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Molecules 2014, 19(4), 4695-4707; doi:10.3390/molecules19044695
Abstract: (‒)-Encecanescin (1) has been isolated from the leaves of Eupatorium aschembornianum. Two conformers are present in the crystal structure as a result of molecular disorder. The structure of 1 was established by 1H- and 13C-NMR spectroscopy in CDCl3 solution using 2D NMR techniques (gHSQC, gHMBC and NOESY). A Monte Carlo random search using molecular mechanics followed by the geometry optimization of each minimum energy structure using density functional theory (DFT) calculations at the B3LYP/6–31G* level and a Boltzmann analysis of the total energies generated accurate molecular models describing the conformational behavior of 1. The three most stable conformers 2–4 of compound 1 were reoptimized at the B3LYP/6-311++G(d,p) level of theory using CHCl3 as a solvent. Correlations between the experimental 1H- and 13C-NMR chemical shifts (δexp) have been found, and the GIAO/B3LYP/6-311++G(d,p) calculated magnetic isotropic shielding tensors (σcalc) for conformers 2 and 3, δexp = a + b σcalc, are reported. A good linear relationship between the experimental and calculated NMR data has been obtained for protons and carbon atoms.
Chromene (2H-1-benzopyran) ring derivatives are often found in natural heterocycles, and some have interesting biological activities . These compounds make up a new family of activators of potassium channels that are useful in the treatment of respiratory diseases as tracheal tissue relaxing agents . One such benzopyran derivative isolated from Ageratina asenii  is (+)-encecanescin, a dimeric chromene with a structure similar to a previously reported compound . Surprisingly, however, the authors found that the available encecanescin crystallized as a racemic mixture.
Later, crystals of (±)-encecanescin were analyzed by X-ray diffraction , revealing that it racemized during crystallization using 1:1 EtOAc/cyclohexane. In addition, it was possible to observe two molecules in the asymmetric unit that differ in geometry around C2. The rings attached to C2 exhibit a half-boat conformation, but differ in the orientation of the gem-dimethyl group present at C2. Our group recently obtained a white solid from Eupatorium aschembornianum by recrystallization using 95:5 hexane/EtOAc , which provided (‒)-encecanescin (1) for the first time as a single crystal, with the structure shown in Figure 1 . However, the molecular crystal also exhibited disorder in the X-ray structure. In the present work, this disorder is explained in terms of two conformers in the solid state, and it is shown that this disorder can be deduced from quantum mechanical calculations.
2. Results and Discussion
(‒)-Encecanescin (1) was isolated using a previously reported protocol . The dimeric structure of compound 1 was confirmed from the high-resolution mass spectra (MS-FAB+), which exhibited a peak at 450.2402 for C28H34O5. The compound was identified using NMR analysis by comparing the results with those previously reported [3,4], with the exception of the resonances for C-3, C-3'; C-4, C-4'; and C-5, C-5', which were reassigned in this study to 127.3, 122.7 and 124.0, respectively, based on gHMBC, gHSQC and NOESY spectra.
2.1. X-ray Crystallography
Crystals of 1 were grown from a 95:5 mixture of n-hexane with ethyl acetate. A crystal cut to the dimensions 0.24 × 0.20 × 0.18 mm was used for X-ray measurements at 293 K using an Enraf-Nonius KappaCCD diffractometer with graphite-monochromated λMo-Ka = 0.71073 Å. The structure was solved by direct methods and refined by full-matrix least-square calculations based on F2. Crystallographic calculations were performed using SHELXL-97 . The details of the crystal structure determinations and refinements are presented in Table 1. The best results were obtained for the disordered model in the crystal, and two conformers (1a, 1b) are found (Figure 1). The rings containing C2 in 1a and 1b show a half-boat conformation but differ in the orientation of the flag atom C2.
|Crystal system, space group||monoclinic, P21/c|
|Unit cell dimensions||a = 11.0930(2) Å alpha = 90°|
|b = 8.4352(2) Å beta = 94.622(11)°|
|c = 27.5559(11) Å gamma = 90°|
|ZCalculated density||40.851 Mg/m3|
|Absorption coefficient||0.061 mm−1|
|Crystal size||0.24 × 0.20 × 0.18 mm|
|Theta range for data collection||2.46° to 27.49°|
|Limiting indices||−14 ≤ h ≤ 13, −10 ≤ k ≤ 10, −35 ≤ l ≤ 28|
|Reflections collected/ unique||14836/5814 [R(int) = 0.0892]|
|Completeness to theta = 27.49||98.7%|
|Refinement method||Full-matrix least-squares on F2|
|Goodness-of-fit on F2||0.918|
|Final R indices||[I > 2sigma(I)] R1 = 0.0671, wR2 = 0.1293|
|R indices (all data)||R1 = 0.2389, wR2 = 0.1772|
|Largest diff. peak and hole||0.160 and −0.166 e.A−3|
2.2. B3LYP Calculations
The theoretical conformational distribution of 1 was obtained by a Monte Carlo random search. A total of 10 minimum energy structures were found within a molecular mechanics energy range of 10 kcal·mol−1. All of these structures were subjected to geometry and energy optimization by density functional theory (DFT) calculations employing the B3LYP/6–31G* basis set. According to these calculations, the original group of 10 structures was reduced to a group of three (within a 0–3 kcal·mol−1 range), as seven conformers appeared as duplicates. These three structures were submitted to geometry reoptimization using DFT calculations at the B3LYP/6-311++G(d,p) level of theory in a CHCl3 solution. Figure 2 shows the total DFT energy in solution, the relative energy and the conformational population of the three optimized conformers of 1 (2, 3 and 4), which account for 99.99% of the conformational population according to the DFT total energy values. Geometry optimizations included a frequency calculation to verify that an energy minimum had been reached. Given that conformer 4 has a relative energy of 2.263 kcal·mol−1, its contribution to the equilibrium (ca. 1.1%) can be neglected.
The selected calculated B3LYP bond lengths, bond angles and torsion angles are given in Table 2. Most of the calculated bonds are slightly longer than the experimental ones, except C(10)-C(5), C(10)-C(9), C(2)-O(1) and C(9)-C(8), which are shorter. The calculated bond angles agree with the experimental values within 1.3°, excluding angles within the rings containing C2, ranging from 5.2° to 35.6°. The largest differences between the X-ray and B3LYP data are in the torsion angles, which vary from 2.5° to 69.6°. The B3LYP calculations accurately reproduce the signs of the torsion angles.
2.3. FTIR and Raman Spectra
The observed and calculated harmonic frequencies of the two conformers (2 and 3) of (‒)-encecanescin (1) and their tentative assignments are presented in Table 3. A comparison of the calculated and experimental frequencies reveals important differences. Two factors may be responsible for the disagreements between the experimental and computed spectra of the studied structures. The first is that the experimental spectrum was recorded for the molecule in the solid state, while the computed spectra correspond to isolated molecules in CHCl3 solution. The second is the fact that the experimental values correspond to anharmonic vibrations, while the calculated values correspond to harmonic vibrations. The overestimation of the computed wavenumbers is quite systematic, and a scaling procedure was used to obtain the predicted frequencies [8,9].
|3043.66||3041 w||3177||15.7||3176||10.4||υC-H Ar|
|2983.39||2974 m||3131||17.8||3131||35.9||υas CH3 methoxy|
|2938.11||2932 w||3106||24.7||3106||23.8||υas CH3 gem|
|1645.95||1644 vw||1651||298.0||1651||3.2||υs HC=CH ring|
|1621.54||1615 m||1599||31.4||1600||26||υs HC=CH ring|
|1578.38||1576 s||1523||155.4||1524||181.6||βC-H Ar+CH3|
|1499.45||1492 w||1493||18.7||1493||23.3||γCH3 methoxy|
|1462 vw||1479||1.8||1479||2.4||ωCH3 methoxy|
|1432.21||1443 vw||1457||2.4||1456||1.1||βring, Ar-O-R|
|1380 m||1382||52.6||1383||49.3||ρHC=CH ring/ωAr-C-H-OCH3|
|1307.86||1303 s||1303||154.2||1303||159.1||υas HC=CH Ar|
|1279 m||1284||35.4||1285||27.8||βAr, ring|
|1239.18||1230 m||1231||33.3||1232||40.8||βHC=CH ring/υas CH3 gem|
|1196 s||1214||158.7||1214||167.4||ρCH3 methoxy, CH3 gem|
|1173.11||1163 m||1178||58.3||1180||89.3||βHC=CH ring, C-H Ar|
|1074 vs||1087||218.5||1087||224.4||ρCH3/υas C-O-C|
|950.96||959 m||967||28.5||967||43.7||υsHC-C(CH3)2-O, CH3-CH-O|
|884.95||894 m||902||55.6||902||55.5||τCH3 gem|
|804.36||801 vw||809||3.1||809||1.1||τAr-O-R, ring|
The abbreviations used are: vs, very strong; s, strong; m, medium; w, weak; vw, very weak; υ, stretching (s, symmetric; as, asymmetric); β, in-plane bending; γ, out-of-plane bending; ω, wagging; ρ, rocking; τ, twisting.
2.4. 1H- and 13C-NMR Spectra
The 1H- and 13C-NMR signals of 1 were assigned based on the observed gHSQC, gHMBC and NOESY correlations in CDCl3  and are listed in Table 4 and Table 5. The gHSQC spectrum (Figure 3) shows cross peaks between the resonances of 1H and those of the 13C atoms to which the protons are attached. The horizontal axis corresponds to the 1H spectrum and the vertical one to the 13C spectrum. On the other hand, in the HMBC spectrum, correlations between the protons or carbons through two and three bonds are observed (Figure 3).
|Atom||δexp||δpred (2)||δpred (3)||σcalc (2)||σcalc (3)|
Starting from the characteristic resonance of the H-15 methoxyl proton (δ 3.67), it was possible to assign the resonance of the sp2 carbon C-7 (δ 157.7) based on its gHMBC correlation with H-15. On the other hand, the signal at δ 6.32 was assigned to H-8 due to its cross peak with CH3O (δ 3.67) in the NOESY plot. Analogously, the methine proton resonating at δ 4.59 produces cross peaks with carbons C-5 (δ 124.0) and C-6 (δ 125.1), thus revealing its position on C-11. The resonance of methyl protons C(12)H3 at δ 1.46 is coupled through two bonds to the carbon C-11. Furthermore, C-7 correlates in the gHMBC through two and three bonds with the protons at δ 6.32 and δ 7.10; therefore, these signals must be assigned to the protons H-8 and H-5, respectively. Similarly, relevant cross peaks were observed for proton H-5 at δ 7.10 through three bonds with the carbons C-4 at δ 122.7 and C-11 at δ 68.6, confirming its assignment to H-5. In addition, the signal at δ 152.8 correlates in the gHMBC through three bonds with the protons H-5 at δ 7.10 and H-4 at δ 6.34; therefore, this signal must be assigned to carbon C-9. The resonances of the methyl protons C(13)H3 and C(14)H3 at δ 1.46 and δ 1.43 are coupled through three bonds to carbon C-3 at δ 127.3. Thus, the signal at δ 5.46 was assigned to H-3 based on its cross peak with C(13)H3 and C(14)H3 and H-4 at δ 6.34 in the NOESY plot. The signal at δ 113.8 correlates in the gHMBC through three bonds to the carbon with the protons H-8 and H-3; therefore, this signal must be assigned to carbon C-10. Accordingly, the remaining carbons C-8 and C-2, resonating at δ 99.2 and δ 78.3, respectively, were assigned based on the gHSQC correlations.
|Atom||δexp||δpred (2)||δpred (3)||σcalc (2)||σcalc (3)|
The relationship between the experimental 13C and 1H chemical shifts (δexp) and the GIAO (gauge-independent atomic orbitals) magnetic isotropic shielding constants (σcalc) calculated for conformers 2 and 3 in CHCl3 are generally linear and are well described by the equation δexp = a + b·σcalc . The slope and intercept of the least-squares correlation line (Figure 4a,b, Table 4 and Table 5) are utilized to scale the GIAO magnetic isotropic shielding constants, σcalc, and to predict the chemical shifts, δpred = a + b·σcalc. The correlations between the experimental chemical shifts and calculated magnetic isotropic shielding constants are generally better for carbon-13 atoms than for protons; however, in this case, the correlations are good for both carbons and protons. This finding can be explained by the absence of hydrogen bonds and other strong interactions that mainly affect outer H atoms. The magnetic isotropic shielding constants confirm the correct assignments of the chemical shifts to the aforementioned atoms.
The infrared spectrum was recorded on a Varian FT-IR spectrometer (Palo Alto city, CA, USA). The Raman spectrum of a crystalline sample was measured using a Thermo Scientific DXR Raman microscope (Madison, WI, USA) equipped with a 532 nm laser with a power of 10 mW and an exposure time of 104 s. High-resolution mass spectroscopy was performed using a JEOL spectrometer (model 102 ASX, Jeol).
Diffraction data were measured using an Enraf-Nonius KappaCCD diffractometer (Nonius, Delft, The Netherlands) with graphite-monochromated λMo-Kα = 0.71073 Å. Frames were collected at T = 293 K ω/φ rotation. The direct methods SHELXS-86 and SIR-2004 were used to solve the structure, and the SHELXL-97 program package was used for refinement and data output. CCDC 996389 for (−)-encecanescin (1) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; [email@example.com]).
The 13C- and 1H-NMR spectra were recorded on an Agilent 400 MR DD2 spectrometer (Santa Clara, CA, USA) operating at 100 MHz for 13C and 400 MHz for 1H. The 13C and 1H chemical shifts were measured in CDCl3 relative to TMS as an internal standard. Typical conditions for the proton spectra were as follows: pulse width of 45°, acquisition time of 2.5 s, FT size of 32 K and digital resolution of 0.3 Hz per point. Typical conditions for the carbon spectra were as follows: pulse width of 45°, FT size of 65 K and digital resolution of 0.5 Hz per point. The number of scans varied from 1200 to 10,000 per spectrum. All proton and carbon-13 resonances were assigned by 1H (NOESY) and 13C (gHSQC, gHMBC), respectively. All 2D NMR spectra were recorded at 298 K on the Agilent 400 MR DD2 spectrometer operating at 100 MHz (13C) and 400 MHz (1H), with a FT size of 2 × 2 K and a digital resolution of 0.3 Hz per point.
Eupatorium aschembornianum leaves were collected in San Juan Tlacotenco, Tepoztlan, Morelos State, México, during August 2007. A specimen from the original collection can be found in “Jorge Espinosa Herbarium-Hortorio” in the Biology Area of Chapingo Autonomous University, with voucher number 1835.
Hexane extract (40 g) from the leaves of E. aschembornianum were chromatographed over silica gel (250 g) with increasing solvent polarity, starting with hexane and increasing the polarity with ethyl acetate. Fractions 17-40 eluted with hexane/EtOAc (95:5) provided a white solid (2.3 g, mp = 148−150 °C) identified as (‒)-encecanescin (1), [α]D 25° (CHCl3, c2.21 g/100 mL): 589 (−0.4), 578 (−0.4), 546 (−0.5), 436 (−0.8), 365 (−1.2). MS-FAB+: observed 450.2402, calculated 450.2406 for C28H34O5. IR (CHCl3): υmax = 3010, 1640, 1385, 1140 cm−1. 1H-NMR (CDCl3): δ = 7.10 (s, H-5, H-5'), 6.34 (d, J = 9.3 Hz, H-4, H-4'), 6.32 (s, H-8, H-8'), 5.46 (d, J = 9.3 Hz, H-3, H-3'), 4.59 (q, J = 6.3 Hz, H-11, H-11'), 3.67 (s, H-15, H-15'), 1.46 (s, H-13, H-13'), 1.43 (s, H-14, H-14'), 1.31 (d, J = 6.3 Hz, H-12, H-12'). The 13C-NMR data (Table 4) correspond to those published for encecanescin , with the exception of the resonances for C-3, C-3'; C-4, C-4'; and C-5, C-5', which were reassigned in this study to 127.3, 122.7 and 124.0, respectively.
3.4. Computational Calculations
The conformational search for 1 was carried out using the Monte Carlo protocol  with the MMFF94 force field as implemented in the Spartan 08 program (Wavefunction, Inc., Irvine, CA, USA). The DFT calculations at the B3LYP/6-31G(d) level of theory [13,14], followed by reoptimization at the B3LYP/6-311++G(d,p)  level using the SMD solvent model , were performed using the Gaussian 09 package . The NMR isotropic magnetic shielding tensors were calculated using the standard gauge-independent atomic orbital (GIAO) approach [11,18] in Gaussian 09.
(−)-Encecanescin (1) has been isolated from leaves of Eupatorium aschembornianum. The structure of 1 was established by X-ray diffraction and characterized by FTIR, Raman and NMR spectroscopy and DFT calculations. The X-ray analysis showed that the molecule is non-planar and is present as a mixture of two conformers in the crystal (2 and 3). Molecular modeling of 1 using the Monte Carlo protocol followed by geometry optimization at the B3LYP 6-31G(d,p) level of theory and a Boltzmann analysis of the total energies confirmed that 2 and 3 are the two most stable conformers of 1. Good correlations between the experimental 1H and 13C chemical shifts in CHCl3 and the GIAO/B3LYP/6-311++G(d,p) calculated magnetic isotropic shielding tensors for both conformers (δexp = a + b·σcalc) confirmed the geometry of 1.
Copies of the room-temperature solid-state FTIR spectra, Raman spectra and calculated vibrational spectra in CHCl3 solution of two conformers (2 and 3) of (−)-encecanescin (1) and a magnification of the gHSQC spectrum of (−)-(1) at 400 MHz in CDCl3 are available. Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/4/4695/s1.
The authors wish to thank the Dirección General de Cómputo y de Tecnologías de Información y Comunicación (DGTIC) at Universidad Nacional Autónoma de México. We are grateful to Marco A. Leyva Ramírez from Departamento de Química, CINVESTAV, Mexico, for technical assistance with the determination of the X-ray diffraction of encecanescin. We thank María C. Zorrilla Cangas from Instituto de Física, UNAM, and Nérida Cuautle Hernández for technical assistance.
Benito Reyes-Trejo participated in design and coordination of the study. Diana Guerra-Ramírez and Holber Zuleta-Prada were responsible for the physical data collection and NMR data acquisition. Rosa Santillán participated in the X-ray data collection and initial refinement. María Elena Sánchez-Mendoza and Jesús Arrieta carried out the isolation and purification of (−)-encecanescin (1). Lino Reyes participated in the design of the theoretical calculations and in preparation of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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