Synthesis of Novel β-Keto-Enol Derivatives Tethered Pyrazole, Pyridine and Furan as New Potential Antifungal and Anti-Breast Cancer Agents

Recently, a new generation of highly promising inhibitors bearing β-keto-enol functionality has emerged. Reported herein is the first synthesis and use of novel designed drugs based on the β-keto-enol group embedded with heterocyclic moieties such as pyrazole, pyridine, and furan, prepared in a one-step procedure by mixed Claisen condensation. All the newly synthesized compounds were characterized by FT-IR, 1H-NMR, 13C-NMR, ESI/LC-MS, elemental analysis, and evaluated for their in vitro antiproliferative activity against breast cancer (MDA-MB241) human cell lines and fungal strains (Fusarium oxysporum f.sp albedinis FAO). Three of the synthesized compounds showed potent activity against fungal strains with IC50 values in the range of 0.055–0.092 µM. The results revealed that these compounds showed better IC50 values while compared with positive controls.

It is emphasized that our products exist exclusively in "3-hydroxy-alk-2-en-1-one" form as confirmed by the spectral data; these tautomeric forms were also confirmed by XRD and the results will appear in due course [17]. However, two products (similar to 5 and 6) were obtained in previous work by others, in the "4-hydroxy-alk-3-en-2-one" tautomeric form under different conditions (NaH, Et2O, reflux) [18].
The β-keto-enol form is greatly favored over the β-diketone form because of the conjugation of the enol with the carbonyl group, and the stability gained, by a strong six-centered intramolecular hydrogen bond. The β-keto-enol interconversion rate (>90%) was determined using the 1 H-NMR integration of signals from the enol =C-H and the ketone CH2. Indeed, according to the NMR spectra, the parent β-diketones exist almost exclusively in the enol form and only a trace of the keto form is seen around 4 ppm. In DEPT-135, a very small negative signal from CH2 was also observed. Finally, crystals of most of the β-keto-enols were isolated from methanol by slow evaporation. Compound 6 was recently reported in a different way [19].

Biological Activities
All synthesized β-keto-enol heterocycles were evaluated for their activity against breast cancer (MDA-MB241) human cell lines using normoxic conditions [20], and against fungal strains (Fusarium oxysporum f.sp albedinis FAO) using the agar diffusion technique (ADT) [21]. It is of note that all products were also tested against three bacterial strains (Echerichia coli, Bacillus subtilis, and Micrococcus luteus), but no significant effect was observed against these organisms.
However, the results of the anticancer and antifungal effects were very significant and are given in Table 1, respectively. Most of these molecules were cytotoxic against breast cancer cell lines in a dose-dependent manner. The activity followed the structure activity relationships (SARs) and showed an interesting dependence on the substitution pattern. Considering the influence of substituent R, it was found that the phenyl residue leads to a stronger growth inhibition [18]. This was especially evident for products 6 and 10. The concentration required to induce the activity (IC50) was more pronounced Scheme 2. Proposed mechanism of the formed products.
It is emphasized that our products exist exclusively in "3-hydroxy-alk-2-en-1-one" form as confirmed by the spectral data; these tautomeric forms were also confirmed by XRD and the results will appear in due course [17]. However, two products (similar to 5 and 6) were obtained in previous work by others, in the "4-hydroxy-alk-3-en-2-one" tautomeric form under different conditions (NaH, Et 2 O, reflux) [18].
The β-keto-enol form is greatly favored over the β-diketone form because of the conjugation of the enol with the carbonyl group, and the stability gained, by a strong six-centered intramolecular hydrogen bond. The β-keto-enol interconversion rate (>90%) was determined using the 1 H-NMR integration of signals from the enol =C-H and the ketone CH 2 . Indeed, according to the NMR spectra, the parent β-diketones exist almost exclusively in the enol form and only a trace of the keto form is seen around 4 ppm. In DEPT-135, a very small negative signal from CH 2 was also observed. Finally, crystals of most of the β-keto-enols were isolated from methanol by slow evaporation. Compound 6 was recently reported in a different way [19].

Biological Activities
All synthesized β-keto-enol heterocycles were evaluated for their activity against breast cancer (MDA-MB241) human cell lines using normoxic conditions [20], and against fungal strains (Fusarium oxysporum f.sp albedinis FAO) using the agar diffusion technique (ADT) [21]. It is of note that all products were also tested against three bacterial strains (Echerichia coli, Bacillus subtilis, and Micrococcus luteus), but no significant effect was observed against these organisms.  21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM,  21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM,  21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM, for compounds 3, 6 and 10 with IC50 values of 21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM, for compounds 3, 6 and 10 with IC50 values of 21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM, for compounds 3, 6 and 10 with IC50 values of 21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM, Molecules 2015, 20, page-page 4 for compounds 3, 6 and 10 with IC50 values of 21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM,  21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity. These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC50 values of 0.055, 0.079, and 0.092 µM, However, the results of the anticancer and antifungal effects were very significant and are given in Table 1, respectively. Most of these molecules were cytotoxic against breast cancer cell lines in a dose-dependent manner. The activity followed the structure activity relationships (SARs) and showed an interesting dependence on the substitution pattern. Considering the influence of substituent R, it was found that the phenyl residue leads to a stronger growth inhibition [18]. This was especially evident for products 6 and 10. The concentration required to induce the activity (IC 50 ) was more pronounced for compounds 3, 6 and 10 with IC 50 values of 21.95, 17.62, and 18.79 µg/mL, respectively. Beside this observation, we also noted the effect of the heterocycle groups for appreciable biological activity.
These structures have also led to unexpected antifungal activity. Indeed, compounds 1, 5, and 9 with methyl in the R position had the most potent activity with IC 50 values of 0.055, 0.079, and 0.092 µM, respectively. This result was better than all the described products. We noted that the substitution of methyl in the R position was essential for this biological activity. The aryl groups strongly decreased the activity. This suggests that the marked bioactivity of the heterocyclic compounds was sensitive to modifications and could be further exploited to determine the structure activity relationship around this novel class of fungal inhibitors. Other structural modifications to these active compounds as antifungal and anti-HIV candidates are currently in progress.

General Information
All commercial reagents were analytical grade (Aldrich, purity >99%, St. Louis, MO, USA). Melting points were measured using a BUCHÏ 510 m.p. apparatus (Oujda, Morocco). 1 H-and 13 C-NMR spectra were performed on a Bruker AC 300 spectrometer (CNRS, Rabat, Morocco) (300 MHz for 1 H and 75.47 MHz for 13 C spectra). JEOL JMS DX-300 mass spectrometer (Rabat, Morocco) was used for the determination of molecular weights. Infrared (IR) spectra were recorded on a Shimadzu infrared spectrophotometer (Oujda, Morocco) using the KBr disc technique. The formazan obtained at the end of the experiment in MTT assays was measured by means of a Perkin Elmer Victor X4 Microplate reader (Brussels, Belgium).

General Procedure for the Synthesis of β-Keto-Enol Heterocycles
To a suspension of sodium (15.21 mmol) in 20 mL of toluene, the appropriate heterocyclic carboxylate (12.01 mmol) in 25 mL of toluene was slowly added; then acetone or aryl methyl ketones (12.01 mmol) in 10 mL of toluene was added at 0˝C. The resulting mixture was stirred at room temperature for two days. The precipitate formed was filtered, washed with toluene, dissolved in water, and neutralized with acetic acid to pH 5. After extraction with CH 2 Cl 2 , the organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The obtained residue was filtered through silica using CH 2 Cl 2 /MeOH as eluant to give the desired products 1-10 as a white solid in 35%-48% yield. β-keto-enol forms were recrystallized from methanol (95%) to obtain target compounds 1-10 which were confirmed by FT-IR, 1 H-NMR, 13 C-NMR, elemental analysis, and mass spectroscopy.  99;H,6.71;N,15.55. Found: C,60.11;H,6.83;N,15.43.

Anticancer Assays
Prepared compounds were screened against breast cancer (MDA-MB241) human cell lines using normoxic conditions [20]. Tests were performed in Angiogenesis and Cancer Research Lab, Institute of Experimental and Clinical Research (UCL, Brussels, Belgium).

Antibacterial and Antifungal Tests
The in vitro antifungal activities were tested by the agar diffusion technique [21] using fungal strains (Fusarium oxysporum f.sp albedinis FAO).
The results were compared with positive controls (benomyl and thiophanate-methyl).The in vitro antibacterial activities were tested using bacterial strains (Echerichia coli, Bacillus subtilis, and Micrococcus luteus).

Conclusions
In summary, we have described the first synthesis of novel β-keto-enols embedded with heterocyclic moieties and the evaluation of their in vitro anticancer and antifungal activities. Most of the compounds showed modest antiproliferative activity against breast cancer (MDA-MB241) human cell lines. Among the synthesized products, compounds 1, 5, and 9 successfully showed the most potent antifungal activity with IC 50 values in the range of 0.055-0.092 µM as compared with positive controls.