Innovative bio-based organic UV-A and blue light filters from Meldrum's acid

Article abstract of DOI:10.3390/molecules25092178

Faced with the ban of some organic UV filters such as octinoxate or avobenzone, especially in Hawaii, it became essential to offer new alternatives that are both renewable and safe for humans and the environment. In this context, a class of bio-based mole

Full text of DOI:10.3390/molecules25092178

molecules
Article
Innovative Bio-Based Organic UV-A and Blue Light
Filters from Meldrum’s Acid
Cédric Peyrot 1 , Matthieu M. Mention 1 , Fanny Brunissen 1 , Patrick Balaguer 2
and Florent Allais 1,
*
1
URD Agro-Biotechnologies Industrielles (ABI), CEBB, AgroParisTech, 51110 Pomacle, France;
cedric.peyrot@agroparistech.fr (C.P.); matthieu.mention@agroparistech.fr (M.M.M.);
fanny.brunissen@agroparistech.fr (F.B.)
IRCM, Inserm, Univ Montpellier, ICM, 208 Avenue des Apothicaires, CEDEX 5, 34298 Montpellier, France;
patrick.balaguer@icm.unicancer.fr
2
*
Correspondence: florent.allais@agroparistech.fr; Tel.: +33-633698126
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Ana Estévez-Braun and Ángel Amesty
Received: 15 April 2020; Accepted: 4 May 2020; Published: 6 May 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Faced with the ban of some organic UV filters such as octinoxate or avobenzone, especially
in Hawaii, it became essential to offer new alternatives that are both renewable and safe for humans
and the environment. In this context, a class of bio-based molecules displaying interesting UV filter
properties and great (photo)stability has been developed from Meldrum’s acid and bio-based and
synthetic p-hydroxycinnamic acids, furans and pyrroles. Moreover, p-hydroxycinnamic acid-based
Meldrum’s derivatives possess valuable secondary activities sought by the cosmetic industry such as
antioxidant and anti-tyrosinase properties. The evaluation of the properties of mixture of judiciously
chosen Meldrum’s acid derivatives highlighted the possibility to modulate secondary activity while
maintaining excellent UV protection. Meldrum’s acid derivatives are not only competitive when
benchmarked against organic filters currently on the market (i.e., avobenzone), but they also do not
exhibit any endocrine disruption activity.
Keywords: photoprotection; bio-based chemicals; Meldrum’s acid; UV-A; blue light; organic UV filters
1. Introduction
While solar light is biologically crucial for life on Earth, significant exposure to radiations can
have negative effects on the skin [
(320–400 nm) penetrate the different skin barriers and lead to the formation of reactive oxygen
species (ROS) [ ], inducing oxidative damage resulting in lipid membrane alteration [ ] or DNA
mutations [ ]. Up until recently, blue light (400–500 nm) irradiation effects were poorly investigated.
1]. It has been widely shown that UV-B (290–320 nm) and UV-A
2
3,4
5,6
Even though its impact on the sleep cycle, memory, and retina is clearly highlighted, its impact on skin
cells is still under examination [7–9].
To protect oneself from these harmful radiations, a wide range of sunscreen lotions and cosmetic
creams are available. Some contain organic filters (i.e., avobenzone, octinoxate, benzophenone,
or octocrylene) that are increasingly criticized by consumers and countries due to their environmental
impact [10
and sale of sunscreen containing avobenzone or octinoxate [13]. These molecules have been the
subject of many studies in the last few years to demonstrate their negative impact on corals [14 16].
–12]. Indeed, Hawaii recently issued a bill, effective on 1 January 2021, against the use
–
Indeed, avobenzone and octinoxate (Scheme 1) are two ingredients contributing to coral bleaching; if
they do not directly kill corals, they put them under significant stress and subject them to increased
mortality levels.
Molecules 2020, 25, 2178; doi:10.3390/molecules25092178
www.mdpi.com/journal/molecules

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Scheme 1. Structures of two common commercial organic filters used in sunscreens.
Moreover, the (photo)degradation of these products in water promotes the formation of chlorinated
phenols well-known to be mostly toxic [17]. Endocrine disruption related to these products has also
been highlighted for several years [18,19] and repeated exposures may induce serious developmental
effects on reproductive organs and the central nervous system. Still, too few alternatives are proposed
to reduce the use of this type of organic filter. The most common are mineral filters such as titanium
dioxide or zinc oxide, which are also widely criticized [20,21]. Indeed, nanoparticles may induce
inflammatory diseases or exacerbate respiratory allergies and asthma [22]. They are also involved
in cardiovascular diseases and promote certain lung cancers [23]. Although the existing UV filters
regulatory lists greatly limits the use of new filters, particularly in China, it is essential to offer new
organic UV-A and UV-B filters able to protect humans against the harmful effects of the sun while
reducing environmental and public health risks.
Conjugated or aromatic compounds are known for their abilities to absorb specific wavelengths,
especially ultraviolet ones [24
furfural derivatives) and less harmful. Lignin-derived phenolic compounds have been recently used
as starting material to develop a new range of UV filters [25 27]. It has been shown that wavelengths
–26]. Some of them are bio-sourced or biodegradable (i.e., phenolic and
–
absorption range can be modulated by modifying the conjugation system through the functionalization
of these phenolics [24,28].
In this study, we focus on the coupling between Meldrum’s acid and various conjugated bio-based
monomers, notably phenolics and furans, using Knoevenagel condensation in order to modulate the UV
absorption band. This synthetic strategy resulted in compounds exhibiting conjugation throughout their
backbone and high steric hinderance on the
as important parameters to obtain anti-UV properties [24
β
position, two factors that have been shown in recent studies
25]. The anti-UV coverage and the photostability
,
under UV irradiation of each compound were evaluated and benchmarked against avobenzone to identify
the most competitive compounds. In addition to the capacity of the synthesized molecules to act as UV
filters, we also investigated whether they could be used to counteract two negative effects of UV radiations
on the human skin, namely the formation of radical ROS and hyperpigmentation. Compounds were thus
assessed for their antiradical and tyrosinase inhibition potentials. Finally, endocrine disruption assays
determined potential health risks associated to these compounds.
2. Results
2.1. Sustainable Synthesis
Meldrum’sacid, whichresultsfromthecondensationofacetoneandmalonicacid, abio-basedoccurring
organic acid, possesses a specific reactivity thanks to its highly acidic proton in
Knoevenagel reaction resulting from the condensation between Meldrum’s acid and an aldehyde is widely
described in the literature and usually does not require the use of a base [29 30]. Different reaction conditions
α of the two esters functions.
,
have been investigated in the presence of ionic liquids [31] or in PEG-400 [32]. Nevertheless, the simplest
and the greenest synthetic procedure remains the reaction in water as the compounds precipitate in water
and can be isolated by a simple filtration, or otherwise they are simply obtained by liquid/liquid extraction,
thus avoiding the use of expensive and waste-generating chromatography (Scheme 2) [33]. In order
to offer renewable and bio-based products, we oriented our choice for aldehydes towards widely used
p-hydroxybenzaldehyde (i.e., vanillin) and furanic compounds (i.e., furfural, obtained by dehydration of

Molecules 2020, 25, 2178
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xyloses). We also chose synthetic aldehydes for a more fundamental purpose (i.e., pyrroles). Through this
straightforward and sustainable synthetic procedure, compounds are obtained in high purity and yield,
similar to those described in the literature (49%–90%) [33].
R
O
O
H2O
O
O
O
+
O
O
R
H
75 °C, 4h
O
O
Scheme 2. Knoevenagel condensation between Meldrum’s acid and aldehyde in water.
Thanks to this simple and sustainable methodology, we populated a library of 14 molecules
presenting different conjugation systems (Scheme 3). All of the chemical structures were confirmed by
1H- & 13C-NMR, and HRMS analyses (see Supporting Information for spectra).
O
O
O
O
O
O
O
O
O
O
Phenolic Series
O
O
O
O
O
O
O
O
O
HO
OH
OH
OH
OH
4, 88%
3, 90%
1, 87%
2, 87%
Furanic Series
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
5, 85%
6, 90%
7, 87%
8, 82%
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
9, 78%
10, 86%
11, 75%
O
Pyrrolic Series
O
O
O
O
O
O
O
O
O
O
O
NH
O
N
NH
12, 90%
13, 72%
14, 57%
Scheme 3. Molecules resulting from the Knoevenagel reaction between Meldrum’s acid and
corresponding aldehydes.

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2.2. UV Properties and Photostability
In order to evaluate the UV properties of each compound, a UV–Vis spectrum at 10
µ
mol.L−1
in ethanol was performed. With regards to the maximum absorption wavelengths obtained for
compounds 1−14 (361–426 nm), their spectra were compared with that of avobenzone (UV-A,
λ
max
=
357 nm) rather than that of octinoxate (UV-B, λ
= 310 nm) (Figure 1A–C).
max
Figure 1. UV–Vis spectra for phenolic series (
A), furanic series (B), and pyrrolic series (C), (Concentration
= 10 µmol.L−1 in ethanol).
Very similar spectra were obtained for the phenolic series with absorbance ranging from 300 nm
to 475 nm (Figure 1A). The wavelength coverage for the simplest phenolic molecule ( ) is close to that
3
of avobenzone, but with a lower absorbance. Substituting the phenol moiety with auxochromes (-OH,
OMe) resulted in a bathochromic shift of the maximum absorbance, leading to a wavelength coverage
that includes part of the UV-A and blue light regions (
moiety ( ) allowed to get a max (361 nm) closer to that of avobenzone (357 nm). Results for the furanic
series (Figure 1B) show a bathochromic shift alike the phenolic series, depending on the electronic
effects of the substituents on the furan moiety. With just one substituent on the aromatic ring (7, 8,
1, 2, and 4). Switching from coumaric (3) to furan
5
λ
and 10), only a small blue shift is observed regardless of the substituent complexity. Introducing a
second group on the furan ring leads to a further shift toward blue light that depends on the nature
of the substituent and its impact on the conjugation. As expected, the presence of a benzene ring
on compound (9) conjugated to the furan moiety further expands the π-system and results in a shift
further into the blue light spectrum, but without a significant change in absorbance. An increase of
the maximal absorbance can be observed with a significant increase of the conjugation on the furan
moiety (11). However, intermediate absorbance values and blue light shifts are obtained with two
unconjugated and not bulky substituents (
pyrrolic moiety were investigated (Figure 1C). Compared to coumaric (
a simple pyrrole ring (12) afforded an absorbance at the frontier between UV-A and blue light regions
= 393 nm). As seen with the previous two series, substitution on the aromatic ring (13) provided
6) (i.e., methyl groups). Finally, the anti-UV properties of the
3
) and furan ( ), the presence of
5
(λ
max
a bathochromic shift, accentuated with a higher conjugation (14), without a significant modification of
the maximal absorbance. The study of the structure–activity relationship on the different aromatic

Molecules 2020, 25, 2178
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moieties highlights the fact that UV properties of such molecules can be fine-tuned by judiciously
adjusting the nature of the substituents and conjugation system.
Beyond the wavelength coverage and absorbance intensity, the photostability (i.e., loss of
absorbance over time of exposure) of molecules induced by UV irradiation is of importance when
looking at potential UV filters. In order to assess the photostability of synthesized molecules, solutions
in ethanol (C = 1 mmol.L−1) were irradiated for 60 min under UV light ( = 300 nm, P = 8.32 W/m2,
λ
stirring, T = 35 ◦C). The absorbance of the resulting solutions was compared to that of the non-irradiated
solutions (t = 0) (Figure 2).
3.5
28.7
3.0
2.5
2.0
1.5
12.4
12.0
11.9
11.4
9.0
1.0
0.5
0.0
6.4
5.1
4.2
3.5
3.5
3.5
3.4
0.6
0.1
Figure 2. Loss of absorbance for phenolic series (blue checked pattern), furanic series (green stripe),
and pyrrolic series (orange), after UV irradiation for 60 min, compared with avobenzone (concentration
= 10 µmol.L−1 in ethanol).
Discussions with various cosmetic manufacturers have led us to set the loss of absorbance limit
to less than 5% in order to properly match commercial UV filters specifications. In such conditions,
the reference (avobenzone) underwent only a slight loss of absorbance (0.6%). When comparing
the simplest structure of each series (
difference between the coumaric moiety (
with a similar loss of absorbance of 3.5%. On the other hand, the introduction of the pyrrole ring (12
3
,
5
, and 12), all three are below the 5% threshold. There is no
3
) and the furanic one ( ) as the two molecules ended up
5
)
drastically reduced the loss of absorbance (0.1%), making 12 a more stable filter than avobenzone (0.6%).
It is noteworthy to mention that an in-depth kinetic study of the photostability of all compounds upon
UV-A/UV-B irradiation, as well as the structural elucidation of the potential photoproducts, will be
performed and reported in due course.
In terms of structure–activity relationship, an increase of substitution on the aromatic rings leads to
lower stability, as highlighted in the phenolic series where the loss of absorbance of the four molecules
studied can be sorted with regards to their substitution on the aromatic ring (coumaric < caffeic <
ferulic < sinapic). For both furanic series (
either with a benzene ring ( and 14) or through a carbon–carbon double bond (11), led to a higher loss
of absorbance. The same conclusion can be drawn for the introduction of a sensitive functional group
such as an ester (10). On the contrary, introduction of a primary alcohol in the furan moiety ( ) did not
result in a higher loss of absorbance as one could have expected with the aforementioned observations,
but in a slightly lower loss of absorbance compared to its alkyl analog ( ). As with absorbance and
9 and 11) and pyrrolic series (14), an increase of conjugation,
9
8
7

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wavelength coverage, photostability of the molecules is highly impacted by the extent of conjugation,
the latter having a negative impact.
2.3. Antiradical Properties
Reactive oxygen species (ROS) resulting from the reduction of molecular oxygen are naturally
produced via biochemical reactions in the body or from exogenous stimulation and play a role in
cell signaling and synaptic plasticity [34]. Despite their natural presence, they can also be harmful
when the organism is not able to regulate their level through endogenous antioxidants. Thereby, high
concentrations of ROS, which can result from overexposure to the sun for example, have been linked
to skin photoaging, inflammations, oxidative stress, or skin carcinogenesis [1]. To limit these harmful
effects, antioxidants are conventionally added to sunscreen formulations. The most commonly used
molecules are butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). If the novel
filters developed herein exhibited antiradical properties in addition to UV absorption, they would
protect the skin from UV/blue light radiation while preventing the negative consequences of such
exposure by regulating the quantity of ROS generated, without having to add any other compound
in the formulation. To determine the potential of the synthesized molecules as antiradicals, their
EC50 value (half maximal effective concentration), defined as the quantity (in nmol) of antiradical
compound needed to reduce 50% of free radicals, was evaluated. The lower the EC50 value, the better
the antiradical. Tests were performed by using DPPH (2,2-diphenyl-1-picrylhydrazyl) as a free radical
and monitoring its disappearance, results are shown in Table 1.
Table 1. EC50 of synthesized molecules for DPPH (2,2-diphenyl-1-picrylhydrazyl) inhibition in ethanol.
Series/References
Compound
EC50 (nmol)
EC50 (mM)
1
2
3
4
3.94 ± 0.20
19.70 ± 1.00
>80
>400
Phenolic series
>80
>400
3.34 ± 0.17
16.70 ± 0.80
5
6
7
>80
>400
a
a
-
-
-
-
a
a
8
9
10
11
>80
>400
Furanic series
a
a
-
-
-
-
a
a
>80
>400
a
a
12
13
14
-
-
-
-
-
-
a
a
Pyrrolic series
References
a
a
BHA
BHT
a
5.74 ± 0.29
28.70 ± 1.45
38.05 ± 1.9
7.61 ± 0.38
No activity observed.
Antiradical properties for the phenolic series have already been described and are in accordance
with those measured [33]. Within the furanic and pyrrolic series, only three derivatives demonstrate
antiradical activity (5, 8, and 11) but with poor EC50 values (>80 nmol). Of all the synthesized
compounds, as expected, only those possessing a phenol moiety exhibited antiradical properties, with
only 1 and 4 being competitive against the two commercial references.
2.4. Tyrosinase Inhibition
In addition of the negative effects cited before, excessive sun exposure can also induce pigment
spots, called age spots, on the exposed skin areas [35]. This hyperpigmentation is due to an abnormal
production of melanin, a pigment present in human skin cells that is initially responsible for tanning
to protect the skin against UV radiations. This pigment is produced from tyrosine by the action of

Molecules 2020, 25, 2178
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tyrosinase during melanogenesis. Pigmentary disorders result mainly from an overproduction of
melanin due to cell degeneration linked to high sun exposure. One way to limit the appearance of
these age spots is to inhibit tyrosinase activity, thus locally reducing the production of melanin and
decreasing pigmentation. Tyrosinase inhibition is widely described in the literature [36–40]. In tubo
inhibition tests can be performed using a fungal tyrosinase to mimic human tyrosinase activity, making
it possible to identify whether the molecules tested can potentially exhibit valuable secondary activity
in addition to their UV filter and antioxidant properties. Kojic acid can be used as a reference in terms
of in tubo tyrosinase inhibition [41]. In order to position our molecules with respect to this reference,
we determined the EC50 value, defined as the concentration (in µM) of product needed to reduce 50%
of the tyrosinase activity (Table 2). The lower the EC50 value, the better the inhibition.
Table 2. EC50 of synthesized molecules for mushroom tyrosinase inhibition.
Series/Reference
Compound
EC50 (µM)
1
2
3
4
17 ± 2
23 ± 3
Phenolic series
106 ± 13
13 ± 2
5
6
7
8
9
10
11
465 ± 56
a
-
-
-
a
a
Furanic series
252 ± 30
a
-
-
a
a
12
13
14
-
-
-
a
Pyrrolic series
Reference
a
Kojic Acid
18 ± 2
a
No activity observed.
Interesting values are obtained for the phenolic series, in particular for compounds
have EC50 very close to that of kojic acid. Some molecules of the furan series exhibit EC50 which is
relatively high (>100 M) or has no activity at all. Pyrrolic series present no activity. Through this
1, 2, and 4 that
µ
preliminary study of tyrosinase inhibition, three molecules were found competitive against kojic acid,
but these results need to be confirmed on cells.
2.5. UV Filter Combinations
As highlighted by the three types of analyses conducted above, some compounds exhibit
remarkable properties and can cover a wide range of wavelengths in the UV-A/blue light regions.
Organic UV filters are usually used in mixtures to afford a wide coverage of wavelengths [42]. In a
similar fashion, we combined specific compounds to study the properties of the resulting mixtures.
One molecule of each series was chosen: compound
tyrosinase inhibition activities; compound from the furanic series for its proximity with avobenzone
UV spectra and stability; and compound 13 from the pyrrolic series for its blue light coverage and high
absorbance. These three compounds allowed the obtention of four different mixtures: (compounds
13), (compounds ), (compounds 13), and (compounds 13), which were
4 from the phenolic series for its antiradical and
8
A
4
+
B
4
+
8
C
8
+
D
4 + 8 +
submitted to the same tests conducted on the individual compounds (i.e., UV properties, loss of
absorbance, antiradical activity, and tyrosinase inhibition).
Spectra obtained for the mixtures are presented in Figure 3. As can be expected when mixing
compounds showing similar UV profiles, combination of the molecules led to increased absorbance and
wider wavelength coverage, allowing protection in the entire UV-A and part of the blue light regions.

Molecules 2020, 25, 2178
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In addition, loss of absorbance upon UV irradiation was decreased. When looking at mixture
for example, UV radiations led only to a very small loss of absorbance (0.1%) while individual molecules
and 13 showed more degradation (6.4% and 3.4% loss of absorbance, respectively). This could
imply a synergic effect between the molecules. All three other mixtures exhibited the same behavior,
leading to higher photostability than their individual components. Only mixture (0.1%) resulted in
better photostability than the reference avobenzone (0.6%), while mixtures , and showed very
A,
4
A
B,
C
D
interesting photostability with 1.7%, 1.4% and 1.9% loss of absorbance, respectively. Combining the
compounds resulted in an impressive impact on the stability toward UV radiations, while conserving
the other properties. Indeed, as shown in Table 3, values of EC50 (DPPH and tyrosinase inhibition)
obtained for the mixtures were in the same order of magnitude as the individual compounds.
1.0
1.9
A
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
A
B
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
B
1.7
C
D
1.4
Av obenzone
0.6
0.1
200
250
300
400
450
500
Wavele3n5g0th (nm)
A
B
C
D
Avobenzone
Figure 3. UV spectra (
A
) and loss of absorbance (
B
) for mixtures
A
,
B
,
C
, and D (individual concentration
of compounds 10 µmol.L−1 in ethanol).
Table 3. EC50 of mixtures for DPPH and tyrosinase inhibition.
DPPH Inhibition EC50
(nmol)
DPPH Inhibition EC50
Tyrosinase Inhibition
Mixture
(µM)
EC50 (µM)
A
B
C
D
3.17 ± 0.16
3.12 ± 0.16
>80
3.15 ± 0.16
15.85 ± 0.80
15.60 ± 0.80
>400
14 ± 2
13 ± 2
a
-
15.75 ± 0.80
24 ± 3
a
No activity observed.
2.6. Endocrine Disruption Assays
In the last years, studies have shed light on the ability of organic UV filters to act as endocrine
disruptors [43 44] whose continuous exposure can cause serious developmental effects on reproductive
organs and the central nervous system [45]. To evaluate potential health risks associated to the
newly synthesized UV filters, their ability to interact with estrogen receptor (ER ), androgen
receptor (AR), and pregnane X receptor (PXR) was evaluated. ER activity is regulated by the steroid
estrogen sex hormone E2 (17 -estradiol). AR is activated by binding with any androgenic hormones,
,
α
α
α
β
and finally PXR is a member of the steroid and xenobiotic sensing nuclear receptors family. All three
receptors were tested in the absence (agonist test) or presence (antagonist test) of their respective
agonist control molecule, E2 (17
and PXR, respectively. Endocrine disruption assays were performed on four molecules—
and 12—as they present the basic structures for phenolic, furfural, 5-hydroxymethylfurfural (HMF),
and pyrrole derivatives. Tests were performed using different concentrations (from 3
10−7 to 3
10−5 M) and we focused on the results for the concentration of synthesized molecules needed to obtain
the previously described UV properties (i.e., 1
10−5 M). Results in Figure 4A show that the four
β-estradiol), R1881 (methyltrienolone), and SR-12813 for ERα, AR,
3, 5, 8,
×
×
×
molecules barely interact with receptors, demonstrating the absence of agonist effects. For antagonist
properties, synthesized molecules were mixed with the agonist control molecule of each receptor.

Molecules 2020, 25, 2178
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As shown in Figure 4B, receptors conserved their specific interactions toward the agonist reference
even in the presence of the synthesized compounds, confirming the absence of antagonistic activities.
These preliminary results demonstrated the innocuousness of these compounds, pushing to seriously
consider them as a replacement for current petroleum-based UV filters.
Figure 4. Receptor activities (%) of estrogen receptor
α
(ER
α
), androgen receptor (AR), and pregnane X
) interactions at 10−5 mol.L−1
receptor (PXR) of synthesized molecules for agonist (
A
) and antagonist (
B
.
3. Conclusions
Herein, the UV properties, photostability, antiradical and tyrosinase inhibition properties of
bio-based organic UV filters deriving from Meldrum’s acid are described. Study of the structure–activity
relationships showed that UV properties of such molecules can be fine-tuned by judiciously playing
with the level of conjugation and congestion on the aromatic moiety. Moreover, some of the compounds
evaluated in this study proved to possess competitive secondary activities such as antiradical and
tyrosinase inhibitory properties. Mixtures of compounds from the different series provided higher UV
properties competitive with the commercial reference (i.e., avobenzone) while retaining the secondary
activities of the individual molecules. Such mixtures can then be perfectly adapted according to the type
of UV protection desired but also to the secondary activity sought for the formulation. Finally, endocrine
disruption assays revealed the innocuousness of the synthesized compounds, making them promising
substitutes for current, toxic fossil-based organic UV filters such as avobenzone.
4. Materials and Methods
Syringaldehyde,
vanillin,
4-hydroxybenzaldehyde,
3,4-dihydroxybenzaldehyde,
3,4,5-trimethoxybenzaldehyde, furfural, 5-(hydroxymethyl)furfural, 4,5-dimethyl-2-furaldehyde, 2,5-
furandicarboxaldehyde, 5-ethyl-2-furaldehyde, tyrosinase from mushroom, and 2,2-di(4-tert-
octylphenyl)-1-picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis, MO, USA).
Meldrum’s acid, pyrrole-2-carboxaldehyde, N-methyl-2-pyrrolecarboxaldehyde, and indole-2-
carboxaldehyde were purchased from TCI (Tokyo, Japan). 5-Acetoxymethyl-2-furanaldehyde,
2-benzofurancarboxaldehyde, and all solvents were purchased from Fischer Scientific (Hampton, NH,
USA). All chemicals were used directly without purification.
1H-NMR spectra were recorded on a Brucker Fourier 300 (300 MHz) (Billerica, MA, USA) and
were calibrated with residual acetone-d6, DMSO-d6, or CDCl3 proton signals at
δ 2.05, 2.50, or 7.26 ppm,
respectively. Data are reported as follows: chemical shift ( ppm), multiplicity (s = singlet, d = doublet,
δ
t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, ddd = doublet of doublets of
doublets, dddd = doublet of doublets of doublets of doublets, and m = multiplet), coupling constant
(Hz), integration, and assignment. 13C-NMR spectra were recorded on a Brucker Fourier 300 (75 MHz)
(Billerica, MA, USA) and were calibrated with acetone-d6, DMSO-d6, or CDCl3 signals at
δ 29.84,
39.52, or 77.16 ppm, respectively. Data are reported as follows: chemical shift ( ppm) and attribution.
δ
All NMR assignments were made using COSY, HMBC, and HSQC spectrum. Melting points were
recorded on a Mettler Toledo MP50 Melting Point System (Greifensee, Switzerland) (heating at 4
◦C/min). High resolution mass spectrometry was performed on an Agilent 1290 system, equipped

Molecules 2020, 25, 2178
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with a PDA UV detector, and a 6545 Q-TOF mass spectrometer (Wilmington, DE, USA). The source
was equipped with a JetStream ESI probe operating at atmospheric pressure.
4.1. General Synthesis Procedure
A mixture of Meldrum’s acid (6.9 mmol, 1 equiv, 1 g) and the corresponding conjugated aldehyde
(6.9 mmol, 1 equiv) in deionized water (10 mL) was stirred at 75 ◦C for 2–4 h. Then, the precipitate
was filtered-off and dried to afford the targeted compounds. In case of no precipitate, the reaction
mixture was extracted with ethyl acetate and the combined organic layers were dried over anhydrous
magnesium sulfate, filtered, and dried in vacuo.
5-[(4-Hydroxy-3,5-dimethoxyphenyl)methyl]-2,2-dimethyl-1,3-dioxane-4,6-dione (
powder (87%). m.p. 164–166 ◦C, UV:
max (EtOH, nm) 405,
CDCl3)
13C-NMR (75 MHz, CDCl3)
1
): filtration led to a yellow
1
λ
ε
(L mol−1.cm−1) 24614. H-NMR (300 MHz,
δ
: 8.33 (s, 1H, H3), 7.73 (s, 2H, H5–50), 6.28 (s, 1H, OH), 3.97 (s, 6H, H8–80), 1.79 (s, 6H, H10–100).
δ
: 164.4 (C1 or C10), 160.8 (C1 or C10), 158.8 (C3), 146.8 (C6–60), 141.6 (C7),
123.6 (C4), 113.0 (C5–50), 110.8 (C2), 104.4 (C9), 56.6 (C8–80), 27.6 (C10–100). HRMS: m/z [M + Na]+
calculated for C15H16O7Na: 331.0794 found: 331.0793
5-(4-Hydroxy-3-methoxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (
powder (87%). m.p. 124–126 ◦C, UV:
max (EtOH, nm) 393,
DMSO-d6)
; 8.58 Hz, 1H, H5), 6.92 (d, J = 8.4 Hz, 1H, H6), 3.81 (s, 3H, H10), 1.72 (s, 6H, H12–120). 13C-NMR
2
): filtration led to a yellow
1
λ
ε
(L mol−1.cm−1) 29453. H-NMR (300 MHz,
δ
: 10.71 (s, 1H, OH), 8.26 (s, 1H, H3), 8.12 (d, J = 2.01 Hz, 1H, H9), 7.78 (dd, J = 2.01 Hz
(75 MHz, DMSO-d6) δ
: 164.0 (C1 or C10), 160.8 (C1 or C10), 158.0 (C3), 154.2 (C7), 147.7 (C8), 132.5 (C5),
123.9 (C4), 118.0 (C9), 116.0 (C6), 110.1 (C2), 104.3 (C11), 56.1 (C10), 27.3 (C12–120). HRMS: m/z [M +
Na]+ calculated for C14H14O6Na: 301.0688 found: 301.0686
5-(4-Hydroxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (
3
): filtration led to a yellow powder (90%).
1
m.p. 198–200 ◦C, UV:
λmax (EtOH, nm) 374,
ε
(L mol−1.cm−1) 29413. H-NMR (300 MHz, DMSO-d6)
δ
: 8.25 (s, 1H, H3), 8.17 (d, J = 8.8 Hz, 2H, H5–50), 6.90 (d, J = 8.8 Hz, 2H, H6–60), 1.72 (s, 6H, H9–90).
13C-NMR (75 MHz, DMSO-d6) : 163.7 (C7), 163.4 (C1 or C10), 160.3 (C1 or C10), 157.1 (C3), 138.0
δ
(C5–50), 123.1 (C4), 115.9 (C6–60), 109.9 (C2), 104.0 (C8), 26.9 (C9–90). HRMS: m/z [M + Na]+ calculated
for C13H12O5Na: 271.0582 found: 271.0582
5-(3,4-Dihydroxybenzyl◦idene)-2,2-dimethyl-1,3-dioxane-4,6-dione (
(EtOH, nm) 398, ε
(L mol−1.cm−1) 26615. 1H-NMR (300 MHz,
max
4): filtration led to a yellow powder
(88%). m.p. 155–156 C, UV:
λ
DMSO-d6)
δ: 10.52 (s, 1H, OH), 9.57 (s, 1H, OH), 8.14 (s, 1H, H3), 7.92 (d, J = 2.4 Hz, 1H, H9), 7.54
(dd, J = 2.1, 8.7 Hz, 2H, H5–50), 6.87 (d, J = 8.1 Hz, 1H, H6), 1.78 (s, 6H, H11–110). 13C-NMR (75 MHz,
DMSO-d6) δ
: 163.5 (C1 or C10), 160.2 (C1 or C10), 157.4 (C3), 153.1 (C7), 145.2 (C8), 131.3 (C5), 123.5
(C4), 120.3 (C9), 115.6 (C6), 109.4 (C2), 103.9 (C10), 26.8 (C11–110). HRMS: m/z [M + Na]+ calculated for
C13H12O6Na: 287.0532 found: 287.0522
5-(2-furanylmethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (
5
): filtration led to brownish powder (85%).
1
m.p. 82–84 ◦C, UV:
λmax (EtOH, nm) 361,
ε
(L mol−1.cm−1) 33809. H-NMR (300 MHz, DMSO-d6)
δ:
8.33 (d, J = 1.2 Hz, 1H, H7), 8.26 (d, J = 3.8 Hz, 1H, H5), 8.12 (s, 1H, H3), 6.96 (ddd, J = 0.6, 1.6, 3.8 Hz,
1H, H6), 1.71 (s, 6H, H9–90). 13C-NMR (75 MHz, DMSO-d6) : 162.7 (C1 or 10), 159.8 (C1 or 10), 152.1
δ
(C7), 149.4 (C4), 139.6 (C3), 127.8 (C5), 115.6 (C6), 107.9 (C2), 104.6 (C8), 27.0 (C9–90). HRMS: m/z [M +
Na]+ calculated for C11H10O5Na: 245.0426 found: 245.0431
5-[(4,5-dimethyl-2-furanyl)methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione (
6
): filtration led to brown powder
(L mol−1.cm−1) 37961. 1H-NMR (300 MHz,
δ
: 8.22 (s, 1H, H5), 7.98 (s, 1H, H3), 2.40 (s, 3H, H8), 2.05 (s, 3H, H9), 1.69 (s, 6H, H11–110).
◦
(90%). m.p. 127–129 C, UV:
λ
max
(EtOH, nm) 400, ε
DMSO-d6)
13C-NMR (75 MHz, DMSO-d6)
δ
: 163.4 (C1 or 10), 161.1 (C1 or 10), 160.5 (C7), 147.8 (C4), 139.1 (C3),
132.3 (C5), 122.7 (C2), 104.6 (C10), 27.3 (C11–110), 12.8 (C8), 9.9 (C9). HRMS: m/z [M + Na]+ calculated
for C13H14O5Na: 273.0739 found: 273.0744

Molecules 2020, 25, 2178
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5-[(5-ethyl-2-furanyl)methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione (
7
): filtration led to brown powder
1
(87%). m.p. 50–52 ◦C, UV:
λmax (EtOH, nm) 374,
ε
(L mol−1.cm−1) 31307. H-NMR (300 MHz, DMSO-d6)
δ
: 8.29 (d, J = 3.8 Hz, 1H, H5), 8.06 (s, 1H, H3), 6.71 (m, 1H, H6), 2.82 (q, J = 7.6 Hz, 2H, H8), 1.70 (s, 6H,
H11–110), 1.25 (t, J = 7.6 Hz, 3H, H9). 13C-NMR (75 MHz, DMSO-d6) : 168.2 (C1 or 10), 162.9 (C1 or 10),
δ
159.9 (C7), 148.4 (C4), 139.0 (C3), 130.3 (C5), 111.9 (C6), 105.4 (C2), 104.2 (C10), 26.9 (C8), 21.5 (C11–110),
11.4 (C9). HRMS: m/z [M + Na]+ calculated for C13H14O5Na: 273.0739 found: 273.0734
5-[[5-(hydroxymethyl)-2-furanyl]methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione (8): filtration led to brown
1
light solid (82%). m.p. 92–94 ◦C, UV:
λmax (EtOH, nm) 374,
ε
(L mol−1.cm−1) 28814. H-NMR (300 MHz,
DMSO-d6) δ: 8.29 (d, J = 3.8 Hz, 1H, H5), 8.07 (s, 1H, H3), 6.80 (d, 1H, J = 3.8 Hz, H6), 5.67 (s, 1H,
OH), 4.58 (s, 2H, H8), 1.71 (s, 6H, H10–100). 13C-NMR (75 MHz, DMSO-d6) : 165.6 (C1 or 10), 162.7
δ
(C1 or 10), 159.8 (C7), 148.7 (C4), 139.3 (C3), 129.3 (C5), 112.6 (C6), 106.6 (C2), 104.4 (C9), 56.3 (C8), 26.9
(C10–100). HRMS: m/z [M + Na]+ calculated for C12H12O6Na: 275.0532 found: 275.0535
5-(2-benzofuranylmethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (
9
): filtration led to yellow powder (78%).
1
m.p. 122–124 ◦C, UV:
λ
max
(EtOH, nm) 386,
ε
(L mol−1.cm−1) 32310. H-NMR (300 MHz, DMSO-d6)
δ:
8.57 (s, 1H, H5), 8.22 (s, 1H, H3), 7.93 (d, J = 7.8 Hz, 1H, H7), 7.72 (d, J = 8.4 Hz, 1H, H10), 7.60 (m, 1H,
H8), 7.40 (t, J = 7.5 Hz, 1H, H9), 1.75 (s, 6H, H13–130). 13C-NMR (75 MHz, DMSO-d6)
δ: 162.3 (C1 or
10), 159.3 (C1 or 10), 156.2 (C11), 149.7 (C4), 139.9 (C3), 129.9 (C8), 127.9 (C6), 124.4 (C9), 124.0 (C7),
122.0 (C5), 112.5 (C10), 112.0 (C2), 104.9 (C12), 27.1 (C13–130). HRMS: m/z [M + Na]+ calculated for
C15H12O5Na: 295.0582 found: 295.0577
5-[[5-[(acetyloxymethyl)-2-furanyl]methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione (10): extraction led to
1
brown oil (86%). UV:
λ
max
(EtOH, nm) 366,
ε
(L mol−1.cm−1) 28728. H-NMR (300 MHz, DMSO-d6)
δ:
8.23 (m, 1H, H5), 8.06 (m, 1H, H3), 6.93 (m,1H, H6), 5.19 (m, 2H, H8), 2.09 (m, 3H, H10), 1.72 (m, 6H,
H12–120). 13C-NMR (75 MHz, DMSO-d6) : 169.9 (C9), 162.5 (C1 or 10), 159.6 (C1 or 10), 158.0 (C7),
δ
149.3 (C4), 139.1 (C3), 128.1 (C5), 115.0 (C6), 108.4 (C2), 104.6 (C11), 57.5 (C8), 27.0 (C12), 20.5 (C10).
HRMS: m/z [M + Na]+ calculated for C14H14O7Na: 317.0637 found: 317.0637
5,50-(2,5-furandiyldimethylidene)bis [2,2-dimethyl-1,3-dioxane-4,6-dione] (11): filtration led to yellow
1
powder (75%). m.p. 204–206 ◦C, UV:
λ
max
(EtOH, nm) 406, 426,
ε
(L mol−1.cm−1) 47642. H-NMR
(300 MHz, DMSO-d6)
DMSO-d6)
δ
: 8.22 (s, 2H, H3), 8.06 (s, 2H, H5), 1.74 (s, 6H, H7–70). 13C-NMR (75 MHz,
: 162.1 (C1 or 10), 159.2 (C1 or 10), 152.9 (C4), 137.5 (C5), 127.4 (C3), 113.9 (C2), 105.1 (C6),
δ
27.2 (C7–70). HRMS: m/z [M + H]+ calculated for C18H16O9: 377.0873 found: 377.0874
5-(1H-pyrrol-2-ylmethy◦lidyne)-2,2-dimethyl-1,3-dioxane-4,6-dione (12): filtration led to a green powder
(EtOH, nm) 393, ε
(L mol−1.cm−1) 40225. 1H-NMR (300 MHz,
max
(90%). m.p. 178–180 C, UV:
λ
Acetone-d6) δ: 8.21 (s, 1H, H3), 7.72 (dddd, J = 0.9, 1.5, 2.4, 3.3 Hz, 1H, H6), 7.35 (s, 1H, H5), 6.59 (dt, J =
2.2, 4.2, Hz, 1H, H7), 1.73 (s, 6H, H9–90). 13C-NMR (75 MHz, DMSO-d6) : 164.7 (C1 or 10), 164.2 (C1 or
δ
10), 143.5 (C3), 133.8 (C6), 131.1 (C5), 129.5 (C4), 114.8 (C7), 104.7 (C8), 27.2 (C9–90). HRMS: m/z [M +
Na]+ calculated for C11H11O4NNa: 244.0586 found: 244.0580
5-(1-methyl-1H-pyrrol-2-ylmethylidene)2,2-dimethyl-1,3-dioxane-4,6-dione (13): filtration led to brown
1
powder (72%). m.p. 102–104◦C, UV:
λmax (EtOH, nm) 401,
ε
(L mol−1.cm−1) 43003. H-NMR (300 MHz,
DMSO-d6) δ: 8.34 (m, 1H, H5), 8.29 (s, 1H, H3), 7.53 (t, J = 2.0 Hz, 1H, H6), 6.43 (ddd, J = 0.8, 2.4, 4.5 Hz,
1H, H7), 3.98 (s, 3H, H8), 1.70 (s, 6H, H10–100). 13C-NMR (75 MHz, DMSO-d6) : 165.0 (C1 or 10), 161.3
δ
(C1 or 10), 140.4 (C3), 137.0 (C6), 129.8 (C4), 128.0 (C5), 112.9 (C7), 104.0 (C9), 103.1 (C2), 34.9 (C8), 27.3
(C10). HRMS: m/z [M + Na]+ calculated for C12H13O4NNa: 258.0742 found: 258.0738
5-(1H-indol-2-ylmethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (14): filtration led to a yellow powder (57%).
1
m.p. 140–142 ◦C, UV:
λmax (EtOH, nm) 416,
ε
(L mol−1.cm−1) 39433. H-NMR (300 MHz, DMSO-d6)
δ
: 11.78 (s, 1H, NH), 8.42 (s, 1H, H3), 7.84 (s, 1H, H5), 7.73 (t, 2H, J = 7.4 Hz, H8–9), 7.40 (t, 1H, J =
7.7 Hz, H7), 7.14 (t, 1H, J = 7.5 Hz, H10), 1.75 (s, 6H, H13–130). 13C-NMR (75 MHz, DMSO-d6)
: 163.0
(C1–10), 161.9 (C1–10), 144.3 (C3), 140.4 (C6), 131.4 (C8), 128.3 (C7), 127.4 (C11), 122.9 (C4), 122.3 (C5),
δ

Molecules 2020, 25, 2178
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121.4 (C10), 113.6 (C9), 106.8 (C2), 104.5 (C12), 26.7 (C13–130). HRMS: m/z [M + Na]+ calculated for
C15H13O4NNa: 294.0742 found: 294.0738
4.2. UV Analysis and Loss of Absorbance
UV–Vis spectra were recorded on a Cary 60 UV–Vis by Agilent (Wilmington, DE, USA) from a
solution of the desired compound at 10
reported in wavelength (nm). Loss of absorbance was obtained by irradiating the solutions for 1 h
into a Rayonet® RPR-200 ( = 300 nm, P = 8.32 W/m2, stirring, T = 35 ◦C) from SNE Ultraviolet Co
(Branford, CT, USA) using 14 RPR-3000A lamps. Absorbance after 1 h was compared to the one before
µ
mol.L−1 in ethanol placed in a 1 cm quartz cuvette and are
λ
irradiation and loss of absorbance was calculated in percentage at the λ
.
max
4.3. DPPH Inhibition
Antiradical activities were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to
determine EC50 values. The test started with the addition of 190 L of DPPH solution (C = 200
M) in ethanol to a well containing 10 L of potential antiradical solution in ethanol (concentrations
from 400 to 12.5 M) [46]. Reaction was carried out in a Multiskan FC system from Thermo Fisher
µ
µ
µ
µ
Scientific (Waltham, MA, USA) and disappearance of the DPPH radicals was monitored at 520 nm
every 5 min for 7.5 h. Use of different concentrations of potential antiradical gave the EC50 value which
is the quantity needed to reduce half the initial population of DPPH radicals.
4.4. Tyrosinase Inhibition
Tyrosinase inhibitor activity was measured by spectrophotometry based on the method presented
by Masamoto et al. [47]. First, 10
µ
L of inhibitor solution at different concentrations (from 5000 to
M) in DMSO were added to a 96-well microplate and mixed with ammonium formate buffer
L, 50 mmol.L−1, pH 6.4). Then, 20 L (0.8 mg.mL−1) of tyrosine in ammonium formate buffer
L of mushroom tyrosinase (5000 U.mL−1 in ammonium formate buffer) were
0.1
(60
µ
µ
µ
were added. Finally, 10
µ
added and the assay mixture was then incubated at 37 ◦C for 10 min. After incubation, the amount of
dopachrome production in the reaction mixture was determined by analysis at 450 nm in a microplate
spectrophotometer. Kojic acid solution at different concentrations was used as a positive control.
The concentration needed for 50% of tyrosinase inhibition (IC50) was determined.
4.5. Endocrine Disruption Assays
The agonistic and antagonistic potentials of Meldrum’s acid derivatives were analyzed following
a literature method in which ER
corresponding reporter cells HELN ER
α
, PXR, and AR transcriptional activities were monitored by using
, HELN AR, and HG5LN PXR cells, respectively [48].
α
Activities were measured in relative light units (RLUs) and 100% of activities were assigned to
the RLU value obtained with 10 nM agonist control (estradiol (E2), R 1881, SR 12813). The sample
(DMSO) was tested as a control without any compound.
5. Patents
F.A., C.P., and M.M.M. filed a patent based on the work described here (Patent application no.
FR1912971).
Supplementary Materials: The following are available online: the 1H and 13C-NMR spectra, UV spectrum, loss of
absorbance study, DPPH assay, and tyrosinase inhibitor assay are provided for every single molecule synthesized
in this work.
Author Contributions: Conceptualization, M.M.M., C.P., and F.A.; methodology, M.M.M. and C.P.; formal
analysis, M.M.M. and C.P.; LC-MS2 analysis, F.B.; endocrine disruption assays, P.B.; data curation, M.M.M. and
C.P.; writing—original draft preparation, M.M.M. and C.P.; writing—review and editing, F.A.; supervision, F.A.;
project administration, F.A.; funding acquisition, F.A. All authors have read and agreed to the published version
of the manuscript.

Molecules 2020, 25, 2178
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Funding: This research was funded by the EU’s Horizon 2020 program, grant number 828753, and the Agence
Nationale de la Recherche, grant number ANR-17-CE07–0046, Grand Reims, Conseil Départemental de la Marne
and Région Grand Est.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of all compounds are available from the authors.
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