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. 2025 Oct 8;33(5):924–938. doi: 10.2174/0109298673372039250614231629

Side Chain Effects on the Lipophilicity-antimicrobial-toxicity Correlation of Greener 4-Alkoxy/Amino-7-Chloroquinolines

Gabriela F Fiss 1,*, Everton P Silva 1, Maria F S Madruga 1, Abraão P Sousa 1, Helivaldo D S Souza 1, Rádamis B Castor 2, Maria H Nascimento 2, Krystyna G Lira 2, Petrônio F Athayde-Filho 1
PMCID: PMC13223418  PMID: 41088934

Abstract

Background

More robust 4-substituted 7-chloroquinolines have been investigated for their diverse properties. However, there is still no systematic study that correlates the effects of the side chain at the 4-position of chloroquine and hydroxychloroquine derivatives with their lipophilicity, antimicrobial and toxicity properties.

Objective

To this end, a cleaner and facile approach was planned to obtain nineteen 4- substituted 7-chloroquinolines, whose influence of the substituent group and side chain extension at the 4-position on their properties was studied.

Methods

4-Alkoxy/amino-7-chloroquinolines were prepared by a nucleophilic aromatic substitution (SNAr) reaction between 4,7-dichloroquinoline and alcohols/amines, evaluated for their in silico ADMET test, in vitro antimicrobial activity against Gram-(+) and Gram-(−) bacteria, and Candida albicans fungus, and in vitro toxicity on Artemia salina larvae.

Results

4-Alkoxy/amino-7-chloroquinolines were obtained in yields ranging from 81 to 100%. The best results showed antimicrobial activity against Pseudomonas aeruginosa for 4-amino-7-chloroquinolines 6-8, with halos greater than 20 mm, and against C. albicans for 4-amino-7-chloroquinolines 1-3, with halos close to 30 mm. A correspondence between Minnow toxicity prediction and in vitro toxicity on A. salina larvae was observed, where compounds 3 and 14, with R = Pent, were both predicted to have high acute toxicity (log LC50 < -0.3) and classified as highly toxic (LC50 < 100 µg mL-1). It seems that increased lipophilicity in the side chain is harmful to A. salina larvae.

Conclusion

Considering the results for compounds 1-3 and 6-8 with greater activity against C. albicans and P. aeruginosa, respectively, especially for 4-amino-7-chloroquinolines 6 and 7, which are slightly toxic on A. salina larvae (LC50 500-1000 µg mL-1), their antimicrobial studies deserve to be continued by the determination of Minimum Inhibitory Concentration (MIC) values.

Keywords: Antibiotics, Artemia salina, 7-chloroquinolines, drug research, fungicides, green chemistry

1. INTRODUCTION

Nitrogen is the most common heteroatom in heterocyclic compounds, which have been used in pharmaceuticals such as antibiotics [1] and antitumor [2]. Considering the most recent commercial data on nitrogenous heterocycles, they were the 32nd most traded product in the world in 2022 [3]. Nitrogenous heterocycles include quinoline and its derivatives. Among the quinoline derivatives, some clinical drugs deserve to be highlighted, such as Bedaquiline, antitubercular, Chloroquine and Hydroxychloroquine, antimalarial, Ciprofloxacin, antibiotic, Pitavastatin, cholesterol reducer, among others [4-8].

Structure-activity relationships in aminoquinolines have suggested that the alkylamino and chloro groups at positions 4 and 7, respectively, are essential for antiplasmodial activity [9]. In another study, the alkyl side chain between three and six carbons in 4-amino-7-chloroquinolines was shown to be more selective and active against malaria [10]. Furthermore, the scientific community has registered several synthetic [11, 12] and supramolecular [13, 14] research on 4-amino-7-chloroquinolines, as well as their antimicrobial properties [15-17].

The variation in the number of carbons in the alkyl chain, i.e., variation in lipophilicity, has been used to modulate microbial resistance mechanisms [18]. It is known that the partition of organic compounds in an octanol:water system is a suitable mimic of the membrane:water interface. Therefore, lipophilicity, which has a direct impact on oral absorption, permeability and toxicity, can be predicted through this partition coefficient, where the measurement of the concentrations of a substance in both phases results in log PO/W (log CO/CW), whose ideal range is between values ​​1 and 3 [19].

The pharmacokinetic phase, which encompasses the Absorption, Distribution, Metabolism and Excretion (ADME) processes, can be drastically affected by the variation in the physicochemical properties of a drug. One of the main physicochemical properties of a micromolecule capable of altering its therapeutic profile is the partition coefficient (P), which expresses the relationship between its hydro/liposolubility profiles. In this context, Lipinski and collaborators [20, 21] proposed a set of predictive parameters, all ideally multiples of five and, therefore, known as the Rule of Five [22]. In addition, Veber and collaborators contributed to the prediction of additional properties for the oral bioavailability profile of drug candidates [23].

According to Lipinski, these rules have been questioned due to the fact that they were proposed at a time when most drugs had been discovered through phenotypic approaches, i.e., based on the observable effects of these drugs on organisms. Currently, the most common approach to drug development is based on the therapeutic target, considering a specific mechanism of action, in silico fragment screening and in vitro tests [24]. Prediction of pharmacokinetic properties is a guide but should not be limiting. Promising compounds that violate one or more parameters must be tested appropriately, considering the whole. Ultimately, the numbers speak and direct us towards more assertive paths.

More robust 4-substituted 7-chloroquinolines have been investigated regarding the influence of the alkyl side chain on their properties [10]. On the other hand, derivatives more similar to chloroquine and hydroxychloroquine [25, 26] have been used as synthetic intermediates, but there is still no systematic study that correlates the effects of the substituent group and alkyl chain at the 4-position on the lipophilicity, antimicrobial and toxicity properties.

4,7-Dichloroquinoline is a very favorable substrate to undergo a nucleophilic aromatic substitution (SNAr) reaction through the 4-position. Thus, 4-substituted 7-chloroquinolines have been prepared from the SNAr reaction of 4,7-dichloroquinoline with different nucleophiles, generally under adverse conditions of high temperature (T > 100°C), long reaction time (t > 12 h), additional solvent or purification step [11]. Therefore, there is still no standardized cleaning method that covers a more extensive series.

Our research group has been engaged in developing processes and/or chemical products that prevent environmental pollution [27-29]. Now, with the aim of studying the influence of the side chain on lipophilicity, antimicrobial and toxicity properties, a cleaner and facile approach was developed to obtain nineteen 4-alkoxy/amino-7-chloroquinolines, which were evaluated for their in silico ADMET test and in vitro antimicrobial activity against Gram-(+) and Gram-(−) bacteria, and Candida albicans fungus. Furthermore, in vitro toxicity on Artemia salina larvae for the most active compounds was required.

2. MATERIALS AND METHODS

2.1. Chemistry

All common reagents were purchased from commercial suppliers and used without further purification. Melting points were measured using QUIMIS equipment, model Q340S23 (Diadema, Brazil). 1H and 13C Nuclear Magnetic Resonance (NMR) spectra were performed at Laboratório Multiusuário de Caracterização e Análise (LMCA-UFPB), which were acquired on Bruker Ascend (Coventry, United Kingdom) and Bruker (Billerica, MA. USA) spectrometers at 400 and 500 MHz, respectively, for 1H, at 298 K, using 5 mm tubes. Infrared (IR) spectra were performed at Laboratório de Síntese Orgânica Medicinal (LASOM-UFPB), which were acquired on a Shimadzu spectrometer (Kyoto, Japan), model IRPrestige-21, using KBr pellets. High-Resolution Mass Spectrometry (HRMS) analyses were performed at LMCA-UFPB, using a Shimadzu HPLC (Kyoto, Japan) coupled to a Bruker MicroTOF II (Billerica, MA, USA) with an electrospray ion (ESI) source, and reported as m/z (relative intensity) for the molecular ion [M+H]+. Acquisition Parameters: Ion Polarity Positive, Capillary 4500 V, End Plate Offset -500 V, Nebulizer 4.0 Bar, Dry Heater 200°C, Dry Gas 8.0 L min-1, Divert Valve Waste.

2.1.1. General Procedure for the Synthesis of 4-amino-7-chloroquinolines (1-12)

A mixture of amine (8 mL) and 4,7-dichloroquinoline (10 mmol, 1.98 g) was maintained under magnetic stirring at 80°C for 4 h (for methylamine/propylamine, at 50°C for 12 h), which was monitored by thin layer chromatography (TLC) in ethyl acetate (AcOEt). Afterwards, the reactional mixture was cooled to room temperature, added to a beaker containing ice water (20 mL) and left in the freezer until freezing point. Then, the precipitate formed was filtered and washed with excess ice water. 4-Amino-7-chloroquinolines (1-12) were obtained in yields ranging from 81 to 100%.

7-Chloro-N-methylquinolin-4-amine (1) in 81% yield (8.1 mmol, 1.56 g); white solid; m.p.: 249-250°C ([30]. 245-246°C); 13C NMR (126 MHz, DMSO-d6): δ = 152.0, 150.9, 148.8, 133.3, 127.5, 124.1, 123.8, 117.4, 98.4 (9 × CAr), 29.2 (CH3) ppm ([12]. (100 MHz, CD3OD): δ = 153.8, 152.6, 149.6, 136.4, 127.7, 126.1, 124.3, 118.9, 99.4 (9 × CAr), 29.9 (CH3) ppm).

7-Chloro-N-propylquinolin-4-amine (2) in 94% yield (9.4 mmol, 2.07 g); white solid; m.p.: 146-148°C ([31]. 148-148.5°C); 13C NMR (126 MHz, CDCl3): δ = 152.1, 149.9, 149.2, 134.9, 128.8, 125.3, 121.0, 117.2, 99.1 (9 × CAr), 45.1 (CH2), 22.2 (CH2), 11.7 (CH3) ppm ([12]. (125 MHz, CD3OD): δ = 152.8, 152.3, 149.7, 136.3, 128.8, 125.9, 124.3, 118.8, 99.6 (9 × CAr), 45.8 (CH2), 22.6 (CH2), 11.9 (CH3) ppm).

7-Chloro-N-pentylquinolin-4-amine (3) in 88% yield (8.8 mmol, 2.18 g); beige solid; m.p.: 119-121°C ([32]. 119-121°C); 1H NMR (500 MHz, DMSO-d6): δ = 8.41 (m, 2H, 2 x HAr), 8.29 (bs, 1H, NH), 7.86 (d, J = 2.1 Hz, 1H, HAr), 7.53 (dd, J = 9.0, 2.3 Hz, 1H, HAr), 6.60 (d, J = 6.3 Hz, 1H, HAr), 3.34 (q, J = 7.3 Hz, 2H, CH2), 1.64 (p, J = 7.4 Hz, 2H, CH2), 1.33 (m, 4H, 2 x CH2), 0.86 (t, J = 7.1 Hz, 3H, CH3) ppm ([32]. (300 MHz, CDCl3): δ = 8.53 (d, J = 5.4 Hz, 1H, HAr), 7.96 (s, 1H, HAr), 7.64 (d, J = 8.9 Hz, 1H, HAr), 7.35 (d, J = 8.9 Hz, 1H, HAr), 6.41 (d, J = 5.3 Hz, 1H, HAr), 5.0 (bs, 1H, NH), 3.25 (m, 2H, CH2), 1.76 (m, 2H, CH2), 1.43 (m, 4H, 2 x CH2), 0.95 (t, J = 7.2 Hz, 3H, CH3) ppm); 13C NMR (126 MHz, CDCl3): δ = 152.5, 147.9, 144.4, 135.5, 125.3, 124.9, 123.7, 116.6, 98.6 (9 × CAr), 42.8 (CH2), 28.8 (CH2), 27.4 (CH2), 22.0 (CH2), 14.0 (CH3) ppm (not found) .

7-Chloro-N-(2-(dimethylamino)ethyl)quinolin-4- amine (4) in 94% yield (9.4 mmol, 2.34 g); beige solid; m.p.: 124-126°C ([8]. 122-124°C); 13C NMR (126 MHz, CDCl3): δ = 152.0, 150.0, 149.1, 134.9, 128.6, 125.3, 121.6, 117.4, 99.2 (9 × CAr), 57.0 (CH2), 45.1 (CH3)2, 39.9 (CH2) ppm ([33]. (101 MHz, DMSO-d6): δ = 151.9, 150.0, 149.1, 133.4, 127.5, 124.1, 124.0, 117.4, 98.7 (9 × CAr), 56.9 (CH2), 45.3 (CH3)2, 40.5 (CH2) ppm).

7-Chloro-N-(2-(diethylamino)ethyl)quinolin-4- amine (5) in 93% yield (9.3 mmol, 2.58 g); beige solid; m.p.: 106-108°C ([34]. 102-103°C); 13C NMR (126 MHz, CDCl3): δ = 152.1, 150.0, 149.1, 134.9, 128.7, 125.3, 121.3, 117.5, 99.3 (9 × CAr), 50.7 (CH2), 46.6 (CH2)2, 39.8 (CH2), 12.1 (CH3)2 ppm ([34]. (50 MHz, CDCl3): δ = 152.0, 149.8, 149.0, 134.6, 128.5, 125.1, 121.1, 117.3, 99.2 (9 × CAr), 50.4 (CH2), 46.3 (CH2)2, 39.6 (CH2), 11.9 (CH3)2 ppm.

N-(2-Aminoethyl)-7-chloroquinolin-4-amine (6) in 93% (9.3 mmol, 2.06 g) yield; white solid; m.p.: 149-151°C ([35]. 140-142°C); 13C NMR (126 MHz, DMSO-d6): δ = 151.9, 150.3, 149.0, 133.3, 127.4, 124.1, 123.9, 117.4, 98.7 (9 × CAr), 46.0 (CH2), 40.0 (CH2) ppm ([35]. (100 MHz, CD3OD): δ = 152.9, 152.5, 149.7, 136.4, 127.6, 126.1, 124.4, 118.9, 99.8 (9 × CAr), 46.3 (CH2), 40.9 (CH2) ppm).

N-(3-Aminopropyl)-7-chloroquinolin-4-amine (7) in 94% yield (9.4 mmol, 2.21 g); white solid; m.p.: 96-98°C ([36]. 96-98°C); 13C NMR (101 MHz, DMSO-d6, 80°C): δ = 151.5, 149.9, 148.8, 133.0, 127.2, 123.5, 123.5, 117.2, 98.3 (9 × CAr), 40.4 (CH2), 40.4 (CH2), 31.1 (CH2) ppm ([9]. (75 MHz, CDCl3): δ = 151.5, 150.0, 148.6, 133.9, 127.6, 124.2, 122.0, 117.1, 97.8 (9 × CAr), 42.8 (CH2), 40.8 (CH2), 29.5 (CH2) ppm).

N-(4-Aminobutyl)-7-chloroquinolin-4-amine (8) in 99% yield (9.9 mmol, 2.47 g); white solid; m.p.: 119-121°C ([36]. 122-124°C); 13C NMR (101 MHz, DMSO-d6, 80°C): δ = 151.5, 149.9, 148.9, 133.0, 127.2, 123.6, 123.5, 117.2, 98.3 (9 × CAr), 42.2 (CH2), 41.0 (CH2), 30.5 (CH2), 25.1 (CH2) ppm ([9]. (75 MHz, CDCl3): δ = 151.9, 150.0, 149.0, 134.5, 128.3, 124.8, 121.6, 117.3, 98.6 (9 × CAr), 43.0 (CH2), 41.4 (CH2), 30.6 (CH2) 25.9 (CH2) ppm).

N-(6-Aminohexyl)-7-chloroquinolin-4-amine (9) in 100% yield (10 mmol, 2.77 g); white solid; m.p.: 130-132°C ([36]. 133-134°C); 13C NMR (126 MHz, DMSO-d6): δ = 151.9, 150.1, 149.1, 133.3, 127.4, 124.1, 123.9, 117.4, 98.5 (9 × CAr), 42.3 (CH2), 41.4 (CH2), 32.9 (CH2), 27.8 (CH2), 26.5 (CH2), 26.2 (CH2) ppm ([9]. (75 MHz, CD3OD): δ = 152.3, 149.6, 136.2, 127.5, 125.8, 124.3, 120.2, 118.7, 99.5 (9 × CAr), 43.9 (CH2), 42.3 (CH2), 33.5 (CH2) 29.3 (CH2), 28.0 (CH2), 27.7 (CH2) ppm).

2-(7-Chloroquinolin-4-ylamino)ethanol (10) in 89% yield (8.9 mmol, 1.98 g); white solid; m.p.: 225-227°C ([37]. 203-205°C); 13C NMR (126 MHz, DMSO-d6): δ = 151.9, 150.2, 149.1, 133.4, 127.4, 124.0, 124.0, 117.4, 98.7 (9 × CAr), 58.7 (CH2), 45.1 (CH2) ppm ([37]. (126 MHz, DMSO-d6): δ = 152.3, 150.7, 149.5, 133.8, 127.9, 124.4, 124.4, 117.9, 99.1 (9 × CAr), 59.2 (CH2), 45.6 (CH2) ppm).

3-(7-Chloroquinolin-4-ylamino)propan-1-ol (11) in 97% yield (9.7 mmol, 2.29 g); white solid; m.p.: 155-157°C ([37]. 149-150°C); 13C NMR (126 MHz, DMSO-d6): δ = 151.9, 150.1, 149.0, 133.4, 127.4, 124.0, 124.0, 117.4, 98.6 (9 × CAr), 58.6 (CH2), 39.6 (CH2), 31.0 (CH2) ppm ([37]. (126 MHz, DMSO-d6): δ = 152.3, 150.6, 149.5, 133.8, 127.8, 124.5, 124.4, 117.9, 99.0 (9 × CAr), 59.0 (CH2), 40.0 (CH2), 31.4 (CH2) ppm).

2-(2-(7-chloroquinolin-4-ylamino)ethylamino)ethanol (12) in 91% yield (9.1 mmol, 2.41 g); white solid; m.p.: 142-144°C ([38]. 139-139.5°C); 13C NMR (101 MHz, DMSO-d6): δ = 151.9, 150.1, 149.0, 133.3, 127.4, 124.0, 124.0, 117.4, 98.7 (9 × CAr), 60.5 (CH2), 51.5 (CH2), 47.3 (CH2), 42.7 (CH2) ppm (not found).

2.1.2. General Procedure for the Synthesis of 4-alkoxy-7-chloroquinolines (13-19)

A mixture of potassium tert-butoxide (10 mmol, 1.12 g), alcohol (6 mL) and 4,7-dichloroquinoline (10 mmol, 1.98 g) was maintained under magnetic stirring at 100°C for 8 h, which was monitored by TLC in AcOEt. Afterwards, the reactional mixture was cooled to room temperature, added to a beaker containing ice water (20 mL) and left in the freezer until freezing point. Then, the precipitate formed was filtered and washed with excess ice water. 4-Alkoxy-7-chloroquinolines (13-19) were obtained in yields ranging from 81 to 99%. Full NMR, IR and HRMS spectra for 4-alkoxy-7-chloroquinolines (14, 18 and 19) are available in Figs. (S1 (817.3KB, pdf) -12 (817.3KB, pdf) ).

7-Chloro-4-propoxyquinoline (13) in 81% yield (8.1 mmol, 1.79 g); white solid; m.p.: 70-72°C ([39]. 77-79°C); 13C NMR (126 MHz, CDCl3): δ = 161.8, 152.6, 149.8, 135.7, 127.9, 126.5, 123.6, 120.0, 101.0 (9 × CAr), 70.2 (CH2), 22.3 (CH2), 10.6 (CH3) ppm (not found).

7-Chloro-4-(pentyloxy)quinoline (14, C14H16ClNO) in 88% yield (8.8 mmol, 2.19 g); Rf: 0.42 (AcOEt); beige solid; m.p.: 39-41°C; 1H NMR (500 MHz, CDCl3): δ = 8.69 (d, J = 5.2 Hz, 1H, HAr), 8.13 (d, J = 8.9 Hz, 1H, HAr), 8.00 (d, J = 2.1 Hz, 1H, HAr), 7.42 (dd, J = 8.9, 2.0 Hz, 1H, HAr), 6.69 (d, J = 5.3 Hz, 1H, HAr), 4.16 (t, J = 6.4 Hz, 2H, CH2), 1.93 (m, 2H, CH2), 1.52 (m, 2H, CH2), 1.43 (m, 2H, CH2), 0.96 (t, J = 7.2 Hz, 3H, CH3) ppm; 13C NMR (126 MHz, CDCl3): δ = 161.8, 152.6, 149.8, 135.7, 127.9, 126.5, 123.6, 120.0, 101.0 (9 × CAr), 68.8 (CH2), 28.6 (CH2), 28.3 (CH2), 22.5 (CH2), 14.1 (CH3) ppm; IR (KBr): ν = 3035 (HAr), 2954, 2927, 2862 (Halkanic), 1612 (C=N), 1581, 1566, 1500, 1469, 1427 (C=C), 1307, 1195, 1157, 1118, 1068 (C-O), 840, 813, 748 (Ar), 644 (C-Cl) cm-1; HRMS (ESI): calcd for C14H17ClNO ([M+H]+) 250.0993, found 250.0993.

2-(7-Chloroquinolin-4-yloxy)ethanol (15) in 86% yield (8.6 mmol, 1.92 g); white solid; m.p.: 122-124°C ([37]. 119-120°C); 13C NMR (126 MHz, DMSO-d6): δ = 161.1, 153.1, 149.2, 134.4, 127.2, 126.1, 124.2, 119.5, 102.1 (9 × CAr), 70.6 (CH2), 59.3 (CH2) ppm ([37]. (126 MHz, DMSO-d6): δ = 161.4, 152.2, 149.4, 135.8, 127.6, 126.5, 123.2, 119.6, 101.5 (9 × CAr), 70.0 (CH2), 60.8 (CH2) ppm).

3-(7-Chloroquinolin-4-yloxy)propan-1-ol (16) in 86% yield (8.6 mmol, 2.04 g); white solid; m.p.: 119-121°C ([37]. 112-115°C); 13C NMR (126 MHz, DMSO-d6): δ = 160.9, 153.1, 149.1, 134.4, 127.2, 126.2, 123.8, 119.4, 102.0 (9 × CAr), 65.7 (CH2), 57.1 (CH2), 31.7 (CH2) ppm ([37]. (126 MHz, DMSO-d6): δ = 161.4, 152.1, 149.1, 135.7, 127.3, 126.3, 123.2, 119.5, 100.7 (9 × CAr), 65.1 (CH2), 58.4 (CH2), 31.7 (CH2) ppm).

4-(7-Chloroquinolin-4-yloxy)butan-1-ol (17) in 99% yield (9.9 mmol, 2.49 g); white solid; m.p.: 150-152°C (not found); 13C NMR (101 MHz, DMSO-d6) δ = 160.8, 153.1, 149.1, 134.3, 127.2, 126.2, 123.7, 119.4, 102.1 (9 × CAr), 68.5 (CH2), 60.3 (CH2), 28.9 (CH2), 25.1 (CH2) ppm ([9]. (75 MHz, CDCl3): δ = 161.6, 152.5, 149.5, 135.5, 127.8, 126.3, 123.3, 119.8, 100.9 (9 × CAr), 68.5 (CH2), 62.5 (CH2), 29.4 (CH2), 25.8 (CH2) ppm).

2-(2-(7-Chloroquinolin-4-yloxy)ethoxy)ethanol (18, C13H14ClNO3) in 82% yield (8.2 mmol, 2.19 g); Rf: 0.42 (AcOEt); white solid; m.p.: 82-84°C; 1H NMR (400 MHz, CDCl3): δ = 8.68 (d, J = 5.3 Hz, 1H, HAr), 8.08 (d, J = 8.9 Hz, 1H, HAr), 8.01 (dd, J = 2.1, 0.4 Hz, 1H, HAr), 7.39 (dd, J = 8.9, 2.1 Hz, 1H, HAr), 6.64 (d, J = 5.3 Hz, 1H, HAr), 4.30 (m, 2H, CH2), 4.00 (m, 2H, CH2), 3.82 (m, 2H, CH2), 3.73 (m, 2H, CH2) ppm; 13C NMR (101 MHz, CDCl3): δ = 161.5, 152.4, 149.6, 135.9, 127.8, 126.6, 123.5, 119.7, 100.9 (9 × CAr), 73.0 (CH2), 69.2 (CH2), 68.1 (CH2), 61.8 (CH2) ppm; IR (KBr): ν = 3228 (O-H), 3055 (HAr), 2947, 2924, 2866 (Halkanic), 1616 (C=N), 1581, 1500, 1431 (C=C), 1311, 1199, 1161, 1126, 1064 (C-O), 837, 821, 767, 748 (Ar), 644 (C-Cl); HRMS (ESI): calcd for C13H15ClNO3 ([M+H]+) 268.0735, found 268.0742.

2-(2-(7-Chloroquinolin-4-yloxy)ethylthio)ethanol (19, C13H14ClNO2S) in 85% yield (8.5 mmol, 2.41 g); Rf: 0.65 (AcOEt); white solid; m.p.: 107-109°C; 1H NMR (500 MHz, CDCl3): δ = 8.73 (d, J = 5.2 Hz, 1H, HAr), 8.13 (d, J = 9.0 Hz, 1H, HAr), 8.03 (d, J = 2.1 Hz, 1H, HAr), 7.46 (dd, J = 8.9, 2.1 Hz, 1H, HAr), 6.73 (d, J = 5.3 Hz, 1H, HAr), 4.38 (t, J = 6.6 Hz, 2H, CH2), 3.83 (t, J = 5.9 Hz, 2H, CH2), 3.10 (t, J = 6.6 Hz, 2H, CH2), 2.88 (t, J = 5.9 Hz, 2H, CH2) ppm; 13C NMR (126 MHz, CDCl3): δ = 161.2, 152.5, 149.9, 136.0, 128.0, 126.9, 123.4, 119.8, 101.1 (9 × CAr), 68.3 (CH2), 61.0 (CH2), 36.0 (CH2), 30.7 (CH2) ppm; IR (KBr): ν = 3182 (O-H), 2939, 2850 (Halkanic), 1612 (C=N), 1573, 1500, 1427 (C=C), 1311, 1276, 1199, 1157, 1122, 1076, 1049, 1018 (C-O/S), 848, 817, 748 (Ar), 644 (C- Cl); HRMS (ESI): calcd for C13H15ClNO2S ([M+H]+) 284.0507, found 284.0516.

2.2. In Silico ADMET Test

The prediction of pharmacokinetic properties, Absorption, Distribution, Metabolism and Excretion (ADME) for 4-alkoxy/amino-7-chloroquinolines 1-19 was carried out through the open-access electronic site SwissADME (http://www.swissadme.ch/). Lipophilicity was deduced from consensus log P (average of all five predictions). The toxicity prediction for 4-alkoxy/amino-7-chloroquinolines 1-19 was carried out through the open-access electronic site pkCSM (https://biosig.lab.uq.edu.au/pkcsm/prediction).

2.3. In Vitro Antimicrobial Evaluation

4-Alkoxy/amino-7-chloroquinolines (1-19) were evaluated for their in vitro antimicrobial activity against six microorganisms from American Type Culture Collection (ATCC), Coleção de Culturas Tropical (CCT) and NEWPROV (NEWP), including Gram-(+) (Bacillus cereus CCT 0198, Enterococcus faecalis NEWP 0012 and Staphylococcus aureus ATCC 25923) and Gram-(−) bacteria (Escherichia coli NEWP 0039 and Pseudomonas aeruginosa NEWP 0027), and Candida albicans (NEWP 0031) fungus, according to Antibiotic Susceptibility Testing by a Standardized Single Disk Method [40] and Clinical and Laboratory Standards Institute (CLSI), document M27-A2 [41]. Methanolic solutions were prepared at a concentration of 2 mg mL-1. Antibiotic controls used against bacteria included Amoxicillin/Clavulanic acid, Ciprofloxacin, Penicillin G, Tetracycline and Vancomycin, and Fluconazole was used as antifungal control. All experiments were performed in triplicate.

2.4. In Vitro Toxicity Evaluation

In vitro toxicity evaluation of 4-amino-7-chloroquinolines (1-3 and 6-8) and 4-alkoxy-7-chloroquinolines (13-14) on Artemia salina larvae was performed according to an adapted method reported by Sousa et al. [42], with some adjustments. In a rectangular aquarium of 3 L capacity and using 20 W light at a height of 20 cm from the water surface, 1 g of A. salina cysts was placed in 1 L of saline solution (30 g L-1, pH ~ 8) and aerated at 27°C for 48 h. The toxicity was evaluated at concentrations of 125, 250 and 375 µg mL-1. To this end, stock solutions were prepared at a concentration of 12.5 mg mL-1 for each compound, from which volumes of 50, 100 and 150 µL were captured, added to tubes and, when necessary, completed with DMSO q.s.p. 150 µL (3%). Next, 100 µL (2%) of Tween 80 was added and completed with saline solution q.s.p. 5 mL. Then, ten healthy A. salina nauplii were placed inside each tube. After 24 h, the number of alive nauplii was counted. Concentration series (125, 250 and 375 µg mL-1) of potassium dichromate solution was used as a positive control. Two negative controls were prepared, one containing only saline solution, and another containing saline solution with 3% DMSO and 2% Tween 80. All experiments were performed in triplicate. Data are expressed in terms of mortality percentage versus concentration sample. The percentage of lethality on A. salina (%LAS) of the samples was determined by the formula %LAS = [(number of dead nauplii in the test − number of dead nauplii in the negative control) / number of alive nauplii in the negative control] × 100 [43]. Average percentages of alive A. salina nauplii in different concentrations were calculated using GraphPad Prism 5.0 (San Diego, CA, USA). Means were significantly different when p ≤ 0.0002*** using One-way analysis of variance and Bonferroni’s Multiple Comparison Test (Fig. S13 (817.3KB, pdf) ). Median lethal concentration (LC50) was estimated using linear regression equation (Table S1 (817.3KB, pdf) ).

3. RESULTS

3.1. Chemistry

Standardized approaches were developed to synthesize twelve 4-amino-7-chloroquinolines (1-12) and seven 4-alkoxy-7-chloroquinolines (13-19) from an SNAr reaction of 4,7-dichloroquinoline with amines and alcohols, respectively (Scheme 1).

Scheme 1.

Scheme 1

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Reagents and reactional conditions to obtain 4-amino-7-chloroquinolines (1-12): (i) amine, 80°C, 4 h (R = Me/Pr, at 50°C for 12 h); Reagents and reactional conditions to obtain 4-alkoxy-7-chloroquinolines (13-19): (ii) alcohol, t-BuOK, 100°C, 8 h.

The novel compounds (14, 18 and 19) were characterized by spectroscopic techniques of IR, and 1H and 13C NMR. Furthermore, high resolution mass analyses were required.

3.2. In Silico ADMET Test

Based on Lipinski's Rule of Five and parameters established by Veber, the ADMET prediction [44] for 4-alkoxy/amino-7-chloroquinolines (1-19) was generated from SwissADME [45] pkCSM [46] programs, which is available in Table 1.

Table 1.

ADMET prediction for 4-alkoxy/amino-7-chloroquinolines (1-19).

Compound, R Lipinski Veber Toxicity
MW (Da) HBD HBA Log P TPSA (Å2) Nrotb Skin Sensitisation Hepato Minnow log LC50
1, Me 192.64 1 1 2.61 24.92 1 No No 0.67
2, Pr 220.70 1 1 3.30 24.92 3 No No 0.19
3, Pent 248.75 1 1 3.98 24.92 5 No Yes -0.33
4, (CH2)2NMe2 249.74 1 2 2.69 28.16 4 No No 1.74
5, (CH2)2NEt2 277.79 1 2 3.35 28.16 6 No Yes 1.41
6, (CH2)2NH2 221.69 2 2 2.05 50.94 3 No No 0.96
7, (CH2)3NH2 235.71 2 2 2.47 50.94 4 No Yes 0.66
8, (CH2)4NH2 249.74 2 2 2.68 50.94 5 No Yes 0.37
9, (CH2)6NH2 277.79 2 2 3.32 50.94 7 No Yes -0.08
10, (CH2)2OH 222.67 2 2 2.14 45.15 3 No No 0.97
11, (CH2)3OH 236.70 2 2 2.56 45.15 4 No Yes 0.67
12, (CH2)2NH(CH2)2OH 265.74 3 3 1.98 57.18 6 No No 2.17
13, Pr 221.68 0 2 3.33 22.12 3 No No -0.14
14, Pent 249.74 0 2 4.01 22.12 5 No No -0.67
15, (CH2)2OH 223.66 1 3 2.18 42.35 3 No No 0.64
16, (CH2)3OH 237.68 1 3 2.50 42.35 4 No No 0.34
17, (CH2)4OH 251.71 1 3 2.80 42.35 5 No No 0.05
18, (CH2)2O(CH2)2OH 267.71 1 4 2.20 51.58 6 No No -0.08
19, (CH2)2S(CH2)2OH 283.77 1 3 2.84 67.65 6 No No -0.11
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Abbreviations: MW: molecular weight; HBD: hydrogen bond donor; HBA: hydrogen bond acceptor; Log P: octanol/water partition coefficient; TPSA: topological polar surface area; nrotb: number of rotatable bonds; Minnow Toxicity: median lethal concentration (LC50) for Flathead Minnows.

Note: Lipinski: MW < 500 Da, HBD ≤ 5, HBA ≤ 10, log p ≤ 5 [22]; and Veber parameters: TPSA ≤ 140 Å2, nrotb ≤ 10 [23].

3.3. In Vitro Antimicrobial Evaluation

In vitro antimicrobial activity of 4-alkoxy/amino-7-chloroquinolines (1-19) was evaluated against three Gram-(+) bacteria, two Gram-(−) bacteria and one yeast fungus. Amoxicillin/Clavulanic acid, Ciprofloxacin, Penicillin G, Tetracycline and Vancomycin were used as antibiotic controls, and Fluconazole as an antifungal control. Zone of inhibition (ZOI) values are available in Table 2.

Table 2.

ZOI (mm) ± SD for 50 µL of 4-alkoxy/amino-7-chloroquinolines (1-19).

Compound, R Bacteria Yeast fungus
Gram-(+) Gram-(−)
B. cereus CCT 0198 E. faecalis NEWP 0012 S. aureus ATCC 25923 E. coli NEWP 0039 P. aeruginosa NEWP 0027 C. albicans NEWP 0031
1, Me - 8.61 ± 0.69 7.58 ± 0.17 13.39 ± 0.00 6.35 ± 0.00 24.63 ± 0.24
2, Pr 11.69 ± 0.61 9.87 ± 0.18 10.97 ± 1.06 14.52 ± 0.50 7.12 ± 0.08 20.10 ± 0.98
3, Pent 17.96 ± 0.31 13.79 ± 0.51 15.40 ± 0.10 12.39 ± 0.38 6.35 ± 0.00 29.59 ± 1.71
4, (CH2)2NMe2 7.67 ± 0.31 - 7.50 ± 0.31 8.32 ± 0.81 6.95 ± 0.36 12.37 ± 0.28
5, (CH2)2NEt2 7.92 ± 1.47 - 7.39 ± 0.45 8.57 ± 0.04 6.75 ± 0.17 8.51 ± 0.63
6, (CH2)2NH2 10.32 ± 0.42 - 7.83 ± 0.75 9.61 ± 0.83 21.88 ± 1.31 8.60 ± 0.18
7, (CH2)3NH2 8.93 ± 0.10 - 7.81 ± 0.52 8.54 ± 0.46 20.91 ± 0.65 7.60 ± 0.26
8, (CH2)4NH2 7.73 ± 0.39 - 6.59 ± 0.21 7.81 ± 0.18 21.90 ± 0.97 6.28 ± 0.07
9, (CH2)6NH2 8.83 ± 1.20 - 8.27 ± 0.25 7.87 ± 0.18 7.37 ± 0.18 6.72 ± 0.05
10, (CH2)2OH - - - 6.68 ± 0.12 - 7.14 ± 0.40
11, (CH2)3OH - - 6.44 ± 0.12 6.61 ± 0.47 7.05 ± 0.62 8.31 ± 0.48
12, (CH2)2NH(CH2)2OH - - 7.55 ± 0.35 6.82 ± 0.12 - 7.78 ± 0.19
13, Pr 9.51 ± 0.45 9.08 ± 0.38 13.73 ± 2.06 7.40 ± 0.11 - 15.90 ± 0.77
14, Pent 8.35 ± 0.04 9.30 ± 0.85 9.64 ± 0.34 - - 14.83 ± 1.35
15, (CH2)2OH - - 7.19 ± 0.57 6.07 ± 0.04 6.70 ± 0.42 6.91 ± 0.17
16, (CH2)3OH 6.62 ± 0.10 8.58 ± 0.43 6.43 ± 0.04 - - 11.71 ± 1.42
17, (CH2)4OH 7.68 ± 0.58 - 8.22 ± 0.40 6.19 ± 0.04 6.93 ± 0.04 12.34 ± 2.35
18, (CH2)2O(CH2)2OH 6.92 ± 0.21 - 7.71 ± 0.22 6.75 ± 0.20 6.71 ± 0.20 7.44 ± 0.06
19, (CH2)2S(CH2)2OH 8.64 ± 0.29 - 6.74 ± 0.29 6.11 ± 0.07 6.04 ± 0.01 13.23 ± 1.06
AMC (30 µg) - - 41.43 ± 2.71 15.30 ± 3.72 19.90 ± 2.14 NA
CIP (5 µg) 23.20 ± 2.11 16.79 ± 2.17 33.49 ± 1.58 25.99 ± 1.22 29.44 ± 2.22 NA
PEN (10 U.I.) - 16.96 ± 0.90 45.11 ± 4.30 - - NA
TET (30 µg) 26.22 ± 2.41 14.97 ± 0.31 30.04 ± 0.99 21.06 ± 0.39 21.06 ± 0.39 NA
VAN (30 µg) 12.15 ± 4.23 16.65 ± 0.36 19.79 ± 1.71 - - NA
FCZ (32 µg) NA NA NA NA NA 32.42 ± 0.00
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Abbreviations: ZOI: Zone of Inhibition; SD: Standard Deviation; AMC: Amoxicillin/Clavulanic acid; CIP: Ciprofloxacin; PEN: Penicillin G; TET: Tetracycline; VAN: Vancomycin; FCZ: Fluconazole; NA: Not Applicable; -: Not Active.

3.4. In Vitro Toxicity Evaluation

Considering the compounds that showed the most relevant antimicrobial activity, an environmental toxicity evaluation was required. Then, in vitro toxicity of 4-amino-7-chloroquinolines (1-3 and 6-8) and 4-alkoxy-7-chloroquinolines (13-14) on Artemia salina larvae was evaluated. A saline solution, and a saline solution containing 3% DMSO and 2% Tween 80, in which the number of dead nauplii was equal to 0.33, were used as negative controls. A potassium dichromate solution was used as a positive control, in which all nauplii died. Average percentages of alive nauplii in different concentrations and median lethal concentration (LC50) values are provided in Tables 3 and 4, respectively.

Table 3.

AP ± SD (%) of alive A. salina nauplii in different concentrations of 4-amino-7-chloroquinolines (1-3 and 6-8) and 4-alkoxy-7-chloroquinolines (13-14).

Compound, R Concentration Series (µg mL-1)
125 250 375
1, Me 68.94 ± 5.97 44.82 ± 5.97 13.79 ± 5.97
2, Pr 6.89 ± 5.97 3.44 ± 5.97 0.00 ± 0.00
3, Pent 3.44 ± 5.97 0.00 ± 0.00 0.00 ± 0.00
6, (CH2)2NH2 75.84 ± 5.97 65.50 ± 5.97 62.05 ± 0.00
7, (CH2)3NH2 95.38 ± 4.00 82.73 ± 0.0 68.94 ± 5.97
8, (CH2)4NH2 91.93 ± 8.69 68.94 ± 5.97 51.71 ± 0.00
13, Pr 6.89 ± 5.97 3.44 ± 5.97 0.00 ± 0.00
14, Pent 6.89 ± 5.97 0.00 ± 0.00 0.00 ± 0.00
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Abbreviations: AP: Average Percentage; SD: Standard Deviation.

Note: Statistics were analyzed using the ANOVA method in a confidence interval with a confidence level of 95%.

Table 4.

LC50 values of 4-amino-7-chloroquinolines (1-3 and 6-8) and 4-alkoxy-7-chloroquinolines (13-14) on A. salina larvae.

Structure Compound, R Log P LC50 (µg mL-1)
graphic file with name CMC-33-5-924-t4-1.jpg 1, Me 2.61 203.87
2, Pr 3.30 << 100
3, Pent 3.98 << 100
6, (CH2)2NH2 2.05 573.09
7, (CH2)3NH2 2.47 777.50
8, (CH2)4NH2 2.68 368.12
graphic file with name CMC-33-5-924-t4-2.jpg 13, Pr 3.33 << 100
14, Pent 4.01 << 100
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Abbreviations: Log P: octanol/water partition coefficient prediction; LC50: lethal concentration value where a substance causes the death of 50% of A. salina larvae.

LC50 values were compared with those recommended in the literature [47], where LC50 values ​​lower than 100 µg mL-1 are classified as highly toxic, between 100 and 500 µg mL-1 are moderately toxic, between 500 and 1000 µg mL-1 are slightly toxic, and above 1000 µg mL-1 are non-toxic.

4. DISCUSSION

According to Scheme 1, amines, including diamines and amino alcohols, and alcohols, including diols, were used as reagents and solvents under mild conditions, with reaction temperature equal to or less than 100°C and time no longer than 12 h. 4-Amino-7-chloroquinolines (1-12) and 4-alkoxy-7-chloroquinolines (13-19) were obtained from precipitation in water in high yields of 81-100% and 81-99%, respectively. Additional steps such as liquid-liquid extraction and/or chromatographic column were not necessary.

In general, 4-alkoxy/amino-7-chloroquinolines have been prepared through the SArN reaction between 4,7-dichloroquinoline and alcohols/amines (Table 5) with the addition of solvent [9, 30, 32, 34, 37, 38] or another reagent [9, 37-39], in high temperature [33, 36, 38], long reaction time [9, 30, 32, 33, 37, 39] or additional purification step [9, 32-34, 36-38].

Table 5.

Some reactional conditions described in the literature to obtain 4-alkoxy/amino-7-chloroquinolines via SNAr reaction between 4,7-dichloroquinoline and alcohols/amines.

Compound, R Additional T (°C) t (h) Separation Step Yield (%) Reference
Reagent Solvent Condition
1, Me - DMSO - 70 216 Precipitation in water 98 [30]
2, Pr - - - Reflux 12 Precipitation in water 80 [31]
3, Pent - EtOH - Reflux 18 Column chromatography - [32]
4, (CH2)2NMe2 - - Sealed tube 110 16 Column chromatography 58 [33]
5, (CH2)2NEt2 - Phenol - 90 11 Column chromatography 35 [34]
6, (CH2)2NH2 - - US - 0,5 Precipitation in water 97 [35]
7, (CH2)3NH2 - - - 80-135 4 Column chromatography 90 [36]
8, (CH2)4NH2 - - - 80-135 4 Column chromatography 75 [36]
9, (CH2)6NH2 - - - 80-135 4 Column chromatography 55 [36]
10, (CH2)2OH - EtOH - Reflux 8 Precipitation in water 84 [37]
11, (CH2)3OH - EtOH - Reflux 8 Precipitation in water 82 [37]
12, (CH2)2NH(CH2)2OH NaI Phenol - Reflux - Distillation with steam 74 [38]
13, Pr HCl (0,5 N) - - Reflux 24 Precipitation in water 66 [39]
14, Pent t-BuOK - - 100 8 Precipitation in water 88 In this work.
15, (CH2)2OH t-BuOK t-BuOH N2(g) 80 16 Liquid-liquid extraction 94 [37]
16, (CH2)3OH t-BuOK t-BuOH N2(g) 80 16 Liquid-liquid extraction 96 [37]
17, (CH2)4OH t-BuOK t-BuOH N2(g) 80 18 Liquid-liquid extraction 66 [9]
18, (CH2)2O(CH2)2OH t-BuOK - - 100 8 Precipitation in water 82 In this work.
19, (CH2)2S(CH2)2OH t-BuOK - - 100 8 Precipitation in water 85 In this work.
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Abbreviations: -: Not Informed; US: Ultrasound Irradiation.

By definition, the key aspect in green chemistry is prevention and reduction [48, 49], such as decreasing the number of reaction steps [50], reactions with green solvents [51, 52] or, preferably, solvent-free [53, 54], safe reaction conditions and short reaction times [55], as well as easy workup processes and high yields. In this work, in compliance with the 1st principle of green chemistry, prevention was achieved, i.e., no additional purification step was required. Moreover, according to principle 5, we avoided the use of solvent, making synthetic processes more economical and environmentally friendly.

In 1H NMR spectra for novel compounds 14, 18 and 19, when compared to the precursor 4,7-dichloroquinoline, the main signals that confirm the obtaining of the products are those attributed to methylene hydrogens, which appear in the ranges of 4.16-1.43, 4.30-3.73 and 4.38-2.88 ppm, respectively. In addition, the permanence of a set of five signals in the aromatic region was observed. For compound 14, a triplet attributed to methyl hydrogens was observed at 0.96 ppm.

In 13C NMR spectra, methylene carbons were observed in the ranges of 68.8-22.5, 73.0-61.8 and 68.3-30.7 ppm for compounds 14, 18 and 19, respectively. For compound 14, a signal attributed to methyl carbon was observed at 14.1 ppm. Finally, nine aromatic carbons were observed in the range of 161.8 to 100.9 ppm. Comparing carbon shifts at position-4 between products and precursor, they appeared at lower fields (161.8-161.2 ppm) due to the higher electronegativity of oxygen compared to chlorine.

Considering IR spectra, the most significant bands were the emergence of alkyl bands at 2954-2850 cm-1, as well as symmetric/asymmetric stretching and in- plane/out-of-plane angular deformations relative to C-O/S at 1311-1006 cm-1. Moreover, for compounds 18 and 19, typical broad bands were observed at 3228 and 3182 cm-1, respectively, relative to O-H.

According to Table 1, all compounds have molecular weights between 192.64 and 283.77 Da, hydrogen bond donors between 0 and 3, and hydrogen bond acceptors between 1 and 4. The partition coefficient log P, a measure of lipo/hydrosolubility ideally no greater than 5, presented values between 1.98 and 4.01. The range between 1 and 3 is optimal for good intestinal absorption, due to the adequate balance between hidrosolubility and permeability. Compounds in the range between 3 and 5 have high permeability, but oral absorption is reduced due to decreased hidrosolubility [18].

In the same way as for Lipinski's Rule of Five, all compounds are in accordance with Veber parameters, with topological polar surface area between 22.12 and 67.65 Å2, and number of rotatable bonds between 1 and 7.

Considering the toxicity prediction, the skin sensitization parameter is expressed as yes or no [46]. All compounds are predicted not to cause skin sensitization, indicating a possible topical use. Hepatotoxicity is a parameter that predicts whether a molecule has the potential to cause liver problems, and its result is also expressed as yes or no [46]. For the 4-amino-7-chloroquinoline series, compounds are predicted to be hepatotoxic when, in the side chain, they have a number of carbon atoms for each polar group equal to or greater than 3, while all 4-alkoxy-7-chloroquinoline series compounds are predicted not to be hepatotoxic.

The Minnow toxicity predicts the median lethal concentration (LC50) for Flathead Minnows, and values below -0.3 are predicted as high acute toxicity [46]. Only 4-alkoxy/amino-7-chloroquinolines 3 and 14, with R = Pent, are both predicted to have high acute toxicity with values of -0.33 and -0.67, respectively.

Analyzing Table 2, 4-amino-7-chloroquinolines 2 and 3 showed the broadest antimicrobial activity, with activity against Gram-(+) and Gram-(−) bacteria, and Candida albicans fungus. More than that, 4-amino-7-chloroquinolines 1-3, with R = alkyl, were the most active against C. albicans, with halos close to 30 mm. Considering the standard deviation, 4-amino-7-chloroquinoline 3 presented a ZOI (31.30 mm) very close to Fluconazole (32.42 mm). According to the World Health Organization (WHO), C. albicans has been ranked and categorized as a fungal pathogen in the critical group, which can cause generalized and invasive infections, and has demonstrated microbial resistance [56].

This same compound, 4-amino-7-chloroquinoline 3, was active against Gram-(+) bacteria, Bacillus cereus and Staphylococcus aureus, with halos greater than 15 mm. Furthermore, 4-amino-7-chloroquinoline 2 was active against the Gram-(−) bacterium Escherichia coli, whose bacterial infection constitutes a neglected tropical disease [57].

On the other hand, the best results against Pseudomonas aeruginosa occurred for 4-amino-7-chloroquinolines 6-8, when R = alkylamino, with halos greater than 20 mm. P. aeruginosa, capable of surviving even in environments with few nutrients, is an opportunistic pathogen, i.e., it rarely causes disease in a healthy immune system, but takes advantage of immunocompromised and burned patients to establish an infection. It typically infects the respiratory, urinary and auditory systems, burns, and can cause bacteremia, a blood infection. Due to its ability to form biofilms and develop multiresistance to antimicrobial and antiseptic agents [58], this microorganism is one of the main causes of hospital infections with a high mortality rate [59].

Regarding Amoxicillin/Clavulanic acid and Tetracycline, 4-amino-7-chloroquinolines 6-8 were equal or more active than these antibiotic controls, while Ciprofloxacin antibiotic control presented a halo close to 30 mm. Although Ciprofloxacin, a fluoroquinolone, covers both Gram-(+) and Gram-(−) bacterial infections, recent studies have raised concerns that fluoroquinolone use may be associated with an increased risk of aortic aneurysm and dissection [60], among other side effects [61]. Therefore, it is essential to search for alternative antimicrobial agents to Ciprofloxacin.

In a previous work, compound 6 (10 mM) inhibited the growth of Penicillium marneffei by 50% [14]. In another study, compound 6 has been evaluated for its in vitro antibacterial activity against E. coli and S. aureus, and antifungal activity against Aspergillus niger and C. albicans, using Penicillin as an antibiotic control and Fluconazole as an antifungal control. Regarding the activity against strains of C. albicans (MTCC 871), E. coli (MTCC 119) and S. aureus (MTCC 96), compound 6 inhibited growth by 76.87, 78.5 and 85.28%, respectively [15].

Finally, the best results for the 4-alkoxy-7-chloroquinoline series were against C. albicans, when R = alkyl, with halos greater than 15 mm. These results were not as impressive as those of the 4-amino-7-chloroquinoline series, but linear alkyl side chains have been shown to be an indicative way of improving antimicrobial activity.

Analogous quinolines had their physicochemical, antifungal and toxicological properties studied. As a result, 2-methylquinoline (log P = 2.45) and 4-ethyl 2-methylquinoline (log P = 3.09) were active against Candida species, with Minimum Inhibitory Concentration (MIC) values ≥ 50 and 25-50 µg mL-1, respectively (Fluconazole, MIC 2-128 µg mL-1). Furthermore, 4-ethyl 2-methylquinoline, the most promising compound, did not show cytotoxic action [62]. These results corroborate the best results observed in the present work, i.e., alkyl chain at 4-position.

Analyzing Table 4, 4-amino-7-chloroquinolines 2 and 3, and 4-alkoxy-7-chloroquinolines 13 and 14, with R = Pr and Pent, respectively, are highly toxic. When lipophilicity decreases, toxicity on A. salina larvae also decreases, as for 4-amino-7-chloroquinoline 1, with R = Me, which is moderately toxic.

On the other hand, 4-amino-7-chloroquinolines 6 and 7 are slightly toxic. Coherently, for 4-amino-7-chloroquinoline 8, which is moderately toxic, both toxicity on A. salina larvae and lipophilicity increased. It seems that increased lipophilicity in the side chain is harmful to A. salina larvae (Fig. 1). These results are in agreement with Minnow toxicity prediction, where compounds 3 and 14, with R = Pent, are both predicted to have high acute toxicity.

Fig. (1).

Fig. (1)

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Lipophilicity-toxicity correlation trend for 4-amino-7-chloroquinolines (1-3 and 6-8) and 4-alkoxy-7-chloroquinolines (13-14). Log P: octanol/water partition coefficient prediction; LC50 (µg mL-1): lethal concentration value where a substance causes the death of 50% of A. salina larvae.

Considering the potential disposal of drugs in the aquatic environment, in vitro toxicity evaluation on A. salina larvae has been used as a possible indicator of ecotoxicity, and may discriminate cytotoxic compounds [63]. Furthermore, 4-ethyl 2-methylquinoline, analogous to the quinolines obtained in the present work, did not show cytotoxicity on Vero cells at concentrations related to its MIC values (25-50 µg mL-1), and a high percentage of viable cells was observed even at the highest concentration tested (100 µg mL-1) [62].

5. STUDY LIMITATIONS

Regarding the limitations of this study, 4-alkoxy/amino-7-chloroquinolines may have more selective molecular targets. However, toxicity studies need to be expanded to include cytotoxicity evaluation, especially for 4-amino-7-chloroquinolines 6 and 7, which are slightly toxic to A. salina larvae, with LC50 values of 573.09 and 777.50 µg mL-1, respectively. Furthermore, antimicrobial studies on different strains, using the broth microdilution method to determine MIC values, should be conducted to provide more robust data.

CONCLUSION

With the aim of investigating the influence of the side chain on lipophilic, antimicrobial and toxicity properties and their correlations, a series of 19 compounds, analogues of chloroquine and hydroxychloroquine, was designed by a cleaner and facile approach, without the use of solvent and additional purification step, in high yields of 81-100%. Thus, 4-alkoxy/amino-7-chloroquinolines were evaluated for their potential as drugs through in silico ADMET test, which do not violate any Lipinski's Rule of Five as well as Veber parameters. The best antimicrobial results occurred for the 4-amino-7-chloroquinoline series against Candida albicans, when R = alkyl, with halos close to 30 mm, and against Pseudomonas aeruginosa, when R = alkylamino, with halos greater than 20 mm. The most promising antimicrobial agents were evaluated for their in vitro toxicity on Artemia salina larvae. At the same time, we can observe a correspondence between Minnow toxicity prediction and in vitro toxicity on A. salina larvae, where compounds 3 and 14, with R = Pent, were both predicted to have high acute toxicity (log LC50 < -0.3) and classified as highly toxic (LC50 < 100 µg mL-1); linear alkyl sides appear to be an assertive way to improve antimicrobial activity. Now, we are motivated to continue antimicrobial studies for compounds 1-3 and 6-8 with greater activity against C. albicans and P. aeruginosa, respectively, by the determination of MIC values, especially for 4-amino-7-chloroquinolines 6 and 7, which are slightly toxic on A. salina larvae (LC50 500-1000 µg mL-1).

ACKNOWLEDGEMENTS

Declared none.

LIST OF ABBREVIATIONS

ADME

Absorption, Distribution, Metabolism and Excretion

HRMS

High-Resolution Mass Spectrometry

ESI

Electrospray Ion

TLC

Thin Layer Chromatography

CLSI

Clinical and Laboratory Standards Institute

AUTHORS’ CONTRIBUTIONS

The authors confirm their contribution to the paper as follows: study conception and design: GFF; data collection: EPS, MFSM, APS, HDSS, RBC, MHN, KGL; draft manuscript: GFF; important reagents: PFA-F. All authors reviewed the results and approved the final version of the manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data and supportive information are available within the article.

FUNDING

This work was financially supported by [Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)] (Grant number 403134/2021-8) and [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)]. Fellowships from CNPq (M.F.S.M., P.F.A.-F.) and CAPES (A.P.S., E.P.S., H.D.S.S.) are acknowledged.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s website along with the published article.

CMC-33-5-924_SD1.pdf (817.3KB, pdf)

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