Esterification of Metronidazole and its Complexation with ? -Cyclodextrin: A Strategy for Enhanced Aqueous Solubility

A synthetic nitro imidazole derivative, metronidazole has strong antibacterial and antiprotozoal properties against a variety of anaerobic bacteria. It was first made available in the 1960s and is still one of the most popular antimicrobial agents because of its wide range of activity, affordability, and effectiveness. The chemical formula for metronidazole (1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole) is C?H?N?O?, and its molecular weight is roughly 171.15 g/mol. Its antimicrobial action depends on the presence of the 5-nitroimidazole ring ^[1-3]. In anaerobic conditions, the nitro group is reduced intracellular, producing reactive intermediates that react with microbial DNA to cause strand breakage and, eventually, cell death. Metronidazole is a prodrug that is highly selective for anaerobic pathogens because it only activates in anaerobic or low oxygen environments. Metronidazole is effective against a wide range of anaerobic bacteria and protozoa. It works especially well against protozoan parasites like Trichomonas vaginalis, Giardia lamblia, and Entamoeba histolytica. Furthermore, it exhibits strong antibacterial action against a number of anaerobic organisms, such as Peptostreptococcus species, Helicobacter pylori, Fusobacterium species, Bacteroides fragilis, and Clostridium difficile. Because of its broad-spectrum antibacterial and antiparasitic properties, metronidazole is frequently used to treat infections of the skin, oral cavity, urogenital tract, and gastrointestinal system ^[4-6]. Metronidazole has a number of drawbacks that impact its overall therapeutic efficacy, despite its extensive clinical use. Its limited water solubility is one of its main disadvantages, which can limit formulation possibilities and lessen its efficacy in specific delivery systems. Furthermore, patient compliance is hampered by its extremely bitter taste, especially in paediatric populations and when using oral liquid preparations. Because of its brief half-life, metronidazole must be taken often in order to maintain therapeutic levels ^[7-8]. Some microbial strains have become resistant to the medication over time, frequently as a result of changes in the nitroreductase enzymes that activate it. Additionally, the medication has a number of adverse effects, such as nausea, a metallic taste, and in rare instances, neurotoxicity. Concerns regarding its possible mutagenicity have also been highlighted by prolonged use, underscoring the need for better formulations or different delivery methods ^[9]. Metronidazole derivatives, especially its amide and ester versions, have been created to get around the original drug's drawbacks and increase its range of therapeutic uses. These derivatives are mainly intended to function as prodrugs, which release the active metronidazole molecule when they are converted chemically or enzymatically within the body. These changes can greatly increase lipophilicity and aqueous solubility, which in turn can boost oral bioavailability and membrane permeability. The harsh taste of metronidazole is a significant compliance concern in paediatric and geriatric groups, where these compounds are particularly helpful in flavour-masked formulations ^[10-12]. Enhancing aqueous solubility and successfully disguising the bitter taste through inclusion complexation with cyclodextrins has also demonstrated promise in increasing patient compliance. Modern methods such as encapsulation technologies and nano formulations are being used to increase metronidazole's overall therapeutic efficacy, reduce systemic side effects, and improve targeted delivery. These novel strategies seek to increase the drug's clinical potential while resolving its physicochemical and pharmacokinetic issues ^[13-15]. A naturally occurring cyclic oligosaccharide, β-cyclodextrin is made up of seven glucose units connected by α-1,4-glycosidic linkages. The distinctive truncated cone-shaped structure of β-cyclodextrin features a hydrophilic exterior and a comparatively hydrophobic interior chamber. It can form inclusion complexes with a wide range of poorly soluble or unstable guest molecules, including vitamins, flavours, and medications, thanks to this structural characteristic ^[16-18]. β-cyclodextrin's main purpose in pharmaceutical applications is to encapsulate drugs within its cavity, improving their solubility, stability, and bioavailability. This molecular encapsulation lessens disagreeable taste or odour, enhances medicine delivery qualities, and shields the guest molecules from deterioration (such as hydrolysis, oxidation, or light). Formulations for oral, parenteral, and topical drug delivery frequently use β-cyclodextrin due to its low toxicity, biocompatibility, and GRAS (Generally Recognized as Safe) status ^[19-21]. In this work, we have prepared metronidazole acetate and metronidazole benzoate by esterifying metronidazole with acetyl and benzoyl chlorides in DCM using pyridine as a catalyst. Further, ester derivatives were molecularly added into β-cyclodextrin using the solvent evaporation method. FT-IR, ¹³C-NMR, and ¹H-NMR spectroscopic techniques were employed to validate these inclusion complexes. Solubility of ester derivatives and their inclusion complexes were analyzed by shake flask method.

Experimental Section

Materials and methods

Analytical standard metronidazole (MTZ) was purchased from Sigma Aldrich Chemicals Pvt. Ltd., Bangalore, India. β-Cyclodextrin (β-CD), acetyl chloride and benzoyl chloride were purchased from S. D. Fine Chem. Ltd., Mumbai, India. Sodium carbonate, sodium sulfate, chloroform, dichloromethane (DCM) ethanol and Pyridine were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. Hydrochloric acid (12N) was purchased from Surya Fine Chem., Ambarnath, India.

Methods of characterization

A Bruker Avance-II spectrophotometer running at 400, 500 MHz, and 100, 125 MHz was used to record ¹H and ¹³C NMR in CDCCl₃ as solvent. Fourier Transform Infra-red (FT-IR) spectra in the region 4000–400 cm^-1 were taken on Shimadzu FTIR 8400 spectrometer.

Synthesis of ester derivatives of Metronidazole

Synthesis of metronidazole acetate and metronidazole benzoate was done as per the procedure given in the article ^[22] with some modifications.

Procedure for the synthesis of metronidazole acetate

A solution of acetyl chloride (1 mL, 0.014 M) in DCM (10 mL) was added drop-wise to a mixture of metronidazole (2 g, 0.011 M), pyridine (1 mL) and DCM (25 mL) under anhydrous condition. Acetyl chloride and metronidazole were taken in 1.2:1 ratio. For 25 hours, the resultant solution was stirred at room temperature. After the solvent was extracted in a vacuo, the residue was stirred for 15 minutes in 25 mL solution of 1M sodium carbonate. The material was then extracted using chloroform (3 x 25 mL). 10% HCl (25 mL) and distilled water (3 x 25 mL) were used to wash the chloroform extract. Metronidazole acetate was obtained by vacuum-removing the solvent after the chloroform extract was dried over anhydrous sodium sulfate.

Figure 1: Synthesis of metronidazole acetate ^[22].

Procedure for the synthesis of metronidazole benzoate

A mixture of benzoyl chloride (1.6 mL, 0.014 M) in DCM (10 mL) was added drop-wise to a mixture of metronidazole (2 g, 0.011 M), pyridine (1 mL) and DCM (25 mL) under anhydrous condition. The resulting solution was stirred at room temperature for 25 hours. The solvent was removed in vacuo and residue obtained was stirred with 1M sodium carbonate solution (25 mL) for 15 min. It was followed by extraction of the material with chloroform (3 x 25 mL). The chloroform extract was wash with 10% HCl (25 mL) and then with distilled water (3 x 25 mL). The chloroform extract was dried over anhydrous sodium sulfate and solvent removed in vacuo to give metronidazole benzoate.

Figure 2: Synthesis of metronidazole benzoate ^[22].

Preparation of inclusion complexes of metronidazole esters with β-cyclodextrin

In an ethanol solvent, inclusion complexes of metronidazole benzoate and metronidazole acetate with β-cyclodextrin were produced. β-cyclodextrin and metronidazole esters were taken in a 1:1 ratio. The guest molecule ester of metronidazole (0.0017 M) in 10 mL ethanol was mixed with a drop-wise addition of a host molecule solution of β-cyclodextrin (0.0017 M) in 10 mL ethanol while being stirred. At room temperature, the mixture was left to stir for two hours. After the stirring, solvent was allowed to evaporate leaving behind inclusion complex.

Solubility measurement

Measurement of solubility of metronidazole acetate, metronidazole benzoate, inclusion complexes of metronidazole acetate and metronidazole benzoate with β-cyclodextrin was carried out by shake flask method as per given in the article ^[23]. There were multiple steps involved in the solubility study. First, two separate 50 mL glass beakers were used to prepare saturation solutions. 10 mL of distilled water were mixed with about 50 mg of pure compound of ester of metronidazole in the first beaker. In the second beaker, 10 mL of distilled water was mixed with an equivalent quantity (50 mg) of the inclusion complex. To allow the solutions to reach equilibrium, both vials were then continuously shaken for 24 hours at room temperature (~25 °C), with a mechanical stirrer. After the stirring period, the mixtures were filtered through Whatman filter paper in order to remove undissolved particles. A 5 mL of sample was taken from each of the resulting clear solutions and allowed to evaporate on a water bath. The obtained dry residue was weighed on digital balance. In order to maintain accuracy, this procedure was carried out three times for every sample. The final solubility value was determined by averaging the three measurements. Solubility of compounds were determined by the formula given as

Solubility (mg/mL) =Amount of dry residue (mg)Volume of solution evaporated (mL)