INTRODUCTION
According to the database of the United States Food and Drug Administration (U.S. FDA), it is estimated that nitrogenous heterocyclic compounds are present in more than half of approved medicines, of which we can highlight those that have the quinoline nucleus, which is structurally diverse and present in a variety of natural products. Numerous studies have identified quinoline and its derivatives as an important class of nitrogenous heterocyclic compounds with activities such as antiproliferative, antimicrobial, leishmanicidal, antiviral, antichagasic, antimalarial, insecticidal, among others (Ferreira et al., 2021).
From structure-activity relationship (REA) studies of quinoline derivatives, it is known that the 4-substituted 7-chloroquinoline nucleus is fundamental for manifestation of bioactivities, with the presence of a side chain, initiated by a substituent in 4-position of the ring, strategic for the intensification of these activities (Segan et al., 2016). Furthermore, the literature has recorded several synthetic (Chiodi; Ishihara, 2023) and supramolecular (Copetti et al., 2020) studies of quinoline derivatives containing the chlorine atom in the 7-position, as well as the investigation of their antimicrobial activities (Behera et al., 2022; Devi et al., 2013; Taramelli et al., 2001).
To combat microbial resistance mechanisms, it is necessary to use molecular modification strategies, such as controlling lipophilicity, for example, which is one of the most important physicochemical characteristics for the study and planning of drugs in terms of bioavailability, permeability and even toxicity (Nogueira, 2009). One of the ways is by changing the number of carbons in the alkyl chain, for example. Lipophilicity can be quantitatively expressed by the partition coefficient (P) of a substance between the organic and aqueous phases. The partition coefficient can be defined by the ratio between the concentrations of the substance in the organic (Corg) and aqueous (Caq) phases in a two-phase system under equilibrium conditions: log P = log Corg/Caq (Barreiro, 2015).
Several research groups, especially those associated with different pharmaceutical industries, have attempted to identify, through the systematic analysis of orally active drug databases, structural properties that can guide the identification of orally active compounds, in the initial stages of the drug development process (Santos et al., 2022). Among these studies, it is worth highlighting the one carried out by Lipinski and collaborators (1997), who, after analysis by the World Drug Index, proposed the so-called Rule of Five, a set of four parameters, with values ??multiple of five, which reflected common structural characteristics of orally active drugs in this database, namely: molecular weight < 500 Da; hydrogen bond donor groups £ 5; hydrogen bond acceptor groups £ 10; log P £ 5 (Lipinski, 2004).
Another independent study carried out by Veber and collaborators (2002) identified the importance of additional properties for the oral bioavailability profile of drugs, such as molecular volume, total polar surface area (TPSA) £ 140 Å and number of rotatable bonds (nrotb) £ 10.
However, although these rules provide an estimate of suitability of the physicochemical and structural properties of new bioactive substances, they must be used with extreme caution, as there are numerous examples of orally active drugs currently on the pharmaceutical market that violate one or more of these requirements, calling into question its real predictive capacity for the oral absorption profile of drugs (Barreiro, 2015).
For the journal PLOS Neglected Tropical Diseases, neglected tropical diseases are defined as a group of chronic infectious diseases that promote poverty and occur mainly in rural and poor urban areas of low- and middle-income countries. They promote poverty due to their impact on child health and development, pregnancy and worker productivity. Under PLOS, bacterial infection caused by the Gram-negative bacterium Escherichia coli is a neglected tropical disease.
More elaborate 4-substituted 7-chloroquinoline derivatives have been studied for side chain variation (Yearick, 2008). However, the most basic nuclei, structurally more similar to chloroquine and hydroxychloroquine, for example, are usually treated only as synthesis intermediates, and their lipophilicity and pharmacological activities are not usually evaluated. In this context, there is still no systematic study about lipophilicity, with or without relation to antimicrobial activity, which investigates the influence of the substituent group in the 4-position, as well as the chain extension.
AIMS
The present work aimed to prepare thirteen 4-substituted 7-chloroquinolines from the reaction of nucleophilic aromatic substitution (SArN) of 4,7-dichloroquinoline with (di)amines, amino alcohols and (di)ols, which were characterized by measuring their melting points and spectroscopy in 1H and 13C nuclear magnetic resonance (NMR). The synthesized molecules were evaluated for their in silico study and in vitro antimicrobial activity against Escherichia coli, a neglected tropical disease.
METHODS
All common reagents were purchased from commercial suppliers and used without further purification. Melting points were measured using QUIMIS equipment, model Q340S23. Melting points were not corrected. 1H and 13C NMR spectra were performed at the Laboratório Multiusuário de Caracterização e Análise (LMCA-UFPB), which were acquired on Bruker spectrometers at 400 and 500 MHz for 1H.
General procedure for synthesis of the compounds 1-10: the mixture of 4,7-dichloroquinoline (10 mmol) and amine (10 mL) was kept under magnetic stirring at 80 °C for 4 h (for the compound 1, at 60 °C for 12 h). Then, the reactional mixture was cooled to room temperature and the precipitate filtered and washed with ice distilled water.
General procedure for synthesis of the compounds 11-13: the mixture of potassium tert-butoxide (10 mmol), 4,7-dichloroquinoline (10 mmol) and alcohol (10 mL), in this order, was kept under magnetic stirring at 100 °C for 8 h. Then, the reactional mixture was cooled to room temperature and the precipitate filtered and washed with ice distilled water.
The in silico study was based on Lipinski and Veber parameters to evaluate the drug-like character of the 4-substituted 7-chloroquinolines 1-13. The prediction of pharmacokinetic properties were carried out through the open access electronic site SwissADME. Lipophilicity were deduced from the log P consensus value (average of five methods).
The compounds 1-13 were assessed for their in vitro antimicrobial activity against Escherichia coli, reference strain NEWP 0039 (NEWPROV), using the antimicrobial susceptibility method through well diffusion in solid media, adapted from Bauer et al. (1966) and following the guidelines established by the Clinical and Laboratory Standards Institute (CLSI). The control antibiotics employed against E. coli included Amoxicillin/Clavulanic acid (AMC), Ciprofloxacin (CIP), Penicillin G (PEN) and Tetracycline (TET).
RESULTS AND DISCUSSION
Regarding the synthetic step, this work began with the preparation of 4-substituted 7-chloroquinolines, which were obtained from the SArN reaction of 4,7-dichloroquinoline with the nucleophiles (di)amines, amino alcohols and (di)ols under heating. The different nucleophiles were used as reagents and solvents. The reactions were monitored by thin layer chromatography (TLC, AcOEt). When necessary, the precipitates were washed with hot hexane to remove the remaining 4,7-dichloroquinoline, without the need for an additional purification step. The compounds 1-13 were obtained in yields of 81 to 100%. All compounds were characterized by measuring their melting points and using 1H and 13C NMR spectroscopic techniques.
An in silico study for the compounds 1-13 was carried out and some pharmacokinetic properties were analyzed, such as absorption, distribution, metabolism and excretion (ADME). From this study, it is possible to predict which compounds may be good drug candidates, taking into account solubility, lipophilicity and toxicity.
All compounds showed adequate pharmacokinetic properties, not violating any Lipinski's Rule of Five. The 4-amino-7-chloroquinolines 1, 4 and 7, with R = NHPr, NH(CH2)2NEt2 and NH(CH2)6NH2, respectively, and the 4-alkoxy-7-chloroquinoline 11, with R = OPr, are the most lipophilic, with log P > 3. Coherently, the compound 10, with R = NH(CH2)2NH(CH2)2OH, is the least lipophilic, with log P < 2.
Among the compounds 2, 8 and 12, which have a hydrocarbon extension equal to two carbons, compound 12, which has oxygen as a substituent in the 4-position, is the most lipophilic. Likewise, for the compounds 5 and 9, which have a hydrocarbon extension equal to three carbons, the compound 9, which has oxygen as a substituent in the 4-position, is the most lipophilic. And, for the compounds 6 and 13, which have a hydrocarbon extension equal to four carbons, the compound 13, which also has oxygen as a substituent in 4-position, is the most lipophilic.
Unlike lipophilicity, hydrocarbon extension does not influence the value of the total polar surface area (TPSA), what does influence is the substituent group in 4-position, as well as the heteroatom within the chain. Therefore, all analyzed compounds are in accordance with Veber's parameters, demonstrating good oral bioavailability.
Finally, the compounds 1-13 were assessed for their in vitro antimicrobial activity against E. coli. Based on the diameter of growth inhibition zones, it was observed that all samples, solubilized in methanol at a concentration of 2 mg.mL-1, inhibited the growth of E. coli, with particular emphasis on the compound 1 (R = NHPr), which exhibited the largest inhibition zones, with an average of 14.5 mm, approaching those displayed by the antibiotic AMC (Table 1).
Table 1 – Antimicrobial activity of the compounds 1-13 against E. coli
Compound | 4-R | Inhibition zonea (mm) |
1 | NHPr | 14.52 ± 0.50 |
2 | NH(CH2)2NH2 | 9.61 ± 0.83 |
3 | NH(CH2)2NMe2 | 8.32 ± 0.81 |
4 | NH(CH2)2NEt2 | 8.57 ± 0.04 |
5 | NH(CH2)3NH2 | 8.54 ± 0.04 |
6 | NH(CH2)4NH2 | 7.81 ± 0.18 |
7 | NH(CH2)6NH2 | 7.87 ± 0.18 |
8 | NH(CH2)2OH | 6.68 ± 0.12 |
9 | NH(CH2)3OH | 6.61 ± 0.47 |
10 | NH(CH2)2NH(CH2)2OH | 6.82 ± 0.12 |
11 | OPr | 7.40 ± 0.11 |
12 | O(CH2)2OH | 6.07 ± 0.04 |
13 | O(CH2)4OH | 6.19 ± 0.04 |
AMCb | _ | 15.30 ± 3.72 |
CIPc | _ | 25.99 ± 1.20 |
PENd | _ | _ |
TETe | _ | 21.06 ± 0.39 |
aThe data are presented as the arithmetic mean ± standard deviation.
bAmoxicillin/clavulanic acid 30 µg, cCiprofloxacin 5 µg, dPenicillin G 10 U.I., eTetracycline 30 µg.
According to the interpretation of data from the in silico study and in vitro evaluation, it seems that the combination of the improvement in the lipophilic character with the amino substituent at 4-position and the alkyl side chain was essential to increase the antimicrobial activity against E. coli. These reports direct us to future research with even more assertive structures.
CONCLUSION
Thirteen 4-substituted 7-chloroquinolines were obtained through a single reaction step, without the need for solvent use, as well as an additional
Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
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