SYNTHESIS AND EVALUATION OF NEW BIS ISATIN MANNICH BASE DERIVATIVES AGAINST ANTIBACTERIAL ACTIVITY

In the year 1841, Erdmann¹ and Laurent² isolated isatin for the first time as a byproduct of oxidizing indigo with nitric and chromic acid.  Isatins, or 1H-indole-2,3-dione, are flexible substrates that can be used to synthesize a wide range of heterocyclic compounds.Isatin's C-3 carbonyl group has a high electrophilic nature.  Because of this, isatins are easily engaged in addition and condensation reactions into 3-substituted oxindoles³ with nucleophiles of the carbanion type. It can be found in a tautomeric form. It is a special molecule with two carbonyl groups at locations 2 and 3 and a nitrogen atom at position 1. It also includes ketocarbonyl and amide groups.  In addition, it has an aromatic ring and an active hydrogen atom linked to either nitrogen or oxygen (Fig.).

Source of isatin

This molecule is present in a variety of plants, including isatis⁴ tinctoria, Calanthe discolor LINDL⁵, and Couroupita guianesis Aubl⁶. As a metabolic product of adrenaline^7-9, it is also present in humans. It is found in fungus species, including Streptomycetes albus¹⁰ and Chetomium glolbusam¹¹. It is also a component of the secretion from the parotid gland of Bufo frogs¹². Coal tar¹³ is another source of it.

SYNTHESIS OF ISATIN

SANDMEYER METHADOLOGY¹⁴

Sandmeyer is the one who created this approach. It is the most traditional and widely applied technique for isatin synthesis. In this case, aniline reacts with chloral hydrate and hydroxylamine hydrochloride in aqueous sodium sulfate to generate isonitrosoacetanilide. This isonitrosoacetanilide, once isolated, reacts with concentrated sulfuric acid to yield isatin in a yield of greater than 75%.

Mannich Reaction¹⁵

In the Mannich reaction, isatin and substituted isatins with formaldehyde yield hydroxymethylisatins; in the absence of an amine, isatin and various amines react with formaldehyde to yield their respective Mannich bases.

Introduction to antibacterial activity^16-17

• An antimicrobial is a substance that either prevents or eliminates germs.  Antimicrobial drugs can be categorized based on the bacteria they mostly target.
• Antibiotics are used to treat bacteria, while antifungals are used to treat fungus.
• The discovery of antibiotics marked a turning point in human history and transformed medical science for the treatment of persistent bacterial, fungal, and parasitic infections.

Causes

Virus – causes dengue, influenza

 Fungi – cause lung and skin infections

 Bacteria – cause cholera

Mechanism

It affects Ionophores' capacity to pass through cell membranes; interferes with the synthesis of cell walls; interferes with the synthesis of DNA, and also interferes with the synthesis of proteins (Fig 2).

METHODOLOGY

The synthesis of designed bis-isatin Mannich base derivatives is depicted below in the scheme.

Synthesis of 5, 5’- Methylene bis(indoline-2,3-dione) (III):

To dissolve 5 grams of 4, 4-Methylenedianiline in 300 milliliters of water, 10 milliliters of conc. HCL was introduced in a 250-milliliter beaker (A). Another 100 ml beaker (B) was filled with 60 ml of distilled water and 18 ml of chloral hydrate.  After that, A and B are combined. Anhydrous sodium sulfate was added to this mixture and stirred until it precipitated. One more 500 ml beaker (C) was filled with 24 g of hydoxylamine hydrochloride, and 300 ml of distilled water was added.  It is heated on a water bath for 45 minutes till the ppt is measured, and then added to the (A+B) solution. Then set aside for filtering for 12 hours, rinsed with water until acid-free, and then dried. A gram of the dried product was taken and, while keeping the temperature between 60 and 800 °C, mixed to 8 milliliters of Conc.H2SO4. After 10 minutes of being held on mantle at 50 ^OC, crushed ice was added to obtain the PPT. Filtered, dried, and recrystallized product was used.

 Synthesis of   bisisatin Mannich base derivatives(V):

A few drops of formaldehyde were added to ethanol together with compound 5,5?-Methylene bis(indoline-2,3-dione) (III, 0.01 mole) and secondary amine (IV, 0.02 mole), which were then swirled for eight hours. Thus, by recrystallizing from an appropriate solvent, the product 5,5'-Methylenebis(indoline-2,3-dione)-1,1-2oamine derivatives were filtered and purified.

PHARMACOLOGICAL EVALUATION:

It has been intended for me to assess the novel bis-isatin Mannich base derivatives due to their diverse biological and pharmacological significance. A sequence of derivatives of the bis-isatin Mannich base for the subsequent biological activity using conventional procedures. Antibacterial activity using the agar well diffusion method, with the zone of inhibition measured in millimeters.

PREPARATION OF BACTERIAL CULTURES OF ASSAY:

Nutrient broth medium was used to subculture the test organisms. The corresponding bacterial strains were added to the tubes holding the sterilized media. Following a 24-hour incubation period at 37±1ºC, they were refrigerated. The stock cultures were preserved. A loop full of culture was transferred to nutrient broth in conical flasks to create bacterial inoculums. Prior to the experiment, the flasks were infected for 48 hours at 37±1ºC.

THE SAMPLE PREPARATION:

In order to prepare the test chemicals for assay, they were dissolved in dimethyl sulfoxide at the necessary concentrations, yielding 50µg/ml, 100µg/ml, and 500µg/ml for assessment, respectively. Ciprofloxacin was prepared as a reference standard at the same quantities as test compounds, with dimethyl sulfoxide serving as the control, for both gram-positive and gram-negative microorganisms.

RESULTS AND DISCUSSION                                 

Antibacterial activity

• The preliminary studies on antibacterial activity of the new bis-isatin Mannich base derivatives have generated some interesting data.  An attempt has been made to interfere with the outcome of the present studies based on this data.
• All the synthesized new bis isatin derivatives were evaluated for antibacterial activity by using standard ciprofloxacin
• All these bis-isatin Mannich base derivatives employed in the antibacterial activity by dissolving in methanol in the required quantity in concentration, making 50 µg/ml, 100 µg/ml, 300µg/ml, respectively, for evaluation of antibacterial activity against gram-positive and gram-negative bacteria.
• Among all these synthesized compounds, the following Ve, Vh, Vc, Vf showed high activity for antibacterial activity when compared with standard ciprofloxacin.

Table 1: Antibacterial activity of Klebsiella pnemoniae

Table 2: Antibacterial activity of Staphylococcus aureus

Table 3: Antibacterial activity of Bacillus subtilis

Table 4: Antibacterial activity of Escherichia coli

Table5: PHYSICAL DATA

Table6:Antibacterial  activity

Fig1.  Keto-enol tatomersim

Fig 2. Mechanism of antibacterial activity

SPECTRAL DATA

IR (KBr cm^-1):   3555.39 (-NH,str), 2725.24(CH-Aliphatic,str), 1619.78(C=O,str),  1007.58(C-O,str).

NMR:1HNMR (CDCl_3,400MHZ):δ(ppm) 3.49(q,alic- CH₂),2.40 (q,alic- CH₂), 4.70(d,ali-                                                CH₂), 3.83(s,ali-CH₂),7.83(t,ar-  CH₂) _,7.79   (d,ar-CH), 7.38(d,ar-CH).

MASS: M⁺¹  peak is observed at 505