Microwave-Assisted Synthesis and Antimicrobial Evaluation of Novel Spiroisoquinoline and Spiropyrido[4,3-d]pyrimidine Derivatives

Bromination of N-substituted homophthalimides and tetrahydropyrido[4,3-d]-pyrimidine-5,7-diones produces 4,4-dibromohomophthalimide and 8,8-dibromo-tetrahydropyrido[4,3-d]pyrimidine-5,7-dione derivatives, respectively, that can be used as precursors for spiro derivatives. The dibromo derivatives react with different binucleophilic reagents to produce several spiroisoquinoline and spirotetrahydropyrido[4,3-d]-pyrimidine-5,7-dione derivatives, respectively. Reaction of the dibromo derivatives with malononitrile produces dicyanomethylene derivatives which react with different binucleophiles to produce new spiro derivatives. All new compounds are prepared by using the usual chemical conditions and microwave assisted conditions. The latter conditions improved the reaction yields, reduced reaction times and ameliorated the effects on the surrounding environment as the reactions are carried out in closed systems. Structures of the newly synthesized compounds are proved using spectroscopic methods such as IR, MS, 1H-NMR and 13C-NMR and elemental analyses. Some of the newly synthesized compounds were tested for their antimicrobial activities, whereby four of them showed moderate activities and the rest showed low or no activities towards the investigated species.


Introduction
Spiro compounds constitute a group of generally less investigated compounds, however, recently growing efforts have been made to synthesize and characterize these compounds. Many spiro compounds possess very promising biological activities as anticancer [1,2], antibacterial [3,4], anticonvulsant [5][6][7], antituberculosis [8], anti-Alzheimer's [9], pain-relief [10,11] and antidermatitis agents [12]. In addition to their medical uses, some spiro-compounds have found other uses in the agricultural and industrial fields. For example, they are used as antifungal agents [13], pesticides [14], laser dyes [15] and electroluminescent devices [16]. Spiro compounds have also been used as antioxidants [17,18]. Our research group is interested in using the microwave technique [3,[19][20][21][22][23][24][25], as it has several advantages over conventional methods of synthesis, such as reduced reaction times, fewer effects on the environment and better reactions. In the present research, we used both the microwave technique as well as conventional methods to prepare some new spiro compounds that were then tested for their antimicrobial activities.

General
Melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on Shimadzu FTIR-8201PC spectrophotometer (Giza, Egypt). 1 H-NMR and 13 C-NMR spectra were recorded on a Varian Mercury 300 MHz or Varian Gemini 200 MHz spectrometers (Giza, Egypt) using TMS as an internal standard and DMSO-d6 as solvent. Microwave reactions were performed with a Millstone Organic Synthesis Unit with touch control terminal (MicroSYNTH, Giza, Egypt) with a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The temperature of the reaction mixture was monitored using a calibrated infrared temperature control under the reaction vessel, and control of the pressure was performed with a pressure sensor connected to the septum of the vessel. Elemental analysis was carried out at the Microanalytical Center of Cairo University, Giza, Egypt.

2-Aryl-4,4-dibromoisoquinoline-1,3-(2H,4H)dione Derivatives 3a,b
A solution of either of 2a (2.37 g, 0.01 mol), 2b (2.51 g, 0.01 mol) or 2c (2.72 g, 0.01 mol) in glacial acetic acid (20 mL) was heated under reflux with bromine (1.1 mL, 3.0 g, 0.02 mole) for 2 h. After cooling, the reaction mixture was poured onto ice-water and the solid that precipitated was filtered off, dried and crystallized from the proper solvent.  (25 mL) and few drops of piperidine for 5 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.
Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 15 min. The reaction mixture was treated similar to method A to obtain compounds 5a-d.    Method A: Each of compounds 3a,b (0.01 mol), was heated under reflux with thiosemicarbazide (0.91 g, 0.01 mol), absolute ethanol (25 mL) and few drops of piperidine for 4 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.

2'-(4-Chlorophenyl
Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 10 min. The reaction mixture was treated similar to method A to obtain compounds 6a,b.     to give 9a,b and 10a,b. Method B: The same reactants of method A were heated in microwave oven at 500 W and 140 °C for 5 min. The reaction mixture was treated similar to method A to obtain compounds 9a,b and 10a,b.     (25 mL) and few drops of piperidine for 2 h. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.

Antimicrobial Screening
The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against the following microorganisms: (a) Gram-negative: Escherichia coli and Pseudomonas putide; (b) Gram-positive: Bacillus subtilis and Streptococcus lactis; (c) Fungi: Aspergillus niger and Penicillium sp.; (d) Yeast: Candida albicans. Media: Three types of specific media were used in this study: Medium 1: Nutrient Medium for bacteria, consisting of (g/L distilled water): peptone, 5 and meat extract, 3. pH was adjusted to 7.0.
For solid media, 2% agar was added. All media were sterilized at 121 °C for 20 min. [27] Proper concentrations of microbial suspensions were prepared from 1 (for bacteria) to 3 (for yeast and fungi)-day-old liquid stock cultures incubated on a rotary shaker (100 rpm). In the case of fungi, five sterile glass beads were added to each culture flask. The mycelia were then subdivided by mechanical stirring at speed No. 1 for 30 min. Turbidity of microorganisms was adjusted with a spectrophotometer at 350 nm to give an optical density of 1.0. Appropriate agar plates were aseptically surface inoculated uniformly by a standard volume (ca. 1 mL) of the microbial broth culture of the tested microorganism, namely E. coli, P. putida, B. subtilis, S. lactis, A. niger, Penicillium sp. and C. albicans. Whatman No. 3 filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at 121 °C. Test compounds were dissolved in 80% ethyl alcohol to give final concentration of 5 μg/mL. The sterile discs were impregnated with the test compounds (5 μg/disc). After the impregnated discs have been air dried, they were placed on the agar surface previously seeded with the organism to be tested. Discs were gently pressed with forceps to insure thorough contact with the media. Three discs were arranged per dish, suitably spaced apart, i.e., the discs should be separated by a distance that is equal to or slightly greater than the sum of the diameters of inhibition produced by each disc alone. Each test compound was conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to permit good diffusion before transferring them to an incubator at 37 °C for 24 h for bacteria and at 30 °C for 72 h for yeast and fungi.