Expeditious and sustainable two-step synthesis of sinapoyl-l-malate and analogues: Towards non-endocrine disruptive bio-based and water-soluble bioactive compounds

Article abstract of DOI:10.1039/d0gc02763d

Faced with the increasing demand from both the cosmetic industries and consumers for bio-based, safe and natural skin products, sinapoyl-l-malate, widely described for its UV protection in plants, appears to be an excellent alternative to substitute chemical filters in sunscreens. Unfortunately, the only synthetic routes described in the literature were not only tedious but also exhibit a strong negative environmental impact, thus seriously limiting the industrialization and commercialization of sinapoyl-l-malate. Herein, a shorter and greener synthetic pathway involving Meldrum's acid opening with unprotected naturally occurring hydroxy acids and its subsequent Knoevenagel-Doebner condensation with biomass-derived p-hydroxybenzaldehydes was designed and developed. This two-step procedure, whom sustainability has been assessed using green metrics (atom economy (AE), process atom economy (PAE), E-factor and LCA), is a great alternative to the already reported procedures and allows the access to sinapoyl-l-malate and several analogs in average to good yield. The study of the anti-UV properties, stability against UV radiation, radical scavenging and antimicrobial activities of the targets revealed attractive properties as photostable UV filters, antioxidants and preservatives. Moreover, the water solubility brought by the free carboxylic acids facilitates the incorporation of these molecules in cosmetic formulations. Finally, their innocuousness toward endocrine disruption was demonstrated.

Full text of DOI:10.1039/d0gc02763d

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ARTICLE
Expeditious and sustainable two-step synthesis of sinapoyl-L-
malate and analogues: towards non-endocrine disruptive bio-
based and water-soluble bioactive compounds
Received 00th January 20xx,
Accepted 00th January 20xx
Cédric PEYROT [a], Matthieu M. MENTION [a], Robin FOURNIER [a], Fanny BRUNISSEN [a], Julien
COUVREUR [a], Patrick BALAGUER [b] and Florent ALLAIS [a]
*
DOI: 10.1039/x0xx00000x
Faced with the increasing demand from both the cosmetic industries and consumers for bio-based, safe and natural skin
products, sinapoyl-L-malate, widely described for its UV protection in plants, appears to be an excellent alternative to
substitute chemical filters in sunscreens. Unfortunately, the only synthetic routes described in the literature were not only
tedious but also exhibit a strong negative environmental impact, thus seriously limiting the industrialization and
commercialization of sinapoyl-L-malate. Herein, a shorter and greener synthetic pathway involving Meldrum’s acid opening
with unprotected naturally occurring hydroxy-acids and its subsequent Knoevenagel-Doebner condensation with biomass-
derived p-hydroxybenzaldehydes was designed and developed. This two-step procedure, whom sustainability has been
assessed using green metrics (atom economy (AE), process atom economy (PAE), E-factor and LCA), is a great alternative to
the already reported procedures and allows the access to sinapoyl-L-malate and several analogs in average to good yield.
The study of the anti-UV properties, stability against UV radiation, radical scavenging and antimicrobial activities of the
targets revealed attractive properties as photostable UV filters, antioxidants and preservatives. Moreover, the water
solubility brought by the free carboxylic acids facilitates the incorporation of these molecules in cosmetic formulations.
Finally, their innocuousness toward endocrine disruption was demonstrated.
mechanism of action and identify, by structural analogy,
components able to protect just as much from UV irradiation11-
14. Nevertheless, being present in very small quantities in plants,
Introduction
Naturally occurring p-hydroxycinnamic acids and their
derivatives - such as p-coumaric acid, ferulic acid, caffeic acid,
sinapic acid, sinapine - represent an abundant and renewable
source of phenolic compounds, rich in structural diversity with
a wide range of high added value applications1-5 . For instance,
p-hydroxycinnamic acids can be used for the preparation of new
bio-based and renewable polymers or as additives to provide
additional properties to the different polymers (antioxidant,
shape memory)6-8. In the form of dimers, they can be used as
non-endocrine disruptive bisphenol A substitutes9, 10. More
often, despite their very valuable biological properties, the use
of phenolic compounds in the medical or cosmetic sectors is
limited due to their very low solubility in water. In Nature, p-
hydroxycinnamic acid derivatives such as sinapoyl-L-malate or
sinapine are readily soluble in water thanks to their free
carboxylic acids and ammonium moieties. Sinapoyl-L-malate,
well-known for its UV filter properties in plants, has been the
subject of multiple studies in order to understand its
and its extraction being too complex and economically
irrelevant, the use of sinapoyl-L-malate at the industrial scale
required the implementation of synthetic pathways. Until now,
only two similar multi-step methodologies based on Steglich
esterification have been reported in the literature for the
synthesis of sinapoyl-L-malate and derivatives15, 16. Although
they provide the target compounds in average yields, these
synthetic methods are tedious to perform, require
protection/deprotection sequences and activation using excess
amount of coupling agent and toxic solvents, thus generating
much waste to dispose of. In 2009, Allais et al.15 described
sinapoyl-L-malate production in 30% overall yield using a 6-step
Steglich-based procedure. Not only the latter requires at least
26 days to be performed (excluding reaction processing time)
due to the first extremely long copper-catalyzed synthesis of
tert-butyl isourea, but also the latter was used in excess to
prepare di-tert-butyl-L-malate, thus producing a stoichiometric
amount of urea to be disposed of (Figure 1A). Moreover, for the
subsequent coupling of sinapic acid with di-tert-butyl-L-malate,
still through Steglich esterification, the phenol moiety had to be
protected via acetylation to prevent its esterification (side
reaction).
a. URD Agro-Biotechnologies Industrielles, CEBB, AgroParisTech, 51110, Pomacle,
France
b. IRCM, Inserm, Univ Montpellier, ICM, 208 avenue des Apothicaires, 34298,
Montpellier cedex 5, France
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/D0GC02763D
ARTICLE
O
O
OH
O
O
OH
Ac2O
Pyridine
(A)
16 h, RT
83%
O
O
O
CO2t-Bu
CO2t-Bu
O
O
O
CO2
H
H
O
O
O
O
O
CO2H
TFA (20 eq)
CH2Cl2
CDI/DMAP
CH2Cl2
OH
OAc
HCl 3M
Acetone
CO2
CO2
O
H
1 d, RT
84%
16 h, RT
93%
16 h, reflux
98%
L-Malic Acid
CH2Cl2
O
O
t-BuOH
CuCl
CO2t-Bu
CO2t-Bu
N
HN
N
OAc
OAc
C
C
Urea
OH
HO
N
Ot-Bu
20 d, RT
64%
2 d, RT
63%
Sinapoyl-L-Malate
Urea
O
(B)
O
O
O
O
CO2
H
H
OH
Syringaldehyde
CO2
CO2
H
O
O
CO2H
O
O
+
CO2
H
CO2H
HO
L-Malic acid
HO
O
O
O
O
O
Meldrum's acid
OH
Sinapoyl-L-Malate
Figure 1: (A) Synthetic pathway published by Allais et al.15; (B) retrosynthetic pathway proposed herein for sinapoyl-L-malate production.
tert-Butanol and acetate protecting groups were then removed recovered in good yield. This synthesis was then benchmarked
in a two-step sequence involving trifluoroacetic acid (TFA) used against the two previous methods. Several green metrics were
in large excess (20 eq), then HCl in acetone. Besides the solvents evaluated for each process. Unfortunately, the lack of details
used for the reactions, several liters of solvents were also regarding the patent did not provide enough information to
necessary (cyclohexane, ethyl acetate, dichloromethane, calculate the data16. However, we were able to compare the
hexane, pyridine, water) for the five column purifications process reported by Allais et al and the one described in this
required to purify every single chemical intermediate. More study15. All these indicators confirmed the significant
recently, a patent described a very similar strategy, still based improvement brought by this novel sinapoyl-L-malate synthetic
on Steglich coupling16. The main differences were (1) the process (Figure 1B). This methodology has then been extended
protection of malic acid with iso-propanol instead of tert- to different sinapoyl malate analogues starting from other
butanol, (2) the in situ acetylation of the free phenol of sinapic renewable organic acids. Finally, the library of compounds was
acid which reduced the synthetic pathway by one step, and (3) screened on different targets of biological activities, notably
the low selective H2SO4-mediated cleavage of the protecting those of interest to cosmetics (i.e., anti-UV, antioxidant, and
groups that also promote the partial hydrolysis of sinapoyl-L- antibacterial).
malate into sinapic acid and L-malic acid, thus explaining the
very low yield (7%) despite the presence of only one column
purification. In this context, with respect to the 12 principles of
Results and Discussion
Green Chemistry, we sought to offer a shorter and greener
synthetic pathway to limit the quantities of solvents while
avoiding protection/deprotection sequences and the use of
coupling agents and catalysts. For this, we have designed and
optimized an expeditious two-step procedure towards sinapoyl-
L-malate involving Meldrum’s acid opening with unprotected
naturally occurring L-malic acid and subsequent Knoevenagel-
Doebner condensation of the resulting malonate monoester
with unprotected biomass-derived p-hydroxybenzaldehydes
(Figure 1B). Using just one chromatography column, sinapoyl-L-
malate and sinapic acid, the reaction by-product which is also a
value-added building block, were readily separated and
Synthetic pathway
Contrary to what was already described in the literature to
obtain p-hydroxycinnamic esters (i.e., esterification of p-
hydroxycinnamic acids), we chose a different strategy and
decided to carry out the opening of Meldrum’s acid with malic
acid to form the corresponding monoester, and then submit the
latter to
a
Knoevenagel-Doebner condensation with
syringaldehyde to access sinapoyl-L-malate (Figure 2). Although
it was well-known that the Knoevenagel-Doebner reaction does
not require the protection of the phenol,1,4 the opening of
Meldrum’s acid in presence of free acid moieties had never
been reported in the literature and remained to be explored.
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DOI: 10.1039/D0GC02763D
H
O
H
N
HO
N
H2
O
HO
O
O
CO2H
O
O
CO2H
X
R2
R2
+
HO
O
HO
O
R1
R3
R4
R3
R4
NH2
R1
R3
R4
H2
O
OH
OH
1 - 4
OH
Aniline
L- Lactic acid (R1 = R2 = H)
1 - 57%
L-Malic acid (R1 = H, R2 = CO2H)
2 - 90%
1a - R3, R4 = H - 28%
1b - R3 = H, R4 = OH - 34%
1c - R3 = H, R4 = OMe - 36%
1d - R3, R4 = OMe - 40%
2a - R3, R4 = H - 62%
R2
R2
R2
2b - R3 = H, R4 = OH - 49%
2c - R3 = H, R4 = OMe - 46%
2d - R3, R4 = OMe - 48%
CO2
H
CO2H
CO2
H
R1
R1
R1
O
O
O
O
O
X'
O
O
CO2
Aniline
H
H
N
L-Tartaric acid diester (R1 = X or X', R2 = CO2H)
4 - 91%
4a - R3, R4 = H - 22%
4b - R3 = H, R4 = OH - 29%
4c - R3 = H, R4 = OMe - 24%
4d - R3, R4 = OMe - 29%
L-Tartaric acid monoester (R1 = OH, R2 = CO2H)
3 - 87%
3a - R3, R4 = H - 30%
3b - R3 = H, R4 = OH - 29%
3c - R3 = H, R4 = OMe - 34%
3d - R3, R4 = OMe - 30%
HO
N
H

R3
R4
R3
R4
R3
R4
OH
OH
1a - 4d
OH
Figure 2: Formation of sinapoyl-L-malate and analogues (1a-4d) through Knoevenagel-Doebner condensation with various p-hydroxybenzaldehydes and malonate derivatives (1-4)
Table 1: Reaction conditions for the synthesis of malonate ester from Meldrum’s (1 eq)
and malic acid
The Knoevenagel-Doebner reaction has been widely used and
described in the literature to access various p-hydroxycinnamic
acids in good yields20-22. Conventionally, it is carried out in the
L-Malic
acid (eq)
1
1
1.5
1
Mono
ester (%)*
74
Malonic
acid (%)*
26
28
27
T
t
Entry
Solvent
presence of pyridine and aniline despite their recognized and
decried toxicity23. Several eco-friendlier alternatives have
recently been described such as the use of ionic liquid to
substitute pyridine24-26, microwave activation to reduce the
reaction time21, 27 or the use of renewable amino-acid to replace
aniline28,29. However, some of these new methods are difficult
to upscale industrially. Recently, Peyrot et al. have proposed a
more eco-responsible and industrially transposable access to
hydroxycinnamic acids requiring ethanol as solvent and proline
as catalyst4. We therefore tested the classic Knoevenagel-
Doebner conditions (Table 2, entry 1) as well as that reported
by Peyrot et al. (Table 2, entry 2). The classic Knoevenagel
conditions (60 °C, 16 h) starting from syringaldehyde and the
90/10 monoester and malonic acid crude mixture from Table 1,
Entry 4 led to sinapoyl-L-malate and sinapic acid in 66% and 17%
conversions, respectively. In presence of ethanol and proline,
the ratio was reversed, leading to sinapoyl-L-malate and sinapic
acid in 16% and 35% conversions, respectively. The majority of
the other by-products (49%) correspond to a total – or partial –
esterification of L-malic acid leading to mono/di-ethyl malonate
of sinapic acid, most likely due to the proline’s acid moiety that
catalyses the esterification. In order to avoid it, the
Knoevenagel-Doebner reaction was carried out in DMF (Table
2, entry 3). Unfortunately, although it resulted in a decrease in
the conversion of these by-products, the conversion in sinapoyl-
L-malate was not improved, worst yet, that of sinapic acid was
promoted. Despite its lesser eco-friendly nature compared to
ethanol/proline, pyridine/aniline was selected as being the
optimal conditions.
(°C)
(h)
1
2
3
4
95
95
95
75
3
1
1
None
None
None
THF
72
73
90
16
10
* Obtained by 1H NMR.
This reaction, which relies on the very specific reactivity of
Meldrum’s acid towards nucleophiles, was first performed
without solvent at 95 °C at which Meldrum’s acid melts and
dissolves the nucleophile, and with strict stoichiometry17-19
(Table 1, entry 1). Under these conditions, the presence of free
acids promoted Meldrum’s acid degradation into malonic acid.
Indeed, after 3 hours of reaction, 74% of monoester and 26% of
malonic acid, resulting from Meldrum’s acid degradation, were
observed by NMR. To avoid, or at least limit, this side-reaction,
we modulated the equivalents of the different reactants, the
reaction time and also used a solvent. Reducing reaction time
did not affect the percentage of monoester as monoester was
obtained in 72% conversion after 1 hour (Table 1, entry 2). An
increase in malic acid equivalents did not significantly change
the monoester conversion either (Table 1, entry 3). We then
tested the reaction in THF at reflux to lower the reaction
temperature and limit the thermally-induced degradation of
Meldrum’s acid. This resulted in a significant increase of the
conversion in monoester (90%) and a concomitant decrease of
the conversion in malonic acid (Table 1, entry 4). In a
sustainability perspective, despite the lower conversion in
monoester, it would be preferable to carry out this step without
solvent. However, in order to obtain the best overall yield,
conditions in Table 1 entry 4 were chosen as the optimal ones.
After completion, the reaction mixture was not purified but
directly subjected to the Knoevenagel-Doebner reaction after
THF removal under vacuo. This method, initially developed and
optimized with L-malic acid as nucleophile, has been extended
to other organic acids (i.e., L-lactic acid and L-tartaric acid) thus
providing access to mono and di-esters analogues (Figure 2).
Table 2: Reaction conditions for the Knoevenagel-Doebner reaction (reaction time = 16
h)
SM
(%)*
66
16
16
SA
(%)*
17
35
67
Other
(%)*
17
49
17
T
Entry
Solvent
Catalyst
(°C)
1
2
3
Pyridine
Ethanol
DMF
Aniline
Proline
Proline
60
95
95
* Obtained by 1H NMR.
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Green Chemistry
This two-step synthetic methodology was then implemented to currently, malic acid is mainly produced from fosVsiielwrAerstiocleuOrcnelinse,
39
DOI: 10.1039/D0GC02763D
various p-hydroxybenzaldehydes (vanillin, syringaldehyde, 3,4- in particular by chemical oxidation of n-butane . Nevertheless,
dihydroxybenzaldehyde and p-hydroxybenzaldehyde) as well as malic acid is a naturally occurring organic acid that can be
different mono-/di-esters deriving from L-lactic and L-tartatric biotechnologically produced40.
acids (Compounds 1-4) to constitute a library of 16 compounds
isolated in acceptable yields (Figure 4, 1a-d, 2a-d, 3a-d & 4a-d).
It is worth mentioning that the majority of the compounds have
been synthesized up to this point by tedious synthesis routes
leading to low yields15,30,31 whereas some have just been
identified in plant extracts32,33. To assess the sustainability of
the synthetic pathway proposed herein, we used several
indicators called green metrics34, which evaluate the
environmental impact of the process as well as its
effectiveness35. We calculated the Atom Economy (AE) and the
Process Atom Economy (PAE), to determine how much of
starting material was directly incorporated to the final product.
The Environmental Factor (E-Factor) allowed to determine the
quantity of all inputs and by-products of the process to
synthesize 1 g of targeted compound (i.e., sinapoyl-L-malate)36.
Figure 3: Environmental impact of the reagents for 2c synthesis
Details on the calculations are available in ESI. As presented in
Several approaches are already described in the literature, such
as production by fermentation processes (bacteria and fungus)
from renewable sugar.41,42 The use of a bio-sourced malic acid
would certainly make possible to provide an even more optimal
process and would significantly reduce the CO2 generated as
well as the global toxicity. Although the synthetic steps have
been largely optimized for the production of this type of
compound, dependence on fossil resources still remains a
limitation in terms of environmental impact reduction. Overall,
this straightforward and sustainable two-step synthetic route
leading to satisfactory yields opens the door to the use of these
molecules in various fields such as cosmetic applications.
Table 3, the two-step synthesis described in this paper allowed
a significant improvement of the Green Metrics compared to
the process of Allais et al., with an increase for both the AE and
the PAE, indicating that more of the starting material was
directly transferred into the product. As for the E-Factor, the
value was reduced 12-fold, the lower number of steps of our
synthetic pathway compared to the one of Allais et al. resulted
to a lesser use of inputs and a lower generation of by-products.
All the green metrics calculated make it possible to confirm the
considerable progress in terms of sustainability for the sinapoyl-
L-malate and analogues production.
Table 3: Green metrics comparison between the synthetic pathway of Allais et al.15 and
the one proposed herein
Cosmetic activities
Green Metrics
Allais et al.
Peyrot et al.
While the main limitation of phenolic compounds in cosmetic or
pharmaceutical applications remains their low solubility in
water, the synthetic route presented above allows access to
compounds with free carboxylic acid(s) and phenol(s), making
them much more hydrophilic. This is particularly the case for
malates and mono-tartrates where the presence of two acid
groups topples the hydrophilic/hydrophobic balance in favour
of solubility in water, allowing the evaluation of numerous
biological properties such as the antioxidant power, the UV
absorption and especially the antibacterial activity of the
compounds.
Atom Economy (%)
Process Atom Economy (%)
E-factor (g/g of product)
29.4
0.5
2809
73.9
36
230
In addition to the green metrics, the Life Cycle Assessment (LCA)
makes possible to study in detail the environmental impact of
each different reagents involved in the synthetic process37. The
analysis was carried out for compound 2c production, following
a standard method described in the ISO 14040-44 norm.
Technical and market information were obtained from
experimental and bibliographic databases and then processed
using openLCA software and EcoInvent 3.4 database. The
environmental impacts were calculated with CML 2001 (from
the University of Leiden)38. Four benchmarks were selected and
the results are shown in Figure 3.
Regarding the indicators, while the ecotoxicity (human, aquatic
and terrestrial) values were very low, the impact on climate was
non-negligible as the production of 1 g of 2c led to the release
of 2.05 kg CO2-eq. However, as seen in Figure 3, all the
indicators were strongly impacted by the presence of malic acid
in the process (parameters details are available in ESI). Indeed,
1. UV filters and stability
Sinapoyl-L-malate is a molecule that plants produce to protect
themselves from the negative effects of UVs. Several studies
have highlighted its action mechanism and structure-activity
relationships, which allowed to determine the key structural
features for a good protection, such as the α,β-unsaturated
ester and the presence of a sterically hindered electron rich
ester moiety, favouring a cis-trans isomerisation upon UV
exposure12-14. However, until now, the lack of efficient and
straightforward synthetic procedure to access this molecule
prevented to produce and study sinapoyl-L-malate analogues.
4 | J. Name., 2012, 00, 1-3
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Figure 4: UV spectra (10 µM in ethanol) of synthesized p-hydroxycinnamic esters (A: lactate series, B: malate series, C: tartrate monomer series, and D: tartrate dimer series)
compared with octinoxate
The library being now available, the absorbance spectra of each 2a-d & 3a-d), the nature of the ester moiety (i.e., lactate, malate
of the molecules in ethanol at 10 µM has been determined. The or tartrate) did not seem to significantly impact the UV
spectra obtained are grouped by series in Figure 4. The properties, as all compounds showed similar wavelength
compounds having mainly absorbance properties in the UV-B coverage throughout the series with absorbance around 0.2,
region, we have chosen to benchmark them against octinoxate, with 1a being the only exception nearing a value of 0.3 (Figure
one of the most common UV-B solar filter in sunscreens. When 4A, B & C). While the presence of caffeic, ferulic or sinapic
looking globally at sinapoyl-L-malate and its analogues (1a-d, moieties resulted in a similar wavelength coverage, the use of a
coumaric structure provided an hypsochromic shift, allowing must also prove stable towards UV in order to ensure its
the corresponding compounds (i.e., 1a, 2a & 3a) to better fit the effectiveness. The stability of each of the compounds was
spectrum of octinoxate, which is also a p-hydroxycinnamic ester therefore evaluated. For this, a solution at 10 µM was irradiated
(i.e., coumarate ester). Only coumaroyl-L-lactate 1a provided an under UV (λ = 300 nm, P = 8.32 W/m², T = 35 °C, 1 hour). The
UV spectrum perfectly fitting that of octinoxate with a better loss of absorbance (LoA) was obtained by comparing the
absorbance compared to the others monophenols (i.e., lactate, resulting absorbance after one hour of irradiation and the initial
malate and tartrate monomer series). The use of L-tartaric acid one. The values are gathered in Figure 5.
allowed the obtention of dimers (4a-d), leading to an expected
significant increase of absorbance levels (Figure 4D). While the
wavelength coverage was not impacted by the introduction of
a second phenol moiety, the increase of absorbance allowed
the coumaric (4a), caffeic (4b) and ferulic (4c) based dimer to
level with octinoxate in the UV-B region (280-315 nm) while
covering part of the UV-A region (315-400 nm). The sinapic-
based dimer (4d) displayed a similar profile only with a
narrower coverage in the UV-B region. Overall, all four coumaric
based compounds (1a, 2a, 3a and 4a) stood out with a coverage
area in line with octinoxate regardless of the ester moiety.
Without exceeding that of octinoxate, the intensity of
absorbance for most of the synthesized monomeric compounds
were similar. The use of dimers allowed to significantly improve
Figure 5: Loss of absorbance for synthesized compounds (orange: lactate series, blue:
malate series, green: tartrate monomer series and red: tartrate dimer series) after 1 h of
UV irradiations, compared to octinoxate (concentration = 10 µM in ethanol)
those properties, both in intensity and wavelength coverage.
Although absorbance is of great importance, a good sun filter
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Under these irradiation conditions, octinoxate showing a 25% series, and as expected, coumaric based molecules did not
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LoA, the acceptable LoA limit was set at 25% in order to select exhibit antioxidant activity. A global trend could be seen in the
the compounds that can compete, or even better, outperform four series, with better activities for caffeic, then sinapic, and
octinoxate. Regarding the lactate series, the four molecules finally ferulic based molecules (caffeic > sinapic > ferulic >>>
exhibited comparable LoA, really close to that of octinoxate coumaric). The nature of the ester had little impact on the
with values between 20 and 26% (Figure 5, compound 1a-d). antioxidant activity with, however, better activities of the
The malate series was less homogeneous as compounds 2a and dimers (4a-d) mostly due to the presence of two free phenols
2d were below the limit (22.5 and 19.1% LoA, respectively), the on those molecules. All compounds presenting antiradical
compound 2c (26.3% LoA) flirted with the acceptable limit activities with a caffeic or sinapic core proved competitive with
whereas 2b was clearly more impacted by UV irradiation (31.5% the references, while the use of a ferulic base allowed only to
LoA). The tartrate series was very satisfying as all of the challenge the properties of BHT. Only compounds 4b, 4c and 4d
compounds had a LoA lower than that of octinoxate. Values exhibited EC50 values lower than all of the references, which
very close to 25% were observed for the tartrate monomer made them promising bio-based substitutes. In addition, as
series (Figure 5, compound 3a-d) while the dimers were clearly described above, they also possess significant UV filter
more stable with an average LoA around 15% (Figure 5, properties as well as good photostability. The dual activity of
compounds 4a-d). From a global point of view, the majority of these molecules makes them the compounds of choice for
the compounds of the library were at least comparable, or even cosmetic formulations, especially sunscreen lotions.
more stable, than octinoxate in terms of stability. If one
considers both the absorbance intensity and the stability,
coumaric analogues matched with the reference in terms of
coverage area and stability. However, the tartrate dimers series
remained the most interesting as they have both greater
absorbance than the reference, wider wavelength coverage,
and a lower loss of absorbance. The UV properties of the
synthesized molecules described in this study lead sinapoyl-L-
malate analogues to be considered as potential candidates to
replace the highly criticized octinoxate in cosmetic
formulations.
2. Antioxidant activity
Figure 6: EC50 of synthesized compounds (orange: lactate series, blue: malate series,
green: tartrate monomer series and red: tartrate dimer series) for DPPH (2,2-diphenyl-
1-picrylhydrazyl) inhibition in ethanol.
Present in the majority of cosmetic products, antioxidants allow
both the stabilization of formulations and the protection of the
skin against damages linked to free radical formation43-45
.
Reactive Oxygen Species (ROS) are formed by biochemical
reactions, within the cell, under the action of UV, ozone or
stress46,47. Their high reactivity induces irreversible cellular
damages which can lead to skin aging acceleration,
inflammation, oxidative stress or even carcinogenesis48,49. It is
therefore essential to add antioxidant compounds to cosmetic
formulations to prevent all of the aforementioned risks. For
this, several antioxidants are conventionally used, such as BHA
(butylated hydroxyanisol), BHT (butylated hydroxytoluene) or
3. Antibacterial activity
Preservatives are also part of the additives commonly used in
cosmetic formulations to prevent the development of
pathogens such as bacteria. For this, parabens have been used
for a long time. Nevertheless, in recent years, their use has
largely diminished in the face of multiple criticisms.
Phenoxyethanol, still widely used today, tends to be more and
more criticized in view of its probable multiple health risks. It is
therefore essential to offer new healthy and renewable
alternatives. In order to pre-screen the conservative activities
of our molecules, we carried out a 24 hours Escherichia coli
growth inhibition test in the presence of a concentration range
for each water-soluble molecule. The growth of bacteria was
monitored by absorbance spectroscopy and the minimum
inhibitory concentration (MIC) and the minimum bactericidal
concentration (MBC) were determined. The MIC and MBC for
Trolox
acid), which are strongly criticized for their probable endocrine
and carcinogenic effects50-52
The withdrawal of these
(6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
.
compounds is imminent and pushes the cosmetic industries to
rethink their formulations. The use of polyvalent molecules,
which can be at the same time UV filter, antioxidants or even
more, is therefore a major asset to limit the number of
compounds present in the formulations while retaining
biological properties. The antiradical properties of the
molecules produced above were assessed and benchmarked
against the EC50 values of the references aforementioned. The
EC50 value (half-maximum effective concentration) was defined
as the amount (in nmol) of antiradical compound required to
reduce 50% of DPPH free radicals. Results are summarized in
Figure 6. From a general point of view, independently of the
each compound are reported in Table
4 and can be
benchmarked against that of phenoxyethanol. Despite the
presence of free acids in the compounds, some of them proved
to be not soluble in the bacteria culture medium (LB broth).
Malate and tartrate monomer series were tested, as well as
compounds 1b and 4b, the caffeic moiety on the molecules
6 | J. Name., 2012, 00, 1-3
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allowing a better water solubility than the others of their we opted to look at the concentration needed tVoiewexAhrtiicbleitOntlhinee
respective series.
previous UV properties (i.e., 1E-05 M). RDesOuI:lt1s0.a10re39p/Dre0sGeCn0t2e7d63iDn
Figure 7.
Table 4: MIC and MBC values of synthesized compounds on Escherichia coli compared
to phenoxyethanol.
MIC
(% w)
MBC
(% w)
Entry
Phenoxyethanol
0.31
*
0.31
0.63
*
0.63
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
*
*
*
*
0.31
0.31
0.31
0.63
0.31
0.31
0.31
0.31
*
0.31
*
*
0.63
0.63
0.63
1.25
0.63
0.63
0.63
0.63
*
0.63
*
*
*Not soluble at 6 or 3%w in LB Broth
Figure 7: Receptor activities (%) of estrogen receptor α (ERα), androgen receptor (AR),
and pregnane X receptor (PXR) for synthesized compounds (A: agonist and B: antagonist
Results were generally comparable. MIC values were found
between 0.63 and 0.31%w and MBC values between 0.63 and interactions) at 10−5 mol.L−1
1.25%w, and competitive to those obtained for phenoxyethanol
.
Part A shows the agonist evaluation. The four molecules did not
show agonistic potential on ERα, AR and PXR. The same
approach was performed for antagonist study where the
selected molecules were put in competition with the reference
ligand for each receptor. Figure 7B shows specific recognition of
the ligand with respect to its receptor despite the presence of
the tested molecules, confirming the absence of antagonistic
activities. These preliminary results confirmed the harmlessness
of these new molecules, allowing to seriously consider them as
substitute for current petro-sourced preservatives, UV filters, or
antioxidant still on the market.
on E. Coli, which is currently used as 0.32%w in cosmetic
formulation. These compounds are thus potential bio-based
alternatives to phenoxyethanol, which complement the range
of biological activities of the compounds presented here.
4. Endocrine disruption
Most of the aforementioned commercial references
(octinoxate, BHT, parabens) are strongly criticized for their
endocrine disrupting effects53,54. In order to replace these
substances, one must offer healthy alternatives for human
health. The absence of interaction between our analogues with
androgen receptor (AR), pregnane X receptor (PXR) and
estrogen receptor α (ERα) was assessed (details are available in
ESI). AR is regulated by interaction with natural androgens like
testosterone and dihydrotestosterone while ERα is controlled
by the estrogenic hormone 17β-estradiol (E2). Finally, PXR takes
part to the xenobiotic receptor family. Agonist and antagonist
tests were performed in reporter cells lines respectively in
absence or presence of respective receptors reference
molecules (E2/ERα, R1881/AR and SR-12813/PXR). Based on
previous work, modification on the p-hydroxycinnamic core
(sinapic, caffeic, ferulic or coumaric) did not present any
endocrine disrupting activity13,55,56. We have therefore
chosen to look at the ester nature impact on the nuclear
receptor interactions. We fixed the sinapic core and thus test
the whole series with four different esters (1d, 2d, 3d, 4d).
Previous UV properties were evaluated at 1E-05 M and
endocrine tests were performed from 3E-07 to 3E-05 M, then
Conclusions
A new sustainable two-step synthetic pathway has been
developed to access sinapoyl-L-malate and analogues without
the need of protection/deprotection sequences nor coupling
agents and metal-catalyst. An optimization of the Knoevenagel-
Doebner conditions adapted to the free acid presence will be
considered subsequently. However, compared to the two
reported multistep procedures, this methodology allowed to
increase the yield while drastically improving atom economy,
process atom economy and E-factor. This synthetic route has
been implemented on various naturally occurring mono- and
diacids and biomass-derived p-hydroxybenzaldehydes to create
a library of 16 p-hydroxycinnamate derivatives bearing free
acids. These molecules, hardly studied until now due to their
tedious access, have demonstrated a wide range of biological
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Green Chemistry
9.
M. Janvier, L. Hollande, A. S. Jaufurally, M. Pernes, R.
activities. The UV filters properties observed for some
molecules highlight their capacity to substitute octinoxate (UV-
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preservative activity on other strains (gram positive, fungus,
yeast) will be the subject of a future publication. In addition, the
absence of endocrine disruption activity for those molecules
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Florent Allais 0000-0003-4132-6210
Cédric Peyrot 0000-0001-8843-6498
Matthieu M. Mention 0000-0002-2309-9870
Fanny Brunissen 0000-0002-0357-4670
Robin Fournier 0000-0003-4788-4782
Julien Couvreur 0000-0002-6532-0668
Patrick Balaguer 0000-0002-3524-3622
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TOC
Straightforward and sustainable access to bioactive sinapoyl malate and analogues through
Meldrum’s acid opening with unprotected hydroxy-acid(s) and subsequent Knoevenagel-
Doebner condensation with p-hydroxybenzaldehydes.