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Article

Synthesis and Antimicrobial Activity of Novel 4-Hydroxy-2-quinolone Analogs

by
Thitiphong Khamkhenshorngphanuch
1,†,
Kittipat Kulkraisri
2,†,
Alongkorn Janjamratsaeng
2,†,
Napasawan Plabutong
3,
Arsa Thammahong
3,
Kanitta Manadee
4,
Sarisa Na Pombejra
4 and
Tanatorn Khotavivattana
5,*
1
Department of General Education, Faculty of Science and Health Technology, Navamindradhiraj University, Bangkok 10300, Thailand
2
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3
Antimicrobial Resistance and Stewardship Research Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
4
Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
5
Center of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2020, 25(13), 3059; https://doi.org/10.3390/molecules25133059
Submission received: 15 June 2020 / Revised: 30 June 2020 / Accepted: 3 July 2020 / Published: 4 July 2020
(This article belongs to the Special Issue Strategies toward Bioactive Natural Product Like-Compounds)
Browse Figures
Review Reports Versions Notes

Abstract

:
Alkyl quinolone has been proven to be a privileged scaffold in the antimicrobial drug discovery pipeline. In this study, a series of new 4-hydroxy-2-quinolinone analogs containing a long alkyl side chain at C-3 and a broad range of substituents on the C-6 and C-7 positions were synthesized. The antibacterial and antifungal activities of these analogs against Staphylococcus aureus, Escherichia coli, and Aspergillus flavus were investigated. The structure-activity relationship study revealed that the length of the alkyl chain, as well as the type of substituent, has a dramatic impact on the antimicrobial activities. Particularly, the brominated analogs 3j with a nonyl side chain exhibited exceptional antifungal activities against A. flavus (half maximal inhibitory concentration (IC50) = 1.05 µg/mL), which surpassed that of the amphotericin B used as a positive control. The antibacterial activity against S. aureus, although not as potent, showed a similar trend to the antifungal activity. The data suggest that the 4-hydroxy-2-quinolone is a promising framework for the further development of new antimicrobial agents, especially for antifungal treatment.

Graphical Abstract

1. Introduction

The infection produced by microorganisms, such as bacteria or fungi, has long been considered as one of the major global health concerns. Recently, the rapid emergence of drug-resistant pathogens has caused a dramatic decrease in the effectiveness of the currently available medications [1]. At present, antimicrobial resistance causes 700,000 deaths annually, and the number is expected to rise to 10 million in 2050 [2]. As a result, the development of a new class of antimicrobial drugs is highly in demand as a complement or replacement to existing antibiotics.
In the search for a new scaffold for antimicrobial drugs, 4-hydroxy-2-quinolone, a class of heterocyclic compound, has shown great potential, as various analogs of 4-hydroxy-2-quinolone including Roquinimex (Linomide), a synthetic immunomodulator [3], have been reported to possess a wide range of biological activities such as antibacterial [4], antifungal [5], anticancer [6], antioxidant [7], and so on. Over the past few decades, several attempts have been made to investigate the structure–activity relationship of the antimicrobial activity of this scaffold; for instance, the derivatives of 4-hydroxy-2-quinolone bearing chalcones [8], pyrimidines [9], pyrazoles [10], and thiadiazole [11] (Figure 1a). Despite the extensive studies on the substituent pattern at the C-3 position, especially with aromatic or heteroaromatic motifs, there is a limited number of studies on the effect of the aliphatic side chain on the antimicrobial activity. According to a study on a similar scaffold such as 4-hydroxycoumarin [12], or quinolone [13,14], it was found that the alkyl side chain plays a crucial role in the antibacterial activity as reflected by the strong correlation between the length of the chain and their bioactivity. In a previous study, it was found that the alkenyl side chain of 4-quinolones can form hydrophobic interactions at a binding pocket within MurE ligase of Mycobacterium tuberculosis [15]. However, the mechanism of action of 4-hydroxy-2-quinolone remains unclear. To date, the effect of substituents on the benzo-carbons of the 2-quinolone core structure has never been thoroughly investigated for the antimicrobial activity; only the 6-nitro [16], 6,7-dimethoxy [17], and 5-hydroxy and/or 6-carboxylic acid [18] analogs were investigated (Figure 1a). According to the development of ciprofloxacin, a 4-quinolone drug popularly used until today, it was found that the introduction of a fluorine atom at the C-6 position dramatically enhanced the antibacterial activity [19]. Moreover, further study revealed that the type of substituents on the C-7 position led to an enormous impact on their antimicrobial properties [20]. However, for the 4-hydroxy-2-quinolone core structure, the only thorough investigation of the structure–activity relationship on the benzo-carbons was reported by Jönsson and group in 2004, regarding their inhibitory effects on autoimmune disorders [21]. On the other hand, the structure–activity relationship for antimicrobial activity remains unclear. Herein, we report the diverted total synthesis of novel 4-hydroxy-2-quinolone analogs bearing an alkyl side chain at C-3 and various types of substituents on C-6 and C-7 positions (Figure 1b). The obtained compounds were then tested for their antibacterial and antifungal activities in order to obtain additional knowledge on the structure–activity relationship of this promising scaffold.

2. Results and Discussions

2.1. Chemical Synthesis

The 4-hydroxy-2-quinolone analogs in this work were synthesized following a three-step protocol starting from isatoic anhydrides with various substituents on the aromatic ring (Figure 2). First, the alkylation of isatoic anhydrides was performed using either methyl iodide or ethyl iodide as the alkylating agents and N,N-diisopropylethylamine (DIPEA) as a base, leading to the formation of N-alkylated isatoic anhydrides (1a–1g) in moderate to excellent yields [22]. Next, β-ketoesters (2a and 2b) bearing long alkyl chains such as nonyl and tridecyl groups were synthesized by the reaction between ethyl potassium malonate and the appropriate acid chlorides mediated by MgCl2 and triethylamine in good yields [23]. Finally, the condensation between 1a–1g and 2a or 2b was achieved using NaH as a base, yielding the analogs of 4-hydroxy-2-quinolone (3a–3j) in relatively low yields [24]. The structures of all the synthesized 4-hydroxy-2-quinolone analogs have been confirmed by several spectroscopic techniques such as 1H NMR, 13C NMR, IR, and HRMS. It is worth noting that these compounds show a characteristic pattern in 13C NMR: A signal at around δ 209 ppm indicating the carbonyl group on the side chain, and a set of signals at 174 and 161 ppm indicating C-4 and C-2 of the quinolone ring, respectively.
In order to gain insight into the reason behind the poor yield of the final step, the reaction between 1d and 2a for the synthesis of 3d was thoroughly investigated (Figure 3). The isolation and characterization of byproducts revealed that, apart from the desired 4-hydroxy-2-quinolone analog 3d, which were obtained in only 9%, two additional products also occurred: 1,4-Dihydroquinoline-3-carboxylate analog 4d, which is a structural isomer of the desired product, and the 2-aminobenzoic acid 5d were obtained in 17% and 51%, respectively. The generation of the two possible isomers, 3d and 4d, has been documented by Coppola and Hardtmann [25], demonstrating that the selectivity of such transformation highly depends on the nature of the methylene species, which is the β-ketoester 2a in this case. The formation of a large amount of 5d could be caused by the hydrolysis of the isatoic anhydride 1d, hence indicating the presence of water in the reaction. Although several attempts were made to exclude the moisture from the reaction, the yield of 3d was not significantly improved.

2.2. Antimicrobial Activity and Structure–Activity Relationship

All the synthesized 4-hydroxy-2-quinolone analogs 3a–3j, as well as the isomer 4d, were screened for their antifungal activity against a pathogenic fungus Aspergillus flavus, as well as the antibacterial activity against a Gram-positive bacteria Staphylococcus aureus and a Gram-negative bacteria Escherichia coli (Table 1). For the antifungal activity, compound 3a with no substituents on the ring exhibited very low inhibition with a half maximal inhibitory concentration (IC50) of 70.97 ± 3.71 µg/mL. The introduction of the substituents on the benzo-carbons led to significant improvement in the potency, especially for the halogenated analogs 3d, 3e, and 3f with up to fivefold enhanced activity. This result is in line with a previous study reported by Huigens and group that the introduction of halogen substituents, especially for bromine, to the quinoline core structure can lead to higher antimicrobial activity, probably due to the metal(II)-dependent mechanism [26].
Replacing the methyl group with the ethyl group at the position R2 (3g) led to a dramatic improvement in the inhibitory activity (IC50 = 9.48 ± 6.17 µg/mL). The same trend was observed for the replacement of the tridecyl with the nonyl group at the position R3 (3h) with the IC50 of 8.45 ± 1.59 µg/mL. This result suggests that an optimum size of these alkyl side chains is required, presumably to fit into the hydrophobic pockets within microbial enzymes, as demonstrated earlier by Bhakta and group [15]. Further enhancement in the antifungal activity was achieved when halogen substituents were introduced at the benzo-carbon positions on the nonyl analog. The IC50 values of the 6-chloro (3i) and 6-bromo (3j) nonyl analogs were 4.60 ± 1.14 and 1.05 ± 1.31 µg/mL, respectively, which is almost 70 times higher than that of 3a. The inhibitory activity of 3j against A. flavus is even greater than amphotericin B, the current fungicidal antifungal agent, with almost two times more effectiveness. Lastly, the 1,4-dihydroquinoline-3-carboxylate analog 4d showed slightly higher activity compared to its corresponding isomer 3d, which may suggest the possibility of further antifungal study on this scaffold. For the antibacterial activity, it seems like these 4-hydroxy-2-quinolone analogs possess almost no activity against both S. aureus and E. coli. Nevertheless, two compounds, 3i and 3j, exhibited significant inhibitory activity against S. aureus. This result correlates well with the antifungal activity in which these two analogs also showed the highest potency. Although the minimum inhibitory concentrations (MICs) of 3i and 3j were unclear, both compounds demonstrated inhibitory effects on the bacterial growth at concentrations between 125–1000 and 125–500 µg/mL, respectively. It was interesting to notice that the high concentration of compounds at 1000 µg/mL gave worse antibacterial results than the medium concentrations (125–500 µg/mL; see Section 2 in the Supporting Information). This phenomenon has been found in some quinolone-class antibiotics such as nalidixic acid. The survival of bacteria at high concentrations of quinolones was explained by a decrease in reactive oxygen species (ROS) level during exceeding quinolone concentrations [27]. The ROS accumulation is a key mechanism of quinolone-mediated lethality [28], and this may relate to the antibacterial activity of 3i and 3j. However, the result of minimum bactericidal concentration (MBC) indicated that both 3i and 3j exhibited a bacteriostatic effect rather than bactericidal activity (see Section 2 in the Supporting Information). Taken together, the data suggest that the synthesized 4-hydroxy-2-quinolone analogs could be further developed as promising antimicrobial agents especially for antifungal treatment. As cellular structures of fungi and bacteria are largely different, based on our antimicrobial results, these newly synthesized compounds may mainly target fungal molecules rather than bacterial ones. This seems to be their outstanding property that is not found in other quinolone drugs, which are primarily used as an antibacterial treatment.

3. Materials and Methods

3.1. General Experimental Procedures

All reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA), TCI Chemicals (Tokyo, Japan), Fluorochem (Hadfield, Derbyshire, UK), and Merck (Darmstadt, Germany). All solvents for column chromatography from RCI Labscan (Samutsakorn, Thailand) were distilled before use. 1H and 13C NMR spectra were recorded on a Bruker Avance (400 MHz) (Bruker, Billerica, MA, USA), country and Jeol Avance (500 MHz) (Jeol Ltd., Tokyo, Japan). Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane or the residual solvent signals in DMSO-d6 or CDCl3 as the internal standard for the NMR spectra. Data are reported as follows: Chemical shift (multiplicity, coupling constants in Hz, integrated intensity, assignment). Abbreviations of multiplicity were as follows: s: Singlet, d: Doublet, t: Triplet, q: Quartet, m: Multiplet, br: Broad. High-resolution mass spectra (HRMS) data were obtained with a Micro-TOF mass spectrometer (Bruker, Billerica, MA, USA). IR spectra were recorded using the Thermo Scientific™ Nicolet™ iS50 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with an ATR module and are reported in wavenumber (cm−1). Reactions were monitored by thin-layer chromatography (TLC) using aluminum Merck TLC plates coated with silica gel 60 F254. Normal-phase column chromatography was performed using silica gel 60 (0.063–0.200 mm, 70–230 mesh ASTM, Merck, Darmstadt, Germany). Melting points were measured using a melting point apparatus (Griffin) and are uncorrected.

3.2. Synthesis of Compounds 1a–1g

Compounds 1a–1g were synthesized using a modified procedure [22]. Substituted isatoic anhydride (1.0 equiv.) was added to a round-bottom flask. DIPEA (2.0 equiv.) and dimethylacetamide (DMAC) were added at room temperature and stirred for 10 min. The flask was fitted with a rubber septum and purged with nitrogen gas. Appropriate alkyl halide (2.0 equiv.) was added dropwise, and the reaction was heated at 45 °C for 2 h and monitored by thin-layer chromatography. Upon completion, the reaction mixture was quenched with H2O. The precipitate was filtered, washed with H2O, and concentrated in vacuo to give the product.
1-Methyl-2H-benzo[d] [1,3] oxazine-2,4(1H)-dione (1a): Isatoic anhydride (2.00 g, 12.3 mmol), methyl iodide (1.60 mL, 24.6 mmol), and DMAC (13 mL) were used to give the title compound (1.75 g, 81% yield) as an off-white solid. 1H NMR (400 MHz, DMSO) δ 8.01 (d, J = 7.7 Hz, 1H), 7.86 (t, J = 7.9 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 3.47 (s, 3H); 13C NMR (101 MHz, DMSO) δ 158.95, 147.71, 142.18, 137.15, 129.28, 123.54, 114.80, 111.49, 31.63 [29].
6-Methoxy-1-methyl-2H-benzo[d] [1,3] oxazine-2,4(1H)-dione (1b): 5-Methoxyisatoic anhydride (290 mg, 1.5 mmol), methyl iodide (186 μL, 3.0 mmol), and DMAC (3 mL) were used to give the title compound (191 mg, 61% yield) as a light-green solid. 1H NMR (400 MHz, DMSO) δ 7.52–7.34 (m, J = 17.7, 8.9 Hz, 3H), 3.83 (s, 3H), 3.45 (s, 3H); 13C NMR (101 MHz, DMSO) δ 158.84, 155.17, 147.52, 136.31, 124.99, 116.54, 112.12, 110.88, 55.80, 31.71 [30].
7-Chloro-1-methyl-2H-benzo[d] [1,3] oxazine-2,4(1H)-dione (1c): 4-Chloroisatoic anhydride (300 mg, 1.5 mmol), methyl iodide (186 μL, 3.0 mmol), and DMAC (2 mL) were used to give the title compound (260 mg, 81% yield) as a white solid. 1H NMR (400 MHz, DMSO) δ 8.00 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.39 (d, J = 8.3 Hz, 1H), 3.46 (s, 3H); 13C NMR (101 MHz, DMSO) δ 158.27, 143.39, 141.89, 131.01, 130.73, 123.64, 114.86, 110.59, 31.91 [31].
1-Methyl-7-nitro-2H-benzo[d][1,3] oxazine-2,4(1H)-dione (1d): 4-Nitroisatoic anhydride (312 mg, 1.5 mmol), methyl iodide (186 μL, 3.0 mmol), and DMAC (2 mL) were used to give the title compound (306 mg, 81% yield) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 8.25 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 1.5 Hz, 1H), 8.06 (dd, J = 8.5, 1.8 Hz, 1H), 3.56 (s, 3H); 13C NMR (101 MHz, DMSO) δ 157.87, 152.43, 147.33, 143.06, 131.13, 117.50, 110.82, 109.84, 32.08 [30].
6-Chloro-1-methyl-2H-benzo[d] [1,3] oxazine-2,4(1H)-dione (1e): 5-Chloroisatoic anhydride (297 mg, 1.5 mmol), methyl iodide (186 μL, 3.0 mmol), and DMAC (2 mL) were used to give the title compound (258 mg, 81% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 2.4 Hz, 1H), 7.82 (dd, J = 8.9, 2.5 Hz, 1H), 7.36 (s, 1H), 3.68 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 157.43, 147.61, 140.72, 137.37, 131.56, 130.16, 115.59, 113.11, 32.20 [32].
6-Bromo-1-methyl-2H-benzo[d] [1,3] oxazine-2,4(1H)-dione (1f): 5-Bromoisatoic anhydride (363 mg, 1.5 mmol), methyl iodide (186 μL, 3.0 mmol), and DMAC (2 mL) were used to give the title compound (368 mg, 96% yield) as a brown solid. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.3 Hz, 1H), 7.85 (dd, J = 8.8, 2.3 Hz, 1H), 7.08 (d, J = 8.9 Hz, 1H), 3.57 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 157.31, 147.59, 141.17, 140.19, 133.20, 117.06, 115.80, 113.42, 32.17 [29].
1-Ethyl-2H-benzo[d] [1,3] oxazine-2,4(1H)-dione (1g): Isatoic anhydride (245 mg, 1.3 mmol), ethyl iodide (240 μL, 3.0 mmol), and DMAC (2 mL) were used to give the title compound (246 mg, 48% yield) as an off-white solid. 1H NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO) δ 145.61, 141.10, 137.23, 129.61, 123.47, 122.62, 114.58, 111.75, 31.67, 11.86 [33].

3.3. Synthesis of Compounds 2a and 2b

Compounds 2a and 2b were synthesized using a modified procedure [23]. Ethyl potassium malonate (2.0 equiv.), MgCl2 (3.0 equiv.), and NEt3 (3.0 equiv.) were added to a round-bottom flask. The flask was fitted with a rubber septum and purged with nitrogen gas, and dry MeCN was then added. The mixture was stirred at room temperature for 2 h, appropriate acid chloride (1.0 equiv.) was then added dropwise at 0 °C, and the mixture was stirred overnight at room temperature. Upon completion, the reaction mixture was extracted with CH2Cl2 and H2O. The combined organic layers were washed with brine, dried over anh. Na2SO4, filtered, and then concentrated in vacuo. The crude mixture was purified by silica gel column chromatography to give the product.
Ethyl 3-oxohexadecanoate (2a): Ethyl potassium malonate (6.30 g, 37.0 mmol), myristoyl chloride (4.80 mL, 17.7 mmol), and MeCN (84 mL) were used to give the title compound (4.24 g, 80% yield) as a light-yellow oil. 1H NMR (400 MHz, CDCl3) δ 4.19 (q, J = 7.1 Hz, 2H), 3.42 (s, 2H), 2.52 (t, J = 7.4 Hz, 2H), 1.65–1.52 (m, 3H), 1.24 (s, 20H), 0.89–0.85 (m, J = 6.6 Hz, 5H); 13C NMR (101 MHz, CDCl3) δ 203.11, 167.43, 89.09, 61.46, 49.45, 43.19, 35.20, 33.97, 32.05, 29.80, 29.77, 29.72, 29.57, 29.48, 29.37, 24.86, 22.81, 14.22 [34].
Ethyl 3-oxododecanoate (2b): Ethyl potassium malonate (1.7 g, 10 mmol), decanoyl chloride (1.6 mL, 7.5 mmol), and MeCN (23 mL) were used to give the title compound (1.271 g, 75% yield) as a light-yellow oil. 1H NMR (400 MHz, CDCl3) δ 4.18 (dt, J = 6.8, 5.4 Hz, 2H), 3.42 (s, 2H), 2.52 (t, J = 7.4 Hz, 2H), 1.60 (dd, J = 21.9, 14.9 Hz, 3H), 1.44–1.12 (m, 14H), 0.87 (t, J = 6.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 61.45, 49.45, 43.18, 35.20, 31.99, 29.53, 29.48, 29.37, 29.19, 29.16, 26.39, 23.62, 22.78, 14.23 [35].

3.4. Synthesis of Compounds 3a–3j, 4d, and 5d

Compounds 3a–3j, 4d, and 5d were synthesized using a modified procedure [24]. Sodium hydride (60% in mineral oil) was added to a round-bottom flask and washed with hexane 3 times. The flask was fitted with a rubber septum and purged with nitrogen gas, and N-alkyl substituted isatoic anhydride (1a–1g), β-ketoester (2a–2b), and dry DMF were then added. The mixture was stirred overnight at room temperature. Upon completion, the reaction mixture was quenched with H2O. The resulting mixture was extracted with ethyl acetate (EtOAc). The combined organic layers were washed with brine, dried over anh. Na2SO4, filtered, and then concentrated in vacuo. The crude mixture was purified by silica gel column chromatography to give the products.
4-Hydroxy-1-methyl-3-tetradecanoylquinolin-2(1H)-one (3a): 1a (354 mg, 1.0 mmol), 2a (596 mg, 1.0 mmol), NaH (60 mg, 1.5 mmol), and DMF (4 mL) were used and purified by silica gel column chromatography (eluent: 5% EtOAc in hexanes) to give the title product (186 mg, 24% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 6.9 Hz, 1H), 7.72–7.66 (m, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.32–7.27 (m, 1H), 3.65 (s, 3H), 3.29 (t, J = 7.4 Hz, 2H), 1.76–1.67 (m, 2H), 1.57 (s, 2H), 1.28 (d, J = 16.6 Hz, 18H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 209.71, 174.28, 161.65, 152.68, 141.81, 134.90, 126.34, 124.24, 122.15, 115.84, 114.29, 57.11, 43.11, 32.08, 29.84, 29.80, 29.72, 29.71, 29.54, 29.51, 29.29, 24.49, 22.84, 14.25; IR (neat): 2916.00, 2848.35, 1651.82, 1624.97, 1590.46, 1557.51, 1502.15, 1463.21, 1427.31, 1409.30, 1339.12, 1303.63, 1280.61, 1251.95, 1198.68, 1171.19, 1061.74, 1039.80, 1030.70, 980.98; HRMS (ESI+): m/z calcd for C24H34NO3Na2+ [M− H + 2Na]+ 430.2329, found 430.2319; Mp: 70.0–70.8 °C.
4-Hydroxy-6-methoxy-1-methyl-3-tetradecanoylquinolin-2(1H)-one (3b): 1b (62 mg, 0.30 mmol), 2a (90 mg, 0.30 mmol), NaH (18 mg, 0.45 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: EtOAc/hexanes = 1:4) to give the title product (21 mg, 22% yield) as a light-green solid. 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 2.8 Hz, 1H), 7.31 (dd, J = 9.2, 2.9 Hz, 1H), 7.25 (d, J = 13.9 Hz, 1H), 3.90 (s, 3H), 3.64 (s, 3H), 3.31 (t, J = 7.4 Hz, 2H), 1.75–1.70 (m, 2H), 1.27 (s, 20H), 0.89 (t, J = 6.7 Hz, 3H); 13C NMR(101 MHz, CDCl3) δ 209.84, 173.65, 161.27, 154.90, 152.80, 136.63, 124.67, 122.63, 115.87, 106.59, 102.05, 89.45, 55.95, 43.11, 32.08, 29.84, 29.80, 29.72, 29.71, 29.54, 29.50, 29.40, 24.49, 22.84, 14.25. IR (neat): 2916.19, 2849.20, 1647.85, 1592.93, 1572.55, 1505.80, 1466.05, 1452.89, 1413.22, 1382.45, 1357.95, 1339.80, 1236.89, 1196.42, 1177.76, 1148.43, 1059.87, 1034.63, 1004.25, 857.67, 821.28; HRMS (ESI+): m/z calcd for C25H36NO4Na2+ [M − H + 2Na]+ 460.2434, found 460.2427; Mp: 67.8–71.5 °C.
7-Chloro-4-hydroxy-1-methyl-3-tetradecanoylquinolin-2(1H)-one (3c): 1c (127 mg, 0.60 mmol), 2a (150 mg, 0.50 mmol), NaH (30 mg, 0.75 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 4% EtOAc in hexanes) to give the title product (26 mg, 12% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 1.2 Hz, 1H), 7.22 (dd, J = 8.6, 1.5 Hz, 1H), 3.61 (s, 3H), 3.27 (t, J = 7.4 Hz, 2H), 1.75–1.65 (m, 2H), 1.26 (s, 20H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 209.63, 173.81, 171.52, 161.49, 142.53, 141.38, 127.71, 122.72, 114.36, 105.86, 105.60, 97.19, 86.07, 43.04, 32.08, 29.83, 29.79, 29.71, 29.68, 29.51, 29.41, 24.43, 22.83, 14.24; IR (neat): 2916.03 2852.38, 1644.46, 1609.69, 1556.11, 1473.10, 1443.16, 1421.20, 1378.16, 1343.22, 1320.45, 1264.80, 1227.80, 1203.14, 1099.25, 1031.70, 966.59, 891.66, 841.66; HRMS (ESI+): m/z calcd for C24H33ClNO3− [M − H]− 418.2154, found 418.2148; Mp: 103.4–103.9 °C.
4-Hydroxy-1-methyl-7-nitro-3-tetradecanoylquinolin-2(1H)-one (3d), ethyl 1-methyl-7-nitro-4-oxo-2-tridecyl-1,4-dihydroquinoline-3-carboxylate (4d), and 2-(methylamino)-4-nitrobenzoic acid (5d): 1d (134 mg, 0.60 mmol), 2a (150 mg, 0.50 mmol), NaH (30 mg, 0.75 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 4% EtOAc in hexanes) to give 3d (18 mg, 9% yield) as a yellow solid, 4d (40 mg, 17% yield) as a yellow-green solid, and 5d (50 mg, 51% yield) as an orange solid, respectively.
3d: 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 8.8 Hz, 1H), 8.17 (d, J = 1.8 Hz, 1H), 8.05 (dd, J = 8.7, 1.8 Hz, 1H), 3.72 (s, 3H), 3.30 (t, J = 7.4 Hz, 2H), 1.71 (d, J = 7.3 Hz, 2H), 1.43–1.37 (m, 2H), 1.26 (s, 18H), 0.88 (t, J = 6.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 210.00, 172.68, 161.20, 141.89, 132.97, 128.08, 120.13, 116.14, 111.27, 110.23, 109.66, 52.31, 46.67, 43.24, 32.07, 29.82, 29.79, 29.68, 29.65, 29.50, 29.45, 24.31, 22.83, 14.24. IR (neat): 2920.29, 2852.03, 1630.31, 1605.71, 1563.83, 1519.60, 1470.98, 1346.51, 1321.48, 1278.93, 1277.83, 1104.60, 1033.57, 986.91, 888.50, 828.10, 813.47; HRMS (ESI+): m/z calcd for C24H33N2O5− [M − H]− 429.2395, found 429.2384; Mp: 156.3–168.4 °C.
4d: 1H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 8.7 Hz, 1H), 8.45 (s, 1H), 8.13 (d, J = 8.7 Hz, 1H), 4.41 (d, J = 7.1 Hz, 2H), 3.86 (s, 3H), 2.87–2.71 (m, 2H), 1.71 (d, J = 7.0 Hz, 2H), 1.44 (d, J = 7.0 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.25 (s, 18H), 0.87 (t, J = 6.7 Hz, 3H); (101 MHz, CDCl3) δ 173.30, 166.95, 154.84, 150.20, 141.36, 129.85, 129.18, 120.11, 117.84, 112.11, 61.87, 35.23, 32.60, 32.02, 29.79, 29.76, 29.73, 29.67, 29.56, 29.44, 29.28, 29.04, 22.79, 14.37. IR (neat): 2917.90, 2852.52, 1726.40, 1597.66, 1518.79, 1496.50, 1464.33, 1384.73, 1345.48, 1214.81, 1135.89, 1105.59, 1037.28, 1022.98, 890.49, 855.73, 825.02; HRMS (ESI+): m/z calcd for C26H38N2O5Na+ [M + Na]+ 481.2678, found 481.2676; Mp: 92.6–95.2 °C.
5d: 1H NMR (400 MHz, DMSO) δ 7.99 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 1.7 Hz, 1H), 7.33–7.27 (m, 1H), 3.46 (s, 1H), 2.91 (s, 3H); 13C NMR (101 MHz, DMSO) δ 168.63, 151.78, 151.39, 133.26, 115.08, 107.65, 104.81, 29.32 [36].
6-Chloro-4-hydroxy-1-methyl-3-tetradecanoylquinolin-2(1H)-one (3e): 1e (159 mg, 0.75 mmol), 2a (150 mg, 0.50 mmol), NaH (30 mg, 0.75 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 5% EtOAc in hexanes) to give the title product (60 mg, 28% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 9.0, 2.5 Hz, 1H), 7.29 (d, J = 3.2 Hz, 1H), 3.66 (s, 3H), 3.31 (t, J = 7.4 Hz, 2H), 1.73 (dt, J = 14.9, 7.5 Hz, 2H), 1.64 (s, 2H), 1.42 (d, J = 8.8 Hz, 2H), 1.28 (s, 16H), 0.91 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 209.76, 173.14, 161.26, 140.24, 134.85, 130.85, 128.03, 125.56, 116.91, 115.86, 65.84, 57.63, 47.37, 43.11, 32.07, 29.83, 29.79, 29.70, 29.68, 29.50, 29.46, 24.39, 22.83, 14.24. IR (neat): 2919.88, 2847.72, 1651.26, 1635.04, 1610.19, 1551.49, 1463.26, 1402.46, 1293.20, 1206.95, 1093.57, 1052.40, 955.38, 897.40, 819.76, 812.24; HRMS (ESI+): m/z calcd for C24H33ClNO3− [M − H]− 418.2154, found 418.2149; Mp: 73.5–80.0 °C.
6-Bromo-4-hydroxy-1-methyl-3-tetradecanoylquinolin-2(1H)-one (3f): 1f (192 mg, 0.75 mmol), 2a (150 mg, 0.50 mmol), NaH (30 mg, 0.75 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 5% EtOAc in hexanes) to give the title product (63 mg, 27% yield) as a light-brown solid. 1H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 2.1 Hz, 1H), 7.74 (dd, J = 9.0, 2.3 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 3.61 (s, 3H), 3.27 (t, J = 7.4 Hz, 2H), 1.70 (dt, J = 14.9, 7.4 Hz, 2H), 1.40 (s, 2H), 1.25 (s, 18H), 0.88 (d, J = 6.5 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 209.71, 173.06, 161.21, 140.61, 137.54, 128.63, 117.29, 116.07, 115.17, 106.28, 61.69, 56.13, 55.98, 43.09, 32.06, 29.82, 29.78, 29.69, 29.67, 29.49, 29.41, 24.37, 22.82, 14.23. IR (neat): 2919.92, 2851.49, 1644.22, 1606.11, 1551.76, 1494.12, 1464.02, 1429.07, 1413.33, 1317.00, 1292.07, 1252.78, 1149.12, 1062.14, 1000.11, 821.17, 807.49; HRMS (ESI+): m/z calcd for C24H33BrNO3− [M − H]− 462.1649, found 462.1657; Mp: 73.2–75.0 °C.
1-Ethyl-4-hydroxy-3-tetradecanoylquinolin-2(1H)-one (3g): 1g (144 mg, 0.75 mmol), 2a (150 mg, 0.50 mmol), NaH (30 mg, 0.75 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 5% EtOAc in hexanes) to give the title product (16 mg, 8% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.24 (dd, J = 8.0, 1.3 Hz, 1H), 7.71–7.62 (m, 1H), 7.31 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 7.3 Hz, 1H), 4.60–4.00 (m, 2H), 3.70–3.02 (m, 3H), 1.70 (dt, J = 23.8, 11.5 Hz, 2H), 1.41–1.10 (m, 23H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 209.71, 174.07, 161.16, 140.88, 134.83, 133.69, 126.53, 123.77, 121.92, 116.00, 114.14, 105.95, 43.16, 37.22, 32.07, 29.83, 29.79, 29.71, 29.51, 29.50, 29.18, 24.41, 22.83, 14.24, 12.94; m/z calcd for C25H37NO3− [M + 3H]+ 402.3008, found 402.3003; Mp: 74.1–75.5 °C.
3-Decanoyl-4-hydroxy-1-methylquinolin-2(1H)-one (3h): 1a (266 mg, 1.50 mmol), 2b (365 mg, 1.50 mmol), NaH (90 mg, 2.25 mmol), and DMF (1 mL) were used and the crude product was purified by silica gel column chromatography (eluent: 5% EtOAc in Hexanes) to give the title product (35 mg, 7% yield) as a light-yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.25–8.08 (m, 1H), 7.65 (d, J = 7.1 Hz, 1H), 7.24 (dd, J = 16.2, 8.1 Hz, 2H), 3.61 (dd, J = 15.7, 1.4 Hz, 3H), 3.25 (d, J = 7.1 Hz, 2H), 1.68 (d, J = 6.2 Hz, 2H), 1.25 (s, 13H), 0.84 (d, J = 6.2 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 209.70, 174.24, 161.64, 141.75, 134.94, 126.31, 122.19, 115.78, 114.32, 105.95, 43.17, 32.07, 29.73, 29.70, 29.54, 29.48, 29.30, 24.46, 22.85, 14.29; m/z calcd for C20H27NO3− [M − H]− 374.1708, found 374.1711; Mp: 65.0–69.1 °C.
6-Chloro-3-decanoyl-4-hydroxy-1-methylquinolin-2(1H)-one (3i): 1e (80 mg, 0.38 mmol), 2b (92 mg, 0.38 mmol), NaH (22.8 mg, 0.57 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 5% EtOAc in Hexanes) to give the title product (31 mg, 22% yield) as an off-white solid. 1H NMR (500 MHz, CDCl3) δ 8.57–7.97 (m, 1H), 7.82–7.43 (m, 1H), 7.53–6.99 (m, 1H), 3.63 (d, J = 13.9 Hz, 3H), 3.34–3.16 (m, 2H), 1.71 (s, 2H), 1.29 (s, 13H), 0.88 (d, J = 6.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 209.80, 173.15, 161.29, 140.23, 134.90, 128.05, 125.58, 116.89, 115.92, 107.49, 106.33, 43.19, 32.08, 29.72, 29.70, 29.51, 29.49, 24.39, 22.86, 14.30; m/z calcd for C20H25ClNO3− [M − H]− 362.1523, found 362.9271; Mp: 79.5–81.0 °C.
6-Bromo-3-decanoyl-4-hydroxy-1-methylquinolin-2(1H)-one (3j): 1f (139 mg, 0.60 mmol), 2b (150 mg, 0.60 mmol), NaH (36 mg, 0.90 mmol), and DMF (1 mL) were used and purified by silica gel column chromatography (eluent: 5% EtOAc in Hexanes) to give the title product (18 mg, 7% yield) as a light-brown solid. 1H NMR (500 MHz, CDCl3) δ 8.40 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.9 Hz, 1H), 3.69 (d, J = 15.0 Hz, 3H), 3.34 (d, J = 7.4 Hz, 2H), 1.78 (d, J = 5.8 Hz, 2H), 1.35 (s, 13H), 0.94 (d, J = 6.8 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 209.76, 173.06, 161.25, 140.61, 137.61, 128.99, 128.64, 117.28, 116.15, 115.22, 106.30, 43.18, 32.08, 29.70, 29.49, 24.83, 24.38, 22.86, 20.96, 14.28; m/z calcd for C20H27BrNO3− [M + H]+ 408.1174, found 408.1335; Mp: 75.0–76.8 °C.

3.5. Evaluation of Antibacterial Activity

The antibacterial activity method was modified from the Manual of Antimicrobial Susceptibility Testing, American Society of Microbiology [37]. The minimum inhibitory concentrations (MICs) of each synthesized quinolone against Staphylococcus aureus (ATCC 6538P) and Escherichia coli (ATCC 25922) were determined using resazurin-96 well plate microdilution assay. Briefly, the quinolone solutions were prepared by dissolving the synthesized compounds in 4% v/v dimethyl sulfoxide (DMSO) in Mueller-Hinton Broth (MHB) to obtain the concentrations of 2000, 1000, 500, 250, 125, and 62.5 µg/mL. Ciprofloxacin, an antibiotic in the fluoroquinolone class, was used as a positive control. The 4% MHB solutions with and without bacteria were also used as experimental controls. The bacterial cultures were prepared by growing each strain in MHB at 37 °C for 3–5 h. Then, bacterial densities were measured by spectrometry at OD 600 nm (Genesys 20, Thermo Scientific, Waltham, MA, USA) and adjusted the cell concentration to 1.15 × 106 cells/mL. To test the antibacterial activity, 50 mL of each prepared quinolone solution was added into each well of 96-well plates, following by addition of 50 mL bacterial solution, so the final concentrations of the tested compounds were 1000, 500, 250, 125, 62.5, and 31.25 µg/mL. Then, the 96-well plates were closed, wrapped with parafilm to prevent the dehydration, and incubated at 37 °C for 21 h. After incubating for 21 h, 30 mL of 0.015 w/v resazurin aqueous solution was added into each well followed by 37 °C incubation for 1 h. The lowest concentrations of the agents that displayed blue color (no color change of resazurin) were assigned as MIC values.
In order to investigate whether the synthesized quinolones could kill the bacteria or not, the minimum bactericidal concentration (MBC) tests were performed by streaking the contents obtained from the wells appearing in a blue color on a Nutrient Agar (NA) medium and incubating at 37 °C for 24 h. The MBC values were defined as the lowest concentrations that the bacteria could not grow on the culture medium.

3.6. Evaluation of Antifungal Activity

The broth microdilution method for non-dermatophyte molds was performed to observe the antifungal activity using 96-well plates in RPMI media according to CLSI M38 (2017) [38]. In addition, 1 × 104 spores of Aspergillus flavus ATCC204304 were utilized and co-incubated with amphotericin B, a fungicidal antifungal agent, in serial dilutions, i.e., 0.125–8 µg/mL (final concentrations), and different quinolone agents dissolved in DMSO in serial dilutions, i.e., 0.39–100 µg/mL (final concentrations), at 37 °C. XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) solution (0.5 mg/mL in PBS) was then added into each well to observe the growth of A. flavus ATCC204304 at 48 h [39]. The plate was further incubated for 15 min at 37 °C and the supernatant was collected to measure the OD at 490 nm using a spectrophotometer (Lambda 1050+ UV/Vis/NIR, PerkinElmer, Waltham, MA, USA). The half maximal inhibitory concentration (IC50) of each quinolone agent was then calculated using linear regression analyses. The experiments were performed in biological triplicates.

4. Conclusions

In summary, ten 3-alkyl-4-hydroxy-2-quinolone analogs (3a–3j), as well as the isomer 4d, were synthesized and fully characterized. Most of the compounds showed substantial antifungal activity against A. flavus. The introduction of substituents, especially for halogens, at C-6 and C-7 led to a significant increase in the antifungal activity. The activity was also enhanced when replacing the tridecyl group at C-3 with the nonyl group. The SAR knowledge obtained from this study might be crucial for the development of new antimicrobial agents based on the 4-hydroxy-2-quinolone privilege scaffold.

Supplementary Materials

The following are available online, spectroscopic data for compounds 1a–1g, 2a, 2b, 3a–3j, 4d, 5d, Figure S1: MIC determination of Ciprofloxacin against S. aureus, Figure S2: MIC determination of 3a−3h against S. aureus, Figure S3: MIC determination of 3i, 3j and 4d against S. aureus, Figure S4: MBC determination of 3i and 3j against S. aureus, Figure S5: MIC determination of Ciprofloxacin against E. coli, Figure S6: MIC determination of 3a−3h Ciprofloxacin against E. coli, Figure S7: MIC determination of 3i, 3j and 4d against E. coli.

Author Contributions

Conceptualization, T.K. (Tanatorn Khotavivattana); Data curation, T.K. (Tanatorn Khotavivattana); Formal analysis, A.T. and S.N.P.; Funding acquisition, T.K. (Thitiphong Khamkhenshorngphanuch) and T.K. (Tanatorn Khotavivattana); Investigation, T.K. (Thitiphong Khamkhenshorngphanuch), K.K., A.J., N.P. and K.M.; Methodology, T.K. (Thitiphong Khamkhenshorngphanuch), A.T., S.N.P. and T.K. (Tanatorn Khotavivattana); Project administration, T.K. (Tanatorn Khotavivattana); Resources, A.T., S.N.P. and T.K. (Tanatorn Khotavivattana); Validation, T.K. (Thitiphong Khamkhenshorngphanuch), A.T., S.N.P. and T.K. (Tanatorn Khotavivattana); Visualization, T.K. (Thitiphong Khamkhenshorngphanuch), A.T., S.N.P. and T.K. (Tanatorn Khotavivattana); Writing—original draft, K.K., A.J. and T.K. (Tanatorn Khotavivattana); Writing—review & editing, T.K. (Thitiphong Khamkhenshorngphanuch), A.T., S.N.P. and T.K. (Tanatorn Khotavivattana). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Thailand Research Fund and the Office of the Higher Education Commission, Research Grant for New Scholar, grant number [MRG6280024] (to T.K.) and Chulalongkorn University, Grants for Development of New Faculty Staff, Ratchadaphiseksomphot Endowment Fund, grant number [DNS62_081_23_014_2] (to T.K.).

Acknowledgments

The authors would like to thank the National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand for the high-resolution mass spectrum (HRMS) analysis.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 3a–3j and 4d are available from the authors.
Molecules 25 03059 g001 550
Figure 1. 4-Hydroxy-2-quinolone analogs: (a) Examples of the structure–activity relationship investigated in previous works; (b) novel 4-hydroxy-2-quinolone analogs bearing alkyl side chain at C-3 and various types of substituents on C-6 and C-7 positions (this work).
Figure 1. 4-Hydroxy-2-quinolone analogs: (a) Examples of the structure–activity relationship investigated in previous works; (b) novel 4-hydroxy-2-quinolone analogs bearing alkyl side chain at C-3 and various types of substituents on C-6 and C-7 positions (this work).
Molecules 25 03059 g001
Molecules 25 03059 g002 550
Figure 2. Synthetic scheme of the 3-step protocol for the preparation of 4-hydroxy-2-quinolone analogs (3a–3j).
Figure 2. Synthetic scheme of the 3-step protocol for the preparation of 4-hydroxy-2-quinolone analogs (3a–3j).
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Molecules 25 03059 g003 550
Figure 3. Synthesis of 3d from the reaction between 1d and 2a, generating 2 additional byproducts, 4d and 5d.
Figure 3. Synthesis of 3d from the reaction between 1d and 2a, generating 2 additional byproducts, 4d and 5d.
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Table
Table 1. Antifungal and antibacterial activity of 3a–3j and 4d.
Table 1. Antifungal and antibacterial activity of 3a–3j and 4d.
CompoundSubstituentsIC50 1 (µg/mL)MIC 2 (µg/mL)
R 1R 2R 3A. flavusS. aureusE. coli
3atridecylHMe70.97 ± 3.71>1000>1000
3btridecyl6-OCH3Me42.15 ± 18.61>1000>1000
3ctridecyl7-ClMe14.72 ± 2.35>1000>1000
3dtridecyl7-NO2Me38.45 ± 5.51>1000>1000
3etridecyl6-ClMe37.08 ± 1.52>1000>1000
3ftridecyl6-BrMe23.19 ± 3.19>1000>1000
3gtridecylHEt9.48 ± 6.17>1000>1000
3hnonylHMe8.45 ± 1.59>1000>1000
3inonyl6-ClMe4.60 ± 1.14*>1000
3jnonyl6-BrMe1.05 ± 1.31**>1000
4d3tridecyl 37-NO2 3Me 323.44 ± 1.63>1000>1000
AMB 4–––1.93 ± 1.04NDND
CIP 5–––ND0.8350.0288
1 Half maximal inhibitory concentration (IC50) of the antifungal activity against A. flavus determined by broth microdilution method, expressed as mean ± SD; 2 Minimum inhibitory concentration (MIC) of the antibacterial activity against S. aureus and E. coli determined by broth microdilution method. All experiments were performed in biological triplicates; 3 The structure is based on the 1,4-dihydroquinoline-3-carboxylate scaffold (see Figure 3); 4 AMB = amphotericin B as a positive control for the antifungal activity; 5 CIP = ciprofloxacin as a positive control for the antibacterial activity; ND = No data. The MIC values of 3i (*) and 3j (**) were unclear; however, both compounds showed inhibitory activities at the concentrations between 125–1000 and 125–500 µg/mL, respectively (Figure S3).

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Khamkhenshorngphanuch, T.; Kulkraisri, K.; Janjamratsaeng, A.; Plabutong, N.; Thammahong, A.; Manadee, K.; Na Pombejra, S.; Khotavivattana, T. Synthesis and Antimicrobial Activity of Novel 4-Hydroxy-2-quinolone Analogs. Molecules 2020, 25, 3059. https://doi.org/10.3390/molecules25133059

AMA Style

Khamkhenshorngphanuch T, Kulkraisri K, Janjamratsaeng A, Plabutong N, Thammahong A, Manadee K, Na Pombejra S, Khotavivattana T. Synthesis and Antimicrobial Activity of Novel 4-Hydroxy-2-quinolone Analogs. Molecules. 2020; 25(13):3059. https://doi.org/10.3390/molecules25133059

Chicago/Turabian Style

Khamkhenshorngphanuch, Thitiphong, Kittipat Kulkraisri, Alongkorn Janjamratsaeng, Napasawan Plabutong, Arsa Thammahong, Kanitta Manadee, Sarisa Na Pombejra, and Tanatorn Khotavivattana. 2020. "Synthesis and Antimicrobial Activity of Novel 4-Hydroxy-2-quinolone Analogs" Molecules 25, no. 13: 3059. https://doi.org/10.3390/molecules25133059

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