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Article

Chalcones and Flavanones Bearing Hydroxyl and/or Methoxyl Groups: Synthesis and Biological Assessments

by
Gonçalo P. Rosa
1,
Ana M. L. Seca
1,2,
Maria do Carmo Barreto
1,
Artur M. S. Silva
2 and
Diana C. G. A. Pinto
2,*
1
cE3c—Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group & University of Azores, Rua Mãe de Deus, 9501-801 Ponta Delgada, Portugal
2
QOPNA & LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
*
Author to whom correspondence should be addressed.
Appl. Sci. 2019, 9(14), 2846; https://doi.org/10.3390/app9142846
Submission received: 20 June 2019 / Revised: 10 July 2019 / Accepted: 14 July 2019 / Published: 17 July 2019
(This article belongs to the Special Issue Biological Activity and Applications of Natural Compounds)
Browse Figures
Versions Notes

Abstract

:
Chalcones and flavanones are isomeric structures and also classes of natural products, belonging to the flavonoid family. Moreover, their wide range of biological activities makes them key scaffolds for the synthesis of new and more efficient drugs. In this work, the synthesis of hydroxy and/or methoxychalcones was studied using less common bases, such as sodium hydride (NaH) and lithium bis(trimethylsilyl)amide (LiHMDS), in the aldol condensation. The results show that the use of NaH was more effective for the synthesis of 2′-hydroxychalcone derivatives, while LiHMDS led to the synthesis of polyhydroxylated chalcones in a one-pot process. During this study, it was also possible to establish the conditions that favor their isomerization into flavanones, allowing at the same time the synthesis of hydroxy and/or methoxyflavanones. The chalcones and flavanones obtained were evaluated to disclose their antioxidant, anticholinesterasic, antibacterial and antitumor activities. 2′,4′,4-Trihydroxychalcone was the most active compound in terms of antioxidant, anti-butyrylcholinesterase (IC50 26.55 ± 0.55 μg/mL, similar to control drug donepezil, IC50 28.94 ± 1.76 μg/mL) and antimicrobial activity. 4′,7-Dihydroxyflavanone presented dual inhibition, that is, the ability to inhibit both cholinesterases. 4′-Hydroxy-5,7-dimethoxyflavanone and 2′-hydroxy-4-methoxychalcone were the compounds with the best antitumor activity. The substitution pattern and the biological assay results allowed the establishment of some structure/activity relationships.

Graphical Abstract

1. Introduction

The (E)-1,3-diphenylpropen-1-ones, best known as chalcones, belong to the flavonoids family and are an important class of natural products across the plant kingdom [1]. Structurally, these compounds contain two aromatic rings, bonded by a three-carbon α,β unsaturated carbonyl bridge (Figure 1), which are synthesized in plants as the C15 key intermediate in the biosynthesis of the other flavonoids [2]. Flavanones are also naturally occurring compounds and are chalcones’ isomeric forms. In fact, the equilibrium between chalcones and flavanones is common in nature and is regulated by chalcone-isomerase [3].
Besides their natural occurrence, both chalcones and flavanones can be obtained synthetically and are often used as the preferred starting material for the synthesis of other polycyclic aromatic compounds [4]. Furthermore, they present great pharmacological potential with a wide variety of biological activities, including antioxidant [5], anticancer [6,7,8], and antimicrobial activities [9,10,11,12], and also the ability to treat cardiovascular diseases and their risk factors [13,14,15,16], among others [17].
On the other hand, cancer, neurodegenerative diseases, oxidative stress-related diseases and multi-resistant bacterial infections are, after cardiovascular diseases, the top four health problems that cause the most victims every year, leading to higher medicine consumption and putting great pressure on the national health systems of many countries [18,19,20,21,22,23,24]. These problems occur either due to a lack of effective medicines to treat diseases, such as neurodegenerative ones, or due to the increasing drug resistance presented by numerous pathogenic bacteria and by some cancers. Therefore, exploring well-known scaffolds as lead compounds will help in the battle against diseases that affect humanity.
Putting together the facts stated above, the synthesis of chalcone-based functionalized derivatives remains a popular research objective. The most common and efficient approach to obtain the chalcone nucleus is the aldol condensation of substituted acetophenones with proper substituted benzaldehydes in the presence of a base, namely sodium or potassium hydroxide [25,26,27,28]. Despite the efficiency of this method, when planning a synthesis some drawbacks should be considered. For instance, the protection of the reagents’ hydroxyl groups should be done previously, the acetophenone hydrogen α acidity should be analyzed, and by-products can be obtained if the bases are also good nucleophilic species [29,30].
In this regard, the objective of this work is to synthesize hydroxy- and/or methoxychalcones by aldol condensation, using the less common bases sodium hydride and lithium bis(trimethylsilyl)amide. Also, this work studies their antioxidant, anticholinesterasic, antibacterial and antitumor activities, aiming to establish some potential medicinal applications. Simultaneously, a structure/activity relationship was established, and the isomeric equilibrium chalcone-flavanone was also studied.

2. Materials and Methods

2.1. General Methods

The 1H, 13C, HSQC and HMBC NMR spectra were measured on Bruker AMC 300 or 500 instruments, operating at 300.13 MHz and 75.47 or 500.13 and 125.75 MHz. Chemical shifts were reported relative to tetramethysilane (TMS) in δ units (ppm) and coupling constants (J) in Hz. Chromatographic purifications were carried out by prep. TLC on silica gel (Merck silica gel 60 F254), the spots being visualized under a UV lamp (at 254 and/or 366 nm). Melting points were determined with a Stuart scientific SPM3 apparatus and are uncorrected. The mass spectra were acquired using ESI(+) with a Micromass Q-Tof 2TM mass spectrometer.

2.2. Synthesis of Chalcones and Flavanones

Synthesis of the compounds described below follows the general scheme outlined in 3.1 (Scheme 1).
2′-Hydroxy-4,4′,6′-trimethoxychalcone1. Compound 1 was synthesized by mixing 2′-hydroxy-4′,6′-dimethoxyacetophenone (661.3 mg, 3.37 mmol) dissolved in a minimum (~15 mL) amount of tetrahydrofuran (THF) with sodium hydride (NaH) (2.5 equivalents), under nitrogen atmosphere at room temperature. After 30 min of stirring, 4-methoxybenzaldehyde (1.2 equivalents) was added to the reaction mixture and allowed to react for 3 h. The product was precipitated from the reaction mixture by pouring onto ground ice and acidifying to pH < 2 with HCl 37%. The solid was filtered and washed with water until pH > 5. The crude product was crystallized from ethanol and the desired compound 1 was obtained (928.1 mg, 88% yield).
2′-Hydroxy-4,4′,6′-trimethoxychalcone1: yellow crystals (ethanol); m.p. 112.4–113.6 °C (Lit. 111–115° [31]). 1H NMR (300 MHz, CDCl3) δ 14.40 (1H, s, 2′-OH), 7.79 (2H, s br, H-α, H-β), 7.56 (2H, d, J = 6.8 Hz, H-2, H-6), 6.92 (2H, d, J = 6.8 Hz, H-3, H-5), 6.11 (1H, d, J = 2.4 Hz, H-3′), 5.96 (1H, d, J = 2.4 Hz, H-5′), 3.92 (3H, s, 6′-OCH3), 3.85 (3H, s, 4′-OCH3), 3.83 (3H, s, 4-OCH3); 13C NMR (75 MHz, CDCl3) δ 192.6 (C=O), 168.4 (C-2′), 166.0 (C-4′), 162.5 (C-6′), 161.4 (C-4), 142.5 (C-β), 130.1 (C-2, C-6), 128.3 (C-1), 125.1 (C-α), 114.4 (C-3, C-5), 106.3 (C-1′), 93.8 (C-3′), 91.2 (C-5′), 55.8 (6′-OCH3), 55.6 (4′-OCH3), 55.4 (4-OCH3); TOF-ESI-MS (+) m/z 315 [M+H]+, 337 [M+Na]+, 353 [M+K]+, 651 [M+Na+M]+.
5,7-Dihydroxy-4′-methoxyflavanone4. The procedure to obtain this compound involved 3 different steps:
(a)
The benzylation of the hydroxyl groups: the 4′ and 6′-hydroxyl groups in the starting material 2′,4′,6′-trihydroxyacetophenone were protected using the methodology described by Figueiredo [32]. Briefly, the 2′,4′,6′-trihydroxyacetophenone (2.7 g, 16.1 mmol), dissolved in a minimum amount of dry dimethylformamide (DMF) (~20 mL), was mixed with K2CO3 (6 equivalents) under constant stirring. Then, benzyl bromide (3 equivalents) was added and the reaction was performed at 150 °C under reflux for 2 h. After that, the reaction mixture was filtered to remove the K2CO3 and the inorganic salts washed with DMF. The filtrate was poured over crushed ice and the mixture acidified to pH < 5 with HCl 20%. The precipitated 4′,6′-dibenzyloxy-2′-hydroxyacetophenone was filtered and crystallized from ethanol (4.75 g, 85% yield).
(b)
The aldol condensation: The synthesis of 4′,6′-dibenzyloxy-2′-hydroxy-4-methoxychalcone 2 was performed by dissolving 4′,6′-benzyloxy-2′-hydroxyacetophenone (1.5566 g) in dried THF and was then mixed with NaH (2.5 equivalents). After 10 min of stirring under nitrogen atmosphere at room temperature, 4-methoxybenzaldehyde (1.2 equivalents) was added. The reaction was finished after 3 h by pouring over crushed ice and addition of HCl 37% to pH < 2. The precipitate was filtered and washed with water, and the crude product was crystallized from acetone to afford 4′,6′-dibenzyloxy-2′-hydroxy-4-methoxychalcone 2 (1.6573 g, 80% yield).
(c)
The benzyl group’s cleavage: The final step was deprotecting the hydroxyl groups at 4′ and 6′ positions by cleavage of the benzyl groups. This procedure was adapted from the method described by Gomes et al. [33]. Briefly, 4′,6′-dibenzyloxy-2′-hydroxy-4-methoxychalcone 2 (567.0 mg) was mixed with 40 mL of a mixture of HCl/Acetic acid (1:10) under stirring at 80 °C during 13 h. The reaction was finished by pouring the mixture over crushed ice, the solid formed was washed with water until pH ~ 5 and then purified by TLC, eluting it twice in CH2Cl2, affording 5,7-dihydroxy-4′-methoxy-flavanone 4 (138.7 mg, 59%).
5,7-Dihydroxy-4′-methoxyflavanone4. pale-yellow crystals (CHCl3); m.p. 191.7–193.3 °C (Lit. 193–194 °C [34]). 1H NMR (300 MHz, CDCl3) δ 12.05 (1H, s, 5-OH), 7.37 (2H, d, J = 8.7 Hz, H-2′, H-6′), 6.95 (2H, d, J = 8.7, H-3′, H-5′), 5.99 (1H, s broad, H-6), 5.98 (1H, s broad, H-8), 5.36 (1H, dd, J = 3.0 and 13.0 Hz, H-2), 3.83 (3H, s, 4′-OCH3), 3.10 (1H, dd, J = 13.0 and 17.2 Hz, H-3a), 2.78 (1H, dd, J = 3.0 and 17.2 Hz, H-3b); 13C NMR (75 MHz, CDCl3) δ 196.2 (C-4), 164.8 (C-7), 164.3 (C-5), 163.3 (C-8a), 160.1 (C-4′), 130.3 (C-1′), 127.8 (C-2,’ C-6′), 114.1 (C-3′, C-5′), 103.1 (C-4a), 96.7 (C-6), 95.5 (C-8), 79.0 (C-2), 55.4 (4′-OCH3), 43.1 (C-3); TOF-ESI-MS (+) m/z 287 [M+H]+, 611 [M+K+M]+.
4′-Hydroxy-5,7-dimethoxyflavanone5. The synthesis of this compound also involved the 3 steps mentioned above for compound 4.
(a)
The benzylation of the hydroxyl groups: the 4-hydroxybenzaldehyde (1.3 g, 10.6 mmol) was dissolved in a minimum of dry dimethylformamide (DMF) (~15 mL), and it was mixed with K2CO3 (3 equivalents) under constant stirring. Then, benzyl bromide (1.5 equivalents) was added, and the reaction was performed at 150 °C under reflux for 2 h. After that, the reaction mixture was filtered to remove the K2CO3 and washed with DMF. The filtrate was poured over crushed ice and HCl 20% added until pH < 5. The precipitated 4-benzyloxybenzaldehyde was filtered and crystallized from ethanol (1.8 g, 78%).
(b)
The aldol condensation: The 2′-hydroxy-4′,6′-dimethoxyacetophenone (898.3 mg) was dissolved in dried THF (~15 mL) and mixed with NaH (2.5 equivalents) at room temperature and under N2 atmosphere. After 10 min, 4-benzyloxybenzaldehyde (1.2 equivalents) was added, and the reaction was finished after 4 h by precipitation over crushed ice acidified with HCl 37% to pH < 2. 4-Benzyloxy-2′-hydroxy-4′,6′-dimethoxychalcone 3 was obtained by crystallization from ethanol (1.433 g, 80%).
(c)
The benzyl group’s cleavage: The chalcone 3 (330.6 mg) was mixed with 30 mL of a mixture of HCl/Acetic acid (1:10) under stirring at 55 °C during 60 h. The reaction was finished by pouring the mixture over crushed ice, the formed solid was washed with water until pH ~5 and purified by TLC using hexane/ethyl acetate (1:1) as eluent, affording 4′-hydroxy-5,7-dimethoxyflavanone 5 as pale-yellow amorphous powder (21.0 mg, 8%).
4′-Hydroxy-5,7-dimethoxyflavanone5: 1H NMR (300 MHz, CDCl3) δ 7.29 (2H, d, J = 8.5 Hz, H-2′, H-6′), 6.86 (2H, d, J = 8.5 Hz, H-3′and H-5′), 6.13 (1H, d, J = 2.3 Hz, H-8), 6.07 (1H, d, J = 2.3 Hz, H-6), 5.34 (1H, dd, J = 3.0 and 12.8 Hz, H-2), 3.86 (3H, s, 5-OCH3), 3.81 (3H, s, 7-OCH3), 3.03 (1H, dd, J = 12.8 and 16.6 Hz, H-3a), 2.78 (1H, dd, J = 3.0 and 16.6 Hz, H-3b); 13C NMR (75 MHz, CDCl3) δ 190.2 (C-4), 166.2 (C-7), 165.2 (C-8a), 162.3 (C-5), 156.4 (C-4′), 130.4 (C-1′), 127.9 (C-2′, C-6′), 115.7 (C-3′, C-5′), 105.8 (C-4a), 93.6 (C-8), 93.1 (C-6), 78.9 (C-2), 56.1 (5-OCH3), 55.6 (7-OCH3), 45.2 (C-3); TOF-ESI-MS (+) m/z 301 [M+H]+, 323 [M+Na]+, 623 [M+Na+M]+.
2′,4′,4-Trihydroxychalcone6and 4′,7-dihydroxyflavanone7. The 2′,4′-dihydroxyacetophenone (226.7 mg) was dissolved in dried toluene and mixed with 10 mL of a 1 mol.dm−3 solution of LiHMDS (6.6 equivalents), under nitrogen atmosphere, at room temperature. After 30 min, 4-hydroxybenzaldehyde (1.2 equivalents) was added, and the reaction was stirred for 5 days. The reaction was finished, poured over crushed ice and acidified to pH < 2 with HCl 37%. The mixture was extracted with CH2Cl2 and purified by TLC, using a mixture of hexane and ethyl acetate (1:1) as eluent (twice). Compounds 2′,4′,4-trihydroxychalcone 6 and 4′,7-dihydroxyflavanone 7 were obtained as yellow amorphous powder, respectively, 18.2 mg (5%) and 8.2 mg (2%). Approximately 80% of the starting acetophenone was also recuperated.
2′,4′,4-Trihydroxychalcone6: 1H NMR (300 MHz, acetone-d6) δ 13.69 (1H, s, 2′-OH), 8.12 (1H, d, J = 8.9 Hz, H-6′), 7.85 (1H, d, J = 15.4 Hz, H-β), 7.77 (1H, d, J = 15.4 Hz, H-α), 7.75 (2H, d, J = 8.6 Hz, H-2, H-6), 6.94 (2H, d, J = 8.6 Hz, H-3, H-5), 6.48 (1H, dd, J = 2.4 and 8.9 Hz, H-5′), 6.38 (1H, d, J = 2.4 Hz, H-3′); 13C-NMR (75 MHz, acetone-d6) δ 192.6 (C=O), 167.6 (C-4′), 166.2 (C-2′), 161.1 (C-4), 145.0 (C-β), 133.2 (C-6′), 131.7 (C-2, C-6), 127.4 (C-1), 118.2 (C-α), 116.8 (C-3, C-5), 114.2 (C-1′), 108.9 (C-5′), 103.7 (C-3′); TOF-ESI-MS (+) m/z 257 [M+H]+, 279 [M+Na]+, 535 [M+Na+M]+, 551 [M+K+M]+.
4′,7-Dihydroxyflavanone7: 1H NMR (300 MHz, acetone-d6) δ 9.53 (1H, s, 7-OH), 8.60 (1H, s, 4′-OH), 7.74 (1H, d, J = 8.6 Hz, H-5), 7.42 (2H, d, J = 8.6 Hz, H-2′, H-6′), 6.91 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.59 (1H, dd, J = 2.3 and 8.6 Hz, H-6), 6.43 (1H, d, J = 2.3 Hz, H-8), 5.46 (1H, dd, J = 2.8 and 13.1 Hz, H-2), 3.06 (1H, dd, J = 13.1 and 16.7 Hz, H-3a); 2.68 (1H, dd, J = 2.8 and 16.7 Hz, H-3b); 13C-NMR (75 MHz, acetone-d6) δ 190.5 (C-4), 165.2 (C-7), 164.5 (C-8a), 158.6 (C-4′), 131.2 (C-1′), 129.4 (C-5), 128.9 (C-2′, C-6′), 116.1 (C-3′, C-5′), 115.1 (C-4a), 111.2 (C-6), 103,6 (C-8), 80.5 (C-2), 44.6 (C-3); TOF-ESI-MS (+) m/z 257 [M+H]+, 279 [M+Na]+, 513 [M+H+M]+, 535 [M+Na+M]+, 551 [M+K+M]+.

2.3. Biological Activities

Compound 2′-hydroxy-4-methoxychalcone 8 was provided by the Laboratory of Organic Chemistry of the University of Aveiro, and the reactional conditions, yield and spectroscopic data are reported by Silva et al. [35,36].

2.3.1. DPPH Scavenging Activity

Antioxidant activity was assayed by the DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay [37]. Serial dilutions of studied or reference compounds (Trolox and quercetin) were carried out in 96-well microplates, at different concentrations, ranging between 0.148 μg/mL and 150 μg/mL in methanol. DPPH dissolved in methanol was added to the microwells, yielding a final concentration of 45 μg/mL, and the absorbance at 515 nm was measured in a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), after 30 min in the dark. In each assay, a control was prepared, in which the sample or standard was substituted by the same amount of solvent. Percentage of antioxidant activity (%AA) was calculated as:
%AA = 100 [1 − (Acontrol − Asample)/Acontrol]
where Acontrol is the absorbance of the control, and Asample is the absorbance of the chalcone/flavanone or standard. All assays were carried out in triplicate and results expressed as EC50, i.e., as the concentration yielding 50% scavenging of DPPH, calculated by interpolation from the %AA vs concentration curve.

2.3.2. ABTS Scavenging Activity

To determine ABTS radical scavenging, the method of Re et al. [38] was adopted. The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12–16 h at room temperature in the dark. The solution was then diluted by mixing 1 mL ABTS solution with the amount of methanol necessary to obtain an absorbance of 0.7 at 734 nm. Serial dilutions of studied or reference compounds (trolox, quercetin) were carried out in 96-well microplates, at different concentrations, ranging between 0.146 μg/mL and 150 μg/mL in methanol. ABTS solution was then added to the microwells, and after 8 min of incubation the absorbance was taken at 405 nm in a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). In each assay, a control was prepared, in which the sample or standard was substituted by the same amount of solvent. Percentage of antioxidant activity (%AA) was calculated as:
(%) = [(Abscontrol − Abssample)]/(Abscontrol)] × 100
where Abscontrol is the absorbance of ABTS radical + methanol; Abssample is the absorbance of ABTS radical + sample/standard. All assays were carried out in triplicate and results expressed as EC50, i.e., as the concentration yielding 50% scavenging of ABTS, calculated by interpolation from the %AA vs concentration curve.

2.3.3. Anticholinesterasic Activity

The assay for measuring AChE and BuChE activity was modified from the assay described by Ellman et al. [39] and Arruda et al. [40]. Briefly, 3 mM 5,5′-dithiobis [2-nitrobenzoic acid] (DTNB, 5 μL), 75 mM acetylthiocholine iodide (ATCI, 5 μL) or butyrylthiocoline iodide (BuTCI, 5 μL), and sodium phosphate buffer 0.1 mol dm−3 (pH 8.0, 110 μL), and sample or standard (quercetin or donepezil) dissolved in buffer containing no more than 2.5% DMSO were added to the wells, and serial dilutions were carried out to obtain concentrations ranging between 0.293 μg/mL and 150 μg/mL (0.098 and 50 μg/mL for galantamine and berberine; 0.010 and 5 μg/mL for donepezil; 0.195 and 100 μg/mL for quercetin), followed by 0.25 U/mL AChE or BuChE (10 μL). The microplate was then read at 415 nm every 2.5 min for 7.5 min in a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For each concentration, enzyme activity was calculated as a percentage of the velocities compared to that of the assay using buffer without any inhibitor. Every experiment was done in triplicate.

2.3.4. Antimicrobial Activity

Antibacterial activity against Gram-positive Bacillus subtilis DSM10 and Micrococcus luteus DSM 20030 and Gram-negative Escherichia coli DSM498 was assessed by the broth microdilution method, as described by De León et al. [41]. The bacteria cultures were developed in nutrient broth (NB) at 30 °C for B. subtilis and M. luteus and at 37 °C for E. coli. Briefly, compounds were added to the microplates at a concentration of 200 μg/mL, and serial dilutions in NB were made until the concentration of 0.391 μg/mL. Then, the starting inoculum (1 × 105 CFU/mL) was added, and the bacterial growth was measured by the increase in optical density at 550 nm with a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), after 24 h of growth (48 h for M. luteus) at the above-mentioned temperatures for each bacterial strain. Penicillin and streptomycin were used as reference compounds. The IC50 was calculated as the concentration of compound that inhibits 50% of bacterial growth by interpolation from the % of growth inhibition vs. concentration curve.

2.3.5. Antitumor Activity

Antitumor activity was determined by the method described in Moujir et al. [42]. A-549 (human lung carcinoma) cell line, obtained from ATCC-LGC (American Type Culture Collection), was grown as a monolayer in DMEM (Sigma) supplemented with 2% fetal bovine serum (Sigma), 1% penicillin–streptomycin mixture (10,000 UI/mL), p-hydroxybenzoic acid (2 × 104 mg/mL) and L-glutamine (200 mM). Cells were maintained at 37 °C in 5% CO2 and 80% humidity in a SANYO CO2 Incubator. Cytotoxicity was assessed using the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] reduction assay. Cell suspension (2 × 104 cells/well) in the lag phase of growth was incubated in a 96-well microplate with the compounds or the standards (colchicine and paclitaxel) dissolved in medium, with the concentrations ranging between 0.195 μg/mL and 200 μg/mL (0.010 μg/mL and 10 μg/mL for standards). After 48 h, MTT was added to the cells, which were then allowed to incubate for 3–4 h, and the optical density was measured using a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 550 nm after dissolving the MTT formazan with DMSO (100 μL). The percentage viability (IC50) was calculated from the % of inhibition vs. concentration curve. All the experiments were repeated three times.

3. Results and Discussion

3.1. Chalcones and Flavanones Synthesis

The compounds obtained in this work were synthesized by aldol condensation between 4′- and/or 6′-substituted 2′-hydroxyacetophenones and either 4-hydroxy or 4-methoxybenzaldehydes, using NaH and LiHMDS (Scheme 1).
Compounds 1 and 3 were synthesized, as far as we could confirm, using for the first time in the aldol condensation the strong non-nucleophilic base NaH, which was used with success in similar condensations [43,44]. The experimental results in the synthesis of chalcones 1–3, with very good yields (~80%) and relatively low reaction times (3 to 4 h), proves that NaH is an effective and efficient base in the aldol condensation when it is desired to obtain non-hydroxylated chalcones, other than at the C-2′ position.
As referred above (Introduction), the hydroxyl groups present in the reagents require protection before the aldol condensation because in the strong basic conditions used, the phenoxide ions can undergo transformation into quinones and consequently prevent the formation of the desired chalcones. So, in order to obtain the desired compounds (the ones bearing hydroxyl and/or methoxyl groups), chalcones 2 and 3 need to be treated with acid to cleave the benzyl groups. The mixture used (HCl/AcOH) is one of the less harsh conditions and, in fact, removed the benzyl groups, but the obtained products were, in fact, flavanones 4 and 5 (Scheme 1). This can be explained by the acidic conditions used and the fact that those conditions favor the chalcones’ isomerization into flavanones [45]. On the other hand, these extra steps, the protection and deprotection of hydroxyl groups, contribute to decrease the overall yield. For example, if we consider the synthesis of flavanones 4 and 5, their precursors’ chalcones 2 and 3 have excellent yields (80%), and the deprotection step, which produces the flavanones, is less efficient. This demonstrates that, due to this step, the flavanones’ overall yield will be below 50%. At the same time, the yields (Scheme 1) allowed the assumption that the deprotection of the ring A hydroxyl groups is easier.
Since our purpose was the synthesis of chalcones bearing hydroxyl and methoxyl groups, taking into consideration that these substituents might improve the activity, and that the synthesis of flavanones 4 and 5 involves three steps, namely, (i) protection of the reagent’s hydroxyl groups, (ii) aldol condensation and (iii) cleavage of the protecting groups, we envisaged the synthesis of the desired chalcones using another base. Knowing that LiHMDS was used in the synthesis of hydroxylated flavones without the protection step and with good results [46,47], we tested its use in the synthesis of chalcone 6 (Scheme 1). The experimental results showed that, using LiHMDS as the base, it is possible to synthesize polyhydroxylated chalcones in a one-pot process. However, although the desired chalcone was obtained, the yield was very low and its isomeric form, flavanone 7, was also obtained. It should be highlighted that this reaction was accomplished at room temperature, controlled by TLC and finished after 5 days, when no more conversion was detected and 80% of the starting acetophenone was recovered. These data suggest that the conversion rate is high and that the reaction does not occur due to a lack of energy. We may suggest that using other sources of energy, such as a microwave, could originate higher yields.
The synthesized compounds’ structures were confirmed by detailed analysis of their 1D and 2D NMR spectra, MS spectra (e.g., in Figures S1–S9, supplementary material) and available literature data; moreover, their purity was confirmed by UHPLC. Herein, chalcones’ and flavanones’ characterization is briefly discussed.
The 1H NMR spectra of compounds 1–3 and 6 present the signal characteristics of chalcone structures: (i) the resonance of the AB system assigned to the olefinic protons; (ii) doublets at δH 7.7–7.9 ppm with J ~ 15 Hz, which confirms the E configuration of the α,β double bond; (iii) the signal at δC ~192 ppm assigned to the carbonyl group; (iv) the singlet at δH 14.4–13.7 ppm assigned to 2′-OH proton signal involvement in a hydrogen bond with the carbonyl oxygen atom; (v) two sets of doublets, with J = 6.8 Hz, at δH 6.9–7.8 ppm, assigned to the protons of the para-substituted aromatic ring B (Figure 1).
The 1H, 13C and HSQC NMR spectra of chalcone 1 also display two sets of doublets at δH 5.9–6.1 ppm, with a meta-coupling constant (J = 2.4 Hz), that exhibit correlation with the signals at 93.8 and 91.2 ppm, which indicates the presence of a tetra-substituted aromatic ring (ring A, Figure 1). The three sets of singlets at δH 3.83–3.92 ppm, showing correlation with three signals at δC 55.4–55.8 ppm, are characteristic of three methoxyl groups. The MS spectrum of chalcone 1 showed a signal at m/z 315 correspondent to [M+H]+, which agrees with the molecular formula C18H18O5. All the spectroscopic data and the melting point are in agreement with previously published data [31], and additionally, the connectivities found in the HMBC NMR spectrum confirm compound 1 as 2′-hydroxy-4,4′,6′-trimethoxychalcone, also named flavokawain A.
The 1H and 13C NMR spectra of chalcone 6 show three sets of signals in the range of δC 6.38–8.12 ppm, characteristic of the a trisubstituted aromatic ring (ring A, Figure 1). The MS spectrum showed a signal at m/z 257 corresponding to [M+H]+, which agrees with the molecular formula C15H12O4. These data and the connectivities found in the HMBC spectrum confirm compound 6 as 2′,4′,4-tri-hydroxychalcone, also known as isoliquiritigenin [48].
Compounds 4, 5 and 7 are flavanones, and this fact is well confirmed by the presence of the signals characteristic of ring C (Scheme 1): two double doublets at δH 2.68–3.10 and δH 2.68–2.78 assigned to the protons H-3, and the double doublet at δH 5.34–5.46 ppm assigned to H-2 in coupling with H-3; the signal at δ 196 ppm characteristic of the carbonyl group (C=O). Two sets of doublets at δH 7.29–7.42 (H-2′ and H-6′) and 6.86–6.95 ppm (H-3′ and H-5′), with orto-coupling constant of J = 8.7 Hz, indicate the presence of a para-substituted aromatic ring (ring B).
The presence of the methoxyl group in the ring B of compound 4 is deduced from the singlet at δ 3.83, correlating with the signal at δC 55.4 ppm. The MS data showed a signal at m/z 287 corresponding to the protonated molecule [M+H]+ that is in accordance with the molecular formula C16H14O5. The spectroscopic data and the melting point are consistent with the published data for isosakuranetin [33,49], and together with the connectivities found in HMBC spectrum, confirm that compound 4 is 5,7-dihydroxy-4′-methoxyflavanone.
The signals found in the 1H and 13C NMR spectra of compound 5 are very similar to the ones found for compound 4. The differences are the non-appearance of the signal at δH 12.05 ppm, which confirms the absence of a 5-OH group; and the appearance of two singlets at δH 3.86 and 3.81 ppm, which means that compound 5 has two methoxyl groups. The location of the two methoxyl groups and the hydroxyl group were confirmed by the connectivities found in the HMBC spectrum of compound 5. All the spectroscopic data are compatible with the structure of 4′-hydroxy-5,7-dimethoxyflavanone, also named naringenin 5,7-dimethyl ether. Although compound 5 is not new, [50,51], to the best of our knowledge, the full spectroscopic data are reported here for the first time.
The quasi-molecular ion at m/z 257, compatible with molecular formula C15H12O4, confirms that compound 7 is an isomeric form of compound 6. The presences of two signals at δH 9.53 and 8.60 ppm are assigned to 7-OH and 4′-OH, respectively, by the connectivities found in the HMBC spectrum. Moreover, the spectroscopic data are identical to that previously published for 4′,7-dihydroxyflavanone, also named liquiritigenin [52].

3.2. Biological Evaluations

The antioxidant, anticholinesterasic, antimicrobial and antitumor activities of the compounds bearing hydroxyl and methoxyl groups were studied, and the results obtained are presented and discussed in the following paragraphs. Compound 8, 2′-hydroxy-4-methoxychalcone, was not synthesized in this work, but was included in the bioactivities study because it is, in structural terms, one of the simplest 2′-hydroxychalcones and can be used as a reference and as a starting point to analyze structure/activity relationships of the compounds that were synthesized.

3.2.1. Antioxidant Activity

In the DPPH (1,1-diphenyl-2-picryl-hydrazyl) scavenging test, the capacity of the compounds to neutralize the DPPH radical via electron transfer is measured [53,54].
Nearly all the compounds had some antioxidant activity, which was concentration-dependent (see Supplementary Materials, Figure S10), except for compounds 4 and 8, which only had a residual activity at the highest concentration tested (150 μg/mL). Chalcone 6, the most active compound, presented higher antioxidant activity than the isomeric flavanone 7 (Table 1), confirming that the conjugated double bond improves the molecule’s ability to scavenge the DPPH radical [55].
The results also confirm that the number of free hydroxyl groups influences antioxidant activity, since compounds with a higher number of these groups present better activity [55], a fact that is also confirmed by the lower activity of the compounds with more methoxyl groups (Figure 2).
A previous work reported that compound 6 presented a low antioxidant activity in the DPPH scavenging test [56] (86.92 ± 0.43% of DPPH inhibition at 150 μg/mL); however, since the maximum concentration used in that work was about six times lower than the maximum tested concentration in the present work, it does not conflict with the results herein reported.
Another test used was the ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) scavenging assay. This assay measures the ability of compounds to neutralize the ABTS radical by radical quenching via hydrogen atom transfer. However, it can also be neutralized by electron transfer on some occasions, resulting in a higher sensitivity of this method when compared with the DPPH assay [53,57]. Again, compound 6 was the one presenting the best activity, with an IC50 of 12.72 μg/mL (Table 1), which is another result that confirms the importance of the hydroxyl groups in the chalcone scaffold for their ability to reduce and/or eliminate free radicals. This conclusion is confirmed by the lower activity of chalcones 1 and 8, which have only one hydroxyl group. Compound 5 presents better activity than 4, which means that the relative position between the hydroxyl and methoxyl groups is more important than the number of hydroxyl groups on the flavanone scaffold. This is confirmed by the results obtained for compound 7, which has two hydroxyl groups, but only one substituent in ring A, and presents lower activity than compounds 4 and 5 (Figure 3). It was observed that the ABTS scavenging activity of the tested compounds is dose-dependent (Figure S11, supplementary material).

3.2.2. Anticholinesterasic Activity

The results for acetylcholinesterase (AChE) inhibitory activity showed that compound 7 is the only one which presents some activity at the maximum concentration tested (47.1 ± 3.6%, at 150 µg/mL). This activity is less pronounced than the inhibition obtained for control compound donepezil (95.2 ± 0.4% at 50 µg/mL), a pure competitive inhibitor of AChE used clinically in early stages of Alzheimer’s disease. The results suggest that the cyclization seems to increase the inhibitory activity of the compound, since compound 7, which is a flavanone, is more active than compound 6, the respective chalcone. Also, the existence of free hydroxyl groups in both A and B rings seems to be important because, out of the three compounds with a flavanone structure (compounds 4, 5 and 7), only the one with a free hydroxyl group in both C-7 and C-4′ (compound 7) shows activity. This is in agreement with the strong activity revealed by quercetin, whose structure also possesses free hydroxyl groups in both rings A and B, like compound 7. So, it can be concluded that the presence of hydroxyl groups in rings A and B of the flavanone scaffold is crucial for the inhibition of AChE activity (Figure 4).
In terms of butyrylcholinesterase inhibition, compound 6 presented excellent activity, with a percentage of inhibition of 96.0 ± 1.1% and IC50 of 26.55 ± 0.55 μg/mL, which are similar values to the ones presented by donepezil (IC50 of 28.94 ± 1.76 μg/mL) and better than quercetin at the same concentrations. This shows that, unlike what happens with acetylcholinesterase, the presence of three hydroxyl groups in the chalcone scaffold highly favors the inhibition of butyrylcholinesterase activity. The results presented by chalcones 1 (12.51 ± 0.82%) and 8 (0%) also confirm that, like with acetylcholinesterase, the presence of methoxyl groups does not confer inhibitory activity to chalcones.
Compound 7 presents an activity very similar to the one observed for acetylcholinesterase inhibition (46.26 ± 1.27%), meaning that it is a dual inhibitor, a much-appreciated feature in the search for compounds with therapeutic potential towards Alzheimer’s disease. The presence of a hydroxyl group in ring B of the flavanone scaffold increases its inhibitory effect, since flavanones 5 (36.30 ± 0.20%) and 7 present higher activity than flavanone 4 (9.00 ± 1.30%), which has a methoxyl group in said position (Figure 4). The compounds inhibited butyrylcholinesterase in a dose-dependent manner (Figure S12, supplementary material).

3.2.3. Antimicrobial Activity

As observable in Table 2, most of the compounds tested inhibited the growth of gram-positive bacteria, but none was effective against the gram-negative strain tested. This inhibition was found to be concentration-dependent (Figures S13 and S14).
The most efficient compounds against Micrococcus luteus were 6 and 7, both with similar IC50, which suggests that cyclization does not influence the toxicity of these molecules against this species. These results are concordant with the literature, since it is described that these compounds presented similar activity against Mycobacterium tuberculosis [58]. Although some compounds tested exhibit an IC50 value greater than 200 μg/mL, the percent of growth inhibition exhibited at the maximum concentration tested allows one to deduce some interesting structure/activity relationships, which are discussed below. The chalcone 1 had no effect against M. luteus, while chalcone 8 presents 41.71% of growth inhibition at 200 μg/mL. These results suggest that the presence of methoxyl groups at ring A reduces its activity against this species. The same effect is observed when comparing the activity of compounds 4 and 5 (Figure 5).
Compound 5 inhibits 43.08% of bacterial growth at 200 μg/mL, while this value is almost double for compound 4 (74.43 ± 1.95%), which confirms that the methoxyl groups at ring A decrease the molecule’s antibacterial effect.
The strongest activity against Bacillus subtilis was displayed by compound 6 (IC50 = 9.33 μg/mL), presenting even better activity than streptomycin (Table 2). The corresponding flavanone 7 has a higher IC50 (20.48 μg/mL), which leads to the conclusion that the α,β unsaturated carbonyl bridge linking ring A to ring B is important in the mode of action of the compounds against this bacterial strain. The molecules with the higher numbers of hydroxyl groups are the ones with better activity, showing that these groups also play an important role in the molecule’s mode of action against B. subtilis. Again, the compounds with two methoxyl groups in meta positioning (1 and 5) were the ones with lower or none antimicrobial activity. Comparing the results from compounds 4 and 7 (both flavanones with two hydroxyl groups), one can conclude that the presence of the methoxyl group in ring B (compound 4) reduces the compound’s activity against this bacterial strain (Figure 6).

3.2.4. Antitumor Activity

Considering the antitumor activity of the studied compounds, it can be noticed that compounds 5 and 8 were the most active, presenting very similar IC50 values. Compounds 1 and 4 showed some activity, although lower than the ones mentioned above (Table 3). However, none of the compounds tested were as active as colchicine or paclitaxel, the reference compounds used (Table 3). It was also observed that the inhibition of tumor cell growth is dose-dependent for all the compounds tested (Figure S15, supplementary material).
Results for the activity of compounds 6 and 7 against the A549 cell line have already been reported in the literature [7,59,60,61]; however, their activity was tested in order to facilitate the comparison of the results, since all the compounds were tested in the same conditions. The results for compound 7 are concordant with the literature, since it is reported that the effects of this compound in A549 cells is limited to the inhibition of cell migration, without any effects on growth or cytotoxicity level [7,60]. Compound 6 did not present any activity, which was unexpected, since it is reported to possess antitumor activity against several cell lines, including A549, by inhibiting proliferation and inducing tumor cell apoptosis [59,61]. This can be explained by the fact that the authors used a much lower cell concentration than the one used in the present work, and it has been proved that lower cell concentrations are correlated with higher activities of the compounds tested [62].
From the results obtained, the only conclusions that can be drawn regarding structure/activity relationships are that neither the α,β unsaturated carbonyl bridge nor the cyclization of chalcone into flavanone have an influence in the inhibition of tumor-cell growth, since both chalcones and flavanones presented interesting activities (Figure 7). Also, the presence of methoxyl groups has some importance in the antitumor effect of the compounds, since compounds 6 and 7, which do not have methoxyl groups, did not present any activity against the A549 cell line (Table 3). The influence of a methoxyl group in synthetic polyphenolic compounds’ antitumor activity was not a surprise because it was previously detected in our group [63].

4. Conclusions

Hydroxylated chalcone/flavanone derivatives were synthesized using the less common bases NaH or LiHMDS. Overall, it was shown that the use of NaH is efficient (~80% yield in 3–4 h) for the synthesis of chalcones not hydroxylated other than at C-2′ position. However, if the desired derivatives are polyhydroxylated chalcones, the use of LiHMDS is preferable, because the conversion rate is good and it is a one-pot procedure that avoids the protection and subsequent deprotection steps, a time-consuming procedure that also significantly decreases the total yield. Although the aim of this work was to obtain the compounds for biological evaluation, we suggest microwave or ultrasound irradiation as a better source of energy to increase the compounds’ yields, an aspect that is important for their future applications.
Some of the compounds synthesized presented interesting results in their bioactivities, with chalcone 6 being the most active compound in terms of antioxidant, anti-butyrylcholinesterase and antimicrobial activity. Its isomer, flavanone 7, showed activity against both acetylcholinesterase and butyrylcholinesterase, which is an interesting result since this dual inhibition is a much-appreciated feature in Alzheimer’s disease therapy. Also, it is interesting that these compounds can be obtained in the one-pot methodology using LiHMDS.
Some important structure/activity relationships were established for all the activities tested, and the most important are highlighted and summarized in Figure 8.
As shown in Figure 8, the free hydroxyl groups are essential to increase antioxidant, anti-butyrylcholinesterase and antimicrobial activities. Not only their presence but also their number increases the compounds’ activity, while methoxyl groups decrease it. The flavanone scaffold increases the acetylcholinesterase inhibitory activity, while for the butyrylcholinesterase inhibition, the chalcone scaffold appears to be better. This is another interesting result because it is known that these compounds are isomers, and in biological systems they can exist in a controlled equilibrium. Regarding the antimicrobial activity, it is possible to detect that the cyclization into flavanone has no effect against the M. luteus strain, whereas against B. subtilis, the flavanone decreases the antibacterial activity. So, it seems that the α,β unsaturated carbonyl bridge linking rings A and B is important to inhibit the growth of the B. subtilis strain. Finally, it can be established that the presence of methoxyl groups in both chalcones and flavanones increases the antitumor activity.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-3417/9/14/2846/s1, Figure S1: 1H NMR spectrum of compound 1; Figure S2: 13C NMR spectrum of compound 1; Figure S3: HSQC NMR spectrum of compound 1; Figure S4: HMBC NMR spectrum of compound 1; Figure S5: ESI(+) Mass spectrum of compound 1; Figure S6: 1H NMR spectrum of compound 4; Figure S7: 13C NMR spectrum of compound 4; Figure S8: HSQC NMR spectrum of compound 4; Figure S9: HMBC NMR spectrum of compound 4; Figure S10: Antioxidant activity (%) presented by the compounds tested at three concentrations in the DPPH assay; Figure S11: Antioxidant activity (%) presented by the compounds tested at three concentrations in the ABTS assay; Figure S12: BuChE inhibitory activity (%) presented by the compounds tested at three concentrations; Figure S13: Inhibition of M. luteus growth (%) of the compounds tested at three concentrations; Figure S14: Inhibition of B. subtilis growth (%) of the compounds tested at three concentrations; Figure S15: Inhibition of A549 cell-line growth (%) of the compounds tested at three concentrations

Author Contributions

Conceptualization and methodology, A.M.L.S., M.C.B. and D.C.G.A.P.; formal analysis and investigation, G.P.R.; writing—original draft preparation, G.P.R.; writing—review and editing, A.M.L.S., M.C.B., A.M.S.S. and D.C.G.A.P.; supervision, A.M.L.S. and M.C.B.

Funding

This research was funded by FCT—Fundação para a Ciência e a Tecnologia, the European Union, QREN, FEDER, COMPETE, by funding the cE3c centre (FCT Unit funding (Ref. UID/BIA/00329/2013, 2015–2018) and UID/BIA/00329/2019) and the QOPNA research unit (project FCT UID/QUI/00062/2019).

Acknowledgments

Thanks are due to the University of Azores and University of Aveiro.

Conflicts of Interest

The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Applsci 09 02846 g001 550
Figure 1. Basic chalcone structure.
Figure 1. Basic chalcone structure.
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Scheme 1. General procedure for the synthesis of chalcones and flavanones.
Scheme 1. General procedure for the synthesis of chalcones and flavanones.
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Figure 2. Structure/activity relationships established for DPPH scavenging activity.
Figure 2. Structure/activity relationships established for DPPH scavenging activity.
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Figure 3. Structure/activity relationships established for ABTS scavenging activity.
Figure 3. Structure/activity relationships established for ABTS scavenging activity.
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Figure 4. Structure/activity relationships established for antioxidant activity.
Figure 4. Structure/activity relationships established for antioxidant activity.
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Figure 5. Structure/activity relationships established for antibacterial effect against M. luteus.
Figure 5. Structure/activity relationships established for antibacterial effect against M. luteus.
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Figure 6. Structure/activity relationships established for antibacterial effect against B. subtilis.
Figure 6. Structure/activity relationships established for antibacterial effect against B. subtilis.
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Figure 7. Structure/activity relationships established for antitumor activity.
Figure 7. Structure/activity relationships established for antitumor activity.
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Figure 8. Summary of the most relevant structure/activity relationships established.
Figure 8. Summary of the most relevant structure/activity relationships established.
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Table
Table 1. Antioxidant activity of the synthesized compounds 1, 4–8.
Table 1. Antioxidant activity of the synthesized compounds 1, 4–8.
Compounds and ReferencesIC50 μg/mL (± SD)
DPPHABTS
1>150>150
4>150140.15 (±0.03) a
5>15085.47 (±0.15) b
626.47 (±0.70) a12.72 (±0.92) c
7>150>150
8>150>150
Trolox7.25 (±0.09) b2.68 (±0.08) d
Quercetin3.01 (±0.03) c0.57 (±0.02) e
In each column, the letters a, b, c, d, and e indicate significant differences (p < 0.05).
Table
Table 2. Antimicrobial activity of synthesized compounds 1, 4–8.
Table 2. Antimicrobial activity of synthesized compounds 1, 4–8.
Compounds and ReferencesIC50 μg/mL (± SD)
M. luteusB. subtilisE. coli
1>200>200>200
4164.89 (±2.97) a76.46 (±4.27) a>200
5>200>200>200
623.78 (±2.04) b9.33 (±0.25) b>200
723.64 (±2.24) b20.48 (±3.13) c>200
8>20074.64 (±3.92) a>200
Penicillin8.47 (±0.55) c0.28 (±0.02) d>40
Streptomycin0.59 (±0.02) d15.86 (±0.21) e14.45 (±1.73)
In each column, the letters a, b, c, d, and e indicate significant differences (p < 0.05).
Table
Table 3. Antitumor activity of the synthesized compounds 1, 4–8.
Table 3. Antitumor activity of the synthesized compounds 1, 4–8.
Compounds and ReferencesIC50 μg/mL (± SD)
A549 Cell Line
1101.23 (±0.42) a
4135.89 (±2.61) b
593.42 (±2.42) c
6>200
7>200
893.31 (±2.45) c
Paclitaxel5.96 (±0.48) d
Colchicine2.78 (±0.71) d
In each column the letters a, b, c, d, and e indicate significant differences (p < 0.05).

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MDPI and ACS Style

Rosa, G.P.; Seca, A.M.L.; Barreto, M.d.C.; Silva, A.M.S.; Pinto, D.C.G.A. Chalcones and Flavanones Bearing Hydroxyl and/or Methoxyl Groups: Synthesis and Biological Assessments. Appl. Sci. 2019, 9, 2846. https://doi.org/10.3390/app9142846

AMA Style

Rosa GP, Seca AML, Barreto MdC, Silva AMS, Pinto DCGA. Chalcones and Flavanones Bearing Hydroxyl and/or Methoxyl Groups: Synthesis and Biological Assessments. Applied Sciences. 2019; 9(14):2846. https://doi.org/10.3390/app9142846

Chicago/Turabian Style

Rosa, Gonçalo P., Ana M. L. Seca, Maria do Carmo Barreto, Artur M. S. Silva, and Diana C. G. A. Pinto. 2019. "Chalcones and Flavanones Bearing Hydroxyl and/or Methoxyl Groups: Synthesis and Biological Assessments" Applied Sciences 9, no. 14: 2846. https://doi.org/10.3390/app9142846

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