Discovery of aromatic components with excellent fragrance properties and biological activities: β-ionols with antimelanogenetic effects and their asymmetric syntheses

Article abstract of DOI:10.1248/cpb.c12-00916

Both enantiomers of dihydro-β-ionol and β-ionol, contained in the aromatic components of Osmanthus flower and of Hakuto peach, were obtained with high optical purity by lipase-catalyzed kinetic resolution of the racemates. It was found that all these enantiomers had different characteristic favorable scents and high antimelanogenetic effects. The absolute configuration and the enantiomer ratios of dihydro-β-ionol in the aromatic components of Osmanthus flower and of Hakuto peach were determined. The asymmetric synthesis of (R)-dihydro-β-ionol, one of the most valuable raw materials for fragrance and flavor, was performed from inexpensive β-ionone via lipase-catalyzed dynamic kinetic resolution followed by reduction.

Full text of DOI:10.1248/cpb.c12-00916

310
Regular Article
Chem. Pharm. Bull. 61(3) 310–314 (2013)
Vol. 61, No. 3
Discovery of Aromatic Components with Excellent Fragrance Properties
and Biological Activities: β-Ionols with Antimelanogenetic Effects and
Their Asymmetric Syntheses
,a
Ryoichi Komaki,* Takashi Ikawa,b Kozumo Saito,b Kazuyo Hattori,a Natsuyo Ishikawa,a
,b
Hidemichi Fukawa,c Masahiro Egi,b and Shuji Akai*
b
a Kanebo Cosmetics Inc.; 5–3–28 Kotobuki-cho, Odawara, Kanagawa 250–0002, Japan: School of Pharmaceutical
c
Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan: and Yukiguni Aguri Co., Ltd.;
1254 Yokotsuka-cho, Numata, Gunma 378–0002, Japan.
Received October 20, 2012; accepted December 3, 2012
Both enantiomers of dihydro-β-ionol and β-ionol, contained in the aromatic components of Osmanthus
flower and of Hakuto peach, were obtained with high optical purity by lipase-catalyzed kinetic resolution of
the racemates. It was found that all these enantiomers had different characteristic favorable scents and high
antimelanogenetic effects. The absolute configuration and the enantiomer ratios of dihydro-β-ionol in the
aromatic components of Osmanthus flower and of Hakuto peach were determined. The asymmetric synthesis
of (R)-dihydro-β-ionol, one of the most valuable raw materials for fragrance and flavor, was performed from
inexpensive β-ionone via lipase-catalyzed dynamic kinetic resolution followed by reduction.
Key words dihydro-β-ionol; antimelanogenetic effect; aromatic component; Osmanthus flower; asymmetric
synthesis; lipase-catalyzed kinetic resolution
It has long been known that spices such as cloves have study, we succeeded in obtaining both enantiomers of ( )-1
antibacterial and antioxidant effects, and they have been used and ( )-2 by enzymatic kinetic resolution of the racemates,
as food preservatives.1–5) It has been reported that one of the and determined the absolute configuration and the enantiomer
main components of cloves, eugenol, is also present in the ratios of 1 contained in the aromatic components of Osman-
essential oils of flowers such as jasmine and rose, and serves thus flower and of Hakuto peach. We clarified the aromatic
as a major aromatic component.6–11) However, research on fra- properties of each enantiomer of 1 and 2 and discovered for
grances has focused only on their aroma, and no attention has the first time that both enantiomers had an antimelanogenetic
been paid to their biological activities. Some flower and leaf effect. These compounds might therefore serve as raw materi-
extracts have antioxidant and antimelanogenetic effects and als for whitening reagents in cosmetics.26) Furthermore, the
have been used as cosmetic raw materials; however, they are asymmetric synthesis of (R)-1, which has an excellent floral
mainly non-volatile compounds and have been studied without scent and is expected to serve as a novel fragrance material
regard to their aromatic properties.12–16)
with high added value, was achieved.
The aromatic components in Osmanthus flower are known
to contain various β-ionone and its derivatives, which have
outstanding scents.17–23) Dihydro-β-ionol 117–21) and β-ionol
Results and Discussion
The bulb-to-bulb distillation of the essential oil of Osman-
222,23) are representative of these compounds. However, their thus absolute (OSM1) obtained from the flowers of Osman-
absolute configurations remained unknown until quite recent- thus fragrans var. thunberghi was conducted under reduced
ly,24) and synthetic racemates [( )-1 and ( )-2] have been used pressure (0.2 kPa). The bath temperature was raised from 50
as raw materials of fragrances.
to 200°C at 50°C intervals. Four distillate fractions were ob-
Based on the above facts, we thought that some of the vola- tained under individual conditions. GC and GC-MS analyses
tile aromatic components obtained from flowers should have of these fractions showed that the distillate (OSM2) obtained
excellent properties in terms of scent and biological activities. at 200°C contained ionone derivatives, namely dihydro-
In addition, it is highly probable that the two enantiomers of β-ionol 1, β-ionol 2, dihydro-β-ionone 3, and β-ionone 4.
an optically active compound will have different aromatic γ-Decalactone 5 was also present as another main ingredient
characters and different biological activities. However, the ar- in the same fraction (Fig. 1). Five fractions, i.e., the above-
omatic components obtained from natural sources are present mentioned four and the distillation residue, were submitted to
in minute amounts, therefore little has been discovered about antimelanogenetic tests using B16 melanoma cells; OSM2 had
the aromatic properties and biological activities of both enan- the strongest antimelanogenetic effect.
tiomers of naturally occurring aromatic components. We have
In order to clarify the antimelanogenetic effects of these
therefore been undertaking a project on the discovery of such components, we investigated the degrees of activity of
compounds from natural essential oils and on the asymmetric the commercial products 3–5 and those of ( )-1 and ( )-
synthesis of sufficient quantities of the natural components 2, obtained by NaBH4 reduction of 3 and 4, respectively.
and their enantiomers to identify their structures and absolute The IC50 values of these compounds are as follows: ( )-1:
configurations, and to evaluate their biological activities. This 0.005mm<IC50<0.050mm, ( )-2: 0.050mm<IC50<0.50mm, 3:
paper reports some results of a study of the aromatic com- 0.050mm<IC50<0.50mm, 4: 0.050mm<IC50<0.50mm, and 5:
ponents in Osmanthus flower and in Hakuto peach.25) In this IC50≥0.50mm.
Next, in order to clarify the differences between the aromat-
ic properties and antimelanogenetic effects of the enantiomers
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: akai@u-shizuoka-ken.ac.jp

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311
Fig. 1. Analysis of the Essential Oil of Osmanthus fragrans var. thunberghi (OSM1) and Distillation Fraction OSM2
(a) Structure of the main components. (b) GC Spectrum of OSM1. (c) GC Spectrum of OSM2.
of ( )-1 and ( )-2, we conducted lipase-catalyzed kinetic
resolutions of ( )-1 and ( )-2. First, the screening of suitable
lipases was carried out based on the reaction of ( )-126) with
various commercial lipases in the presence of vinyl acetate in
i-Pr2O at room temperature. We found that Alcaligenes sp. li-
pase (Meito QLM) showed very high enantioselectivity. Thus,
(S)-1 (49%, 96% ee) and (R)-6 (45%, 94% ee) were obtained
by reaction for 4.5h (E value = 136). Burkholderia cepacia
lipase (Roche Diagnostics, CHIRAZYME L-1), Burkholderia
sp. lipase (Toyobo LIP), Burkholderia cepacia lipase (Amano
PS-C), and Candida antarctica lipase B (Roche Diagnostics,
CHIRAZYME L-2) also gave high enantioselectivities (E
Chart 1. Lipase-Catalyzed Kinetic Resolution of ( )-1
value=38 to 72). (R)-6 was then hydrolyzed with K2CO3 in
MeOH to obtain (R)-1 (83%, 94% ee) (Chart 1).
For the kinetic resolution of ( )-2,27) Candida antarctica li- amounts.
pase B was found to be suitable for providing (S)-2 (45%, 99%
ee) and (R)-7 (46%, 99% ee). The alkaline hydrolysis of (R)-7 compounds showed that (S)-1 had a woody ambery scent and
then produced (R)-2 (94%, 99% ee) (Chart 2). a powdery floral aroma of violet and cassis. We also found
Sensory evaluation of the aromatic properties of these
GC analysis using a chiral column (Beta DEX™ 325, that (R)-1 had an outstanding high-class musky leathery scent
30m×0.25mm, Spelco) showed that (S)-1 and (R)-1 were pres- and also had a powdery floral aroma of violet and cassis. In
ent in the volatile aromatic components of Osmanthus flower contrast, (S)-2 had a floral, ambery, and woody scent, whereas
in a ratio of 9:1. It was found that (S)-1 and (R)-1 were also (R)-2 had a floral, fruity, and woody scent.
present in the aromatic components of Hakuto peach in a ratio
These compounds showed very high antimelanogenetic
of 1:10.28) However, it is not possible at present to determine effects in tests using B16 melanoma cells (Table 1). In par-
the absolute configuration and content ratio of 2 in these natu- ticular, (S)-1 and (R)-1 had powerful antimelanogenetic effects
ral aromatic components because it is present in very small equivalent to or better than that of a typical antimelanogenetic

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Vol. 61, No. 3
Chart 2. Preparation of Optically Active (R)-1, (R)-2, and (S)-2
optically pure (R)-1.
Table 1. Antimelanogenetic Effects of (S)-1, (R)-1, (S)-2, (R)-2, and N-
Phenylthiourea (PTU), Examined Using B16 Melanoma Cells
Conclusion
Compound
IC50 value (mm)
Both enantiomers of dihydro-β-ionol 1 and of β-ionol 2,
contained in the aromatic components of Osmanthus flower
and of Hakuto peach, were synthesized, and the absolute con-
figuration and enantiomer ratios of 1 in these natural aromatic
components were determined. It was found that (S)-1, (R)-1,
(S)-2, and (R)-2 had different characteristic favorable scents,
which would be applicable in the fragrance, flavor, cosmetics,
and food industries.
(S)-1
(R)-1
(S)-2
(R)-2
PTU
0.051
0.11
0.34
0.26
0.15
compound, N-phenylthiourea (PTU).
(R)-1 has excellent value as a fragrance raw material be-
It is also worth mentioning that (S)-1, (R)-1, (S)-2, and (R)-
cause its scent is high-class and superior to that of (S)-1, and 2 were all found to show high antimelanogenetic effects. In
also because it has a very high antimelanogenetic effect. We particular, (R)-1 is expected to find use as a fragrance raw
examined another method for the asymmetric synthesis of (R)- material with high added value because it has superior floral
1. We already reported the lipase-catalyzed dynamic kinetic aromatic properties and also because it has a remarkable anti-
resolution of ( )-2, which is readily and quantitatively avail- melanogenetic effect. Although the supply of high-purity (R)-1
able from the less expensive β-ionone 4 rather than dihydro-β- from natural sources is very limited, the asymmetric synthe-
ionone 3, to give (R)-7 (79%, >99% ee).27) The chemoselective sis of (R)-1 from inexpensive 4, which we developed in this
reduction of the external double bond of (R)-2, which was study, will provide the possibility of mass production of (R)-1
obtained from (R)-7 in almost quantitative yield, was therefore once further improvements have been made to the synthesis.
studied to produce (R)-1. After intensive trials, the catalytic Because the synthetic product (R)-1 is identical to the natural
hydrogenation of (R)-2 using 5% Pd/C (20wt%) in MeOH product, it has high value as a safe raw material for fragrance
produced a mixture (77% total yield) of (R)-1 and some other and flavor.
reduced products. (R)-1 (99% ee) was the largest component,
constituting 46% of the product mixture, and therefore, its
Experimental
General All reactions were carried out under an argon or
yield was calculated as 35% from (R)-2. Tetrahydro-β-ionol29)
and dihydro-α-ionol26) were also found in the mixture as nitrogen (or hydrogen for hydrogenation) atmosphere in an ov-
minor components (Chart 2). Although this method still needs en-dried flask, containing a stirring-bar, with a rubber septum
improvement in terms of its chemoselectivity, we believe that or with an inlet adaptor with a three-way stopcock. Candida
the asymmetric synthesis of (R)-1 from relatively inexpensive antarctica lipase B was gifted by Roche Diagnostics K.K.,
4 will open up a new pathway to the practical synthesis of Japan, and Lipase QLM was obtained from Meito Sangyo,

March 2013
313
Co., Ltd., Japan. Pd/C (5%) was purchased from Wako Pure 23.3, 24.7, 28.6, 32.7, 35.0, 39.8, 39.9, 68.8, 127.0, 136.8. IR
Chemical Industries, Ltd. MeOH was used as a solvent (CHCl3) cm−1: 3612. High resolution (HR)-MS m/z: 197.1937
without further purification, and all other reagents were pur- [Calcd for C13H25O (M+H)+: 197.1905].
chased from commercial sources (Aldrich, TCI, Wako, Kanto,
(R)-4-(2,6,6-Trimethylcyclohex-1-enyl)butan-2-ol [(R)-1]
Kishida and Nacalai) and used without further purification. A mixture of (R)-6 (0.47g, 2.0mmol), obtained above, and
All reactions were monitored using thin-layer chromatogra- K2CO3 (1.4g, 10mmol) in MeOH (8mL) was refluxed at 90°C
phy on glass-backed silica gel 60 F254, 0.2-mm plates (Merck) for 5h. The reaction mixture was concentrated under reduced
or GC-MS performed using an Agilent Technologies 6890N pressure and filtered through a Celite pad. The filtrate was
(GC) combined with a JEOL JMS-AMSUN (MS) or JMS- evaporated and purified by column chromatography on silica
Q1000GC K9 (MS). GC analysis was performed using Agilent gel (hexane–EtOAc=5:1) to provide (R)-1 (0.34g, 83%) as
Technologies 7890N (GC) equipped with a column (DB-WAX, a colorless oil [14% recovery of (R)-6]. The optical purity of
60m×0.25mm) in an oven (65 to 120°C, 3°C/min and then (R)-1 was determined to be 94% ee by HPLC analysis using
120 to 245°C, 5°C/min) or with a chiral column (Beta DEX™ the procedure described for (S)-1. [α]D26 −3.7 (c=0.17, CHCl3).
1
325, 30m×0.25mm, Spelco) in an oven (40 to 85°C, 2°C/min Its H-, 13C-NMR and IR data were in good agreement with
and then 85°C for 8h) with a flame ionization detector. Flash those of (S)-1. HR-MS m/z: 197.1933 [Calcd for C13H25O (M+
chromatography was performed with silica gel 60n, spherical H)+: 197.1905].
1
neutral (40–50µm), purchased from Kanto Chemical Co. H-
Lipase-Catalyzed Kinetic Resolution of (±)-2 (Chart 2,
and 13C-NMR spectra were recorded on a JEOL JMN-A500 or Method A) A mixture of ( )-227) (2.0g, 10mmol), Candida
ECA-500 (1H: 500MHz, 13C: 125MHz) instrument with chem- antarctica lipase B (6.0g, 300wt%), and vinyl acetate (2.0mL,
ical shifts reported in ppm relative to the residual protons of 21mmol) in MeCN (80mL) was stirred at room temperature
deuterated solvents or the internal standard tetramethylsilane.
for 6.5h and filtered using a Celite pad. The filtrate was con-
An essential oil from Osmanthus fragrans var. thunberghi centrated under reduced pressure, and the residue was purified
flowers, produced in the Zhuang Zu Autonomous Region of by column chromatography on silica gel (hexane–EtOAc=
Guang Xi Provence, People’s Republic of China, was pur- 10:1) to afford (S)-β-ionol (S)-2 (0.90g, 45%, 99% ee) and
chased from Toyotama International Inc. and used as OSM1 (R)-O-acetyl-β-ionol (R)-7 (1.1g, 46%, 99% ee). The optical
without further modification. Bulb-to-bulb distillation of this purity of (S)-2 was determined by HPLC analysis using a
product was performed under reduced pressure (0.2kPa). The chiral column (Daicel CHIRALCEL OD-H; hexane–i-PrOH=
bath temperature was raised from 50 to 200°C at 50°C inter- 99.9:0.1).
vals, and four distillate fractions were obtained under indi-
vidual conditions. The fraction obtained at a bath temperature CHCl3) [ref.30) [α]D24 −8.0 (c=0.158, CHCl3)]. 1H-NMR
of 200°C was used as OSM2.
(500MHz, CDCl3) δ: 0.98 (3H, s), 0.99 (3H, s), 1.31 (3H, d,
(S)-β-Ionol [(S)-2].30) A colorless oil. [α]D26 −7.8 (c=0.60,
Hakuto peaches (Prunus persica var. vulgaris), produced J=6.5Hz), 1.43–1.45 (2H, m), 1.57–1.62 (2H, m), 1.66 (3H, s),
in Okayama Prefecture, Japan, were put into a Tedlar® gas 1.97 (2H, t, J=6.5Hz), 4.34–4.39 (1H, m), 5.49 (1H, dd, J=6.5,
sampling bag (purchased from GL Sciences Inc.), and the 16.0Hz), 6.05 (1H, d, J=16.0 Hz).
aroma was collected for 13d using MonoTrap DSC18, DCC18,
(R)-O-Acetyl-β-ionol [(R)-7] A colorless oil. The 1H-
RSC18, and RCC18 (purchased from GL Sciences Inc.). The NMR data were in good agreement with those reported in the
MonoTraps were immersed in Et2O (2mL) and ultrasonic ir- literature.27)
radiation was performed for 15min. The ether phases were
filtered, and nitrogen gas was bubbled into the filtrate to con- 0.56g, 2.4mmol), obtained using the reported method,27) and
centrate it to 5µL. K2CO3 (0.84g, 6.1mmol) in MeOH (8mL) was stirred at room
(R)-β-Ionol [(R)-2]30) A mixture of (R)-7 (>99% ee,
Lipase-Catalyzed Kinetic Resolution of (±)-1 (Chart temperature for 2h. The reaction mixture was treated similar-
1) A mixture of ( )-126) (49g, 0.25mol), vinyl acetate (16g, ly to the preparation of (R)-1. The crude product was purified
0.19mmol), and lipase QLM (6.0g, 12wt%) in i-Pr2O (0.63L) by column chromatography on silica gel (hexane–EtOAc=
was stirred under a nitrogen atmosphere at room tempera- 5:1) to provide (R)-2 (0.45g, 94%, 99% ee) as a colorless oil.
ture for 4.5h and filtered using a Celite pad. The filtrate was The optical purity of (R)-2 was determined by HPLC analysis
concentrated under reduced pressure, and the residue was using a chiral column (Daicel CHIRALCEL OD-H; hexane–i-
purified by column chromatography on silica gel (hexane– PrOH=99.9:0.1). [α]D26 +8.7 (c=0.66, CHCl3) [ref.30) [α]D27 +7.6
1
EtOAc=30:1) to afford (S)-4-(2,6,6-trimethylcyclohex-1- (c=0.176, CHCl3)]. Its H-NMR data were in good agreement
enyl)butan-2-ol [(S)-1] (24g, 49%, 96% ee) and (R)-4-(2,6,6- with those of (S)-2.
trimethylcyclohex-1-enyl)butan-2-yl acetate [(R)-6] (26g, 45%,
(R)-4-(2,6,6-Trimethylcyclohex-1-enyl)butan-2-ol [(R)-1]
94% ee). The optical purity of (S)-1 was determined by HPLC (Chart 2) (R)-2 (194mg, 1.0mmol), 5% Pd/C (39mg,
analysis using a chiral column (Daicel CHIRALCEL OD) 20wt%), and MeOH (1.0mL) were placed in a test tube with
after esterification with 3,5-dinitrobenzoyl chloride in pyri- a rubber septum, and the tube was evacuated and backfilled
dine, and that of (R)-6 was dedused from the optical purity of with hydrogen gas. This treatment was repeated twice. The re-
(R)-1, derived from (R)-6 (vide infra).
action mixture was stirred under ambient pressure (balloon) of
(S)-4-(2,6,6-Trimethylcyclohex-1-enyl)butan-2-ol [(S)-1] hydrogen at room temperature (ca. 20°C) for 6h. The reaction
A colorless oil. [α]D26 +4.1 (c=0.19, CHCl3 for 96% ee). mixture was filtered using a membrane filter (Kanto Chemical
1H-NMR (500MHz, CDCl3) δ: 0.98 (6H, s), 1.21 (3H, d, Co., Ltd., polytetrafluoroethylene (PTFE), pore size 0.2µm),
J=6.5Hz), 1.39–1.42 (2H, m), 1.45–1.58 (4H, m), 1.59 (3H, s), and the filtrate was concentrated under reduced pressure. The
1.89 (2H, t, J=6.0Hz), 1.92–1.98 (1H, m), 2.10–2.16 (1H, m), residue was purified by flash column chromatography on silica
3.76–3.82 (1H, m). 13C-NMR (125MHz, CDCl3) δ: 19.5, 19.8, gel (hexane–EtOAc=10:1) to provide a mixture (153mg, 77%

314
Vol. 61, No. 3
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total yield), in which (R)-1 was found as the largest compo-
nent. Tetrahydro-β-ionol29) and dihydro-α-ionol26) were also
found in the mixture as minor products. The GC, GC-MS,
1H-, 13C-NMR, and HPLC analysis of the product mixture
showed that (R)-1 constituted 46% of the mixute (35% yield
from (R)-2) and that its optical purity was 99% ee. The
above mentioned flash column chromatography also provided
tetrahydro-β-ionone (30mg, 15%).
Preparation of B16 Melanoma Cells Cryopreserved B16
cells were revived, and subcultures were prepared using 10%
fetal bovine serum and Dulbecco’s modified Eagle’s medium
(DMEM). Culturing was performed in an incubator (5% CO2,
37°C).
Melanin Inhibition Tests B16 melanoma cells were seed-
ed on 24-well plates at 3×104 (mL/well), and cultured for 48h
on the next day in a medium (1mL) containing various con-
centrations of the experimental samples dissolved in dimethyl
sulfoxide. The amounts of intracellular melanin in all the cells
were then measured.
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Measurement of Intracellular Melanin After remov-
ing the medium supernatant, the cells pasted on the wells
were washed in 1mL of phosphate-buffered saline 1 (PBS).
A 0.25% trypsin–ethylenediaminetetraacetic acid solution
(100 µL) was added to the cells, and the cells were allowed
to stand at 37°C in 5% CO2 for 1min. The cells exfoliated by
trypsinization in 1mL of PBS were collected and centrifuged
at 1000rpm at 4°C for 5min. After removing the superna-
tant, the cell pellets were rinsed twice in PBS. The PBS was
removed, the cells were suspended in 110µL of 1n sodium
hydroxide, and the cell suspension was incubated at 100°C
for 30min. This suspension (80µL) was transferred to a 96-
well plate, the absorbance at 405nm was measured using a
micro-plate reader, and the intracellular melanin content was
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property and biological activity of each enantiomer of 1. See: Na-
kayama Y., Abe S., Yamaguchi Y., Sakurai K., 55th Symposium on
the Chemistry of Terpenes, Essential Oils, and Aromatics, Tsukuba,
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28) Dihydro-β-ionol 1 is known to be present in volatile aromatic com-
ponents of Hakuto peach, see: Osaki K., Yukawa C., Zhong X.,
Iwabuchi H., Foods and Food Ingredients Journal of Japan, 44–60
(2001).
Acknowledgements Soda Aromatic Co., Ltd. is thanked
for fruitful discussions on this work. Roche Diagnostics
K.K., Japan, Amano Enzyme Inc., Toyobo Co., Ltd., and
Meito Sangyo Co., Ltd. are thanked for the generous gifts of
lipases. This work was supported by Adaptable & Seamless
Technology Transfer Program through Target-driven R&D
(AS231Z03022D) from Japan Science and Technology Agency.
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