Enzymatic Synthesis of Austroeupatol Esters with Enhanced Antiprotozoal Activity

Abstract

Austroeupatol, the principal diterpene isolated from the invasive shrub Austroeupatorium inulifolium, holds promise for structural diversification and biological assessment of its derivatives due to its abundant availability and high yield isolation. We propose an efficient enzymatic synthesis of a series of austroeupatol esters derived from aliphatic and heterocyclic carboxylic acids. Systematic optimization of reaction parameters, including enzyme type and quantity, acylating agent amount, solvent, and temperature, was conducted. Thermomyces lanuginosus lipase in cyclohexane at 55 °C, yielded esters with favorable conversion rates. Through enzymatic catalysis, mono- and diacylated derivatives were obtained, with a diacylation–monoacylation ratio influenced by temperature and acylating agent amount. The antiprotozoal activity of austroeupatol and all synthesized derivatives was evaluated, observing that acylation improved it. The 19-valeroyl, 19-indolylpropyl, and 19-octyl derivatives were the most potent compounds against Trypanosoma cruzi and Leishmania infantum, highlighting this approach as a valuable method for synthesizing austroeupatol derivatives as potential antiparasitic agents.

Neglected tropical diseases (NTD) are a group of illnesses that affect the most vulnerable populations of the world, with a high socioeconomic impact and very limited resources destined for their control and eradication.¹ Chagas’ disease is a NTD caused by the protozoan Trypanosoma cruzi.² It is transmitted to humans through the bite of infected triatomine bugs or ingestion of food or beverages contaminated with their excreta.^3,4 It can also be transmitted via nonvectorial routes, such as congenital path, infected blood transfusion, or organ transplantation.⁵⁻⁷ It is estimated that 6–7 million people are infected, mostly in Latin America, where triatomine bugs are found. Recently, the disease has spread to nonendemic areas due to the migration of infected people and transmission by nonvectorial routes, the United States, Canada, Europe, Australia, and Japan being the most affected regions outside Latin America, with almost 400 000 infected individuals.⁸ Leishmaniasis is a NTD caused by several species of protozoa from the genus Leishmania carried by phlebotomine sandflies.¹ It is endemic to Asia, the Middle East, North Africa, East Africa, the Mediterranean, and South and Central America, with over 12 million infected people.⁹ There are three forms of the disease depending on the Leishmania species: visceral, cutaneous, and mucocutaneous leishmaniasis.¹⁰ Visceral leishmaniasis, the most severe form of the disease, is caused by Leishmaniainfantum, Leishmaniadonovani, and Leishmaniatropica.¹¹ Although drugs are available for Chagas’ disease and leishmaniasis treatment, they are associated with several drawbacks such as limited efficacy, high toxicity, low patient compliance, and drug resistance.^12,13 Thus, the development of new therapeutic agents to combat Chagas’ disease and leishmaniasis is urgently required. Natural products have a significant role in the development of drugs for human use, the antimalarial sesquiterpene lactone artemisinin being a notable example used for parasitic diseases treatment.¹⁴ Labdane diterpenes are natural compounds widely distributed among plants, fungi, insects, and marine organisms. They have shown several biological activities, including antimicrobial, algicidal, anti-inflammatory, antitubercular, cytotoxic, and cardiovascular. Some labdane diterpenes have also shown promising activity against human pathogenic protozoa such as T. cruzi, Leishmania sp. and Plasmodium sp.^15,16 Despite the promising biological activities of several natural compounds, they can seldom be directly employed for clinical use. In this sense, the semisynthesis of derivatives is often carried out to enhance the activity and selectivity and improve the physicochemical and pharmacokinetic properties of natural lead molecules.¹⁷ For example, masking hydrophilic groups by acylation is a common strategy to increase the lipophilicity of compounds and enhance their permeability.¹⁸

Enzymes offer highly sustainable and efficient synthetic alternatives to current existing chemical methods, and they have emerged in recent decades as ideal catalysts for synthetic transformations under mild reaction conditions. In addition, enzymes are capable of accelerating a huge number of biotransformations with high levels of selectivity and broad substrate specificity.¹⁹⁻²¹ The use of hydrolases and acyltransferases is a convenient tool for the acylation of natural compounds because it allows the semisynthesis of derivatives under mild conditions, avoiding the need for protection/deprotection steps and the use of strong conditions that could affect the integrity of these compounds.^22,23 Besides, enzymatic-catalyzed reactions take place with high regio- and enantioselectivity.^24,25

Austroeupatol (1) is the major labdane diterpene isolated from the aerial parts of Austroeupatorium inulifolium (Kunth) R.M. King & H. Rob. (Asteraceae), an invasive shrub that is mainly distributed in the warm areas of South America and South Asia.^26,27 The antibacterial activity of this natural compound and its semisynthetic derivatives has previously been studied;^28,29 however, no other biological activities related to austroeupatol or any semisynthetic derivatives have been reported so far. Thus, considering that austroeupatol is obtained in high yield from an easily available natural source and taking into account the antiprotozoal activity of some labdane diterpenoids, we aimed to synthesize austroeupatol esters by enzymatic catalysis and assess the antiprotozoal activity of this natural compound and its derivatives against Trypanosoma cruzi and Leishmania infantum.

In a first experiment, we assessed the performance of Candida antarctica lipase B (CALB), lipozyme from Thermomyces lanuginosus (TLIM), and lipozyme from Rhizomucor miehei (RMIM) in acylating austroeupatol (0.03 mmol) with octanoic acid (acylating agent:substrate ratio, A/S = 1) (Scheme 1). The reactions were carried out in 2 mL of cyclohexane, DIPE, or acetonitrile at 55 °C and 200 rpm using an enzyme:substrate ratio (E/S) of 10:1. After a 24 h incubation period, reactions showing austroeupatol conversion on TLC were further analyzed by HPLC to quantify the percentage conversion (Supporting Information (SI) Table S1).

Scheme 1. Lipase-Catalyzed Acylation of Austroeupatol (1) with Octanoic Acid.

Although austroeupatol is more soluble in DIPE and acetonitrile, no significant catalytic activity was observed using these solvents. By contrast, a significant acylation of austroeupatol was observed employing TLIM and RMIM in cyclohexane: 85 and 52% of conversion, respectively. Because a layer of water molecules is crucial for maintaining the tridimensional structure and activity of lipases, higher catalytic activity is expected in hydrophobic solvents that preserve the enzyme hydration layer.³⁰ Considering these results, TLIM combined with cyclohexane was selected to continue the optimization process due to its higher conversion percentage.

In a second experiment, we evaluated the influence of temperature (55 and 40 °C), enzyme:substrate ratio (mg) (E/S = 1:1, 5:1, and 10:1) and acylating agent:substrate ratio (mmol) (A/S = 1:1 and 3:1). An aliquot was withdrawn at 24, 48, 72, and 96 h and analyzed by HPLC to evaluate the progress of the reaction. More than 70% of conversion was achieved within the first 24 h in all experimental conditions.

Monoacylated and diacylated derivatives of austroeupatol were obtained in all cases, and no triacylated derivative was detected. The reaction curves (Figure 1) indicate that the diacylated derivative is formed at the expense of the monoacylated product. According to NMR analysis, a shift in the 19-H signals from 3.7 to 4.3 and 4.5 to 4.8 ppm suggests that the first acylation step takes place at the least sterically hindered position at C19-OH while a shift in the 3-H signal from 3.8 to 4.6 ppm indicates that the second acylation step takes place at C3-OH.

Figure 1.

Reaction curves of austroeupatol acylation (0.031 mmol, 10 mg) with octanoic acid using TLIM in cyclohexane (2 mL). A/S = acylating agent:substrate ratio (mmol/mmol). E/S = enzyme:substrate ratio (mg/mg).

Esterification at this position may be favored over C2-OH due to an intramolecular hydrogen bond between the hydroxy group at C-3 and the oxygen at C-19. The temperature had a significant influence on the acylation position: reactions carried out at 40 °C showed preferential acylation of austroeupatol at 19-OH, with a low conversion of the monoacylated derivative into the diacylated one. By contrast, reactions carried out at 55 °C showed a significant conversion of monoacylated austroeupatol into the diacylated product, the effect being more pronounced when higher E/S and A/S ratios were employed. To obtain sufficient amounts of both monoacylated and diacylated derivatives for antiprotozoal activity evaluation, we selected an experimental condition where the diacylated/monoacylated ratio (D/M) was close to 1. The D/M obtained for each E/S at 55 °C using three parts of octanoic acid is shown in Figure 2. The D/M = 1 was achieved between 24 and 48 h using an E/S = 10/1. However, because using smaller amounts of enzyme is always more convenient, we decided to carry out austroeupatol esterification with a E/S ratio of 5 despite extending the reaction time to 48 h.

Figure 2.

Diacylated:monoacylated product ratio (D/M) for each enzyme:substrate ratio (E/S) in the esterification of austroeupatol (0.031 mmol) with octanoic acid (0.093 mmol) using TLIM in cyclohexane (2 mL) at 55 °C.

Once we established the optimal reaction conditions (cyclohexane as solvent, E/S = 5/1, A/S = 3/1, 55 °C, 48 h), we performed the esterification of austroeupatol using some linear aliphatic carboxylic acids (Scheme 2). Taking into account previous works that indicate that the lipase activity in the acylation reactions is related to the chain length of the fatty acid, we assayed with chains of 2, 5, 12 and 16 carbon atoms. In addition, we also evaluated thioctic acid and 3-indolylpropanoic acid, considering that they present interesting frameworks found in numerous biologically active compounds. The conversion percentages are indicated in Scheme 2. All compounds were identified by NMR spectroscopy and MS spectrometry.

Scheme 2. Preparation of Austroeupatol Derivatives.

In exploring the potential antiprotozoal activity, austroeupatol and the synthesized compounds underwent testing against T. cruzi epimastigotes and L. infantum promastigotes at 25 μM. Austroeupatol exhibited low growth inhibition activity against T. cruzi epimastigotes (24.9%), and it was inactive against L. infantum promastigotes. Acylation of austroeupatol at C-19 OH with valeric acid (4) significantly improved its antiprotozoal activity against both parasites, with a growth inhibition of 93.5% for T. cruzi epimastigotes and 94.1% for L. infantum promastigotes. The monoacylated octyl ester (2a) also exhibited a high inhibitory effect on T. cruzi as well, with a growth inhibition of 99.5%. Comparing monoacylated and diacylated octyl (2a, 2b) and lauryl (5a, 5b) esters, it can be seen that acylation at both C-19 and C-3 OH decreases the antiprotozoal activity compared to the monoacylated derivative. On the other hand, the acetyl (3) and palmitoyl (6a, 6b) esters exerted a low growth inhibition activity against both parasites. These results suggest that esterification of austroeupatol at C-19 improves its antiprotozoal activity, with potency affected by the length of the acyl chain. The indolylpropanoic derivative (8) showed a high activity against L. infantum promastigotes, with an inhibition of 91.5%, and it was less active against T. cruzi (64.3%). The thioctic acid derivative (7) showed moderate activity against both T. cruzi and L. infantum (58.5 and 47.7%, respectively). In general, the synthesized esters displayed higher inhibitory activity against T. cruzi than Leishmania sp. Thus, we decided to test the compounds against the intracellular form of the parasite. Vero cells were infected with T. cruzi trypomastigotes expressing β-galactosidase. After incubation of infected cells with austroeupatol and its esters (10 μM), the growth inhibition activity was determined by a spectrophotometric method. As Table 1 shows, austroeupatol exhibited low inhibitory activity against amastigotes (28.0%), while the valeryl (4) and octyl monoacylated (2a) derivatives were the most active compounds with a growth inhibition of 99.11% and 100%, respectively. The indolylpropanoic (8) and thioctic acid (7) derivatives also showed high inhibitory activity against amastigotes, with growth inhibitions of 98.74% and 95.24%, respectively. Those compounds showing activity more than 90% of growth inhibition were then tested at a range of concentrations from 0.1 to 15 μM to estimate the inhibitory concentration 50% (IC₅₀). Infected Vero cells were then treated with increasing concentrations of the most active compounds to determine the half-maximal inhibitory concentration (IC₅₀) of each derivative. Results are shown in Table 1. To determine the selectivity of the most active austroeupatol esters, the cytotoxicity of the compounds was tested on Vero cells by the MTT method. The thioctic (7) and indolylpropanoic acid (8) derivatives demonstrated significant cytotoxicity (<10 μM), while the octyl (2a) and valeryl (4) esters were approximately half as toxic to Vero cells compared to T. cruzi amastigotes (Table 1). Thus, the octyl (SI = 3.2) and valeryl (SI = 2.7) derivatives were the most selective compounds among the synthesized austroeupatol esters.

Table 1. Antiprotozoal Activity and Selectivity of Austroeupatol and Esters Derivatives.

	Leishmania infantum	Trypanosoma cruzi
	promastigotes	epimastigotes	amastigotes		Vero cells
compd	growth inhibition at 25 μM (%)	growth inhibition at 25 μM (%)	growth inhibition at 10 μM (%)	IC50 (μM)	CC50 (μM)
1	0	24.9	28 ± 0.26	>15	85.38
2a	57.1	99.5	100 ± 0.00	7.03 ± 0.43	18.12 ± 1.05
2b	16.4	27.4	9.79 ± 8.06	n.d.	n.d.
3	15.7	21.6	22.67 ± 0.26	n.d.	n.d.
4	94.1	93.5	99.11 ± 0.91	8.5 ± 0.22	19.34 ± 0.24
5a	29.1	69.7	12.54 ± 2.47	n.d.	n.d.
5b	2.4	46.8	4.73 ± 0.39	n.d.	n.d.
6a	0	24.9	2.98 ± 9.37	n.d.	n.d.
6b	0	31.8	2.06 ± 1.82	n.d.	n.d.
7	47.7	58.5	95.24 ± 1.43	2.06 ± 0.04	<10
8	91.5	64.3	98.74 ± 1.69	2.26 ± 0.12	<10
benznidazole				0.94 ± 0.8	>384
glucantime	20.0 ± 0.9a

^aIC₅₀

In summary, we report here the enzymatic synthesis of 10 new compounds by direct acylation of austroeupatol, the major labdane diterpene isolated from Austroeupatorium inulifolium. The enzymatic procedure constitutes an excellent alternative to conventional chemical methods, which involve the use of hazardous reagents and harsh reaction conditions. The use of enzymes as biocatalysts makes this process more sustainable, less risky, and with less environmental impact. Among the evaluated enzymes, the lipase from the fungus Thermomyces lanuginosus (TLIM) gave the best results. This adds an additional advantage, taking into account its lower cost compared to other commercial lipases. In addition, the enzymatic catalyst lead to the synthesis of mono- and diacylated derivatives, with a diacylation–monoacylation ratio influenced by temperature and acylating agent amount.

On the other hand, the antiprotozoal activity of austroeupatol and all synthesized derivatives against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigores and amastigotes was evaluated. The acylation of austroeupatol significantly improved the antiparasitic activity. While austroeupatol presented almost no activity, the 19-valeroyl (4) and 19-indolylpropyl (8) were the most active compounds against both parasites. The 19-octyl (2a) and 19-indolylpropyl (8) derivatives almost completely inhibited the proliferation of amastigote form of T. cruzi, the clinically relevant form of parasite. These findings emphasize the significance of natural products diversification to generate new compounds for the development of more efficient and safe antiparasitic agents.

Glossary

Abbreviations

NTD

neglected tropical diseases

CAL B

Candida antarctica lipase B

TLIM

lipozyme from Thermomyces lanuginosus

RMIM

lipozyme from Rhizomucor miehei

E/S

enzyme:substrate ratio

A/S

acylating agent:substrate ratio

D/M

diacylated:monoacylated product ratio

IC₅₀

half-maximum inhibitory concentration

CC₅₀

half-maximum cytotoxic concentration

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00070.

• Materials and methods, synthesis and spectral data for compounds 1–8, biological evaluation, and ¹H and ¹³C NMR spectra of all compounds (PDF)

Author Contributions

Conceptualization, methodology, and formal analysis: O.G.E., A.E.B., E.A., G.A., V.P.S. and. G.G.L; writing-original draft preparation: O.G.E and G.G.L; writing-review and editing: O.G.E., A.E.B., G.A., V.P.S. and. G.G.L. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.