Synthesis and antimicrobial activity evaluation of new norfloxacine-azole hybrids

Chemistry

For the synthesis of the target hybrids, the strategy depicted in Schemes 1 and 2 was chosen.

Scheme 1

Reagents and conditions: (i) BrCH₂CO₂Et in DMF, TEA, 24 h, rt. (method 1) or BrCH₂CO₂Et, EtOH, TEA, 15 min, 200 W (method 2); (ii) H₂NNH₂, chloroform, 24 h, rt. (method 1) or 15 min 80 W MW irradiation MW (method 2); (iii) KOH, CS₂, EtOH-H₂O, 10 h, reflux (method 1) or 20 min 150 W MW irradiation MW (method 2); (iv) amine, DMF, HCHO, 24 h, rt.

Scheme 2

 

The use of microwave irradiation provided an efficient and green synthetic approach with dramatically reduced reaction times and improved yields [23]. Norfloxacin and ethyl bromoacetate were subjected to condensation in dimethylformamide (DMF) yielding product 1. The synthesis of compound 1 was achieved in a moderate yield (74%) in DMF with a reaction time of 24 h by the procedure reported by Zsoldos-Mádyet and co-workers. On the other hand, a similar reaction under microwave irradiation afforded compound 1 in good yield after 10 min. The treatment of compound 1 with hydrazine hydrate under the conventional [23] and microwave-assisted conditions afforded hydrazide 2. Compared to conventional thermal heating in chloroform, the microwave-irradiation technique without solvent decreased the reaction time from 24 h to 15 min, and the yield was increased from 78% to 94%. The given structure of 2 is fully consistent with the spectral data. In particular, the mass spectral fragmentation confirms the proposed structure.

The cyclization of hydrazide 2 with carbon disulfide was achieved in ethanol under conventional heating and also microwave-irradiated conditions yielding 1,3,4-oxadiazole 3. The use of microwave irradiation resulted in an increased yield from 81% to 97%. However, the most striking effect of the irradiation was the decrease of the reaction time from 10 h to 20 min. In the ¹H NMR spectrum of compound 3, the signals derived from the hydrazide function disappeared and a new signal due to -NH proton on the 1,3,4-oxadiazole ring is seen at 9.23 ppm as a D₂O-exchangeable singlet. The C=S stretching band is observed at 1256 cm⁻¹ in the IR spectrum. The LC MS and elemental analysis data also support the proposed structure.

The treatment of hydrazide 2 with isothiocyanates furnished the corresponding products 5a–c (Scheme 2). The IR spectra of derivatives 5a,b show an absorption band at 1265 cm⁻¹ (for 5a) and 1250 cm⁻¹ (for 5b) indicating the presence of a C=S double bond. Furthermore, the IR and ¹H NMR spectra of 5a–c exhibit signals for three NH protons (exchangeable with D₂O), while no signal derived from an -NH₂ group is observed. The treatment of 5a–c with a base resulted in cyclization to 1,2,4-triazole derivatives 6a–c. The reaction was investigated in ethanol-water under classical heating conditions and under microwave irradiation conditions. The progress of the reactions was monitored by thin layer chromatography (TLC). With the use of the microwave irradiation, the yield was improved to 94–97% and the reaction time for complete consumption of the starting material was decreased from 15–18 h to a remarkable 25 min. The optimal microwave power in terms of yield and product stability was found to be 120 W in a closed vessel. A decrease in microwave power resulted in lower yields and longer reaction times. In an attempt to optimize the reaction conditions for the irradiation, solvents were also screened. The best results were obtained in a mixture of methanol and water. In the ¹H NMR spectra of compounds 6a,b, a singlet characteristic for the -SH group is recorded at 13.88 ppm, while the NH proton on a 1,2,4-triazol ring of 6c resonates at 11.89 ppm. The IR spectra of compounds 6a,b exhibit absorption bands originating from the -SH function at 2823–2824 cm⁻¹. In addition, compounds 6a–c gave a mass fragmentation pattern and elemental analysis data consistent with the assigned structures. The one-pot three-component synthesis of compounds 4a,b, 7a–g and 8a–c was achieved by the amination of compounds 3 and 6a–c with amines in the presence of formaldehyde in DMF (conventional method) or without solvent (irradiation method). These structures merge azine, azole and norfloxacin units into a single molecule in an attempt to obtain antimicrobial agents with improved properties (Scheme 2) [24], [25]. Initially, to optimize the conditions for this three-component reaction, compound 7a was selected as the model product, and the model reaction was performed in a polar solvent, in the absence of solvent and in the presence of a Lewis or Bronsted acid catalyst such as p-TSA, FeCl₃, InCl₃ and HCl. The solvent-free reaction with HCl as a catalyst is the fastest method yielding the desired product 7a after 18 min at 100 W with the yield of 86%.

Biological activity

All compounds were screened for their antimicrobial activities in vitro [26], [27] and the results are presented in Tables 1 and 2. The antimicrobial drugs norfloxacin, ampicillin, streptomycin and fluconazole were used as reference drugs. The results presented in Table 1 reveal that most of the synthesized fluoroquinolones effectively inhibit the growth of all tested microbial strains in vitro except Candida albicans (Ca) and Saccharomyces cerevisiae (Sc). Compound 1, which is a norfloxacin derivative carrying an ethoxycarbonylmethyl function on the piperazine ring shows the best minimum bactericidal concentration (MBC) values with <0.041 μg/mL, exhibiting equal strong antimicrobial efficacy in comparison with norfloxacin, and stronger activity than ampicillin. This compound displays moderate antifungal activity on Ca and Sc. Compounds 2 and 3, containing a hydrazide group or a 1,3,4-oxadiazole ring attached to the norfloxacin core with a methylene linker shows good MBC values in the range of 0.24–15.6 μg/mL. Table 2 reveals that compounds 2 and 3 inhibit the activity of Escherichia coli (Ec), Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef) equally to norfloxacin. Compounds 2 and 3 demonstrate better antibacterial activity than the reference drug ampicillin. Moreover, with the minimum inhibitory concentration (MIC) values of 0.24 μg/mL, these compounds exhibit a better mycobacterial inhibition on Mycobacterium smegmatis (Ms) than the standard drug streptomycin (MBC 4 μg/mL).

On the other hand, compounds 4a and 4b show excellent MBC values of 0.24 μg/mL on Ec and Ms. Compound 5a shows little activity. By contrast, the analogue 5b is highly active with the MBC values of 0.24 μg/mL that are equal to those of norfloxacin. Many compounds are more active against Ms than the standard drug streptomycin. Strong activities of 6a and 6b against several microbial strains should be noted. Several derivatives 7 and 8 also show respectable activities in comparison to the properties of the standard drugs.

Thus, most of the newly synthesized compounds exhibit strong antimicrobial efficacy in comparison with norfloxacin, and these compounds are more potent than the reference drug ampicillin. Activity of some compounds is stronger than activity of streptomycin against Ms. Comparison of our previous [24], [27], [28] and current data shows that when the fluorophenylene linker is present between the norfloxacin core and the azole unit, the antimicrobial activity decreases compared with the structure without the linker.

Synthesis

All chemicals were purchased from Fluka Chemie AG (Buchs, Switzerland). Melting points were determined in open capillaries on a Buchi B-540 melting point apparatus and are uncorrected. Progress of the reactions was monitored by TLC on silica gel 60 F254 aluminum sheets. The mobile phase was ethyl acetate and UV light was used for the detection. IR spectra were recorded in potassium bromide pellets using a Perkin-Elmer 1600 series Fourier transform-infrared (FT-IR) spectrometer. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded in dimethyl sulfoxide (DMSO)-d₆ on a Bruker Avance II 400 spectrometer. Elemental analysis was performed on a Costech Elemental Combustion System CHNS-O elemental analyzer. The mass spectra were obtained using a Quattro LC-MS (70 eV) spectrometer. Microwave-assisted reactions were performed in a CEM Discovery synthesis reactor.

7-(4-(2-Ethoxy-2-oxoethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1)

Method 1 [29] A mixture of compound 1 (25 mmol) and sodium bicarbonate (10 mmol) in dry DMF was treated dropwise at 0–5°C with ethyl bromoacetate (3.74 mL, 15 mmol). Then, the mixture was stirred for 24 h at room temperature. The progress of the reaction was monitored by TLC. The precipitate was removed by filtration and the solution was extracted with chloroform. The extract was dried with sodium sulfate and concentrated under reduced pressure. The residue was crystallized from acetone to give the title compound as a white solid.

Method 2 A solution of norfloxacin (10 mmol) and sodium ethoxide (10 mmol) in dry ethanol was irradiated in a microwave reactor in closed vessel at 100 W for 4 min. Then, ethyl bromoacetate (15 mmol) was added dropwise at 0–5°C, and the mixture was irradiated for an additional 15 min. The crude product was collected by filtration and crystallized from acetone.

Yield 74% (method 1), 98% (method 2); mp 215°C; IR (υ_max, cm⁻¹): 3055, 1735, 1625, 1614, 1254; ¹H NMR: δ 1.41 (t, J=8 Hz, 3H), 1.62 (t, J=8 Hz, 3H), 2.70 (s, 4H), 2.94 (s, 4H), 3.53 (s, 2H), 4.31 (q, J=8 Hz, 2H), 4.79 (q, J=8 Hz, 2H), 7.39 (d, J=8 Hz, 1H), 8.12 (d, J=12.7 Hz, 1H), 9.16 (s, 1H), 15.49 (s, 1H). LC-MS. Calcd for C₂₀H₂₄FN₃O₅, [M+H+Na]⁺: m/z 429.17. Found: m/z 429.25.

7-[4-(2-Hydrazino-2-oxoethyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2)

Method 1 [29] A mixture of compound 1 (10 mmol), hydrazine hydrate (30 mL) and chloroform (30 mL) was stirred at room temperature for 24 h. After treatment with ethanol, the resultant white solid was filtered off and crystallized from DMSO/H₂O (1:10).

Method 2 A mixture of compound 1 (1 mmol) and hydrazine hydrate (6 mL) was irradiated in a microwave reactor in a closed vessel with pressure control at 80 W for 15 min. The solid product obtained was crystallized from DMSO/H₂O (1:10).

Yield 78% (method 1), 94% (method 2); mp 253–256°C; IR (υ_max, cm⁻¹): 3403, 3307, 3210, 3058, 1706, 1685, 1626, 1267; ¹H NMR: δ 1.42 (t, J=8 Hz, 3H), 2.67 (s, 4H), 3.02 (s, 2H), 3.36 (s, 4H), 4.25 (bs, 2H), 4.59 (q, J=8 Hz, 2H), 7.16 (d, J=4 Hz, 1H), 7.90 (d, J=12.2 Hz, 1H), 8.95 (s, 1H), 8.99 (s, 1H), 15.35 (s, 1H). LC-MS. Calcd for C₁₈H₂₂FN₅O₄, [M+Na]⁺: m/z 414.17. Found: m/z 414.14.

1-Ethyl-6-fluoro-4-oxo-7-{4-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]piperazin-1-yl}-1,4-dihydroquinoline-3-carboxylic acid (3)

Method 1 A solution of KOH (10 mmol) in water was added to a solution of compound 2 (10 mmol) in ethanol and the mixture was heated under reflux 10 h in the presence of CS₂ (20 mmol), then cooled to room temperature and acidified to pH 6 with 37% HCl. Upon cooling in the refrigerator, the resultant solid was crystallized from acetone to give a white solid.

Method 2 A mixture of compound 2 (10 mmol), KOH (10 mmol) and CS₂ (20 mmol) in ethanol was irradiated in a microwave reactor in a closed vessel with pressure control at 150 W for 20 min. After cooling to room temperature, the mixture was acidified to pH 6 with 37% HCl. Upon cooling in the refrigerator, the resultant solid was filtered off and crystallized from acetone.

Yield 81% (method 1), 97% (method 2); mp 199–200°C; IR (υ_max, cm⁻¹): 3198, 3036, 1694, 1656, 1256; ¹H NMR: δ 1.41 (bs, 3H), 3. 16 (bs, 4H), 3.43 (m, 4H), 3.65 (s, 2H), 4.60 (s, 2H), 7.20 (s, 1H), 7.92 (d, J=12 Hz, 1H), 8.96 (s, 1H), 9.23 (s, 1H), 15.30 (s, 1H); ¹³C NMR: δ 176.7, 168.5, 166.5, 165.6, 151.9 and 154.4 (d, J=248 Hz), 149.1, 144.9 (d, J=10 Hz), 137.6, 120.2, 111.6 (d, J=12 Hz), 107.6, 106.5 (d, J=10 Hz), 61.0, 52.2, 52.0, 50.3, 49.6, 48.0, 14.9; LC MS: m/z 433.3, [M]⁺. Anal. Calcd for C₁₉H₂₀FN₅O₄S: C, 52.65; H, 4.65; N, 16.16. Found: C, 52.69; H, 4.71; N, 16.08.

General method for the synthesis of compounds 4a,b; 7a-e and 8a,b

Method 1 A secondary amine (10 mmol) was added to a solution of compound 3 (10 mmol) (for 4a,b), 6a (10 mmol) (for 7a–e) or 6c (10 mmol) (for 8a,b) in DMF containing HCl (50% mmol) and the mixture was stirred at room temperature in the presence of formaldehyde (37%, 30 mmol) for 24 h. Then, the solvent was removed under reduced pressure and the residue of the product was crystallized from DMF/H₂O (1:3).

Method 2 A mixture of a secondary amine (1 mmol), compound 6a (1 mmol) (for 7a–e) or 6c (1 mmol) (for 8a,b), HCl (50% mmol) and formaldehyde (37%, 3 mmol) was irradiated in a microwave reactor in a closed vessel with pressure control at 100 W for 7 min. The solid product was crystallized from DMF/H₂O (1:3).

General methods for synthesis of compounds 5a–c

Method 1 An isothiocyanate (20 mmol) in dry DMF was added dropwise to a solution of compound 2 (10 mmol) in dry DMF. The mixture was stirred at room temperature for 24 h, then poured into ice water. The resultant precipitate was filtered off and crystallized from an appropriate solvent.

Method 2 The mixture of an isothiocyanate (20 mmol) and compound 2 (10 mmol) was irradiated in a microwave reactor in a closed vessel with pressure control at 150 W for 5 min. The resultant solid was crystallized from an appropriate solvent.

General methods for synthesis of compounds 6a–c

Method 1 A mixture of 2N NaOH (25 mL) and compound 5 in ethanol was heated under reflux for 18 h, then cooled to room temperature and neutralized to pH 7 with 37% HCl. The resultant white solid was filtered off, washed with water and crystallized from acetone.

Method 2 A mixture of compound 5 (10 mmol) and 2N NaOH (25 mL) was irradiated in a microwave reactor in a closed vessel with pressure control at 120 W for 25 min, then cooled to room temperature and neutralized to pH 7 with 37% HCl. The white solid formed was filtered off, washed with water and crystallized from acetone.

Microorganisms

The test microorganisms were obtained from the Hifzissihha Institute of RefikSaydam (Ankara, Turkey): Escherichia coli (E. coli) ATCC35218, Yersinia pseudotuberculosis (Y. pseudotuberculosis) ATCC911, Pseudomonas aeruginosa (P. aeruginosa) ATCC43288, Enterococcus faecalis (E. faecalis) ATCC29212, Staphylococcus aureus (S. aureus) ATCC25923, Bacillus cereus (B. cereus) 709 Roma, Mycobacterium smegmatis (M. smegmatis) ATCC607, Candida albicans (C. albicans) ATCC60193 and Saccharomyces cerevisiae (S. cerevisia) RSKK 251. All newly synthesized compounds were weighed and dissolved in DMSO to prepare stock solution of 20.0 μg/mL.

Minimum inhibitory concentration (MIC) assay

The antimicrobial effects of the substances were tested quantitatively in broth media by using double microdilution to determine the MIC values (μg/mL). The antibacterial and antifungal assays were performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI, USA) at pH 7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI, USA) at pH 7.0, respectively. The microdilution test plates were incubated for 18–24 h at 35°C. Brain Heart Infusion broth (BHI) (Difco, Detriot, MI, USA) was used for Ms, and the incubation period was 48–72 h at 35°C [26]. Norfloxacin (10 μg), ampicillin (10 μg), streptomycin (10 μg) and fluconazole (5 μg) were used as reference antibacterial and antifungal drugs. DMSO with dilution of 1:10 was used as the solvent control.

Minimum bactericidal concentration (MBC) assay

The MBC assay [27] was performed in sterile 2.0-mL microfuge tubes against the test bacteria cultured overnight in MH broth. Serial dilutions of test compounds at different concentrations ranging from 0 to 125 mg/mL were prepared in the MH broth. To the test compound, 100 mL of overnight cultured bacterial suspension was added to reach a final concentration of 1.5×10⁸ cfu/mL (equal to 0.5 McFarland standard) and the mixture was incubated at 37°C for 24 h. After the incubation, MBC was determined by sampling 10 mL of suspension from the tube onto the MH agar plate and the mixture was incubated for 24 h at 37°C to observe the growth of the test organism. MBC is the lowest concentration of the test compound required to kill a particular bacterial strain. All experiments were carried out in duplicates and mean values are reported.