IJCRR

IJCRR - 13(21), November, 2021

Pages: 65-72

Date of Publication: 09-Nov-2021

Synthesis and in vitro Antibacterial, Antitubercular and Cytotoxicity Evaluation of Lomefloxacin Derivatives

Author: Gurunani Gulshan, Agrawal Kapil, Walde Sheelpriya, Ittadwar Abhay

Category: Healthcare

Abstract:Introduction: The fluoroquinolones antibacterial agents are one of the fastest growing groups of drugs in recent years. The various side chains on it can be altered and the resulting analogues are evaluated for their anti-microbial and antitubercular properties. Most of these agents are substituted at the 7 positions by nitrogen heterocycles. Lomefloxacin at C-7, which represents a site amenable to significant modification. Objective: Based on evidence of research results and in search of new bioactive molecules in the fluoroquinolones, a of Nsubstituted piperazinyl quinolones have been designed, synthesized, characterized and evaluated for their antibacterial activity and antitubercular activity. Method: A series of 2-((5-chloro-1, 3, 4-thiadiazol-2yl) thio)-1-(4-subs.) ethanone (4a\?4j)were prepared by diazotization of amines (3a-3j) in concentrated HCl in the presence of Cu-powder. The reaction of(4a-4j) with piperazinyl quinolone (lomefloxacin) in DMF yield (5a-5j). The synthesized compounds were evaluated against some Gram-positive and Gram-negative bacterias and antitubercular activity against Mtb WT H37Rv. Result: The structure of the synthesized compound was confirmed by their IR, 1HNMR, data. The antibacterial data revealed that all substituted derivatives (5a to 5j), are found to be least active against Gram-positive and Gram-negative organisms. Among all of the tested compounds,5b(Lomefloxacin derivative) exhibited excellent antitubercular activity against Mtb WT H37Rv (MIC0.8 µg/ml) which is comparable to that of standard. (MIC 0.8 µg/ml) Conclusion: Although the nature of the C-7 substituent is known to enhance quinolone activity in bacteria but results of the present study reveal that the synthesized derivative shows significant antitubercular property but poor antibacterial activity.

Keywords: Antibacterial activity, Antitubercular activity, Fluoroquinolone, Lomefloxacin, N-piperazinyl quinolone, Synthesis

Full Text:

Introduction

Fluoroquinolones, a major class of antibiotics, are under clinical development. The antibacterial activity of Fluoroquinolones is due to the inhibition of bacterial enzymes; DNA-gyrase and topoisomerase IV. They have potent activity, rapid bactericidal effects, and a low prevalence of resistance development.¹The fluoroquinolones exert certain adverse effects, have restricted activity against Grampositivepathogens and methicillin-resistant Staphylococcus aureus (MRSA).²Therefore, there is a need of synthesizing novel quinolones with better activity profile, pharmacokinetics, and acceptability, to overcome the limitations of existing drugs.³ Most of the quinolone antibacterial research has been focused on substitution at the C-7  as it is the most adaptable site for chemical change.C-7 position is an area that determines potency and target preference and also controls the pharmacokinetic properties of the drugs, with basic nitrogen.^4-6The most commonly found substitution at the C-7 position is a five- or six-membered ring. For example, aminopyrrolidine substituent at C-7 in trovafloxacinandgemifloxacinandPiperazine substitution at the C-7 position in norfloxacin, ciprofloxacin, pefloxacin, pefloxacin, ofloxacin, amifloxacin, fleroxacin, lomefloxacin, sparfloxacin, difloxacin, enoxacin, enrofloxacin, levofloxacin, marbofloxacin, and orbifloxacin which has triggered a wide range of clinically useful fluoroquinolone antibacterial agents.^7-17(Figure 1) The site near the C-7 substituent is regarded as the domain for drug–enzyme interaction and the cell permeability.^18-21 The piperazine moiety of 7-piperazinyl quinolones possesses enough structural flexibility to allow product optimization. In the present study, we have aimed to achieve a better antimicrobial profile at a lower concentration, by preparing [(7-(4--(5-substituted-benzoylthio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazine-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid](5a to 5j) derivatives and have been evaluated for its in vitro anti-bacterial and anti-tubercular effect. (Figure 2).

Materials and Methods

Materials

All the chemicals, reagents, and solvents used in this research were bought from E Merck Ltd, Loba chemicals Ltd, Sigma-Aldrich Ltd., Spectrochem Ltd., Hi-media, and Rankem Chemicals Ltd. Mumbai, India. Solvents used were dried and purified as and when required. The melting points reported were uncorrected and were determined in open capillaries using Thiele's melting point apparatus and measured in (°C). The yields of synthesized compounds were mentioned in tables along with respective physical constants. The FT-IR spectra were obtained Shimadzu FTIR spectrophotometer and values were measured in cm⁻¹(potassium bromide disks). ¹HNMR and ¹³C NMR were recorded at 400MHz and 100MHz respectively on a Bruker AM spectrometer, IISc Bangalore, and chemical shifts are expressed as δ (ppm) with tetramethylsilane as an internal standard. The FAB / EIMS mass spectra were recorded on Autospec Mass spectrometer, IICT, Hyderabad.

Methods

General Procedure for Synthesis of 2(a–j) (Figure 3)

Synthesis of substituted/unsubstituted phenacyl bromide 2(a–j)

0.1 mol of substituted/un-substituted acetophenones 1(a–j) were taken in the two-necked round bottom flask, suitable anhydrous solvents (ether, acetone, methanol, chloroform) was added with anhydrous AlCl3. The reaction condition was kept up either in cold or at room temperature and bromine (0.09mol) was added with stirring. Mixtures 2(a–j) were acquired as colourless to brown to shining crystals. The product was washed twice with appropriate solvents and recrystallized from methanol to get lachrymatory crystals.^22-23 Liquefying point ranges of 2a–j; R = H, Cl, Br, F, NO2, CH3, OCH3,  NH2, OH, C6H5; 48-50°, 90–92°, 110–112°, 46-48, 96–98°, 52–54°,  72–74°, 80–84°, 102-104, 98-102 respectively (50–74 %).

General Procedure for Synthesis of3(a–j). (Figure 3)

Synthesis of 2-((amino-1,3,4-thiadiazol-2yl)thio)-1-(4-subst.) ethanone  3 (a–j).

The 2-amino-5- mercapto-1,3,4-thiadiazole (0.1mol) was suspended in 15 ml of water and 80% potassium hydroxide (0.1 mol) was added. This solution was de-colorized with activated charcoal, followed by the addition of 32 mL of ethanol and stirred rapidly with 2 (a–j) (0.1mol). The reaction mixture was cooled for 40 minutes and it added 200 mL of cold water. It is then filtered to obtain the solid product and washed with ether and water. The 3(a–j) were obtained (Scheme 1), with 54–68% yield and melting point (80–108^oC). ^24-25

General Procedure for Synthesis of4(a–j). (Figure 3)

Procedure for Synthesis of 2-((5-chloro-1, 3, 4-thiadiazol-2yl) thio)-1-(4-subs.)ethanone4 (a–j)

Triturated 2-((amino-1,3,4-thiadiazol-2yl) thio)-1-(4-subs.) ethanone  3(a–j) (30 mmol) with sodium nitrite (60 mmol). The triturate was introduced in the ice-cooled (0–5^oC) mixture of 15 ml water and 30 ml concentrated HCl with stirring in the presence of copper powder. The product was refluxed for 1 hour at 75⁰C and cool. Then the mixture was extracted thrice with dry chloroform (75 ml). The combined extracts of chloroform were washed with a sodium bicarbonate solution. Then the solution was dried over sodium sulphate followed by evaporation under reduced pressure. Finally,  recrystallization of the product was done using ethanol to yield 2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subst) ethanone 4 (a–j) (Scheme 1). The compound was purified by column chromatography with methanol: chloroform (1:9) as mobile phase²⁶, m.p. 85–110^oC (48–60%).

General Procedure for Synthesis of 5(a–j). (Figure 3)

Synthesis of 1-subst.-6-fluoro-8-subst.-7-(3-subst.-4-(5-subst.((2-oxo-2-(p-subst.)ethyl)thio)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid  5(a–j).

A combination of equimolar quantities of compound 2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subs.)ethanone4(a–j) and piperazinyl fluoroquinolone (sparfloxacin), along with sodium-bicarbonate in 10 ml dimethyl-formamide was refluxed on an oil bath at 140–160^oC for hrs. After cooling the reaction mixture, 10ml of cold water was added to it. The precipitated product was filtered and washed with water. The product was then subjected to recrystallization using a blend of dimethylformamide and water to yield (5a-j) compounds.^27-28 (Scheme 1). The Physicochemical results are shown in (Table 1)

Antibacterial Activity

Preliminary in vitro antibacterial activity was employed by the broth micro-dilution technique.  Antibacterial Activity was examined against two Gram-negative microorganisms, Pseudomonas aeruginosa and Escherichia coli, and two Gram-positive microorganisms, Staphylococcus aureus and Bacillus subtilis. The test compounds and reference drugs (Sparfloxacin and Rifampicin) were prepared in Mueller-Hinton agar medium by two-fold serial dilutions. The required concentrations of 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 µg/ml was obtain by Progressive double dilutions with agar.  The Petri plates were inoculated with 1–5 × 10⁴ colonies forming units (CFU/ml) and incubated at 37⁰C for 18 hours.²⁹The results are presented in (Table 2).

Anti-tubercular Activity

In vitro screening for anti-mycobacterial was performed by utilizing M. tuberculosis virulent H37Rv strain. The broth dilution assay for each drug for determination of  MIC was determined by using the frozen culture of Middlebrook 7H9 broth supplemented with 10% ADC (albumin dextrose catalase) and 0.2% glycerol. It is used as inoculum with dilution in broth to 2 × 10⁵CFU/ml. In the assay, for the accommodation of compounds U-tubes were used in 0.1, 0.5, 1.5, 2.5, 05, 7.5, 10, 12.5, 15, 17.5 and 20 mg/ml dilutions.^30-31 The results are presented in (Table 2).

In-vitro cytotoxic study

Estimation of cell viability

Conversion of MTT [(3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrasodium bromide)] to dark blue formazan crystals due to the presence of living cells, was employed for estimation of cell viability. Colourimetric analysis was used for the estimation of MTT cleaved to the viable cells.The solution of compounds under investigation in DMSO was diluted to achieve test concentrations. The DMSO content was maintained below 0.1% in all the aliquots under investigation. The cultured Hep-G2 normal liver-cell lines were added in plates with 96 wells and then preserved with variable dilutions of investigational compounds in DMSO, at 37^oC in a carbon dioxide incubator for four days. Further, the MTT reagent was instilled into the wells and incubated for four hours, and then the dark blue formazan developed was allowed to dissolve in DMSO and the colourimetric absorbance was read at 550 nm. The IC₅₀value was estimated by graph plotted between percentage cells inhibited versus concentrations.³² The findings are provided in (Table 2).

Results

Spectral Data of synthesized compound

1-ethyl-6,8-difluoro-7-(3-methyl-4-(5-((2-oxo-2-phenylethyl)thio)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5a)

IR (KBr) cm^-1: 3422(carboxylic, O-H str.), 2943(Ar. C-H str.), 2856(Ali. CH_2, C-H str.), 1716(carboxylic, C=O str.), 1642(ketonic, C=O str.), 1588(Imine, C=N str.), 1320(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.52(s, carboxylic, 1H, OH), 9.02(s, 1H, H2-quinoline), 7.56-7.94(m, 5H, Ar.), 7.58(s, 1H, H5-quinoline), 4.92(s, 2H, CH₂), 4.64(q, 2H, NCH₂CH₃), 2.92-3.50(m, 7H, piperazinyl), 1.38(t, 3H, NCH₂CH₃), 1.29 (s, 3H, piperazinyl CH₃). 

7-(4-(5-((2-(4-chlorophenyl)-2-oxoethyl)thio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5b)

IR (KBr) cm^-1: 3445(carboxylic, O-H str.), 3005(Ar. C–H str.), 2852(Ali. CH_2, C-H str.), 1725(carboxylic, C=O str.), 1656(ketonic, C=O str.), 1583(Imine, C=N str.), 1307(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.55(s, carboxylic, 1H, OH), 8.91(s, 1H, H2-quinoline), 7.60-7.92(m, 4H, Ar.), 7.42(s, 1H, H5-quinoline), 4.84(s, 2H, CH₂), 4.57(q, 2H, NCH₂CH₃), 2.94-3.47(m, 7H, piperazinyl), 1.46(t, 3H, NCH₂CH₃), 1.33(s, 3H, piperazinyl CH₃); ¹³C-NMR (DMSO-?6) ?ppm: 198, 180, 161, 158, 148, 144, 130, 118, 110, 68, 40, 18; MS: m/z = 619 [M⁺];CHN calcd;C₂₇H₂₄ClF₂N₅O₄S₂;C, 52.30; H, 3.90; N, 11.29; Found C, 52.34; H, 3.90; N, 11.30.

7-(4-(5-((2-(4-bromophenyl)-2-oxoethyl)thio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5c)

IR (KBr) cm^-1: 3444(carboxylic, O-H str.), 3009(Ar. C-H str.), 2850(Ali. CH_2, C-H str.), 1728(carboxylic, C=O str.),  1625(ketonic, C=O str.), 1554(Imine,  C=N str.), 1310(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.58(s, carboxylic, 1H, OH), 8.93(s, 1H, H2-quinoline), 7.61-7.94(m, 4H, Ar.), 7.44(s, 1H, H5-quinoline), 4.80(s, 2H, CH₂), 4.59(q, 2H, NCH₂CH₃), 2.93-3.48(m, 7H, piperazinyl), 1.48(t, 3H, NCH₂CH₃), 1.31(s, 3H, piperazinyl, CH₃); ¹³C-NMR (DMSO-?6) ?ppm: 199, 181, 165, 160, 149, 128, 120, 108, 70, 42, 17.

5.4 7-(4-(5-((2-(4-fluorophenyl)-2-oxoethyl)thio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5d)

IR (KBr) cm^-1: 3454(carboxylic, O-H str.), 2926(Ar. C-H str.), 2853(Ali. CH_2, C-H str.), 1726(carboxylic, C=O str.),  1658(ketonic, C=O str.), 1584(Imine, C=N str.), 1327(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.68(s, carboxylic, 1H, OH), 8.94(s, 1H, H2-quinoline), 7.98-8.02(m, 4H, Ar.), 7.78(s, 1H, H5-quinoline), 4.59(s, 2H, CH₂), 4.42(q, 2H, NCH₂CH₃), 2.50-3.50(m, 7H, piperazinyl), 1.48(t, 3H, NCH₂CH₃), 1.25(s, 3H, piperazinyl CH₃); ¹³C-NMR(DMSO-?6) ?ppm: 197, 173, 148, 133, 117, 107, 98, 74, 48, 38, 37; MS: m/z = 602 [M⁺]; CHN calcd; C₂₇H₂₄F₃N₅O₄S₂; C, 53.72; H, 4.01; N, 11.60; Found C, 54.12; H, 3.98; N, 11.94.

7-(4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5e)

IR (KBr) cm^-1: 3441(s, carboxylic, O-H str.), 3006(Ar. C-H str.), 1374(Ali. CH_2, C-H str.), 1728(carboxylic, C=O str.), 1624(ketonic, C=O str.), 1547(Imine, C=N str.), 1311(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.56(s, 1H, carboxylic, OH), 8.92(s, 1H, H2-quinoline), 7.74-7.92(m, 4H, Ar.), 7.47(s, 1H, H5-quinoline), 4.83(s, 2H, CH₂), 4.65(q, 2H, NCH₂CH₃), 2.92-3.47(m, 7H, piperazinyl), 1.47(t, 3H, NCH₂CH₃), 1.38(s, 3H, piperazinyl CH₃); ¹³C-NMR (DMSO-?6) ?ppm: 192, 183, 165, 155, 147, 138, 129, 113, 74, 47, 11; MS : m/z = 630 [M⁺];CHN calcd;C₂₇H₂₄F₂N₆O₆S₂;C, 51.42; H, 3.84; N, 13.33; Found C, 51.40; H, 3.85; N, 13.30.

1-ethyl-6,8-difluoro-7-(3-methyl-4-(5-((2-oxo-2-(p-tolyl)ethyl)thio)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5f)

IR (KBr) cm^-1: 3427(s, carboxylic, O-H str.), 2934(Ar. C-H str.), 2854(Ali. CH_2, C-H str.), 1710(carboxylic, C=O str.),  1623(ketonic, C=O str.), 1576(Imine, C=N str.), 1298(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.06(s, 1H, carboxylic, OH), 8.84(s, 1H, H2-quinoline), 7.54-7.68(m, 4H, Ar.), 7.38(s, 1H, H5-quinoline), 4.64(s, 2H, CH₂), 4.32(q, 2H, NCH₂CH₃), 2.83-3.27(m, 7H, piperazinyl), 2.37(s, 3H, tolyl), 1.32(t, 3H, NCH₂CH₃), 1.19(s, 3H, piperazinyl CH₃); ¹³C- NMR (DMSO-?6) ?ppm: 188, 179, 158, 147, 135, 128, 116, 108, 68, 35, 16, 7.

7-(4-(5-((2-(4-methoxyphenyl)-2-oxoethyl)thio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5g)

IR (KBr) cm^-1: 3445(carboxylic, O-H str.), 3011(Ar. C-H str.), 2851(Ali. CH_2, C-H str.), 1728(carboxylic, C=O str.), 1634(ketonic, C=O str.), 1581(Imine, C=N str.), 1310(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.83(s, carboxylic, 1H, OH), 9.04(s, 1H, H2-quinoline), 7.14-7.98(m, 4H, Ar.), 7.84(s, 1H, H5-quinoline), 4.71(s, 2H, CH₂), 4.37(q, 2H, NCH₂CH₃), 3.83(s, 3H, methoxyl), 2.92-3.67(m, 7H, piperazinyl), 1.31(t, 3H, NCH₂CH₃), 1.11(s, 3H, piperazinyl CH₃); ¹³C- NMR (DMSO-?6) ?ppm: 191, 184, 168, 156, 138, 129, 112, 108, 60, 38, 18, 11; MS: m/z = 617 [M+1];CHN calcd;C₂₈H₂₇F₂N₅O₅S₂;C, 54.62; H, 4.42; N, 11.38; Found C, 54.64; H, 4.40; N, 11.37.

7-(4-(5-((2-(4-aminophenyl)-2-oxoethyl)thio)-1,3,4-thiadiazol-2-yl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5h)

IR (KBr) cm^-1: 3452(carboxylic, O-H str.), 3367(Ar. NH₂, N-H str.), 3027(Ar. C-H str.), 2851(Ali. CH_2, C-H str.), 1736(carboxylic, C=O str.), 1628(ketonic, C=O str.), 1580(Imine, C=N str.), 1327(ethylic, C-H str.); ¹H-NMR (DMSO-?6) ?ppm: 12.88(s, carboxylic, 1H, OH), 9.12(s, 1H, H2-quinoline), 7.86(s, 1H, H5-quinoline), 6.83-7.72(m, 4H, Ar.), 6.31(s, 2H, Ar. NH₂), 4.72(s, 2H, CH₂), 4.32(q, 2H, NCH₂CH₃), 3.02-3.62(m, 7H, piperazinyl), 1.29(t, 3H, NCH₂CH₃), 1.14(s, 3H, piperazinyl CH₃); ¹³C- NMR (DMSO-?6) ?ppm: 197, 187, 172, 151, 133, 123, 117, 107, 67, 35, 16; MS: m/z = 601 [M+1];CHN calcd;C₂₇H₂₆F₂N₆O₄S₂; C, 53.99; H, 4.36; N, 13.99; Found C, 54.02; H, 4.37; N, 13.98.

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