Ammonium chloride catalyzed synthesis of novel Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitriles and evaluation of their antimicrobial and anti-breast cancer activities

Background Indolinone and spiro-indoline derivatives have been employed in the preparation of different important therapeutic compounds required for treatment of anticonvulsants, antibacterial, Antitubercular, and anticancer activities. Schiff bases have been found to possess various pharmacological activities such as antitubercular, plant growth inhibiting, insecticsidal, central nerve system depressant, antibacterial, anticancer, anti-inflammatory, and antimicrobial. Mannich bases have a variety of biological activities such as antibacterial and antifungal activities. Results In this study, a green, rapid and efficient protocol for the synthesis of a new series of Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives using ammonium chloride as a very inexpensive and readily available reagent. The prepared compounds were assessed in vitro for their antimicrobial activity. Also, the cytotoxic activity of the prepared compounds was assessed in vitro against human cells line MCF7 breast cancer. Conclusion Good activity was distinguished for Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitriles, with some members recorded higher antimicrobial and anti-breast cancer activities.Graphical abstract Novel Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitriles


Background
The development of eco-friendly and environmentally benign catalytic systems is one of the main themes of modern organic synthesis. Ammonium chloride (NH 4 Cl) is a very inexpensive and readily available catalyst; it has been reported as a catalyst for the synthesis of various heterocyclic compounds (Shaabani et al. 2003;Dabiri et al. 2009;Fortenberrya et al. 2013;Foroughifarab et al. 2011;Maleki and Salehabadi 2010;Shaabani et al. 2008;Hussein 2015). There are many bioactive molecules which possess various heteroatoms such as nitrogen, sulfur and oxygen, always taken the attention of chemists over the years mainly because of their biological significance.
Pyrano derivatives have well-known biological effects, such as analgesic and anti-inflammatory activities (El-Zohry et al. 2008a). Indolinone and spiro-indoline derivatives have possessed broad-spectrum therapeutic activities such as anticonvulsants (Ragavendran et al. 2007;Azam et al. 2009;Sridhar et al. 2002), antibacterial (Rahman et al. 2004;Singh and Luntha 2009;Olomola and Bada 2009), Antitubercular (Sriram et al. 2005), and anticancer activities (Vine et al. 2007;Solomon et al. 2009;Wee et al. 2009). Schiff bases are important compounds owing to their wide range of biological activities and industrial applications. They have been found to possess various pharmacological activities such as antitubercular (Kascheres 2003), plant growth inhibiting (Simunek and Machácek 2010), insecticsidal (Shams et al. 2011;Boyd 1988), central nerve system (CNS) depressant (Drews 2000), antibacterial (Maren 1976;Supuran and Scozzafava 2000), anticancer (Simunek et al. 2007), anti-inflammatory, and antimicrobial (Abbate et al. 2004;Abdel-Mohsen and Hussein 2014). Moreover, Mannich bases are reported to show a variety of biological activities, such as antibacterial and antifungal activities (Singare and Ingle 1976;Huneck et al. 1993;Hussein et al. 2015a). Based on these prior observations, we postulated that a Schiff base containing both indoline and pyran pharmacophores could be very effective for antimicrobial and anticancer activity. In this paper and as a consequence of our previous work on the green synthesis of different spiroheterocyclic (Hussein 2013;Hussein and El-Khawaga 2012;Hussein 2012;El-Zohry et al. 2008b, c, 2009, and bioactive heterocyclic compounds (Hussein et al. 2015b; Hussein and Abdel-Monem 2011), we investigated a novel green and efficient protocol that was developed for the synthesis of some Schiff bases (5a-l) by the condensation of spiro[indoline-3,4′-pyran]-3′carbonitrile derivatives (3a-c) with aromatic aldehydes (4a-d) using ammonium chloride (10 mol%) in refluxing ethanol as shown in Scheme 2 and Table 1. The antimicrobial and cytotoxic properties of the prepared compounds were screened.

Effect of the reaction conditions
In our initial study, we tried to optimize the model procedure mentioned above by detecting the efficiency of different reaction conditions in the absence and presence of catalysts, such as AcOH, MeOH, EtOH, DMF/AcOH, EtOH/AcOH, EtOH/Et 3 N, EtOH/piperidine, dioxane/ NH 4 Cl, DMF/NH 4 Cl, MeOH/NH 4 Cl, and EtOH/NH 4 Cl (Scheme 3).
In each case, the reactants (10 mmol) were allowed together in 10 mL solvent at reflux temperature for 2 h. In the absence of catalyst, the reaction proceeded with comparatively lower reaction yield (Table 2, entries 1-3). DMF/AcOH, EtOH/AcOH, EtOH/Et 3 N and EtOH/ piperidine can push the reaction towards the formation of product in yields of 52, 61, 71, and 71 %, respectively (Table 2, entries 4-7). In the presence of ammonium chloride (NH 4 Cl) the reaction was possible and the product (5a) was obtained in good yields. Ammonium chloride was used in different reaction media such as dioxane, DMF, methanol and ethanol (Table 2, entries 8-11). The best results were obtained when NH 4 Cl was used as catalyst in ethanol as reaction medium, which provided a yield of 92 %.

Evaluation of catalytic activity of ammonium chloride
To determine the appropriate concentration of the catalyst used, we investigated the model reaction at different concentrations of NH 4 Cl (5, 10, 15, 20, and 25 mol%). The product was formed in 80, 92, 92, 89, and 85 % yield, respectively (Table 3). This indicates that 10 mol% NH 4 Cl is sufficient to carry out the reaction smoothly.
The structures of the isolated new products 5a-l were deduced by analyzing their physical and spectroscopic data, such as the data obtained using IR, 1 H NMR, and 13 C NMR spectroscopy. Taking 5a as an example, broad absorption band at 3356 cm −1 for OH group, sharp absorption band at 2210 cm −1 for CN group, and two absorption band at 1735, 1620 cm −1 for two C=O groups were observed in the IR spectrum with absence of absorption bands at 3350, 3260 cm −1 which corresponding to NH 2 group. The 1 H NMR spectrum showed the presence of triplet and quartet signals at 1.28, and 3.85 for ethyl protons, as well as, four singlet signals at δ = 2. 28, 5.30, 8.15, and 10.38 ppm for the methyl, methylene, methane, and OH protons, respectively. In the 13 C NMR spectrum, the quaternary spiro carbon typically appeared at δ = 48.9 ppm. The nitrile and two carbonyl carbons resonated at 117.4, 164.4, and 178.5 ppm, respectively.

Biological activity Antimicrobial activity
In view of biological significance, it was studied the synthesized some spiro-indoline derivatives as previous, to get the activities of the potent compounds and evaluated their potential in vitro as antibacterial, antifungal and antitumor activities.
Antimicrobial activities of all the synthesized Schiff bases 5a-l were done by cup-plate agar diffusion method. The compounds were prepared in DMSO and evaluated them for their in vitro antibacterial and antifungal activities against Bacillus subtilis and Fusarium moniliforme respectively. The bacterial isolate was grown on nutrient agar (37 °C, 24 h) the fungus was grown on potato dextrose agar plates (26 °C, 48-72 h). The results were noted  (Table 4).
All the synthesized compounds 3a-c and 5a-l were tested for in vitro antibacterial activity by inhibition zone method against the reference compound amoxicillin (20 mm). It has been observed that all the compounds tested showed mild to moderate activity against tested bacterium but 5f, 5g and 3a. The antifungal activity of the compounds was studied with F. Moniliforme. The results are summarized in Table 4. Fluconazole has been used as reference for inhibitory activity (18 mm) against fungi and some tested compounds showed lesser activity to standard against the tested fungi. While the others showed no antifungal activities against the fungus.

In vitro anticancer activity
Antitumor activities were found moderate effective as screened for in vitro cytotoxicity activity against human cancer cells line MCF7 breast cancer (Table 5). Although the positive impact of each of the synthesized compound conducted toxicity in cells, some lost IC 50 in the concentrations used. Viewing of the results, the IC 50 required was higher than that of the reference compound (3.8 µg/ mL) used in the analysis. There are no significant differences between the results of the synthetic chemical compounds compared to the reference compound, where statistically significant differences is the numerical value, and therefore all synthesized compounds located with reference drug in one hand.
It is worth mentioning, that the curve of the compound 5c only showed clear a straight line. A high concentration compared to the control has shown. Theoretically an expectation of the IC 50 may be located on the curve. Statistically the range of lethal concentrations IC 50 may be at about 70 µg/mL, concentration that's when kills ninety percent of the living cells.

Conclusion
The authors have developed a green, rapid and efficient protocol for the synthesis of a new series of Schiff bases from spiro[indoline-3,4′-pyran]-3′-carbonitrile derivatives using ammonium chloride as a very inexpensive and readily available reagent. The prepared compounds were assessed in vitro for their antibacterial activity against B. subtilis as well as antifungal activity against F. moniliforme. Also, the cytotoxic activity of the prepared compounds was assessed in vitro against human cells line MCF7 breast cancer.

Antibacterial activity
The newly synthesized spiro-indoline derivatives 3a-c and 5a-l were screened for their antibacterial activity against bacterial isolate namely B. subtilis by inhibition zone method against the reference compound amoxicillin (20 mm). The bacterial subcultures (18-24 h grown) were added to sterilize nutrient agar medium and shaken thoroughly to ensure uniform distribution of organism throughout the medium. In sterilized Petri dishes containing about 20 mL of the medium, wells were made with a sterile cork borer and were filled with 0.1 mL of respective solution. Then, the Petri dishes were kept for incubation in an inverted position for 24-48 h at 37 °C in an incubator. When growth inhibition zones were developed, diameter (in mm) was measured and compared with that of amoxicillin.

Antifungal activity
The newly synthesized spiro-indoline derivatives 3a-c and 5a-l were screened for their antifungal activity against fungus F. moniliforme at the concentration levels of 50 µg/mL by inhibition zone method. Fluconazole has been used as reference for inhibitory activity (18 mm) against fungi. To the sterilized potato dextrose agar medium, subculture of fungus were added and shaken thoroughly to ensure uniform distribution and incubated for 72 h. Then, this was poured into sterilized and labeled Petri dishes and allowed to solidify. Wells were made in each plate by a cork borer. Each well was filled with 0.1 mL of test solution and the other with respective concentrations of standard dilutions. The plates were left 2-3 h for diffusion and incubated at 37 °C for 24 h. The diameter of the zones of growth inhibition was measured and compared with that of standard. The solutions of required concentration (50 μg/mL) of test compounds were prepared by dissolving the compounds in DMSO.

Anticancer activity
Breast cancer cell line (MCF7) as human tumor was used in this study. The cytotoxicity was measured in vitro for the newly synthesized compounds assay using the method of Philip et al. (1990). The in vitro anticancer. Screening was done by the pharmacology unit at Pharmacology unit, Cancer biology department, the National Cancer Institute, Cairo University. Cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the compound(s) to allow attachment of cell to the wall of the plate. Tested compounds were dissolved in dimethyl sulfoxide (DMSO). Different concentrations of the compound under test (0.0, 5.0, 12.5, 25.0 and 50.0 µg/mL) were added to the cell monolayer. Triplicate wells were prepared for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 h at 37 °C and in atmosphere of 5 % CO 2 . After 48 h. cells were fixed, washed and stained for 30 min with 0.4 % (W/V) SRB dissolved in 1 % acetic acid. Excess unbound dye was removed by four washes with 1 % acetic acid and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve for breast tumor cell line after the specified time. The molar concentration required for 50 % inhibition of cell viability (IC 50 ) was calculated and compared to the reference drug Doxorubicin (CAS, 25316-40-9). The surviving fractions were expressed as means and the results are given in Table 5.
Authors' contributions HFA-S analyzed the data and shared in experimental section; HAE analyzed the data, shared in the experimental section and shared in writing the manuscript; AHA performed the biological activity and shared in writing manuscript. EMH designed the research, shared in the experimental work, shared in writing the manuscript, and revised the manuscript. All authors read and approved the final manuscript.
Author details 1 Department of Chemistry, Faculty of Applied Sciences, Umm Al-Qura University, Makkah, Saudi Arabia. 2 Department of Chemistry, Faculty of Science (Girl's), Al-Azhar University, Cairo, Egypt. 3 Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza, Egypt. 4 Department of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt.