Synthetic 2-Nitrocinnamates: Investigation of the Antifungal Action against <i>Candida</i> Species

Gomes do Nascimento, Lázaro; Castro de Morais, Mayara; Klidenberg de Oliveira Júnior, José; de Oliveira Lima, Edeltrudes; de Sousa, Damião Pergentino

doi:https://doi.org/10.1155/2023/8525145

Journal of Chemistry

On this page

Abstract Introduction Results Discussion Materials and Methods Conclusion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 8525145 | https://doi.org/10.1155/2023/8525145

Synthetic 2-Nitrocinnamates: Investigation of the Antifungal Action against Candida Species

Lázaro Gomes do Nascimento,¹Mayara Castro de Morais,¹José Klidenberg de Oliveira Júnior,²Edeltrudes de Oliveira Lima,²and Damião Pergentino de Sousa¹

Academic Editor: Vinod Kumar Tiwari

Received12 Nov 2022

Revised05 Mar 2023

Accepted09 Mar 2023

Published03 Apr 2023

Abstract

Derivatives of cinnamic acid have several pharmacological actions, such as antimicrobial activity. Therefore, in the present study, a series of fourteen alkyl and aryl derivatives from (E)-2-nitrocinnamic acid were obtained using Fischer esterification, nucleophilic substitution with halides, and Mitsunobu reaction. Esters were evaluated for antifungal activity against several strains of Candida spp. using nystatin as a positive control. Among the synthetic derivatives obtained, nine are compounds unprecedented in the literature. The characterization of chemical structures was carried out using the techniques of IR, ¹H-NMR and ¹³C-NMR spectroscopy, and high-resolution mass spectrometry. Isopropyl 2-nitrocinnamate (4) was the compound that showed the best antifungal activity (MIC = 513.52 μM) against all fungal strains, followed by compound perillyl 2-nitrocinnamate (14) with MIC = 390.99–781.98 μM.

1. Introduction

The Candida genus includes more than 200 species of human pathogens. Among the most important are Candida albicans, Candida tropicalis, and Candida krusei. The Candida species are opportunistic pathogens and the leading cause of death from fungal infections in the world, representing a serious threat to public health [1–3]. Changes in the host’s defense mechanisms, invasive medical procedures, and failures in the anatomical barrier, as well as the indiscriminate use of broad-spectrum antimicrobials, are some of the factors that favor infection with these microorganisms [4, 5].

In recent decades, researchers have been giving greater attention to natural compounds derived from plants due to their less potential to cause toxicity in mammals, as well as for their easy occurrence in nature [6]. As an example, there is a wide distribution of esters of cinnamic acid and its derivatives in plants such as cereals, vegetables, oilseeds, fruits, and some drinks such as tea and coffee [7].

According to the literature, it is known that derivatives of cinnamic acid, including natural compounds, have several pharmacological actions, such as antimicrobial [8–11], anti-Mycobacterium tuberculosis [12], anticancer [13], anti-inflammatory [14], and antiparasitic [15, 16], and induce the proliferation of neural progenitor cells [17]. In addition, it was found that cinnamic acids and their ester derivatives also inhibit the growth of some species of fungi, such as Candida albicans and Aspergillus niger [18]. In another study carried out by Narasimhan et al. [19], the derivatives, such as methyl cinnamate, ethyl cinnamate, propyl cinnamate, butyl cinnamate, and isobutyl cinnamate that were tested, showed antifungal properties against Candida albicans and Aspergillus niger, with emphasis on isobutyl cinnamate which showed biological activity equal to the reference compound, salicylic acid.

Compounds containing nitro groups (-NO₂) are known to have activities against microorganisms, including antifungal [20] and antiparasitic [15]. These findings can be corroborated by the work of Lima et al. [21], in which the introduction of nitro groups contributed to the improvement of the antifungal effect when comparing methyl cinnamate with methyl 2-nitrocinnamate. Although cinnamic acid and its cinnamate derivatives have shown inhibitory activity against some human pathogenic fungi [19], few reports have been found in the investigation of the activity of 2-nitrocinnamic esters against these fungi. Thus, the objective of the present work is to evaluate the bioactivity of fourteen esters derived from (E)-2-nitrocinnamic acid against C. albicans ATCC 90028, C. albicans LM 106, C. tropicalis ATCC 13803, C. tropicalis LM 31, C. krusei LM 13, C. krusei LM 08, C. parapsilosis LM 14, and C. parapsilosis LM 02, including investigating the influence of the chemical characteristics of the substituent groups on the biological activity.

2. Results

2.1. Chemistry

Fourteen compounds were prepared by Fischer esterification, nucleophilic substitution with halides, and Mitsunobu reaction. Nine new compounds were obtained. Substrate (2-nitrophenyl) acrylate was maintained for all esters. The biological activities associated with the structural changes in the compounds in their side chain, such as an increase in the alkyl chain, structural ramifications, and addition of aromatic rings were evaluated using R = methyl (1), ethyl (2), propyl (3), isopropyl (4), butyl (5), 2-methoxyethyl (6), pentyl (7), decyl (8), 4-chlorobenzyl (9), benzhydryl (10), 4-hydroxyphenethyl (11), 4-methoxybenzyl (12), naphthalen-2-ylmethyl (13), and perillyl (14) groups (see Scheme 1).

Products were obtained in low to high yields using three synthetic methods. The structural characterization of the compounds was performed by spectroscopic and spectrometric techniques. The IR spectra showed absorption bands between 2850 and 3000 cm⁻¹ referring to the C-H sp³ stretching; signals between 3000 and 3100 cm⁻¹ are from C-H sp² stretch. C=O stretching bands in the range of 1750–1730 cm⁻¹ are the characteristic of ester carbonyls. The bands at 1636 and 1432 cm⁻¹ confirm the presence of aromatic rings.

In the ¹H-NMR spectra, chemical shifts showed that derivatives have six hydrogens in common, four belonging to the aromatic ring and two olefinic hydrogens to the lateral carbon chain. In view of the common signals for all analogs, the most unprotected hydrogen signal in the spectrum was that of olefinic hydrogen, which presented itself in the form of a doublet close to δH 8.08 ppm, coupled to neighboring hydrogen that presents a signal in the form of a doublet around δH 6.33 ppm; the configuration of the double bond is trans since the value of the coupling constant (J) is close to 16 Hz. In addition, there is a signal in the form of a doublet with an integral for hydrogen close to δH 8.01 ppm with J of 9 Hz referring to hydrogen in ortho to the NO₂ group in the aromatic ring and a multiplet with an integral for three hydrogens approximately at δH 7.44 ppm referring to three hydrogens of the aromatic ring. The ¹³C-NMR spectra showed chemical shifts from cinnamic derivatives with nine carbons in common. A signal close to δC 165.0 ppm is attributed to a carbonyl, signals from 130.2 to 133.6 are attributed to aromatic carbons, and a characteristic signal from carbon is linked to nitrogen at 148.2 (C-N). Furthermore, there is a signal around δC 140.0 ppm belonging to the most unprotected olefinic carbon, and a signal close to δC 120.3 ppm is attributed to the most protected olefinic carbon.

2.2. Biological Activity

The antifungal activity of 2-nitrocinnamates (1–14) was evaluated against eight strains of Candida: C. albicans ATCC 90028, C. albicans LM 106, C. tropicalis ATCC 13803, C. tropicalis LM 31, C. krusei LM 13, C. krusei LM 08, C. parapsilosis LM 14, and C. parapsilosis LM 02. The results were defined as the minimum inhibitory concentration (MIC) capable of inhibiting fungal growth in the wells and visually observed in comparison to the control. The values are shown as an arithmetic mean of two repetitions of the MIC values, as determined by the microdilution method. Furthermore, the activities of the compounds were compared to those of the positive control, nystatin (Table 1).

3. Discussion

The results of the antifungal test revealed that the tested compounds exhibited various antifungal potency, which depends on the structural variation of substituents. Among the fourteen derivatives, compounds 2, 4, 6, 13, and 14 showed the best inhibitory activity against the strains of fungi. These findings may be related to the presence of the substituent in the aromatic ring of the cinnamate, nitro group (-NO₂), since it was found that methyl 2-nitrocinnamate was more active than methyl cinnamate with MIC = 128 μg/mL compared to all strains of C. albicans tested (ATCC 76645, LM 106, and LM 23) [21].

Isopropyl 2-nitrocinnamate (4) had MIC = 513.52 μM, and among linear chain compounds, it showed the best antifungal activity against all strains, followed by perillyl 2-nitrocinnamate (14) with MIC = 390.99–781.98 μM. These results suggest that isopropyl or perillyl substituents may contribute to improving interaction with the cell’s active site, promoting greater inhibitory activity. These results are in agreement with those of the study by Khatkar et al. [22], in which butyl p-coumarate was more active than the standard drug (fluconazole) used in the antifungal test against the strain of C. albicans MTCC 227.

When analyzing decyl 2-nitrocinnamate (8) (without activity), it was found that the compound did not inhibit fungal growth. This result shows that the addition of very long carbon chains can result in loss of biological activity due to high lipophilicity. Indeed, variation in the carbon chain length of substituents can result in a decrease or loss of antifungal activity, such as propyl 2-nitrocinnamate (3) (MIC = 1088.25–2176.50 μM) with butyl 2-nitrocinnamate (5) (without activity). There is the absence of growth inhibition by derivative 5. These findings corroborate the work of Narasimhan et al. [19], in which isopropyl cinnamate and isobutyl cinnamate branches reduced activity against Candida albicans when compared with respective linear chain cinnamates, propyl cinnamate, and butyl cinnamate.

To assess whether aromatic substituents with electron donor and withdrawal groups alter the MIC, esters 9, 11, and 12 were compared. 4-chlorobenzyl 2-nitrocinnamate (9) (MIC = 402.87, 805.74, 1611.48, and 3222.96 μM) showed a better spectrum of action, inhibiting all tested strains, while 4-methoxybenzyl 2-nitrocinnamate (12) did not show activity in any of the tested strains. These findings suggest that electron-withdrawing groups appear to improve antifungal activity in aromatic derivatives. In a study carried out by Khatkar et al. [22], it was shown that when the electron-withdrawing group is added to the phenyl substituent of phenyl p-coumarate, its activity tends to improve. In addition, Lima et al. [21] found that aromatic electron-withdrawing substituents (Cl and -NO₂) in cinnamic esters improve the inhibitory activity of compounds against Candida strains. While aromatic methoxyl in the para position of the ring was not important for the bioactivity of compound 12, unlike the hydroxyl substituent, which contributes to 4-hydroxybenzyl 2-nitrocinnamate (11) activity. This fact may be due to the interaction of phenolic hydroxyl with the cells of microorganisms, performing interactions with their membrane components. The nitro substituent in the ortho position may have been relevant for better bioactivity, as well as substituents with an aromatic ring.

When analyzing diphenyl substituents, esters 10 and 13 were compared, noting that benzhydryl 2-nitrocinnamate (10) has a lower spectrum of action than naphthalen-2-ylmethyl 2-nitrocinnamate (13). Ester 10 was active only against five strains of Candida, while derivative 13 was active against all strains (MIC = 383.99–767.98 μM). This result seems to be related to the arrangement of substituting rings, as well as their spatial volume which can contribute to the steric impediment and hinder the molecule’s interaction with the active site.

Analyzing perillyl derivative 2-nitrocinnamate (14), it was found that the ester inhibited the growth of the eight strains tested with MIC = 390.99–781.98 μM, showing greater inhibitory action than compounds with aromatic substituents. This result may be related to its terpenic substructure since several monoterpenes have antifungal activities [23].

4. Materials and Methods

4.1. Chemistry

All reagents were of commercial quality purchased by Sigma Aldrich. The ¹H and ¹³C-NMR and IR signals attributing to (E)-2-nitrocinnamic acid analogs were compared with previously published data. The IR spectra were obtained on a prestige-21 FTIR spectrometer (Shimadzu, Kiyoto, Japan) using KBr tablets. The ¹H and ¹³C-NMR spectra were obtained on a MERCURY-VARIAN 200 MHz and 50 MHz spectrometer and a VNMRS-VARIAN 500 MHz and 125 MHz spectrometer. The mass spectra were performed on an Ultraflex II TOF/TOF spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a high-performance solid-state laser (λ = 355 nm) and reflector. The stereochemistry of the compounds was determined from spectroscopic data in which there was a coupling constant for hydrogens 7 and 8 of the double bond at 15.8 Hz, confirming stereochemistry (E). Chemical changes have been reported in relation to the peak of the deuterated chloroform solvent (CDCl₃).

4.1.1. General Procedure for Obtaining Compounds 1–6

Concentrated sulfuric acid (H₂SO₄) (0.5 mL) was added to a mixture of (E)-2-nitrocinnamic acid (0.5 g; 2.59 mmols) and alcohol (100 mL). The resulting mixture was stirred at reflux for 3–5 h and monitored by thin-layer chromatography. Excess alcohol was evaporated under reduced pressure, and the crude product was diluted with chloroform (20 mL) and washed with water (20 mL). The aqueous phase was extracted with chloroform (3 × 20 mL), and the combined organic phase was treated with 5% NaHCO₃, dried over anhydrous sodium sulfate (Na₂SO₄), filtered, and evaporated under reduced pressure. The pure product was obtained by column chromatography with silica gel in a hexane system: EtOAc (8 : 2) [24].

(1) Methyl 2-Nitrocinnamate (1). White solid (464 mg; 2.23 mmols), 86.10% yield; MP: 97–98°C (lit. 71°C, [1]); TLC (9 : 1 hexane/EtOAc), R_f = 0.44; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.10 (d, J = 16.0 Hz, 1H), 8.01 (d, J = 10.0 Hz, 1H), 7.65–7.54 (m, 3H), 6.35 (d, J = 16.0 Hz, 1H), and 3.81 (s, 3H); ¹³C-NMR (50 MHz, CDCl₃) δ_C 166.3, 148.4, 140.3, 133.7, 130.6, 130.4, 129.2, 125.0, 122.9, and 52.1; and IR ѵ_max (KBr, cm⁻¹) 3090, 2953, 1717, 1636, 1522, and 1196 [25].

(2) Ethyl 2-Nitrocinnamate (2). Brown solid (491 mg; 2.22 mmols), 85.71% yield; MP: 38–40°C (lit. 40°C, [2]); TLC (9 : 1 hexane/EtOAc), R_f = 0.46; ¹H-NMR (500 MHz, CDCl₃) δ_H 8.09 (d, J = 15.0 Hz, 1H), 8.02 (d, J = 10.0 Hz, 1H), 7.66–7.62 (m, 3H), 6.35 (d, J = 15.0 Hz, 1H), 4.28 (q, J = 7.5 Hz, 2H), and 1.33 (t, J = 7.5 Hz, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ_C 165.7, 148.3, 139.7, 133.4, 130.6, 130.2, 129.1, 124.8, 123.3, 60.9, and 14.2; and IR ѵ_max (KBr, cm⁻¹) 3090, 2953, 1717, 1636, 1522, and 1196 [25].

(3) Propyl 2-Nitrocinnamate (3). Brown solid (595 mg; 2.53 mmols), 98.94% yield; MP: 50–52°C; TLC (9 : 1 hexane/EtOAc), R_f = 0.46; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.10 (d, J = 16.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.65–7.49 (m, 3H), 6.36 (d, J = 16.0 Hz, 1H), 4.17 (t, J = 8.0 Hz, 2H), 1.70 (sex, J = 6.0 Hz, 2H), and 0.98 (t, J = 8.0 Hz, 3H); ¹³C-NMR (50 MHz, CDCl₃) δ_C 165.8, 148.2, 139.8, 133.5, 130.6, 130.2, 129.1, 124.8, 123.3, 66.5, 21.9, and 10.4; and IR ѵ_max (KBr, cm⁻¹) 3078, 2970, 1713, 1639, 1524, and 1168 [25].

(4) Isopropyl 2-Nitrocinnamate (4). Brown oil (602 mg; 2.56 mmols), 98.80% yield; TLC (9 : 1 hexane/EtOAc), R_f = 0.44; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.06 (d, J = 16.0 Hz, 1H), 8.0 (d, J = 6.0 Hz, 1H), 7.64–7.52 (m, 3H), 6.33 (d, J = 16.0 Hz, 1H), 5.10 (sept, J = 6.0 Hz, 1H), and 1.30 (d, J = 6.0 Hz, 6H);¹³C-NMR (50 MHz, CDCl₃) δ_C 165.2, 148.2, 139.4, 133.4, 130.5, 130.1, 129.0, 124.8, 123.7, 68.3, and 21.8; and IR ѵ_max (KBr, cm⁻¹) 3075, 2980, 1705, 1638, 1518, and 1206 [25].

(5) Butyl 2-Nitrocinnamate (5). Brown oil (567 mg; 2.27 mmols), 88.03% yield; TLC (9 : 1 hexane/EtOAc), R_f = 0.47; ¹H-NMR (500 MHz, CDCl₃) δ_H^8.09 (d, J = 15.0 Hz, 1H), 8.02 (d, J = 10.0 Hz, 1H), 7.64–7.53 (m, 3H), 6.35 (d, J = 15.0 Hz, 1H), 4.22 (t, J = 10.0 Hz, 2H), 1.68 (quin, J = 5.0 Hz, 2H), 1.43 (sex, J = 5.0 Hz, 2H), and 0.95 (t, J = 10.0 Hz, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ_C 165.8, 148.3, 139.7, 133.4, 130.6, 130.2, 129.1, 124.8, 123.3, 64.8, 30.6, 19.1, and 13.7; and IR ѵ_max (KBr, cm⁻¹) 3099, 2961, 1717, 1639, 1526, and 1180 [25].

(6) Methoxyethyl 2-Nitrocinnamate (6). Brown oil (420 mg; 1.67 mmols), 64.44% yield; TLC (9 : 1 hexane/EtOAc), R_f = 0.48; ¹H-NMR (500 MHz, CDCl₃) δ_H 8.06 (d, J = 16.0 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.63–7.48 (m, 3H), 6.37 (d, J = 16.0 Hz, 1H), 4.34 (t, J = 5.0 Hz, 2H), 3.36 (t, J = 5.0 Hz, 2H), and 3.37 (s, 3H);¹³C-NMR (125 MHz, CDCl₃) δ_C 165.6, 148.2, 140.2, 133.4, 130.3, 130.2, 129.0, 124.8, 122.7, 70.3, 63.8, and 58.9; IR ѵ_max (KBr, cm⁻¹) 3072, 2930, 1719, 1643, 1526, and 1180; and EMAR (MALDI) calculated for C₁₄H₁₇NO₄ [M+Na]⁺: 274.0890; found 274.0931.

4.1.2. General Procedure for the Synthesis of Compounds 7–13

To a mixture of (E)-2-nitrocinnamic acid (0.5 g; 2.59 mmols), acetone 31.4 mL, and triethylamine (1.4 mL; 10.36 mmols; 3.8 eq.), alkyl or aryl halides (3.89 mmos; 1.5 eq.) were added. The resulting mixture was stirred at reflux for 12–52 h and monitored by thin-layer chromatography. At the end of the reaction, excess acetone was evaporated under reduced pressure and the crude product was diluted with chloroform (20 mL) and washed with water (20 mL). After separating the organic phase, the aqueous phase was extracted with chloroform (3 × 20 mL), and the combined organic phase was dried over anhydrous sodium sulfate (Na₂SO₄), filtered, and evaporated under reduced pressure. The pure product was obtained by column chromatography with silica gel in a hexane system: EtOAc (7 : 3) [26, 27].

(1) Pentyl 2-Nitrocinnamate (7). Yellow oil (287.8 mg; 1.09 mmols), 42.23% yield; TLC (9 : 1 hexane/EtOAc), R_f = 0.49; ¹H-NMR (500 MHz, CDCl₃) δ_H 8.06 (d, J = 15.8 Hz, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.62–7.48 (m, 3H), 6.33 (d, J = 15.8 Hz, 1H), 4.18 (t, J = 6.8 Hz, 2H), 1.71–1.65 (m, 2H), 1.37–1.30 (m, 4H), and 0.88 (t, J = 5.7 Hz, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ_C 165.8, 148.3, 139.7, 133.4, 130.5, 130.2, 129.0, 124.8, 123.3, 65.0, 28.3 28.0, 22.2, and 13.8; IR ѵ_max (KBr, cm⁻¹) 3073, 2957, 1717, 1638, 1526, and 1179 [1]; and EMAR (MALDI) calculated for C₁₄H₁₇NO₄ [M+Na]⁺: 286.1050; found 286.1050.

(2) Decyl 2-Nitrocinnamate (8). Brown solid (443.4 mg; 1.33 mmols), 51.35% yield; MP: 29–30°C; TLC (9 : 1 hexane/EtOAc), R_f = 0.53; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.09 (d, J = 16.0 Hz, 1H), 8.02 (d, J = 6.0 Hz, 1H), 7.65–7.48 (m, 3H), 6.36 (d, J = 16.0 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 1.69 (quin, J = 6.0 Hz, 2H), 1.41–1.25 (m, 14H), and 0.86 (t, J = 6.0 Hz, 3H); ¹³C-NMR (50 MHz, CDCl₃) δ_C 165.8, 148.2, 139.7, 133.4, 130.6, 130.2, 129.1, 124.8, 123.3, 65.1, 31.8, 29.5, 29.5, 29.2, 29.2, 28.6, 25.9, 22.6, and 14.1; IR ѵ_max (KBr, cm⁻¹) 3073, 2926, 1717, 1639, 1528, and 1177; and EMAR (MALDI) calculated for C₁₆H₁₂ClNO₄ [M+Na]⁺: 356.1838; found 356.1837.

(3) 4-Chlorobenzyl 2-Nitrocinnamate (9). Yellow solid (429 mg; 1.35 mmols), 52.12% yield; MP: 115–117°C; TLC (1 : 1 hexane/EtOAc), R_f = 0.33; ¹H-NMR (500 MHz, CDCl₃) δ_H 8.16 (d, J = 15.0 Hz, 1H), 8.04 (d, J = 10.0 Hz, 1H), 7.65–7.53 (m, 3H), 7.40–7.35 (m, 4H), 6.39 (d, J = 15.0 Hz, 1H), and 5.23 (s, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ_C 165.4, 148.3, 140.7, 134.2, 134.2, 133.5 130.4, 130.4, 129.7, 129.1, 128.8, 124.9, 122.7, and 65.8; IR ѵ_max (KBr, cm⁻¹) 3063, 2949, 1701, 1639, 1526, and 1190; and EMAR (MALDI) calculated for C₁₆H₁₂ClNO₄ [M+Na]⁺: 342.0420; found 342.0425.

(4) Benzhydryl 2-Nitrocinnamate (10). Yellow solid (256.5 mg; 0.71 mmols) 27.41% yield; MP: 58–60°C; TLC (9 : 1 hexane/EtOAc), R_f = 0.30; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.27 (d, J = 15.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.64–7.52 (m, 3H), 7.42–7.32 (m, 10H), 7.06 (s, 1H), and 6.49 (d, J = 15.8 Hz, 1H); ¹³C-NMR (50 MHz, CDCl₃) δ_C 164.7, 148.1, 140.7, 139.9, 133.5, 130.4, 130.3, 129.1, 128.5, 128.4, 128.0 126.5, 124.9, 122.9, and 77.4; IR ѵ_max (KBr, cm⁻¹) 3086, 2970, 1713, 1634, 1518, 1493, 1342, and 1171; and EMAR (MALDI) calculated for C₂₂H₁₁NO₄ [M+Na]⁺: 382.1055; found 382.1059.

(5. 4-Hydroxyphenethyl 2-Nitrocinnamate (11). Yellow solid (58.05 mg; 0.188 mmols), 72.22% yield; TLC (9 : 1 hexane/EtOAc), R_f = 0.31; ¹H-NMR (500 MHz, CDCl₃) δ_H 8.09 (d, J = 15.8 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.62–7.49 (m, 3H), 7.10 (d, J = 8.3 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 6.32 (d, J = 15.8 Hz, 1H), 5.46 (s, 1H), 4.37 (t, J = 7.0 Hz, 2H), and 2.92 (t, J = 7.0 Hz, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ_C 165.1, 154.4, 148.2, 140.1, 133.4, 130.4, 130.2, 130.1, 129.6, 129.0, 124.8, 123.0, 115.4, 65.7, and 34.2; IR ѵ_max (KBr, cm⁻¹) 3335, 3030, 2957, 1682, 1636, 1516, and 1209; EMAR (MALDI) calculated for C₁₇H₁₅NO₅ [M+Na]⁺: 336.080; found 336.051.

(6) 4-Methoxybenzyl 2-Nitrocinnamate (12). Yellow solid (487 mg; 1.55 mmols), 59.84% yield; MP: 65–68°C; TLC (9 : 1 hexane/EtOAc), R_f = 0.29; ¹H-NMR (500 MHz, CDCl₃) δ_H 8.13 (d, J = 16.0 Hz, 1H), 8.01 (d, J = 5.0 Hz, 1H), 7.64–7.51 (m, 3H), 7.35 (d, J = 10.0 Hz, 2H), 6.90 (d, J = 10.0 Hz, 2H), 6.38 (d, J = 15.5 Hz, 1H), 5.20 (s, 2H), and 3.80 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ_C 165.5, 159.7, 148.2, 140.2, 133.4, 130.4, 130.2, 130.1, 129.0, 127.8, 124.8, 123.0, 113.9, 66.4, and 55.2; IR ѵ_max (KBr, cm⁻¹) 3076, 2957, 1715, 1638, 1524, and 1169; and EMAR (MALDI) calculated for C₁₇H₁₅NO₅ [M+Na]⁺: 336.2954; found 336.2953.

(7) Naphthalen-2-Ylmethyl 2-Nitrocinnamate (13). Yellow solid (215 mg; 0.64 mmols), 24.90% yield; MP: 83–85°C; TLC (9 : 1 hexane/EtOAc), R_f = 0.30; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.20 (d, J = 16.0 Hz), 8.03 (d, J = 8.0 Hz, 1H), 7.89–7.83 (m, 3H), 7.63–7.58 (m, 4H), 7.54–7.48 (m, 3H), 6.43 (d, J = 16.0 Hz, 1H), and 5.44 (s, 2H); ¹³C-NMR (50 MHz, CDCl₃) δ_C 165.6, 148.2, 140.5, 133.5, 133.1, 133.1, 133.1, 130.4, 130.3, 129.1, 128.4, 128.0, 128.0, 127.7, 127.4, 126.3, 125.8, 124.8, 122.8, and 66.8; IR ѵ_max (KBr, cm⁻¹) 3057, 2959, 1707, 1636, 1341, and 1179; and EMAR (MALDI) calculated for C₂₀H₁₅NO₄ [M+Na]⁺: 356.0899; found 356.0896.

4.1.3. Procedure for Synthesis of Compound 14

A mixture of (E)-2-nitrocinnamic acid (0.1 g; 0.52 mmols) and perillyl alcohol (0.082 mL; 0.52 mmols) in tetrahydrofuran (THF) (1.73 mL) was left at 0°C for 30 minutes with magnetic stirring. Next, triphenylphosphine (TPP) (0.14 g; 0.52 mmols) and dialkyl azodicarboxylate (DEAD) (0.1 mL; 0.52 mmols) were added; the mixture was left for five minutes under stirring at 0°C and for 48 h at the temperature environment and was monitored by thin-layer chromatography. At the end of the reaction, the solvent was evaporated on a rotary evaporator and the crude product was diluted with ethyl acetate (10 mL) and washed with water (10 mL). After separating the organic phase, the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The combined organic phase was treated with 5% NaHCO₃ and saturated sodium chloride solution (NaCl), dried with Na₂SO₄, filtered, and evaporated under reduced pressure. The pure product was obtained by column chromatography with silica gel in a hexane system: EtOAc (9 : 1) [28].

(1) Perillyl 2-Nitrocinnamate (14). Yellow solid (70 mg; 0.21 mmols), 40.38% yield; MP: 76–77°C; TLC (1 : 1 hexane/EtOAc), R_f = 0.42; ¹H-NMR (200 MHz, CDCl₃) δ_H 8.12 (d, J = 16.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.63–7.54 (m, 3H), 6.38 (d, J = 16.0 Hz, 1H), 5.80–5.83 (m, 1H), 4.71 (s, 2H), 4.61 (s, 2H), 2.28–2.14 (m, 2H), 2.14–2.12 (m, 1H), 2.05–1.98 (m, 2H), 1.98–1.96 (m, 2H), and 1.71 (s, 3H); ¹³C-NMR (50 MHz, CDCl₃) δ_C 165.6, 149.5, 148.2, 140.0, 133.5, 132.4, 130.5, 130.2, 129.1, 126.1, 124.9, 123.1, 108.7, 68.9, 40.7, 30.4, 27.2, 26.4, and 20.7; IR ѵ_max (KBr, cm⁻¹) 3078, 2922, 1707, 1638, 1518, and 1180; and EMAR (MALDI) calculated for C₁₉H₂₁NO₄ [M+Na]⁺: 350.1368; found 350.1367.

4.2. Biological Evaluation

4.2.1. In Vitro Antifungal Assay

The 14 compounds selected were checked for antifungal activity against strains of Candida albicans (ATCC 90028 and LM 106), C. krusei (LM 13 and LM 08), C. tropicalis (ATCC 13803 and LM 31), and Candida parapsilosis (LM 14 and LM 02). The strains were acquired from the MICOTECA of the Mycology Laboratory, Department of Pharmaceutical Sciences (DCF), Health Science Center (CCS) of the Federal University of Paraíba. All strains were kept on Sabouraud dextrose agar (SDA) and brain heart infusion broth (BHI) at a temperature of 4°C and were used for the assays for 24–48 h in SDA/ABHI incubated at 35 ± 2°C. The microorganism suspension was prepared according to McFarland standard 0.5 to obtain 1–5 × 10⁶ CFU/mL [29–32]. The standard drug, nystatin (yeast), was used as a control.

4.2.2. Determination of the Minimum Inhibitory Concentration (MIC)

MIC was determined using the broth microdilution method, using 96-well U-shaped plates. In each well, 100 μL of the liquid medium from the Roswell Park Memorial Institute (RPMI) doubly concentrated with 100 mL of the product solution was added to the first row of plate wells. Thus, through serial dilution, concentrations of 1024 μg/mL to 2 μg/mL were obtained for the tested compounds and drug, with a higher concentration in the first row of the plate and a lower concentration in the last. Subsequently, 10 μL of inoculum was added to the wells in each column of the plate and was also made for the culture medium with nystatin; the plates were left to incubate at 37°C for 24–48 h. For each strain, MIC was defined as the lowest concentration capable of inhibiting fungal growth in the wells and was visually observed in comparison with the control. All tests were performed in duplicate, and the results were expressed as an arithmetic mean of the MIC values obtained in both tests [23, 33, 34].

5. Conclusion

Among the tested alkyl compounds, 4 showed a better spectrum of action against all evaluated Candida strains, suggesting that the presence of the isopropyl group potentiates antifungal activity. Ester 14 showed good inhibition against the tested strains, indicating the importance of the terpenic substructure to enhance bioactivity. Thus, structural changes in these compounds can contribute to the development of new antifungal agents.

Data Availability

All data used to support the findings of the study (¹³C-NMR, IR, and ¹H-NMR spectra) are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

LGN and MCM conducted the synthesis of compounds and wrote the manuscript. JKOJ and EOL performed the biological tests. DPS edited the manuscript. All authors have read and approved the final manuscript.

Acknowledgments

This research was funded by the Brazilian agencies, namely, the National Council for Scientific and Technological Development (421285/2016-8) and Higher Education Personnel Improvement Coordination (CAPES).

Supplementary Materials

The supplementary material contains the nuclear magnetic resonance of hydrogen and carbon thirteen spectra, infrared spectra, and high-resolution mass spectra of the prepared compounds. (Supplementary Materials)

References

M. A. Pfaller, D. R. Andes, D. J. Diekema, D. L. Horn, A. C. Reboli, and C. Rotstein, “Epidemiology and outcomes of invasive candidiasis due to non-albicans species of Candida in 2.496 patients: data from the prospective antifungal therapy (PATH) registry 2004-2008,” PLoS One, vol. 9, no. 7, Article ID 101510, 2014.
View at: Publisher Site | Google Scholar
D. K. Matthaiou, T. Christodoulopoulou, and G. Dimopoulos, “How to treat fungal infections in ICU patients,” BMC Infectious Diseases, vol. 15, no. 205, 2015.
View at: Publisher Site | Google Scholar
P. G. Pappas, C. A. Kauffman, D. R. Andes et al., “Clinical practice guideline for the management of candidiasis: 2016, update by the infectious diseases society of America,” Clinical Infectious Diseases, vol. 62, no. 4, pp. 1–50, 2016.
View at: Publisher Site | Google Scholar
L. S. Barbedo and D. B. G. Sgarbi, “Candidíase,” Jornal Brasileiro de DST, vol. 22, pp. 22–38, 2010.
View at: Google Scholar
A. M. Bergold and S. Georgiadis, “Novidades em fármacos antifúngicos: uma revisão,” Visão Acadêmica, vol. 5, no. 2, pp. 159–172, 2004.
View at: Publisher Site | Google Scholar
D. E. Wedge and N. D. Camper, in Biologically Active Natural Products in Agrochemicals and Pharmaceuticals, CRC Press, Boca Raton, FL, USA, 2000.
M. N. Clifford, “Chlorogenic acids and other cinnamates-nature, occurrence, dietary burden, absorption and metabolism,” Journal of the Science of Food and Agriculture, vol. 80, no. 7, pp. 1033–1043, 2000.
View at: Publisher Site | Google Scholar
D. De vita, L. Friggeri, F. D. D'auria et al., “Activity of caffeic acid derivatives against Candida albicans biofilm,” Bioorganic and Medicinal Chemistry Letters, vol. 24, no. 6, pp. 1502–1505, 2014.
View at: Publisher Site | Google Scholar
J. Fu, K. Cheng, Z. Zhang, R. Fang, and H. Zhu, “Synthesis, structure and structure-activity relationship analysis of caffeic acid amides as potential antimicrobials,” European Journal of Medicinal Chemistry, vol. 45, no. 6, pp. 2638–2643, 2010.
View at: Publisher Site | Google Scholar
M. Sova, “Antioxidant and antimicrobial activities of cinnamic acid derivatives,” Mini-Reviews in Medicinal Chemistry, vol. 12, no. 8, pp. 749–767, 2012.
View at: Publisher Site | Google Scholar
F. Bisogno, L. Mascoti, C. Sanchez et al., “Structure-antifungal activity relationship of cinnamic acid derivatives,” Journal of Agricultural and Food Chemistry, vol. 55, no. 26, pp. 10635–10640, 2007.
View at: Publisher Site | Google Scholar
J. D. Guzman, P. N. Mortazavi, T. Munshi et al., “2-Hydroxysubstituted cinnamic acids and acetanilides are selective growth inhibitors of Mycobacterium tuberculosis,” MedChemComm, vol. 5, no. 1, pp. 47–50, 2014.
View at: Publisher Site | Google Scholar
P. De, M. Baltas, and F. Bedos-belval, “Cinnamic acid derivatives as anticancer agents - a review,” Current Medicinal Chemistry, vol. 18, no. 11, pp. 1672–1703, 2011.
View at: Publisher Site | Google Scholar
B. Debnath, S. Samanta, K. Roy, and T. Jha, “QSAR study on some p-arylthio cinnamides as antagonists of biochemical ICAM-1/LFA-1 interaction and ICAM-1/JY-8 cell adhesion in relation to anti-inflammatory activity,” Bioorganic and Medicinal Chemistry, vol. 11, no. 8, pp. 1615–1619, 2003.
View at: Publisher Site | Google Scholar
B. Zhang, C. Lv, W. Li et al., “Ethyl cinnamate derivatives as promising high-efficient acaricides against Psoroptes cuniculi: synthesis, bioactivity and structure-activity relationship,” Chemical and Pharmaceutical Bulletin, vol. 63, no. 4, pp. 255–262, 2015.
View at: Publisher Site | Google Scholar
E. Otero, S. M. Robledo, S. Díaz et al., “Synthesis and leishmanicidal activity of cinnamic acid esters: structure-activity relationship,” Medicinal Chemistry Research, vol. 23, no. 3, pp. 1378–1386, 2014.
View at: Publisher Site | Google Scholar
T. Yabe, H. Hirahara, N. Harada et al., “Ferulic acid induces neural progenitor cell proliferation in vitro and in vivo,” Neuroscience, vol. 165, no. 2, pp. 515–524, 2010.
View at: Publisher Site | Google Scholar
S. Mohammadzadeh, M. Shariatpanahi, M. Hamedi, R. Ahmadkhaniha, N. Samadi, and S. N. Ostad, “Chemical composition, oral toxicity and antimicrobial activity of Iranian propolis,” Food Chemistry, vol. 103, no. 4, pp. 1097–1103, 2007.
View at: Publisher Site | Google Scholar
B. Narasimhan, D. Belsare, D. Pharande, V. Mourya, and A. Dhake, “Esters, amides and substituted derivatives of cinnamic acid: synthesis, antimicrobial activity and QSAR investigations,” European Journal of Medicinal Chemistry, vol. 39, no. 10, pp. 827–834, 2004.
View at: Publisher Site | Google Scholar
B. Tebbets, D. Stewart, S. Lawry et al., “Identification and characterization of antifungal compounds using a Saccharomyces cerevisiae Reporter Bioassay,” PLoS One, vol. 7, no. 5, Article ID 36021, 2012.
View at: Publisher Site | Google Scholar
T. C. Lima, A. R. Ferreira, D. F. Silva, E. O. Lima, and D. P. De Sousa, “Antifungal activity of cinnamic acid and benzoic acid esters against Candida albicans strains,” Natural Product Research, vol. 32, no. 5, pp. 572–575, 2018.
View at: Publisher Site | Google Scholar
A. Khatkar, A. Nanda, P. Kumar, and B. Narasimhan, “Synthesis, antimicrobial evaluation and QSAR studies of p-coumaric acid derivatives,” Arabian Journal of Chemistry, vol. 10, no. 2, pp. 3804–3815, 2017.
View at: Publisher Site | Google Scholar
V. L. G. Rehder, A. L. M. Machado, C. Delarmelina et al., “Composition and antimicrobial activity of essential oils from aromatic plants used in Brazil,” Brazilian Journal of Microbiology, vol. 35, no. 4, pp. 275–280, 2004.
View at: Publisher Site | Google Scholar
A. Khatkar, A. Nanda, P. Kumar, and B. Narasimhan, “Synthesis, antimicrobial evaluation and qsar studies of gallic acid derivatives,” Arabian Journal of Chemistry, vol. 10, no. 2, pp. 2870–2880, 2017.
View at: Publisher Site | Google Scholar
D. C. Dibakar and M. Paul, “Indirect olefination of o-aminobenzaldehy,” Journal of Chemical Research, vol. 2006, no. 2, pp. 102-103, 2006.
View at: Publisher Site | Google Scholar
P. Boeck, M. M. Sá, B. S. D. Souza et al., “A simple synthesis of kaurenoic esters and other derivatives and evaluation of their antifungal activity,” Journal of the Brazilian Chemical Society, vol. 16, no. 6B, pp. 1360–1366, 2005.
View at: Publisher Site | Google Scholar
M. C. de Morais, E. de Oliveira Lima, Y. Perez-Castillo, and D. P. de Sousa, “Synthetic cinnamides and cinnamates: antimicrobial activity, mechanism of action, and in silico study,” Molecules, vol. 28, no. 4, p. 1918, 2023.
View at: Publisher Site | Google Scholar
D. I. Batovska, T. Kishimoto, V. S. Bankova, Z. Kamenarska, and M. Ubukata, “Synthesis of Some phenylpropanoid monoglycerides via the Mitsunobu protocol,” Molecules, vol. 10, no. 3, pp. 552–558, 2005.
View at: Publisher Site | Google Scholar
F. Hadacek and H. Greger, “Testing of antifungal natural products: methodologies, comparability of results and assay choice,” Phytochemical Analysis, vol. 11, no. 3, pp. 137–147, 2000.
View at: Publisher Site | Google Scholar
R. Cleland and E. Squires, “Evalution of new antimicrobials “in vitro” and in experimental animal infections,” in Lorian VMD. Antibiotics in Laboratory Medicine, pp. 739–788, Willians and Wilkins, Philadelphia, PA, USA, 1991.
View at: Google Scholar
R. M. P. Antunes, E. O. Lima, M. S. V. Pereira et al., “Atividade antimicrobiana “in vitro” e determinação da concentração inibitória mínima (CIM) de fitoconstituintes e produtos sintéticos sobre bactérias e fungos leveduriformes,” Revista Brasileira de Farmacognosia, vol. 16, no. 4, pp. 517–524, 2006.
View at: Google Scholar
I. C. M. Freire, A. L. A. L. Pérez, A. M. R. Cardoso et al., “Atividade antibacteriana de óleos essenciais sobre Streptococcus mutans e,” Revista Brasileira de Plantas Medicinais, vol. 16, pp. 372–377, 2014.
View at: Publisher Site | Google Scholar
F. B. Holetz, G. L. Pessini, N. R. Sanches, D. A. G. Cortez, C. V. Nakamura, and B. P. Dias Filho, “Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases,” Memorias do Instituto Oswaldo Cruz, vol. 97, no. 7, pp. 1027–1031, 2002.
View at: Publisher Site | Google Scholar
P. J. Houghton, M. J. Howes, C. C. Lee, and G. Steventon, “Uses and abuses of in vitro tests in ethnopharmacology: visualizing an elephant,” Journal of Ethnopharmacology, vol. 110, no. 3, pp. 391–400, 2007.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2023 Lázaro Gomes do Nascimento 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.

PDF Download Citation

Download other formats

Order printed copies