative configuration of 2 could not be determined by

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the NOESY experiment either. Using a similar method of GIAO NMR calculations

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for 1, the four conformers of 1S*, 7R*, 10S*-2a, 1S*, 7R*, 10R*-2b, 1S*, 7S*, 10S*-

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2c and 1S*, 7S*, 10R*-2d were further confirmed by calculation of the mPW1PW91/6-311+G(d,p) level in CDCl3 . The calculated

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C NMR chemical shifts

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of 1S*, 7R*, 10S*-2a (R2 =0.9983), showed the best correlation of the four

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diastereomers, and led to the assignment of the relative configuration of 2 as 1S*, 7R*,

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10S* (Fig. 5). Moreover, the DP4+ analysis of the four possible conformers gave 100% probability for the configuration of 1S*, 7R*, 10S* (Fig. 5). The absolute configuration of 2 was also determined by comparing experimental and calculated ECD spectra for 1S, 7R, 10S-2 and 1R, 7S, 10R-2. The ECD spectrum showed that the experimental ECD curve matched well with calculated curve of 2 with 1S, 7R, 10S configuration (Fig. 5). Therefore, the absolute configuration of 2 was defined as 1S, 7R, 10S. The molecular formula of 3 was assigned as C15 H20O 3 from its HRESIMS ion peak at m/z 271.1311 [M + Na]+, (calcd for C15 H20 O3Na, 271.1305). The NMR data indicate that 3 was structurally similar to compound 1, except that a hydroxyl group at

Journal Pre-proof C-1 in 1 was replaced with a double bond in 3. Also, comparison of the 1 H NMR (Table 1) and 13 C NMR data (Table 2) showed an additional hydroxyl group at C-8 in compound 3. This was confirmed via the HMBC correlations (Fig. 2) from H3 -14/C-1, C-9, H-6/C-8, H-9/C-8, and H-7/C-8. The relative configuration of 3 was determined to be similar to that of 1 based on quantum chemical calculations. Thus, NMR calculations of two conformers (7R*, 8S*-3a and 7R*, 8R*-3b) were performed by comparing experimental and calculated

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H and

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C chemical shifts. The small

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difference linear correlation with 3b (R2 =0.9976) and 3a (R2 =0.9975) was obtained between the experimental and the calculated 13 C NMR (Fig. 6). In addition, the DP4+

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probability analysis also confirmed that the most probable relative configuration of 3

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was 7R*, 8R* with 96.97% probability (Fig. 6). The overall pattern of the calculated

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ECD curve for the (7R, 8R)-3 was consistent with the experimental ECD curve of 3, allowing the absolute configuration of 3 as 7R, 8R (Fig. 5).

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Compound 4 was assigned the molecular formula C15 H22 O3 based on the

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HRESIMS data (m/z 251.1650 [M + H]+, calcd for C15 H23O3 , 251.1642). The NMR

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data (Tables 1 and 2) indicated that 4 was structurally similar to 3, except for replacement of the terminal double bond with the methyl group and hydroxyl group at C-11, and the absence of a hydroxyl group at C-8 in 4. This was corroborated by the HMBC correlations (Fig. 2) of H3 -12/C-7, C-11, C-13. The NOESY experiment did not afford any useful correlations for the elucidation of the configuration of the two adjacent chiral centers (C-7 and C-11) in the flexible chain. In order to establish the relative configuration of 4, the theoretical NMR calculations of the two possible conformers 7R*, 11S*-4a and 7R*, 11R*-4b were predicted. The results showed that the linear correlation coefficients (R2 = 0.9986) of the two conformers were the same between experimental and calculated data (Fig. S11). The application of DP4+

Journal Pre-proof probability confirmed isomer 7R*, 11R*-4b as the most probable isomer (DP4+ probability, 100%) (Fig. S11). The calculated ECD spectrum of 7R, 11R-4 configuration gave the similar Cotton effects by comparing with the experimental ECD spectrum (Fig. 5). Thus, the absolute configuration of 4 was confirmed as 7R, 11R. Compound 5 gave a molecular formula of C15 H24O3 from its HRESIMS data at m/z 253.1809 [M + H]+ (calcd for C15 H25 O3 253.1798). The analysis of the 1 H and

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C

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NMR data (Tables 1 and 2) suggested that 5 had an analogous structural skeleton to 4. The only difference was that an additional double bond at C-1 and C-10 in 5.

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Comparison of the NMR data of 5 with that of 3-oxoguai-4-ene-11,12-diol showed a

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same structure, and its configuration had not been accurately determined in literature

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[22]. The relative configuration of 5 could be determined by the NOESY spectrum in combination with theoretical NMR calculations. In NOESY spectrum, the significant

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correlation of H-1/H-7, and no correlation of H-1/H3 -14 indicated that these protons

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(H-1, H-7 and H-10) were in the same side. However, the configuration at C-11 was

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impossible to determine by NOESY experiment. The major two conformers of (1S*, 7R*, 10S*, 11S*)-5a and (1S*, 7R*, 10S*, 11R*)-5b were applied to verify the relative configuration of 5 based on the GIAO NMR calculations. According to the calculations result, the correlation coefficients R2 of the two conformers were identical between experimental and calculated

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C NMR chemical shifts (Fig. S12).

The DP4+ probability further predicted the conformers (1S*, 7R*, 10S*, 11R*)-5b with 100% probability (Fig. S12). In order to determine the absolute configuration of 5, the ECD calculation for (1S, 7R, 10S, 11R)-5 and (1R, 7S, 10R, 11S)-5 were performed using TDDFT (Fig. 5). The theoretical ECD curve of (1S, 7R, 10S, 11R)-5

Journal Pre-proof matched well with the experimental ECD curve of 5. Hence, the absolute configuration of 5 was assigned as 1S, 7R, 10S, 11R. Compounds 1−5 were evaluated for their cytotoxicity in vitro against two human hepatocellular carcinoma, HepG2 and Hep3B cells. In the primary assay, all compounds showed no obvious inhibitory activities on HepG2 and Hep3B cells under the tested concentration. 4. Conclusions

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In the present study, five new guaiane-type sesquiterpenoids were isolated from the roots of the D. genkwa. The gross structures were elucidated on the basis of

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comprehensive spectroscopic analyses. The relative configurations were fully

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elucidated through GIAO NMR calculations in combination with the advanced

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statistical method DP4+ probability. By comparing between experimental and TDDFT-calculated ECD spectra, the absolute configurations of 1-5 were established.

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All compounds were tested for the cytotoxicity in vitro against Hep3B and HepG2

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cell lines. However, they did not show obvious inhibitory activities.

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ORCID

Xiao-Xiao Huang: 0000-0003-0099-1118 Shao-Jiang Song: 0000-0002-9074-2467 Acknowledgements This project was funded by the National Nature Science Foundation of China (81872766 and 81573319), the Young and Middle-aged Science and Technology Innovative Talent Support Program of Shenyang City (RC180250) and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University (ZQN2018006).

Journal Pre-proof Conflict of interest The authors declare no competing financial interest. References [1] N.V. Minh, B.S. Han, H. Y. Choi, J.S. Byun, J.S. Park, W.G. Kim, Genkwalathins A and B, new lathyrane-type diterpenes from Daphne genkwa. Nat. Prod. Res. 32 (2017) 1782–1790. [2] R. Wang, J.Y. Li, H.Y. Qi, Y.P. Shi, Two new tigliane diterpene esters from the flower buds of

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Daphne genkwa, J. Asian Nat. Prod. Res. 15 (2013) 502–506.

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[3] C.P. Jiang, X. He, X.L. Yang, S.L. Zhang, H. Li, Z.J. Song, Anti-rheumatoid arthritic activity of flavonoids from Daphne genkwa, Phytomedicine 21 (2014) 830–837.

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[4] T. Okunishi, T. Umezawa, M. Shimada, Isolation and enzymatic formation of lignans of

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Daphne genkwa and Daphne odora, J. Wood. Sci. 47 (2001) 383–388.

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[5] L.Z. Li, S.J. Song, P.Y. Gao, F.F. Li, L.H. Wang, Q.B. Liu, Neogenkwanines A–H: daphnanetype diterpenes containing 4,7 or 4,6-ether groups from the flower bud of Daphne genkwa,

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RSC Adv. 5 (2015) 4143–4152.

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[6] Z.J. Zhan, C.Q. Fan, J. Ding, J.M. Yue, Novel diterpenoids with potent inhibitory activity against endothelium cell HMEC and cytotoxic activities from a well-known TCM plant

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Daphne genkwa, Bioorg. Med. Chem. 13 (2005) 645–655. [7] B.S. Han, K.S. Kim, Y.J. Kim, H. Y. Jung, Y.M. Kang, K.S. Lee, M.J. Sohn, Daphnane diterpenes from Daphne genkwa activate Nurr1 and have a neuroprotective effect in an animal model of Parkinson’s disease, J. Nat. Prod. 79 (2016) 1604–1609. [8] B.Y. Park, B.S. Min, S.R. Oh, J.H. Kim, K. H. Bae, H. K. Lee, Isolation of flavonoids, a biscoumarin and an amide from the flower buds of Daphne genkwa and the evaluation of their anti-complement activity, Phytother. Res. 20 (2010) 610–613. [9] K.K. Bang, C.Y. Yun, C. Lee, Q.H. Jin, J.W. Lee, S.H. Jung, Melanogenesis inhibitory daphnane diterpenoids from the flower buds of Daphne genkwa, Bioorg. Med. Chem. Lett. 23 (2013) 3334–3337.

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derivative from Rhododendron brachycarpum and its structural assignment via a protocol

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combining NMR and DP4 plus application, Phytochemistry 133 (2016) 45–50. [16] M.W. Lodewyk, D.J. Tantillo, Prediction of the structure of nobilisitine A using computed

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Daphne aurantiaca and their potential anti-inflammatory activity, J. Nat. Prod. 73 (2010)

Journal Pre-proof Table caption Table 1. 1 H NMR data (600 MHz, J in Hz) of compounds 1-5 in CDCl3 (δ in ppm).

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C NMR data (150 MHz) of compounds 1-5 in CDCl3 (δ in ppm).

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Table 2.

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Table 1. 1 H NMR data (600 MHz, J in Hz) of compounds 1-5 in CDCl3 (δ in ppm). position 1 2 6 7 8 9

1

2

a 2.45 (1H, d, 18.2) b 2.63 (1H, d, 18.2) a 2.51 (1H, dd, 11.8, 19.2) b 2.82 (1H, d, 19.2) 2.89 (1H, m) 1.55 (2H, m)

a 2.24, (1H, d, 17.7) b 2.99, (1H, d, 17.7) a 2.62 (1H, m) b 2.74 (1H, m) 2.71 (1H, m) a 1.57 (1H, m) b 1.65 (1H, m) a 1.70 (1H, m) b 2.21 (1H, m)

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a 1.73 (1H, m) b 2.18 (1H, m) 2.28 (1H, m) a 4.95 (1H, s) b 5.06 (1H, s) 4.15 (2H, brs)

14 15

0.77 (3H, d, 7.1) 1.66 (3H, s)

10 12

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a 4.72 (1H, s) b 4.77 (1H, s) 1.76 (3H, s)

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1.39 (3H, s) 1.69 (3H, s)

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Overlapping signals are expressed as o.

3

4

1.10 (3H, s)

5 3.10 (1H, m) a 2.03 (1H, d, 18.8) b 2.53 (1H, dd, 7.0, 18.8) a 2.19 (1H, dd, 12.4, 19.2) b 2.83 (1H, d, 19.2) 1.90 (1H, m) a 1.25 (1H, m) b 2.08 (1H, m) a 1.69 (1H, m) b 1.85 (1H, m) 2.12 (1H, m) 1.14 (3H, s)

a 3.45 (1H, d, 10.9) b 3.59 (1H, d, 10.9) 1.84 (3H, s) 1.78 (3H, s)

a 3.50 (1H, d, 11.0) b 3.66 (1H, d, 11.0) 0.61 (3H, d, 7.1) 1.64 (3H, s)

2.94 (2H, brs)

2.87 (2H, brs)

a 2.72 (1H, o) b 3.05 (1H, m) 2.83 (1H, m) 4.15 (1H, o)

a 2.58 (1H, m) b 3.02 (1H, m) 1.98 (1H, m) a 1.49 (1H, m) b 1.92 (1H, m) a 2.27 (1H, m) b 2.53 (1H, m)

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a 2.57 (1H, m) b 2.72 (1H, o)

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a 5.00 (1H, s) b 5.21 (1H, s) a 4.15 (1H, d, 12.8) b 4.25 (1H, d, 12.8) 1.86 (3H, s) 1.78 (3H, s)

Journal Pre-proof C NMR data (150 MHz) of compounds 1-5 in CDCl3 (δ in ppm).

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4 131.9 40.2 205.4 137.7 169.1 28.6 42.2 28.6 33.5 136.1 75.3 18.9 68.5 24.0 8.5

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3 131.8 40.8 204.4 139.0 166.4 29.7 45.8 70.6 43.3 130.4 149.5 115.0 66.1 24.3 8.6

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2 84.8 48.7 205.7 140.0 170.7 35.5 42.9 29.2 39.7 75.3 150.6 109.4 20.3 27.6 8.4

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1 83.1 51.5 205.1 139.0 170.8 36.8 38.3 30.8 31.0 40.1 154.8 108.8 65.5 14.5 8.0

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position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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Table 2.

5 45.8 41.5 208.7 138.0 175.5 34.0 43.6 25.9 37.0 35.6 75.1 20.0 67.9 12.3 8.2

Journal Pre-proof Figure caption Fig. 1. The structures of compounds 1-5. Fig. 2. Key HMBC correlations of 1, 2, 3, and 4, the key 1 H−1 H COSY correlations of 1, 3, and 4, and Key NOESY correlation of 5. Fig. 3. The NMR calculation results of four plausible conformers at the mPW1PW91/6-311+G(d,p) level with CPCM in CDCl3 solvent. (A) Linear correlations between the scaled calculated and experimental 13 C NMR chemical shifts for compound 1. (B) DP4+ probability of 1 H and13 C NMR chemical shifts of 1. Fig. 4. Calculated and experimental ECD spectra of 1-5.

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Fig. 5. The NMR calculation results of four plausible conformers at the mPW1PW91/6-311+G(d,p) level with CPCM in CDCl3 solvent. (A) Linear correlations between the scaled calculated and experimental 13 C NMR chemical shifts

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for compound 2. (B) DP4+ probability of 1 H and 13 C NMR chemical shifts of 2.

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Fig. 6. The NMR calculation results of two plausible conformers at the mPW1PW91/6-311+G(d,p) level with CPCM in CDCl3 solvent. (A) Linear

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correlations between the scaled calculated and experimental 13 C NMR chemical shifts

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for compound 3. (B) DP4+ probability of 1 H and

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C NMR chemical shifts of 3.

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Fig. 1. The structures of compounds 1-5.

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Fig. 2. Key HMBC correlations of 1, 2, 3, and 4, the key 1 H−1 H COSY correlations of 1, 3, and 4,

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and Key NOESY correlation of 5.

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Fig. 3. The NMR calculation results of four plausible conformers at the mPW1PW91/6311+G(d,p) level with CPCM in CDCl3 solvent. (A) Linear correlations between the scaled 1

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C NMR chemical shifts for compound 1. (B) DP4+ probability of

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calculated and experimental

H and13 C NMR chemical shifts of 1.

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Fig. 4. Calculated and experimental ECD spectra of 1-5.

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Fig. 5. The NMR calculation results of four plausible conformers at the mPW1PW91/6-

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311+G(d,p) level with CPCM in CDCl3 solvent. (A) Linear correlations between the scaled calculated and experimental 13

C NMR chemical shifts for compound 2. (B) DP4+ probability of

H and C NMR chemical shifts of 2.

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1

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Fig. 6. The NMR calculation results of two plausible conformers at the mPW1PW91/6-311+G(d,p)

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level with CPCM in CDCl3 solvent. (A) Linear correlations between the scaled calculated and experimental 13 C NMR chemical shifts for compound 3. (B) DP4+ probability of 1 H and 13 C NMR

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chemical shifts of 3.