Tetrahydrobenzofuran-6(2H)-one Neolignans from Ocotea

Article Cite This: J. Nat. Prod. 2018, 81, 1968−1975

pubs.acs.org/jnp

Tetrahydrobenzofuran-6(2H)‑one Neolignans from Ocotea heterochroma: Their Platelet Activating Factor (PAF) Antagonistic Activity and in Silico Insights into the PAF Receptor Binding Mode Catalina Rozo-Lugo,*,†,‡ Luis Enrique Cuca-Suárez,† Thomas J. Schmidt,§ and Ericsson Coy-Barrera*,‡

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†

Laboratorio de Investigación en Productos Naturales Vegetales, Departamento de Química, Universidad Nacional de Colombia, Ciudad Universitaria, 111321, Bogotá D.C., Colombia ‡ Laboratorio de Química Bioorgánica, Departamento de Química, Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, 250247, Cajicá, Colombia § Institut für Pharmazeutische Biologie und Phytochemie (IPBP), University of Münster, PharmaCampus, Corrensstraße 48, D-48149, Münster, Germany S Supporting Information *

ABSTRACT: Three new tetrahydrobenzofuran-6(2H)-one-type neolignans, heterochromins A−C (1−3), along with a bicyclo[3.2.1]octane neolignan, cinerin C (4), were isolated from an ethanol extract from the leaves of Ocotea heterochroma, a native plant growing in the Colombo−Ecuadorian region of the Andes. The chemical structures of 1−3 were elucidated by spectroscopic methods. The platelet activating factor (PAF) antagonistic activity was tested in vitro for these compounds. Additionally, their binding mode to the PAF receptor was studied by molecular docking and molecular dynamics simulations in order to rationalize such activity. Heterochromin A (1) was found to be a potent PAF antagonist with a favorable molecular profile for interacting with the PAF receptor binding site.

T

he genus Ocotea in Colombia is the largest taxon of the plant family Lauraceae. Despite other Ocotea species being located typically in low-altitude regions, an endemic plant from the Colombo−Ecuadorian Andes is O. heterochroma Mez & Sodiro, which is particularly interesting since it grows above 2600 m in altitude on the Bogota plateu. A previous study has led to the isolation of a friedelane triterpene, a diarylbutane lignan, a furofuran lignan, and a bicyclo[3.2.1]octane neolignan from an ethanol extract derived from the fruits of O. heterochroma.1 Benzofuran and bicyclo[3.2.1]octane-related neolignans have been widely reported from different Lauraceae species, particularly from the genus Ocotea, and they have a valuable structural diversity occurring in different parts of the plant.2−4 These metabolites have been demonstrated to exhibit platelet activating factor (PAF)antagonistic activity, a cellular factor responsible for platelet aggregation and thrombus formation, which are related to particular cardiovascular disorders as well as other pathologies involving inflammatory processes.5 Several compounds of © 2018 American Chemical Society and American Society of Pharmacognosy

natural origin have been evaluated as PAF antagonists through in vitro and in vivo models, and, among them, the benzofuranrelated structures (e.g., kadsurenone, 5) are the most widely studied neolignans.6 However, there is a lack of information related to the mode of action of this type of compound for interacting within the PAF receptor binding site. As part of our research on Lauraceous neolignans, we describe herein the isolation of three new tetrahydrobenzofuran-6(2H)-one-type neolignans (1−3), heterochromins A−C, along with a known bicyclo[3.2.1]octane neolignan (cinerin C, 4) from an ethanol extract obtained from the leaves of O. heterochroma by chromatographic methods. The structures of the new compounds were fully elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1 and 2 were determined by electronic circular dichroism (ECD) Received: March 2, 2018 Published: September 6, 2018 1968

DOI: 10.1021/acs.jnatprod.8b00189 J. Nat. Prod. 2018, 81, 1968−1975

Journal of Natural Products

Article

CHCl3-soluble fraction. This fraction was purified by conventional (VLC, flash CC, and preparative TLC) and instrumental (semipreparative HPLC) chromatographic techniques to isolate three new tetrahydrobenzofuran-6(2H)-one neolignans (1−3) as well as a known bicyclo[3.2.1]octane neolignan (cinerin C, 4). Compound 1 was isolated as a pale yellow oil. HRESIMS suggested a molecular formula of C23H30O7 with nine degrees of unsaturation (m/z 419.2077 [M + H]+; calcd for C23H31O7+, 419.2070). The presence of an α,β-unsaturated ketone was revealed by the IR absorption at 1647 cm−1. The 1 H NMR spectrum showed signals for 30 protons including two homotopic protons within a tetrasubstituted aromatic ring (δH 6.78, s, 2H, H-2,H-6), five methoxy groups [three attached to an aromatic ring (δH 3.86, s, 3H, OCH3−C-4; δH 3.85, s, 6H, OCH3−C-3,C-5), one at an olefinic carbon (δH 3.8, s, 3H, OCH3−C-3′), and one at an aliphatic quaternary carbon (δH 3.03, s, 3H, OCH3−C-5′)], an allyl substituent (δH 5.08, m, 2H, H-9′; δH 5.76, m, 1H, H-8′; δH 2.19, m, 1H, H-7′a, and δH 2.78, m, 1H, H-7′b), a methyl (δH 1.19, d, 3H, H-9), a methylene (δH 1.60, dd, 1H, H-6′a; δH 2.25, dd, 1H, H-6′b), and three methines (δH 5.13, d, 1H, H-7; δH 2.74, dq, 1H, H-8; δH 2.66, ddd, 1H, H-1′). The 13C NMR spectrum indicated the

measurements in combination with time-dependent density functional theory (TD-DFT) calculations. Molecular docking and molecular dynamics simulations were also used in order to describe their binding mode to the PAF receptor.

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RESULTS AND DISCUSSION Leaves of O. heterochroma were extracted with 96% EtOH by percolation. The resulting crude extract was fractionated by liquid−solid extraction in a Soxhlet apparatus, affording a Table 1. 1H and

13

C NMR Spectroscopic Data of Compounds 1−3 (CDCl3, 600 MHz)a 1

position

δC, type

1 2 3 4 5 6 7

135.8, C 102.9, CH 155.4, C 137.7, C 155.4, C 102.9, CH 94.3, CH

8 9 1′

43.8, CH 17.4, CH3 40.9, CH

2′ 3′ 4′ 5′ 6′

194.9, C 133.0, C 163.3, C 84.7, C 29.3, CH2

7′

34.3, CH2

8′ 9′

136.1, CH 117.4, CH2

OCH3-3 OCH3-4 OCH2O3,4 OCH3-4 OCH2O3,4 OCH3-5 OCH3-3′ OCH3-5′

56.3, CH3 61.0, CH3

61.0, CH3

56.3, CH3 60.4, CH3 50.9, CH3

2

δH (J in Hz)

HMBC

δC, type

6.61, s

1, 3, 4, 7

6.61, s 5.13, d (2.1)

1, 5, 4, 7 2, 9, 4′, 5′

134.9, C 99.9, CH 149.1, C 134.8, C 143.8, C 105.3, CH 93.9, CH

2.74, dq (7.4, 2.2) 1.19, d (7.5) 2.66, ddd (12.4, 8.4, 4.1)

1, 4′, 6′ 7, 5′ 3′, 5′, 8′

44.0, CH 17.2, CH3 40.9, CH

δC, type

6.55, d, (1.4)

1, 3, 4, 7

6.56, d (1.5) 5.11, d (1.6)

1, 5, 4, 7 2, 9, 4′, 5′

2.70, dq (4.8) 1.17, d (7.5) 2.65, ddd (12.8, 8.2, 3.8)

1, 8, 4′, 6′ 7, 5′ 3′, 5′, 8′

42.8, CH 11.4, CH3 40.8, C

1.59, dd (13.9, 12.8) (α) 2.23, dd (13.9, 4.3) (β) 2.20, m 2.78, m 5.75, m 5.04, m 5.09, m

8, 2′, 4′, 7′

194.8, C 133.2, C 161.5, C 85.8, C 29.0, CH2

9′, 6′

34.2, CH2

1′, 7′, 9′ 7′, 8′

136.1, CH 117.3, CH2

101.7, CH2

5.97, d (0.4)

3,4

101.7, CH2 56.8, CH3 60.3, CH3 50.9, CH3

5.97, d (0.4)

3,4

3.90, s 3.82, s 3.02, s

5 3′ 5′

1.60, dd (13.9, 12.4) (α) 2.25, dd (13.9, 4.3) (β) 2.78, m 2.19, m 5.76, m 5.08, m

8, 2′, 4′, 7′

9′, 6′

34.2, CH2

1′, 7′, 9′ 7′

136.1, CH 117.4, CH2

3.86, s 3.85, s

3 4

3.86, s 3.83, s 3.03, s

HMBC

134.9, C 100.3, CH 149.2, C 134.9, C 143.6, C 105.5, CH 93.9, CH

194.9, C 133.0, C 163.2, C 84.6, C 29.4, CH2

3.85, s

δH (J in Hz)

3 δH (J in Hz)

HMBC

6.47, d (0.7)

1, 3, 4, 7

6.48, d (0.6) 5.11, d (0.8) 2.70, m 0.57, d (7.4) 2.65, m

1, 5, 4, 7 7, 2, 9, 4′, 5′ 1, 8, 4′, 6′ 1, 7, 5′ 3′, 5′, 8′

1.66, dd (14.2, 12.4) (α) 2.29, dd (14.2, 4.3) (β) 2.15, m 2.81, m 5.80, m 5.08, m

8, 2′, 4′, 7′

9′, 6′

101.7, CH2

5.98, d (0.8)

3,4

101.7, CH2 56.9, CH3 60.3, CH3 52.3, CH3

5.98, d (0.8)

3,4

3.91, s 3.83, s 3.39, s

5 3′ 5′

7′

4

5 3′ 5′

a

Chemical shifts are given in ppm; assignments were confirmed by gCOSY and gHSQCad experiments. 1969



Article

3.00 vs 3.12), and C-5′ (δC 80.9 vs 77.6).7,9 The absolute configuration of 1 was then established by ECD measurements (Figure 2a) through analysis of the corresponding Cotton effects (CEs). According to the exciton chirality method,10 the ECD spectrum showed a positive exciton couplet (i.e., positive first CE at longer wavelength and corresponding negative CE at shorter wavelength), which corresponded clearly to a righthanded helical orientation of the two main chromophores, which would occur in stereoisomers R-configured at C-7 (Figure 2b). The experimental ECD spectrum of 1 was also compared with its theoretical spectrum simulated by a TDDFT calculation (Figure 2a). The experimental curve with intense negative and positive CEs at 275 and 310 nm, respectively, coincided very well with the simulated spectrum for 1, so that the assignment of the absolute configuration of 1 was unambiguous. With the C-7 absolute configuration in hand, the other centers were defined by the above-mentioned chiral relationships. Therefore, compound 1, heterochromin A, was designated as (7R,8S,5′R,1′S)-1′-allyl-3′,5′-dimethoxy-8methyl-7-(3,4,5-trimethoxyphenyl)-7,1′,5′,6′-tetrahydrobenzofuran-2′(2H)-one. The 1H and 13C NMR data of compound 2 (m/z 403.1768 [M + H]+; calcd for C22H27O7+, 403.1757) were found to be similar to those of compound 1 (Table 1), but involving a modification in the substitution pattern of the aromatic ring corresponding to a 5-methoxypiperonyl substituent [δH 5.97, d (0.4 Hz), 2H, OCH2O−C-3, C-4); δH 3.90, s, 3H, OCH3−C5)] instead of a 3,4,5-trimethoxyphenyl moiety. The other signals observed were comparable to those of compound 1 and supported the same chiral relationships (trans-C-7−C-8, pseudo-equatorial orientation of allyl group, and a β-OCH3 at C-5′). The ECD spectrum exhibited a similar positive exciton couplet to that of compound 1. Therefore, compound 2 (heterochromin B) was designated as (7R,8S,5′R,1′S)-1-allyl3′,5′-dimethoxy-7-(3,4-methylenedioxy-5-methoxy)-8-methyl7,1′,5′,6′-tetrahydrobenzofuran-2′(2H)-one. Compound 3 was found to be structurally quite similar to compound 2. However, the chemical shift and coupling constant of the methyl group at C-9 [δH 0.57, 3H, d (7.4 Hz), CH3-9] suggested a cis relationship between C-7 and C-8 instead of a trans relationship as in 1 and 2.7 In addition, the

presence of a ketone carbonyl group (δC 194.9), an aromatic ring (δC 135.8, 102.9, 155.4, 137.7, 155.4, 102.9), two oxygenated aliphatic carbons (δC 94.3, 84.7), a methyl group (δC 17.4), and four olefinic carbons (δC 163.3, 133, 136.1, 117.4); the last one was found to be the typical signal of a terminal methylene.7,8 All proton signals were assigned to their attached carbons by the gHSQCad experiment (Table 1). In addition, fragment assignments were established by 1H−1H gCOSY and gHMBCad correlations (Figure 1) such as H-2

Figure 1. Key HMBC correlations for compound 1.

and H-6 [δH 6.61 (2H, s)] to C-3, C-5 (δC 155.4), and C-7 (δC 94.3) for the aryl group connectivity; H3-9 [δH 1.19 (3H, d)] to C-5′ (δC 84.7) and C-6′ (δC 29.3); H-7 [δH 5.13, (1H, d)] to C-4′ (δC 137.7), C-5′ (δC 84.7), and C-9 (δC 17.4) for a tetrahydrobenzofuran-6(2H)-one moiety; H-6′a [δH 1.60 (1H, dd)] to C-7′ (δC 34.3) and C-2′(δC 194.9); and H-6′b [δH 2.25 (1H, dd)] to C-2′ (δC 194.9) and C-4′ (δC 137.7) for the allyl substituent connectivity. The above-mentioned spectroscopic data suggested that compound 1 has a similar structure to those of furocyclohexenone-related neolignans previously found in species of the plant family Lauraceae such as mirandin A (isolated from Nectandra miranda)7 and armenins A and B (isolated from Aniba sp.).8 A trans relationship between C-7 and C-8 was determined by the 1H NMR chemical shift of the methyl group [δH 1.19, 3H, d (7.5 Hz), CH3-9].8 The coupling constant between H-6′a [δH 1.60, 1H, dd (13.9, 12.4 Hz)] and H-1′ [δH 2.66, 1H, ddd (12.4, 8.4, 4.1 Hz)] indicated the allyl group to have a pseudo-equatorial orientation. A trans relationship between the methoxy group at C-5′ and the methyl group at C-8 was determined by 1H and 13C NMR chemical shifts in comparison to those reported for mirandin A (trans) and B (cis) for H-8 (δH 2.65 vs 2.16), OCH3−C-5′ (δH

Figure 2. (a) ECD spectrum of compound 1: experimental (blue line), TD-DFT simulated (red line). (b) Exciton chirality method applied to compound 1 for defining the absolute configuration at C-7. 1970



Article

Figure 3. Box-plot-based distribution of results obtained from molecular docking for the test compounds 1−5: (a) affinity and (b) RMSD values. GB = ginkgolide B. 1

H and 13C NMR chemical shifts for the methoxy group at C5′ [δH 3.39, 3H, s; δC 52.3 for 3 vs δH 3.02, 3H, s; δC 50.9 for 1] suggested a 1,3-trans relationship between the aryl−C-7 and the C-5′ OCH3 group.7 All other chiral relationships were found to be identical. The absolute configuration of this compound was not determined since suitable ECD measurements could not be recorded due to the limited amount isolated. Therefore, heterochromin C (3) was designated as rel-(7R,8R,5′R,1′S)-1-allyl-3′,5′-dimethoxy-7-(3,4-methylenedioxy-5-methoxy)-8-methyl-7,1′,5′,6′-tetrahydrobenzofuran2′(2H)-one. A previous study has described certain neolignans as promising PAF-antagonist agents.11 Data (as IC50 values in μM), compared to a positive control, ginkgolide B (IC50 0.93 μM), supporting kadsurenone (5) (having a dihydrobenzofuran-6(2H)-one moiety) as the most active neolignan (IC50 0.15 μM), even more potent than the positive control. Due to the close structural similarity between the new compounds and 5, the PAF-antagonistic activity was evaluated for compounds 1 and 2. Neolignan 3 could not be evaluated due to an insufficient amount being available. Compounds 1 and 2 exhibited IC50 values of 0.23 ± 0.02 and 0.39 ± 0.02 μM, respectively. The results indicated that compound 1 is more active than 2, probably by the influence of the trimethoxyphenyl moiety in 1. The other isolated neolignan, compound 4, having a bicyclo[3.2.1]octane moiety, was found to be less active (IC50 1.09 μM) than neolignans 1 and 2. Taking into account the above-mentioned considerations, ginkgolide B, cinerin C (4), and kadsurenone (5) were included into the present molecular docking calculations for comparing and validating the following computational results of compounds 1−3 in order to analyze/rationalize their potential as PAF antagonists. In order to assess the interaction of compounds 1−3, a molecular docking protocol was implemented using a homology model of the PAF receptor. This model was completed structurally using a reported procedure, according to the specifications of alignment and refinement already defined.12 AM1-based optimized structures were docked into the PAF receptor binding site, and affinity values of the best-docked poses were obtained from six replicates. In Figure 3a the scattering of these calculated affinity values is depicted. The results for 1−3 and 5 exhibited the highest reproducibility of the affinity calculations, indicating a good performance for these docking predictions. Compound 1 showed a similar affinity to that of ginkgolide B,

whereas 2 and 3 both showed a lesser affinity than that of this control substance. The substitution pattern in the aromatic ring was found to be an important structural feature in the affinity exhibited by these compounds at the PAF receptor binding site. Thus, the trimethoxyphenyl moiety (as in 1) showed a better affinity than the 5-methoxy-3,4-methylenedioxyphenyl moiety (as in 2 and 3). In addition, an important influence on the docking results was observed for the chiral relationship between C-7 and C-8 as observed in previous work,6,11 since 3 (cis relationship) could be predicted to have a lesser affinity than 2 (trans relationship), implicating a lower reproducibility. The structurally related kadsurenone (5) (a well-known PAF-antagonist substance) was found to have a similar affinity to 1 and ginkgolide B. Cinerin C (4) revealed a H-bond contact between OCH3−C-5′ and the HIS-249 residue (2.5 Å), exhibiting lower affinity and convergence. Root-mean-square deviations (RMSD in Å) among replicates of the test compounds (see Figure 3b) indicated that 1, 2, and 5 gave the best convergence. Thus, the bestdocked ligand pose inside the binding site was reproducible when the docking calculation was repeated several times, authenticating therefore the predictions for 1 and 2. Interactions within the binding site of the PAF receptor were analyzed for the best-docked compounds. In this context, the tetrahydrobenzofuran-6(2H)-one-type compounds 1 and 2 exhibited different interactions with the binding site. The binding mode for 1, 2, and 5 within the PAF receptor after docking was also analyzed by a two-dimensional (2D)residual interactions diagram. In Figure 4a and b are presented the π−π stacked and π-cation interactions between the trimethoxyphenyl moiety of 1 and the PHE98 and HIS248 residues, respectively. Also, the methyl group (CH3-9) exhibited a π-alkyl interaction with the HIS249 and PHE245 residues, and the C-1′-alyllic substituent showed nonpolar interactions with the VAL105, PHE241, and LEU197 residues. A lipophilic allyl or propyl side chain at the C-1′ position has been described as an important structural feature to contribute to PAF receptor binding.6 Compound 2 (see Figure 4c and d) displayed a similar interaction profile in comparison to that of 1, which might be expected, since these structurally related compounds are quite similar. However, the presence of the trimethoxyphenyl group in 1 seems to favor carbon−hydrogen bond interactions with the CYS173, PHE174, and GLY94 residues, involving a π-alkyl interaction with the PHE245 residue as well. These former interactions could explain the 1971



Article

Figure 4. Docked models (3D and 2D) of PAF receptor complexes with compounds 1, 2, and 5. Close-up view of the binding site of PAF receptor (as green cartoon) showing selected interacting residues (as gray sticks) with docked compounds (as gray sticks): (a) heterochromin A (1), (c) heterochromin B (2), (e) kadsurenone (5); 2D-residual interaction diagrams of (b) heterochromin A (1), (d) heterochromin B (2), and (f) kadsurenone (5). Interacting residues are shown as colored circles depending on the interactions (as colored dashed lines). (g) Interaction types according to the 2D-residual interaction diagrams after docking simulations.

higher affinity of 1 in comparison to that of 2. On observing the interaction profile of the PAF-antagonist kadsurenone (5) (Figure 4e and f), PHE98, HIS248, PHE245, HIS249, CYS173, and PHE174 were found to be common contact residues between 5 and 1; in fact, the methoxy group at position C-4 in 1 indicated the same interactions as that of 5, demonstrating the importance of methoxy groups in the aryl

substitution pattern. Other strong interactions were found to be present in the 1:PAF receptor complex, so these details could rationalize the best affinity observed for compound 1, which is in agreement with its good PAF-antagonistic activity. Ligand:protein stabilities for the complexes between 1 and 2 and the PAF receptor were also analyzed through molecular dynamics, by monitoring time-dependent geometric properties. 1972



Article

Figure 5. Results after MD simulations of the best docked poses of compounds 1 and 2 within the PAF receptor binding site. (a) RMS deviations of the Cα atoms of the whole protein: apo-PAF receptor (blue line), 1:PAF receptor (red line), and 2:PAF receptor (green line) systems during the 5000 ps MD simulations. (b) RMS fluctuations of the residue of the whole protein: apo-PAF receptor (blue line), 1:PAF receptor (red line), and 2:PAF receptor (green line) complexes by MD simulations. (c) Radius of gyration (RoG) of the Cα atoms of the whole protein: apo-PAF receptor (blue line), 1:PAF receptor (red line), and 2:PAF receptor (green line) systems during the 5000 ps MD simulations.

generate a decrease in flexibility. Other residues within helix Vbordering regions (i.e., 180−190 and 228−242) also showed differences between both complexes and the apo-PAF receptor. Since these residues are located close to some binding site residues, this fluctuation could be explained by a receptor reaccommodation effect, acting as pivot regions, promoted by the interactions with 1 and 2. Nevertheless, the evolution of PAF receptor packing level occurs during the simulation time, evaluated though radius of gyration (RoG) (Figure 5c), since the 2:PAF receptor complex was found to be similar to that of the apo-PAF receptor. Thus, this interaction does not affect the receptor packing, but, in contrast, the 1:PAF receptor complex generated a greater receptor deformation that would lead to an inactivation of the PAF receptor and, thereby, for example, promote a platelet aggregation inhibition. In summary, results suggested that compound 1 might inactivate the PAF receptor particularly by the presence of the trimethoxyphenyl moiety, the methyl group at C-9, and the allyl group at C-1′, through π−π stacked, π-cation, π-alkyl, and nonpolar interactions with PHE98, HIS248, PHE245, PHE241, and HIS249 residues. These structural features should therefore be retained in future studies to develop further PAF antagonists based on tetrahydrobenzofuranonelike compounds.

RMSD values were used to evaluate the structural stability of the receptor frame by measuring the distance between different positions (in Å) that a set of atoms exhibited through time. Therefore, after independent molecular dynamics simulations for complexes of this receptor with 1 and 2 as ligands for 5 ns, the receptor underwent several shifts upon binding to compounds 1 and 2 promoted by a reasonable perturbation of its conformations (Figure 5a). However, the ligand−enzyme complexes exhibited a similar evolution in the conformational changes, but their RMSD values were found to be higher than in the apo-PAF receptor. During tracking of the dynamic movements, both complexes reached stability after about 2000 ps, but the 1:PAF receptor complex exhibited an almost constant evolution along the simulation time. This behavior was found to be different from that of the 2:PAF receptor complex, indicating that its structural stability seems to be a two-step process. The first one is a stabilizing state between 850 and 1650 ps (RMSD value ca. 0.8 Å) prior to a final state at 2000 ps (RMSD value ca. 1.4 Å) to remain almost unchanged.13 This final conformationally stabilized state exhibited different RMSD values for the 1:PAF receptor and 2:PAF receptor complexes (1.2 vs 1.4 Å, respectively), indicating that 1 exerted more stability than 2 on interacting at this receptor. In order to analyze the fluctuations of the atomic positions in each residue of the receptor, the RMSF (root mean square fluctuation) values were also considered to examine the flexibility and secondary structure of the PAF receptor under interaction with the ligands.14,15 The 1:PAF receptor and 2:PAF receptor complexes exhibited similar behavior (see Figure 5b) in almost whole PAF receptor sequence, but they showed explicit differences in some regions, such as the 39−63 and 70−94 residues, corresponding to the first intra- (I1) and extracellular (E1) loops (β-twists) of the PAF receptor, respectively. In addition, binding site residues showed slight differences in the RMSF values (i.e., HIS188, PHE245, and GLN252), but the RMSF values of the 1:PAF receptor complex generally exhibited lower values in comparison to those of the 2:PAF receptor complex and apo-PAF receptor. This behavior explains the better affinity observed in the docking results for the 1:PAF receptor complex since it would

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded using a Jasco P-2000ST digital polarimeter. UV and ECD spectra were acquired on a Thermo Fisher Scientific Genesys 10S spectrophotometer and a Jasco J-710 spectrometer, respectively. The IR spectra were recorded on a Thermo Nicolet 6700 FT-IR spectrometer. 1H and 13C NMR as well as one-dimensional (DEPT 135) and two-dimensional (1H−1H gCOSY, gHMQCad, and gHMBCad) experiments were recorded in an Agilent DD2 600 MHz spectrometer using CDCl3 as solvent. All shifts are given in δ (ppm) using the signal of tetramethylsilane as reference. All coupling constants (J) are given in Hz. ESIMS and HRESIMS data were obtained on a Shimadzu LCMS2020 single quadrupole mass spectrometer and a Bruker micro-QToF II spectrometer, respectively. A Shimadzu Prominence reversed-phase HPLC equipped with a Shimadzu Premier C18 column (100 mm × 4.6 mm, 5 μm) with a diode-array detector coupled to the single quadrupole mass spectrometer was used. Trifluoroacetic acid (TFA)/Milli-Q 0.005% 1973



Article

H2O (A) and CH3CN (B) were used as mobile phases. Mobile phases for semipreparative RP-HPLC were MeOH (A) and TFA 0.005% (B) in isocratic elution (A:B, 70:30) using a Luna C18 (150 mm × 10 mm, 5 μm) column monitored at 270 nm. Thin-layer chromatography (TLC) using silica gel 60 F254 TLC plates (Merck) and mobile phases comprising solvent mixtures of petroleum ether, n-hexane, toluene, EtOAc, i-PrOAc, Me2CO, MeOH, and HCOOH were used. Plates after TLC development were observed under UV light (254 and 365 nm) and derivatized using I2 vapor and Hannessian’s reagent (aqueous solution of ammonium molybdate, cerium sulfate, and H2SO4). Silica gel 60 (0.063−0.200 mm) (Merck), silica gel 60 (0.04−0.063 mm) (Merck), and silica gel 60 HF254 (Merck) were used for column chromatography (CC), flash chromatography (flash CC), and vacuum liquid chromatography (VLC), respectively. Plant Material. Ocotea heterochroma was collected in Colombia along the Cota-Tabio road in July 2010, and the species was classified by the biologist Adolfo Jara. A voucher specimen has been deposited at Herbario Nacional Colombiano (COL) under collection number COL 563467. Extraction and Isolation. A crude extract (185 g) of O. heterochroma leaves was obtained from 2140 g of dry leaves by percolation using 96% ethanol. A portion (80 g) of ethanol extract was fractionated through a solid−liquid extraction in a Soxhlet apparatus using separately petroleum ether, CHCl3, AcOEt, and MeOH until exhaustive extraction. The resulting CHCl3-soluble fraction was further fractioned by VLC using petroleum ether/EtOAc mixtures in gradient elution, obtaining 21 fractions (F1 to F21). Fraction F7 (324 mg) was fractionated by flash CC using petroleum ether/EtOAc (9:1) as mobile phase, affording four fractions. Fraction F7.3 (92.9 mg) was purified using flash CC and preparative TLC, yielding 1 (17.6 mg) and 4 (7.8 mg). Fraction F4 (35.3 mg) was separated using flash CC with toluene/i-PrOAc/MeOH (9:1:0.4) as mobile phase to obtain three fractions. From fraction F4.2 (14.3 mg), using CC and toluene/i-PrOAc/MeOH (95:5:0.4) for elution, was obtained fraction F4.2.2 (8 mg), which was further separated by semipreparative RP-HPLC into 2 (3.1 mg) and 3 (0.5 mg). Heterochromin A (1): light yellow liquid; [α]25D +35 (c 0.06, CHCl3); UV λmax (MeOH) (log ε) 207 (4.65), 265 (4.28) nm; ECD λmax (c 0.02, MeOH) nm (Δε) 275 (−7.70), 286 (0), 310 (+13.6); IR νmax (film) 2934 (CH), 1647 (CO) cm−1; 1H (600 MHz) and 13C (125 MHz) NMR, see Table 1; ESIMS (positive mode) m/z 419 [M + H]+; HRESIMS positive mode) m/z 419.2077 [M + H]+ (calcd for C23H31O7, 419.2070). Heterochromin B (2): light yellow liquid; [α]25D +41 (c 0.03, CHCl3); UV λmax (MeOH) (log ε) 209 (4.89), 262 (4.33) nm; ECD λmax (c 0.01, MeOH) nm (Δε) 265 (−4.31), 289 (0), 306 (+5.72); IR νmax (film) 2948 (CH), 1658 (CO) cm−1; 1H (600 MHz) and 13C (125 MHz) NMR, see Table 1; ESIMS (positive mode) m/z 403 [M + H]+; HRESIMS (positive mode) m/z 403.1768 [M + H]+ (calcd for C22H27O7, 403.1757). Heterochromin C (3): light yellow liquid; [α]25D +12 (c 0.01, CHCl3); UV λmax (MeOH) (log ε) 210 (4.25), 275 (3.78) nm; 1H (600 MHz) and 13C (125 MHz) NMR, see Table 1; ESIMS (positive mode) m/z 403 [M + H]+; HRESIMS (positive mode) m/z 403.1764 [M + H]+ (calcd for C22H27O7, 403.1757). Time-Dependent Density Functional Theory Calculations for Compound 1. The ECD spectrum of compound 1 was recorded and related to its theoretical spectrum simulated by TD-DFT calculations. To this end, a three-dimensional (3D) molecular model of 1 was optimized geometrically using the MMFF94X force field. Compound 1 was then subjected to a conformational search using the low mode molecular dynamics method and the MMFF94X force field, as implemented in MOE. After energy minimization of the resulting six low-energy conformers with Gaussian W03 using the B3LYP density functional and a 6-31G(d,p) basis set, the populations of the resulting conformers were calculated using their energy differences and the Boltzmann equation. They were thus estimated to represent 95% of the overall conformational equilibrium. TD-DFT calculations were then performed for the first two conformers using the same density functional and basis set as above and considering the

21 lowest-energy electronic transitions for each conformer. The resulting rotatory strength vectors were converted into a simulated ECD spectrum for each molecule species as described previously.16 The individual spectra were averaged according to the obtained equilibrium percentages, which resulted in the simulated spectrum shown in Figure 2. Molecular Docking. A conformational random search using the Merck Molecular Force Field (MMFF), without geometric restrictions, included in SPARTAN software (Spartan 14v114 (2013) Wavefunction, Inc., Irvine, CA, USA), was performed with a 500-conformers limit. Energetically most-stable conformers were MMFF-optimized to be used into the docking protocol. Docking calculations were performed with the Autodock/Vina (1.1.2) plug-in for PyMOL (1.3r2) under a Python 2.5.2 environment for Windows.17 The structure of the PAF receptor was modeled homologically from the G-protein (GPCR) imported from the Protein Data Bank RCSB (PDB code: 1L9H),18 following the same reported procedure, according to the specifications of alignment and refinement already stated.12 Docking calculations were then performed between the minimized ligand through a cube (dimensions 24 × 24 × 24 Å, grid spacing 0.375 Å) located in the geometric center (coordinates 60.3, 12.3, 22.0) of the reported binding pocket present in the PAF receptor. Residues HIS248 and HIS249 (in helix VI) and HIS188 (in helix V) were involved in this binding site for the phosphocholine moiety of PAF.12,19 Docking poses were classified according to their docking score (such as the free energy or affinity). Each calculation was performed in six replicates. Ginkgolide B was used as control. 2D-Residual interaction diagrams were visualized on Discovery Studio 2016 Visualizer Client (Biovia, San Diego, CA, USA). Molecular Dynamics Simulations. Molecular dynamics (MD) simulations were run in Gromacs 5.0.5 on a Ubuntu 12.04 server. The ligand best pose from docking and a crystallized structure were employed as input for dynamics simulations of complexes and standalone proteins, respectively. Ligands were prepared by adding hydrogen atoms and the corresponding charges using the AM1BCC charge scheme in UCSF Chimera. Subsequently, ligand topologies were generated automatically with ACPYPE script. Protein topologies were obtained in Gromacs using the Amber 99SB force field, and the TIP3P water model was implemented. Solvation was performed in a triclinic box using a margin distance of 1.0 nm. Addition of 0.1 M NaCl to complexes and proteins was carried out by randomly replacing water molecules until neutrality was achieved. The systems were energy-minimized by 2000 steps of the steepest descent method. NVT equilibration at 310 K for 50 ps, followed by NPT equilibration during 500 ps using the Parrinello−Rahman method at 1 bar as reference, was conducted on systems using position restraints. Finally, solute position restraints were released and a production run for 5 ns was performed. The temperature and pressure were kept constant at 310 K and 1 bar, respectively. Coordinates were recorded in a 1 fs time step. Electrostatic forces were calculated using the particle-mesh Ewald method. Periodic boundary conditions were used in all simulations, and covalent bond lengths were constrained by the LINCS algorithm. Inhibition of PAF-Induced Aggregation Assay. Compounds 1 and 2 were evaluated in vitro for their anti-PAF activity following the previously reported procedure to assess PAF antagonism.20

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00189. 1

H NMR and 13C NMR spectra of new compounds 1−3

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Article

AUTHOR INFORMATION

Corresponding Authors

*Tel: +57 313 211 6935. E-mail: [email protected]. co (C. Rozo-Lugo). *Tel: +57 1 6500000 x 3270. E-mail: ericsson.coy@unimilitar. edu.co (E. Coy-Barrera). ORCID

Ericsson Coy-Barrera: 0000-0002-3553-9749 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Universidad Nacional de Colombia and Universidad Militar Nueva Granada for the financial support. Part of this work was performed in collaboration within the Research Network Natural Products against Neglected Diseases (ResNet NPND, www.resnentnpnd.org).

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REFERENCES

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Tetrahydrobenzofuran-6(2H)-one Neolignans from Ocotea

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