Synthesis, Characterization and Antimicrobial Evaluation of Some New Schiff, Mannich and Acetylenic Mannich Bases Incorporating a 1,2,4-Triazole Nucleus

A series of Schiff and Mannich bases derived from 4-amino-5-(3-fluoro-phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione were synthesized. The alkylation of 4-phenyl-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with propargyl bromide afforded the corresponding thiopropargylated derivative which upon treatment with the appropriate secondary amines in the presence of CuCl2 furnished the desired acetylenic Mannich bases. The synthesized compounds were characterized on the basis of their spectral (IR, 1H- and 13C-NMR) data and evaluated for their biological activities. Some of the compounds were found to exhibit significant antimicrobial activity.


OPEN ACCESS
The presence of fluorine in organic molecules often results in unexpected biological activity, which is rationalized as being due to their higher lipholicity which enhances the rate of penetration and transport of the drug to an active site [7,8]. Furthermore, the incorporation of a Schiff base moiety within the 1,2,4-triazole ring gave compounds with enhanced biological activities [9,10]. On the other hand, Mannich bases of 1,2,4-triazoles have gained importance due to their biological properties such as anticancer, antifungal, anti-inflammatory and antimalarial activities [11][12][13][14][15]. Among these, some 1,2,4-triazole Mannich bases incorporating N-methylpiperazine or morpholine moieties were recently found to possess antimicrobial activity [16][17][18]. In view of these facts and in an attempt to design and synthesize some novel fluorine-containing 1,2,4-triazoles with improved biological activity, a new series of Schiff, Mannich and acetylenic Mannich bases bearing fluorophenyl-1,2,4-triazole moieties were synthesized with a view to explore their potential as better antibacterial and antifungal agents.

Chemistry
The reaction sequences employed for the synthesis of title compounds are shown in Schemes 1-3.
The key intermediate 4-amino-5-(3-fluorophenyl)-2H-1,2,4-triazole-3-thione (1) was synthesized in 85% yield by the fusion of 3-fluorobenzoic acid with thiocarbohydrazide for 20-25 min (Scheme 1). The resulting triazole 1 was identical to that previously obtained via multi-steps synthesis [19]. The structure of the triazole 1 was confirmed by IR, 1 H-NMR, 13 C-NMR, and elemental analysis. In the IR spectrum of compound 1, the NH group of the triazole ring was observed at 3227-3373 cm −1 , thus confirming the formation of the aminotriazole. The absorption band observed at 1284 cm −1 could be attributed to the C=S group. The 1 H-NMR spectrum of compound 1 showed two singlets at δH 5.82 and 13.80 ppm and a multiplet at δH 7.32-7.93 ppm corresponding to NH2 and NH protons, as well as the phenyl protons, respectively. The condensation of the aminotriazole 1 with 4-fluorobenzaldehyde and/or 3,4-difluorobenzaldehyde in the presence of a catalytic amount of hydrochloric acid gave Schiff bases 2a and 2b in good yields (Scheme 2).
All Schiff bases displayed IR, 1 H-and 13 C-NMR absorptions and elemental analyses consistent with the assigned structures. In the IR spectra of compounds 2a and 2b, the most characteristic absorptions were observed at 3288-3315 cm −1 (N-H), 1604-1614 cm −1 (C=N) and 1280-1298 cm −1 (C=S). Lack of resonances attributable to NH2 protons and the appearance of a sharp H-C=N group singlet at δH 9.79-9.89 in their 1 H-NMR spectra agreed with the formation of Schiff bases. The 13 C-NMR signals at δc 160.55-161.86 ppm were due to the azomethine-carbon. Moreover, the C=S group resonated at δC 164.34-165.91 ppm, thus confirming the presence of Schiff bases 2a and 2b in the thione form. Mannich reactions on 3,4,5-trisubstituted-1,2,4-triazole which exist as thiol-thione tautomers gave the new Mannich bases 3a-3f, 4a-4b, and 5a-5b via aminomethylation of the endocyclic nitrogen (N-2) of the triazole ring with formaldehyde and the appropriate secondary amine in ethanol (Scheme 3).The structural assignments of Mannich bases 3a-3f, 4a-4b and 5a-5b were based on their elemental analysis and spectral (IR, 1 H-NMR and 13 C-NMR) data. In the 1 H-NMR spectra of compounds 3a-3f, the N-CH2-N protons resonated as singlet at δH 5.14-5.30 ppm integrating for two protons. The -CH2-O-CH2 protons corresponding to the morpholine ring resonated as a triplet at δH 3.59 ppm (J = 4.4 Hz) in 3a and at 3.65 ppm (J = 4.6 Hz) in 3b, while the -CH2-N-CH2protons of the morpholine ring resonated as a triplet at δH 2.77 ppm (J = 4.4 Hz) and 2.82 ppm (J = 4.6 Hz), respectively. The methyl protons of 3e and 3f appeared as singlets at δH 2.12 and 2.16 ppm, respectively.
In addition, the 1 H-NMR spectrum of compound 4a showed a characteristic singlet integrating for four protons at δH 5.19 ppm attributed to two N-CH2-N groups ( Figure 1) which appeared as a multiplet at δH 5.11-5.30 in the 1 H-NMR spectrum of compound 5b (Figure 2).    The disappearance of the NH stretch at 3345 cm −1 in the IR spectrum of compound 9 and appearance of the characteristic C≡C and ≡C-H bands at 2145 and 3290 cm −1 , respectively, confirmed the formation of thiopropargylated triazole 9. In the 1 H-NMR spectrum of compound 9, a triplet corresponding to the ≡C-H group was observed at δH 2.26 ppm and a doublet at δH 4.01 ppm integrating for two protons of SCH2 group and a peak at δC 79.85 ppm due to C≡C in the 13 C-NMR spectrum confirmed the formation of compound 9. The IR, 1 H-NMR, 13 C-NMR and elemental analysis data of compound 9 was in agreement with the assigned structure. The acetylenic Mannich bases 10a-10c were synthesized in one pot multi-component Mannich reaction involving the thiopropargylated triazole 9, formaldehyde, CuSO4 and the appropriate secondary amine in refluxing dioxane. The structures of the newly synthesized acetylenic Mannich bases 10a-10c have been established on the basis of their elemental analysis, IR, 1 H-NMR, and 13 C-NMR data. The IR spectra of compounds 10a-10c showed characteristic C≡C group bands at 2148-2152 cm −1 . In addition, their 1 H-NMR spectra showed two singlets at δH 3.43-3.46 and 4.01-4.05 ppm characteristic for C-CH2-N and SCH2 groups, respectively. The 1 H-NMR spectrum of compound 10a gave signals at δH 2.81 and 3.62 ppm characteristic for NCH2 and OCH2 morpholine protons, respectively. Moreover, the 1 H-NMR spectrum of compound 10c gave a characteristic singlet at δH 2.24 ppm integrating for three protons of NCH3 group which resonated at 42.35 ppm in its 13 C-NMR spectrum.

Antibacterial and Antifungal Activity
Both antimicrobial studies were assessed by minimum inhibitory concentration (MIC) assays carried out by the broth dilution method [20][21][22]. MIC is the highest dilution of a compound which shows clear fluid with no development of turbidity.
The antibacterial and antifungal screening revealed that some of the tested compounds showed good to excellent activity at 4-62 showed comparatively good activity against Gram positive bacterial strains at MIC 16-31.25 μg/mL and excellent activity towards fungal strains at MIC 4-8 μg/mL. The Mannich bases 3a and 3b bearing a morpholine moiety showed excellent antibacterial activity against all bacterial strains and good activity against fungal species at 16-31.25 μg/mL. On the contrary, compounds 3c and 3d possessing a piperidine exhibited good to moderate antibacterial activity but lost the activity against the tested fungal species.
The remaining compounds were found to be active at higher concentrations, e.g., 62.5 and 125 mg/mL. It was therefore concluded that the presence of a morpholine and/or piperazine moiety, in addition to 3-fluorophenyl groups, was essential for high antibacterial and antifungal activities in these compounds. The results of antibacterial and antifungal screening of the newly prepared Schiff, Mannich and acetylenic Mannich bases, expressed as MIC values, are summarized in Table 1.

General Information
Melting points were determined on a Melt-temp apparatus and are uncorrected. The 1 H-and 13 C-NMR spectra were recorded on a Bruker AC-400 NMR spectrometer operating at 400 MHz for 1 H-NMR, 100 MHz for 13 C-NMR. Compounds were dissolved in DMSO-d6 and chemical shifts were referenced to TMS ( 1 H-and 13 C-NMR). The IR spectra were measured as potassium bromide pellets using a Perkin-Elmer 1430 series FT-IR spectrometer. The elemental analyses were performed by the microanalysis unit at the Faculty of Science, Cairo University.

General Procedure for the Synthesis of Schiff Bases 2a-2b
A mixture of compound 1 (10 mmol) and the appropriate benzaldehyde derivative (10 mmol) was refluxed in ethanol (30 mL) containing HCl (1 mL) for 6 h. The solution was cooled and a yellow solid appeared. The obtained precipitate was filtered and recrystallized from ethanol to afford the desired product.

General Procedure for Preparation of Acetylenic Mannich Bases 10a-10c
To a stirring solution of compound 9 (5 mmol) in dioxane (25 mL) was added cuprous chloride (0.0025 g) and the mixture was heated for a few min, then paraformaldehyde (5 mmol) and the appropriate secondary amine (5 mmol) were added. The mixture was heated at 90 °C for four h. After cooling, the mixture was filtered then poured onto ice water (100 mL). The residue was extracted with chloroform (3 × 25 mL) and purified on a column of silica gel using ethylacetate-hexane (1:3).  (colony forming unit/mL) and incubated at 37 °C for 24 h. The growth control consisting of media and media with DMSO at the same dilutions as used in the experiments was employed.

Conclusions
New Schiff, Mannich and acetylenic Mannich bases containing 1,2,4-triazole and fluorophenyl moieties were successfully synthesized. Antimicrobial activity screening revealed that some of the tested compounds exhibited good antibacterial and antifungal activities. The combination of three biologically potent units, namely Schiff base, morpholine/piperazine and 1,2,4-triazole in one framework is essential for significant antimicrobial activity.