HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 2, 2014, pp. 453-464
Received, 17th October, 2013, Accepted, 9th January, 2014, Published online, 20th January, 2014.
DOI: 10.3987/COM-13-12865

■ Identification, Synthesis and Photo-protection Evaluation of Arylthiazole Derivatives as a Novel Series of Sunscreens

Guoliang Li, Yundong He, Wenbo Zhou, Peng Wang, Yong Zhang, Weiguang Tong, Haigang Wu, Mingyao Liu, Xiyun Ye,* and Yihua Chen*

Institute of Biomedical Sciences and School of Life Sciences, Shanghai Key Laboratory of Regulatory Biology, East China Normal University, 500 Dongchuan Rd., Minhang, Shanghai 200241, China

Abstract

A novel series of aryl thiazole derivatives have been designed, synthesized and evaluated in preventing keratinocyte cell (HaCaT) from UVB exposure induced cellar damage. The structure-activity relationship (SAR) was discussed from chemical modification of amide, aryl-thiazole rings and the linker. Preliminary data suggested that four compounds (5a, 11a, 13c and 17) effectively protected Keratinocytes cell from UVB induced cell death compare with positive control polydatin. More importantly, compound 5a significantly protected the dorsal skin of BALB/c-nu mice against UVB-induced decrustation in vivo. The in vitro and in vivo data for these aryl thiazole derivatives suggest further studies for their potential use as photo-protection agents as well as sunscreen candidates.

Excessive exposure to ultraviolet radiation (UV) is believed to induce various skin diseases such as sunburn, photo-aging and photo-immunosuppression.1-3 Moreover, skin oncology research has demonstrated that acute or chronic UV exposure is responsible for photo-carcinogenesis as one of the key factors in the process of skin cancer.4-6 Commercial sunscreens have been used since 1928 as one of the most effective and prevalent methods of photo-protection, which have still played a major role in decreasing UV radiation and skin cancer occurrence until today.7 Among UV light, UVB (290-320 nm) is a predominant and most harmful component, which was regarded as main reasons for the most severe damages.8,9 As a result, more concern should be taken to develop sunscreen products capable of preventing UVB exposure. Up to date, FDA has approved a series of UVB organic sunscreen filters, which include aminobenzoates, cinnamates, salicylates, octocrylene and ensulizole.10 However, several striking weaknesses such as photoallergic reaction or estrogen-like side effects restrict the popularity of these sunscreens.11 In addition, a limited number of sunscreen products can’t meet our growing demand. Thus discovering and developing more effective and safer sunscreens that minimize damage from UVB radiation is still a crucial goal of our research.
In this study, a series of arylthiazole derivatives were identified as a novel class of sunscreens for their potent protective activity against UVB-induced damage. By screening our chemical library with high structural diversity which was derived from ChemBridge company as well as set up in our laboratory, we found compound 1 (Figure 1), an arylthiazole derivative exerted commendable ability to protect keratinocytes cell from UVB-induced damage. Arylthiazole derivatives were reported to show various biological activities such as antitumor,12 anti-tubercular,13 anti-virulence,14 accelerating neuronal differentiation.15 But whether these arylthiazole compounds showed the function of reversing or blocking UVB-induced damage or not still remained unclear. Therefore, a series of novel arylthiazole derivatives based on compound 1 were designed, synthesized and their activity were assessed for their photo-protection effects. The most potent compound 5a was validated through cell viability assay in vitro and skin damage experiment in vivo. Compound 5a effectively protected UVB-induced skin damage of nude mice, suggesting it was a novel potential photo-protection agent or a promising sunscreen candidate.

Synthesis of 2-(4-pyridinyl)thiazole derivatives (4a−d, 5a−d, 6a−c, 8, 9a−c). Compounds 4a−d, 5a−d, 6a−c, 8 and 9a−c were prepared using the methods outlined in Scheme 1. Compounds 2a−d16 were hydrolyzed to corresponding acids 3a−d, compound 3b was further coupled with arylamines in the presence of EDC·HCl and HOBt to provide compounds 4a−d. Coupling of acid 3b with a variety of amino acid esters gave compounds 5a−d, while 6a−c were afforded based on 3a and 3c−d coupled with glycine ethyl ester hydrochloride. In addition, compound 2b can also be reduced under lithium aluminium hydride to give compound 7 in a high yield, which was then reacted with methyl bromoacetate in the presence of sodium hydride at ambient temperature to generate intermediate 8, which was hydrolyzed and then coupled with various amines to afford compounds 9a−c.

Synthesis of 2-phenylthiazole derivatives (11a−f). To synthesize compounds 11a−f, a series of commercial or prepared substituted thiobenzamides were selected to react with ethyl 2-bromo-3-oxobutanoate to afford corresponding esters,16 which were then hydrolyzed by lithium hydroxide monohydrate to obtain compound 10a−f, then coupled with glycine ethyl ester hydrochloride under EDC·HCl and HOBt conditions to give target compounds 11a−f in a satisfactory yield.

Synthesis of compound 13a-c. Intermediates 12a−c were prepared using standard palladium-catalyzed cross-coupling reaction followed by oxidation with selenium dioxide.17,18 Compounds 12a−c were subsequently coupled with glycine ethyl ester hydrochloride to give target compounds 13a−c, as shown in Scheme 3.

Synthesis of compound 15 and 17. Compounds 15 and 17 were afforded through intermediates 1419 and 16,20 which were hydrolyzed, and then coupled with glycine ethyl ester hydrochloride to give compounds 15 and 17.

Structure-activity relationship of arylthiazole derivatives
To investigate the photo-protection activities of these arylthiazole derivatives, polydatin15 and BP-3 (2-hydroxy-4-methoxy-benzophenone)16 which showed photo-protection and sunscreen activities were selected as positive controls. For demonstration of structure-activity relationships, a series of compounds containing arylthiazole skeleton derived from compound 1 were evaluated for their photo-protective effects against 30 mJ/cm2 of UVB exposure which was appropriate to induce about 50% HaCaT cell death. The strategic modification was shown in Figure 1. Firstly, piperidinyl group of compound 1 was replaced by alkyl or alkenyl amines with the retention of 2-(2-ethylpyrdinyl)thiazole scaffold. Unfortunately the result indicated that protective effects of these compounds were significantly lost (data not shown). Secondly, substitutive anilines with different electron-donating or electron-withdrawing properties were selected to afford compounds 4a-d based on coupling with the 2-(2-ethylpyrdinyl)thiazole scaffold of the hit compound 1. Examination of the assay result, compound 4c with introduction of 3-methoxyphenyl group showed mild protection against UVB exposure as compound 1, though the protection effect was weaker than positive controls polydatin and BP-3 (Figure 3). These results suggested that more diverse substitution would be explored to increase activities. Subsequently a variety of chainlike amino acid esters with different length were chosen to couple with 2-(2-ethylpyrdinyl)thiazole carboxylic acid 3b because amino acid ligands usually contributed to potent protection against UVB induced damage.17 Interestingly, the length of chain between amino and terminal carboxyl group of amino acid demonstrated extremely influences toward protective activity. When n = 1, compound 5a provided best protective effect among them (5a−d) and superior to positive controls polydatin and BP-3 in the same concentration, while the potency was decreased when increasing chain length (5a >5b >5c >5d). Thus compound 5a was chosen for subsequent optimizations (Figures 1, 2).

The following chemical modifications of compound 5a were divided into four parts. As listed in Figure 2 and Figure 3, the first part was that the substitution in pyridinyl group, alkyl substitutions (6b−c) in this position as well as hydrogen atom (6a) were explored, which showed that a suitable small alkyl group was crucial for maintaining the activity. For example, when the position was not substituted (6a), the activity was decreased compared to 5a, while if the substitution was a large group such as butyl group (6c), it showed toxicity against the HaCat cell instead of protection. Our interest was then turned over other parts of compound 5a. Next, when the linker amide of 5a was investigated for changing to ether 8 with similar length, the activity was not increased, and replacement of ester with amide moiety in the terminal of compound 8 was also proven to decrease the activities of those compounds (9a−c). Further pyridinyl group was substituted by other biomimetic aryl groups such as substituted phenyl ring (11a−f), which resulted in the discovery of 11a with almost equal activity to compound 5a, while other substituted phenyl ring either with electron-donating or electron-withdrawing groups showed no increase in activity. These results suggested that pyridinyl group was not the necessary moiety, and other aromatic groups might lead to the bioactive compounds. Thus pyridinylthiazole group was further replaced by other heteroaromatic groups such as phenylfuran (13a), phenylthiophene (13b), phenyloxazole (13c), phenylisoxazole (15) and phenylthiazole (17). The result showed these changes were tolerant, since most of them showed good photo-protection against UVB exposure except 13a, which showed severe cytotoxicity against HaCaT cell (Figure 3). In conclusion, the introduction of amino acid moiety to the arylthiazole skeleton and the existence of amide other than ether as a linker group were necessary for retaining photo-protection activities of these compounds. Replacement of thiazole with oxazole moiety that possessed similar electron configuration was tolerant, whose activity was in line with compound 5a.
With good potency against UVB induced damage, compounds 5a, 11a, 15 and 17 were selected for comprehensive comparison. Among these candidates, compound 5a was found to show lower cytotoxicity, better photo-protection effect against UV radiation, and more favourable physical characters especially water solubility, and was subsequently chosen for further evaluation in vitro and in vivo.

In order to detect the toxicity of compound 5a against HaCaT cell, a viability test experiment was carried out and the result suggested that inferior to 500 μM, compound 5a had very weak effects on the viability of HaCaT cell, and the viability of HaCaT cell was maintained more than 75% (Figure 4A). Irradiation with 30 mJ/cm2 of UVB resulted in half of cell death compared with the non-irradiated group while treatment with compound 5a reduced HaCaT cell death induced by UVB-irradiation in a dose-dependent manner and the proportions of cell death were 35.0 ± 7.0%, 23.7 ± 6.2% and 8.1 ± 6.8% at 25, 50, 100 μM of 5a, respectively, compared with the non-irradiated group. Those results showed that the photo-protection activity of compound 5a was directly correlated with its treatment dose suggesting its potential use as a sunscreen (Figure 4B). Furthermore, more cell death was observed after UVB irradiation compared with the non-irradiated groups and compound 5a dose-dependently reduced HaCaT cell death by photograph (Figure 4C). To further investigate the mechanisms of compound 5a, the free radical scavenging capacity and UV absorption spectra of compound 5a were assessed. We found that compound 5a did not have radical scavenging activity but potently absorbed the UVB radiation at wavelength 200-400 nm (Figure S2 and Figure S3). Moreover, we compared the photo-protective effect of compound 5a with a widely known UV absorber BP-3 (2-hydroxy-4-methoxy-benzophenone).16. As shown in Figure S1C, both 5a and BP-3 have the photo-protective effect on HaCaT cells and there is no significant difference between them at the indicated concentrations. Taken together, our results indicated that 5a is an effective sunscreen candidate.

Compound 5a protects BALB/c-nu mice against UVB-induced desquamation and epidermis thickness
Compound 5a was next investigated its protective effect from UVB induced skin damages in BALB/c-nu mice. For in vivo studies, the ointment containing 5a (1.0 mM) was applied on the right side of BALB/c-nu mice dorsal skin once every 12 h while the left side as a control was treated with ointment without compound 5a. One hour later, the animals were irradiated with Bio-Sun system at wavelength 312 nm for UVB (360 mJ/cm2) irradiation once a day for 7 days. The results were listed in Figure 5A and Figure 5B. Figure 5A clearly demonstrated that 360 mJ/cm2 UVB irradiation resulted in severe desquamation whereas treatment with 1.0 mM 5a attenuated those skin damages induced by UVB irradiation. As shown in Figure 5B, the epidermis thickness of nude mice dorsal skin was incrassate after UVB exposure and the average thickness of epidermis of UVB-irradiated mice was 86.8 ± 8.4 μm which was decreased to 37.5 ± 3.7 μm after treatment with 1.0 mM 5a twice a day. Altogether, these data suggested that compound 5a effectively protect against UVB-induced skin damage in vivo.

CONCLUSION
In this study, a novel class of arylthiazole derivatives were designed and synthesized and then their effect on UVB-induced cellular damage were evaluated by measuring cell viability. The relationship between their structure and photo-protection activity was concluded. The photo-protection activity of potent compound 5a was studied at BALB/c-nu mice model. Both in vivo and in vitro experiments suggested that compound 5a had an excellent photo-protective activity on UVB-induced damage. Further studies to identify the detail mechanism of photo-protective activity of compound 5a are in process.

EXPERIMENTAL
All of the reagent and solvents used in synthesis experiments were purchased from Sigma-Aldrich and TCI Co. Ltd, and when necessary, were purified and dried by standard methods before use.
The 1H NMR Spectra were recorded with a Bruker Avance 300 MHz spectrometer instruments, using TMS as an internal standard. Chemical shifts are expressed in parts per million (ppm). Mass spectra were obtained in ESI mode via Shimadzu LCMS spectrometer. TLC and preparative thin-layer chromatography were performed on silica gel GF/UV 254, and the chromatography were performed on silica gel ( 200-300 mesh) visualized under UV light at 254 nm and 365 nm. Unless otherwise a special note, the progress of reaction was detected by TLC and protected under nitrogen. High-resolution mass data of 5a, 8, 11a, 13c, 15 and 17 were obtained on a Micromass Q-Tof UltimaTM spectrometer.
N-(Ethoxycarbonylmethyl)-2-(2-ethyl-4-pyridinyl)-4-methyl-5-thiazolecarboxamide (5a). The title compound was prepared according to 4a except using glycine ethyl ester hydrochloride instead of aniline. Yield: 44%: 1H NMR (300 MHz, CDCl3): δ 8.62 (d, J = 5.1 Hz, 1H), 7.67 (s, 1H), 7.57 (d, J = 5.1 Hz, 1H), 6.71 (br s, 1H), 3.73–3.68 (m, 5H), 2.90 (q, J = 7.2 Hz, 2H), 2.75 (s, 3H), 2.70 (t, J = 5.7 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 169.90, 165.42, 165.11, 161.56, 156.92, 150.39, 140.11, 127.22, 118.88, 118.06, 62.15, 42.17, 31.62, 17.67, 14.35, 13.98. ESI-HRMS Calculated for [C16H19N3O3S + H]+: 334.1225, found: 334.1227.
Methyl 2-((2-(2-ethyl-4-pyridinyl)-4-methyl-5-thiazolyl)methoxy)acetate (8). To a solution of compound 7 (1.22 g, 5.2 mmol) in 10 mL of DMF was added sodium hydride (60%, 312 mg, 7.8 mmol) at 0 °C with stirring for 10 min, then methyl 2-bromoacetate (0.73 mL, 7.8 mmol) was added in at the same temperature. The resultant mixture was sirring at room temperature for 2 h. The reaction was quenched with ice water and extracted with EtOAc. The organic layer was washed with water and brine in twice and then dried with anhydrous Na2SO4. After concentrated by a rotary evaporator, the products was purified by silica gel column chromatography (petroleum ether/EtOAc = 1/1) to give compound 8 (908 mg, 57% yield). 1H NMR (300 MHz, CDCl3): δ 8.58 (d, J = 5.1 Hz, 1H), 7.66 (s, 1H), 7.55 (d, J = 5.1 Hz, 1H), 4.81 (s, 2H), 4.16 (s, 2H), 3.79 (s, 3H), 2.88 (q, J = 7.2 Hz, 2H), 2.49 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). ESI-HRMS Calculated for [C15H18N2O3S + H ]+: 307.1116, found: 307.1096.
N-(Ethoxycarbonylmethyl)-2-phenyl-4-methyl-5-thiazolecarboxamide (11a). The title compound was prepared according to 5a except using compound 10a instead of 3b. Yield 63%: 1H NMR (300 MHz, CDCl3): δ 7.94–7.92 (m, 2H), 7.46–7.44 (m, 3H), 6.39 (br s, 1H), 4.27 (q, J = 6.9 Hz, 2H), 4.21 (d, J = 4.8 Hz, 2H), 2.77 (s, 3H), 1.32 (t, J = 6.9 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 170.01, 167.98, 161.99, 156.82, 133.08, 131.09, 129.28, 127.00, 125.44, 62.08, 42.15, 17.74, 14.37. ESI-HRMS Calculated for [C15H16N2O3S + Na]+: 327.0779, found: 327.0777.
N-(Ethoxycarbonylmethyl)-2-phenyl-4-methyl-5-oxazolecarboxamide (13c). The title compound was prepared according to 5a except using compound 12c instead of 3b. Yield 63%: 1H NMR (300 MHz, CDCl3): δ 8.08–8.05 (m, 2H), 7.49–7.47 (m, 3 H), 6.81 (br s, 1H), 4.28 (q, J = 6.9 Hz, 2H), 4.22 (d, J = 4.8 Hz, 2H), 2.56 (s, 3H), 1.32 (t, J = 6.9 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 170.02, 160.66, 158.49, 144.92, 138.87, 131.60, 129.15, 127.17, 126.60, 62.02, 41.12, 14.38, 13.31. ESI-HRMS Calculated for [C15H16N2O4 + Na]+: 311.1008 , found: 311.1003.
N-(Ethoxycarbonylmethyl)-3-phenyl-5-methyl-4-isooxazolecarboxamide (15). The title compound was prepared according to 5a except using 5-methyl-3-phenyl-4-isoxazolecarboxylic acid instead of 3b. Yield 44%: 1H NMR (300 MHz, CDCl3): δ 7.66–7.63 (m, 2H), 7.54–7.52 (m, 3H), 5.95 (br s, 1H), 4.16 (q, J = 7.2 Hz, 2H), 4.03 (d, J = 5.4 Hz, 2H), 2.74 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 174.78, 169.41, 161.81, 160.47, 130.72, 129.39, 129.30, 128.16, 110.70, 61.76, 41.52, 14.32, 13.28. ESI-HRMS Calculated for [C15H16N2O4 + Na]+: 311.1008, found: 311.0997.
N-(Ethoxycarbonylmethyl)-2-phenyl-4-thiazolecarboxamide (17). The title compound was prepared according to 5a except using 2-(2-ethyl-4-pyridinyl) thiazole-4-carboxylic acid instead of 3b. Yield 27%: 1H NMR (300 MHz, CDCl3): δ 9.04 (br s, 1H), 8.02 (d, J = 7.2 Hz, 2H), 7.80 (d, J = 15.3 Hz,), 7.73–7.68 (m, 1H), 7.61–7.56 (m, 2H), 7.05 (d, J = 15.3 Hz, 1H), 4.12 (q, J = 7.2 Hz, 2H), 4.00 (d, J = 2.0 Hz, 2H), 3.33 (s, 2H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 169.88, 166.14, 165.17, 161.01, 150.93, 150.45, 140.00, 124.98, 118.92, 118.04, 61.89, 41.52, 31.68, 14.39, 14.07. ESI-HRMS Calculated for [C15H17N3O3S + H]+: 320.1069, found: 320.1072.

ACKNOWLEDGEMENTS
This work was supported by the grants from National Basic Research Program of China (2012CB910404), National Major Scientific and Technological Special Project for "Significant New Drugs Development" (2013ZX09507001), National Natural Science Foundation of China (81202407), The Science and Technology Commission of Shanghai Municipality (11DZ2260300), Research Fund for the Doctoral Program of Higher Education of China (20120076120029) and the Fundamental Research Funds for the Central Universities (78210048).

References

1. G. Halliday, Mutat. Res., 2005, 571, 107. CrossRef
2. M. Ichihashi, M. Ueda, A. Budiyanto, T. Bito, M. Oka, M. Fukunaga, K. Tsuru, and T. Horikawa, Toxicology, 2003, 189, 21. CrossRef
3. N. Puizina-Ivic, Acta Dermatovenerol. Alp. Panonica Adriat., 2008, 17, 47.
4. F. de Gruijl, H. van Kranen, and L. Mullenders, J. Photochem. Photobiol. B, 2001, 63, 19. CrossRef
5. E. Gonzaga, Am. J. Clin. Dermatol., 2009, 10 Suppl 1, 19. CrossRef
6. K. Rass and J. Reichrath, Adv. Exp. Med. Biol., 2008, 624, 162. CrossRef
7. D. Sambandan and D. Ratner, J. Am. Acad. Dermatol., 2011, 64, 748. CrossRef
8. H. Ryu, C. Kim, J. Kim, J. Chung, and J. Kim, J. Invest. Dermatol., 2010, 130, 1095. CrossRef
9. S. Wu, L. Wang, A. Jacoby, K.Jasinski, R. Kubant, and T. Malinski, Photochem. Photobiol., 2010, 86, 389. CrossRef
10. A. Chen and H. Lim, In Lifestyle of Medicine, 2 edition: J. Rippe, Ed.; CRC Press, 2010, 965.
11. M. Palm and M. O'Donoghue, Dermatol. Ther., 2007, 20, 360. CrossRef
12. Y. Lu, C. Li, Z. Wang, J. Chen, M. Mohler, W. Li, J. Dalton, and D. Miller, J. Med. Chem., 2011, 54, 4678. CrossRef
13. X. Lu, B. Wan, S. Franzblau, and Q. You, Eur. J. Med. Chem., 2011, 46, 3551. CrossRef
14. N. Desroy, F. Moreau, S. Briet, G. Le Fralliec, S. Floquet, L. Durant, V. Vongsouthi, V. Gerusz, A. Denis, and S. Escaich, Bioorg. Med. Chem., 2009, 17, 1276. CrossRef
15. H. Wurdak, S. Zhu, K. Min, L. Aimone, L. Lairson, J. Watson, G. Chopiuk, J. Demas, B. Charette, R. Halder, E. Weerapana, B. Cravatt, H. Cline, E. Peters, J. Zhang, J. Walker, C. Wu, J. Chang, T. Tuntland, C. Cho, and P. Schultz, Proc. Natl. Acad. Sci. U S A, 2010, 107, 16542. CrossRef
16. M. Sierra, V.Beneton, A. Boullay, T. Boyer, A. Brewster, F. Donche, M. Forest, M. Fouchet, F. Gellibert, D. Grillot, M. H. Lambert, A. Laroze, C. Le Grumelec, J. Linget, V. Montana, V. Nguyen, E. Nicodeme, V. Patel, A. Penfornis, O. Pineau, D. Pohin, F. Potvain, G. Poulain, C. Ruault, M. Saunders, J. Toum, H. Xu, R. Xu, and P. Pianetti, J. Med. Chem., 2007, 50, 685. CrossRef
17. J. Yano, T. Denton, M. Cerny, X. Zhang, E. Johnson, and J. Cashman, J. Med.Chem., 2006, 49, 6987. CrossRef
18. B. Travis, M. Sivakumar, G. Hollist, and B. Borhan, Org. Lett., 2003, 5, 1031. CrossRef
19. P. Maloney, D. Parks, C. Haffner, A. Fivush, G. Chandra, K. Plunket, K. Creech, L. Moore, J. Wilson, M. Lewis, S. Jones, and T. Willson, J. Med. Chem., 2000, 43, 2971. CrossRef
20. R. Rzasa, M. Kaller, G. Liu, E. Magal, T. Nguyen, T. Osslund, D. Powers, V. Santora, V. Viswanadhan, H. Wang, X. Xiong, W. Zhong, and M. Norman, Bioorg. Med. Chem., 2007, 15, 6574. CrossRef