Antiprotozoal action of synthetic cinnamic acid analogs

Ana Paula de Azevedo dos Santos Saara Nery Fialho Daniel Sol Sol de Medeiros Ana Fidelina Gómez Garay Jorge Alfonso Ruiz Diaz Maria Celeste Vega Gómez Carolina Bioni Garcia Teles Leonardo de Azevedo Calderon About the authors

Abstract

INTRODUCTION

Leishmaniasis, Chagas disease, and malaria cause morbidity globally. The drugs currently used for treatment have limitations. Activity of cinnamic acid analogs against Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum was evaluated in the interest of identifying new antiprotozoal compounds.

METHODS

In vitro effects of analogs against L. braziliensis, L. infantum chagasi, T. cruzi, and P. falciparum, and hemolytic and cytotoxic activities on NCTC 929 were determined.

RESULTS

Three analogs showed leishmanicidal and tripanocidal activity. No antiplasmodial, hemolytic, or cytotoxic activity was observed.

CONCLUSIONS

Antiprotozoal activity of analogs against L. infantum braziliensis, L. infantum chagasi, and T. cruzi was demonstrated.

Keywords:
Bioactivity; Cinnamic acid; Leishmaniasis; Synthetic

The World Health Organization (WHO) lists 17 Neglected Tropical Diseases (NTDs), most of which are caused by protozoa. These diseases cause serious health problems, affecting more than one billion people, most of who live in extreme poverty in tropical and subtropical regions. These diseases are widespread, with an incidence of over 10% in the world population, and yet, their treatments are highly toxic and sometimes ineffective11. World Health Organization. Urbanization: The Weekly Epidemiological Record. Geneva: 2017. (Revised September 2017). Available from: Available from: http://www.who.int/wer/2017/en/ . Accessed: October 15, 2017.
http://www.who.int/wer/2017/en/...
, presumably due to lack of attention from the scientific community.

Among these, leishmaniasis is responsible for high morbidity and mortality rates, with an average of 12 million infected people, and about 350 million living in at-risk areas in 98 countries on five continents11. World Health Organization. Urbanization: The Weekly Epidemiological Record. Geneva: 2017. (Revised September 2017). Available from: Available from: http://www.who.int/wer/2017/en/ . Accessed: October 15, 2017.
http://www.who.int/wer/2017/en/...
. During the chemotherapeutic treatment of leishmaniasis, pentavalent antimonials (Sb+5), pentamidines, and amphotericin B are used. However, these treatments have their limitations, including high cost, exclusive venous administration, cardiac alterations, hepatic and renal toxicity, and the emergence of strains resistant to treatment22. Elmahallawy EK, Sampedro MA, Rodriguez-Granger J, Hoyos-Mallecot Y, Agil A, Navarro MJM, et al. Diagnosis of leishmaniasis. J Infect Dev Ctries. 2014;8(8):961-72..

Trypanosoma cruzi is a flagellate protozoan, responsible for causing Chagas disease. Fourteen million people are infected with T. cruzi worldwide, causing approximately 7000 deaths per year, with more than 25 million people living in at-risk areas11. World Health Organization. Urbanization: The Weekly Epidemiological Record. Geneva: 2017. (Revised September 2017). Available from: Available from: http://www.who.int/wer/2017/en/ . Accessed: October 15, 2017.
http://www.who.int/wer/2017/en/...
. Nifurtimox and benznidazole, the currently available treatment options, are relatively toxic, poorly effective in the chronic phase of the disease, and their prolonged administration encourages patient evasion leading to the emergence of resistant strains33. Bermudez J, Davies C, Simonazzi A, Real JP, Palma S. Current drug therapy and pharmaceutical challenges for Chagas disease. Act Trop. 2016;56:1-16..

In this group of diseases that pose public health challenges, malaria, caused by Plasmodium falciparum, stands out, especially in endemic areas such as Africa and Asia, where it is responsible for high rates of morbidity and mortality11. World Health Organization. Urbanization: The Weekly Epidemiological Record. Geneva: 2017. (Revised September 2017). Available from: Available from: http://www.who.int/wer/2017/en/ . Accessed: October 15, 2017.
http://www.who.int/wer/2017/en/...
. The development of resistance of these parasites to major therapeutic compounds, artemisinin and quinine derivatives, makes the search for new bioactive molecules important, especially those studied from a biodiversity perspective44. Mita T, Tanabe K. Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance. Jpn J Infect Dis. 2012;65(6):465-75..

Investing in the discovery of new molecules may have solutions in biodiversity. In this context, plants of the genus Piper L. (Piperaceae) are composed of components such as: alkaloids, flavonoids, arylpropanoids, terpenoids, phenylpropanoids, lignans, and cinnamic acid; which are characterized by antifungal, antioxidant, antiplasmodial, and trypanocidal bioactivity55. Regasini LO, Cotinguiba F, Passerini GD, Bolzani VS, Cicarelli RMB, Kato MJ, et al. Trypanocidal activity of Piper arboreum and Piper tuberculatum (Piperaceae). Rev Bras Farmacogn. 2009;19(1b):199-203.,66. Ferreira MGPR, Kayano AM, Silva-Jardim I, Da Silva TO, Zuliani JP, Facundo VA, et al. Antileishmanial activity of 3-(3,4,5-trimethoxyphenyl) propanoic acid purified from Amazonian Piper tuberculatum Jacq., Piperaceae, fruits. Rev Bras Farmacogn . 2010;20(6):1003-6.,77. Sharma P. Cinnamic acid derivatives: A new chapter of various pharmacological activities. J Chem Pharm Res. 2011;3(2):403-23.. Among these compounds, cinnamic acid (CinAc) was selected as a model in this study’s search for synthetic analogs with trypanocidal and antimalarial action66. Ferreira MGPR, Kayano AM, Silva-Jardim I, Da Silva TO, Zuliani JP, Facundo VA, et al. Antileishmanial activity of 3-(3,4,5-trimethoxyphenyl) propanoic acid purified from Amazonian Piper tuberculatum Jacq., Piperaceae, fruits. Rev Bras Farmacogn . 2010;20(6):1003-6.,77. Sharma P. Cinnamic acid derivatives: A new chapter of various pharmacological activities. J Chem Pharm Res. 2011;3(2):403-23.. By using this leader molecule we intend to enable the development of more effective drugs with low toxicity, thus presenting a new alternative for treating such diseases.

Synthetic analogs are derivatives of CinAc, isolated from the plant P. tuberculatum Jacq. These compounds (Figure 1) were synthesized by Sigma (Sigma-Aldrich).

FIGURE 1:
Analogous synthetic derivatives of cinnamic acid.

The analogs were diluted in dimethylsulfoxide-DMSO (Sigma-Aldrich) at concentrations at or below 0.6%. The drugs pentamidine, benznidazole, and artemisinin were used as negative controls (100% death) for Leishmanias spp., T. cruzi, and P. falciparum, respectively.

L. braziliensis (MHOM/BR/75/M2904) and L. infantum chagasi (MCAN/ES/92/BNC 83) promastigotes were maintained in vitro in RPMI 1640 medium (Sigma-Aldrich) supplemented with 25 mM HEPES (Sigma-Aldrich), 21 mM sodium bicarbonate (Sigma-Aldrich), 11 mM glucose (Sigma-Aldrich), 40 μg/mL of 10% gentamycin (v/v), and 10% previously inactivated fetal bovine serum (FBS) (Sigma-Aldrich). The parasites were kept in a BOD incubator at 26 ºC, and subcultured every five days.

Trypanosoma cruzi epimastigotes, “CL-B5” strain, were grown in liver infusion tryptose culture medium (LIT, Sigma-Aldrich) supplemented with 10% FBS; cultures were kept in a BOD incubator at 24 °C for weekly subculturing.

Chloroquine-resistant and mefloquine-sensitive P. falciparum (W2) strain derived from the Indochin III/CDC strain were cultured in vitro in human red blood cells with 2% hematocrit, diluted in RPMI 1640 culture medium supplemented with Albumax (Thermo Fisher Scientific). The cultures were maintained in desiccators at 37 °C in a gasogenic mixture containing 5% O2, 5% CO2, and 90% N2.

L. braziliensis and L. infantum chagasi promastigotes were plated at 1.5×106 parasites/mL in 96-well microplates, incubated at 26 °C for 72 h with varied concentrations (1260-9.8 μM) of the analogs. Subsequently, a 3 mM solution of alamarBlue (Sigma-Aldrich) was added and incubated at 26 °C for 4 h. Absorbance was monitored at 570 and 600 nm using a Synergy H1 (Biotek) multimodal spectrophotometer. Positive controls (parasites with no treatment) and negative controls in pentamidine, at varied concentrations (9-0.14 μM), and DMSO (0.6%) were used in order for the assay to be effective.

T. cruzi epimastigotes were plated at 2.5×105 parasites/mL on microplates and incubated at 24 °C for 72 h in serial concentrations of the analogs (1,260-9.8 μM). Subsequently, a solution of 200 μM chlorophenolRed-β-D-galactopyranoside (CPRG) was added followed by incubation at 37 °C for 4 h; absorbance was monitored using a Synergy H1 multimodal spectrophotometer (Biotek) at 570 nm88. Vega C, Roló M, Martínez-Fernández AR, Escario JA, Gómez-Barrio A. A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing-galactosidase. Parasit Research. 2005;95(4):296-8.. Parasites incubated with culture medium were considered as the growth control; reference drug benznidazole (100-1.56 μM) was used as a negative control.

P. falciparum cultures (W2) were treated with 5% sorbitol (Sigma-Aldrich), for synchronization in young trophozoites; parasitemia (0.05%) was determined by optic microscopy using a Rapid Panoptic kit (Laborclin, BR). Analogs were added to microplates at varied concentrations (1,260-9.8 μM), and infected red cells (positive control) and varied concentrations (0.17-0.0026 μM) of Artemisinin (negative control) were used; the activity was evaluated for 48 h at 37 °C.

The antimalarial evaluation was performed using SYBR (SYBR Green I, Thermo Fiser). RBCs were washed with 1× PBS and centrifuged at 700 g for 10 min. Then, the supernatant was discarded and 100 μL of a SYBR Green I solution (0.001% v/v in lysis buffer) was added; the contents were transferred to microplates containing 100 μL of 1x PBS. The plates were incubated for 30 min at room temperature; fluorescence was obtained at an excitation of 485 nm and an emission of 535 nm99. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique forhigh-throughput antimalarial drug screening. Antimicrob Agents Chemother. 2004;48(5):1803-6..

The cytotoxicity was assessed using NCTC 929 fibroblasts in an alamarBlue assay. Cells were plated in microplates with approximately 2.5×104 cells/well and incubated with varied concentrations of the analogs (1,260-9.8 μM) for 48 h in an incubator at 37 °C in a humid atmosphere containing 5% CO2. Subsequently, a 3 mM solution of alamarBlue was added followed by incubated at 37 °C for 4 h. Absorbance was monitored at 570 and 600 nm using a Synergy H1 (Biotek) multimodal spectrophotometer. The 50% cytotoxic concentration for cell growth (CC50) in the presence of the analogs and the control drugs was determined and compared with cells grown only in culture medium. The activity of the molecules tested was estimated by calculating the percentage of viable cells.

The 50% inhibitory concentration of parasite (IC50) and CC50 was determined by dose-response curves based on non-linear regression, applying the formula y = A1 + (A2-A1)/(1+10^((LOGx0-x)*p)), with determination of the level of significance at p < 0.05. The program Origin (OriginLab Corporation, Northampton, MA 01060, USA) was used. Standard deviation was calculated using MSExcel software. All experiments were performed in triplicates.

The selectivity index (SI) of samples was obtained by calculating the ratio between CC50 and IC50. Values > 10 were considered nontoxic, while substances with values below ten were considered toxic1010. Bézivin C, Tomasi S, Lohézic-Le FD, Boustie J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine. 2003;10(6-7):499-503..

Another way to assess the safety of a drug is to measure its hemolytic activity. Hemolytic assays were performed at varied concentrations (1,260-9.8 μM); 180 μL of erythrocytes (human) were added with 1% hematocrit in microplates with a U-bottom. The plates were incubated for 30 min at 37 °C with agitation after every 5 min. They were then centrifuged for 10 min. The supernatant was analyzed using a microplate spectrophotometer (Biochrom model: Expert plus) at 540 nm. Saponin (0.05%) was used as a positive control for hemolysis.

Chemical compounds isolated from plant extracts were studied because of their proven in vitro antiprotozoal activity, including the molecules derived from CinAc1111. Glaser J, Schultheis M, Hazra S, Hazra B, Moll H, Schurigt U, et al. Antileishmanial lead structures from nature: Analysis of structure-activity relationships of a compound library derived from caffeic acid bornyl ester. Molecules. 2014;19(2):1394-410.. Among the synthetic analogs of CinAc, E5 presented the lowest IC50 value against L. infantum chagasi (212.75 μM); when compared to L. braziliensis (IC50 340.43 μM). When tested against T. cruzi, IC50 was 1071.5 μM. Another analog, I9, presented IC50 value of 248.56 μM against L. braziliensis; against L. infantum chagasi and T. cruzi, I9 showed IC50 values ranging between 582 and 723 μM. Analog G7 showed inhibitory concentration against L. infantum chagasi (IC50 642.9 μM) and against T. cruzi (IC50 667.11 μM). Analogs A1, B2, C3, D4, and F6 were not effective at a concentration of 1,260 μM (Table 1). The analogs, at concentration of 1,260 μM (the highest concentration tested), presented no antimalarial, hemolytic, or cytotoxic activity; thus, selectivity index calculations were based on this concentration.

TABLE 1:
Leishmanicidal, trypanocidal, antiplasmodial, and cytotoxic evaluation of CinAc synthetic analogs.

Analogs E5 (L. infantum chagasi) and I9 (L. braziliensis) presented the best IC50 of 212.7 and 248.5 μM, respectively. When we analyzed the leishmanicidal action among parasite species in relation to the treatment control pentamidine, greater efficacy was obtained against L. braziliensis (IC50 0.76 μM) than against L. infantum chagasi (IC50 1.4 μM).

Studies have described the fractionation of biomonitoring of Valeriana wallichii chloroform extract, which resulted in two active CinAc derivatives against L. major promastigotes with IC50 of 48.8 μM1111. Glaser J, Schultheis M, Hazra S, Hazra B, Moll H, Schurigt U, et al. Antileishmanial lead structures from nature: Analysis of structure-activity relationships of a compound library derived from caffeic acid bornyl ester. Molecules. 2014;19(2):1394-410.. In this context, 3-(3,4,5-trimethoxyphenyl) propanoic acid obtained naturally from Piper tuberculatum Jacq, also presented activity against L. amazonensis promastigotes after 96 h hours of treatment66. Ferreira MGPR, Kayano AM, Silva-Jardim I, Da Silva TO, Zuliani JP, Facundo VA, et al. Antileishmanial activity of 3-(3,4,5-trimethoxyphenyl) propanoic acid purified from Amazonian Piper tuberculatum Jacq., Piperaceae, fruits. Rev Bras Farmacogn . 2010;20(6):1003-6. with IC50 at the concentration of 145 μg/mL (608.63 µM). In another study, synthetic derivatives of CinAc (3,4,5-trimethoxicinnamic acid) were effective at the concentration of 2 mg/mL (8.39 mM) against promastigotes of L. amazonensis, causing death of 92% of the parasites when treated for 96 h1212. Cury TAC, Yoneda JS, Zuliani JP, Soares AM, Stábeli RG, Calderon LA, et al. Cinnamic acid derived compounds loaded into liposomes: antileishmanial activity, production standardization and characterization. J Microencapsul. 2015;32(5):467-77.. The results of this study corroborate the results presented here, where the tests against Leishmania promastigotes were performed for 72 h and the maximum concentration was 1,260 μM.

The CinAc derivatives were inactive against the W2 (CQ-resistant) P. falciparum strain (IC50 > 1,260 μM). However, we expected high activity for this class since CinAc isolated from K. africana (β-hydroxycinnamic) showed IC50 ranging from 53.84 to 6.71 μM against other P. falciparum strains (W2, W2mef, CAM10, and SHF4)1313. Zofou D, Tene M, Tane P, Titanji VP. Antimalarial drug interactions of compounds isolated from Kigelia africana (Bignoniaceae) and their synergism with artemether, against the multidrug resistant W2mef Plasmodium falciparum strain. Parasitol Res. 2012;110(2):539-44.. The main mechanism of antiplasmodial action of CinAc class is the decline in energy production (ATP) due to inhibition of lactate transporters, changes in mitochondrial respiration, and reduction of translocation of carbohydrates and amino acids in parasitized red blood cells. Previous studies have frequently suggested that structural modifications of CinAc may alter its effectiveness, thus, reducing its antiplasmodial activity1414. Pérez BC, Teixeira C, Figueiras M, Gut J, Rosenthal PJ, Gomes JRB, et al. Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials. Eur J Med Chem. 2012;54:887-99..

Derivatives of natural products of plant origin exhibiting trypanocidal activity have been considered valid alternatives for control of parasites1515. Mejía-Parra JIJ, Pérez-Araujo MA, Roldán-Rodríguez J, Rojas-Idrogo C, Kato MJ, Delgado-Paredes GE. Actividad tripanocida de Piper solmsianum C. DC. sobre formas epimastigota y tripomastigota de Trypanosoma cruzi. Rev Cubana Med Trop. 2016;68(3):217-232.. In this context, studies performed with the species P. arboreum and P. tuberculatum, using the hexane extracts obtained from leaves against T. cruzi epimastigotes, resulted in an IC50 of 13.3 μg/mL for P. arboreum and 17.2 μg/mL for P. tuberculatum55. Regasini LO, Cotinguiba F, Passerini GD, Bolzani VS, Cicarelli RMB, Kato MJ, et al. Trypanocidal activity of Piper arboreum and Piper tuberculatum (Piperaceae). Rev Bras Farmacogn. 2009;19(1b):199-203.. Among the CinAc analogs tested, G7 showed the best trypanocidal activity, exhibiting IC50 of 667.1 μM and selectivity index of 1.9.

The results demonstrated that synthetic analogs of CinAc have antiprotozoal activity against L. braziliensis and L. infantum chagasi promastigotes, and T. cruzi epimastigotes. However, prior to performing the in vitro and in vivo tests, it is suggested that the compounds undergo structural modifications in order to obtain a more satisfactory selectivity index. The analogs tested are promising in the development of new molecules with greater effectiveness and fewer side effects.

Acknowledgement

The authors express their gratitude to Amy Grabner for the English review of the present manuscript, Malaria and Leishmaniasis Bioassay Platform (PBML) - FIOCRUZ-RO and Federal University of Rondônia for the opportunity of carrying out the research.

REFERENCES

  • 1
    World Health Organization. Urbanization: The Weekly Epidemiological Record. Geneva: 2017. (Revised September 2017). Available from: Available from: http://www.who.int/wer/2017/en/ Accessed: October 15, 2017.
    » http://www.who.int/wer/2017/en/
  • 2
    Elmahallawy EK, Sampedro MA, Rodriguez-Granger J, Hoyos-Mallecot Y, Agil A, Navarro MJM, et al. Diagnosis of leishmaniasis. J Infect Dev Ctries. 2014;8(8):961-72.
  • 3
    Bermudez J, Davies C, Simonazzi A, Real JP, Palma S. Current drug therapy and pharmaceutical challenges for Chagas disease. Act Trop. 2016;56:1-16.
  • 4
    Mita T, Tanabe K. Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance. Jpn J Infect Dis. 2012;65(6):465-75.
  • 5
    Regasini LO, Cotinguiba F, Passerini GD, Bolzani VS, Cicarelli RMB, Kato MJ, et al. Trypanocidal activity of Piper arboreum and Piper tuberculatum (Piperaceae). Rev Bras Farmacogn. 2009;19(1b):199-203.
  • 6
    Ferreira MGPR, Kayano AM, Silva-Jardim I, Da Silva TO, Zuliani JP, Facundo VA, et al. Antileishmanial activity of 3-(3,4,5-trimethoxyphenyl) propanoic acid purified from Amazonian Piper tuberculatum Jacq., Piperaceae, fruits. Rev Bras Farmacogn . 2010;20(6):1003-6.
  • 7
    Sharma P. Cinnamic acid derivatives: A new chapter of various pharmacological activities. J Chem Pharm Res. 2011;3(2):403-23.
  • 8
    Vega C, Roló M, Martínez-Fernández AR, Escario JA, Gómez-Barrio A. A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing-galactosidase. Parasit Research. 2005;95(4):296-8.
  • 9
    Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique forhigh-throughput antimalarial drug screening. Antimicrob Agents Chemother. 2004;48(5):1803-6.
  • 10
    Bézivin C, Tomasi S, Lohézic-Le FD, Boustie J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine. 2003;10(6-7):499-503.
  • 11
    Glaser J, Schultheis M, Hazra S, Hazra B, Moll H, Schurigt U, et al. Antileishmanial lead structures from nature: Analysis of structure-activity relationships of a compound library derived from caffeic acid bornyl ester. Molecules. 2014;19(2):1394-410.
  • 12
    Cury TAC, Yoneda JS, Zuliani JP, Soares AM, Stábeli RG, Calderon LA, et al. Cinnamic acid derived compounds loaded into liposomes: antileishmanial activity, production standardization and characterization. J Microencapsul. 2015;32(5):467-77.
  • 13
    Zofou D, Tene M, Tane P, Titanji VP. Antimalarial drug interactions of compounds isolated from Kigelia africana (Bignoniaceae) and their synergism with artemether, against the multidrug resistant W2mef Plasmodium falciparum strain. Parasitol Res. 2012;110(2):539-44.
  • 14
    Pérez BC, Teixeira C, Figueiras M, Gut J, Rosenthal PJ, Gomes JRB, et al. Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials. Eur J Med Chem. 2012;54:887-99.
  • 15
    Mejía-Parra JIJ, Pérez-Araujo MA, Roldán-Rodríguez J, Rojas-Idrogo C, Kato MJ, Delgado-Paredes GE. Actividad tripanocida de Piper solmsianum C. DC. sobre formas epimastigota y tripomastigota de Trypanosoma cruzi Rev Cubana Med Trop. 2016;68(3):217-232.

  • Financial Support: This work was supported by Coordination of Improvement of Higher Level Personnel (CAPES) and Institute of Epidemiology of Western Amazonia (INCT-EpiAMO).

Publication Dates

  • Publication in this collection
    Nov-Dec 2018

History

  • Received
    23 Dec 2017
  • Accepted
    05 June 2018
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