Synthesis, Characterization and Pharmacological Studies of Some Substituted Fluoroquinolones

Introduction

The first clinically useful quinolone was naldixic acid, discovered by Lesher and co-workers in 1962, which was generated from chloroquine, an antimalarial agent (1). It was active against some Gram negative bacteria and had limited usefulness because of its high protein binding (approximately 90%) and little half life (about 1.5 h) (6). Unfortunately, bacteria could develop a rapid resistance to this agent (7, 11). Flumequine was the first fluoroquinolone which was patented in 1973, after that many fluoroquinolones have been patented and are still used today, including norfloxacin (1978), pefloxacin (1979), enoxacin (1980), fleroxacin (1981), ciprofloxacin (1981) and ofloxacin (1982) (1). An advantage of these compounds over previous ones is their broad spectrum. A big revolution was made in 1980 ís when an analog of naldixic acid, enoxacin was derived with significantly increased spectrum of activity against Gram negative or Gram positive bacteria (8). An antioxidant is a molecule capable of inhibiting or preventing the oxidation of other molecules. Antioxidants are substances that may protect cells from the damaging effects of oxygen radicals, highly reactive chemicals that play a part in atherosclerosis, some forms of cancer and reperfusion injuries (4). Free radicals are created when cells use oxygen to generate energy. These by-products are generally reactive oxygen species (ROS) such as super oxide anion, hydroxyl radical and hydrogen peroxide that result from the cellular redox process. At low or moderate concentrations, (ROS) exert beneficial effects on cellular responses and immune function but at high levels, free radicals and oxidants generate oxidative stress, a deleterious process that can damage cell structures, including lipids, proteins, and DNA (9). Oxidative stress plays a major part in the development of chronic and degenerative ailments such as cancer, autoimmune disorders, rheumatoid arthritis, cataract, aging, cardiovascular and neurodegenerative diseases, (14, 9). The human body has several mechanisms to counteract oxidative stress by producing antioxidants, which are either naturally produced in situ, or externally supplied through foods and/or supplements. These antioxidants act as free radical scavengers by preventing and repairing damages caused by ROS, and therefore can enhance the immune defense and lower the risk of cancer and degenerative diseases (9). In recent years, there is an increasing interest in finding antioxidant phytochemicals, because they can inhibit the propagation of free radical reactions, protect the human body from diseases (12).

Methodology 

Synthesis of derivative FI-ETH Equimolar amounts of 3-chloro-4-fluoroaniline (i), and diethylethoxymethylene malonate (ii) were condensed at 145⁰C to get 3-chloro-4-fluoroanilinomethylene malonic diethyl ester (iii), which was then cyclized by heating at 250⁰C in diphenyl ether to get ethyl 7-chloro-6-fluoro-4-oxo1, 4-dihydroquinoloine-3-carboxylate (iv).   Whole reaction mixture change into the semisolid mass having white to pale yellow appearance and washed with acetone to get almost white solid recrystillised using DMF as solvent. Showed in scheme 1 fig. I.

Synthesized derivatives (FII-SUL, FIII-ATY, FIV-BZO, and FV-BZY) are synthesized by this following method. ethyl 7-chloro-6-fluoro-4-oxo1, 4-dihydroquinoloine-3-carboxylate (iv) 0.01 mol was added to 10 ml. of DMF, followed by addition of N-1 position (R) (0.01 mol). The reaction mixture was heated to dissolve the 3-acid which was partially soluble in cold condition, and then anhydrous potassium carbonate (0.02 mol) was added to the reaction mixture. The whole reaction mixture was heated to 120⁰ – 140⁰C and stirred for 5-8 hrs. then, the reaction mixture was poured onto the crushed ice or ice cold water, washed with cold water to remove DMF and potassium carbonate if any, the solid (v) obtained was recrystillised from acetone to get the derivative. Showed in scheme 2 fig. II. All synthesized derivatives checked by TLC technique using adsorbent silica gel G of Merck specialties Pvt. Ltd., Mumbai and each derivatives are isolate & purified by column chromatography using silica gel mesh size 60-120 of Merck specialties Pvt. Ltd., Mumbai.

In-vitro antibacterial activity (5)

Agar- Well Diffusion Method: Petriplates containing 20 ml Muller Hinton medium were seeded with 24 hr culture of bacterial strains. Wells were cut and 20 μl of the given sample (of different concentrations) were added. The plates were then incubated at 37°C for 24 hours. The antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the well. Ofloxacin were used as a positive control.

In-vitro antioxidant activity by

DPPH Free Radical Scavenging Assay (2, 10)

The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free radical-scavenging activity of the sample Different concentrations (50 – 250 µg/ml) of each sample were added, at an equal volume, to 90% methanolic solution of DPPH (100 μM). After 15 min at room temperature, the absorbance was recorded at 517 nm. The experiment was repeated for three times. Ascorbic acid were used as standard controls. IC50 values denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals.

 Hydrogen Peroxide Radical Scavenging Activity (4)

The ability of the sample (FI-ETH, FII-SUL, FIII-ATY, FIV-BZO, and FV-BZY) to scavenge hydrogen peroxide was determined. A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). The concentration of hydrogen peroxide was determined by absorption at 230 nm using a spectrophotometer. Sample (50 – 250 µg/ml) in distilled water, and 1 ml added to  2.4 ml. of 0.1 M phosphate buffer solution and a hydrogen peroxide solution (600 µl, 40 mM). The absorbance of hydrogen peroxide at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage of hydrogen peroxide scavenging by the extracts and standard compounds was calculated as follows: % Scavenged [H₂O₂] = [(Ao − A1)/Ao] × 100 where Ao was the absorbance of the control and A1 was the absorbance in the presence of the sample and standard.

Nitric Oxide Assay (13)

The procedure is based on the principle that, sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (5 mM), in phosphate-buffered saline, was mixed with different concentrations (50 – 250 µg/ml) of each sample dissolved in methanol and incubated at room temperature for 30 min. After the incubation period, 1.5 ml incubated solution was removed and diluted with 1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% N-1-naphthyethylenediamine dihydrochloride) was added. The absorbance of the chromophore formed was read at 546 nm. Rutin was used as standard.

Reducing Power Assay (3)

The reducing power assay of sample was determined. Different amounts of each sample (50 -800 μg/ ml) in 1 ml of methanol were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferrocyanide [K₃Fe(CN)₆] (2.5 ml, 1%). The mixture was incubated at 50^oC for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture to stop the reaction, which was then centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃

(0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid used as a standard.

Spectral Analysis

All synthesized derivatives done characterized by ¹H NMR, MASS, and FT-IR.

FI-ETH: FT-IR (cm^-1)

1445 (C-N stretch of quinolines having C=C-N), 1044 (C-F), 751 (C-Cl), 1650 (C=C-C=O), 1733 (C=C-COOR), 1618 (N-H bending).

MS (m/z)

269 (M⁺) C₁₂H₉ClFNO₃ calculated value: C= 53.53, H= 3.35, Cl= 13.20, F= 7.05, N= 5.2, O= 17.84; 270 (M⁺) Found value: C= 53.33, H= 3.33, Cl= 13.14, F= 7.03, N= 5.18, O= 17.77.

⁺H NMR (dmso d₆ 300 MHz) δ ppm= 7.06 (1H, s, J= 7.021), 7.46 (1H, s, J= 7.424), 4.15 (1H, s, J= 4.133), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J= 4.353), 8.0 (1H, s, J= 7.954).

FII-SUL: FT-IR (cm^-1)

1450 (C-N stretch of quinolines C=C-N), 1044 (C-F), 637 (C-Cl), 1635 (C=C-C=O), 1733 (C=C-COOR), 3325 (aromatic C-H), 2972 (C-H alkane), 1380 (SO₂ Symmetric), 1274 (SO₂ Asymmetric).

MS (m/z)

423 (M⁺), C₁₉H₁₅ClFNO₅S calculated value: C= 53.90, H= 3.54, Cl= 8.39, F= 4.49, N= 3.30, O= 18.91, S= 7.5; 422 (M⁺) Found value: C= 53.77, H= 3.53, Cl= 8.37, F= 4.48, N= 3.30, O= 18.86, S= 7.56.

⁺H NMR (dmso d₆ 300 MHz) δ ppm= 7.06 (1H, s, J= 7.021), 7.46 (1H, s, J= 7.424), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J= 4.353), 8.0 (1H, s, J= 7.954), 7.3 (2H, m, J= 7.414), 7.71 (2H, m, J= 7.734), 2.28 (3H, m, J= 2.281).

FIII-ATY: FT-IR (cm^-1)

1425 (C-N stretch of quinolines C=C-N), 1044 (C-F), 659 (C-Cl), 1649 (C=C-C=O), 1733 (C=C-COOR), 2973 (C-H alkane).

MS (m/z)

311 (M⁺), C₁₄H₁₁ClFNO₄ calculated value: C= 54.01, H= 3.5, Cl= 11.41, F= 6.1, N= 4.5, O= 20.5; 311 (M⁺) Found value: C= 54.01, H= 3.5, Cl= 11.41, F= 6.1, N= 4.5, O= 20.5.

⁺H NMR (dmso d₆ 300 MHz) δ ppm= 7.83 (1H, m, J= 7.844), 7.54 (1H, m, J= 7.615), 8.75 (1H, s, J= 8.728), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J= 4.353), 2.27 (3H, s, J= 2.299).

FIV-BZO: FT-IR (cm^-1)

1466 (C-N stretch of quinolines C=C-N), 1030 (C-F), 618 (C-Cl), 1690 (C=C-C=O), 3099 (aromatic C-H).

MS (m/z)

373 (M⁺) C₁₉H₁₃ClFNO₃ calculated value: C= 61.12, H= 3.48, Cl= 9.51, F= 5.09, N= 3.75, O= 17.15; 373 (M⁺) Found value: C= 61.12, H= 3.48, Cl= 9.51, F= 5.09, N= 3.75, O= 17.15.

⁺H NMR (dmso d₆ 300 MHz) δ ppm= 7.83 (1H, m, J= 7.844), 7.54 (1H, m, J= 7.615), 8.75 (1H, s, J= 8.728), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J= 4.353), 1.13 (3H, m, J= 1.056), 4.4 (2H, m, J= 4.353), 7.79 (2H, m, J= 7.812), 7.34 (2H, m, J= 7.386), 7.57 (1H, s, J= 7.601).

FV-BZY: FT-IR (cm^-1)

1494 (C-N stretch quinolines having C=C-N), 881 (C-F), 658 (C-Cl), 1655 (C=C-C=O), 3423 (Aromatic C-H), 2969 (C-H alkane).

MS (m/z)

359 (M⁺) C₁₉H₁₅ClFNO₃ calculated value: C= 63.50, H= 4.17, Cl= 9.88, F= 5.29, N= 3.89, O= 13.37; 359 (M⁺) Found value: C= 63.50, H= 4.17, Cl= 9.88, F= 5.29, N= 3.89, O= 13.37.

⁺H NMR (dmso d₆ 300 MHz) δ ppm= 7.23 (1H, s, J= 7.299), 7.97 (1H, m, J= 7.973), 4.26 (2H, m, J= 4.253).

Figure 1: Scheme 1: Click here to View figure

 

Figure 2: Scheme 2: Click here to View figure

 

Table 1: Physico-chemical properties of the Derivatives

Derivatives	Substituents R	Melting Point	Yield (%)	Rf	Solvent System
FI-ETH	H	2500-2530C	80	0.64	Ethyl Acetate:Benzene:Acetone 5:2:2
FII-SUL	C7H7ClO2S	2610-2660C	70	0.9	Ethyl Acetate:Benzene:Acetone 4:2:2
FIII-ATY	C2H3ClO	2430-2480C	67	0.82	Ethyl Acetate:Benzene:Acetone 5:2.5:2
FIV-BZO	C7H5ClO	2560-2600C	65	0.64	Ethyl Acetate:Chloroform:Acetone 5:2:2
FV-BZY	C7H7Cl	2570-2610C	62	0.48	Ethyl Acetate:Hexane 4:1

---

This work is licensed under a Creative Commons Attribution 4.0 International License.