Synthesis of Sparfloxacin derivatives as an antibacterial, antimycobacterial agents with cytotoxicity investigation

 

Gulshan Gurunani¹*, Kapil Agrawal², Sheelpriya Walde^1,2, Abhay Ittadwar³

¹Department of Pharmaceutical Chemistry, Gurunanak College of Pharmacy, Nagpur, 440026, India.

²Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist. Dhule, 425405, India.

³Principal, Gurunanak College of Pharmacy, Nagpur, 440026, India.

 

ABSTRACT:

Based on earlier pieces of evidence of N-piperazinyl fluoroquinolone, and in search of new bioactive molecules from the fluoroquinolone class, the derivates of N-substituted piperazinyl quinolones were synthesized. A series of 2-((amino-1,3,4-thiadiazol-2yl)thio)-1-(4-subst.) (3a–j) were used for diazotization of amines in concentrated hydrochloric acid in the presence of Cu-powder, resulting into 2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subst.)ethanone (4a-j). The reaction of (4a-j) with piperazinyl quinolone in dimethylformamide resulted (5a-j). The structure of synthesized compounds was confirmed by their spectral analysis. The compounds are screened against Staphylococcus aureus, Bacillus subtilis(Gram positive)and Escherichia coli, Pseudomonas aeruginosa,(Gram negative) and mycobacterium tuberculosis. The findings revealed moderate activity against Gram-positiveand poorly active against Gram-negative bacteria. Results indicated that halogenated analogs with nitro substitution (5b, 5e, and 5j) derivatives revealed antibacterial and antimycobacterial activity. The results advocate the need for further exploration of such derivatives, coupled with their preclinical and clinical investigation.

 

 

 

INTRODUCTION:

Fluoroquinolones (FQ), the broad-spectrum antimicrobial agents, have been used to treat a variety of bacterial infections from six decades and are found clinically effective against tuberculosis, meningeal, respiratory, osteoarticular, urogenital, and urinary tract infections, because of their extraordinarily good diffusion.^1-3 During the 1960’sNalidixic acid (Fig. 1) is the progenitor derivative of the FQ class of anti-microbials. It was in the synthesis of chloroquine discovered as by-product. It is the first generation of quinolone anti-microbials.⁴

 

 

Fluoroquinolones (FQ), the broad-spectrum antimicrobial agents, have been used to treat a variety of bacterial infections from six decades and are found clinically effective against tuberculosis, meningeal, respiratory, osteoarticular, urogenital, and urinary tract infections, because of their extraordinarily good diffusion.^1-3 During the 1960’sNalidixic acid (Fig. 1) is the progenitor derivative of the FQ class of anti-microbials. It was in the synthesis of chloroquine discovered as by-product. It is the first generation of quinolone anti-microbials.⁴ The antibacterial activity of 4-quinolones is greatly increased by the addition of 6-fluoro and 7-piperazinyl groups hence named fluoroquinolones.⁵

 

 

Fluoroquinolones have the unique property by binding to the enzyme-DNA complex to inhibit the DNA synthesis directly.

 

 

 

 

Figure 1 Discovery of Nalidixic acid

 

In gram-positive and gram-negative bacteria, FQ stabilizes DNA strand breaks which are created by the enzymes like topoisomerase IV and DNA gyrase results in bacterial DNA impairment and cell decease.^6-8

 

The substitution at C-6 by fluorine atom on quinolone has significantly enhanced antibacterial and anti-tubercular activity. Because of fluorine at C-6 it increases cell penetration (1–70 times) as well as gyrase inhibition (2–17 times). Modifications at the C-7 position developed the most effective compounds. The substituent at C-7 position controlled the level of solubility, cell permeability, antibacterial activity and pharmacokinetics. The  substituents at position C-7 are azabicyclic, pyridyl, pyrrolidinyl, piperidinyl, and piperazinyl moieties (Figure 2)are in clinical use. ^9-12

 

Figure 2 General structures of fluoroquinolones

 

A piperazine substituted fluoroquinolone shows enhanced activity against gram-negative and pyrrolidine ring substituted fluoroquinolone shows improved activity against gram - positive microorganisms separately. The substitution of 1,2-dihydropyrrolizines groups at the C-7 position is found to be more potent against gram-positive microorganisms and have increased the lipophilicity. The replacement at C-8 influenced the pharmacokinetics, antibacterial spectrum and adverse effects.¹¹Newly synthesized compounds showing enhanced antibacterial^12-13, antifungal^14-15, antitumor ^16-17, antiviral ¹⁸, anti-HIV-1 integrase¹⁹ and anti-HCV-NS3 helicase activities are due to modifications in the structure of quinolone at the different postions.²⁰ [C-5, C-6, C-7, C-8 and N-1]

Countless FQ have been endorsed for the treatment of bacterial diseases. Notwithstanding, there is a obligation to find new substituted derivatives to conquer the budding issue of bacterial and multidrug resistance.^21-22 Recently few antibacterial FQ have found to show unwanted adverse effects like cardiac and liver toxicity due to grepafloxacin and trovafloxacin respectively.¹

 

The first broad-spectrum quinolones; Norfloxacin and Ciprofloxacin have poor tissue penetration and low serum levels. The chemical change at C-7 position in FQ is considered as most adaptable site that can determine target preferences and effectiveness^23–28. Therefore, in the present research work, newly synthesized derivatives (5a–j) have been assessed for its in-vitro antibacterial and anti-tubercular effects.  

 

MATERIAL AND METHODS:

Materials:

All the solvents, chemicals as starting materials were buy from chemical suppliers (Himedia, Merck India, Loba chemicals, Scientific Research and Spectrochem).  FTIR spectrophotometer (Shimadzu) is used for recording IR spectra  and values are estimated in cm⁻¹. ¹HNMR was recorded at 400MHz and¹³C NMR at 100MHz from IISc Bangalore on a Bruker AM spectrometer and values are estimated in δ ppm. C, H, N investigation for percentage analysis was acted in a Thermo Scientific Flash 2000 Organic Elemental Analyzer. Autospec Mass spectrometer at IICT, Hyderabad was used for recording FAB/EIMS mass spectra

 

Methods:

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

0.1 mol of substituted/un-substituted acetophenones 1(a–j) were taken in two-necked round bottom flask,  suitable anhydrous solvents (ether, aceton, methanol, chloroform) wasadded with anhydrous AlCl₃.The temperature of reaction was set up below 8⁰C (in cold) and bromine (0.09mol) was mixed dropwise with continuous stirring. Mixtures 2(a–j) were acquired as colorless to brown to shiningcrystals. The product was washed with anhydrous  solvents and recrystallized from methyl alcohol to obtain lachrymatory crystals.^29-30

 

Liquefying point ranges  (2a–j); R = H, F, Cl, Br, NO_2, CH₃, OCH₃, NH_2, OH, C₆H₅; 48-50°, 46-48, 90–92°, 110–112°, 96–98°, 52–54°,72–74°, 80–84°, 102-104, 98-102 respectively (50–74%).

 

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

The 5-amino-1,3,4-thiadiazole-2thiol (0.1mol) were suspended in water (15ml) and potassium hydroxide (80%) (0.1mol) was added. This solution was de-colorized with activated charcoal, followed by addition of 32mL of ethanol and stirred rapidly with 2 (a–j) (0.1mol). The product of reaction  was cooled for 40 minute and to it furthur added 200mL of cold water. It is then filtered to obtainthe solid product and washed with ether and water. The3(a–j) were obtained (Scheme 1), with 54–68% yieldand melting point (80–108^oC).³¹

 

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

Triturated 2-((amino-1,3,4-thiadiazol-2yl)thio)-1-(4-subst.) ethanone 3(a–j) (30mmol) with sodium nitrite (60 mmol). The triturate was introduced in the ice-cooled (0–5^oC) mixture of 15ml water and 30ml concentrated HCl with stirring with 0.1g Cu powder as a catalyst. The product wasrefluxed for 60 min. at 75⁰C and cool. Then the solution was extracted by using dry chloroform (3 x 75ml). The combined solution mixture of chloroform was washed with NaHCO₃ solution. Then the extracted chlorofom solution was dried over Na₂SO₄. The solvent was evaporated under reduced pressure. Finally, recrystallization of product was done using ethanol to yield 2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subst.)ethanone4 (a–j) (Scheme 1). The compound was purified by column chromatography with methanol : chloroform (1:9) as mobile phase, m.p. 85–110^oC (48–60%).³²

 

Synthesis of 5-amino-7-[4-(5-(substituted-benzoylthio)-1, 3, 4-thiadiazol-2yl)-3, 5-dimethylpiperazin-1yl]-1-cyclopropyl-6, 8-difluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid 5(a–j).

2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subst.)ethanone 4(a–j), piperazinyl fluoroquinolone (sparfloxacin) and sodium-bicarbonate each 10mmol was refluxed  with 10ml dimethyl-formamide on oil bath  at 140–160^oC for 20hrs. After cooling the reaction mixture, added 10ml water to obtain the precipitate . The precipitated product was filtered followed by washing with water. The solid was recrystallized using a mixture of DMF and H₂O to obtained (5a-j) compounds.³³ (Scheme 1).

Antibacterial Activity:

The in vitro antibacterial activity was determined by broth micro-dilution technique against Gram-negative and Gram-positive bacteria (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Bacillus subtilis). In Mueller-Hinton agar medium by two-fold serial dilution method the test compounds and reference standard Sparfloxacin were prepared. The required concentrations of 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 50.0 and 100.0µg/ml were obtained by Progressive double dilutions with agar.  The petri plates were inoculated and incubated at 37⁰C for 18 hours.^34-35

Anti-mycobacterial Activity:

In vitro screening for anti-mycobacterial test was assessed utilizing M. tuberculosis virulent H37Rv strain by broth dilution assay. The determination of MIC was done for all derivatives by using Middlebrook 7H9 broth supplemented with 10% ADC (albumin dextrose catalase) and 0.2% glycerol frozen culture. It is used as inoculum with dilution in broth to 2 × 10⁵ cfu/ml. In the process, U-tubes were used to keep 0.8, 1.6, 3.12, 6.25, 12.5, 25.0, 50.0 and 100.0µg/ml dilutions.^36-38

 

Estimation of cell viability:

Conversion of MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra sodium bromide)] to dark blue formazan crystals due to presence of living cells, was employed for estimation of cell viability. Colorimetric analysis was used for 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 livercell lines were added in plates with 96 wells, and then preserved with variable dilutions of investigational compounds in DMSO, at 37^oC in carbon dioxide incubator for four days. Further, the MTT reagent were instilled into the wells. The instilled wells were incubated for four hours, and then the dark blue formazan developed was allowed to dissolve in DMSO and the colorimetric absorbance was read at 550nm. The IC₅₀value was estimated by graph plotted between percentage cells inhibited versus concentrations.³⁹ The findings are provided in table 1.

 

RESULT AND DISCUSSION:

Table 1: Antibacterial, anti-mycobacterial activity, cytotoxicity and selectivity index of the compounds 5(a–j).

^{a  :-}MIC: Minimum inhibitory concentration (in µg/ml)

^b :-As a bacteria; SA:- S. aureus (Gram positive), BS:- B. subtilis(Gram positive); EC:- E. Coli (Gram negative), PA:-P. aeruginosa(Gram negative)

^CM. TB:- Mycobacterium Tuberculosis       ^{d :-}  IC₅₀ inhibition concentration   *:- As reference pure drug:- SPAR: Sparfloxacin; RIP: rifampicin.

^e PI:- Protective index  (ratio of IC₅₀/MIC value)

 

Spectral data of synthesized derivatives:

Spectral Data of 5-amino-7-[4-(5-(benzoylthio)-1, 3, 4-thiadiazol-2yl)-3, 5-dimethylpiperazin-1yl]-1-cyclopropyl-6, 8-difluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (5a)

FT-IR (cm^-1): 3452 (OH str., carb. acid), 3367 (NH str., Ar. amine), 2933(Ar. alkyl str.), 2852 (Ali. alkyl str.), 1714 (C=O str., carb. acid), 1656 (C=O str, ketonic), 1590 (C=N, str Imine), 1373 (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 13.14 (s, 1H, OH), 7.92 (s, 1H, ), 7.33-7.45 (m, 5H, Ar.), 5.42 (s, 2H, NH₂), 4.28 (s, 2H, CH₂), 3.12-3.22 (m, 6H, piperazinyl) and (1H, CH, ∆), 1.22-1.44 (m, 4H, ∆), 1.14 (s, 6H, dimethylpiperazine).

 

Spectral data of 5-amino-7-[4-({5-(4-chlorobenzoylthio)-1, 3, 4-thiadiazol-2yl}-3, 5-dimethylpiperazin-1yl]-1-cyclopropyl-6, 8-difluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (5b)

FT-IR (cm^-1): 3461 (OH str., carb. acid), (NH str., Ar. amine), 2937 (Ar. alkyl str.), 2850 (Ali. alkyl str.), 1732 (C=O str., carb. acid), 1644(C=O str, ketonic), 1574 (C=N, str Imine), 1320 (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 12.78 (s, 1H, OH), 8.72 (s, 1H), 7.90–8.04 (m, 4H, Ar.), 6.58 (s, 2H, NH₂), 4.87 (s, 2H, CH₂), 4.22 and 3.10-3.86 (m, 1H, CH,  ∆ and 6H, piperazinyl), 1.10-1.32 (m, 4H, ∆), 1.16 (s, 6H, dimethylpiperazine); ¹³C- NMR (DMSO-𝑑6) 𝛿ppm: 196, 178, 164, 141, 130, 131, 114, 109, 52, 45, 33, 11, 7; MS: m/z = 662 [M+1];CHN calculated; C₂₉H₂₇ClF₂N₆O₄S₂;C, 52.68; H, 4.14; N, 12.71; Found C, 52.71; H, 4.16; N, 12.73.

 

Spectral data of 5-amino-7-[4-({5-(4-bromobenzoylthio)-1, 3, 4-thiadiazol-2yl}-3, 5-dimethylpiperazin-1yl]-1-cyclopropyl-6, 8-difluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (5c)

FT-IR (cm^-1): 3456c (OH str., carb. acid), 3375 (NH str., Ar. amine), 2932 (Ar. alkyl str.), 2855 (Ali. alkyl str.), 1733 (C=O str., carb. acid), 1652 (C=O str, ketonic), 1564 (C=N, str Imine), 1321(C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 13.10 (s, 1H, OH), 8.68 (s, 1H), 7.68-7.98 (m, 4H, Ar.), 6.46 (s, 2H, NH₂), 4.52 (s, 2H, CH₂), 4.10 and 3.12-3.88 (m, 1H, CH, ∆ and 6H, piperazinyl), 1.12-1.34 (m, 4H, ∆), 1.11 (s, 6H, dimethylpiperazine); ¹³C- NMR (DMSO-𝑑6) 𝛿ppm: 194, 173, 161, 139, 128, 112, 74, 52, 48, 32, 10, 7.

 

Spectral data for 5-amino-1-cyclopropyl-6,8-difluoro-7-[4-{5-(4-fluorobenzoylthio)-1,3,4-thiadiazol-2yl}-3,5-dimethylpiperazin-1yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5d)

FT-IR (cm^-1): 3442 (OH str., carb. acid), 3377 (NH str., Ar. amine), 2922 (Ar. alkyl str.), 2851 (Ali. alkyl str.), 1724(C=O str., carb. acid), 1601 (C=O str, ketonic), 1581 (C=N, str Imine), 1320 (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 15.01 (s, 1H, OH), 8.78 (s, 1H), 7.90-8.15 (m, 4H, Ar.), 6.98 (s, 2H, NH₂), 4.55 (s, 2H, CH₂), 3.36-4.01 (m, 1H, CH, ∆ and 6H, piperazinyl), 2.59-2.97 (m, 4H, ∆), 1.59 (s, 6H, dimethylpiperazine); ¹³C- NMR (DMSO-𝑑6) 𝛿ppm: 190, 166, 153, 140, 133, 129, 108, 77, 41, 29, 8; MS : m/z = 644 [M⁺]; CHN calculated; C₂₉H₂₇F₃N₆O₄S₂; C, 54.03; H, 4.22; N, 13.04; Found; C, 54.11; H, 4.21; N, 12.98.

 

Spectral data for 5-amino-1-cyclopropyl-7-[3,5-dimethyl-4-(5-(4-nitrobenzoylthio)-1,3,4-thiadiazol-2yl}piperazin-1yl]--6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5e)

FT-IR (cm^-1): 3480 (OH str., carb. acid), 3388 (NH str., Ar. amine), 3040 (Ar. alkyl str.), 2850 (Ali. alkyl str.), 1708(C=O str., carb.  acid), 1625  (C=O str, ketonic), 1548 (C=N, str Imine), 1338  (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 13.45 (s, 1H, OH), 8.62 (s, 1H, ), 7.74-7.88 (m, 4H, Ar.), 6.51 (s, 2H, NH₂), 4.33 (s, 2H, CH₂), 4.12 and 3.23-3.70 (m, 1H, CH, ∆ and 6H, piperazinyl), 1.10-1.39 (m, 4H, ∆), 1.14 (s, 6H, dimethylpiperazine); ¹³C-NMR (DMSO-𝑑6) 𝛿ppm: 190, 174, 163, 144, 139, 112, 76, 59, 48, 39, 31, 16, 10; MS : m/z = 671 [M⁺];CHN calculated:C₂₉H₂₇F₂N₇O₆S₂;C, 51.86; H, 4.05; N, 14.60; Found C, 51.88; H, 4.03; N, 14.62.

 

Spectral data for 5-amino-1-cyclopropyl-7-[3,5-dimethyl-4-(5-(4-methylbenzoylthio)-1,3,4-thiadiazol-2yl}piperazin-1yl]- -6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5f)

FT-IR (cm^-1): 3456 (OH str., carb. acid), 3323 (NH str., Ar. amine), 2967 (Ar. alkyl str.), 2848 (Ali. alkyl str.), 1711 (C=O str., carb. acid), 1664 (C=O str, ketonic), 1530 (C=N, str Imine), 1342  (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 12.40 (s, 1H, OH), 7.90 (s, 1H, ), 6.68-7.27 (m, 4H, Ar.), 6.12 (s, 2H, NH₂), 4.16 (s, 2H, CH₂), 3.92 and 3.19-3.54 (m, 1H, CH, ∆ and 6H, piperazinyl), 2.34 (s, 3H, Ar. methyl), 1.21-1.38 (m, 4H, ∆) 1.10 (s, 6H, dimethylpiperazine); ¹³C- NMR(DMSO-𝑑6) 𝛿ppm: 188, 168, 159, 140, 135, 107, 72, 47, 44, 35, 27, 12, 6.

Spectral data for 5-amino-1-cyclopropyl-6,8-difluoro-7-[4-{5-(4-methoxybenzoylthio)-1,3,4-thiadiazol-2yl}-3,5-dimethylpiperazin-1yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5g)

FT-IR (cm^-1): 3465 (OH str., carb. acid), 3328 (NH str., Ar. amine), 2972 (Ar. alkyl str.), 2854 (Ali. alkyl str.), 1717  (C=O str., carb. acid), 1630 (C=O str, ketonic), 1585 (C=N, str Imine), 1347 (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 12.56 (s, 1H, OH), 8.42 (s, 1H), 7.14-7.38 (m, 4H, Ar.), 6.23 (s, 2H, NH₂), 4.24 (s, 2H, CH₂), 3.64 and 3.19-3.29 (m, 1H, CH, ∆ and 6H, piperazinyl), 3.83 (s, 3H, methoxy), 1.20-1.44 (m, 4H, ∆), 1.04(s, 6H, dimethylpiperazine); ¹³C-NMR (DMSO-𝑑6) 𝛿ppm: 192, 170, 161, 144, 130, 113, 84, 68, 40, 32, 25, 15, 9; MS : m/z = 657 [M+1];CHN calculated;C₃₀H₃₀F₂N₆O₅S₂;C, 54.87; H, 4.60; N, 12.80; Found C, 54.88; H, 4.61; N, 12.84.

 

Spectral data for 5-amino-7-[4-({5-(4-aminobenzoylthio)-1, 3, 4-thiadiazol-2yl}-3, 5-dimethylpiperazin-1yl]-1-cyclopropyl-6, 8-difluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (5h)

FT-IR (cm^-1): 3469 (OH str., carb. acid), 3334 (NH str., Ar. amine), 2975 (Ar. alkyl str.), 2850 (Ali.  Alkyl str.), 1721(C=O str., car b. acid), 1620  (C=O str, ketonic), 1541(C=N, str Imine), 1351 (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 12.62 (s, 1H, OH), 8.43 (s, 1H), 7.14-7.36 (m, 4H, Ar.), 6.12 (s, 2H, NH₂), 4.64 (s, 2H, CH₂), 3.98 and 3.24-3.33 (m, 1H, CH, ∆ and 6H, piperazinyl), 1.21-1.45 (m, 4H, ∆), 1.12 (s, 6H, dimethylpiperazine); ¹³C- NMR (DMSO-𝑑6) 𝛿ppm: 195, 171, 162, 143, 132, 111, 89, 72, 56, 31, 18, 09; MS: m/z = 642 [M+1];CHN calculated; C₂₉H₂₉F₂N₇O₄S₂;C, 54.28; H, 4.57; N, 15.28; Found C, 54.30; H, 4.58; N, 15.31.

 

Spectral data for 5-amino-1-cyclopropyl-6,8-difluoro-7-[4-{5-(4-hydroxybenzoylthio)-1,3,4-thiadiazol-2yl}-3,5-dimethylpiperazin-1yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5i)

FT-IR (cm^-1): 3546 (OH str., phenolic), 3427 (OH str., carb. acid), 3337 (NH str., Ar. amine), 3010 (Ar. alkyl str.), 2852 (Ali. alkyl str.), 1728 (C=O str., car b. acid), 1662  (C=O str, ketonic), 1549 (C=N, str Imine), 1364(C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 12.64 (s,  1H, OH), 8.54 (s, 1H), 6.88-7.37 (m, 4H, Ar.), 6.24 (s, 2H, NH₂), 5.34 (s, 1H, phenolic OH), 4.58 (s, 2H, CH₂), 4.10 and 3.44-3.78 (m, 1H, CH, ∆ and 6H, piperazinyl), 1.28-1.46 (m, 4H, ∆), 1.12 (s, 6H, dimethylpiperazine); ¹³C-NMR (DMSO-𝑑6) 𝛿ppm: 198, 174, 165, 147, 138, 111, 92, 74, 56, 38, 19, 11; MS : m/z = 643 [M+1];CHN calculated;C₂₉H₂₈F₂N₆O₅S₂;C, 54.20; H, 4.39; N, 13.08; Found C, 54.23; H, 4.40; N, 13.13.

 

Spectral data for7-(4-(5-(([1,1'-biphenyl]-4-carbonyl)thio)-1,3,4-thiadiazol-2-yl)-3,5-dimethylpiperazin-1-yl)-5-amino-1-cyclopropyl-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5j)

 

FT-IR (cm^-1): 3462 (OH str., carb. acid), 3377 (NH str., Ar. amine), 2922 (Ar. alkyl str.), 2851 (Ali.  Alkyl str.), 1724 (C=O str., car b. acid), 1601 (C=O str, ketonic), 1581  (C=N, str Imine), 1383 (C-H str., ∆); ¹H-NMR (𝛿ppm) (DMSO-𝑑6): 13.02 (s, 1H, OH), 8.70 (s, 1H, ), 7.98–8.20 (m, 4H, Ar.), 7.38-7.77 (m, 5H, Ar.), 5.50 (s, 2H, NH₂), 4.10 (s, 2H, CH₂), 2.89-3.33 (m, 1H, CH, ∆ and 6H, piperazinyl), 1.24-1.39 (m, 4H, ∆), 1.10 (s, 6H, dimethylpiperazine); ¹³C- NMR (DMSO-𝑑6) 𝛿ppm: 196, 171, 162, 128, 108, 105, 78, 40, 38, 35, 30, 7; MS: m/z = 703 [M+1]; CHN calculated; C₃₅H₃₂F₂N₆O₄S₂; C, 59.81; H, 4.59; N, 11.96; Found C, 60.10; H, 4.64; N, 11.98.

 

Chemistry:

The p-substituted phenacyl bromide 2 (a–j) formation mechanisms carried out by substitution acetophenone, acetic acid and liquid bromine, reaction mechanism followed via acetic acid mediated H⁺ ions rather than lewis acid. Probably this reaction mechanism followed by bromination of the methyl group and it can be restricted to monosubstitution, when the reaction is carried out in acidic media by using anhydrous AlCl₃as a catalyst. The synthesis of 2-((amino-1,3,4-thiadiazol-2yl)thio)-1-(4-subst.) ethanone 3(a–j) was carried out by reacting substituted/un-substituted phenacyl bromide 2(a–j) and 5-amino-1,3,4-thiadiazole-2-thiol, through de-hydro-bromination mechanisms. In the reaction alkali hydroxyl ion abstract the mercapto proton not the amino proton, because mercapto group having strong electron density center on sulphur rather than amino nitrogen atom although nitrogen have strong electronegative element than sulphur but electron density more on sulphur hence, the probability to abstract the proton of mercapto group by hydroxyl ion is more where finding more electron density center. Compounds 3(a–j), by diazotization of amines 2-((amino-1,3,4-thiadiazol-2yl)thio)-1-(4-subst.) ethanone were directly converted to 2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subst.)ethanone4(a–j) followed by chlorination, which obeyed Sandmeyer reaction. This reaction is considered to be more convenient method for introducing a halogen substituent at the desired position of an aromatic ring. The 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) was based on nucleophilic aromatic substitution reaction mechanism involving substrate as 2-((5-chloro-1, 3, 4-thiadiazol-2yl)thio)-1-(4-subst.)ethanone(4a–j) and FQ (sparfloxacin) in presence sodium bicarbonate and N, N’dimethyl formamide, (DMF) which seemed to be suitable route to execute the synthesis of title compounds.^40-42

Antibacterial Activity:

The synthesized compounds 5 (a-j) explored In-vitro antibacterial activity against two gram positive and two-gram negative strains i.e. Staphylococcus aureus, Bacillus subtilis and Escherichia coli, Pseudomonas aeruginosa respectively to determine the MICs, which gave profound conclusion about the most active analogues. As per results depicted in compounds, classified the active and non-active analogues based on screened standard drug’s MICs. According to that, 1.33 to 2.50 categorized highly active; 2.50-10.0 considered improved to moderate active and >10.0 deemed to be least active if compared with that of standards sparfloxacin. Largely, most of the synthesized derivatives were found progressive to moderate active against the gram-positive bacterial strains, except unsubstituted (5a) (R = H) analogues of sparfloxacin. Finally, (1.00 to 1.89 µg/ml) in 5b (-Cl), 5e (-NO₂) & 5j (-C₆H₅) of sparfloxacin, found most active against gram negative bacterial strains.⁴³

 

Anti-mycobacterial Activity:

The derivatives were screened for In-vitro antitubercular against H₃₇Rv strain, using micro alamar blue reagent, in this assay, blue colour converted into red colour indicate the test compound would be non-active up to that particular concentration. This assay has conducted based on BACTEC radiometric method. Microplate alamar blue assay (MABA) has certain advantages over conventional disc diffusion method.⁴⁴ In this assay, eight double-fold serially diluted concentrations taken between 0.8 to 100 µg/ml, in which 5b and 5j substituted to sparfloxacin were found excellent activity (0.8 µg/ml) which is comparable to that of standards (0.8 µg/ml). However, rest of the derivatives were found MIC (6.25-25 µg/ml) which showed moderate to least active as compared to standard first line Rifampicin.^44-45

 

In-vitro toxicity study:

All the potent antitubercular agents were subjected to toxicity cell viability study with the help of MTT assay on Hep-G2 normal liver cell line. The value of PI is unit less and higher value considered to most efficient agent. The compound 8j (-C₆H₅substituted derivative) found same efficient agent compared to reference as its PI value was 75.00. Similarly 8g and 8h (-OCH₃; -NH₂substituted derivatives) shown 31.25 and 40.62, indicated more toxic agents. Hence these derivatives have not secured the safe candidate, as the PI value is very low. The compound 8j was the most efficient agent i.e. higher potency with same toxicity profile as compared to reference Rifampicin.⁴⁶

 

CONCLUSIONS:

The synthesis of 5(a–i) by conventional synthetic method with good yield were synthesized. These synthesized compounds were confirmed by using several spectral analysis. The synthesized compounds were screened for anti-bacterial and anti-mycobacterial activity, and it was concluded that there is a strong resemblance in the activities exhibited between 5b, 5e, and 5j derivatives, sparfloxacin and rifampicin. However, the activities reflected were slightly weaker than those of the reference standards, when compared to the other derivatives, which revealed very weak activity. Altogether, the findings advocate need of further investigation in this domain, which may result into novel synthetic compounds revealing potential therapeutic effect.

 

ACKNOWLEDGMENTS:

The authors express ardent gratitude to the executives, Management, Gurunanak college of Pharmacy, Nagpur, India for giving important offices to do this examination work. The creators might likewise want to recognize Relife drug Ltd., Nagpur, for giving blessing tests of Sparfloxacin.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Owens, R. C., Jr.; Ambrose, P. G., ‘Clinical use of the fluoroquinolones’, The Medical clinics of North America, 2000; 84(6): 1447-69.

2.      Smith, A.; Pennefather, P. M.; Kaye, S. B.; Hart, C. A., ‘Fluoroquinolones: place in ocular therapy’, Drugs, 2001; 61(6):747-61.

3.      Naeem A.; Badshah S. L.; Muska M.; Ahmad N.; Khan K., ‘The current case of quinolones: synthetic approaches and antibacterial activity’ Molecules, 2016; 21(4):87–90.

4.      Ball, P. ‘Quinolone generations: natural history or natural selection?’ J. Antimicrob. Chemother., 2000; 46 (TOPIC T1):17-24.

5.      Rakesh Patel, Anil Bhandari. Spectrofluorimetric Estimation of Some Fluoroquinolones. Asian J. Pharm. Ana. 2017; 7(4): 235-238.

6.      Mitscher L. A., ‘Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial agents’, Chem Rev, 2005; 105(2):559–592.

7.      Aldred K. J.; Kerns R. J.; Osheroff N., ‘Mechanism of quinolone action and resistance’ Biochem, 2014; 53(10):1565–1574.

8.      K Girija, Divya Chacko. Eco-Friendly Synthesis and Biological Evaluation of Some Novel N-Substituted Piperazinyl Fluoro Quinolone Derivatives. Research J. Pharm. and Tech.2, July-Sept 2009; (3):498-501.

9.      Peterson, L. R. ‘Quinolone Molecular Structure Activity Relationships: What We Have Learned about Improving Antimicrobial Activity’ Clinical Infectious Diseases, 2001; 33(s3):S180–S186.

10.   Soni K., ‘Fluoroquinolones: chemistry and action – a review’, Indo Global J Pharm Sci; 2012;  2(1): 43–53.

11.   Mentese M. Y.; Bayrak H.; Uygun Y.; Mermer A.; Ulker S.; Karaoglu S. A.; Demirbas N.,. ‘Microwave assisted synthesis of some hybrid moleculesderivated from norfloxacin and investigation of their biological activities’ Eur J Med Chem; 2013; 67:230–242.

12.   Mallikarjuna Gouda M, Somashekar shyale, Putta Rajesh Kumar, SM Shanta kumar. Analytical Method Development, Validation studies of a Fluoroquinolone chemotherapeutic antibiotic and its Characterization studies. Research J. Pharm. and Tech., March 2011; 4(3):433-436.

13.   Reuman M.; Daum S. J.; Singh B.; Wentland M. P.;  Perni R. B.; Pennock P.; Carabateas P.; M.; Gruett M. D., ‘Saindane MT, Dorff PH, Coughlin SA, Sedlock DM, Rake JB, Lesher GY: Synthesis and antibacterial activity of some novel 1-substituted 1,4-dihydro-4-oxo-7-pyridinyl-3-quinolinecarboxylic acids. Potent antistaphylococcal agents’ J Med Chem; 1995; 38(14):2531–2540.

14.   Emami S.; Shafiee A.; Foroumadi A, ‘Quinolones: Recent structural and clinical developments’ Iran J Pharm Res; 2005; 4:123–136.

15.   Abuo-Rahma G. E. D.; Sarhan H. A.; Gad G. F. M. ‘Design, synthesis, antibacterial activity and physicochemical parameters of novel N-4-piperazinylderivatives of norfloxacin’ Bioorg Med Chem; 2009; 17(11):3879–3886.

16.   Abdel-Aziza A. A. M.; Asiri Y. A.; Al-Agamy M. H. M.; ‘Design, synthesis and antibacterial activity of fluoroquinolones containing bulky arenesulfonyl fragment: 2D-QSAR and docking study, Eur J Med Chem; 2011; 46(11):5487–5497.

17.   Zhang L.; Kumar K. V.; Geng R. X.; Zhou C. H.; ‘Design and biological evaluation of novel quinolone-based metronidazole derivatives as potentCu 2+ mediated DNA-targeting antibacterial agents’, Bioorg Med Chem Lett; 2015; 25:3699–3705.

18.   Wang Y.; Damu G. L. V.; Lv J. S.; Geng R. X.; Yang D. C.; Zhou C. H.; ‘Design, synthesis and evaluation of clinafloxacin triazole hybrids as a newtype of antibacterial and antifungal agents’, Bioorg Med Chem Lett; 2012; 22:5363–5366.

19.   Rajasekaran N.; Anbalagan M.; Kumar N. R.; Panneerselvam P.; ‘Synthesis and antimicrobial studies of novel 7-(N-4-substituted sulfonamide)-6-fluoroquinolone derivatives’, Int J Med Chem Analysis, 2012; 2: 50–56.

20.   Fang K. C.; Chen Y. L.; Sheu J. Y.; Wang T. C.; Tzeng C. C.; ‘Synthesis, antibacterial, and cytotoxic evaluation of certain 7-substituted norfloxacin derivatives’ J Med Chem; 2000; 43(20):3809–3812.

21.   Shou K. J.; Li J.; Jin Y.; Lv Y. W.; ‘Design, synthesis, biological evaluation, and molecular docking studies of quinolone derivatives as potentialantitumor topoisomerase I inhibitors’ Chem Pharm Bull; 2013; 61(6):631–636.

22.   Richter S.; Parolin C.; Palumbo M.; Palu G.; ‘Antiviral properties of quinolone-based drugs’, Curr Drug Targets Infect Disord,; 2004; 4(2):111–116.

23.   Sato M.; Motomura T.; Aramaki H.; Matsuda T.; Yamashita M.; Ito Y.; Kawakami H.; Matsukari Y.; Watanabe W.; Yamataka K.; Ikeda S.; Kodama E.; Matsuoka M.; Shinkai H.; ‘Novel HIV-1 integrase inhibitors derived from quinolone antibiotics’ J Med Chem; 2006; 49(5):1506–1508.

24.   Foroumadi A.; Emami S.; Hassanzadeh A.; ‘Synthesis and antibacterial activity of N-(5-benzylthio-1,3,4-thiadiazol-2- yl) and N-(5-benzylsulfonyl-1,3,4-thiadiazol-2-yl)piperazinyl quinolone derivatives’, Bioorganic and Medicinal Chemistry Letters, 2005; 15(20):4488–4492.

25.   Sanchez J. P.; Domagala J. M.; Hagen S. E.; ‘Quinolone antibacterial agents. Synthesis and structure-activity relation- ships of 8-substituted quinoline-3-carboxylic acids and 1,8- naphthyridine-3-carboxylic acids’, Journal of Med Chem., 1988; 31(5):983–991.

26.   Chu D. T. W.; Fernandes P. B.; Claiborne A. K.; ‘Synthesis and structure-activity relationships of novel aryl-fluoroquinolone antibacterial agents,’ J. Med. Chem.; 1985; 28(11):1558–1564.

27.   Shen L. L.; Mitscher L. A.; Sharma P. N.; ‘Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug-DNA binding model,’ Biochemistry, 1989; 28(9):3886–3894.

28.   Jacoby G. A.; ‘Mechanisms of resistance to quinolones’, Clin Infect Dis; 2005; 41(S2) S120–S126.

29.   Li X. Z.; ‘Quinolone resistance in bacteria: emphasis on plasmid-mediated mechanisms, Int J Antimicrob Agents; 2005; 25(6):453–463.

30.   Bauer A. W.; Kirby W. M.; Sherris J. C.; ‘Turck M: Antibiotic susceptibility testing by a standardized single disk method’, Am J Clin Pathol; 1966; 45(4):493-496.

31.   R. T. Morrison and R. N. Boyd, Organic Chemistry, Pearson Education, New Delhi, India, 2004.

32.   Organic Syntheses, Coll, R. M. Cowper, L. H. Davidson, and A. H. Blatt, Eds., vol. 2, pp. 480–481, JohnWiley&Sons, NewYork, NY, USA, 1943.

33.   Organic Syntheses, Coll, G. H. Coleman, G. E. Honeywell, and A. H. Blatt, Eds., vol. 2, pp. 443–445, John Wiley & Sons, New York, NY, USA, 1943.

34.   Foroumadi, A.; Rineh, A.; Emami, S.; Siavoshi, F.; Massarrat, S.; Safari, F.; Rajabalian, S.; Falahati, M.; Lotfali, E.; Shafiee, A.; ‘Synthesis and anti-Helicobacter pylori activity of 5-(nitroaryl)-1,3,4-thiadiazoles with certain sulfur containing alkyl side chain’, Bioorg. Med. Chem. Lett., 2008; 18 (11):3315-3320.

35.   Sahu Sudeep, Dey Tathagata, Khaidem Somila, Y Jyothi. Microwave Assisted Synthesis of Fluoro-Pyrazole Derivatives for Antiinflammatory Activity. Research J. Pharm. and Tech. March 2011; 4(3):413-419.

36.   Foroumadi, A.; Emami, S.; Pournourmohammadi, S.; Kharazmi, A.; Shafiee, A., ‘Synthesis and in vitro leishmanicidal activity of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)-5-substituted-1, 3, 4-thiadiazole derivatives’, Eur. J. Med. Chem., 2005; 40 (12):1346-1350.

37.   Foroumadi, A.; Mansouri, S.; Kiani, Z.; Rahmani, A., Synthesis and in vitro antibacterial evaluation of N-[5-(5-nitro-2-thienyl)-1, 3, 4-thiadiazole-2-yl] piperazinyl quinolones. Eur. J. Med. Chem., 2003; 38 (9):851-854.

38.   R. S. Kalkotwar, R. B. Saudagar. Design, Synthesis and anti microbial, anti-inflammatory, Antitubercular activities of some 2,4,5-trisubstituted imidazole derivatives. Asian J. Pharm. Res. 3(4): Oct. - Dec.2013: 159-165.

39.   Goto S.; Sakamoto H.; Ogawa M.; ‘Bactericidal activity of cefazolin, cefoxitin, and cefmetazole against Escherichia coli and Klebsiella pneumoniae’, Chemotherapy, 1982; 28(1):18–25.

40.   Suling W. J.; Seitz L. E.; Pathak V.; ‘Antimycobacterial activities of 2,4-diamino-5-deazapteridine derivatives and effects on mycobacterial dihydrofolate reductase’, Antimicrobial Agents and Chemotherapy, 2000; 44(10):2784–2793.

41.   Kumud Gupta, Surendra Nath Pandeya, Ashish Kumar Pathak, Anuradha Gupta, Synthesis and Antibacterial Activity of Ciprofloxacin Derivatives. Research J. Pharm. and Tech. 4(2): February 2011:308-314.

42.   Palled M. S.., Lohar V. R., Kalekar M. C., Patil P. B., Bhat A. R. Synthesis of 1,2,3,4-[ 1-N- Methyl Benzimidazole] 3-Phenyl Substituted Thiazolidine, 4-One and Carbohydrazide Derivatives for Antimicrobial Activity. Research J. Pharm. and Tech; Feb. 2012; 5(2):249-252.

43.   Yajko D. M.; Madej J. J.; Lancaster M. V.; ‘Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis’, Journal of Clinical Microbiology, 1995; 33(9):2324–2327.

44.   Sriram D.; Yogeeswari P.; Reddy S. P.; ‘Synthesis of pyrazinamideMannich bases and its antitubercular properties’, Bioor and Med Chem Lett, 2006; 16(8):2113–2116.

45.   Sriram D.; Yogeeswari P.; Reddy S. P.; ‘Synthesis of pyrazinamide mannich bases and its antitubercular properties’, Bioorg Med Chem Lett, 2006; 16(8):2113–2116.

46.   Agrawal K. M.; Talele G. S.; ‘Synthesis and antibacterial, antimycobacterial and docking studies of novel N-piperazinyl fluoroquinolones’, Medicinal Chemistry Research, 2013; 22(2):818–831.

 

 

 

Accepted on 25.02.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4359-4366.