Structure-Odor Activity Studies on Derivatives of Aromatic and Oxygenated Monoterpenoids Synthesized by Modifying p-Cymene

Article abstract of DOI:10.1021/acs.jnatprod.9b00339

Thymoquinone was recently reported as having a unique pencil-like odor and being the impact compound for the cedar-like and cedar wood-based product smell such as pencils. The compounds thymol and carvacrol are structurally related odorants commonly found in plants and foods such as thyme and oregano, also having a significant contribution to their overall aroma. However, a systematic elucidation of the sensory properties in this class of oxygenated, aromatic monoterpenoids has not been carried out. To close this gap and gain new insights into structure-odor relationships leading to pencil-like and woody odors, 19 structurally related derivatives of p-cymene starting from thymol and carvacrol were synthesized and characterized. The compounds had odor thresholds ranging from 2.0 ng/L air to 388.8 ng/L air, being lowest for thymol and carvacrol and highest for thymohydroquinone. The compounds smelled mostly thyme-like, oregano-like, and pencil-like with phenolic, earthy, and medicinal variations in their odor character, which could be successfully linked to structural motifs.

Full text of DOI:10.1021/acs.jnatprod.9b00339

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Structure−Odor Activity Studies on Derivatives of Aromatic and
Oxygenated Monoterpenoids Synthesized by Modifying p‑Cymene
Linda Schreiner,§ Johannes Bauer,§ Eva Ortner, and Andrea Buettner*
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ABSTRACT: Thymoquinone was recently reported as having a unique pencil-like odor and being
the impact compound for the cedar-like and cedar wood-based product smell such as pencils. The
compounds thymol and carvacrol are structurally related odorants commonly found in plants and
foods such as thyme and oregano, also having a significant contribution to their overall aroma.
However, a systematic elucidation of the sensory properties in this class of oxygenated, aromatic
monoterpenoids has not been carried out. To close this gap and gain new insights into structure−
odor relationships leading to pencil-like and woody odors, 19 structurally related derivatives of p-
cymene starting from thymol and carvacrol were synthesized and characterized. The compounds
had odor thresholds ranging from 2.0 ng/L air to 388.8 ng/L air, being lowest for thymol and
carvacrol and highest for thymohydroquinone. The compounds smelled mostly thyme-like,
oregano-like, and pencil-like with phenolic, earthy, and medicinal variations in their odor character,
which could be successfully linked to structural motifs.
alternative remedy in the treatment of antibiotic-resistant
bacteria.15−17
onoterpenes are a natural class of compounds
Mcomprising 10 carbon atoms. They are of great
diversity, as they can consist of acyclic, monocyclic, or bicyclic
structures and unsaturated, saturated, or aromatic structural
elements, as well as being decorated with different functional
groups, yielding alcohols, aldehydes, or ketones. By adding an
oxygen function, the molecules are commonly called
monoterpenoids. Monoterpenes and monoterpenoids are
naturally synthesized by many plants, including conifers and
often have pharmacological effects, such as antifungal,
antibacterial, or antioxidant activities.1 Furthermore, they are
of interest for the perfume and food industry due to their
aroma properties.2
Thymol (1), with its thyme-like, rosemary-like, ethereal
smell and its structural isomer carvacrol (2), with an oregano-
like, wood-like, and pencil-like odor, are monoterpenoids and
well known for their occurrence in various aromatic plants such
as thyme and oregano, thereby representing the main
components of the essential oil.3 They can also be found in
basil,4 eyebright,5 black cumin,6 and seafennel.7 In addition,
thymol is present in lettuce,8 whereas carvacrol can be found in
savory2,9 and majoram.2,9 The essential oils of aromatic plants
containing thymol and carvacrol as well as the pure substances
themselves are used as flavoring agents for food, e.g., baked
goods, chewing gums, beverages, or hard candy.10 Further-
more, both compounds show antimicrobial activity and can
therefore act as food preservatives.11−14 Owing to their
antioxidative, antibacterial, and anticancer activity, both
compounds are considered with regard to medical applications,
either as auxiliary agents in cancer treatment or as an
Thymol and carvacrol are hydroxylated derivatives of p-
cymene (3). p-Cymene itself is also a common aroma-active
compound in essential oils having a wood-like, citrus-like odor,
and can be found in numerous plants such as thyme and
horsemint, as well as cinnamon, cypress, eucalyptus, or
turpentine (Table S1, Supporting Information).2,18 All three
molecules share the following core moiety: a benzene ring
alkylated at C-1 and C-4 with a methyl and an isopropyl group,
respectively. This structural similarity can also be found in the
corresponding benzoquinone thymoquinone (4). Thymoqui-
none is one of the main components in black cumin (Nigella
sativa L.) seed oil and has been reported to exert many positive
physiological properties19 relating to, for example, hepatopro-
tective,20 anti-inflammatory,21 antioxidant,22 and anti-
cancer23,24 effects. Despite its potential for medical purposes,
thymoquinone is an odor-active molecule with a unique
medicinal, pencil- and leather-like odor, thereby being a natural
molecule that had been reported for the first time to elicit this
odor impression. Apart from that, we recently found that
thymoquinone is an important odorous constituent of cedar
and pine wood.25,26 Thymol, carvacrol, and p-cymene, on the
other hand, have been reported with various smell descriptors
Received: April 12, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
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1
Figure 1. Chemical structures of compounds 1−19. The numbering (1−7) corresponds to the H NMR data in Tables S1−S4, Supporting
Information.
such as wood-like, leaf-like (for thymol),27 spicy (for carvacrol
and thymol),27,28 fuel-like (for p-cymene), and thyme-like,
oregano-like (for carvacrol and thymol).29
carvacrol methyl ether, O-acetylthymol, and O-acetylcarvacrol
as well as thymohydroquinone dimethyl ether. Those
substances were found, inter alia, in thyme,34 oregano,35
willowleaf yellowhead,36 aya-pana,37 seafennel,7 and lettuce8
(detailed information, cf. Table S1, Supporting Information).
Despite knowledge of the presence of those substances as
constituents in natural plants, there has been no comprehen-
sive data provided regarding their sensory properties. Likewise,
no further oxygenated aromatic monoterpenoids had been
described. Thus, the aim of the present study was to synthesize
structurally related derivatives and set up a database providing
detailed sensory and analytical data for these substances.
Therefore, 19 structurally related derivatives of thymol and
carvacrol were synthesized, and their odor qualities and odor
thresholds in air were determined. Furthermore, the MS data
and retention indices on two chromatographic capillaries of
different polarity as well as NMR spectra were compiled. The
data generated will facilitate future identification of molecules
belonging to this substance class and contribute to the analysis
of structure−odor relationships in general, but also with regard
to woody smells.
From our numerous studies in the field of smell research it
becomes evident that, despite decades of intense research in
odorant discovery and on structure−smell activity relation-
ships, there still is a lot to be unveiled. Nowadays, odorants are
still discovered, commonly those that are very odor potent, yet
hard to detect in nature due to their occurrence in traces or in
complex substance mixtures. This is especially true in the case
of terpenoid and sesquiterpenoid substances, as these
molecules tend to be buried in complex mixtures of structurally
related, but less odorous molecules, bringing about special
challenges in chromatographic separation and unequivocal
mass spectrometric detection. An example of this kind was our
recent discovery of mustakone as a potent odorant in
frankincense that required sophisticated chromatographic
separation and enrichment steps in the course of structural
identification of this substance.30 Such studies demonstrate the
need for establishing systematic databases in a bottom-up
approach to ease future discovery of odor molecules.
According to this concept, an extended database in a series
of recent studies comprising, among others, acyclic mono-
terpenes and their oxygenated derivatives had been estab-
lished.31−33 Using a systematic approach for odor threshold
determination and for the recording of the respective smell
descriptors it was demonstrated that structurally related
monoterpenes are often similar in smell (e.g., citrus-like smells
for linalool and its derivatives) with some of them being highly
odor-active. Accordingly, this concept was extended in the
present study to the investigation of structurally related
substances of thymol, carvacrol, p-cymene, and thymoquinone.
We hypothesized that targeted synthesis of related compounds
might reveal further still unknown odorants, potentially even
with related odor qualities, namely, wood- or pencil-like smells.
Structurally related substances that had previously been
reported as natural compounds are thymol methyl ether,
RESULTS AND DISCUSSION
■
Syntheses. Nineteen compounds (Figure 1) were inves-
tigated, and 15 of these compounds were synthesized following
general synthetic procedures (cf. Figures S1−S6, Supporting
Information). Thymol (1), carvacrol (2), p-cymene (3), and
thymocatechol (5) were obtained from commercial sources.
Thymoquinone (4) was synthesized by catalytic oxidation of
either thymol (1) or carvacrol (2) using Co(II)salen as catalyst
and molecular oxygen as oxidant (Figure S4, Supporting
Information).38 The advantage of this reaction compared to
stoichiometric oxidations is the decrease of the amount of
chemicals used as well as the high yield. The thymo-1,2-
quinone (6) was also successfully obtained via oxidation
(Figure S6, Supporting Information). To this end, NaIO4 was
B
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Table 1. Retention Indices (RI), Odor Thresholds in Air (OT, Median, Range, and Factor between Highest and Lowest OT),
and Odor Qualities of the Investigated Monoterpenoids
b
d
RI on
OT (ng/L air)
a
c
f
e
no.
odorant
DB-FFAP DB-5
N
median
range
factor (high/low)
odor qualities
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
thymol
carvacrol
p-cymene
thymoquinone
thymocatechol
1950
1984
1200
2825
nd
nd
2818
1462
1482
2222
1709
2252
1667
1700
2222
2749
2752
2807
2811
1293
1303
1030
1257
nd
6
6
9
4
2
2
8
6
6
6
6
4
4
4
9
9
9
4
4
2.0
2.7
43.2
24.3
nd
0.7−10.8
0.3−10.8
21.6−345.6
1.4−345.6
nd
16
32
16
256
nd
nd
640
32
8
thyme-like, rosemary-like, ethereal
oregano-like, wood-like, pencil-like
wood-like, citrus-like
medicinal, pencil-like, leather-like
g
h
thyme-like, ethereal
h
thymo-o-quinone
nd
nd
nd
thyme-like, pencil-like and ethereal
smoke-like, pencil-like
thymohydroquinone
thymol methyl ether
carvacrol methyl ether
p-methoxythymol
p-methoxycarvacrol
thymohydroquinone dimethyl ether
O-acetylthymol
O-acetylcarvacrol
di-O-acetyl-thymohydroquinone
p-acetylthymol
p-acetylcarvacrol
2-hydroxy-4-methyl-acetophenone
3-hydroxy-4-methyl-acetophenone
1562
1232
1241
1482
1414
1490
1349
1368
1664
1676
1656
1500
1521
388.8
10.8
8.1
8.1
8.1
1.4−345.6
2.7−86.4
2.7−21.6
2.7−43.2
2.7−172.8
10.8−43.2
2.7−10.8
2.7−43.2
0.7−691.2
10.8−1728
2.7−86.4
2.7−21.6
21.6−172.8
thyme-like, rosemary-like, medicinal
pencil-like, bergamot-like, oregano-like
oregano-like, pencil-like, resin-like
oregano-like, pencil-like, resin-like
earthy, moldy
thyme-like, ethereal-like, rosemary-like
oregano-like, thyme-like
pencil-like, medicinal
16
64
4
32.4
8.1
4
12.2
172.8
43.2
21.6
5.4
16
1024
160
32
8
phenolic, leather-like, faecal
phenolic, rubber-like
plastic-like, rubber-like
32.4
8
smoke-like, ham-like, phenolic
a
b
c
Numbers chosen according to structural relation and used within the whole publication. RI = retention index. N = number of panelists
determining the odor thresholds in air. OT = odor threshold in air determined according to the literature.28−30 Odor qualities determined by the
d
e
f
g
trained sensory panel. Factor of variation between highest and lowest individual value. nd = not determined due to instability of the compounds.
Due to instability only determined directly after synthesis by two trained panelists.
h
Table 2. Odor Thresholds (OT) of All Panelists of Thymol, Carvacrol, and Its Derivatives in Air Determined by GC-O
b
c
OT in ng/L air
panelists
no.
odorant
geometric mean P1-A
P2-A
P3-A
P4-A
P5-A
P6-A P1−B P2−B
P3−B
P4−B
a
1
2
3
4
7
8
9
10
11
12
13
14
15
16
17
18
19
thymol
carvacrol
1.9
0.7
2.7
2.7
2.7
1.4
10.8
2.7
n.i
n.i.
n.i.
86.4
5.4
172.8
43.2
n.i.
n.i.
n.i.
345.6
n.i.
21.6
n.i.
0.7
2.7
21.6
43.2
n.i.
2.7
2.7
5.4
5.4
n.i
n.i.
n.i.
21.6
n.i.
43.2
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
172.8
n.i.
10.8
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
172.8
n.i.
345.6
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
345.6
864
86.4
n.i.
n.i.
n.i.
43.2
n.i.
864
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
172.8
43.2
21.6
n.i.
3.0
0.3
10.8
172.8
1.4
691.2
2.7
21.6
2.7
2.7
43.2
2.7
2.7
345.6
43.2
2.7
10.8
43.2
5.4
p-cymene
thymoquinone
thymohydro-quinone
thymol methyl ether
carvacrol methyl ether
p-methoxythymol
p-methoxycarvacrol
thymohydro-quinone dimethyl ether
O-acetylthymol
O-acetylcarvacrol
di-O-acetylthymo- hydroquinone
p-acetylthymol
74.1
18.2
172.3
9.6
43.2
n.i.
5.4
10.8
5.4
345.6
345.6
3456
10.8
10.8
10.8
5.4
21.6
10.8
21.6
691.2
1728
21.6
5.4
432
10.8
10.8
43.2
43.2
43.2
10.8
43.2
43.2
43.2
10.8
21.6
21.6
7.6
10.8
13.6
25.7
6.4
5.4
10.8
10.8
5.4
n.i.
n.i.
n.i.
21.6
n.i.
n.i.
n.i.
n.i.
43.2
10.8
2.7
n.i.
n.i.
n.i.
86.4
345.6
5.4
9.1
2.7
93.3
90.8
13.6
6.4
0.7
43.2
43.2
2.7
p-acetylcarvacrol
2-hydroxy-4-methyl-acetophenone
3-hydroxy-4-methyl-acetophenone
5.4
43.2
n.i.
43.2
21.6
172.8
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
a
b
c
n.i. = not investigated (by the respective panelist). OT geometric mean values determined in air by GC-O. Panelists investigated the compounds
partially two times (A and B); in total the values of panelists 1−6 (P1−P6) in investigations A and B are displayed.
added to thymocatechol (5). As both substances readily
decompose, especially when being dissolved or during gas
chromatography, sensory properties and mass spectra could
not be determined. In these cases, two trained panelists
smelled the pure substances directly after synthesis to gain an
immediate odor impression. Thymohydroquinone (7) was
obtained by reducing thymoquinone with elemental Zn and
HOAc.
droquinone dimethyl ether (12) were generated applying a
Wiliamson etherification on thymol (1), carvacrol (2), and
thymohydroquinone (7) with CH3I as methylation agent
(Figure S1, Supporting Information). The syntheses of the
mono methyl ethers were conducted with KOH as base. For
the synthesis of thymohydroquinone dimethyl ether (12) the
reaction was carried out stepwise using the stronger base
potassium tert-butoxide (Figure S5, Supporting Information).
The acetylated substances O-acetylthymol (13), O-acetylcar-
vacrol (14), and di-O-acetylthymohydroquinone (15) were
Thymol methyl ether (8), carvacrol methyl ether (9), p-
methoxythymol (10), p-methoxycarvacrol (11), and thymohy-
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Figure 2. Odor thresholds for thymol and carvacrol derivatives shown as median value of all panelists.
formed by reaction with (Ac)2O (Figure S2, Supporting
Information).
be noted that although the panel size might seem to be small,
numerous studies investigating OT values in air consisted of
only 1−3 panelists, as the method is time-consuming.41,43,44
The OTs ranged from 2.0 ng/L air to 388.8 ng/L air (Table
1). A graphical overview is provided in Figure 2. Thereby, the
lowest OTs were obtained for thymol (1, 2.0 ng/L air) and
carvacrol (2, 2.7 ng/L air), followed by 2-hydroxy-4-
methylacetophenone (18, 5.4 ng/L air). Their acetates 13
and 14 and their mono methyl ethers 8 and 9 as well as the
mono methyl ethers of thymohydroquinone 10 and 11 showed
comparable OTs of around 10 ng/L air. The OTs of p-cymene
(3), O-acetylcarvacrol (14), 3-hydroxy-4-methylacetophenone
(19), p-acetylthymol (16), p-acetylcarvacrol (17), thymohy-
droquinone dimethyl ether (12), and thymoquinone (4)
ranged between 21.6 ng/L air and 43.2 ng/L air. The highest
OTs were obtained for di-O-acetylthymohydroquinone (15)
and thymohydroquinone (7) with 172.8 and 388.8 ng/L air,
respectively. The latter two molecules have a higher degree of
p-oxygenation, resulting in a more hydrophilic character and
being the likely reason for their higher OTs in comparison to
thymoquinone (4) and thymohydroquinone dimethyl ether
(12), which are comparable in structure but comprise more p-
located lipophilic groups. The nasal mucosa is lipophilic in
nature; accordingly, it has been hypothesized that more
lipophilic substances might more easily interact with the nasal
mucosa, and other groups stated that, accordingly, more
lipophilic substances have enhanced odor potency.45,46 On the
other hand, one needs to keep in mind that odorous molecules
first need to pass an aqueous mucus layer, and may,
furthermore, undergo biotransformation at the periphery
prior to interaction with the receptor sites.47 Accordingly,
without any factual biochemical and nasal and olfactory tissue
studies, such considerations remain mostly speculative. With
the sole exception of the higher OTs in the case of
For further changes of the structural motifs related to the
carbon skeletons of 1, 2, and 7, a Fries rearrangement reaction
was applied. First, p-acetylthymol (16) and p-acetylcarvacrol
(17) were obtained via the readily formed phenolic esters,
followed by the Fries rearrangement,39 which converted the
phenolic esters into hydroxyacetophenones (Figure S3,
Supporting Information). Based upon steric considerations,
acylation in the para-position was preferred. When adjusting
the conditions to favor the o-acetylated products by using a
nonpolar solvent (cyclohexane) and increased temperature (80
°C) the substances 2-hydroxy-4-methylacetophenone (18) and
3-hydroxy-4-methylacetophenone (19) were obtained. Steric
and electronic effects led to ipso-substitution instead of the
common reaction pathway. All the compounds were
synthesized in high purity, as was verified by GC-MS and
NMR measurements.
Determination of Odor Thresholds (OTs). For the
determination of odor thresholds in air, a state-of-the-art gas
chromatography−olfactometry (GC-O) method was chosen.40
The use of GC-O allows for the separation of possible odor-
active impurities accompanying the target compound during
gas chromatography so that such potential impurities cannot
influence the results of smell characterization. Furthermore, the
determination of OTs in air rules out any influence of matrix
constituents, as it would be the case when OTs are determined,
e.g., in water, oil, or cellulose.41 OT values in air were
calculated both as median (cf. Table 1) and as geometric mean
(cf. Table 2). In the following, mainly the median values will
be discussed, since both the geometric mean and the
corresponding median values showed the same overall
tendency in OTs. This approach is proposed by Lorber et
al.42 in the case of a limited panel size. Nevertheless, it has to
D
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Figure 3. Overview of the odor qualities of the synthesized compounds (n.d.: not determined, due to instability of the compounds).
thymohydroquinone (7) and di-O-acetylthymohydroquinone
(15), the remaining substances showed relatively similar OTs.
It is especially interesting to note that there was no clear
relationship between odor threshold value and the different
structural motifs that were present in the p-cymene derivatives.
Neither an acetyl, hydroxy, or methoxy group nor having two
or one substituents (p-positioned) influenced the OT values of
the resulting molecules. Likewise, it had no effect on the OT
values whether the molecule was a structurally modified
derivative of thymol (hydroxy group in ortho-position relative
to the isopropyl group) or of carvacrol (hydroxy group in meta-
position relative to the isopropyl group).
In the present study, the compounds were always modified
in either position 1 or 3 regarding the substitution pattern in p-
cymene (3) (hydroxy groups in ortho- and meta-positions with
regard to the methyl and isopropyl groups) or in combined
patterns, implying substituents occurring in 1,4-para-locations.
Only in the case of thymocatechol (5) and thymo-o-quinone
(6) did the compounds have a 1,2-disubstitution relative to the
methyl group and hence were ortho to each other. In
accordance with catechol and benzo-1,2-quinone, both
substances were unstable and decomposed readily.48 There-
fore, an attempt to synthesize further derivatives of compounds
1, 2, and 7 substituted in 1,2-locations to the methyl group
might be challenging, even though the sensory properties of
such unstable intermediates would be of interest.
Odor threshold single values (Table 2) were only slightly
variable between individual panelists. To provide a better
visualization of the breadth of the range covered by the
individual values, calculated factors between the highest and
lowest individual value are provided for each substance in
Table 1. Generally, this factor was in the range of 16 (typically
ranging from 4 to 64), which might appear to be high but is, in
comparison to other findings of our group on interindividual
variation in smell sensitivity to terpenoid and sesquiterpenoid
substances, surprisingly low.30,33 The highest factors, however,
between individual extremes were obtained for di-O-acetylth-
ymohydroquinone (factor 1024) and thymohydroquinone
(factor 640). Such deviations in odor thresholds between
individuals have repeatedly been reported.42,49 Humans differ
in their individual olfactory receptor patterns; in view of this, it
is noteworthy that the olfactory receptor repertoire is, generally
speaking, one of the most diverse in the human genome.50,51
Based on individual expression patterns and levels, the
activation of one or several receptors by one or several
odorants can be divergent between individuals, which might be
one of the reasons for the observed threshold variations.
Furthermore, odorants may be biotransformed (e.g., by nasal
enzymes) before reaching the respective receptor site.47,52 This
biotransformation can, inter alia, occur in the course of
cytochrome P450 metabolization and has previously been
reported to be relevant for phenolic compounds.53 For both
substances with highest extremes between individuals, namely,
di-O-acetylthymohydroquinone (15) and thymohydroquinone
(7), the same panelist (P-3A) perceived the substance with
very high OTs, which might indicate that this panelist is more
insensitive to the substances because of the receptor
disposition.
In general, the OT values from the present study correspond
well with those of other terpenes. The OTs determined in air
of α-pinene, myrcene, and limonene,54 as well as (+)-(4S)-
carvone,55 were also in the range of 5−450 ng/L air. On the
other hand, comparable phenolic structures such as alkylated
phenols or guaiacol derivatives had OT values between around
0.05 ng/L air and 502 ng/L air.43,56 Thereby, monomethyla-
tion of phenols in the meta-position showed the lowest OTs
with below 0.5 ng/L air, whereas additional methyl groups as
well as a prolongation of the alkyl group increased the OTs,
leading to values in the range of those of the investigated
substances (2−500 ng/L air).43
Odor threshold information on the investigated substances
is rare. Matrix-based odor thresholds are only reported for
carvacrol, thymol, and p-cymene.57−59 Moreover, no literature
odor threshold values in air are known. OTs in water have
been reported in a study on mango volatiles,57 with values of
2.29 and 1.7 ppb for carvacrol and thymol, respectively. In
another study targeting the characterization of odor-active and
physiologically active compounds in thyme, oregano, and
majoram,60 odor thresholds in water were determined for
thymol and carvacrol, as well as for p-cymene. In that study,
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thymol has been reported with the lowest OT in water with a
value of 0.08 μg/L water, followed by carvacrol with 5 μg/L
water and p-cymene with 5.1 μg/L water. Our results of 2.0
ng/L air for thymol, 2.7 ng/L air for carvacrol, and 43.2 ng/L
air for p-cymene align well in rank order. In contrast to that, a
further study on sensory thresholds of phenolic compounds
reports the OTs of carvacrol and thymol in sunflower oil with
31 mg/kg for carvacrol and 124 mg/kg for thymol, thus being
ranked in reverse order. Since a determination of odor
thresholds is matrix-dependent and can differ in several orders
of magnitude between the applied methods, these discrep-
ancies are not surprising.61
Determination of Odor Qualities. In contrast to the
determined OTs, where the differences in structures had little
effect on the respective odor thresholds, the structural
modifications in the investigated molecules apparently
influenced their smell character. For a better comparison, all
descriptors selected by the trained panel during sensory
evaluation of the compounds are provided in Table 1.
The unsubstituted p-cymene (3) was primarily described as
smelling wood-like and citrus-like. Most of the substances
showed similar odor impressions with thyme-, oregano-, and
pencil-like smells. Nevertheless, it became evident that the
substances can be clustered into groups according to their
common olfactory properties (cf. Figure 3). Generally, the
panelists reported that thymol (1), carvacrol (2), thymol
methyl ether (8), carvacrol methyl ether (9), O-acetylthymol
(13), O-acetylcarvacrol (14), p-methoxythymol (10), and p-
methoxycarvacrol (11) predominantly elicited odors that were
reminiscent of aromatic plants. The substances smelled mainly
thyme-, rosemary-, oregano-, and bergamot-like, at times
accompanied by pencil-like, resin-like, and ethereal notes.
Moreover, the substances can be subgrouped, according to
their core structures, into thymol and its derivatives thymol
methyl ether (8), O-acetylthymol (13) comprising substances
with a substitution in meta-position to the isopropyl group, and
carvacrol and its derivatives carvacrol methyl ether (9) and O-
acetylcarvacrol (14) with substitution with a hydrophilic group
in ortho-position to the isopropyl group. The compounds with
the thymol core structure elicited smells that were dominated
by a clear thyme-like impression, whereas those with a
carvacrol core moiety tended to smell oregano-like. This
corresponds with the fact that the essential oil of thyme is
dominated by thymol, while the essential oil of oregano has a
higher carvacrol content.3 It is interesting to note that p-
methoxythymol (10) and p-methoxycarvacrol (11) have
substitutions in both meta- and ortho-position relative to the
isopropyl group; these compounds were perceived with smell
that appeared to be rather a blend of oregano-like, pencil-like,
and resin-like odors.
and di-O-acetylthymohydroquinone (15), which all have an
oxygen atom ortho to the methyl group. Yet, the odor qualities
of these substances are accompanied by a pencil-like odor.
Accordingly, the proximity of the substituents to the isopropyl
group appears to be related to more pencil-like odors, which is
also the case for the p-methoxythymol (10) and p-
methoxycarvacrol (11). An exception was observed for the
thymohydroquinone dimethyl ether (12), which was reported
with an earthy, moldy odor, despite its substitution with an
electronegative group in the para-position.
In the case of 2-hydroxy-4-methylacetophenone (18), 3-
hydroxy-4-methylacetophenone (19), p-acetylthymol (16),
and p-acetylcarvacrol (17), the characteristic aromatic plant-
like and pencil-like odors were not perceivable; instead, plastic-
like, rubber-like, smoky, phenolic, and leather-like notes
characterized the smell of these substances. The common
structural element in those substances is an acetyl group being
located para to either the hydroxy (16, 17) or the methyl
group, giving the respective ipso-isomers (18, 19). In a study
on the odor properties of alkylated phenols, various alkylated
phenolic compounds were rated with similar attributes as
smelling leather-like (e.g., 3-ethylphenol), medicinal (e.g., o-
cresol), fecal (e.g., p-cresol), or rubber-like (4-isopropylphe-
nol).43 Moreover, various derivatives of guaiacol are charac-
terized by smoke-, ham-, and plastic-like odors,56 which appear
to be, consequently, common odor qualities in the class of
phenolic substances, as previously observed, and now being
confirmed by further derivatives. In addition, a plastic-like
smell is reported for acetophenone, 1-hexen-3-one, and 1-
octen-3-one64 as well as for various mono- or diunsaturated
carbonyl compounds.65 The common substitution pattern is
the α,β-unsaturated carbonyl group, which is also present in
the synthesized compounds 18, 19, 16, and 17 and could give
a hint of plastic-like odors.
The synthesized products with a 1,2-substitution pattern,
thymocatechol (5) and thymo-o-quinone (6), respectively,
were, as mentioned above, unstable, and gas chromatographic
analyses could not be performed. In order to still get some
insights into their smell properties, the following approach was
applied: directly after synthesis, two panelists evaluated the
immediately perceived odor qualities, coupled with immediate
NMR experiments that confirmed the successful synthesis and
high purity of the obtained compounds. The smell impressions
reported by the panelists were thyme-like, ethereal for
thymocatechol (5) and thyme-like, pencil-like, and ethereal
for thymo-o-quinone (6). This indicates that the modified 1,2-
substitution pattern also leads to olfactorily interesting
molecules that are related to pencil-like odors and provide
further insights into structure−odor relationships of oxy-
genated, aromatic monoterpenoids.
Thymohydroquinone (7), thymoquinone (4), and di-O-
acetylthymohydroquinone (15), which have electronegative
functional groups in positions 1 and 4, elicited pencil-like
smells, accompanied by smoky and medicinal notes. A smoky
odor is known for guaiacol and guaiacol dervivatives such as 4-
methylguaiacol, 2,6-dimethoxyguaiacol, and plenty of other
alkylated and alkenylated guaiacols.56,62 In the case of a
medicinal odor, previous studies showed that phenols
substituted in an ortho-position elicit this odor impression
regardless of the substituent being a halogen or an alkyl
group.43,63 This specific substitution pattern of an oxygenated
functional group and an alkyl group in an ortho-position can
also be found in thymohydroquinone (7), thymoquinone (4),
In summary, the investigations on structure−odor relations
showed that the different substituents clearly influence the
odor character of the target molecules of the present study. We
could show that the odor quality changed from woody, citrus-
like (unsubstituted p-cymene) to thyme- and oregano-like (for
the monosubstituted molecules). The position of the hydro-
philic group and the isopropyl group also had an impact on the
odor quality: a substitution in the meta-position resulted in a
thyme-like smell, whereas the substitution in ortho-position to
the isopropyl group yielded oregano-like odors. In the case of
p-methoxythymol (10) and p-methoxy-carvacrol (11), dis-
ubstitution in p-position led to comparable odor qualities,
which is likely due to the fact that both compounds share a
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and a Gerstel CIS 3 injection system (Gerstel, Duisburg, Germany).
The samples were injected automatically with a total volume of 2 μL.
The analyses were conducted using the same capillaries and
temperature programs as described above with a flow rate of 1.0
mL/min. Mass spectra were generated in the electron impact mode
(EI-MS) at 70 eV.
Retention indices (RI) for all compounds were calculated based on
a series of homologous n-alkanes (C6−C31) according to van den
Dool and Kratz.66
substitution motif in the meta- and ortho-position. In addition
to the prevalent oregano-like impression, those two substances
were additionally described as smelling pencil- and resin-like.
In the case of thymohydroquinone (7), thymoquinone (4),
and di-O-acetylthymohydroquinone (15), disubstitution in the
p-position likewise provoked pencil-like smells, which was
replaced by more plastic-like, rubber-like smells for the
acetophenone derivatives. Whereas differences in odor
qualities could be assigned, the structural modifications did,
however, not influence the odor thresholds to a larger extent.
Thus, we provide with this study comprehensive analytical
and sensory data, comprising routes for synthesis, retention
indices, NMR and mass spectra, odor thresholds in air, and
odor qualities, on derivatives of thymol and carvacrol. The data
generated further extends common knowledge on substances
that elicit woody, aromatic, and pencil-like odors. Further
studies on the interaction of those substances with the specific
odorant receptors will help to fully resolve the structure−odor
relationships.
Panelists. Panelists were trained volunteers from the Chair of
Aroma and Smell Research of the Friedrich-Alexander-University
̈
Erlangen-Nurnberg (Erlangen, Germany) and the Fraunhofer
Institute for Process Engineering and Packaging IVV (Freising,
Germany), exhibiting no known illness at the time of examination and
with audited olfactory function. In preceding weekly training sessions,
the assessors were trained prior to participation in the experiments for
at least half a year to correctly recognize, identify, and name the odor
qualities of reference substances and natural products according to an
in-house developed odor language. Moreover, no anosmia of the
chosen assessors was known and they showed no clearly deviating
olfactory sensitivity.
Odor Quality Determination. The characteristic odor qualities
of all synthesized compounds (except nos. 5 and 6 due to instability)
were evaluated following a systematic procedure for sensory analysis.
Therefore, the substances were dropped onto a filter paper and put
into glass vessels. The purity of all compounds was ascertained by 1H
NMR and GC-MS analyses prior to the sensory experiments. All
synthesized compounds were further checked for olfactory purity
using GC-O. The substances were presented to the trained sensory
panel (cf. panelists) in covered glass vessels coded with a random
three-digit number in a sensory room, at 21 °C. The panel consisted
of four males and six females in the age range of 23 to 33 years. First,
the panelists were asked to name the individual odor qualities of the
compounds based on the in-house odor language and weekly training
sessions (cf. 2.5), and then the characteristic attributes for each
substance were chosen in consensus.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Unless otherwise stated, all
solvents and reagents were purchased from commercial sources
̈
(Sigma-Aldrich, VWR, Grussing GmbH Analytica, Acros, Carl Roth,
Fluka); they were analytically pure and used without further
purification. All solvents were freshly distilled prior to use. All
reactions requiring anhydrous conditions were carried out under a
nitrogen atmosphere, and the solvents were dried before use to
remove moisture using appropriate drying solvents. For column
chromatography silica gel with a size of 40−60 μm, purchased from
Carl Roth, was used. Thin-layer chromatography (TLC) was
conducted on silica gel 60 F254 TLC plates from Macherey-Nagel
(Duren, Germany) with a pore diameter of 60 Å.
̈
Odor Threshold Values. Odor thresholds in air were determined
by GC-O using (E)-dec-2-enal as internal standard.40,43,67 A stock
solution of the target substances and the internal standard in known
concentrations (1000 μg/mL) was prepared in CH2Cl2 and diluted
stepwise 1:1 (v:v). Every dilution was then injected into the GC-O
system and analyzed by each panelist. The thresholds were
determined by 4−6 panelists (four male, two female), with each
experiment being conducted at least once by each panelist. GC-O
analyses for RI determination were performed on DB-5 and DB-FFAP
capillaries as described in the section Gas Chromatography−
Olfactometry and Gas Chromatography−Mass Spectrometry (GC-
MS). The purity of all commercially available and synthesized
All reactions were monitored by TLC using cyclohexane and
EtOAc (5:1) as separation agents. The visualization of the reaction
components was achieved using UV fluorescence (254 nm) and
KMnO4 stain. If not stated otherwise, column chromatography over
silica gel 60 was carried out for purification. The yields reported here
are those obtained after purification.
1H NMR spectra were recorded in CDCl3 on Avance 360 and
Avance 600 spectrometers (Bruker Biospin, Rheinstetten, Germany)
at room temperature operated at 360 or 600 MHz with TMS as
internal standard. Chemical shifts (δ) are quoted in parts per million
(ppm) calibrated to TMS (1H). Coupling constants (J) were
measured in hertz (Hz). The following abbreviations are used to
describe multiplicities: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet. The identity of all intermediates and synthetic
products was additionally determined by GC-MS in EI-mode as
described in the following.
1
compounds was ensured by H NMR and GC-MS analyses prior to
the GC-O experiments. All synthesized compounds were further
checked using GC-O for potential odor-active impurities during the
sniffing process to exclude any interferences.
Ethics Statement. The study was conducted in agreement with
the Declaration of Helsinki. The study (registration number
180_16B) was approved by the Ethical Committee of the Medical
Gas Chromatography−Olfactometry and Gas Chromatog-
raphy−Mass Spectrometry (GC-MS). GC-FID/O analyses were
performed with a Trace GC Ultra (Thermo Fisher Scientific GmbH,
Dreieich, Germany) housing a DB-FFAP or a DB-5 fused silica
capillary both with dimensions 30 m × 0.32 mm, 0.25 μm (both
Agilent J&W, Santa Clara, CA, USA). The samples were injected
manually by the cold on-column technique at 40 °C with an injection
volume of 2 μL. After 2 min, the oven temperature was raised at 10
°C/min to a final temperature of 240 °C (DB-FFAP) or 280 °C (DB-
5). The final temperature was held for 10 min. The flow rate of the
carrier gas helium was 2.5 mL/min. At the end of the capillary
column, the effluent was split into a ratio of 1:1 (by volume) into an
FID and a sniffing port using two deactivated uncoated fused silica
capillaries (50 cm × 0.32 mm) of the same length. The FID and the
sniffing port were held at 250 °C, respectively.
̈
̈
Faculty, Friedrich-Alexander Universitat Erlangen-Nurnberg. In-
formed consent was obtained from all subjects participating in the
study.
Syntheses. The synthetic procedures are general procedures. The
reader is referred to the Supporting Information for a detailed
description.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00339.
■
sı
1
General synthetic routes and H NMR spectroscopic
GC-MS analyses were performed using an Agilent MSD quadru-
pole system (GC 6890 and MSD 5973, Agilent Technologies,
Waldbronn, Germany), equipped with a Gerstel MPS 2 autosampler
data of all synthesized compounds, information on
previous identification sources, mass spectrometric data
G
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AUTHOR INFORMATION
Corresponding Author
Andrea Buettner − Aroma and Smell Research, Friedrich-
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Alexander-Universitat Erlangen-Nurnberg (FAU), 91054
Erlangen, Germany; Department of Sensory Analytics,
Fraunhofer Institute for Process Engineering and Packaging
(IVV), 85354 Freising, Germany; orcid.org/0000-0002-
6205-5125; Email: andrea.buettner@ivv.fraunhofer.de
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Alexander-Universitat Erlangen-Nurnberg (FAU), 91054
Erlangen, Germany; Department of Sensory Analytics,
Fraunhofer Institute for Process Engineering and Packaging
(IVV), 85354 Freising, Germany
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Johannes Bauer − Aroma and Smell Research, Friedrich-
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Erlangen, Germany
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Eva Ortner − Department of Sensory Analytics, Fraunhofer
Institute for Process Engineering and Packaging (IVV), 85354
Freising, Germany
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b00339
Author Contributions
§L. Schreiner and J. Bauer contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the members of our working group for their
participation in the sensory analyses. This study was carried
out within the framework of the Campus of the Senses, a joint
endeavor of the Fraunhofer Institutes for Process Engineering
and Packaging IVV and Integrated Circuits ISS, together with
́
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Friedrich-Alexander-Universitat Erlangen-Nurnberg as an
academic partner. The Campus of the Senses initiative is
financially supported by the Bavarian Ministry of Economic
Affairs, Regional Development and Energy (StMWi) and the
Fraunhofer Society.
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