Synthesis and tyrosinase inhibitory activities of 4-oxobutanoate derivatives of carvacrol and thymol

Article abstract of DOI:10.1016/j.bmcl.2018.11.013

Carvacrol (1) and thymol (2) were converted to their alkyl 4-oxobutanoate derivatives (7–20) in three steps, and evaluated for tyrosinase inhibitory activity. The compounds showed structure-dependent activity, with all alkyl 4-oxobutanoates, except 7 and 20, showing better inhibitory activity than the precursor 4-oxobutanoic acids (5 and 6). In general, thymol derivatives exhibited a higher percent inhibitory activity than carvacrol derivatives at 500 μM. Derivatives containing three-carbon and four-carbon alkyl groups gave the strongest activity (carvacrol derivatives 9–12, IC₅₀ = 128.8–244.1 μM; thymol derivatives 16–19, IC₅₀ = 102.3–191.4 μM).

Full text of DOI:10.1016/j.bmcl.2018.11.013

Accepted Manuscript
Synthesis and tyrosinase inhibitory activities of 4-oxobutanoate derivatives of
carvacrol and thymol
Nicholas Brotzman, Yiming Xu, Allison Graybill, Alexander Cocolas, Andrew
Ressler, Navindra P. Seeram, Hang Ma, Geneive E. Henry
PII:
S0960-894X(18)30878-3
https://doi.org/10.1016/j.bmcl.2018.11.013
BMCL 26124
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
30 August 2018
2 October 2018
7 November 2018
Please cite this article as: Brotzman, N., Xu, Y., Graybill, A., Cocolas, A., Ressler, A., Seeram, N.P., Ma, H., Henry,
G.E., Synthesis and tyrosinase inhibitory activities of 4-oxobutanoate derivatives of carvacrol and thymol,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.013
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Synthesis and tyrosinase inhibitory activities of 4-oxobutanoate derivatives of
carvacrol and thymol
Nicholas Brotzmana, Yiming Xub, Allison Graybilla, Alexander Cocolasa, Andrew Resslera,
Navindra P. Seeramb, Hang Mab, Geneive E. Henrya,*
aDepartment of Chemistry, Susquehanna University, 514 University Avenue, Selinsgrove, PA 17870, USA
bBioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences,
College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
*Corresponding author: henry@susqu.edu, 570-372-4222
Abstract
Carvacrol (1) and thymol (2) were converted to their alkyl 4-oxobutanoate derivatives (7-20) in three
steps, and evaluated for tyrosinase inhibitory activity. The compounds showed structure-dependent
activity, with all alkyl 4-oxobutanoates, except 7 and 20, showing better inhibitory activity than the
precursor 4-oxobutanoic acids (5 and 6). In general, thymol derivatives exhibited a higher percent
inhibitory activity than carvacrol derivatives at 500 M. Derivatives containing three and four carbon
alkyl groups gave the strongest activity (carvacrol derivatives 9-12, IC50 = 128.8-244.1 M; thymol
derivatives 16-19, IC50 = 102.3-191.4 M).
Keywords: Carvacrol, thymol, alkyl 4-oxobutanoates, tyrosinase inhibition.
The major components of oregano and thyme essential oils are the isomeric monoterpene phenols,
carvacrol (1) and thymol (2). These phenols exhibit a wide range of biological and pharmacological
effects including anticancer, anti-inflammatory, antibacterial, antifungal, anticholinesterase, insecticidal,
and antioxidant activities.1-3 Several derivatives of carvacrol and thymol have been synthesized and show
similar or enhanced activity relative to the parent phenols. Ester and carbamate derivatives of both
compounds showed increased antifungal4 and anticholinesterase5 activities, respectively. Some Schiff
base derivatives exhibited similar or better antioxidant activity compared to thymol and ascorbic acid.6
Heterocyclic derivatives of thymol containing pyridazinone,7 pyridone,8 and oxadiazole9 moieties gave
moderate to significant antibacterial activity when compared to standard antibiotics. Similarly, oxadiazole
and thiadiazole derivatives of carvacrol displayed significant enhancement of insect growth regulation in
a moth species,10 and moderate improvement in antioxidant activity11 relative to carvacrol.
Tyrosinase is the rate-limiting enzyme for the biosynthesis of melanin. Although melanin serves to protect
the skin from UV damage, overproduction can lead to skin defects such as melasma, freckles and age
spots. As a catechol oxidase, tyrosinase is also implicated in the undesirable browning of fruit. Thus,
tyrosinase inhibitors are important in the medical, agricultural and cosmeceutical fields, owing to their
ability to mitigate fruit browning and skin defects arising from melanin overproduction.12-13 The
tyrosinase inhibitory activity of carvacrol and thymol have not been well documented. Satooka indicated
that thymol affects the redox processes involved in dopachrome, and subsequently melanin formation, but
does not directly affect the tyrosinase enzyme activity.14 However, diesters of carvacrol and thymol
incorporating glycolic acid and benzoic or cinnamic acid moieties have shown moderate to potent
tyrosinase inhibitory activity when compared with kojic acid,15-16 a commercially used skin whitening
agent. In addition, a diester containing a succinoyl moiety coupled with two kojic acid units gave a better
than 2-fold increase in inhibitory activity than kojic acid.17 Based on the improvements in tyrosinase

inhibitory activities upon structural modification of carvacrol, thymol and kojic acid, it was envisioned
that alkyl 4-oxobutanoate derivatives of carvacrol and thymol would give enhancement in activity relative
to the parent phenols.
Fig. 1: Synthesis of alkyl 4-oxobutanoate derivatives of carvacrol and thymol
4-Oxobutanoate derivatives of carvacrol and thymol were synthesized in three steps (Fig. 1). Carvacrol
(1) and thymol (2) were converted to their corresponding ethyl ethers by modification of the procedure
reported by Silva et al.18 Treatment of acetonitrile solutions of the phenols with sodium methoxide and
bromoethane afforded ethyl carvacrol (3) and ethyl thymol (4) in 60% and 93% yields, respectively. The
yields and spectroscopic data for compounds 3 and 4 were in good agreement with literature data.18 With
the ethyl ethers in hand, it was anticipated that the 4-oxobutanoic acid moiety could be introduced to the
carvacrol/thymol core by Friedel-Crafts acylation with succinic anhydride and aluminum chloride, as
reported for butyl thymol.7 However, attempts to synthesize the 4-oxobutanoic acids using succinic
anhydride were unsuccessful in our hands. The use of succinoyl chloride, followed by acidic work up
gave the 4-oxobutanoic acids 5 and 6 in 68% and 81% yields, correspondingly. While the bis-carvacrol or
bis-thymol -diketone could have been obtained as a product of the reaction, there was no evidence of its
formation. The carvacrol and thymol 4-oxobutanoic acids were each converted to methyl, ethyl, propyl,
allyl, butyl, crotyl and benzyl 4-oxobutanoates by O-alkylation, using cesium carbonate and the
corresponding alkyl bromides or iodides in acetonitrile.19 The fourteen new alkyl 4-oxobutanoate
derivatives of carvacrol and thymol (7-20) were characterized by NMR, IR and HRMS analyses.
Tyrosinase converts L-tyrosine to dopaquinone, which is subsequently converted to dopachrome. The
tyrosinase inhibitory assay measures the amount of dopachrome produced in the presence of test
compounds.20 In order to determine structure-activity correlations, compounds 1-20 were evaluated at 500
M for inhibitory activity against mushroom tyrosinase (Table 1). Carvacrol (1) and thymol (2) gave
comparable data, with percent inhibition of 35.7 and 29.4%, respectively. For carvacrol there was a 1.6
fold increase in activity for the ethyl ether derivative (3) and a 2.1 fold decrease in activity for the 4-
oxobutanoic derivative (5). However, there was no significant difference in activity between thymol and
its ethyl ether (4) and 4-oxobutanoic acid (6) derivatives. The similar activity observed for thymol and its
ethyl ether derivative is in contrast to a previous study which suggested that the methyl ether derivative of
thymol had a lower inhibitory effect than thymol.14 The carvacrol and thymol alkyl 4-oxobutanoates
showed better inhibitory activities than the precursor 4-oxobutanoic acids, except for the methyl ester
derivative of carvacrol, 7, and the benzyl ester derivative of thymol, 20. In general, the activity was
2

enhanced with an increase in the carbon chain length, with esters containing three and four carbon alkyl
groups showing the greatest inhibitory activity (%Inhibition = 72.8-100%). The only exception is the
carvacrol allyl 4-oxobutanoate (10), which showed significantly lower inhibitory activity (58.2%) than the
corresponding propyl 9 (85.6%), butyl 11 (82.3%) and crotyl 12 (72.8%) derivatives. The benzyl ester
derivatives showed significantly lower percent inhibition than their three and four carbon counterparts (13
= 47.0%; 20 = 31.3%).
IC50 data were obtained for alkyl 4-oxobutanoates (9-12 and 15-19) showing greater than 51% inhibition
at 500 µM (Table 1). Kojic acid (IC50 = 21.8 M), was used as a positive control. The compounds
showed moderate inhibitory activity, with IC50 values ranging from 122.8 to 244.1 M for the carvacrol
series (9-12) and 102.3-212.3 M for the thymol series (15-19). Despite the anomalous percent inhibition
of the carvacrol allyl 4-oxobutanoate (10), it showed the highest inhibitory activity among the carvacrol
derivatives (IC50 = 128.8 M). For thymol, the crotyl derivative 19 had the highest inhibitory activity
(IC50 = 102.3 M). While the precise mechanism of action of the 4-oxobutanoates is unknown, the
increase in inhibitory activity for the three and four carbon derivatives and decrease for the benzyl group
and smaller alkyl groups may indicate interaction with the hydrophobic pocket of the tyrosinase enzyme,
12-13 with optimal binding interactions occurring with three and four carbon alkyl groups.
In conclusion, fourteen alkyl 4-oxobutanoate derivatives of carvacrol and thymol were synthesized and
evaluated for their tyrosinase inhibitory effects using mushroom tyrosinase. Although all the alkyl 4-
oxobutanoate derivatives showed lower inhibitory activity than kojic acid, the correlations between the
structure of the alkyl side chain and tyrosinase inhibitory activity are evident. Esters containing three or
four carbon atoms in the alkyl chain were the most effective. Further structural modifications of the
carvacrol and thymol derivatives could potentially lead to compounds with enhanced tyrosinase inhibitory
activities.
Table 1: Tyrosinase inhibitory activities of carvacrol (1)/thymol (2) and their derivatives 3-20.
Compound
Carvacrol (1) Thymol (2) Carvacrol Thymol
%Inhibition
35.75.4
57.52.5
16.94.0
31.76.2
50.92.1
85.66.6
58.22.3
82.32.1
72.83.3
47.09.0
%Inhibition
29.43.7
28.85.6
30.72.7
45.52.1
72.05.5
78.82.4
IC50 (M) IC50 (M)
Parent 1/2
Ethyl ether 3/4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4-Oxobutanoic acid 5/6
Methyl 4-oxobutanoate 7/14
Ethyl 4-oxobutanoate 8/15
Propyl 4-oxobutanoate 9/16
Allyl 4-oxobutanoate 10/17
Butyl 4-oxobutanoate 11/18
Crotyl 4-oxobutanoate 12/19
Benzyl 4-oxobutanoate 13/20
Kojic acid (IC50 = 21.81.7 M)
212.31.7
217.94.3 154.03.7
100.046.7 128.81.9 125.63.5
1003.9
99.38.0
31.37.1
176.52.8 191.42.4
244.13.6 102.3.1.8
n.d.
n.d.
% Inhibition (500 M); n.d. = not determined (IC50 >500 M)
Acknowledgments
We are grateful to Susquehanna University for financial support. The JEOL ECZ 400S NMR
spectrometer was acquired by a grant to Susquehanna University from the National Science Foundation
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(NSF:MRI CHE 1625340). The Waters SYNAPT G2-S QTOFMS system used for HRMS data is located
in the RI-INBRE core facility at the University of Rhode Island. The instrument was obtained by a grant
(# P20GM103430) from National Center for Research Resources (NCRR), a component of the National
Institutes of Health (NIH).
Supplementary data
1
Experimental procedures, tyrosinase inhibitory assay and data (HRMS, IR, H and 13C NMR) data for
compounds 5-20 are available.
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Highlights



Fourteen alkyl 4-oxobutanoate derivatives of carvacrol and thymol were synthesized.
Derivatives show structure-dependent activity against tyrosinase enzyme.
Derivatives containing three and four carbon alkyl groups were most active.
7