Pterosin Sesquiterpenoids from Pteris cretica as Hypolipidemic Agents

Article pubs.acs.org/jnp

Pterosin Sesquiterpenoids from Pteris cretica as Hypolipidemic Agents via Activating Liver X Receptors Xiangkun Luo,†,⊥ Chanjuan Li,†,⊥ Pan Luo,† Xin Lin,† Hang Ma,‡ Navindra P. Seeram,‡ Ching Song,§ Jun Xu,*,† and Qiong Gu*,† †

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China ‡ Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States § School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, People’s Republic of China S Supporting Information *

ABSTRACT: Four new pterosin sesquiterpenoids (1−4), a new entkaurane diterpenoid (17), and 18 known compounds were isolated from the aerial parts of Pteris cretica L. The structures of the isolates were elucidated based on spectroscopic data analysis, and their absolute configurations were determined by comparison of experimental and calculated electronic circular dichroism spectra. The compounds were evaluated for lipid-lowering effects in 3T3-L1 adipocytes. Compounds 4, 8, 17, and 22 were more potent than the positive control, berberine, in decreasing triglycerides activity, with compound 4 exerting the most potent activity. Compound 4 activated LXRα/β in a HEK 293T cellbased reporter gene assay. Molecular dynamic simulations revealed that compound 4 activates liver X receptors (LXRs) through hydrogen bonding with the residues of LXRα/β, suggesting that compound 4 reduces total triglycerides through the regulation of LXRα/β.

by n-BuOH. Chromatography of the EtOAc fraction afforded compounds 1−23. The molecular formula of compound 1, a colorless oil, was C21H30O8, obtained from HRESIMS ion at m/z 409.18576 [M − H]− (calcd 409.18679). The IR spectrum (Figure S1, Supporting Information) displayed signals for −OH, −CO, and aromatic CC. The 1H NMR data (Table 1) revealed four methyl singlets at δH 1.05, 1.20, 2.67, and 2.51 ppm. A low-field hydrogen singlet at δH 7.37, together with the six aromatic carbon signals, suggested the presence of a characteristic pentasubstituted benzene moiety. The remaining 1H NMR signals were consistent with a C14 pterosin sesquiterpenoid and a β-glucopyranose unit.3,4 A carbon chemical shift at δC 104.4 (d) with the corresponding anomeric proton chemical shift at δH 4.33 (1H, d, J = 7.8 Hz) indicated the presence of a βconfigured glucoside. The 13C NMR data of 1 (Table 1) showed 15 distinct carbon signals excluding the carbon signals corresponding to the glucosyl moiety. These carbon signals (Table 1) also supported the presence of a pentasubstituted aromatic moiety, including a low-field signal at 211.6 ppm (C O), which is typical of C-1 of a pterosin-type sesquiterpenoid

Pteris cretica L. (Pteridaceae) is a traditional Chinese medicinal plant, widely distributed in the southwest and southern regions of China. Historically, plants from the Pteris genus have been used for heat-clearing, inducing diuresis, and reducing edema, as antibacterial agents, and for the treatment of diabetes.1 While members of the Pteris genus are reported to produce sesquiterpenoids and diterpenoids,2 the systematic phytochemical investigation of P. cretica has not yet been reported. Hyperlipidemia can cause cardiovascular and other metabolic disorders. To search for hypolipidemic agents, ca. 20 herbal extracts were screened for their effects in murine 3T3-L1 adipocytes, among which P. cretica showed the most potent antihyperlipidemic activity. Bioassay-guided fractionation of this extract resulted in identifying four new pterosin sesquiterpenoids (1−4), a new ent-kaurane diterpenoid (17), 12 known sesquiterpenoids (5−16), and six known ent-kaurane diterpenoids (18−23). Compound 4 showed the most potent lipidlowering effect in 3T3-L1 adipocytes. Mechanism studies indicated that the antihyperlipidemic activity of compound 4 involves activation of LXRα/β (LXR = liver X receptor).

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RESULTS AND DISCUSSION An aqueous suspension of a 95% EtOH extract of the air-dried aerial plants of P. cretica was partitioned with EtOAc followed © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 17, 2016

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DOI: 10.1021/acs.jnatprod.6b00558 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H and 13C NMR Chemical Shifts (δ) of Compounds 1−4 1a no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′

δH (J in Hz)

4.76, s 7.37, s

1.05, 1.20, 2.51, 3.15, 3.94, 3.68, 2.67,

s s s t (8.0) m overlap s

4.33, 3.21, 3.37, 3.28, 3.29, 3.87, 3.67,

d (7.8) dd (8.9, 7.8) m m m dd (11.9, 1.9) overlap

2a δC 211.6, 52.4, 77.5, 126.1, 146.5, 138.1, 138.6, 131.1, 153.8, 20.8, 23.5, 21.5, 30.3, 69.1,

C C CH CH C C C C C CH3 CH3 CH3 CH2 CH2

14.1, CH3 104.4, 75.1, 78.2, 71.6, 78.0, 62.7,

3a

δH (J in Hz)

7.79, s

1.38, 2.59, 3.24, 3.78,

s s t (6.9) t (6.9)

5.29, d (11.7) 5.11, d (11.7)

δC

δH (J in Hz)

203.4, 76.1, 202.3, 125.5, 149.2, 149.1, 140.7, 136.6, 139.7,

C C C CH C C C C C

21.5, 21.4, 33.4, 62.0,

CH3 CH3 CH2 CH2

56.6, CH2

2.83, m 5.19, d (6.5) 7.52, s

1.20, 2.51, 3.11, 3.69,

d (7.5) s t (7.3) t (7.3)

5.09, d (11.8) 5.04, d (11.8)

4a δC 210.3, 49.7, 70.4, 128.9, 147.1, 139.3, 139.5, 132.6, 155.6,

C CH CH CH C C C C C

10.7, 21.4, 32.6, 62.4,

CH3 CH3 CH2 CH2

56.9, CH2

δH (J in Hz) 2.79, m 5.20, d (6.6) 7.66, s

1.19, 4.78, 3.02, 3.65,

d (7.5) s t (7.4) t (7.4)

2.66, s

δC 210.4, 49.7, 70.4, 124.1, 148.8, 137.8, 138.3, 133.2, 155.2,

C CH CH CH C C C C C

10.7, 63.5, 32.1, 62.0,

CH3 CH2 CH2 CH2

13.9, CH3

CH CH CH CH CH CH2

a1

H NMR measured at 400 MHz, 13C NMR measured at 100 MHz and obtained in pyridine-d5 with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

skeleton.4 The HMBC cross-peaks of H-1′ (δH 4.33, 1H, d, J = 7.8 Hz) with C-14 (δC 69.1, t) demonstrated that the βglucopyranose unit was connected to C-14 (Figure 1). The 2D structure of 1 was the same as that of the known compound (3S)-pteroside D.5 However, the specific rotation of 1 was [α]20D +50 (c 0.1, MeOH), which was opposite to that of (3S)pteroside D.5 The experimental electronic circular dichroism (ECD) spectrum of 1 showed a positive Cotton effect at 328 (+1.84) nm, which was consistent with the calculated ECD of (3R)-1 (Figure 2). The experimental ECD spectrum of 1 was similar to that of (3R)-pterosin D,6 but opposite to that of (3S)-pterosin D.6 Therefore, the structure of 1 was assigned as (3R)-pteroside D. The molecular formula of compound 2 is C14H16O5, determined from the positive HRESIMS ion at m/z 265.10703 [M + H]+ (calcd 265.10705). The IR spectrum exhibited absorptions at 3430 cm−1 (−OH), 1662 cm−1 (−C O), and 1587 cm−1 (phenyl). The NMR data (Table 1), together with the COSY spectrum, indicated the presence of two methyls (δH 1.38, 2.59), a hydroxyethyl group, a hydroxymethyl group, a pentasubstituted benzene moiety, and two carbonyl groups (δC 203.4, 202.3). The above data suggested that this compound was also a pterosin-type sesquiterpenoid. Compared to the known compound pterosin N,4 compound 2 had an additional carbonyl group and CH2OH with one less methyl and methylene groups. The HMBC cross-peaks of CH3 (δH 1.38, s) with the two carbonyl carbons at δC 203.4 and 202.3 and the oxygenated tertiary carbon at δC 76.1 indicated that the CH3 group was located at C-2 with two carbonyl groups positioned at C-1 and C-3. The hydroxymethyl group substituted at C-7 was deduced from the

HMBC cross-peaks of H-15 with C-6 and C-8. Thus, the 2D structure was deduced as shown. The calculated ECD spectrum of the (2R)-enantiomer was consistent with the experimental ECD spectrum (Figure 2) of 2. This is the first reported pterosin sesquiterpenoid containing two carbonyl groups. Compound 2 was given the trivial name pterosone A. Compound 3, a colorless oil, had the molecular formula C14H18O4 as established via the (+)-HRESIMS ion at m/z 251.12729 [M + H]+ (calcd 251.12779). The IR spectrum exhibited absorptions corresponding to −OH (3384 cm−1), −CO (1694 cm−1), and aromatic (1599 cm−1) functionalities. The 1H and 13C NMR data (Table 1) of compound 3 showed marked similarities with the known pterosin C,4 except for resonances of an additional hydroxymethyl (δH 5.09, 5.04) group and the absence of one methyl signal. Detailed 2D NMR analysis (Figures S21−24, Supporting Information) revealed the presence of a HOCH2-15 group in 3. Therefore, compound 3 was identified as 15-hydroxymethylpterosin C. The ECD spectrum was used to define the absolute configuration of compound 2. It was reported that the ECD spectrum of pterosin C depends mainly on C-3, independent of the C-2 configuration.6 Furthermore, (3R)- and (3S)-pterosins show negative and positive Cotton effects at 320 nm, respectively.6 Therefore, on the basis of a positive Cotton effect at 320 nm in the experimental ECD spectrum, a (3S)-configuration (Figure 2) was assigned to compound 3. The conformation of pterosin C-type compounds can be deduced from the 3J2,3 value.6 The chemical shift of H-3 (δH 5.19, d, J = 6.5 Hz) in 3 was similar to that of (2R,3S)-pterosin C (δH 5.09, d, J = 6.5 Hz). Thus, compound 3 was defined as (2R,3S)-7-hydroxymethylpterosin B



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Figure 1. 1H−1H COSY (bold lines) and HMBC (→) of compounds 1−4.

Compound 4, a colorless oil, had the same molecular formula as that of 3, as deduced from HREIMS data. The NMR data of 4 (Table 1) were similar to those of 3. Analysis of HMBC data (Figure S31, Supporting Information) revealed that the hydroxymethyl group was positioned at C-5. Compared to 3, compound 4 has a similar positive specific rotation ([α]20D +24) and a positive Cotton effect at 329 nm. Thus, the structure of compound 4 was defined as (2R,3S)-5-hydroxymethylpterosin C. Compound 17 had a molecular formula of C20H34O4, as established by the HREIMS ion at m/z 338.2450 [M]+ (C20H34O4, calcd for 338.2452). The IR spectrum of 17 suggested the presence of −OH (3380 cm−1) groups. The NMR data (Table 2) combined with the molecular formula, C20H34O4, revealed that compound 17 was a tetracyclic diterpenoid. Furthermore, the DEPT spectrum exhibited signals of four methyl groups (δC 38.1, 23.6, 22.2, 20.4), seven methylenes (δC 57.1, 55.1, 54.5, 50.8, 45.3, 34.9, 20.5), four methines (δC 68.5, 64.1, 61.3, 56.0), three quaternary carbons (δC 43.3, 42.1, 36.5), and two oxygenated tertiary carbons (δC 80.5, 76.2). Compounds 17 and 22 have similar

Figure 2. Experimental ECD spectra (190−400 nm) and TDDFTcalculated ECD spectra for compounds 1−4.

C. This was confirmed by the calculated ECD spectrum (Figure 2). C



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(2R,3S)-pterosin C (7),4 (2S,3S)-pterosin C (8),4 2-hydroxypterosin C (9),12 pterosin T (10),12 pterosin S (11),13 pterosin T (12),12 (2S,3S)-pterosin U (13),14 jamesonin (14), 13 pterosin L (15), 4 pterosin D (16), 4 2β,15αdihydroxy-ent-kaur-16-ene (18),15 2β,6β,15α-trihydroxy-entkaur-16-ene (19),16 creticoside A (20),17 creticoside B (21),17 2β,6β,16α-trihydroxxy-ent-kaurane (22),11 and substanz D (23)17 by comparing their spectroscopic data with reported data. Lipid-Lowering Activity of the Isolates in 3T3-L1 Cells. High triglyceride (TG) level is a risk factor of cardiovascular and other metabolic disorders such as atherosclerosis, obesity, and diabetes. The 3T3-L1 cell, which recapitulates the differentiation and lipogenesis of a preadipocyte, is a wellestablished model for evaluating a compound with lipidlowering property in vitro. Triglycerides are used as indicators of intracellular accumulation of lipids suggesting adipogenesis. Using this cell model, the 23 isolates along with berberine, the positive control, were evaluated at 10 μM. Compounds 4, 8, 17, and 22 were more potent than berberine in reducing TG levels (Figure 4). These four compounds decreased TG levels via a

Table 2. 1H and 13C NMR Chemical Shifts (δ) of Compound 17b no.

δH (J in Hz)

δC

no.

1α 1β 2α

2.48, d (11.5) 1.10, m 4.30, m

50.8, CH2

1.88, 1.66, 1.95, 1.89,

3α 3β 4

2.09, m 1.63, m

55.1, CH2

11α 11β 12α 12β 13

5β

1.21, m

61.3, CH

2.25, 2.11, 2.13, 1.65,

6α 7α 7β 8 9β 10

4.20, m 2.19, m 1.94, m

68.5, CH 54.5, CH2

14α 14β 15α 15β 16 17

1.45, s

76.2, C 22.2, CH3

42.1, C 56.0, CH 43.3, C

18 19 20

1.65, s 1.29, s 1.19, s

38.1, CH3 23.6, CH3 20.4, CH3

64.1, CH

36.5, C

1.07, m

δH (J in Hz) m m m m

δC 20.5, CH2 34.9, CH2 80.5, C

m m m m

45.3, CH2 57.1, CH2

b1

H NMR measured at 400 MHz, 13C NMR measured at 100 MHz and obtained in pyridine-d5 with TMS as internal standard. Assignments were supported with HSQC and HMBC NMR spectra.

NMR data except for the H-13 in 22 being replaced by a 13OH group. The HMBC cross-peaks of H-17 (δH 1.45) with C13 (δC 80.5) and H-15 (δH 2.13) with C-13 (δC 80.5) suggested that the hydroxy group was located at C-13. Therefore, compound 17 was identified as shown. The absolute configuration of the 13,16-diol unit was established using Snatzke’s method.7−9 For tert/tert vic-diols, the ECD Cotton effect appears between 300 and 400 nm in the presence of dimolybdenum tetraacetate [Mo2(OAc)4]. When the O−C− C−O torsion angle of the vic-diol unit is negative, a negative sign of the Cotton effect (CE) at around 310 and 400 nm should be observed. The experimental ECD spectrum induced by [Mo2(OAc)4] (IECD) of 17 showed negative bands at around 315 and 383 nm, and around 350 nm a negative CE (Figure 3), which is in agreement with literature observations.9

Figure 4. Evaluation of the lipid-lowering effect of the isolates in 3T3L1 adipocytes. 3T3-L1 preadipocytes were treated with 10 μM of test compounds on day 0 and day 3 for 3 days during the differentiation. The levels of intracellular TG were determined using a commercial triglycerides (TG) assay kit. Data are represented as mean ± SD of three independent experiments (*p < 0.05 vs Veh, **p < 0.01 vs Veh).

concentration-dependent pattern in 3T3-L1 cells (Figure 5A). The lipid-lowering effects of 4, 8, 17, and 22 were confirmed by microscopy with Oil Red O staining (Figure 5B). Effects of Compound 4 on LXRα/β. LXRα and LXRβ are two isoforms of the liver X receptor, which belong to the nuclear hormone receptor superfamily.18 LXRs act as oxysterol sensors, regulating cholesterol and lipid metabolism.19 The LXRα isoform is mainly expressed in the liver, intestine, adipose tissue, and macrophages, while LXRβ is present ubiquitously in organs and tissues. LXRs are regarded as promising drug targets for the treatment of atherosclerosis, hyperlipidemia, and many other metabolic syndromes.20 Herein, the isolates were assayed for their effects on LXR using a cell-based dual-luciferase reporter gene system. The transfection of the HEK 293T cell line is widely used for the study of LXR activation. Therefore, hLXRα or rLXRβ as receptor plasmids, together with hRXRα, a firefly luciferase reporter gene containing LXR response elements, and a renilla luciferase reporter gene were cotransfected into HEK 293T cells and exposed to the isolates. GW3965 was used as a positive control. As shown in Figure 6, GW3965 activated LXRα and LXRβ. Compound 4 showed even stronger activation of LXRα/β than GW3965 at 10 μM.

Figure 3. Key 1H−1H COSY (bold lines) and HMBC (→) crosspeaks of compound 17 and IECD of compound 17.

Thus, the O−C−C−O dihedral angle of the 13,16-diol moiety was determined to be negative. Among the four possible stereoisomers of 17, stereoisomers I and IV satisfied the requirements for a negative dihedral angle (Figure S41, Supporting Information). In the NOESY spectrum of 17 (Figure S40, Supporting Information), cross-peaks of H-2/H319/H3-20, H-6α/H3-20, and H-9/H-15β/H3-17 revealed that 2-OH, 6-OH, and CH3-17 are β-oriented. Thus, the structure of 17, pterosone B, was defined as 2β,6β,13α,16α-tetrahydroxyent-kaurane. The known compounds were characterized as (2R,3S)pteroside C (5),10 pterosin C 14-O-β-D-glucoside (6),11 D



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Figure 6. Effect of the isolates on LXRα (A) and LXRβ (B). HEK 293T cells were cotransfected with pGL3/(DR-4)-c-fos-FF-luc, pSG5/ hLXRα or pSG5/rLXRβ, pSG5/hRXRα, and pCMV/R-luc as internal normalization. After 10 h, the cells were treated with compounds. GW3965 served as the positive control, and 0.1% DMSO was the vehicle control. Luminescence measurement was processed 20 h after sample treatments. The results are expressed as relative firefly luciferase activity normalized to the renilla luciferase activity (fold change compared to vehicle control). Data are represented as mean ± SD of three independent experiments (*p < 0.05 vs Veh, **p < 0.01 vs Veh).

Figure 5. Lipid-lowering effects of the four best inhibitors. (A) Compounds 4, 8, 17, and 22 decreased the TG amounts in 3T3-L1 cells in a concentration-dependent manner. (B) Lipid accumulation of 3T3-L1 adipocytes was visualized by Oil Red O staining. Pictures were taken on day 6 with 4× magnification. UD: undifferentiated cells. Control: differentiated cells without compounds. Data are represented as mean ± SD of three independent experiments (*p < 0.05 vs Veh, **p < 0.01 vs Veh).

Cytotoxicity Assay. To investigate whether the lipidlowering activity is caused by cytotoxicity, an MTT assay was performed in HEK 293T cell lines. As shown in Figure 7, all 23 compounds showed near 100% cell viability relative to vehicletreated control cells at 10 μM. Molecular Dynamic (MD) Simulation. To investigate the interaction of 4 with LXRs, molecular docking and MD simulations were performed using MOE and AMBER 12, respectively. The structures of GW3965, LXRα, and LXRβ were obtained from the RCSB Protein Data Bank (PDB code: 3IPQ, 1PQ6). Missing residues were fixed via the homology modeling module. Compound 4 was docked into the ligandbinding domain of LXRα or LXRβ. The 20 best poses were kept for analysis, and the complex with the best binding mode was chosen for 20 ns MD simulations. Cluster analyses were carried out by the PTRAJ module of AMBER 12. All 10 000 frames of each trajectory were analyzed and divided into three clusters. The representative structures of the clusters that cover most of the trajectories are shown in Figure 8. Compound 4 formed two hydrogen bonds with LXRα at Ala261 and Phe257 residues. It also interacted with LXRβ at Phe257 and His421 residues. According to the crystal structure (PDB code: 3IPQ, 1PQ6),21 His421 forms an electrostatic interaction with Trp443 and therefore holds the AF2 helix in the active position to keep LXRβ in an active conformation. Therefore, the hydrogen bond formed between compound 4 and His421 may play a key role in the activation of LXRβ. To better illustrate the interaction between compound 4 and LXRs, the last 15 ns MD trajectories were taken for binding

Figure 7. Cell viability of compounds 1−23 in HEK 293T cell lines. Data are represented as mean ± SD of three independent experiments.

Figure 8. Optimal binding modes of LXRs/4 and LXR/GW3965 complexes. (A) LXRα/4; (B) LXRα/GW3965; (C) LXRβ/4; (D) LXRβ/GW3965. E



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semipreparative RP-HPLC with 40% aqueous MeOH as the solvent. Fraction F4 was chromatographed over Sephadex LH-20 with MeOH as eluent and further purified by semipreparative HPLC with 14% MeCN−H2O to give 17 (10.2 mg), 14 (10.6 mg), and 13 (7.8 mg). (3R)-Pteroside D (1): colorless oil; [α]20D +48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (3.20), 259 (2.83), 291 (2.06) nm; ECD (MeOH) λmax (Δε) 211 (+3.79), 227 (−3.45), 298 (−2.17), 329 (+2.02) nm; IR (KBr) νmax 3425, 2924, 1693, 1656, 1624, 1603, 1461, 1377, 1322, 1081, 899, 781 cm−1; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Table 1; HRESIMS m/z 409.18576 [M − H]− (calcd for C21H29O8, 409.18679). Pterosone A (2): colorless oil; [α]20D −17 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.79), 241 (4.09) nm; ECD (MeOH) λmax (Δε) 244 (+0.7) nm; IR (KBr) νmax 3430, 2962, 2295, 2856, 1744, 1710, 1662, 1587, 1445, 1301, 1261, 1195, 1092, 903 cm−1; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Table 1; HRESIMS m/z 265.10703 [M + H]+ (calcd for C14H17O5, 265.10705). (2R,3S)-7-Hydroxymethylpeterosin C (3): colorless oil; [α]20D +26 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.21), 257 (3.86), 302 (3.06) nm; ECD (MeOH) λmax (Δε) 207 (+4.93), 226 (−5.23), 297 (−3.83), 326 (+3.06) nm; IR (KBr) νmax 3384, 2970, 2934, 2881, 1694, 1599, 1453, 1328, 1229, 1127, 1038, 921 cm−1; 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Table 1; HRESIMS m/z 251.12729 [M + H]+ (calcd for C14H19O4, 251.12779). (2R,3S)-5-Hydroxymethylpterosin C (4): colorless oil; [α]20D +24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.28), 257 (3.93), 301 (3.13) nm; ECD (MeOH) λmax (Δε) 208 (+2.71), 225 (−3.97), 298 (−3.34), 330 (+2.63) nm; IR (KBr) νmax 3422, 2963, 2912, 1693, 1623, 1424, 1374, 1261, 1091, 1035, 803 cm−1; 1H NMR (methanold4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Table 1; HREIMS m/z 250.1203 [M]+ (calcd for C14H18O4, 250.1200). Pterosone B (17): white powder; [α]20D −15 (c 0.2, MeOH); IR (KBr) νmax 3380, 3003, 2937, 1657, 1445, 1231, 1146, 1042, 941 cm−1; 1 H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz) data, see Table 2; HREIMS m/z 338.2450 [M]+ (calcd for C20H34O4, 338.2452). ECD Computational Methods. The ECD spectrum were calculated according to reported methods.22 Briefly, the ECD spectra of compounds 1−4 and 17 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(d,p) level in MeOH. The computational ECD data were fitted in the SpecDis software package.23 Determination of Absolute Configuration of the 1,2-Diol Unit of 17. A solution of 17 (0.5 mg) in anhydrous DMSO (1 mL) was mixed with Mo2(OAc)4. The ECD spectrum of the mixture (molar ratio ca. 1:1.2 diol/Mo2(OAc)4) was measured immediately after mixing, and the time course was monitored until it reached a stable phase (about 15 min after mixing). The inherent ECD spectrum was subtracted. The relation between the absolute configuration of the 1,2-diol moiety of 17 and the sign of the O−C−C−O dihedral angle was estimated as previously reported.9 Cell Culture. Cells (HEK 293T, 3T3-L1) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2. Adipocyte differentiation was carried out on 3T3-L1.24 Briefly, 2 days after full confluence (day 0), the complete culture medium was changed to differentiation medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/ mL streptomycin, 2 μg/mL insulin, 100 ng/mL dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 ng/mL biotin) for 3 days. Then (day 3), differentiation medium was switched to postdifferentiation medium (DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 μg/mL insulin) for an additional 3 days. At day 6, cells were used for further analysis. Test

energy calculations and energy decompositions. The binding energies of compound 4 with LXRα and LXRβ are −16.36 and −18.57 kcal/mol, respectively (Table S1, Supporting Information). Binding energy decomposition (Figure S43, Supporting Information) demonstrated the energy contribution of all residues. His421 and Trp443 are among the top three energetically pivotal residues, which further proved the stabilization effect of compound 4 on the AF2 helix during the activation of LXRs. In summary, four new pterosin sesquiterpenoids (1−4), a new ent-kaurane diterpenoid (17), and 18 known compounds were isolated from the aerial parts of P. cretica. The isolates were evaluated for triglyceride-lowering effects in 3T3-L1 adipocytes. Compounds 4, 8, 17, and 22 showed significant triglyceride-lowering activity, with compound 4 exerting the most significant inhibitory activity. The HEK 293T cell-based reporter gene assay indicated that compound 4 activated LXRα/β. MD simulations demonstrated that compound 4 activated the LXRs through hydrogen bond formation with the Ala261 and Phe257 residues of LXRα or Phe257 and His421 residues of LXRβ indicating that compound 4 reduced triglyceride levels through activating LXRα/β.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a PerkinElmer 341 polarimeter. ECD data were collected on an Applied Photophysics Chirascan spectrometer. UV spectra were recorded using a Shimadzu UV2450 spectrophotometer. IR spectra were determined using a Bruker Tensor 37 infrared spectrophotometer. The 1D and 2D NMR were recorded on a Bruker AVANCE400 NMR spectrometer with tetramethylsilane (TMS) as an internal reference. A Shimadzu LCMS-IT-TOF system was used to measure HRESIMS spectra. HREIMS spectra were measured on a MAT95XP high-resolution mass spectrometer. A Shimadzu instrument (LC20AT) was used to perform semipreparative HPLC separation with a Zorbax SB-C18 column (250 × 9.4 mm, 5 μm). Plant Material. The aerial parts of P. cretica L. (7.6 kg) were collected at Longli County of the Qiannan Buyi and Miao Autonomous Prefecture of Guizhou Province, People’s Republic of China, in July 2014. The sample was identified by Dr. Qingwen Sun from Guiyang College of Traditional Chinese Medicine, and a voucher specimen (GZQSY357) has been deposited at the School of Pharmaceutical Science, Sun Yat-sen University. Extraction and Isolation. The aerial parts of P. cretica were dried at room temperature, powdered, and extracted with 95% EtOH to afford an extract (329 g) that was partitioned with EtOAc (4 × 4 L), nBuOH (3 × 3 L), and H2O. The EtOAc fraction (110 g) was chromatographed on a silica gel column eluting with CH2Cl2−MeOH (100:0 to 0:100) to obtain seven fractions (A−G). Fraction D (20 g) was subjected to silica gel column chromatography using petroleum ether−EtOAc (3:2) as eluent to give compound 18 (1.6 g). Fraction E (5 g) was purified using an MCI gel column with MeOH−H2O (40− 100%) as eluting solvent to give compounds 19 (12.7 mg), 20 (22.6 mg), 21 (24.7 mg), 22 (16.8 mg), and 23 (28.3 mg). Fraction F (19 g) was subjected to semipreparation HPLC using an RP-C18 column eluting with a step gradient of MeOH−OH from 50% to 100% to afford five subfractions (F1−F5). Fraction F1 was further separated using semipreparative HPLC with 45% MeOH−H2O as the solvent to produce compounds 7 (20.7 mg), 8 (10.9 mg), and 16 (3.7 mg). Fraction F2 was subjected to semipreparative HPLC eluting with 17% CH3CN−H2O and further purified through semipreparative HPLC eluting with 45% MeOH−H2O to give 2 (2.8 mg), 3 (5.1 mg), 9 (3.5 mg), and 11 (41.0 mg). Fraction F3 was chromatographed on a silica gel column with CH2Cl2−MeOH (20:1, 9:1) and further purified by semipreparative HPLC using 20% MeCN−H2O as the solvent to afford 4 (5.4 mg), 12 (14.6 mg), 10 (6.6 mg), and 16 (7.1 mg). Compounds 1 (5.5 mg), 5 (6.4 mg), and 6 (3.2 mg) were obtained by F



Article

potential and assigning the GAFF force field parameters.33 For the protein receptors, the AMBER ff12SB force field was used.34,35 The ligand−receptor complexes were neutralized and solvated in an octahedral box of TIP3P36 water molecules with solvent layers of 10 Å between the box edges and solute surface. The SHAKE37,38 algorithm was used to restrict all covalent bonds involving hydrogen atoms. The particle mesh Ewald (PME) method39 was performed to treat longrange electrostatic interactions. Each ligand−receptor system was subjected to three steps of minimization followed by heating and equilibration (see Experimental Section in the Supporting Information). The root-mean-square deviations (RMSDs) of the complexes were calculated using the PTRAJ module (Figure S42, Supporting Information). Trajectories were analyzed using the average linkage algorithm to produce three clusters using the pairwise RMSD between frames as a metric comparing the atoms named CA. Binding Mode and Binding Energy Calculations. The calculation simulations were performed with the AMBER 12 program using the AMBER ff12SB force field. The MM/PBSA method40 in the Amber Tools suite was used to calculate the binding energies for each receptor−ligand complex (see Experimental Section in the Supporting Information).

compounds dissolved in DMSO were supplemented at indicated concentrations throughout the course of differentiation. Berberine (Sigma-Aldrich, St. Louis, MO, USA) served as a positive control, and DMSO (< 0.1%) served as vehicle control. Triglyceride Assay. 3T3-L1 cells were harvested and washed twice with cold phosphate-buffered saline (PBS) (pH 7.4) at the end of differentiation. The cell lytes were used for determination of intracellular triglycerides using a commercial TG assay kit (Jiancheng Bioengineering Institution, Nanjing, China) according to the manufacturer’s protocol. Results are presented as the relative TG content compared to the negative control cells. Oil Red Staining. Oil Red O staining was performed using the reported methods.25 A 0.35% Oil Red O stock solution in isopropyl alcohol was diluted with ddH2O (3:2 v/v) and then filtrated via a 0.22 μm filter (Millipore). The cells were fixed with 4% (v/v) formaldehyde for 1 h at room temperature, and the fixed cells were stained with freshly prepared Oil Red O (ORO) solution for 30 min at 60 °C and washed three times with water. Pictures of cells stained with ORO were taken using an Olympus CKX41 microscope and camera. Transfection and Luciferase Assay. The effects of compounds on LXR were assayed according to the reported method.26 Briefly, the HEK 293T cells (3 × 104 cell/well) were plated (96-well plates) and allowed to attach over 12 h. Receptor plasmids pSG5/hLXRα or pSG5/rLXRβ together with pSG5/hRXRα, firefly luciferase reporter plasmid pGL3/(DR-4)-c-fos-FF-luc, and renilla luciferase reporter plasmid pCMV/R-luc were cotransfected into HEK 293T cells using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer’s instructions.27 After incubation for 10 h, cells were treated with the test compounds at indicated concentrations. LXR agonist GW3965 was used as the positive control. Luminescence was measured after 20 h. The data are presented as relative firefly luciferase activity normalized to the renilla luciferase activity (fold change compared to vehicle control). Cytotoxicity Assay. All the isolates were assayed for cytotoxicity in HEK 293T cells. HEK 293T cells were inoculated in 96-well plates with DMEM medium containing 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. The cells were treated with the compounds (10 μM) for 24 h in triplicate. MTT (20 μL; 2.5 mg/mL) was added to each well and incubated for 4 h, after which 100 μL of DMSO was added to each well. The absorbance of wells was recorded at 492 nm after 20 min of shaking. The inhibition percentage of test compounds against 293T cell lines was calculated with the following formula: survival rate (%) = (Aexperimental group − Ablank)/(Acontrol group − Ablank) × 100%. Docking. The MMFF9428 force field was applied to minimize the initial structures to yield the lowest energy 3D conformations. The structures of GW3965, LXRα, and LXRβ were obtained from RCSB Protein Data Bank (PDB code: 3IPQ, 1PQ6). The ligand in the binding pocket was removed. Homology modeling was applied to fix the missing residues using Molecular Operating Environment 2015.10 (MOE, Chemical Computing Group Inc., Montreal, Canada). The endogenous ligand position was selected as the binding site. The protein was protonated at pH 7.4 and assigned with the AMBER 12/ EHT force field using MOE. Default parameters were used for docking. Triangle Matcher placement and GBVI/WSA dG refinement were used. The docked poses were scored using built-in scoring functions. The 20 best poses were kept for binding mode analysis. The complex with the best binding mode was kept for further molecular dynamics simulation. Molecular Dynamics Simulation. MD simulations were performed using AMBER 12. The initial coordinates of compound 4 were obtained through molecular docking. The structures of LXRα and LXRβ obtained from the RCSB Protein Data Bank (PDB code: 3IPQ, 1PQ6) were fixed through homology modeling. The PMEMD module of AMBER 1231 was used to perform GPU-based MD simulations29,30 on LXRα/compound 4, LXRα/GW3965, LXRβ/ compound 4, and LXRβ/GW3965. The Hartree−Fock method of the Gaussian 0932 program was applied to calculate the partial atomic charges of the ligands. This is based on the 6-31G(d) basis set. The Antechamber program was used for fitting the restricted electrostatic

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00558. The IR, MS, and NMR spectra of compounds 1−4 and 17, molecular dynamics simulation and binding energy calculations of 4, and four configurations of 17 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax (J. Xu): +86-20-39943023. E-mail: junxu@ biochemomes.com. *Tel/Fax (Q. Gu): +86-20-39943077. E-mail: guqiong@mail. sysu.edu.cn. ORCID

Jun Xu: 0000-0002-1075-0337 Qiong Gu: 0000-0001-6011-3697 Author Contributions ⊥

X. K. Luo and C. J. Li contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by the National High-Tech R&D Program of China (863 Program) (2012AA020307), the Guangdong Province Frontier and Key Technology Innovation Program (2015B010109004), and the National Natural Science Foundation of China (81173470, 81473138).

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REFERENCES

(1) Yang, D. M.; Xing, F. W. Chinese Wild Plant Resources 2010, 29, 29−32. (2) Ge, X.; Ye, G.; Li, P.; Tang, W. J.; Gao, J. L.; Zhao, W. M. J. Nat. Prod. 2008, 71, 227−231. (3) Kuraishi, T.; Murakami, T.; Taniguchi, T.; Kobuki, Y.; Maehashi, H.; Tanaka, N.; Saiki, Y.; Chen, C. M. Chem. Pharm. Bull. 1985, 33, 2305−2312. (4) Fukuoka, M.; Yoshihira, K.; Natori, S.; Mihashi, K.; Nishi, M. Chem. Pharm. Bull. 1983, 31, 3113−3128. G



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(5) Kuroyanagi, M.; Fukuoka, M.; Yoshihira, K.; Natori, S. Chem. Pharm. Bull. 1979, 27, 592−601. (6) Kuroyanagi, M.; Fukuoka, M.; Yoshihira, K.; Natori, S. Chem. Pharm. Bull. 1979, 27, 731−741. (7) Di Bari, L.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. J. Org. Chem. 2001, 66, 4819−4825. (8) Frelek, J.; Ruśkowska, P.; Suszczyńska, A.; Szewczyk, K.; Osuch, A.; Jarosz, S.; Jagodziński, J. Tetrahedron: Asymmetry 2008, 19, 1709− 1713. (9) Górecki, M.; Jablońska, E.; Kruszewska, A.; Suszczyńska, A.; Urbańczyk-Lipkowska, Z.; Gerards, M.; Morzycki, J. W.; Szczepek, W. J.; Frelek, J. J. Org. Chem. 2007, 72, 2906−2916. (10) Fukuoka, M.; Kuroyanagi, M.; Yoshihira, K.; Natori, S. Chem. Pharm. Bull. 1978, 26, 2365−2385. (11) Murakami, T.; Maehashi, H.; Tanaka, N.; Satake, T.; Kuraishi, T.; Komazawa, Y.; Saiki, Y.; Chen, C. M. Yakugaku Zasshi 1985, 105, 640−648. (12) Tanaka, N.; Satake, T.; Takahashi, A.; Mochizuki, M.; Murakami, T.; Saiki, Y.; Yang, J. Z.; Chen, C. M. Chem. Pharm. Bull. 1982, 30, 3640−3646. (13) Satake, T.; Murakami, T.; Saiki, Y.; Chen, C. M.; Gomez, P. L. D. Chem. Pharm. Bull. 1984, 32, 4620−4624. (14) Murakami, T.; Satake, T.; Chen, C. M. Chem. Pharm. Bull. 1975, 23, 936−939. (15) Chen, C. M.; Murakami, T. Tetrahedron Lett. 1971, 16, 1121− 1124. (16) Hakamatsuka, T.; Tanaka, D.; Namatame, Y.; Wada, H.; Tanaka, N. Nat. Med. 1997, 51, 278−280. (17) Satake, T.; Murakami, T.; Saiki, Y.; Chen, C. M. Chem. Pharm. Bull. 1983, 31, 3865−3871. (18) Nomura, S.; Endo-Umeda, K.; Aoyama, A.; Makishima, M.; Hashimoto, Y.; Ishikawa, M. ACS Med. Chem. Lett. 2015, 6, 902−907. (19) Harasiuk, D.; Baranowski, M.; Zabielski, P.; Chabowski, A.; Gorski, J. Cell. Physiol. Biochem. 2015, 35, 1095−1106. (20) Hu, B.; Unwalla, R. J.; Goljer, I.; Jetter, J. W.; Quinet, E. M.; Berrodin, T. J.; Basso, M. D.; Feingold, I. B.; Nilsson, A. G.; Wilhelmsson, A.; Evans, M. J.; Wrobel, J. E. J. Med. Chem. 2010, 53, 3296−3304. (21) Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert, M. H.; Miller, A. B.; Moore, J.; McKee, D. D.; Moore, L.; Nichols, J.; Parks, D.; Watson, M.; Wisely, B.; Willson, T. M. J. Biol. Chem. 2003, 278, 27138−27143. (22) Cui, H.; Xu, B.; Wu, T. Z.; Xu, J.; Yuan, Y.; Gu, Q. J. Nat. Prod. 2014, 77, 100−110. (23) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision E.01; Gaussian, Inc.: Wallingford, CT, 2009. (24) Zeng, X. Y.; Zhou, X.; Xu, J.; Chan, S. M.; Xue, C. L.; Molero, J. C.; Ye, J. M. Biochem. Pharmacol. 2012, 84, 830−837. (25) Chen, Y. C.; Zeng, X. Y.; He, Y.; Liu, H.; Wang, B.; Zhou, H.; Chen, J. W.; Liu, P. Q.; Gu, L. Q.; Ye, J. M.; Huang, Z. S. ACS Chem. Biol. 2013, 8, 2301−2311. (26) Cronican, A. A.; Fitz, N. F.; Pham, T.; Fogg, A.; Kifer, B.; Koldamova, R.; Lefterov, I. Biochem. Pharmacol. 2010, 79, 1310−1316. (27) Fukuchi, J.; Song, C.; Ko, A. L.; Liao, S. Steroids 2003, 68, 685− 691.

(28) Halgren, T. A. J. Comput. Chem. 1996, 17, 490−519. (29) Salomon-Ferrer, R.; Götz, A. W.; Poole, D.; Le Grand, S.; Walker, R. C. J. Chem. Theory Comput. 2013, 9, 3878−3888. (30) Gotz, A. W.; Williamson, M. J.; Xu, D.; Poole, D.; Le Grand, S.; Walker, R. C. J. Chem. Theory Comput. 2012, 8, 1542−1555. (31) Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Swails, J.; Goetz, A. W.; Kolossváry, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wolf, R. M.; Liu, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M. J.; Cui, G.; Roe, D. R.; Mathews, D. H.; Seetin, M. G.; Salomon-Ferrer, R.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER 12; University of California: San Francisco, 2012. (32) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09; Gaussian Inc.: Wallingford, CT, 2009. (33) Mukherjee, G.; Patra, N.; Barua, P.; Jayaram, B. J. Comput. Chem. 2011, 32, 893−907. (34) Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Proteins: Struct., Funct., Genet. 2006, 65, 712−725. (35) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179−5197. (36) Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. J. Mol. Graphics Modell. 2006, 25, 247−260. (37) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977, 23, 327−341. (38) Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 13, 952− 962. (39) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089. (40) Hou, T.; Wang, J.; Li, Y.; Wang, W. J. Chem. Inf. Model. 2011, 51, 69−82.

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