INTRODUCTION:

Isatin is considered as one of the non-homogeneous cyclic compounds, which is named according to IUPAC as 1H-indole-2,3-dione. It is an indole derivative containing two ketone groups, composed of a pyrrole ring attached to a benzene ring, having the formula shown in (Figure 1). It was first synthesized in 1841 by researchers Erdmann and Laurent through the oxidation of indigo dye in the presence of chromic acid and nitric acid ^1,2.

Figure 1. Chemical formula of Isatin (1H-indole-2,3-dione)

Since its discovery, a lot of research has been conducted to study its composition and chemical properties. Biological and industrial applications of isatin have also been acheived, where isatin and its derivatives have been used in many medicines synthesis, such as Thiosemicarbazide, which has been used in the treatment of Human Immunode-ficiency Virus (HIV effect)^{3,4,5 -11}. Isatin has various biological effects such as antibacterial effect, antioxidant effect, central nervous system ,depressant effect, sedative effect, anticonvulsant effect, anti-cancer effect, and anti-HIV effect ^3,4,6-12. The presence of several active sites in isatin makes it susceptible to several chemical reactions. The two carbonyl groups in it, especially the one at carbonyl carbon, make it contribute in many condensation reactions, such as the Schiff reaction with primary aromatic amines. Isatin can also participate in N-alkylation and N-acylation reactions due to the presence of the secondary amine group. ^13,14,15. A Mannich reaction that linked the triazole rings on the nitrogen atom to Isatin produced several of Isatin and triazole derivatives. The effectiveness of these compounds as antibacterial agents was evaluated against ampicillin and nystatin, which were used as a standard. The synthesized Mannich bases showed higher activity against Gram-negative bacteria comparing to Gram-positive bacteria ^16-24.

Mannich reaction requires nonenolizable aldehydes or ketone, primary or secondary amine and enolizable carbonyl compound, according to the mechanism as in scheme 1.

Scheme 1. Mechanism for preparing Mannish bases in alkaline medium

MATERIALS AND METHODS:

Materials:

All reagents and chemicals were acquired from commercial sources with analytical grade.Isatin(BHD-99%) Gatifloxacin (Miamed Pharmaceutiacl Industry). Butanol (Panreac Sintesis-99%). Chloroform (Merck- ACS,PhEur).Ammonia (Merck-99%). Methanol (Merck -99%) .Dichloromethane (Panreac Sintesis- 99.8%). Acetonirtile (Merck-HPLC analysis).

Apparatus used:

Round bottom flask with joint 100 ml, Condenser, Electric heater, Ultrasound sonicator, Eppendorf tubes, TLC f254 plates, Becher 100-150-50ml, Flask 150-250 ml, Micropipette 10-100-1000 µl, glass plates TLC f254 1ml, Rotary evaporator, Filtration paper, Filter funnels, Melting Point Apparatus with a Microscope.

Synthesis of Mannich base from Isatin:

The synthesis process was carried out according to the following steps:1mmol (0.147g) of Isatin was weighed in a round bottom flask and dissolved with a minimal amount of methanol. Then 1mmol (0.804g) of Gatifloxacin sesquihydrate was weighed in a flask and dissolved using a dichloromethane catalyst. Then, Gatifloxacin was gradually added to the methanolic solution of Isatin, and then the round bottom flask was placed on the sonicator after completing the addition of Gatifloxacin according to the equation as follows below, the reaction was monitored via TLC plates from the zero-moment using a mobile phase butanol/methanol/ ammonia (3.45 ml: 50 μl: 1.5 ml), where the reaction reached equilibrium after 3 hours from the zero-moment.The filtrate was evaporated using a rotary evaporator and a precipitate was obtained, which was purified by recrystallizing it using chloroform solution finally obtaining a yellow, amorphous powder is optained.

Determination of the melting point of the compound:

The melting point of the compound was measured using a DMP100 Melting Point Apparatus with a Microscope..

Characterization of the compound by spectroscopic methods:

Analysis of the compound via HPLC-UV:

The Purity of the synthesized compound was confirmed through HPLC-UV analysis at a λmax=400 nm using a mobile phase of methanol 70%/Acetonitrile 30% with a flow rate of 0.9 ml/min, C18 (Octyldecylsilane).

Analysis of the compound via FT-IR spectroscopy:

IR spectral data (American, Bruker).

Analysis of the compound via ¹H-NMR, ¹³C-NMR spectroscopy:

NMR spectral data were recorded at 400 MHz for 1H- NMR and 101 MHz for 13C -NMR. The spectra were obtained in DMSO-d6(Avance, Bruker,), with chemical shifts referenced to tetramethylsilane (TMS) (δ = 0.00 ppm). The NMR recordings were performed at room temperature.

RESULT:

Synthesis and purification results:

Monitoring the reaction using TLC Chromatography:

Through TLC plates, it was shown that the reaction occurred and a new product was formed after reaction between Isatin and Gatifloxacin, which gave a yellow spot, where the spots represent the following order (Isatin, reaction filtrate, Gatifloxacin) at the point of equilibrium (Figure 6).

Purification of the Compound:

Figure 2. Verifying the purity of the synthesized Compound via HPLC-UV analysis

Results of characterization of the compound using appropriate spectroscopic methods

FT-IR infrared spectroscopy for the Compound

Figure 3. FT-IR spectrum

A. Synthesized Compound B.Gatifloxacin:

¹H-NMR spectroscopy:

¹H NMR (400 MHz, DMSO-d6) δ 1.01 – 1.09 (m, 9H), 3.39 – 3.50 (m, 8H), 4.47 – 4.53 (d, J = 4.7 Hz, 5H), 6.42 – 6.51 (m, 1H), 6.67 – 6.74 (d, J = 8.4 Hz, 1H), 7.05 – 7.09 (s, 2H), 7.12 – 7.21 (tt, J = 1.3, 8.2 Hz, 1H), 7.49 – 7.56 (m, 1H)

Figure 4. ¹H-NMR spectrum of the proton atom

A. Synthesized Compound. B.Comparison of ¹H-NMR spectrum between Isatin and the synthesized compound

¹³C-NMR spectroscopy

Figure 5. ¹³C-NMR spectrum of the Carbon atom of synthesized Compound

Physical and chemical properties of synthesized Compound:

Physical and chemical properties of synthesized Compound is shown in Table 1. The compound is a yellow powder . It is highly soluble in methanol and ethanol solutions, and. it was charred before reaching its expected melting point (Decomposition).

Table 1. Physical and chemical properties

Color	Yellow powder
Solubility	Highly soluble in methanol ,ethanol
Solubility	Insoluble in chloroform ,ethyl acetate
Melting point	It was charred before reaching its expected melting point
Melting point	(Decomposition)

DISCUSSION:

The results of TLC showed the presence of reactants. The compound was purified by recrystallization using chloroform, and a pure compound was obtained, which was a yellow, amorphous powder. The Rf of the manufactured compound was 0.29 while the Rf of Isatin was 0.89 and the Rf of Gatifloxacin was 0.18.The Purity was confirmed through HPLC-UV analysis at a λmax=400 nm using a mobile phase of methanol 70%/Acetonitrile 30% with a flow rate of 0.9 ml/min, C18 (Oc-tyldecylsilane) Column (Figure 2).

As for the characterization of the Compound and ensuring its structure, spectro-scopic analysis using FT-IR spectroscopy showed the appearance of a new absorption band at wavenumber 1319.07 cm-1 due to the presence of a new third amide group (Figure3),that wasn`t presented in Gatifloxacin which got a change in a position No.7 (Figure3). This change was later confirmed by NMR spectroscopy

The results of the ¹H-NMR spectrum showed the following chemical shifts for the Compound, Figure 11 that the number of protons is 28, which matches the assumed structure of the synthetic compound, where there is a disappearance of the peak at posi-tion 11.28, belonging to the proton of the amide group of Isatin, which proves the com-pletion of the reaction. And the difference in the ¹H-NMR spectrum between Isatin and the synthesized compound is shown (Figure4).

The results of the ¹³C-NMR spectrum atom showed the following peaks for the Compound (Figure 5). The NMR spectrum of the carbon atom also shows that the number of carbon atoms is 27, identical to the formula of the manufactured compound. There were multiple significant peaks, the first peak identifies the ketone group of Isatin at 171, the second peak identifies the amide group of Isatin at 170.6, and the final peak identifies the carboxylic acid group of Gatifloxacin at 200. With the most notable peak being at 81.28 which represents a carbon bridge that links Isatin and Gatifloxacin, so the success the condensation reaction is confirmed.

CONCLUSION:

In this study, a new Mannich base compound was synthesized by reacting Isatin with Gatifloxacin by a Sonicator with using DCM as a catalyst. After purification through recrystallization, a Yellow, amorphous compound was obtained with a yield of 50%. Its purity was tested through HPLC-UV analysis.The FT-IR, ¹H-NMR, ¹³C-NMR analysis indicated the formation of the target compound. The potential uses and effects of this novel compound will be investigted in vitro in upcoming tests.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

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Received on 15.03.2024 Modified on 12.06.2024

Research J. Pharm. and Tech 2024; 17(10):4851-4855.

DOI: 10.52711/0974-360X.2024.00746