Oxonitrogenated Derivatives of Eremophilans and Eudesmans: Antiproliferative and Anti-Trypanosoma cruzi Activity

Cancer is one of the most important causes of death worldwide. Solid tumors represent the vast majority of cancers (>90%), and the chemotherapeutic agents used for their treatment are still characterized by variable efficacy and toxicity. Sesquiterpenes are a group of natural compounds that have shown a wide range of biological activities, including cytotoxic and antiparasitic activity, among others. The antiproliferative activity of natural sesquiterpenes, tessaric acid, ilicic acid, and ilicic alcohol and their semisynthetic derivatives against HeLa, T-47D, WiDr, A549, HBL-100, and SW1573 cell lines were evaluated. The effect of the compounds on Trypanosoma cruzi epimastigotes was also assessed. The selectivity index was calculated using murine splenocytes. Derivatives 13 and 15 were the most antiproliferative compounds, with GI50 values ranging between 5.3 (±0.32) and 14 (±0.90) μM, in all cell lines tested. The presence of 1,2,3-triazole groups in derivatives 15–19 led to improvements in activity compared to those corresponding to the starting natural product (3), with GI50 values ranging between 12 (±1.5) and 17 (±1.1) μM and 16 being the most active compound. In relation to the anti-T. cruzi activity, derivatives 7 and 16 obtained from tessaric acid and ilicic acid were among the most active and selective compounds with IC50 values of 9.3 and 8.8 µM (SI = 8.0 and 9.4), respectively.


Introduction
Nature constitutes an important resource for the discovery of new bioactive compounds of interest. Plants are constituted by about 250,000 species, of which only about The Asteraceae family, also known as Compositae, includes a large number o comprising approximately 1700 genera and 24,000 species [6]. It is the largest plan in the Argentine Republic, with 227 genera (five of which are endemic) and abo species [7]. This family includes food, medicinal, ornamental and industrial plants, and toxic plants.
Many species belonging to this family are known for their economic importa ther for their use in human nutrition (such as seeds and vegetables), ornamental i and for their pharmaceutical or industrial use, due to the presence of a large num secondary metabolites [8].
The species Tessaria absinthioides Hook et Arm DC and Flourensia oolepis Bla longing to the Asteraceae family, are characterized by secondary metabolites of a nature, mainly sesquiterpenes with eremophilan and eudesman nuclei. In p works, we have reported improvements in the activity of sesquiterpene lactones from Ambrosia, Gaillardia, Parthenium, and oxygenated and oxo-nitrogenated der obtained from NPs [9,10].
In this work, we report 16 new compounds derived from natural sesquitepe lated from T. absinthioides and F. oolepis and their antiproliferative and trypanocid ity. This situation determined that the interest in natural products increased oriented to the search for chemical diversity and the obtention of new "leaders" [3]. Currently, natural products (NP) constitute a strategic starting point for the discovery of new drugs, since they exhibit a wide range of pharmacophores and a large number of pre-established chiral centers, and they allow interaction with a wide variety of proteins and biological targets. An example is rosuvastatin, which is widely used to reduce high cholesterol levels and whose structure is based on the pharmacophore of the natural product mevastatin (Figure 1) produced by the fungus Penicillium citrinum [4].
According to a study carried out by Newman and Cragg between 1981 and 2019 [5], 60% of the new chemical entities identified are natural products, semi-synthetic analogs, or synthesized compounds based on their pharmacophores.
The Asteraceae family, also known as Compositae, includes a large number of plants comprising approximately 1700 genera and 24,000 species [6]. It is the largest plant family in the Argentine Republic, with 227 genera (five of which are endemic) and about 1400 species [7]. This family includes food, medicinal, ornamental and industrial plants, weeds, and toxic plants.
Many species belonging to this family are known for their economic importance, either for their use in human nutrition (such as seeds and vegetables), ornamental interest, and for their pharmaceutical or industrial use, due to the presence of a large number of secondary metabolites [8].
The species Tessaria absinthioides Hook et Arm DC and Flourensia oolepis Blake, belonging to the Asteraceae family, are characterized by secondary metabolites of a terpene nature, mainly sesquiterpenes with eremophilan and eudesman nuclei. In previous works, we have reported improvements in the activity of sesquiterpene lactones isolated from Ambrosia, Gaillardia, Parthenium, and oxygenated and oxo-nitrogenated derivatives obtained from NPs [9,10].
In this work, we report 16 new compounds derived from natural sesquitepenes isolated from T. absinthioides and F. oolepis and their antiproliferative and trypanocidal activity.

Chemistry
The eremophilan tessaric acid (1) and the eudesmans ilicic acid (2) and ilicic alcohol (3) were isolated from Tessaria absinthioides and Flourensia oolepis, respectively ( Figure 2). In preliminary tests of bioactivity, these natural sesquiterpenes did not show cytotoxic activity but oxygenated derivatives did [11]. Therefore, these NPs have been used as starting materials for the preparation of a series of oxo-nitrogenated products. These modifications led to derivatives with an improvement in the activity. Derivatives 4-19 were prepared in this way ( Figure 3). Multicomponent and cycloaddition reactions are convergent procedures of high synthetic utility that combine chemical and atomic efficiency. Within the second type, the azidealkyne Huisgen cycloaddition is a powerful tool to prepare 1,2,3-triazoles in a simple way [12]. Multicomponent reactions (MCR) are convergent reactions in which three or more starting materials react to form a product where essentially all or most of the atoms contribute to the newly formed product. In MCRs, a product is assembled according to a cascade of elementary chemical reactions. Therefore, there is an equilibrium network of reaction that eventually leads to an irreversible step that produces the product. In this work, a series of oxy-nitrogenated derivatives were prepared, with compounds 4-15 by In preliminary tests of bioactivity, these natural sesquiterpenes did not show cytotoxic activity but oxygenated derivatives did [11]. Therefore, these NPs have been used as starting materials for the preparation of a series of oxo-nitrogenated products. These modifications led to derivatives with an improvement in the activity. Derivatives 4-19 were prepared in this way ( Figure 3).

Chemistry
The eremophilan tessaric acid (1) and the eudesmans ilicic acid (2) and ilicic alcohol (3) were isolated from Tessaria absinthioides and Flourensia oolepis, respectively ( Figure 2). In preliminary tests of bioactivity, these natural sesquiterpenes did not show cytotoxic activity but oxygenated derivatives did [11]. Therefore, these NPs have been used as starting materials for the preparation of a series of oxo-nitrogenated products. These modifications led to derivatives with an improvement in the activity. Derivatives 4-19 were prepared in this way ( Figure 3). Multicomponent and cycloaddition reactions are convergent procedures of high synthetic utility that combine chemical and atomic efficiency. Within the second type, the azidealkyne Huisgen cycloaddition is a powerful tool to prepare 1,2,3-triazoles in a simple way [12]. Multicomponent reactions (MCR) are convergent reactions in which three or more starting materials react to form a product where essentially all or most of the atoms contribute to the newly formed product. In MCRs, a product is assembled according to a cascade of elementary chemical reactions. Therefore, there is an equilibrium network of reaction that eventually leads to an irreversible step that produces the product. In this work, a series of oxy-nitrogenated derivatives were prepared, with compounds 4-15 by Multicomponent and cycloaddition reactions are convergent procedures of high synthetic utility that combine chemical and atomic efficiency. Within the second type, the azidealkyne Huisgen cycloaddition is a powerful tool to prepare 1,2,3-triazoles in a simple way [12]. Multicomponent reactions (MCR) are convergent reactions in which three or more starting materials react to form a product where essentially all or most of the atoms contribute to the newly formed product. In MCRs, a product is assembled according to a cascade of elementary chemical reactions. Therefore, there is an equilibrium network of reaction that eventually leads to an irreversible step that produces the product. In this work, a series of oxy-nitrogenated derivatives were prepared, with compounds 4-15 by the Ugi reaction and 16-19 by Huisgen cycloadditions. It should be noted that in all the proposals the stereochemistry of the natural product remained intact.
Derivatives 4-9 were obtained by the Ugi reaction using 1 as carboxylic acid and acetone as a source of carbonyl group. Aniline (compounds 4, 6, and 8) and benzylamine (compounds 5, 7, and 9) were used as amines varying the corresponding isocyanides (Scheme 1). the Ugi reaction and 16-19 by Huisgen cycloadditions. It should be noted that in all the proposals the stereochemistry of the natural product remained intact. Derivatives 4-9 were obtained by the Ugi reaction using 1 as carboxylic acid and acetone as a source of carbonyl group. Aniline (compounds 4, 6, and 8) and benzylamine (compounds 5, 7, and 9) were used as amines varying the corresponding isocyanides (Scheme 1).

Scheme 1. Synthesis of compounds 4-9.
Derivatives 10-15 were obtained by the Ugi reaction using 2 as carboxylic acid and acetone as a source of the carbonyl group. Aniline (compounds 10, 12, and 14) and benzylamine (compounds 11, 13, and 15) were used as amines varying the corresponding isocyanides (Scheme 2). Synthetic organic chemistry has aroused great interest in 1,2,3-triazoles in the development of new biologically active molecules [12]. The triazole moiety is not found in nature, but 1,2,3-triazole nuclei can form the basis of small molecule pharmaceuticals. Molecules containing this heterocyclic core have been reported to have anti-HIV, antimicrobial, antiallergic, antifungal, and antitumor activities [13]. One of the most popular reactions within the click chemistry paradigm is the Cu(I)-catalyzed 1,3-dipolar Hüisgen cy-Scheme 1. Synthesis of compounds 4-9.
Derivatives 10-15 were obtained by the Ugi reaction using 2 as carboxylic acid and acetone as a source of the carbonyl group. Aniline (compounds 10, 12, and 14) and benzylamine (compounds 11, 13, and 15) were used as amines varying the corresponding isocyanides (Scheme 2). the Ugi reaction and 16-19 by Huisgen cycloadditions. It should be noted that in all the proposals the stereochemistry of the natural product remained intact. Derivatives 4-9 were obtained by the Ugi reaction using 1 as carboxylic acid and acetone as a source of carbonyl group. Aniline (compounds 4, 6, and 8) and benzylamine (compounds 5, 7, and 9) were used as amines varying the corresponding isocyanides (Scheme 1).

Scheme 1. Synthesis of compounds 4-9.
Derivatives 10-15 were obtained by the Ugi reaction using 2 as carboxylic acid and acetone as a source of the carbonyl group. Aniline (compounds 10, 12, and 14) and benzylamine (compounds 11, 13, and 15) were used as amines varying the corresponding isocyanides (Scheme 2). Synthetic organic chemistry has aroused great interest in 1,2,3-triazoles in the development of new biologically active molecules [12]. The triazole moiety is not found in nature, but 1,2,3-triazole nuclei can form the basis of small molecule pharmaceuticals. Molecules containing this heterocyclic core have been reported to have anti-HIV, antimicrobial, antiallergic, antifungal, and antitumor activities [13]. One of the most popular reactions within the click chemistry paradigm is the Cu(I)-catalyzed 1,3-dipolar Hüisgen cy- Synthetic organic chemistry has aroused great interest in 1,2,3-triazoles in the development of new biologically active molecules [12]. The triazole moiety is not found in nature, but 1,2,3-triazole nuclei can form the basis of small molecule pharmaceuticals. Molecules containing this heterocyclic core have been reported to have anti-HIV, antimicrobial, antiallergic, antifungal, and antitumor activities [13]. One of the most popular reactions within the click chemistry paradigm is the Cu(I)-catalyzed 1,3-dipolar Hüisgen cycloaddition of alkynes and azides. This reaction proceeds with great efficiency and selectivity in aqueous media and produces triazole fractions [14]. Our first objective was to obtain azide 20 using alcohol 3 as a starting material (Scheme 3).
Molecules 2022, 27, x FOR PEER REVIEW 5 of 18 cloaddition of alkynes and azides. This reaction proceeds with great efficiency and selectivity in aqueous media and produces triazole fractions [14]. Our first objective was to obtain azide 20 using alcohol 3 as a starting material (Scheme 3).

Antiproliferative Activity
In vitro antiproliferative activity was evaluated using the protocol of the National Cancer Institute (NCI) after 48 h of drug exposure using the sulforhodamine B (SRB) assay. The results, expressed as GI50 values, are shown in Table 1 and Figure 4 [15].
The data revealed that almost all the synthesized oxo-nitrogen derivatives were more active than the natural products (1-3); compounds 6-12 and 14 showed moderate to good activity with GI50 values ranging between 17 (±5.6) and 64 (±1.5) μM, in all cell lines. On the other hand, derivatives 13 and 15 were the most active, with GI50 values that ranged between 5.3 (±0.32) and 14 (±0.90) μM.
The presence of 1,2,3-triazole groups in derivatives 16-19 led to improvements in activity values compared to those corresponding to the starting natural product (3), with 16 being the most active compound and with GI50 values that ranged between 12 (±1.5) and 17 (±1.1) μM. Coupling of the azide 20 and various commercial alkynes, under Huisgen conditions, gave the 1,2,3-triazoles 16-19 in good yields (Scheme 4).
Molecules 2022, 27, x FOR PEER REVIEW 5 of 18 cloaddition of alkynes and azides. This reaction proceeds with great efficiency and selectivity in aqueous media and produces triazole fractions [14]. Our first objective was to obtain azide 20 using alcohol 3 as a starting material (Scheme 3).

Antiproliferative Activity
In vitro antiproliferative activity was evaluated using the protocol of the National Cancer Institute (NCI) after 48 h of drug exposure using the sulforhodamine B (SRB) assay. The results, expressed as GI50 values, are shown in Table 1 and Figure 4 [15].
The data revealed that almost all the synthesized oxo-nitrogen derivatives were more active than the natural products (1-3); compounds 6-12 and 14 showed moderate to good activity with GI50 values ranging between 17 (±5.6) and 64 (±1.5) μM, in all cell lines. On the other hand, derivatives 13 and 15 were the most active, with GI50 values that ranged between 5.3 (±0.32) and 14 (±0.90) μM.
The presence of 1,2,3-triazole groups in derivatives 16-19 led to improvements in activity values compared to those corresponding to the starting natural product (3), with 16 being the most active compound and with GI50 values that ranged between 12 (±1.5) and 17 (±1.1) μM.

Antiproliferative Activity
In vitro antiproliferative activity was evaluated using the protocol of the National Cancer Institute (NCI) after 48 h of drug exposure using the sulforhodamine B (SRB) assay. The results, expressed as GI 50 values, are shown in Table 1 and Figure 4 [15]. n.e.: not evaluated.
The results are expressed as 50% growth inhibition (GI50) ± SD and are the mean of at least two independent experiments.   (Table 1).
The data revealed that almost all the synthesized oxo-nitrogen derivatives were more active than the natural products (1-3); compounds 6-12 and 14 showed moderate to good activity with GI 50 values ranging between 17 (±5.6) and 64 (±1.5) µM, in all cell lines. On the other hand, derivatives 13 and 15 were the most active, with GI 50 values that ranged between 5.3 (±0.32) and 14 (±0.90) µM.
The presence of 1,2,3-triazole groups in derivatives 16-19 led to improvements in activity values compared to those corresponding to the starting natural product (3), with 16 being the most active compound and with GI 50 values that ranged between 12 (±1.5) and 17 (±1.1) µM.
The results are expressed as 50% growth inhibition (GI 50 ) ± SD and are the mean of at least two independent experiments.

Anti-Trypanosoma cruzi Activity
The natural sesquiterterpenes tessaric acid (1), ilicic acid (2), and ilicic alcohol (3) as well as their derivatives were evaluated against T. cruzi epimastigotes. The effect of the compounds on the parasite is shown in Figure 5.
The tessaric acid derivative 7 presented a SI of 8.0 on T. cruzi epimastigotes. The other derivatives obtained from this sesquiterpene presented SI values in the range of 0.3 to 4.8. In relation to the triazole-type oxonitrogenated derivatives obtained from ilicic alcohol, compound 16 was the one that presented the highest selectivity index on epimastigotes (SI = 9.4), while derivatives 17 and 18 were less selective on this parasite (SI of 2.7 and 2.1, respectively). Derivative 11, obtained from ilicic acid, was the one with the highest selectivity over epimastigotes in comparison to the other compounds (SI = 12.6) ( Table 3).
Since these tests were conducted with T. cruzi epimastigotes, it will be necessary to perform further studies with the clinically more relevant form, intracellular amastigotes, to obtain a better estimate of these compounds' usefulness against this parasite.   The tessaric acid derivative 7 presented a SI of 8.0 on T. cruzi epimastigotes. The other derivatives obtained from this sesquiterpene presented SI values in the range of 0.3 to 4.8. In relation to the triazole-type oxonitrogenated derivatives obtained from ilicic alcohol, compound 16 was the one that presented the highest selectivity index on epimastigotes (SI = 9.4), while derivatives 17 and 18 were less selective on this parasite (SI of 2.7 and 2.1, respectively). Derivative 11, obtained from ilicic acid, was the one with the highest selectivity over epimastigotes in comparison to the other compounds (SI = 12.6) ( Table 3).
Since these tests were conducted with T. cruzi epimastigotes, it will be necessary to perform further studies with the clinically more relevant form, intracellular amastigotes, to obtain a better estimate of these compounds' usefulness against this parasite.

General
Unless otherwise stated, all solvents were purified by standard techniques. Reactions requiring anhydrous conditions were performed under an argon atmosphere. Anhydrous magnesium sulfate was used for drying solutions. Reactions were monitored by thin layer chromatography (TLC) on silica gel plates (60 F254) and were visualized with UV light, 2.5% phosphomolybdic acid in ethanol, or vanillin with acetic and sulfuric acid in ethanol with heating. Purification was performed by column chromatography (CC) on silica gel (230-400 mesh) using n-hexane and ethyl acetate gradient as the solvent. 1 H-NMR spectra were recorded on a Bruker (Fallanden, Switzerland) 200, or 600 MHz; 13 C-NMR spectra were recorded at 50 and 125 MHz; and chemical shifts were reported relative to internal Me 4 Si (δ = 0). Melting points were determined by using an Electrothermal IA9000 melting point apparatus (Essex, UK); the results are reported in degrees Celsius and are uncorrected. Optical rotations were recorded in a 343 Perkin Elmer polarimeter (Waltham, MA, USA). High-resolution ESI mass spectra were obtained from a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, a RF-only hexapole ion guide, and an external electrospray ion source. HRMS spectra were obtained on a Micromass AutoSpec mass spectrometer (Waters Co., Milford, MA, USA) (see Supplementary Materials).

Plant Material
The species used in the present work were Tessaria absinthioides Hook et Arm (Asteraceae) and Flourensia oolepis Baker (Asteraceae). All specimens were collected in

Isolation of Tessaric Acid from T. absinthioides
The aerial parts of T. absinthioides (2.0 kg) were extracted at room temperature with EtOH (3 × 48 h), and the ethanolic extracts obtained were concentrated under reduced pressure. The residue was suspended in 2 L of 10% sodium bicarbonate and extracted with CH 2 Cl 2 (4 × 0.5 L). The aqueous fraction was acidified with 10% HCl and partitioned using CH 2 Cl 2 (4 × 0.5 L). The CH 2 Cl 2 extracts from this operation were combined for subsequent fractionation. This procedure was performed as described in the literature [16].
Under these conditions, 6 g of tessaric acid (1) were obtained, identified by spectroscopic and spectrometric methods (NMR, IR, and HRMS or HRMS-ES).

Isolation of Ilici Alcohol and Ilicic Acid from F. oolepis
The aerial parts of F. oolepis (2.0 kg) were extracted at room temperature with n-hexane for 24 h, carrying out a degreasing process. After filtering, the residue obtained was extracted with EtOAc for another 24 h. The organic extract was divided into two parts, and both were taken to dryness, leaving two residues of 20 g (Fraction A) and 30 g (Fraction B) each [17].
The 30 g fraction was taken up in ethyl ether. Subsequently, a partition was made with a 10% aqueous solution of sodium bicarbonate (3 × 200 mL). The aqueous solution was acidified with 0.1 M hydrochloric acid solution (acid added to litmus paper turn) and then liquid/liquid partitioned with ethyl ether (4 × 150 mL). Fraction A (20 g) was subjected to separation by column chromatography with hexane-ethyl acetate mixtures of increasing polarity.
In this process, 4.6 g of ilicic acid (2) were obtained from Fraction B and 3 g of ilicic alcohol (3) from Fraction A, which represents a yield of 2.3 g of ilicic acid and 1.5 g of ilicic alcohol per kg of dry plant.

Chemistry
The following is the general procedure for the Ugi reaction. A solution of acetone (1.2 eq, 0.472 mmol), and amine (aniline or benzylamine) (1 eq, 0.393 mmol) in methanol (0.7 mL, 0.5 M) were stirred for 3 h. After that, natural acid (1 eq, 0.393 mmol) and isocyanide (1.1 eq, 0.4323 mmol) were added. The reaction mixture was stirred for 24 h. The solvent was removed under reduced pressure, and the crude reaction mixture was purified by flash chromatography on silica gel to afford the desired product, Schemes 2 and 3.
Preparation of derivative 4: This compound was prepared by general procedure for the Ugi reaction, using 1 as an acid, aniline, and tert-butyl-isocyanide to obtain 92 mg of 4 (51% yield), as an amorphous solid.
Preparation of derivative 5: This compound was prepared by general procedure for the Ugi reaction, using 1 as an acid, benzylamine, and tert-butyl-isocyanide to obtain 40 mg of 5 (42% yield), as an amorphous solid.
Preparation of derivative 6: This compound was prepared by general procedure for the Ugi reaction, using 1 as an acid, aniline, and benzylisocyanide to obtain 115 mg of 6 (61% yield), as an amorphous solid.
Preparation of derivative 7: This compound was prepared by general procedure for the Ugi reaction, using 1 as an acid, benzylamine, and benzylisocyanide to obtain 120 mg of 7 (65% yield), as an amorphous solid.
Preparation of derivative 8: This compound was prepared by general procedure for the Ugi reaction, using 1 as an acid, aniline, and cyclohexyl isocyanide to obtain 90 mg of 8 (46% yield), as an amorphous solid.
Preparation of derivative 9: This compound was prepared by general procedure for the Ugi reaction, using 1 as an acid, benzylamine, and cyclohexyl isocyanide to obtain 82 mg of 9 (42% yield), as an amorphous solid.
Preparation of derivative 10: This compound was prepared by general procedure for the Ugi reaction, using 2 as an acid, aniline, and tert-butyl-isocyanide to obtain 120 mg of 10 (65% yield), as an amorphous solid.
Preparation of derivative 11: This compound was prepared by general procedure for the Ugi reaction, using 2 as an acid, benzylamine, and tert-butyl-isocyanide to obtain 140 mg of 11 (76% yield), as an amorphous solid.
Preparation of derivative 12: This compound was prepared by general procedure for the Ugi reaction, using 2 as an acid, aniline, and benzylisocyanide to obtain 113 mg of 12 (68% yield), as an amorphous solid.
Preparation of derivative 13: This compound was prepared by general procedure for the Ugi reaction, using 2 as an acid, benzylamine, and benzylisocyanide to obtain 110 mg of 13 (71% yield), as an amorphous solid.
Preparation of derivative 14: This compound was prepared by general procedure for the Ugi reaction, using 2 as an acid, aniline, and cyclohexyl isocyanide to obtain 92 mg of 14 (47% yield), as an amorphous solid.
Preparation of the derivative 15: This compound was prepared by general procedure for the Ugi reaction, using 2 as an acid, benzylamine, and cyclohexyl isocyanide to obtain 80 mg of 15 (50% yield), as an amorphous solid.
The following is the general procedure for the Huisgen reaction. A solution of 50 mg (1eg, 0.2 mmol) of the azide 20 and 1.2 eq of alkyne, in 2 mL (0.1 M) in a mixture of ethanol and water 1:1, then 12 mg (0.3 eq, 0.06 mmol) of sodium ascorbate and 3 mg (0.1 eq, 0.02 mmol) of sulfate CuSO 4 ·5H 2 O, was stirred for 24 h at room temperature. The solvent was removed under reduced pressure, and the crude reaction mixture was purified by flash chromatography on silica gel to afford the desired product.
Preparation of derivative 16: This compound was prepared by general procedure for the Huisgen reaction, using 20 as a starting material and 1-dodecyne as an alkyne to obtain 49 mg of 16 (60% yield), as like a colorless oil.
Preparation of derivative 17: This compound was prepared by general procedure for the Huisgen reaction, using 20 as a starting material and 1,6-heptadiyne as an alkyne to obtain 27 mg of 17 (41% yield), as like a colorless oil.
Preparation of derivative 18: This compound was prepared by general procedure for the Huisgen reaction, using 20 as starting material and 3-phenyl-1-propyne as an alkyne to obtain 39 mg of 18 (55% yield), as like a colorless oil.

Spectroscopic and Physical Data
Compound  calculated with respect to untreated control cells (C) at each level of drug concentrations based on the difference in OD at the start time (T 0 ) and at the end of drug exposure (T), according to NCI formulas. Therefore, if T is greater than or equal to T 0 , the calculation is 100 × ((T − T0)/(C − T 0 )). If T is lower than T 0 , denoting cell death, the calculation is 100 × ((T − T 0 )/(T 0 )). The effect is defined as the growth percentage, where 50% growth inhibition (GI 50 ) represents the concentration at which PG is +50. Based on these calculations, a PG value of 0 corresponds to the number of cells present at the beginning of drug exposure, while negative PG values denote net cell death.

Parasites
Trypanosoma cruzi epimastigotes (RA strain, from discrete typing unit (DTU) VI) were grown in a biphasic medium. Cultures were maintained by weekly passages at 28 • C [18].

In Vitro Anti-Trypanosoma cruzi Assay
The growth inhibition of T. cruzi epimastigotes was evaluated by a ( 3 H) thymidine uptake assay as previously described [19]. Natural sesquiterpenes and derivatives were evaluated at concentrations ranging from 1.5 to 50 µg/mL. Benznidazole (Active Pharmaceutical Ingredient) was used as positive control (Elea). The percentage of inhibition was calculated as 100 = {((cpm of treated parasites)/(cpm of untreated parasites)) × 100}.

Cytotoxicity on Primary Cell Culture
The cytotoxicity of the compounds was evaluated on splenocytes obtained from Balb/c mouse (1.5 × 10 5 ) [20,21] and incubated with different drug dilutions (5-200 µg/mL) as previously described [9]. Cell death was determined by flow cytometry using a BD FACSaria II cytometer. Cells incubated only with drug vehicle were used as 100% viability control, and death percentage was calculated according to the following formula: The concentration capable of causing 50% cell death (CC 50 ) was determined using a non-linear regression approach.

Statistical Analysis
The results are presented as means ± SD. GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA) was employed to carry out calculations. The results account for three to four independent experiments.

Conclusions
In this investigation, sesquiterpenes were the starting material for their transformation into various oxygenated and oxo-nitrogenated derivatives by chemical reactions directed at primary hydroxyls or acid groups. Our strategy was to obtain new derivatives, including functionalities such as diamides and 1,2,3-triazoles. The natural sesquiterpenes tessaric acid, ilicic acid, and ilicic alcohol did not show antiproliferative activity nor trypanocidal activity, but most of the oxonitrogenated derivatives showed activity against the solid tumor cell lines evaluated and against T. cruzi. The results obtained showed that from abundant and easily accessible natural products, the introduction of new functional groups improves their activity, so they can be used as scaffolds for the synthesis of new active molecules.