Balsacones D-I, dihydrocinnamoyl flavans from Populus balsamifera buds

Article abstract of DOI:10.1016/j.phytochem.2013.12.018

A phytochemical investigation of an ethanolic extract from Populus balsamifera L. buds resulted in the isolation and characterization of twelve new flavan derivatives consisting of six pairs of enantiomers. Structures of (+) and (-)-balsacones D-I were elucidated based on spectroscopic data (1D and 2D NMR, MS) and their absolute configurations were established using X-ray single crystal diffraction analysis and ECD computational calculations. Antibacterial activity and cytotoxicity of all purified enantiomers were evaluated in vitro against Staphylococcus aureus and human skin fibroblast cells, respectively.

Full text of DOI:10.1016/j.phytochem.2013.12.018

Phytochemistry 100 (2014) 141–149
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Balsacones D-I, dihydrocinnamoyl flavans from Populus balsamifera buds
⇑
François Simard, Jean Legault, Serge Lavoie, André Pichette
Laboratoire d’Analyse et de Séparation des Essences Végétales (LASEVE), Département des sciences fondamentales, Université du Québec à Chicoutimi, Québec G7H 2B1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2013
Received in revised form 25 November 2013
Available online 30 January 2014
A phytochemical investigation of an ethanolic extract from Populus balsamifera L. buds resulted in the
isolation and characterization of twelve new flavan derivatives consisting of six pairs of enantiomers.
Structures of (+) and (ꢀ)-balsacones D-I were elucidated based on spectroscopic data (1D and 2D
NMR, MS) and their absolute configurations were established using X-ray single crystal diffraction
analysis and ECD computational calculations. Antibacterial activity and cytotoxicity of all purified
enantiomers were evaluated in vitro against Staphylococcus aureus and human skin fibroblast cells,
respectively.
Keywords:
Populus balsamifera L.
Salicaceae
Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Bud
Flavan
Dihydrochalcone
Balsacone
Chiral separation
Circular dichroism
Antibacterial activity
Staphylococcus aureus
Computational calculations
Introduction
Populus species possess antibacterial activity similar to that of pop-
lar-type propolis (Vardar-Ünlü et al., 2008). Antioxidant properties
The genus Populus includes nearly 30 species distributed mostly
throughout the northern hemisphere (Dickmann, 2001). Buds from
several Populus species are covered with an exudate (Eckenwalder,
1996; Greenaway and Whatley, 1990) that has attracted growing
interest in the scientific community since it has been identified
as a known plant source of propolis (Bankova et al., 2002; Salatino
et al., 2011). Propolis is a bee product that has been used in folk
medicines around the world for the treatment of wounds and
burns, sore throat, stomach ulcers, etc. (Bankova, 2005; Salatino
et al., 2011). Several studies about propolis have established simi-
larities between the compositions of poplars buds exudate (PBE)
and what is sometimes referred to as poplar-type propolis. For
example, hydroxycinnamates and their derivatives, which are
characteristic constituents of PBE from the entire genus Populus
(Chen et al., 2009; Tsai et al., 2006), have been identified many
times in propolis (Salatino et al., 2011). Other species-specific com-
ponents of Populus, such as dihydrochalcones present in PBE from
section Tacamahaca, have also been described in propolis (Christov
et al., 2006). On the other hand, only a handful of studies about
biological and pharmacological properties of PBE are available.
Vardar-Ünlü et al. demonstrated that bud extracts from different
were also detected for Populus nigra bud extract (Dudonné et al.,
2011). Close relations between the chemical compositions of pop-
lar type propolis and PBE suggest that PBE might be an interesting
untapped source for the discovery of new bioactive compounds.
The exudate covering buds from Populus balsamifera L. (section
Tacamahaca) has a strong balsam smell in the spring. Amongst
others, North American aboriginals used tincture or poultice of P.
balsamifera buds as dermatological and gastrointestinal aids as
well as for the treatment of chronic rheumatism (Moermann,
1998). Earlier studies established that buds are rich in phenolic
compounds, such as simple phenols and phenolic acids (Isidorov
and Vinogorova, 2003; Mattes et al., 1987; Pearl and Darling,
1968b; Reichardt et al., 1990; Sentsov et al., 1997), phenolic glyco-
sides (Pearl and Darling, 1968b), hydroxycinnamic acids and their
esters (Greenaway et al., 1992a,b; Greenaway and Whatley, 1990;
Isidorov and Vinogorova, 2003; Pearl and Darling, 1968a), flavones
and flavanones (Isidorov and Vinogorova, 2003; Kurkin et al., 1990;
Sentsov et al., 1997), chalcones and dihydrochalcones (Greenaway
et al., 1992a, 1989; Greenaway and Whatley, 1990; Kurkin et al.,
1990). However, little work has been conducted on their biological
and pharmacological potential. Recently, the LASEVE team identi-
fied three new hydroxycinnamylated dihydrochalcones from an
ethanolic extract of P. balsamifera buds (Lavoie et al., 2013). Balsa-
cone A, B and C, were found active against Staphylococcus aureus
⇑
Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545 5012.
E-mail address: andre_pichette@uqac.ca (A. Pichette).
0031-9422/$ - see front matter Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.12.018

142
F. Simard et al. / Phytochemistry 100 (2014) 141–149
with MIC values ranging from 3.1 to 6.3 lM. Continuing the search
for new antibacterial compounds, the ethanol extract of buds from
P. balsamifera has been further investigated. The isolation and
structural elucidation of 12 new flavan derivatives which have
been isolated as 6 pairs of enantiomers is reported in the present
paper. The antibacterial activity and the cytotoxicity of these com-
pounds were also evaluated against S. aureus and human skin
fibroblast cells respectively.
COSY
HMBC
NOESY
MS
O
OH
HO
O
OH
271
Results and discussion
Fig. 2. HMBC, COSY, NOESY and MS key data for identification of compound 1.
Structural determination
at dH 2.84 (2H, t, J = 8.1 Hz, H-700) and 3.24 (2H, m, H-800) and a
AA0BB0M system at dH 2.06 (1H, m, H-3a), 2.19 (1H, m, H-3b),
2.68 (1H, m, H-4a), 2.78 (1H, ddd, J = 17.0, 5.9, 2.4 Hz, H-4b) and
5.07 (1H, dd, J = 11.0, 2.0 Hz, H-2). Also, the presence of a chelated
hydroxyl proton was detected based on the signal at dH 13.82 (1H,
s, 7-OH) on the 1D 1H spectrum. The HMBC correlations of dC 205.2
(C-900) with H-700 and H-800 and of dC 31.2 (C-700) with H-200/H-600 sug-
gested that the two methylenes were linked to a ketone and to the
monosubstituted aromatic ring resulting in a dihydrocinnamoyl
moiety. The HMBC correlations of dC 158.3 (C-40) with H-30/H-50
and of dC 132.7 (C-10) with H-20/H-60 and H-2 indicated that the
1,4 disubstituted aromatic ring was hydroxylated and branched
to the AA0BB0M system. The six remaining carbon resonances at
dC 163.2 (C-5), 96.1 (C-6), 166.0 (C-7), 105.7 (C-8), 159.7 (C-9)
and 102.0 (C-10) were assigned to a third aromatic ring. This third
aromatic ring was also attached to the AA0BB0M spin system as
shown by the HMBC correlation of C-5 with H-4a and H-4b which
suggested it might be forming the ring A of a flavan skeleton
hydroxylated in position 5, 7 and 40. The proton signal at dH 6.02
(1H, s, H-6) implied the presence of a pentasubstituted ring A to
Fractionation of P. baslamifera bud ethanolic extract by liquid–
liquid extraction, silica gel column chromatography and reversed
phase flash chromatograhy followed by semi-preparative HPLC
purification yielded compounds 1–6 (Fig. 1).
Balsacone D (1) was isolated as a yellow powder and its
molecular formula was established as C24H22O5 based on the
[M+Na]+ quasimolecular ion peak at m/z 413.1374 in the HRMS
spectrum. The IR spectrum showed bands at 3347 and
1615 cmꢀ1, due to phenol and carbonyl functions, respectively.
The 13C and DEPT-135 NMR spectra afforded signals accounting
for 20 carbons, i.e. one carbonyl, four oxygenated quaternary aro-
matic carbons, six sp2 and one oxygenated sp3 methines, four aro-
matic quaternaries and four methylenes. Detail analysis of 1D 1H,
2D-COSY and HSQC experiments (Fig. 2) showed resonances for a
1,4 disubstituted aromatic ring at dH 7.35 (2H, d, J = 8.5 Hz, H-20/
H-60) and 6.85 (2H, d, J = 8.5 Hz, H-30/H-50) and a mono-substituted
aromatic ring at dH 6.91 (2H, d, J = 7.3 Hz, H-200/H-600), 7.16 (2H, t,
J = 7.3 Hz, H-300/H-500) and 7.10 (1H, t, J = 7.3 Hz, H-400). Two other
spin systems could be observed in the COSY, i.e. two methylenes
R1
R1
4''
4''
1''
1''
7''
7''
OH
OH
4'
O
O
4'
8''
8''
9''
9''
1
1
8
5
1'
8
5
1'
3
HO
O
HO
O
7
7
9
2
9
2
10
10
6
3
6
4
4
O
O
R2
R2
(-)-1 (2S) R1 = R2 = H
(+)-1 (2R) R1 = R2 = H
(-)-2 (2S) R1 = OCH3, R2 = H
(-)-3 (2S) R1 = OH, R2 = CH3
(+)-2 (2R) R1 = OCH3, R2 = H
(+)-3 (2R) R1 = OH, R2 = CH3
OH
OH
4'
4'
1
O
1
O
8
8
1'
1'
HO
HO
9
9
4''
4''
7
6
2
3
7
6
2
3
8''
8''
9''
9''
1''
10
1''
10
OH
OH
5
5
7''
4
7''
4
O
OH
O
OH
(-)-4 (2S,3R)
R1
(+)-4 (2R,3S)
R1
4''
4''
1''
1''
7''
9''
7''
9''
O
OH
O
OH
4'
4'
8''
8''
1
1
8
5
1'
8
5
1'
HO
O
HO
O
7
7
2
3
2
3
9
9
10
10
6
6
OH
OH
4
4
OH
OH
1
(-)- (2R,3S) R1 = H
(+)- (2S,3R) R = H
5
6
5
6
1
(-)- (2R,3S) R1 = OCH3
(+)- (2S,3R) R = OCH3
Fig. 1. Structures of 1–6.

F. Simard et al. / Phytochemistry 100 (2014) 141–149
143
which the dihydrocinnamoyl unit would be attached. The presence
of a peak at m/z 271 on the mass spectrum (APCI) representing the
fragment obtained by the typical retro Diels–Alder fragmentation
reaction of flavonoids (Pelter et al., 1965) confirmed the attach-
ment of the dihydrocinnamoyl moiety to the A ring. A W-coupling
between C-900 and H-6 was also present in the HMBC spectrum.
From these observations, only two possibilities can be considered
for the position of the dihydrocinnamoyl moiety, i.e. C-6 and C-8.
The 2D-NOESY correlations of H-800 with H-2 and H-20/H-60 con-
firmed that the dihydrocinnamoyl moiety was attached at C-8
and therefore the resonance at dH 6.02 could be assigned to H-6.
Assignments of positions 5, 7, and 9 were determined using HMBC
correlations of C-5 and C-7 with H-6 and of C-5 and C-9 with H-4a
and H-4b. The signal at dC 102.0 was assigned as C-10 based on its
HMBC correlations with H-3b and therefore, the resonance at dC
105.7 was assigned as C-8, the only remaining position. Based on
the above evidence, balsacone D (1) was characterized as 8-(dihy-
drocinnamoyl)-5,7,40-trihydroxyflavan.
Balsacone E (2) was isolated as a yellow powder and its molecu-
lar formula was established as C25H24O6 based on the [M+H]+ quasi-
molecular ion peak at m/z 421.1656 in the HRMS spectrum. The IR
spectrum had bands at 3400 and 1622 cmꢀ1, due to phenol and car-
bonyl functions, respectively. The 1H and 13C NMR spectra showed
the presence of the same 5,7,40-trihydroxyflavan skeleton as for
compound 1 but with a different substituent at C-8. Analysis of 1D
1H, 2D-COSY and HSQC experiments showed signals for a 1,4 disub-
stituted aromatic ring at dH 6.79 (2H, d, J = 8.7 Hz, H-200/H-600) and
6.72 (2H, d, J = 8.7 Hz, H-300/H-500) and two methylenes at 2.77 (2H,
m, H-700) and 3.19 (2H, m, H-800). The HMBC correlations of dC
205.4 (C-900) with H-700 and H-800 and of dC 30.3 (C-700) with H-200/
H-600 suggested that the two methylenes were linked to a ketone
and to the 1,4-disubstituted aromatic ring. The HMBC correlations
of dC 158.7 (C-400) with H-300/H-500 and dH 3.74 (400-O-CH3) established
that the 1,4-disubstituted aromatic ring was methoxylated. It was,
therefore, concluded that the C-8 substituent was a 4-methoxydihy-
drocinnamoyl moiety. The 2D NOESY correlations of H-800 with dH
5.08 (1H, dd, J = 10.7, 2.3 Hz, H-2) and 7.37 (1H, d, J = 8.5 Hz, H-20/
H-60) confirmed that the 4-methoxydihydrocinnamoyl moiety was
in C-8. From the above evidence, balsacone E (2) was characterized
as 8-(4-methoxydihydrocinnamoyl)-5,7,40-trihydroxyflavan.
Balsacone F (3) was isolated as a yellow powder and its molec-
ular formula was established as C25H24O6 based on the [M+Na]+
quasimolecular ion peak at m/z 443.1477 in the HRMS spectrum.
The IR spectrum showed bands at 3363 and 1617 cmꢀ1, due to phe-
nol and carbonyl functions, respectively. The 1H and 13C NMR spec-
troscopic data were closely related to 2 suggesting that 3 was an
isomer. Indeed, after detailed analysis of NMR spectra, the only dif-
ference accounted for was the position of the methoxy group,
which was determined as C-5 by the HMBC correlation of dC
164.7 (C-5) with dH 3.89 (3H, s, OCH3). Based on the above evi-
dence, balsacone F (3) was characterized as 8-(4-hydroxydihydro-
cinnamoyl)-7,40-dihydroxy-5-methoxyflavan.
Balsacone H (5) was obtained as a yellow powder and its molec-
ular formula was established as C24H22O6 based on the [M+K]+
quasimolecular ion peak at m/z 445.1055 in the HRMS spectrum.
The IR spectrum showed bands at 3450 and 1632 cmꢀ1, due to phe-
nol and carbonyl functions, respectively. Details analysis of the 1D
1H, 2D-COSY and HSQC showed the presence of the same 3,5,7,40-
tetrahydroxyflavan skeleton and dihydrocinnamoyl unit as in 4.
However, the 1H NMR and 13C spectra of 5 shows discrepancies
from those of 4. These discrepancies were attributed to the posi-
tion of attachment of the dihydrocinnamoyl unit which was pro-
posed to be C-8 instead of C-6. The C-8 attachment was
confirmed by the HMBC correlations of dC 158.7 (C-9) with dH
4.81 (1H, d, J = 8.4 Hz, H-2) and of dC 163.3 (C-5) and 166.1 (C-7)
with dH 6.05 (1H, s, H-6). Based on the above evidence, balsacone
H
(5) was characterized as 8-(dihydrocinnamoyl)-3,5,7,40-
tetrahydroxyflavan.
Balsacone I (6) was obtained as a yellow powder and its molec-
ular formula was established as C25H24O7 based on the [M+Na]+
quasimolecular ion peak at m/z 459.1431 in the HRMS spectrum.
The IR spectrum showed bands at 3337 and 1614 cmꢀ1, due to phe-
nol and carbonyl functions, respectively. The 1H and 13C NMR spec-
tra were almost identical to those of compound 5, but with an
additional methoxy group at dH 3.74 (3H, s, OCH3) and dC 55.3
(OCH3). This group was assigned at 400 from the HMBC correlation
between dC 158.7 (C-400) and the methoxy protons. 2D-NOESY cor-
relations of dH 7.36 (2H, d, J = 8.6 Hz, H-20/H-60) with 2.76 (2H, t,
J = 7.9 Hz, H-700) and dH 3.16 (2H, m, H-800) further confirmed that
the (4-methoxydihydrocinnamoyl moiety was located at C-8.
Based on the above evidence, balsacone I (6) was characterized
as 8-(4-methoxydihydrocinnamoyl)-3,5,7,40-tetrahydroxyflavan.
Measurement of the optical rotation of compounds 1–6 yielded
[a] values of 0. Furthermore, no Cotton Effect (CE) was observed
d
on their ECD spectra suggesting that they were racemic. Chiral ana-
lytical HPLC confirmed the presence of two compounds in equal
amounts (Fig. S-25 to S-30) and chiral semi-preparative HPLC puri-
fication yielded the (+) and (ꢀ) enantiomers for the six racemic
mixtures (1–6). Crystals of (ꢀ)-1 were obtained after drying an
(isopropyl alcohol) IPA solution under a N2 stream. Single-crystal
X-ray diffraction analysis allowed us to determine that the C-2
absolute configuration of (ꢀ)-1 was S (Fig. 3). The absolute config-
uration of other purified enantiomers was determined by compar-
ing their experimental ECD spectra with the calculated ones
obtained using the time dependent density functional theory
(TDDFT) method (Fig. 4). Comparison of the experimental ECD
spectrum of (ꢀ)-1 with calculated ECD spectrum of (2S)-1 (Fig. 4)
further confirmed its 2S absolute configuration. Indeed, the calcu-
lated ECD spectrum showed a positive CE at 214 nm and two neg-
ative CEs at 238 and 271 nm which were in good agreement with
the experimental spectrum (Fig. 4). It is interesting to note that
X-ray diffraction analysis (Fig. 3) and conformational calculations
(Fig. S-31) both showed that the pyran ring of (ꢀ)-1 had a half-
chair conformation with P-helicity. The negative 1Lb band CE
(288 nm) observed on the experimental ECD spectrum of (ꢀ)-1
was, therefore, consistent with the helicity rule established for
the chroman chromophore (Kurtán et al., 2012; Slade et al.,
2005). The experimental ECD spectrum of (+)-1 showed CEs of
opposite signs, i.e. one negative at 227 nm and two positive at
239 and 288 nm, thus confirming its 2R absolute configuration.
In the case of (ꢀ)-2 and (ꢀ)-3, the positive CE at 224 nm and the
two negative CEs at 239 and 289 nm, observed on their experimen-
tal ECD spectrum, correlated well with the calculated ECD spectra
of 2S-2 and 2S-3, respectively (Fig. 4). The absolute configurations
of (+)-2 and (+)-3 were determined to be 2R based on the negative
and the two positive CEs observed on both ECD spectrum at 224,
239 and 289 nm, respectively. For compound 4, a relative trans
2,3 configuration with the hydroxyphenyl and the hydroxyl in
Balsacone G (4) was obtained as a yellow powder and its molec-
ular formula was established as C24H22O6 based on the [M+Na]+
quasimolecular ion peak at m/z 429.1330 in the HRMS spectrum.
The IR spectrum had bands at 3300 and 1628 cmꢀ1, due to phenol
and carbonyl functions, respectively. Details analysis of 1D 1H, 2D-
COSY, HSQC and HMBC showed the presence of a 3,5,7,40-tetra-
hydroxyflavan skeleton and a dihydrocinnamoyl moeity. HMBC
correlations of dC 161.8 (C-9) with dH 4.80 (1H, d, J = 7.5 Hz, H-2)
and 5.97 (1H, s, H-8) indicated that position C-8 on the 3,5,7,40-tet-
rahydroxyflavan skeleton is protonated. Also, a W-coupling be-
tween dC 205.6 (C-900) and H-8 was observed on the HMBC
spectrum indicating that the dihydrocinnamoyl unit was attached
at C-6. Based on the above evidence, balsacone G (4) was charac-
terized as 6-(dihydrocinnamoyl)-3,5,7,40-tetrahydroxyflavan.

144
F. Simard et al. / Phytochemistry 100 (2014) 141–149
A potential biosynthetic pathway for racemic mixtures 1, 2
and 3 could consist of a non-stereospecific cyclization between
C-20-OH and C-700 in compounds 12, 11 and 10, respectively
(Fig. 5). Formation of racemic mixtures 5 and 6 could then be
explained by a trans-hydroxylation in position 3 of the racemic
mixtures 1 and 2, respectively. Additionally, non-stereospecific
intramolecular cyclization between C-40-OH and C-700 in com-
pound 12 followed by
a trans-hydroxylation in position 3
would explain the formation of racemic mixture 4 (Fig. 5).
Recently, a pair of flavonol racemates and a flavonol racemic
mixture having similar structures to compounds 1–6 were iso-
lated from chinese propolis (Sha et al., 2009). The fact that they
also appear as racemic mixtures in the propolis extract suggests
that their biosynthesis might be related to that of compounds
1–6.
Antibacterial activity and cytotoxicity of enantiomers pairs from 1–6
Antibacterial activity of pairs of enantiomers 1–6 was evalu-
ated against S. aureus after 24 h of incubation and the results
were expressed as minimal inhibitory concentration (MIC).
Chloramphenicol and gentamycin were used as positive controls
and showed MIC of 0.94 and 0.02
(ꢀ)-1 and (+)-1 possess higher activity than the other pairs of
enantiomers tested (2–6), with a MIC of 6.25 M for both com-
lM, respectively. Compounds
Fig. 3. Single crystal X-ray structure of 1a.
l
pounds. No difference was observed between the activity of the
(+) and the (ꢀ) enantiomers for the six pairs evaluated. Com-
pounds (ꢀ)-4, (+)-4, (ꢀ)-5, (+)-5, (ꢀ)-6 and (+)-6 were found ac-
the axial position of the half chair pyran ring was established based
on the J2–3 coupling constant of 7.5 Hz observed, in its 1H NMR
spectrum. Compound (+)-4 was assigned to having a 2R,3S absolute
configuration, since its ECD spectrum was well correlated with the
calculated ECD spectrum of (2R,3S)-4. The experimental ECD spec-
trum of (ꢀ)-4 was the opposite to that of (+)-4 and the absolute
configuration of (ꢀ)-4 was, therefore, determined to be 2S,3R. In
this case, the results obtained from ECD calculations conducted
for 2R,3S-4 indicated that the helicity rule established for flavan-
3-ols (Slade et al., 2005) was not applicable for the determination
of the absolute configuration of compound 4. The J2–3 coupling
constant of 8.4 Hz observed in the 1H NMR spectra of 5 and 6 also
indicated a 2,3-trans relative configuration. The absolute configu-
rations of (ꢀ)-5 and (ꢀ)-6 were established as 2R,3S, based on good
agreement of the respective experimental ECD spectrum with the
calculated ECD spectrum for (2R,3S)-5 and (2R,2S)-6 (Fig. 4). The
experimental ECD spectrum of (+)-5 and (+)-6 showed CEs of oppo-
site sign to those present in spectrum of (ꢀ)-5 and (ꢀ)-6 indicating
a 2S,3R absolute configuration.
tive with MIC ranging from 25 to 50
lM, while compounds (ꢀ)-
2, (+)-2, (ꢀ)-3 and (+)-3 were inactive at a concentration of
50 M. These results suggest that an hydroxyl group at position
l
4 (compounds (ꢀ)-5 and (+)-5) or a methoxy group at position
400 (compounds (ꢀ)-2 and (+)-2) decrease antibacterial activity.
Previously reported antibacterial activity of balsacones A (10),
B (11) and C (12) (Lavoie et al., 2013) on the same S. aureus
strain (MIC ranging between 3.1 and 6.3) are similar to the
activities obtained for compounds (ꢀ)-1 and (+)-1. Finally, puri-
fied enantiomers cytotoxicity was evaluated on human skin
fibroblasts WS1. Etoposide was used as
(IC50 = 23 3). All compounds were not cytotoxic at a concentra-
tion of 50 M.
a positive control
l
Concluding remarks
Proposed biosynthetic pathway
Isolation of racemic mixtures from plant extracts is rather
uncommon (Finefield et al., 2012). In this work, 6 new com-
pounds were isolated as racemic mixtures from an ethanolic ex-
tract of buds from P. balsamifera. Chiral HPLC allowed the
separation of these mixtures to obtain 12 new flavan derivatives
consisting of 6 pairs of enantiomers. Since it is rather exceptional
to be able to obtain both enantiomers of closely related chiral
compounds, the 12 new balsacones constitute a unique library
of compounds. This library can be especially useful to study rela-
tions between chiral structures and their spectral data. ECD spec-
troscopy, for example, can be used to establish useful rules for the
determination of the absolute configuration of the chroman ring
in natural products. The library also contains compounds having
toxicity towards S. aureus bacteria with MIC ranging between
Recently, it was proposed that the biosynthesis of balsacones A
(10), B (11) and C (12) proceeded by cinnamylation of dihydroch-
alcones 7, 8 and 9, respectively (Fig. 5), which were also identified
in the P. balsamifera buds ethanolic extract (Lavoie et al., 2013). The
presence of compounds 10–12 in the extract and the fact that com-
pounds 1–6 were isolated as racemic mixtures suggested that the
later might be artifacts from the extraction process which was
conducted under conditions of EtOH reflux. Indeed, the presence
of 1–6 could be the result of an intramolecular addition of the phe-
nol function in 20 or 40 on the alkene of 10, 11 or 12. To rule out this
possibility, buds were extracted in cold EtOH for a shorter period of
time. The resulting extract was submitted to fractionation using re-
versed phase flash chromatography. The resulting fractions were
analyzed using HPLC–MS–SIM selecting ions at m/z 301, 391,
407, 421 and 437. As shown in Fig. 6, the presence of 1–6 could
be confirmed in fraction II. This result suggest that 1–6 originated
rather from the buds.
6.25 and 50 lM. Further in vitro study of the antibacterial activity
of the compounds of the library is in progress in the LASEVE lab-
oratory to establish a clearer relation between the structures and
absolute configuration of the isolated compounds and their anti-
bacterial activity.

F. Simard et al. / Phytochemistry 100 (2014) 141–149
145
Fig. 4. Experimental ECD spectra for compounds 1–6 and calculated (TDDFT at the B3LYP/6-311G(2d,2p) level) EDC spectra of compounds (2S)-1, (2S)-2, (2S)-3, (2R,3S)-4,
(2R,3S)-5 and (2R,3S)-6.
All spectra were acquired in acetone-d6 and chemical shifts were
reported in ppm (d) relative to TMS. High resolution electrospray
ionisation mass spectra were recorded on an Agilent Technology
6210 TOF MS system. APCI MS (positive mode) spectrum were ob-
tained with an Agilent 1100 series HPLC equipped with an Agilent
G1946 VL Mass Selective Detector in the positive mode at 70 eV.
The column used for HPLC-APCI MS analysis was an Inerstil Prep
Experimental
General
Melting points were measured with a Mettler Roledo MP70
melting point system (60–350 °C, 5 °C/min, uncorr.). Optical rota-
tions were obtained at the sodium D line (589 nm) on a Rudolph
Research Analytical Autopol IV automatic polarimeter. Absorption
UV–Vis spectra were recorded using an Agilent 8453 diode-array
spectrophotometer while ECD spectra were measured on a Jasco
J-815 ECD spectrometer. FTIR spectra were acquired on a Perkin-
Elmer SpectrumOne (neat, thin films on NaCl plates). The 1D and
2D NMR spectra (1H–1H COSY, NOESY, HSQC and HMBC) were ob-
tained using an Avance 400 Bruker spectrometer (400.13 MHz for
1H, 100.61 MHz for 13C spectra) equipped with a 5 mm QNP probe.
ODS column (6 ꢁ 250 mm, 10
HPLC–MS–SIM analysis were conducted using a Zorbax Eclips
m) selecting ions at m/z 301,
lm) at a flow rate of 1 ml/min.
XDB-C18 column (4.6 ꢁ 250 mm, 5
l
391, 407, 421 and 437 at 1 ml/min on the same instrument.
Semi-preparative HPLC was carried out on an Agilent 1100 HPLC
series with an Inerstil Prep ODS column (20 x 250 mm, 10
a flow rate of 10 ml/min and Chiral semi-preparative HPLC with
tris-(3,5-dimethylphenyl)carbamoyl amylose chiral coated
lm) at
a

146
F. Simard et al. / Phytochemistry 100 (2014) 141–149
5'
3
O
OH
R1
4
4'
3'
2
1
R2
6'
1'
O
OH
O
R1
cinnamylation
8
R2
Non-stereospecific
2'-OH-7'' cyclization
9
5
9''
8''
6''
5''
2'
7
6
1, 2 and 3
O
O
7''
H
OH
1''
2''
10 R1 = OH, R2 = CH3
11 R1 = OCH3, R2 = H
12 R1 = R2 = H
Trans hydroxylation
5 and 6
7 R1 = OH, R2 = CH3
8 R1 = OCH3, R2 = H
9 R1 = R2 = H
3''
4''
OH
Non-stereospecific
4'-OH-7'' cyclization
HO
O
OH
Trans hydroxylation
4
OH
O
Fig. 5. Proposed biosynthetic pathway.
Fig. 6. HPLC–MS–SIM chromatogram trace of fraction II obtained from a cold solvent extract.
column (RegisPack™, 10 ꢁ 250 mm, 5
l
m) at a flow rate of 4 ml/
was extracted 5 times (5 h each time) with EtOH–H2O (95:5, V/
V) 2.5 L, under conditions of reflux and the extracts were com-
bined. After evaporation of the solvent using a rotary evaporator,
the residue was suspended in MeOH and washed with hexanes.
The dried MeOH residue (380.4 g) was suspended in Et2O and ex-
tracted with H2O. The dried Et2O phase (355.1 g) was submitted
to three successive fractionation steps: (1) silica gel CC (CHCl3–
MeOH, 40:1, 20:1, 10:1, 0:1); (2) silica gel CC (CHCl3–MeOH,
40:1, 30:1, 20:1, 10:1, 0:1); (3) C18 flash chromatography
(MeOH–H2O, 65:35, 70:30, 72:28, 0:100). The third fractionation
step afforded 5 fractions (A–E). Fraction A (19.3 g) was suspended
in a CHCl3–EtOAc mixture (10:1, 100 ml) and filtrated. The filtrate
was recovered, dried, and the residue (5.7 g) was submitted to sil-
ica gel CC (CHCl3–EtOAC, 5:1, 4:1, 3:1, 2:1, 1:1, followed by MeOH)
to afford 9 subfractions (A1–A9). A1 (261.2 mg) was purified by
semi-preparative HPLC (CH3CN–H2O, 50:50) to yield racemic mix-
ture 1 (TR = 33.3 min, 58.3 mg) which was purified by chiral semi-
preparative HPLC (Hexane-IPA, 92:8) to afford (ꢀ)-1 (TR = 21.9 min,
15.9 mg) and (+)-1 (TR = 23.2 min, 18.5 mg). A2 (152.5 mg) was
submitted to semi-preparative HPLC (CH3CN–H2O, 50:50) to obtain
racemic mixture 2 (TR = 31.2 min, 18.3 mg) which was purified by
chiral semi-preparative HPLC (hexane-IPA, 92:8) to afford (ꢀ)-2
(TR = 25.9 min, 8.6 mg) and (+)-2 (TR = 28.7 min, 6.1 mg). A3
min. Reversed phase flash chromatography was conducted on a
Biotage Flash+ system using a C18 40iM cartridge (carbon loading
17%, 135 g) from Silicycle Inc. (Ville de Québec, Québec, Canada).
Reagents and analytical grade solvents were purchased from
VWR International (Ville Mont-Royal, Québec, Canada) and were
used as received. Ultra pure silica gel (40–63
lm) and glass TLC
plates (40–63 m with F254 indicator) were supplied by Silicycle
l
Inc. (Ville de Québec, Québec, Canada).
Plant material
Buds from different trees of P. balsamifera were collected in
March 2005 in the Chicoutimi region, Québec, Canada. The plant
was authenticated by Mr. Patrick Nadeau (Université du Québec à
Chicoutimi) and a voucher specimen (No. 499678) was deposited
at the Louis-Marie Herbarium of Université Laval, Québec City, Qué-
bec, Canada. After collection, buds were kept frozen until extraction.
Extraction and isolation
Buds (1036 g) previously immersed in li. N2 were coarsely
ground using an electrical blender. The resulting crude powder

F. Simard et al. / Phytochemistry 100 (2014) 141–149
147
Table 2
(119.7 mg) was applied to a semi-preparative HPLC (MeOH–H2O,
13C NMR spectroscopic data of compounds 1–6 in acetone-d6 (100 MHz).
80:20 ? 100:0, 30 min) to yield racemic mixture 3 (TR = 15.9 min,
30.0 mg) which was purified by chiral semi-preparative HPLC (hex-
ane-IPA, 90:10) to obtain (ꢀ)-3 (TR = 26.7 min, 6.5 mg) and (+)-3
(TR = 29.1 min, 6.1 mg). A4 (545.0 mg) was subjected to two
semi-preparative HPLC purification steps [(1) MeOH–H2O, 75:25;
(2) CH3CN–H2O, 50:50] to obtain racemic mixture 4 (TR = 22.8 min,
15.2 mg) which was submitted to chiral semi-preparative HPLC
(hexane-IPA, 85:15) to yield (ꢀ)-4 (TR = 32.5 min, 3.4 mg) and
(+)-4 (TR = 40.7 min, 2.5 mg). A7 (244.9 mg) was subjected to
semi-preparative HPLC (MeOH–H2O, 60:40) to obtain racemic mix-
ture 5 (TR = 44.5 min, 32.0 mg) which was purified by chiral semi-
preparative HPLC (hexane-IPA, 90:10) to afford (ꢀ)-5 (TR = 25.8 -
min, 12.2 mg) and (+)-5 (TR = 30.0 min, 12.6 mg). Fractions A8
(90.1 mg) and A9 (118.9 mg) were both subjected to semi-prepara-
tive HPLC (CH3CN–H2O, 40:60) and further purified by another
semi-preparative HPLC (MeOH–H2O, 65:35) to obtain racemic mix-
ture 6 (TR = 25.6 min, 11.2 mg) which was further purified by chiral
semi-preparative HPLC (hexane-IPA, 85:15) to yield (ꢀ)-6
(TR = 13.0 min, 1.5 mg) and (+)-6 (TR = 15.7 min, 1.2 mg).
No.
dc, mult.
1
2
3
4
5
6
2
3
4
5
6
7
8
9
79.9, d
29.1, t
20.4, t
163.2, s
96.1, d
166.0, s
105.7, s
159.7, s
102.0, s
132.7, s
79.9, d
29.1, t
20.4, t
163.1, s
96.0, d
166.0, s
105.7, s
159.7, s
102.0, s
132.7, s
79.8, d
29.1, t
20.3, t
164.7, s
92.8, d
166.1, s
106.1, s
158.7, s
102.7, s
132.6, s
83.3, d
67.5, d
27.7, t
165.0, s
105.4, s
160.6, s
95.1, s
161.8, s
101.2, s
130.6, s
83.8, d
67.1, d
29.3, t
163.3, s
96.5, d
166.1, s
105.4, s
158.7, s
101.3, s
130.1, s
83.8, d
67.1, d
29.4, t
164.0, s
96.5, d
165.8, s
105.1, s
158.7, s
101.5, s
130.3, s
10
10
20/60
30/50
40
128.9, d 129.0, d 128.8, d 129.4, d 129.9, d 130.0, d
116.2, d 116.2, d 116.2, d 115.9, d 116.1, d 116.1, d
158.3, s
142.3, s
129.1, d 129.9, d 129.9, d 129.3, d 129.0, d
129.0, d 114.4, d 115.8, d 129.2, d 129.0, d
126.4, d 158.7, s
31.2, t
46.1, t
205.2, s
–
158.4, s
134.1, s
158.4, s
132.7, s
158.2, s
142.9, s
158.4, s
142.2, s
158.4, s
134.1, s
129.9, d
114.4, d
100
200/600
300/500
400
156.2, s
30.4, t
46.6, t
205.8, s
56.3, q
–
126.7, d 126.4, d 158.7, s
700
30.3, t
46.4, t
205.4, s
–
31.4, t
46.5, t
205.6, s
–
31.2, t
45.9, t
205.2, s
–
30.6, t
46.3, t
205.4, s
–
800
900
5-O–CH3
400-O–CH3
–
55.4, q
–
–
55.3, q
Detection of compounds 1–6 in a cold solvent extract
Buds previously immersed in li. N2 were coarsely ground using
an electrical blender. The resulting crude powder (25.0 g) was ex-
tracted three times (30 min each time) with EtOH (150 ml) on a re-
ciprocal shaker at room temperature. Extracts were combined and
evaporated to dryness (2.3 g). Part of the dry residue (500 mg) was
submitted to reversed phase flash chromatography (MeOH–H2O,
50:50, 60:40, 70:30, 80:20, 90:10, 100:0) yielding 7 fractions
(I–VII). Fraction II (97.5 mg) was submitted to HPLC–MS–SIM
analysis.
(+)-Balsacone D [(+)-1]
Yellow powder;
½
a 2D1
ꢂ
+15.5 (c 0.16, Me2CO), ECD (c
e239 = +1.8, e288 = +2.4; UV,
1.3 ꢁ 10ꢀ3 M, MeOH):
D
e227 = ꢀ1.1,
D
D
IR, 1H, 13C NMR, APCIMS and ESIMS data were the same as (ꢀ)-1.
(ꢀ)-Balsacone E [(ꢀ)-2]
Yellow powder; ½a D22
ꢂ
ꢀ9.8 (c 0.1, Me2CO), ECD (c 1.2 ꢁ 10ꢀ3 M,
MeOH):
D
e224 = +3.8,
D
e239 = ꢀ1.6,
D
e289 = ꢀ1.9; UV (MeOH) kmax
(loge): 206 (4.1), 292 (3.9), 330 (3.3); IR mmax 3400, 1622, 1512,
1440, 1424, 1373, 1243, 1220, 1147, 1086, 1030, 989, 9 55, 897,
827, 678, 598, 569, 535 cmꢀ1; for 1H and 13C NMR spectroscopic
Physical data of new compounds
data, see Tables 1 and 2; APCIMS: m/z 421 [M+H]+ (100), 301
(ꢀ)-Balsacone D [(ꢀ)-1]
(42); HRESIMS: m/z 421.1656 [M+H]+ (calcd. for C25H25O6
,
+
Yellow powder; mp 186–188; ½a D22
ꢂ
ꢀ17.4 (c 0.25, Me2CO); ECD
e239 = ꢀ2.0, e288 = ꢀ2.6;
421.1646).
(c 1.3 ꢁ 10ꢀ3 M, MeOH):
D
e227 = +4.0,
D
D
UV (MeOH) kmax (loge): 210 (4.2), 293 (4.1), 330 (3.5); IR mmax
3347, 1615, 1517, 1424, 1372, 1337, 1220, 1145, 1085, 988, 956,
899, 831, 699 cmꢀ1; for 1H and 13C NMR spectroscopic data, see
Tables 1 and 2; APCIMS: m/z 391 [M+H]+ (100), 271 (60); HRESIMS:
m/z 413.1374 [M+Na]+ (calcd. for C24H22O5Na+, 413.1359).
(+)-Balsacone E [(+)-2]
Yellow powder;
½
a 2D2
+13.4 (c 0.09, Me2CO), ECD (c
ꢂ
1.2 ꢁ 10ꢀ3 M, MeOH):
D
e224 = ꢀ2.6,
De239 = +2.4, De289 = +2.7; UV,
IR, 1H, 13C NMR, APCIMS and ESIMS data were the same as (ꢀ)-2.
Table 1
1H NMR spectroscopic data of compounds 1–6 in acetone-d6 (400 MHz).
No.
dH ( J in Hz)
1
2
3
4
5
6
2
3a
3b
4a
4b
6
5.07, dd (11.0, 2.0)
2.06, m
2.19, m
2.68, m
2.78, ddd (17.0, 5.9, 2.4)
6.02, s
5.08, dd (10.7, 2.3)
2.11, m
2.19, m
2.70, m
2.79, m
6.03, s
5.06, dd (10.8, 2,1)
2.06, m
2.19, m
2.66, m
2.72, m
6.10, s
4.80, d (7.5)
4.09, td (7.0, 5.5)
–
2.55, dd (16.1, 8.1)
2.88, dd (16.1, 5.3)
–
4.81, d (8.4)
4.15, td (8.8, 5.9)
–
2.59, dd (16.0, 9.0)
3.02, dd (16.2, 5.7)
6.05, s
4.79, d (8.4)
4.15, td (8.8, 5.8)
–
2.59, dd (16.2, 9.0)
3.03, dd, (16.2, 5.9)
6.05, s
8
–
–
–
5.97, s
7.24, d (8.6)
6.84, d (8.6)
7.29, m
7.29, m
7.18, m
3.00, t (7.7)
3.43, t (7.7)
–
–
–
20/60
30/50
200/600
300/500
400
7.35, d (8.5)
6.85, d (8.5)
6.91, d (7.3)
7.16, t (7.3)
7.10, t (7.3)
2.84, t (8.1 Hz)
3.24, m
13.82, s
–
–
7.37, d (8.5)
6.88, d (8.5)
6.79, d (8.7)
6.72, d (8.7)
–
2.77, m
3.19, m
13.81, s
3.74, s
7.35, d (8,6)
6.87, d (8,6)
6.71, d (8,6)
6.64, d (8,6)
–
2.75, m
3.20, m
13.92, s
–
7.34, d (8.4)
6.85, d (8.4)
6.89, d (7.2)
7.15, t (7.2)
7.09, t (7.2)
2.84, t (7.9)
3.22, m
13.76, s
–
–
7.36, d (8.6)
6.87, d (8.6)
6.78, d (8.6)
6.71, d (8.6)
–
2.76, t (7.9)
3.16, m
13.83, s
700
800
7-OH
400-O–CH3
5-O–CH3
–
–
3.74, s
–
–
3.89, s

148
F. Simard et al. / Phytochemistry 100 (2014) 141–149
(ꢀ)-Balsacone F [(ꢀ)-3]
100 K. Crystal data were: C24H22O5, M = 390.42, monoclinic, space
group P21, a = 8.42583(14) Å, b = 24.2381(4) Å, c = 9.38693(15) Å,
b = 104.9617(9)°, Z = 4,
V = 1852.05(5) Å3, = 1.400 g/cm3,
(CuK total reflections 104988, independent
) = 0.797 mmꢀ1
reflections 7175 (Rint 0.038%), final indices [I > 2 (I)]
R1 = 0.0351, wR2 = 0.0943, absolute structure parameter = 0.03
(11). The structure was solved by direct methods SHELXS-97
(Sheldrick, 2008) and refined with full-matrix least-squares calcu-
lations on F2 using SHELX-97 (Sheldrick, 2008). Crystallographic
data for compound (ꢀ)-1 were deposited at the Cambridge Crystal-
lographic Data Center (Deposition No. CDCC 919719). Copies of
these data can be obtained free of charge via www.ccdc.cam.a-
c.uk/conts/retrieving.html or from the Cambridge Crystallographic
Data Center, 12, Union Road, Cambridge, CB2 1EZ, UK. (fax: +44
1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
Yellow powder;
½
a 2D1
ꢂ
ꢀ10.0 (c 0.09, Me2CO), ECD (c
1.2 ꢁ 10ꢀ3 M, MeOH):
D
e227 = +5.0, e289 = ꢀ1.1; UV
D
e239 = ꢀ1.3,
D
q
(MeOH) kmax (loge): 224 (4.4), 287 (4.2), 327 (3.4); IR mmax 3364,
2922, 2851, 1701, 1618, 1589, 1516, 1443, 1372, 1215, 1143,
1113, 1001, 969, 849, 831, 700, 613, 596, 569, 540 cmꢀ1; For 1H
and 13C NMR spectroscopic data, see Tables 1 and 2; APCIMS: m/
z 421 [M+H]+ (100), 301 (89); HRESIMS: m/z 443.1477 [M+Na]+
(calcd. for C25H24O6Na+, 443.1465).
l
a
,
R
r
(+)-Balsacone F [(+)-3]
Yellow powder;
½
a 2D3
ꢂ
+12.1 (c 0.09, Me2CO); ECD (c
e239 = +2.0, e289 = +1.6; UV,
1.2 ꢁ 10ꢀ3 M, MeOH):
D
e227 = ꢀ2.0,
D
D
IR, 1H, 13C NMR, APCIMS and ESIMS data were the same as (ꢀ)-3a.
(ꢀ)-Balsacone G [(ꢀ)-4]
Yellow powder;
½
a 2D2
D
ꢂ
ꢀ43.5 (c 0.03, Me2CO), ECD (c
e238 = +3.0,
e290 = ꢀ3.8; UV
Conformational analysis, geometrical optimization and ECD
calculation
1.2 ꢁ 10ꢀ3 M, MeOH):
e225 = +3.7,
D
D
(MeOH) kmax (loge): 210 (4.6), 290 (4.6), 327 (3.7); IR mmax 3300,
2923, 1698, 1628, 1599, 1517, 1430, 1368, 1246, 1146, 1089,
988, 921, 832, 750, 700, 560, 522 cmꢀ1; for 1H and 13C NMR spec-
troscopic data, see Tables 1 and 2; APCIMS: m/z 407 [M+H]+ (100),
271 (5); HRESIMS: m/z 429.1330 [M+Na]+ (calcd. for C24H22O6Na+,
429.1309).
Conformational distributions for 1–6 were obtained through
stochastic Monte Carlo guided conformational searches at the
molecular mechanics (MMFF) level of theory using the Spartan’10
V1.1.0 software package (Wavefunction, Inc., Irvine, CA, 2010).
These conformers were subjected to geometry optimizations by
the density functional theory (DFT) method at B3LYP/6-31G(d) le-
vel on the Gaussian 09 software (Frisch et al., 2010). Selected opti-
mized conformers (>5% of Boltzmann distribution) were then
subjected to the TDDFT calculation on Gaussian 09 for 40 excited
states at B3LYP/6-311G(2d,2p) level. MeOH solvent effects were ta-
ken into account using PCM model. Excitation energy (in nm) and
rotatory strength in (velocity form in 1040 erg-esu-cm/Gauss) were
thus obtained for the different states. The ECD spectra were simu-
lated from these data by normalization, Boltzmann averaging and
overlapping Gaussian functions for each transition according to:
(+)-Balsacone G [(+)-4]
Yellow powder;
½
a 2D3
+66.2 (c 0.03, Me2CO), ECD (c
ꢂ
1.2 ꢁ 10ꢀ3 M, MeOH):
D
e225 = ꢀ3.6,
D
e238 = ꢀ3.8,
De290 = +4.9;
UV, IR, 1H, 13C NMR, APCIMS and ESIMS data were the same as
(ꢀ)-4.
(ꢀ)-Balsacone H [(ꢀ)-5]
Yellow powder; ½a D25
ꢂ
ꢀ38.2 (c 0.2, Me2CO), ECD (c 1.2 ꢁ 10ꢀ3 M,
MeOH):
D
e226 = +3.2,
D
e230 = +2.7,
D
e239 = ꢀ0.4,
D
e287 = ꢀ4.0; UV
(MeOH) kmax (loge): 226 (4.4), 291 (4.2), 327 (3.6); IR mmax 3450,
2091, 1632, 1518, 1497, 1450, 1429, 1378, 1223, 1147,
1062 cmꢀ1; for 1H and 13C NMR spectroscopic data, see Tables 1
and 2; APCIMS: m/z 407 [M+H]+ (100), 271 (46); HRESIMS: m/z
445.1055 [M+K]+ (calcd. for C24H22O6K+, 445.1048).
A
X
1
1
D
EiÞ=ð2
r
Þꢂ2
D
EiRieꢀ½ðEꢀ
pffiffiffiffiffiffiffiffiffi
D
eðEÞ ¼
2:297 ꢁ 10ꢀ39
2
pr
i
where
r
is the width of the band at 1/e height and
D
Ei and Ri are the
excitation energies and rotatory strengths for transition i, respec-
tively.
r
= 0.15 eV and Rvel have been used in this work.
(+)-Balsacone H [(+)-5]
Yellow powder; ½a D25
ꢂ
+36.9 (c 0.2, Me2CO), ECD (c 1.2 ꢁ 10ꢀ3 M,
Antibacterial activity
MeOH):
D
e226 = ꢀ2.2,
D
e230 = ꢀ2.6,
De239 = +0.9, De287 = +4.7; UV,
IR, 1H, 13C NMR, APCIMS and ESIMS data were the same as (ꢀ)-5.
(ꢀ)-Balsacone I [(ꢀ)-6]
Antibacterial activity against S. aureus was evaluated using the
microdilution method as reported previously (Banfi et al., 2003;
Lavoie et al., 2013). The lowest concentration at which no growth
was observed after 24 h of incubation was defined as the minimum
inhibitory concentration (MIC). Gentamycin and chloramphenicol
were used as positive controls.
Yellow powder;
½
a 2D5
ꢂ
ꢀ16.2 (c 0.02, Me2CO), ECD (c
e240 = ꢀ0.6, e288 = ꢀ2.3;
0.57 ꢁ 10ꢀ3 M, MeOH):
D
e226 = +2.7,
D
D
UV(MeOH) kmax (loge): 217 (4.4), 291 (4.3), 325 (3.8); IR mmax
3337, 2924, 1614, 1512, 1425, 1373, 1223, 1177, 147, 1103,
1060, 1031, 971, 933, 829 cmꢀ1; for 1H and 13C NMR spectroscopic
data, see Tables 1 and 2; APCIMS: m/z 437 [M+H]+ (100), 301 (56);
HRESIMS: m/z 459.1431 [M+Na]+ (calcd. for C25H24O7Na+,
459.1414).
Cytotoxicity
Cytotoxicity was evaluated against human fibroblast cell line
WS1 using the resazurin reduction test reported previously (Bellila
et al., 2011; O’Brien et al., 2000). Cytotoxicity was expressed as the
concentration inhibiting 50% of cell growth (IC50). Etoposide was
used as a positive control.
(+)-Balsacone I [(+)-6]
Yellow powder;
½
a 2D5
+35.0 (c 0.02, Me2CO), ECD (c
ꢂ
0.57 ꢁ 10ꢀ3 M, MeOH):
D
e226 = ꢀ2.6,
De240 = +1.69, De288 = +4.1;
UV, IR, 1H, 13C NMR, APCIMS data were the same as (ꢀ)-6; HRE-
SIMS: m/z 437.1586 [M+H]+ (calcd. for C25H25O7+, 437.1595).
Acknowledgments
X-ray crystal structure analysis of (ꢀ)-balsacone D (ꢀ)-1
This project was supported by a FQRNT fonds forestier 02 Grant,
NSERC (doctoral scholarship to FS) and by Fonds de recherche nat-
ure et technologies (doctoral scholarship to FS). This research has
been enabled by the use of computing resources provided by West-
Grid and Compute/Calcul Canada. The authors would like to thank
Diffraction intensity data were collected by a Bruker smart dif-
fractometer equipped with an APEX II CCD detector employing
graphite monochromated Cu
Ka radiation (k = 1.54178 Å) at

F. Simard et al. / Phytochemistry 100 (2014) 141–149
149
Dapprich, S., Daniels, A.D., Farkas, O., Foresman, J.B., Ortiz, J.V., Cioslowski, J.,
Fox, D.J., 2010. Gaussian 09, Revision C.01, Gaussian, Inc., Wallingford, CT.
Greenaway, W., Whatley, F.R., 1990. Resolution of complex mixtures of phenolics in
poplar bud exudate by analysis of gas chromatography—mass spectrometry
data. J. Chromatogr. A 519, 145–158.
Greenaway, W., May, J., Whatley, F.R., 1989. Flavonoid aglycones identified by gas
chromatography–mass spectrometry in bud exudate of Populus balsamifera. J.
Chromatogr. 472, 393–400.
Greenaway, W., English, S., Whatley, F.R., 1992a. Relationships of
Populus ꢁ acuminata and Populus ꢁ generosa with their parental species
examined by gas chromatography–mass spectrometry of bud exudates. Can. J.
Bot. 70, 212–221.
Greenaway, W., May, J., Scaysbrook, T., Whatley, F.R., 1992b. Compositions of bud
and leaf exudates of some Populus species compared. Z. Naturforsch. 47, 329–
334.
F. Otis and N. Voyer from PROTEO and Université Laval for their
help in recording circular dichroism spectra and C. Dusseault for
its technical assistance in the evaluation of antibacterial activity
and cytotoxicity.
Appendix A. Supplementary data
Supplementary data associated with this article (1H, 13C NMR
and chiral HPLC chromatograms of racemic mixtures) can be
found, in the online version, at http://dx.doi.org/10.1016/
j.phytochem.2013.12.018.
Isidorov, V.A., Vinogorova, V.T., 2003. GC–MS analysis of compounds extracted from
buds of Populus balsamifera and Populus nigra. Z. Naturforsch. [C] 58, 355–360.
Kurkin, V.A., Zapesochnaya, G.G., Braslavskii, V.B., 1990. Flavonoids of the buds of
Populus balsamifera. Chem. Nat. Compd. 26, 224–225.
References
Kurtán, T., Antus, S., Pescitelli, G., 2012. Electronic CD of Benzene and other
Aromatic Chromophores for Determination of Absolute Configuration.
Comprehensive Chiroptical Spectroscopy. John Wiley & Sons, Inc. (pp. 73–114).
Lavoie, S., Legault, J., Simard, F., Chiasson, É., Pichette, A., 2013. New antibacterial
dihydrochalcone derivatives from buds of Populus balsamifera. Tetrahedron Lett.
54, 1631–1633.
Mattes, B.R., Clausen, T.P., Reichardt, P.B., 1987. Volatile constituents of balsam
poplar: the phenol glycoside connection. Phytochemistry 26, 1361–1366.
Moermann, D.E., 1998. Native American Ethnobotany. Timber Press, Inc..
O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the alamar blue
(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.
Eur. J. Biochem. 267, 5421–5426.
Pearl, I.A., Darling, S.F., 1968a. Studies on the barks of the family Salicaceae—xix:
continued studies on the hot water extractives of Populus balsamifera bark.
Phytochemistry 7, 1851–1853.
Pearl, I.A., Darling, S.F., 1968b. Studies on the barks of the family Salicaceae—XX:
variations in the hot water extractives of Populus balsamifera bark.
Phytochemistry 7, 1855–1860.
Pelter, A., Stainton, P., Barber, M., 1965. The mass spectra of oxygen heterocycles. II.
The mass spectra of some flavonoids. J. Heterocycl. Chem. 2, 262–271.
Reichardt, P.B., Bryant, J.P., Mattes, B.R., Clausen, T.P., Chapin, F.S., Meyer, M., 1990.
Winter chemical defense of Alaskan balsam poplar against snowshoe hares. J.
Chem. Ecol. 16, 1941–1959.
Salatino, A., Fernandes-Silva, C.C., Righi, A.A., Salatino, M.L.F., 2011. Propolis
research and the chemistry of plant products. Nat. Prod. Rep. 28, 925–936.
Sentsov, M.F., Braslovskii, V.B., Kurkin, V.A., Zapesochnaya, G.G., Bakulin, V.T.,
Pravdivtseva, O.E., 1997. Comparative study of the component composition of
buds of some Populus L. species with HPLC method. Rastitel’nye Resursy 33, 51–
56.
Sha, N., Guan, S.-H., Lu, Z.-Q., Chen, G.-T., Huang, H.-L., Xie, F.-B., Yue, Q.-X., Liu, X.,
Guo, D.-A., 2009. Cytotoxic constituents of Chinese propolis. J. Nat. Prod. 72,
799–801.
Sheldrick, G.M., 2008. A short history of SHELX. Acta Crystallogr. A 64, 112–122.
Slade, D., Ferreira, D., Marais, J.P.J., 2005. Circular dichroism, a powerful tool for the
assessment of absolute configuration of flavonoids. Phytochemistry 66, 2177–
2215.
Banfi, E., Scialino, G., Monti-Bragadin, C., 2003. Development of a microdilution
method to evaluate Mycobacterium tuberculosis drug susceptibility. J.
Antimicrob. Chemother. 52, 796–800.
Bankova, V., 2005. Recent trends and important developments in propolis research.
Evid. Based Complement. Altern. Med. 2, 29–32.
Bankova, V., Popova, M., Bogdanov, S., Sabatini, A.G., 2002. Chemical composition of
European propolis: expected and unexpected results. Z. Naturforsch. [C] 57,
530–533.
Bellila, A., Tremblay, C., Pichette, A., Marzouk, B., Mshvildadze, V., Lavoie, S., Legault,
J., 2011. Cytotoxic activity of withanolides isolated from Tunisian Datura metel
L. Phytochemistry 72, 2031–2036.
Chen, F., Liu, C.-J., Tschaplinski, T.J., Zhao, N., 2009. Genomics of secondary
metabolism in Populus: interactions with biotic and abiotic environments.
Crit. Rev. Plant Sci. 28, 375–392.
Christov, R., Trusheva, B., Popova, M., Bankova, V., Bertrand, M., 2006. Chemical
composition of propolis from Canada, its antiradical activity and plant origin.
Nat. Prod. Res. 20, 531–536.
Dickmann, D.I., 2001. An overview of the genus Populus. In: Dickmann, D.I.,
Isebrands, J.G., Eckenwalder, J.E., Richardson, J. (Eds.), Poplar Culture in North
America. NRC Research Press, Ottawa, pp. 1–42.
Dudonné, S., Poupard, P., Coutière, P., Woillez, M., Richard, T., Mérillon, J.-M., Vitrac,
X., 2011. Phenolic composition and antioxidant properties of poplar bud
(Populus nigra) extract: Individual antioxidant contribution of phenolics and
transcriptional effect on skin aging. J. Agric. Food Chem. 59, 4527–4536.
Eckenwalder, J.E., 1996. Systematics and evolution of Populus. In: Stettler, R.F.,
Bradshaw, H.D., Jr., Heilman, P.E., Hinckley, T.M. (Eds.), Biology of Populus and
Its Implications for Management and Conservation. NRC Research Press,
Ottawa, Canada, pp. 7–32.
Finefield, J.M., Sherman, D.H., Kreitman, M., Williams, R.M., 2012. Enantiomeric
natural products: occurrence and biogenesis. Angew. Chem. Int. Ed. 51, 4802–
4836.
Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R.,
Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M.,
Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada,
M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T.,
Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr., J.A., Peralta, J.E.,
Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N.,
Keith, T., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C.,
Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E.,
Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E.,
Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L.,
Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J.,
Tsai, C.-J., Harding, S.A., Tschaplinski, T.J., Lindroth, R.L., Yinan, Y., 2006. Genome-
wide analysis of the structural genes regulating defense phenylpropanoid
metabolism in Populus. New Phytol. 172, 47–62.
Vardar-Ünlü, G., Silici, S., Ünlü, M., 2008. Composition and in vitro antimicrobial
activity of Populus buds and poplar-type propolis. World J. Microbiol.
Biotechnol. 24, 1011–1017.