p-ISSN 1693-5683; e-ISSN 2527-7146 29 Vol. 19, No. 1, May 2022, pp. 29-33 Research Article Characterization of 1H NMR and 13C NMR Spectrometry and Antibacterial Activities of 4-tert-Butylbenzoyl Amoxicillin Hadi Barru Hakam Fajar Siddiq*, Dewi Rashati, Siti Nur Azizah Departement of Pharmacy, Akademi Farmasi Jember, Jember, Indonesia https://doi.org/10.24071/jpsc.002183 J. Pharm. Sci. Community, 2022, 19(1), 29-33 Article Info ABSTRACT Received: 27-10-2019 Revised: 10-04-2021 Accepted: 23-03-2022 *Corresponding author: Hadi Barru Hakam Fajar Siddiq email: hakamfajar@gmail.com Keywords: 4-tert-butylbenzoyl amoxicillin; Bacillus subtilis; Escherichia coli; spectrometry; Staphylococcus aureus; The synthesis of 4-tert-butylbenzoyl amoxicillin has been done by reacting amoxicillin with 4-tert-butylbenzoyl chloride. The product was characterized using proton and carbon-13 spectrometry. Antibacterial activity of 4-tert-butylbenzoyl amoxicillin against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus was tested using paper disk diffusion method. Results showed that 4-tert- butylbenzoyl amoxicillin had a specific spectrum activity. Antibacterial test of 4-tert-butylbenzoyl amoxicillin showed clear zones around the disc paper for Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The study results indicated 4-tert-butylbenzoyl amoxicillin has antimicrobial properties but is categorized as a less-effective antibiotic agent based on the response to bacterial growth inhibition. INTRODUCTION Pathogenic enteric bacteria are a common bacteria that infect the digestive tract of both animals and humans. Many of these bacteria come from contaminated food and water. These bacteria are a group of gram-negative and gram- positive rods that are widely bred in clinical laboratories and most commonly cause gastrointestinal diseases. Families including pathogenic enteric bacteria that frequently contaminate food include several genera, including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis) (Siddiq et al., 2018). Infectious disease is a threat to human survival and has become the third leading cause of death in the world (Dzen et al., 2003). Prevention of infectious diseases is generally done by administering antibiotics. Inappropriate use of antibiotics such as inaccurate indications of use, free use by the public, as well as inappropriate dosage and duration of administration will cause new problems, namely increased bacterial resistance to antibiotics (Dzen et al., 2003). The incidence of bacterial resistance continues to increase in various parts of the world. However, the increase was accompanied by a downward trend in the development of new antibiotics (Buntaran, 2007; Finch and Hunter, 2006). To overcome this problem, efforts are needed to develop new antibiotics (Spellberg et al., 2004; Yoneyama et al., 2006). Amoxicillin is a penicillin derivative whose structure is similar to ampicillin, with the difference in the presence of a hydroxy group at the position of the benzene rings. Some of the benefits of amoxicillin compared to ampicillin is the absorption of the drug in the digestive tract that is more complete so that plasma levels are higher. Maximum blood levels are achieved within 1 hour after oral application (Soekardjo and Siswandono, 2000). On the one hand, amoxicillin is not effective against some bacteria including E. coli (Krisnaningsih et al., 2005) and S. aureus (Shituu et al., 2011). Therefore, modification of amoxicillin compounds is needed that can improve its performance or reduce antibiotic resistance in bacteria. On the other hand, 4-tert-butylbenzoyl amoxicillin compound has been synthesized and its physical and chemical properties http://issn.pdii.lipi.go.id/issn.cgi?daftar&1180428136&1&& http://issn.pdii.lipi.go.id/issn.cgi?daftar&1465346481&1&& https://e-journal.usd.ac.id/index.php/JFSK/index https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.24071/jpsc.002183 Research Article Journal of Pharmaceutical Sciences and Community Characterization of 1H NMR and 13C NMR Spectrometry... 30 Siddiq et al. J. Pharm. Sci. Community, 2022, 19(1), 29-33 characterized including organoleptic, pH, melting point, Rf value on thin layer chromatography, maximum wavelength on UV- Vis spectrophotometry, and functional groups using IR spectrophotometry (Siddiq et al., 2018). In this research, further characterization was done in the form of structural characterization using proton and carbon-13 spectrometry. Then, the antibacterial activity was tested against E. coli, S. aureus, and B. subtilis. Figure 1. 1H NMR spectrum of 4-tert-butylbenzoyl amoxicillin compound. Table 1. 1H NMR 4-tert-butylbenzoyl amoxicillin data, chemical shift, multiplicity, H amount, and Group Chemical Shift (ppm) Multiplicity H amount Group 1,099 s 3 -CH3 1,169 s 3 -CH3 1,286 d 2 -CH2- 1,525 s 1 -CH- 4,640 t 1 R-CO-O-CH- 5,607 d 1 R-CO-NH- 6,695 d 2 Ar-CH 7,269 d 2 Ar-H 7,501 q 3 Ar-H 7,865 q 3 Ar-H 8,381 t 2 R-CO-NH2- 8,528 d 1 R-CO-NH- 9,386 s 1 R-COOH Journal of Pharmaceutical Sciences and Community Characterization of 1H NMR and 13C NMR Spectrometry... Research Article 31 Siddiq et al. J. Pharm. Sci. Community, 2022, 19(1), 29-33 Figure 2. 13C NMR spectrum of 4-tert-butylbenzoyl amoxicillin compound METHODS Research Materials The equipment used in this research is the 1H NMR and 13C NMR spectrometers (JNM- ECZ500R/S1), Petri dish, calipers, autoclaves (Westlab), incubators, micropipettes, inoculation needles OSE, Bunsen burner, laminar air flow (stainless steel Indiamart), colony counters, and tools laboratory glassware. The materials used in this study were E. coli bacteria, S. aureus bacteria, B. subtilis bacteria, amoxicillin trihydrate (pharmaceutical grade), 4-tert-butylbenzoyl amoxicillin synthesized, ethanol p.a. (Merck), re- distilled water, nutrient agar and nutrient broth, and paper discs. Synthesis of 4-tert-butylbenzoyl amoxicillin compound After 28.8 mmol of amoxicillin was added with 100 mL tetrahydrofuran and 20 mL, the re- distilled mixture was stirred at 0-5 °C, then 2N potassium hydroxide solution was added. Next, the pH was adjusted between 6.8 to 7.2, followed by adding 25 mmol of 4-tert-butylbenzoyl chloride in 40 ml of tetrahydrofuran drop by drop. The mixture was left for several hours at room temperature while stirring occasionally. Next, the tetrahydrofuran in the mixture was evaporated and the solids obtained were dissolved in 300 mL re-distilled water. In the next step, the solution was extracted using 250 mL ethyl acetate. The water phase was separated and in the ethyl acetate phase, as much as 250 mL ethyl acetate was added and cooled. The ethyl acetate in the solution then was evaporated. To remove the remaining ethyl acetate, the solid obtained was dissolved in methanol and then evaporated again to dryness (Siddiq et al., 2018). Characterization of 1H NMR and 13C NMR spectrometry of 4-tert-butylbenzoyl amoxicillin compound A number of dissolved synthesized 4-tert- butylbenzoyl amoxicillin compounds were analyzed using `1H NMR and 1C NMR spectrometry with DMSO-d6 solvent. Research Article Journal of Pharmaceutical Sciences and Community Characterization of 1H NMR and 13C NMR Spectrometry... 32 Siddiq et al. J. Pharm. Sci. Community, 2022, 19(1), 29-33 Figure 3. Reaction synthesis 4-tert-butylbenzoyl amoxicillin Figure 4. Clear zone diameter of bacteria by 4-tert- butylbenzoyl amoxicillin compound Antibacterial activity test of 4-tert- butylbenzoyl amoxicillin compound The antibiotic activity test method was the diffusion method using 6 mm diameter paper discs (Kirby-Bauer method). Each compound with a different concentration of 100% (w/v), 50% (w/v), 25% (w/v), 12.5% (w/v) was measured as much as 20 µL and dropped on paper discs until saturated (Ningsih, 2013). The bacterial suspension used was as much as 100 µL to enter into NA media in a sterile Petri dish and then left until it solidified. The saturated disc paper was placed on the surface of the NA media. The media was incubated for 24 hours at 37 °C. Antibacterial activity testing was done in triplicate (Saraswati, 2015). Antibiotic activity is said to be positive if an inhibitory zone is formed in the form of a clear zone around the disc paper. Inhibition zone measurement is done by reducing the diameter of the resistance with the diameter of the paper disc (Hermawan, 2007). RESULTS AND DISCUSSION The results of the 1H NMR spectrum characterization of the 4-tert-butylbenzoyl amoxicillin compound are shown in Figure 1 and Table 1. The 13C NMR spectrum of the 4-tert- butylbenzoyl amoxicillin compound is shown in Figure 2. Based on spectrometric data of 1H NMR and 13C NMR, the 4-tert-butylbenzoyl amoxicillin compounds were formed by nucleophilic substitution reaction between the hydroxyl group in amoxicillin phenol compound and carbonyl 4-tert-butylbenzoyl chloride compound. The reaction that occurs is shown in Figure 3. The next step was testing the antibacterial activity. Tests were conducted using paper disk diffusion method on E. coli, S. aureus, and B. subtilis through clear zone observations and the results are shown in Figure 4. The mechanism of bacterial inhibition is the destruction of the bacterial cell wall. Gram negative bacteria (E. coli and S. aureus) have a thin layer of peptidoglycan on the cell wall and are surrounded by lipoproteins, lipopolysaccharides, phospholipids, and several proteins. Meanwhile, the gram-positive bacteria (B. subtilis) cell walls are composed of tissue with many pores and a thick layer of peptidoglycans and surrounded by a layer of ketoic acid. In Figure 4, gram-negative bacteria are more easily inhibited by the 4-tert-butylbenzoyl amoxicillin compound, because it has a thinner layer of peptidoglycan. HO H C NH2 C O NH N S CH CH3 CH3 C O HO O + CH3 CH3 H3C C O Cl O HC NH2 C O H N N S CH H3C CH3 C O OH O CH3 H3C CH3 C O Amoxicillin 4t-butylbenzoyl chloride 4t-butylbenzoyl Amoxicillin Journal of Pharmaceutical Sciences and Community Characterization of 1H NMR and 13C NMR Spectrometry... Research Article 33 Siddiq et al. J. Pharm. Sci. Community, 2022, 19(1), 29-33 In general, the response to inhibition of bacterial growth of 4-tert-butylbenzoyl amoxicillin compound is still less effective in providing inhibition of bacterial growth. According to Greenwood (1995) responses to bacterial growth inhibition can be classified as presented in Table 2. Table 2. Classification of bacterial growth inhibition Clear zone diameter (mm) Bacterial growth response > 20 Strong 16 – 20 Medium 10 – 15 Weak < 10 Less effective CONCLUSIONS The 4-tert-butylbenzoyl amoxicillin compound has a specific spectrum for the 1H NMR and 13C NMR test results, so that the mechanism of nucleophilic substitution reaction between the hydroxyl group in the amoxicillin phenol compound and the carbonyl compound 4- tert-butylbenzoyl chloride can be explained. The 4-tert-butylbenzoyl amoxicillin compound forms a clear zone against E. coli, S. aureus, and B. subtilis bacteria and belongs to the less effective category based on the response to bacterial growth inhibition. ACKNOWLEDGEMENTS Authors thank to Kemenristekdikti for providing funding assistance in this research and Jember Pharmacy Academy for providing facilities for this research. REFERENCES Buntaran, L., 2007. Infeksi nosokomial: menggantung harapan pada antibiotik anyar, Farmacia. 6(11).46-47. 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