Synthesis and Evaluation of 1,3 Di-Substituted Schiff, Mannich Bases and Spiro Isatin Derivatives

Schiff bases of isatin with aminothiazole, its N-mannich bases and Spiro isatin derivatives were synthesized. Their chemical structures were confirmed by Infrared, 1H-Nuclear Magnetic Resonance data and elemental analysis. Antimicrobial evaluation was performed by the agar diffusion method against four pathogenic bacteria and two pathogenic fungi. Anti-inflammatory activity was tested by carragenin-induced rat paw edema and compounds were evaluated for analgesic action by the acetic acid-induced writhing method; Compounds Aa, Ab and A5, A6 were found to be active against bacteria and fungi. The compounds A3, A6, Aa and Ab showed anti-inflammatory activity, having a percentage protection value of 34.69, 32.65, 38.77 and 36.73 as compared with that of indomethacin, with % protection of 46.93. Similarly, the compounds Aa, Ab and A6 showed analgesic activity, with % protection of 67.51, 64.78 and 49.81 as compared with the standard with % protection of 79.56.


INTRODUCTION
Isatin (1-H indole 2-3 dione) are synthetically versatile substances that are employed for the synthesis of a large variety of heterocyclic compounds, including some drugs. Over the years, the molecules with isatin show diversified biological activities, e.g. antibacterial, [1] anticonvulsants, [2] antiviral [3] and antitubercular [4] activities. Schiff and mannich bases of isatin have shown good antimicrobial activities against gram-negative bacteria and fungi. [3] A number of Spiro compounds derived from isatin are important pharmacophores and exhibit promising biological activities, [5] such as analgesic, fungicidal and anti-inflammatory activity. Thiazole and its derivatives are reported to show good antibacterial, anti-inflammatory and analgesic [6] activity. Hence, it was thought worthwhile to synthesize new Schiff, mannich and Spiro isatin derivatives SHIMADZU FTIR-8300, spectrophotometer, Japan. The H-NMR spectra were recorded on a Bruker Advance-400 MHz spectrometer, USA. Purity of the compounds was checked by Thin layer chromatography For analgesic, anti-inflammatory activity studies, adult healthy albino rats (Swiss strain) of either sex weighing 80-120 g were used. All the animals were maintained under standard conditions and had access to pelleted animal feed and water. The study protocol was approved by the Institutional Animal Ethics Committee.

Synthesis [7] of 3(1'-3'Thiazole-2-yl Imino) 1-3 Dihydro-2H Indole-2-One (A)
Equimolar quantity of isatin and 2-amino thiazole was taken in a round bottom flask, dissolving the content with a sufficient amount of ethanol and two to three drops of glacial acetic acid was added. The content was refluxed for 4-5 h and then poured into ice cold water. The solid obtained was filtered, dried and recrystallized from ethanol.

Antimicrobial activity
The in vitro antimicrobial activity [9] was carried out against 24-h-old cultures of microorganisms by the cup-plate method. Compound A 1 -A 6 and A a , A b were tested against four pathogenic bacteria, Pseudomonas mirebelis, Pseudomonas auroginosa, Escherichia coli and Staphylococcus aureus. The antifungal activity was tested against Aspergillus niger and Candida albicans. Ampicillin and cotrimoxazole was taken as an internal standard for antibacterial and antifungal activity, respectively. The compound was tested at a concentration of 100 µ/ml in DMF against all organisms. The zone of inhibition was compared with the standard drug after 24-h incubation at 37°C for antibacterial and 48 h at 25°C for antifungal activity.

Analgesic activity
The analgesic activity [10] was evaluated by the acetic acidinduced writhing test [6] using adult Swiss albino mice of either sex. In this method, mice are made to writhe by a simple intraperitonial injection of 0.6% v/v aqueous acetic acid (0.1 ml/kg). Test substances were administered 30 min before the injection of acetic acid. The number of writhes (full extension of hind paws) was recorded.

Anti-inflammatory activity
The anti-inflammatory activity [10] was evaluated by rat paw edema. Edema represents the early phase of inflammation and carragenin-induced paw edema is the simplest and most widely used method [8] for studying the antiinflammatory activity of the chemical compounds. This method is based on the plethysmographic measurement of carragenin-induced acute rat paw edema. For this study, Wister rats of either sex, weighing between 80 and 100 g were divided into 10 groups of five animals each. Group1 serves as a solvent control, and received 0.5% Carboxy Methyl Cellulose in normal saline orally. Group 2 received indomethacin (10 mg/kg) in solvent as a standard. Groups 3-10 received test drugs at a dose of 100 mg/kg orally. These drugs were administered 1 h before the injection of an irritant, carragenin. After 1 h, all the animals were injected subcutaneously with a suspension of carragenin in CMC solution (0.1 ml) to the left hind paw in the subplantar region and the paw volume was measured immediately. After 3 h, the paw volume was measured for the control, standard and test groups. Percentage inhibition of paw volume was calculated.

RESULTS AND DISCUSSION
The structures of newly synthesized compounds were elucidated by IR, 1 H-NMR and elemental analysis and are reported as follows: The IR spectrum of A in KBr (in cm        Evaluation of 1,3 Di-substituted schiff, mannich bases and spiro isatin Values are expressed as mean ± SE (n = 6). *P<0.05, **P<0.01 and ***P<0.001 compared with vehicle control (ANOVA followed by Dunnet's t-test).  The characterization of synthesized compounds is given in Table 1. Analytical data of the compounds supports the proposed structures.
Results of antimicrobial activity, analgesic activity and antiinflammatory activity are shown in Tables 2-4, respectively. The results of the antimicrobial activity revealed that the compounds A 3 , A 5 , A 6 , A a and A b were found to be active against bacteria and fungi. The compounds A 3 , A 6   Values are expressed as mean ± SE (n = 6). *P<0.05, **P<0.01 and ***P<0.001 compared with vehicle control (ANOVA followed by Dunnet's t-test).
Thus, it is observed that the synthesized compounds showed promising antimicrobial, analgesic and antiinflammatory activities. Further studies are needed to discover new novel compounds of this class with profound activities.