Design, synthesis and identification of novel molecular hybrids based on naphthoquinone aromatic hydrazides as potential trypanocide and leishmanicidal agents

Abstract

In pursuit of potential agents to treat Chagas disease and leishmaniasis, we report the design, synthesis, and identification novel naphthoquinone hydrazide-based molecular hybrids. The compounds were subjected to in vitro trypanocide and leishmanicidal activities. N’-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)-3,5-dimethoxybenzohydrazide (13) showed the best performance against Trypanosoma cruzi (IC₅₀ 1.83 µM) and Leishmania amazonensis (IC₅₀ 9.65 µM). 4-Bromo-N’-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzohydrazide (16) exhibited leishmanicidal activity (IC₅₀ 12.16 µM). Regarding trypanocide activity, compound 13 was low cytotoxic to LLC-MK2 cells (SI = 95.28). Furthermore, through molecular modeling studies, the cysteine proteases cruzain, rhodesain and CPB2.8 were identified as the potential biological targets.

Key words
Neglected diseases; Trypanocide activity; Leishmanicidal activity; Lawsone; Naphthoquinone hydrazide hybrids

INTRODUCTION

Neglected diseases (NTD) are a distinct group of transmissible diseases that occur in tropical and subtropical regions of the world. They are especially common in low-incoming populations that affect more than a billion people and cost emerging economies billions of dollars every year (Warusavithana et al. 2022, WHO 2020).

Leishmaniasis and Chagas disease, caused by the kinetoplastid parasites Leishmania spp and Trypanosoma cruzi, respectively, are public health problems in countries where these diseases are prevalent. They are considered to be within the most relevant group of neglected tropical diseases (Nicolás-Hernández et al. 2023, Silva-Jardim et al. 2014).

Chagas disease occurs in 21 countries in Latin America and is estimated that 8 million people are infected worldwide (Vásconez-González et al. 2023). T. cruzi enters the human bloodstream mainly through wounded skin or mucous membranes with infected faeces from triatomine insects. This infection in humans is characterized by three distinct clinical phases as following: 1) acute, which causes fever and edema at infection point (chagoma), 2) generally asymptomatic intermediate phase and, 3) chronic phase. Almost 30% of infected patients develop the chronic phase, which may lead to clinical manifestations such as cardiac, gastrointestinal, and cardiodigestive forms of Chagas disease (Abras et al. 2022). More than 50% of patients who enter the chronic form die within 7 months to 2 years of the initial symptoms (Abras et al. 2022, Nasim & Qureshi 2023, Salas et al. 2008, Silva-Jardim et al. 2014, WHO 2017).

Leishmaniasis, caused by protozoan parasites of the genus Leishmania, is a vector-borne infection that can occur in tropical and subtropical regions of the world. Depending on the region, the disease is spread by different species of insects of the genus Phlebotomus or Lutzomyia (Venkatesan et al. 2023). Clinical expressions depend on the Leishmania species. Visceral forms of leishmaniasis are responsible for most deaths and the most common manifestation are skin lesions (de Castro et al. 2013, Olivier et al. 2005, Singh et al. 2023).

Sadly, currently there are no vaccines available to prevent these infections. The main option to treat these diseases is chemotherapy using pentavalent antimonials, amphotericin B, miltefosine, paromomycin, and pentamidine. However, these medications are associated with various adverse effects (Herwaldt 1999, Silva-Jardim et al. 2014, Verdan et al. 2023). Therefore, there is a high demand for new efficient and safer drug scaffolds to treat Chagas disease and leishmaniasis (Jorge et al. 2023, Rycker et al. 2023).

Among the potential medicinal chemistry tools that can circumvent this issue, there is the molecular hybridization strategy for drug discovery. This approach is based on the design of new chemical entities by the fusion of bioactive molecular fragments derived from known bioactive molecules (Burkner et al. 2023, Pedroso et al. 2023, Lourenço et al. 2023, Wang et al. 2023). Usually, it is essential to verify the synergistic characteristics of the two fragments in order to plan the new target molecular entity (Dong et al. 2023, Fershtat & Makhova 2017, Fraga 2009, Langdon et al. 2010, Lazar et al. 2004, Wang et al. 2023, Viegas-Junior et al. 2007).

Hence, examining molecular frameworks to design novel antiparasitic agents preferably endowed with multitarget features, the 1,4-naphthoquinone skeletons emerged as promising starting material candidates for developing new bioactive hybrid agents (Navarro-Tovar et al. 2023). The reason for this is that the literature describes, for example, lawsone, α-lapachone, β-lapachone (Figure 1) and their congeners as having diverse biological properties, including trypanocide and leishmanicidal activities (Cardoso et al. 2017, Dantas et al. 2017, da Silva et al. 2013, Guimarães et al. 2013, Rani et al. 2022, Naujorks et al. 2015).

Figure 1
Molecular structure of lawsone and analogous.

Consequently, hydrazide-bearing molecules and similar scaffolds such as N’-(1, 4-naphtho-quinone-2-yl) isonicotinohydrazide (I), benzohydrazones (II), and quinoline-piperazine propionic acid hydrazones (III) are also reported as important compounds endowed with trypanocide, antibacterial, antiamoebic, antimalarial, anticancer, antiviral, anti-inflammatory, antiatherosclerotic and antifungal activities (Bouhadir et al. 2017, Caffrey et al. 2002, Hu et al. 2023, Kavitha et al. 2015, Kumar et al. 2017, Reddy et al. 2013)

Considering molecular hybridization strategy, we have come across only two reports in the literature in which lawsone (Figure 1) was subjected to a reaction with aromatic hydrazide to form C-2 substituted bioactive naphthoquinones (Dudley et al. 1969, Hu et al. 2023, Kavitha et al. 2015). As part of our research interest in synthesis and biological evaluation of heterocyclic compounds (Botteselle et al. 2021, dos Santos et al. 2021, 2022, Franco et al. 2021, Moraes et al. 2023, Rafique et al. 2020, Scheide et al. 2020, Veloso et al. 2021, Vitor et al. 2021), we decided to prepare naphthoquinone aromatic hydrazide-based molecular hybrids, using a simple, inexpensive and reliable protocol. These new frameworks of N’-(1,4-naphtho-quinone-2yl) hydrazide (Figure 3 ) were tested as potential trypanocide and leishmanicidal drug prototypes.

Figure 2
Hydrazine linker in bioactive molecules.

Figure 3
Targeted naphthoquinone aromatic hydrazide-based molecular hybrids.

RESULTS AND DISCUSSION

Figure 4 shows the synthetic route designed to achieve the fused molecules of naphthoquinone hydrazides. Initially, we selected substituted benzoic acid starting materials (1-4) that were subjected to simple esterification reactions with methanol under acid catalysis. The respective esters formed (5-8) were necessary to generate the aromatic hydrazides (9-12) of the aimed parent by treatment with hydrazine hydrate (Rodrigues et al. 2016). The key coupling step was then followed by the reaction of lawsone (Figure 1) with hydrazides (da Silva et al. 2013) to give the four corresponding target hybrids (13-16).

Figure 4
Synthetic route to prepare hybrids 13-16.

All compounds were characterized using high-resolution mass spectrometry (HRMS), Fourier transform infrared (FTIR) and, ¹³C and ¹H nuclear magnetic resonance (NMR) spectroscopies. The compound 13, for example, analyzed as C₁₉H₁₇N₂O₅ [M+H]⁺, showed a molecular ion peak at m/z 353.1059 in its high-resolution mass spectrum. The relevant features of its ¹H NMR spectrum comprised the presence of two singlets at δ 9.49 and 10.71 corresponding to hydrogens bonded to the azide group. Furthermore, two doublets were recorded at δ 7.94 and δ 8.02, two multiplets at δ 7.77 and δ 7.85, as well as a singlet at δ 5.67 due to the aromatic and olefinic protons of the naphthoquinone ring. A doublet at δ 7.08 and a singlet at δ 6.72 were attributed to the aromatic ring of the hydrazide moiety. Hydrogens of methoxy groups showed signals at δ 3.80. The appearance of absorptions at δ 182.6, δ 181.4 and δ 168.3 corresponding to three carbonyls along with the presence of methylene carbons at δ 102.1 and δ 148.9 as confirmed by ¹³C (DEPT) spectra additionally corroborated the assigned structure.

The synthesized naphthoquinone hydrazide hybrids were evaluated for their in vitro activities against epimastigote forms T. cruzi and promastigote L. amazonensis and the results are registered in Table I.

Table I
LogP, logS, trypanocidal and leishmanicidal activities of compounds tested against epimastigote T. cruzi and promastigote L. amazonensis forms.

Compound	logPa	logSb	cIC50 (µM) ± SD T. cruzi	cIC50(µM) ± SD L. amazonensis
13	2.02	-4.11	1.83 ± 0.36	9.65 ± 0.36
14	2.14	-4.13	114.10 ± 9.12	>100
15	1.50	-4.03	117.63 ± 10.96	>100
16	2.69	-4.88	86.89 ± 7.00	12.16 ± 0.92
Benznidazoled	0.49	-2.06	8.80 ± 0.4	-
Amphotericin Bd	-0.39	-5.37	-	0.065

• Values represent the mean ± S.D. of duplicate determination. ^aLogP, octanol/water partition coefficient measured by SwissADME (Daina et al. 2017); ^bLogS expressed as log (g/100 g water) measured by SwissADME (Daina et al. 2017); ^c IC₅₀ is the concentration of the compound that is required for 50% inhibition in vitro. ^d Positive controls.

Although scaffolds 14-16 were inactive against the epimastigote T. cruzi, this result is not considered insignificant when comparing these activities with the parent lawsone (IC₅₀ 410 µM) (Muñoz et al. 2006). On the other hand, compound 13 (IC₅₀ 1.83 µM) revealed a higher trypanocide activity than the reference drug benznidazole (IC₅₀ 8.80 µM). An analysis of the structure-activity relationship (SAR) of the synthesized hybrids disclosed the dependence of trypanocide activity on the nature and pattern of substitution at the aromatic ring of the azide segment. Interestingly, comparing the 3,5-dimethoxy substitute compound (13) with the 3,4,5-trimethoxy substitute compound (14) (Table I), the latter significantly decreased its activity, suggesting a possible requirement to locate the molecules during interaction with biological receptors. Studies have stated that electronic and structural properties are important factors in the interaction between trypanocide quinone derivatives and biological receptors (Molfetta et al. 2005).

The results of in vitro antiproliferative assays in promastigote L. amazonensis showed that compounds 14 and 15 (IC₅₀ >100) were inactive (Table I). However, compounds 13 (IC₅₀ 9.65 µM) and 16 (IC₅₀ 12.16 µM) displayed moderate leishmanicidal activity. According to Muñoz and coworkers (31), the enhancement of the trypanocide and leishmanicidal activities of derivatives related to hydrazides, such as various disubstituted aromatic hydrazones, depends on the groups attached to each side of the molecules. Furthermore, the authors mentioned that molecules designed to be used as antiparasitic can be simultaneously active against Trypanosoma and Leishmania, since the parasites share proteins with very similar active site (Muñoz et al. 2006).

Biopharmaceutical aspects are also crucial to be measured in drug candidates. In particular, logP values are vastly discussed in medicinal chemistry since the postulation of the rule of five. This parameter analysis the solubility profile of a compound in an immiscible biphasic system of lipids and water (Bhal 2007, Brown 2012). Considering the physiological environment, this aspect is of great importance, as it can provide information on whether a compound will penetrate the cell membrane or be easily solubilized in an aqueous medium such as blood. In the study of rule of five, Lipinski et al. (1997) investigated the biopharmaceutical aspects of a set of standard drugs. Between them, a result of clogP ≤ 5 was postulated as being necessary for an ideal prototype. Meanwhile, logS are directly related with the solubility in water of a molecule and values close to −4 were defined as standard for a drug candidate (Sander 2001). All tested compounds had logS values close to −4 and logP ≤ 5, following the postulated characteristics (Table I). It is also important to highlight that Benznidazole presented a logS value considerably greater than -4, which represents a violation of what is postulated for biopharmaceutical aspects of a drug. Therefore, compound 13 is not only more active than Benznidazole for the epimastigote form of T. cruzi, but it is also more viable considering its potential for greater bioavailability.

Cytotoxicity of all hybrids (13-16) was determined using the MTT LLC-MK2 cell assay (Rodrigues et al. 2014) to determine whether the observed activities are due to their trypanocide and leishmanicidal efficacy or cytotoxicity (Table II).

Table II
.Cytotoxicity of naphthoquinone hydrazide hybrids on LLC-MK2 cells and their selectivity index (SI).

Compound	LLC-MK2 CC50 (µM) ± SD	SI T. cruzi (epimastigote)	SI L. amazonensis (promastigote)
13	174.37 ± 18.52	95.28	18.06
14	269.08 ± 44.05	2.35	> 2.69
15	152.29 ± 18.95	1.29	> 1.52
16	237.36 ± 29.62	2.73	19.52

• CC₅₀ = concentration of the compound that causes death in 50% of viable cells in the culture medium after 96 h of exposition. IC₅₀ = minimum inhibition concentration to inhibit 50% of the protozoa in its culture medium. SI = CC₅₀ LLC-MK2 / IC₅₀ of epimastigote or promastigote forms.

As is evident, compound 13 was not cytotoxic for LLC-MK2 cells (SI = 95.28) with respect to trypanocide activity and, although this index was lower for leishmanicidal activity (SI = 18.06), it is still attractive if we take into account the possibility of generating new derivatives with some skeletal modification. For compound 16, it was nearly twice as cytotoxic against Leishmania versus LLC-MK2 cells (SI = 19.52) (Table II) and appears to have potential for the development of new bioactive halogen analogues.

For drug development, target identification is crucial to follow-up studies, aiding medicinal chemistry efforts (Schenone et al. 2013). In the context of neglected diseases such as Leishmaniasis and Chagas disease, the target-based strategy has been the dominant tool over the last decades due to the advances in molecular biology and urgency to discover new effective drugs (Lourenço et al. 2023, Gilbert 2013). Therefore, to deeply analyze the influence of different substituents of tested compounds on the antiparasitic action, it is necessary to identify their potential pharmacological targets. Although the results of antiparasitic activity for this set of compounds are unprecedented, previous studies in the literature may shed light on the investigation of potential biological targets behind their pharmacological effects.

A previous study investigated the ability to inhibit the cysteine proteases cruzaine and rhodesain by a set of compounds based on 1,4-naphthoquinone core (Silva et al. 2021). Interestingly, CR-70 was active against these proteases and had a very similar structure to those tested in our study. (Figure 5). Considering that structurally similar molecules commonly have similar biological activities (Martin et al. 2002), we performed docking and molecular dynamics simulations using cruzain and rhodesain as proteins. In addition, to select the ligands used in molecular modeling studies, we used the Activity cliff theory as a tool. Activity cliffs (ACs) are of particular interest in structure-activity relationship (SAR) analysis and compounds optimization. ACs are defined as pairs or groups of molecules marked by their high structural similarity but large difference in potency (Stumpfe et al. 2019). Between the tested compounds, 13 and 15 can be considered as an AC for both antileishmanial and anti-T. cruzi activities. Therefore, these compounds were selected to better understanding a potential SAR for this set of antiparasitic molecules.

Figure 5
Chemical structure of compound CR-70.

The differences in the potency of compounds 13 and 15 were confirmed by the distinct binding mode positions at cruzain active site. Compound 15 only occupied one hydrophobic pocket of the protein biding site (Figure 6a). As a result, this compound mainly made hydrophobic interactions with the amino acid residues (Figure 6b). However, the replacement of a nitro by a methoxy group, as well as the addition of a second methoxy group in R1 resulted in a greater interaction with the amino acid residues of the active site. Between them, the hydrogen bond, considered as a strong interaction, with Asp161 stood out (Figure 6c). Furthermore, 13 occupied the entire active site of cruzain (Figure 6d), explaining its high antiparasitic activity demonstrated by in vitro assays.

Figure 6
Binding mode positions of compounds 15 (a) and 13 (c), and 2D representations of these compounds (b and d) at the biding site of cruzain (PBD ID: 1AIM).

The same molecular scenario was identified considering the interactions of the tested compounds with rhodesain. Although the binding modes of 15 and 13 were similar (Figures 7a and 7c), the types of intermolecular interactions with the protein’s amino acid residues were notably different. Likewise, hydrophobic interactions were the only ones made between compound 15 and the rhodesain binding site. (Figures 7b). In contrast, compound 13 demonstrated hydrophobic interactions as well as a hydrogen bond with Gln19 (Figure 7d). Combined, these results reinforce the better in vitro results of compound 13 and explain the potency differences between this AC.

Figure 7
Binding mode positions of compounds 15 (a) and 13 (c), and 2D representations of this compounds (b and d) at the biding site of rhodesain (PBD ID: 6EXQ).

Cruzain and rhodesain are cysteine proteases widely expressed in T. cruzi. However, the compounds have also been tested and proven to be active against promastigotes forms of L. amazonensis. This fact may be related to the expression of other cysteine proteases by Leishmania parasite such as CPB which are structurally very similar to cruzain and rhodesain. It is important to highlight that both mentioned proteins are L-type cathepsins, resulting in similar inhibition capacity by the prototypes (Lourenço et al. 2023, Rebello et al. 2010). CPB is a virulence factor and plays an important role for parasite survival in mammalian host cells. Although it is more expressed in amastigotes, some previous works have demonstrated the presence of CPB on the surface of the promastigote form. Therefore, this cysteine protease could be a potential target for molecular hybrids based on naphthoquinone aromatic hydrazide.

Analyzing the output results of compounds 13 and 15, a difference in type of molecular interactions with amino acids residues was again observed. Both compounds had occupied all the pockets of the active site of CPB2.8 (Figures 8a and 8c). However, compound 13 showed two hydrogen bonds with Gly191 and Gln144 as well as hydrophobic interactions (Figure 8d). Meanwhile, compound 15 made only hydrophobic interactions with the amino acid residues (Figure 8b).

Figure 8
Binding mode positions of compounds 15 (a) and 13 (c), and 2D representations of this compounds (b and d) at the biding site of CPB2.8.

The calculated binding free energy values reflected the strongest intermolecular interactions made by compound 13 on cruzain, rhodesaine and CPB2.8. Between the results, the lowest binding-free energy was obtained by the interaction between 13 and the proteins cruzain and rhodesaine (-1.25 and -0.94 kcal·mol−¹, respectively). The results are 3.2 and 2.93-fold higher than those demonstrated for compound 15 (Table III). The most promising results for T. cruzi cysteine proteases also reinforce the in vitro results since compound 13 was more active against epimastigote forms. Furthermore, binding free energy values from simulations using CPB2.8 as the protein also demonstrate a more stable interaction with 13 by 2.27-fold (Table III). Combined, the molecular modelling approaches pointed out that cysteine proteases may be the potential targets for naphthoquinone aromatic hydrazide-based molecular hybrids and demonstrated the importance of a polar substituent on R1.

Table III
Calculated energy values for the interaction of 13 and 15 with cruzain, rhodesain and CPB2.8

E nzyme	Binding-free energy a
13
Cruzain	-1.25
Rhodesaine	-0.94
CPB2.8	-0.68
15
Cruzain	-0.39
Rhodesaine	-0.32
CPB2.8	-0.30

• ^aAll energy values are expressed in kcal·mol−¹.

CONCLUSIONS

Based on the molecular hybridization strategy, we have designed, synthesized, and identified novel molecular hybrids based on naphthoquinone aromatic hydrazides (13-16). Among them, compound 13 showed antileishmanial activity and a higher trypanocidal activity than standard benznidazole, also being non-cytotoxic to LLC-MK2 cells. This representative scaffold suggested a dependence of electronic and structural molecular features during the interaction with the biological receptors Trypanosoma and Leishmania. Although compound 16 demonstrated lower leishmanicidal activity than standard, it could serve as a molecular basis for the planning of additional bioactive halogen derivatives. Through molecular modelling studies, cysteine proteases were pointed as a potential target behind the antiparasitic action and the importance of a polar substituent on R1 was confirmed, Consequently, we assume that our findings substantiate the potential of the present approach and will inspire complementary studies on the discovery of novel antiparasitic drug prototypes related to these molecular frameworks.

EXPERIMENTAL SECTION

Chemistry

All reagents were analytical grade and were used without further purification. Chromatographic purification was performed on silica gel (Merck, 100-200 mesh) and analytical thin layer chromatography (TLC) was performed on silica gel 60-F₂₅₄. ^{The 1H} NMR (300 MHz) and ¹³C NMR (75 MHz) spectra were measured with a Bruker Avance DPXT-300 spectrometer with CDCl₃ and DMSO-d₆ as solvents and recorded in ppm relative to the internal tetramethylsilane standard (TMS). The ¹H NMR spectra are reported as follows: ppm (multiplicity, coupling constant J/Hz, number of protons). Multiplicity is abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), br (broad signal). The coupling constants (J) are quoted in Hertz and recorded to the nearest 0.1 Hz. High resolution mass spectrometry (HRMS) was performed on a UFLC Shimadzu LC-20AD apparatus, with and IES-Q-QTOF-microTOF III detector (Bruker Daltonics) in chemical ionisation positive ion mode (m/z 120-1200). Samples were prepared with 0.1 g/mL (methanol/ water 7:3) and injected 1μL, using elution gradient water (phase A) and acetonitrile (phase B), both with 1% acetic acid, 50% isocratic method and a running time of 3 min. The infrared spectra were recorded on a Bomen FT-IR-MB100FT-IR spectrometer and reported as wavenumbers (cm^-1).

General procedure for synthesis of esters (5-8)

Substituted carboxylic acids (1-4) (1.1 mmol) previously solubilized in dry methanol (30 mL) were added to a 50 ml round bottom flask, followed by dropwise addition of H₂SO₄ (1.3 mL). The reaction mixture was then stirred and heated under reflux for 4 h. After this period, the TLC analysis indicated the complete consumption of the starting material. The excess of solvent was removed by rotoevaporation, and the mixture was neutralized with sodium bicarbonate. The organic layer was extracted with ethyl acetate (3 x 20 mL), washed with water (3 x 25 mL) and, dried in MgSO₄. The drying agent was removed by filtration and the solvent was removed by rotoevaporation to give the product as a colorless oil.

General procedure for synthesis of hydrazides (9-12)

Substituted esters (5-8), resulting from acids (1-4), respectively, (1.3 mmol) along with hydrazine, were added to a 25 ml round bottom flask. The mixture was stirred at room temperature overnight. After this period, the reaction mixture was poured into a beaker containing chipped ice. The precipitated mixture was then subjected to simple filtration, washed with distilled cold water (30 mL) and left to dry at room temperature.

General procedure for the synthesis of derivatives of hydrazides of lawsone (13-16)

Acylhydrazides (9-12), resulting from esters (5-8), respectively, (1.1 mmol) and 1,4-hydroxy-naphthoquinone (1.1 mmol) were added to a 25 ml round bottom flask containing acetic acid 80% (20 mL). The reaction mixture was kept under stirring overnight and monitored by TLC analysis. After this period, the product was filtered, washed with hexane (50 mL) and, recrystallised in ethanol.

N’-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-3,5-dimethoxybenzohydrazide (13) (Supplementary Material - Figure S1)

This compound was obtained from hydrazide 9. Yield 51%. Orange solid. m.p. = 187-195 ° ^C. ¹H NMR (300 MHz, DMSO-d ₆) δ 3.80 (s, 6H), 5.67 (s, 1H), 6.72 (s, 1H), 7.08 (d, J = 1.8 Hz, 2H), 7.77 (m, 1H), 7.85 (m, 1H), 7.94 (d, J = 7.0 Hz, 1H), 8.02 (d, J = 7.0 Hz, 1H), 9.49 (br, 1H), 10.71 (s, 1H). ¹³C NMR (75 MHz, DMSO-d ₆,) δ 55.9 (2CH₃), 102.2 (CH), 104.5 (CH), 105.8 (CH), 125.9 (CH), 126.3 (CH), 130.9 (C), 132.9 (C), 133.2 (C), 134.4 (CH), 135.5 (CH), 148.9 (C), 160.9 (C), 165.3 (C), 181.4 (C=0), 182.8 (C=O). FT-IR (KBr, cm^-1) ν_max 3289-3412 (NH), 1666-1681 (C=O), 1457-1553 (C=C). C₁₉H₁₇N₂O₅ [M+H]⁺ 353.1131. Found 353.1123.

N’-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-3,4,5-trimethoxybenzohydrazide (14) (Figure S2)

This compound was obtained from hydrazide 10. Orange solid. Yield 55%. mp = 154.2-163.1 ¹H NMR (300 MHz, DMSO-d ₆) δ 3.73 (s, 3H), 3.84 (s, 6H), 6.17 (s, 1H), 7.26 (s, 1H), 7.82 (m, 1H), 7.82 (m, 1H), 7.95 (dd, J = 7.3 Hz; J = 1.5 Hz, 1H), 8.0 (dd, J = 7.3 Hz; J = 1.5 Hz, 1H), 10.45 (s, 1H), 11.69 (br, 1H). ¹³C NMR (75 MHz, DMSO-d ₆) δ 56.5 (2CH₃), 60.5 (CH₃), 105.4 (CH), 111.4 (CH), 125.8 (CH), 126.3 (CH), 128.0 (C), 131.0 (C), 132.3 (CH), 133.6 (CH), 134.8 (CH), 140.9 (C), 153.1 (C), 160.0 (C), 165.8 (C), 181.7 (C=O), 185.1 (C=O). FT-IR (KBr, cm^-1) _max 3445-3512 (NH), 1644-1677 (C=O), 1459-1588 (C=C). C₂₀H₁₈N₂O₆K [M+K]⁺ 421.0790. Found 421.1602.

N’-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-nitrobenzohydrazide (15) (Figure S3)

This compound was obtained from hydrazide 11. Orange solid. Yield 52%. ¹H NMR (300 MHz, DMSO-d ₆) δ 5.78 (s, 1H), 7.80 (m, 1H), 7.83 (m, 1H), 7.94 (d, J = 6.9 Hz, 1H), 8.02 (d, J = 6.9 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.36 (1d, J = 8.7 Hz, H), 9.57 (s, 1H), 11.07 (s, 1H). ¹³C NMR (75 MHz, DMSO-d ₆) δ 102.4 (CH), 124.2 (CH), 125.9 (CH), 126.3 (CH), 129.6 (C), 130.9 (CH), 132.9 (CH), 133.2 (C), 135.4 (CH), 138.2 (C), 148.7 (C), 150.0 (C), 164.4 (C), 181.3 (C=O), 182.7 (C=O). FT-IR (KBr, cm^-1) ν_max 3451-3516 (NH), 1644-1782 (C=O), 1459-1592 (C=C). C₂₀H₁₉N₂O₆ [M+H]⁺ 338.0771. Found 338.0762.

4-bromo-N’- (1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzohydrazide (16) (Figure S4)

This compound was obtained from hydrazide 12. Brown solid. Yield 55%. ¹H NMR (300 MHz, DMSO-d ₆) δ 5.71 (s, 1H), 7.77 (m, 1H), 7.77 (m, 1H), 7.87 (m, 1H), 7.95 (d, ³ J = 6 Hz, 1H), 8.02 (d, ³ J = 6 Hz, 1H), 9.50 (s, 1H), 10.82 (s, 1H). ¹³C NMR (75 MHz, DMSO-d ₆) δ 102.2 (CH), 125.9 (CH), 126.3 (CH), 126.4 (CH), 130.0 (C), 130.9 (CH), 132.0 (C), 132.1 (CH), 133.1 (CH), 133.2 (C), 135.4 (C), 148.8 (C), 165.0 (C), 181.4 (C=O), 182.7 (C=O). FT-IR (KBr, cm^-1): ν_max 3451-3516 (NH), 1644-1782 (C=O), 1459-1592 (C=C). C₁₇H₁₂BrN₂O₆ [M+H]⁺ 371.0025. Found 371.0031.

Biological assays

Parasites and cell cultures

Antiparasitic activity experiments were carried out with the Y strain of Trypanosoma cruzi (epimastigotes) and promastigotes of Leishmania amazonensis (WHOM/BR/75/JOSEFA strain). The forms of epimastigotes were grown in liver infusion tryptose (LIT) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco Invitrogen, Grand Island, NY, USA), kept at 28 ° C and maintained by weekly transfers. The promastigote forms of L. amazonensis were maintained in culture at 25 ° C with weekly transfers to fresh Warren’s medium supplemented with 10% FBS. To evaluate the cytotoxicity of the compounds, LLCMK2 cells (Macaca mulatta epithelial kidney cells) were maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco Invitrogen), pH 7.4, supplemented with 2 mM L-glutamine, 10% FBS, and 50 mg.L^-1 gentamicin at 37 ° C in a humidified atmosphere of 5% CO₂.

Antiproliferative Activity against Epimastigote Forms (Trypanosoma cruzi)

Epimastigotes (1 × 10⁶ parasites.mL^-1) in the exponential growth phase (96 h) were harvested and incubated in the presence of LIT supplemented with 10% FBS added or not to increase concentrations of drug candidates. Subsequently, the incubated organisms at 28 ° C in 96-well flat bottom plates were counted on a Neubauer hemocytometer under light microscopy. IC₅₀ (concentration that inhibited 50% parasite growth) was determined by regression analysis of the data.

Antiproliferative activity against promastigote forms (Leishmania amazonensis)

Promastigotes (1 × 10⁶ cells.mL^-1) in the exponential growth phase (48 h cultures) were inoculated in a 96-well plate in the absence or presence of different concentrations of drug candidates. The activity against promastigote forms was evaluated after 72 h using the XTT method, which consists of incubation of cultures in the presence of a combination of the 2,3-Bis- (2-Methoxy-4-Nitro-5-Sulfophenyl) -2H-Tetrazolium-5-Carboxanilide compound (XTT, Sigma) and the electron coupling reagent phenazine methosulfate (PMS, Sigma). After parasite treatment, 100 L of the parasites, 100 µL of the mixture of XTT (0.5 mg.mL^-1) and PMS (0.06 mg.mL^-1) was added to each well, the plate was incubated for 4 h protected from light at 28 ° C and the absorbance measured at 450 nm in a microplate reader (Bio Tek – Power Wave XS). By comparing the absorbance of the control untreated parasites with those of the treated ones, the inhibitory activity was determined. IC₅₀ (concentration that inhibited 50% parasite growth) was determined by regression analysis of the data.

Cytotoxicity Assay

To evaluate the cytotoxicity of the compounds, the MTT assay was applied as previously described (Muñoz et al. 2006). This colorimetric assay is based on the ability of viable mitochondria to convert MTT, a water-soluble salt of tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), into an insoluble, purple-colored formazan precipitate. LLCMK2 cells were collected from confluent cultures, seeded in 96-well plates, and incubated at 37 ° C in a humid atmosphere of 5% CO2. After 24 h, the medium was replaced with new DMEM that contained concentrations of compounds ranging from 3.7 to 73.6 µM. After 96 h of incubation, cells were washed in PBS and 50 µL of MTT (2 mg.mL^-1) was added to each well. The formazan crystals were solubilized in DMSO and absorbance was read at 570 nm on a microplate reader (Bio Tek – Power Wave XS). The concentration that decreased 50% of the absorbance value observed in the control represented the CC₅₀ (cytotoxic concentration for 50% of the cells).

Molecular modelling studies

Compounds 13 and 15 were drawn using the program MarvinSketch 16.9.5 (ChemAxon Ltd., Budapest, Hungary). The structure optimization was carried out through PM7 semiempirical method incorporated in the software MOPAC2016 and the definition of charges was made considering a pH of 7.4.

To investigate the potential mechanism of action of these compounds, molecular docking simulations were carried out using cruzain, rhodesain and CPB2.8 as proteins. The 3D structures of cruzain, rhodesain were obtained from the Protein Data Bank (PDB ID: 1AIM and 6EXQ, respectively) (Gillmor et al. 1997, Giroud et al. 2018). Meanwhile, the three-dimensional structure of rCPB2.8 was obtained based on the homology modelling methodology using the Swiss-Model program (Daina et al. 2017). The homology modelling assay followed the procedures described in literature (Lourenço et al. 2023) using papain-like cysteine protease obtained from the Protein Data Bank (PDB ID: 1F2A) as a layout (Brinen et al. 2000) and the primary structure of rCPB2.8 as the target sequence. The molecular docking simulations was carried out using AutoDockVINA software (Trott & Olson 2010). The grid box used for research was large enough to encompass the target binding site and the crystallographic ligand was deleted before each simulation. Also, the systems of protein and tested compound were subjected to a dock preparation using AutoDockVINA software in which the charges were added through AMBER ff14SB method for standard residues and AM1-BCC method for other residues.

For each protein, the output result of docking simulations with the lowest score value was selected for an optimization of binding mode. The geometry optimizations were made with GROMACS 2018 package and CHARMM force field (Vanommeslaeghe et al. 2010, Zoete et al. 2011). SwissParam Server was used to obtain ligand topology and solvent properties were mimetic based on TIP3P water model (Zoete et al. 2011). A cubic box was built, and its dimensions determined to ensure a space of 1.2 nm between the protein and the walls of the box. The system charges were neutralized with the addition of ions in the physiological condition (0.15µM) and the energy optimization steps were carried out using the steepest descent followed by the conjugate gradient algorithm. The convergent criterion was a maximum of 50 N force on the atoms. Geometry optimization of the solvated system was performed using the steepest descent algorithm, followed by equilibration simulations with nVT and nPT. To perform them, temperature was maintained at 300°K coupling the system to a V-rescale thermostat (0.1 ps) and, using Parinello-Rahman coupling algorithm, the pressure was kept constant at 1 bar. Unrestrained simulations were performed for 1000 ps and the stabilization of all systems were monitored by RMSD and RMSF graphs (Figure S5). After stabilization, the binding free energy was calculated through GROMACS 2018 package considering the short range Columbic and Lennard-Jones interaction energies between compounds and the surroundings. Intermolecular interactions were investigated using the 2D representation provided by LigPlot program (Wallace et al. 1995) and the occupation at the binding site was analyzed using 3D representations using UCSF Chimera.

ACKNOWLEDGMENTS

The authors acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brazil (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001, Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia – Brazil (FUNDECT – MS) grant n° 266/2022 (SIAFEM 32184 – DPL), FUNDECT-MS (204/2022 – AB). SS and JR would like to acknowledge CNPq (316687/2023-5, 309975/2022-0, 315399/2020-1, 404172/2023-7, and 405655/2023-1). The authors declare that the research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest.

SUPPLEMENTARY MATERIAL

Figure S1-S6.

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Publication Dates

• Publication in this collection
  13 May 2024
• Date of issue
  2024

History

• Received
  5 Apr 2023
• Accepted
  6 July 2023