Journal of Pharmaceutical Negative Results

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 9  |  Issue : 1  |  Page : 39--43

Triazole-chalcones: Lack of antibacterial, anti-candida, and anti-dengue virus activities


Michelli dos Santos1, Felipe Rocha Da Silva Santos1, Ariane Coelho Ferraz2, Jéssica Tauany Andrade1, Karina Marjorie Silva Herrera1, William Gustavo Lima1, Lailah Horácio Sales Pereira3, Marina Goulart da Silva4, José Augusto Ferreira Perez Villar4, José Carlos de Magalhães2, Alex Gutterres Taranto5, Luciana Lara dos Santos3, Jamária Adriana Pinheiro Soares-Martins6, Jaqueline Maria Siqueira Ferreira1,  
1 Laboratory of Medical Microbiology, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil
2 Department of Bioprocess Engineering, Laboratory of Molecular and Cellular Biology, Alto Paraopeba Campus, Federal University of São João del-Rei, Ouro Branco, MG, Brazil
3 Laboratory of Molecular Biology, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil
4 Laboratory of Organic Synthesis and Nanostructures, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil
5 Laboratory of Pharmaceutical Chemistry, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil
6 Milwaukee Area Technical College, Milwaukee, WI, USA

Correspondence Address:
Jaqueline Maria Siqueira Ferreira
Laboratory of Medical Microbiology, Central-West Campus Dona Lindu, Federal University of São João del-Rei. 400 Sebastiao Gonçalves Coelho Street, Chanadour, Divinopolis, MG
Brazil

Abstract

Background: The need to identify new antimicrobial drugs that present different mechanisms of action is urgent. The variety of biological properties of chalcones give them a potential therapeutic compounds. Chalcones' synthetic manipulations are investigated worldwide in search of more powerful and efficient drugs to several infectious diseases' treatment. Objective: The purpose of this work was to evaluate the potential antibacterial, antifungal, and anti-dengue virus (DENV) of new ten triazole chalcones. Materials and Methods: Triazole chalcones were used to determine its antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis), Gram-negative bacteria (Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and Pseudomonas aeruginosa) species, and anti-Candida activity (Candida albicans) by the broth microdilution assay. The anti-DENV activity was evaluated by cell viability quantification after viral infection by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Results: Although Triazole chalcones have shown a low cytotoxicity concentration (with CC50of 47.21–192.51 μg/mL), these compounds did not exhibit antimicrobial activity against Gram-negative and Gram-positive bacteria, C. albicans neither DENV, even in the highest concentration tested. Triazole chalcones had no activity against all tested microorganisms, except for Gram-positive S. epidermidis bacteria when a poor antimicrobial effect was observed (125 μg/mL). Conclusion: The results suggest that triazole chalcones investigated in this study are not promising antimicrobial agents.



How to cite this article:
Santos Md, Da Silva Santos FR, Ferraz AC, Andrade JT, Silva Herrera KM, Lima WG, Sales Pereira LH, da Silva MG, Perez Villar JA, de Magalhães JC, Taranto AG, dos Santos LL, Pinheiro Soares-Martins JA, Siqueira Ferreira JM. Triazole-chalcones: Lack of antibacterial, anti-candida, and anti-dengue virus activities.J Pharm Negative Results 2018;9:39-43


How to cite this URL:
Santos Md, Da Silva Santos FR, Ferraz AC, Andrade JT, Silva Herrera KM, Lima WG, Sales Pereira LH, da Silva MG, Perez Villar JA, de Magalhães JC, Taranto AG, dos Santos LL, Pinheiro Soares-Martins JA, Siqueira Ferreira JM. Triazole-chalcones: Lack of antibacterial, anti-candida, and anti-dengue virus activities. J Pharm Negative Results [serial online] 2018 [cited 2019 Sep 24 ];9:39-43
Available from: http://www.pnrjournal.com/text.asp?2018/9/1/39/239508


Full Text



 Introduction



Chalcones (benzylideneacetophenones or 1,3-diaryl-2 -propen-1-ones), also known as α-β-unsaturated ketones, are a secondary metabolites' class found in plants. They are precursors of flavonoids and isoflavonoids.[1] Chalcones have a chemical structure composed of a central core and two aryl rings linked by an enone.[2] This natural compounds' class has been widely studied due to its easy chemical handling, simple synthesis process, and its potential pharmacological activities as antineoplastic,[3] antiretroviral,[4] anti-inflammatory,[5] antioxidant,[6] antibacterial,[7] and antifungal,[8] among others.[9]

In recent decades have been being a continuous effort to search for new antimicrobial drugs that present different mechanisms from those already existing, as an answer to the global threat of bacterial and fungal resistance.[10] Furthermore, viral diseases continue to be a concern due to the lack of available vaccines or treatments. Dengue virus (DENV), an arbovirus of the Flaviviridae family of great clinical importance, causes approximately 390 million infections and about 20,000 deaths per year.[11]

Considering the relevance of the aspects presented, the aim of this study was to evaluate the antibacterial, anti-Candida, and anti-DENV activities of new ten triazole chalcones.

 Material and Methods



Chemistry

Triazole chalcones were synthesized as anteriorly described,[12],[13] and its chemical structures are shown in [Figure 1]. The compounds were diluted in dimethyl sulfoxide (DMSO) and stored at −20°C in the concentration of 10 mg/mL until the use.{Figure 1}

Microorganisms

The microorganisms used in this study were obtained from the American Type Culture Collection (ATCC) and were kindly provided by the Osvaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil). Antibacterial assays were conducted using six bacterial strains: (i) two Gram-positive bacteria: Staphylococcus aureus ATCC 29213 and Staphylococcus epidermidis ATCC 12228; (ii) four Gram-negative bacteria: Klebsiella pneumoniae ATCC 43816, Enterobacter cloacae ATCC 23355, Pseudomonas aeruginosa ATCC 27853, and Acinetobacter baumannii ATCC 19606. For antifungal assays, Candida albicans ATCC 18804 was employed.

Antibacterial assay

Antibacterial activity against Gram-positive and Gram-negative bacteria was evaluated by broth microdilution method described at M07-A9 document of Clinical and Laboratory Standards Institute (CLSI),[14] with minor modifications.[15] Triazole chalcones were diluted in Mueller-Hinton Broth (Himedia, India) at 1.95–250 μg/mL concentration range. Chloramphenicol (Sigma-Aldrich, Brazil), gentamicin (Inlab, Brazil), and amoxicillin (Germed, Brazil) were used as positive control. Assays were performed in triplicate and repeated three times.

Antifungal assay

To evaluate antifungal activity against C. albicans, was used the broth microdiluition method described at M27-A3 document,[16] with minor modifications.[17] Triazole chalcones at 3.91–500 μg/mL concentration range were diluted on Sabouraud Dextrose Broth (Himedia, India). Ketoconazole (Pharma Nostra, Brazil) and nystatin (Pharma Nostra, Brazil) were used as positive control. Assays were performed in triplicate and repeated three times.

Cell line and virus

Baby hamster kidney cells (BHK-21) (ATCC CCL-10, USA) were cultured in Dulbecco's Modified Eagle's Minimum Medium (Cultilab, Brazil) with 5% fetal bovine serum and 0.3% penicillin-streptomycin-amphotericin solution (10,000 U/mL + 10 mg/mL + 2 mg/mL, respectively) (Sigma-Aldrich, USA). Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2.

The DENVserotype 2 (DENV-2) used in the antiviral assays was kindly provided by Dr. Erna Geessien Kroon of Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Cytotoxicity assay

The cytotoxic concentration to 50% of cells (CC50) was evaluated by colorimetric method 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described by Roy et al.[18] with minor modifications. Cells were cultured in 96-well plates and treated with different concentrations of Triazole chalcones (3.15–400 μg/mL), followed by incubation at 37°C for 48 h. After, MTT solution (5 mg/mL) was added to each well, and plates were incubated at 37°C for 90 min. Then, 100 μL of DMSO was added to each well to solubilize formazan crystals. The absorbance was measured in a microplate reader at 540 nm and the CC50 was calculated as the compound concentration required to reduce cell viability by 50%.[19] Assays were performed in triplicate and results were expressed as a mean of three independent experiments.

Antiviral assay

The antiviral activity of triazole chalcones was evaluated as described by Flechas et al.,[20] with minor modifications. Antiviral assays were performed using confluent cultures of BHK-21 cells in 96-well plates infected with DENV-2 at multiplicity of infection of 0.1 in the presence of equal and lower CC50 concentrations of the compounds. The plates were incubated for 5 days at 37°C in a humidified atmosphere of 5% CO2. Then, a MTT solution (5 mg/mL) was added to each well as previously described. The effective antiviral concentration (EC50) was calculated as the protective concentration of 50% of infected cells treated with triazole chalcones. The percentage of viral protection was calculated as described by Hidari et al.[21]

 Results



It was found that triazole chalcones have been exhibited no antibacterial activity against different pathogenic species with minimum inhibitory concentration (MIC) above 250 μg/mL [Table 1]. Triazole chalcones did not inhibit the growth of Gram-negative bacteria (K. pneumoniae, E. cloacae, P. aeroginosa, and A. baumannii) at various concentrations (1.95–250 μg/mL) unlike they observed with the positive controls gentamicin (MIC 0.49 μg/mL) and chloramphenicol (MIC ranged from 3.9 μg to 125 μg/mL). Similar results were obtained for Gram-positive bacteria (S. aureus). On the other hand, the triazole chalcones TC02, TC03, and TC04 exhibited a low antibacterial activity against S. epidermidis (MIC at 125 μg/mL). However, amoxicillin, used as a positive control against Gram-positive bacteria, had a MIC ranged from 0.2 to 3.9 μg/mL [Table 1]. The Triazole chalcones did not exhibit antifungal activity against C. albicans when compared to positive controls (ketoconazole at 31.25 μg/mL and nystatin at 4 μg/mL).{Table 1}

The triazole chalcones exhibit low cytotoxicity in BHK-21 cells, with CC50 ranging from 47.21 μg/mL to 192.51 μg/mL. The antiviral activity of Triazole chalcones was evaluated against DENV-2; however, none of our compounds were able to reduce the cytopathic effect caused by viral infection in BHK-21 cells.

 Discussion



The chalcones are secondary plant metabolites and bioenergetic precursors of flavonoids and isoflavonoids. These compounds are considered as important natural subgroup that show promising biological properties usually attributed to α-β-unsaturated ketone group and chemical substitutions of the two aryl rings.[22] Structural changes have been done in the original structure of chalcones, mainly on the aryl rings and enone linker to reduce cytotoxicity and improve biological activity.[23] These changes were made replacing the aryl rings with different heterocyclic structures such as 1,2,3-triazole, thiophene, thiazole, imidazole, benzothiophene, indole, chromene, and chromanone.[23]

Nitrogenous heterocyclic structures such as 1,2,3-triazoles have been studied in medicinal chemistry due to its strong resistance to metabolic degradation and presence of three nitrogen atoms in its structure, which enable the formation of hydrogen bonds between these molecules and another biological compounds.[24]

Therefore, the objective of this study was to synthesize and evaluate the antibacterial, antifungal, and antiviral effects of 1,2,3-triazole chalcones. The antibacterial activity was represented as the MIC required to inhibit the growth of Gram-positive and Gram-negative bacteria.[10] According to Mbaveng et al.,[25] an antimicrobial activity is considered as significant when MIC is below 10 μg/mL, moderate when is 10 μg/mL et al.[26] also synthetized triazole chalcones and studied their antibacterial activity against Gram-negative bacteria (Escherichia coli, P. aeruginosa, and Salmonella typhi) and Gram-positive bacteria (Streptococcus pyogenes and S. aureus). However, these authors found promising antibacterial activity with higher potency than antibiotics used, especially with chalcones showing a fluoride atom linked to a phenyl triazole group conjugated with quinolones.[26]

The antifungal activity of triazole chalcones against C. albicans was evaluated; however, our results showed that triazole chalcones exhibited no promising antifungal activity. On the other hand, Yin et al.[27] synthesized α-triazole chalcones and evaluated its action against several pathogenic microorganisms, including C. albicans and Candida mycoderma, demonstrating that these compounds exhibited antimicrobial activity against all the species tested. In this case, Bis-pyrrolidyl chalcone showed antifungal activity against C. mycoderma at MIC concentration of 4 μg/mL, which was similar to positive control (fluconazole at 4 μg/mL). Authors suggested that the antifungal activity of Bis-pyrrolidyl could be related to the presence of bulky nitrogen groups directly linked to the aromatic ring of the chalcone core.

Bioactive molecules derived from chalcones are important components in therapeutic studies focused on the discovery of new antimicrobial drugs, as studies showing antiviral activity of these molecules against hepatitis C virus [28] and human immunodeficiency virus (HIV).[4] To looking for new antiviral compounds, it is necessary to evaluate first their cytotoxicity in vitro in a safe way. Therefore, in this study, triazole chalcones presented a low cytotoxicity in BHK-21 cells what suggests that these compounds may have a low nephrotoxicity. However, an EC50 of Triazole chalcones could not be determined in this study against DENV-2. In contrast, other studies have been reveled high activity anti-DENV of natural chalconas. Srivarangkul et al.[11] have isolated chalcones from apple leaves (Syzygium samarangense) and evaluated their antiviral activity against DENV-2. The chalcone F5Y (2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone) showed a CC50 of 48.87 μM and EC50 of 5.09 μM, whereas the chalcone F8Y (2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone) presented a CC50 >100 μM and EC50 of 31 μM. These compounds exhibited great antiviral activity but have not already investigated against DENV.

These in vitro analyses have been shown that 1,2,3-triazole chalcones have not antibacterial, anti-Candida, and anti-DENV promising effects under the experimental conditions employed.

 Conclusion



Our results showed a lack of antimicrobial activity of triazole-chalcones against several pathogenic species of bacteria, C. albicans and DENV -2. Nevertheless, more studies about the relationship between the structure of triazole-chalcones and their antimicrobial activity are still required to understand and enhance their pharmacological effects. Molecular modifications of these compounds should be encouraged, with the aim of potentiating the antimicrobial effect.

Acknowledgments

The authors are grateful for the support provided by Brazilian Council for Development of Science and Technology (CNPq) (UNIVERSAL 446997/2014-5), A. G. Taranto is grateful to CNPq's fellowship (305117/2017-3) and Minas Gerais Foundation for the Support of Science (FAPEMIG). We thank UFSJ/PPGCS for the availability of instrumental analytical support.

Financial support and sponsorship

Brazilian Council for Development of Science and Technology (CNPq) and Minas Gerais Foundation for the Support of Science (FAPEMIG).

Conflicts of interest

There are no conflicts of interest.

References

1Fong HY, Abd Malek SN, Yee HS, Karsani SA. Helichrysetin induces DNA damage that triggers JNK-mediated apoptosis in Ca ski cells. Pharmacogn Mag 2017;13:607-12.
2Tajuddeen N, Isah B, Suleiman A, Van Heerden FR, Ibrahim MA. The chemotherapeutic potential of chalcones against leishmaniases: A review. Int J Antimicrob Agents 2017;17:30220.
3Mahapatra DK, Bharti SK, Asati V. Anti-cancer chalcones: Structural and molecular target perspectives. Eur J Med Chem 2015;98:69-114.
4Rizvi SU, Siddiqui HL, Johns M, Detorio M, Schinazi RF. Anti-HIV-1 and cytotoxicity studies of piperidyl-thienyl chalcones and their 2-pyrazoline derivatives. Med Chem Res 2012;21:3741-9.
5Israf DA, Khaizurin TA, Syahida A, Lajis NH, Khozirah S. Cardamonin inhibits COX and iNOS expression via inhibition of p65NF-kappaB nuclear translocation and lkappa-B phosphorylation in RAW 264.7 macrophage cells. Mol Immunol 2007;44:673-9.
6Aoki N, Muko M, Ohta E, Ohta S. C-geranylated chalcones from the stems of Angelica keiskei with superoxide-scavenging activity. J Nat Prod 2008;71:1308-10.
7Wei ZY, Chi KQ, Yu ZK, Liu HY, Sun LP, Zheng CJ, et al. Synthesis and biological evaluation of chalcone derivatives containing aminoguanidine or acylhydrazone moieties. Bioorg Med Chem Lett 2016;26:5920-5.
8Lahtchev KL, Batovska DI, Parushev SP, Ubiyvovk VM, Sibirny AA. Antifungal activity of chalcones: A mechanistic study using various yeast strains. Eur J Med Chem 2008;43:2220-8.
9Mahapatra DK, Bharti SK. Therapeutic potential of chalcones as cardiovascular agents. Life Sci 2016;148:154-72.
10Chu WC, Bai PY, Yang ZQ, Cui DY, Hua YG, Yang Y, et al. Synthesis and antibacterial evaluation of novel cationic chalcone derivatives possessing broad spectrum antibacterial activity. Eur J Med Chem 2018;143:905-21.
11Srivarangkul P, Yuttithamnon W, Suroengrit A, Pankaew S, Hengphasatporn K, Rungrotmongkol T, et al. A novel flavanone derivative inhibits dengue virus fusion and infectivity. Antiviral Res 2018;151:27-38.
12da Silva GD, da Silva MG, Souza EM, Barison A, Simões SC, Varotti FP, et al. Design and synthesis of new chacones substituted with azide/triazole groups and analysis of their cytotoxicity towards HeLa cells. Molecules 2012;17:10331-43.
13Evangelista FC, Bandeira MO, Silva GD, Silva MG, Andrade SN, Marques DR, et al. Synthesis and in vitro evaluation of novel triazole/azide chalcones. Med Chem Res 2016;26:27-43.
14Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard. 9th ed. Pennsylvania: Wayne; 2012. p. M07-A9.
15Ugur AR, Dagi HT, Ozturk B, Tekin G, Findik D. Assessment of in vitro antibacterial activity and cytotoxicity effect of Nigella sativa oil. Pharmacogn Mag 2016;12:471-4.
16Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. 3rd ed., Vol. 28. Pennsylvania: Wayne; 2008. p. M27-A3.
17Filho JF, Roldi LL, Fiorot RG, Andrade JT, Aleixo ÁA, Carvalho RS, et al. Synthesis, in vitro antifungal activity and molecular modeling studies of new mannich bases derived from lawsone. J Braz Chem Soc 2016;27:2127-40.
18Roy S, Chaurvedi P, Chowdhary A. Evaluation of antiviral activity of essential oil of Trachyspermum ammi against Japanese encephalitis virus. Pharmacognosy Res 2015;7:263-7.
19Samarghandian S, Boskabady MH, Davoodi S. Use of in vitro assays to assess the potential antiproliferative and cytotoxic effects of saffron (Crocus sativus L.) in human lung cancer cell line. Pharmacogn Mag 2010;6:309-14.
20Flechas MC, Ocazionez RE, Stashenko EE. Evaluation of in vitro antiviral activity of essential oil compounds against dengue virus. Pharmacogn J 2017;10:55-9.
21Hidari KI, Ikeda K, Watanabe I, Abe T, Sando A, Itoh Y, et al. 3-O-sulfated glucuronide derivative as a potential anti-dengue virus agent. Biochem Biophys Res Commun 2012;424:573-8.
22Kunthalert D, Baothong S, Khetkam P, Chokchaisiri S, Suksamrarn A. A chalcone with potent inhibiting activity against biofilm formation by nontypeable Haemophilus influenzae. Microbiol Immunol 2014;58:581-9.
23Ayati A, Esmaeili R, Moghimi S, Oghabi Bakhshaiesh T, Eslami-S Z, Majidzadeh-A K, et al. Synthesis and biological evaluation of 4-amino-5-cinnamoylthiazoles as chalcone-like anticancer agents. Eur J Med Chem 2018;145:404-12.
24Dalvie DK, Kalgutkar AS, Khojasteh-Bakht SC, Scott obach R., O'Donnell JP. Biotransformation reactions of five-membered aromatic heterocyclic rings. Chem Res Toxicol 2002;15:269-99.
25Mbaveng AT, Sandjo LP, Tankeo SB, Ndifor AR, Pantaleon A, Nagdjui BT, et al. Antibacterial activity of nineteen selected natural products against multi-drug resistant gram-negative phenotypes. Springerplus 2015;4:823.
26Faidallah HM, Girgis AS, Tiwari AD, Honkanadavar HH, Thomas SJ, Samir A, et al. Synthesis, antibacterial properties and 2D-QSAR studies of quinolone-triazole conjugates. Eur J Med Chem 2018;143:1524-34.
27Yin BT, Yan CY, Peng XM, Zhang SL, Rasheed S, Geng RX, et al. Synthesis and biological evaluation of α-triazolyl chalcones as a new type of potential antimicrobial agents and their interaction with calf thymus DNA and human serum albumin. Eur J Med Chem 2014;71:148-59.
28Mateeva N, Eyunni SV, Redda KK, Ononuju U, Hansberry TD 2nd, Aikens C, et al. Functional evaluation of synthetic flavonoids and chalcones for potential antiviral and anticancer properties. Bioorg Med Chem Lett 2017;27:2350-6.