Synthesis, Characterization and Preliminary Biological Evaluation of New 3 and 4-nitro Isoindoline-1, 3-dione/phthalimide Analogues

Aims: To synthesize new nitroisoindoline-1,3-diones analogues and evaluate their preliminary biological activities Methodology: New isoindoline-1,3-diones analogues were synthesized by coupling phthalic anhydride derivatives with appropriate aromatic amines. Newly synthesized heterocyclic compounds were evaluated for their in vitro antibacterial activity against gram-positive bacterial strains and gram-negative bacterial strains. They were also tested for their in vitro antifungal activity against fungi strains. Determination of the preliminary antibacterial and antifungal activity were investigated using agar-dilution method. The structures of newly synthesized analogues were elucidated by 1 H and 13 C-NMR techniques. Results: Bioassay indicated that some of the newly synthesized isoindoline-1,3-dione analogues shows moderate biological activities. Conclusion: Newly or antifungal agents on modifications.


INTRODUCTION
Phthalimides belong to community of cyclic imides which are obtained by various organic synthetic processes, generally using phthalic anhydride as the precursor. Phthalimide is an important drug structural unit. Norcantharimide 1, phthalimide 2, N-substituted norcantharimide 3 are some examples of isoindole derivatives containing imide (-CO-N(R)-CO-) functional group [1] (Fig. 1).
Cyclic imides such as phthalimides possess structural features which confer biologically potent and pharmaceutically very useful. Some marketed pharmaceutical products of isoindoline are reported in Table 1.
So, considering the efficacy of N-substituted derivatives of phthalimides, we decided to synthesize 8 new N-aryl phthalimides. All of them were tested for biological activities because the literature did not record such evaluation of these compounds against gram positive bacterial strains, gram negative bacterial strains and antifungal activity against fungi strains.

MATERIALS AND METHODS
All chemicals required for the synthesis were purchased from commercial suppliers and were used without any further purification. Reactions were monitored by TLC analysis using silica gel plates (eluents petroleum ether -AcOEt in various proportions). Compounds were visualized by UV irradiation. 1 H and 13 C NMR spectra of all compounds were recorded on NMR spectrometer (600, 300 and 75 MHz). Chemical shifts are given in parts per million from TMS or deuterated solvent (DMSO-d 6 , chloroform-d) as internal standard.

Isoindoline-1, 3-dione Analogues 3a-d and 4a-d; general procedure
The target compounds (3a-d and 4a-d) were synthesized by the condensation of an appropriate phthalic anhydride with an appropriate primary aromatic amine in refluxing glacial acetic acid for 2-3 hrs. The progress of the reaction was monitored using TLC. This reaction was then quenched in cold water. The crude product was filtered and washed several times with water and then dried.

RESULTS AND DISCUSSION
The starting compound named 3-nitro phthalic anhydride (1a) was converted into 3-nitro-2-(mtolyl) isoindoline-1,3-dione (3a) by the reaction with m-toluidine (2a) in glacial acetic acid for 2-3 hrs. The compounds (3b-d and 4a-d) were synthesized by using the same reaction conditions. The chemical structures of all newly synthesized compounds were confirmed by both elemental and spectral data.

Antibacterial and Antifungal Activity
All newly synthesized heterocyclic compounds were evaluated for their in vitro antibacterial activity against gram-positive bacterial strain (S. aureus) and gram-negative bacterial strain (E. Coli). They were also tested for their in vitro antifungal activity against fungi (A. brasiliensis) strain.
Determination of the preliminary antibacterial and antifungal activity were investigated using agardilution method. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around discs in mm (Table 3). Results show that compounds 3a and 3b show antimicrobial activity against E.Coli whereas compounds 3b and 4a shows antimicrobial activity against A.Brasiliensis. Compounds 3c, 3d did not comply specification requirements and compound 4c did not show any antimicrobial activity upto 1 g/100 μl.    Determination of the preliminary antibacterial and antifungal activity were investigated using agardiffusion method (Broth Dilution Method). It is one of the non-automated in vitro bacterial susceptibility tests. This classic method yields a quantitative result for the number of antimicrobial agents that is needed to inhibit growth of specific microorganisms. It is carried out in tubes. The main advantage of the 'Broth Dilution Method' for MIC determination lies in the fact that it can readily be converted to determine the MIC as well.

Scheme. Synthesis of new nitroisoindoline-1,3-dione analogues (3a-d and 4a-d)
Both the zones of inhibition (mm) and minimum inhibitory/fungicidal concentration (MIC) (μg/ml) of the investigated compounds were recorded and compared with Gentamycin, Ampicillin, Chloramphenicol, Ciprofloxacin, Norfloxacin as antibacterial and Nystatin, Greseofulvin as antifungal reference medications. DMSO was used as vehicle to get desired concentration of drugs to test upon Standard bacterial strains.
The investigation of antibacterial screening data is summarized in Table 4. Results show that MIC value less than that of standard drug were considered promising, results reveal that compound 4b exhibited higher activity (100 μg/mL) against S. Aureus while compound compound 4d possessed pronounced activity against E. coli.

CONCLUSION
It is concluded that new isoindoline-1,3-diones analogues were synthesized by coupling phthalic anhydride derivatives with appropriate aromatic amines.
Newly synthesized heterocyclic compounds were evaluated for their in vitro antibacterial activity against gram-positive bacterial strain and gram-negative bacterial strain. Newly Synthesized compounds show moderate antibacterial and antifungal activities.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It's not applicable.

ETHICAL APPROVAL
It's not applicable.