Amides from Porcelia ponderosa Roots Display Efficacy In Vitro Against Intracellular Amastigotes of Trypanosoma cruzi

ABSTRACT

In the present work, the MeOH extract from roots of Porcelia ponderosa R.E. Fries (Annonaceae) was subjected to several chromatographic techniques to afford three chemically related amides: N‐trans‐p‐coumaroyltyramine (1), N‐trans‐caffeoyltyramine (2), and N‐trans‐feruloyltyramine (3). Isolated compounds were characterized by NMR and MS spectral analysis. This is the first report of compounds 1–3 in P. ponderosa, while 1 is a previously unreported metabolite from the Porcelia genus. Previous studies reported the effects of amides 1–3 on the inhibition of sterol 14‐α‐demethylase (CYP51), a key enzyme in the life cycle of Trypanosoma cruzi, using an in silico approach. However, no experimental evidence was conducted to prove their effects against the parasite. Based on these data and isolation of amides 1–3 from roots of P. ponderosa, these compounds were tested in vitro against the intracellular amastigotes of T. cruzi. Compounds 1 and 2, containing p‐coumaroyl and caffeic moieties, displayed potent activity with EC₅₀ values of 6.3 ± 1.2 and 3.4 ± 0.6 µM, respectively, a similar efficacy observed to the standard drug benznidazole (EC₅₀ of 5.5 ± 2.2 µM). Otherwise, compound 3, with a feruloyl moiety, showed moderate activity (EC₅₀ of 14.0 ± 0.3 µM). These results indicated that p‐coumaric and caffeoyl moieties play an important role in the potency against amastigotes of T. cruzi. Otherwise, simplified derivatives of 1–3 such as p‐coumaric, caffeic and ferulic acids as well as tyramine, showed to be inactive against amastigotes (EC₅₀ > 150 µM) reinforcing the importance of condensation of these free acids and tyramine for the efficacy of amides 1–3. Considering the cytotoxicity against murine fibroblasts, compounds 1–3 displayed CC₅₀ values higher than 200 µM, and SI values higher than 31.7, 55.6, and 14.3, respectively. These findings highlight a safe profile and the antiparasitic potential of amides 1 and 2, which could be used as promising hit compounds for the design of new drug candidates for Chagas disease.

Amides from Porcelia ponderosa display potent activity against amastigotes from Trypanosoma cruzi.

1. Introduction

Neglected tropical diseases (NTDs) are a group of approximately 20 diverse infectious conditions that primarily affect the poorest and most vulnerable populations in tropical and subtropical areas of the world [1, 2]. The World Health Organization (WHO) preconized a roadmap that establishes several political approaches to eradicate at least one of these diseases on the globe [3]. Included in this group, Chagas disease (CD) is a parasitic disease caused by the hemoflagellate Trypanosoma cruzi, which affects seven million people worldwide, mostly in Latin America, and causes 10 000 deaths annually by heart and related complications [4, 5]. Actually, the therapy for CD is based on two drugs, nifurtimox and benznidazole, which pose a restricted efficacy in the chronic phase of the disease and severe side effects [6, 7, 8]. Due to the lack of treatment options for CD, it is necessary to search for new molecules with potent activity against the intracellular amastigotes, the clinically relevant parasitic form, especially during the chronic phase. Considering this aspect, plant‐derived metabolites represent an interesting source for the discovery of new bioactive entities.

Phytochemical investigations of Annonaceae species have led to the identification of a wide array of bioactive compounds, including acetogenins, flavonoids, alkaloids, and terpenoids with immunosuppressive, antineoplastic, cytotoxic, antimicrobial, and anti‐inflammatory effects [9]. In the context of the discovery of antiparasitic natural products, our research group has demonstrated the effects of different acetogenins and fatty acids from Porcelia macrocarpa, acting especially against Leishmania infantum and Trypanosoma cruzi [10, 11, 12, 13, 14, 15]. More recently, we reported the occurrence of γ‐lactones from Porcelia ponderosa with potent activity in vivo against Schistosoma mansoni [16]. As part of our continuous studies with P. ponderosa, we report here the isolation of three chemically related amides: N‐trans‐p‐coumaroyltyramine (paprazine, 1), N‐trans‐caffeoyltyramine (2), and N‐trans‐feruloyltyramine (3) from its roots. Recently, the inhibitory potential of compounds 1–3 against sterol 14‐α‐demethylase (CYP51), a key enzyme in the life cycle of Trypanosoma cruzi, was evaluated in silico [17]. These results indicated a superior binding affinity of compound 2, in comparison with compounds 1 and 3, to CYP51, making it a promising bioactive compound against T. cruzi. However, no experimental approaches were conducted, indicating that in vitro studies are necessary to validate their efficacy as antitrypanosomal agents. Considering the isolation of 1–3 form roots of P. ponderosa, in the present work, the in vitro activity of these compounds against amastigote forms of T. cruzi was evaluated, in addition to a cytotoxicity in mammalian cells aiming at the determination of selectivity index. Furthermore, the simplified compounds p‐coumaric, caffeic, and ferulic acids, as well as tyramine, were tested to evaluate their effects against T. cruzi amastigotes.

2. Results and Discussion

Chromatographic fractionation of the MeOH extract from roots of P. ponderosa afforded three amides: N‐trans‐p‐coumaroyltyramine (1), N‐trans‐caffeoyltyramine (2), and N‐trans‐feruloyltyramine (3), as shown in Figure 1. Structures of 1–3 were identified by analysis of their ¹H NMR and MS spectral data and comparison with those previously reported in the literature [18, 19, 20]. This is the first occurrence of amides 1–3 in P. ponderosa, whereas compound 1 is a previously unreported compound from the Porcelia genus.

FIGURE 1.

Amides 1–3 isolated from P. ponderosa roots.

Previous studies reported the biological effects of amides 1–3 including antimicrobial, antidiabetic, antioxidant, hypoglycemic, hypotensive, neuroprotective, and alpha‐glucosidase inhibitory potential [21, 22, 23, 24, 25, 26], but no information regarding their effects against T. cruzi has been previously reported. On the other hand, a recent work described that amides 1–3 can be considered possible bioactive metabolites against T. cruzi since these compounds, as evidenced using in silico analysis, can act as potential inhibitors of 14‐α‐demethylase (CYP51) protease from the parasite [17]. Additionally, compounds 1–3 were subjected to virtual ADME screening, suggesting reduced toxicity [17]. However, considering that no experimental assays were performed to validate their potential against T. cruzi and the isolation of amides 1–3 from roots of P. ponderosa, the effects against intracellular amastigotes of T. cruzi were evaluated in vitro.

Light microscopy studies revealed a significant reduction in the number of intracellular parasites and infected macrophages when compared to untreated parasites (control group). As shown in Table 1, compounds 1–3 induced no mammalian cytotoxicity to mammalian NCTC cells at the highest tested concentration (CC₅₀ > 200 µM). Compounds 1 and 2 displayed high effectiveness against intracellular amastigotes of T. cruzi with EC₅₀ values of 6.3 ± 1.2 and 3.4 ± 0.6 µM, respectively, similar to the potency benznidazole with benznidazole (EC₅₀ = 5.5 ± 2.2 µM). The determination of the selectivity index (SI), given by the ratio between the mammalian toxicity and the activity against intracellular amastigotes of T. cruzi, resulted in values higher than 31.7 and 55.6 for compounds 1 and 2, respectively. On the other hand, compound 3 displayed moderate potency with an EC₅₀ of 14.0 ± 0.3 µM and SI > 14.3. In addition, the treatment with compounds 1–3 in the intracellular amastigote form demonstrated the integrity of the host cells after treatment, as well as the complete elimination of the parasites.

TABLE 1.

Anti‐T. cruzi activity (amastigotes) and mammalian cytotoxicity (NCTC cells) for amides 1–3 and simplified derivatives p‐coumaric, caffeic, and ferulic acids, and tyramine.

compound	EC50 ± SD (µM)	CC50 (µM)	SI
1	6.3 ± 1.2	>200	>31.7
2	3.4 ± 0.6	>200	>55.6
3	14.0 ± 0.3 *	>200	>14.3
p‐Coumaric acid	>150	>200	—
Caffeic acid	>150	>200	—
Ferulic acid	>150	>200	—
Tyramine	>150	>200	—
Benznidazole	5.5 ± 2.2	>200	>36.4

Note: CC50 and EC50 values were calculated from dose–response sigmoid curves. Data are presented as mean ± SEM from duplicate samples (n = 2 per concentration), with each experiment performed at least twice independently. Error bars represent ± SD. Statistical significance among groups was determined using one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. The p value is indicated as p < 0.05 and was considered statistically significant. All analyses were conducted using GraphPad Prism 5.

Abbreviations: CC50, 50% cytotoxic concentration in NCTC L929 cells; EC50, 50% effective concentration against intracellular amastigotes of T. cruzi (Y strain); SD, standard deviation; SI, selectivity index.

^* p < 0.05 compared to benznidazole.

Due to the occurrence of the chemically related amides 1–3, it was possible to suggest some considerations regarding their structures and their anti‐T. cruzi activities. Initially, it was observed that the presence of p‐coumaroyl and caffeoyl moieties plays an important role in the detected antitrypanosomal potential, especially when comparing the EC₅₀ values of compounds 1 and 2 with that determined for the standard drug benznidazole. However, on comparing the EC₅₀ values of compounds 1 and 2 to that obtained for compound 3, the effectiveness against the amastigotes was reduced when the hydroxy group at the C‐8 position was methylated. However, these differences caused no alteration in the mammalian toxicity to NCTC cells, with CC₅₀ values of compounds 1–3 higher than 200 µM.

Considering the different antitrypanosomal potencies of the tested compounds, simplified derivatives of compounds 1–3 were obtained, resulting in p‐coumaric, caffeic, and ferulic acids as well as tyramine, as shown in Figure 2. As observed in Table 1, all free carboxylic acids and tyramine were shown to be inactive at higher tested concentrations (EC₅₀ > 150 µM) against the intracellular amastigotes of T. cruzi. Therefore, the obtained data reinforce the importance of condensation of these free acids and tyramine, which, associated with the presence of p‐coumaroyl and caffeoyl moieties, appears to be essential for the biological activity observed of amides 1–3.

FIGURE 2.

Molecular simplification of amides 1–3 to give p‐coumaric, caffeic, and ferulic acids (red moiety) and tyramine (blue moiety).

Moreover, the Drugs for Neglected Diseases initiative (DNDi) recommends EC₅₀ values against intracellular amastigote forms below 10 µM, as well as a selectivity index higher than 10, both values as criteria for the selection of new hit compounds for Chagas disease [27]. Accordingly, compounds 1 and 2 fulfilled these criteria, displaying potent activity against the intracellular amastigotes and high selectivity indexes (Figure 1).

3. Conclusion

In this study, three chemically related amides 1–3 were isolated from the roots of Porcelia ponderosa (Annonaceae), and their effects against the clinically relevant form of T. cruzi (amastigotes) were demonstrated, corroborating the previous in silico findings. Compounds 1 and 2 showed high potency and lack of mammalian toxicity, with similar EC₅₀ values to the standard drug benznidazole. Additionally, when simplified compounds were evaluated, free acids (p‐coumaric, caffeic, and ferulic) and tyramine were shown to be inactive, corroborating the importance of condensed product (amide) for the antiparasitic activity. Besides, all the compounds tested showed no cytotoxicity to fibroblasts, resulting in high selectivity indexes, similar to the standard drug. Thus, based on the obtained results, the amides 1 and 2 could be considered promising scaffolds for future hit‐to‐lead optimization studies.

4. Experimental

4.1. General Experimental Procedure

Chromatographic procedures employed silica gel 60 (0.063–0.2 mm; Macherey‐Nagel) for column chromatography and silica gel F₂₅₄ plates (Merck) for thin‐layer chromatography (TLC). Semi‐preparative HPLC was carried out using an Ultimate 3000 system (Dionex, Sunnyvale, CA, USA), equipped with a quaternary pump, photodiode array (PDA) detector, and a reversed‐phase RP‐C₁₈ column (Phenomenex, 250 × 10.0 mm, 5 µm). NMR spectra were acquired on a Bruker AC‐200 or on a Varian INOVA spectrometer operating at 200 or 500 MHz for ¹H nucleus, respectively. Methanol‐d₄ or acetone‐d₆ (Merck) was used as a solvent. ESI‐HRMS spectra were recorded on a MicrOTOF‐QII (Bruker Daltonics) and Q Exactive (Thermo Fisher Scientific) instruments. All used solvents and reagents, including p‐coumaric, caffeic, and ferulic acids and tyramine, were purchased from commercial sources (Merck and Sigma) and were used without further purification.

4.2. Plant Material

Roots of P. ponderosa were collected in the city of Tarauaca, a region of Amazonian rainforest in Acre State/Brazil (8°10'02.1''S 70°53'01.0''W) in March 2020. The plant material was identified by Prof. Dr. Maria C. Souza (Acre Federal University, Brazil), and a voucher has been deposited at the Herbarium of the Federal University of Acre, under number CFCZS M.C.Souza 791, and received a registration code at SisGen A4123E4.

4.3. Extraction and Isolation

Dried and milled roots of P. ponderosa (497 g) were defatted with hexane and sequentially exhaustively extracted with MeOH. Evaporation of the solvent under reduced pressure afforded 26.1 g of MeOH extract. Part of this extract (25.0 g) was suspended in MeOH, acidified with 1% HCl (pH 2), and extracted with CH₂Cl₂. The hydroalcoholic phase was basified with NH₄OH 25% (pH 10) and extracted using CH₂Cl₂. The organic phase afforded, after elimination of the solvent under reduced pressure, 726 mg of the alkaloidal fraction. Part of this material (500 mg) was chromatographed over silica gel, eluted with increasing amounts of EtOAc in hexane (up to 100%), to give 10 groups (A–J). Group F (14.5 mg) was chromatographed over silica gel, eluted with CHCl₃:MeOH:NH₄OH (9:1:0.5 to 8:2:0.5), to give compound 1 (5.3 mg). Group I (22.1 mg) was purified by RP‐HPLC using a mobile phase of MeOH:H₂O containing 0.1% formic acid, yielding compounds 2 (2.4 mg) and 3 (3.0 mg).

N‐trans‐p‐coumaroyltyramine [(E)‐3‐(7‐hydroxyphenyl)‐N‐(6’‐hydroxyphenethyl)acrylamide, 1]. White amorphous powder (purity 98% by HPLC). ESI‐HRMS m/z 284.1291 [M + H]⁺ (calcd for C₁₇H₁₈NO₃, 284.1286); ¹H NMR (methanol‐d₄, 500 MHz): δ/ppm 7.42 (d, J = 15.7 Hz, H‐3), 7.38 (d, J = 8.7 Hz, H‐5/H‐9), 7.03 (d, J = 8.5 Hz, H‐4‘/H‐8‘), 6.77 (d, J = 8.7 Hz, H‐6/H‐8), 6.70 (d, J = 8.5 Hz, H‐5‘/H‐7‘), 6.36 (d, J = 15.7 Hz, H‐2), 3.44 (t, J = 7.4 Hz, H‐1‘), 2.73 (t, J = 7.4 Hz, H‐2‘).

N‐trans‐caffeoyltyramine [(E)‐3‐(7,8‐dihydroxyphenyl)‐N‐(6’‐hydroxyphenethyl)acrylamide, 2]. White amorphous powder (purity 99% by HPLC). ESI‐HRMS m/z 300.1239 [M + H]⁺ (calcd for C₁₇H₁₈NO₄, 300.1235); ¹H NMR (methanol‐d₄, 200 MHz) δ/ppm 7.37 (d, J = 15.7 Hz, H‐3), 7.06–6.98 (m, H‐9/H‐4’/H‐8’), 6.89 (dd, J = 8.2 and 2.0 Hz, H‐5), 6.77–6.68 (m, H‐6/H‐5’/H‐7’), 6.32 (d, J = 15.7 Hz, H‐2), 3.44 (t, J = 7.8 Hz, H‐1’), 2.77 (t, J = 7.8 Hz, H‐2’).

N‐trans‐feruloyltyramine [(E)‐3‐(7‐hydroxy‐8‐methoxyphenyl)‐N‐(6’‐hydroxyphenethyl)acrylamide, 3]. White amorphous powder (purity 99% by HPLC). ESI‐HRMS m/z 314.1388 [M + H]⁺ (calcd for C₁₈H₂₀NO₄,  314.1392); ¹H NMR (acetone‐d₆, 200 MHz) δ 7.44 (d, J = 15.7 Hz, H‐3), 7.13 (d, J = 1.7 Hz, H‐9), 6.81 (d, J = 8.1 Hz, H‐6), 7.03 (d, J = 8.4 Hz, H‐4’/H‐8’), 7.01 (dd, J = 8.1 and 1.7 Hz, H‐5), 6.73 (d, J = 8.4 Hz, H‐5’/H‐7’), 6.49 (d, J = 15.7, H‐2), 3.83 (s, 8‐OCH ₃), 3.45 (t, J = 7.8 Hz, H‐1’), 2.71 (t, J = 7.8 Hz, H‐2’).

4.4. Animals

Female BALB/c mice were utilized to obtain peritoneal macrophages for intracellular experiments [28]. The mice were housed in sterilized boxes with absorbent material and received water and food ad libitum. This project received approval from the Ethics Committee of the Instituto Adolfo Lutz (Project CEUA 05/2018), in accordance with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences.

4.5. Determination of the 50% Effective Concentration (EC₅₀) Against Intracellular Amastigotes of Trypanosoma cruzi

Intracellular amastigote forms of T. cruzi were obtained from the infection of peritoneal macrophages from BALB/c mice and applied to 16 well Nunc plates (Thermo Fisher Scientific) at 5 × 10⁴ macrophages/well to adhere and incubate at 37°C and 5% of CO₂. After the adherence of macrophages on Nunc plates, amastigotes were added at a ratio of 10:1, incubated for 24 h, and washed, and then it was incubated with compounds 1–3 for 48 h at 37°C and 5% of CO₂. After the end of the incubation, the plates were fixed with MeOH, stained with Giemsa, and examined under a light microscope. The EC₅₀ was achieved in 200 cells, using the infection index. Untreated cells were used as a negative control [28, 29].

4.6. Determination of Cytotoxicity Against Mammalian Cells

The 50% cytotoxic concentration (CC₅₀) was assessed using NCTC L929 cell lines (RRID:CVCL_0462). Mammalian cells were added in 96‐well plates at a concentration of 6 × 10⁴ cells/well in medium containing 10% of FBS and incubated with compounds 1–3 for 48 h at 37°C with 5% CO₂. Cellular viability was determined by the MTT assay (20 µL of MTT). After 4 h, 80 µL of SDS (10% w/v) was added, and the plates were incubated for 24 h before measuring absorbance at 570 nm using a spectrophotometer. The selectivity index was calculated as the ratio of CC₅₀ to EC₅₀ [14].

4.7. Statistical Analysis

The CC₅₀ and EC₅₀ values were determined using dose–response sigmoid curves. Statistical significance between samples was assessed using p‐values obtained through one‐way ANOVA followed by Tukey's multiple comparison test. All analyses were performed using GraphPad Prism 5 software. The samples were tested in duplicate, and each experiment was repeated at least twice.

Author Contributions

Carlos Henrique Dos Santos: isolation, identification, and writing. Mariana B. Abiuzi: bioactive evaluation. Beatriz A. De Andrade: bioactive evaluation. Erica V. C. Levatti: bioactive evaluation. Mariana H. Chaves: identification and methodology. Andre G. Tempone: bioactive evaluation, methodology and supervision. João Henrique G. Lago: identification, methodology, and supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71025‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.