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)

RESULT AND DISCUSSION

Spectroscopic Characterization   

Two ester derivatives, such as metronidazole acetate and metronidazole benzoate, and their inclusion complexes with β-cyclodextrin were characterized by FT-IR, ¹H-NMR, and ¹³C-NMR spectroscopic analysis. Results of FT-IR analysis are shown in Figure 3. In β-cyclodextrin, the IR frequencies for C-O, C-O-C, C-H, and O-H were observed at 1037.70, 1149.57, 2924.09, and 3323.14 cm?¹, respectively. In the case of metronidazole acetate, the band 1056.99 cm?¹ was observed for the C-N bond. The IR frequency at 1253.73 cm?¹ is associated with the stretching frequency of the C-O bond. For the C=N bond, the IR frequency was observed at 1467.83 cm?¹. Similarly, for the N-O bond frequency observed at 1525.69 cm?¹. The carbonyl group of ester was observed at 1737.86 cm?¹. The IR frequency for the aliphatic C-H bond was observed at 3120.82 cm?¹. In metronidazole benzoate, the stretching frequency at 1105.21 cm?¹ corresponded. The band at 1508.33 cm?¹ is associated with the C-O bond. For the aromatic C=C band observed at 1585.49 cm?¹. The band of C=O of the ester group was observed at 1718.58 cm?¹. The band at 3001.24 cm?¹ is associated with the C-H bond. From the IR spectra of inclusion complexes, it was observed that the spectra were a combination of both host (β-cyclodextrin) and guest (ester derivatives of metronidazole). After comparing spectra of ester derivatives with β-cyclodextrin, it was found that the IR frequency of the carbonyl group of the ester and the nitro group moved slightly towards higher frequency. In metronidazole acetate, C=O of ester and N-O were found at 1737.86 and 1525.69 cm?¹, while the same group frequencies were observed at 1745.58 and 1531.48 cm?¹, respectively, in their inclusion complexes. Similarly, in metronidazole benzoate, C=O and N-O group frequencies were found at 1718.58 and 1508.33 cm?¹, whereas in inclusion complexes, frequencies were observed at 1726.29 and 1529.55 cm?¹, respectively. This shifting of IR bands towards higher frequency might be due to Van der Waals interaction between guests (ester derivatives of metronidazole) with the host (β-cyclodextrin). This study confirms the synthesis of inclusion complexes was done successfully.

Figure 3: FT-IR spectra of β-cyclodextrin, metronidazole acetate, metronidazole benzoate, inclusion complex of metronidazole acetate with β-cyclodextrin and inclusion complex of metronidazole benzoate with β-cyclodextrin. Results of ¹H-NMR spectroscopic analysis are shown in Figure 4. Comprising seven D-glucopyranose units connected by α-1,4-glycosidic linkages, β-cyclodextrin is a cyclic oligosaccharide with a torus-shaped (truncated cone) structure. The protons of the seven glucose units in β-cyclodextrin are represented by various signals in its ¹H NMR spectrum. The anomeric H-1 (~4.8–5.1 ppm) and the H-3 and H-5 protons (~3.6–3.9 ppm) were important signals that indicate the hydrophobic cavity and were sensitive to the presence of guests. Between 3.3 and 3.8 ppm, the remaining protons (H-2, H-4, and H-6) were visible. The development of inclusion complexes was indicated by changes in the chemical shifts of H-3 and H-5. In metronidazole acetate, the CO-CH₃ and C=C-CH₃ proton chemical shifts appeared at 2.02 and 2.52 ppm, respectively. Chemical shifts for CH₂-O and CH₂-N appeared at 4.59 and 4.42 ppm, respectively. Aromatic-H chemical shift was observed at 7.95 ppm. The chemical shift at 7.28 ppm corresponded to solvent CHCl₃ In the case of metronidazole benzoate, 2.46 ppm corresponded to C=C-CH₃. Chemical shifts for CH₂-O and CH₂-N appeared at 4.71 and 4.67 ppm, respectively. The chemical shift for the proton at the meta-position of the phenyl ring was observed at 7.42 ppm, whereas the proton at the para-position was observed at 7.57 ppm. The chemical shift at 7.96 ppm was associated with the proton at the para-position. The chemical shift for the proton of the imidazole ring (H-C=C) was found at 7.91 ppm. The ¹H NMR spectra of the inclusion complex of metronidazole acetate with β-cyclodextrin showed distinct up field shifts in the H-3 and H-5 protons of β-cyclodextrin, indicating that the metronidazole moiety had entered the hydrophobic cavity. Some metronidazole acetate protons, especially the aromatic and aliphatic ones, may have shifted concurrently due to the shielding action inside the cavity. These changes in chemical composition validated the formation of a host–guest combination in the inclusion complex of metronidazole acetate with β-cyclodextrin. The ¹H NMR spectra of the inclusion complex of metronidazole benzoate with β-cyclodextrin often show up field shifts (around 3.6–3.9 ppm) in the H-3 and H-5 protons of β-cyclodextrin due to the hydrophobic benzoate ring being inserted into the cavity. The aromatic protons of metronidazole benzoate, which normally showed up at 7.3–8.0 ppm, simultaneously moved slightly up field, indicating shielding within the β-CD cavity. These changes validated the formation of a stable host–guest inclusion complex.

Figure 4: ¹H-NMR spectra of β-cyclodextrin, metronidazole acetate, metronidazole benzoate, inclusion complex of metronidazole acetate with β-cyclodextrin and inclusion complex of metronidazole benzoate with β-cyclodextrin. The findings of ¹H-NMR spectroscopic analysis are shown in Figure 5. The β-cyclodextrin's ¹³C NMR spectra revealed distinctive peaks for each of the six different carbon atoms (C1–C6) in each glucose unit. Around 102–105 ppm, the anomeric carbon (C1) was seen downfield, whereas C2, C3, and C5 were seen between 72 and 76 ppm. At roughly 81–83 ppm, C4 was visible, while at 60–62 ppm, the principal hydroxyl carbon (C6) was observed up field. When guests interacted with C3 and C5, their chemical shifts, which face the inner cavity, were observed to modestly alter, indicating their participation in the creation of inclusion complexes. All of the carbon atoms in metronidazole acetate were easily identified by their chemical shifts in the recorded ¹³C NMR spectrum. Successful acetylation was indicated by the appearance of the ester carbonyl carbon at about 170 ppm. The signals from the imidazole ring carbons at roughly 151 ppm (C=N), 138 ppm, and 122 ppm corresponded to the ring's aromatic carbons. The methyl group connected to the imidazole ring reverberated at about 14 ppm, while the methylene carbon next to the ring appeared at about 45 ppm. The methylene carbon next to the acetate group was observed at about 62 ppm. Near 20 ppm, the acetate methyl carbon produced a peak. These chemical shift values supported the successful synthesis of metronidazole acetate and validated its structure. Metronidazole benzoate's ¹³C NMR spectra revealed well-defined peaks that matched each of the molecule's carbon atoms. In order to confirm ester production, the benzoate group's ester carbonyl carbon showed up downfield at about 167 ppm. With the quaternary carbon of the ring showing up around 135 ppm, the carbons of the benzene ring displayed signals between 128 and 133 ppm. About 151 ppm, 139 ppm, and 134 ppm of imidazole ring carbons, respectively, were detected. The imidazole side chain's methyl carbon was found at about 14 ppm, whereas the methylene carbon next to the imidazole ring was visible at about 46 ppm. The methylene carbon next to the benzoate group was found at about 62 ppm. Successful esterification was confirmed by these chemical changes, which were in line with the predicted structure of metronidazole benzoate. To verify the development of the inclusion complex between metronidazole acetate and β-cyclodextrin, the ¹³C NMR spectra were captured and examined. Around 171 ppm, the acetate group's carbonyl carbon emerged, suggesting that the ester functionality was unaffected. With minor inclusion-related alterations, the imidazole ring carbons displayed signals at roughly 152 ppm, 133 ppm, and 102 ppm, which were comparable to the free drug. Methyl and methylene carbons were found at 14 and 60 ppm, respectively. The inner cavity carbons in the β-cyclodextrin portion, especially C3 and C5, showed slight up field or downfield shifts, indicating interaction with the guest molecule. The effective incorporation of metronidazole acetate into the β-cyclodextrin cavity was facilitated by these modifications in chemical shifts. The molecular encapsulation was confirmed by recording and analyzing the ¹³C NMR spectrum of the inclusion complex of metronidazole benzoate with β-cyclodextrin. The benzoate group's ester carbonyl carbon showed up at about 165 ppm, suggesting that the ester bond was undamaged. With the quaternary carbon close to 134 ppm, the benzene ring carbons had maxima at 129.61 and 133 ppm. At roughly 151 ppm, 134 ppm, and 129.32 ppm, the imidazole ring carbons were detected, whereas the methylene and methyl carbons showed up at 60 ppm and 14 ppm, respectively. For β-cyclodextrin cavity carbons, particularly C3 and C5, slight changes in chemical shift were observed, supporting the host-guest molecule interaction. These spectral shifts helped the inclusion complex form successfully.

Figure 5: ¹³C-NMR spectra of β-cyclodextrin, metronidazole acetate, metronidazole benzoate, inclusion complex of metronidazole acetate with β-cyclodextrin and inclusion complex of metronidazole benzoate with β-cyclodextrin.Results of solubility measurement are shown in Table 1. When combined with β-cyclodextrin, metronidazole acetate's solubility increased to 9.26 mg/mL, surpassing its solubility of 7.76 mg/mL when used alone. The solubility of metronidazole benzoate was 7.42 mg/mL; however, its inclusion complex with β-cyclodextrin showed a significantly higher solubility of 9.23 mg/mL. This demonstrates that β-cyclodextrin uses inclusion complexation to boost the drug's water solubility.

Table 1: Results of solubility measurement.