NPC

2011
Vol. 6
No. 0
1-4

Natural Product Communications

Solid State Structure and Absolute Configuration of

Filifolinol Acetate
Marcelo A. Muoza, Alejandro Urzab, Javier Echeverrab, Brenda Modakb and
Pedro Joseph-Nathanc,*
a

Instituto de Ciencias Qumicas, Universidad Austral de Chile, Casilla 567, Valdivia, Chile
Facultad de Qumica y Biologa, Universidad de Santiago de Chile, Santiago, Chile
c
Departamento de Qumica, Centro de Investigacin y de Estudios Avanzados del Instituto Politcnico
Nacional, Apartado 14-740, Mxico, D. F., 07000 Mxico
b

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.

12

Filifolinol (1) [1], the parent molecule of a series of 3Hspiro-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-3Hspiro[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].

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 g/mL,
close to that of the ribavirine control (EC50 14.8 g/mL)
[5].

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].

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 g/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].

7a

R'O2C

2
4

1
2
3
4
5
6

11
8

10

OR

R = H, R = Me
R = Seneciate, R = Me
R = H, R = H
R = Ac, R = Me
R = i-Valerate, R = Me
R = Bz, R = Me

2 Natural Product Communications Vol. 6 (0) 2011

Muoz 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

-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

O1-C2-C8-C9

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 alphaaxial 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 Spartan04 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

EMMFFa

pMMFFb

E0c,d

H298e,f

G298d

pDFTg

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

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 zeropoint 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 E298 values equal to H298 in all conformations. g DFT
population in % calculated from G298 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 Gaussian03
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

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

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).

General: The melting point was determined on a FisherJohns 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
L of 100% atom-D CDCl3 and placed in a BaF2 cell with
a path length of 100 m. 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 113115C.
Single crystal X-ray diffraction analysis of filifolinol
acetate (4): Data were collected on a Bruker-Nonius
CAD4 diffractometer equipped with CuK radiation ( =
1.54184 ) at 293(2) K in the -2 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) , b = 13.163(1) , c = 15.162(3) ,
V = 1905.5(5) 3, Z = 4, = 1.21 mg/mm3, (CuK) =
0.700 mm-1, total reflections = 1430, unique reflections
1339 (Rint 0.27%), observed reflections 762, final R
indices [I>2(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

mechanics force field implemented in the Spartan04
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

Muoz et al.
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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