The involvement of xanthone and (E)-cinnamoyl chromophores for the design and synthesis of novel sunscreening agents

Article abstract of DOI:10.3390/ijms22010034

Excessive UV exposure contributes to several pathological conditions like skin burns, er-ythema, premature skin aging, photodermatoses, immunosuppression, and skin carcinogenesis. Effective protection from UV radiation may be achieved with the use of sunscreens containing UV filters. Currently used UV filters are characterized by some limitations including systemic absorp-tion, endocrine disruption, skin allergy induction, and cytotoxicity. In the research centers all over the world new molecules are developed to improve the safety, photostability, solubility, and absorption profile of new derivatives. In our study, we designed and synthesized seventeen novel molecules by combining in the structures two chromophores: xanthone and (E)-cinnamoyl moiety. The ultraviolet spectroscopic properties of the tested compounds were confirmed in chloroform solutions. They acted as UVB or UVA/UVB absorbers. The most promising compound 9 (6-meth-oxy-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(2,4-dimethoxyphenyl)acrylate) absorbed UV radiation in the range 290–369 nm. Its photoprotective activity and functional photostability were further evaluated after wet milling and incorporation in the cream base. This tested formulation with compound 9 possessed very beneficial UV protection parameters (SPFin vitro of 19.69 ± 0.46 and UVA PF of 12.64 ± 0.32) which were similar as broad-spectrum UV filter tris-biphenyl triazine. Additionally, compound 9 was characterized by high values of critical wavelength (381 nm) and UVA/UVB ratio (0.830) thus it was a good candidate for broad-spectrum UV filter and it might protect skin against UVA-induced photoaging. Compound 9 were also shown to be photostable, non-cytotoxic at con-centrations up to 50 μM when tested on five cell lines, and non-mutagenic in Ames test. It also possessed no estrogenic activity, according to the results of MCF-7 breast cancer model. Addition-ally, its favorable lipophilicity (miLogP = 5.62) does not predispose it to penetrate across the skin after topical application.

Full text of DOI:10.3390/ijms22010034

International Journal of
Molecular Sciences
Article
The Involvement of Xanthone and (E)-Cinnamoyl
Chromophores for the Design and Synthesis of Novel
Sunscreening Agents
2,
Justyna Popiół 1 , Agnieszka Gunia-Krzyzak
*
, Karolina Słoczyn´ska 1 , Paulina Koczurkiewicz-Adamczyk 1,
˙
Kamil Piska 1 , Katarzyna Wójcik-Pszczoła 1 , Dorota Zelaszczyk
, Anna Krupa 3 , Paweł Zmudzki
,
2
4
˙
˙
Henryk Marona 2 and Elzbieta Pe˛kala
˙
1
1
Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College,
Medyczna 9, 30-688 Kraków, Poland; justyna.popiol@uj.edu.pl (J.P.); karolina.sloczynska@uj.edu.pl (K.S.);
paulina.koczurkiewicz@uj.edu.pl (P.K.-A.); kamil.piska@uj.edu.pl (K.P.);
katarzynaanna.wojcik@uj.edu.pl (K.W.-P.); elzbieta.pekala@uj.edu.pl (E.P.)
Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30-688 Kraków, Poland; dorota.zelaszczyk@uj.edu.pl (D.Z.);
2
˙
henryk.marona@uj.edu.pl (H.M.)
3
4
Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30-688 Kraków, Poland; a.krupa@uj.edu.pl
Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College,
Medyczna 9, 30-688 Kraków, Poland; pawel.zmudzki@uj.edu.pl
*
Correspondence: agnieszka.gunia@uj.edu.pl; Tel.: +48-126-205-576
Abstract: Excessive UV exposure contributes to several pathological conditions like skin burns,
erythema, premature skin aging, photodermatoses, immunosuppression, and skin carcinogenesis.
Effective protection from UV radiation may be achieved with the use of sunscreens containing UV
filters. Currently used UV filters are characterized by some limitations including systemic absorption,
endocrine disruption, skin allergy induction, and cytotoxicity. In the research centers all over the
world new molecules are developed to improve the safety, photostability, solubility, and absorption
profile of new derivatives. In our study, we designed and synthesized seventeen novel molecules by
combining in the structures two chromophores: xanthone and (E)-cinnamoyl moiety. The ultraviolet
spectroscopic properties of the tested compounds were confirmed in chloroform solutions. They acted
as UVB or UVA/UVB absorbers. The most promising compound 9 (6-methoxy-9-oxo-9H-xanthen-2-
yl)methyl (E)-3-(2,4-dimethoxyphenyl)acrylate) absorbed UV radiation in the range 290–369 nm. Its
photoprotective activity and functional photostability were further evaluated after wet milling and
incorporation in the cream base. This tested formulation with compound 9 possessed very beneficial
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
˙
Citation: Popiół, J.; Gunia-Krzyzak,
A.; Słoczyn´ska, K.; Koczurkiewicz-
Adamczyk, P.; Piska, K.; Wójcik-
˙
Pszczoła, K.; Zelaszczyk, D.; Krupa,
˙
A.; Zmudzki, P.; Marona, H.; et al.
The Involvement of Xanthone and (E)-
Cinnamoyl Chromophores for the De-
sign and Synthesis of Novel
Sunscreening Agents.
Int. J. Mol. Sci. 2021, 22, 34. https://
dx.doi.org/10.3390/ijms22010034
UV protection parameters (SPFin vitro of 19.69
±
0.46 and UVA PF of 12.64
±
0.32) which were similar
Received: 20 November 2020
Accepted: 18 December 2020
Published: 22 December 2020
as broad-spectrum UV filter tris-biphenyl triazine. Additionally, compound 9 was characterized by
high values of critical wavelength (381 nm) and UVA/UVB ratio (0.830) thus it was a good candidate
for broad-spectrum UV filter and it might protect skin against UVA-induced photoaging. Compound
9 were also shown to be photostable, non-cytotoxic at concentrations up to 50 µM when tested on
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
five cell lines, and non-mutagenic in Ames test. It also possessed no estrogenic activity, according to
the results of MCF-7 breast cancer model. Additionally, its favorable lipophilicity (miLogP = 5.62)
does not predispose it to penetrate across the skin after topical application.
Keywords: UV absorption; UV filters; UV radiation; photoprotective activity; xanthone; (E)-cinnamoyl
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
1. Introduction
Ultraviolet radiation (UVR) detected on Earth significantly affects all living organisms
including humans. Apart from positive effects of the reasonable UVR exposure, e.g.,
vitamin D production, several pathological conditions like skin burns, erythema, premature
Int. J. Mol. Sci. 2021, 22, 34. https://dx.doi.org/10.3390/ijms22010034
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 34
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skin aging, photodermatoses, immunosuppression, and skin carcinogenesis were identified
as results of extensive UVR exposure [1–3]. In order to provide effective protection from
UV radiation, sunscreen preparations containing UV filters are recommended. Currently
used UV filters are characterized by many drawbacks such as interrupting of endocrine
system, skin allergic reactions, and cytotoxicity [2,4–6]. Further attempts are made all over
the world to discover novel molecules which would provide effective UV protection with
minimum adverse side effects.
One of the strategies in the development of new UV filter molecules is the modification
of well-known sunscreens by means of multiplying the chromophore and by incorporation
of additional auxochromes or alkyl groups. These modifications are intended to improve
safety, photostability, solubility, and absorption profile of new derivatives. For example,
the molecule of ethylhexyl triazone approved in the late 1990s contains three molecules
of PABA (para-aminobenzoic acid) linked to a triazine ring. The chromophore used in
triplicate allowed to obtain broad absorption profile and high molecular weight that
prevent penetration through the skin [7,8]. Bino et al. focused on the modification of
2-phenyl-1H-benzimidazole-5-sulfonic acid by incorporating additional functional groups
in phenyl and benzimidazole rings to obtain molecules able to act as UV absorbers and
inhibitors of radical species formation [9]. In our recent study, we designed new UV
filters based on the structure of 3-benzylidene camphor. In our compounds, the camphor
fragment was replaced with the imidazolidine-2,4-dione moiety with the aim of improving
safety, the incorporation of allyl group contributed to the extension of the UV absorption,
whereas 2-ethoxy-2-oxoethyl substituents in the imidazolidine ring improved the solubility
of molecules [10].
Cinnamates are widely used in cosmetics, not only as UV filters or UV absorbers but
also as skin and hair conditioners, antioxidants, perfuming, masking, and antimicrobial in-
gredients [11]. UV filters that are (E)-cinnamoyl derivatives have been approved in selected
countries all over the world: octinoxate (INCI: Ethylhexyl methoxycinnamate, EHMC), amilox-
ate (INCI: Isoamyl p-methoxycinnamate), octocrylene, cinoxate, diethanolamine methoxycin-
namate, 2,5-diisopropyl methyl cinnamate, and isopentyl trimethoxycinnamate trisilox-
ane [12,13]. Octinoxate (Figure 1a) is ethylhexyl ester of 4-methoxycinnamic acid, its
structure allows electron delocalization required for absorbance in UVB region and is
typical for UV filters—constitutes disubstituted aromatic molecule with electron releasing
(-OCH3) and electron accepting (ester group) substituents. Electron releasing group is
in para position to the carbonyl group which is additionally conjugated with a double
bond [14]. Octocrylene (Figure 1b) is ethylhexyl ester of 2-cyano-3,3-diphenylacrylic acid,
this compound is characterized by relatively low extinction coefficient but has an excellent
photostability and is used as photostabilizer of other UV filters [15]. Absorption range
of cinnamates is narrow, they provide protection only in UVB region, moreover EHMC
upon irradiation undergoes trans-cis isomerization which results in absorption loss [16].
Therefore, the aim of our study was to design new potential UV filters by modifications
of (E)-cinnamic acid derivatives to obtain new, safe, broad absorbing and photostable
compounds. We decided to combine (E)-cinnamoyl moiety with another chromophore—
xanthone skeleton.
Xanthone (dibenzo-γ-pyrone) possesses conjugated double bond system and several
potential substitution positions. Coupling with an appropriate auxochromic groups and
additional chromophores gives the opportunity to select substances not only with the
intended photoprotectiveness in a wide UV range but also with a satisfactory safety profile.
Xanthone derivatives have been subject of multidirectional research for many years. A
lot of them have a favorable biological and pharmacological activity profile, including
among other antioxidant [17], antimicrobial [18], anticancer [19
effect [21]. Moreover, xanthone derivatives have also recently been of interest in the field of
cosmetics chemistry. Mangiferin (2- -D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-
,20], and anti-inflammatory
β
9-one, Figure 2) and its derivatives are of particular interest, its cosmetic application is the
subject of many patents claims [22]. Moreover in in vivo study mangiferin was shown to

Int. J. Mol. Sci. 2021, 22, 34
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inhibit UVB-induced length and depth of skin wrinkles. Additionally, mangiferin improves
the photostability of avobenzone and reduces its phototoxic potential [23,24].
Figure 1. The chemical structures of octinoxate (INCI: Ethylhexyl methoxycinnamate) (a) and octocrylene (b).
In the present study, we designed and synthesized novel derivatives combining in the
structures the two above mentioned chromophores: xanthone and (E)-cinnamoyl moiety
(Figure 3). We intended to obtain compounds characterized by lipophilicity predisposing
for affinity to stratum corneum rather than penetrating into deeper layers of the skin. Apart
from UV absorption properties, we aimed to incorporate the most promising compounds
into cosmetic formulation to evaluate their photoprotective activity and photostability
in the conditions of future use. Additionally, with in vitro methods we assessed their
safety profile in mutagenic and estrogenic activity assays. We also performed tests of
their cytotoxicity in keratinocytes and skin fibroblasts as well as in hepatocytes, astrocytes
and cardiomyoblasts.
Figure 2. The chemical structure of mangiferin.
Figure 3. The design concept of the novel UV filters: combination of xanthone and (E)-cinnamoyl chromophores.
2. Results and Discussion
2.1. Chemistry
Chemical structures of tested compounds were shown in Table 1 and the route of their
synthesis was presented in Schemes 1, 2, 3.

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Table 1. Chemical structures and lipophilicity parameters of tested compounds.
Compound
1
R1
Position of R2
R2
miLogP *
RM0 **
H
4-
6.16
5.02
2
H
4-
6.38
5.28
3
4
6-OCH3
6-OCH3
4-
4-
6.19
6.42
5.66
5.06
5
6
7
6-OCH3
6-OCH3
6-OCH3
4-
4-
4-
5.57
5.77
5.79
5.10
4.96
5.06
8
9
6-OCH3
6-OCH3
2-
2-
5.82
5.62
6.15
4.64
10
11
6-OCH3
6-OCH3
2-
2-
5.41
5.39
4.53
4.50
12
6-OCH3
2-
7.10
5.62

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Table 1. Cont.
Position of R2
Compound
13
R1
R2
miLogP *
RM0 **
7-Cl
2-
2-
2-
6.44
4.85
5.18
4.19
14
15
H
H
6.14
5.24
16
17
H
H
3-
4-
5.24
5.21
4.54
4.25
* calculated LogP values by Molispiration on-line tool, ** relative lipophilicity parameter determined
by reversed-phase thin-layer chromatography.
Scheme 1. General route of synthesis of compounds 1–13.
Scheme 2. General route of synthesis of compound 14.
Scheme 3. General route of synthesis of compounds 15–17.

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Synthesis of starting materials for preparation of compounds
1–13, i.e., 4-(bromomethyl)-
9H-xanthen-9-one [25], 2-(bromomethyl)-6-methoxy-9H-xanthen-9-one [26], 5-(bromomethyl)-
3-methoxy-9H-xanthen-9-one [19] and 2-(bromomethyl)-7-chloro-9H-xanthen-9-one [27
]
was performed according to previously published procedures. These compounds were
further used in the reaction with appropriate derivatives of (E)-cinnamic acid in DMF
in the presence of anhydrous K2CO3 as a proton acceptor resulting in obtaining of com-
pounds
1
–
13 (Scheme 1). Substrates for synthesis of compounds 14–17, i.e., 2-, 3-, or
4-hydroxy-9H-xanthen-9-one [28
–
31] and 2-(3-bromopropoxy)-9H-xanthen-9-one [32] were
also synthesized according to available literature. 2-(3-Bromopropoxy)-9H-xanthen-9-one
was further used in the reaction with (E)-4-methoxycinnamic acid to synthesize compound
14 (Scheme 2). 2-, 3-, or 4-hydroxy-9H-xanthen-9-ones were used in the reaction with
(E)-cinnamoyl chloride to synthesize compounds 15–17 (Scheme 3). Chemical structures
and purity of final compounds were confirmed by spectral analysis (NMR, LC/MS/MS).
2.2. Lipophilicity Evaluation
The relative lipophilicity parameters (RM0) of the tested compounds were determined
using reversed-phase thin-layer chromatography (RP-TLC) according to previously pub-
lished procedures [33,34]. The investigations were carried on RP-18-coated aluminum
sheets with mobile phase consisting of diverse concentrations of methanol and potassium
phosphate buffer (pH = 7.4). The experimental data showed a linear correlation between
RM values and the concentration of methanol, the obtained correlation coefficients ranged
from 0.956 to 0.996 (Table S2 Supplementary Materials). The relative lipophilicity values
(RM0) of the tested compounds, calculated as extrapolations of RM for 0% methanol concen-
trations, ranged from 4.19 to 6.15 (Table 1). We also calculated LogP values by Molispiration
on-line tool [35] and provided results in Table 1. The calculated values of miLogP ranged
from 5.21 to 7.10.
Previous studies showed that lipophilicity was an important parameter of the molecule
affecting the possibility of skin permeation. Compounds with logP of 4–5 were character-
ized by poor permeation rate across the skin, while compounds with more hydrophilic
nature (logP = 0–2.5) were predisposed to effective skin permeation after topical applica-
tion [36]. Similarly, according to formal guidance for the testing of cosmetic ingredients,
substances with logP
≥
4 were characterized by very low dermal absorption [37]. Based on
the performed lipophilicity evaluation, reported here UV filters would not penetrate across
the skin after topical application.
2.3. Ultraviolet Spectroscopic Properties
The results of studies on the ultraviolet spectroscopic properties of the tested com-
pounds and reference UV filters were presented in Table 2. This type of screening using
low-concentrated compound solutions is very useful in the initial phase of searching for
new UV filter candidates and is aimed at identification of structures for more advanced tests.
Among presented group of (E)-cinnamoyl xanthone derivatives, all tested compounds
might be characterized as ultraviolet radiation absorbers. UV spectroscopic properties of
some of them surpass commercially available UV filters such us 3-(4-methyl)benzylidene
camphor (4-MBC), octocrylene (OCT), octinoxate (EHMC) and avobenzone (BMDM).
Compound
of 6-methoxy group in xanthone skeleton additionally chloro substituent is located in
ortho position of phenyl ring of trans-cinnamoyl residue. Compound with chlorine
atom in para position was characterized by much higher molar extinction coefficient in
comparison to the compound , whereas compound with both chlorine atom in para
position and 6-methoxy group in xanthone skeleton showed not only higher and
1 showed the weakest absorption properties. This compound is deprived
2
1
4
ε
max
‹E1,1
›
value than compounds
1
–
3
but also bathochromic shift by 6 nm. Ultraviolet
spectroscopic properties of compounds and were very similar which indicated that
position of methoxycinnamoyl substituent in xanthone ring did not significantly affect their
UV absorption properties. The max of these compounds (300 and 303 nm, respectively),
mean
7
8
λ

Int. J. Mol. Sci. 2021, 22, 34
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similarly to EHMC, was located in UVB region, additionally their
ε
max, ‹E1,1
›
and
mean
absorption range were much higher in comparison to the reference UVB filters (Figure
4). E(1,1) value as well as molecular mass are often used to present UV filters properties.
Reported here (E)-cinnamoyl xanthone derivatives possessed high molar mass, which is
desirable from modern UV filters, therefore despite much higher
ε
for compounds 7
max
and 8 in comparison to the EHMC and 4-MBC, their E(1,1) values were similar.
Table 2. Ultraviolet spectroscopic properties of the tested xanthone derivatives and reference UV
filters obtained in chloroform solutions.
Absorption
Range (nm)
E(1,1) (λmax)
(g L cm
−1
−1
)
Compound
λ
(nm)
ε
max
(M cm
‹E1,1›
mean
max
−1
−1
)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
290–352
290–351
290–342
290–341
290–350
290–342
290–346
290–348
290–369
290–355
290–354
290–340
290–359
290–375
290–355
290–345
290–351
290–328
290–347
290–337
290–391
290
15,311
25,688
27,690
36,895
24,118
25,749
39,002
38,128
392
657
658
948
580
619
937
916
774, 766
694, 682
898
463
633
69
290
290
296
290
290
300
303
299, 329
301, 326
304
299
312
300
290
290
290
300
306
309
357
133
168
202
221
168
342
314
360
307
326
112
243
214
188
235
120
221
141
266
520
34,534, 34,177
31,005, 30,469
33,330
21,409
26,590
28,360
38,152
36,410
20,100
22,053
14,330
659
1115
1064
587
867
396
858
1158
4-MBC
OCT
EHMC
BMDM
24,907
35,939
Figure 4. UV-absorption spectra of tested compounds and reference UV filters in chloroform solutions.
E(1,1)—extinction at 1% concentration and 1 cm optical pathlength.

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Absorption spectra of compounds
9
and 10 with two methoxy groups in phenyl ring
of cinnamoyl residue respectively in 2,4- and 3,4- position differed from other derivatives
in the tested group demonstrated two peaks of one in UVB and one in UVA II region,
λ
max
additionally the values of
ε
were equally high at both wavelengths which was well
max
reflected in the high ‹E1,1
›
value, especially for compound 9. Ultraviolet absorption
mean
curve of compound 9 was presented in Figure 4.
Compounds 15 17 without alkyl or alkoxy linker between xanthone moiety and
–
(E)-cinnamoyl residue showed different ultraviolet spectroscopic properties than other
compounds. The higher values of molar extinction coefficient were observed at 290 nm
moreover E(1,1) value for compounds 15 (1115) and 16 (1064) with methyl cinnamate sub-
stituent in position 2- or 3- of xanthone ring were comparable to avobenzone. Despite high
E(1,1) values, these compounds were characterized by relatively low ‹E1,1
›
parameters.
mean
This was due to the fact that the absorbance was very high at the initial wavelengths
but gradually decreased as the wavelength increased (Figure 4). Compound 16 might be
considered as a strong UVB absorber.
2.4. Photoprotective Activity
The most promising ultraviolet spectroscopic properties showed (E)-cinnamoyl deriva-
tives of 6-metoxy-2-methyl-9H-xanthen-9-one, compounds
8 and 9. They were subjected to
further tests of photoprotective activity. These compounds were poorly soluble in common
solvents used in cosmetic products, such as ethanol, polysorbates, triacetin, glycerol, or
polyoxyethylene glycols. Thus, the selection of an appropriate solvent that would enable
us to achieve the desired concentration of the UV filter in the final formulation, turned out
to be impossible. This prompted us to search for a more advanced technological approach
to incorporate these insoluble compounds into a cosmetic formulation. For that reason,
the tested compounds were wet milled in a high-energy ball milling process. The milling
procedure was carried out in the presence of triacetin, and finally a concentrated paste of
each of the tested compounds was formed. This paste could be easily combined with the
cream base.
Finally, the formulations loaded with 12.5% (w/w) of compounds 8 or 9 were tested
using an in vitro method, recommended for the assessment of photoprotective parameters
in cosmetics products [38]. The absorbance of the samples spread onto PMMA (polymethyl-
methacrylate) plates with a rough surface, imitating the physical characteristic of the skin
was measured with the SPF-290AS Analyzer. This device was equipped with an integrating
sphere which collects light scattered by the sample.
Further studies aimed to compare the photoprotective activity of the compounds
. The results were presented in Table 3 and Figure 5. Compound ((E)-4-methoxy-
cinnamoyl xanthone derivative) showed high absorption of UVB radiation. The value of
SPFin vitro of a 12.5% formulation was 14.69 2.25 and exceeded the SPFin vitro coefficient,
8
and
9
8
±
which can be achieved at the maximum allowable concentration by a number of UV protec-
tive substances, such as octocrylene, octinoxate, enzacamene, ensulizole, or iscotrizinol [39].
Moreover compound 8 was also active in UVA II region, UVA PF reached 4.92 ± 0.78.
Table 3. Photoprotective activity of compounds 8 and 9 obtained in 12.5% (w/w) cosmetic formulation.
Compound
SPFin vitro ± SD
UVA PF ± SD
λ (nm)
c
UVA/UVB Ratio
8
9
14.69 ± 2.25
4.92 ± 0.78
345
381
0.329
0.830
19.69 ± 0.46
12.64 ± 0.32
Results are presented as mean
scans were performed.
±
SD from two independent experiments, at each determination six

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Figure 5. UV absorption spectra of compounds
8
and
9
obtained in cosmetic formulation at 12.5%
(w/w) cosmetic formulation applied on polymethylmethacrylate plates. MPF—monochromatic
protection factor.
Compound
9 possessed an additional methoxy group in 2- position of phenyl ring
of (E)-cinnamoyl residue. This additional auxochrome caused significant changes in UV
absorption properties as compared to compound
8
. Compound
9
in a 12.5% formulation
0.32. These values were
showed SPFin vitro coefficient of 19.69 0.46 and UVA PF of 12.64
±
±
similar to tris-biphenyl triazine, which is a broad-spectrum UV-filter and was approved
for the use in cosmetic products within the European Union in 2014. Couteau et al. tested
this compound with a similar in vitro method using 50 mg of the formulation instead of
32 mg applied to the PMMA plate in the present study. The obtained values of SPFin vitro
and UVA-PF were respectively 21.85 ± 2.70 and 9.64 ± 0.75 [40].
Differences between compound
parameters that characterize sunscreen formulations such as critical wavelength (
8
and
9
were also found in two other important
c) and
λ
UVA/UVB ratio. Critical wavelength is the wavelength for which the section under the
integrated optical density curve starting at 290 nm is equal to 90% of the integrated section
between 290 to 400 nm [41] and is in the range from 320 nm which means that UVA
radiation is not absorbed to 389 nm when UVA radiation is absorbed to the same extent
as UVB [42]. Critical wavelength and UVA/UVB ratio in final sunscreen product should
be respectively at least 370 nm and 1/3 (0.33333) to provide a minimum UVB and UVA
protection. For compound
0.329, respectively, which indicated that it provided little protection against UVA radiation
and could be considered as a UVB filter. For compound these parameters were 381 nm
and 0.830, respectively. These findings indicated that compound was a good candidate for
broad-spectrum UV filter and provided sufficient spectrum of sunscreen product (required
in EU) without other UV filters in formulation [41]. Additionally, compound might protect
8 critical wavelength and UVA/UVB ratio were 345 nm and
9
9
9
skin against UVA-induced photoaging, because its critical wavelength exceeded 380 [43].
2.5. Photostability Studies
In order to evaluate functional photostability of compounds
formulations containing tested compounds applied on PMMA plates were irradiated with
solar light simulator. The changes in SPFin vitro, UVA PF, and UVA/UVB ratio following
8 and 9, thin layer of
λ
c
1 h irradiation corresponding to natural sunlight were investigated. Results were presented

Int. J. Mol. Sci. 2021, 22, 34
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in Figure 6 and Table 4. The irradiation of formulation with compound
8
caused the change
in the shape of absorption curve-slight hypsochromic shift and hypochromic effect were
observed. The decrease of SPFin vitro and UVA PF by 23.95 and 32.8%, respectively, was
observed. The value of critical wavelength shifted by 10 nm from 345 nm to 335 nm,
additionally the UVA/UVB ratio decreased from 0.339 to 0.201. It indicated that irradiation
mainly contributed to decrease in protection in the UVA region. The decrease of SPFin vitro
was comparable to that showed by EHMC. In previous study, formulation containing
EHMC was irradiated in the same conditions and % of Initial SPFin vitro was 80.83 [10].
Figure 6. UV absorption spectra of compounds
8 and 9 obtained in pre-irradiation conditions and 1 h post-irradiation with
solar light simulator conducted at 500 W/m2 in 12.5% (w/w) cosmetic formulations.
Table 4. The changes in photoprotective activity of compounds 8 and 9 after irradiation with solar light simulator at 500 W/m2.
λ (nm)
UVA/UVB Ratio
c
% of Initial
SPFin vitro
% of Initial
UVA PF
Compound
Pre-Irradiation
Post-Irradiation
Pre-Irradiation
Post-Irradiation
8
9
76.05
89.03
67.20
82.75
345
381
335
380
0.339
0.830
0.201
0.784
Results are presented as means from two independent experiments, at each determination six scans were performed.
Compound 9 was characterized by much higher photostability. None hypsochromic
shift was observed, the value of critical wavelength changed only by 1 nm. The decrease
in SPFin vitro and UVA PF was 10.97 and 17.25%, respectively. Based on the fact that the
photoprotective activity parameters did decrease by more than 20%, compound
considered as a photostable UV filter [44 45]. It is worth mentioning that for avobenzone
which is most widely used UVA absorber in Europe, USA, Australia, and Japan the decrease
9 might be
,
of UVA PF in cosmetic formulation reaches 41% [46]. Thus, compound
alternative for avobenzone.
9
may be a good
The changes in photoprotective activity upon irradiation of compounds
8
and most
9
likely resulted from the photoisomerization process. Both compounds, similarly to EHMC,
are (E)-cinnamate derivatives. The main photochemical reaction for these compounds is
E-Z (trans-cis) isomerization. This photoreaction takes place upon irradiation of UV filters
containing unsaturated bonds and is considered to be a very efficient way of dispersing the
absorbed energy [16
which results in UV absorption loss (30–50%) because the resulting cis isomer of EHMC
has almost twice lower extinction coefficient [16]. Compound in comparison to the 8,
,47]. In the case of EHMC, both isomers form a photostationary state
9
was characterized by higher photostability. It probably was results of the presence of an
additional methoxy group in ortho position of phenyl ring.

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2.6. Safety Assessment
Compound , due to its broad-spectrum UV absorbance and excellent photostability,
9
and compound 16, a strong UVB absorber with the most promising properties within
the group of compounds with methyl cinnamate substituent were subjected to in vitro
preliminary safety assessment. The cytotoxicity of
9 and 16 was evaluated using two
cell lines of human origin: keratinocytes (HaCaT) and skin fibroblasts (BJ). Additionally,
hepatocytotoxicity, neurocytotoxicity and cardiocytotoxicity were tested. As reference
standards, four commercially available UV filters were used including cinnamic acid
derivatives (octocrylene (OCT) and octinoxate (EHMC)) as well as 3-(4-methyl)benzylidene
camphor (4-MBC), and benzophenone-1 (BP-1). To investigate the metabolic activity
of cells incubated in the presence of tested compounds, a MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. Additionally, estrogenic
activity of compound
9 was examined in preliminary indirect model involving MCF-
7 breast cancer cell line, while UV filter benzophenone-2 (BP-2) was used as positive
control. Additionally, mutagenicity of compounds
microplate Ames assay.
8, 9, and 10 was evaluated in vitro using
2.6.1. Cytotoxicity Assessment
The cytotoxicity of compounds
9 and 16 was tested on five cell lines: human ker-
atinocytes, human skin fibroblast, human liver cancer, rat cardiomyoblast and mouse astro-
cytes. The two cell lines delivered from human skin cells: keratinocytes and fibroblasts were
chosen based on the fact that skin cells are mostly exposed to topically applied cosmetic
formulations. However, some known UV-filters (e.g., octinoxate and benzophenone-3)
were proved to cross skin barrier and penetrate to systemic blood stream after topical
application [5] so we used further cell lines: human liver cancer, rat cardiomyoblast, and
mouse astrocytes to investigate potential systemic hepatocytotoxicity, cardiocytotoxicity,
and neurocytotoxicity of the tested compounds, respectively.
The viability of two different types of skin cells grown in presence of the highest
administered concentration was presented in Figure 7a,b. The cytotoxicity was examined
after 24 h of incubation with compounds
Results of cytotoxicity study on human keratinocytes indicated that compounds
16, and EHMC in the range of tested concentration (2–50 M) were deprived of cytotoxic
activity. A viability of HaCaT cells at the highest tested concentration (50 M) was above
9
and 16 and reference UV filters (EHMC, OCT).
9
and
µ
µ
78%. In contrast, the viability of human keratinocytes incubated in the presence of reference
filters significantly decreased (less than 55%). A similar dependence was observed also in
BJ fibroblast model, where compounds 9 and 16 did not significantly reduce cell viability
(>80% of control), while incubation with EHMC and OCT resulted in 70.90% and 63.47%
viable cells, respectively.
The results obtained after incubation of HepG2 cells, mouse astrocytes and rat car-
diomyoblasts with the compounds
administered (50 M) concentration were presented in Figure 7c–e. Results of cytotoxicity
on HepG2 hepatocellular cells indicated that compounds and 16 were safe in the range of
tested concentrations—viability of cells at 50 M concentration was 82.79% and 94.26%,
respectively. Viability of HepG2 cells incubated with EHMC and OCT was 68.87% and
72.75%, respectively. Compounds and 16 were also deprived of cytotoxic activity against
9 and 16, octinoxate, and octocrylene at the highest
µ
9
µ
9
mouse astrocytes, the viability of cells was higher than 80%. However reference control
decreased cellular viability to 59%. Results obtained after incubation of rat cardiomy-
oblasts with compounds
9 and 16 at 50 µM concentration indicated cell viability at the
level of 57.02% and 71.25%, respectively, for EHMC and OCT viability was 46.25% and
42.12%, respectively.
2.6.2. In Vitro Effect on MCF-7 Cells—Preliminary Indirect Model of Estrogenic Activity
Altering of normal function of endocrine system may lead to a variety of health
problems including reproductive impairments as well as female and male cancers. Some

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currently used UV filters (e.g., benzophenones and benzylidene camphor derivatives) were
proved to influence estrogenic system [48]. Thus, we used preliminary indirect model
involving MCF-7 breast cancer cell line to investigate estrogenic activity of compound
9. In this indirect model, potential estrogenic activity may be concluded based on the
observation of increased cell proliferation after binding of the tested compound with the
estrogen receptor [48]. We also examined some commercially available UV filters (EHMC,
OCT, and BP-2). The obtained results were presented in Figure 8. Proliferation analyses
indicated lack of stimulation effect of tested compounds in MCF-7 breast cancer model.
In contrast, UV filter BP-2, stimulated proliferation by over 60%, which was statistically
significant. This preliminary evaluation indicated lack of estrogenic activity of compound 9.
Figure 7. Viability of human keratinocytes HaCaT (
a
) and human skin fibroblast (BJ) (
b
) HepG2 cells (
c), mouse astrocytes
(d
) rat cardiomyoblasts H9c2 ( ) incubated for 24 h in the presence of compounds
e
9
and 16, EHMC, and OCT at 50 µ
M. Cell
viability was determined using MTT assay. Each experiment was repeated in triplicate. Graphs represent the number of
viable cells expressed as percent of control (cells not treated with compounds) ± SD.
Figure 8. Proliferation rate (
pound , EHMC, and OCT showed lack of proliferation-stimulating effect. BP-2 increased MCF-7
proliferation by over 60% (*—data significantly different from control, p < 0.05).
±
SD) of MCF-7 cells incubated with tested compounds at 5 µM. Com-
9

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2.6.3. Mutagenic Activity
Mutagenic activity is one of the most important endpoints for risk assessment of chem-
ical compounds including drugs and candidate drugs. In the present study, mutagenicity
of test compounds
format mutagenicity assay showed that test compounds
8
and
9
was evaluated in vitro using microplate Ames assay. Microplate
and did not induce more than
8
9
a twofold induction over the baseline or a dose dependent response, demonstrating the
absence of mutagenic activity (Table 5).
Table 5. Mutagenic evaluation of test compounds in the Ames microplate format (MPF) assay.
Positive Wells per Microplate
Salmonella typhimurium
TA98
TA100
TA1535
TA1537
Compound
8
Concentration (mM)
FIB *
0.1
0.2
0.5
0.1
0.2
0.5
0.5
1.1
1.0
0.4
0.2
0.5
6.4
0.1
1.2
1.3
0.8
0.7
0.8
3.4
0.7
0.4
0.3
0.1
0.8
0.7
14.7
0.7
1.0
0.7
1.0
0.3
1.0
48.0
9
Positive control **
* FIB—fold induction over baseline (baseline = mean zero-dose control + 1 SD); SD—standard
deviation. ** Positive controls: 2-nitrofluorene (2-NF) at 2 g/mL (TA98); 4-nitroquinoline-N-oxide
(4-NQO) at 0.1 g/mL (TA1535); 9-aminoacridine
g/mL (TA100); N4-aminocytidine (N4-ACT) at 100
µ
µ
µ
(9-AAc) at 15 µg/mL (TA1537).
2.7. Structure-Activity Relationship Studies
(E)-cinnamoyl xanthone derivatives absorbed UV radiation in chloroform solutions in
the range of 290–375 nm with max 290–329, depending on the position of (E)-cinnamoyl
λ
moiety in xanthone skeleton and on the presence, type and position of auxochrome in both
xanthone and cinnamoyl moiety. The presence of methoxy group in position 6 of xanthone
ring contributed to significant improvement of the ultraviolet spectroscopic properties,
compounds
1
and
2
without 6-methoxy substituent showed worse UV absorption potential
and . It seemed more
when compared to their 6-methoxy analogues, compounds
3
4
favorable to introduce a methoxy group than a chlorine atom into the phenyl ring of the
cinnamoyl residue because it did not contribute to the increase of extinction coefficient,
but the higher values of ‹E1,1
›
was observed for compounds
5
and
The effect of the presence of two methoxy groups in the phenyl ring of the cinnamoyl
residue was very favorable—it contributed to the appearance of additional as well
7 than 3 and 4.
mean
λ
max
as extension of the UV absorption range into the UVA region and increase of ‹E1,1
›
mean
value. Moreover, an additional methoxy group in “ortho” position caused improvement of
photoprotective activity and functional photostability (compound vs. ). Introduction of
further methoxygroup in meta position did not result in improvement of UV absorption
properties (compounds 10 and 11). In compounds 13 the position of (E)-cinnamoyl
substituent in xanthone skeleton did not affect ultraviolet absorption properties. On the
other hand, in compounds 15 17 the influence of the position of (E)-cinnamoyl substituent
8
9
1–
–
was significant. Based on the obtained values of extinction coefficient and ‹E1,1
most favorable was substitution in position 3 of xanthone ring (compound 16).
›mean, the

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3. Materials and Methods
3.1. Chemistry
All reagents used in synthesis of compounds were commercially available of at least
97% purity. Solvents were commercially available materials of reagent grade. Melting
points (mp) were uncorrected and were determined using a Buchi SMP-20 apparatus (Buchi
Labortechnik, Flawil, Switzerland). The 1HNMR (300 MHz) were obtained in CDCl3 with a
Varian Mercury-VX 300 NMR spectrometer (Varian Inc., Palo Alto, CA, USA) using TMS as
an internal standard. Spectral data includes chemical shifts in ppm, multiplicities, constant
couplings in Hz, number of protons, protons’ positions. Multiplicities are abbreviated as
follow: s (singlet), d (doublet), dd (doublet of doublets), ddd (double doublet of doublets),
ddt (double doublet of triplets), t (triplet), quint (quintet), and m (multiplet). The LC/MS
system consisted of an Acquity UPLC system (Waters Corporation, Milford, MA, USA)
coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem
quadrupole). All the LC/MS analyses were carried out using an Acquity UPLC BEH C18,
1.7
µ
m, 2.1
×
100 mm2 column. A flow rate of 0.3 mL/min and a gradient of (5–95)% B over
10 min and then 100% B over 2 min was used. Eluent A: water/0.1% HCOOH; eluent B:
acetonitrile/0.1% HCOOH. LC/MS data were obtained by scanning the first quadrupole in
0.5 s in a mass range from 50 to 1000 Da; 8 scans were added to produce the final spectrum.
3.1.1. Preparation of Substrates for Syntheses
Substrates used in the synthesis of compounds 1–17 were obtained according to pre-
viously published procedures. Their physicochemical data was consistent with available
literature. The first step was the modified Ullmann’s condensation [49] of the commercially
available substrates, i.e., appropriate sodium salts of 2-chlorobenzoic acid reacted with
cresol, 2- or 4-methoxyphenol or phenol (also as sodium salts). The condensation was per-
◦
formed at the temperature of 200–210 C in paraffin oil with the catalytic amount of Cu and
Cu2O. Then by cyclization (2 to 4 h) of condensation products using concentrated sulfuric
acid obtained a corresponding 9H-xanthen-9-ones: 4-methyl-9H-xanthen-9-one [25,50]
6-chloro- or 7-chloro-2-methyl-9H-xanthen-9-one [27], 3-chloro-5-methyl-9H-xanthen-9-
one [19], 3-chloro-9H-xanthen-9-one [51]. 2- and 4-Hydroxy-9H-xanthen-9-one [28 31] were
,
,
products of demethylation in cyclization process. 3-Methoxy- [30], 6-methoxy-2-methyl-
9H-xanthen-9-one [52] and 3-methoxy-5-methyl-9H-xanthen-9-one [19] were obtained by
methanolysis of appropriate 3-chloro and 6-chloro substituted 9H-xanthen-9-ones. Then
appropriate methyl derivatives of 9H-xanthen-9-one were subject to bromination with
N-bromosuccinimide in the presence of benzoyl peroxide catalyst in tetrachloride [19,25,26].
3-Hydroxy-9H-xanthen-9-one was obtained by demethylation of 3-metoxy-9H-xanthen-9-
one using 80% H2SO4 [53]. 2-(3-Bromopropoxy)-9H-xanthen-9-one was prepared in the
two-step reaction from 2-hydroxy-9H-xanthen-9-one using 3-chloropropanol and phospho-
rus tribromide [32] (Schemes S1 and S2 Supplementary Materials).
3.1.2. Preparation of Compounds 1–13
A mixture of appropriate bromo-substituted xanthone derivative (0.01 mol), appro-
priate (E)-cinnamic acid derivative (0.011 mol), and potassium carbonate (0.007 mol) was
stirred in DMF (20 mL) for 8 to 10 h at room temperature. Then the mixture was poured
into ice and obtained solid was filtered and washed with 5% sodium bicarbonate solution
and water. After drying crude product was crystallized from toluene: heptan (2/1) with
the addition of silica gel. The yield of reaction was 64 to 76%.
3.1.3. Preparation of Compound 14
A mixture of 2-(3-bromopropoxy)-9H-xanthen-9-one (0.01 mol), trans-4-methoxycinnamic
acid (0.011 mol), and potassium carbonate (0.007 mole was stirred in DMF (20 mL) for 8 to
10 h at room temperature. Then the mixture was poured into ice and obtained solid was
filtered and washed with 5% sodium bicarbonate solution and water. After drying crude

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product was crystallized from toluene: heptan (2/1) with the addition of silica gel. The
yield of reaction was 63%.
3.1.4. Preparation of Compounds 15–17
A mixture of appropriate 2-, 3- or 4-hydroxy-9H-xanthen-9-one derivative (0.01 mol),
0.015 mole of (E)-cinnamoyl chloride (0.015 mol), and potassium carbonate (0.007 mol) was
stirred in DMF (20 mL) for 6 to 8 h at room temperature. Then the mixture was poured into
ice and obtained solid was filtered and washed with 5% sodium bicarbonate solution and
water. After drying crude product was crystallized from toluene: heptan (2/1) with the
addition of silica gel. The yield of reaction was 64 to 70%.
3.1.5. Physiochemical Properties of Tested Compounds
(9-oxo-9H-xanthen-4-yl)methyl (E)-3-(2-chlorophenyl)acrylate (
white solid (yield 64%), MW = 390.82, mp 156–157 C, H NMR (300 MHz, CDCl3)
1
) was obtained as
◦
1
δ
8.40–8.30 (m, 2H), Ar-H1, Ar-H8, 8.20 (dd, J = 16.0, 0.7 Hz, 1H, =CH-), 7.86 (ddd, J = 7.3,
1.7, 0.8 Hz, 1H, Ar-H3), 7.76 (ddd, J = 8.7, 7.1, 1.8 Hz, 1H, Ar-H6), 7.67–7.52 (m, 2H, Ar-H7,
Cyn-H6), 7.51–7.35 (m, 3H, Ar-H2, Cyn-H3, Cyn-H4), 7.35–7.20 (m, 2H, Ar-H5, Cyn-H5),
6.52 (d, J = 16.0 Hz, 1H, -CH=), 5.67 (s, 2H, -CH2-), ESI-MS (m/z): [M + H]+ calcd. for
C23H15ClO4, 391.82, found, 391.11 100%.
(9-oxo-9H-xanthen-4-yl)methyl (E)-3-(4-chlorophenyl)acrylate (
2
) was obtained as
white solid (yield 66%), MW = 390.82, mp 177–178 ◦C, 1H NMR (300 MHz, CDCl3)
δ
8.34
(dt, J = 8.0, 1.6 Hz, 2H, Cyn-H2, Cyn-H6), 7.83 (dd, J = 7.3, 1.7 Hz, 1H, Ar-H8), 7.79–7.61
(m, 2H, Ar-H1, =CH-), 7.54 (dd, J = 8.5, 1.1 Hz, 1H, Ar-H3), 7.50–7.26 (m, 6H, Ar-H7,
Ar-H6, Ar-H5, Ar-H2, Cyn-H3, Cyn-H5), 6.49 (d, J = 16.0 Hz, 1H, -CH=), 5.63 (s, 2H, -CH2-),
ESI-MS (m/z): [M + H]+ calcd. for C23H15ClO4, 391.82, found, 391.11 98.46%.
(6-methoxy-9-oxo-9H-xanthen-4-yl)methyl (E)-3-(2-chlorophenyl)acrylate (
3
) was ob-
tained as white solid (yield 69%), MW = 420.85, mp 160–162 C, 1H NMR (300 MHz, CDCl3)
8.32 (dd, J = 8.0, 1.8 Hz, 1H, Ar-H1), 8.27–8.10 (m, 2H, =CH-, Ar-H7), 7.81 (ddd, J = 7.3, 1.7,
◦
δ
0.8 Hz, 1H, Ar-H3), 7.66–7.56 (m, 1H, Ar-H8), 7.48–7.19 (m, 4H, Ar-H2, Cyn-H3, Cyn-H4,
Cyn-H5), 7.00–6.89 (m, 2H, Ar-H5, Cyn-H6), 6.52 (d, J = 16.0 Hz, 1H, -CH=), 5.64 (s, 2H,
-CH2-), 3.94 (s, 3H, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for C24H17ClO5, 421.85, found,
421.15 98.02%.
(6-methoxy-9-oxo-9H-xanthen-4-yl)methyl (E)-3-(4-chlorophenyl)acrylate (
4
) was ob-
tained as white solid (yield 66%), MW = 420.85, mp 202–203 C, 1H NMR (300 MHz, CDCl3)
8.33 (dd, J = 8.0, 1.8 Hz, 1H, Ar-H1), 8.25 (d, J = 8.8 Hz, 1H, Cyn-H5), 7.81 (dd, J = 7.4, 1.8
◦
δ
Hz, 1H, Ar-H2), 7.72 (d, J = 16.0 Hz, 1H, =CH-), 7.46 (d, J = 8.5 Hz, 2H, Cyn-H2, Cyn-H6),
7.40 (d, J = 7.6 Hz, 1H, Ar-H8), 7.36 (d, J = 3.1 Hz, 1H, Cyn-H3), 7.34 (s, 1H, Ar-H5), 6.98 (d,
J = 2.4 Hz, 1H, Ar-H7), 6.96–6.91 (m, 1H, Ar-H3), 6.50 (d, J = 16.0 Hz, 1H, -CH=), 5.63 (s,
2H, -CH2-), 3.94 (s, 3H, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for C24H17ClO5, 421.85,
found, 421.15 100%.
(6-methoxy-9-oxo-9H-xanthen-4-yl)methyl (E)-3-(2-methoxyphenyl)acrylate (
obtained as white solid (yield 68%), MW = 416.43, mp 164–165 ◦C, 1H NMR (300 MHz,
CDCl3) 8.32 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H1), 8.27–8.21 (m, 1H, Ar-H8), 8.09 (d, J = 16.2 Hz,
1H, =CH-), 7.85–7.79 (m, 1H, Cyn-H6), 7.51 (dd, J = 7.7, 1.7 Hz, 1H, Ar-H3), 7.42–7.31 (m, 2H,
Ar-H2, Cyn-H4), 6.99–6.88 (m, 4H, Ar-H7, Ar-H5, Cyn-H3, Cyn-H5), 6.62 (d, J = 16.1 Hz
5) was
δ
,
1H, -CH=), 5.62 (s, 2H, -CH2-), 3.93 (s, 3H, -OCH3), 3.87 (s, 3H, -OCH3), ESI-MS (m/z): [M
+ H]+ calcd. for C25H20O6, 417.43, found, 417.16 100%.
(6-methoxy-9-oxo-9H-xanthen-4-yl)methyl (E)-3-(3-methoxyphenyl)acrylate (
obtained as white solid (yield 67%), MW = 416.43, mp 153–154 ◦C, 1H NMR (300 MHz,
CDCl3) 8.32 (dd, J = 8.0, 1.8 Hz, 1H, Ar-H1), 8.31–8.18 (m, 1H, Ar-H8), 7.81 (ddd,
6) was
δ
J = 7.4, 1.6, 0.8 Hz, 1H, Ar-H3), 7.74 (d, J = 16.0 Hz, 1H, =CH-), 7.44–7.33 (m, 1H, Cyn-
H4), 7.33–7.22 (m, 1H, Ar-H2), 7.12 (ddt, J = 7.6, 1.5, 0.7 Hz, 1H, Cyn-H6), 7.04–6.88 (m, 4H,
Ar-H5, Ar-H7, Cyn-H2, Cyn-H5), 6.51 (d, J = 16.0 Hz, 1H, -CH=), 5.62 (s, 2H, -CH2-), 3.93

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(s, 3H, -OCH3), 3.81 (s, 3H, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for C25H20O6, 417.43,
found, 417.09 100%.
(6-methoxy-9-oxo-9H-xanthen-4-yl)methyl (E)-3-(4-methoxyphenyl)acrylate (
obtained as white solid (yield 71%), MW = 416.43, mp 167–168 ◦C, 1H NMR (300 MHz,
CDCl3) 8.33 (dd, J = 8.0, 1.7 Hz, 1H, Ar-H1), 8.25 (dd, J = 8.6, 0.7 Hz, 1H, Ar-H8), 7.82
7) was
δ
(dd, J = 7.4, 1.8 Hz, 1H, Ar-H7), 7.74 (d, J = 16.0 Hz, 1H, =CH-), 7.49 (d, J = 8.8 Hz, 2H,
Cyn-H2, Cyn-H6), 7.39 (t, J = 7.7 Hz, 1H, Ar-H2), 6.98 (d, J = 2.4 Hz, 1H, Ar-H3), 6.95 (s, 1H,
Ar-H5), 6.90 (d, J = 8.8 Hz, 2H, Cyn-H3, Cyn-H5), 6.40 (d, J = 15.9 Hz, 1H, -CH=), 5.62 (s,
2H, -CH2-), 3.94 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for
C25H20O6, 417.43, found, 417.20 100%.
(6-methoxy-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(4-methoxyphenyl)acrylate (
obtained as white solid (yield 71%), MW = 416.43, mp 169–171 ◦C, 1H NMR (300 MHz,
CDCl3) 8.38–8.33 (m, 1H, Ar-H1), 8.24 (d, J = 8.9 Hz, 1H, Ar-H8), 7.77–7.72 (m, 1H,
8) was
δ
Ar-H3), 7.69 (d, J = 15.9 Hz, 1H, =CH-), 7.51–7.43 (m, 3H, Ar-H4, Cyn-H6, Cyn-H2), 6.96 (d,
J = 2.4 Hz, 1H, Ar-H5), 6.93 (d, J = 2.4 Hz, 1H, Ar-H7), 6.91–6.83 (m, 2H, Cyn-H3, Cyn-H5),
6.40–6.32 (m, 1H, -CH=), 5.33 (s, 2H, -CH2-), 3.93 (s, 3H, -OCH3), 3.82 (s, 3H, -OCH3),
ESI-MS (m/z): [M + H]+ calcd. for C25H20O6, 417.43, found, 417.23 99.14%.
(6-methoxy-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(2,4-dimethoxyphenyl)acrylate (9)
◦
was obtained as white solid (yield 73%), MW = 446.46, mp 194–195 C, 1H NMR (300 MHz,
CDCl3) 8.40–8.34 (m, 1H, Ar-H1), 8.25 (d, J = 8.9 Hz, 1H, Cyn-H5), 7.95 (d, J = 16.1 Hz,
δ
1H, =CH-), 7.75 (dd, J = 8.6, 2.3 Hz, 1H, Ar-H3), 7.45 (t, J = 8.4 Hz, 2H, Ar-H4, Ar-H8), 6.95
(dd, J = 8.9, 2.4 Hz, 1H, Ar-H7), 6.89 (d, J = 2.4 Hz, 1H, Ar-H5), 6.56–6.45 (m, 2H, Cyn-H3,
-CH=), 6.44 (d, J = 2.4 Hz, 1H, Cyn-H6), 5.33 (s, 2H, -CH2-), 3.94 (s, 3H, -OCH3), 3.86 (s, 3H,
-OCH3), 3.83 (s, 3H, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for C26H22O7, 447.46, found,
447.20 98.69%.
(6-methoxy-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(3,4-dimethoxyphenyl)acrylate (10
)
◦
was obtained as white solid (yield 76%), MW = 446.46, mp 189–190 C, 1H NMR (300 MHz,
CDCl3) 8.41–8.34 (m, 1H, Ar-H1), 8.25 (d, J = 8.9 Hz, 1H, Ar-H8), 7.74 (dd, J = 8.6, 2.3 Hz
δ
,
1H, Ar-H3), 7.68 (d, J = 15.9 Hz, 1H, =CH-), 7.51–7.42 (m, 1H, Ar-H7), 7.15–7.02 (m, 2H,
Ar-H4, Ar-H5), 7.00–6.92 (m, 1H, Cyn-H5), 6.92–6.81 (m, 2H, Cyn-H2, Cyn-H6), 6.37 (d,
J = 15.9 Hz, 1H, -CH=), 5.34 (s, 2H, -CH2-), 3.96–3.85 (m, 9H, -OCH3, -OCH3, -OCH3).,
ESI-MS (m/z): [M + H]+ calcd. for C26H22O7, 447.46, found, 447.14 96.99%.
(6-methoxy-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(2,3,4-trimethoxyphenyl)acrylate (11
)
◦
was obtained as white solid (yield 68%), MW = 476.48, mp 188–190 C, 1H NMR (300 MHz,
CDCl3) 8.39 (dd, J = 2.3, 0.6 Hz, 1H, Ar-H1), 8.26 (d, J = 8.9 Hz, 1H, Ar-H8), 7.74 (dd,
δ
J = 8.6, 2.3 Hz, 1H, Ar-H3), 7.65 (d, J = 15.9 Hz, 1H, =CH-), 7.48 (d, J = 8.6 Hz, 1H, Ar-H7),
7.29–7.23 (m, 1H, Ar-H4), 7.01–6.93 (m, 1H, Cyn-H2), 6.90 (d, J = 2.4 Hz, 1H, Ar-H5), 6.76 (s,
1H, Cyn-H6), 6.42 (d, J = 15.9 Hz, 1H, -CH=), 5.35 (s, 2H, -CH2-), 3.94 (s, 3H, -OCH3), 3.88
(d, J = 2.0 Hz, 9H, -OCH3, -OCH3, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for C27H24O8,
477.48, found, 477.18 100%.
(6-methoxy-9-oxo-9H-xanthen-2-yl)methyl (E)-2,3-diphenylacrylate (12) was obtained
◦
as white solid (yield 72%), MW = 462.50, mp 171–172 C, 1H NMR (300 MHz, CDCl3)
δ ppm
8.30 (d, J = 2.34 Hz, 1H, Ar-H1), 8.24 (d, J = 8.79 Hz, 1H, Ar-H8), 7.89 (s, 1H, =CH-), 7.65 (dd,
J = 8.50, 2.05 Hz, 1H, Ar-H3), 7.33–7.44 (m, 4H, Ar-H4, Cyn-H3, Cyn-H5, Ph-H4), 7.11–7.27
(m, 5H, Cyn-H4, Ph-H2, Ph-H3, Ph-H2, Ph-H6), 7.03–7.08 (m, 2H, Cyn-H2, Cyn-H6), 6.94
(dd, J = 8.79, 2.34 Hz, 1H, Ar-H7), 6.87 (d, J = 2.34 Hz, 1H, Ar-H5), 5.35 (s, 2H, -CH2-), 3.92
(s, 3H, -OCH3), ESI-MS (m/z): [M + H]+ calcd. for C30H22O5, 463.50, found, 463.22 98.64%.
(7-chloro-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(4-methoxyphenyl)acrylate (13) was
obtained as white solid (yield 68%), MW = 420.85, mp 160–161 ◦C, 1H NMR (300 MHz,
CDCl3) δ 8.38–8.26 (m, 2H, Ar-H8, Ar-H1), 7.80 (dd, J = 8.7, 2.3 Hz, 1H, Ar-H3), 7.76–7.62
(m, 2H, Ar-H5, =CH-), 7.58–7.42 (m, 4H, Ar-H4, Ar-H6, Cyn-H2, Cyn-H6), 6.96–6.85 (m, 2H,
Cyn-H3, Cyn-H5), 6.37 (d, J = 15.9 Hz, 1H, -CH=), 5.34 (s, 2H, -CH2-), 3.84 (s, 3H, -OCH3),
ESI-MS (m/z): [M + H]+ calcd. for C24H17ClO5, 421.85, found, 421.08 98.32%.

Int. J. Mol. Sci. 2021, 22, 34
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3-((9-oxo-9H-xanthen-2-yl)oxy)propyl (E)-3-(4-methoxyphenyl)acrylate (14) was ob-
◦
1
tained as white solid (yield 63%), MW = 430.46, mp 139–140 C, H NMR (300 MHz,
CDCl3) 8.39–8.29 (m, 1H, Ar-H8), 7.77–7.68 (m, 2H, Ar-H1, Ar-H7), 7.65 (d, J = 16.0 Hz,
δ
1H, =CH-), 7.53–7.41 (m, 4H, Ar-H3, Ar-H5, Cyn-H2, Cyn-H6), 7.41–7.29 (m, 2H, Ar-H4,
Ar-H6), 6.94–6.83 (m, 2H, Cyn-H3, Cyn-H5), 6.39–6.26 (m, 1H, -CH=), 4.42 (t, J = 6.2 Hz,
2H, -CH2-O), 4.23 (t, J = 6.1 Hz, 2H, O-CH2-), 3.82 (s, 3H, -OCH3), 2.24 (quint, J = 6.2 Hz,
2H, -CH2-), ESI-MS (m/z): [M + H]+ calcd. for C26H22O6, 431.46, found, 431.25 100%.
9-oxo-9H-xanthen-2-yl cinnamate (15) was obtained as white solid (yield 70%), MW = 342.35
,
mp 162–164 ◦C, 1H NMR (300 MHz, CDCl3)
δ
ppm 8.35 (dd, J = 7.91, 1.47 Hz, 1H, Ar-H8),
8.09–8.13 (m, 1H, Ar-H1), 7.92 (d, J = 15.82 Hz, 1H, =CH-), 7.71–7.79 (m, 1H, Ar-H6), 7.62
(dd, J = 6.45, 2.93 Hz, 2H, Cyn-H2, Cyn-H6), 7.55–7.58 (m, 2H, Ar-H4, Ar-H5), 7.52 (d,
J = 7.62 Hz, 1H, Ar-H3), 7.42–7.47 (m, 3H, Ar-H7, Cyn-H3, Cyn-H5), 7.37–7.42 (m, 1H,
Cyn-H4), 6.67 (d, J = 15.82 Hz, 1H, -CH=), ESI-MS (m/z): [M + H]+ calcd. for C22H14O4,
343.35, found, 343.12 100%.
9-oxo-9H-xanthen-3-yl cinnamate (16) was obtained as white solid (yield 64%), MW = 342.35
,
mp 173–174 ◦C, 1H NMR (300 MHz, CDCl3)
δ
ppm 8.39 (d, J = 8.79 Hz, 1H, Ar-H1), 8.35
(dd, J = 7.91, 1.47 Hz, 1H, Ar-H8), 7.93 (d, J = 16.41 Hz, 1H, =CH-), 7.58–7.77 (m, 3H, Ar-H6,
Cyn-H2, Cyn-H6), 7.36–7.53 (m, 6H, Ar-H4, Ar-H7, Ar-H5, Cyn-H3, Cyn-H4, Cyn-H5), 7.22
(dd, J = 8.79, 2.34 Hz, 1H, Ar-H2), 6.66 (d, J = 15.82 Hz, 1H, -CH=), ESI-MS (m/z): [M + H]+
calcd. for C22H14O4, 343.35, found, 343.12 100%.
9-oxo-9H-xanthen-4-yl cinnamate (17) was obtained as white solid (yield 67%), MW = 342.35
,
mp 192–193 ◦C, 1H NMR (300 MHz, CDCl3) δ 8.40–8.29 (m, 1H, Ar-H8), 8.29–8.21 (m, 1H,
Ar-H1), 8.00 (d, J = 16.0 Hz, 1H, =CH-), 7.76–7.54 (m, 4H, Ar-H6, Ar-H3, Cyn-H3, Cyn-H5),
7.52–7.33 (m, 6H, Ar-H7, Ar-H5, Ar-H2, Cyn-H2, Cyn-H6, Cyn-H4), 6.79 (d, J = 16.0 Hz,
1H, -CH=), ESI-MS (m/z): [M + H]+ calcd. for C22H14O4, 343.35, found, 343.12 100%.
3.2. Evaluation of Lipophilicity Parameter RM0
Reversed-phase thin-layer chromatography (RP-TLC) was performed on aluminum
sheets 20
×
20 cm2 coated with silica gel RP-18 F254 (Merck, Darmstadt, Germany). The
mobile phases constituted mixtures of methanol and 100 mM potassium-phosphate buffer
(pH = 7.4), with the methanol content between 95 and 65% (v/v). The tested compounds
were dissolved separately in chloroform (1 mg/mL) and 5 µL of solutions were transferred
on the sheets. Chromatographic chambers were saturated with mobile phase for 1 h at room
temperature before developing the sheets. The spots were observed in UV light. For each
concentration of methanol Rf values were calculated and mean values are reported from two
independent experiments (Table S1 Supplementary material). RM values were calculated
from the experimental Rf using equation: RM = log(1/Rf
material). The calculated values of RM were extrapolated for 0% methanol concentration
(Table 1) [33,34].
−
1) (Table S2 Supplementary
3.3. Ultraviolet Spectroscopy
Spectra with a scan range of 290–400 nm were recorded in 30 µM chloroform solutions,
in 1 cm path length, 1.5 mL quartz cuvettes on a U-2800 double beam spectrophotometer
(Hitachi, Tokyo, Japan) controlled by UV Solution version 2.2 software. The molar extinc-
tion coefficient at maximum absorption (εmax) of tested compounds was determined in
chloroform as the slope of the linear regression of absorbance vs. concentration of tested
compound (from 2.5 to 30 µM). E(1,1) coefficient was calculated from the formula:
ꢂ ꢃ
ꢀ
ꢁ
g
10
L
L
ꢂ
ꢃ
E1,1 = ε
×
× 1[cm]
g
mol
mol cm
M
‹E1,1
›
value is a mean value of the specific extinction over the spectral range from
mean
290 to 400 nm.

Int. J. Mol. Sci. 2021, 22, 34
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3.4. Photoprotective Activity
3.4.1. Preparation of Simple Cosmetic Formulations
To assess the activity of compounds
8
and
9
as UV filters, they were incorporated
into a macrogol cream. This cream was made of PEG 400 and PEG 1500 mixed at 70 ◦C in
1:1 (w/w) ratio. Then, two concentrates, containing 50% (w/w) of the tested compounds
8
or 9 were prepared with the aim to combine them with this macrogol cream. First, the
tested compounds were mixed with triacetin in 1:1 (w/w) ratio. Each mixture was wet
milled using a high-energy planetary ball mill (Pulverisette 7 classic line, Fritsch, Germany).
The total milling time was 12 h, but to avoid the overheating of the sample, the milling
procedure was performed in cycles, i.e., the milling periods of 20 min were followed by the
pause periods of 10 min. The rotation speed of the solar disc was kept at 400 rpm. After
such processing, the concentrates in the form of a white paste were obtained. Then, each of
◦
these concentrates was mixed with the macrogol cream at 70 C in three wt. ratios, namely
1:3, 1:5.25 or 1:9.
3.4.2. In Vitro Photoprotection Study
In vitro photoprotective activity of compounds
8 and 9 was evaluated according to
EN ISO 24443:2012 [38] with slight modifications. 32.5 mg of formulation with tested
compound was spread onto roughened polymethylmethacrylate plate (PMMA) purchased
from Schonberg GmbH (Hamburg, Germany) with the application area 25 cm2 and 5
µm
roughness value to simulate the skin surface. After 30 min in the dark in vitro absorbance
of samples was measured from 290 to 400 nm with 1 nm steps by reflectance spectropho-
tometry. SPF-290AS Analyser (Solar Light Company, Glenside, PA, USA) equipped with
integrating sphere coupled with a WinSPF version 4.4 software was used. For each sample
two plates were prepared, absorbance measures were performed on 6 different positions of
the plate. The results were expressed as an average of data from 12 scans.
3.5. Photostability Study
To evaluate functional photostability of compounds
8 and 9 PMMA plates with tested
compounds was irradiated with solar light simulator (Suntest CPS+, Atlas, Linsengericht,
Germany) equipped with an optical filter cutting off wavelengths shorter than 290 nm and
IR-block filter to neutralize thermal effects. The light source emission was maintained at
500 W/m2 and irradiation procedure was conducted for 1 h which corresponds to cumula-
tive dose of ultraviolet radiation 218 kJ/m2 and is consistent with previous studies [10
,54].
The UV absorption spectrum of the samples and photoprotective activity parameters were
analysed post-irradiation and compared with pre-irradiation results. All analyses were
performed in duplicates.
3.6. In Vitro Viability Assessment
Skin fibroblast BJ (ATCC CRL 2522), keratinocytes cell line HaCaT (Sigma Aldrich)
human liver cancer HepG2 cell line (ATCC HB8065), rat cardiomyoblast H9c2 (ATCC
CRL1446), and mouse astrocytes (ATCC, CRL2541) were cultured in standard conditions
◦
(37 C, 5% CO2, 95% humidity) in appropriate culture medium (according to manufacturer
procedure), supplemented with 10% fetal bovine serum (FBS, Gibco) and antibiotics (1%
streptomycin/penicillin mixture; Sigma-Aldrich). Cells were seeded at density of 2 × 104
(BJ, HaCaT) on 96-well plates. After 24 h cells were treated with increasing doses of
compounds and incubated for additional 24 h. Following the incubation, MTT reagent
(Sigma-Aldrich, Darmstadt, Germany) was added to each well and after 4 h incubation, the
medium was aspirated, and the formazan produced in the cells appeared as dark crystals
in the bottom of the wells. Next, DMSO (dissolving solution) was added to each well. Then,
the absorbance of solution was determined at 570 nm (A570) on plate reader (Spectra iD3
Max, Molecular Devices; San Jose, CA, USA). Viability (% of control) was determined by
dividing A570 of experimental wells by of A570 of control wells
cytotoxicity tests were subjected to one-way analysis of variance, followed by Dunnett’s
×
100%. Data from the

Int. J. Mol. Sci. 2021, 22, 34
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test using GraphPad Prism 7.0 Software (GraphPad Software Inc., San Diego, CA, USA).
Values of p < 0.05 were considered to be statistically significant.
3.7. Estrogenic Activity
Estrogenic activity of tested compounds was investigated by study of their influence
on estrogen-dependent MCF-7 breast cancer cell line proliferation. Cells were seeded
into 24-wells plates in density of 40,000 per well. After 24 h, cells were washed twice
with PBS and fresh, estrogen- and phenol red-free medium (DMEM low-glucose; 2.5%
Charcoal Stripped FBS; 2 mM glutamine; Sigma Aldrich, Darmstadt, Germany) was added.
Then cells were incubated with tested compounds and benzophenone-2 (BP-2; Sigma
Aldrich, Darmstadt, Germany) to final concentration of 5 µM. After 120 h incubations,
cells proliferation was determined with crystal violet assay. Briefly, cells were washed
with PBS and fixed with 3.7% formaldehyde. Then, crystal violet solution was added for
10 min. After incubation, solution was removed, and cells were washed five times with
PBS. Crystal violet was extracted from cells using destaining solution (1.33% citric acid,
1.09% sodium citrate in water/methanol (1:1) solution) and absorbance of the solution was
determined at 570 nm (A570). Proliferation rate was determined by dividing the A570 of
experimental wells by the A570 of control wells × 100%.
3.8. Mutagenicity Assay Procedure
Test compounds mutagenic effects were evaluated using the Ames microplate format
(MPF) mutagenicity assay with Salmonella typhimurium TA98, TA100, TA1535 and TA1537
strains (Xenometrix, Allschwil, Switzerland). These strains meet the requirements of
the Organization for Economic Cooperation and Development (OECD) guideline 471 for
testing of chemicals [55]. Culture media, positive control chemicals, and tester strains were
provided in the kit. TA100 and TA1535 strains are used to detect mutagens that cause
base-pair substitutions, while TA98 and TA1537 detect frameshift mutations.
MPF assay is a modification of the traditional Salmonella test [56,57]. During the
assay, bacteria are exposed to a test compound in a medium containing histidine to support
cell divisions. Then, the cultures are diluted using indicator medium which lacks the
required amino acid and selects for prototrophic reversion, and aliquoted into 48 wells of
a 384-well plate. Within two days, bacteria that have undergone reversion to amino acid
prototrophy will form colonies. The indicator medium turns yellow as the pH drops as
a result of catabolic activity of revertant bacteria that grow in the absence of the required
amino acid [58].
In the present study bacterial cultures were grown overnight (16 h) in growth medium
and then exposed to test compounds (8 and 9
) in 24-well plates for 90 min at 37 ◦C with
constant shaking. Compounds were tested at final concentrations of 0.1, 0.2, and 0.5 mM.
After preincubation, the cultures were diluted in the indicator medium and the contents of
each 24-well culture were transferred to 48 wells on a 384-well plate. Upon completion of 48
h incubation period, yellow revertant wells were counted for each dose and compared with
the number of spontaneous revertants obtained with the negative control. The standard
positive controls for the Ames MPF assay were 2-nitrofluorene (2-NF) at 2
4-nitroquinoline-N-oxide (4-NQO) at 0.1 g/mL (TA100); N4-aminocytidine (N4-ACT) at
100 g/mL (TA1535) and 9-aminoacridine (9-AAc) at 15 g/mL (TA1537), whereas pure
µg/mL (TA98);
µ
µ
µ
DMSO was used as a negative control. Each dose was done in triplicate [58–61].
Two criteria were applied to evaluate mutagenicity assay results such as the fold
increase in the number of positive wells over the solvent control baseline (FIB), and the
dose dependent response. FIB was calculated by dividing the mean number of positive
wells at each dose by the solvent control at baseline (mean of negative control plus 1 SD).
When an induction of more than twofold relative to the baseline at more than one dose
with a dose-response was observed the sample was considered positive, whereas when no
response >2 times the baseline and no dose-response was stated the sample was classified
as negative in the assay [57–59].

Int. J. Mol. Sci. 2021, 22, 34
20 of 22
4. Conclusions
We designed and synthetized 17 novel structures containing two chromophores:
xanthone and (E)-cinnamoyl. Among the presented group of compounds, there were
potential UVA and UVB filters, their λmax was in the range from 290 to 329 nm, additionally
compounds were characterized by high extinction coefficient and favorable lipophilic-
ity predisposing to cumulate in stratum corneum. The most promising compound in
the tested series was compound
dimethoxyphenyl)acrylate). It showed two peaks of
II region. After milling, it was loaded into a simple cream base at the concentration of
12.5% and showed very beneficial photoprotective properties (SPFin vitro 19.69 0.46, UVA
PF 12.64 0.32), which were comparable to tris-biphenyltiazine. Moreover, high value
of critical wavelength (381 nm) indicated that compound was a good candidate for a
broad-spectrum UV filter that may protect skin against UVA-induced photoaging. Com-
pound in high concentration (50 M) was deprived of cytotoxic activity toward human
keratinocytes, human skin fibroblasts, mouse astrocytes and hepatoma cells. Compound
showed no estrogenic activity in MCF-7 breast cancer model. It also showed no mutagenic
activity, the performed Ames test indicated that compound was not base substitution or
9
((6-methoxy-9-oxo-9H-xanthen-2-yl)methyl (E)-3-(2,4-
λmax, one in UVB and one in UVA
±
±
9
9
µ
9
9
frameshift mutagen. According to the results of performed research it can be concluded,
that in the group of xanthone derivatives new UV filters may be identified.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-00
67/22/1/34/s1.
Author Contributions: Conceptualization, E.P. and H.M.; methodology, J.P., A.G.-K., K.S., P.K.-A.,
˙
K.P., K.W.-P., D.Z., A.K., H.M., and E.P.; validation, E.P. and H.M.; formal analysis, E.P. and A.G.-K.;
˙
˙
investigation, J.P., A.G.-K., K.S., P.K.-A., K.P., K.W.-P., D.Z., A.K., and P.Z.; writing—original draft
˙
preparation, J.P., A.G.-K., K.S., P.K.-A., K.P., K.W.-P., D.Z., and A.K.; writing—review and editing, J.P.
and A.G.-K.; supervision, E.P. and H.M.; project administration, E.P.; funding acquisition, A.G.-K.,
K.S., and E.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Polish National Science Centre, grant number 2016/21/B/
NZ7/01756.
Data Availability Statement: Data is contained within the article and supplementary material.
Conflicts of Interest: The authors declare no conflict of interest.
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