Synthesis of ionones and carvone analogues: Olfactory properties and preliminary toxicity assays

Article abstract of DOI:10.1016/S0223-5234(00)00181-1

Vilsmeier reagents react with α/β-ionones and carvone to produce aldehydes 7-11 in a one-step procedure. The indene derivative 11, which came from the double iminoalkylation of carvone and ring closure with the elimination of dimethylamine, was practically odourless, while all the others had peculiar odours which were very different from the starting material. The cytotoxicity data of 9 and 10, which are the most promising potential perfume ingredients, are also reported.

Full text of DOI:10.1016/S0223-5234(00)00181-1

Eur. J. Med. Chem. 35 (2000) 797−803
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001811/FLA
Original article
Synthesis of ionones and carvone analogues:
olfactory properties and preliminary toxicity assays
Maria Anzaldia, Enzo Sottofattoria, Fabiola Dusattib, Margherita Ferrob, Marcella Panic, Alessandro
Balbia*
aDipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Viale Benedetto XV, 3 - 16132 Genova, Italy
bDipartimento di Medicina Sperimentale, Sezione di Patologia Generale, Via Leon Battista Alberti, 2 - 16132 Genova, Italy
cDipartimento di Chimica e Chimica Industriale, Via Dodecaneso 31, 1 - 16146 Genova, Italy
Received 28 December 1999; revised 21 February 2000; accepted 24 February 2000
Abstract – Vilsmeier reagents react with a/ꢀ-ionones and carvone to produce aldehydes 7–11 in a one-step procedure. The indene derivative
11, which came from the double iminoalkylation of carvone and ring closure with the elimination of dimethylamine, was practically odourless,
while all the others had peculiar odours which were very different from the starting material. The cytotoxicity data of 9 and 10, which are
the most promising potential perfume ingredients, are also reported. © 2000 Éditions scientifiques et médicales Elsevier SAS
ionone analogues / carvone analogues / odoriferous substances / aldehydes / cytotoxicity / Vilsmeier reaction
1. Introduction
Various substances resulting from a combination of
conjugate or non-conjugate double bonds with the carbo-
nyl group in correlation with the natural pattern of the
2,6,6-trimethyl or 1-methyl-4-isopropyl substituents have
been isolated or have been prepared and studied for their
olfactory properties. Among them are comprised famous
fragrances of high impact such as menthones, carvones,
ionones and damascones.
A very large number of cyclohexane and cyclohexen-
ecarboxaldehydes have been synthesised. Some of them
showed outstanding odoriferous properties like the simple
2,4-dimethyl-3-cyclohexene carboxaldehyde 1 (Cyclal-
Givaudan) [2] or the more complex 4-(4-methyl-3-penten-
1-yl)-3-cyclohexene carboxaldehyde 2 (Myrac aldehyde-
IFF) [2, 3] and its hydroxy derivative 3 (Lyral-IFF) [3]
(figure 2).
Aldehydes and ketones have played a primary role in
perfumery and continue to be one of the leading choices
in perfume composition [1]. While ketones are widely
represented in nature, the majority of aldehyde deriva-
tives come from synthesis. Moreover, among the mono-
cyclic terpenes, aldehydes occur in very low concentra-
tions in essential oils and are seldom used. On the other
hand, ketones having the p-menthane or (trimethylcyclo-
hexenyl)alkenone skeletons A and B are commercially
relevant as fragrance and flavour compounds (figure 1).
Figure 1. p-Menthane and (trimethylcyclohexenyl)alkenone
skeletons A and B.
Figure 2. Compounds 1–3.
* Correspondence and reprints balbi@unige.it

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M. Anzaldi et al. / Eur. J. Med. Chem. 35 (2000) 797–803
Figure 3. Aldehydes 4 and 5.
Only a few can be correlated to p-menthane A and
generally do not possess noticeable olfactory properties.
To the best of our knowledge only aldehydes 4 and 5 are
reported to be used as perfuming agents [4] (figure 3).
Moreover, among the numerous aldehydes which can
be correlated to the structure B, only Ambra aldehyde 6,
which comes from the degradation of Ambrein, has been
studied and synthesised for its outstanding marine-ozone
odor [5] (figure 4).
Figure 5. Synthesis of compounds 7 and 8.
To obtain more insight regarding how the introduction
of an aldehyde into structures A and B influences the
odour, we planned to formylate the commercially avail-
able α- and ꢀ-ionones and carvone.
changed carefully in order to improve separately the yield
of each compound obtained. In fact, the Vilsmeier formyl-
ation of carvone afforded the mixture of the expected
formylcyclohexadiene
9
together
with
the
2-chlorobenzaldehyde 10 and the indene derivative 11
(figure 6).
2. Chemistry
Modifying the reaction time resulted in improved
yields of each compound. Any attempt to further increase
the yields, such as changing the temperature and the
molar rate from the typical procedure, failed. We specu-
late that this may be due to the formation of other
(poly)formylated products which come from the more
active position of intermediate 12 and 13. These by-
products end up in an unworkable thick brownish mixture
(see figure 7 and Experimental protocols).
The reaction pathways depicted in figure 7 account for
the formation of the indene derivative 11 and can give an
explanation to the critical reaction. Carvone undergoes
iminoalkylation to give cation 12, which is the source of
9 after hydrolysis. Deprotonation of 12 affords 13, which
may be further iminoalkylated to give species 14, which
affords 15 by ring closure with elimination of dimethyl-
amine. Finally, benzaldehyde 10 comes from the oxida-
tive aromatization of 9.
Vilsmeier reagents have been applied in numerous
synthetic transformations of acyclic and cyclic carbonyl
compounds to give several distinct types of products
whose nature prevalently depends on the substituents.
Among them mono- and polyformylated products are the
usual outcome [6]. When α- and ꢀ-ionones were reacted
with
the
Vilsmeier
reagent
N,N-dimethyl-
(diethyl)formamide/phosphorus oxychloride (VR), the
expected ꢀ-chlorovinylaldehydes 7 were obtained to-
gether with the unprecedented enaminones 8 [7] (figure
5). The latter, on the other hand, besides having an
interesting odour, have proven to be useful key interme-
diates in synthesising new short retinoid-like compounds
[8]. We were also able to obtain compounds 7 or 8
separately, thus improving the yields, simply by modify-
ing the workup (see figure 5 and Experimental protocols).
Transfer of the reaction method from the ionones to the
carvone was successful, but some conditions needed to be
Figure 4. Ambra aldehyde 6.
Figure 6. Synthesis of compounds 9–11.

M. Anzaldi et al. / Eur. J. Med. Chem. 35 (2000) 797–803
799
MTT-formazan precipitate by mitochondrial dehydroge-
nase enzymes.
Finally, the evaluation of total protein content (TPC)
was used as a measure of surviving cells still attached to
the monolayer support [13]. Sodium dodecylsulphate
(SDS) was used as positive control.
4. Results and discussion
4.1. Olfactory properties
Table I summarises the olfactory properties and poten-
tial use of the aldehyde derivatives 7–11.
Natural α-ionone possesses a sweet-floral note remi-
niscent of violets while ꢀ-ionone, reminiscent of cedar-
wood, changing to violet upon dilution. These descriptors
are completely lost in 7a, revealing instead a bitter, tarry,
unpleasant note. In 7b a sweet-floral note recalling that of
ꢀ-ionone remains, but is stronger, more ‘medicinal’ and
woody. In sharp contrast, 8a possesses a weak but
persistent fragrance of interesting and pleasant character:
an iron-like tonality occurring in orris oil accompanied by
a vanillic note and a ‘poudrée’ subnote, which are the
typical olfactory notes of red lipsticks having orris and
vanilla components. On the other hand, 8b has a fruity
note, reminescent of cooked prunes and tagetes oil and
then evolves into a saffron and ionone-like note.
The formylcyclohexadiene 9 exhibits an extremely
strong but pleasant bitter note with a woody fragrance,
evolving towards a typical quinoline note. The character-
istics of the odour change completely in 2-chloro-
benzaldehyde 10, which has a strong, very attractive,
fruity note recalling the tagetes oil with undertones of
dried fruit.
Figure 7. Synthesis of compound 11.
The formation of 11 accounts for further iminoalkylation
on the unactivated double bond of the isopropenyl group.
This second step, which leads to intermediate 14, has to
be highly stereoselective, since only isomer E is able to
give 15 by ring closure. That the thermodynamic equi-
librium was in favour of the E isomer has been supported
by the yield increase of 11 with reaction time. Analogous
equilibration in favour of the E form was observed in the
Vilsmeier formylation of limonene [9].
All new compounds were characterised by their el-
1
emental analyses and by IR, H- and 13C-NMR spectra
(see Experimental protocols). The structure of 11 was
also secured by X-ray crystallographic analysis [10].
3. Toxicology
The incorporation of an aldehyde group in the ‘natural’
pattern of the α- and ꢀ-ionones completely changed the
odour of 7a–b. We, at the moment, cannot state whether
this is due to the concomitant presence of chlorine. On the
other hand, the substitution of that chlorine with a
dialkylamino group gave 8a–b, providing a pleasant and
well-balanced note in which the ‘medicinal’, tarry and
bitter notes disappeared.
The presence of chlorine together with an aldehyde
group seems to have played well in the p-menthane
pattern. In fact 9 showed a note, which is not comparable
to other existing notes and whose strength relies on the
concentration; while 10, on the other hand, which with
only one extra double bond becomes aromatic, possesses
a characteristic fragrance which could well apply to both
flowery and fruity compositions.
To evaluate the cytotoxicity of compounds 9 and 10,
the human keratinocyte NCTC 2544 cell line was used.
We applied three in vitro tests, which are increasingly
used as cost-effective and rapid methods in the pre-
screening phase in the development of new drugs and
other chemicals [11].
One of the most widely used cytotoxicity assays
(NRU) is based on the ability of viable cells to incorpo-
rate and bind neutral red, which is a vital, weakly
cationic, dye; it penetrates cell membranes by non-ionic
passive diffusion and concentrates in the lysosomes,
where it binds to various groups of the lysosomal matrix
by electrostatic hydrophobic bonds [12].
In the MTT assay, the yellow MTT tetrazolium [2-(4,5-
dimethyl-2-thiazolyl)3,5-dipheny-2H-tetrazolium
bro-
mide] is reduced in viable cells to a purple coloured

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M. Anzaldi et al. / Eur. J. Med. Chem. 35 (2000) 797–803
Table I. Olfactory properties of compounds 7–11.
Compound aStrength Evolution
Persistence P. after 24 h Quality
Potential use
7a
5
long lasting
good
sufficient
Harsh, bitter, tarry, unpleasant Special use (e.g. tarry sham-
poo). Fixative
7b
4
long lasting
medium
sufficient
Penetrating, sharp, similar to Pharmaceutical use. Top note.
ꢀ-ionone but more woody and
medicinal
8a
8b
9
2
2
6
long lasting
good
good
low
Powdery, vanillic, a reminder Lipsticks, powder cosmetics.
of orris oil
Fixative
towards saffron note
low
Fruity, cooked prunes, a re- Top or body note for fruity or
minder of tagetes oil
Bitter, woody
spicy compositions
towards Quinoline note good
good
Not comparable to existing
notes. According to concentra-
tion can be used as top, body or
end note
10
11
4
towards fruity notes
good
adequate
Flowery, fruity, a reminder of Flowery and fruity composi-
dried fruits
tions. Top or body note.
Too weak for an evaluation
a 1 = very weak, 2 = weak, 3 = moderate, 4 = strong, 5 = very strong, 6 = extremely strong. The olfactory evaluation was carried out on pure
compounds and on the same compounds diluted in ethanol 1 to 10.
4.2. Cytotoxicity
comparing the IC50s (table II) it is clear that 10 has an
intrinsic cytotoxicity which is one order of magnitude
lower than that of 9. Additionally, 9 has the same IC50 of
SDS, therefore this compound should be considered as a
strong irritant. Nonetheless, the IC10s (concentration
inhibiting viability by 10%) of 9 and SDS (table II)
demonstrate that, at micromolar concentrations, only
10% of the cells die.
Compounds 9 and 10 were selected for a preliminary
study of cytotoxicity and the results obtained by means of
three different in vitro assays are reported as dose–
response curves in figure 8. Table II shows the IC50
(concentration inhibiting viability by 50%) obtained from
those curves. All three assays gave curves having very
similar patterns. It is evident that, at low concentrations,
10 is much less cytotoxic than 9, whereas the opposite is
true at concentrations higher than 5 mM. Moreover, by
Therefore, this in vitro evaluation suggests that 10 is
safer than 9, and 9 should be used in formulations at low
Figure 8. Comparative cytotoxicity of compound 9 (■) and 10 ([) in NCTC 2544 human keratinocytes as detected with (a) NRU,
(b) MTT, (c) TPC assays. The values represent the mean ± SEM of the four experiments in quadruplicate.

M. Anzaldi et al. / Eur. J. Med. Chem. 35 (2000) 797–803
801
Table II. Cytotoxicity of 9, 10 and sodium dodecylsulphate (SDS)
Microanalysis Laboratory in our Institute and the results
were within ± 0.4% of theoretical values. For spectral
data of 7–8 see [7].
in NCTC 2544 human keratinocytes evaluated as IC50 and IC10.
IC50 (mM)
Compound
9
10
NRU test
0.123
3.2
MTT test
0.12
TPC test
0.119
6.1.1. Vilsmeier reaction on α- and ꢀ-ionones. General
procedure
1.5
2.5
Phosphorous oxychloride (50.0 mmol; 4.57 mL) was
added dropwise, within 15 min at 0 °C, to 3.87 mL of
N,N-dimethyl(diethyl)formamide in a two-necked flask
protected from atmospheric moisture and efficiently stirred
with a magnetic bar. A solution of α/ꢀ-ionones
(25.0 mmol) in 3 mL of dimethylformamide was dropped
into the above Vilsmeier reagent, cooled at –20 °C, left to
stir while the temperature was left to rise to 0 °C for a
total of 45 min and finally poured onto crushed ice. The
aqueous layer was separated and treated as further de-
scribed for each compound.
SDS
0.132
0.133
0.133
IC10 (mM)
9
10
SDS
0.022
0.1
0.07
0.03
0.1
0.047
0.05
0.038
0.103
concentrations. However, conditions of use, chemical and
physical properties and levels of absorption should also
be taken into consideration.
6.1.1.1. 3-Chloro-5-(2,6,6-trimethyl-
2-cyclohexen-1-yl)-2,4-pentadienal 7a
5. Conclusion
The aqueous layer was carefully neutralized with 10%
sodium hydroxide (pH control) and allowed to stand
overnight at room temperature. The colourless oil, which
was easily separated out from the aqueous phase, was
purified by column chromatography on silica gel, eluting
with toluene, giving 7a in 25% yield. IR (film): ν 2 900,
2 840, 1 665, 1 615, 1 575, 1 160 cm–1 [7].
In summary, the introduction of a formyl group, even if
accompanied by chlorine or a dialkylammino group, has
once more proven to furnish odorants with unusual notes
that could stimulate new creations in perfumery and
aromatisation.
Regarding the cytotoxicity tests, we thought it inter-
esting to evaluate the cytotoxicity of our molecule by
means of three in vitro assays that test different end-
points in order to assess its toxic potential in a non-animal
system. It is clear that these tests cannot replace the
complete safety appraisal of a cosmetic ingredient, but
they give an indication of the degree of potential toxicity
for further in vivo investigations or more specific in vitro
tests (i.e. contact irritancy and hypersensitivity).
6.1.1.2. 3-Chloro-5-(2,6,6-trimethyl-
1-cyclohexen-1-yl)-2,4-pentadienal 7b
By using the above procedure, compound 7b was
finally purified by column chromatography as a colour-
less oil in 28% yield. IR (film): ν 2 920, 2 860, 1 670,
1 610, 1 590, 1 570, 1 170 cm–1 [7].
6.1.1.3. 3-Dimethylamino-5-(2,6,6-
trimethyl-2-cyclohexen-1-yl)-2,4-pentadienal 8a
The aqueous layer was alkalinized with 30% sodium
hydroxide and allowed to stand overnight at room tem-
perature. The resulting water/oil mixture was extracted
with chloroform, and the combined organic layer dried on
magnesium sulphate and evaporated to give an oil which
solidified on standing, giving 8a as an already pure pale
yellow powder in 40% yield. m.p. 147 °C from n-hexane
[7].
6. Experimental protocols
6.1. Chemistry
Melting points were determined with Fisher-Johns
apparatus and are uncorrected. The IR spectra were
recorded in chloroform or in potassium bromide disks on
a Perkin-Elmer 398 spectrometer. The 1H- and 13C-NMR
spectra were recorded on a Varian Gemini 200 (200 MHz,
1H; 50 MHz, 13C) spectrometer in deuteriochloroform
solutions with tetramethylsilane as the internal standard
(δ = 0). The purity of all compounds was checked by
thin-layer chromatography on silica gel 60-F-254 pre-
coated plates and the spots were located in UV light or by
vanillin in sulfuric acid. Elemental analyses were per-
formed on a Carlo Erba 1106 Elemental Analyser in the
6.1.1.4. 3-Diethylamino-5-(2,6,6-
trimethyl-2-cyclohexen-1-yl)-2,4-pentadienal 8b
The aqueous layer, worked as above, gave a thick oil
which was purified by column chromatography on silica
gel, eluting with ethyl acetate, giving 8b as a thick yellow
oil in 28% yield. IR (film): ν 2 900, 1 620, 1 540, 1 430,
1 380, 1 310, 1 260, 1 200, 1 180 cm–1 [7].

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M. Anzaldi et al. / Eur. J. Med. Chem. 35 (2000) 797–803
6.1.2. Vilsmeier reaction
on Carvone. General procedure
6.1.2.3. 7-Chloro-3,6-
dimethyl-1H-indene-2-carbaldehyde 11
A solution of 10 g (66.6 mmol) of Carvone in 5 mL of
dimethylformamide (DMF) was added at 20 °C over
5 min to the Vilsmeier reagent, which was prepared by
adding phosphorus oxychloride (6.04 mL; 66.6 mmol)
over 5 min to a stirring 5.10 mL (66.6 mmol) of DMF at
0 °C and allowing the mixture to continue stirring at
20 °C for 20 min. The mixture of Carvone and Vilsmeier
reagent was stirred at 20 °C from 1–3.5 h, crushed ice
was then added and stirred for an additional 20 min. The
aqueous layer was separated and basified with a solution
of sodium hydroxide and stirred overnight. The red oil,
which separated during the night, was extracted with
chloroform (3 × 60 mL) and the combined organic layers
were washed with water (2 × 70 mL), dried over anhy-
drous magnesium sulfate and evaporated to give a thick
dark red oil. Separation by column chromatography on
silica gel (200 g) using toluene, toluene/ethyl acetate
(1:1) and ethyl acetate as eluents gave 9, 10 and 11.
Toluene provided compounds 9 and 10, toluene/ethyl
acetate gave the unreacted Carvone and ethyl acetate
afforded indene derivative 11. Elution with ethanol gave
a thick brownish unworkable oil.
By use of the general method, the reaction mixture was
allowed to stir for 3.5 h at 20 °C. The final thick dark red
oil was separated by column chromatography on silica
gel. The elution with ethyl acetate afforded 11 in 25%
yield as yellow crystals. M.p. 165 °C from cyclohexane.
1
IR (KBr) ν 2 910, 1 640, 1 600, 1 440 cm–1; H-NMR
(200 MHz, CDCl3) 2.46 (3H, s, CH3), 2.53 (3H, s, CH3),
3.64 (2H, s, CH2), 7.30 (1H, d, CH, J = 7.7 Hz), 7.33 (1H,
d, CH, J = 7.7 Hz), 10.20 (1H, s, CHO); 13C-NMR
(50 MHz, CDCl3) 11.43 (CH3), 20.53 (CH3), 36.39
(CH2), 120.20 (CH), 130.43 (CH), 131.45 (C), 137.72
(C), 139.66 (C), 143.25 (C), 144.43 (C), 155.76 (C),
187.32 (CHO).
6.2. Biological methods
6.2.1. Chemicals
Dulbecco’s modified Eagle medium (DMEM), (MTT)
tetrazolium salt, sodium dodecylsulphate (SDS) were
purchased from Sigma (St Louis, MO, USA); L-glutamine
and neutral red were from ICN Biomedicals Inc. (Costa
Mesa, CA, USA); Trypsine-EDTA was fom Gibco BRL
(Paisley, Scotland); foetal calf serum was from Mascia
Brunelli (Milan, Italy); the protein assay dye reagent was
from BioRad Laboratories GmbH (Munchen, Germany);
cell culture flasks and 96-well plates were from Costar
(Cambridge, UK).
6.1.2.1. 2-Chloro-6-isopropenyl-3-
methyl-cyclohexa-1,3-dienecarbaldehyde 9
By use of the general procedure, the reaction mixture
was allowed to stir for 2 h at 20 °C. Separation by column
chromatography on silica gel of the final thick dark red
oil gave 9 in 18% yield as yellow oil. IR (film) ν 2 910,
6.2.2. Cell cultures
1
1 660, 1 550, 890 cm–1; H-NMR (200 MHz, CDCl3): δ
Normal human keratinocyte cell line NCTC 2544
(kindly furnished by Prof. Marinovich, Institute of Phar-
macological Sciences, Milan, Italy) grown in DMEM
medium supplemented with 10% foetal calf serum and
2% L-glutamine at 37 °C in a humidified atmosphere of
5% CO2/95% air.
The medium was changed every 2–3 days and, when
the original flask was approximately 80% confluent, the
cells were subcultured by trypsin-EDTA digestion.
1.72 (3H, s, CH3), 1.97 (3H, s, CH3), 2.46 (2H, m, CH2),
3.45 (1H, dd, CH), 4.59 (1H, s, CH2), 4.74 (1H, s, CH2),
6.24 (1H, m, CH), 10.20 (1H, s, CHO); 13C-NMR
(50 MHz, CDCl3) 19.61 (CH3), 21.63 (CH3), 27.71
(CH2), 37.59 (CH), 111.83 (CH2), 132.05 (C), 132.61
(C), 132.83 (CH), 143.37 (C), 149.28 (C), 190.75 (CHO).
6.1.2.2. 2-Chloro-6-
isopropenyl-3-methyl-benzaldehyde 10
6.2.3. Treatments
The final thick dark red oil, obtained after 3 h of
reaction time, and chromatographed on silica gel eluting
with toluene provided 10 in 14% yield as yellow oil. IR
(film) ν 2 910, 1 660, 1 600, 1 570, 1 440, 1 360 cm–1;
1H-NMR (200 MHz, CDCl3) 2.06 (3H, s, CH3), 2.42
(3H, s, CH3), 4.83 (1H, s, CH2), 5.24 (1H, s, CH2), 7.09
(1H, d, CH, J = 7.72 Hz), 7.36 (1H, d, CH, J = 7.72 Hz)
10.40 (1H, s, CHO); 13C-NMR (50 MHz, CDCl3) 20.52
(CH3), 24.92 (CH3), 117.26 (CH2), 127.81 (CH), 131.85
(C), 134.87 (CH), 135.96 (C), 137.40 (C), 144.30 (C),
146.12 (C), 192.24 (CHO).
Cells were seeded 24 h before treatment in 96-well
plates at 4 × 104 cells/well (keratinocytes) in order to
obtain semi-confluent cultures. Cells were exposed for
3 h to the medium containing various concentrations
(0.025–50 mM) of 9 and 10 dissolved in 1.25% dimeth-
ylsulphoxide (final concentration, DMSO). Positive con-
trols received SDS (0.01–0.2 mM) and controls for sol-
vent were carried out. After that time, the medium was
removed and the plates were prepared for the NRU, MTT
and TPC assays.

M. Anzaldi et al. / Eur. J. Med. Chem. 35 (2000) 797–803
803
6.2.4. Neutral red uptake
Acknowledgements
Cells were exposed to a medium containing 50 µg/mL
of neutral red dye and processed according to the Boren-
freund et al. method [14]. After 3 h incubation at 37 °C,
the cells were washed twice in PBS and fixed according
to the procedure described by Riddell et al. [15]. The
plates were then left at room temperature for 10 min and
the absorbance of the extracted neutral red dye was read
at 550 nm wavelength in a Uniskan II microplate reader
(Labsystems, Helsinki, Finland).
We wish to thank Dr Luigi Rigano (via Calabria
5-20158 Milan) for the olfactory evaluations. Financial
support was provide by CNR and MURST (Rome).
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6.2.7. Statistical analysis
All experiments were performed at least four times
using four wells for each concentration of the tested
agents. Results were expressed as percentage of viability
compared to control. Linear regression analysis was used
to compute the concentration needed to reduce absor-
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