Neolignans and Norlignans from Insect Medicine Polyphaga plancyi and Their Biological Activities

1 Introduction

Insects are a special group of existence on the earth. Facing the changing environment, they have strong adaptability, productivity and continuity. They seem small and insignificant, but the largest number of living beings on the planet, and their presence has created a huge molecular libraries [1, 2]. The tenacious vitality of insects is highly likely to be related to their unique molecular mechanisms [2], which provides us a confidence to track their bioactive molecules.

In China, the insect Polyphaga plancyi Bolivar has been used for promoting blood circulation [3]. Whereas, so far there have few reports about its chemical composition [4]. Previous studies revealed the significance of structurally novel non-peptide small molecules (NPSMs) [5-11], inspiring our further interest on insect chemistry. As far as the title species was concerned, eight new compounds and two known compounds belonging to neolignans and norlignans were isolated in this study (Fig. 1). Chiral HPLC separation was used to afford optically active isomers. Subsequent absolute configuration clarification was aided by electronic circular dichroism (ECD) calculations. In addition, the biological activities of all the new compounds were assessed using multiple assays.

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2 Results and Discussion

2.1 Structure Elucidation of the Compounds

Plancyin A (1) has the molecular formula C₁₈H₁₈O₆ deduced from analysis of its HRESIMS ([M−H]⁻, m/z 329.1040, calcd 329.1031), ¹³C NMR, and DEPT spectra, indicating 10 degrees of unsaturation. The ¹H NMR spectrum (Table 1) of 1 indicates the presence of three aromatic/olefinic protons at δ_H 6.63 (brs, H-2), 6.82 (brs, H-4), 6.90 (brs, H-6), indicating the presence of a 1, 3, 5-trisubstituted benzene ring. The ¹³C NMR and DEPT spectra show 9 carbons ascribed to two methylene, three sp² methine, and four quaternary carbons (one carbonyl, three olefinic including one oxygenated). Based on the molecular formula, we judged that compound 1 is a completely symmetrical structure. The ¹H–¹H COSY spectrum (Fig. 2) shows correlations of H-7/H-8. The HMBC correlations of H-7/C-2, C-6, C-9 (δ_C 176.9), H-8/C-1, and H-4/C-2, C-3 (δ_C 158.8), C-6 allow to establish half the structure fragment of 1. Taken together, the structure of 1 was established as shown.

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Plancyin B (2) has a molecular formula of C₁₉H₂₀O₆ derived from its HREIMS (m/z 343.1193 [M−H]⁻, calcd for 343.1187), ¹³C NMR, and DEPT spectra, having 10 degrees of unsaturation. The ¹H and ¹³C NMR data of 2 resemble those of 1, differing in that the additional existence of a methyl in 2. The HMBC correlation of H-10′ (δ_H 3.65, s)/C-9′ (δ_C 175.2) indicates the connection position of the methyl group as shown. With these in hand, the structure of 2 was readily determined.

Plancyin C (3) possesses a molecular formula C₁₈H₁₈O₆ (10 degrees of unsaturation) deduced from its negative HRESIMS at m/z 329.1036 [M−H]⁻ (calcd for 329.1031) and NMR data. The ¹H NMR spectrum (Table 1) indicates the presence of seven aromatic/olefinic protons. The ¹³C NMR and DEPT spectra display 18 carbons, of which twelve are olefinic carbons belonging to two phenyl groups. Besides, the residual signals include three methylene, one sp³ methine, and two quaternary carbons. The ¹H–¹H COSY spectrum (Fig. 2) shows correlations of H-4/H-5/H-6, H-7/H-8 and H-7′/H-8′. The HMBC correlations of H-7/C-2, C-6, C-9 (δ_C 177.7), H-8/C-1 (δ_C 142.5), in consideration of the chemical shift of C-1, indicate the presence of phenylpropanoid (part A). The HMBC correlations of H-7′/C-2′, C-6′, C-9′ (δ_C 177.4) and H-8′/C-1′ (δ_C 143.9) allow to establish the structure fragment of another phenylpropanoid (part B). In addition, the chemical shift of C-5′ and the HMBC correlations of H-4′, H-6′/C-8 (δ_C 55.3) indicate that parts A and B in 3 are linked via C-8–C-5′. Taken together, the data enable assignment of the planar structure of 3. The lack of an optical rotation indicates that 3 is racemic. Separation by using chiral HPLC yielded two enantiomers, whose absolute configurations at stereogenic centers were assigned using computational methods. The ECD spectrum of (8S)-3 is correlated well with the experimental spectrum of (+)-3, leading to the unambiguous assignment of the absolute configurations at the stereogenic centers in (+)-3 as 8S (Fig. 3).

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Plancyin D (4) has the molecular formula C₁₉H₂₀O₆ (10 degrees of unsaturation), based on analysis of its HREIMS, ¹³C NMR, and DEPT spectra. The ¹H and ¹³C NMR data of 4 resemble those of 3, differing in that the additional existence of a methyl in 4. The HMBC correlation of H-10′ (δ_H 3.63, s)/C-9′ (δ_C 175.2) indicates the connection position of the methyl group as shown. Thus, the planar structure of 4 was determined. For the structure like 3, it is challenging to clarify the stereochemistry at C-8. Separation of 4 by chiral HPLC afforded (+)-4 and (−)-4, whose absolute configurations were further determined by ECD calculations and comparison (Supplementary material) with the data of 3, allowing (+)-4 to be 8S.

Plancyin E (5) has the molecular formula C₁₇H₁₈O₄ (9 degrees of unsaturation), based on analysis of its HREIMS and NMR data. Inspection of these NMR data disclosed that the structure of 5 extremely resemble that of 3, differing in that a carboxyl group of C-8 is absent gaining support from the HMBC correlations of H-8/C-1, C-4′, C-6′ and the chemical shift of C-8 (δ_C 39.0). Therefore, the structure of 5 was constructed.

Plancyin F (6) possesses a molecular formula C₁₅H₁₄O₄ deduced from its negative HRESIMS and NMR data, indicating 9 degrees of unsaturation. The ¹H NMR spectrum of 6 shows two ABX systems indicated by the signals at δ_H 7.15 (d, J = 1.8 Hz, H-2), and 6.86 (d, J = 8.1 Hz, H-5), 7.13 (dd, J = 8.1, 1.8 Hz, H-6) and δ_H 7.05 (d, J = 1.9 Hz, H-2′), 6.80 (d, J = 8.1 Hz, H-5′), and 7.01 (dd, J = 8.1, 1.9 Hz, H-6′), suggesting the presence of two 1, 2, 4-trisubstituted benzene rings. In addition, there exists a signal at δ_H 3.53 (s, 2H, H-7) and a methyl signal at δ_H 2.28 (s, 3H, H-7′). The ¹³C NMR and DEPT spectra display 15 carbons ascribed to one methyl, one methylene, six sp² methine, seven quaternary carbons (one carbonyl, six sp² including two oxygenated). The ¹H–¹H COSY spectrum (Fig. 4) gives cross peaks of H-5/H-6 and H-5′/H-6′, in conjunction with the HMBC correlations of H-7/C-2, C-6, C-8 (δ_C 177.3), and the chemical shift of C-4 (δ_C 154.0), revealing the west part of 6. The HMBC correlations of H-7′/C-1′, C-2′, C-6′ and H-6′/C-4′ (δ_C 152.7) allow to establish the structure fragment of the east part. Two parts are connected via C-3–C-3′ aided by the HMBC correlations of H-2/C-3′ and H-2′/C-3. The structure of 6 was therefore identified.

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Plancyin G (7) has the molecular formula C₂₀H₁₈O₈ (12 degrees of unsaturation) deduced from its HRESIMS and NMR data. The ¹H NMR spectrum of 7 shows the presence of two doublets at δ_H 7.59 and δ_H 6.37 both with J value of 15.9 Hz, indicating the trans relationship of H-7 and H-8, two ABX systems characteristic of signals at δ_H 7.31 (d, J = 1.7 Hz, H-2), 6.78 (d, J = 8.3 Hz, H-5), and 7.06 (dd, J = 8.3, 1.7 Hz, H-6) and δ_H 7.43 (d, J = 1.7 Hz, H-2′), 6.74 (d, J = 8.3 Hz, H-5′), and 7.08 (dd, J = 8.3, 1.7 Hz, H-6′), and two methyl signals respectively at δ_H 3.96 (s, H-10) and δ_H 3.69 (s, H-10′). The ¹³C NMR and DEPT spectra display 20 carbons ascribed to two methyl, nine sp² methine, nine quaternary carbons (two carbonyl, seven sp² including five oxygenated). The architecture of 7 was constructed mainly based on the HMBC spectrum (Fig. 4). In the HMBC spectrum, the correlations of H-7/C-2, C-6, C-9 (δ_C 170.8), H-7′/C-2′, C-6′, C-9′ (δ_C 167.7) indicate the presence of two phenylpropanoid parts, the correlations of H-5, H-10/C-3 (δ_C 150.5), H-5′, H-10′/C-3′ (δ_C 148.9) and ROESY correlations H-2/H-10, H-2′/H-10′ indicate the position of methoxyl, respectively. Further, a pivotal ROESY correlation of H-8/H-7′ in consideration of the chemical shift of C-8′ (δ_C 139.9) suggests the position of ester oxygen and the presence of E-configurated olefinic protons (H-7′ and H-8′), in accordance with the requirement of degrees of unsaturation. Taken together, these data enable assignment of the structure of 7.

Plancyin H (8) possesses a molecular formula C₁₈H₁₆O₆ (11 degrees of unsaturation) deduced from its HRESIMS and NMR data. The ¹H NMR spectrum indicates the presence of seven aromatic/olefinic protons. The ¹³C NMR and DEPT spectra display 18 carbons, of which twelve are olefinic carbons belonging to two phenyl groups. Besides, the residual signals include one methylene, three sp³ methine, and two quaternary carbons (carbonyl). The HMBC correlations (Fig. 4) of H-7/C-2, C-6, C-9 (δ_C 178.3), H-8/C-1, and the ¹H–¹H COSY correlation of H-7/H-8 indicate the substructure of C-1–C-7–C-8–C-9 (part A). The structure fragment C-1′–C-7′–C-8′–C-9′ (part B) was established the HMBC correlations of H-7′/C-2′, C-6′, C-9′ (δ_C 178.4), H-8′/C-1′, and the ¹H–¹H COSY correlation of H-7′/H-8′. Further, the ¹H–¹H COSY correlation of H-8/H-8′ and the HMBC correlations of H-5/C-7′, H-7/C-8′, and H-7′/C-8 suggests two parts are connected via C-6–C-7′ and C-8–C-8′. The ¹H–¹H COSY correlations of H-4/H-5, H-5′/H-4′, H-6′, and HMBC correlations of H-5/C-1, C-3 (δ_C 156.6) and H-5′/C-1′, C-3′ (δ_C 158.4) indicate the replacement of benzene ring. Taken together, the planar structure of 8 was established as shown. There are three chiral centers in 8, the ROESY correlations (Fig. 4) of H-8/H-7′ and H-8′/H-2′, H-6′ evidently imply the relative configuration of 8. Compound 8 is racemic, chiral HPLC separation afforded (+)-8 and (−)-8, whose absolute configurations was further determined as 8R, 7′S, 8′R for (+)-8 and 8S, 7′R, 8′S for (−)-8 by ECD comparison (Fig. 5).

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Compound 9 was determined to have the molecular formula C₁₆H₁₆O₆ (9 degrees of unsaturation) on the basis of HRESIMS and NMR data. The ¹H NMR spectrum shows an ABX system with signals resonated at δ_H 6.53 (d, J = 1.6 Hz, H-2′), 6.69 (d, J = 8.0 Hz, H-5′), and 6.50 (dd, J = 8.0, 1.6 Hz, H-6′). Two aromatic protons at δ_H 6.58 (s, H-2) and δ_H 6.31 (s, H-5) suggest the presence of a 1, 2, 4, 5-tetrasubstituted benzene ring. The ¹³C NMR and DEPT spectra indicate 16 carbons ascribed to two oxygenated methylene, seven methine (5 sp² and 2 sp³), and seven quaternary carbons (4 oxygenated). The ¹H–¹H COSY spectrum shows correlations of H-5′/H-6′; H-7/H-8, and H-7′/8′. The HMBC correlations of H-7′/C-1, C-1′, C-2′, C-6 and C-6′ indicate that the two phenyl groups are connected via C-7′. The HMBC correlations of H-8/C-1, H-2/C-7, H-7/C-8′, and H-8′/C-1′, C-6 suggest the structural motif of an isochroman. Taken together, the planar structure of 9 was determined as shown. There are two chiral centers in 9, the ROESY spectrum (Fig. 4) of 9 displays the correlation of H-7′/H-8. We found that the NMR data of compound 9 are exactly same as those of periplanol A [20]. However, the absolute configuration of periplanol A has not been determined yet. To clarify the absolute configuration of 9, chiral HPLC separation was first conducted due to it is racemic. The absolute configuration of each enantiomer of racemic 9 was assigned by using ECD calculations. The ECD spectrum (Fig. 6) of (7R, 7′S)-9 agrees well with the experimental one of (−)-9, leading to the unambiguous assignment of the absolute configurations at the stereogenic centers in (−)-9 as 7R, 7′S and (+)-9 as 7S, 7′R (Tables 2 and 3).

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The known compound (10) was identified as 5–5′ dehydrodi-3-(4-hydroxy-3-methoxyphenyl) propionic acid by comparing its NMR data with those in the literature [21].

Proteins or peptides are commonly considered to be the active substances in the insects. However, the chemical profiling and biological role of nonpeptidal small molecules in the insects remains largely unknown. In this study, ten lignan derivatives were characterized. It is evident that compounds 5, 6, and 9 are diverse norlignans and there exist four types of connection pattern for the ten lignans, adding new facets for the insect derived natural products.

2.2 Biological Evaluation

To explore the mechanism underlying the antifibrotic effect of the compounds, we first examined whether the compounds affected TGF-β1–induced activation of the marker genes in NRK-52e cells (40 μM, data not shown). As presented in Fig. 7, compound 1 reduces three marker genes, especially for fibronectin and α-SMA in TGF-β1–induced NRK-52e cells (Fig. 7a). Compound 7 significantly inhibits three marker genes (Fig. 7b). Besides, Western bolt assay shows that compounds 1 and 7 decrease the protein expression with a dose-dependent manner in TGF-β1-induced NRK-52e cells (Fig. 8a, b). Next, we assessed the expression of Smad2/3 phosphorylation in NRK-52e cells. As presented in Fig. 9, compound 1 inhibits Smad3 phosphorylation, but does not affect TGF-β1–induced Smad2 phosphorylation (Fig. 9a). Compound 7 decreases both Smad3 and Smad2 phosphorylation level, and the effect for Smad2 appears to be more potent than Smad3 at 40 μM (Fig. 9b). Although more investigation are needed, our current study provides evidence that compounds 1 and 7 can play potential roles in the therapy of renal fibrosis by the disruption of Smad activation.

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In addition, all the new compounds were evaluated for their inhibitory activities against human cancer cells (K562, A549, and Huh7), EV71, ROCK2, JAK3, DDR1, and coagulation. Compounds (+)-3 and (−)-3 show cytotoxicity against K562, A549, and Huh-7 cell lines with IC₅₀ values ranging from 16.6 to 67.7 µM (Table 4). In contrast, compounds (+)-3 and (−)-3 also exhibit inhibitory activity against EV71 with IC₅₀ values of 23.2 μM and 27.1 μM, respectively. It was found that compound 6 exhibits potent inhibitory activities toward these kinases with IC₅₀ values of 9.17 μM for JAK3 and 27.1 μM for ROCK2. Compounds (+)-9 and (−)-9 exhibit inhibitory activities against JAK3 and DDR1 kinases (Table 5). In addition, partial compounds exhibit negligible inhibitory effect on coagulant (Tables 6, 7 and 8).

3 Experimental Section

3.1 General Experimental Procedures

Optical rotations were recorded on a Horiba SEPA-300 polarimeter. UV spectra were measured on a Shimadzu UV-2401PC spectrometer. CD spectra were determined on a Chirascan instrument. NMR spectra were recorded on a Bruker AV-600 MHz spectrometer, with TMS as an internal standard. ESIMS and HRESIMS were collected on an Agilent 1290 UPLC/6540 Q-TOF spectrometer. C-18 silica gel (40–60 μm; Daiso Co., Japan), MCI gel CHP 20P (75–150 μm, Mitsubishi Chemical Industries, Tokyo, Japan) and Sephadex LH-20 (Amersham Pharmacia, Uppsala, Sweden) were used for column chromatography. Semi-preparative HPLC was underwent on an Agilent 1200 liquid chromatograph with an YMC-Pack ODS-A column (250 × 10 mm, i.d., 5 μm, flow rate: 3 mL/min) and a Daicel Chiralpak (IC, 250 mm × 4.6 mm, i.d., 5 μm, flow rate: 1 mL/min).

3.2 Insect Material

The specimen of Polyphaga plancyi was purchased from Henan Province, China, in November 2014, and identified by Prof. Da-Rong Yang at Kunming Institute of Zoology, Chinese Academy of Sciences. A voucher specimen (CHYX-0593) is deposited at the School of Pharmaceutical Sciences, Shenzhen University Health Science Center, People's Republic of China.

3.3 Extraction and Isolation

The whole bodies of P. plancyi (50 kg) were extracted under reflux with 70% EtOH (300 L, 4 h, 3 h, 3 h) to give a crude extract (6.32 kg), which was suspended in water followed by successive partition with petroleum ether and EtOAc to afford an EtOAc soluble extract. This extract (127 g) was divided into six parts (Frs. A–F) by using a MCI gel CHP 20P column eluted with gradient aqueous MeOH (10%–100%). Fr. B (8.1 g) was gel filtrated over Sephadex LH-20 (aqueous MeOH, 80%) to afford 3 parts (Frs. B1–B3). Compound 9 (2.6 mg, t_R = 19.2 min) was obtained from Fr. B3 (0.40 g) by semi-preparative HPLC (MeCN/H₂O, 13%, flow rate: 3 mL/min). Fr. C (12.0 g) was gel filtrated over Sephadex LH-20 (aqueous MeOH, 80%) to afford 5 parts (Frs. C1–C5). Fr. C2 (5.51 g) was submitted to a RP-18 column eluted with gradient aqueous MeOH (15–50%) to yield 8 subfractions (Frs. C2.1–C2.8). Fr. C3 (2.45 g) was submitted to a RP-18 column eluted with gradient aqueous MeOH (15–50%) followed by semi-preparative HPLC (MeCN/H₂O, 29%, flow rate: 3 mL/min) to produce 4 (1.3 mg, t_R = 28.0 min). Fr. C4 (1.71 g) was gel filtrated over Sephadex LH-20 (MeOH) followed by semi-preparative HPLC (MeCN/H₂O, 21%, flow rate: 3 mL/min) to produce 8 (2.6 mg, t_R = 27.2 min). Fr. D (8.7 g) was gel filtrated over Sephadex LH-20 (aqueous MeOH, 80%) to afford 6 parts (Frs. D1–D6). Further, Fr. D5 (1.23 g) was gel filtrated over Sephadex LH-20 (MeOH) followed by semi-preparative HPLC (MeCN/H₂O, 29%, flow rate: 3 mL/min) to produce 3 (22.5 mg, t_R = 11.6 min), 10 (2.5 mg, t_R = 12.3 min), 7 (2.1 mg, t_R = 19.2 min), 6 (2.8 mg, t_R = 28.4 min), and 5 (5.5 mg, t_R = 34.0 min). Fr. D6 (1.01 g) was gel filtrated over Sephadex LH-20 (MeOH) followed by semi-preparative HPLC (MeCN/H₂O, 29%, flow rate: 3 mL/min) to produce 1 (12.5 mg, t_R = 12.2 min) and 2 (3.8 mg, t_R = 34.5 min). Notably, compounds 3, 4, 8, and 9 were isolated as racemates, which were subjected to chiral HPLC to yield (+)-3 (10.1 mg, t_R = 8.3 min) and (−)-3 (10.5 mg, t_R = 11.6 min) (n-hexane/ethanol, 85:15, flow rate: 1 mL/min), (+)-4 (0.7 mg, t_R = 10.4 min) and (−)-4 (0.5 mg, t_R = 15.8 min) (n-hexane/ethanol, 80:20, flow rate: 1 mL/min), (+)-8 (1.4 mg, t_R = 12.5 min) and (−)-8 (1.1 mg, t_R = 14.6 min) (n-hexane/ethanol, 90:10, flow rate: 1 mL/min), (+)-9 (1.2 mg, t_R = 8.9 min) and (−)-9 (1.1 mg, t_R = 14.3 min) (n-hexane/ethanol, 77:23, flow rate: 1 mL/min), respectively.

3.4 Compound Characterization

Plancyin A (1): Yellowish solid; UV (MeOH) λ_max (log ε) 288 (3.82), 255 (4.02), 211 (4.45) nm; ESIMS m/z 329 [M−H]⁻, HRESIMS m/z 329.1040 [M−H]⁻ (calcd for C₁₈H₁₇O₆, 329.1031). ¹H and ¹³C NMR data, see Table 1.

Plancyin B (2): Yellowish solid; UV (MeOH) λ_max (log ε) 291 (3.76), 256 (4.00), 214 (4.54) nm; ESIMS m/z 343 [M−H]⁻, HRESIMS m/z 343.1193 [M−H]⁻ (calcd for C₁₉H₁₉O₆, 343.1187). ¹H and ¹³C NMR data, see Table 1.

Plancyin C (3): Yellowish solid; {[α]_D²⁵ + 49.5 (c 0.17, MeOH); CD (MeOH) Δε₁₉₅ − 27.87, Δε₂₀₈ + 15.93; (+)-3}; {[α]_D²⁵ − 58.0 (c 0.16, MeOH); CD (MeOH) Δε₁₉₅ + 18.55, Δε₂₀₈ − 11.62; (−)-3}; UV (MeOH) λ_max (log ε) 278 (3.77), 205 (4.80) nm; ESIMS m/z 329 [M−H]⁻, HRESIMS m/z 329.1036 [M−H]⁻ (calcd for C₁₈H₁₇O₆, 329.1031). ¹H and ¹³C NMR data, see Table 1.

Plancyin D (4): Yellowish solid; {[α]_D²⁵ + 86.7 (c 0.07, MeOH); CD (MeOH) Δε₁₉₇ − 39.04, Δε₂₀₉ + 15.41; (+)-4}; {[α]_D²⁵ − 86.5 (c 0.05, MeOH); CD (MeOH) Δε₁₉₅ + 33.86, Δε₂₀₈ − 17.87; (−)-4}; UV (MeOH) λ_max (log ε) 277 (3.53), 205 (4.60) nm; ESIMS m/z 343 [M−H]⁻, HRESIMS m/z 343.1178 [M−H]⁻ (calcd for C₁₉H₁₉O₆, 343.1187). ¹H and ¹³C NMR data, see Table 2.

Plancyin E (5): Yellowish solid; UV (MeOH) λ_max (log ε) 277 (3.60), 217 (4.24), 205 (4.60) nm; ESIMS m/z 285 [M−H]⁻, HRESIMS m/z 285.1133 [M−H]⁻ (calcd for C₁₇H₁₇O₄, 285.1132). ¹H and ¹³C NMR data, see Table 2.

Plancyin F (6): Yellowish solid; UV (MeOH) λ_max (log ε) 291 (3.72), 208 (4.44) nm; ESIMS m/z 257 [M−H]⁻, HRESIMS m/z 257.0816 [M−H]⁻ (calcd for C₁₅H₁₃O₄, 257.0819). ¹H and ¹³C NMR data, see Table 2.

Plancyin G (7): Yellowish solid; UV (MeOH) λ_max (log ε) 323 (4.39), 204 (4.39) nm; ESIMS m/z 385 [M−H]⁻, HRESIMS m/z 385.0917 [M−H]⁻ (calcd for C₂₀H₁₇O₈, 385.0929). ¹H and ¹³C NMR data, see Table 3.

Plancyin H (8): Yellowish solid; {[α]_D²⁵ + 34.9 (c 0.11, MeOH); CD (MeOH) Δε₁₉₈ + 29.58, Δε₂₃₀ − 5.48; (+)-8}; {[α]_D²⁵ − 29.6 (c 0.14, MeOH); CD (MeOH) Δε₁₉₈ − 37.08, Δε₂₃₇ + 2.03; (−)-8}; UV (MeOH) λ_max (log ε) 280 (3.53), 221 (4.15), 205 (4.52) nm; ESIMS m/z 327 [M−H]⁻, HRESIMS m/z 327.0873 [M−H]⁻ (calcd for C₁₈H₁₅O₆, 327.0874). ¹H and ¹³C NMR data, see Table 3.

Periplanol A (9): Yellowish solid; {[α]_D²⁵ + 36.1 (c 0.04, MeOH); CD (MeOH) Δε₁₉₇ − 6.41, Δε₂₀₆ + 11.24, Δε₂₉₂ + 2.06; (+)-9}; {[α]_D²⁵ − 12.8 (c 0.41, MeOH); CD (MeOH) Δε₁₉₆ + 7.91, Δε₂₀₄ − 12.17, Δε₂₉₄ − 2.08; (−)-9}; UV (MeOH) λ_max (log ε) 507 (2.58), 474 (2.59), 290 (3.65), 206 (4.36); ESIMS m/z 303 [M−H]⁻, HRESIMS m/z 303.0863 [M−H]⁻ (calcd for C₁₆H₁₅O₆, 303.0874). ¹H and ¹³C NMR data, see Table 3.

3.5 Biological Evaluation for Kidney Fibrosis

3.5.1 Cell Culture and Cytotoxicity Assay

NRK-52e cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin in 37 ℃ with atmosphere of 5% CO₂. The growing cells were seeded at 1 × 10⁴ cells per well in 96-well culture plates for 24 h. Cells were exposed to the compounds with different concentrations (2.5, 5, 10, 20, 40 μM) for 48 h. The equal volume of DMSO was used as the solvent control. CCK-8 solution (10% v/v) was added and incubated for another 2 h. Light absorbance of the solution was measured at 450 nm (Epoch 2; BioTek Instruments, Inc.).

3.5.2 Western Blot Assay

Protein expression was analyzed by Western blot analysis as described previously [12]. The concentration of all the compounds was 40 μM for primary screening and 10, 20, 40 μM for dose-dependent experiments. The primary antibodies as follows: Anti-Fibronectin antibody [IST-9] (#ab6328; Abcam), COL1A1 Antibody (#84336; Cell Signaling Technology), α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb (#19245; Cell Signaling Technology), Smad3 (C67H9) Rabbit mAb (#9523, Cell Signaling Technology), Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb (#9520, Cell Signaling Technology), Smad2 (D43B4) XP® Rabbit mAb (#5339, Cell Signaling Technology), Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb (#3108, Cell Signaling Technology), GAPDH (D16H11) XP® Rabbit mAb (#5174, Cell Signaling Technology). Cell pellets were collected and resuspended in RIPA lysis buffer (containing 0.1 mM PMSF). GW: GW788388 (Medchemexpree, HY-10326) [13].

3.6 Biological Evaluation for Human Cancer Cells (K562, A549, and Huh7), ROCK2, JAK3, and DDR1

Compounds 1–9 were evaluated for their inhibitory effects against human cancer cells (K562, A549, and Huh7) [14], ROCK2 [15], JAK3 [16], and DDR1 [4] as previously described methods.

3.7 Biological Evaluation for EV71

Compounds 1–9 were evaluated for their EV71 inhibitory activities in vitro. VERO cells were plated into 384-well plate at a suitable density and allowed to adhere prior to addition of varying concentrations of drugs. Then the cells were covered with enteroviruses EV71 which was diluted at fresh culture medium. After that, the cells were incubated at 37 ℃ for a further 72 h. Then discard the old medium, add fresh medium containing CCK-8, and incubated in 37 ℃ for 2 h. The A450 was then measured with an Envision Plate Reader (PerkinElmer).

3.8 Anticoagulant Assay

The prothrombin time (PT) and thrombin time (TT) was determined with a coagulometer (TECO MC-4000, Germany). All the reagents were purchased from TECO GmbH (Germany). PT reagent, TT reagent, and normal human plasma was reconstituted in 4 mL (or 1 mL for plasma) of distilled water, according to the instructions of the manufacturer. The compounds were dissolved to 2 mM in DMSO at various concentrations. Then use 20 mM Tris–HCl pH 7.4 (including 5% Tween 80) diluted to 200 µM to be measured. For the PT and TT assays, 5 μL samples (or 10 μL for TT) were mixed with 45 μL (or 90 μL for TT) of normal human plasma and incubated for 2 min at 37 ℃; 100 μL of PT (or 50 μL of TT) reagent was then added and the clotting time was recorded.

3.9 Platelet Aggregation Assay

Turbidometric measurements of platelet aggregation inhibition were performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation, Havertown, PA, USA) according to Born's method [17, 18]. The present study was approved by the Research Ethics Committee of Kunming Institute of Botany, Chinese Academy of Sciences. The blood from the rabbits by ear central arter puncture, were anticoagulated with 3.8% sodium citrate (9:1, v/v). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared shortly after blood collection by spinning the sample at 180 g for 10 min at 22 ℃. The PRP was carefully removed and the remaining blood centrifuged at 2400×g for 10 min to obtain PPP. The centrifuge temperature was maintained at 22 ℃. Platelet counts were adjusted by the addition of PPP to the PRP to achieve a count of 250 × 10⁹/L. Platelet aggregation studies were completed within 3 h of preparation of PRP. Immediately after preparation of PRP, 250 μL was transferred into each of the test tubes, with 250 μL PPP set as a control. Before addition of inducers, the compounds were incubated with PRP at 37 ℃ for 5 min. Final concentration of agonist was: arachidonic acid (AA) 0.5 mM as positive reference. Percentage inhibition by the compounds was calculated according to the formula:

where A: maximum change of turbidity in DMSO added, B: maximum change of turbidity in sample added.

3.9.1 Computational Methods

Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan'14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program package [19], the conformational search generated low-energy conformers within a 10 kcal/mol energy was finished by software Conflex 7. Geometry optimizations of compounds 3, 8, and 9 were carried out at the DFT/B3LYP/6-311G (d, p) level. The calculated ECD spectra were determined by using Gaussian 09 software employing the TDDFT-B3LYP functional and the 6-311G (d, p) basis sets. ECD calculations further were conducted at the B3LYP SCRF (PCM)/6-311G (d, p) level in MeOH.

Notes

Acknowledgements

This study was supported by the National Science Fund for Distinguished Young Scholars (81525026) and Shenzhen Government's Plan of Science and Technology (JCYJ20170412110504956).

Compliance with Ethical Standards

Conflicts of interest

The authors declare that they have no conflict of interest.