A New Fluorinated Tyrosinase Inhibitor from a Chemically Engineered Essential Oil

Article abstract of DOI:10.1021/acscombsci.6b00004

The generation of fluorinated essential oils as a source of bioactive compounds is described. Most of the components of the natural mixtures were altered, leading to the discovery of a new fluorinated tyrosinase inhibitor.

Full text of DOI:10.1021/acscombsci.6b00004

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Letter
A new fluorinated tyrosinase inhibitor
from a chemically engineered essential oil
Paula García, Mario Oscar Salazar, I. Ayelen Ramallo, and Ricardo L. E. Furlan
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00004 • Publication Date (Web): 04 May 2016
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A new fluorinated tyrosinase inhibitor from a chemically engineered es-
sential oil
Paula García,‡ Mario O. Salazar,‡ I. Ayelen Ramallo and Ricardo L. E. Furlan*
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Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,
2000, Suipacha 531, Rosario, Argentina.
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Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR, CONICET-UNR)
Ocampo y Esmeralda, 2000, Rosario, Argentina.
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KEYWORDS: diversification, essential oil, fluorination, tyrosinase, bioactive compound
Abstract. The generation of fluorinated essential
oils as a source of bioactive compounds is de-
scribed. Most of the components of the natural
mixtures were altered leading to the discovery of
a new fluorinated tyrosinase inhibitor.
Fluorine is one such element, the incorpora-
tion of which into a molecule can modulate phys-
icochemical properties such as pKa, lipophilicity,
hydrogen bonding and electrostatic interactions,
as well as metabolic stability (oxidative metabo-
lism, hydrolytic metabolism, in vivo racemiza-
tion).11 The strategic use of fluorine substitution in
drug design has led to the production of some of
Natural products are biologically validated
starting points for the development of new drugs.
They are the outcome of a long evolution process
that has resulted in a unique assortment of skele-
tons with high affinity for biomolecules.1
the key drugs available on the market.12, The
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average proportion of fluorine in drugs is signifi-
cantly higher than in natural products.14 Natural
organofluorines represent less than 1% of the nat-
urally occurring organohalogens.15
Essential oils (EOs) are natural multi-
component systems2 composed of low-molecular
weight lipophilic compounds derived from differ-
ent biosynthetic pathways.3 In general their pro-
duction by plants is diversity oriented, with the
generation of complex mixtures of compounds
that have the potential to regulate plant–insect
and plant–mammal interactions. This bioactive
volatilome is now emerging as a novel potential
source of interesting lead structures for drug dis-
covery.3
Considering that (a) small molecule natural
products have had a significant impact on drug
discovery, (b) 20-25% of drugs in the pharmaceu-
tical pipeline contain at least one fluorine atom,11
and (c) organofluorine compounds are virtually
absent as natural products,16 it becomes interest-
ing to evaluate the effect of fluorination on the
biological properties of natural mixtures of small
molecules such as EOs (Figure 1).
Several approaches have been proposed to in-
crease the diversity of natural product mixtures
such us combinatorial biosynthesis4 and related
techniques.5 Chemically engineered extracts
(CEEs) represent alternative sources of molecules
for the search of new bioactive compounds based
on natural skeletons.6-9 In this strategy, natural
mixtures are chemically altered through reactions
directed towards the incorporation of molecular
fragments or elements that are relevant for bioac-
tivity and rarely found in secondary metabolites.10
Figure 1. Diversification of essential oil mixtures by
fluorination to generate libraries of biologically active
compounds.
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For EOs diversification we selected Selectfluor
pal Component Analysis (PCA). The score plot
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as a highly reactive fluorinating reagent that is
safe, nontoxic, stable, and easy to handle.17-20 Se-
lectfluor can introduce fluorine atoms into mole-
cules by reaction with double bonds, aromatic
rings, and through the transformation of carbon–
hydrogen bonds to carbon–fluorine bonds at satu-
rated secondary and tertiary carbon sites.21 Ac-
cording to the Dictionary of Natural Products,22
78% of the essential oil constituents include at
least one non-aromatic carbon–carbon double
bond in their structures, 22% contain at least one
aromatic group, and 84% contain the types of C-
H bonds that may be reactive. In addition, Se-
lecfluor has been applied to the fluorination of
other functional groups23 that are present in es-
sential oil components such as enols (6.8%) and
alkynes (2.5%). The fluorination of essential oils
has not been reported to date.
showed discrimination between two groups by
PC1 and PC2: one corresponding to the FEOs
(Figure 2, red triangles) and the other corre-
sponding to the EOs (Figure 2c, blue circles). Sim-
1
ilarly, PCA of H NMR spectra of the 24 mixtures
showed discrimination between two groups by
PC1, PC2 and PC3 (Fig. S1 in Supporting Infor-
mation).
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The success of the CEEs approach lies in the
power of numbers: in order to increase the chanc-
es of generating a bioactive compound, it is im-
portant to produce many compounds that incor-
porate the desired chemical feature or element. A
series of 12 essential oils was fluorinated by reac-
tion with Selectfluor in refluxing acetonitrile, and
changes in chemical composition and bioactivity
were evaluated. Incorporation of fluorine into the
EOs constituents was confirmed by 19F NMR anal-
ysis, with new peaks appearing between -97 ppm
and -197 ppm as expected for aromatic fluorine,
α-fluoroketones and α-fluoroenones.
Figure 2. Box and whiskers plot for a) percentage of
peaks that disappeared from the chromatograms be-
cause of the reaction (blue) and that appeared in the
chromatograms after the reaction (red), b) number of
peaks from EOs (blue) and FEOs (red) detected in the
GC-MS chromatograms. c) Score plot of PCA of GC-MS
data: EOs (blue circles) and FEOs (red triangles).
The effect of the reaction on the biomolecular
properties of the mixtures was evaluated by com-
paring the tyrosinase inhibitory properties of the
fluorinated and the non-fluorinated mixtures. The
discovery of tyrosinase inhibitors is attractive due
to their potential applications in cosmetic, medic-
inal and agricultural industries. This enzyme ca-
talyses the production of melanin and other pig-
ments by oxidation of L-tyrosine.24 Various der-
matological disorders such as melasma, age spots
and sites of actinic damage, arise from an exces-
sive level of epidermal pigmentation.25 Addition-
ally, the browning observed in vegetables and
fruits after harvest is associated with tyrosinase
activity, which produces a less attractive appear-
ance and loss in nutritional quality.26
The impact of the reaction over the chemical
composition of the mixtures was evaluated by GC-
MS, showing that most of the EO components
were transformed by the reaction, expanding the
chemical diversity of the mixtures. At least 60 %
of the peaks observed in the chromatograms of
EOs disappeared after the reaction, and at least
88% of the peaks present in the gas chromato-
grams of the resulting fluorinated essential oils
(FEOs) were absent in the chromatogram of the
precursor EO (Figure 2a). The average number of
major compounds detected in the mixtures in-
creased from 37 to 155 due to the fluorination re-
action (Figure 2b). This suggests that, on average,
four products were generated from each natural
precursor.
The tyrosinase inhibition properties of the
mixtures were surveyed by TLC bioautography,27 a
technique particularly well suited for the analysis
Changes in the composition of the mixtures
were also evident from GC-MS coupled to Princi-
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of mixtures.28 This methodology allows the evalu-
ation of inhibitory properties of a sample devel-
O
OCH3
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Selectfluor
oped onto a TLC plate that is covered with a gel
that contains enzyme and substrate. When ap-
plied to tyrosinase, fluorination was observed to
enhance the inhibitory properties of two mix-
tures: the FEOs from Ocimum basilicum L., Lami-
naceae (FOB) and Artemisia dracunculus L.,
Asteraceae (FAD) showed intense inhibition spots
that were absent in the non-fluorinated EOs. A
follow-up microplate assay29 showed that the IC50
values for O. basilicum oil decreased from 278.4 ±
1.38 µg/mL to 174.4 ± 1.45 µg/mL after fluorina-
tion. Similar results were obtained for A. dra-
cunculus, in which IC50 decreased from 232.2 ± 1.19
µg/mL to 125.5 ± 1.56 µg/mL.
ACN, 82°C, 16 hs
F
OCH3
+
CH3
2
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F
3
Scheme 1. Proposed synthesis of 4-allyl-4-
fluorocyclohexa-2,5-dienone (1) from methyl chavicol
(2) with Selectfluor through para-fluoro cation (3).
The main bioactive compound in both mix-
tures was identified as 4-allyl-4-fluorocyclohexa-
2,5-dienone (1, Scheme 1). The identity of this
compound was established by NMR (1H, 19F and
13C NMR), IR, and HRMS analyses. This fluorinat-
ed derivative could have been formed from the
inactive natural component methyl chavicol (2,
Scheme 1) that is present in both O. basilicum and
of A. dracunculus EOs.30 This was confirmed by
fluorination of pure 2 using the same reaction
protocol previously employed for the EOs. The
TLC-bioactivity profile of the reaction showed the
generation of the same bioactivity spot that was
previously detected in O. basilicum and of A. dra-
cunculus FEOs.
The inhibitory potency of the fluorinated com-
pound 1 (IC50 59.14 ± 1.15 µM) was found to be
similar to that of the known tyrosinase inhibitor
kojic acid (IC50 42.16 ± 1.04 µM). Under these ex-
perimental conditions the natural precursor of 1,
methyl chavicol (2) was inactive (IC50 >1000 µM).
In summary, the generation of chemically en-
gineered extracts through fluorination is de-
scribed for the first time. Chemical diversification
of a series of EOs led to the transformation of
most of the components of the starting mixtures,
producing fluorinated mixtures of expanded di-
versity (four-fold increase in the number of com-
pounds). Fluorination increased tyrosinase inhibi-
tion in two mixtures. The use of a straightforward
bioautographic assay enabled the identification of
a minor fluorinated compound with similar inhib-
itory properties to kojic acid, generated in the
mixture from a natural inactive precursor.
It is interesting to note that compound 1 was a
minor constituent of both bioactive fluorinated
mixtures. Although comprising only 0.6% of the
total peak area of the chromatogram of O. basili-
cum FEO, and 0.5% total peak area of the chro-
matogram of A. dracunculus FEO (Fig. S2 in Sup-
porting Information), this active component was
easily identified in the bioautography assay.
Compound 1 could result from an addition-
elimination process initiated with the generation
of a para-fluoro cation (3, Scheme 1) from 2. Re-
lease an alkyl cation from the phenolic oxygen
would thus result in the formation of the ob-
ASSOCIATED CONTENT
Supporting Information. Detailed experimental
procedures and spectroscopic data (PDF). This
material is available free of charge via the Internet
at http://pubs.acs.org.
served
(Scheme 1).31
4-allyl-4-fluorocyclohexa-2,5-dienone
AUTHOR INFORMATION
Corresponding Author.
*Phone: +54-341-4237868 ext 753.
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*E-mail: furlan@iidefar-conicet.gob.ar
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O
OCH3
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Selectfluor
ACN, 82°C, 16 hs
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OCH3
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CH3
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