Abstract
Aim: To synthesize novel chloroquine analogues and evaluate them for antimicrobial and cytotoxic potential. Methods: Novel analogues were synthesized from chloroquine by nucleophilic substitution reaction at the 4-amino position. Results: Analogue CS1 showed maximum antimicrobial potential (30.3 ± 0.15 mm zone) against Pseudomonas aeruginosa and produced a 19.2 ± 0.21 mm zone against Candida albicans, while CS0 produced no zone at the same concentration. Analogue CS9 has excellent cytotoxic potential (HeLa cell line), showing 100% inhibition (IC50 = 8.9 ± 1.2 μg/ml), compared with CS0 (61.9% inhibition at 30 μg/ml). Conclusion: These synthesized chloroquine analogues have excellent activity against different microbial strains and cervical cancer cell lines (HeLa) compared with their parent molecule.
Keywords: : cervical cancer, chloroquine, cytotoxic assay, quinoline, sulfonamides
GRAPHICAL ABSTRACT
Plain language summary
Summary points.
Novel chloroquine analogues were synthesized by nucleophilic substitution reaction at the 4-amino position.
These analogues were characterized using nuclear magnetic resonance and Fourier transform infrared spectroscopic techniques.
The antimicrobial result showed that the new analogues have good antimicrobial activity, while chloroquine was not active against any strain.
In the cytotoxicity study, a few analogues had improved activity compared with chloroquine.
Docking results also support in vitro findings due to good binding affinity.
The emergence of resistance and reduced antibiotic efficacy against diverse types of lethal diseases have posed a critical need for discovering and developing novel efficient compounds for their treatment [1]. Bacterial infections were determined to be responsible for 7.7 million fatalities globally, and nearly 15 million losses of life annually will be anticipated by 2030 due to different cancers [2]. This seriously threatens the global healthcare system [3,4]. In recent decades, medicinal chemists have used the most time- and cost-effective method to develop new drugs for the market through slight changes in the structure of available drugs [5].
In drug research, N-based heterocyclic-based aromatic ligands, particularly quinoline derivatives, are the most versatile in terms of pharmacotherapy and include compounds with antimicrobial (ciprofloxacin) 1, antitumor (neratinib) 2, kinase inhibitor (bosutinib) 3 and antimalarial (chloroquine) 4 activities, as explained in Figure 1 [6,7]. Some other common quinolines have antimalarial potential, such as tafenoquine, chloroquine, mefloquine and amodiaquine [8–12]. Quinoline analogues has good antiplasmodial activity with nitrogen at the fourth position [13]. Moreover, quinoline-based drugs treat various infections with both Gram-positive and Gram-negative bacteria [14]. Additionally, quinoline derivatives successfully suppress an enzyme (kinase) that is a target in cancer research, including cervical cancer, according to another study [15]. In light of the significance of the quinoline nucleus, researchers have synthesized several other quinoline analogs that are promising chemotherapeutic agents [16].
Figure 1.
Summary of some marketed drugs having a quinoline core, active against various diseases.
Chloroquine derivatives are widely recognized for their antimalarial action but have an extensive spectrum of pharmacological effects, including anti-inflammatory and anticancer properties [17]. The aromatic chloroquine urea derivatives are active against cancer cells at low concentrations [18]. Chloroquine derivatives 7-chloro-N-(3-(4-(7-(trifluoromethyl)quinolin-4-yl)piperazin-1-yl)propyl)quinolin-4-amine and {3-[4-(7-chloro-quinolin-4-yl)-piperazin-1-yl]-propyl}-(7-trifluoromethyl-quinolin-4-yl)-amine emerged as the most active derivatives against MCF7 cell lines when compared with chloroquine [19]. Due to its potential antimicrobial and anti-inflammatory properties, the sulfonamide functional group has critical applications in medicinal and synthetic organic chemistry. Medicinal chemistry primarily focuses on developing novel sulfonamides [20]. Several physiologically active chemicals, including SO2NH, have applications as antimicrobial, antithyroid and carbonic anhydrase inhibitors [15,21]. Sulfonamides can be effective against microbial infection and cervical cancer cell lines (HeLa) due to their inhibition potential against topoisomerase and kinase enzymes [15]. Some important sulfonamides available in the market with antibacterial potential include sulfasalazine, sulfisoxazole, sulfamerazine, sulfadimethoxine, sulfamethazine, sulfadiazine and sulfamethoxazole [21].
These discoveries have sparked our drug research by strengthening our understanding that appropriate sulfonamide based on the well-known chloroquine scaffold might result in molecules with desirable therapeutic properties. The present study used novel synthetic analogues with substitution at 4-amino positions from chloroquine and formed sulfonamide. These analogues were evaluated in vitro for their antimicrobial and cytotoxic potential.
Materials & methods
General
Methanol, acetonitrile, n-hexane and ethyl acetate (purity ≥99.5%) were purchased from Merck KGaA (Darmstadt, Germany). Bayer HealthCare Pharmaceuticals LLC (Karachi, Pakistan), gifted chloroquine (N4-(7-chloroquinolin-4-yl)-N1, N1-diethyl pentane-1,4-diamine). All the sulfonyl chlorides (purity ≥98%) utilized in this study were purchased from Alfa Aesar by Thermo Fisher Scientific (MA, USA). All synthetic chloroquine analogues’ melting points were initially confirmed using the melting point apparatus (Gallenkamp, Stuart digital MP [SMP-10] apparatus, Cambridge, UK), using capillary tubes. Merck silica gel (60–120 mesh) was used to purify chloroquine analogs. TLC and a UV lamp were used to determine each product’s retention factor (Rf) value. Fourier-transform infrared spectroscopy was employed to identify the presence of a new functional group. NMR spectroscopic investigations were performed on a AvanceTM III HD NanoBay 400 MHz NMR Spectrometer (MA, USA), at COMSATS University Islamabad, Abbottabad Campus Pakistan, using deuterated chloroform CDCl3 and dimethylsulfoxide (DMSO) as the solvent [15].
Statistical analysis
The standard deviation and 50% lethal dose (LD50) values were calculated using Microsoft® Excel® 2010 (Microsoft Corp., WA, USA) [22]. The results (percentage inhibition and IC50 value) of chloroquine analogs were analyzed using SoftMax® Pro (Molecular Devices, CA, USA) [23].
General synthetic scheme
Initially, 5 mmol chloroquine (CS0) was added to 100 ml of acetonitrile in a reaction flask, then refluxed until dissolved. After that, potassium bicarbonate (5 mmol) was added as a base, and reflux continued for 1 h. Finally, the appropriate sulfonyl chloride (5 mmol) was added to the reaction flask and stirred until completion of the reaction at 60–70°C, as shown in Figure 2.
Figure 2.
Synthetic scheme and structures of synthesized chloroquine analogues.
(A) General scheme for analogues synthesis. (B) Synthesis of novel chloroquine analogs CS1–9: CS1(i), CS1,(ii) CS2, (iii) CS3,(iv) CS4,(v) CS5, (vi) CS6, (vii) CS7, (viii) CS8, (ix) CS9.
Purification
Column chromatography was used to purify the product based on polarity using hexane and ethyl acetate as a solvent. TLC was used for the initial confirmation of product formation. Dried purified compounds were further used for spectroscopic characterization and biological investigation.
Synthesis
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)benzenesulfonamide (CS1)
In a round-bottomed reaction flask, acetonitrile, N4-(7-chloroquinolin-4-yl)-N1 and N1-diethylpentane-1,4-diamine were added, then refluxed until the drug was dissolved. Potassium bicarbonate was added as a base after 30 min, and reflux continued for 1 h. Finally, benzene sulfonyl chloride was added. Stirring continued at 60°C until the reaction was completed (as confirmed by TLC). The flask was cooled, and filter paper was used for filtration; the filtrate was dried at 40°C under reduced pressure, and the desired product was separated by column chromatography.
4-chloro-N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)benzenesulfonamide (CS2)
In a round-bottomed flask, acetonitrile and N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane were added. Stirring with reflux continued for 2 h. Potassium bicarbonate was added to the reaction flask, and reflux continued for 1 h. After that, chlorobenzene sulfonyl chloride was added to the reaction flask, and stirring was continued for 56 h at 65°C. After the reaction, the filtrate was dried and the product purified using solvent (n-hexane and ethyl acetate) via column chromatography.
N-(4-(N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl) sulfamoyl)phenyl)acetamide (CS3)
For preparing the reaction mixture, acetonitrile and N4-(7-chloroquinolin-4-yl)-N1, N1-diethylpentane were added to a round-bottomed flask. Reflux continued for 1 h. As a base, potassium bicarbonate was used, and reflux continued for 1 h; then N-acetyl sulfonyl chloride was added to the reaction flask and stirring continued for the next 50 h at 63°C. The material was filtered from the flask and the filtrate dried under reduced pressure. Column chromatography was used to purify the product using n-hexane and ethyl acetate as a solvent.
2N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)-4-methylbenzenesulfonamide (CS4)
Acetonitrile and N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane were added to a predried reaction flask, then stirring was performed at 42°C for the next 2 h. After that, potassium bicarbonate was used as a base in the reaction flask, and reflux continued for 1 h. Finally, 5 mmol of 4-methyl benzene sulfonyl chloride was added to the reaction flask, and reflux continued for 68 h at 70°C. After that, the reaction material was filtered in a beaker. The reaction mixture was dried under reduced pressure and purified using n-hexane and ethyl acetate by column chromatography.
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)methanesulfonamide (CS5)
Acetonitrile was added to a 250-ml round-bottomed flask, then N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane was added to the flask. The mixture was refluxed for 2 h and then potassium bicarbonate was used as a base. The reaction reflux was further continued for 1 h. Methane sulfonyl chloride was added to the reaction flask for substitution, and stirring continued for 48 h at 60°C. After completion of the reaction, the reaction material was filtered. The slurry was made using silica gel and purified using column chromatography.
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)-4-fluorobenzenesulfonamide (CS6)
In a flask, acetonitrile and N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine were added, then refluxed till dissolved. Potassium bicarbonate was added, and the reaction continued under reflux for 1 h. Finally, 4-fluorobenzene sulfonyl chloride was added to the reaction flask, and stirring continued at 60°C until the formation of product. Filter paper was used for filtration, and the filtrate was dried at 40°C under reduced pressure; the column chromatography technique was used to separate the desired product.
4-bromo-N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)benzenesulfonamide (CS7)
First, N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine and acetonitrile were added to a round-bottomed flask, then refluxed until it dissolved. After that, potassium bicarbonate was added, and the reflux continued for 1 h. For substitution, 4-bromobenzene sulfonyl chloride was added in the reaction flask and stirring continued at 60°C until the completion of the reaction. The flask was cooled and then filter paper was used for filtration. Column chromatography was used to separate the desired product from the reaction mixture.
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)-4-isopropylbenzenesulfonamide (CS8)
Acetonitrile as a solvent was added to the reaction flask, then N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine was added and refluxed until dissolved. Potassium bicarbonate was used as a base in the reaction. Reflux again continued for 1 h. Lastly, 4-isopropylbenzene sulfonyl chloride was added in the reaction flask, and stirring continued at 60°C until the completion of the reaction. The filtrate was dried at 40°C under reduced pressure. The column chromatography technique was used to separate the purified product, using n-hexane and ethyl acetate as solvents.
4-(tert-butyl)-N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)benzenesulfonamide (CS9)
In a predried round-bottomed flask, acetonitrile and N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine were added. Reflux was performed until the dissolution of the drug. After that, potassium bicarbonate was added to the reaction flask, and reflux continued for 1 h. Finally, 4(tert-butyl)benzene sulfonyl chloride was added in the reaction flask and stirring continued at 60°C until completion of the reaction. For the separation of the product, column chromatography technique was used, with n-hexane and ethyl acetate as solvents.
Antibacterial assay
In this study, the disc diffusion method was used to examine the antibacterial activity of the synthesized chloroquine analogs (CS1–9) on two Gram-positive (Staphylococcus aureus, American Type Culture Collection [ATCC] #6538 and Bacillus subtilis ATCC #6633) and two Gram-negative bacterial strains (Escherichia coli ATCC #15224 and Pseudomonas aeruginosa ATCC #9721). For sample preparation at 2 mg/ml concentration, DMSO was utilized. Chloroquine was utilized for the comparison of results. DMSO was used as a negative control. The mean value and standard deviation from triplicate data from the experiment were computed. Cefixime was used as a standard drug. The zone of inhibition was measured in mm [15,22].
Antifungal assay
The procedure described previously (Islam et al.), with slight modification, was used to test the antifungal activity of synthesized chloroquine analogues against two strains, Candida albicans (ATCC #9002) and Candida parapsilosis (ATCC #7330) by measuring their inhibition zones [15]. Before the experiment, synthetic analogues and chloroquine samples were prepared at 2 mg/ml in DMSO. The equipment was sterilized using an autoclave. Chloroquine (CS0) was used to compare results, and the negative control was DMSO. The zone of inhibition was measured in mm. The experiment was performed in triplicate. The mean value and standard deviation were calculated. Clotrimazole was used as a standard drug [15,22].
Toxicity assay (brine shrimp lethality assay)
The ability of the newly synthesized chloroquine-based analogs to kill brine shrimp was evaluated using the technique established by Olowa and Nuneza, with minor modifications. Artemia salina eggs (INVE AQUACULTURE, INC. UT, USA) were incubated in artificial seawater for 24–48 h in a two-compartment tray (45 g sea salt dissolved in 1 l of water) [15,24]. Test samples were employed at four different concentrations: 50, 25, 12.5 and 6.25 μg/ml. We used 1% DMSO in seawater as a negative control. The mean value was used throughout the experiment, which was done in triplicate [24].
Cytotoxicity assay
For the cytotoxicity assay, 96-well plates were used to measure the cytotoxic activity of all synthesized chloroquine analogs using the 3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) colorimetric test by following predefined protocols, using the HeLa cervical cancer cell line. The experiment was performed in triplicate. HeLa cells from passage numbers 7, 9, 11, 12, 16 and 17 were used in the study. The IC50 of analogs whose inhibition values were above 50% were evaluated. The results of new analogs were compared with those obtained with chloroquine. Doxorubicin was used as a standard [23].
Molecular docking studies
For calculation of the binding energy, structures were drawn in ChemDraw® Professional v.15.0, and the crystal structure of a targeted protein (Protein Data Bank [PDB] ID: 1PFK) was retrieved from the PDB (www.rcsb.org/). Based on the Swiss target prediction report, analogs were docked against 1PFK using Molecular Operating Environment (MOE, developer Chemical Computing Group, Montreal, Canada) software to know the binding affinity. All synthesized chloroquine analogs’ physicochemical and pharmacokinetic properties were predicted online via www.swissadme.ch [15].
Results
Chemistry
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)benzenesulfonamide (CS1)
The purified product yield of CS1 was 19% and the melting point was 47–48°C. Rf value = 0.25. IR (cm-1): 1335 (S=O). 1H NMR (400 MHz, CDCl3) δ: 8.46 (d, J = 5.5 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.97 (s, 1H), 7.75 (d, J = 2.3 Hz, 1H), 7.80–7.41 (m, 2H), 7.27 (dd, J = 9.1, 2.3 Hz, 1H), 7.24 (s, 1H), 6.51 (d, J = 5.6 Hz, 1H), 4.10 (m, 1H), 3.56 (ddd, J = 10.3, 6.2, 1.9 Hz, 1H), 3.25 (s, 1H), 2.36–2.24 (m, 2H), 2.15 (d, J = 7.6 Hz, 1H), 2.01 (dtd, J = 7.9, 4.2, 2.1 Hz, 1H), 1.75 (s, 1H), 1.85–1.64 (m, 2H), 1.62–1.59 (m, 2H), 1.40 (s, 1H), 1.38–1.35 (m, 2H), 1.23 (d, J = 22.1 Hz, 1H), 1.23 (d, J = 6.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ: 153.37, 150.38, 150.35, 134.81, 134.73, 129.26, 128.01, 127.19, 126.99, 126.83, 124.21, 120.91, 104.73, 77.48, 77.16, 76.84, 55.84, 55.40, 34.45, 31.76, 29.82, 25.55, 18.99, 14.16 p.p.m.
4-chloro-N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)benzenesulfonamide (CS2)
We obtained 18% purified product from the reaction mixture using column chromatography and Rf = 0.50. The melting point of CS2 was 48–50°C. IR (cm-1): 1370 (S=O). 1H NMR (400 MHz, CDCl3) δ: 8.37 (d, J = 6.1 Hz, 1H), 8.31 (dd, J = 17.6, 7.6 Hz, 1H), 8.12–8.02 (m, 2H), 8.02–7.67 (m, 2H), 7.42 (d, J = 8.3 Hz, 1H), 7.39–7.26 (m, 2H), 7.12 (s, 1H), 6.53 (d, J = 6.1 Hz, 1H), 6.47 (d, J = 6.4 Hz, 1H), 4.14 (p, J = 6.5 Hz, 1H), 4.04 (td, J = 9.7, 6.3 Hz, 1H), 3.85 (s, 1H), 3.73 (t, J = 7.8 Hz, 1H), 3.15 (h, J = 7.9 Hz, 1H), 2.39 (m, 1H), 2.17 (m, 1H), 1.91–1.68 (m, 2H), 1.40 (s, 1H), 1.36 (s, 1H), 1.31 (s, 3H), 0.83 (dt, J = 21.5, 7.7 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ: 154.34 C, 148.52 C, 138.39 C, 137.73 C, 133.79 C, 129.57 C, 128.55 C, 127.14 C, 126.57 C, 125.41 C, 125.11 C, 103.87 C, 56.74 C, 55.51 C, 47.42 C, 34.09 C, 25.51 C, 19.04 C, 14.09 2C.
N-(4-(N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)sulfamoyl)phenyl)acetamide (CS3)
Using column chromatography, we obtained 22% purified product, with Rf = 0.14. The melting point of the product was 52–54°C. IR (cm-1): 1370 (S=O). 1H NMR (400 MHz, CDCl3) δ: 8.43 (d, J = 5.5 Hz, 1H), 7.88 (d, J = 2.2 Hz, 1H), 7.78–7.58 (m, 2H), 7.28 (dd, J = 2.2, 8.9 Hz, 1H), 6.37 (d, J = 5.5 Hz, 1H), 5.51 (d, J = 7.4 Hz, 1H), 3.66 (p, J = 6.2 Hz, 1H), 3.43 (s, 2H), 3.15 (q, J = 7.2 Hz, 1H), 2.53 (q, J = 7.1 Hz, 4H), 2.50–2.41 (m, 2H), 2.15 (s, 1H), 1.76–1.53 (m, 4H), 1.27 (d, J = 6.3 Hz, 3H), 1.09–0.91 (m, 7H). 13C NMR (101 MHz, CDCl3) δ: 169.47 C, 151.72 C, 149.29 C, 149.10 C, 142.54 C, 134.81 C, 134.36 C, 128.32 C, 128.02 C, 124.97 C, 121.57 C, 119.31 C, 117.32 C, 99.13 C, 52.44 C, 48.30 C, 46.78 C, 34.35 C, 23.55 C, 20.13 C, 14.11 C, 11.03 2C.
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)-4-methylbenzenesulfonamide (CS4)
Purified product yield calculated after column chromatography was 12% and Rf value = 0.29. The melting point was 54–55°C. IR (cm-1): 1354 (S=O). 1H NMR (400 MHz, CDCl3) δ: 8.42 (d, J = 5.8 Hz, 1H), 8.03 (d, J = 9.5 Hz, 2H), 7.29 (dd, J = 9.1, 2.3 Hz, 4H), 6.52 (d, J = 5.8 Hz, 1H), 4.14 (m, 1H), 4.01 (td, J = 9.7, 6.1 Hz, 1H), 3.65 (ddd, J = 10.0, 7.1, 2.5 Hz, 4H), 2.36–2.27 (m, 4H), 2.06 (ddt, J = 12.4, 6.5, 3.4 Hz, 3H), 1.89–1.67 (m, 4H), 1.26 (d, J = 6.0 Hz, 3H), 1.03 (t, J = 8.0 Hz, 5H). 13C NMR (101 MHz, CDCl3) δ: 153.87 C, 148.59 C, 148.50 C, 142.53 C, 138.56 C, 135.68 C, 129.02 C, 128.27 C, 126.99 C, 126.65 C, 124.66 C, 120.20 C, 104.29 C, 77.48 C, 77.16 C, 76.84 C, 56.31 C, 55.46 C, 55.45 C, 34.26 C, 25.52 C, 21.49 C, 19.47 C, 19.01 C, 14.09 2C.
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)methanesulfonamide (CS5)
The purified product yield was 19%, the Rf value = 0.26 and the melting point was 53–56°C. IR (cm-1): 1343 (S=O). 1H NMR (400 MHz, DMSO-d6) δ: 9.04 (d, J = 8.1 Hz, 1H), 8.75 (d, J = 9.2 Hz, 1H), 8.51 (dd, J = 4.0, 7.3 Hz, 1H), 7.98 (d, J = 2.1 Hz, 1H), 7.72 (dd, J = 2.1, 9.1 Hz, 1H), 4.36 (s, 5H), 4.16–4.07 (m, 1H), 3.07–3.04 (m, 3H), 2.54 (d, J = 1.7 Hz, 1H), 2.44 (s, 8H), 1.89–1.61 (m, 4H), 1.30 (d, J = 6.4 Hz, 3H), 1.15 (td, J = 2.1, 7.2 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ: 155.39 C, 143.44 C, 138.99 C, 138.49 C, 127.13 C, 126.58 C, 119.43 C, 115.84 C, 99.27 C, 50.91 C, 49.76 C, 46.68 C, 46.65 C, 40.13 2C, 32.31 C, 20.53 C, 19.95 C, 8.89 2C (aliphatic).
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)-4-fluoro benzene sulfonamide (CS6)
A 17% purified yield was obtained using column chromatography, and the Rf value was 0.18 using TLC. The melting point was 57–58°C. IR (cm-1): 1335 (S=O), 1190 (C-F), 782 (C-Cl). 1H NMR (400 MHz, CDCl3) δ: 8.40 (d, J = 6.0 Hz, 2H), 8.11–8.02 (m, 4H), 7.91–7.76 (m, 2H), 7.32 (dd, J = 2.1, 9.2 Hz, 2H), 7.19–7.09 (m, 1H), 3.21 (q, J = 7.2 Hz, 1H), 2.59–2.26 (m, 2H), 2.22–1.95 (m, 2H), 1.89–1.69 (m, 3H), 1.50–1.16 (m, 9H), 1.12–0.78 (m, 3H). 13C NMR (101 MHz, CDCl3) δ: 154.01 1C, 136.07 1C, 134.67 1C, 132.51 1C, 129.52 1C, 126.91 2C, 125.68 1C, 124.76 2C, 119.61 1C, 116.29 1C, 116.07 1C, 103.80 1C, 56.40 1C, 55.35 1C, 41.97 1C, 33.99 2C, 29.68 1C, 25.38 2C, 18.86 2C, 14.08 2C.
4-bromo-N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl) benzene sulfonamide (CS7)
The purified product yield of the product was 19%, and its Rf value was 0.17. The melting point of the purified product (CS7) was 50–52°C. IR (cm-1): 1370 (S=O), 782 (C-Cl), 704 (C-Br). 1H NMR (400 MHz, CDCl3) δ: 8.46–8.43 (m, 2H), 7.99 (d, J = 9.1 Hz, 1H), 7.90 (dd, J = 2.3, 8.2 Hz, 3H), 7.84 (d, J = 9.0 Hz, 2H), 7.77–7.68 (m, 1H), 7.62–7.47 (m, 1H), 7.35–7.26 (m, 2H), 6.37 (d, J = 5.5 Hz, 2H), 5.62 (d, J = 7.4 Hz, 2H), 2.63 (d, J = 7.2 Hz, 6H), 1.65 (tt, J = 3.5, 8.5 Hz, 4H), 1.21 (s, 4H), 1.06 (s, 6H). 13C NMR (101 MHz, CDCl3) δ: 151.64 1C, 150.69 1C, 149.36 1C, 134.87 1C, 134.34 1C, 132.35 1C, 128.31 2C, 128.16 1C, 126.69 1C, 125.05 2C, 123.94 1C, 121.79 1C, 117.33 1C, 104.68 1C, 52.40 1C, 48.32 1C, 46.79 2C, 34.23 1C, 23.19 1C, 20.19 1C, 18.84 1C, 10.58 2C.
N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl)-4-isopropyl benzene sulfonamide (CS8)
The purified product yield of CS8 was 28%, its Rf value was 0.28 and its melting point was 53–55°C. IR (cm-1): 1354 (S=O), 780 (C-Cl). 1H NMR (400 MHz, CDCl3) δ: 8.46 (d, J = 5.4 Hz, 1H), 8.27–8.11 (m, 1H), 8.28–7.98 (m, 1H), 8.07 (d, J = 20.5 Hz, 1H), 7.90 (d, J = 2.2 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 19.3 Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 6.38 (d, J = 5.5 Hz, 1H), 5.44 (d, J = 7.3 Hz, 1H), 3.68 (p, J = 6.4 Hz, 1H), 3.05 (s, 2H), 2.61–2.41 (m, 6H), 2.21–1.97 (m, 2H), 1.82–1.75 (m, 1H), 1.70–1.51 (m, 3H), 1.26 (dd, J = 6.6, 22.9 Hz, 4H), 1.02 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ: 151.85 1C, 149.23 1C, 149.17 1C, 139.07 1C, 134.77 1C, 128.56 1C, 127.00 1C, 126.92 1C, 124.98 1C, 121.47 1C, 117.39 1C, 117.32 1C, 99.14 1C, 52.45 1C, 48.28 1C, 46.79 2C, 35.49 1C, 34.33 1C, 23.67 1C, 23.51 1C, 21.45 1C, 20.17 1C, 10.97 2C.
4-(tert-butyl)-N-(7-chloroquinolin-4-yl)-N-(5-(diethylamino)pentan-2-yl) benzene sulfonamide (CS9)
We obtained 23% purified yield using the column chromatography technique and the Rf value was 0.20. The melting point of analog CS9 was 50–51°C. IR (cm-1): 1369 (S=O), 770 (C-Cl). 1H NMR (400 MHz, CDCl3) δ: 8.43 (s, 1H), 7.89 (s, 1H), 7.88 (d, J = 2.2 Hz, 2H), 7.84–7.20 (m, 5H), 6.36 (s, 1H), 2.65 (d, J = 7.2 Hz, 6H), 1.72–1.64 (m, 4H), 1.34–1.25 (m, 12H), 1.05 (s, 7H). 13C NMR (101 MHz, CDCl3) δ: 151.71 1C, 151.49 1C, 149.50 1C, 148.88 1C, 136.50 1C, 134.91 1C, 128.43 1C, 128.12 1C, 126.02 1C, 125.04 1C, 121.92 1C, 117.33 1C, 98.99 1C, 77.35 1C, 77.03 1C, 76.72 1C, 52.24 1C, 48.31 1C, 46.59 1C, 34.02 1C, 31.20 1C, 31.08 1C, 31.03 1C, 29.43 1C, 20.16 1C, 14.17 1C, 10.36 2C.
Novelty of compounds
To confirm the novelty of the compounds, we used online tools such as www.swisssimilarity.ch/ and www.chemspider.com/. It was confirmed that our compounds were novel, and these chloroquine analogs have not previously been reported for these studies. Their data were unavailable on these sources on 18 April 2023 [15].
Antibacterial assay
The newly synthesized chloroquine analogs showed good antibacterial potential. The analog CS1 has an excellent activity of 21.5 mm and CS8 revealed a 15.6-mm inhibitory zone against S. aureus, while minor activity was shown by analog CS6 (a 7.2-mm zone of inhibition). Analog CS1 (the most potent) also showed the highest activity against P. aeruginosa (a 30.3-mm zone of inhibition), followed by analog CS7 with a 15.3-mm zone of inhibition. Against the E. coli strain, the maximum zone of inhibition was again shown by CS1 (24.1 mm). In contrast, the minor zone was shown by analog CS6 (8.2 mm). Analog CS5 produced a 14.2-mm zone against B. subtilis. CS0 did not have activity against any bacterial strain at the same concentration, as shown in Table 1.
Table 1.
In vitro antimicrobial activity of chloroquine analogs.
| No | Sample | Staphylococcus aureus | Pseudomonas aeruginosa | Escherichia coli | Bacillus subtilis | Candida albicans | Candida parapsilosis |
|---|---|---|---|---|---|---|---|
| 1 | CS1 | 21.5 ± 0.15 | 30.3 ± 0.15 | 24.1 ± 0.26 | 13.4 ± 0.10 | 19.2 ± 0.21 | 11.4 ± 0.42 |
| 2 | CS2 | 10.6 ± 0.25 | 11.4 ± 0.21 | 15.2 ± 0.25 | 12.4 ± 0.35 | 13.3 ± 0.32 | 13.5 ± 0.26 |
| 3 | CS3 | – | – | 8.3 ± 0.10 | 9.1 ± 0.12 | 7.2 ± 0.12 | – |
| 4 | CS4 | 14.3 ± 0.20 | 11.2 ± 0.12 | 13.3 ± 0.44 | 12.3 ± 0.31 | 10.5 ± 0.21 | 11.2 ± 0.06 |
| 5 | CS5 | 9.4 ± 0.26 | 9.4 ± 0.31 | 11.2 ± 0.47 | 14.2 ± 0.12 | 12.1 ± 0.10 | 13.1 ± 0.15 |
| 6 | CS6 | 7.2 ± 0.15 | 11.1 ± 0.46 | 8.2 ± 0.21 | 9.2 ± 0.38 | 10.0 ± 0.32 | 11.3 ± 0.21 |
| 7 | CS7 | 12.5 ± 0.32 | 15.3 ± 0.12 | 12.3 ± 0.97 | 13.3 ± 0.20 | 11.1 ± 0.07 | 11.0 ± 0.55 |
| 8 | CS8 | 15.6 ± 0.17 | 14.4 ± 0.31 | 11.9 ± 0.53 | 11.4 ± 0.53 | 12.5 ± 0.26 | 11.1 ± 0.59 |
| 9 | CS9 | 12.3 ± 0.20 | 12.1 ± 0.61 | 10.2 ± 0.26 | 11.2 ± 0.60 | 11.2 ± 0.15 | 8.2 ± 0.49 |
| 10 | CS0 | – | – | – | – | – | – |
| 11 | DMSO | – | – | – | – | – | – |
| 12 | Cefixime | 20.3 ± 0.14 | 18.3 ± 0.22 | 17.3 ± 0.32 | 18.6 ± 0.56 | – | – |
| 13 | Clotrimazole | – | – | – | – | 22.1 ± 0.16 | 19.3 ± 0.18 |
The experiment was performed in triplicate.
The zone of inhibition was measured in mm.
Cefixime and clotrimazole were used as a positive control, while DMSO was used a negative control.
Antifungal assay
As shown in Table 1, the chloroquine analogs also exhibited good activity against C. albicans and C. parapsilosis. Against the C. albicans strain, a 19.2-mm zone of inhibition was shown by analog CS1. The smallest zone of inhibition was shown by analog CS3 (7.2 mm). The synthetic chloroquine analog CS2 showed a 13.5-mm zone of inhibition against C. parapsilosis, while the smallest zone of inhibition against the same strain was displayed by analog CS9 (8.2 mm).
Toxicity assay (brine shrimp lethality assay)
A brine shrimp lethality assay was performed to investigate the lethality potential of the chloroquine analogs. Analog CS1 was the most potent, with 100% activity at 50 μg/ml and LD50 = 2.78 μg/ml, while the most negligible inhibition was shown by analog CS3 (16.7% lethality). Our seven analogs’ activity was more than that of the parent molecule, CS0 (73.3%). This confirms that substitution at the amino position improves that activity, as shown in Table 2.
Table 2.
Toxicity assay (brine shrimp lethality activity) and cytotoxicity activity of chloroquine analogs against the HeLa cell line.
| No | Sample | % mortality | LD50 (μg/ml) | % Inhibition at 30 μg/ml | IC50 ± SD (μg/ml) |
|---|---|---|---|---|---|
| 1 | CS1 | 100 | 2.78 | 95.4 | 13.0 ± 0.4 |
| 2 | CS2 | 36.7 | 29.9 | ||
| 3 | CS3 | 16.7 | 11.1 | ||
| 4 | CS4 | 100 | 3.2 | 94.9 | 13.1 ± 0.3 |
| 5 | CS5 | 80 | 17.32 | 59.6 | 26.8 ± 1.8 |
| 6 | CS6 | 100 | 4.7 | 100 | 11.6 ± 0.5 |
| 7 | CS7 | 86.6 | 10.7 | 100 | 14.2 ± 0.8 |
| 8 | CS8 | 100 | 13.01 | 100 | 12.6 ± 0.9 |
| 9 | CS9 | 96.6 | 17.32 | 100 | 8.9 ± 1.2 |
| 10 | CS0 | 73.3 | 21.3 | 61.9 | 24.0 ± 0.3 |
| 11 | DMSO | – | – | – | – |
| 12 | Doxorubicin | 100 | 3.1 | 100 | 0.9 ± 0.14 |
The experiment was performed in triplicate.
Doxorubicin was used as a positive control.
Cytotoxicity assay
All synthesized chloroquine-based sulfonamides showed excellent anticancer activity against the HeLa cell line. Analogs CS6, CS7, CS8 and CS9 exhibited 100% inhibition at 30 μg/ml against the HeLa cell line in the MTT assay compared with CS0 (61.9%). Analog CS9 has an excellent IC50 value (8.9 μg/ml), as shown in Table 2.
Discussion
Compounds with quinoline nuclei have well-known pharmacologically active properties used to treat various lethal diseases, such as malaria and cancer [25]. In this study biologically active chloroquine analogues were synthesized with substitution at 4-amino positions and formed sulfonamides. A nucleophilic substitution reaction was carried out at the 4-amino position of chloroquine, and hydrogen was replaced with sulfonamide formation. HCl was produced as a by-product [26]. The synthesized analogues were confirmed using the TLC procedure by calculating the Rf value difference and melting point difference. Fourier-transform IR spectroscopy confirmed a new functional group (i.e., sulfonamide). The purified analogs were further characterized through carbon and proton NMR spectroscopy. The synthesized analogs were dissolved in chloroform-d and DMSO [27]. With the help of the accessible online chemical databases www.swisssimilarity.ch/ and www.chemspider.com/, it was confirmed that these compounds have not previously been reported [28,29].
These chloroquine analogues were investigated in vitro for their antibacterial significance against four Gram-positive and Gram-negative strains and two fungal strains. The results were compared with the parent drug (i.e., chloroquine) used in synthesis. The antimicrobial assay measured the zone of inhibition (in mm) to calculate the inhibitory effect of the analogs against specific strains. The newly synthesized chloroquine analogs have excellent antibacterial activity, as described above in Table 1. Analog CS1 exhibited a 21.5-mm inhibition zone against the bacterium S. aureus, a 30.3-mm zone of inhibition against P. aeruginosa and a 24.1-mm inhibition zone against E. coli. Analog CS5 produced a 14.2-mm inhibitory zone against B. subtilis [30,31]. These results are promising as all the samples exhibited activity compared with the parent molecule. Cefixime was used as a standard drug, which showed a 20.3-mm zone against S. aureus, an 18.3-mm zone against P. aeruginosa, a 17.3-mm zone against E. coli and an 18.6-mm zone against B. subtilis. Other studies also confirm that our chloroquine analogs have an excellent average zone of inhibition, and chloroquine was not active at the same concentration [27,32].
In antifungal screening against two fungal strains, chloroquine analog CS1 showed a 19.2-mm inhibitory zone against C. albicans and CS2 produced a 13.5-mm zone against C. parapsilosis, as described in Table 1. These synthesized analogs showed good average antifungal activity compared with the inactive parent molecule (chloroquine) at the same concentration and in another study [33]. Thus it can be concluded that all the sulfonamides are sufficiently active against the tested strains compared with their original molecule (chloroquine). In toxicity screening using the brine shrimp lethality assay, the chloroquine analogs showed better mortality data, as shown in Table 2. Synthesized analogs CS1 and CS4 revealed 100% mortality at 30 μg/ml concentration. Analog CS1 showed excellent activity, with LD50 2.78 μg/ml, compared with CS0 (73.3%) and other studies [34,35].
Cervical cancer is the fourth most common type of cancer in women worldwide [36]. The novel chloroquine analogs showed good-to-excellent activity against the HeLa cell line. Chloroquine analog CS9 revealed excellent (100%) activity, with an IC50 value of 8.9 μg/ml, compared with CS0 (61.9%) and other studies [37–39]. As per the predicted data, the newly synthesized analogs have good bioavailability scores (0.55) and lipophilic values. As presented in Table 4, analog CS5 can pass the blood–brain barrier, and this analog could be further investigated as a lead molecule in treating brain disorders. Analog CS8 does not cross the blood–brain barrier but has high gastrointestinal absorption. Analog CS1 also has good gastrointestinal absorption and a molecular weight of less than 500 g/mol. It also did not cross the blood–brain barriers and was processed for docking studies. The Swiss target prediction predicted that CS1 has the highest potential against family-A G-protein-coupled receptors and enzymes.
Table 4.
Pharmacokinetic properties of chloroquine analogs.
| Pharmacokinetic properties | CS1 | CS2 | CS3 | CS4 | CS5 | CS6 | CS7 | CS8 | CS9 |
|---|---|---|---|---|---|---|---|---|---|
| GI absorption | High | Low | Low | Low | High | Low | Low | High | Low |
| BBB permeant | No | No | No | No | Yes | No | No | No | No |
| P-gp substrate | No | No | No | No | No | Yes | Yes | Yes | Yes |
| CYP1A2 inhibitor | No | No | No | No | No | No | No | No | No |
| CYP2C19 inhibitor | Yes | No | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| CYP2C9 inhibitor | Yes | Yes | Yes | Yes | Yes | No | No | Yes | No |
| CYP2D6 inhibitor | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| CYP3A4 inhibitor | Yes | Yes | Yes | Yes | Yes | No | No | Yes | Yes |
| Log Kp (skin permeation) | -5.24 cm/s | -5.01 cm/s | -6.17 cm/s | -5.07 cm/s | -5.97 cm/s | -5.02 cm/s | -4.72 cm/s | -5.12 cm/s | -4.91 cm/s |
BBB: Blood–brain barrier; GI: Gastrointestinal; P-gp: P-glycoprotein.
Further docking studies using MOE software confirmed that analogue CS1 has an affinity of -6.4780 kcal/mol against PDB ID 1PFK (crystal structure of the complex of phosphofructokinase) due to interaction with aspartic acid, as shown in Figure 3 & Table 3. The structure–activity relationships showed that due to substitution at the 4-amino position, chloroquine analogues exhibited better antimicrobial potential than their parent molecule (i.e., chloroquine), an antimalarial drug. Furthermore, with substitution at the benzene ring attached with O=S=O, the antimicrobial activity decreased, while in the case of cytotoxic activity by addition of tertiary butyl (analog CS9) with benzene ring attached with O=S=O, it has maximum cytotoxic potential as compared with chloroquine. These analogues can be further investigated in antimicrobial and anticancer drug research [40].
Figure 3.
Docking studies.
(A) Target prediction of analog CS1. (B). Analog CS1 binding affinity with Protein Data Bank ID 1PFK.
Table 3.
The binding affinity of chloroquine analogs.
| Docking results against Protein Data Bank ID 1PFK | |
|---|---|
| Analog/drug | Affinity (kcal/mol) |
| CS1 | -6.4780 |
| CS2 | -5.1431 |
| CS3 | -5.6393 |
| CS4 | -5.9502 |
| CS4 | -6.2490 |
| CS5 | -5.8908 |
| CS6 | -6.2473 |
| CS7 | -6.9568 |
| CS8 | -6.4193 |
| CS9 | -6.0407 |
| Doxorubicin | -5.0226 |
PDB ID 1PFK is the crystal structure of the complex of Escherichia coli phosphofructokinase with its reaction products.
Results were obtained using Molecular Operating Environment (MOE) software.
PDB: Protein Data Bank.
Conclusion
In the present paper, the newly synthesized chloroquine analogues showed excellent activity against different microbial strains and the HeLa cell line due to substitution at the 4-amino position compared with their parent molecule, chloroquine. Analogue CS1 was the most active against all microbial strains compared with standard drugs due to sulfonamide formation. The chloroquine molecule was inactive in antimicrobial screening. In the cytotoxicity assay against the HeLa cell line, analogs CS1 and CS9 had excellent activity; that is, 1.8- and 2.5-times more than the parent molecule, chloroquine. These analogues can be further processed for advanced studies and maybe a drug candidate in future.
Acknowledgments
The authors acknowledge COMSATS University, Islamabad, Pakistan, and HEJ Research Institute, University of Karachi, Pakistan, for providing excellent research facilities.
Financial disclosure
The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Competing interests disclosure
The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Writing disclosure
No writing assistance was utilized in the production of this manuscript.
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