Synthesis of 1,2,3-Triazole Benzophenone Derivatives and Evaluation of in vitro Sun Protection, Antioxidant Properties, and Antiproliferative Activity on HT-144 Melanoma Cells

Maria C. F. Dias Bianca L. de Sousa Marisa Ionta Róbson R. Teixeira Thiago Q. Goulart Guilherme Á. Ferreira-Silva Eduardo J. Pilau Marcelo H. dos Santos About the authors

Abstract

Benzophenones display several biological activities, including antioxidant, anticancer, and photoprotective. Furthermore, antioxidants can minimize both ultraviolet absorption and tumor development. In the present investigation, a series of twenty-six 1,2,3-triazole-benzophenone derivatives were synthesized and had their antioxidant, anticancer, and photoprotective effects evaluated. For the compounds synthesis, 4,4’-dihydroxybenzophenone (1a) and 2,4-dihydroxybenzophenone (1b) were propargylated, affording the alkynes bis(4-(prop-2-yn-1-yloxy))benzophenone (2a) and (2-hydroxy-4-(prop-2-yn-1-yloxy))benzophenone (2b), respectively. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between the compounds 2a/2b and several benzyl azides gave the 1,2,3-triazole-benzophenone derivatives with yields ranging from 35 to 95%. The 1,2,3-triazole-benzophenone derivatives at the concentration of 0.2 μg mL-1 (a no-cytotoxic concentration) exhibited a solar protection factor (SPF) comparable to positive control benzophonen-3 (BP-3). Concerning their antioxidant and cytotoxic effects, the derivatives from 1b showed high in vitro antioxidant effects as well as cytotoxicity against A549 (lung carcinoma), MCF-7 (breast carcinoma), and HT-144 (metastatic melanoma) cell lines, without significant cytotoxicity to a non-cancerous cell line. Derivatives 19, 20, and 24 induced cell death and cell cycle arrest at G1/S in HT-144 melanoma cells.

Keywords:
benzophenone; 1,2,3-triazole; CuAAC reaction; antioxidant; photoprotective; cytotoxicity


Introduction

Benzophenones are a class of relevance to medicinal chemistry and industrial fields.11 Surana, K.; Chaudhary, B.; Diwaker, M.; Sharma, S.; Med. Chem. Commun. 2018, 9, 1803. These substances exhibit a wide spectrum of biological activities including antibacterial,22 Aranda, M. I. R.; Gomez, G. A. T.; de Barros, M.; dos Santos, M. H.; de Oliveira, L. L.; Pena, J. L.; Moreira, M. A. S.; Front. Microbiol. 2019, 10, 490. anti-human immunodeficiency virus (HIV),33 Song, Z.; Wang, P.; Huang, S.; Wang, C.; Wang, R.-R.; Yang, L.-M.; Zhen, Y.; Liu, K.; Zheng, Y.-T.; Ma, X.; Med. Chem. 2016, 13, 398. antioxidant,44 Skorkowska, A.; Maciejska, A.; Pomierny, B.; Krzyżanowska, W.; Starek-Świechowicz, B.; Bystrowska, B.; Broniowska, Ż.; Kazek, G.; Budziszewska, B.; Neurotoxic. Res. 2020, 37, 683. and antitumor.55 Ionta, M.; Ferreira-Silva, G. A.; Niero, E. L.; Costa, E. D. M.; Martens, A. A.; Rosa, W.; Soares, M. G.; Machado-Santelli, G. M.; Lago, J. H. G.; Santos, M. H.; Molecules 2015, 20, 12804.,66 Sales, L.; de Sousa, G. R.; Ferreira-Silva, G.; Castro-Gamero, A. M.; Ionta, M.; de Oliveira, J. C.; Anticancer Drugs 2016, 28, 298. Moreover, the role of the benzophenones and their derivatives as photoprotective agents has been well documented77 Suzuki, T.; Kitamura, S.; Khota, R.; Sugihara, K.; Fujimoto, N.; Ohta, S.; Toxicol. Appl. Pharmacol. 2005, 203, 9.

8 Rodriguez-Gomez, R.; Zafra-Gomez, A.; Dorival-Garcia, N.; Ballesteros, O.; Navalon, A.; Talanta 2015, 134, 657.
-99 Liu, W.; Wei, D.; Liu, Q.; Du, Y.; Chemosphere 2016, 154, 491. and some of them have been incorporated in sunscreen formulations.88 Rodriguez-Gomez, R.; Zafra-Gomez, A.; Dorival-Garcia, N.; Ballesteros, O.; Navalon, A.; Talanta 2015, 134, 657.,1010 Barbosa, T. C.; Nascimento, L. E. D.; Bani, C.; Almeida, T.; Nery, M.; Santos, R. S.; Menezes, L. R. O.; Zielinska, A.; Fernandes, A. R.; Cardoso, J. C.; Jager, A.; Jager, E.; Sanchez-Lopez, E.; Nalone, L.; Souto, E. B.; Severino, P.; Toxics 2019, 7, 51.

Hydroxybenzophenones, such as 2,4-dihydroxy-benzophenone, 2,2’,4,4’-tetrahydroxy-benzophenone, and their derivatives, have a high ability to absorb and dissipate ultraviolet (UV) radiations, UVA and UVB.77 Suzuki, T.; Kitamura, S.; Khota, R.; Sugihara, K.; Fujimoto, N.; Ohta, S.; Toxicol. Appl. Pharmacol. 2005, 203, 9. Excessive skin exposure to UVB radiation is related to photo-aging due to the degradation of important structures of the dermal extracellular matrix, such as elastin and collagen.1111 Liyanaarachchi, G. D.; Samarasekera, J. K. R. R.; Mahanama, K. R. R.; Hemalal, K. D. P.; Ind. Crops Prod. 2018, 111, 597. These effects promoted by UVB exposure can be minimized by antioxidant agents, which include different types of substances.1212 Craythorne, E.; Al-Niami, F.; Medicine 2017, 45, 429.,1313 Watson, M.; Holman, D. M.; Maguire-Eisen, M.; Semin. Oncol. Nurs. 2016, 32, 241.

Phenolic compounds are an important class of antioxidants, particularly hydroxy benzophenones.1414 Navarro, M.; Arnaez, E.; Moreira, I.; Quesada, S.; Azofeifa, G.; Wilhelm, K.; Vargas, F.; Chen, P.; Foods 2019, 8, 384.,1515 Nguyen, L. H.; Venkatraman, G.; Sim, K. Y.; Harrison, L. J.; Phytochemistry 2005, 66, 1718. For instance, it has been reported1616 Yamaguchi, F.; Ariga, T.; Yoshimura, Y.; Nakazawa, H.; J. Agric. Food Chem. 2000, 48, 180. that garcinol, a natural polyisoprenylated and phenolic benzophenone extracted from the fruit peels of Garcinia indica, has high antioxidant activity being three times more potent towards 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical than DL-α-tocopherol. In another investigation, Doriguetto et al.1717 Doriguetto, A. C.; Martins, F. T.; Ellena, J.; Salloum, R.; dos Santos, M. H.; Moreira, M. E. C.; Schneedorf, J. M.; Nagem, T. J.; Chem. Biodiversity 2007, 4, 488. evaluated the antioxidant activity of 2,2’,4-trihydroxybenzophenone and compared its effect to the parent benzophenone. The 2,2’,4-trihydroxybenzophenone was more active than benzophenone and this superior antioxidant activity was linked to the presence of the ortho-hydroxy groups. The authors1717 Doriguetto, A. C.; Martins, F. T.; Ellena, J.; Salloum, R.; dos Santos, M. H.; Moreira, M. E. C.; Schneedorf, J. M.; Nagem, T. J.; Chem. Biodiversity 2007, 4, 488. also reported that these groups increase the hydrogen donation power due to the electronic delocalization of the unpaired electron of the generated phenoxy radicals.

Natural antioxidants protect the organism from cellular damage resulting from an excess of free radicals, which are responsible for inducing oxidative stress. Oxidative stress, in turn, is associated with many pathological conditions including cancer.1818 Lushchak, V. I.; Chem.-Biol. Interact. 2014, 224, 164.,1919 Poprac, P.; Jomova, K.; Simunkova, M.; Kollar, V.; Rhodes, C. J.; Valko, M.; Trends Pharmacol. Sci. 2017, 38, 592. Indeed, oxidative stress has an influence on tumor development and progression by modulating different biological processes including proliferation, survival, and invasion.2020 Pisoschi, A. M.; Pop, A.; Eur. J. Med. Chem. 2015, 97, 55.,2121 Sosa, V.; Moline, T.; Somoza, R.; Paciucci, R.; Kondoh, H.; LLeonart, M. E.; Ageing Res. Rev. 2013, 12, 376.

Cancer is a leading cause of death worldwide. In 2018, there were 18 million new cancer cases and an increase of 21.6 million is estimated by 2030.2222 Saidi, L.; Rocha, D. H. A.; Talhi, O.; Bentarzi, Y.; Nedjar-Kolli, B.; Bachari, K.; Paz, F. A. A.; Helguero, L. A.; Silva, A. M. S.; ChemMedChem 2019, 14, 1041. Therefore, it is imperative to identify new substances that effectively could improve therapeutic propose for cancer.2323 Gholampour, M.; Ranjbar, S.; Edraki, N.; Mohabbati, M.; Firuzi, O.; Khoshneviszadeh, M.; Bioorg. Chem. 2019, 88, 102967. In the last years, extensive efforts have been made to find new, less toxic, and effective therapeutic drugs for cancer treatment.

Among the various strategies that have been used by the pharmaceutical industry to obtain new drugs, it stands out the use of click chemistry. Considering the products that can be obtained via the click chemistry approach, it should be mentioned substances bearing the 1,2,3-triazole ring, a well-recognized pharmacophoric group.2424 Hou, W.; Zhang, G.; Luo, Z.; Su, L.; Xu, H.; Chem. Biol. Drug Des. 2019, 93, 473.,2525 Mareddy, J.; Suresh, N.; Kumar, C. G.; Kapavarapu, R.; Jayasree, A.; Pal, S.; Bioorg. Med. Chem. Lett. 2017, 27, 518. The 1,2,3-triazole fragment is of great importance in medicinal chemistry and it has been involved in the synthesis of several heterocyclic compounds with important biological activities, including antibacterial,2626 Torres, L. M. F. C.; Almeida, M. T.; Santos, T. L.; Marinho, L. E. S.; de Mesquita, J. P.; da Silva, L. M.; dos Santos, W. T. P.; Martins, H. R.; Kato, K. C.; Alves, E. S. F.; Liao, L. M.; de Magalhaes, M. T. Q.; de Mendonca, F. G.; Pereira, F. V.; Resende, J. M.; Bemquerer, M. P.; Rodrigues, M. A.; Verly, R. M.; Colloids Surf., B 2019, 177, 94. antifungal,2626 Torres, L. M. F. C.; Almeida, M. T.; Santos, T. L.; Marinho, L. E. S.; de Mesquita, J. P.; da Silva, L. M.; dos Santos, W. T. P.; Martins, H. R.; Kato, K. C.; Alves, E. S. F.; Liao, L. M.; de Magalhaes, M. T. Q.; de Mendonca, F. G.; Pereira, F. V.; Resende, J. M.; Bemquerer, M. P.; Rodrigues, M. A.; Verly, R. M.; Colloids Surf., B 2019, 177, 94. for the treatment of Alzheimer’s disease,2727 Di Pietro, O.; Alencar, N.; Esteban, G.; Viayna, E.; Szałaj, N.; Vazquez, J.; Juarez-Jimenez, J.; Sola, I.; Perez, B.; Sole, M.; Unzeta, M.; Munoz-Torrero, D.; Luque, F. J.; Bioorg. Med. Chem. 2016, 24, 4835. anticancer,2828 Banerji, B.; Chandrasekhar, K.; Sreenath, K.; Roy, S.; Nag, S.; Saha, K. D.; ACS Omega 2018, 3, 16134.,2929 Yamada, M.; Takahashi, T.; Hasegawa, M.; Matsumura, M.; Ono, K.; Fujimoto, R.; Kitamura, Y.; Murata, Y.; Kakusawa, N.; Tanaka, M.; Obata, T.; Fujiwara, Y.; Yasuike, S.; Bioorg. Med. Chem. Lett. 2018, 28, 152. and antioxidant.3030 Saraiva, M. T.; Kruger, R.; Baldinotti, R. S. M.; Lenardao, E. J.; Luchese, C.; Savegnago, L.; Wilhelm, E. A.; Alves, D.; J. Braz. Chem. Soc. 2016, 27, 41.,3131 Tan, W.; Li, Q.; Li, W.; Dong, F.; Guo, Z.; Int. J. Biol. Macromol. 2016, 82, 404.

The biological properties exhibited by both benzophenones and compounds containing the 1,2,3-triazole fragments, among them photoprotective, cytotoxic, and antioxidant, prompted us to design and synthesize new benzophenones bearing 1,2,3-triazole fragments aimed to find compounds with relevant biological effects. Herein, we describe the synthetic steps involved in the preparation of these new 1,2,3-triazole-benzophenone derivatives and the results related to the evaluation of their photoprotective, antioxidant, and cytotoxic activities.

Results and Discussion

The synthetic strategy used to prepare the 1,2,3-triazole-benzophenone derivatives is depicted in Figure 1. For their synthesis, it was required the preparation of several benzyl azides, which was carried out from the corresponding commercially available benzyl bromides and using procedures that have been described in the literature.3232 Dias, M. C. F.; Gularte, T. Q.; Teixeira, R. R.; Santos, J. A. N.; Pilau, E. J.; Mendes, T. A. O.; Demuner, A. J.; dos Santos, M. H.; J. Braz. Chem. Soc. 2019, 30, 97.

Figure 1
Reagents and conditions: (i) sodium azide (4.00 equiv.), DMSO, r.t., 2 h, 80-90% yield; (ii) propargyl bromide (2.40 equiv.), K2CO3 (4.00 equiv.), acetone, reflux, 24 h, 83%; (iii) propargyl bromide (1.20 equiv.), K2CO3 (2.00 equiv.), acetone, reflux, 24 h, 61%; (iv) CuSO4∙5H2O (0.400 equiv.), sodium ascorbate (0.800 equiv.), DCM/H2O (1:1 v/v), r.t., 6 h, 42-70%; (v) CuSO4∙5H2O (0.200 equiv.), sodium ascorbate (0.400 equiv.), DCM/H2O (1:1 v/v), r.t., 6 h, 35-95%.

The synthesis of the benzophenone derivatives containing 1,2,3-triazole fragments was performed in two steps. The first one corresponded to the propargylation reaction of 4,4’-dihydroxybenzophenone (1a) and 2,4-dihydroxybenzophenone (1b), producing the terminal alkynes bis(4-(prop-2-yn-1-yloxy))benzophenone (2a) and (2-hydroxy-4-(prop-2-yn-1-yloxy))benzophenone (2b) in 83 and 68% yields, respectively. Next, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also known as click reaction, between the alkynylated compounds 2a/2b and benzyl azides afforded the 1,2,3-triazole-benzophenone derivatives 4-29 (Figure 1) with yields in the range of 35 to 95%. All the derivatives were characterized by infrared (IR) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopies as well as high-resolution mass spectrometry (HRMS). In the 1H NMR spectra, the hydrogen atoms of the methylene groups attached to nitrogen or oxygen were noticed as singlets within 5.42-5.61 and 5.20-5.25 ppm ranges. The signals for the hydrogens present in the triazolic rings were observed as singlets within 7.44-8.32 ppm range. The carbon chemical shifts are compatible with the structures of the compounds. Further, in the IR spectra, expected bands for functional groups were noticed. Finally, the molecular formulas of the triazolic derivatives were confirmed by HRMS analysis. The spectroscopic and spectrometric data of the 1,2,3-triazole-benzophenone derivatives are available in the Supplementary Information.

Once prepared, the compounds were submitted to biological assays to assess their photoprotective, antioxidant, and cytotoxic effects.

In vitro sun protection factor (SPF) evaluation

The 1,2,3-triazolic-benzophenone derivatives 2a, 2b, 4-7, 10, 11, 13, 15, 17-28 were evaluated regarding their in vitro SPF at 0.2 μg mL-1, a no-cytotoxic concentration. These compounds were selected since they were soluble in ethanol, the solvent used to conduct the assays. All compounds showed SPF approximately equal to 5.00 (Table 1). According to the Brazilian legislation (National Health Surveillance Agency (ANVISA) Resolution,3434 Agencia Nacional de Vigilancia Sanitaria (ANVISA); Resolucao - RDC No. 30, de 1 de junho de 2012, Aprova o Regulamento Técnico Mercosul sobre Protetores Solares em Cosméticos e Dá Outras Providências; Ministerio da Saude: Brasilia, Brazil, 2012, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0030_01_06_2012.html#:~:text=Aprova%20o%20Regulamento%20T%C3%A9cnico%20Mercosul,Regulamento%20aprovado%20pelo%20Decreto%20n, accessed in October 2020.
http://bvsms.saude.gov.br/bvs/saudelegis...
RDC number 30, June 2012), the evaluated compounds have low photoprotective activity since the determined SPF values were smaller than or equal to 6.0. Statistical analyses were performed for comparison between the SPF values of triazolic derivatives and benzophenone-3 (BP-3). Only compound 18 presented statistical difference as compared to the positive control BP-3 (p < 0.05).

Table 1
Solar protection factor (SPF) values (at the concentration of 0.2 µg mL-1), molar absorptivity (ε), molecular weight (MW) and MlogP for the benzophenone derivatives. Benzophenone-3 (BP-3) was used as positive control

Brazilian legislation (ANVISA Resolution,3535 Agencia Nacional de Vigilancia Sanitaria (ANVISA); Resolucao - RDC No. 48, de 16 de marco de 2006, Aprova o Regulamento Técnico sobre Lista de Substâncias que Não Podem Ser Utilizadas em Produtos de Higiene Pessoal, Cosméticos e Perfumes; Ministerio da Saude: Brasilia, Brazil, 2006, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2006/rdc0048_17_03_2006.html#:~:text=Fica%20proibido%20o%20uso%20em,ou%20t%C3%B3xicas%20para%20a%20reprodu%C3%A7%C3%A3o, accessed in October 2020.
http://bvsms.saude.gov.br/bvs/saudelegis...
RDC number 48, 2006) also requires, for sunscreen efficacy and safety, the following attributes for compounds to be used in sun protection products: (i) UV light absorption in the range 280-400 nm; (ii) molar absorptivity (ε) above 1044 Skorkowska, A.; Maciejska, A.; Pomierny, B.; Krzyżanowska, W.; Starek-Świechowicz, B.; Bystrowska, B.; Broniowska, Ż.; Kazek, G.; Budziszewska, B.; Neurotoxic. Res. 2020, 37, 683. M-1 cm-1; (iii) low water solubility. The compounds 2a, 2b, 4-7, 10, 11, 13, 15, 17-28 presented UV absorption near to 290 nm (see Supplementary Information, Figure S113). As can be noticed in Table 1, the molar absorptivities of triazolic derivatives are in agreement with the legislation. Water solubility can be associated with MlogP (logP calculated by the Moriguchi method using MedChem Designer© 3.1.0.30 software).3333 MedChem Designer, version 3.1.0.30; Simulations Plus, Inc, Lancaster, USA, 2011. We found MlogP values between 3.54 and 5.28 to triazolic derivatives and 4.20 to BP-3. These values indicated that compounds present water solubility comparable to BP-3. Taking together, the data show that the properties of all benzophenone derivatives synthesized in this work are in consonance with Brazilian legislation3434 Agencia Nacional de Vigilancia Sanitaria (ANVISA); Resolucao - RDC No. 30, de 1 de junho de 2012, Aprova o Regulamento Técnico Mercosul sobre Protetores Solares em Cosméticos e Dá Outras Providências; Ministerio da Saude: Brasilia, Brazil, 2012, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0030_01_06_2012.html#:~:text=Aprova%20o%20Regulamento%20T%C3%A9cnico%20Mercosul,Regulamento%20aprovado%20pelo%20Decreto%20n, accessed in October 2020.
http://bvsms.saude.gov.br/bvs/saudelegis...
,3535 Agencia Nacional de Vigilancia Sanitaria (ANVISA); Resolucao - RDC No. 48, de 16 de marco de 2006, Aprova o Regulamento Técnico sobre Lista de Substâncias que Não Podem Ser Utilizadas em Produtos de Higiene Pessoal, Cosméticos e Perfumes; Ministerio da Saude: Brasilia, Brazil, 2006, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2006/rdc0048_17_03_2006.html#:~:text=Fica%20proibido%20o%20uso%20em,ou%20t%C3%B3xicas%20para%20a%20reprodu%C3%A7%C3%A3o, accessed in October 2020.
http://bvsms.saude.gov.br/bvs/saudelegis...
concerning photoprotective substances.

The penetrability of compounds used in sunscreen formulations into the skin is affected by their molecular weights. Compounds presenting molecular weights below 500 Daltons can easily penetrate the skin’s barrier and be absorbed.3636 Heo, S.; Hwang, H. S.; Jeong, Y.; Na, K.; Carbohydr. Polym. 2018, 195, 534.

Benzophenones typically used as UV filters and absorbers, such as BP, BP-1, and BP-3, have low molecular weight (below 500 Daltons) and, as a result, can diffuse through the skin and accumulate in the body. The diffusion of benzophenone can make the skin more susceptible to exposure to UV radiation. Besides, the aforementioned benzophenones are endocrine disruptors and their presence inside the body can interfere with several biological processes.3737 Krause, M.; Frederiksen, H.; Sundberg, K.; Jorgensen, F. S.; Jensen, L. N.; Norgaard, P.; Jorgensen, C.; Ertberg, P.; Juul, A.; Drzewiecki, K. T.; Skakkebaek, N. E.; Andersson, A. M.; Environ. Int. 2018, 110, 51. Thus, the increase in the molecular weight of benzophenone derivatives prevents their penetration through the skin. The triazole derivatives 5-7, 10, and 11 showed molecular weight values greater than 500 Daltons (Table 1). In addition, the derivatives of hydroxylated benzophenones showed molecular weight values similar to compounds patented and currently used in the market in the composition of cosmetic products intended to protect the skin against the deleterious effects of UV light radiation.3838 do Nascimento, L. F.; dos Santos, E. P.; de Aguiar, A. P.; Rev. Virtual Quim. 2014, 6, 190.

Antioxidant activity

Antioxidant activity of the 1,2,3-triazole-benzophenone derivatives was determined considering their ability to react with the stable radical DPPH. Just compounds soluble in ethanol, the solvent used to conduct the assays, were tested. This spectrophotometric method is based on the reduction of DPPH by an antioxidant compound, resulting in a colorless solution. As a consequence, the absorbance decreases.3939 Sousa, M. S. B.; Vieira, L. M.; de Lima, A.; Braz. J. Food Technol. 2011, 14, 202. The antioxidant effect was expressed as the Trolox equivalent antioxidant capacity (TEAC) and the values are shown in Table 2.

Table 2
Trolox equivalent antioxidant capacity (TEAC) for 1,2,3-triazole-benzophenone derivatives. The TEAC is related to the antioxidant capacity of a given substance, as compared to the standard Trolox

The 4,4’-dihydroxybenzophenone derivatives 2a, 5, 8, 13, 17, 18 showed low TEAC, being 5 and 17 the compounds that displayed lower values, compared to 2a, for both experimental times. The derivatives 8, 13, and 18 presented greater antioxidant activity than 2a, with no statistical difference among the 1,2,3-triazoles.

The results in Table 2 show that the propargylated derivative 2b presented superior antioxidant activity (almost 5.8-fold higher) than 2a. This difference can be rationalized by the fact that derivative 2b still has a free phenolic hydroxyl group in its structure, while these groups in 2a were both propargylated. Rice-Evans et al.4040 Rice-Evans, C. A.; Miller, N. J.; Paganga, G.; Free Radicals Biol. Med. 1996, 20, 933. reported that the presence of free phenolic hydroxyl groups contributes to the antioxidant activity. The presence of these groups at the 3’ and 4’ positions of luteolin, a naturally-occurring flavonoid, contributes about 25% to the antioxidant activity, as described by the authors, presenting a TEAC value of 2.1 ± 0.05 mmol L-1. When the hydroxyl group was absent at the 3’ position, the TEAC value decreased to approximately 1.45 ± 0.08 mmol L-1.4040 Rice-Evans, C. A.; Miller, N. J.; Paganga, G.; Free Radicals Biol. Med. 1996, 20, 933.

The presence of the chelatogenic hydroxyl group in the 2,4-dihydroxybenzophenone derivatives plays an important role in the antioxidant activity. However, only the presence of free hydroxyl group does not necessarily assure significant antioxidant activity, since 1b and most triazole derivatives of 2b showed lower TEAC values compared to 2b.

It should be mentioned that the introduction of the triazole fragments into the benzophenone framework, in most cases, did not positively influence the antioxidant activity. However, triazolic derivative 19 was the only compound that showed no statistical difference when compared to 2b, considering both experimental times.

Comparing derivatives that have the same benzyl substituent group, we noticed that the presence of the para-iodine benzyl group influenced antioxidant activity to the same degree, with derivatives 8 and 27 presenting no statistical difference. In contrast, derivatives containing fluorine showed significant differences in their TEAC values. Considering the 4,4’-dihydroxybenzophenone triazole derivatives bearing fluorine, compound 13 was more reactive towards DPPH than 5, indicating that the presence of two fluorine atoms favors the activity. The 2,4’-dihydroxybenzophenone triazole derivative 24, presenting two fluorine atoms, showed 62% (after two hours) and 24% (after four hours) reduction in TEAC value as compared to 13. The compound 26, a derivative that also has fluorine atoms in its structure, presented a better antioxidant effect than 17.

Compounds 20, 21, 22, 24, and 28, which are derivatives of 2b, showed lower TEAC values after two hours when compared to 1b. However, 20, 22, and 24 increased their antioxidant activities by approximately 246, 143, and 62%, respectively, after two more hours of reaction.

The low values of TEAC for the 2,4-dihydroxybenzo-phenone derivatives compared to those reported in the literature4141 Liu, H.-T.; Ho, Y.-S.; Food Sci. Hum. Wellness 2018, 7, 134.,4242 Wright, J. S.; Johnson, E. R.; DiLabio, G. A.; J. Am. Chem. Soc. 2001, 123, 1173. for phenolic compounds are directly associated with the formation of intramolecular hydrogen bonds between the neighboring OH and carbonyl groups of benzophenone, which leaves hydrogen less available to be donated to the DPPH radical. Thus, the antioxidant activity is directly related to the hydrogen transfer capacity, and the dissociation of O-H bond is an important factor in determining the efficacy of an antioxidant. The weaker the O-H bond, the faster the reaction with DPPH free radical.

The derivatives 13, 20, 22, 23, 24, and 26 showed statistical differences in TEAC values calculated after two and four hours of reaction (Figure 2). These results suggest that for these compounds, the hydrogen transfer reaction to the DPPH radical occurs following slow kinetics. For example, comparing compounds 23 and 2b (Table 2), it can be noticed that after 4 h of reaction the TEAC value of 23 is about 20% smaller than the TEAC value of 2b. Using the DPPH method, Brand-Williams et al.4343 Brand-Williams, W.; Cuvelier, M. E.; Berset, C.; LWT - Food Sci. Technol. 1995, 28, 25. evaluated the antioxidant activity of several phenolic compounds. They also found remarkable differences concerning the kinetics of the reactions between DPPH and compounds under evaluation. The phenolic compounds ascorbic acid, isoascorbic acid, and isoeugenol achieved a steady-state quickly.4343 Brand-Williams, W.; Cuvelier, M. E.; Berset, C.; LWT - Food Sci. Technol. 1995, 28, 25.

Figure 2
Influence of the reaction time on TEAC values for 1,2,3-triazole benzophenone derivatives and compounds 1b, 2a, and 2b. ***p < 0.005 and *p < 0.05 according to t-test.

In the next step, the cytotoxicity of the 1,2,3-triazole derivatives (4-18 and 19-29) was evaluated against three human tumor cell lines A549 (lung carcinoma), MCF-7 (breast carcinoma), and HT-144 (metastatic melanoma). The benzophenones used in chemical design (1a and 1b) and precursors of synthesized substances (2a and 2b) were included in the cell viability assays.

Cytotoxicity evaluation

Cell viability was assessed after 48 h treatment and the substances were screened at 40 µmol L-1. The viability rates were not significantly altered in cell cultures treated with the substances 1a, 2a, and 4-18 compared to control cultures (data not shown), indicating that these compounds had no cytotoxic activity on tested tumor cell lines at 40 µmol L-1. By contrast, triazole derivatives obtained from 1b displayed cytotoxic activity against tumor cells when assayed at the same conditions (40 µmol L-1 for 48 h). The viability rates observed for MCF-7 cultures treated with substances 19-21, 24, and 26 were lower compared to 1b. All these substances were more efficient in reducing cell viability (p < 0.05) than 1b, and substances 19 and 26 were the most actives (Figure 3). In A549 cultures, there was a significant reduction in cell viability in samples treated with 19-21, 23, 24, 26, and 28. The substances 19, 21, and 24 displayed higher cytotoxicity on A549 cells than other compounds, and their effects were much more pronounced than 1b (Figure 3). Regarding HT-144 cells, reduced viability rates were observed in almost all treated samples in comparison to controls. Only substances 22 and 26 had no cytotoxic activity on melanoma cells. The substances 19, 20, and 24 were the most active compared to other derivatives of the series or 1b (Figure 3).

Figure 3
Cell viability determined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay after 48 h of treatment with 1b, 2b and triazoles 19-29. ***Significant difference (p < 0.001) from DMSO group according to ANOVA followed by Dunnet’s post-test. #: significant difference (p < 0.05) from precursor (2b) according to ANOVA followed by Dunnet’s post-test. DMSO and DMEM are the negative controls; cisplatin is the positive control.

We further determined the IC50 values (concentration capable of inhibiting 50% of cell viability) for the most active compounds (Table 3). Both assayed substances 19 and 26 against MCF-7 cells displayed IC50 values lower than those found for cisplatin, a powerful cytotoxic agent and used as positive control. Besides, these substances showed a better selectivity profile compared to cisplatin that presented higher cytotoxicity toward normal cells than 19 and 26. These findings are very promising considering that breast cancer is the second most prevalent cancer worldwide. Despite advances in diagnostic and treatment modalities, breast cancer remains the major cause of cancer deaths among women.4444 Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.; Jemal, A.; Ca-Cancer J. Clin. 2018, 68, 394. The high mortality rate for breast cancer is closely associated with complexity of the disease and intrinsic or acquired resistance to available drugs.4545 AlFakeeh, A.; Brezden-Masley, C.; Curr. Oncol. 2018, 25, S18. Further studies will be addressed to evaluate the underlying mechanism related to cytotoxic activity of the substances 19 and 26 on estrogen-positive MCF-7 breast cancer cells.

Table 3
IC50 values determined from MTS assay. Cell cultures were treated with different substances for 48 h

Even though the IC50 values found for substances 19, 21 and 24 on A549 cells have been upward to that observed for cisplatin, these compounds significantly reduced viability in A549 cultures compared to control groups. Furthermore, these substances were much less cytotoxic against normal cells (CCD-1059Sk) than tumor cells. These findings suggest that these triazoles might be useful in future clinical application, supporting further studies to evaluate better their antitumor activity on lung adenocarcinoma cells. Platinum-based drugs are commonly used to treat lung cancer patients; however, these substances are highly toxic and trigger several side-effects including nephrotoxicity. Indeed, carboplatin, an analogue of cisplatin, were developed especially to minimize toxic effects caused by the cisplatin.4646 Weiss, R. B.; Christian, M. C.; Drugs 1993, 46, 360. In the present study we found that triazole derivatives 19, 21 and 24 were effective to reduce cell viability in adenocarcinoma A549 cells and further studies will be conducted to evaluate the mechanism of action associated with cytotoxic activity of these substances on A549 cells and their antitumor potential in pre-clinical studies.

We showed that the substances 19, 20 and 24 significantly reduced viability rate in melanoma cultures, a type of skin cancer highly aggressive.4747 Napolitano, S.; Brancaccio, G.; Argenziano, G.; Martinelli, E.; Morgillo, F.; Ciardiello, F.; Troiani, T.; Cancer Treat. Rev. 2018, 69, 101. In view of that, we decided to further explore the effects of these substances on HT-144 cells that harbor BRAF V600E mutation,4848 Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Wooster, R.; Stratton, M. R.; Futreal, P. A.; Nature 2002, 417, 949.,4949 Abbotts, R.; Jewell, R.; Nsengimana, J.; Maloney, D. J.; Simeonov, A.; Seedhouse, C.; Elliott, F.; Laye, J.; Walker, C.; Jadhav, A.; Grabowska, A.; Ball, G.; Patel, P. M.; Newton-Bishop, J.; Wilson, D. M.; Madhusudan, S.; Oncotarget 2014, 5, 3273. a punctual mutation frequently found in melanoma. BRAF gene encodes rapidly accelerated fibrosarcoma (RAF) protein, a member of mitogen-activated protein kinase (MAPK) family. RAF/MEK/ERK (RAF/mitogen-activated protein/extracellular-signal-regulated kinase) signaling pathway regulates important biological processes including proliferation and survival.500 5. McCubrey, J. A.; Steelman, L. S.; Chappell, W. H.; Abrams, S. L.; Wong, E. W. T.; Chang, F.; Lehmann, B.; Terrian, D. M.; Milella, M.; Tafuri, A.; Stivala, F.; Libra, M.; Basecke, J.; Evangelisti, C.; Martelli, A. M.; Franklin, R. A.; Biochim. Biophys. Acta, Mol. Cell Res. 2007, 1773, 1263.,5151 Cseh, B.; Doma, E.; Baccarini, M.; FEBS Lett. 2014, 588, 2398. BRAF mutation in melanoma is direct or indirectly associated to ERK and protein kinase B (AKT) hyper activation leading to uncontrolled cell proliferation, survival and tumor progression.5252 Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.; Project, C. G.; Jones, C. M.; Marshall,C. J.; Springer, C. J.; Barford, D.; Marais, R.; Cell 2004, 116, 855.,5353 Maurer, G.; Tarkowski, B.; Baccarini, M.; Oncogene 2011, 30, 3477.

We performed cell cycle analysis to investigate whether the viability reduction previously observed in HT-144 cell cultures treated with substances 19, 20 and 24 could be associated to their ability of inhibiting cell cycle progression and/or to induce cell death. Increased G0/G1 populations were observed in all cell cultures treated (Figure 4). Besides, there was a significant reduction in G2/M populations in samples treated with substances 19 and 24. Taken together, the data indicate that these compounds inhibited cell cycle progression of HT-144 cells at G1 phase. G1/S transition is positively regulated by cyclin-dependent kinases (CDKs) 4/6 and 2, which are activated respectively by cyclin D and cyclin E.5454 Lapenna, S.; Giordano, A.; Nat. Rev. Drug Discovery 2009, 8, 547. As mentioned before, the high proliferative ability of melanoma cells is closely associated to ERK activation that, in turn, induces high expression of cyclin D1.5555 Daveri, E.; Valacchi, G.; Romagnoli, R.; Maellaro, E.; Maioli, E.; Evidence-Based Complementary Altern. Med. 2015, 2015, 545838.

Figure 4
Representative histograms obtained by flow cytometry and quantitative analysis. HT-144 cell cultures were treated for 48 h with different substances at 40 μmol L-1. The different cell populations are shown in brown (SubG1); pink (G0/G1); green (S); blue (G2/M).

We demonstrated, for the first time, that hydroxylated benzophenone derivatives containing 1,2,3-triazole fragments inhibit cell cycle arrest at G1 in melanoma cells. It has been reported5656 Farooq, S.; Hussain, A.; Qurishi, M. A.; Hamid, A.; Koul, S.; Anti-Cancer Agents Med. Chem. 2018, 18, 1531. that cycle arrest at G1/S transition in MCF-7 cells was induced by 7-hydroxycoumarin triazoles. Similarly, tetrahydro-[1,2,4]triazolo[3,4-a]isoquinoline chalcones promoted cell cycle arrest of MCF-7 cells at G1 phase. Begnini et al.5757 Begnini, K. R.; Duarte, W. R.; da Silva, L. P.; Buss, J. H.; Goldani, B. S.; Fronza, M.; Segatto, N. V.; Alves, D.; Savegnago, L.; Seixas, F. K.; Collares, T.; Biomed. Pharmacother. 2017, 91, 510. also observed cell cycle arrest when treated triple negative breast cancer cells (MDA-MB-231) with 7-chloroquinoline-1,2,3-triazoyl carboxamides. A recent study5858 Osorio, L. S.; Ionta, M.; Demuner, A. J.; de Sousa, B. L.; Ferraz, G. O.; Varejao, E. V. V.; Ferreira-Silva, G. A.; Pilau, E. J.; Silva, E.; dos Santos, M. H.; J. Braz. Chem. Soc. 2020, 31, 2500. demonstrated that 1,2,3-triazole derivatives of hydnocarpic acid induced cell cycle arrest at G1/S transition in non-small cell lung cancer A549 cells.

In addition to its ability to inhibit cell cycle progression, substance 19 induced cell death as demonstrated by increasing of the sub-G1 population. Interestingly, this same substance displayed a significant antioxidant activity in DPPH assay. Many studies5959 Trachootham, D.; Alexandre, J.; Huang, P.; Nat. Rev. Drug Discovery 2009, 8, 579.

60 Kruk, J.; Duchnik, E.; Asian Pac. J. Cancer Prev. 2014, 15, 561.
-6161 Zhang, W.; Shu, X. O.; Li, H.; Yang, G.; Cai, H.; Ji, B. T.; Gao, J.; Gao, Y. T.; Zheng, W.; Xiang, Y. B.; J. Natl. Cancer Inst. 2012, 104, 1173. have demonstrated that antioxidant agents may be useful to prevent tumorigenesis. On the other hand, several studies6262 Chikara, S.; Nagaprashantha, L. D.; Singhal, J.; Horne, D.; Awasthi, S.; Singhal, S. S.; Cancer Lett. 2018, 413, 122. have showed that many phytochemical substances with antioxidant property inhibit proliferation and induce cell death in cancer cells by increasing oxidative stress. The metabolic conditions of cancer cells are different from normal cells. Cancer cells have a high metabolism and produce elevated levels of reactive oxygen species (ROS), and therefore cancer cells rely on a robust endogenous antioxidant system to maintain ROS levels below the cytotoxic threshold.5959 Trachootham, D.; Alexandre, J.; Huang, P.; Nat. Rev. Drug Discovery 2009, 8, 579. Antioxidant agents may interfere with metabolic-redox in cancer cell and consequently contribute for inducing cancer cell death.6363 Bansal, A.; Simon, M. C.; J. Cell Biol. 2018, 217, 2291.

Our findings showed that 1,2,3-triazole-benzophenone derivatives have antitumor potential and support further studies to evaluate deeply their molecular targets in HT-144 cells. These substances might turn out to be help in a future clinical application for melanoma treatment which therapeutic proposals are limited due to intrinsic and acquired drug resistance.

Conclusions

A series of twenty-six 1,2,3-triazole-benzophenone derivatives were obtained in two steps from commercially available 4,4’-dihydroxybenzophenone and 2,4-dihydroxybenzophenone. The 1,2,3-triazoles were obtained with yields ranging from 35 to 95%. The compounds were evaluated with regard to their photoprotective, antioxidant, and cytotoxicity in vitro activities. The 1,2,3-triazole derivatives showed efficacy comparable to the commercial product BP-3 at 0.2 μg mL-1. The derivatives 2b and 19 displayed a significant antioxidant activity. Moreover, the substance 19 efficiently reduced viability in tumor cell lines (A549, MCF-7, and HT-144) and did not show significant cytotoxicity on a non-cancerous cell line (CCD-1059Sk). We also demonstrated that derivatives 19, 20, and 24 induced cell cycle arrest at G1 in HT-144 cells. It is important to highlight that at 0.2 μg mL-1, concentration used to photoprotective assays, represents a no-cytotoxic concentration. Thus, taken together, the data show that 1,2,3-triazole derivatives obtained from hydroxybenzophenones may be considered promising prototypes for the development of new drugs with the dual antioxidant and anticancer effect.

Experimental

Generalities

All reagents and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Vetec (Duque de Caxias, RJ, Brazil) and were used without further purification. Analytical thin layer chromatography (TLC) analysis was conducted on aluminum-backed precoated silica gel plates (Macherey-Nagel DC-Fertigfolien ALUGRAM® Xtra SIL G/UV254, Düren, Germany) using different solvent systems. TLC plates were visualized using UV light (λ = 254 nm) and potassium permanganate solution.

Flash column chromatography was performed with silica gel (70-230 mesh, Macherey-Nagel, Düren, Germany). 1H and 13C NMR spectra were recorded on a Varian Mercury 300 instrument (Varian, Palo Alto, CA, USA) at 300 and 75 MHz, respectively, using CDCl3 or dimethyl sulfoxide (DMSO-d6) as solvents.

1H NMR data are presented as follows: chemical shift (δ) in ppm, multiplicity, the number of hydrogens, and coupling constant (J) values in hertz (Hz). Multiplicities are shown as the following abbreviations: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet). For fluorine-containing derivatives, the multiplicity of some carbon signals are described along with J values in hertz. Melting points (mp) were recorded on the MQAPF-302 equipment (Microquímica Equipamentos, Palhoça, SC, Brazil) and were not corrected. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were obtained using Varian 660-IR (Varian, Palo Alto, CA, USA) equipped with GladiATR scanning from 4000 to 500 cm-1. The HRMS analysis was performed using the Impact II Brucker UHR-QqTOF (ultra-high resolution qq time of flight) mass spectrometry (Bruker Daltonics Corporation, Bremen, Germany). The acquisition software used was Otof Control and Hystar software package (Bruker Daltonics) and equipped with an electrospray source operating in negative ionization mode.

Synthesis

Synthesis of bis(4-(prop-2-yn-1-yloxy))benzophenone (2a)

In a previous study,3232 Dias, M. C. F.; Gularte, T. Q.; Teixeira, R. R.; Santos, J. A. N.; Pilau, E. J.; Mendes, T. A. O.; Demuner, A. J.; dos Santos, M. H.; J. Braz. Chem. Soc. 2019, 30, 97. we reported the production of 2a. Potassium carbonate (3.87 g, 28.0 mmol) and propargyl bromide (1.50 mL) were added to a stirred solution of 4,4’-dihydroxybenzophenone (1.50 g, 7.00 mmol) in acetone (50 mL). The reaction mixture was refluxed for 24 h. The reaction was quenched with ice-water/CH2Cl2 (dichloromethane (DCM)). The phases were separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The extracts were combined, and the resulting organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to provide crude compound 2a. After purification by silica gel flash column chromatography, eluted with ethyl acetate-DCM-hexane 3:1:3 v/v, compound 2a was obtained as a yellow solid with 83% yield (1.68 g, 5.79 mmol). The structure of compound 2a is supported by the following data.

TLC: Rf = 0.60 (ethyl acetate-DCM-hexane 3:1:3 v/v); mp 91.3-92.2 °C; IR (ATR) ν / cm-1 3219, 2971, 2112, 1578, 1558, 1503, 1267; 1H NMR (300 MHz, CDCl3) δ 2.57 (s, 2H), 4.77 (s, 4H), 7.05 (d, 4H, J 8.3 Hz), 7.80 (d, 4H, J 8.3 Hz); 13C NMR (75 MHz, CDCl3) δ 55.9, 76.1, 76.6, 114.4, 131.3, 132.2, 160.7, 194.3; HRMS m/z, calculated for C19H15O3 [M + H]+: 291.1016, found: 291.1016.

Synthesis of (2-hydroxy-4-(prop-2-yn-1-yloxy))benzophenone (2b)

Potassium carbonate (2.58 g, 18.7 mmol) and propargyl bromide (1.00 mL) were added to a stirred solution of 2,4-dihydroxybenzophenone (1.50 g, 7.00 mmol) in acetone (50 mL). The reaction mixture was refluxed for 24 h. After this time, the reaction was quenched with ice-water/CH2Cl2. The phases were separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The extracts were combined, and the resulting organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to provide crude compound 2b. After purification by silica gel flash column chromatography, eluted with hexane-ethyl acetate 2:1 v/v, compound 2b was obtained as a yellow solid with 61% yield (1.44 g, 5.69 mmol). The structure of compound 2b is supported by the following data.

TLC: Rf = 0.79 (ethyl acetate-DCM-hexane 3:1:3 v/v); mp 70.8-71.9 °C; IR (ATR) ν / cm-1 3301, 3284, 2120, 1620, 1594, 1573, 1502, 1444, 1381, 1343, 1278, 1249, 1221, 1167, 1111, 1019, 909, 693; 1H NMR (300 MHz, CDCl3) δ 2.58 (t, 1H, J 2.4 Hz), 4.74 (d, 2H, J 2.1 Hz), 6.48 (dd, 1H, J1 2.7 Hz and J2 9.0 Hz), 6.61 (d, 1H, J 2.4 Hz), 7.48 (dd, 2H, J 1.5, 8.4 Hz), 7.52 (s, 1H), 7.56 (d, 1H, J 6.3 Hz), 7.64 (dd, 2H, J 1.5, 8.4 Hz), 12.63 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 55.9, 76.4, 102.3, 107.6, 113.7, 128.3, 128.8, 131.6, 135.3, 138.1, 163.9, 166.0, 200.1; HRMS m/z, calculated for C16H13O3 [M + H]+: 253.0859, found: 253.0855.

Synthesis of target compounds 4-18

The synthesis of triazoles was performed according to our previous study.3232 Dias, M. C. F.; Gularte, T. Q.; Teixeira, R. R.; Santos, J. A. N.; Pilau, E. J.; Mendes, T. A. O.; Demuner, A. J.; dos Santos, M. H.; J. Braz. Chem. Soc. 2019, 30, 97. A round bottom flask (50 mL) was charged with bis(4-(prop-2-yn-1-yloxy))benzophenone (2) (1.00 equiv.), benzyl azide (4.00 equiv.), sodium ascorbate (0.800 equiv.), dichloromethane (1.50 mL), distilled water (1.50 mL), and CuSO4·٥H2O (0.400 equiv.). The resulting reaction mixture was vigorously stirred at room temperature for 6 h and the progress of the reaction was monitored by TLC analysis. The reaction was quenched by addition of saturated sodium bicarbonate aqueous solution (15 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 20 mL). The organic extracts were combined, and the resulting organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide crude compounds. Compounds 4-18 were purified by silica gel flash column chromatography eluted with ethyl acetate-DCM-hexane 3:1:3 v/v. Structures of the synthesized compounds are supported by the following data.

Bis(4-(1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzo-phenone (4)

Yield: 66% (0.190 g, 0.269 mmol); white solid; mp 144-145 °C; TLC: Rf = 0.18 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3091, 2159, 2017, 1639, 1559, 1506, 1247; 1H NMR (300 MHz, CDCl3) δ 5.25 (s, 2H), 5.54 (s, 4H), 7.02 (d, 4H, J 8.4 Hz), 7.26-7.38 (m, 10H), 7.56 (s, 2H), 7.75 (d, 4H, J 8.4 Hz); 13C NMR (75 MHz, CDCl3) δ 54.3, 62.1, 114.2, 122.8, 128.1, 128.9, 129.2, 131.1, 132.2, 134.2, 143.9, 161.4, 194.3; HRMS m/z, calculated for C33H29N6O3 [M + H]+: 557.2296, found: 557.2322.

Bis(4-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (5)

Yield: 65% (0.190 g, 0.303 mmol); white solid; mp 139-140 °C; TLC: Rf = 0.10 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3137, 1639, 1599, 1225; 1H NMR (300 MHz, CDCl3) δ 5.25 (s, 4H), 5.51 (s, 4H), 7.03 (t, 4H, J 8.7 Hz), 7.09 (s, 4H), 7.25-7.30 (m, 4H), 7.56 (s, 2H), 7.74 (d, 4H, J 8.7 Hz); 13C NMR (75 MHz, CDCl3) δ 53.5, 62.1, 114.2, 116.2 (d, J 21.5 Hz), 122.7, 130.2 (d, J 3.2 Hz), 130.4 (d, J 8.5 Hz), 131.0, 132.2, 144.0, 161.4, 162.9 (q, J 246.9 Hz), 194.3; HRMS m/z, calculated for C33H27F2N6O3 [M + H]+: 593.2107, found: 593.2109.

Bis(4-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (6)

Yield: 53% (0.170 g, 0.272 mmol); white solid; mp 163-164 °C; TLC: Rf = 0.10 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3158, 2935, 2364, 1978, 1638, 1600, 1249; 1H NMR (300 MHz, CDCl3) δ 5.25 (s, 4H), 5.51 (s, 4H), 7.02 (dd, 4H, J 9.0, 2.3 Hz), 7.22 (dd, 4H, J 8.7, 2.2 Hz), 7.35 (dd, 4H, J 8.7, 2.2 Hz), 7.57 (s, 2H), 7.77 (dd, 4H, J 9.0, 2.3 Hz); 13C NMR (75 MHz, CDCl3) δ 53.6, 62.0, 114.2, 122.7, 129.4, 129.5, 131.1, 132.2, 132.8, 134.9, 144.1, 161.4, 194.3; HRMS m/z, calculated for C33H27Cl2N6O3 [M + H]+: 625.1516, found: 625.1513.

Bis(4-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (7)

Yield: 71% (0.248 g, 0.347 mmol); white solid; mp 182-183 °C; TLC: Rf = 0.13 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3158, 2935, 2360, 1982, 1638, 1600, 1225; 1H NMR (300 MHz, DMSO-d6) δ 5.24 (s, 4H), 5.60 (s. 4H), 7.16 (d, 4H, J 8.7 Hz), 7.27 (d, 4H, J 8.1 Hz), 7.57 (d, 4H, J 8.1 Hz), 7.57 (s, 2H), 7.69 (d, J 8.7 Hz); 13C NMR (75 MHz, DMSO-d6) δ 53.6, 62.1, 121.9, 125.4, 130.6, 130.7, 132.2, 132.3, 135.8, 143.0, 144.2, 161.7, 194.6; HRMS m/z, calculated for C33H27Br2N6O3 [M + H]+: 713.0506, found: 713.0515.

Bis(4-(1-(4-iodobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (8)

Yield: 42% (0.055 g, 0.062 mmol); white solid; mp 190-191 °C; TLC: Rf = 0.18 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3102, 2163, 1734, 1634, 1601, 1582, 1278; 1H NMR (300 MHz, DMSO-d6) δ 5.25 (s, 4H), 5.58 (s, 4H), 7.12 (d, 4H, J 8.1 Hz), 7.17 (d, 4H, J 8.6 Hz), 7.67 (d, 4H, J 8.6 Hz), 7.74 (d, 4H, J 8.1 Hz), 8.32 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 52.7, 61.8, 94.9, 114.9, 125.4, 130.6, 130.7, 132.2, 136.2, 138.0, 142.9, 161.7, 193.6; HRMS m/z, calculated for C33H27I2N6O3 [M + Na]+: 809.0228, found: 809.0202.

Bis(4-(1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (9)

Yield: 55% (0.165 g, 0.282 mmol); white solid; mp 186-187 °C; TLC: Rf = 0.32 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3099, 2921, 1970, 1639, 1601, 1258; 1H NMR (300 MHz, CDCl3) δ 2.28 (s, 6H), 5.24 (s, 4H), 5.56 (s, 4H), 7.02 (d, 4H, J 8.4 Hz), 7.20-7.33 (m, 8H), 7.44 (s, 2H), 7.75 (d, 4H, J 8.4 Hz); 13C NMR (75 MHz, CDCl3) δ 18.9, 52.5, 62.1, 114.3, 122.6, 126.7, 129.3, 129.5, 131.0, 131.1, 132.2, 136.9, 143.7, 161.4, 194.3; HRMS m/z, calculated for C35H33N6O3 [M + H]+: 585.2609, found: 585.2609.

Bis(4-(1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (10)

Yield: 42% (0.126 g, 0.215 mmol); white solid; mp 130-131 °C; TLC: Rf = 0.25 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3122, 2944, 2165, 1976, 1640, 1596, 1236; 1H NMR (300 MHz, CDCl3) δ 2.34 (s, 6H), 5.25 (s, 4H), 5.50 (s, 4H), 7.02 (d, 4H, J 8.7 Hz), 7.06-7.09 (m, 4H), 7.17 (d, 2H, J 7.5 Hz), 7.26 (t, 2H, J 8.4 Hz), 7.56 (s, 2H), 7.75 (d, 4H, J 8.7 Hz); 13C NMR (75 MHz, CDCl3) δ 21.3, 54.3, 62.1, 114.2, 122.7, 125.2, 128.9, 129.0, 129.6, 131.1, 132.2, 134.2, 139.0, 143.8, 161.4, 194.3; HRMS m/z, calculated for C35H33N6O3 [M + H]+: 585.2609, found: 585.2600.

Bis(4-(1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (11)

Yield: 59% (0.177 g, 0.302 mmol); white solid; mp 134-135 °C; TLC: Rf = 0.18 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3031, 2917, 2364, 2022, 1976, 1640, 1599, 1246; 1H NMR (300 MHz, CDCl3) δ 2.35 (s, 6H), 5.24 (s, 4H), 5.49 (s, 4H), 7.02 (d, 4H, J 8.1 Hz), 7.20 (s, 4H), 7.53 (s, 2H), 7.75 (d, 4H, J 8.1 Hz); 13C NMR (75 MHz, CDCl3) δ 21.2, 54.1, 62.1, 114.2, 122.7, 128.2, 129.8, 131.0, 131.3, 132.2, 138.9, 143.8, 161.4, 194.3; HRMS m/z, calculated for C35H33N6O3 [M + H]+: 585.2609, found: 585.2604.

Bis(4-(1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (12)

Yield: 54% (0.181 g, 0.280 mmol); white solid; mp 171-172 °C; TLC: Rf = 0.10 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3101, 2351, 1976, 1642, 1599, 1528, 1246; 1H NMR (300 MHz, CDCl3) δ 5.30 (s, 4H), 5.66 (s, 4H), 7.03 (d, 4H, J 8.7 Hz), 7.56-7.61 (m, 4H), 7.67 (s, 2H), 7.76 (d, 4H, J 8.7 Hz), 8.17 (s, 2H), 8.24 (d, 4H, J 7.2 Hz); 13C NMR (75 MHz, CDCl3) δ 53.3, 62.0, 114.3, 122.9, 123.9, 130.4, 131.2, 132.2, 133.9, 136.4, 144.5, 148.6, 161.4, 194.3; HRMS m/z, calculated for C33H27N8O7 [M + H]+: 647.1997, found: 647.1995.

Bis(4-(1-(2,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (13)

Yield: 57% (0.185 g, 0.294 mmol); white solid; mp 180-181 °C; TLC: Rf = 0.10 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3140, 2886, 2009, 1978, 1638, 1600, 1259; 1H NMR (300 MHz, CDCl3) δ 5.26 (s, 4H), 5.56 (s, 4H), 6.85-6.92 (m, 4H), 7.03 (d, 4H, J 8.7 Hz), 7.26-7.36 (m, 2H), 7.70 (s, 2H), 7.76 (d, 4H, J 8.7 Hz); 13C NMR (75 MHz, CDCl3) δ 47.3 (d, J 3.8 Hz), 62.0, 104.5 (t, J 25.2 Hz), 112.2 (dd, J 21.5, 3.8 Hz), 114.2, 117.8 (dd, J 14.9, 3.8 Hz), 122.8, 131.1, 131.8 (dd, J 9.9, 4.8 Hz), 132.2, 144.0, 161.4, 162.1 (dd, J 52.1, 12.0 Hz), 194.3; HRMS m/z, calculated for C33H25F4N6O3 [M + H]+: 629.1919, found: 629.1911.

Bis(4-(1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (14)

Yield: 60% (0.193 g, 0.310 mmol); white solid; mp 176-177 °C; TLC: Rf = 0.21 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3098, 2943, 1970, 1640, 1599, 1257; 1H NMR (300 MHz, CDCl3) δ 5.27 (s, 4H), 5.68 (s, 4H), 7.03 (d, 4H, J 8.7 Hz), 7.21-7.34 (m, 6H), 7.43 (d, 2H, J 8.7 Hz), 7.67 (s, 2H), 7.75 (d, 4H, J 8.7 Hz); 13C NMR (75 MHz, CDCl3) δ 51.5, 62.0, 114.3, 123.1, 127.6, 129.9, 130.4, 130.5, 131.1, 132.2, 132.2, 133.5, 143.8, 161.4, 194.3; HRMS m/z, calculated for C33H27Cl2N6O3 [M + H]+: 625.1516, found: 625.1505.

Bis(4-(1-(2,6-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (15)

Yield: 57% (0.204 g, 0.294 mmol); white solid; mp 187-188 °C; TLC: Rf = 0.33 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3094, 2354, 1976, 1642, 1600, 1256; 1H NMR (300 MHz, CDCl3) δ 5.24 (s, 4H), 5.87 (s, 4H), 7.03 (d, 4H, J 8.7 Hz), 7.25-7.33 (m, 2H), 7.41 (d, J 8.1 Hz), 7.62 (s, 2H), 7.75 (d, 4H, J 8.7 Hz); 13C NMR (75 MHz, CDCl3) δ 49.1, 62.1, 114.3, 122.7, 128.9, 129.9, 131.1, 131.2, 132.2, 136.8, 143.4, 161.4, 194.3; HRMS m/z, calculated for C33H24Cl4N6NaO3 [M + H]+: 717.3833, found: 717.0584.

Bis(4-(1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (16)

Yield: 51% (0.295 g, 0.413 mmol); white solid; mp 166-167 °C; TLC: Rf = 0.24 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3096, 2366, 1976, 1640, 1596, 1232; 1H NMR (300 MHz, CDCl3) δ 5.28 (s, 4H), 5.68 (s, 4H), 7.03 (d, 4H, J 8.1 Hz), 7.20-7.29 (m, 6H), 7.33 (d, 2H, J 7.8 Hz), 7.64 (d, 4H, J 8.1 Hz), 7.68 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 53.9, 62.1, 114.3, 123.1, 123.5, 128.3, 130.4, 130.5, 131.1, 132.2, 133.3, 133.9, 143.8, 161.4, 194.3; HRMS m/z, calculated for C33H26Br2N6NaO3 [M + H]+: 737.3952, found: 737.0310.

Bis(4-((1-(4-trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (17)

Yield: 58% (0.100 g, 0.139 mmol); white solid; mp 178-179 °C; TLC: Rf = 0.11 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3131, 2186, 1976, 1640, 1600, 1251; 1H NMR (300 MHz, CDCl3) δ 5.27 (s, 4H), 5.61 (s, 4H), 7.02 (d, J 8.7 Hz), 7.38 (d, 4H, J 8.4 Hz), 7.63 (d, 6H, J 9.6 Hz), 7.75 (d, 4H, J 8.4 Hz); 13C NMR (75 MHz, CDCl3) δ 53.6, 62.0, 114.2, 122.9, 123.7 (q, J 270.8 Hz), 126.2 (q, J 7.5 Hz), 128.3, 131.1 (q, J 18.6 Hz), 132.2, 138.3, 144.3, 161.3, 194.3; HRMS m/z, calculated for C35H27F6N6O5 [M + H]+: 725.1942, found: 725.1936.

Bis(4-((1-(4-trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (18)

Yield: 58% (0.209 g, 0.302 mmol); white solid; mp 161-162 °C; TLC: Rf = 0.13 (ethyl acetate-DCM-hexane 3:1:3 v/v); IR (ATR) ν / cm-1 3109, 2608, 1605, 1598, 1246; 1H NMR (300 MHz, CDCl3) δ 5.27 (s, 4H), 5.53 (s, 4H), 7.03 (d, 4H, J 7.8 Hz), 7.23 (d, 4H, J 8.3 Hz), 7.32 (d, 4H, J 8.3 Hz), 7.60 (s, 2H), 7.76 (d, 4H, J 7.8 Hz); 13C NMR (75 MHz, CDCl3) δ 53.4, 62.0, 114.2, 120.3 (q, J 256.5 Hz), 121.6, 122.8, 129.6, 131.1, 132.2, 133.0, 144.2, 149.5, 161.4, 194.3; HRMS m/z, calculated for C35H27F6N6O3: 693.2043 [M + H]+, found: 693.2024.

Synthesis of target compounds 19-29

A round bottom flask (50 mL) was charged with 2-hydroxy-4-(prop-2-yn-1-yloxy)benzophenone (2b) (1.00 equiv.), benzyl azide (1.20 equiv.), sodium ascorbate (0.400 equiv.), dichloromethane (1.50 mL), distilled water (1.50 mL), and CuSO4∙5H2O (0.200 equiv.). The resulting reaction mixture was vigorously stirred at room temperature for 1 h and the progress of the reaction was monitored by TLC analysis. The reaction was quenched by addition of saturated sodium bicarbonate aqueous solution (15 mL). The layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 20 mL). The organic extracts were combined, and the resulting organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide crude compounds. Compounds 19-29 were purified by silica gel flash column chromatography eluted with hexane-ethyl acetate 2:1 v/v. Structures of the synthesized compounds are supported by the following data.

2-Hydroxy-(4-((1-benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (19)

Yield: 78% (0.172 g, 0.446 mmol); white solid; mp 98-99 °C; TLC: Rf = 0.33 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3137, 2923, 2856, 2364, 2159, 2021, 1618, 1596, 1573, 1497, 1340, 1252, 1161, 1111, 1049, 1001, 978, 805, 679, 619; 1H NMR (300 MHz, CDCl3) δ 5.21 (s, 2H), 5.54 (s, 2H), 6.46 (dd, 1H, J 2.1, 8.9 Hz), 6.59 (s, 1H), 7.28 (dd, 1H, J 2.2, 6.8 Hz), 7.36-7.38 (m, 3H), 7.45-7.53 (m, 4H), 7.57 (s, 1H), 7.61 (d, 2H, J 6.9 Hz), 12.64 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 54.2, 62.0, 102.0, 107.4, 113.4, 122.8, 122.9, 128.1, 128.3, 129.1, 131.5, 134.2, 135.3, 135.4, 138.0, 164.5, 166.0, 200.0; HRMS m/z, calculated for C23H20N3O3 [M + H]+: 386.1499, found: 386.1497.

2-Hydroxy-(4-((1-(3-methyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (20)

Yield: 89% (0.212 g, 0.531 mmol); white solid; mp 77-78 °C; TLC: Rf = 0.47 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3138, 3058, 2923, 2366, 1618, 1574, 1501, 1445, 1341, 1254, 1189, 1162, 1113, 1003, 910, 699; 1H NMR (300 MHz, CDCl3) δ 2.34 (s, 3H), 5.22 (s, 2H), 5.50 (s, 2H), 6.46 (dd, 1H, J 2.1, 9.0 Hz), 6.59 (s, 1H), 7.08 (d, 2H, J 6.6 Hz), 7.17 (d, 1H, J 7.5 Hz), 7.26 (t, 1H, 7.5 Hz), 7.46-7.54 (m, 4H), 12.63 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 21.1, 54.3, 62.1, 102.1, 107.5, 113.5, 122.8, 122.9, 125.2, 128.3, 128.9, 129.0, 129.6, 131.5, 134.2, 135.4, 138.1, 139.1, 143.4, 164.6, 166.1, 200.1; HRMS m/z, calculated for C24H22N3O3 [M + H]+: 400.1656, found: 400.1657.

2-Hydroxy-(4-((1-(4-chloro)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (21)

Yield: 71% (0.177 g, 0.422 mmol); white solid; mp 87-88 °C; TLC: Rf = 0.30 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3138, 3058, 3028, 2923, 2366, 1618, 1574, 1501, 1406, 1445, 1341, 1253, 1225, 1161, 1188, 1111, 1049, 1002, 978, 909, 801, 734, 698, 615; 1H NMR (300 MHz, CDCl3) δ 5.22 (s, 2H), 5.51 (s, 2H), 6.46 (dd, 1H, J 2.1, 9.0 Hz), 6.59 (s, 1H), 7.15 (d, 2H, J 8.1 Hz), 7.35 (d, 2H, J 8.1 Hz), 7.46-7.54 (m, 4H), 7.57 (s, 1H), 7.61 (d, 2H, J 7.8 Hz), 12.64 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 53.6, 62.1, 102.1, 107.4, 113.6, 122.8, 122.9, 128.3, 128.8, 129.4, 129.5, 131.6, 132.8, 134.9, 138.1, 143.6, 164.5, 166.0, 200.1; HRMS m/z, calculated for C23H20ClN3O3 [M + H]+: 420.1109, found: 420.1109.

2-Hydroxy-(4-((1-(3-nitro)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (22)

Yield: 73% (0.186 g, 0.422 mmol); white solid; mp 127-128 °C; TLC: Rf = 0.24 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3143, 3058, 2966, 2933, 1700, 1619, 1597, 1574, 1492, 1341, 1253, 1225, 1188, 1161, 1111, 1002, 909, 801, 734, 698, 615; 1H NMR (300 MHz, CDCl3) δ 5.25 (s, 2H), 5.66 (s, 2H), 6.46 (dd, 1H, J 2.1, 9.0 Hz), 6.58 (s, 1H), 7.45-7.62 (m, 8H), 7.69 (s, 1H), 8.16 (s, 1H), 8.21 (d, 1H, J 7.5 Hz), 12.62 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 53.2, 62.0, 102.1, 107.4, 113.9, 123.0, 123.1, 123.9, 128.3, 128.8, 130.4, 131.6, 133.7, 135.5, 136.4, 138.0, 143.9, 148.5, 164.5, 166.0, 200.1; HRMS m/z, calculated for C23H19N4O5 [M + H]+: 431.1349, found: 431.1353.

2-Hydroxy-(4-((1-(4-bromo)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (23)

Yield: 95% (0.261 g, 0.562 mmol); white solid; mp 121-122 °C; TLC: Rf = 0.41 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3143, 3058, 2929, 1700, 1618, 1597, 1574, 1558, 1502, 1445, 1344, 1253, 1225, 1189, 1169, 1112, 1003, 909, 732; 1H NMR (300 MHz, CDCl3) δ 5.22 (s, 2H), 5.49 (s, 2H), 6.46 (dd, 1H, J 2.3, 8.7 Hz), 6.59 (s, 1H), 7.15 (d, 2H), 7.46-7.63 (m, 9H), 12.64 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 53.6, 62.1, 102.1, 107.4, 113.6, 122.8, 122.9, 123.1, 128.3, 128.8, 129.7, 131.6, 132.4, 133.3, 138.1, 143.6, 164.6, 166.1, 200.1; HRMS m/z, calculated for C23H19BrN3O3 [M + H]+: 464.0604, found: 464.0601.

2-Hydroxy-(4-((1-(2,4-difluoro)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (24)

Yield: 46% (0.116 g, 0.401 mmol); white solid; mp 108-109 °C; TLC: Rf = 0.32 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3151, 3099, 2923, 1621, 1595, 1573, 1505, 1445, 1429, 1349, 1342, 1281, 1260, 1184, 1159, 1121, 1095, 1047, 1004, 979, 936, 910, 846, 788, 696, 654, 629; 1H NMR (300 MHz, CDCl3) δ 5.22 (s, 2H), 5.56 (s, 2H), 6.47 (dd, 1H, J 2.0, 9.0 Hz), 6.60 (s, 1H), 6.88-6.92 (m, 2H), 7.26-7.36 (m, 1H), 7.46-7.56 (m, 4H), 7.62 (d, 2H, J 6.9 Hz), 7.67 (s, 1H), 12.63 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 47.3, 62.1, 102.2, 104.4 (q, J 25.6 Hz), 107.4, 112.3, 113.5, 123.0, 128.3, 128.8, 131.6, 131.7 (q, J 5.0 Hz), 135.4, 138.1, 143.5, 164.6, 166.1, 200.1; HRMS m/z, calculated for C23H18F2N3O3 [M + H]+: 422.1310, found: 422.1309.

2-Hydroxy-(4-((1-(4-trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (25)

Yield: 94% (0.253 g, 0.558 mmol); white solid; mp 132-133 °C; TLC: Rf = 0.28 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3137, 3087, 2926, 2366, 2016, 1618, 1597, 1573, 1503, 1445, 1419, 1322, 1252, 1159, 1111, 1065, 1017, 920, 808, 701; 1H NMR (300 MHz, CDCl3) δ 5.23 (s, 2H), 5.61 (s, 2H), 6.47 (d, 1H, J 8.7 Hz), 6.59 (s, 1H), 7.38 (d, 2H, J 8.1 Hz), 7.45-7.56 (m, 4H), 7.60-7.62 (m, 4H), 7.65 (s 1H), 12.61 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 53.6, 62.1, 102.2, 107.4, 113.6, 122.9, 126.1 (q, J 3.6, 7.5 Hz), 128.2, 128.3, 128.8, 131.5, 135.4, 138.1, 143.8, 164.5, 166.1, 200.1; HRMS m/z, calculated for C24H19F3N3O3 [M + H]+: 454.1373, found: 454.1374.

2-Hydroxy-(4-((1-(4-trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (26)

Yield: 93% (0.261 g, 0.556 mmol); white solid; mp 97-98 °C; TLC: Rf = 0.28 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3135, 2923, 2852, 2364, 2163, 1702, 1619, 1597, 1508, 1445, 1342, 1250, 1217, 1157, 1111, 1002, 918, 808, 699, 598; 1H NMR (300 MHz, CDCl3) δ 5.22 (s, 2H), 5.54 (s, 2H), 6.45 (dd, 1H, J 1.8, 9.0 Hz), 6.59 (s, 1H), 7.21 (d, 2H, J 8.3 Hz), 7.31 (d, 2H, J 8.3 Hz), 7.44-7.55 (m, 4H), 7.59 (s, 2H), 7.62 (s, 1H), 12.60 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 53.4, 62.1, 102.2, 107.4, 113.6, 120.4 (q, J 249.5 Hz), 121.5, 122.8, 128.3, 128.8, 129.6, 131.5, 133.1, 135.4, 149.5, 164.4, 166.1, 200.0; HRMS m/z, calculated for C24H19F3N3O4 [M + H]+: 470.1322, found: 470.1318.

2-Hydroxy-(4-((1-(4-iodo)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (27)

Yield: 78% (0.231 g, 0.452 mmol); white solid; mp 144-145 °C; TLC: Rf = 0.28 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3141, 3060, 2922, 2853, 2360, 1734, 1618, 1595, 1574, 1501, 1486, 1445, 1402, 1341, 1252, 1188, 1112, 977, 935, 796, 757, 699, 619, 598, 532, 472; 1H NMR (300 MHz, CDCl3) δ 5.20 (s, 2H), 5.46 (s, 2H), 6.44 (d, 1H, J 9.0 Hz), 6.59 (s, 1H), 7.00 (d, 2H, J 8.3 Hz), 7.43-7.54 (m, 4H), 7.59 (d, 3H, J 8.3 Hz), 7.68 (d, 2H, J 8.4 Hz), 12.61 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 53.7, 62.0, 94.7, 102.1, 107.4, 113.5, 122.9, 128.3, 128.8, 129.9, 131.6, 134.0, 135.4, 138.1, 138.3, 143.6, 164.6, 166.1, 200.1; HRMS m/z, calculated for C23H19IN3O3 [M + H]+: 512.0465, found: 512.0452.

2-Hydroxy-(4-(1-(piridin-2-ylmethyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (28)

Yield: 68% (0.156 g, 0.404 mmol); white solid; mp 103-104 °C; TLC: Rf = 0.45 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 3141, 3052, 2924, 2852, 1752, 1621, 1549, 1574, 1500, 1441, 1345, 1257, 1223, 1164, 1113, 1047, 1051, 997, 976, 935, 910, 838, 751, 697, 617, 596, 535, 466; 1H NMR (300 MHz, CDCl3) δ 5.23 (s, 2H), 5.65 (s, 2H), 6.46 (dd, 1H, J 1.5, 9.0 Hz), 6.60 (s, 1H), 7.22 (d, 1H, J 8.1 Hz), 7.27 (d, 1H, J 7.5 Hz), 7.44-7.55 (m, 4H), 7.61 (d, 2H, J 7.0 Hz), 7.69 (t, 1H, J 7.7 Hz), 7.84 (s, 1H), 8.58 (br, 1H), 12.62 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 55.7, 62.1, 102.1, 107.5, 113.5, 122.6, 123.5, 123.7, 128.3, 128.8, 131.5, 135.4, 137.4, 138.1, 143.3, 149.8, 154.1, 164.6, 166.1, 200.1; HRMS m/z, calculated for C23H19IN3O3 [M + H]+: 387.1451, found: 387.1453.

2-Hydroxy-(4-(1-(benzo[d][1,3]dioxol-5-ylmethyl)-1H-1,2,3-triazol-4-yl)methoxy)benzophenone (29)

Yield: 35% (0.088 g, 0.228 mmol); white solid; mp 144-145 °C; TLC: Rf = 0.38 (hexane-ethyl acetate 2:1 v/v); IR (ATR) ν / cm-1 2922, 2852, 1620, 1597, 1575, 1502, 1491, 1445, 1342, 1248, 1189, 1162, 1113, 1035, 1003, 923, 801, 741, 699, 615, 598, 532, 465; 1H NMR (300 MHz, CDCl3) δ 5.21 (s, 2H), 5.42 (s, 2H), 5.96 (s, 2H), 6.46 (dd, 1H, J 2.4, 9.0 Hz), 6.59 (s, 1H), 6.75 (s, 1H), 6.79 (s, 2H), 7.45-7.63 (m, 4H), 7.55 (s, 1H), 7.61 (d, 2H, J 7.8 Hz), 12.63 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 54.2, 62.1, 101.5, 102.2, 107.4, 108.5, 108.6, 113.5, 122.1, 122.6, 127.8, 128.3, 128.8, 131.5, 135.4, 138.1, 143.4, 148.2, 148.3, 164.6, 166.1, 200.1; HRMS m/z, calculated for C24H20N3O5 [M + H]+: 430.1397, found: 430.1397.

Biological assays

In vitro sun protection factor

Sun protection factor of each compound (2a, 2b, 4-7, 10, 11, 13, 15, 17-28) was determined using the UV absorbance method in a microdilution plate reader (Thermo Scientific, Vantaa, Finland). Solutions of compounds were prepared at 0.2 µg mL-1 using ethanol as solvent. Ethanol was used as blank. Each experiment was performed in triplicate. Absorbance was observed in the range of 290-320 nm, with intervals of 5 nm, according to Mansur’s method.6464 Mansur, M. C. P. P. R.; Leitao, S. G.; Cerqueira-Coutinho, C.; Vermelho, A. B.; Silva, R. S.; Presgrave, O. A. F.; Leitao, A. A. C.; Leitao, G. G.; Ricci-Junior, E.; Santos, E. P.; Rev. Bras. Farmacogn. 2016, 26, 251. The SPF determination is the correlation between the erythemogenic effect (EE) and the radiation intensity (I) at each wavelength (λ) (Table 4), adjusted according to equation 1. The correction factor (CF) is 10 and Abs (λ) is the sample spectrophotometric absorbance value at λ.

(1) Spectrophotometric SPF = CF × 290 320 EE λ × I λ × Abs λ
Table 4
Correlation between the erythemogenic effect (EE) and the radiation intensity (I) at each wavelength (λ)6464 Mansur, M. C. P. P. R.; Leitao, S. G.; Cerqueira-Coutinho, C.; Vermelho, A. B.; Silva, R. S.; Presgrave, O. A. F.; Leitao, A. A. C.; Leitao, G. G.; Ricci-Junior, E.; Santos, E. P.; Rev. Bras. Farmacogn. 2016, 26, 251.

Determination of antioxidant activity (AC)

In vitro evaluations of the AC were performed through the DPPH method using the DPPH solution at 0.1 mmol L-1. The standard antioxidant used in all TEAC determinations was the synthetic antioxidant Trolox at concentrations ranging from 20 to 200 µmol L-1. At first, solutions at 2 mg mL-1 of 1b, 2a, 2b, 5, 8, 13, 17-24, 26-28 were prepared using ethanol. From this solution, dilutions (1, 0.5, 0.25, 0.125 and 0.062 mg mL-1) were made. The DPPH (175 µL) was added to the sample (25 µL) and absorbance was measured at 517 nm in a microdilution plate reader (Thermo Scientific, Vantaa, Finland) after 2 and 4 h of incubation at room temperature (25 ± 2 °C) in the dark. The TEAC was obtained by plotting the absorbance (517 nm) as a function of Trolox solution concentrations. The DPPH TEAC was expressed as µmol Trolox mL-1.

Cell lines and treatment schedule

Cell lines derived from human cancer were used in this study: A549 (lung carcinoma), MCF-7 (breast carcinoma), and HT-144 (metastatic melanoma). Fibroblast derived from normal skin (CCD-1059Sk) also was examined. The cell cultures were maintained in Dulbecco’s modified Eagle’s minimum essential medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Vitrocell, Campinas, Brazil). Cells were grown in a 37 °C humidified incubator containing 5% CO2. Compounds were solubilized in DMSO to obtain a stock solution (20 mmol L-1); subsequently, new dilutions were performed in culture medium immediately before the treatment. The final concentration of DMSO did not exceed 0.8% (v/v).

Cell viability analysis

Cells were seeded into 96-wells plate at a density of 5 × 103 cells (A549 and CCD-1059Sk) or 1 × 1044 Skorkowska, A.; Maciejska, A.; Pomierny, B.; Krzyżanowska, W.; Starek-Świechowicz, B.; Bystrowska, B.; Broniowska, Ż.; Kazek, G.; Budziszewska, B.; Neurotoxic. Res. 2020, 37, 683. cells (HT-144 and MCF-7). The cell cultures were treated with different compounds (2a, 2b, 4-29) at 40 µmol L-1 for 48 h to screen the most active substances. Cell viability was determined by MTS (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) colorimetric assay using CellTiter 96® AQueous Non-Radiactive Cell Proliferation Assay Kit (Promega Corporation, Madison, WI, USA) according to manufacturer’s instructions. The MTS was added to the samples (10% v/v in culture medium) and analysis was performed after 4 h in a spectrophotometric plate reader at 490 nm. Relative viability was calculated according to equation 2. The experiments were conducted in triplicate wells and repeated twice. Data are presented as the mean ± standard deviation (SD).

(2) Cell viability % = Ab sample × 100 / Ab control

where Ab is the absorbance value.

Further, the IC50 values of the most promising substances were determined. Cell cultures were treated for 48 h with compounds at different concentrations (10, 20, 40, 80, and 160 µmol L-1) and the IC50 values were determined from non-linear regression using GraphPad Prism.6565 GraphPad PrismR, version 5.0; GraphPad Software, San Diego, CA, USA, 2007. Cisplatin was used as a positive control at the same concentrations of the tested compounds.

Cell cycle analysis

Cell cycle progression analysis was performed according to Azevedo-Barbosa et al.6666 Azevedo-Barbosa, H.; Ferreira-Silva, G. A.; Silva, C. F.; de Souza, T. B.; Dias, D. F.; de Paula, A. C. C.; Ionta, M.; Carvalho, D. T.; Toxicol. In Vitro 2019, 59, 150. Cells were seeded into 35 mm at 2 × 1055 Ionta, M.; Ferreira-Silva, G. A.; Niero, E. L.; Costa, E. D. M.; Martens, A. A.; Rosa, W.; Soares, M. G.; Machado-Santelli, G. M.; Lago, J. H. G.; Santos, M. H.; Molecules 2015, 20, 12804. density. After adhesion (24 h) the cells were treated with the benzophenone-1,2,3-triazole derivatives for 48 h at 40 μmol L-1. After that, the cells were collected by enzymatic digestion (trypsin-ethylenediamine tetraacetic acid (EDTA) solution (Sigma-Aldrich, St. Louis, MO, USA)) and fixed in ethanol at 4 °C (75% in PBS (phosphate buffered saline)) for 30 min. The samples were incubated for 1 h with a solution containing PBS, RNAse (1.5 mg mL-1, Sigma-Aldrich, St. Louis, MO, USA) and propidium iodide (90 μg mL-1). The analysis was performed on a flow cytometer (Guava Mini EasyCyte, 8HT) using GuavaSoft 2.7 software.6767 GuavaSoft, version 2.7; EMD Millipore Corporation, Hayward, CA, USA, 2013. The data presented refer to the mean ± SD of three independent experiments performed in triplicate.

Statistical analysis

Data were expressed as the mean ± SD. Analysis of variance (ANOVA) and Tukey’s post-test was used to compare SPF and TEAC values to benzophenone-triazole derivatives. The t-test was used to compare TEAC values after two and four hours. ANOVA followed by Tukey’s and Dunnett’s post-test was used to cell viability. The software used was GraphPad Prism® 5.0.6565 GraphPad PrismR, version 5.0; GraphPad Software, San Diego, CA, USA, 2007.

Supplementary Information

Supplementary information (IR, 1H and 13C NMR and HRMS spectra) is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported by FAPEMIG, CAPES, FINEP and CNPq. The authors would like to thank Professor Paulo C. Stringheta (Department of Food Technology, University Federal of Viçosa) for the acquisition of reagents to antioxidant test.

References

  • 1
    Surana, K.; Chaudhary, B.; Diwaker, M.; Sharma, S.; Med. Chem. Commun. 2018, 9, 1803.
  • 2
    Aranda, M. I. R.; Gomez, G. A. T.; de Barros, M.; dos Santos, M. H.; de Oliveira, L. L.; Pena, J. L.; Moreira, M. A. S.; Front. Microbiol. 2019, 10, 490.
  • 3
    Song, Z.; Wang, P.; Huang, S.; Wang, C.; Wang, R.-R.; Yang, L.-M.; Zhen, Y.; Liu, K.; Zheng, Y.-T.; Ma, X.; Med. Chem. 2016, 13, 398.
  • 4
    Skorkowska, A.; Maciejska, A.; Pomierny, B.; Krzyżanowska, W.; Starek-Świechowicz, B.; Bystrowska, B.; Broniowska, Ż.; Kazek, G.; Budziszewska, B.; Neurotoxic. Res. 2020, 37, 683.
  • 5
    Ionta, M.; Ferreira-Silva, G. A.; Niero, E. L.; Costa, E. D. M.; Martens, A. A.; Rosa, W.; Soares, M. G.; Machado-Santelli, G. M.; Lago, J. H. G.; Santos, M. H.; Molecules 2015, 20, 12804.
  • 6
    Sales, L.; de Sousa, G. R.; Ferreira-Silva, G.; Castro-Gamero, A. M.; Ionta, M.; de Oliveira, J. C.; Anticancer Drugs 2016, 28, 298.
  • 7
    Suzuki, T.; Kitamura, S.; Khota, R.; Sugihara, K.; Fujimoto, N.; Ohta, S.; Toxicol. Appl. Pharmacol. 2005, 203, 9.
  • 8
    Rodriguez-Gomez, R.; Zafra-Gomez, A.; Dorival-Garcia, N.; Ballesteros, O.; Navalon, A.; Talanta 2015, 134, 657.
  • 9
    Liu, W.; Wei, D.; Liu, Q.; Du, Y.; Chemosphere 2016, 154, 491.
  • 10
    Barbosa, T. C.; Nascimento, L. E. D.; Bani, C.; Almeida, T.; Nery, M.; Santos, R. S.; Menezes, L. R. O.; Zielinska, A.; Fernandes, A. R.; Cardoso, J. C.; Jager, A.; Jager, E.; Sanchez-Lopez, E.; Nalone, L.; Souto, E. B.; Severino, P.; Toxics 2019, 7, 51.
  • 11
    Liyanaarachchi, G. D.; Samarasekera, J. K. R. R.; Mahanama, K. R. R.; Hemalal, K. D. P.; Ind. Crops Prod. 2018, 111, 597.
  • 12
    Craythorne, E.; Al-Niami, F.; Medicine 2017, 45, 429.
  • 13
    Watson, M.; Holman, D. M.; Maguire-Eisen, M.; Semin. Oncol. Nurs. 2016, 32, 241.
  • 14
    Navarro, M.; Arnaez, E.; Moreira, I.; Quesada, S.; Azofeifa, G.; Wilhelm, K.; Vargas, F.; Chen, P.; Foods 2019, 8, 384.
  • 15
    Nguyen, L. H.; Venkatraman, G.; Sim, K. Y.; Harrison, L. J.; Phytochemistry 2005, 66, 1718.
  • 16
    Yamaguchi, F.; Ariga, T.; Yoshimura, Y.; Nakazawa, H.; J. Agric. Food Chem. 2000, 48, 180.
  • 17
    Doriguetto, A. C.; Martins, F. T.; Ellena, J.; Salloum, R.; dos Santos, M. H.; Moreira, M. E. C.; Schneedorf, J. M.; Nagem, T. J.; Chem. Biodiversity 2007, 4, 488.
  • 18
    Lushchak, V. I.; Chem.-Biol. Interact. 2014, 224, 164.
  • 19
    Poprac, P.; Jomova, K.; Simunkova, M.; Kollar, V.; Rhodes, C. J.; Valko, M.; Trends Pharmacol. Sci. 2017, 38, 592.
  • 20
    Pisoschi, A. M.; Pop, A.; Eur. J. Med. Chem. 2015, 97, 55.
  • 21
    Sosa, V.; Moline, T.; Somoza, R.; Paciucci, R.; Kondoh, H.; LLeonart, M. E.; Ageing Res. Rev. 2013, 12, 376.
  • 22
    Saidi, L.; Rocha, D. H. A.; Talhi, O.; Bentarzi, Y.; Nedjar-Kolli, B.; Bachari, K.; Paz, F. A. A.; Helguero, L. A.; Silva, A. M. S.; ChemMedChem 2019, 14, 1041.
  • 23
    Gholampour, M.; Ranjbar, S.; Edraki, N.; Mohabbati, M.; Firuzi, O.; Khoshneviszadeh, M.; Bioorg. Chem. 2019, 88, 102967.
  • 24
    Hou, W.; Zhang, G.; Luo, Z.; Su, L.; Xu, H.; Chem. Biol. Drug Des. 2019, 93, 473.
  • 25
    Mareddy, J.; Suresh, N.; Kumar, C. G.; Kapavarapu, R.; Jayasree, A.; Pal, S.; Bioorg. Med. Chem. Lett. 2017, 27, 518.
  • 26
    Torres, L. M. F. C.; Almeida, M. T.; Santos, T. L.; Marinho, L. E. S.; de Mesquita, J. P.; da Silva, L. M.; dos Santos, W. T. P.; Martins, H. R.; Kato, K. C.; Alves, E. S. F.; Liao, L. M.; de Magalhaes, M. T. Q.; de Mendonca, F. G.; Pereira, F. V.; Resende, J. M.; Bemquerer, M. P.; Rodrigues, M. A.; Verly, R. M.; Colloids Surf., B 2019, 177, 94.
  • 27
    Di Pietro, O.; Alencar, N.; Esteban, G.; Viayna, E.; Szałaj, N.; Vazquez, J.; Juarez-Jimenez, J.; Sola, I.; Perez, B.; Sole, M.; Unzeta, M.; Munoz-Torrero, D.; Luque, F. J.; Bioorg. Med. Chem. 2016, 24, 4835.
  • 28
    Banerji, B.; Chandrasekhar, K.; Sreenath, K.; Roy, S.; Nag, S.; Saha, K. D.; ACS Omega 2018, 3, 16134.
  • 29
    Yamada, M.; Takahashi, T.; Hasegawa, M.; Matsumura, M.; Ono, K.; Fujimoto, R.; Kitamura, Y.; Murata, Y.; Kakusawa, N.; Tanaka, M.; Obata, T.; Fujiwara, Y.; Yasuike, S.; Bioorg. Med. Chem. Lett. 2018, 28, 152.
  • 30
    Saraiva, M. T.; Kruger, R.; Baldinotti, R. S. M.; Lenardao, E. J.; Luchese, C.; Savegnago, L.; Wilhelm, E. A.; Alves, D.; J. Braz. Chem. Soc. 2016, 27, 41.
  • 31
    Tan, W.; Li, Q.; Li, W.; Dong, F.; Guo, Z.; Int. J. Biol. Macromol. 2016, 82, 404.
  • 32
    Dias, M. C. F.; Gularte, T. Q.; Teixeira, R. R.; Santos, J. A. N.; Pilau, E. J.; Mendes, T. A. O.; Demuner, A. J.; dos Santos, M. H.; J. Braz. Chem. Soc. 2019, 30, 97.
  • 33
    MedChem Designer, version 3.1.0.30; Simulations Plus, Inc, Lancaster, USA, 2011.
  • 34
    Agencia Nacional de Vigilancia Sanitaria (ANVISA); Resolucao - RDC No. 30, de 1 de junho de 2012, Aprova o Regulamento Técnico Mercosul sobre Protetores Solares em Cosméticos e Dá Outras Providências; Ministerio da Saude: Brasilia, Brazil, 2012, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0030_01_06_2012.html#:~:text=Aprova%20o%20Regulamento%20T%C3%A9cnico%20Mercosul,Regulamento%20aprovado%20pelo%20Decreto%20n, accessed in October 2020.
    » http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0030_01_06_2012.html#:~:text=Aprova%20o%20Regulamento%20T%C3%A9cnico%20Mercosul,Regulamento%20aprovado%20pelo%20Decreto%20n
  • 35
    Agencia Nacional de Vigilancia Sanitaria (ANVISA); Resolucao - RDC No. 48, de 16 de marco de 2006, Aprova o Regulamento Técnico sobre Lista de Substâncias que Não Podem Ser Utilizadas em Produtos de Higiene Pessoal, Cosméticos e Perfumes; Ministerio da Saude: Brasilia, Brazil, 2006, available at http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2006/rdc0048_17_03_2006.html#:~:text=Fica%20proibido%20o%20uso%20em,ou%20t%C3%B3xicas%20para%20a%20reprodu%C3%A7%C3%A3o, accessed in October 2020.
    » http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2006/rdc0048_17_03_2006.html#:~:text=Fica%20proibido%20o%20uso%20em,ou%20t%C3%B3xicas%20para%20a%20reprodu%C3%A7%C3%A3o
  • 36
    Heo, S.; Hwang, H. S.; Jeong, Y.; Na, K.; Carbohydr. Polym. 2018, 195, 534.
  • 37
    Krause, M.; Frederiksen, H.; Sundberg, K.; Jorgensen, F. S.; Jensen, L. N.; Norgaard, P.; Jorgensen, C.; Ertberg, P.; Juul, A.; Drzewiecki, K. T.; Skakkebaek, N. E.; Andersson, A. M.; Environ. Int. 2018, 110, 51.
  • 38
    do Nascimento, L. F.; dos Santos, E. P.; de Aguiar, A. P.; Rev. Virtual Quim. 2014, 6, 190.
  • 39
    Sousa, M. S. B.; Vieira, L. M.; de Lima, A.; Braz. J. Food Technol. 2011, 14, 202.
  • 40
    Rice-Evans, C. A.; Miller, N. J.; Paganga, G.; Free Radicals Biol. Med. 1996, 20, 933.
  • 41
    Liu, H.-T.; Ho, Y.-S.; Food Sci. Hum. Wellness 2018, 7, 134.
  • 42
    Wright, J. S.; Johnson, E. R.; DiLabio, G. A.; J. Am. Chem. Soc. 2001, 123, 1173.
  • 43
    Brand-Williams, W.; Cuvelier, M. E.; Berset, C.; LWT - Food Sci. Technol. 1995, 28, 25.
  • 44
    Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.; Jemal, A.; Ca-Cancer J. Clin. 2018, 68, 394.
  • 45
    AlFakeeh, A.; Brezden-Masley, C.; Curr. Oncol. 2018, 25, S18.
  • 46
    Weiss, R. B.; Christian, M. C.; Drugs 1993, 46, 360.
  • 47
    Napolitano, S.; Brancaccio, G.; Argenziano, G.; Martinelli, E.; Morgillo, F.; Ciardiello, F.; Troiani, T.; Cancer Treat. Rev. 2018, 69, 101.
  • 48
    Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Wooster, R.; Stratton, M. R.; Futreal, P. A.; Nature 2002, 417, 949.
  • 49
    Abbotts, R.; Jewell, R.; Nsengimana, J.; Maloney, D. J.; Simeonov, A.; Seedhouse, C.; Elliott, F.; Laye, J.; Walker, C.; Jadhav, A.; Grabowska, A.; Ball, G.; Patel, P. M.; Newton-Bishop, J.; Wilson, D. M.; Madhusudan, S.; Oncotarget 2014, 5, 3273.
  • 0
    5. McCubrey, J. A.; Steelman, L. S.; Chappell, W. H.; Abrams, S. L.; Wong, E. W. T.; Chang, F.; Lehmann, B.; Terrian, D. M.; Milella, M.; Tafuri, A.; Stivala, F.; Libra, M.; Basecke, J.; Evangelisti, C.; Martelli, A. M.; Franklin, R. A.; Biochim. Biophys. Acta, Mol. Cell Res. 2007, 1773, 1263.
  • 51
    Cseh, B.; Doma, E.; Baccarini, M.; FEBS Lett. 2014, 588, 2398.
  • 52
    Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.; Project, C. G.; Jones, C. M.; Marshall,C. J.; Springer, C. J.; Barford, D.; Marais, R.; Cell 2004, 116, 855.
  • 53
    Maurer, G.; Tarkowski, B.; Baccarini, M.; Oncogene 2011, 30, 3477.
  • 54
    Lapenna, S.; Giordano, A.; Nat. Rev. Drug Discovery 2009, 8, 547.
  • 55
    Daveri, E.; Valacchi, G.; Romagnoli, R.; Maellaro, E.; Maioli, E.; Evidence-Based Complementary Altern. Med. 2015, 2015, 545838.
  • 56
    Farooq, S.; Hussain, A.; Qurishi, M. A.; Hamid, A.; Koul, S.; Anti-Cancer Agents Med. Chem. 2018, 18, 1531.
  • 57
    Begnini, K. R.; Duarte, W. R.; da Silva, L. P.; Buss, J. H.; Goldani, B. S.; Fronza, M.; Segatto, N. V.; Alves, D.; Savegnago, L.; Seixas, F. K.; Collares, T.; Biomed. Pharmacother. 2017, 91, 510.
  • 58
    Osorio, L. S.; Ionta, M.; Demuner, A. J.; de Sousa, B. L.; Ferraz, G. O.; Varejao, E. V. V.; Ferreira-Silva, G. A.; Pilau, E. J.; Silva, E.; dos Santos, M. H.; J. Braz. Chem. Soc. 2020, 31, 2500.
  • 59
    Trachootham, D.; Alexandre, J.; Huang, P.; Nat. Rev. Drug Discovery 2009, 8, 579.
  • 60
    Kruk, J.; Duchnik, E.; Asian Pac. J. Cancer Prev. 2014, 15, 561.
  • 61
    Zhang, W.; Shu, X. O.; Li, H.; Yang, G.; Cai, H.; Ji, B. T.; Gao, J.; Gao, Y. T.; Zheng, W.; Xiang, Y. B.; J. Natl. Cancer Inst. 2012, 104, 1173.
  • 62
    Chikara, S.; Nagaprashantha, L. D.; Singhal, J.; Horne, D.; Awasthi, S.; Singhal, S. S.; Cancer Lett. 2018, 413, 122.
  • 63
    Bansal, A.; Simon, M. C.; J. Cell Biol. 2018, 217, 2291.
  • 64
    Mansur, M. C. P. P. R.; Leitao, S. G.; Cerqueira-Coutinho, C.; Vermelho, A. B.; Silva, R. S.; Presgrave, O. A. F.; Leitao, A. A. C.; Leitao, G. G.; Ricci-Junior, E.; Santos, E. P.; Rev. Bras. Farmacogn. 2016, 26, 251.
  • 65
    GraphPad PrismR, version 5.0; GraphPad Software, San Diego, CA, USA, 2007.
  • 66
    Azevedo-Barbosa, H.; Ferreira-Silva, G. A.; Silva, C. F.; de Souza, T. B.; Dias, D. F.; de Paula, A. C. C.; Ionta, M.; Carvalho, D. T.; Toxicol. In Vitro 2019, 59, 150.
  • 67
    GuavaSoft, version 2.7; EMD Millipore Corporation, Hayward, CA, USA, 2013.

Publication Dates

  • Publication in this collection
    01 Mar 2021
  • Date of issue
    Mar 2021

History

  • Received
    23 July 2020
  • Accepted
    27 Oct 2020
Sociedade Brasileira de Química Instituto de Química - UNICAMP, Caixa Postal 6154, 13083-970 Campinas SP - Brazil, Tel./FAX.: +55 19 3521-3151 - São Paulo - SP - Brazil
E-mail: office@jbcs.sbq.org.br