In Vitro Cytotoxic and Leishmanicidal Activity of Isolated and Semisynthetic ent-Pimaranes from Aldama arenaria

• Abstract
• Introduction
• Results and Discussion
• Conclusion
• Material and Methods
• Plant material
• Extraction and isolation of ent-pimara-8(14),15-dien-19-oic acid (1) and ent-8(14),15-pimaradien-3β-ol (2) from the roots of A. arenaria
• Obtaining the Derivatives of ent-pimara-8(14),15-dien-19-oic Acid (1) and ent-8(14),15-pimaradien-3β-ol (2)
• Structural determination of pimaranes
• Evaluation of In Vitro Cytotoxic Activity
• Evaluation of in vitro leishmanicidal activity in promastigote forms
• In vitro cytotoxicity assessment for fibroblast and macrophage cell lines
• Evaluation of in vitro infection of macrophages with L. amazonensis
• Contributorsʼ Statement
• References

Abstract

Two pimaranes ent-pimara-8(14),15-dien-19-oic acid (1) and ent-8(14),15-pimaradien-3β-ol (2), isolated from Aldama arenaria, and six semi-synthetic derivatives methyl ester of the ent-pimara-8(14),15-dien-19-oic acid (3), ent-pimara-8(14),15-dien-19-ol (4), acetate of ent-pimara-8(14),15-dien-19-ol (5), ent-pimara-8(14),15-dien-19-ol succinic acid (6), acetate of ent-8(14),15-pimaradien-3β-ol (7), ent-8(14),15-pimaradien-3β-ol succinic acid (8) were evaluated in vitro for their cytotoxic activities to childhood leukemia cell lines and leishmanicidal activity against the parasite Leishmania amazonensis. Among these compounds, 1 to 6 presented moderate cytotoxic activity, with compound 4 being the most active (GI₅₀ of 2.6 µM for the HL60 line) and the derivatives 7 and 8 being inactive. Against the parasite Leishmania amazonensis, the most promising derivative was the acetate of ent-pimara-8(14),15-dien-19-ol (5), with EC₅₀ of 20.1 µM, selectivity index of 14.5, and significant reduction in the parasite load. Pimarane analogues 1, ent-pimara-8(14),15-dien-19-oic acid, and 2, ent-8(14),15-pimaradien-3β-ol, presented different activities, corroborating the application of such molecules as prototypes for the design of other derivatives that have greater cytotoxic or leishmanicidal potential.

Introduction

Aldama arenaria (Baker) E. E. Schill. & Panero, classified previously as Viguiera arenaria (Baker), Asteraceae, is a species found in the center-east of the state of São Paulo [1], [2]. It has aromatic underground organs and can be chemically characterized by the presence of diterpene compounds of the pimarane type, class of secondary metabolites with numerous described biological activities, and sesquiterpene lactones [3], [4], [5]. Studies with isolated and semi-synthetic compounds of this species showed inhibitory action of the contraction of the vascular smooth muscle, in vitro activity against the protozoan Trypanosoma cruzi, capacity to reduce the influx of extracellular Ca²⁺ and consequent induction of hypotension in normotensive rats, vasorelaxant action, antibacterial, schistosomicidal and antiproliferative activity [6], [7], [8], [9], [10], [11], [12], [13]. As a result, and continuing the research project of our working group, we evaluated pimaranes 1, 2 and six semi-synthetic derivatives.

Among the different types of cancers, leukemias comprise about 2.4% of incidents in the world, and statistics show the emergence of 437 033 new cases in 2018. It is one of the most common cancers, involving clonal neoplastic proliferation of immature cells or blasts of the hematopoietic system. It has two main subtypes identified on the basis of malignancy: lymphoid cells (B cells and T cells) or myeloid cells (granulocytic, erythroid, and megakaryocytic cells); and whether the disease is initially chronic or acute. In 2018 alone, when added together, all types of leukemia caused around 309,006 deaths worldwide. In Brazil, as in developed countries, cancer among children and adolescents aged 1 to 19 years represents the leading cause of death from diseases (8% of the total). Currently, mainly due to the significant progress in the treatment of cancer in childhood, around 80% of children and adolescents can be cured, if diagnosed early and treated in specialized centers [14].

They are usually more aggressive cancers, with few treatment options and, thus, poor prognosis. The main therapy for childhood leukemia is chemotherapy, but other treatments include transplantation, specific drugs, immunotherapy, surgery, and radiotherapy. Side effects such as hair loss, mouth sores, loss of appetite, diarrhea, nausea, and vomiting are commonly observed [15]. Some drugs widely used in the treatment of these patients come from a plant species, such as: vincristine, etoposide, and teniposide [16], [17], [18]. These worrying data show that we must always look for new, innovative, more efficient, and less aggressive drugs, to ensure an ever greater chance of cure and survival for patients.

Leishmaniasis is a disease typical of poor countries, which affects more than 12 million people worldwide. It is a Neglected Tropical Disease (NTD) transmitted by different species of infected sandflies (genus Phlebotomus) and is defined as a set of parasitic diseases caused by protozoa of the genus Leishmania. It is known that more than 90 species of sandflies transmit these parasites, and it is estimated that, annually, there are 0.7 to 1 million new cases, of which 20 to 30 thousand result in deaths. Medications available for the treatment of leishmaniasis are few and have serious side effects and resistance, such as pentavalent antimonials. In the case of visceral Leishmania, other drugs can be used, such as liposomal amphotericin B and oral miltefosine, but these also present problems, such as high cost and teratogenic effect [19], [20]. In Brazil, this disease is directly related to geographic regions, and the most common species are Leishmania (Vianna) braziliensis and Leishmania amazonensis [21].

In its biological cycle, the parasite has two morphological forms: promastigote, a flagellate form found in the female of the insect vector (invertebrate host), and amastigote, present in mammalian hosts. The amastigote forms are intracellular parasites commonly found in macrophages and are located in the phagolysosome or parasitophorous vacuole, where they multiply [22].

Knowing the high impact that these diseases have on society and the importance of studying substances isolated from plant species, this study aimed to evaluate in vitro isolated and semi-synthetic pimaranes of A. arenaria ([Fig. 1]) against the leishmanicidal [Leishmania amazonensis parasite] and cytotoxic activities to childhood leukemia cell lines: myeloid (K562), promyelocytic (HL60), acute T-cell (JURKAT, MOLT-4), Burkitt lymphoma (RAJI and RAMOS), B lymphoid leukemia (NALM-6), and acute lymphocytic leukemia (REH).

Results and Discussion

The yield obtained for the dichloromethane extract of the roots (DE) of A. arenaria in the turbo extraction was 14.1% (m/m, 42,36 g), a yield higher than that observed by Ambrosio et al. (2004) in their ultrasound extraction [3]. This extract presented the compounds 1 and 2 as major compounds by gas chromatography coupled to a mass spectrometry detector (GC-MS). Isolation of pimaranes 1 and 2 from DE by flash chromatography was efficient, with yields of 15.99 and 7.54% (m/m, 3,52 g and 1,66 g, respectively) and purity greater than 95%, respectively, proving to be a fast, appropriate, and economic technique.

The characterization of pimaranes 1 and 2 was performed by comparing the spectroscopic data obtained with those described in the literature, and served as a reference to confirm the structures of semi-synthetic derivatives 3 to 8 ([Tables 1], [2], and [3]) [10], [11], [23], [24], [25], [26], [27].

The fragmentation pattern obtained in the mass spectra of these compounds is characteristic of pimaranes, presenting as base peak the fragments of m/z 121.10, 135.10 and 257.2 [28].

Regarding the synthesis of pimarane derivatives, the proposed synthetic routes were considered easy to perform and satisfactory, as their yields ranged from 80 to 100% ([Fig. 2] and [3]).

The pimaranes 1 (ent-pimara-8(14),15-dien-19-oic acid), 2 (ent-8(14),15-pimaradien-3-β-ol alcohol), 4 (ent-pimara-8(14),15-dien-19-ol alcohol), and 7 (derived in C-3 ent-8(14),15-pimaradien-3β-acetoxy) were described in the species A. arenaria and Gnaphalium gaudichaudianum and biologically explored for antibacterial, vasorelaxant, trypanocidal, and cytotoxic activities [6], [7], [8], [9], [10], [11], [12], [13], [29].

Derivatives 3 and 4, obtained by microbial transformation, were evaluated for their capacity to inhibit vascular smooth muscle contraction, its spasmolytic effects, and its anticariogenic activities [23], [30]. No activities were described for derivatives 5, 6, and 8.

The cytotoxic activity of pimaranes 1 to 8 was evaluated in vitro for cell lines myeloid leukemia (K562), promyelocytic leukemia (HL60), acute T-cell leukemia (JURKAT, MOLT-4), Burkitt lymphoma (RAJI and RAMOS), B lymphoid leukemia (NALM-6), and acute lymphocytic leukemia (REH). [Table 4] shows the GI₅₀ values (concentration necessary to inhibit 50% of cell growth) obtained for the different samples and different lines. Vincristine was used as a reference chemotherapeutic drug.

Pimaranes with different functional groups at C19 (1, 3, 4, 5, and 6) had similar cytotoxicity values, with GI₅₀ ranging from 2.6 ± 0.5 to 11.5 ± 0.1 µM. Derivative 4, ent-pimara-8(14),15-dien-19-ol, was the most promising and presented GI₅₀ of 2.6 ± 0.5 µM for the HL60 cell line.

Among the molecules with different substituents in C3 (2, 7, and 8), the compound 2 was the most active of all, with GI₅₀ ranging from 4.2 ± 1.0 to 7.8 ± 0.7 µM.

Among the evaluated pimaranes, derivatives 7 and 8 were the least active, with EC₅₀ > 15 µM; in this case, the addition of an acetate function or a succinate radical in C3 significantly reduced the cytotoxic activity for all lines tested, compared to 2.

The main objective of antineoplastic chemotherapy is to exterminate cancer cells; however, this cannot always be done in a directional and selective way, almost always damaging healthy cells. This is because these drugs act in a non-specific way, harming both normal and malignant cells. The combination of several chemotherapeutic agents and their efficiency has been shown to be a beneficial tool for the treatment of malignant tumors, with cure rates of 75 to 90% in different types of cancer [31].

Additionally, compound 4 was evaluated for B lymphoid leukemia (NALM-6), Burkitt lymphoma (RAMOS), acute T-cell leukemia (MOLT-4), myeloid leukemia (K562), promyelocytic leukemia (HL60), and acute lymphocytic leukemia (REH) lines to verify whether this molecule had selectivity ([Table 5]).

The results obtained showed that compound 4 does not present any selectivity when evaluated for these other lines, with EC₅₀ > 19 µM.

These data indicate moderate action of pimarane derivatives 1 to 6 isolated from A. arenaria and semi-synthetic ones, with a concentration-dependent profile and little selectivity among the evaluated lines.

Leishmanicidal activity was evaluated in vitro against the promastigote forms of L. amazonensis (MHOM/BR/73/M2269). About 20 described species can be associated with clinical manifestations in humans [32].

The initial screening was performed with pimaranes (1 to 8) at concentrations of 50 and 100 µg/mL ([Fig. 4]).

This assay revealed that only pimaranes 1, 2, 3, 5, and 7 were able to decrease the cell viability of the parasites at rates lower than 30%, showing mortality of promastigote parasites in more than 82% for some samples. These samples were evaluated against parasites at different concentrations (0.15 to 660 µM), resulting in sigmoidal concentration curves versus parasite cell viability ([Fig. 5]).

Through sigmoidal regression analysis of these data, it was possible to calculate the effective concentration needed to inhibit 50% of cell growth (EC₅₀) for pimaranes 1, 2, 3, 5, and 7 ([Table 6]).

Among the most active samples, compound 5 was the most promising derivative, with an EC₅₀ from 16.02 to 24.82 µM. When we compare its activity with its precursor, pimarane 1, we see an increase in action due to the addition of an acetate in C19. In the case of derivative 7, we can see a less pronounced action when this substituint is positioned in C3.

Comparing the mean EC₅₀ values obtained, we conclude that only derivative 5 has a potential leishmanicidal action in vitro ([Fig. 6]).

The cytotoxicity of these molecules was evaluated in fibroblast cells (L929) and in macrophages and compared by the selectivity index, determined as the ratio between the CC₅₀ for macrophages and the EC₅₀ for L. amazonensis. A selectivity greater than 100 times is expected for pathogens, however, such higher rates are quite uncommon [32] ([Table 7]).

Among the pimaranes evaluated, only acetate 5 showed potential action against parasite L. amazonensis, with a selectivity index greater than 10, compared to the macrophage and low cytotoxicity regarding the L929 line.

In vitro infection was performed with compound 5 in macrophages extracted from the femur and tibia of Black mice in a 24-well plate, at four concentrations from 4.73 to 37.82 µM. The micrographs obtained from the infection ([Fig. 7]) showed that the coverslips containing only the infected macrophages (control) had a high degree of infection, represented by the circumferences with a diameter of less than 5 µM (arrowhead in [Fig. 7 a]). In [Fig. 7 b], one can observe healthy macrophages and the absence of intracellular parasites. The photomicrographs of the coverslips can be seen in [Fig. 7].

By counting the numbers of macrophages, infected macrophages, and amastigote parasites, it was possible to plot the graphs that are shown in [Fig. 8] to [10].

Assessing the number of amastigotes per 100 macrophages, we verified a small reduction in the number of infected macrophages at the highest concentrations (18.91 and 37.82 µM), compared to the untreated control group. In this case, there was no significant difference in infection between the two highest concentrations. This profile is also observed when we verify the percentage of infection ([Fig. 8]).

Regarding the infectivity index, we can infer that pimarane 5 contributed to the reduction of the parasite load when evaluated at the highest concentrations ([Fig. 10]), reducing macrophage infection by more than 50% compared to the untreated control. This inhibition is quite significant for a patient, because macrophage cells are the main cells infected by parasites of the genus Leishmania, playing a crucial role in the initial immune response to infections.

The results of the in vitro evaluation of macrophage infection allow us to conclude that pimarane 5 was active against L. amazonensis amastigotes at concentrations 18.91 and 37.82 µM.

Conclusion

This study has shown that pimaranes isolated from A. arenaria and their semi-synthetics derivatives have in vitro cytotoxic action in childhood leukemia lines and leishmanicidal activity when evaluated for the L. amazonensis line.

Among the pimaranes evaluated, the alcohol 4, ent-pimara-8(14),15-dien-19-ol, presented greater cytotoxicity, with an GI₅₀ of 2.6 for the promyelocytic leukemia (HL60) cell line, without showing any specific selectivity. The most promising pimarane against L. amazonensis parasites was the acetate from ent-pimara-8(14),15-dien-19-ol (5), with an EC₅₀of 20.1 µM and selectivity index of 14.5.

Pimarane analogues ent-pimara-8(14),15-dien-19-oic acid (1) and ent-8(14),15-pimaradien-3β-ol (2) presented different activities, indicating that these precursor molecules can be considered prototypes for the planning of other derivatives that have greater cytotoxic or leishmanicidal potential. Additionally, other in vitro and in vivo studies are needed to unravel their mechanisms of action and toxicities.

Material and Methods

Plant material

Plant material was collected from Ecological Station of Itirapina, São Paulo, Brazil (22°14′S, 47°51′W) in its natural state. The identification was carried out by the botanist Mara Angelina Galvão Magenta, and the exsiccate was deposited in the herbarium of the “Luiz de Queiroz” School of Agriculture – ESALQ-USP, under number 111 847 and study permits from CGEN, process no. 010 216/2012 – 0 and SisGen A7C6E5C. This matrix plant provided seeds used for the production of 20 seedlings that were cultivated for 2 years in the experimental field of CPQBA/UNICAMP and processed for the production of extracts.

Extraction and isolation of ent-pimara-8(14),15-dien-19-oic acid (1) and ent-8(14),15-pimaradien-3β-ol (2) from the roots of A. arenaria

Fresh roots of A. arenaria were ground and extracted in an Ultra Turrax (IKA) disperser at room temperature with dichloromethane (1 g/10 mL) for 5 min, followed by filtration and re-extraction of the plant residue with two more portions of dichloromethane. The pooled extracts were evaporated under reduced pressure, providing the crude dichloromethane extract (DE).

DE (22 g) was fractionated in a medium pressure liquid chromatograph (CombiFlash Rf+/Teledyne ISCO) in a chromatographic column containing 220 g of silica gel 60 (High Performance Gold, spherical particle 20 – 40 µm and pore size of 60 Å), gradient of hexane and ethyl acetate with mobile phase, flow rate of 50 mL/min, and wavelength of 254 nm. 1020 fractions of 18 mL each were collected. The resulting fractions were analyzed by thin layer chromatography (TLC), pooled according to similarity and evaporated under reduced pressure. The pooled fraction enriched in pimarane 1 was crystallized in dichloromethane in a freezer, filtered, and vacuum dried. CG-MS ions (m/z) [relative intensity, %]: 302.20 [34]; 123.10 [34]; 121.10 [100]; 91.10 [40]; 79.10 [33]. ¹H NMR data: δ= 0.66 (s, 3H, 10-CH₃), 1.01 (s, 3H, 13-CH₃), 1.27 (s, 3H, 4-CH₃), 4.91 (dd, 1H), 4.96 (dd, 1H), 5.16 (s, 1H), 5.72 (dd, 1H). ¹³C NMR: δ = 184.3 (C-19), 147.2 (C-15), 137.9 (C-8), 128.0 (C-14), 112.9 (C-16), 56.1 (C-5), 50.5 (C-9), 44.0 (C-4), 39.2 (C-1), 39.2 (C-10), 38.5 (C-13), 37.9 (C-3), 35.8 (C-7), 36.4 (C-12), 29.3 (C-17), 29.2 (C-18), 24.1 (C-6), 19.6 (C-11), 19.2 (C-2), 13.8 (C-20).

To obtain pimarane 2, fractions from the DE flash chromatography containing large amounts of pimarane alcohol were pooled and methylated with the reagent TMSD (trimethylsilyl diazomethane). Because of the similar Rf values, this reaction is necessary for the conversion of the acid 1 to the correspondent ester ensuring the isolation of alcohol. The reaction remained under stirring for 2 h. After completion of the reaction, confirmed by TLC, the sample was vacuum dried. This sample enriched in pimarane 2 (4.9 g) was fractionated again in a medium pressure liquid chromatograph in a chromatographic column containing 80 g of silica gel 60 (High Performance Gold, spherical particle 20 – 40 µm and pore size of 60 Å), gradient of hexane and ethyl acetate with mobile phase, flow rate of 10 mL/min, and wavelength of 254 nm. 322 fractions of 15 mL each were collected. The resulting fractions were analyzed by TLC, pooled according to similarity and vacuum evaporated. The pooled fraction enriched in compound 2 was solubilized at heat (45 °C) and then crystallized in methanol at room temperature. The crystallisate from pimarane 2 was centrifuged and washed with cold methanol. After separating the supernatant, the precipitate was vacuum dried. GC-MS ions (m/z) [relative intensity, %]: 288.20 [8]; 270.20 [11]; 255.20 [17]; 135.10 [100]; 91.10 [22]. ¹H NMR: δ = 0.83 (s, 3H, 4-CH₃), 1.02 (s, 3H, 4- CH₃), 1.07 (s, 3H, 10-CH₃), 1.17 (s, 3H, 13-CH₃), 3.3 (dd, 1H), 4.89 (dd, 1H), 4.96 (dd, 1H), 5.16 (d, 1H), 5.74 (dd, 1H). ¹³C NMR: δ = 147.3(C-15), 137.9 (C-8), 128.1 (C-14), 112.8 (C-16), 79.2 (C-3), 54.1 (C-5), 51.2 (C-9), 39.0 (C-4), 38.6 (C-13), 38.1 (C-10), 37.1 (C-1), 35.7 (C-7), 35.7 (C-12), 29.4 (C-17), 28.4 (C-18), 27.5 (C-2), 22.1 (C-6), 19.1 (C-11), 15.7 (C-19), 14.7 (C-20).

Obtaining the Derivatives of ent-pimara-8(14),15-dien-19-oic Acid (1) and ent-8(14),15-pimaradien-3β-ol (2)

Methyl ester of ent -pimara-8(14),15-dien-19-oic acid (3): 502.9 mg of pimarane 1 was weighed into a 50 mL flask and 10 mL of a mixture of dichloromethane and methanol (9 : 1) was added. 2.50 mL of the TMSD reagent (trimethylsilyl diazomethane) was added to the reaction medium and the system was left under stirring for 2 h. After completion of the reaction, confirmed by TLC, the sample was vacuum dried. GC-MS ions (m/z) [relative intensity, %]: 316.20 [15]; 257.20 [14]; 180.10 [26]; 121.10 [100]; 91.10 [18].

ent -pimara-8(14),15-dien-19-ol (4): In an inert atmosphere, 50 mL of anhydrous ethyl ether and 200.6 mg of lithium and aluminum hydride were added to a 250 mL flask. In another container, 503.0 mg of derivative 3 were solubilized in 5 mL of anhydrous ethyl ether. The solubilized derivative 3 was then added slowly to the reaction medium. The system remained under heating at 40 °C and stirring for 1 h. After completion of the reaction, confirmed by TLC, 10 mL of a mixture of ethyl ether and distilled water (1 : 1) were added, followed by 2 mL of distilled water. The reaction medium was filtered under cotton, and anhydrous sodium sulfate was added to the ether phase. The organic phase was filtered again and vacuum dried. GC-MS ions (m/z) [relative intensity, %]: 288.30 [6]; 257.20 [100]; 135.10 [20]; 121.10 [21]; 91.10 [26].

Acetate of ent -pimara-8(14),15-dien-19-ol (5): In an inert atmosphere, 150.5 mg of derivative 4 and 40 mL of dichloromethane were added to a 125 mL flask. A further 2 mL of pyridine, 103.7 mg of the 4-dimethylaminopyridine (DMAP) catalyst, and 1.3 mL of acetic anhydride were added to the reaction medium. The system remained at room temperature and under stirring for 1 h. After completion of the reaction, confirmed by TLC, 25 mL of distilled water and 1 M hydrochloric acid solution were added until pH 3. The reaction medium was partitioned with 50 mL of dichloromethane. The extraction was repeated two more times, grouping the organic phases. The organic phase was then dried with anhydrous sodium sulfate, filtered and vacuum dried. GC-MS ions (m/z) [relative intensity, %]: 330.30 [18]; 257.20 [91]; 135.10 [100]; 93.10 [38]; 43.10 [41].

ent -pimara-8(14),15-dien-19-ol succinic acid (6): In a 125 mL flask and inert atmosphere, 141.5 mg of derivative 4 and 40 mL of dichloromethane were added. A further 4 mL of pyridine, 205.5 mg of the 4-dimethylaminopyridine (DMAP) catalyst, and 3.05 g of succinic anhydride were added to the reaction medium. The system remained at room temperature and under stirring for 78 h. After completion of the reaction, confirmed by TLC, 1 M hydrochloric acid solution was added until pH 3. The reaction medium was partitioned with 50 mL of dichloromethane. The extraction was repeated three more times, grouping the organic phases. The organic phase was then dried with anhydrous sodium sulfate, filtered and vacuum dried, and recrystallized in dichloromethane at room temperature. GC-MS ions (m/z) [relative intensity, %]: 288.20 [5]; 257.2 [100]; 135.10 [21]; 121.10 [24]; 91.10 [28].

Acetate of ent -8(14),15-pimaradien-3 β -ol (7): In a 125 mL flask and inert atmosphere, 240.8 mg of pimarane 2 and 75 mL of dichloromethane were added. Additional 3.2 mL of pyridine, 173.4 mg of the 4-dimethylaminopyridine (DMAP) catalyst, and 2.10 mL of acetic anhydride were added to the reaction medium. The system remained at room temperature and under stirring for 1 h. After completion of the reaction, confirmed by TLC, 1 M hydrochloric acid solution was added until pH 2. The reaction medium was partitioned with 100 mL of dichloromethane. The extraction was repeated two more times, grouping the organic phases. The organic phase was then dried with anhydrous sodium sulfate, filtered and vacuum dried, and recrystallized in dichloromethane at room temperature. GC-MS ions (m/z) [relative intensity, %]: 330.30 [8]; 255.20 [29]; 135.10 [100]; 119.10 [23]; 43.10 [31].

ent -8(14),15-pimaradien-3 β -ol succinic acid (8): 262.0 mg of pimarane 2 was solubilized in 80 mL of dichloromethane in a 250 mL flask. A further 7.5 mL of pyridine, 380.5 mg of the 4-dimethylaminopyridine (DMAP) catalyst, and 8.01 g of succinic anhydride were added to the reaction medium. The system remained at room temperature and under stirring for 144 h. After completion of the reaction, confirmed by TLC, 200 mL of distilled water were added and the medium acidified with 1 M hydrochloric acid until pH 2. The reaction medium was partitioned with 100 mL of dichloromethane. The extraction was repeated two more times, grouping the organic phases. The organic phase was then dried with anhydrous sodium sulfate, filtered and vacuum dried, and recrystallized in dichloromethane at room temperature. GC-MS ions (m/z) [relative intensity, %]: 288.30 [7]; 255.20 [16]; 135.10 [100]; 121.10 [14]; 91.10 [21].

Structural determination of pimaranes

The structural determination of pimaranes 1 to 8 was carried out by the analysis of ¹H and ¹³C NMR spectroscopic data, mass spectra, and data described in the literature [19], [20], [21], [22], [29].

Evaluation of In Vitro Cytotoxic Activity

The cytotoxicity of pimaranes 1 to 8 was evaluated in vitro in childhood leukemia cell lines from ATCC (American Type Culture Collection), namely: myeloid leukemia (K562), promyelocytic leukemia (HL60), acute T-cell leukemia (JURKAT, MOLT-4), Burkitt lymphoma (RAJI and RAMOS), B lymphoid leukemia (NALM-6), and acute lymphocytic leukemia (REH),

To assess the cytotoxic potential, samples diluted in dimethyl sulfoxide (100 mg/mL) were used. This first dilution was at the maximum concentration of 0.1% DMSO, and, in each well, the concentration reached a maximum of 0.03%, not representing cytotoxicity. Cell viability was assessed by the MTT method (3-[4,5-dimethyl-triazol-2-yl]-2,5-diphenyltetrazolium bromide) in 96-well plates [33].

Cells were cultured in appropriate plastic bottles with Dulbeccoʼs Modified Eagleʼs Medium (DMEM), supplemented with 10% inactivated fetal bovine serum (FBS) for one hour at 56 °C, 10 mM of 4-(2-acid) hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.5 g · L⁻¹ of sodium bicarbonate, 1% of penicillin G (100 U/mL), 100 mg/mL of streptomycin, and 50 µg/mL of amphotericin B in an oven with 5% CO₂ atmosphere at 37 °C. Before the experiments, the number of viable cells was determined by the trypan blue exclusion method.

The cells were plated using serial dilution and the epMotion 5070 equipment (Eppendorf, Vaudaux, Schonenbuch, Switzerland), which distributed 2 × 10⁴ cells per well (96-well plates) and added the substances in different concentrations (0.01 to 1000 µg/mL) for 48 h at 37 °C and 5% CO₂. The chemotherapy drug vincristine was used as a positive standard control. After the treatment period, the culture medium was removed by centrifugation and discarded, 100 µL of MTT solution (0.5 mg/mL) was added, and the plate was incubated again for 4 h. After this period, the medium was removed and the formazan precipitate was dissolved into 100 µl DMSO per well. The plate was then analyzed in a microplate reader at 540 nm (Bio-Tek Power Wave XS) for optical density reading. The control group of untreated cells, incubated with the growth medium only, was considered as 100% of viable cells for calculation of dose-response curves and, consequently, to calculate the GI₅₀ (concentration needed to inhibit 50% of cell growth) using sigmoidal regression in the Graphpad Prism 5.0 software for Windows (GraphPad Software), according to the equation: Relative cell viability = (sample absorbance/control absorbance) × 100. The samples were evaluated in triplicate, and the results were expressed as mean ± standard deviation by Excel 2016 software (Microsoft).

Evaluation of in vitro leishmanicidal activity in promastigote forms

Promastigote of L. amazonensis (strain MHOM/BR/73/M2269) kindly donated by Prof. Dr Silvia Uliana (University of São Paulo, Brazil) were cultured in 25 mL flask with 5 mL of medium 199 (Sigma-Aldrich) supplemented with 50 units/mL penicillin, 0.1 mM adenine, 50 µg/mL streptomycin, pH 7.4, 40 mM HEPES, 0.0001% biotin, 0.0005% hemin, and 10% fetal bovine serum (FBS-Vitrocell).

The samples were diluted in dimethyl sulfoxide (10 mg/mL, maximum final concentration of 0.07%) and submitted to the biological activity test according to the MTT method (3-[4,5-dimethyl-triazol-2-yl]-2,5-diphenyltetrazolium bromide) in 96-well plates [19], [34], [35]. In this procedure, the culture of L. amazonensis containing 2.5 × 10⁶ promastigote parasites in logarithmic phase and maintained in culture medium 199 was evaluated against the sample in different concentrations (50 to 100 µg/mL), for 24 hours. After this period, 30 µL of an MTT solution (3-[4,5-dimethyl-triazol-2-yl-2,5-diphenyltetrazolium, 5 mg/mL) was added to each well and the plates were incubated in the oven at 26 °C (2 hours). To lyse the cells, 30 µL of SDS (20% sodium dodecyl sulfate) was added, and the absorbance was read in a microplate reader (595 nm). Results were expressed as percentages of cell viability compared to the group of untreated parasites (control, equivalent to 100%).

Defining the most promising samples, cell mortality above 20%, cell viability curves were drawn with at least 6 concentrations and at least three independent assays. Using sigmoidal regression in the Origin software, it was possible to calculate the effective concentration for a 50% reduction in cell viability (EC₅₀) for each sample.

In vitro cytotoxicity assessment for fibroblast and macrophage cell lines

The fibroblast culture maintained with 20 mL of complete RPMI medium was transferred to falcon tube, washing with complete RPMI medium. The solution was centrifuged at 1000 g and 4 °C for 5 minutes. The supernatant was discarded and an additional 10 mL of RPMI medium was added. The number of cells was determined in a Neubauer chamber, with 2 × 10⁵ cells per well, and the samples were evaluated according to the MTT method described above.

The macrophages used for cytotoxic evaluation were differentiated from bone marrow precursor cells extracted from the tibia and femur of mice. These cells were cultured in 7.5 cm diameter plates containing 10 mL R2020 medium, modified from RPMI Lonza medium, containing: RPMI, 20% fetal bovine serum, 20% L929 fibroblast supernatant and gentamicin, and kept in an oven at 37 °C with 5% CO₂ atmosphere. After 4 days, another 5 mL of R2020 medium was added and, in 7 days, the macrophages had already gone through the differentiation process and were ready for plating. Macrophages adhered to the bottom of the plate were removed with a sterile cell scraper (Corning), collected in RPMI medium, centrifuged at 1000 G for 5 minutes at 4 °C, and resuspended in R105 medium (RPMI + 10% FBS + 5% of L929 supernatant). The number of cells was determined in a Neubauer chamber, with 2 × 10⁵ cells per well in the cytotoxic analysis and 5 × 10⁵ cells in the Leishmania infection process, according to the MTT method.

Evaluation of in vitro infection of macrophages with L. amazonensis

To assess the viability of intracellular amastigote parasites after exposure to the pimaranes under study, macrophage cells were infected with L. amazonensis promastigotes. Macrophages were derived from bone marrow precursor cells obtained from C57BL/6 J mice provided by the “Multidisciplinary Center for Biological Investigation on Laboratory Animal Science” at the University of Campinas (CEMIB-Unicamp). Briefly, 5 × 10⁵ macrophages were cultured on coverslips in 24-well plates and infected for 24 h before being exposed to the test sample. Infection schemes followed multiplicities of infection (MOI) equivalent 5 to 10 stationary phase promastigotes per macrophage. An untreated group was used as a negative control. Infections were analyzed under immersion with an optical microscope, considering three parameters: total percentage of infected cells; number of parasites per 100 macrophages; and infectivity index, which combines the two previous parameters (infectivity index = percentage of infected macrophages multiplied by the average number of amastigotes for 100 macrophages).

Contributorsʼ Statement

Data collection, design of the study, statistical analysis, analysis and interpretation of the data: ASS Oliveira and VL Garcia; biologic activities assays: GG Conrado, N Grazzia, DC Miguel, G Frianchi Júnior; critical revision of the manuscript: ASS Oliveira, VL Garcia and DC Miguel.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was supported by CNPq (process #010216/2012-0) and FAPESP (process #2010/51454-3). We thank the Itirapina Ecological Station authorizing plant collection. We also thank Professor Mara Angelina Galvão Magenta for carrying out species identification, and Espaço da Escrita – Pró-Reitoria – UNICAMP for the language services provided. Conrado GG, thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, n. 1 481 071) and Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM, n. 002/2015) for scholarships. Grazzia N, received a CAPES-Demanda Social Scholarship.

• References

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  PubMedSearch in Google Scholar
• 22 Kedzierski L, Sakthianandeswaren A, Curtis JM, Andrews PC, Junk PC, Kedzierska K. Leishmaniasis: current treatment and prospects for new drugs and vaccines. Curr Med Chem 2009; 16: 599-614
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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• 25 Ansell SM, Pegel KH, Taylor DAH. Diterpenes from the timber of 20 Erythroxylum species. Phytochemistry 1993; 32: 953-959
  CrossrefPubMedSearch in Google Scholar
• 26 Mihashi S, Yanagisawa I, Tanaka O, Shibata S. Further study on the diterpenes of Aralia spp. Tetrahedron Lett 1969; 21: 1683-1686
  CrossrefPubMedSearch in Google Scholar
• 27 Jung HA, Lee EJ, Kim JS, Kang SS, Lee JH, Min BS, Choi JS. Cholinesterase and BACE1 inhibitory diterpenoids from Aralia cordata . Arch Pharm Res 2009; 32: 1399
  CrossrefPubMedSearch in Google Scholar
• 28 Adams RP. Identification of Essential oil Components by Gas Chromatography/Mass Spectrometry. 4nd edition. Carol Stream, Illinois: Allured Publishing Corporation USA; 2007: 804
  Search in Google Scholar
• 29 Meragelman TL, Silva GL, Mongelli E, Roberto RG. Ent-Pimarane type diterpenes from Gnaphalium gaudichaudianum . Phytochemistry 2003; 62: 569-572
  CrossrefPubMedSearch in Google Scholar
• 30 Severiano ME, Simão MR, Porto TS, Martins CHG, Veneziani RCS, Furtado NAJC, Arakawa NS, Said S, Oliveira DCR, Cunha WR, Gregorio LE, Ambrosio SR. Anticariogenic properties of ent-pimarane diterpenes obtained by microbial transformation. Molecules 2010; 15: 8553-8566
  CrossrefPubMedSearch in Google Scholar
• 31 De Almeida VL, Leitão A, Reina LDCB, Montanari CA, Donnici CL, Lopes MTP. Câncer e agentes antineoplásicos ciclo-celular específicos e ciclo-celular não específicos que interagem com o DNA: Uma introdução. Quim Nova 2005; 28: 118-129
  CrossrefPubMedSearch in Google Scholar
• 32 Grimaldi jr. G, David JR, McMahon-Pratt D. Identification and distribution of New World Leishmania species caracterized by serademe analysis using monoclonal antibodies. Am J Trop Med Hyg 1987; 36: 270-287
  CrossrefPubMedSearch in Google Scholar
• 33 Mosmann TJ. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. Immunol Methods 1983; 65: 55-63
  CrossrefPubMedSearch in Google Scholar
• 34 Katsuno K, Burrows JN, Duncan K, van Huijsduijnen RH, Kaneko T, Kita K, Mowbray CE, Schmatz D, Warner P, Slingsby BT. Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat Rev Drug Discov 2015; 14: 751-758
  CrossrefPubMedSearch in Google Scholar
• 35 Miguel DC, Yokoyama-Yasunaka JKU, Andreoli WK, Mortara RA, Uliana SRB. Tamoxifen is effective against Leishmania and induces a rapid alkalinization of parasite phorous vacuoles harbouring Leishmania (Leishmania) amazonensis amastigotes. J Antimicrob Chemother 2007; 60: 526-534
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 23 August 2021

Accepted after revision: 28 November 2021

Accepted Manuscript online:
28 November 2021

Article published online:
10 February 2022

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Magenta MAG. Viguiera Kunth (Asteraceae, Heliantheae) na América do Sul e sistemática das espécies do Brasil [PhD thesis]. São Paulo: University of São Paulo; 2006
  PubMedSearch in Google Scholar
• 2 Schilling EE, Panero JL. A revised classification of subtribe Helianthinae (Asteraceae: Heliantheae) II. Derived lineages. Bot J Linn Soc 2011; 167: 311-331
  CrossrefPubMedSearch in Google Scholar
• 3 Ambrosio SR, Schorr K, Da Costa FB. Terpenoids of Viguiera arenaria (Asteraceae). Biochem Syst Ecol 2004; 32: 221-224
  CrossrefPubMedSearch in Google Scholar
• 4 Oliveira TS, Bombo AB, Oliveira ASS, Garcia VL, Appezzato-da-Glória B. Seasonal variation of the essential oil from two Brazilian native Aldama La Llave (Asteraceae) species. An Acad Bras Cienc 2016; 88: 1899-1907
  CrossrefPubMedSearch in Google Scholar
• 5 Reveglia P, Cimmino A, Masi M, Nocera P, Berova N, Ellestad G, Evidente A. Pimarane diterpenes: Natural source, stereochemical configuration, and biological activity. Chirality 2018; 30: 1115-1134
  CrossrefPubMedSearch in Google Scholar
• 6 Ambrosio SR, Tirapelli CR, Bonaventura D, De Oliveira AM, Da Costa FB. Pimarane diterpene from Viguiera arenaria (Asteraceae) inhibit rat carotid contraction. Fitoterapia 2002; 73: 484-489
  CrossrefPubMedSearch in Google Scholar
• 7 Ambrosio SR, Tirapelli CR, Da Costa FB, Oliveira AM. Kaurane and pimarane-type diterpenes from the Viguiera species inhibit vascular smooth muscle contractility. Life Sci 2006; 79: 925-933
  CrossrefPubMedSearch in Google Scholar
• 8 Ambrosio SR, Arakawa NS, Esperandim VR, Albuquerque S, Da Costa FB. Trypanocidal activity of pimarane diterpenes from Viguiera arenaria (Asteraceae). Phytother Res 2008; 22: 1423-1425
  CrossrefPubMedSearch in Google Scholar
• 9 Tirapelli CR, dos Anjos Neto Filho M, Bonaventura D, Melo MC, Ambrosio SR, de Oliveira AM, Bendhack LM, da Costa FB. Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats. J Pharm Pharmacol 2008; 60: 453-459
  CrossrefPubMedSearch in Google Scholar
• 10 Porto TS, Rangel R, Furtado NAJC, De Carvalho TC, Martins CHG, Veneziani RCS, Da Costa FB, Vinholis AHC, Cunha WR, Heleno VCG, Ambrosio SR. Pimarane-type diterpenes: Antimicrobial activity against oral pathogens. Molecules 2009; 14: 191-199
  CrossrefPubMedSearch in Google Scholar
• 11 Porto TS, Furtado NAJC, Heleno VCG, Martins CHG, Da Costa FB, Severiano ME, Silva NA, Veneziani RSC, Ambrosio SR. Antimicrobial ent-pimarane diterpenes from Viguiera arenaria against Gram-positive bacteria. Fitoterapia 2009; 80: 432-436
  CrossrefPubMedSearch in Google Scholar
• 12 Porto TS, da Silva Filho AA, Magalhães LG, dos Santos RA, Furtado NAJC, Arakawa NS, Said S, de Oliveira DC, Gregório LE, Rodrigues V, Veneziani RC, Ambrósio SR. Fungal transformation and schistosomicidal effects of pimaradienoic acid. Chem Biodivers 2012; 9: 1465-1474
  CrossrefPubMedSearch in Google Scholar
• 13 Oliveira ASS, Imamura PM, Ruiz ALTG, Appezzato-da-Glória B, de Oliveira T, Garcia VL. Antiproliferative activity from Aldama arenaria (Baker) E.E. Schill. & Panero. Bol Latinoam Caribe Plant Med Aromat 2021; 20: 51-60
  CrossrefPubMedSearch in Google Scholar
• 14 Brazilian National Cancer Institute – INCA. Accessed August 01, 2020 at: http://www.inca.gov.br
  PubMed
• 15 Ministério da Saúde, Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA). Livro: Ministério da Saúde. Estimativa 2020: Incidência de câncer no Brasil, Edição Anual. Rio de Janeiro, Brasil: Instituto Nacional de Câncer José Alencar Gomes da Silva; 2019
  Search in Google Scholar
• 16 Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J Nat Prod 2020; 83: 770-803
  Search in Google Scholar
• 17 Lee KH, Xiao Z. Podophyllotoxin and Analogs. In: Cragg GM, Kingston DGI, Newman DJ. eds Anticancer Agents from Natural Products. Boca Raton: Taylor and Francis; 2012: 95-122
  Search in Google Scholar
• 18 Kingston DGI. Taxol and its Analogs. In: Cragg GM, Kingston DGI, Newman DJ. eds Anticancer Agents from Natural Products. Boca Raton: Taylor and Francis; 2012: 123-175
  Search in Google Scholar
• 19 Alcântara LM, Ferreira TCS, Gadelha FR, Miguel DC. Challenges in drug discovery targeting TriTryp diseases with an emphasis on leishmaniasis. Int J Parasitol Drugs Drug Resist 2018; 8: 430-439
  CrossrefPubMedSearch in Google Scholar
• 20 De Souza ROMA, Pereira VLP, Muzitano MF, Falcão CAB, Rossi-Bergmann B, Filho EBA, Vasconcellos MLAA. High selective leismanicidal activity of 3-hydroxy-2-methylene-3-(4-bromophenyl)propanenitrile and analogous compounds. Eur J Med Chem 2007; 42: 99-102
  CrossrefPubMedSearch in Google Scholar
• 21 Benchimol JL, Gualandi FC, Barreto DCS, Pinheiro LA. Leishmaniasis: Historical configuration in Brazil with an emphasis on the visceral disease, from the 1930s to the 1960s. Bol Mus Para Emílio Goeldi Ciênc hum 2019; 14: 611-626
  PubMedSearch in Google Scholar
• 22 Kedzierski L, Sakthianandeswaren A, Curtis JM, Andrews PC, Junk PC, Kedzierska K. Leishmaniasis: current treatment and prospects for new drugs and vaccines. Curr Med Chem 2009; 16: 599-614
  CrossrefPubMedSearch in Google Scholar
• 23 Severiano ME, Simão MR, Ramos HP, Parreira RLT, Arakawa NS, Said S, Furtado NAJC, Oliveira DCR, Gregório LE, Tirapelli CR, Veneziani RCS, Ambrosio SR. Biotransformation of ent-pimaradienoic acid by cell cultures of Aspergillus niger . Bioorg Med Chem 2013; 21: 5870-5875
  CrossrefPubMedSearch in Google Scholar
• 24 Matsuo A, Nakayama M, Hayashi S, Yamasaki K, Kasai R, Tanaka O. (−)-Thermarol, a new ent-pimarane-class diterpene diol from Jungermannia thermarum (Liverwort). Tetrahedron Lett 1976; 28: 2451-2454
  CrossrefPubMedSearch in Google Scholar
• 25 Ansell SM, Pegel KH, Taylor DAH. Diterpenes from the timber of 20 Erythroxylum species. Phytochemistry 1993; 32: 953-959
  CrossrefPubMedSearch in Google Scholar
• 26 Mihashi S, Yanagisawa I, Tanaka O, Shibata S. Further study on the diterpenes of Aralia spp. Tetrahedron Lett 1969; 21: 1683-1686
  CrossrefPubMedSearch in Google Scholar
• 27 Jung HA, Lee EJ, Kim JS, Kang SS, Lee JH, Min BS, Choi JS. Cholinesterase and BACE1 inhibitory diterpenoids from Aralia cordata . Arch Pharm Res 2009; 32: 1399
  CrossrefPubMedSearch in Google Scholar
• 28 Adams RP. Identification of Essential oil Components by Gas Chromatography/Mass Spectrometry. 4nd edition. Carol Stream, Illinois: Allured Publishing Corporation USA; 2007: 804
  Search in Google Scholar
• 29 Meragelman TL, Silva GL, Mongelli E, Roberto RG. Ent-Pimarane type diterpenes from Gnaphalium gaudichaudianum . Phytochemistry 2003; 62: 569-572
  CrossrefPubMedSearch in Google Scholar
• 30 Severiano ME, Simão MR, Porto TS, Martins CHG, Veneziani RCS, Furtado NAJC, Arakawa NS, Said S, Oliveira DCR, Cunha WR, Gregorio LE, Ambrosio SR. Anticariogenic properties of ent-pimarane diterpenes obtained by microbial transformation. Molecules 2010; 15: 8553-8566
  CrossrefPubMedSearch in Google Scholar
• 31 De Almeida VL, Leitão A, Reina LDCB, Montanari CA, Donnici CL, Lopes MTP. Câncer e agentes antineoplásicos ciclo-celular específicos e ciclo-celular não específicos que interagem com o DNA: Uma introdução. Quim Nova 2005; 28: 118-129
  CrossrefPubMedSearch in Google Scholar
• 32 Grimaldi jr. G, David JR, McMahon-Pratt D. Identification and distribution of New World Leishmania species caracterized by serademe analysis using monoclonal antibodies. Am J Trop Med Hyg 1987; 36: 270-287
  CrossrefPubMedSearch in Google Scholar
• 33 Mosmann TJ. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. Immunol Methods 1983; 65: 55-63
  CrossrefPubMedSearch in Google Scholar
• 34 Katsuno K, Burrows JN, Duncan K, van Huijsduijnen RH, Kaneko T, Kita K, Mowbray CE, Schmatz D, Warner P, Slingsby BT. Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat Rev Drug Discov 2015; 14: 751-758
  CrossrefPubMedSearch in Google Scholar
• 35 Miguel DC, Yokoyama-Yasunaka JKU, Andreoli WK, Mortara RA, Uliana SRB. Tamoxifen is effective against Leishmania and induces a rapid alkalinization of parasite phorous vacuoles harbouring Leishmania (Leishmania) amazonensis amastigotes. J Antimicrob Chemother 2007; 60: 526-534
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material