Journal of Chemistry
Volume 2019 (2019), Article ID 3408315, 9 pages
https://doi.org/10.1155/2019/3408315

Research Article

Synthesis, Antibacterial Evaluation, and QSAR of Caffeic Acid Derivatives

Marianna O. Araújo,¹ Hilzeth L. Freire Pessoa,² Andressa B. Lira,¹ Yunierkis P. Castillo,³ and Damião P. de Sousa1,4

¹Postgraduate Program in Natural and Synthetic Bioactive Products, Federal University of Paraíba, João Pessoa, PB, Brazil
²Department of Molecular Biology, Federal University of Paraíba, João Pessoa, PB, Brazil
³Escuela de Ciencias Físicas y Matemáticas, Universidad de Las Américas, Quito, Ecuador
⁴Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, PB, Brazil

Correspondence should be addressed to Damião P. de Sousa; damiao_desousa@yahoo.com.br

Received 28 November 2018; Revised 10 January 2019; Accepted 27 January 2019; Published 12 February 2019

Academic Editor: Sevgi Kolaylı

Copyright © 2019 Marianna O. Araújo 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.

Abstract.

The present study evaluates the antibacterial effects of a set of 16 synthesized caffeic acid ester derivatives against strains of Staphylococcus aureus and Escherichia coli, as well as discusses their structure-activity relationship (SAR). The antibacterial assays were performed using microdilution techniques in 96-well microplates to determine minimal inhibitory concentration (MIC). The results revealed that five of the compounds present strong to optimum antibacterial effect. Of the sixteen ester derivatives evaluated, the products with alkyl side chains, as propyl caffeate (3), butyl caffeate (6), and pentyl caffeate (7), presented the best antibacterial activity with MIC values of around 0.20 μM against Escherichia coli and only butyl caffeate (6) showing the same MIC against Staphylococcus aureus. For products with aryl substituents, the best MIC results against the tested strain of Escherichia coli were 0.23 µM for (di-(4-chlorobenzyl)) caffeate (13) and 0.29 µM for diphenylmethyl caffeate (10) and all were less active against the Staphylococcus aureus strain. Preliminary quantitative structure-activity relationship (QSAR) analyses confirmed that certain structural characteristics, such as a median linear carbon chain and the presence of electron withdrawal substituents at the para position of the aromatic ring, help potentiate antibacterial activity.

1. Introduction

Bacterial diseases are a global health problem and are prevalent in developing and low-income countries [1, 2], causing 13 to 17 million deaths annually, equivalent to 25% of global deaths and 45% for developing countries [3]. Due to the bacterial resistance of Staphylococcus aureus to methicillin and vancomycin [4] and Escherichia coli to β-lactams [5], there is an urgent need for discovery and development of new classes of antimicrobial compounds to treat bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for high hospital infection rates [6, 7]. These infections range from simple acnes, boils, and abscesses to more severe forms such as pneumonia, meningitis, endocarditis, and septicemia [8, 9]. β-Lactams are not always fully effective against strains of MRSA [10]. Vancomycin is an alternative; however, there are already case reports of intermediate [11] and full resistance to the drug [12].

Escherichia coli is active in the human intestinal tract [13]; it participates in vitamin K₂ synthesis and in defense against pathogenic bacteria [14]. Escherichia coli can cause severe food poisoning and more severe conditions such as peritonitis [15], meningitis, and urinary infections [16]. In E. coli strains, production of extended-spectrum β-lactamases (ESBL) [17] hydrolyzes most penicillins, cephalosporins, and monobactams (aztreonam), the cephamycins and carbapenems being among the few exceptions [18]. E. coli strains are resistant to aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol, and sulfamethoxazole-trimethoprim [19]. In the most serious infections involving ESBL, carbapenems are indicated [20], but there are already reports in the literature of resistance to carbapenems [21, 22].

Many studies have investigated the bioactivity of caffeic acid derivatives finding that some of these analogues possess remarkable activities, with potential as anti-inflammatory [23–27], antiviral [28], antiatherosclerotic [29], anti-HIV [30], antioxidant [31, 32], immunomodulatory [33], antitumor [34–37], neuroprotective [38], antifungal [39], and antibacterial agents [40]. Caffeic acid phenethyl ester (CAPE) or phenethyl caffeate is considered one of the most important derivatives of caffeic acid and has several various biological activities, such as antibacterial action [41–43]. It is found in propolis extract and occurs in many plants [41]. Due to the problems associated with treatment of bacterial infections, such as drug-resistant strains, various studies have been conducted to discover new and safer antibacterial agents. In the present study, synthesis and antibacterial activity evaluations of 16 caffeic acid derivatives against two representative strains Staphylococcus aureus (ATCC-25619) and Escherichia coli (ATCC-2536) were performed.

2. Chemicals

All of the chemical products used during synthesis were from Sigma-Aldrich. ¹H-NMR (200 and 50 MHz) and ¹³C-NMR (500 and 125 MHz) spectra were, respectively, recorded on Varian Mercury and Varian-RMN-System spectrometers. Chemical shifts (δ) are expressed in parts per million (ppm) using TMS as an internal standard. Spin multiplicities are given as s (singlet), brs (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), quint (quintet), sext (sextet), sept (septet), and m (multiplet). High-resolution mass spectrometry was carried out using an Ultraflex II TOF/TOF spectrometer with a high performance solid state laser (λ = 355 nm) and a reflector using the MALDI (matrix-assisted laser desorption ionization) technique. Column adsorption chromatography (CC) was performed on silica gel (Merck 60, 230–400 mesh); analytical TLC was performed on precoated silica gel plates (Merck 60 F₂₅₄). FTIR spectra were recorded on a Bruker FTIR spectrometer, Vertex 70 model, using KBr pellets.

3. Materials and Methods

3.1. Synthesis of Compounds 1–8 for Fischer’s Esterification

A mixture of caffeic acid (0.25 g, 1.39 mmol) and alcohol (50 ml) was heated under reflux in the presence of sulfuric acid (0.4 ml) until completion of the reaction (5–21 hours) and verified by a single spot in TLC. The alcohol was then removed under reduced pressure, and the solution was diluted with 20 ml of water. The product was extracted with ethyl acetate (15 ml). The organic phase was neutralized successively with 5% sodium bicarbonate and water, dried over anhydrous sodium sulfate, and filtered. After evaporation under reduced pressure, this phase yielded the ester derivatives [44].

3.2. Reaction with Alkyl and Aryl Halides for Compounds 9–14

3.2.1. Synthesis of Compounds 9 and 10

Caffeic acid (0.2 g, 1.11 mmol) in acetone (13.5 ml) was heated under reflux in the presence of triethylamine (0.6 ml) and halide (1.14 mmol) until complete reaction (44–120 hours), which was verified by a single spot in TLC. The solvent was then removed under reduced pressure, and the solution was diluted with 20 ml of water. The product was extracted with ethyl acetate (20 ml). The organic phase was treated with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (eluent: hexane-ethyl acetate and an increasing polarity gradient) [45, 46].

3.2.2. Synthesis of Compounds 11–14

A mixture of caffeic acid (0.2 g, 1.11 mmol) and potassium carbonate (0.17 g, 1.22 mmol) was dissolved in 5.5 mL of dimethylformamide in the presence of halide (1.11 mmol). The mixture was stirred at room temperature, until complete reaction (27–50 hours), which was verified by a single spot in TLC. The solvent was then removed under reduced pressure, and the solution was diluted with 15 ml of water. The product was extracted with ethyl acetate (10 ml). The organic phase was neutralized successively with 5% sodium bicarbonate (10 ml), washed with brine (10 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by using a chromatographic column with hexane and ethyl acetate eluents in an increasing polarity to isolate the ester derivatives [47].

3.2.3. Synthesis of Compounds 15 and 16 for the Mitsunobu Reaction

A mixture of caffeic acid (0.1 g, 0.55 mmol) and alcohol (0.55 mmol) was dissolved in 1.85 ml tetrahydrofuran. The reaction mixture was stirred under magnetic stirring at 0°C for about 30 minutes. Afterwards, di-isopropyl azodicarboxylate (0.11 ml, 0.55 mmol) and triphenylphosphine (0.15 g, 0.55 mmol) were added as esterification agents, with continuous stirring at room temperature for about 46–50 hours, which was verified by a single spot in TLC. The solvent was then removed under reduced pressure, and the solution was diluted with 10 ml of water. The product was then extracted with ethyl acetate (10 ml). The organic phase was neutralized successively with saturated sodium bicarbonate, washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was then purified on a silica gel column (eluent: hexane-ethyl acetate, 7 : 3) [48].

(1) Methoxyethyl Caffeate (5). Brown liquid, 61.64% yield; IR υmax (KBr, cm⁻¹): 3319, 3071, 2945, 1689, 1600 and 1442, 1272 and 1176; ¹H-NMR (DMSO-d₆, 200 MHz): δH 3.27 (3H; s), 3.56 (2H; t; J = 4.8 Hz), 4.22 (2H; t; J = 4.8 Hz), 6.28 (1H; d; J = 16.0 Hz), 6.76 (1H; d; J = 8.0 Hz), 7.01 (1H; dd; J = 8.0 Hz, 2.0 Hz), 7.06 (1H; d; J = 2.0 Hz), 7.47 (1H; d; J = 16.0 Hz); ¹³C-NMR (DMSO-d₆, 50 MHz): δC 58.2, 63.0, 69.9, 113.8, 114.9, 115.8, 121.6, 125.5, 145.5, 145.6, 148.5, and 166.7 [49]; HRMS (MALDI) calculated for C₁₂H₁₄O₅ [M+H]⁺: 239.0919, found 239.1599.

(2) Diphenylmethyl Caffeate (10). Brown liquid, 27.29% yield; IR υmax (KBr, cm⁻¹): 3370, 3059, 2970, 1702, 1606 and 1448, 1271 and 1149; ¹H-NMR (DMSO-d₆, 200 MHz): δH 6.43 (1H; d; J = 16.0 Hz), 6.77 (1H; d; J = 8.0 Hz), 6.90 (1H; s), 7.06 (1H; dd; J = 8.0 Hz, 2.0 Hz), 7.10 (1H; d; J = 2.0 Hz), 7.47–7.28 (10H; m), 7.59 (1H; d; J = 16.0 Hz), [44]; ¹³C-NMR (DMSO-d₆, 50 MHz): δC 76.1, 113.7, 115.0 115.8, 121.7, 125.5, 126.6, 127.8, 128.6, 140.8, 145.6, 146.0, 148.6, and 165.6 [50]; HRMS (MALDI) calculated for C₂₂H₁₈O₄ [M+Na]⁺: 369.1103, found 369.1168.

(3) 4-Chlorobenzyl Caffeate (11). Yellow amorphous solid, 26.43% yield; IR υmax (KBr, cm⁻¹): 3338, 3053, 2964, 1677, 1600 and 1435, 1258 and 1175, 1011; ¹H-NMR (DMSO-d₆, 200 MHz): δH 5.18 (2H; s), 6.33 (1H; d; J = 16.0 Hz), 6.76 (1H; d; J = 8.0 Hz), 7.02 (1H; dd; J = 8.0 Hz, 2.0 Hz), 7.07 (1H; d; J = 2.0 Hz), 7.44 (4H; brs), 7.53 (1H; d; J = 16.0 Hz); ¹³C-NMR (DMSO-d₆, 50 MHz): δC 64.5, 113.6, 114.9, 115.8, 121.6, 125.5, 128.5, 129.9, 132.6, 135.5, 145.6, 145.8, 148.5, and 166.3 [51]; HRMS (MALDI) calculated for C₁₆H₁₃ClO₄ [M]⁺: 304.0502, found 304.3855.

(4) (Di-(4-chlorobenzyl)) Caffeate (13). White amorphous solid, 11.82% yield; IR υmax (KBr, cm⁻¹): 3332, 3059, 2976, 1695, 1606 and 1442, 1277 and 1169, 1011; ¹H-NMR (DMSO-d₆, 200 MHz): δH 5.16 (2H; s), 5.19 (2H; s), 6.41 (1H; d; J = 16.0 Hz), 7.00 (1H; d; J = 8.4 Hz); 7.10 (1H; d; J = 2.0 Hz); 7.14 (1H; s), 7.49–7.43 (8H; m), 7.56 (1H; d; J = 16.0 Hz); ¹³C-NMR (DMSO-d₆, 50 MHz): δC 64.6, 68.9, 113.7, 114.8, 115.0, 121.2, 127.2, 128.4, 128.5, 129.5, 129.9, 132.4, 132.7, 135.4, 136.0, 145.2, 147.0, 148.8, and 166.3 [51, 52]; HRMS (MALDI) calculated for C₁₆H₁₃ClO₄ [M+H]⁺: 429.0660, found 429.0667.

(5) (Di-(4-methoxybenzyl)) Caffeate (14). Orange amorphous solid, 36.98% yield; IR υmax (KBr, cm⁻¹): 3344, 3059, 2831, 1702, 1620 and 1435, 1188 and 1112; ¹H-NMR (DMSO-d₆, 200 MHz): δH 3.74 (3H; s), 3.74 (3H; s), 5.07 (2H; s); 5.11 (2H; s), 6.36 (1H; d; J = 15.8 Hz), 6.94–6.86 (5H; m), 7.02 (1H; dd, J = 8.0 Hz, 2.0 Hz), 7.10 (1H; s), 7.22 (2H; d; J = 8.6 Hz), 7.35 (2H; d; J = 8.6 Hz), 7.51 (1H; d; J = 16.0 Hz); ¹³C-NMR (DMSO-d₆, 50 MHz): δC 55.1, 55.2, 65.4, 69.6, 113.6, 113.8, 113.9, 114.6, 115.1, 121.3, 127.1, 128.4, 128.9, 129.8, 130.3, 144.9, 147.2, 149.2, 159.1, 159.3, and 166.5 [52, 53]; HRMS (MALDI) calculated for C₂₅H₂₄O₆ [M]⁺: 420.1573, found 420.0234.

(6) 4-Methylbenzyl Caffeate (16). Yellow amorphous solid, 50.95% yield; IR υmax (KBr, cm⁻¹): 3332, 3027, 2976, 1677, 1600 and 1435, 1271 and 1163; ¹H-NMR (DMSO-d₆, 200 MHz): δH 2.29 (3H; s), 5.13 (2H; s), 6.30 (1H; d; J = 16.0 Hz), 6.75 (1H; d; J = 8.0 Hz), 7.00 (1H; dd; J = 8.2 Hz, 2.0 Hz), 7.06 (1H; d; J = 2.0 Hz), 7.18 (2H; d; J = 8.0 Hz), 7.29 (2H; d; J = 8.0 Hz), 7.50 (1H; d; J = 16.0 Hz); ¹³C-NMR (DMSO-d₆, 50 MHz): δC 21.9, 65.3, 113.8, 114.9, 115.8, 121.6, 125.5, 128.3, 129.1, 133.5, 137.4, 145.5, 145.6, 148.6, and 166.5 [50]; HRMS (MALDI) calculated for C₁₇H₁₆O₄ [M]⁺: 284.1049, found 284.4357 (Scheme 1).

Scheme 1: Synthesis of the caffeic acid derivatives: (a) ROH, H₂SO₄, reflux; (b) Et₃N, RX, acetone, reflux; (c) DMF, K₂CO₃, RX, room temperature; (d) ROH, THF, TPP, DIAD, 0°C to room temperature.

4. Antibacterial Assay

The microbiological testing used single strains of Staphylococcus aureus (ATCC-25619) and Escherichia coli (ATCC-2536). The strains belonged to the Bacterioteca of the Laboratory of Toxicological Tests, Institute of Research in Drugs and Medications, Paraíba Federal University. The strains were maintained in an appropriate culture medium, BHI (Difco Laboratories/France/USA), and stored at 4°C and 35°C. The microorganism suspension was prepared according to McFarland tube 0.5, corresponding to approximately 10⁸ CFU·ml⁻¹ [54, 55].

Culture Medium. The antifungal activity assays were performed in brain heart infusion (BHI) broth liquid medium (Difco Laboratories/France/USA), which was prepared and used according to the manufacturer’s instructions.

Determination of the Minimum Inhibitory Concentration (MIC). The MIC values were determined by the microdilution method [54] using 96-well “U”-shaped microtiter plates in duplicate. In each plate well, 100 μl of twice concentrated BHI liquid medium was added, except the first line which received 160 μl. Then, 40 μl of product solution (also doubly concentrated) was placed in the first row of the plate wells. By serial dilution (ratio of two), concentrations from 200–12.5 μg/ml were obtained, such that in the first line of the plate was the highest concentration and in the latter lower concentration. Finally, another 100 μl of the BHI medium was added to all wells, and 10 μl of inoculum was added to the wells in each plate column that referred specifically to a strain. The same was also done in the culture medium with the bacterial drug chloramphenicol (100 mg/ml). The plates were incubated at 37°C for 24 h for further reading; this was performed with the addition of 10 μl of a 0.01% (w/v) solution of resazurin (Sigma), a colorimetric metabolic activity indicator. For each strain, the MIC was defined as the lowest concentration capable of inhibiting bacterial growth in the wells, as visually observed compared with the control. All of the tests were performed in duplicate, and the results were expressed as a geometric mean of the MIC values obtained from both tests [56].

5. 3D-QSAR Modeling

Otherwise noted, default parameters were employed for all software. One initial 3D conformer per compound was generated with OpenEye Omega [57]. Each compound’s initial conformation was subjected to 200 cycles (100 ps each) of quenched molecular dynamics simulations with Open3DAlign [58] using an implicit solvent model. Molecules alignment was also performed with Open3DAlign by means of its mixed algorithm considering multiple conformers for the template and candidate compounds.

Bioactivities were converted to pIC₅₀ values (), and QSAR models were separately trained for Escherichia coli and Staphylococcus aureus using Open3DQSAR [59]. The box step for the grid was set to 0.5 Å, and van der Waals (VDW) and electrostatic molecular interaction fields (MIFs) were computed. The dielectric constant was set to 4. Grid points providing VDW values larger than 10⁴ kcal/mol were removed from both MIFs. Values of the grid exceeding 30 kcal/mol or below −30 kcal/mol were set to 30 kcal/mol and −30 kcal/mol, respectively, for both MIFs. In addition, all grid points with an absolute value lower than 0.05 were set to zero, and variables with standard deviation lower than 0.1 were excluded from the calculations. Variables spanning up to four levels were also excluded. After these variables filtering steps, those remaining in the dataset were scaled according to the BUW algorithm as implemented in Open3DQSAR.

We considered up to 10 principal components (PCs) for models training. The optimal number of PCs considered in the final model was selected according to the r² value of the leave-one-out (LOO) cross validation of the model (q²LOO). With this optimal number of PCs, variables were further grouped using the Smart Region Definition procedure implemented in Open3DQSAR. The grouped variables were subjected 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 the previous step remained on the dataset. Finally, given the limited number of available samples, the PLS model was recomputed and its quality was evaluated in 100 cycles of LMO cross validation.

6. Results and Discussion

6.1. Antibacterial Activity and Structure-Activity Relationship (SAR)

In the current work, a set of sixteen caffeic acid ester derivatives were evaluated for their in vitro antibacterial activity against strains of Staphylococcus aureus and Escherichia coli. Methyl caffeate (1), ethyl caffeate (2), propyl caffeate (3), isopropyl caffeate (4), methoxy-ethyl caffeate (5), butyl caffeate (6), pentyl caffeate (7), isopentyl caffeate (8), decyl caffeate (9), diphenylmethyl caffeate (10), 4-chlorobenzyl caffeate (11), 4-methoxybenzyl caffeate (12), (di-(4-chlorobenzyl)) caffeate (13), (di-(4-methoxybenzyl)) caffeate (14), benzyl caffeate (15), and 4-methylbenzyl caffeate (16) each with differing substitutions on the ester side chain (R₁) and the hydroxyl of the para position of the caffeic ring (R₂) were tested. The results were expressed as minimal inhibitory concentration (MIC) in μM. The structure-activity relationship (SAR) study verified which chemical structures influenced the antibacterial activity. In addition, a quantitative structure-activity relationship (QSAR) was performed to verify contributions of steric and electrostatic factors towards the Escherichia coli and Staphylococcus aureus activity.

The results obtained in this study revealed that five compounds presented strong to optimum antibacterial activity; the compounds were found to be more active against the Escherichia coli strain. Of the sixteen ester derivatives evaluated, propyl caffeate (3), butyl caffeate (6), and pentyl caffeate (7) were found to exhibit the best antibacterial activity of the products with alkyl side chains, with an MIC value of 0.2 μM against the Escherichia coli strain and only butyl caffeate (6) showed the same MIC for the Staphylococcus aureus strain, whereas of the products with aryl side chains, the best MIC results against the Escherichia coli strain were 0.23 µM for (di-(4-chlorobenzyl)) caffeate (13) and 0.29 µM for diphenylmethyl caffeate (10), both being less active against Staphylococcus aureus. Table 1 summarizes the in vitro susceptibilities of the two Escherichia coli and Staphylococcus aureus strains against all of the test compounds.

Table 1: Minimal inhibitory concentration (MIC) values (expressed in µM) of the compounds 1–16.

Compounds
MIC (µM)

Staphylococcus aureus (ATCC-25619)
Escherichia coli (ATCC-2536)

1
0.52
0.52

2
0.48
0.48

3
0.45
0.22

4
0.45
0.45

5
0.84
0.84

6
0.21
0.21

7
0.40
0.20

8
0.80
0.40

9
0.62
0.62

10
0.58
0.29

11
1.31
1.31

12
0.67
0.33

13
0.93
0.23

14
0.95
0.48

15
0.74
0.37

16
1.41
0.70

Chloramphenicol
0.31
0.31

Of the screened ester derivatives, certain structural features were observed that may increase antibacterial activity, giving information on functional groups that might be important to antibacterial effect, such as alkyl ester side chain, especially a median linear carbon chain, benzyl radicals with two aromatic rings without substituents, and electron-withdrawal substituents in the para position such as chloro.

Data analyses revealed that introduction of alkyl substituents as short chains and the presence of a heteroatom and long (10 carbon atoms) or branching chains resulted in decreased antibacterial effect. For example, butyl caffeate (6: butyl group) was found to be the most bioactive compound for both strains (MIC = 0.20 μM), whereas methyl caffeate (1; methyl group), ethyl caffeate (2; ethyl group), isopropyl caffeate (4; isopropyl group), methoxyethyl caffeate (5; methoxyethyl group), isopentyl caffeate (8; isopentyl group), and decyl caffeate (9; decyl group) presented increased MICs (decreased activities). This reduction in antibacterial activity probably occurs due to steric hindrance caused by the presence of bulky groups and also poorly lipophilic short-chain groups. Activity achieved a maximum for the esters with a median linear carbon chain conferring lipophilicity to the molecules.

The compounds presenting aryl radicals resulted in decreased antibacterial effect, such as aromatic ring substituents with electron donor groups in the para position such as methyl and methoxy or with electron withdrawal groups such as chloro (except compound (13) which was active against E. coli). Those without substituents also resulted in decreased antibacterial effect, except compound (10) which was active against E. coli. The presence of an electron-withdrawing group such as di-(4-chlorobenzyl) caffeate (13; di-(4-chlorobenzyl) group) provided more activity against the Escherichia coli strain, which probably occurred due to the para chloro substituent in the caffeic ring, which was the only difference compared to 4-chlorobenzyl caffeate (11; chlorobenzyl group), and presented similarity to the reference drug having two chloro substituents. Compound (10) presented good results against Escherichia coli due to the presence of bulky groups without substituents with two aromatic rings, such as with diphenylmethyl caffeate (10; diphenylmethyl group); this was compared to benzyl caffeate (15; benzyl group), which presented a better bond to its target.

The antimicrobial activity of caffeic acid derivatives has been reported in other studies [43, 60–65]. For example, Merkl et al. [66] investigated the antibacterial activity of a series of caffeic acid esters (methyl, ethyl, propyl, and butyl caffeate) against E. coli DMF 7503. All of the esters screened showed potent antibacterial activity against the test strain, principally propyl and butyl caffeate, with MIC values of 5.00 mM. The MIC values of the other caffeic acid esters tested were roughly 10.00 mM [67]. In the study of Meyuhas et al. [42], the phenethyl caffeate exhibited antibacterial activity against S. aureus (48 mM) and E. coli (254 mM). This compound has structural similarity to ester 15, which showed strong antibacterial action (Table 1). The ester derivatives of caffeic acid were prepared using short linear carbon chain radicals, such as methyl and ethyl caffeate, and from them, compounds synthesized with (4-chloro-benzyl) radicals on the two hydroxyls of the meta and para positions of the caffeic ring. The antibacterial activities of the compounds were evaluated against E. coli and S. aureus; the esters, methyl and ethyl caffeates, showed activity for both strains, while their by-products were only active for the E. coli strain. Derivatives thus prepared are promising candidates for treating bacterial pathologies, and their test results corroborate those already published in the literature.

6.2. Quantitative Structure-Activity Relationship (QSAR) Studies

The statistical parameters of the 3D-QSAR models obtained for the E. coli and S. aureus inhibitory activities are summarized in Table 2. As can be seen, both models have good statistical parameters. Furthermore, for both E. coli and S. aureus, bioactivity is mainly sterically driven. Also, given the lower number of PCs included in the S. aureus model and its statistical parameters, it can be considered statistically more robust.

Table 2: Statistical parameters of the 3D-QSAR models for the antibacterial activity against E. coli and S. aureus.

E. coli
S. aureus

No. PCsa
4
2

R2b
0.95
0.86

q²LOOc
0.70
0.76

q²LMOd
0.68
0.74

VDW contrib.e
77%
73%

Ele. contrib.f
23%
27%

aNumber of PCs in the final model. bCoefficient of determination. cLOO cross-validation performance. dLMO cross-validation performance. e, fContribution of VDW and electrostatic factors to bioactivity, respectively.

6.2.1. Scaffold

Bioactivity is mostly steric-guided. Models show 77% and 73% contributions of the steric factor to the Escherichia coli and Staphylococcus aureus activities, respectively (Figure 1).

Figure 1

6.2.2. Electrostatic Molecular Interaction Field

The electrostatic contribution to bioactivity shows that certain substitutions at R₂ can improve bioactivity. In general, the electronegative molecular interaction field next to the O-R₂ bond indicates a positive contribution to bioactivity of substitutions at this position. This positive contribution cannot be exploited when R₂ = H. This later observation holds for both Escherichia coli and Staphylococcus aureus. Figures 2 and 3 summarize the electrostatic contribution to bioactivity in both strains.

(a)

(b)

Figure 2: Electrostatic molecular interaction field for compounds 11 and 13 in Escherichia coli.

(a)

(b)

Figure 3: Electrostatic molecular interaction field for compounds 11 and 13 in Staphylococcus aureus.

The comparisons of compounds 11 and 13 and of compounds 12 and 14 reveal some interesting details. In the case of Escherichia coli, there is an electronegative molecular interaction field close to the 4-chlorobenzyl substitution at R₂, indicating that benzyl groups with electronegative para-substitutions can have an additional positive contribution to bioactivity against this strain. In contrast, substitutions at R₂ such as 4-methoxybenzyl present in compound 14 are unable to increase the bioactivity of R₂-substituted compounds. This can be clearly exemplified for compounds 12 and 14.

The lack of any additional electrostatic molecular interaction field surrounding R₂ in the Staphylococcus aureus model indicates that no increase in the anti-S. aureus bioactivity could be obtained with specific substitutions at R₂.

6.2.3. Steric Molecular Interaction Field

The analysis of the steric molecular interaction field shows that there is a steric favorable region around R₁ in both E. coli and S. aureus models. Compounds with small aliphatic substituents at R₁ are favored, while too bulky or too long substituents such as long aliphatic chains and aromatic substitutions are disfavored at this position. This effect is more pronounced in the Staphylococcus aureus model where there is a large unfavorable steric region close to R₁. For example, the R₁ substituent of compound 6 completely falls into the steric favorable region defined by both strains models. On the other hand, the 4-methylbenzyl substituent of compound 16 falls beyond the steric favorable region in Escherichia coli and in the steric unfavorable region defined by the Sthaphylococcus aureus model. In addition, bulky substitutions at R₂ (such as those present in compound 14) decrease anti-E. coli bioactivity. The influence of the steric factor in bioactivity is summarized in Figures 4 and 5.

(a)

(b)

Figure 4: Steric contribution to Escherichia coli bioactivity for compounds 6 and 16.

(a)

(b)

Figure 5: Steric contribution to Staphylococcus aureus bioactivity for compounds 6 and 16.

Considering all these observations, for dual Escherichia coli and Staphylococcus aureus bioactivity, small aliphatic substitutions are essential at R₁. Furthermore, basing on the available data, substitutions at R₂ could be beneficial for dual bioactivity. This last hypothesis should be confirmed by testing more compounds substituted at R₂ with small groups.

Our QSAR studies confirmed the biological activity of the tested compounds, connecting with chemical structures for (di-(4-chlorobenzyl)) caffeate (13), through the electronegative electrostatic molecular interaction field at R₂ as against Escherichia coli and for butyl caffeate (6), due to the small aliphatic radical completely falling into the sterically favorable region as defined by both strain models. In contrast to these, 4-methylbenzyl caffeate (16) exceeded the sterically favorable regions of these models and presented the worst bacterial activity against the tested strains.

7. Conclusion

The present study investigated the antibacterial activity of sixteen caffeic acid ester derivatives against a Gram-positive Staphylococcus aureus strain and a Gram-negative Escherichia coli strain. Based on the findings of the present investigation, we conclude that the propyl, butyl, pentyl (di-(4-chlorobenzyl)), and diphenylmethyl caffeate derivatives present the highest antibacterial activity against the Escherichia coli strain; only butyl caffeate was active against the Staphylococcus aureus strain. Further, the QSAR study demonstrated that small aliphatic substituents in R₁ (butyl caffeate) and substituents in the aromatic ring, with electron withdrawal groups in the para position in R₂ ((di-(4-chlorobenzyl)) caffeate), result in bioactive compounds. This preliminary study revealed that certain structural features are important for antibacterial action. Additional studies should be performed to optimize antimicrobial activity in this class of compounds for eventual synthesis of new antibacterial agents.

Data Availability

The data used to support the findings of this study have been deposited in the Federal University of Paraíba repository at https://repositorio.ufpb.br/jspui/handle/tede/9074.

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).

Supplementary Materials

Spectroscopy data of known compounds. ¹³C-NMR and ¹H-NMR spectra of the unpublished compounds: methoxyethyl caffeate, diphenylmethyl caffeate, 4-chlorobenzyl caffeate, (di-(4-chlorobenzyl)) caffeate, (di-(4-methoxybenzyl)) caffeate, and 4-methylbenzyl caffeate. (Supplementary Materials)

References

I. C. Bygbjerg, “Double burden of noncommunicable and infectious diseases in developing countries,” Science, vol. 337, no. 6101, pp. 1499–1501, 2012.

R. J. Coker, B. M. Hunter, J. W. Rudge, M. Liverani, and P. Hanvoravongchai, “Emerging infectious diseases in southeast Asia: regional challenges to control,” The Lancet, vol. 377, no. 9765, pp. 599–609, 2011.

L. Wannmacher, “Uso indiscriminado de antibióticos e resistência microbiana: uma guerra perdida?” Uso racional de medicamentos:temas selecionados, vol. 1, no. 4, 2004.

A. Breves, C. A. C. Miranda, C. Flores, I. d. Filippis, and M. M. Clementino, “Methicillin- and vancomycin-resistantStaphylococcus aureusin health care workers and medical devices,” Jornal Brasileiro de Patologia e Medicina Laboratorial, vol. 51, no. 3, pp. 143–152, 2015.

S. Shaikh, J. Fatima, S. Shakil, S. M. D. Rizvi, and M. A. Kamal, “Antibiotic resistance and extended spectrum beta-lactamases: types, epidemiology and treatment,” Saudi Journal of Biological Sciences, vol. 22, no. 1, pp. 90–101, 2015.

A. I. Fattom, G. Horwith, S. Fuller, M. Propst, and R. Naso, “Development of StaphVAX, a polysaccharide conjugate vaccine against S. aureus infection: from the lab bench to phase III clinical trials,” Vaccine, vol. 22, no. 7, pp. 880–887, 2004.

E. A. Palombo and S. J. Semple, “Antibacterial activity of Australian plant extracts against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE),” Journal of Basic Microbiology, vol. 42, no. 6, pp. 444–448, 2002.

S. Das, C. Lindemann, B. C. Young et al., “Natural mutations in aStaphylococcus aureusvirulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation,” in Proceedings of the National Academy of Sciences, vol. 113, no. 22, pp. E3101–E3110, 2016.

J. A. Lindsay and M. T. G. Holden, “Staphylococcus aureus: superbug, super genome?” Trends in Microbiology, vol. 12, no. 8, pp. 378–385, 2004.

L. C. Gelatti, R. R. Bonamigo, A. P. Becker, and P. A. d’Azevedo, “Staphylococcus aureus resistentes à meticilina: disseminação emergente na comunidade,” Anais Brasileiros de Dermatologia, vol. 84, no. 5, pp. 501–506, 2009.

G. A. Oliveira, A. M. Dell’Aquila, R. L. Masiero et al., “Isolation in Brazil of nosocomial Staphylococcus aureus with reduced susceptibility to vancomycin,” Infection Control & Hospital Epidemiology, vol. 22, no. 7, pp. 443–448, 2015.

S. Chang, D. M. Sievert, J. C. Hageman et al., “Infection with vancomycin-ResistantStaphylococcus aureusContaining thevanAResistance Gene,” New England Journal of Medicine, vol. 348, no. 14, pp. 1342–1347, 2003.

M. Rajilic-Stojanovic, H. Smidt, and W. M. De Vos, “Diversity of the human gastrointestinal tract microbiota revisited,” Environmental Microbiology, vol. 9, no. 9, pp. 2125–2136, 2007.

S. Hudault, J. Guignot, and A. L. Servin, “Escherichia coli strains colonising the gastrointestinal tract protect germfree mice against Salmonella typhimurium infection,” Gut, vol. 49, no. 1, pp. 47–55, 2001.

A. Sabrá, “ECEP, ECET, ECEA, ECEH, ECEI, ECAD: a E. coli revisitada no contexto da diarréia aguda,” Jornal de Pediatria, vol. 77, no. 1, pp. 5–7, 2002.

J. B. Kaper, J. P. Nataro, and H. L. T. Mobley, “Pathogenic Escherichia coli.,” Nature Reviews Microbiology, vol. 2, no. 2, pp. 123–140, 2004.

K. Bush and G. A. Jacoby, “Updated functional classification of -lactamases,” Antimicrobial Agents and Chemotherapy, vol. 54, no. 3, pp. 969–976, 2009.

D. M. Livermore, “Defining an extended-spectrum β-lactamase,” Clinical Microbiology and Infection, vol. 14, no. 1, pp. 3–10, 2008.

D. L. Paterson, “Recommendation for treatment of severe infections caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs),” Clinical Microbiology and Infection, vol. 6, no. 9, pp. 460–463, 2000.

D. L. Paterson, W.-C. Ko, A. Von Gottberg et al., “Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum -lactamases,” Clinical Infectious Diseases, vol. 39, no. 1, pp. 31–37, 2004.

M. Gonzalez-Padilla, J. Torre-Cisneros, F. Rivera-Espinar et al., “Gentamicin therapy for sepsis due to carbapenem-resistant and colistin-resistant Klebsiella pneumoniae,” Journal of Antimicrobial Chemotherapy, vol. 70, no. 3, pp. 905–913, 2014.

R. Moayednia, D. Shokri, S. Mobasherizadeh, A. Baradaran, S. M. Fatemi, and A. Merrikhi, “Frequency assessment of β-lactamase enzymes in Escherichia coli and Klebsiella isolates in patients with urinary tract infection,” Journal of Research in Medical Sciences, vol. 1, no. 19, pp. S41–S45, 2014.

F. M. da Cunha, D. Duma, J. Assreuy et al., “Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties,” Free Radical Research, vol. 38, no. 11, pp. 1241–1253, 2009.

S. Juman, N. Yasui, K. Ikeda et al., “Caffeic acid phenethyl ester suppresses the production of pro-inflammatory cytokines in hypertrophic adipocytes through lipopolysaccharide-stimulated macrophages,” Biological and Pharmaceutical Bulletin, vol. 35, no. 11, pp. 1941–1946, 2012.

Y.-J. Lee, P.-H. Liao, W.-K. Chen, and C.-C. Yang, “Preferential cytotoxicity of caffeic acid phenethyl ester analogues on oral cancer cells,” Cancer Letters, vol. 153, no. 1-2, pp. 51–56, 2000.

Z. Orban, N. Mitsiades, T. R. Burke, M. Tsokos, and G. P. Chrousos, “Caffeic acid phenethyl ester induces leukocyte apoptosis, modulates nuclear factor-kappa B and suppresses acute inflammation,” Neuroimmunomodulation, vol. 7, no. 2, pp. 99–105, 2000.

T. Toyoda, T. Tsukamoto, S. Takasu et al., “Anti-inflammatory effects of caffeic acid phenethyl ester (CAPE), a nuclear factor-κB inhibitor, onHelicobacter pylori-induced gastritis in Mongolian gerbils,” International Journal of Cancer, vol. 125, no. 8, pp. 1786–1795, 2009.

M. R. Fesen, K. W. Kohn, F. Leteurtre, and Y. Pommier, “Inhibitors of human immunodeficiency virus integrase,” in Proceedings of the National Academy of Sciences, vol. 90, no. 6, pp. 2399–2403, 1993.

K. Natarajan, S. Singh, T. R. Burke, D. Grunberger, and B. B. Aggarwal, “Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B,” in Proceedings of the National Academy of Sciences, vol. 93, no. 17, pp. 9090–9095, 1996.

C.-n. Xia, H.-b. Li, F. liu, and W.-x. Hu, “Synthesis of trans-caffeate analogues and their bioactivities against HIV-1 integrase and cancer cell lines,” Bioorganic & Medicinal Chemistry Letters, vol. 18, no. 24, pp. 6553–6557, 2008.

A. Russo, R. Longo, and A. Vanella, “Antioxidant activity of propolis: role of caffeic acid phenethyl ester and galangin,” Fitoterapia, vol. 73, no. 1, pp. S21–S29, 2002.

H. Yang, Y. Dong, H. Du, H. Shi, Y. Peng, and X. Li, “Antioxidant compounds from propolis collected in Anhui, China,” Molecules, vol. 16, no. 4, pp. 3444–3455, 2011.

J. H. Park, J. K. Lee, H. S. Kim et al., “Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice,” International Immunopharmacology, vol. 4, no. 3, pp. 429–436, 2004.

Y.-J. Chen, M.-S. Shiao, M.-L. Hsu, T.-H. Tsai, and S.-Y. Wang, “Effect of caffeic acid phenethyl ester, an antioxidant from propolis, on inducing apoptosis in human leukemic HL-60 cells,” Journal of Agricultural and Food Chemistry, vol. 49, no. 11, pp. 5615–5619, 2001.

C.-P. Chuu, H.-P. Lin, M. F. Ciaccio et al., “Caffeic acid phenethyl ester suppresses the proliferation of human prostate cancer cells through inhibition of p70S6K and Akt signaling networks,” Cancer Prevention Research, vol. 5, no. 5, pp. 788–797, 2012.

H. P. Lin, S. S. Jiang, and C. P. Chuu, “Caffeic acid phenethyl ester causes p21(Cip1) induction, akt signaling reduction, and growth inhibition in PC-3 human prostate cancer cells,” PLoS One, vol. 7, no. 2, pp. 1–12, 2012.

T. Nagaoka, A. H. Banskota, Y. Tezuka, I. Saiki, and S. Kadota, “Selective antiproliferative activity of caffeic acid phenethyl ester analogues on highly liver-metastatic murine colon 26-L5 carcinoma cell line,” Bioorganic & Medicinal Chemistry, vol. 10, no. 10, pp. 3351–3359, 2002.

A. Ilhan, M. Iraz, A. Gurel, F. Armutcu, and O. Akyol, “Caffeic acid phenethyl ester exerts a neuroprotective effect on CNS against pentylenetetrazol-induced seizures in mice,” Neurochemical Research, vol. 29, no. 12, pp. 2287–2292, 2004.

J. Breger, B. B. Fuchs, G. Aperis, T. I. Moy, F. M. Ausubel, and E. Mylonakis, “Antifungal chemical compounds identified using a C. elegans pathogenicity assay,” PLoS Pathogens, vol. 3, no. 2/e18, pp. 168–178, 2007.

A. Kujumgiev, V. Bankova, A. Ignatova, and S. Popov, “Antibacterial activity of propolis, some of its components and their analogs,” Pharmazie, vol. 48, no. 10, pp. 785–6, 1993.

G. Murtaza, S. Karim, M. R. Akram et al., “Caffeic acid phenethyl ester and therapeutic potentials,” BioMed Research International, vol. 2014, pp. 1–9, 2014.

S. Meyuhas, M. Assali, M. Huleihil, and M. Huleihel, “Antimicrobial activities of caffeic acid phenethyl ester,” Journal of Molecular Biochemistry, vol. 4, pp. 21–31, 2015.

C. Velazquez, M. Navarro, A. Acosta et al., “Antibacterial and free-radical scavenging activities of Sonoran propolis,” Journal of Applied Microbiology, vol. 103, no. 5, pp. 1747–1756, 2007.

M. O. De Farias, T. C. Lima, A. L. A. L. Pérez et al., “Antifungal activity of ester derivatives from caffeic acid against Candida species,” International Journal of Pharmacy & Pharmaceutical Research, vol. 7, no. 1, pp. 151–159, 2016.

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.

W. Li, N. Li, Y. Tang et al., “Biological activity evaluation and structure-activity relationships analysis of ferulic acid and caffeic acid derivatives for anticancer,” Bioorganic & Medicinal Chemistry Letters, vol. 22, no. 19, pp. 6085–6088, 2012.

H. Shi, D. Xie, R. Yang, and Y. Cheng, “Synthesis of caffeic acid phenethyl ester derivatives, and their cytoprotective and neuritogenic activities in PC12 cells,” Journal of Agricultural and Food Chemistry, vol. 62, no. 22, pp. 5046–5053, 2014.

J. G. Handique, D. Mahanta, A. Devi, and M. P. Boruah, “Synthesis and electrochemical behavior of some dendritic polyphenols as antioxidants,” Letters in Organic Chemistry, vol. 10, no. 1, pp. 53–59, 2013.

P. Xiao, S. Zhang, H. Ma, A. Zhang, X. Lv, and L. Zheng, “Stereoselective synthesis of caffeic acid amides via enzyme-catalyzed asymmetric aminolysis reaction,” Journal of Biotechnology, vol. 168, no. 4, pp. 552–559, 2013.

J. T. Sanderson, H. Clabault, C. Patton et al., “Antiproliferative, antiandrogenic and cytotoxic effects of novel caffeic acid derivatives in LNCaP human androgen-dependent prostate cancer cells,” Bioorganic & Medicinal Chemistry, vol. 21, no. 22, pp. 7182–7193, 2013.

J. Zhang, L. X. Xu, X. S. Xu, B. W. Li, R. Wang, and J. J. Fu, “Synthesis and effects of new caffeic acid derivatives on nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages,” International Journal of Clinical and Experimental Medicine, vol. 7, no. 4, pp. 1022–1027, 2014.

N.-G. Li, R. Wang, Y.-P. Tang et al., “Design, synthesis and biological study of novel NO-donor-caffeic acid hybrids as potential anti-atherosclerotic drug candidates,” Letters in Drug Design & Discovery, vol. 8, no. 6, pp. 550–557, 2011.

K. D. Kim, M. H. Song, E. K. Yum, O. S. Jeon, Y. W. Ju, and M. S. Chang, “2,4-Dihydroxycinnamic esters as skin depigmenting agents,” Bulletin of the Korean Chemical Society, vol. 30, no. 7, pp. 1619–1621, 2009.

R. Cleeland and E. Squires, “Evaluation of new antimicrobials “in vitro” and in experimental animal infections,” in Antibiotics in Laboratory Medicine, V. M. D. Lorian, Ed., Lippincott Williams & Wilkins, Baltimore, MD, USA, 1991.

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.

P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, USA, 1994.

P. C. D. Hawkins, A. G. Skillman, G. L. Warren, B. A. Ellingson, and M. T. Stahl, OMEGA, “Santa Fe, NM: OpenEye Scientific Software”, 1997, http://www.eyesopen.com.

P. Tosco, T. Balle, and F. Shiri, “Open3DALIGN: an open-source software aimed at unsupervised ligand alignment,” Journal of Computer-Aided Molecular Design, vol. 25, no. 777, 2011.

P. Tosco and T. Balle, “Open3DQSAR: a new open-source software aimed at high-throughput chemometric analysis of molecular interaction fields,” Journal of Molecular Modeling, vol. 17, no. 1, pp. 201–208, 2010.

C. Balachandran, V. Duraipandiyan, N. A. Al-Dhabi et al., “Antimicrobial and antimycobacterial activities of methyl caffeate isolated from solanum torvum swartz. Fruit,” Indian Journal of Microbiology, vol. 52, no. 4, pp. 676–681, 2012.

J. Fu, K. Cheng, Z.-m. Zhang, R.-q. Fang, and H.-l. 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.

V. D. Gangan, L. Jazly, C. T. Chakraborty et al., “Ethyl caffeate ether derivatives as future potential drug,” Journal of Pharmacy Research, vol. 8, no. 6, pp. 818–821, 2014.

M. Kartal, S. Yıldız, S. Kaya, S. Kurucu, and G. Topçu, “Antimicrobial activity of propolis samples from two different regions of Anatolia,” Journal of Ethnopharmacology, vol. 86, no. 1, pp. 69–73, 2003.

I. K. Lee, K. H. Kim, S. U. Choi, J. H. Lee, and K. R. Lee, “Phytochemical constitutents of Thesium chinense Turcz and their cytotoxic activities in vitro,” Natural Product Scienses, vol. 15, no. 4, pp. 246–249, 2009.

A. Uzel, K. y. Sorkun, Ö. Önçağ, D. Çoğulu, Ö. Gençay, and B. r. Sali˙h, “Chemical compositions and antimicrobial activities of four different Anatolian propolis samples,” Microbiological Research, vol. 160, no. 2, pp. 189–195, 2005.

R. Merkl, I. Hrádková, V. Filip, and J. Šmidrkal, “Antimicrobial and antioxidant properties of phenolic acids alkyl esters,” Czech Journal of Food Sciences, vol. 28, no. 4, pp. 275–279, 2010.

L. Jazly, V. D. Gangan, C. T. Chakraborty, A. V. Tamhankar, J. J. Kadam, and S. Bhalekar, “Synthesis and antibacterial activity of novel Caffeic acid ester derivatives,” Journal of Pharmacy Research, vol. 1, no. 10, pp. 942–946, 2013.