The Search for New Antibacterial Agents among 1,2,3-Triazole Functionalized Ciprofloxacin and Norfloxacin Hybrids: Synthesis, Docking Studies, and Biological Activity Evaluation

Abstract

Among all modern antibiotics, fluoroquinolones are well known for their broad spectrums of activity and efficiency toward microorganisms and viruses. However, antibiotic resistance is still a problem, which has encouraged medicinal chemists to modify the initial structures in order to combat resistant strains. Our current work is aimed at synthesizing novel hybrid derivatives of ciprofloxacin and norfloxacin and applying docking studies and biological activity evaluations in order to find active promising molecules. We succeeded in the development of a synthetic method towards 1,2,3-triazole-substituted ciprofloxacin and norfloxacin derivatives. The structure and purity of the obtained compounds were confirmed by ¹H NMR, ¹³C NMR, ¹⁹F NMR, LC/MS, UV-, IR- spectroscopy. Docking studies, together with in vitro research against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Candida albicans NCTC 885-653 revealed compounds in which activity exceeded the initial molecules.

1. Introduction

As the COVID-19 pandemic progresses, the task of finding a perfect form of therapy for this disease is becoming increasingly challenging. Clinicians need to make daily decisions and prescribe existing medicines, even when the data about their activities, in this case, are absent. Throughout the world, medicinal chemists are searching for effective remedies among existing antibiotics and antivirals.

Investigations have been made to analyze antibiotic prescribing practices for patients with COVID-19. According to the authors of [1], a combination of beta-lactams and macrolides or fluoroquinolones are reported in most cases.

Among these groups of antibiotics, fluoroquinolones are known as broad-spectrum synthetic antimicrobials. Their development started in the early 1960s, with the nalidixic acid synthesis; since then, four generations of highly efficient fluoroquinolones have been created [2] (Figure 1).

General fluoroquinolone structures contain a bicyclic system that is substituted at the N-1 position, has a carboxyl group at position 3, an oxo group at position 4, a fluorine atom at the 6-position, and a substituent (often nitrogen-based heterocyclic moiety) at C-7 [3]. Different structural modifications through a wide range of synthetic approaches are possible in each of the above-mentioned positions, mainly aimed at changing pharmacodynamics and pharmacokinetics of the initial molecules [4,5,6]. Therefore, such a vast family of synthetic antibiotics have evolved because of the attractiveness of fluoroquinolones from the viewpoint of chemists.

Moreover, novel molecules of fluoroquinolones were created in order to cope with the problem of resistance to antibiotics [7,8], and to increase the effectiveness along with decreasing the side effects. In this way, the concept of a hybrid design of medicines, which utilizes several pharmacophores and unites them in one molecule, appears to be of high interest.

Recently, the authors of [9] reported that the fluoroquinolones—levofloxacin and moxifloxacin—are the most commonly used antimicrobial agents for the treatment of chronic obstructive pulmonary disease and community-acquired pneumonia. Noteworthy, they not only reveal wide antimicrobial activity, but also possess antiviral action against vaccinia virus, papovavirus, CMV, VZV, HSV-1, HSV-2, HCV, and HIV [10].

It is no wonder that, through the COVID-19 pandemic, scientists have turned their attention towards fluoroquinolones as possible medicines for treatment, and there are already some promising results. The authors of [11] conducted an in silico molecular docking study, and demonstrated for the first time the ability of ciprofloxacin and moxifloxacin to interact with the COVID-19 main protease. Another group [12] described low antiviral activity potency of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) against SARS-CoV-2 and MERS-CoV.

According to the above-mentioned information, we chose fluoroquinolones, namely norfloxacin and ciprofloxacin, as the objects for investigation and creation of new hybrid drugs.

We should note that norfloxacin and ciprofloxacin have become popular starting molecules, as many recent studies used them to synthesize new derivatives. They represent the second generation of fluoroquinolones, and the first ones with the fluorine atom at C-6. However, while they were widely used in clinical practices, the problem of antibiotic resistance has erupted, and led to the creation of third- and fourth-generation of fluoroquinolones.

However, norfloxacin and ciprofloxacin are still utilized in medicinal chemistry, and modifications of their structures often provide inspiring results. Research for new antibacterials mainly focuses on the C-7 position. First, substituents here are important for the spectrum and potency of fluoroquinolone action. Moreover, chemical modifications at C-7 possess influence on pharmacokinetic properties and cell permeability of molecules. Additionally, paper [13] shows that the C-7 substituent has a great impact on the interaction with the target enzyme DNA gyrase.

According to the literature data, nitrogen in piperazine ring can be modified with such heterocyclic moieties as phenylthiazole [13], thiazolidine [14], quinazoline [15], thiadiazole [16,17], pyrimidine [18,19], dithienylethene [20], and 1,2,4-triazole [21]. Azole functionalized hybrids revealed promising levels of antibacterial activity [22], which led us to assume the effectiveness of 1,2,3-triazole moiety utilization for hybrid modification of fluoroquinolones.

It is also worth mentioning that our previous experience in quinolones chemistry [23,24,25] gave a powerful background for the planning stage of this research.

Therefore, the main purpose of our scientific team was to synthesize novel 1,2,3-triazole substituted derivatives of fluoroquinolones norfloxacin and ciprofloxacin, and to use docking and in vitro studies to find new hybrid molecules with antimicrobial activity.

2. Materials and Methods

2.1. Chemicals and Apparatus

The starting fluoroquinolones were commercially available; they, as well as the solvents, were purchased from Sigma Aldrich, St. Louis, MO 68178, The United States, and were used without further purification.

All NMR spectra were recorded on a Varian MR-400 spectrometer with standard pulse sequences operating at 400 MHz for ¹H NMR, 101 MHz for ¹³C NMR, and 376 MHz for 19F NMR. For all NMR spectra, DMSO-d₆ was used as a solvent. Chemical shift values are referenced to residual protons (δ 2.49 ppm) and carbons (δ 39.6 ppm) of the solvent as an internal standard. Elemental analysis was performed on a EuroEA-3000 CHNS-O analyzer. Melting points were determined with a Buchi B-520 melting point apparatus. LC/MS spectra were recorded on an ELSD Alltech 3300 liquid chromatograph equipped with a UV detector (λmax 254 nm), API-150EX mass-spectrometer, and using a Zorbax SB-C18 column, Phenomenex (100 × 4 mm) Rapid Resolution HT cartridge 4.6 × 30 mm, 1.8-Micron. Elution started with 0.1 M solution of HCOOH in water and ended with 0.1 M solution of HCOOH in acetonitrile, using a linear gradient at a flow rate of 0.15 mL/min and an analysis cycle time of 25 min. IR spectra in KBr pellets were recorded on a Perkin–Elmer 298 spectrophotometer in KBr pellets. UV/Vis spectra of 0.01 mmol solutions in 2%DMSO/MeOH or 0.08%DMSO/MeOH were recorded on a Specord 200 spectrometer (Supplementary Materials).

2.2. Synthesis of 7-(4-(5-Amino-1-R-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-R-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 1b, c, 2d-l (General Method)

To a solution of quinolone (1a, 2a) (10 mmol) in DMSO (10 mL) and MeOH (5 mL), substituted azide (12 mmol) and sodium methoxide in methanol (30 mmol) were added. The reaction mixture was refluxed overnight. After completion of the reaction (detected by TLC), the reaction mixture was cooled. Then water was added and the mixture was acidified with 0.1 М HCl. The solid product was collected by filtration, washed with water, and dried and purified by recrystallization with butanol. Yield after recrystallization was 37–68%.

2.2.1. 7-(4-(5-Amino-1-(p-tolyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 1b

Yield 0.34 g (65%). Mp 250-255C. ¹H NMR spectrum ppm, δ 8.54 (s, 1H), 7.84 (d, J = 13.6 Hz, 2H), 7.44 (d, J = 10.5 Hz, 6H), 7.08 (s, 2H), 6.57 (s, 2H), 4.63 (s, 1H), 4.35 (s, 2H), 3.85 (s, 1H), 2.41 (s, 5H), 1.35 (s, 3H). ¹³C NMR (100 MHz, DMSO) δ 175.76, 166.65, 161.39, 152.43, 149.98, 147.87, 145.52, 144.76, 137.60, 137.43, 134.35, 130.09, 123.78, 123.47, 120.41, 113.30, 113.07, 109.77, 105.19, 49.64, 49.60, 48.93, 46.48, 21.07, 14.31. LC/MS m/z (I_rel, %): 520.1 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3419 (OH, st), 2923, 2855 (C=C, st), 1622 (C=O carboxylic, st), 1581 (C=O amide, st), 1492 (C=C phenyl, st), 1255 (C-O, st), 1014 (C=C, δ). UV/Vis spectrum (0.08% DMSO/MeOH), λ_max nm (ε): 280(0.014), 332 (0.004). Calc C, 60.11; H, 5.04; F, 3.66; N, 18.87; O, 12.32. Found C, 60.11; H, 5.05; F, 3.67; N, 18.85; O, 12.32.

2.2.2. 7-(4-(5-Amino-1-(4-bromophenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 1c

Yield 0.30 g (58%). Mp 300-304C. ¹H NMR spectrum ppm, δ 15.34 (s, 1H), 8.96 (s, 1H), 7.96 (d, J = 13.2 Hz, 1H), 7–7.58 (m, 2H), 7.52–7.43 (m, 2H), 7.26 (d, J = 7.2 Hz, 1H), 6.65 (s, 1H), 4.61 (d, J = 7.1 Hz, 2H), 3.44 (s, 2H), 1.43 (t, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO) δ 175.76, 166.65, 161.39, 152.16, 149.98, 147.87, 145.52, 144.78, 137.43, 135.36, 132.80, 124.99, 123.78, 120.41, 119.78, 113.30, 113.07, 109.77, 105.14, 49.62, 48.93, 46.48, 14.31. LC/MS m/z (I_rel, %): 524.2 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3400 (OH, st), 2922 (C=C, st), 1628 (C=O carboxylic, st), 1501 (C=O amide, st), 1466, 1447 (C=C phenyl, st), 1256 (C-O, st), 1014 (C=C, δ). UV/Vis spectrum (2%DMSO/MeOH), λ_max nm (ε): 277 (0.051), 314 (0.012), 327 (0.012). Calc C, 57.36; H, 4.43; F, 7.26; N, 18.73; O, 12.22. Found C, 57.34; H, 4.47; F, 7.28; N, 18.81; O, 12.10.

2.2.3. 7-(4-(5-Amino-1-phenyl-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2d

Yield 0.34 g (66%). Mp 258-260C. ¹H NMR spectrum ppm, δ 15.20 (s, 1H), 8.67 (s, 1H), 7.94 (d, J = 13.1 Hz, 1H), 7.69–7.52 (m, 5H), 6.65 (s, 1H), 4.66 (s, 2H), 3.82 (td, J = 7.2, 3.7 Hz, 3H), 3.46 (d, J = 6.1 Hz, 4H), 1.39–1.29 (m, 2H), 1.19 (dd, J = 6.5, 3.9 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 176.36, 176.33, 165.87, 161.20, 154.22, 151.74, 147.98, 146.86, 146.81, 145.01, 144.91, 139.15, 134.68, 129.81, 129.22, 124.30, 124.28, 121.77, 121.75, 118.85, 118.77, 111.10, 110.88, 106.76, 106.66, 106.62, 49.70, 35.89, 7.58. LC/MS m/z (I_rel, %): 518.0 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3390 (OH, st), 2921, 2854 (C=C, st), 1623 (C=O carboxylic, st), 1512 (C=O amide, st), 1445 (C=C phenyl, st), 1254 (C-O, st), 1014 (C=C, δ). UV/Vis spectrum (0.08% DMSO/MeOH), λ_max nm (ε): 276 (0.082), 312 (0.022), 326 (0.021). Calc C, 60.34; H, 4.67; F, 3.67; N, 18.95; O, 12.37. Found C, 60.35; H, 4.62; F, 3.70; N, 18.99; O, 12.34.

2.2.4. 7-(4-(5-Amino-1-(2-methylphenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2e

Yield 0.20 g (37%). Mp 215-220C. ¹H NMR spectrum ppm 1H NMR (400 MHz, DMSO-d6) δ 15.19 (s, 1H), 8.67 (s, 1H), 7.94 (dd, J = 13.1, 7.0 Hz, 1H), 7.62 (t, J = 9.4 Hz, 1H), 7.58–7.40 (m, 2H), 7.37 (d, J = 7.8 Hz, 1H), 6.48 (s, 1H), 4.63 (d, J = 42.7 Hz, 2H), 3.83 (s, 4H), 3.47 (s, 3H), 2.54 (s, 2H), 2.07 (s, 2H), 1.35 (q, J = 6.9 Hz, 2H), 1.19 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.34, 152.56, 150.11, 147.86, 145.61, 144.90, 144.78, 138.81, 136.40, 133.41, 130.69, 130.12, 126.88, 124.25, 123.92, 120.90, 112.88, 112.65, 110.19, 106.65, 106.59, 49.64, 49.60, 46.48, 35.75, 17.35, 8.34. LC/MS m/z (I_rel, %): 548.1 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3427 (OH, st), 2923, 2854 (C=C, st), 1628 (C=O carboxylic, st), 1511 (C=O amide, st), 1470,1447 (C=C phenyl, st), 1256 (C-O, st), 1013 (C=C, δ). UV/Vis spectrum (0.08%DMSO/MeOH), λ_max nm (ε): 277 (0.047), 327 (0.011). Calc C, 61.01; H, 4.93; F, 3.57; N, 18.45; O, 12.04. Found C, 61.01; H, 4.96; F, 3.55; N, 18.49; O, 11.99.

2.2.5. 7-(4-(5-Amino-1-(4-ethylphenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2f

Yield 0.20 g (68%). Mp 258-262C. ¹H NMR spectrum ppm, δ 15.21 (s, 1H), 8.68 (s, 1H), 7.96 (d, J = 13.1 Hz, 1H), 7.63 (d, J = 7.4 Hz, 1H), 7.48 (q, J = 8.4 Hz, 3H), 6.59 (s, 1H), 4.66 (s, 1H), 3.87–3.80 (m, 2H), 3.46 (s, 3H), 2.72 (q, J = 7.6 Hz, 2H), 2.54 (s, 5H), 1.34 (t, J = 6.6 Hz, 2H), 1.29–1.12 (m, 5H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.39, 152.56, 150.11, 147.86, 144.90, 144.76, 141.21, 138.81, 134.91, 128.75, 124.53, 123.78, 120.90, 112.88, 112.65, 110.19, 106.65, 106.59, 49.64, 49.60, 46.48, 35.75, 28.14, 15.38, 8.34. LC/MS m/z (I_rel, %): 546.2 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3374 (OH, st), 2960, 2926 (C=C, st), 1619 (C=O carboxylic, st), 1520 (C=O amide, st), 1445 (C=C phenyl, st), 1254 (C-O, st), 1013 (C=C, δ). UV/Vis spectrum (0.08% DMSO/MeOH), λ_max nm (ε): 281 (0.078), 318 (0.022), 331 (0.021). Calc C, C, 61.64; H, 5.17; F, 3.48; N, 17.97; O, 11.73. Found C, 61.64; H, 5.15; F, 3.45; N, 18.00; O, 11.75.

2.2.6. 7-(4-(5-Amino-1-(4-ethylphenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2g

Yield 0.24 g (43%). Mp 260-262C. ¹H NMR spectrum ppm, δ 15.20 (s, 1H), 8.68 (s, 1H), 7.95 (d, J = 13.1 Hz, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.53–7.45 (m, 2H), 7.20–7.12 (m, 2H), 6.52 (s, 1H), 4.66 (s, 2H), 3.85 (s, 2H), 3.82 (dd, J = 7.4, 4.0 Hz, 2H), 3.46 (d, J = 6.0 Hz, 3H), 2.54 (s, 4H), 1.34 (d, J = 6.8 Hz, 2H), 1.18 (q, J = 5.7, 4.2 Hz, 2H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.39, 159.03, 152.56, 150.11, 147.86, 144.90, 144.78, 144.68, 138.81, 130.70, 126.09, 123.85, 120.90, 115.27, 112.88, 112.65, 110.19, 106.65, 106.59, 55.33, 49.64, 49.60, 46.48, 35.75, 8.34. LC/MS m/z (I_rel, %): 548.2 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3435 (OH, st), 2923, 2855 (C=C, st), 1621 (C=O carboxylic, st), 1523 (C=O amide, st), 1468, 1448 (C=C phenyl, st), 1246 (C-O, st), 1021 (C=C, δ). UV/Vis spectrum (0.08%DMSO/MeOH), λ_max nm (ε): 281 (0.145), 318 (0.046), 329 (0.045). Calc C, 59.23; H, 4.79; F, 3.47; N, 17.91; O, 14.61. Found C, 59.20; H, 4.78; F, 3.49; N, 17.98; O, 14.56.

2.2.7. 7-(4-(5-Amino-1-(4-(methylthio)phenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2h

Yield 0.28 g (49%). Mp 280-285C. ¹H NMR spectrum ppm, δ 15.22 (s, 1H), 8.68 (s, 1H), 7.96 (d, J = 13.2 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.50 (q, J = 8.7 Hz, 4H), 6.62 (s, 2H), 4.66 (s, 1H), 3.83 (s, 4H), 3.45 (s, 3H), 1.34 (d, J = 6.7 Hz, 2H), 1.19 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.39, 152.56, 150.11, 147.86, 144.90, 144.77, 138.81, 137.25, 134.23, 128.42, 123.78, 123.55, 120.90, 112.88, 112.65, 110.18, 106.65, 106.59, 49.64, 49.60, 46.48, 35.75, 15.49, 8.34. LC/MS m/z (I_rel, %): 564.1 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3445 (OH, st), 2923, 2852 (C=C, st), 1620 (C=O carboxylic, st), 1520 (C=O amide, st), 1469 (C=C phenyl, st), 1256 (C-O, st), 1016 (C=C, δ). UV/Vis spectrum (2%DMSO/MeOH), λ_max nm (ε): 281 (0.062), 318 (0.014), 331 (0.013). Calc C, 57.54; H, 4.65; F, 3.37; N, 17.40; O, 11.35; S, 5.69. Found C, 57.50; H, 4.65; F, 3.38; N, 17.47; O, 11.30; S, 5.70.

2.2.8. 7-(4-(5-Amino-1-(3-(methylthio)phenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2i

Yield 0.32 g (57%). Mp 226-232C. ¹H NMR spectrum ppm, δ 15.20 (s, 1H), 8.68 (s, 1H), 7.95 (d, J = 13.1 Hz, 1H), 7.63 (d, J = 7.4 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.44 (d, J = 11.7 Hz, 2H), 7.35 (d, J = 7.9 Hz, 1H), 6.70 (s, 1H), 4.66 (s, 3H), 3.84 (s, 4H), 3.46 (s, 3H), 1.34 (t, J = 6.8 Hz, 2H), 1.19 (s, 2H). ¹³C NMR (75 MHz, DMSO-d6) δ 175.67, 165.78, 161.39, 152.56, 147.86, 144.95, 138.81, 138.46, 136.62, 128.73, 125.26, 123.94, 121.43, 121.03, 120.90, 112.88, 112.65, 110.18, 106.65, 49.64, 49.60, 46.48, 35.75, 15.61, 8.34. LC/MS m/z (I_rel, %): 564.2 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3444 (OH, st), 2921, 2852 (C=C, st), 1620 (C=O carboxylic, st), 1507 (C=O amide, st), 1469,1446 (C=C phenyl, st), 1255 (C-O, st), 1016 (C=C, δ). UV/Vis spectrum (0.08% DMSO/MeOH), λ_max nm (ε): 280 (0.016), 316 (0.006). Calc C, C, 57.54; H, 4.65; F, 3.37; N, 17.40; O, 11.35; S, 5.69. Found C, C, 57.54; H, 4.65; F, 3.35; N, 17.90; O, 11.32; S, 5.64.

2.2.9. 7-(4-(5-Amino-1-(4-bromophenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2j

Yield 0.35 g (61%). Mp 275-280C. ¹H NMR spectrum ppm, δ 15.20 (s, 1H), 8.68 (s, 1H), 7.96 (d, J = 13.0 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 8.3 Hz, 2H), 6.71 (s, 1H), 4.65 (s, 2H), 3.83 (s, 4H), 3.45 (s, 3H), 2.44 (s, 2H), 1.34 (d, J = 7.0 Hz, 2H), 1.19 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.39, 152.56, 147.86, 144.78, 138.81, 135.36, 132.80, 124.99, 123.78, 119.78, 112.88, 112.65, 110.18, 106.65, 49.62, 46.48, 35.75, 8.34. LC/MS m/z (I_rel, %): 598.1 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3391 (OH, st), 2920, 2854 (C=C, st), 1627 (C=O carboxylic, st), 1515 (C=O amide, st), 1443 (C=C phenyl, st), 1258 (C-O, st), 1017 (C=C, δ). UV/Vis spectrum (2%DMSO/MeOH), λ_max nm (ε): 283 (0.048), 315 (0.012), 331 (0.011). Calc C, 52.36; H, 3.89; Br, 13.40; F, 3.19; N, 16.44; O, 10.73. Found C, 52.35; H, 3.85; Br, 13.42; F, 3.15; N, 16.47; O, 10.77.

2.2.10. 7-(4-(5-Amino-1-(5-fluoro-2-methylphenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2k

Yield 0.27 g (50%). Mp 278-282C. ¹H NMR spectrum ppm, δ 15.21 (s, 1H), 8.68 (s, 1H), 7.96 (d, J = 13.1 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.54 (dd, J = 6.8, 2.5 Hz, 1H), 7.49–7.41 (m, 1H), 7.39 (d, J = 8.8 Hz, 1H), 6.64 (s, 1H), 4.66 (s, 2H), 3.83 (s, 4H), 3.46 (s, 3H), 2.33 (d, J = 2.1 Hz, 3H), 1.34 (d, J = 6.9 Hz, 2H), 1.19 (d, J = 4.5 Hz, 2H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.34, 152.56, 147.86, 145.42, 138.81, 131.74, 131.65, 129.18, 124.12, 120.90, 113.63, 113.41, 112.88, 112.65, 110.18, 108.25, 107.99, 106.65, 49.64, 49.60, 46.48, 35.75, 17.46, 8.34. LC/MS m/z (I_rel, %): 550.2 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3393 (OH, st), 2922, 2853 (C=C, st), 1627 (C=O carboxylic, st), 1509 (C=O amide, st), 1470,1446 (C=C phenyl, st), 1255 (C-O, st), 1020 (C=C, δ). UV/Vis spectrum (0.08% DMSO/MeOH), λ_max nm (ε): 281 (0.055), 318 (0.016), 329 (0.015). Calc C, C, 59.01; H, 4.59; F, 6.91; N, 17.84; O, 11.65. Found C, C, 59.01; H, 4.55; F, 6.97; N, 17.88; O, 11.59.

2.2.11. 7-(4-(5-Amino-1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid 2l

Yield 0.29 g (49%). Mp 222-228C. ¹H NMR spectrum ppm, δ 15.14 (s, 1H), 8.67 (s, 1H), 7.99–7.91 (m, 4H), 7.91–7.83 (m, 2H), 7.62 (d, J = 7.3 Hz, 1H), 6.81 (s, 2H), 4.64 (s, 1H), 3.82 (s, 4H), 3.46 (s, 3H), 1.34 (d, J = 5.9 Hz, 2H), 1.18 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ 175.67, 165.78, 161.39, 152.56, 150.11, 147.86, 144.90, 144.77, 138.81, 135.82, 127.40, 127.08, 123.78, 123.47, 112.88, 112.65, 110.18, 106.60, 40.43, 40.15, 39.87, 39.59, 39.31, 39.04, 38.76, 35.75, 8.34. LC/MS m/z (I_rel, %): 586.1 [M+H]⁺ (100). IR spectrum (KBr), ν, cm⁻¹: 3412 (OH, st), 2922, 2853 (C=C, st), 1626 (C=O carboxylic, st), 1508 (C=O amide, st), 1469, 1445 (C=C phenyl, st), 1256 (C-O, st), 1017 (C=C, δ). UV/Vis spectrum (2%DMSO/MeOH), λ_max nm (ε): 281 (0.027). Calc C, 55.39; H, 3.96; F, 12.98; N, 16.75; O, 10.93. Found C, 55.35; H, 3.99; F, 12.95; N, 16.80; O, 10.92.

2.3. Docking Studies

The software package for docking studies was the same as in our previous studies [26,27], namely: AutoDock 4.2; MGL Tools 1.5.6 program; Avogadro program, Discovery Studio Visualizer program. The output formats of the receptor and ligand data were converted to a PDBQT format. The antibacterial targets were as follows: Staphylococcus aureus DNA Gyrase PDB ID: 2XCR; Mycobacterium tuberculosis topoisomerase II PDB ID:5BTL; Streptococcus pneumoniae topoisomerase IV PDB ID: 4KPF from the Protein Data Bank (PDB). The same docking parameters were determined as in our previous paper.

Type IIA topoisomerases cleave and repair DNA to regulate DNA topology and are a major target for antibacterials, but do not have a well-developed structural basis for understanding the mechanism of action of the medicine. Recent studies have reported a crystalline structure of a new class of broad-spectrum antibacterial agents in combination with DNA gyrase and Staphylococcus aureus DNA, demonstrating a new method of inhibition that bypasses fluoroquinolone resistance in this clinically important drug target. The inhibitor connects DNA and a temporary non-catalytic double-axis pocket on the GyrA dimeric region, which is located close to the active sites and binding sites of fluoroquinolone. In the inhibitor complex, the active site appears to be ready to cleave DNA with a single metal ion located between the TOPRIM domain (topoisomerase/primase) and the cleaving phosphate. This development gives a new idea on the new mechanisms of action and the possibility of creating a platform for the design of medicines based on the structures of a new class of antibacterial agents, relative to the clinically tested and conformationally flexible class of enzymes [28].

Fluoroquinolone antibacterials have DNA gyrase as a target and are critical agents used to stop the progression of multidrug-resistant tuberculosis; however, resistance to fluoroquinolones is constantly increasing, and new ways to circumvent resistance are needed. Analysis of the structure of complexes in the range from 2.4 to 2.6 Å shows that the truly low susceptibility of Mycobacterium tuberculosis (Mtb) to fluoroquinolones correlates with a decrease in contacts involved in additional stabilization of the target molecule complex through the overlapping cation Mg²⁺ interaction of fluoroquinolone and gyrase.

Stability analysis using purified components showed a clear relationship between the reversibility of the ternary complex and the inhibitory activity reported with cultured cells. Taken together, the data obtained indicate that the stability of the fluoroquinolone/DNA interaction is a major determinant of fluoroquinolone activity and that the fragments added to the C-7 position of the various quinolone scaffolds have no advantage in contacting the enzyme. These concepts point to new approaches to the development of quinolone class compounds that have increased efficacy against Mtb and the ability to overcome resistance [29].

As part of the synthesis program and study of biological properties of new antibacterial agents from the group of fluoroquinolones and their focus on topoisomerase IV Streptococcus pneumoniae—a crystallographic model of complexes with 7,8-substituted fluoroquinolones (with limited content of C-7) was selected in complex with topoisomerase IV S. pneumoniae, with the DNA binding site, consisting of 18 common pairs—E-site [30].

All selected crystallographic models contain ligands based on the quinoline framework, which will predetermine the emergence of affinity for the studied molecules in this series.

2.4. Antibacterial Activity

2.4.1. Method of Double Serial Dilutions

The macro method of double serial dilutions [31,32] was used in the course of the study, which is regulated by EUCAST (v11.0 2021) and the international standard ISO 20776-1: 2006 “Clinical laboratory testing and in vitro diagnostic test systems—Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices—Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases”).

Solutions of the synthesized compounds in DMF were prepared at a concentration of 1 mg/mL.

According to WHO recommendations, the following test strains were used: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Candida albicans NCTC 885-653.

The antimicrobial properties were studied by performing two dilutions of test samples in 2 mL of meat peptone broth (MPB medium №1) (a total of 10 tubes). A separate pipette was used for each dilution. After that, 0.2 mL of microbial suspension of each test strain with the appropriate number of microbial cells was added to each tube. For inoculation, a microbial suspension equivalent to 0.5, according to the McFarland standard, diluted 100 times in nutrient broth, was used. Afterward, the concentration of the microorganism in it, approximately 10 CFU/cc. 0.5 cc of inoculum, was added to each tube containing 0.5 cc of test samples of appropriate dilutions, and in one tube of 0.5 cc of nutrient broth, without test substances as a ‘negative control’. The final concentration of the microorganism in each tube will reach the required—approximately 5 × 10 CFU/cc. The inoculum should be introduced into test tubes with dilutions of the test samples no later than 15 min from the moment of its preparation.

Additionally, the control was prepared: 2 tubes with 2 mL of used medium in each—control of medium; 2 tubes with 2 mL of used medium with 0.2 mL of microbial suspension of each test strain—growth control of test microorganisms.

The tubes were placed in a thermostat for 18–24 h. The results were determined visually by the presence or absence of turbidity. The concentration of the compound in the last tube with a clear medium (not visible to the naked eye growth of the test strain) corresponded to the MIC. The control of the test microorganism growth should be observed; medium control must be sterile.

Evaluation of the results determined that the presence of microorganism growth was performed visually according to EUCAST and ISO 20776-1:2006, examining tubes with cultures in transmitted light.

Experiments with C. albicans were performed by double dilution of test samples in 2 mL of dextrose broth Sabouraud (a total of 10 tubes). A separate pipette was used for each dilution. After that, 0.2 mL of the microbial suspension of the test strain with the appropriate number of microbial cells was added to each tube. Additionally, the control was prepared: 2 tubes with 2 mL of used medium in each—control of medium; 2 tubes with 2 mL of used medium with 0.2 mL of suspension of the test strain—growth control.

The tubes were placed in a thermostat for 18–24 h. The results were determined visually by the presence or absence of turbidity. The concentration of the compound in the last tube with a clear medium (no visible to the naked eye growth of the test strain) corresponded to the MIC. The control of the growth should be observed; medium control must be sterile. The obtained data were analyzed by variation statistics. The significance level p ≤ 0.05 was accepted.

2.4.2. Agar Diffusion Method

The antimicrobial activity of the compounds synthesized was studied in vitro by the method of diffusion into agar (the method of wells), the principle of which was to measure the zone of growth retardation of microorganisms [31,32].

The antimicrobial activity was determined immediately after preparation of samples. The studies were performed under aseptic conditions. Pure reference cultures were used as test cultures: Gram-positive culture of Staphylococcus aureus ATCC 25923, as well as Gram-negative culture of Escherichia coli ATCC 25922 and culture of yeast-like fungi Candida albicans ATCC 885-653. In addition, clinical strains of Staphylococcus aureus, Escherichia coli, and Candida albicans were used in the experiment.

One-day suspensions of bacterial microorganisms in physiological solution were used. The microbial load was 107 microbial cells in 1 mL of nutrient medium. Molten agar gel (bottom layer, V = 10 mL) was used as the non-inoculated medium for the Petri dishes mounted on a horizontal surface, and meat-peptone agar (V = 14–15 mL) was used as the inoculated medium. Sterile metal thin-walled cylinders (diameter—8.0 ± 0.1 mm, height—10.0 ± 0.1 mm) were used to form the holes. Prior to introduction into the wells, the test substances were dissolved in dimethyl sulfoxide (DMSO).

The results were recorded by measuring the growth retardation zone of microorganisms. Measurements were performed with an accuracy of 1 mm, focusing on the complete absence of visible growth. The value of the zone of growth retardation of pure DMSO was subtracted from the results of the analyses of the model samples. The antimicrobial activity of experimental samples was evaluated by the diameter of the growth retardation zone of microorganisms: sample; growth retardation zones with a diameter of 11–15 mm were evaluated as low sensitivity of the culture to the concentration of the active antimicrobial substance; zones of growth retardation with a diameter of 16–25 mm—as an indicator of the sensitivity of the strain of the microorganism to the test sample; the growth retardation zone, the diameter of which exceeded 25 mm, indicates a high sensitivity of microorganisms.

3. Results

3.1. Synthesis

Previous investigations of our scientific team [33] resulted in the creation of an effective one-step procedure towards novel 7-(4-(2-cyanoacetyl)piperazin-1-yl)-1-R-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acids 1a, 2a (Figure 2).

This reaction can proceed both in DMF solution and without it. However, the first option gave the target products with higher purity and better yield. Moreover, the two-step approach is possible utilizing cyanoacetic and CDI to synthesize intermediate N-(сyanoacetyl)imidazole for the reaction with starting fluoroquinolones. However, according to the obtained results, the one-step procedure depicted above appeared to be much more suitable.

Continuing this investigation, first, we synthesized substituted azides according to known methods [34]. Secondly, they were utilized under traditional reaction conditions with sodium methoxide as a catalyst in reactions with 7-(4-(2-cyanoacetyl)piperazin-1-yl)-1-R-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acids 1a, 2a (Figure 3).

According to the described procedure, novel norfloxacin and ciprofloxacin derivatives 1b,c, 2d-l were successfully obtained with 37–68% yield and their structures were confirmed by ¹H NMR, ¹³C NMR, ¹⁹F NMR, LC/MS, UV-, IR- spectroscopy (

Table 1. Melting points, elemental analyses, and yields for compounds 1b,c, 2d-l.

Compound Number	R2	M.p., °C	Molecular Formula, M. w.	Yield, %
1b	p-Me	250–255	C26H26FN7O4 519.54	65
1c	p-Br	300–304	C25H23BrFN7O4 523.49	58
2d	H	258–260	C26H24FN7O4 517.52	66
2e	o-Me	215–220	C27H26FN7O4 531.54	37
2f	p-Et	258–262	C28H28FN7O4 545.58	68
2g	p-OMe	260–262	C27H26FN7O5 547.55	43
2h	p-SMe	280–285	C27H26FN7O4S 563.62	49
2i	m-SMe	226–232	C27H26FN7O4S 563.62	57
2j	p-Br	275–280	C26H23BrFN7O4 596.42	61
2k	2-Me-5-F	278–282	C27H25F2N7O4 549.54	50
2l	p-CF3	222–228	C27H23F4N7O4 585.52	49

).

3.2. Docking Studies

Based on the results of the molecular docking, we calculated the scoring function, indicating the enthalpy contribution to the value of the free binding energy (Affinity DG) for the best conformation positions; the values of the free binding energy and binding constants (EDoc kcal/mol and Ki (uM micromolar) for a definite conformational position of the ligand, which allowed us to evaluate the stability of complexes formed between ligands and the corresponding targets (

Table 2. The values of affinity DG, free binding energy, and binding coefficients for the best conformational positions of the test compounds in combination with biotargets (PDB ID: 2XCR, 5BTL, 4KPF).

Molecule	2XCR			5BTL			4KPF
	Affinity DG, kcal/mol	EDoc kcal/mol	Ki µM Micromolar	Affinity DG, kcal/mol	EDoc kcal/mol	Ki µM Micromolar	Affinity DG, kcal/mol	EDoc kcal/mol	Ki µM Micromolar
1b	−9.4	−4.96	233.07 µM	−9.0	−4.90	255.07 µM	−9.5	−4.30	700.35 µM
1c	−9.4	−5.25	141.96 µM	−10.8	−5.08	187.55 µM	−9.1	−4.77	316.56 µM
2d	−9.8	−4.16	891.54 µM	−8.6	−5.24	144.69 µM	−9.3	−4.45	547.64 µM
2e	−9.6	−4.51	498.52 µM	−10.6	−6.51	16.79 µM	−9.6	−5.03	204.77 µM
2f	−10.4	−4.96	230.68 µM	−8.7	−5.06	195.51 µM	−9.1	−4.73	341.42 µM
2g	−9.8	−5.54	87.25 µM	−9.6	−4.41	581.77 µM	−9.2	−4.13	934.16 µM
2h	−9.1	−5.66	70.49 µM	−8.8	−4.90	256.42 µM	−9.2	−4.65	388.76 µM
2i	−9.3	−4.78	312.12 µM	−8.7	−5.32	125.18 µM	−9.0	−4.69	366.76 µM
2j	−9.6	−4.60	423.83 µM	−9.5	−5.16	165.25 µM	−8.4	−4.01	1150 µM
2k	−9.8	−5.05	197.24 µM	−9.4	−5.45	101.76 µM	−8.5	−4.00	1170 µM
2l	−9.9	−5.24	144.73 µM	−8.8	−5.92	45.87 µM	−9.7	−3.99	1191 µM
Ciprofloxacin	−7.2	−5.10	183.79 µM	−7.5	−5.51	91.69 µM	−7.4	−5.38	113.52 µM
Norfloxacin	−7.2	−4.30	708.28 µM	−7.8	−5.25	142.92 µM	−7.4	−4.78	315.73 µM

). The thermodynamic probability of such binding is confirmed by the negative values of the scoring functions.

3.3. Antibacterial Activity Evaluation

Investigation of the synthesized compounds antimicrobial activity at the first stage was performed in vitro against next test strains of bacterial and fungal cultures: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Candida albicans NCTC 885-653.

The method of double serial dilution was used in the study. Solutions of the synthesized compounds in DMF were prepared at a concentration of 1 mg/mL.

According to the obtained results, the compound 2d (Figure 4) showed a rather wide range of bactericidal activity against all tested test strains (MIC = 15.6 μg/mL). The activity of other compounds was at the control level (MIC = 125 μg/mL).

As a continuation of this research, we decided to check the synthesized molecule potency utilizing the diffusion in agar method and hospital strains. The results against reference and clinical strains of S. aureus and E. coli are represented in

Table 3. Activity of the synthesized compounds measured by the agar diffusion method.

Compound	Growth Retardation Zone, mm
	Reference Strains			Clinical Strains
	S. aureus АТСС 25923	E. coli АТСС 25922	C. albicans АТСС 885-653	S. aureus	E. coli	C. albicans
1b	34.3 ± 1.8 p = 0.0089	23.8 ± 1.6 p = 0.7521	30.1 ± 1.5 p = 0.0030	25.8 ± 1.4 p = 0.5903	18.7 ± 1.2 p = 0.0569	24.6 ± 1.5 p = 0.1984
1c	34.4 ± 1.4 p = 0.0044	40.4 ± 1.9 p = 0.0008	17.1 ± 1.8 p = 0.2443	27.9 ± 1.7 p = 0.2495	29.3 ± 1.5 p = 0.1185	14.7 ± 1.2 p = 0.0298
2d	28.8 ± 1.9 p = 0.2158	26.5 ± 1.8 p = 0.5553	25.1 ± 1.3 p = 0.0720	24.4 ± 1.7 p = 0.9736	22.3 ± 1.5 p = 0.4811	19.4 ± 1.4 p = 0.5773
2e	33.1 ± 1.8 p = 0.0171	33.4 ± 1.4 p = 0.0075	16.8 ± 1.3 p = 0.1367	27.6 ± 1.7 p = 0.2886	27.4 ± 1.7 p = 0.3458	18.2 ± 1.4 p = 0.3242
2f	36.1 ± 1.7 p = 0.0028	35.4 ± 1.7 p = 0.0038	15.8 ± 1.4 p = 0.0758	28.8 ± 1.7 p = 0.1574	26.3 ± 1.9 p = 0.5665	13.2 ± 1.3 p = 0.0123
2g	35.7 ± 1.8 p = 0.0042	35.4 ± 1.4 p = 0.0021	22.4 ± 1.3 p = 0.4254	26.4 ± 1.3 0.4416	29.6 ± 1.5 p = 0.1001	19.6 ± 1.8 p = 0.6688
2h	36.3 ± 2.6 p = 0.0108	29.3 ± 1.4 p = 0.1077	20.7 ± 1.9 p = 0.9454	30.3 ± 1.6 p = 0.0622	22.3 ± 1.7 p = 0.5044	18.7 ± 1.6 p = 0.4457
2i	29.2 ± 1.5 p = 0.1121	32.2 ± 1.3 p = 0.0140	16.6 ± 2.2 p = 0.2446	25.3 ± 1.6 p = 0.7338	20.1 ± 1.4 p = 0.1547	11.5 ± 1.2 p = 0.0033
2j	35.7 ± 1.67 p = 0.0034	28.9 ± 1.7 p = 0.1726	16.3 ± 1.7 p = 0.1150	29.2 ± 1.8 p = 0.1372	21.2 ± 1.6 p = 0.2786	13.1 ± 1.3 p = 0.0115
2k	33.8 ± 1.4 p = 0.0064	42.7 ± 2.1 p = 0.00001	18.3 ± 1.6 p = 0.4096	27.3 ± 1.3 p = 0.2790	33.4 ± 1.3 p = 0.0079	16.4 ± 1.7 p = 0.1466
2l	17.5 ± 1.8 p = 0.0572	32.3 ± 1.6 p = 0.0207	18.6 ± 1.8 p = 0.5051	14.1 ± 1.4 p = 0.0036	21.8 ± 1.6 p = 0.3987	13.1 ± 1.1 p = 0.0083
Control	24.2 ± 1.8	24.7 ± 1.6	20.5 ± 1.34	24.3 ± 1.7	24.4 ± 1.8	20.9 ± 1.6

Note. р—level of significance of differences from control (Student’s t-test for independent variables).

. Growth retardation zone exceeded 25 mm, which corresponds to the high sensitivity of microorganisms towards the tested compounds.

4. Discussion

Existing fluoroquinolones possess several positions in their structures that can be easily modified from the chemical viewpoint. Such modifications lead to changes in pharmacokinetics and pharmacodynamics of the starting molecules, as previously shown by authors [4,5,6].

We introduced a substituent (nitrogen based heterocyclic 1,2,3-triazole moiety) at C-7 of norfloxacin and ciprofloxacin, this position is very important for the interaction with enzyme DNA gyrase and, therefore, for the biological activity of the molecule according to the structure–activity relationship studies [13].

Moreover, in vitro investigations of antibacterial activity of earlier synthesized 7-(4-(2-cyanoacetyl)piperazin-1-yl)-1-R-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acids revealed the potency of these scaffolds. Together with docking studies, such results led us to the idea of further research in this direction, namely, inserting of heterocyclic moiety at C-7 of norfloxacin and ciprofloxacin.

The synthetic approach was chosen because of the suitability of ‘click chemistry’ concept particularly for substituted 1,2,3-triazoles synthesis [35] and resulted in target 7-(4-(5-amino-1-R-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)-1-R-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acids with moderate yields.

As can be seen from the obtained docking results, scoring functions do not always correlate with the values of free energies and binding constants. Among the tested molecules, the leader compound with respect to Staphylococcus aureus PDB gyrase DNA ID: 2XCR is molecule 2h with EDoc values of −5.66 kcal/mol, Affinity DG −9.1 kcal/mol, Ki 70.49 µM micromolar. The leader in relation to topoisomerase II Mycobacterium tuberculosis PDB ID: 5BTL is molecule 2l with EDoc values of −5.92 kcal/mol, Affinity DG −8.8 kcal/mol, Ki 45.87 µM micromolar. Prior to Streptococcus pneumoniae topoisomerase IV, the most active compound is molecule 2f with EDoc values of −4.73 kcal/mol, Affinity DG −9.1 kcal/mol, Ki 341.42 µM micromolar. According to the results, it was also found that in all of the calculated values of the studied molecules exceed the reference medicine ciprofloxacin (Table 2).

Altogether, the improvement of affinity, according to the docking studies, can be obtained by the preservation of the fluorine atom in the sixth position and the piperazine system in the seventh position with subsequent substitution by the 1,2,3-triazole ring, additional saturation of molecules, with both donor and acceptor substituents in the aryl molecular moiety.

The next stage of molecular docking was a detailed analysis of the geometric location of the compounds in the active sites of selected topoisomerases, in order to provide a full understanding of the molecules fragments that are involved in binding to biotargets, to get clear guidelines for the rational design of future antibacterials.

The bond between the fluorine atom at the 6-position of the quinoline framework and the amino acid residue of glutamine Glu561 promotes the formation of the complex of molecule 2h with the DNA gyrase of Staphylococcus aureus. The glutamine residue additionally stabilizes the complex with the quinoline cycle due to the π-anionic interaction. The formation of hydrogen bonds is facilitated by the presence of oxygen and hydrogen atoms of the carboxyl group in the 3-position of the quinoline cycle and the amino acid residues of arginine Arg517, glycine Gln541, and alanine Ala540, respectively. The aspartic acid residue Asp643 forms a π-amide bond with the aryl moiety. The quinoline framework is also involved in the formation of the π–π bond with the Tyr557 tyrosine fragment. Alkyl interactions contribute to additional stabilization of the complex among the methyl sulfonyl substituent, phenyl moiety, and lysine residue Lys564 (Figure 5).

The formation of molecule 2l complex with Mycobacterium tuberculosis topoisomerase II is facilitated by hydrogen bonds that occur between the oxygen atoms of the carboxyl group in position 3, and the carbonyl group in position 4 of the quinoline framework, with arginine residues Arg98 and lysine Lys49, respectively. Hydrogen bonds are also formed between the carbonyl linker, which binds 1,2,3-triazole and piperazine rings, the Nitrogen atom of the 1,2,3-triazole moiety to the amino acid residues histidine His85, serine Ser91, His87, and Asp586, respectively (Figure 6).

Fluorine atoms form hydrogen and halogen bonds with amino acid residues Lys484, Asp 586, Glu459. Hydrocarbon bond and Alk interaction occur due to the piperazine ring and serine residues Ser91 and valine Val51. π-Anionic and π–π interactions are formed in the presence of 1,2,3-triazole and phenyl nuclei with His87.

The complex of molecule 2f with topoisomerase IV Streptococcus pneumoniae is formed with the participation of the fluorine atom in the 6-position of the quinoline cycle and the glycine residue Gln90 in the form of a halogen bond. Hydrogen bonds are formed in the presence of oxygen atoms of the carboxyl group and carbonyl in the third and fourth positions of the quinoline ring with amino acid residues Asn264, Ser107, Trp92. Hydrogen bonding is also observed between the proton of the amino group of the 1,2,3-triazole ring with a serine fragment of Ser80. Hydrocarbon binding occurs between the piperazine ring and the aspartic acid fragment Asp93.

Intermolecular π-cationic and π-σ interactions are formed between the quinoline framework and the phenyl fragment with Lys93 and Val40 residues. Π-Alk and Alk interactions between the alkyl group and the 1,2,3-triazole ring with Ala84, Val40, Arg28, His74, and Pro99 residues contribute to additional stabilization (Figure 7).

Given the detailed analysis of the geometric location in the binding sites, the formations between them of a number of intermolecular interactions, negative values of scoring functions, free binding energy, and calculated values of binding constants, we conclude that the studied molecules have affinity to selected biotargets (PDB ID: 2XCR, 5BTL, 4KPF).

This fact was proven by the antibacterial activity investigations, which have shown high sensitivity of microorganisms toward the tested compounds.

5. Conclusions

Novel ciprofloxacin and norfloxacin hybrid derivatives were synthesized and their biological activities were investigated in silico and in vitro. The structure and purity of the obtained compounds were confirmed by ¹H NMR, ¹³C NMR, ¹⁹F NMR, LC/MS, UV-, and IR- spectroscopy.

The molecular docking studies for the obtained hybrid compounds showed affinity on the level of ciprofloxacin and norfloxacin. The obtained data contributed to the rational design of novel antibacterials, namely the important structural moieties were revealed:

• Quinoline heterocycle and a fluorine atom in position 6;
• Carbonyl and carboxyl fragments in the third and fourth positions of quinoline, which are involved in additional stabilization of the target molecule complex through the Mg²⁺ cation;
• Substitution in the seventh position of the quinoline framework by pharmacophores of heterocyclic and aromatic structures (triazole, piperazine, and phenyl fragments);
• Additional saturation of molecules with donor and acceptor substituents in the aromatic inclusions of the designed molecules also enhance the activity.

The antibacterial activity research showed antimicrobial and antifungal activities at the reference level for the double dilution method and exceeded control for the agar well diffusion method.

The obtained results appeared to be prospective in the course of new antimicrobial creation.