INTRODUCTION
Numerous species, including bacteria, viruses, fungi and parasites, are the cause of countless infectious disorders. These microorganisms can be transmissible or non-transmissible and can be transmitted between humans, insects or plants and agents that transmit infections to any hosts are referred to as disease vectors or intermediaries. There are countless microorganisms in our bodies, most of which are beneficial or even harmless. Sometimes these are the cause of fatal diseases, including typhus, smallpox, cholera, tuberculosis, plague, typhoid fever, pneumonia and others (MERCER, 2021).
New biologically active organic compounds have been developed in response to the growing increase in drug-resistant bacteria for the identification and treatment of life-threatening human diseases (SHARMA et al., 2021). Targeting microbial pathogens, which are the leading cause of infectious diseases and the second leading cause of death worldwide (ADEBOYE; OYELEKE; AGBOLUAJE, 2022), low production cost, structural diversity and many uses for active chemicals, natural products are seen as a highly promising alternative (GOLKAR; BAGASRA; PACE, 2014).
The species Piper nigrum L. (WANG et al., 2021), Piper longum (GUPTA et al. , 2022), P. chaba (MOHAPATRA et al., 2015), P. guineense (JULIANI et al., 2013) and P. sarmentosum (HUSSAIN et al., 2009) are the sources of piperine, which is chemically an alkaloid with the molecular formula C17H19NO3 and which, due to the biological properties and beneficial pharmacological activities that the various analogues or derivatives have, such as anti-leishmanial action (PEIXOTO et al., 2021), larvicidal (TANTAWAY et al., 2020) and antimycotic (TRINDADE et al., 2020) action, has attracted the attention of national (SOUZA JR et al., 2021) and international (KHAN et al., 2017) research in the scientific development of fine chemistry.
Trindade et al. (2020) used piperine, through a reaction between potassium piperate and ?-chloroester intermediates, to produce new chemical products. The results showed that the synthesized compounds had an antifungal effect against Candida and Aspergillus yeasts, with MICs ranging from 256 to 1024 g.mL-1. The use of piperine as a natural source for the development of new drugs has a positive influence on reducing toxicity, as shown in this and other studies (PEIXOTO et al., 2021; SOUZA JUNIOR et al., 2021; KHAN et al., 2017).
In view of this, it is clear that the development of drugs that act directly on problems that are so important to the health of the population has a major impact. In this sense, this work presents the synthesis of an organic molecule derived from piperine using planned synthetic procedures and low-cost reagents, generating possibilities of numerous benefits for human health, with the potential to become new alternatives in medicinal chemistry, through the development of new drugs active against pathogenic microorganisms.
AIMS
To develop a new diester derived from piperine, using potassium piperate in the synthesis of a biologically active molecular congener.
METHODS
Piperine extraction
500 mL of 95% ethanol was added to 100 g of finely powdered black pepper. The mixture was then extracted in a Soxhlet apparatus for 2 hours. After extraction, the solvent was concentrated in a rotaevaporator. A 10% alcoholic solution of KOH was added to the residue obtained, in a quantity of 100 mL. The precipitated material was filtered. A small amount of water was added to the remaining alcoholic solution until the medium became cloudy. The alcoholic solution was left overnight, resulting in yellow precipitate needles. The solid obtained was washed with a small amount of cold water, yielding 3.5 g of piperine.
Potassium piperate
A reflux was carried out for about 20 hours with 3 g (10.5 mmol) of piperine and 30 mL of a 20% alcoholic solution of KOH, with a capacity of 50 mL. Once the reaction was complete, the product was filtered, washed with ethanol and dried, obtaining a brown granular solid with a yield of 2.3 g (85.5%).
Preparation of benzyl alcohol
To a solution of benzaldehyde (2.12 g, 20 mmol) in 20 mL of EtOH, NaBH4 (0.907 g, 24 mmol) was added in parts. The resulting suspension was stirred at room temperature until complete. A saturated NH4Cl solution (10 mL) was then added and the resulting phase was extracted with dichloromethane (3 x 10 mL). The combined organic phases were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. 1.8 g (83.33%) of a colorless liquid was obtained, which was used in the next step without further purification.
Preparation of benzyl 2-chloroacetate
2-chloroacetyl chloride (1.35 g, 12 mmol) solubilized in 5 mL of dichloromethane was slowly added to a mixture of benzyl alcohol (1.08 g, 10 mmol) and Et3N (1.65 mL, 12 mmol) previously solubilized in 25 mL of dichloromethane in a 50 mL round-bottomed flask cooled in an ice bath to a temperature of 0 °C. When the addition was complete, the ice bath was removed and the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was then taken to the separatory funnel, washed with a 10% HCl solution (3 x 25 mL), a 10% NaHCO3 solution (3 x 25 mL), brine (3 x 25 mL) and dried in sodium sulphate. The solvent was evaporated at reduced pressure to obtain a yellowish liquid. The compound was purified through a chromatographic column using silica gel as the stationary phase and a hexane:ethyl acetate (99:1) solvent mixture as the mobile phase. 1.43 g (78%) of a colorless liquid was obtained.
2-(benzyloxy)-2-oxoethyl piperate
DMF (10 mL) was added to a mixture of potassium piperate (0.220 g, 0.86 mmol) and benzyl 2-chloroacetate (0.131 g, 0.713 mmol) contained in a 50 mL round-bottomed flask. The reaction mixture was heated to 80°C and stirred until the alkyl halide was completely consumed (as monitored by TLC). After the reaction was complete, the mixture was cooled, ice-cold water was added and the reaction mixture was extracted with ethyl acetate (3x 20 mL), washed with brine (3 x 25 mL) and dried over sodium sulphate. The solvent was evaporated at reduced pressure and a precipitate was formed. The crude product was recrystallized in ethanol/water (8:2). 0.183 g (70.38 %) of orange solid was obtained.
In vivo bological study
The tests for this study were carried out at the Research Laboratory: Antibacterial and Antifungal Activity of Bioactive Natural and Synthetic Products of the Department of Pharmaceutical Sciences/Health Sciences Center/Federal University of Paraíba: March 2023.
This molecule was subjected to biological tests to assess its antimicrobial activity on strains of yeast-like fungi and filamentous fungi. The antimicrobial used as a control was fluconazole/fungi (Sigma-Aldrich/Merck/Brazil). The product was weighed and duly solubilized in 150 µL (3%) of dimethyl sulphoxide (DMSO) and 100 µL (2%) of Tween 80 added, completing the final volume with sterile distilled water q.s.p. 5 mL. In this way, the initial product concentration of 1024 µg/mL was obtained and serially diluted to 16 µg/mL (CLEELAND; SQUIRES, 1991; NASCIMENTO et al., 2007; PEREIRA et al., 2014).
RESULTS AND DISCUSSION
To synthesize the target molecule, potassium piperate was obtained, benzyl alcohol was obtained by reducing benzyl aldehyde, followed by an esterification reaction with chloroacetyl chloride to obtain the ?-chloro-ester, and then a nucleophilic substitution reaction with potassium piperate to obtain the final diester.
In the first stage, piperine was extracted from black pepper, using a methodology well established in the literature and in the research group of the Bioenergy and Organic Synthesis Research Laboratory (LPBS) (SOUZA et al., 2021). Piperine was successfully extracted, obtaining a yield of 7.0g of piperine (3.5%) from the alcoholic extraction.
The extract obtained was recrystallized in ethanol and characterized via 1H NMR. The melting point (128-129ºC) and spectrometric data (1H) of the isolated product were identical to those reported in the literature (TRINDADE et al., 2020). Its structure was confirmed by infrared (IR) and hydrogen magnetic resonance (1H NMR) spectral data.
The infrared spectrum shows the vibrational stretching of the conjugated amide carbonyl (C-1) in the 1633 cm-1 region. The aliphatic C-H stretching signals appear in the 2939-2862 cm-1 region. Asymmetric and symmetric stretching vibrations of the methylenedioxide group (O-CH2-O) were observed at 1251 and 1134 cm-1, respectively. The C=C stretching absorptions of the aromatic ring were observed at 1583-1490 cm-1, while the C=C trans olefinic stretching vibrations were observed at 1612 cm-1.
The 1H NMR spectrum shows a singlet at ? 5.96 referring to the methylenic protons of the methylenedioxy group (O-CH2-O). The signals observed in the ? 7.38-6.42 region refer to aromatic and olefinic protons. In the ? 3.58-1.58 region, signals characteristic of the hydrogens of the piperidine ring are observed. The characterization of these compounds is already well discussed in the literature, as in the articles by Souza et al. (2021) and Trindade et al. (2020).
Potassium piperate was obtained from the hydrolysis reaction of piperine with a 20% alcoholic solution of KOH under reflux for 20 hours. The reaction yield was 85.5%, in the form of a brown granular solid, which was characterized using infrared spectroscopy and 1H NMR, using D2O as the solvent.
From the 1H NMR spectrum, it was possible to observe the main characteristic signals of this compound, confirming the structure and absence of impurities in the piperate. The structure was elucidated by the presence of a singlet at ? 5.88 for the two methylenic protons (O-CH2-O) of the methylenedioxide group. In the region of ? 7.05-6.00 it was possible to observe signals involving olefinic and aromatic hydrogens.
Benzyl alcohol was obtained from the reduction of benzaldehyde using NaBH4. The reduction of aldehyde is traditionally carried out using combustible solvents such as ethanol, 2-propanol and methanol, despite the fact that it is unstable in any of them due to solvolysis (VANITHA et al., 2020). Next, benzyl 2-chloroacetate was obtained from the esterification reaction of benzyl alcohol with 2-chloroacetyl chloride in the presence of triethylamine as a base and using the solvent dichloromethane under stirring at room temperature for 20 hours, with a yield of 78%.
In the 1H NMR spectrum of benzyl 2-chloroacetate, singlet signals related to the methylene protons CH2O and CH2Cl were identified, located at ? 5.22 and 4.09, respectively. The aromatic protons appear at ? 7.37. In the carbon magnetic resonance spectrum (13C NMR) it was possible to observe a signal at ? 167.17 referring to the carbon of the carbonyl of the ester group. In the aliphatic region there are two signals for the methylenic carbons: one for CH2O at ? 67.89 and the other for CH2Cl at ? 40.90.
In the last step of the synthetic route, the piperine derivative was prepared from the nucleophilic substitution reaction between potassium piperate and benzyl 2-chloroacetate using DMF as the solvent at a temperature of 80 °C for 1 hour. The crude product was re-crystallized using a solvent mixture of EtOH/water (8:2) and was obtained in 70% yield.
In the 1H NMR spectrum of the compound derived from piperine, three characteristic signals present in the structures were shown in the form of singlets: a singlet for two methylene protons (H-13) at ? 4.75, a singlet for two methylene protons (H-15) at ? 5.22-5.14, and finally a singlet for two methylene protons referring to the methylenedioxy group (H-12) at ? 5.99. The protons present in the aromatic ring and the olefinic protons were observed in the region 7.50-6.02 ppm.
For the 13C NMR spectrum for the compound, the carbon of the carbonyl (C-14) corresponding to the ester of the benzyl portion was characterized at approximately 168 ppm, and the carbon of the carbonyl (C-1) corresponding to the ester conjugated to the ?,? unsaturation was characterized at approximately 166 ppm. The compound showed signals characteristic of methylenic carbons: a signal in the region of 60 ppm referring to C-13, a signal in the region of 101 ppm referring to C-12 (methylenedioxy) and finally, a signal in the region of 67 ppm referring to C-15.
The PEB0 molecule had an inhibitory effect on the growth of filamentous fungi, including Candida albicans (ATCC-76485), C. albicans (LM_72), C. tropicalis (ATCC-13803) and Trichophyton rubrum (ATCC-28188). The MIC of this molecule was 512 ?g/mL.
CONCLUSION
The synthetic route used to synthesize the piperine derivative from potassium piperate and benzyl 2-chloroacetate proved to be simple and effective. The final product was obtained in 70% yield. The chemical structure of this product was determined using infrared spectroscopy, hydrogen and carbon NMR techniques.
The synthesized compound was sent to the pharmacological research laboratory, where it was evaluated for four strains of fungi, namely Candida albicans (ATCC-76485), C. albicans (LM_72), C. tropicalis (ATCC-13803) and Trichophyton rubrum (ATCC-28188), showing antimicrobial potential through pharmacological screening tests for all the microorganisms tested with a MIC of 512 ?g/mL.
ACKNOWLEDGMENT
We would like to thank the Federal University of Paraíba, the Research Laboratory: Antibacterial and Antifungal Activity of Bioactive Natural and Synthetic Products of the Department of Pharmaceutical Sciences/Health Sciences Center, as well as the funding agencies Capes, CNPq and Fapesq/PB, for financing the research.
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