Molecules 2014, 19(4), 4695-4707; doi:10.3390/molecules19044695

Article
Molecular Disorder in (‒)-Encecanescin
Benito Reyes-Trejo 1,*, Diana Guerra-Ramírez 1, Holber Zuleta-Prada 1, Rosa Santillán 2, María Elena Sánchez-Mendoza 3, Jesús Arrieta 3 and Lino Reyes 4,*
1
Laboratorio de Productos Naturales, Área de Química, Departamento de Preparatoria Agrícola AP 74 Oficina de Correos Chapingo, Universidad Autónoma Chapingo, Km. 38.5 Carretera México-Texcoco, Texcoco 56230, Estado de México, Mexico; E-Mails: dg_bonita33@yahoo.com.mx (D.G.-R.); hozuleta_13@hotmail.com (H.Z.-P.)
2
Departamento de Química, CINVESTAV-IPN, Apdo. Postal 14-740, México D.F. 07000, Mexico; E-Mail: rsantill@cinvestav.mx
3
Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, Colonia Santo Tomás, Delegación Miguel Hidalgo, México D.F. 11340, Mexico; E-Mails: mesmendoza@hotmail.com (M.E.S.-M.); jearrval@yahoo.com.mx (J.A.)
4
Departamento de Química Orgánica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Delegación Coyoacán D.F. 04510, Mexico
*
Authors to whom correspondence should be addressed; E-Mails: benijovi@yahoo.com.mx (B.R.-T.); linoj@unam.mx (L.R.); Tel.: +55-513-31108 (ext. 5760) (B.R.-T.); Fax: +55-595-952-1617 (B.R.-T.); Tel./Fax: +55-562-23802 (L.R.).
Received: 20 January 2014; in revised form: 3 April 2014 / Accepted: 4 April 2014 /
Published: 15 April 2014

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 24 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.
Keywords:
encecanescin; DFT calculations; X-ray diffraction; FTIR and NMR spectra

1. Introduction

Chromene (2H-1-benzopyran) ring derivatives are often found in natural heterocycles, and some have interesting biological activities [1]. 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 [2]. One such benzopyran derivative isolated from Ageratina asenii [3] is (+)-encecanescin, a dimeric chromene with a structure similar to a previously reported compound [4]. Surprisingly, however, the authors found that the available encecanescin crystallized as a racemic mixture.

Later, crystals of (±)-encecanescin were analyzed by X-ray diffraction [5], 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 [6]. 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.

Molecules 19 04695 g001 200
Figure 1. Atom numbering for (‒)-encecanescin (1). The segments of atoms C1, C2, C3, C4, C9, C10 and the methyl groups at C2 are disordered in the crystal and two conformers (1a and 1b labeled with A) are present.

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Figure 1. Atom numbering for (‒)-encecanescin (1). The segments of atoms C1, C2, C3, C4, C9, C10 and the methyl groups at C2 are disordered in the crystal and two conformers (1a and 1b labeled with A) are present.
Molecules 19 04695 g001 1024

2. Results and Discussion

(‒)-Encecanescin (1) was isolated using a previously reported protocol [6]. 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 [7]. 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.

Table Table 1. Crystal data and structure refinement for (‒)-encecanescin (1).

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Table 1. Crystal data and structure refinement for (‒)-encecanescin (1).
Empirical formulaC28H34O5
Formula weight450.2402
Temperature293(2) K
Wavelength0.71073 Å
Crystal system, space group monoclinic, P21/c
Unit cell dimensionsa = 11.0930(2) Å alpha = 90°
b = 8.4352(2) Å beta = 94.622(11)°
c = 27.5559(11) Å gamma = 90°
Volume2570.07(14) Å3
ZCalculated density40.851 Mg/m3
Absorption coefficient0.061 mm−1
F(000)724
Crystal size0.24 × 0.20 × 0.18 mm
Theta range for data collection2.46° to 27.49°
Limiting indices−14 ≤ h ≤ 13, −10 ≤ k ≤ 10, −35 ≤ l ≤ 28
Reflections collected/ unique14836/5814 [R(int) = 0.0892]
Completeness to theta = 27.4998.7%
Refinement methodFull-matrix least-squares on F2
Data/restraints/parameters 5814/216/379
Goodness-of-fit on F20.918
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.

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Figure 2. Conformational distribution of (‒)-encecanescin (1).

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Figure 2. Conformational distribution of (‒)-encecanescin (1).
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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.

Table Table 2. Selected bond lengths (Å), bond angles and torsion angles (°) for (−)-encecanescin (1) determined by X-ray diffraction and B3LYP calculations at the 6-311++G(d,p) level of theory.

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Table 2. Selected bond lengths (Å), bond angles and torsion angles (°) for (−)-encecanescin (1) determined by X-ray diffraction and B3LYP calculations at the 6-311++G(d,p) level of theory.
Parameter123
Bond length
C(10)-C(5)1.401(15)1.400261.4005
C(10)-C(4)1.436(15)1.455591.4568
C(10)-C(9)1.482(16)1.402251.4032
C(4)-C(3)1.310(10)1.337931.3386
C(3)-C(2)1.38(2)1.513631.5129
C(2)-C(13)1.446(15)1.527961.53535
C(2)-C(14)1.451(16)1.537261.52713
C(2)-O(1)1.486(13)1.463341.46937
O(1)-C(9)1.36(2)1.364141.36453
C(9)-C(8)1.44(2)1.392921.39302
C(5)-C(6)1.377(4)1.389311.39071
C(6)-C(7)1.396(4)1.409471.41067
C(3')-C(2')1.494(4)1.513631.51290
C(2')-C(13')1.521(4)1.527961.53535
C(2')-C(14')1.521(4)1.537261.52713
Bond angle
C(5)-C(10)-C(4)126.7(9)124.31413124.28231
C(5)-C(10)-C(9)113.3(13)118.07731118.01906
C(4)-C(10)-C(9)112.7(14)117.54869117.66232
C(3)-C(4)-C(10)122.6(8)120.34825120.33249
C(4)-C(3)-C(2)125.4(10)121.02151121.32649
C(3)-C(2)-C(13)123.9(16)111.64217110.71254
C(3)-C(2)-C(14)76.0(9)110.62884111.60611
C(13)-C(2)-C(14)157.0(19)111.16920111.19836
C(3)-C(2)-O(1)115.7(14)110.60774110.50808
C(13)-C(2)-O(1)75.0(8)104.5417108.01913
C(14)-C(2)-O(1)108.6(10)108.03583104.57564
C(9)-O(1)-C(2)118.6(13)118.74609119.00702
O(1)-C(9)-C(8)119.7(11)117.56225117.58616
O(1)-C(9)-C(10)123.7(17)121.35132121.26106
C(3')-C(2')-C(13')111.4(3)110.60783110.71254
C(3')-C(2')-C(14')111.2(3)111.56751111.60611
C(13')-C(2')-C(14')111.0(3)111.18802111.19836
C(13)-C(2)-O(1)106.7(3)107.99372108.01913
Dihedral angle
C(4)-C(3)-C(2)-C(13)80.0(17)140.8387993.83749
C(4)-C(3)-C(2)-C(14)−112.9(12)−94.753−141.72175
C(13)-C(2)-O(1)-C(9)−115.3(12)−156.22747−84.38296
C(14)-C(2)-O(1)-C(9)88.5(17)85.28967157.09634
C(13')-C(2')-C(3')-C(4')97.5(3)94.5522493.83749
C(14')-C(2')-C(3')-C(4')−138.1(3)−141.02837−141.72175
C(13')-C(2')-O(1')-C(9')−88.3(3)−85.26823−84.38296
C(14')-C(2')-O(1')-C(9')153.8(2)156.2661157.09634

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].

Table Table 3. Experimental and calculated (B3LYP/6-311G(d,p)) vibrational frequencies of (−)-encecanescin (1).

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Table 3. Experimental and calculated (B3LYP/6-311G(d,p)) vibrational frequencies of (−)-encecanescin (1).
RamanIRexpIRcalcINTIRcalcINTProposed assignment
123
3043.663041 w317715.7317610.4υC-H Ar
2983.392974 m313117.8313135.9υas CH3 methoxy
2938.112932 w310624.7310623.8υas CH3 gem
1645.951644 vw1651298.016513.2υs HC=CH ring
1621.541615 m159931.4160026υs HC=CH ring
1578.381576 s1523155.41524181.6βC-H Ar+CH3
1499.451492 w149318.7149323.3γCH3 methoxy
1462 vw14791.814792.4ωCH3 methoxy
1432.211443 vw14572.414561.1βring, Ar-O-R
1380 m138252.6138349.3ρHC=CH ring/ωAr-C-H-OCH3
1362.521360 m137212.6137426.1γC-H
1307.861303 s1303154.21303159.1υas HC=CH Ar
1279 m128435.4128527.8βAr, ring
1239.181230 m123133.3123240.8βHC=CH ring/υas CH3 gem
1196 s1214158.71214167.4ρCH3 methoxy, CH3 gem
1173.111163 m117858.3118089.3βHC=CH ring, C-H Ar
1114.621123 vs1145625.31145615.6υasC-O-C
1093 m1104100.6109937.7τCH3-C-O-C-CH3
1074 vs1087218.51087224.4ρCH3as C-O-C
1029 m104839.8104844.3υCH3-CH,CH3-O
1013 s103388.9103377.6υasAr-O-CH3/τHC-CH3
950.96959 m96728.596743.7υsHC-C(CH3)2-O, CH3-CH-O
884.95894 m90255.690255.5τCH3 gem
804.36801 vw8093.18091.1τAr-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 [10] 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).

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Figure 3. gHMBC and gHSQC spectra of (‒)-encecanescin (1) in CDCl3.

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Figure 3. gHMBC and gHSQC spectra of (‒)-encecanescin (1) in CDCl3.
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Table Table 4. Carbon-13 chemical shifts (δ, ppm) in CDCl3 and calculated GIAO nuclear magnetic shielding (σcal) for (−)-encecanescin (1). The predicted GIAO chemical shifts were computed from the linear equation δexp = a + b·σcalc with a and b determined from the fit to the experimental data.

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Table 4. Carbon-13 chemical shifts (δ, ppm) in CDCl3 and calculated GIAO nuclear magnetic shielding (σcal) for (−)-encecanescin (1). The predicted GIAO chemical shifts were computed from the linear equation δexp = a + b·σcalc with a and b determined from the fit to the experimental data.
Atomδexpδpred (2)δpred (3)σcalc (2)σcalc (3)
C(2)78.380.780.298.4798.84
C(3)127.3124.1124.552.5352.00
C(4)122.7124.9124.351.6852.19
C(5)124.0124.4123.952.2252.58
C(6)125.1126.3125.750.2750.70
C(7)157.7158.8157.615.8517.03
C(8)99.296.295.782.0782.46
C(9)152.8153.6154.521.3220.25
C(10)113.8113.4114.063.9163.12
C(11)68.668.569.3111.29110.30
C(12)23.224.724.5157.67157.66
C(13)28.329.527.0152.57155.04
C(14)28.126.829.5155.41152.43
C(15)55.353.853.7126.87126.85
C(2')78.380.780.298.4798.84
C(3')127.3124.5124.552.1652.00
C(4')122.7125.0124.351.6452.19
C(5')124.0123.7123.953.0152.58
C(6')125.1125.0125.751.6150.70
C(7')157.7157.8157.616.9617.03
C(8')99.295.795.782.5882.46
C(9')152.8153.3154.521.6720.25
C(10')113.8113.9114.063.2863.12
C(11')68.668.869.3110.98110.30
C(12')23.224.424.5157.92157.66
C(13')28.327.027.0155.15155.04
C(14')28.129.629.5152.41152.43
C(15')55.353.753.7126.98126.85
A173.81173.68
B−0.946−0.946
r20.99860.9986

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.

Table Table 5. Experimental chemical shifts (δexp, CDCl3) vs. the isotropic magnetic shielding tensors (σcalc) from the GIAO/B3LYP/6-311++G(d,p) calculations for encecanescin (1); δexp = a+b·σcalc: (a) 13C (a = 173.81; b = −0.946; r2 = 0.9986) and (b) 1H (a = 32.101; b = −1.0124; r2 = 0.9952).

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Table 5. Experimental chemical shifts (δexp, CDCl3) vs. the isotropic magnetic shielding tensors (σcalc) from the GIAO/B3LYP/6-311++G(d,p) calculations for encecanescin (1); δexp = a+b·σcalc: (a) 13C (a = 173.81; b = −0.946; r2 = 0.9986) and (b) 1H (a = 32.101; b = −1.0124; r2 = 0.9952).
Atomδexpδpred (2)δpred (3)σcalc (2)σcalc (3)
H(3)5.465.445.4426.3426.34
H(4)6.346.256.3925.5425.33
H(5)7.107.347.2124.4624.46
H(8)6.326.156.0525.6425.69
H(11)4.594.684.6927.0827.15
H(12)1.311.261.2930.4630.78
H(13)1.461.691.2930.0430.77
H(14)1.431.411.6430.3230.39
H(15)3.673.483.6828.2828.22
A32.07630.141
B−1.0114−0.9376
r20.99560.9956

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 [11]. 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.

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Figure 4. Experimental chemical shifts (δexp, CHCl3) vs. the isotropic magnetic shielding tensors (σcalc, weighed by taking into account the Boltzmann distribution) from the GIAO/B3LYP/6-311++G(d,p) calculations for encecanescin (1); δexp = a + b·σcalc: (a) 13C (a = 173.70; b = −0.956; r2 = 0.998) and (b) 1H (a = 31.16; b = −0.987; r2 = 0.997).

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Figure 4. Experimental chemical shifts (δexp, CHCl3) vs. the isotropic magnetic shielding tensors (σcalc, weighed by taking into account the Boltzmann distribution) from the GIAO/B3LYP/6-311++G(d,p) calculations for encecanescin (1); δexp = a + b·σcalc: (a) 13C (a = 173.70; b = −0.956; r2 = 0.998) and (b) 1H (a = 31.16; b = −0.987; r2 = 0.997).
Molecules 19 04695 g004 1024

3. Experimental

3.1. General

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; [deposit_reply@ccdc.cam.ac.uk]).

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.

3.2. Materials

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.

3.3. Methods

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 [3], 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 [12] 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) [15] level using the SMD solvent model [16], were performed using the Gaussian 09 package [17]. The NMR isotropic magnetic shielding tensors were calculated using the standard gauge-independent atomic orbital (GIAO) approach [11,18] in Gaussian 09.

4. Conclusions

(−)-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.

Supplementary Materials

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.

Acknowledgements

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.

Author Contributions

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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  • Sample Availability: A sample of (−)-encecanescin (1) is available from the authors.
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