Solid State Structure and Absolute Configuration of Filifolinol Acetate Marcelo A. Muñoza, Alejandro Urzúab, Javier Echeverríab, Brenda Modakb and Pedro Joseph-Nathanc,* aInstituto de Ciencias Químicas, Universidad Austral de Chile, Casilla 567, Valdivia, Chile

bFacultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile

cDepartamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, México, D. F., 07000 México pjoseph@nathan.cinvestav.mx

Received: January 19th, 2011; Accepted: April 2nd, 2011

Careful reevaluation of the 1H and 13C NMR spectroscopic data of filifolinol acetate (4) led to the reassignment of the C-10 and C-11 signals, as well as the gem-dimethyl signals. Single crystal X-ray analysis provided an independent structural confirmation of 4, and comparison of the experimental vibrational circular dichroism spectrum with calculations performed using density functional theory provided the absolute configuration of this 3H-spiro-1-benzofuran-2,1’-cyclohexane and related molecules. Keywords: Heliotropium filifolium, filifolinol acetate, crystal structure, absolute configuration, vibrational circular dichroism, density functional theory calculations. Filifolinol (1) [1], the parent molecule of a series of 3H-spiro-1-benzofuran-2,1’-cyclohexanes, was isolated from the cuticle of Heliotropium filifolium, together with filifolinyl senecionate (2) [2]. The structures of these unusual spiro natural products of mixed biogenesis were obtained through extensive NMR spectroscopic studies. Further re-examination of the minor cuticle components of the plant allowed the isolation of four additional related compounds: 3’-hydroxy-2’,2’,6’-trimethyl-3H-spiro[1-benzofuran-2,1’-cyclohexane]-5-carboxylic acid (3), filifolinyl acetate (4), filifolinyl isopentanoate (5), and filifolinyl benzoate (6) [3]. Compounds 3-6 were identified by their spectroscopic analogies with filifolinol (1), and confirmation of their structures and identical stereochemistry was achieved by chemical correlation with 1. Compound 3 was methylated with ethereal CH2N2 to afford filifolinol (1), and compounds 4, 5 and 6 were obtained by acylation of filifolinol (1) using the appropriate acyl chlorides [3].

This group of compounds shows interesting biological properties. Regarding antibacterial activity, compounds 1 and 3-6 were evaluated as antimicrobial agents against Gram-positive and Gram-negative bacteria. All proved to be inactive against the latter, but some exhibited significant activity against Gram-positive microorganisms like Bacillus subtilis, B. cereus, Micrococcus luteus and Staphylococcus aureus, in fact comparable with or higher than commercial antibiotics like chloramphenicol, ampicillin and tetracycline [3].

O

R'O2COR

2

34

6

8 91011

127a

1 R = H, R’ = Me2 R = Seneciate, R’ = Me3 R = H, R’ = H4 R = Ac, R’ = Me5 R = i-Valerate, R’ = Me6 R = Bz, R’ = Me

Regarding antiviral activity, filifolinol (1) and filifolinyl senecionate (2) were tested against five virus species of importance in human diseases, whereby 1 was the most active compound, especially against polio virus, but with a close relationship between antiviral doses/cytotoxic doses [4]. In addition, filifolinol (1) showed antiviral activity against Hantavirus with an EC50 value of 15.8 µg/mL, close to that of the ribavirine control (EC50 14.8 µg/mL) [5]. The antiviral activity against infectious pancreatic necrosis virus (IPNV) was evaluated for filifolinol (1) and filifolinyl senecionate (2). Only 2 showed an excellent inhibition effect for viral replication at a non-toxic concentration with an EC50 of 160 µg/mL and a cytotoxic concentration required to reduce cell viability by 50% up to 400 mg/mL. The activity was ascribed to the inhibition of the synthesis of viral RNA, but not at the level of protein expression [6].

NPC Natural Product Communications 2011 Vol. 6 No. 0 1 - 4

2 Natural Product Communications Vol. 6 (0) 2011 Muñoz et al.

Table 1: Selected torsion angles (in deg) of 4 in the solid state, and for its four low energy conformers.

Torsion angle X-ray 4a 4b 4c 4d

O1-C2-C8-C9 -169.5 -169.0 -169.2 -62.8 -62.2

O1-C2-C12-C11 173.0 171.7 172.5 62.0 61.7

C2-C8-C9-C10 50.3 50.8 50.2 -52.7 -53.3

C8-C9-C10-C11 -51.1 -50.9 -50.4 57.3 57.3

C9-C10-C11-C12 54.6 53.3 53.4 -56.4 -56.1

C10-C11-C12-C2 -58.1 -56.3 -56.9 53.7 53.5

C11-C12-C2-C8 57.9 56.5 57.1 -52.5 -52.7

C12-C2-C8-C9 -53.8 -53.5 -53.5 50.5 51.2

C4-C5-C=O 166.9 179.8 0.0 179.9 -0.2

Due to the significant antiviral properties of filifolinol (1) and related molecules, and in order to gain insights to the mechanisms of action of this series of compounds, independent structural verification, conformational evaluation and determination of absolute configuration is of relevance. The 1H and 13C NMR spectra of 4 were measured again and evaluated in detail with the aid of NOESY, gHSQC and gHMBC plots, which resulted in the reassignment of both the 1H and 13C NMR peaks of the methylene signals at C-10 and C-11, as well as the axial and equatorial methyl groups of the gem-dimethyl arrangement at C-8. All remaining NMR assignments were in agreement with published values [3]. An independent structural confirmation of filifolinol acetate (4) was obtained from a single crystal X-ray diffraction study. The orthorhombic P212121 crystal structure could be refined to a discrepancy index of 4.8%, the details of this study being summarized in the Experimental Section. The solid state structure, represented in Figure 1, shows the six-member ring in an almost ideal chair conformation with torsion angles as given in Table 1, the acetyloxy group at C9 in the alpha-axial orientation and the secondary methyl group at C12 in an alpha-equatorial disposition. Filifolinol acetate (4) is a good candidate for vibrational circular dichroism (VCD) studies since, due to its rigid molecular structure, a small number of final conformers can be anticipated, thereby simplifying the overall density functional theory (DFT) calculations. In fact, the VCD methodology has proved to be very powerful to establish the absolute configuration of natural products [7], in particular for the study of rigid mono- [8,9] and sesquiterpenes [10,11]. Thus, the absolute configuration of 4 was determined from a study in which the experimental spectrum was compared with the theoretical curve obtained by DFT calculation at the B3LYP/DGDZVP level of theory. In order to calculate the VCD spectrum of 4, the atom coordinates derived from the single crystal X-ray study were fed into the Spartan’04 software. The molecular

Figure 1: Single crystal X-ray structure of filifolinol acetate (4). Table 2: Molecular mechanics relative energy, molecular mechanics population, DFT thermochemical parameters, and DFT population for the four minimum energy conformers of filifolinol acetate (4).

conformer ΔEMMFFa pMMFF

b ΔE0c,d ΔH298

e,f ΔG298d pDFT

g

4a 0.19 41.64 0.00 0.07 0.00 62.40

4b 0.00 57.64 0.02 0.00 0.33 35.74

4c 3.11 0.30 2.20 2.32 2.20 1.52

4d 2.91 0.42 2.02 1.56 3.09 0.34

a Molecular mechanics energy of conformers obtained from a systematic search, in kcal/mol relative to 4b with EMMFF = 64.081 kcal/mol . b Molecular mechanics population in %. c Sum of electronic and zero-point DFT B3LYP/DGDZVP energies in kcal/mol relative to conformer 4a calculated at 298 K and 1 atm. d For conformer 4a the absolute values are E0 = -1153.654350 au and G298 = -1153.710077 au. e For conformer 4b the absolute values are E298 = -1153.629698 au and E298 + H298 = -1153.628754 au. f ΔE298 values equal to ΔH298 in all conformations. g DFT population in % calculated from ΔG298 values.

structure built following this methodology was subjected to a systematic conformational search using the molecular mechanics force field (MMFF). This search algorithm consisted of the systematic change of cyclic and acyclic dihedral angles at fixed steps (usually 120 degrees) in order to obtain all relevant conformations, which in the case of 4 provided eight conformers in a 10 kcal/mol energy gap. The first two low energy models account for 99.3% of the conformational distribution, the third and fourth models being 2.9 and 3.1 kcal/mol, respectively, above the global minimum (Table 2). These four models were submitted to DFT single point calculations using the B3LYP/DGDZVP level of theory to provide the values shown in Table 1, followed by further optimization at 298 K using the same level of theory with the Gaussian’03 software (Gaussian Inc, Wallingford, CT 06492, USA), providing their free energy values. The use of this basis set and functional has shown a good balance between computer time and VCD spectral accuracy [9]. The two lower energy molecular models, which now account (Table 2) for 98.1% of the overall conformational distribution, differ only in the disposition of the carbomethoxyl group and show the cyclohexane ring in an almost ideal chair conformation (O1 and O9 in equatorial and axial positions, respectively), as seen in Figure 2.

Absolute configuration of filifolinol acetate Natural Product Communications Vol. 6 (0) 2011 3

Figure 2: Molecular models for the two low energy conformers of filifolinol acetate (4). Furthermore, the conformation of the global minimum model closely resembles that of 4 in the solid state, as can be deduced from a torsion angle comparison using the values given in Table 1. The third and forth molecular models have the cyclohexane ring in the opposite chair conformation (O1 and O9 in axial and equatorial positions, respectively) and with energetically equivalent carbomethoxyl group dispositions. The IR and VCD frequencies were thus calculated only for the two lower energy conformers using DFT at the B3LYP/DGDZVP level of theory.

Figure 3: Comparison of the experimental (top) and DFT//B3LYP/DGDZVP calculated VCD spectra of filifolinol acetate (4).

Calculated VCD spectra of these two conformers were combined in a single weighted data file according to the Boltzmann conformational population (Table 2) derived from their relative free energy values. The combined data were then in silico compared with the experimental VCD data, using the CompareVOA software (BioTools Co, Jupiter, FL 33458, USA), which provided an anharmonicity factor [9] of 0.983, and then plotted as Lorentzian bands with half-widths of 6 cm-1 and compared with the experimental VCD spectrum, as shown in Figure 3. The similarity of the experimental and theoretical plots allows the conclusion to be drawn that filifolinol acetate (4) is correctly represented as the 2S,9S,12R enantiomer. Experimental

General: The melting point was determined on a Fisher-Johns apparatus and is uncorrected. NMR spectra were determined from a 99.8% atom-D CDCl3 solution containing internal TMS on a Varian Mercury 300 spectrometer, while IR and VCD spectra were measured using a BioTools-BOMEM ChiralIR FT-VCD spectrophotometer equipped with dual photoelastic modulation. A sample of 7.2 mg of 4 was dissolved in 150 μL of 100% atom-D CDCl3 and placed in a BaF2 cell with a path length of 100 μm. Data were acquired at a resolution of 4 cm-1 during 4 h, and the baseline correction was achieved by subtracting the spectrum of the solvent. Compound 4 was available from a previous study [3], where it is described as oil. Crystals could be obtained by slow evaporation from MeOH and showed a mp of 113-115°C. Single crystal X-ray diffraction analysis of filifolinol acetate (4): Data were collected on a Bruker-Nonius CAD4 diffractometer equipped with CuKα radiation (λ = 1.54184 Å) at 293(2) K in the ω-2θ scan mode. Unit cell refinements using 25 machine centered reflections were made using the CAD4 Express v2.0 software. The data were C20H26O5, M = 346.41, orthorhombic, space group P212121, a = 9.548(1) Å, b = 13.163(1) Å, c = 15.162(3) Å, V = 1905.5(5) Å3, Z = 4, ρ = 1.21 mg/mm3, μ(CuKα) = 0.700 mm-1, total reflections = 1430, unique reflections 1339 (Rint 0.27%), observed reflections 762, final R indices [I>2σ(I)] R1 = 4.8%, wR2 = 9.7%, The structure was solved by direct methods using the SIR2004 program included in the WinGX v1.70.01 crystallographic software package. For the structural refinement, the non-hydrogen atoms were treated anisotropically, and the hydrogen atoms, included in the structure factor calculation, were refined isotropically. Crystallographic data (excluding structure factors) have been deposited at the Cambridge Crystallographic Data Centre (No. 819708). Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 IEZ, UK. Fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk. DFT calculations: The conformational search for 4 was started with a Monte Carlo protocol using the molecular

4 Natural Product Communications Vol. 6 (0) 2011 Muñoz et al.

mechanics force field implemented in the Spartan’04 software package (Wavefunction Inc, Irvine, CA 92612, USA) considering a 10 kcal/mol energy cutoff above the global minimum energy value. All structures were submitted to single point energy calculations at the B3LYP/6-31G(d) level of theory using the same software. The relevant conformers, selected according to their energy range, were further optimized with the Gaussian 03W software package (Gaussian Inc, Wallingford, CT 06492, USA) using the B3LYP hybrid functional and the DGDZVP basis set, and then the IR and VCD frequencies

were calculated at the same level of theory. The theoretical VCD spectrum was generated by weighting the individual VCD spectra according to their free energy values using a Boltzmann distribution. Frequencies were scaled using an anharmonicity factor of 0.983 estimated using the CompareVOA software (BioTools Co, Jupiter, FL 33458, USA), and plotted as Lorentzian bands with half-widths of 6 cm-1. On average, the calculations for each conformer required 21.6 h when using a personal computer operated at 3.0 GHz with 4 Gb RAM.

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