Discovery of novel UV-filters with favorable safety profiles in the 5-arylideneimidazolidine-2,4-dione derivatives group

molecules

Article

Discovery of Novel UV-Filters with Favorable Safety

Proﬁles in the 5-Arylideneimidazolidine-2,4-dione

Derivatives Group

1

2

Justyna Popiół ^1,2, Agnieszka Gunia-Krzyz˙ak ^2,*, Kamil Piska , Dorota Zelaszczyk

,

˙

Paulina Koczurkiewicz ¹, Karolina Słoczyn´ska ¹, Katarzyna Wójcik-Pszczoła ¹, Anna Krupa ³

,

5

6

7

Agata Kryczyk-Poprawa ⁴, Ewa Zesławska

˙

, Wojciech Nitek , Paweł Zmudzki ,

˙

Henryk Marona ²and Elz˙bieta Pe˛kala ¹

1

Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College,

Medyczna 9, 30-688 Krakow, Poland; justyna.popiol@uj.edu.pl (J.P.); kamil.piska@uj.edu.pl (K.P.);

paulina.koczurkiewicz@uj.edu.pl (P.K.); karolina.sloczynska@uj.edu.pl (K.S.);

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 Krakow, Poland; dorota.zelaszczyk@uj.edu.pl (D.Z.);

henryk.marona@uj.edu.pl (H.M.)

2

3

4

5

6

7

Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian

University Medical College, Medyczna 9, 30-688 Krakow, Poland; a.krupa@uj.edu.pl

Department of Inorganic and Analytical Chemistry, Faculty of Pharmacy, Jagiellonian University Medical

College, Medyczna 9, 30-688 Krakow, Poland; agata.kryczyk@uj.edu.pl

Department of Chemistry, Institute of Biology, Pedagogical University of Cracow, Podchora˛z˙ych 2,

30-084 Krakow, Poland; ewa.zeslawska@up.krakow.pl

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland;

wojciech.nitek@uj.edu.pl

Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College,

Medyczna 9, 30-688 Krakow, Poland; pawel.zmudzki@uj.edu.pl

*

Correspondence: agnieszka.gunia@uj.edu.pl; Tel.: +48-12-6205576

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 22 May 2019; Accepted: 19 June 2019; Published: 24 June 2019

Abstract: Eﬀective protection from the harmful eﬀects of UV radiation may be achieved by

using sunscreens containing organic or inorganic UV ﬁlters. The number of currently available

UV ﬁlters is limited and some of the allowed molecules possess limitations such as systemic

absorption, endocrine disruption properties, contact and photocontact allergy induction, and

low photostability. In the search for new organic UV ﬁlters we designed and synthesized a

series consisting of 5-benzylidene and 5-(3-phenylprop-2-en-1-ylidene)imidazolidine-2,4-dione

(hydantoin) derivatives. The photoprotective activity of the tested compounds was conﬁrmed

in methanol solutions and macrogol formulations. The most promising compounds possessed

similar UV protection parameter values as selected commercially available UV ﬁlters. The compound

diethyl 2,2⁰-((Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4g

was characterized as an especially eﬃcient UVA photoprotective agent with a UVA PF of

6.83

0.05 and favorable photostability. Diethyl 2,2⁰-((Z)-4-(4-methoxybenzylidene)-2,5-dioxo-

imidazolidine-1,3-diyl)diacetate (3b) was the most promising UVB-ﬁlter, with a SPF_{in vitro}of 3.07 0.04

)

±

and very good solubility and photostability. The main photodegradation products were geometric

isomers of the parent compounds. These compounds were also shown to be non-cytotoxic at

concentrations up to 50 µM when tested on three types of human skin cells and possess no estrogenic

activity, according to the results of a MCF-7 breast cancer model.

Keywords: UV-filters; UV radiation; 5-arylidenehydantoin derivatives; photoprotection; safety evaluation

Molecules 2019, 24, 2321; doi:10.3390 /molecules24122321

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1. Introduction

Sunscreens are necessary to protect the skin against the acute and chronic consequences of

ultraviolet radiation (UVR). The main active ingredients of sunscreens called UV-ﬁlters are inorganic

blockers which reﬂect or scatter UVR or organic absorbers. Organic UV-ﬁlters act as chemical energy

converters—after absorption of photons the molecule is excited to a higher energy state, then the

absorbed energy is dissipated by the emission of photons or heat and molecule returns to the ground

state [1,2].

In the European Union sunscreen products are classiﬁed as cosmetics, but in the USA, Australia

and Canada they are recognized as over-the-counter (OTC) drugs, thus requirements for their safety

and eﬃcacy are comparable with those of other dermatological drugs [3,4]. Modern organic UV-ﬁlter

molecules are expected to be not only UV-absorbers with high extinction coeﬃcients, but they must

also be photostable, safe and readily soluble in cosmetics solvents. It is estimated that process

of commercialization of a new UV-ﬁlter takes about 10 years [5,6]. Typical exposure to UV-ﬁlters

is very high because of their concentration in sunscreens which reaches 10% for some organic

UV-ﬁlters. Moreover, the product is applied on the entire surface of the skin. Some UV-ﬁlters or

their metabolites were detected in the blood [7], urine [8,9] and milk of nursing mothers [10], what

indicates that these compounds penetrate into the bloodstream. More concerns appeared after several

adverse eﬀects of currently used UV-ﬁlters were recognized. Many of them may cause endocrine

disruption [11–13] and adversely aﬀect the viability of nerve cells, which may contribute to the

development of neurodegenerative diseases [14]. Additionally, benzophenone-3 showed cytogenetic

eﬀect on human lymphocytes increasing micronuclei and chromosomal aberrations frequencies [15].

The other limitations of currently available UV-ﬁlters are related to contact and photocontact allergy

induction [16,17] and unsatisfactory photostability [18,19]. One of the most popular UVB-ﬁlter,

octinoxate (INCI: ethylhexyl methoxycinnamate, EHMC), upon ultraviolet irradiation undergoes

a photoisomerisation process, and the resulting Z-isomer has a lower extinction coeﬃcient, which

results in a signiﬁcant reduction in its photoprotectiveness [20]. Moreover, it was shown that the

photolysis products of EHMC are more toxic to the mammalian cells than EHMC alone. Low

photostablity also characterises an UVA-ﬁlter—avobenzone (INCI: butyl methoxy-dibenzoylmethane).

After irradiation its ability to absorb UVA is signiﬁcantly reduced, additionally, it was shown that

arylglyoxals, the main products of avobenzone photodegradation, are strong sensitizers [21]. Due to

their unsatisfactory safety proﬁle according to the European Commission regulations, in the latest

years two UV-ﬁlters—4-aminobenzoic acid (PABA) and 3-benzylidene camphor—have been banned

from use in cosmetics [22,23].

Considering the disadvantages of currently available UV-ﬁlters,

for new UV-ﬁlters remains an important issue. The list of UV ﬁlters allowed

in cosmetic products within the European Union is still expanding. In 2014

the search

2,4,6-tris([1,1’-biphenyl]-4-yl)-1,3,5-triazine (INCI: trisbiphenyl triazine) was authorized [24],

whereas in 2018 2,2’-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (INCI:

methylene bisbenzotriazolyltetramethylbutylphenol nano) was added to the list [25]. Moreover, several

scientiﬁc papers on this topic were published recently. Researchers have focused on modiﬁcations

of currently used compounds which represent derivatives of benzophenone, dibenzoylomethane,

benzotriazole, phenylbenzimidazole and cinnamic acid [26–30].

Derivatives of 5-arylideneimidazolidine-2,4-dione, depending of the presence and location of the

substituents on the phenyl or imidazolidine rings exhibit diverse biological activity. Their ability to

modulate cancer eﬄux pumps and the melanogenesis process, antimicrobial, and antiproliferative

activity, as well as

α

1-adrenoceptor antagonistic properties were reported [31–35]. Their ultraviolet

absorbing properties were also described [36

,

37], which prompted us to synthesize and evaluate the

photoprotective activity of a new series of 5-arylideneimidazolidine-2,4-dione (5-arylidenehydantoin)

derivatives. For further characterization of the most promising compounds we also performed tests

on their photostability and safety. We designed the compounds on the basis of the structure of

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3-benzylidenecamphor, in which the camphor fragment was replaced with a hydantoin moiety. We

anticipated that this structural change would positively aﬀect the safety proﬁle of the molecules

(Figure 1). We also planned several further modiﬁcations such as introduction of substituents in the

phenyl ring and/or hydantoin fragment as well as extension of the linker by adding an allyl group in

order to modify the physicochemical and photoprotective properties of the tested compounds.

Figure 1. Design concept of the tested series: camphor was exchanged with hydantoin, while the

benzylidene moiety remained unchanged or was extended.

2. Results and Discussion

2.1. Chemistry

Synthesis and chemical structures of the tested compounds are shown in Scheme 1. These

compounds were obtained from appropriately substituted benzaldehyde (compounds 1a–1e, 2a–2e,

3a–3e, 5a) or cinnamaldehyde (compounds 4f–4h) and hydantoin.

Scheme 1. Synthesis and structures of tested compounds. Reagents and Conditions: (i) Knoevenagel

condensation CH₃COONa/CH₃COOH; (ii) ClCH₂COOEt, K₂CO₃, TEBA, acetone; (iii) EtOH/H₂O,

NaOH; (iv) BrCH₂COOEt, K₂CO₃, DMF.

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In the ﬁrst step, appropriate 5-arylideneimidazolidine-2,4-diones (5-arylidenehydantoins,

compounds 1a 1h) were obtained by Knoevenagel condensations. Physicochemical analyses were

performed to conﬁrm their structures and purity. Compounds 1a 1h served for further syntheses, while

compound 1e was also tested for its photoprotective properties and safety. Compounds 1a 1h were

used in reactions with ethyl chloroacetate or ethyl bromoacatate to synthesize substituted on nitrogen(s)

mono- (compounds 2a 2e) or diesters (compounds 3a 3e and 4f 4h), respectively. Additionally,

–

compound 2a was subjected to hydrolysis in order to obtain the corresponding acid (compound

5a). Chemical structures and purity of ﬁnal compounds were conﬁrmed by spectral analysis (NMR,

LC/MS/MS).

2.2. X-ray Crystallographic Studies

The molecular geometries in the crystal structures of 3b and 4g, together with the atom numbering

schemes, are shown in Figure 2.

(a)

(b)

Figure 2. The molecular geometries of (a) 3b and (b) 4g, with the atom numbering schemes.

Displacement ellipsoids are drawn at the 30% probability level.

Both molecules contain a double bond between the C5 and C14 atoms. The crystal

structures conﬁrm the Z arrangement at this bond. The molecule of 4g possesses an additional

double bond between the C15 and C16 atoms, and at this bond the E arrangement is observed.

The angles between the planes of the hydantoin and aromatic rings are 72.68(4)^◦and 9.21(7)^◦

for 3b and 4g, respectively, which implies that the 5-benzylidenehydantoin fragment of 3b is

not planar, while the 5-cinnamylidenehydantoin fragment of 4g is almost planar. We have

previously determined crystal structures of 5-benzylidene-2-selenohydantoin derivatives, wherein the

5-benzylidene-2-selenohydantoin fragment is almost planar [38,39]. Considering that, we have carried

out a search in the Cambridge Structural Database (CSD) [40] for 5-benzylidenehydantoin (20 structures

found), 5-benzylidene-2-thiohydantoin (10 structures found) and 5-benzylidene-2-selenohydantoin

(two structures found) fragments. Most of the crystal structures deposited in the CSD possess

an almost planar benzylidene(thio/seleno)hydantoin moiety (Figure S1). The non-planarity of the

benzylidenehydantoin fragment is very seldom observed in crystal structures, while for thio- and

seleno-hydantoins it is not observed at all.

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The CSD includes no crystal structures containing cynnamylidenehydantoin moiety. We have

determined earlier the crystal structures containing similar fragment, namely cinnamylidene rhodanine

moiety [41], this fragment was also almost planar like in 4g.

Both investigated molecules possess the same 2-ethoxy-2-oxoethyl substituents at the nitrogen

atoms (N1 and N3). There are some diﬀerences in the mutual orientation of these substituents in

both molecules (Figure S2). The substituents at the N3 atom diﬀer mainly in conformation of the

ethyl group. The C11-O6-C12-C13 torsion angle has values of

88.0(2)^◦in 4g. Signiﬁcant diﬀerences are visible in the conformation of the substituent at the N1 atom.

The torsion angle C5-N1-C6-C7 has values of

65.5(1)^◦and 67.8(2)^◦for 3b and 4g, respectively, what

−

101.5(5)^◦(for occupancy A) in 3b and

−

implies opposite orientation of these substituents.

The crystal packing is determined by C-H···O intermolecular interactions (Table S1). In the 3b

two molecules, related by the inversion center, create dimers by C8-H8A···O4, C14-H14···O1 and

C16-H16···O1 intermolecular interactions (Figure 3a), while in 4g the are also observed dimers, but

created by other contacts, namely C14-H14···O4 and C16-H16···O4 (Figure 3b).

(a)

(b)

Figure 3. The intermolecular interactions of two molecules of (a) 3b and (b) 4g. Dashed lines indicates

hydrogen bonds. The disordered fragment of 3b is depicted only for major occupancy.

2.3. Ultraviolet Spectroscopic Properties

Among tested compounds there are both UVA and UVB absorbers (

ultraviolet absorbing properties are presented in Table 1 and Figure S3. Compounds 2a

to the group of ethyl esters of (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetic acid, absorb UV

radiation with diﬀerent range, _maxand _maxdepending on the presence and position of the auxochrome.

λ_maxfrom 295 to 379 nm). Their

–2e, which belong

λ

ε

They show higher molar absorption coeﬃcients and wider absorption ranges than commercially

used UV ﬁlters such as 4-methylbenzylidene camphor (4-MCB), ethylhexyl methoxycinnamate

or octocrylene. The presence of an additional N-alkyl substituent in the imidazolidine ring in

compounds 3b

hypochromic eﬀect (

–

3e contributes to a hypsochromic shift by 15 nm (in compound 3a by 21 nm) as well as a

_maxdecreased almost two-fold) when compared to N-monosubstituted derivatives

ε

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(compounds 2a

of 5-benzylideneimidazolidine-2,4-dione are slightly weaker UV absorbers than N-monosubstituted

derivatives, but their still surpass octocrylene. The of compounds 2a and 3a without

–2e) which is consistent with former ﬁndings [42]. Thus N,N-bis-substituted derivatives

ε

λ

max

substituents on the phenyl ring is located in the UVB region (316 and 295 nm, respectively), while the

of other 5-benzylideneimidazolidine-2,4-dione derivatives with methoxy groups is shifted to the

λ

max

UVA II or UVA I region.

Table 1. Ultraviolet spectroscopic properties of the tested 5-arylideneimidazolidine-2,4-dione

derivatives and reference UV ﬁlters obtained in methanol solutions.

Compound

1e

Chemical Structure

λ

_max[nm]

ε

max

[M⁻¹cm⁻¹

]

Absorption Range [nm]

341

28440

290–384

2a

2b

2c

316

334

336

28460

31920

35610

290–345

290–372

290–376

2d

2e

349

344

30700

30740

290–394

290–381

3a

3b

3c

295

318

321

13980

16310

17010

290–335

290–362

290–367

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Table 1. Cont.

Compound

3d

Chemical Structure

λ

_max[nm]

ε

max

[M⁻¹cm⁻¹

]

Absorption Range [nm]

333

17310

290–374

3e

4f

329

354

379

14200

41477

54996

290–380

295–389

290–400

O

N

O

4g

4h

370

34987

290–400

5a

316

299

27310

25550

290–346

290–332

4-MBC

Octocrylene

302

13650

290–349

EHMC

309

358

22990

36820

290–339

290–393

Avobenzone

The tested (Z)-5-((E)-3-phenylallylidene)imidazolidine-2,4-dione derivatives 4f

–4h show the

highest molar absorption coeﬃcients among the tested series (especially compounds 4f and 4g).

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Elongation of the unsaturated spacer caused a signiﬁcant bathochromic shift in comparison to the

corresponding benzylidene analogues. Their UV absorption ranges and

of avobenzone, which is one of the most commonly used UV ﬁlters.

λ

max

are very similar to those

2.4. Photoprotective Activity

Absorption spectra and thus the photoprotective properties of UV-ﬁlters depend on the solvent

used [43], hence to investigate the functional absorbing eﬃcacy of the tested compounds and reference

UV-ﬁlters (octocrylene, EHMC, 4-MBC, and avobenzone) they were tested in macrogol formulations

(F1–F3) at 2% (w/w) concentration.

The composition of formulations and concentration of tested compounds were based on their

limited solubility. N-monosubstituted compounds 2a–2e were insoluble at desirable concentrations

in the typical solvents used in cosmetic formulations such as alcohols, triacetin, surfactants, and

co-surfactants. To dissolve compounds 2a and 2b before incorporation to the formulation DMSO: Tween

20 (1:9 w/w) was used, for less soluble 2c–2e it was necessary to use DMSO alone as solvent. Incorporation

of an additional 2-ethoxy-2-oxoethyl substituent in the imidazolidine ring of N-mono-substituted

5-arylidene-2,4-imidazolidinediones signiﬁcantly improved the solubility of the compounds so it

was possible to eliminate DMSO from the formulation and use the solvent system triacetin:Tween 20

(1:9 w/w) to dissolve compounds 3a–3e and 4f–4h before incorporation into the formulation. Thus the

detailed composition of the formulations is as follows: F1–Tween 20 43.2%, PEG-400 25%, PEG-1500

25%, Triacetin 4.8%, tested compound 2%; F2–Tween 20 43.2%, PEG-400 25%, PEG-1500 25%, DMSO

4.8%, tested compound 2%; F3–PEG-400 25%, PEG-1500 25%, DMSO 48%, tested compound 2%.

Considering the disadvantages of currently reported macrogol formulations and the low concentration

achieved for the tested compounds (2%), we plan to work on cosmetic emulsions containing the most

promising derivatives at higher concentrations as well as required excipients including preservatives,

antioxidants, etc. This research will be carried out in the near future.

The results of studies on the photoprotective activity of the tested compounds are

presented in Table 2 and Figure 4.

compounds can be considered as potential UV-ﬁlters.

(Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetic acid (compounds 2a

the highest value of SPF_{in vitro}, which is comparable to EHMC SPF_{in vitro}. This is related to the fact

that the of 2a is located in the UVB region. On the other hand, the of compounds 2b 2e

2e) region due to presence of electron donating groups

in the phenyl ring, thus these compounds besides higher SPF_{in vitro}than octocrylene, show higher

The performed study indicate that all tested

In the group of ethyl esters of

2e), compound 2a shows

–

λ

max

λ

max

–

is shifted to UVA II (2b, 2c) or UVA I (2d,

eﬃciency in UVA absorption (UVA PF from 4.29

excluding avobenzone.

±

0.11 to 4.77

±

0.24) than 2a and reference UV-ﬁlters

N,N-bis-substituted 5-benzylideneimidazolidine-2,4-dione derivatives 3a

as slightly weaker UV-ﬁlters. Their SPF_{in vitro}values range from 1.84 0.02 to 3.07

values are found for compounds 3a (2.97 0.45) and 3b (3.07 0.04), which are lower than for EHMC

and 4-MBC, but higher than for octocrylene. Additionally, compounds 3b 3e provides higher UVA

–

3e may be characterized

±

0.04. The highest

±

–

protection in comparison to the reference UV-ﬁlters excluding avobenzone and range from 2.17

±

0.11

for 3e to 2.59 ± 0.23 for 3b.

Among (Z)-5-((E)-3-phenylallylidene)imidazolidine-2,4-dione derivatives 4f

–

4g there are strong

potential UVA-ﬁlters. Their UVA PF values range from 4.28

found for compounds 4f and 4g. The absorption curve of 4f is very similar to that of avobenzone, thus

4f seems to be a good candidate as a novel alternative to avobenzone in photoprotective formulations.

±

0.02 to 8.43

±

1.35. The best eﬃcacy is

Compound 4g shows lower UVA PF, despite higher molar absorption coeﬃcient at its

λ

max

when

compared to 4f and avobenzone, which is probably caused by crystallization of the compound

from solvents during formulation. The signiﬁcantly higher UVA PF values of these compounds in

comparison to 5-benzylideneimidazolidine-2,4-dione are associated with the presence of an additional

conjugated double bond. The extension of the π-system contributes to the bathochromic shift and

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higher extinction coeﬃcient, thus UVA or broad spectrum UV-ﬁlters have higher molecular weight

than UVB ﬁlters [43].

Table 2. Photoprotective activity of tested compounds and reference UV-ﬁlters obtained in 2% (w/w)

macrogel formulations (F1^*, F2^#, F3^ˆ).

Compound

2a^#

Chemical Structure

SPF_{in vitro}

UVA PF

λ

UVA/UVB Ratio

0.35 ± 0.00

c

4.79 ± 0.02

2.40 ± 0.01

339

363

367

2b^#

2c^ˆ

3.20 ± 0.07

3.16 ± 0.05

4.29 ± 0.11

4.68 ± 0.02

1.20 ± 0.00

1.36 ± 0.01

2d^ˆ

2e^ˆ

2.00 ± 0.11

2.50 ± 0.08

4.48 ± 0.38

4.77 ± 0.24

386

379

2.59 ± 0.09

1.95 ± 0.02

3a^*

2.97 ± 0.45

1.29 ± 0.09

331

0.16 ± 0.02

3b^*

3c^*

3.07 ± 0.04

2.49 ± 0.08

2.59 ± 0.23

2.46 ± 0.01

366

370

0.84 ± 0.08

1.05 ± 0.03

3d^*

1.84 ± 0.02

2.35 ± 0.03

383

1.63 ± 0.01

3e^*

4f^*

1.87 ± 0.08

2.51 ± 0.17

2.17 ± 0.11

8.43 ± 1.35

382

381

1.43 ± 0.02

2.58 ± 0.06

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Table 2. Cont.

Compound

4g^*

Chemical Structure

SPF_{in vitro}

UVA PF

λ

UVA/UVB Ratio

c

1.68 ± 0.04

6.83 ± 0.05

391

4.50 ± 0.32

4h^*

1.94 ± 0.04

2.87 ± 0.16

4.28 ± 0.02

1.20 ± 0.03

389

327

2.58 ± 0.01

0.12 ± 0.01

O

Octocrylene^*

O

CN

EHMC^*

4-MBC^*

4.96 ± 0.33

5.59 ± 0.27

1.34 ± 0.05

1.08 ± 0.1

321

316

0.05 ± 0.01

0.00 ± 0.00

Avobenzone^*

2.23 ± 0.03

7.56 ± 0.19

381

2.49 ± 0.03

Results are presented as mean

±

SD from two independent experiments, at each determination six scans were

performed. ^*F1–Tween 20 43.2%, PEG-400 25%, PEG-1500 25%, Triacetin 4.8%, tested compound 2%; ^#F2–Tween 20

43.2%, PEG-400 25%, PEG-1500 25%, DMSO 4.8%, tested compound 2%; ^ˆF3–PEG-400 25%, PEG-1500 25%, DMSO

48%, tested compound 2%.

Figure 4. UV absorption spectra of tested compounds and reference UV ﬁlters obtained in macrogol

formulation at 2% (w/w) concentration applied on polymethylmethacrylate plates. MPF–Monochromatic

Protection Factor.

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The impact of the presence and position of alkoxy groups in the phenyl ring is also visible when

another important parameters that characterize sunscreen formulations such as critical wavelength (

λ

c

)

and UVA/UVB ratio are compared. Compounds like 2a

, 3a, 4f without substituents in the phenyl ring

show the lowest value of and UVA/UVB ratio in comparison to their analogues with alkoxy groups.

λ

c

These values are increasing when substituents in the 4- (compounds 2b, 3b, 4g) and 3,4- (compounds

2e 3e) positions are present and are the highest when electron donating groups are in the 2,4- position

,

(compounds 2d, 3d).

2.5. Photostability Studies

2.5.1. Photostability in Methanol Solutions

The photostability of the tested compounds in methanol solutions was investigated by

the measure of the changes in the and in the area under curve (AUC) upon irradiation.

λ

max

Results are presented in Table S2 and Figure S4. After irradiation of both mono- and diester

5-(Z)-benzylideneimidalidine-2,4-dione derivatives a bathochromic shift and slight hypochromic eﬀect

are observed. In the group of monoesters with methoxy substituents on the phenyl ring (compounds

2b

found. In the case of compound 2a without a phenyl ring substituent, only a 1 nm shift of

almost a 30% decrease of AUC are found. After irradiation of diester derivatives 3a 3e, bathochromic

shifts from 6 to 11 nm are observed. The photostability of 3-phenylallylidene derivatives 4f 4h is

reduced in comparison to the benzylidene derivatives substituted on the phenyl ring (compounds

2b 2e 3b 3e). Although no shifts are observed, the decrease of AUC reaches to 40.9% in the case

–

2e)

λ

max

shifts from 5 to 8 nm and decreases of the area under the curve from 0.96 to 4.33% are

λ_maxand

–

,

–

of the non-substituted derivative 4f. For compounds 4g and 4h with methoxy groups in the para or

ortho position, the decrease of AUC is 19.9 and 1.5%, respectively. In comparison to the reference

UV-ﬁlters, the tested compounds, excluding 4f, show better photostability than EHMC, for which a

36.7% decrease of AUC is detected.

The analysis of chromatograms and mass spectra of selected compounds obtained after appropriate

irradiation suggests that changes in absorption curves result from photoisomerisation processes taking

place in solution, which are a very eﬃcient way of dispersing the absorbed energy that aﬀects many

UV ﬁlters, e.g., cinnamic acid derivatives and benzylidene camphor derivatives [44] (Figure S5).

In post-irradiation chromatograms the additional peak with the same mass as tested compound

appears which indicates that the observed photoproduct is a geometric isomer. The percentage of

photoisomer formation range from 22.02 to 36.03%. In general, the tested compounds may be regarded

as photostable in methanol solutions.

2.5.2. Photostability in Cosmetic Formulations

After irradiation of thin layer of formulations applied on PMMA plates, their photoprotective

parameters were determined and compared with the pre-irradiation results. The study gives an

overview on functional photostability of the tested compounds. It is an important test, because

the photostability of some compounds in low concentrated solutions may meaningfully diﬀer from

results obtained for higher concentrations used in sunscreen products [45]. According to the previous

studies, formulations whose SPF do not decrease more than 20% after irradiation are considered

photostable [46,47]. In the present study both UVB and UVA-absorbers were tested, so the changes in

SPF_{in vitro}, UVA PF and AUC after irradiation are taken into account as a measure of photostability.

The tested were performed for formulations described in photoprotective activity section, the results

are presented in Table 3 and Figure 5.

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Table 3. The changes in photoprotective activity of tested compounds and reference UV-ﬁlters after

irradiation with solar light simulator at 500 W/m². In the experiments appropriate formulations (F1^*,

F2^#, F3^ˆ) were used.

Compound

% of Initial SPF_{in vitro}

% of Initial UVA PF

% of Initial AUC

2a^ˆ

2a^#

33.25

55.49

36.51

57.19

44.37

75.69

56.00

83.31

80.46

72.69

88.73

89.92

81.28

62.24

86.29

82.22

91.05

80.83

37.87

68.96

24.13

54.31

35.83

51.45

38.99

103.50

87.04

84.08

86.59

97.66

87.10

25.98

34.39

62.38

101.39

88.76

46.72

62.42

35.28

59.55

34.13

58.87

41.99

88.19

84.67

76.50

85.29

94.01

79.59

27.92

51.98

69.19

90.89

78.45

2b^ˆ

2b^#

2c^ˆ

2d^ˆ

2e^ˆ

3a^*

3b^*

3c^*

3d^*

3e^*

4f^*

4g^*

4h^*

4-MBC^*

EHMC^*

Results are presented as means from two independent experiments, at each determination six scans were performed.

^*F1–Tween 20 43.2%, PEG-400 25%, PEG-1500 25%, Triacetin 4.8%, tested compound 2%; ^#F2–Tween 20 43.2%,

PEG-400 25%, PEG-1500 25%, DMSO 4.8%, tested compound 2%; ^ˆF3–PEG-400 25%, PEG-1500 25%, DMSO 48%,

tested compound 2%.

Figure 5. UV absorption spectra of tested compounds and 4-MBC pre-irradiation and after 1 hour

irradiation with solar light simulator conducted at 500 W/m²obtained in 2% (w/w) macrogol formulations.

Compounds 3a–3e in the macrogol formulation show the highest photostability–they may be

considered as photostable UV-absorbers. The decrease of SPF_{in vitro}from 10.1 to 19.5% was observed.

The percentage of initial UVA PF and AUC values range from 103.5 to 87.0% and 94.0 to 79.6%

respectively. Especially compound 3d is characterized by functional photostability very similar to that

of 4-MBC which is considered a photostable UV-ﬁlter [48]. The analysis of chromatograms and mass

Molecules 2019, 24, 2321

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spectra of selected compounds indicates that in macrogol formulations the compounds behave as in

methanol solutions. In the post-irradiation chromatogram of compound 3b, 32.1% of photoisomer

is observed while 28.4% is detected in methanol solution. Additionally, a new photoproduct with

2.4% relative peak area appears. Similarly, in the post-irradiation chromatogram of compound 3c

a photoproduct with 2.8% relative peak area is detected. In the chromatogram of compound 3d no

additional peaks besides the photoisomer are found.

,

Interesting results are obtained for compounds 2a–2e. These compounds are insoluble in the

solvent system selected for compounds 3a 3e, thus it was necessary to dissolve them in DMSO before

–

incorporation into the formulation. The functional photostability of these compounds is drastically

reduced in comparison to the results obtained in methanol solutions. The decrease of tested parameters

is from 41.1% for 2d and reaches 75.9% for 2b. This was probably due to evaporation of the solvent

from the formulation and thus the precipitation of the compound. To conﬁrm that the reason of the low

functional photostability of compounds 2a–2e was their precipitation, additional tests for compounds

2a and 2b in formulations containing a reduced amount of DMSO (from 48 to 4.8%) were carried out.

The results indicates an improvement of the functional photostability by 31.1% for 2a and by 30.2% for

2b. Additionally, the analysis of post-irradiation chromatograms of 2a shows that despite signiﬁcant

changes in the reduction of photoprotection parameters between two formulations diﬀering in the

content of DMSO, the percentage of photoisomer formation is very similar for both formulations (45%

vs 48%). These ﬁndings encourage us to search for another, better solvent system for compounds

2a–2e.

Photostability evaluation of compounds 4f–4h in macrogel formulations indicates that as in

methanol solutions they are not photostable. The decrease of their AUC was from 30.8% for 4h and

reached 72.1% for 4f. In the corresponding post-irradiation chromatograms, beside photoisomers, the

peaks of photodegradation products are also detected. The percentage of photoproducts content ranges

from 11.0% for 4h to 22.9% for 4f. This result does not discredit compounds 4f–4h. Low photostability

is also associated with other UV-ﬁlters, namely avobenzone and ethylhexyl methoxycinnamate, but it

can be improved by adding, for example, triplet-state quenchers to the formulations [20,49].

There are many factors that contribute to the photostability of a potential UV-ﬁlter molecule.

These compounds are from diﬀerent chemical groups, their photobehaviour depends on the structure

and presence of functional groups. High photostability is a feature of molecules with intramolecular

hydrogen bonds such as hydroxyphenyltriazine derivatives. In this class of compounds after absorption

of a photon a tautomeric transfer of a H-atom to an N-atom is observed and then the absorbed energy

is lost as thermal energy (vibronic relaxation) [6]. Compounds containing unsaturated bonds (such as

derivatives of 5-arylidenehydantoin, camphor and cinnamic acid) after absorbing a photon undergo a

photoisomerisation process. This chemical transformation is a desirable way to lose absorbed energy.

It is most preferably when the cis and tans forms have similar absorption curve shapes and extinction

coeﬃcients but forming a photoisomer often results in a signiﬁcantly lower absorption intensity [20].

2.6. Preliminary Safety Assessment

The safety proﬁle of selected compounds (1e, 2d, 2e, 3b, 4g, 4f and 5a) was evaluated. A

cytotoxicity assay in a unique skin panel model, containing three types of cells that are involved in the

structural organization of human skin was performed. Additionally, considering the possibility of

absorption of the compounds into the bloodstream and reports about adverse eﬀects on viability of

nerve cells caused by some UV-ﬁlters [14], cytotoxicity against hepatocytes (HepG2) and neuroblastoma

cell lines (SH-SY5Y) was evaluated. As reference standards 4-MBC and/or BP-1 were used. A MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to investigate metabolic

activity of cells incubated in the presence of the analyzed compounds. Estrogenic activity of compounds

was also determined, where the UV-ﬁlter benzophenone-2 (BP-2) exhibiting estrogenic activity was used

as a positive control. Our preliminary safety assessment of the compounds also included evaluation of

their metabolic stability. Skin not only is the major protective environmental barrier, but also is involved

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in the biosynthesis of endogenous compounds, detoxiﬁcation of xenobiotics and also participates in

the activation of pro-toxic substances [50]. As a consequence, metabolism studies are becoming a

consideration in the development of new cosmetic ingredients. The xenobiotic metabolising enzymes

within the epidermis in general represent the same classes as liver, although their levels are lower

and activities are weaker than those found in liver [51]. On the other hand xenobiotics such as some

cosmetic ingredients can enter into systemic circulation through the epidermis and dermis being

exposed to liver enzymes [52]. Therefore, we used rodents liver microsomes in this study to assess the

rate of stability of selected potential sunscreens. Additionally the mutagenic activity of two selected

compounds (3b, 4g) was examined.

2.6.1. Skin Panel Model

The cytotoxicity was examined after 24 h incubation of the cells with compounds. Results after

incubation at the higher administrated concentration are presented in Figure 6. Cytotoxicity studies

on human keratinocytes and human skin ﬁbroblasts showed that all tested compounds except the

references 4-MBC and BP-1 are deprived of cytotoxic activity in the range of tested concentrations

(2–50

µ

M), and the viability of BJ cells and HaCaT is

≥

87% and 89%, respectively (Figure 6a,b).

68% at the highest

Cytotoxicity studies on human primary dermal melanocytes indicate a cell viability

tested concentration except for BP-1 and 4-MCB, where the viability was <43% and 36% respectively

≥

(Figure 6c).

*

(a)

(b)

(d)

(c)

(e)

Figure 6. Viability of human keratinocytes HaCaT (

primary dermal melanocytes ( ), HepG2 cells ( ) and SH-SY5Y cells (

compounds at 50 M for 24 hours. Cell viability was determined using MTT assay. Each experiment

a), human skin ﬁbroblast (BJ) (b) cell lines, human

c

d

e) incubated in the presence of

µ

was repeated in triplicate. Graphs represent the number of viable cells expressed as percent of control

(cells not treated with compounds) ± SEM (p < 0.05).

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2.6.2. Hepatocytotoxicity and Neurocytotoxicity Studies

Results after incubation of HepG2 cells and SH-SY5Y cells with the tested compounds and reference

compound 4-MBC at the highest (50 M) concentration are presented in Figure 6. Cytotoxicity studies

µ

on HepG2 hepatocellular tumor cells indicate that the tested 5-arylidene imidazolidine-2,4-dione

derivatives are safe at the concentrations used. In the MTT test, the viability of HepG2 cells at the

highest concentration is >75%. For reference compounds used in the test, the observed viability of cells

was <32%. Cytotoxicity studies on SH-SY5Y cells indicate the viability of cells >75% at the highest

tested concentration with exclusion of 4-MCB, where viability was <31% (Figure 6e).

2.6.3. Estrogenic Activity

Proliferation analyses indicated lack of stimulation eﬀect of tested compounds in MCF-7 breast

cancer model. In contrast, BP-2 stimulated proliferation by over 60%, what was statistically signiﬁcant.

Hence, lack of estrogenic activity of compounds was concluded. Results are presented in Figure 7.

Figure 7. Proliferation rate of MCF-7 cells incubated with tested compounds at 5 µM. Compounds

showed lack of proliferation-stimulating eﬀect. BP-2 increased MCF-7 proliferation by over 60%

(p < 0.05).

2.6.4. In Vitro Stability of Compounds in Mouse Liver Microsomes

Stability and direction of biotransformation of hydantoin derivatives in mouse liver

microsomes depend on the structure of the side chains.

was evaluated by determining the half-life (t_1/2) and intrinsic clearance (Cl_int) of the

compounds upon incubation (Table 4). In the group of studied structures the

The metabolic stability

most stable compound was 5a (((Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetic acid))

with t_1/2higher than 23 hours and the value of in vitro intrinsic clearance Cl_int

below

2,2⁰-((Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate) with a

half-life of 3 min and value of Cl_intover 300 L/mg/min. Other compounds’ stability remained

1

µL/mg/min.

The least stable compound was proved to be 4g (diethyl

µ

at a moderate level, most of them being slightly less stable than the reference BP-2.

We observed two directions of hydantoin derivatives’ microsomal biotransformation (Table 4).

One is O-dealkylation of compounds with alkoxy substituents on the phenyl ring (1e

while the second is ester hydrolysis of diester derivatives 3b and 4g to monoesters.

, 2c–e, 3b and 4g)

Microsomal cytochrome P450 enzymes catalyze the oxidative O-dealkylation of xenobiotics,

cleaving methoxy or ethoxy groups in aromatic rings vulnerable to this reaction [53 54]. This reaction

requires the presence of NADPH. We observed O-dealkylation as a biotransformation reaction in case

of all tested structures bearing the methoxy (1e 2d, 2e, 3b and 4g) or ethoxy (2c) side chains. Such

results are consistent with the previous reports regarding the biotransformation of structurally similar

compounds [53 55]. Apart from CYP450 isoforms other drug metabolizing enzymes are also present in

,

microsomal preparations including carboxylesterases [56] which mainly catalyze the hydrolysis of

diverse endogenous and xenobiotic esters. Carboxylesterases (CEs) are present in microsomal fractions

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of various tissues, including liver, intestine and skin [57]. CEs play crucial roles in the metabolism

of various ester xenobiotics including drugs [56] and cosmetic ingredients such as parabens [58 59],

,

or salicylates [60]. On the contrary to CYP450 enzymes no catalytic cofactors are involved in the

CEs-catalyzed reactions [59]. Therefore the interpretation of CEs contribution to microsomal stability

of compounds requires using additional blank incubations deprived not only NADPH regenerating

systems but also microsomes [59]. In our study all tested esters of hydantoin(di)acetic acid derivatives

(2c–2e, 3b, 4g) were stable in buﬀer at pH 7.4 in the absence of microsome solution showing no sign of

spontaneous hydrolysis after 60 min incubation at 37 ^◦C (data not showed). Hydantoin derivative

1,3-diacetate diesters (3b and 4g) were hydrolysed to monoesters (parent compound losing 28 Da,

Table 4) in the presence of microsomes and independently of the presence of a NADPH-regenerating

system. The hydantoin 3-acetate monoesters derivatives 2c–2e did not hydrolyzed under the same

conditions. Therefore the most probable reaction is formation of 3-acetate monoesters from 3b and 4g

by mouse liver CEs. Formation of monoester from diester by mammalian liver microsomal CEs was

recently reported for di(2-ethylhexyl) phthalate [61].

Table 4. In vitro stability (t_1/2and Cl_int) of compounds 1e, 2c–2e, 3b, 4g, 5a and reference substance

benzophenone-2 (BP-2) in mouse liver microsomal system. Metabolites formation in the presence and

absence of NADPH; compounds elemental composition changes and probable transformation pathways.

Metabolite Formation in the

Presence (P) or/and Absence

(A) of NADPH

Elemental

Composition

Change

Cl_int

(µL/mg/min)

Probable

Transformation

[M + H⁺] Da

t

(min)

1

2

Compound

1e

2c

2d

2e

249

319

335

61

33

15

41

14

52

59

21

M1 P

−14

−28

−14

−28

−14

−28

−14

O-dealkylation

hydrolysis

O-dealkylation

hydrolysis

O-dealkylation

M1 ^a

M1^a

P

M1 P

M1 P and A

M2 P

M1 P and A

M2 P

3b

391

12

75

4g

417

3

328

5a

BP-2

247

1386

66

0.6

13

ND

NT

ND–not detected; NT–not tested.

2.6.5. Mutagenic Activity

In the present study an Ames MPFTM Penta 2 microplate format mutagenicity assay (Xenometrix,

Allschwil, Switzerland) was used to evaluate mutagenicity of two selected compounds (3b and 4g

)

exhibiting the most promising photoprotective potential. Growth, exposure and indicator media,

liver S9 fraction, S9-NADP, S9-G-6-P, positive control chemicals as well as Salmonella typhimurium and

Escherichia coli strains were included in the kit from Xenometrix. According to the obtained results,

there were no doses of test compounds 3b and 4g with more than a 2-fold induction over the baseline,

and a dose dependent response was not observed both in the absence and presence of metabolic

activation (Table S3). Therefore, compounds 3b and 4g were clearly non-mutagenic in the absence and

presence of metabolic activation.

2.7. Structure-Activity Relationship Studies

Monoethyl esters of (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetic acid (N-mono

substituted derivatives, compounds 2a

range of 290–394 nm with _max316–349 nm depending on the presence and position of the auxochrome.

An additional N-alkyl substituent in imidazolidine ring in compounds 3b 3e caused a hypsochromic

shift by 15–21 nm. Introduction of methoxy group(s) in the phenyl ring caused a shift of from

–2e) absorbed UV radiation in methanol solutions in the

λ

–

λ

max

the UVB region for compounds 2a and 3a to the UVA region for 2b

unsaturated spacer between the phenyl and hydantoin rings in compounds 4f

–

2e and 3b

–

3e. Elongation of the

4h contributed to the

–

signiﬁcant bathochromic shift when compared to the appropriate benzylidene analogues leading to the

Molecules 2019, 24, 2321

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extension of the UV absorption range into the UVA region. These compounds were also characterized

with the highest molar absorption coeﬃcients among the tested series.

The tested compounds, especially 2a–2e, were diﬃcult to dissolve in the proposed macrogol

formulations. In order to obtain a 2% formulation it was necessary to use DMSO because common

cosmetic solvents were not able to dissolve the compounds. However, we noticed that an additional

2-ethoxy-2-oxoethyl substituent in the imidazolidine ring (as in compounds 3a–3e) caused a signiﬁcant

improvement of the solubility so that the use of DMSO in those formulations was not necessary.

Tests performed in formulations proved that compounds without methoxy substituents in the

phenyl ring were characterized as UVB ﬁlters while the presence of some methoxy groups shifted

the UV-protection properties into the UVA region which was observed as an increase of the UVA PF

value. Moreover, compounds without a substituent in the phenyl ring showed the lowest values of

critical wavelength (λ_c) and UVA/UVB ratio in comparison to their analogues possessing alkoxy groups.

Values of λ and UVA/UVB ratio increased with the following kind of substitution in the phenyl ring:

c

no substitution < 4-methoxy < 3,4-dimethoxy < 2,4-dimethoxy.

The currently reported compounds were shown to be less cytotoxic than commercially available

UV-ﬁlters at concentrations up to 50 µM in tested cell lines (human epidermal keratinocytes HaCaT,

human skin ﬁbroblasts BJ, human primary epidermal melanocytes, human hepatoma cells HepG2,

human neuroblastoma cell line SH-SY5Y). The results suggest that exchange of camphor with hydantoin,

as presented in our design concept (Figure 1) caused an improvement of the safety proﬁle.

3. Materials and Methods

3.1. Chemistry

2-Methoxycinnamalaldehyde, 4-methoxycinnamalaldehyde, ethyl chloroacetate and ethyl

bromoacetate were purchased from Sigma–Aldrich Chemie (Darmstadt, Germany), hydantoin

was obtained from TCI Chemicals (Tokyo, Japan), TEBA, 3,4-dimethoxybenzaldehyde and

cinnamalaldehyde were purchased from Alfa Aesar (Kandel, Germany), 4-ethoxybenzaldehyde

were purchased from Acros Organics (Geel, Belgium) whereas the reagents benzaldehyde,

4-methoxybenzaldehyde and 2,4-dimethoxybenzaldehyde were provided by Fluoro Chem (Hadﬁeld,

England). Triacetin was obtained from ICN Biomedicals, Inc. (Irvine, CA, USA), PEG-400 and PEG-1500

were purchased from Merck (Darmstadt, Germany). 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 ¹H-NMR (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), bs

(broad singlet), d (doublet), dd (doublet of doublets), ddd (double doublet of doublets), t (triplet), 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

µm,

2.1 100 mm 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% HCO₂H; 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 (Z)-5-benzylidene- and (Z)-5-((E)-3-phenylallylidene)imidazolidine-2,4-diones

(compounds 1a-1h)

Starting materials and compound 1e were synthesized by means of previously published

procedures [62]. Glacial acetic acid (100 mL) was mixed with anhydrous sodium acetate (33.3 g) in

round-bottom ﬂask. Then hydantoin (10.1 g, 0.11 mole) was added and mixture was heated under

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reﬂux. After achieving boiling point, the appropriate aldehyde (0.1 mole) was added and the mixture

was heated for the next 4–8 h. After cooling the mixture was ﬁltered and the obtained solid was washed

well with water. Dried crude product was crystallized from ethanol or ethanol/acetic acid (1:1).

3.1.2. Preparation of Ethyl (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetates (compounds

2a–2e)

The appropriate (Z)-5-beznylideneimidazolidine-2,4-dione (0.01 mole) was mixed with acetone

(100 mL) and potassium carbonate (4 g). Then TEBA (0.3 g, 0.001 mole) was added and the mixture was

heated under reﬂux with stirring. To the mixture ethyl chloroacetate (1.22 g, 0.01 M) in acetone (40 mL)

was added dropwise and the mixture was heated for 4 h. Then the reaction mixture was ﬁltered, the

ﬁltrate was evaporated under reduced pressure and the obtained crude product was recrystallized

from ethanol.

3.1.3. Preparation of Diethyl 2,2⁰-((Z)-4-benzylidene-2,5-dioxoimidazolidine-1,3-diyl) Diacetates and

Diethyl 2,2⁰-((Z)-2,4-dioxo-5-((E)-3-phenylallylidene)imidazolidine-1,3-diyl)diacetates (compounds

3a-3e, 4f-4h)

The appropriate (Z)-5-beznylideneimidazolidine-2,4-dione or (Z)-5-((E)-3-phenylallylidene)-

imidazolidine-2,4-dione (0.01 mole) was mixed with potassium carbonate (8 g), then DMF (30 mL) was

added. After dissolving the imidazolidine-2,4-dione derivative, ethyl bromoacetate (0.02 M, 3.34 g)

was added dropwise. The mixture was stirred at room temperature, while the progress of a reaction

was monitored by TLC (chloroform:acetone:methanol 5:1:0.1). After about 8 h the mixture was poured

into ice. Obtained solid was collected by ﬁltration. After drying crude product was crystallized

from ethanol.

3.1.4. Preparation of (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetic acid (5a)

Ethyl (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetate (0.01 M) was mixed with ethanol

(50 mL) then NaOH (0.4 g, 0.01 M) dissolved in distilled water was added. The mixture was heated on

a water bath. After hydrolysis the solvent was distilled oﬀ. The obtained oily residue was dissolved in

distilled water then it was treated with 16% sulfuric acid with the aim of separating the free acid. The

crude product was crystallized from 50% ethanol.

3.1.5. Physicochemical Properties of Tested Compounds

(Z)-5-Benzylideneimidazolidine-2,4-dione (1a) was obtained as yellow solid (yield 74%), M = 188.19, mp

222–224 ^◦C, ESI-MS (m/z): [M + H]⁺calcd. for C₁₀H₈N₂O₂, 189.19, found, 189.01 100%. Physicochemical

data for 1a were previously published by Martínez-López et al. [63] CAS: 3775-01-7, CAS for Z

isomer: 74805-60-0.

(Z)-5-(4-Methoxybenzylidene)imidazolidine-2,4-dione (1b) was obtained as yellow solid (yield 72%),

M = 218.21, mp 251–252 ^◦C, ESI-MS (m/z): [M + H]⁺calcd. for C₁₁H₁₀N₂O₃, 219.21, found, 219.11

100%. Physicochemical data for 1b were previously published by Luo et al. [64] CAS: 5349-42-8, CAS

for Z isomer: 108402-52-4.

(Z)-5-(4-Ethoxybenzylidene_◦)imidazolidine-2,4-dione (1c) was obtained as yellow solid (yield 81%),

M = 232.24, mp 229–231 C, ESI-MS (m/z): [M+H]⁺calcd. for C₁₂H₁₂N₂O₃, 233.24, found, 233.13

97.83%. Physicochemical data for 1c were previously published by Thenmozhiyal et al. [65] CAS:

6325-66-2, CAS for Z isomer: none.

(Z)-5-(2,4-Dimethoxybenzylidene)imidazolidine-2,4-dione (1d) was obtained as yellow solid (yield 82%),

M = 248.24, mp 238–240 ^◦C, ESI-MS (m/z): [M + H]⁺calcd. for C₁₂H₁₂N₂O₄, 249.24, found, 249.15 100%.

Physicochemical data for 1d were previously published by Thenmozhiyal et al. [65] CAS: 91559-39-6,

CAS for Z isomer: 1312438-16-6.

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(Z)-5-(3,4-Dimethoxybenzylidene)imidazolidine-2,4-dione (1e) was obtained as yellow solid (yield 69%),

M = 248.42, mp 279–280 ^◦C, ESI-MS (m/z): [M + H]⁺calcd. for C₁₂H₁₂N₂O₄, 249.42, found, 249.08

100%. Physicochemical data for 1e were previously published by Luo et al. [64] CAS: 10040-91-2, CAS

for Z isomer: 140894-76-4.

(Z)-5-((E)-3-Phenylallylidene)imidazolidine-2,4-dione (1f) was obtained as dark yellow solid (yield 73%),

M = 214.22, mp 272–274 ^◦C, ¹H-NMR (DMSO-d₆) δ 11.09 (s, 1H, -NH-), 10.66 (s, 1H, -NH-), 7.54–7.44

(m, 2H, Ar-H3, Ar-H5), 7.44–7.20 (m, 4H, =CH-, Ar-H2, Ar-H4, Ar-H6), 6.86 (dd, J = 29.2, 15.7 Hz, 1H,

-CH=), 6.23 (dd, J = 11.9, 0.8 Hz, 1H, -CH=), ESI-MS (m/z): [M + H]⁺calcd. for C₁₂H₁₀N₂O₂, 215.12,

found, 215.13 100%. Physicochemical data for 1f were previously published by Lamiri et al. [66] CAS:

66835-63-0, CAS for Z,E isomer: 137920-57-1.

(Z)-5-((E)-3-(4-Methoxyphenyl)allylidene)imidazolidine-2,4-dione (1g) was obtained as brown solid (yield

82%), M = 244.25, mp 289–291 ^◦C, ¹H-NMR (DMSO-d₆)

δ 11.02 (s, 1H, -NH-), 10.57 (s, 1H, -NH-), 7.43

(d, J = 8.7 Hz, 2H, Ar-H2, Ar-H6), 7.11 (dd, J = 15.5, 11.8 Hz, 1H, =CH-), 6.95 (d, J = 8.8 Hz, 2H, Ar-H3,

Ar-H5), 6.86 (d, J = 15.5 Hz, 1H, -CH=), 6.21 (dd, J = 11.8, 0.7 Hz, 1H, -CH=), 3.76 (s, 3H, O-CH₃),

ESI-MS (m/z): [M + H]⁺calcd. for C₁₃H₁₂N₂O₃, 245.25, found, 245.16 100%. CAS: 664353-78-0, CAS for

Z,E isomer: none.

(Z)-5-((E)-3-(2-Methoxyphenyl)allylidene)imidazolidine-2,4-dione (1h) was obtained as brown solid (yield

64%), M = 244.25, mp 260–263 ^◦C, ¹H-NMR (DMSO-d₆)

δ 11.05 (s, 1H, -NH-), 10.65 (s, 1H, -NH-), 7.55

(dd, J = 7.8, 1.7 Hz, 1H, Ar-H6), 7.33–7.16 (m, 2H, Ar-H4, -CH=C), 7.10 (d, J = 15.7 Hz, 1H, -CH=),

7.05–6.90 (m, 2H, Ar-H3, Ar-H5), 6.24 (d, J = 11.5 Hz, 1H, =CH-), 3.82 (s, 3H, -OCH3), ESI-MS (m/z): [M

+ H]⁺calcd. for C₁₃H₁₂N₂O₃, 245.25, found, 245.10 98.85%. CAS for E,E isomer: 1776969-29-9, CAS for

Z,E isomer: none.

Ethyl (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetate (2a) was obtained as white solid (yield 63%)

M = 274.28, mp 169–171 ^◦C, ¹H-NMR (chloroform-d)

δ 8.55 (s, 1H, -NH), 7.49–7.44 (m, 1H, Ar-H4),

7.44–7.30 (m, 4H, Ar-H2, Ar-H3, Ar-H5, Ar-H6), 6.79 (s, 1H, -CH=), 4.37 (s, 2H, -CH₂-), 4.24 (q,

J = 7.2 Hz, 2H, -CH₂-), 1.35–1.18 (m, 3H, -CH₃), ESI-MS (m/z): [M + H]⁺calcd. for C₁₄H₁₄N₂O₄, 275.28,

found, 275.20 96.84% CAS: 463317-70-6 CAS for Z isomer: 1416719-77-1.

Ethyl (Z)-2-(4-(4-methoxybenzylidene)-2,5-dioxoimidazolidin-1-yl)acetate (2b) was obtained as white solid

(yield 52%), M = 304.30, mp 178–179 ^◦C, ¹H-NMR (chloroform-d)

δ 8.34 (s, 1H, -NH), 7.45–7.34 (m, 2H,

Ar-H2, Ar-H6), 7.01–6.90 (m, 2H, Ar-H3, Ar-H5), 6.76 (s, 1H, -CH=), 4.37 (s, 2H, N-CH₂), 4.24 (q, J = 7.1

Hz, 2H, -CH₂-), 3.85 (s, 3H, -OCH₃), 1.29 (t, J = 7.1 Hz, 3H, -CH₃) ESI-MS (m/z): [M + H]⁺calcd. for

C₁₅H₁₆N₂O₅, 305.30, found, 305.17 99,31%. CAS: 463317-69-3 CAS for Z isomer: 2247638-41-9.

Ethyl (Z)-2-(4-(4-ethoxybenzylidene)-2,5-dioxoimidazolidin-1-yl)acetate (2c) was obtained as white solid

(yield 61%), M = 318.33, mp 181–182 ^◦C, ¹H-NMR (chloroform-d)

δ 8.66–8.60 (m, 1H, -NH), 7.48–7.34

(m, 2H, Ar-H2, Ar-H6), 6.98–6.87 (m, 2H, Ar-H3, Ar-H5), 6.75 (s, 1H, -CH=), 4.37 (s, 2H, N-CH₂), 4.23

(q, J = 7.2 Hz, 2H, -CH₂-), 4.07 (q, J = 7.0 Hz, 2H, -CH₂-), 1.43 (t, J = 7.0 Hz, 3H, -CH₃), 1.36–1.19 (m, 3H,

-CH₃) ESI-MS (m/z): [M + H]⁺calcd. for C₁₆H₁₈N₂O₅, 319.33, found, 319.20 99.02%.

Ethyl (Z)-2-(4-(2,4-dimethoxybenzylidene)-2,5-dioxoimidazolidin-1-yl)acetate (2d) was obtained as light

yellow solid (yield 56%), M = 334.33, mp 197–198 ^◦C, ¹H-NMR (chloroform-d)

δ 7.98 (s, 1H, -NH),

7.31–7.21 (m, 1H, Ar-H6), 6.75–6.68 (m, 1H, -CH=), 6.61–6.47 (m, 2H, Ar-H3, Ar-H5), 4.34 (s, 2H, -CH₂-),

4.23 (q, J = 7.1 Hz, 2H, -CH₂-), 3.93 (s, 3H, -OCH₃), 3.85 (s, 3H, -OCH₃), 1.34–1.21 (m, 3H, -CH3) ESI-MS

(m/z): [M + H]⁺calcd. for C₁₆H₁₈N₂O₆, 335.33, found, 335.37 100%.

Ethyl (Z)-2-(4-(3,4-dimethoxybenzylidene)-2,5-dioxoimidazolidin-1-yl)acetate (2e) was obtained as white

solid (yield 48%), M = 334.33, mp 198–200 ^◦C, ¹H-NMR (chloroform-d)

δ 8.41 (s, 1H, -NH), 7.10–7.00

(m, 1H, Ar-H6), 6.97–6.87 (m, 2H, Ar-H2, Ar-H5), 6.75 (s, 1H, -CH=), 4.36 (s, 2H, N-CH₂), 4.23 (q,

J = 7.1 Hz, 2H, -CH₂-), 3.93 (s, 3H, 3-OCH₃), 3.92 (s, 3H, 4-OCH₃), 1.28 (t, J = 7.2 Hz, 3H, -CH₃), ESI-MS

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(m/z): [M + H]⁺calcd. for C₁₆H₁₈N₂O₆, 335.33, found, 335.15 100%. CAS: 463317-68-2 CAS for Z

isomer: none.

Diethyl 2,2⁰-((Z)-4-benzylidene-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3a) was obtained as white solid

(yield 81%), M = 360.37, mp 47–49 ^◦C, ¹H-NMR (chloroform-d)

δ 7.39–7.30 (m, 3H, Ar-H3, Ar-H4,

Ar-H5), 7.26–7.19 (m, 2H, Ar-H2, Ar-H6), 7.01 (s, 1H, -CH₂=), 4.37 (s, 2H, -CH₂-), 4.23 (q, J = 7.2 Hz,

2H, -CH₂-), 4.20 (s, 2H, -CH₂-), 3.95 (q, J = 7.1 Hz, 2H, -CH₂-), 1.28 (t, J = 7.2 Hz, 3H, -CH₃), 1.09 (t,

J = 7.1 Hz, 3H, -CH₃). ESI-MS (m/z): [M + H]⁺calcd. for C₁₈H₂₀N₂O₆, 361.37, found, 361.27 96.74%.

Diethyl 2,2⁰-((Z)-4-(4-methoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3b) was obtained as

white solid (yield 79%), M = 390.39, mp 74–76 ^◦C, ¹H-NMR (chloroform-d)

δ 7.24–7.13 (m, 2H, Ar-H2,

Ar-H6), 6.98 (s, 1H, -CH=), 6.95–6.84 (m, 2H, Ar-H3, Ar-H5), 4.38 (s, 2H, -CH₂-), 4.30–4.18 (m, 4H,

-CH₂-, -CH₂-), 4.01 (q, J = 7.1 Hz, 2H, -CH₂-), 3.83 (s, 3H, O-CH₃), 1.30 (t, J = 7.2 Hz, 3H, -CH₃), 1.12 (t,

J = 7.1 Hz, 3H, -CH₃), ESI-MS (m/z): [M + H]⁺calcd. for C₁₉H₂₂N₂O₇, 391.39, found, 391.17 100%.

Diethyl 2,2⁰-((Z)-4-(4-ethoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3c) was obtained as light

yellow solid (yield 84%), M = 404.42, mp 44–46 ^◦C, ¹H-NMR (chloroform-d)

δ 7.22–7.11 (m, 2H, Ar-H2,

Ar-H6), 6.97 (s, 1H, -CH=), 6.95–6.82 (m, 2H, Ar-H3, Ar-H5), 4.37 (s, 2H, N1-CH₂), 4.30–4.15 (m, 4H,

N3-CH₂, -CH₂-), 4.02 (dq, J = 12.6, 7.1 Hz, 4H, -CH₂-, -CH₂-), 1.42 (t, J = 7.0 Hz, 3H, -CH₃), 1.34–1.21

(m, 3H, -CH₃), 1.17–1.04 (m, 3H, -CH₃), ESI-MS (m/z): [M + H]⁺calcd. for C₂₀H₂₄N₂O₇, 405.42, found,

405.26 100%.

Diethyl 2,2⁰-((Z)-4-(2,4-dimethoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3d) was obtained

as white solid (yield 75%), M = 420.42, mp 88–90 ^◦C, ¹H-NMR (chloroform-d)

δ 7.05 (dt, J = 8.1, 0.7 Hz,

1H, Ar-H6), 6.94 (s, 1H, -CH=), 6.54–6.41 (m, 2H, Ar-H3, Ar-H5), 4.36 (s, 2H, -CH₂-), 4.29–4.16 (m, 4H,

-CH₂-, -CH₂-), 4.00 (qd, J = 7.1, 0.6 Hz, 2H, -CH₂-), 3.82 (s, 3H, 4-OCH₃), 3.79 (s, 3H, 2-OCH₃), 1.34–1.21

(m, 3H, -CH₃), 1.12 (t, J = 7.1, 0.6 Hz, 3H, -CH₃), ESI-MS (m/z): [M + H]⁺calcd. for C₂₀H₂₄N₂O₈, 421.42,

found, 421.22 100%.

Diethyl 2,2⁰-((Z)-4-(3,4-dimethoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (3e) was obtained as

light yellow solid (yield 68%), M = 420.42, mp 80–82 ^◦C, ¹H-NMR (chloroform-d)

δ 6.97 (s, 1H, -CH=),

6.90–6.78 (m, 2H, Ar-H5, Ar-H6), 6.76 (d, J = 1.8 Hz, 1H, Ar-H2), 4.38 (s, 2H, -CH₂-), 4.32–4.16 (m, 4H,

-CH₂-,-CH₂-), 4.01 (qd, J = 7.2, 0.6 Hz, 2H, -CH₂-), 3.90 (s, 3H, -OCH₃), 3.86 (s, 3H, -OCH₃), 1.33–1.25

(m, 3H, -CH₃), 1.18–1.05 (m, 3H, -CH₃). ESI-MS (m/z): [M + H]⁺calcd. for C₂₀H₂₄N₂O₈, 421.42, found,

421.22 100%.

Diethyl 2,2⁰-((Z)-2,4-dioxo-5-((E)-3-phenylallylidene)imidazolidyne-1,3-diyl)diacetate (4f) was obtained as

yellow solid (yield 84%), M = 386.40, mp 99–100 ^◦C, ¹H-NMR (DMSO-d₆)

δ 7.57–7.49 (m, 2H, Ar-H2,

Ar-H6), 7.49–7.25 (m, 3H, Ar-H3, Ar-H4, Ar-H5), 7.18–7.00 (m, 2H, -CH=, -CH-), 6.66–6.53 (m, 1H,

=CH-), 4.92 (s, 2H, -CH₂-), 4.35 (d, J = 4.1 Hz, 2H, -CH₂-), 4.22–4.06 (m, 4H, -CH₂-, -CH₂-), 1.26–0.98

(m, 6H, -CH₃, -CH₃), ESI-MS (m/z): [M + H]⁺calcd. for C₂₀H₂₂N₂O₆, 387.40, found, 387.19 100%.

Diethyl 2,2⁰-((Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4g) was

obtained as yellow solid (yield 89%), M = 416.43, mp 134–135 ^◦C, ¹H-NMR (DMSO-d₆)

δ 7.55–7.42 (m,

2H, Ar-H2, Ar-H6), 7.12–6.86 (m, 4H, -CH=, =CH-, Ar-H3, Ar-H5), 6.55 (d, J = 11.9 Hz, 1H, -CH=), 4.90

(s, 2H, -CH₂-), 4.35 (s, 2H, -CH₂-), 4.13 (qd, J = 7.1, 3.7 Hz, 4H, -CH₂-, -CH₂-), 3.77 (s, 3H, O-CH₃), 1.19

(t, J = 7.1 Hz, 3H, -CH₃), 1.13 (t, J = 7.1 Hz, 3H, -CH₃). ESI-MS (m/z): [M + H]⁺calcd. for C₂₁H₂₄N₂O₇,

417.43, found,417.16 100%.

Diethyl 2,2⁰-((Z)-4-((E)-3-(2-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4h) was

obtained as yellow solid (yield 75%), M = 416.43, mp 134–135 ^◦C, ¹H-NMR (chloroform-d)

δ 7.38 (dd,

J = 7.7, 1.7 Hz, 1H, Ar-H6), 7.36–7.18 (m, 2H, -CH=, -CH=), 7.17–7.03 (m, 1H, Ar-H4), 7.00–6.75 (m, 2H,

=CH-, Ar-H5), 6.69 (dd, J = 11.0, 0.6 Hz, 1H, Ar-H3), 4.72 (s, 2H, -CH₂-), 4.36 (s, 2H, -CH₂-), 4.24 (qd,

J = 7.2, 6.1 Hz, 4H, -CH₂-, -CH₂-), 3.89 (s, 3H, 2-OCH₃), 1.36–1.11 (m, 6H, -CH₃, -CH₃), ESI-MS (m/z):

[M + H]⁺calcd. for C₂₁H₂₄N₂O₇, 417.43, found, 417.29 97.93%.

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(Z)-2-(4-Benzylidene-2,5-dioxoimidazolidin-1-yl)acetic acid (5a) was obtained as white solid (yield 49%),

M = 246.22, mp 215–217 ^◦C, ¹H-NMR (DMSO-d₆)

10.91 (s, 1H, -OH), 7.69–7.59 (m, 2H, -NH, Ar-H4),

δ

7.47–7.28 (m, 4H, Ar-H1, Ar-H2, Ar-H5, Ar-H6), 6.58 (s, 1H, -CH=), 4.19 (s, 2H, -CH₂-)., ESI-MS

(m/z): [M + H]⁺calcd. for C₁₂H₁₀N₂O₄, 247.22, found, 247.16 100%. CAS: 857796-52-2 CAS for Z

isomer: 850636-92-9.

3.2. Crystal Structures of 3b and 4g

Crystals suitable for an X-ray structure analysis were obtained from ethanol by slow evaporation

of the solvent at room temperature. Data for single crystals were collected using the Oxford Diﬀraction

SuperNova four circle diﬀractometer (Agilent, Wroclaw, Poland), equipped with the Mo (0.71073 Å)

Kα radiation source and graphite monochromator. The phase problems were solved by direct methods

using SIR-2014 program [67]. All non-hydrogen atoms were reﬁned anisotropically using weighted

full-matrix least-squares on F2. Reﬁnement and further calculations were carried out using SHELXL

program [68]. The hydrogen atoms bonded to carbons were included in the structure at idealized

positions and were reﬁned using a riding model with Uiso(H) ﬁxed at 1.2 Ueq of C and 1.5 Ueq for

methyl groups. The fragment O6-C12-C13 in 3b is disordered. The occupancy factors of these atoms

after reﬁnement are 0.797 and 0.203 for the major (A) and minor (B) components, respectively. For

molecular graphics ORTEP [69] and MERCURY [70] programs were used.

3b: C₁₉H₂₂N₂O₇, Mr = 390.38, crystal size = 0.21

×

0.44

×

0.77 mm3, monoclinic, space group C2/c,

a = 22.7269(5) Å, b = 12.7839(2) Å, c = 13.7085(6) Å,

β

= 106.579(2)^◦, V = 3817.3(1) Å3, Z = 8, T = 130(2) K,

14040 reﬂections collected, 4450 unique reﬂections (Rint = 0.0163), R1 = 0.0360, wR2 = 0.0887 [I > 2

and R1 = 0.0417, wR2 = 0.0929 [all data].

σ(I)]

4g: C₂₁H₂₄N₂O₇, Mr = 416.42, crystal size = 0.17 x 0.23 x 0.79 mm3, monoclinic, space group I2/a,

a = 17.7480(2) Å, b = 8.8899(1) Å, c = 27.1816(3) Å,

= 96.619(1)^◦, V = 4116.31(8) Å3, Z = 8, T = 130(2)

β

K, 27541 reﬂections collected, 4942 unique reﬂections (Rint = 0.0202), R1 = 0.0435, wR2 = 0.1031 [I >

2σ(I)] and R1 = 0.0526, wR2 = 0.1094 [all data].

CCDC 1889259-1889260 contain the supplementary crystallographic data. These data can be

obtained free of charge from The Cambridge Crystallographic Data Centre via ww w .ccdc.cam.ac.uk /

data_request /cif.

3.3. Ultraviolet Spectroscopy

Spectra with a scan range of 290–400 nm were recorded in 50 or 25 µM methanol 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 extinction coeﬃcient at maximum

absorption (ε_max) of tested compounds was determined in methanol as the slope of the linear regression

of absorbance vs. concentration of tested compound (from 10 to 50 µM or 2.5 to 30 µM).

3.4. Photoprotective Activity

3.4.1. Cosmetic Formulations

Tested compounds and commercial UV ﬁlters as reference standards were incorporated to a

neutral macrogel formulation in the concentration of 2%. Prior to the incorporation, compounds 3a–3e

and 4f–4h and reference UV-ﬁlters were dissolved in a triacetin:Tween 20 (1:9) solvent system. For

compounds 2a–2e it was necessary to use DMSO. The ﬁnal composition of the macrogol formulations

is presented in the photoprotective activity section.

3.4.2. In Vitro Photoprotection Study

In vitro photoprotection study was performed according to EN ISO 24443:2012 [71] with slight

modiﬁcations. Formulation with tested compound (32.5 mg) was applied on a polymethylmethacrylate

plate (PMMA) with the application area 25 cm²and 5

µm roughness value to simulate the skin surface

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(Schonberg GmbH, Hamburg, Germany). As a reference a PMMA plate treated with approximately

15 L of glycerin was used. Measurements were carried out by reﬂectance spectrophotometry with

µ

SPF-290AS Analyser (Solar Light Company, Glenside, PA, USA) equipped with integrating sphere

controlled by WinSPF version X.X. software. For each sample two plates were prepared, absorbance

measures were performed on 6 diﬀerent positions of the plate from 290 to 400 nm with 1 nm steps. The

results were expressed as an average of data from 12 scans.

3.4.3. Photostability Study

The photostability evaluation of compounds was performed both in methanol solutions and in

macrogol formulations. Irradiation of samples according to previous studies [29,72] was conducted

for 1 h at 500 W/m²(cumulative dose of ultraviolet radiation 218 kJ/m²) 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 UV absorption spectrum of

the samples were analysed post-irradiation and compared with pre-irradiation results. UPLC-MS/MS

analyses were also performed.

Methanol solutions were tested at concentration 25 or 50 µM in quartz cuvettes with the PTFE

stopper to prevent evaporation or leaking of the solvent. Macrogol formulations (2% w/w) were

tested on PMMA plates (1.3 mg/cm²). After post-irradiation measure of UV absorption, exposed and

unexposed plates were washed with 5 mL of methanol, then mixtures were ﬁltered and analysed by

UPLC-MS/MS. All analyses were performed in duplicates.

3.5. Skin Panel Model

Primary Epidermal Melanocytes (PCS-200-013, ATCC; Manassas, VA, USA), human epidermal

keratinocyte cell line HaCaT (CLS Cell Lines Service; Eppelheim, Germany) and human skin ﬁbroblast

cell line BJ (CRL-2522, ATCC,) were used in the study. Primary Epidermal Melanocytes were cultured

in Dermal Cell Basal Medium (ATCC) supplemented with Adult Melanocyte Growth Kit Components

(ATCC), HaCaT and BJ cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM; New

York, NY, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA).

Cell maintenance was performed in a humidiﬁed incubator at 37 ^◦C with 5% CO₂. Cells were passage

when the culture has reached approximately 80 to 90% conﬂuence. Cells were seeded at density of

6

×

10³on 96-well plates. Following overnight culture, the cells were then treated with increasing

doses of compounds and incubated for 24 h. Following cell exposure to each drug for 24 h, 10

µL

MTT reagent (Sigma Aldrich, Darmstadt, Germany) was added to each well. After 4 hours incubation

(37 ^◦C, 5% CO₂), 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 wells. Then

the optical density (OD) of solution was determined at 570 nm on micro plate reader (Spectra iD3 Max,

Molecular Devices; San Jose, CA, USA). Viability (% of control) was determined by dividing A₅₇₀of

experimental wells by of A₅₇₀of control wells × 100%.

3.6. Hepatocytotoxicity and Neurocytotoxicity Studies

For hepatotoxicity and neurotoxicity assays a human neuroblastoma cell line—SH-SY5Y

(CRL-2266™, ATCC) and a human hepatocellular carcinoma cell line–HepG2, respectively have

been used. Cells were seeded on 96-well plates at an initial density of 10,000 cells per well. Cells were

cultured in standard conditions for 24 h and then study compounds were administrated at diﬀerent

concentrations (10–50 µM) for another 24 h. After incubation time cells viability was measured with

MTT assay. For this purpose culture medium has been replaced for the fresh one and MTT reagent

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) has been added at the concentration

0.5 mg/mL. Cells were incubated with MTT reagent at 37 ^◦C for 4 h. Then formazan crystals have been

dissolved in DMSO solution and the absorbance has been measured at 570 nm. Experiments were

conducted in triplicates. Values represent mean percent of viability (in the percent of control).

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3.7. Estrogenic Activity

To determine estrogenic activity of tested compounds their inﬂuence on estrogen-dependent

MCF-7 breast cancer cell line proliferation was investigated. Cells were seeded into 24-wells plates

in density of 40,000 per well. After 24 h incubation, 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. Than cells were incubated with tested

compounds or benzophenone-2 (BP-2; Sigma Aldrich, Darmstadt, Germany), which is a UV ﬁlter

exhibiting estrogenic activity, 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 minutes. After incubation, solution

was removed and cells were washed ﬁve times with PBS. Crystal violate 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 A₅₇₀of experimental wells by the A₅₇₀of control wells × 100%.

3.8. In Vitro Stability of Compounds in Mouse Liver Microsomes

Mouse liver microsomes (MLMs) were used to investigate the phase I metabolism of selected

compounds (2c, 2d, 3b, 4g, 5a) and the reference substance BP-2. Samples composed of test compound

(20 M), MLMs (0.8 mg/mL) and potassium phosphate buﬀer (100 mM, pH 7.4) were preincubated

µ

prior to addition of NADPH-regenerating system. NADPH-regenerating system contained NADP,

glucose-6-phosphate, glucose-6-phosphate dehydrogenase and potassium phosphate buﬀer (100 mM,

pH 7.4). Control incubations were performed in the absence of the NADPH-regenerating system [73

and in case of esters (2c 2d 3b and 4g) without the mouse liver microsomes solution to determine

spontaneous hydrolysis [59]. Incubations were conducted at 37 ^◦C for at least 15 min up to 60 min.

The reactions were stopped by the addition of 250 L ice-cold methanol containing internal standard

PTX (20 M). The mixture was then centrifuged. Supernatant analysis was performed using UPLC/MS

]

,

µ

(Waters Corporation). The assays were repeated two times. The in vitro half times (t_1/2) for test

compounds were determined from the slope of the linear regression of ln % parent compound

remaining versus incubation time. The calculated t_1/2was incorporated into the following equation to

obtain intrinsic clearance: (Cl_int) = (volume of incubation [

t_1/2[74].

µL]/ protein in the incubation [mg])

×

0.693 /

3.9. Ames Assay

The assay is a liquid microplate modiﬁcation of the traditional Salmonella test [75,76]. During

the procedure, bacteria are exposed to diﬀerent concentrations of a test compound for 90 min in a

medium containing suﬃcient histidine (S. typhimurium) or tryptophan (E. coli) to support cell divisions.

Then, the cultures are diluted in pH indicator medium lacking histidine or tryptophan and aliquoted

into 48 wells of a 384-well plate. Within 48 hours, cells that have undergone reversion to amino acid

prototrophy will grow into colonies. Bacterial metabolism reduces pH of the medium, changing the

color of that well from purple to yellow (https://ww w .xenometrix.ch [77]).

The following strains were used in the study: S. typhimurium TA98, TA100, TA1535, TA1537

and E. coli wp2 uvrA[pKM101]. TA100, TA1535 and E. coli strains are used for the detection of base

substitution mutations, whereas TA98 and TA1537 are utilized for the detection of frameshift mutations.

S. typhimurium strains have GC base pairs, whereas E. coli strain has AT base pair at their primary

reversion site and detect certain oxidizing mutagens, cross-linking agents and hydrazines.

The test procedure provided by Xenometrix (https://www.xenometrix.ch [77]) and described

earlier was followed [78

–

80]. Brieﬂy, the bacterial strains were grown overnight in exposure medium

◦

and exposed to test compounds 4g and 3b in 24-well plates for 90 min at 37 C with agitation in

the absence (–S9) or presence (+S9) of 4.5% phenobarbital/ β-naphthoﬂavone-induced rat liver S9.

Molecules 2019, 24, 2321

24 of 29

Compounds 4g and 3b were dissolved in DMSO and tested at ﬁnal concentrations of 0.1, 0.2 and

0.5 mM. After the preincubation, the cultures were diluted in the indicator medium and the contents of

each 24-well culture were distributed into 48 wells on a 384-well plate and incubated further for 48 h at

37 ^◦C without agitation. After the exposure, the number of wells containing bacteria was scored by eye

for yellow wells. Positive and negative controls were included in the assay, and all doses were done

in triplicate.

Positive controls used for the MPF protocol were 2-nitroﬂuorene (2-NF) at 2

–S9); 4-nitroquinoline-N-oxide (4-NQO) at 0.1 g/mL (TA100, –S9); N4-aminocytidine (N4-ACT) at

100 g/mL (TA1535, –S9); 9-aminoacridine (9-AAc) at 15 g/mL (TA1537, -S9); 4-NQO at 2 g/mL

(E.coli uvrA[pKM101], –S9); 2-aminoanthracene (2-AA) at 0.5 g/mL (TA98, + S9); 2-AA at 1.25 g/mL

(TA100, + S9); 9-AA at 2.5 g/mL (TA1535 and TA1537, + S9); 2-aminoﬂuorene (2-AF) at 400 g/mL

(E.coli uvrA[pKM101], + S9). Pure DMSO was used as the negative control.

µg/mL (TA98,

µ

To evaluate the test results the following criteria were used: the fold increase in the number of

positive wells over the solvent control baseline (FIB), and the dose dependency. The fold increase of

revertants relative to the solvent control was determined by dividing the mean number of positive

wells at each dose by the solvent control at baseline. The solvent control at baseline was deﬁned as

the mean number of positive wells in the solvent control plus one standard deviation (SD). When an

increase of more than 2-fold relative to the baseline at more than one dose with a dose–response is

observed the sample is classiﬁed as positive, whereas when no response >2 times the baseline and no

dose–response is stated the sample is negative [76–80].

4. Conclusions

In summary all tested compounds may be considered as potential UVA or

UVB ﬁlters.

than those of selected commercially available UV-ﬁlters.

2,2⁰-((Z)-2,4-dioxo-5-((E)-3-phenylallylidene)-imidazolidyne-1,3-diyl)diacetate) shows the best UVA

photoprotective properties among the tested series with a UVA PF of 8.43 1.35 but its photostability

Their absorption parameters are comparable or more favorable

Compound 4f (diethyl

±

is unsatisfactory. Compound 2a (ethyl (Z)-2-(4-benzylidene-2,5-dioxoimidazolidin-1-yl)acetate)

shows the best UVB photoprotective properties within the tested series with a SPF_{in vitro}

of 4.79

±

0.02, but it is only slightly soluble in the formulations used.

Considering

the other features of modern UV-ﬁlters such as good solubility in cosmetic solvents and

high photostability, the most favorable UVA-ﬁlter seems to be compound 4g (diethyl

2,2⁰-((Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate) with a UVA

PF of 6.83

is compound 3b (diethyl 2,2⁰-((Z)-4-(4-methoxybenzylidene)-2,5-dioxoimidazolidine-1,3-diyl)-

diacetate) with SPF_{in vitro}of 3.07 0.04, characterized by very good solubility and photostability.

The preliminary safety assessment of the compounds is also promising. Tested compounds at high

concentrations (50 M) are non-toxic toward human skin cells, hepatoma and neuroblastoma cells and

±

0.05 and better photostability than compound 4f. The most promising potential UVB-ﬁlter

±

µ

possess no estrogenic activity. We may conclude that neither test compounds 3b and 4g nor their

potential metabolites are base substitution or frameshift mutagens. Their metabolic stability is various

and depends on substituents in phenyl and imidazolidine ring.

Supplementary Materials: The following are available online at http://www.mdpi.com /1420-3049 /24 /12 /2321 /

s1, Figure S1: The distribution of the angle values between the planes of hydantoin and aromatic rings

in crystal structures containing a 5-benzylidenehydantoin (hyd), 5-benzylidene-2-thiohydantoin (S-hyd) or

5-benzylidene-2-selenohydantoin (Se-hyd) fragment retrieved from the CSD. Figure S2: The overlap of hydantoin

rings of 3b (carbon atoms in grey) and 4g (carbon atoms in green). The disordered fragment of 3b is depicted only

for major occupancy (

in methanol solutions (for 2a–2e

Figure S4: UV absorption spectra of tested compounds and EHMC obtained pre-irradiation and 1 hour after

irradiation with solar light simulator conducted at 500 W/m2 in 25 M (4g) or 50 M (2d, 3b, EHMC) methanol

solutions. Figure S5: The chromatograms and mass spectra of methanol solutions of compounds 3b and 4g

pre-irradiation ( ) and 1 hour after irradiation ( ) with solar light simulator conducted at 500 W/m2. Table S1:

A

). Figure S3: UV-absorption spectra of tested compounds and reference UV ﬁlters obtained

,

3a 3e, octocrylene and EHMC at 50 M, for 4f 4h and avobenzone at 25 M).

–

µ

–

µ

A

B

Molecules 2019, 24, 2321

25 of 29

Parameters of intermolecular interactions in the crystal structures of 3b and 4g. Table S2: UV absorption changes

after 1h irradiation at 500 W/m²of methanol solution of tested compounds and reference UV-ﬁlters. Table S3:

Mutagenic activity of compounds 4g and 3b tested with the Ames assay.

Author Contributions: Data curation, J.P. and A.G.-K.; Formal analysis, J.P.; Funding acquisition, A.G.-K., K.S.

˙

and E.P.; Investigation, J.P., K.P., D.Z., P.K., K.S., K.W.-P., A.K., E.Z., W.N. and P.Z.; Methodology, J.P., K.P., D.Z.,

˙

P.K., K.S., K.W.-P., A.K., A.K.-P., E.Z., W.N. and P.Z.; Supervision, H.M. and E.P.; Validation, E.P.; Visualization, J.P.;

Writing—Original Draft, J.P. and A.G.-K.; Writing—Review and Editing, J.P., A.G.-K. and E.Z.

˙

Funding: This research was funded by the POLISH NATIONAL SCIENCE CENTRE, grant number

2016/21/B/NZ7/01756.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Discovery of novel UV-filters with favorable safety profiles in the 5-arylideneimidazolidine-2,4-dione derivatives group

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