Design, Antileishmanial Activity, and QSAR Studies of a Series of Piplartine Analogues

Nóbrega, Flávio R.; Silva, Larisse V.; Bezerra Filho, Carlos da Silva M.; Lima, Tamires C.; Castillo, Yunierkis P.; Bezerra, Daniel P.; Lima, Tatjana K. Souza; Sousa, Damião P. de

doi:https://doi.org/10.1155/2019/4785756

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Research Article | Open Access

Volume 2019 | Article ID 4785756 | https://doi.org/10.1155/2019/4785756

Design, Antileishmanial Activity, and QSAR Studies of a Series of Piplartine Analogues

Flávio R. Nóbrega,¹Larisse V. Silva,²Carlos da Silva M. Bezerra Filho,¹Tamires C. Lima,³Yunierkis P. Castillo,⁴Daniel P. Bezerra,⁵Tatjana K. Souza Lima,²and Damião P. de Sousa¹

Academic Editor: Andrea Trabocchi

Received14 Oct 2018

Accepted17 Dec 2018

Published08 Jan 2019

Abstract

Piplartine is an alkamide found in different Piper species and possesses several biological activities, including antiparasitic properties. Thus, the aim of the present study was to evaluate a series of 32 synthetic piplartine analogues against the Leishmania amazonensis promastigote forms and establish the structure-activity relationship and 3D-QSAR of these compounds. The antileishmanial effect of the compounds was determined using the MTT method. Most compounds were found to be active against L. amazonensis. Among 32 assayed derivatives, compound (E)-(−)-bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate exhibited the most potent antileishmanial activity (IC₅₀ = 0.007 ± 0.008 μM, SI > 10), followed by benzyl 3,4,5-trimethoxybenzoate (IC₅₀ = 0.025 ± 0.009 μM, SI > 3.205) and (E)-furfuryl 3-(3,4,5-trimethoxyphenyl)-acrylate (IC₅₀ = 0.029 ± 0.007 μM, SI > 2.688). It was found that the rigid substituents contribute to increasing antiparasitic activity against L. amazonensis promastigotes. The presence of the unsaturated heterocyclic substituent in the phenylpropanoid chemical structure (furfuryl group) resulted in a bioactive derivative. Molecular simplification of benzyl 3,4,5-trimethoxybenzoate by omitting the spacer group contributed to the bioactivity of this compound. Furthermore, bornyl radical appears to be important for antileishmanial activity, since (E)-(−)-bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate exhibited the most potent antileishmanial activity. These results show that some derivatives studied would be useful as prototype molecules for the planning of new derivatives with profile of antileishmanial drugs.

1. Introduction

Leishmaniasis is a tropical disease prevalent in 98 countries and has high impact on South America, Africa, and Asia (especially India). This illness is caused by protozoan parasites of the Leishmania genus and is transmitted to humans by the bite of infected female phlebotomine sandflies. There are at least twenty species of Leishmania that cause clinical manifestations in humans, and they can occur in three different forms: (i) visceral leishmaniasis, (ii) cutaneous leishmaniasis, and (iii) mucocutaneous leishmaniasis. The most severe form is visceral leishmaniasis, characterized by prolonged fever, hepatomegaly, splenomegaly, substantial weight loss, and progressive anemia, and if left untreated, it can be fatal in 95% of cases [1–3].

The drugs currently available for treatment of leishmaniasis are pentavalent antimonials glucantime, bis-amidines (pentamidine and stilbamidine), miltefosine, and amphotericin B. Unfortunately, all these drugs have several limitations, such as low efficacy, toxicity to the liver and heart, elevated cost, and parasite resistance. For these reasons, new and more efficient therapeutic approaches are needed for prevention and treatment of leishmaniasis [1]. Natural products provide a virtually unlimited source of inspiration for new, powerful and selective drug leads. It is estimated that 20,000 plant species have antiparasitic activities. A wide variety of secondary metabolites have antileishmanial activity, namely, alkaloids, triterpenes, terpenoids, chalcones, saponins, glycosides, acetogenins, and flavonoids [4–6].

Piperaceae family, also known as pepper family, comprises over 1,000 species distributed pantropically. The Piper genus is the most diverse of the Piperaceae family and produces a number of structural classes (lignans, phenylpropanoids in the form of amides, and other derivatives of phenylpropanoids) with several biological activities, including antileishmanial activity [7–9]. In a study [7], antileishmanial activity of n-hexane, ethyl acetate, acetone, and methanol extracts from Piper cubeta fruits and Piper retrofractum stem bark was investigated against Leishmania donovani promastigotes. Among these substances, piplartine demonstrated the highest leishmanicidal activity, with an IC₅₀ value of 7.5 μM. Furthermore, this molecule was 3 times more potent than positive control pentamidine (IC₅₀ = 25 μM) [7, 8].

Piplartine (1) (Figure 1), also known as piperlongumine, is an alkamide found in large quantities in long pepper (Piper longum L.). In addition to antileishmanial and trypanocidal activities, this alkamide has been also reported as having other pharmacological activities, including antitumor, cytotoxic, antinociceptive, antiplatelet aggregation, and antimetastatic activities [10]. Thus, the goals of the present study were to synthesize a series of 32 piplartine analogues and evaluate the structure-activity relationship among these derivatives against Leishmania amazonensis promastigotes.

2. Results and Discussion

2.1. Chemistry and Antileishmanial Activity of Compounds 5–36

For this study, a series of 32 analogues of 1 (Scheme 1) were synthesized, preserving the (E)-3-(3,4,5-trimethoxyphenyl)-acryloyl moiety on cinnamic esters and amides and changing this moiety on benzoic esters by removing the ethylene group between carbons 2 and 3. Side-chain modifications were also evaluated changing the radical R to methyl (5), ethyl (6), propyl (7), iPr (8), butyl (9), pentyl (10), decyl (11), 2-methoxyethyl (12), 4-methoxybenzyl (13), phenylethyl (14), 4-methylphenylethyl (15), carvacryl (16), CHPh₂ (17), furfuryl (18), eugenyl (19), (−)-bornyl (20) and piperonyl (21) on cinnamic esters, and butyl (22), N,N-diethyl (23), octyl (24), cyclohexyl (25) benzyl (26), pyrrolidyl (27), 4-methylbenzyl (28), 4-methoxybenzyl (29), 4-fluorobenzyl (30), 4-chlorobenzyl (31), 4-bromobenzyl (32) 2,4-dimethoxybenzyl (33), and 3,4-dimethoxybenzyl (34) on amides. The benzoic esters synthetized were 2-methoxyethyl-3,4,5-trimethoxybenzoate (35) and benzyl 3,4,5-trimethoxybenzoate (36) [8–12].

To synthesize the analogues, the following starting materials were used: 3,4,5-trimethoxycinnamic acid (2); 3,4,5-trimethoxybenzoic acid (3); 3′,4′,5′-trimethoxyacetophenone (4); and ROH to esters and primary or secondary amines to amides. Most products were obtained in high yields, and the reactions (a, b, c, d, e, and f) were performed in one step (Scheme 1).

The antileishmanial potential of all piplartine analogues (5–36) against the strains IFLA/BR/1967/PH8 L. amazonensis was evaluated using the MTT method. The experiments were performed against promastigote forms, that is the proliferative form found in the invertebrate host, and the tests compounds were assayed at following concentrations: 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 µg/ml. The antileishmanial activity was assessed as IC₅₀ and expressed in µM. IC₅₀ values of the piplartine analogues are summarized in Table 1.

Most compounds tested against the promastigote forms of L. amazonensis were bioactive (6–13, 15, 17, 18, 20, 21, 23, 25, 27, 28, and 36). Among the 32 assayed derivatives, compound 20 (IC₅₀ = 0.007 ± 0.008 μM) exhibited the best antileishmanial activity, followed by 36 (IC₅₀ = 0.025 ± 0.009 μM), 18 (IC₅₀ = 0.029 ± 0.007 μM), and 21 (IC₅₀ = 0.042 ± 0.011 μM). For compounds 31, 33, and 34, their IC₅₀ cannot be determined. Amphotericin B, standard drug, exhibited an IC₅₀ value of 0.0015 μM.

The statistical parameters of the obtained 3D-QSAR model as well as superposition of the 3D structures from which it was derived are summarized in Figure 2. In this figure are also presented the general scaffold of the compounds under study, the plot of the predicted vs. observed pIC₅₀ values for the training set, and the results of LOO cross-validation procedures. The obtained statistical parameters show that the developed model is accurate and robust. This is supported by the high values of q² obtained during the LOO and LMO cross-validation experiments. Regarding the influence of the steric and electrostatic factors on bioactivity, it was found that both contribute almost equally to explain the observed bioactivity.

In general, compounds containing O in the X position (see Figure 2) were more active than those containing NH at the same position. This effect can be clearly observed for compounds 17 and 23 whose only difference is the presence of NH (17) vs. O (23) at position X, compound 23 being 6.5-fold more active than 17. This can be due to electrostatic fields surrounding this position, as shown in Figure 3(a). These fields indicate that negatively charged substituents are favored at position X, which is the case of the compounds with O at this position. This observation can be generalized to all the compounds in the dataset. A special case is compound 29 that lacks the double bond spacer. In terms of electrostatic potential, the O atom at the X position remains close to the electronegative field while the lack of the spacer places the trimethoxyphenyl group in a region where an electronegative MIF is present. Thus, compounds without the spacer will have higher bioactivity compared to those containing the double bond spacer. Finally, the electropositive potential observed around position R correlates with the favorable influence of aromatic substitutions at this position.

(a)

(b)

(c)

We also analyzed the influence of the steric molecular interaction fields. These fields are summarized in Figure 3(b), and it can be seen that the substituents attached to the R position are surrounded by a steric favorable region beyond which a steric unfavorable potential is located. In consequence, bulky substituents at R are favorable for bioactivity. Para-substituted phenyl rings are tolerated, whereas meta- and ortho-substituted rings decrease bioactivity. The steric effect for different groups at R is depicted in Figure 3(c) for compounds 27, 10, and 21.

From Figure 3, it can be seen that compound 27 lies entirely within the steric favorable region, while compounds 10 and 21 bear substituents which protrude into the unfavorable steric region. In addition to 27, the other two most active compounds (29 and 25) are also substituted at R with groups completely falling within the steric favorable region. On the other hand, large substituents such as those present in compounds 9 and 26 reach the steric unfavorable region, which reduce their bioactivity. To avoid falling within the steric unfavorable region, if rings are used at R, no more than one methyl group should link them to X. Furthermore, nonbulky groups at R such as aliphatic substitutions lead to nonoptimal bioactivity because of their inability to completely occupy the steric favored region. The structure-activity relationship (SAR) derived from these analyses is summarized in Figure 3(c).

2.2. Anti-Leishmania Activity of Compounds 5–36

Pentavalent antimonials, in use for more than six decades, are still the first-line of treatment for visceral leishmaniasis, also known as kala-azar black fever. However, this therapeutic option presents several limitations, including side effects, the need for daily injections, and drug resistance, and requires a long treatment regimen. Thus, developing and testing of new compounds with leishmanicidal activity is of paramount importance to improve disease treatment and control. Several medicinal plant secondary metabolites have shown interesting antileishmanial activities, including alkaloids, steroids, terpenoids, phenolic acids, and phenylpropanoids [13–15].

The chloroform extract of Valeriana wallichii rhizomes was investigated to identify the structures responsible for its antileishmanial activity. A bioassay-guided fractionation was undertaken and resulted in (−)-bornyl caffeate, α-kessyl alcohol, two cinnamic acid derivatives, and four valtrates. All these compounds exhibited antileishmanial activity against L. major promastigotes, with IC₅₀ values varying between 0.8 and 48.8 µM. Furthermore, some compounds were more potent than the positive control miltefosine (IC₅₀ = 36.2 µM) [16].

A phytochemical study guided by the antileishmanial activity of the crude extract from the stem bark of Tecoma mollis yielded seven phenylpropanoid glycosides. These secondary metabolites were tested against the promastigote forms of L. donovani, and pentamidine and amphotericin B were used as positive controls. The compounds acteoside, luteoside A, and luteoside B shared the highest IC₅₀ values: 30.08, 15.07, and 6.71 µg/ml, respectively. The IC₅₀ values of the positive controls were 0.34 µg/ml to amphotericin B and 1.31 µg/ml to pentamidine. The obtained results also revealed that ortho-dihydroxy phenyl group (catechol moiety) was important to the antileishmanial activity [17].

Piplartine (1), a plant metabolite, is classified as an alkamide and contains in its structure a 3,4,5-trimethoxycinnamic acid (2) moiety and a δ-lactam ring. Both 1 and 2, used as starting materials to synthesize some piplartine analogues, were tested against IFLA/BR/1967/PH8 L. amazonensis. The IC₅₀ values found were 179 ± 0.05 μg/ml (0.564 μM) for 1 and 145 μg/ml (0.608 μM) for 2 [18, 19]. In addition, piplartine (1) was tested against promastigote forms of L. infantum and L. amazonensis and reached IC₅₀ values of 7.9 and 3.3 μM, respectively (positive control: amphotericin B, IC₅₀ = 0.2 μM). These results demonstrate the potential antileishmanial activity of piplartine and 3,4,5-trimethoxycinnamic acid, justifying the choice of these compounds as starting materials for the planning of new derivatives with higher antileishmanial activities [20].

2.3. Structure-Activity Relationship (SAR)

The assayed compounds in this study were divided into three groups: analogues 5–21 (cinnamic esters), analogues 22–34 (amides), and analogues 35–36 (benzoic esters). Previous studies have investigated different compounds containing the 3,4,5-trimethoxyphenyl ring in order to understand its contribution to the biological activity [21]. For example, in a study performed by Dong-Jun et al., the authors showed that 3,4,5-trimethoxyphenyl moiety at the N-1 position of the β-lactam-azide derivatives was crucial for the antiproliferative activity against MGC-803, MCF-7, and A549 cancer cell lines [22]. Furthermore, anticancer compounds such as colchicine, steganacin, phenstatin, podophyllotoxin, combretastatin, and other synthetic analogues of these compounds share a 3,4,5-trimethoxyphenyl moiety as a common structural feature [23]. Thus, herein we preserved the (E)-3-(3,4,5-trimethoxyphenyl)-acryloyl moiety on cinnamic esters and amides and changed this moiety on benzoic esters (without the spacer group in the chemical structure).

Analysis on the structure-activity relationship revealed that, in general, an acrylate moiety on cinnamic esters appears be more important for improving antileishmanial activity when compared to the acrylamide moiety on amides. This effect can be clearly observed comparing compounds 13 (IC₅₀ = 0.101 ± 0.031 μM) and 29 (IC₅₀ = 0.662 ± 0.188 μM), whose only difference is the replacement of the OH group 13 by NH 29 at this position, being compound 13 6.6-fold more active than 29.

The chemical structure of ester 36 (IC₅₀ = 0.025 ± 0.009 µM) differs from other compounds by a molecular simplification of the (E)-3-(3,4,5-trimethoxyphenyl)-acryloyl moiety with removal of the ethylene group (carbons 2 and 3) and another simplification by changing from phenylethyl radical to benzyl. This structural modification contributed to increase the antileishmanial activity, since compound 36 (IC₅₀ = 0.02 ± 0.01 µM) was more active than the ester 14 (IC₅₀ = 0.975 ± 0.241). In addition, the comparison can also be made with amide 26 (IC₅₀ = 0.734 ± 0.252 µM), a structural analogue containing the same benzyl radical attached to the (E)-3-(3,4,5-trimethoxyphenyl)-acrylamide moiety [12, 19, 24, 25]. Similarly, ester 36 showed more potent antileishmanial activity than amide 26.

The ester 20 (IC₅₀ = 0.007 ± 0.008 µM) containing the monoterpenic substructure bornyl attached on the (E)-3-(3,4,5-trimethoxyphenyl)-acryloyl moiety yielded the best IC₅₀ value, near the positive control amphotericin B (IC₅₀ = 0.0015 µM). This result demonstrates the importance of this radical on antileishmanial activity and can be a prototype for the planning of new derivatives with higher antileishmanial activities. In an earlier study, a series of compounds based on caffeic acid bornyl ester was synthesized and tested in vitro against L. major, L. donovani promastigotes, and L. major amastigotes to investigate the SAR. The results showed that the bornyl moiety was important for the antileishmanial activity. The most active compounds (IC₅₀ values varying between 15.6 and 50.2 μM) were all esters of borneol, namely caffeic acid (−)-bornyl ester, caffeic acid isobornyl ester, cinnamic acid (−)-bornyl ester, and phenyl propanoic acid (−)-bornyl ester [1].

Esters 14 (IC₅₀ = 0.975 ± 0.241 μM), 16 (IC₅₀ = 0.513 ± 0.065 μM), and 19 (IC₅₀ > 1.041 μM), containing aromatic groups, decreased the antileishmanial activity when compared with some amides (28, IC₅₀ = 0.109 ± 0.010 µM). However, the esters 17 (IC₅₀ = 0.083 ± 0.016 µM) and 20 (IC₅₀ = 0.007 ± 0.008 µM) gave betters results, demonstrating that the biological effect of these changes is not easily predictable [9, 12]. The introduction of methyl groups contributes to increase lipophilicity. It was observed that the methyl group of the ester 15 (IC₅₀ = 0.125 ± 0.035 μM) in the para-position on the phenylethyl moiety was the determinant change to improve the antileishmanial activity when compared with ester 14 (IC₅₀ = 0.975 ± 0.24 μM) lacking this group [9, 12].

The introduction of methylene groups in general increases the lipophilicity, providing lower IC₅₀ values. Table 1 shows that the antileishmanial activity of esters 5 (IC₅₀ = 0.641 ± 0.052 µM), 6 (IC₅₀ = 0.257 ± 0.041 µM), and 7 (IC₅₀ = 0.09 ± 0.032 µM) was inversely proportional to the length of the carbon chain attached to (E)-3-(3,4,5-trimethoxyphenyl)-acrylate. For esters 9 (IC₅₀ = 0.149 ± 0.025 µM) and 10 (IC₅₀ = 0.133 ± 0.014 µM), the IC₅₀ values have increased compared to 7, demonstrating that from four carbons the antileishmanial activity begins to decrease, with exception of ester 11 (IC₅₀ = 0.076 ± 0.009 µM) that even with decyl radical provided IC₅₀ near the ester 7 (IC₅₀ = 0.09 ± 0.032 µM) [12, 26].

Branches on side-chains R of esters 8 (IC₅₀ = 0.194 ± 0.04 µM) and 17 (IC₅₀ = 0.083 ± 0.016 µM) did not provide better results compared to esters of saturated chains. However, the aromatic rings of ester 17 were determinant to improve the antileishmanial activity when compared to ester 8. The exchange of side-chain R, from butyl in ester 9 (IC₅₀ = 0.149 ± 0.025 µM) to methoxyethyl in ester 12 (IC₅₀ = 0.076 ± 0.022 µM), reduced the IC₅₀ value. On ester 18 (IC₅₀ = 0.029 ± 0.007 µM), the furfuryl radical reduced the molecular flexibility and, compared to ester 12, improved the antileishmanial activity. A molecular simplification approach was tried on ester 35 (IC₅₀ = 0.656 ± 0.049 µM) compared to ester 12, but did not provide better results. However, the presence of oxygen on the methoxyethyl group on side-chain R of ester 36 (IC₅₀ = 0.025 ± 0.009 µM) contributed to increase the antileishmanial activity [12, 24, 25].

The increase of stiffness on side-chain R of ester 21 (IC₅₀ = 0.042 ± 0.011 µM), containing two atoms of oxygen of the dioxolane group fused on benzyl, increased the antileishmanial activity when compared to ester 13 (IC₅₀ = 0.101 ± 0.031 µM) with 4-methoxylbenzyl radical. Comparing the saturated side-chain R on amides 22 (IC₅₀ = 0.577 ± 0.080 µM) and 24 (IC₅₀ = 0.564 ± 0.061 µM) with the six-membered ring on amide 25 (IC₅₀ = 0.117 ± 0.027 µM), it was observed that the replacement of saturated side-chains R by rigid groups increased the antileishmanial activity also on amides [12, 24, 25]. Compound 23 (IC₅₀ = 0.091 ± 0.015 µg/ml) with a tertiary amide provides better result compared to 27 (IC₅₀ = 0.353 ± 0.080 µM).

Replacing the methyl groups attached to the aromatic ring with the methoxy group dramatically decreased the antileishmanial activity in series of amide analogues, and this reduction was dependent on the number of substituted groups. For example, compound 28 (R₃ = CH₃) exhibited an IC₅₀ value of 0.109 ± 0.010 μM, whereas compounds 29 (R₃ = OCH₃), 33 (R₁ and R₃ = OCH₃), and 34 (R₁ and R₃ = OCH₃) revealed IC₅₀ values of 0.662 ± 0.188, >1.033 and > 1.033, respectively. Similarly, the addition of halogens atoms (Br, F or Cl) to the aromatic ring of amide series also reduced the antileishmanial activity, since compounds 30 (R₃ = Br, IC₅₀ = 1.137 ± 0.252 μM), 31 (R₃ = Br, IC₅₀ > 1.108 μM), and 32 (R₃ = Br, IC₅₀ = 0.486 ± 0.043 μM) showed weak or no activity.

3. Experimental

3.1. Chemical Characterization and Reagents

The ¹H and ¹³C NMR and IR spectral data were recorded, and their signals were compared with published data. For compounds 18, 19, 21, 32, 33, 34, and 35, the spectrometric methods LS-MALDI TOF/TOF or ESI-TOF were used to confirm the chemical structures. The preparation and structural characterization data of compounds 5–9, 10–12, 14–17, 22, 23, 26, 28, 29, and 31 were published in a previous article [21].

All used reagents were of reagent grade and purchased from Sigma Aldrich. The following equipments were used: Irprestige-21 Shimadzu Fourier Transform spectrophotometer (Shimadzu, Kyoto, Japan) for IR, and Varian Mercury spectrometer (Palo Alto, CA, USA) 200 MHz for ¹H NMR and 50 MHz for ¹³C NMR. The standard for chemical shifts was the CDCl₃ or TMS solvent peak. For HR-MS, the LS-MALDI TOF/TOF apparatus was used equipped with a high-performance laser (λ = 355 nm) and a reflector operated by the software FlexControl 2.4 (Bruker Daltonics G, bsH, Bremen, Germany), and microOTOF operated by software Bruker Compass DataAnalysis 4.0 (Bruker Daltonics G, bsH, Bremen, Germany). Melting points were measured on an equipment Tecnal PFM-II, 220 V. To measure the optical activity of 20 and of starting material (−)-borneol a digital polarimeter P-2000 (Jasco) was used after standardization with 98% anhydrous methanol.

3.2. General Procedure for Preparation of Compound 35

To a solution of 0.25 g of carboxylic acid in 250 ml of ROH under stirring, 0.5 ml of 96% (v/v) H₂SO₄ were added and solution was refluxed for 3 h. After removing of half ROH under reduced pressure, the solution was diluted in 10 ml of water, and product was extracted with ethyl acetate. Then, the product was successively washed with 5% (w/v) NaHCO₃ and water and dried with anhydrous Na₂SO₄. Finally, the solvent was evaporated to yield the pure compound [27–29].

3.3. 2-Methoxyethyl 3,4,5-trimethoxybenzoate (35)

Yield 91%; brown solid; MM: 270.11 g/mol; m.p.: 32–35°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.31 (s, 2H), 4.51–4.40 (m, 2H), 3.89 (s, 9H), 3.77–3.66 (m, 2H), 3.40 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.0, 152.7, 142.1, 124.9, 106.8, 70.4, 64.0, 60.7, 58.8, 56.0; IR νmax (KBr, cm⁻¹): 2941, 2839, 1715, 1589, 1504, 864; microOTOF: m/z [M + Na]⁺ 293.3107 (calcd. for C₁₃H₁₈O₆: 293.1103).

3.4. General Procedure for Preparation of Compound 13

In a 50 ml round-bottom flask, 0.42 mmol (0.1 g) of 3,4,5-trimethoxycinnamic acid were dissolved in 5.0 ml of acetone. Then, 1.68 mmol (0.22 ml) of Et₃N and 0.43 mmol of alkyl halide were added in the same solution, and the reaction mixture was refluxed for 48 h. The acetone was removed under reduced pressure, and the content of the reactions was diluted in 10 ml of ethyl acetate and 10 ml of water. The products were extracted thrice with 10 ml of ethyl acetate. Afterwards, organic phases were successively washed with 5% (w/v) NaHCO₃ and water, dried over anhydrous Na₂SO₄, and filtered. The product was purified via a silica gel 60 column chromatography (mobile phase: hexane-ethyl acetate (1 : 1)) [30].

3.4.1. (E)-4-Methoxybenzyl 3-(3,4,5-trimethoxyphenyl)-acrylate (13)

Yield 77.4%; white solid; MM: 358.14 g/mol; m.p.: 110–111°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.61 (d, J = 16 Hz, 1H), 7.34 (d, J = 2.0 Hz, 2H), 6.90 (d, J = 2.0 Hz, 2H), 6.73 (s, 2H), 6.37 (d, J = 16 Hz, 1H), 5.17 (s, 2H), 3.85 (s, 9H), 3.80 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.8; 159.7; 153.5; 145.0; 140.2; 130.2; 129.9; 128.2; 117.3; 114.0; 105.3; 66.3; 61.0; 56.2; 55.3; IR νmax (KBr, cm⁻¹): 2999, 2939, 2837, 1709, 1636, 1034, 1005, 1584, 1506, 825, 609; LS-MALDI TOF/TOF m/z [M]⁺ 358.1353 (calcd. for C₂₀H₂₂O₆: 358.1416).

3.5. General Procedure for Preparation of Compounds 18–21

To a solution of 0.1 g (0.42 mmol) of 3,4,5-trimethoxycinnamic acid dissolved in 6.3 ml of CH₂Cl₂, 0.54 mmol of ROH and 0.015 g of DMAP were added. After 5 min of stirring, 0.1 g (0.54 mmol) of DCC was also added. Stirring was continued overnight at room temperature. The solvent was removed under vacuum, and the remaining content was diluted in 10 ml of ethyl acetate and 10 ml of water. The product was extracted with 10 ml ethyl acetate (three times), and the organic phases were successively washed with 5% (w/v) NaHCO₃ and water, dried over anhydrous Na₂SO₄, and filtered. After removing ethyl acetate under vacuum, the products were purified on a silica gel 60 column chromatography (mobile phase: hexane-ethyl acetate).

3.5.1. (E)-Furfuryl 3-(3,4,5-trimethoxyphenyl)-acrylate (18)

Yield 79%; brown oil; MM: 318.11 g/mol; ¹H NMR (200 MHz, CDCl₃): δ_H 7.58 (d, J = 16.0 Hz, 1H), 7.43–7.37 (m, 1H), 6.69 (s, 2H), 6.45–6.16 (m, 3H), 5.15 (s, 2H), 3.81 (s, 6H), 3.83 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.4, 153.3, 149.5, 145.3, 143.3, 140.0, 129.7, 116.7, 110.7, 110.6, 105.5, 60.8, 58.0, 56.0; IR νmax (KBr, cm⁻¹): 2997, 2939, 2839, 1713, 1634, 1003, 1582, 1504, 827; LS-MALDI TOF/TOF m/z [M]⁺ 318.1103 (calcd. for C₁₇H₁₈O_6: 318.1103).

3.5.2. (E)-Eugenyl 3-(3,4,5-trimethoxyphenyl)-acrylate (19)

Yield 86%; white solid; MM: 384.16 g/mol; m.p.: 170–172°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.77 (d, J = 16.0 Hz, 1H), 7.00 (d, 1H, J = 6.0 Hz), 6.83–6.73 (m, 4H), 6.58 (d, J = 16.0 Hz, 1H), 6.08–5.83 (m, 1H), 5.18–5.02 (m, 2H), 3.88 (s, 3H), 3.86 (s, 6H), 3.80 (s, 3H), 3.37 (d, J = 6.0 Hz, 2H); ¹³C NMR (50 MHz, CDCl₃): δ_C 165.0, 153.4, 151.0, 146.4, 140.2, 139.0, 137.9, 137.0, 129.7, 122.6, 120.6, 116.3, 116.1, 112.7, 105.3, 60.9, 56.0, 55.8, 40.1; IR νmax (KBr, cm⁻¹): 3067, 2968, 2939, 2839, 1722, 1630, 1034, 1009, 1582, 1508, 824; LS-MALDI TOF/TOF m/z [M]⁺ 384.1575 (calcd. for C₂₂H₂₄O₆: 384.1573).

3.5.3. (E)-(−)-Bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate (20)

Yield 87%; white solid; MM: 374.21 g/mol; m.p.: 100–102°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.54 (d, J = 16.0 Hz, 1H), 6.73 (s, 2H), 6.34 (d, J = 16.0 Hz, 1H), 4.98 (d, J = 8.0 Hz, 1H), 3.85 (s, 9H), 2.47–2.28 (m, 1H), 2.05–1.96 (m, 1H), 1.67 (d, J = 4.0 Hz, 1H), 1.37–1.15 (m, 3H), 1.07–0.95 (m, 1H), 0.90 (s, 3H), 0.84 (s, 6H); ¹³C NMR (50 MHz, CDCl₃): δ_C 167.2, 153.4, 144.2, 139.9, 130.0, 118.0, 105.1, 79.9, 60.9, 56.1, 48.9, 47.9, 44.9, 36.9, 28.1, 27.3, 19.7, 18.9, 13.6; IR νmax (KBr, cm⁻¹): 2953, 2839, 1697, 1634, 1026, 1007, 1582, 1504, 831; = −5.5° (c 0.001, MeOH), RSD: 4.3% [31].

3.5.4. (E)-Piperonyl-3-(3,4,5-trimethoxyphenyl)-acrylate (21)

Yield 30%; white solid; MM: 372.12 g/mol; m.p.: 123–126°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.61 (d, J = 16.0 Hz, 1H), 6.93–6.76 (m, 3H), 6.74 (s, 2H), 6.36 (d, J = 16.0 Hz, 1H), 5,95 (s, 2H), 5,12 (s, 2H), 3,86 (s, 9H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.8, 153.4, 147.9, 147.7, 145.1, 140.1, 129.9, 129.8, 122.4, 117.2, 109.1, 108.3, 105.2, 101.2, 66.4, 61.0, 56,2; IR νmax (KBr, cm⁻¹): 3067, 2926, 2849, 1703, 1630, 1038, 1005, 1582, 1502, 854; LS-MALDI TOF/TOF m/z [M + H]⁺ 373.1288 (calcd. for C₂₀H₂₀O₇ + H: 373.1268).

3.6. General Procedure for Preparation of Amides

To a solution of 0.1 g of 3,4,5-trimethoxycinnamic in 0.84 ml of DMF in a round-bottom flask, 0.06 ml (0.42 mmol) of trimethylamine was added. The solution was cooled in an ice water bath and 0.42 mmol of amine were added, followed by a solution of 0.42 mmol of BOP (0.84 ml) in CH₂Cl₂. After stirring for 30 min, the reaction continued at room temperature for 3 h. CH₂Cl₂ was removed under vacuum, and the remaining solution was diluted in 10 ml of ethyl acetate and 10 ml of water. The product was extracted with 10 ml of ethyl acetate (three times), and the organic phases were washed successively with 1 N HCl, water, 1 M NaHCO₃, and water, dried over anhydrous Na₂SO₄, filtered, and evaporated. The product was purified on a silica gel 60 column chromatography (mobile phase: hexane-ethyl acetate (1 : 1)).

3.6.1. (E)-N-Octyl-3-(3,4,5-trimethoxyphenyl)-acrylamide (24)

Yield 62.4%; white solid; MM: 349.23 g/mol; m.p.: 133–134°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.50 (d, J = 16.0 Hz, 1H), 6.69 (s, 2H), 6.33 (d, J = 16.0 Hz, 1H), 6.00 (s, 1H), 3.83 (s, 9H), 3.34 (q, J = 6.0 Hz, 2H), 1.62–1.44 (m, 2H), 1.31–1.22 (m, 10H), 0.84 (t, J = 6.0 Hz, 3H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.0, 153.4, 147.6, 140.7, 139.5, 130.6, 120.3, 104.7, 61.0, 56.1, 39.9, 31.9, 29.7, 29.4, 27.1, 22.7, 14.2; IR νmax (KBr, cm⁻¹): 3290, 3003, 2955, 2924, 2853, 1655, 1622, 1580, 1504, 827 [32].

3.6.2. (E)-N-Cyclohexyl-3-(3,4,5-trimethoxyphenyl)-acrylamide (25)

Yield 59%; white solid; MM: 319.18 g/mol; m.p.: 320–321°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.49 (d, J = 16.0 Hz, 1H), 6.68 (s, 2H), 6.34 (d, J = 16.0 Hz, 1H), 5.90 (s, 1H), 3.83 (s, 3H), 3.81 (s, 6H), 2.01–1.05 (m, 11H); ¹³C NMR (50 MHz, CDCl₃): δ_C 165.0, 153.4, 140.5, 139.3, 130.6, 120.7, 104.8, 61.0, 56.1, 48.4, 33.3, 25.6, 24.9; IR νmax (KBr, cm⁻¹): 3287, 3003, 2928, 2853, 1663, 1612, 1582, 1508, 822 [33].

3.6.3. (E)-1-(Pyrrolidine-1-yl)-3-(3,4,5-trimethoxyphenyl)-prop-2-en-1-one (27)

Yield 72%; white solid; MM: 291.15 g/mol; m.p.: 233–235°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.59 (d, J = 16.0 Hz, 1H), 6.72 (s, 2H), 6.59 (d, J = 16,0 Hz, 1H), 3.86 (s, 6H), 3.84 (s, 3H), 3.68–3.51 (m, 4H), 2.07–1.79 (m, 4H), ¹³C NMR (50 MHz, CDCl₃): δ_C 164.8, 153.4, 142.0, 139.5, 131.0, 118.0, 105.1, 61.0, 56.2, 46.8, 46.2, 26.2, 24.4; IR νmax (KBr, cm⁻¹): 3049, 2963, 2939, 2839, 1645, 1605, 1580, 1504, 820 [29].

3.6.4. (E)-N-(4-Fluorobenzyl)-3-(3,4,5-trimethoxyphenyl)-acrylamide (30)

Yield 87%; white solid; MM: 345.14 g/mol; m.p.: 195–196°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.57 (d, J = 16.0 Hz, 1H), 7.30–7.19 (m, 2H), 6.99 (t, J = 8.0 Hz, 2H), 6.69 (s, 2H), 6.39 (d, J = 14.0 Hz, 1H), 4.50 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 6H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.0, 164.7, 159.8, 153.4, 141.5, 139.6, 134.1, 130.4, 129.6, 129.5, 119.8, 115.8, 115.4, 105.0, 61.0, 56.1, 43.2; IR νmax (KBr, cm⁻¹): 3291, 3012, 2959, 2932, 2839, 1651, 1616, 1580, 1508, 829 [34].

3.6.5. (E)-N-(4-Bromobenzyl)-3-(3,4,5-trimethoxyphenyl)-acrylamide (32)

Yield 99.8%; white solid; MM: 405.6 g/mol; m.p.: 360–363°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.57 (d, J = 16.0 Hz, 1H), 7.43 (d, J = 8.00 Hz, 2H), 7.17 (d, J = 8.00 Hz, 2H), 6.70 (s, 2H), 6.45–6.15 (m, 2H), 4.49 (d, J = 6.0 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 6H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.0, 153.4, 141.6, 139.7, 137.4, 131.8, 13.,4, 129.5, 121.4, 119.7, 105.0, 61.0, 56.2, 43.2; IR νmax (KBr, cm⁻¹): 3292, 3044, 2957, 2932, 2835, 1651, 1614, 1582, 1506, 824; LS-MALDI TOF/TOF m/z [M]⁺ 405.0558 (calcd. for C₁₉H₂₀BrNO₄: 405.0576).

3.6.6. (E)-N-(2,4-Dimethoxybenzyl)-3-(3,4,5-trimethoxyphenyl)-acrylamide (33)

Yield 23%; white solid; MM: 387.17 g/mol; m.p.: 235–238°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.50 (d, J = 16,0 Hz, 1H), 7.20 (d, J = 8,0 Hz, 1H), 6.67 (s, 2H), 6.42 (s, 1H), 6.41–6.23 (m, 3H), 4.46 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H), 3.82 (s, 6H), 3.80 (s, 3H), 3,76 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): δ_C 165.6, 160.6, 158.6, 153.4, 140.8, 139.4, 130.6, 130.6, 120.4, 118.8, 104.9, 104.0, 98.6, 61.0, 56.1, 55.4, 39.1; IR νmax (KBr, cm⁻¹): 3283, 3040, 3001, 2932, 2837, 1651, 1605, 1585, 1506, 820; LS-MALDI TOF/TOF m/z [M]⁺ 387.1674 (calcd. for C₂₁H₂₅NO₆: 387.1682).

3.6.7. (E)-N-(3,4-Dimethoxybenzyl)-3-(3,4,5-trimethoxyphenyl)-acrylamide (34)

Yield 75%; white solid; MM: 387.17 g/mol; m.p.: 170–173°C; ¹H NMR (200 MHz, CDCl₃): δ_H 7.48 (d, J = 16.0 Hz, 1H); 6.80–6.64 (m, 3H); 6.61 (s, 2H), 6.41 (d, J = 16.0 Hz, 1H), 4.38 (d, J = 6.0 Hz, 2H), 3.76 (s, 3H), 3.73 (s, 3H), 3.71 (s, 9H); ¹³C NMR (50 MHz, CDCl₃): δ_C 165.9, 153.2, 148.9, 148.2, 140.8, 139.3, 130.7, 130.3, 120.1, 120.0, 111.0, 104.8, 60.8, 55.9, 43.5; IR νmax (KBr, cm⁻¹): 3370, 3061, 2961, 2926, 2853, 1670, 1585, 1502, 827; LS-MALDI TOF/TOF m/z [M + Na]⁺ 410.1579 (calcd. for C₂₁H₂₅NO₆ + Na: 410.1580).

3.7. General Procedure for Preparation of Compound 36

To a 5 ml round-bottom flask with 0.1 g (0.47 mmol) of 3′,4′,5′-trimethoxyacetophenone in 0.1 ml (0.94 mmol) of benzylic alcohol, the solution of 0.007 g of CuBr (0.047 mmol) in 0.07 ml (0.94 mmol) of pyridine was added, and then the solution of 0.025 mmol BF₃·Et₂O in 0.5 ml of chlorobenzene was added dropwise. The mixture was then stirred at 130°C open air for 10 h. After cooling at room temperature, chlorobenzene was removed under reduced pressure. The remaining content was diluted in 10 ml of ethyl acetate and 10 ml of water. The product was extracted with 10 ml of ethyl acetate (three times), and the organic phase was successively washed with 5% (w/v) NaHCO₃ and water, dried over anhydrous Na₂SO₄, and filtered. After the removal of ethyl acetate under vacuum, the product was purified on a silica gel 60-column chromatography (mobile phase: hexane-ethyl acetate) [35].

3.7.1. Benzyl 3,4,5-trimethoxybenzoate (36)

Yield 56%; yellow oil; MM: 307.12 g/mol; ¹H NMR (200 MHz, CDCl₃): δ_H 7.37-7.27 (m, 5H), 7.25 (s, 2H), 5.28 (s, 2H), 3.81 (m, 6H), 3.80 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): δ_C 166.1, 153.0, 142.3, 136.1, 128.7, 128.3, 128.2, 125.2, 107.0, 66.9, 61.0, 56.3; IR νmax (KBr, cm⁻¹): 2999, 2939, 2837, 1713, 1589, 1502, 864 [36].

3.8. Leishmania Culture Conditions

Leishmania amazonensis promastigote forms (IFLA/BR/67/PH8) were cultivated at 26°C in Schneider’s medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Gaithersburg, MD, USA) and the antibiotic gentamicin (40 µg/ml) (Schering–Plough, Rio de Janeiro, Brazil).

3.9. MTT Assay

The antileishmanial activity on promastigote forms of L. amazonensis was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method (Amaresco, Ohio, USA). The promastigotes of L. amazonensis were distributed in a 96-well plate (1 × 10⁶ cells) and treated with different concentrations of piplartine derivatives (5–36) (400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 μg/ml) diluted in Schneider’s medium (Sigma) supplemented with 10% FBS. The plates were incubated for 72 hours at 34°C. After the incubation period, 10 μl of a 5 mg/ml MTT solution in PBS was added to each well. After treatment, the plate was incubated for additional 4 hours, and at the end 50 μl of a solution of 10% sodium dodecyl sulphate (Sigma Chemical-st. Louis, MO, USA) was added and left overnight to allow dissolution of the crystals of formazan [37]. Finally, the absorbance reading was performed using a spectrophotometer (Biotek model Elx800) at 540 nm. The Schneider medium was used as negative control, and amphotericin B, a reference drug for treatment of visceral leishmaniasis, was used as positive control [37]. The IC₅₀ values were defined as the concentration of each compound that reduced the absorbance of treated cells by 50% when compared with the cell control. All assays were performed in triplicate.

3.10. Alamar Blue Assay

To assess the cytotoxicity against normal cells, the cell line MRC-5 (human lung fibroblast) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured as recommended by the ATCC. The cell culture was tested for mycoplasma using Mycoplasma Stain Kit (Sigma-Aldrich) to validate the use of cells free from contamination. The cell viability was measured colorimetrically using the alamarBlue assay [38]. Shortly, compounds 18, 20, and 36 (dissolved in DMSO and diluted in the RPMI-1640 medium at a range of eight different concentrations from 0.19 to 25 μg/ml) were added to each well and incubated for 72 h. Doxorubicin (Sigma-Aldrich) was used as the positive control. Four hours before the end of incubation, 20 μl of a stock solution (0.312 mg/ml) of resazurin (Sigma-Aldrich) was added to each well. Absorbance was measured at 570 nm and 600 nm using the SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The half maximal inhibitory concentration (IC₅₀) values with 95% confidence intervals were obtained by nonlinear regression using GraphPad Prism (Intuitive Software for Science; San Diego, CA, USA).

4. Methods

The first step for modeling was obtaining the initial 3D structure of each compound using the OpenEye’s Omega software [39]. Fragments for 3D structures generation were obtained with MAKEFRAGLIB, which is a part of the OMEGA suite. The Merck Molecular Force Field (MMFF94s) excluding the Coulomb interactions (mmff94s_NoEstat) was employed for fragment generation. Fragments with 4.0 kcal above the global minimum as well as those with a RMS distance lower than 0.1 from any conformer in the database were discarded. Default parameters were used for generating one 3D conformation per compound with OMEGA. Conformational exploration and alignment of the compounds were performed with the Open3DALIGN software [40]. A conformational library of each compound was obtained through quenched molecular dynamics (QMD) conformational searches that employed TINKER with an implicit solvent model [41]. Two hundred molecular dynamic simulations of length 100 ps were performed for each compound. The remaining parameters for the QMD in Open3DALIGN were kept to their default values. Structural alignment took place was performed using a method that combines atom- and pharmacophore-based alignments as implemented in Open3DALIGN.

3D-QSAR models were developed with the Open3DQSAR suite [42], which performs partial least squares (PLS) regression models from molecular interaction fields (MIF). Unless otherwise noted, default parameters were employed for Open3DQSAR. The input to Open3DQSAR is a set of aligned conformers of the dataset with associated bioactivities. The best alignment produced by Open3DALIGN in the previous step was used for 3D-QSAR models generation. In this study, compounds 19, 26, 31, and 32 were removed from the modeling process because of their undetermined IC₅₀. The bioactivity of the remaining compounds was transformed to pIC₅₀ values (pIC₅₀ = −log₁₀IC₅₀), provided that IC₅₀ values are expressed in molar units.

A grid was constructed around the aligned molecules in such a way that its box exceeded in 5 Å the largest molecule and grid spacing was set to 0.5 Å. Steric and electrostatic molecular mechanics of MIFs were computed using the Merck force field (MMFF94). For the computation of both MIFs, an alkyl carbon (charge +1) was used as probe. Before model construction, variables were filtered to discard those with van der Waals energies above 10⁴ kcal/mol. Corresponding points on the electrostatic MIF were also removed. For both MIFs, energies greater than 30 kcal/mol or lower than −30 kcal/mol were set to 30 kcal/mol and −30 kcal/mol, respectively. Also values of energy between −0.05 and 0.05 kcal/mol were set to 0 in the two MIFs, and all variables with standard deviation lower than 0.1 were discarded. All variables spanning up to four levels were removed from both fields. As a final filter, variables were scaled employing the AUTO option of Open3DQSAR.

Initial PLS models considered 10 principal components. The optimal number of principal components (PCs) was selected based on the value of the Leave One Out cross-validation R² (q²). Variables were then grouped using the Smart Region definition procedure implemented in Open3DQSAR taking into account the previously identified optimal number of PCs. The grouped variables were subject to a selection procedure according to the Fractional Factorial Design using Leave Many Out (LMO) cross validation with 20 runs and 5 groups. Only selected variables in previous step were kept on the dataset. After these variable selection procedures, the PLS model was recomputed considering the optimal number of PCs and validated using the LOO and LMO strategies.

5. Conclusion

We synthesized a series of 32 piplartine analogues. The preliminary bioassay revealed that the most prominent compounds had rigid rings as substituents. Compound 18 contains a furfuryl portion attached to the (E)-3-(3,4,5-trimethoxyphenyl)-acryloyl moiety. This modification significantly improved the bioactivity. Molecular simplification of 36 in relation to other derivatives formed an analogue with better biological activity against Leishmania. Furthermore, bornyl radical appears to be important for the bioactivity, given that compound 20 revealed the strongest antileishmanial activity. Together, these results show that compounds 18, 20, and 36 are promising lead compounds in the development of new options of pharmacotherapeutic drugs for leishmaniasis.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Copyright © 2019 Flávio R. Nóbrega et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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