INTRODUCTION
High blood pressure is a chronic condition that affects over 30% of the adult population, which amounts to more than one billion individuals worldwide, according to the Pan American Health Organization (2022). From a medical standpoint, hypertension is considered the primary risk factor for various cardiovascular diseases, such as arteriosclerosis, coronary disease, stroke, peripheral artery disease, and heart failure. Additionally, hypertension is a complex condition influenced by genetic, epigenetic, environmental, and social factors, as mentioned in the study by BARROSO et al., (2021).
Regarding the treatment of high blood pressure, the initial approach involves nonpharmacological therapies, including dietary modifications (adopting a healthy, low-sodium diet), weight loss, limiting alcohol consumption, smoking cessation, and regular physical activity. These measures are recommended both for controlling hypertension in its early stages and as preventive strategies. The introduction of drug therapy is considered when non-pharmacological therapies fail to achieve the desired results. Currently, there are five classes of available antihypertensive medications, including diuretics (DIU), calcium channel blockers (BCC), angiotensin-converting enzyme inhibitors (IECA), angiotensin II receptor blockers (BRA), and beta-blockers (BB) (WHELTON et al., 2018).
Angiotensin-converting enzyme inhibitors (ACE inhibitors) have a well established presence in the market for over four decades and demonstrate significant efficacy in the treatment of arterial hypertension (ZAMAN; OPARIL; CALHOUN, 2002). Among them, captopril, enalapril, and lisinopril are widely recognized as first-line medications for the treatment of hypertension and myocardial infarction (THOMOPOULOS; PARATI; ZANCHETTI, 2014). These pharmaceutical agents work by inhibiting the angiotensin-converting enzyme (ACE), responsible for converting angiotensin I into angiotensin II. Angiotensin II is a potent substance that causes blood vessel constriction and stimulates the release of aldosterone, which also has vasoconstrictive effects. Furthermore, these medications reduce the degradation of bradykinin, a natural substance in the human body that promotes blood vessel dilation (HOFFMAN, 2001). However, it has been observed that prolonged use of these drugs can lead to a range of side effects, such as headaches, fatigue, dry cough, elevated blood potassium levels, abnormally low blood pressure, and renal dysfunction (BAKRIS, 2009). It is believed that the adverse effects associated with commercial ACE inhibitors are related to their lack of selectivity, as their nonspecific action leads to the gradual accumulation of bradykinin in patients' bodies (CABALLERO, 2020).
The angiotensin-converting enzyme (ACE) is a dipeptidyl-carboxy-peptidase composed of two similar catalytic domains (nECA and cECA) (FUCHS et al., 2008). Structurally, these domains adopt an ellipsoid shape crossed by a long and deep cleft at the active site, where ?-helices predominate, and the binding cavity is covered by a lid containing the catalytic zinc (Zn2+), located at the center of four subsites (S2, S1, S1', and S2'). However, it is important to emphasize that the pharmacological profile, sensitivity, and specificity of ligands vary depending on the domain in question (CABALLERO, 2020). In vivo studies with RXP407, a selective inhibitor of the nECA domain, have shown more efficient cleavage of angiotensin I in the cECA domain (COTTON et al., 2002). This suggests that the cECA domain plays an essential role in blood pressure regulation, while the nECA domain exhibits greater specificity for AcSDKP, a tetrapeptide involved in the control of hematopoietic stem cell differentiation and proliferation (NATESH et al., 2004). Furthermore, it has been observed that the presence of both the pseudo-proline moiety and tryptophan in the structure of RXPA330 significantly contributes to selectivity for the cECA domain, as these groups do not effectively fit into the nECA domain due to unfavorable interactions with residues present in the nECA domain (GEORGIADIS et al., 2004).
Given the above, the importance of conducting new studies focused on drug design becomes evident, especially due to the increasing use of ACE inhibitors for oral hypertension treatment. Therefore, the aim of this study is to develop antihypertensive drugs through the synthesis of innovative derivatives of 1,2,4-oxadiazolin-5-ones. These derivatives will be pre-planned through in silico analyses (molecular docking) with the purpose of improving inhibitor specificity and reducing side effects. This approach aims to contribute to more effective treatments and greater patient adherence.
GOALS
General
• To develop new derivatives of 1,2,4-oxadiazolin-5-ones as potential antihypertensive drugs, utilizing in silico studies (molecular docking), and assess their ability to inhibit ACE (Angiotensin-Converting Enzyme) through in vitro assays.
Specific
• Utilize molecular docking to guide the synthesis of new derivatives.
• Synthesize novel derivatives of 1,2,4-oxadiazolin-5-one.
• Perform the functionalization of the synthesized 1,2,4-oxadiazolin-5-ones through a 1,3-dipolar cycloaddition reaction between terminal alkynes and azides.
• Conduct the amidification of compounds resulting from functionalization via cycloaddition with the methyl ester of tryptophan.
• Evaluate target-ligand interactions in silico through molecular docking simulations.
• Conduct in vitro tests to assess the potential inhibition of the synthesized compounds on ACE (Angiotensin-Converting Enzyme).
METHODOLOGY
Chemistry
The synthesis of the new derivatives of 1,2,4-oxadiazole-5-ones (11a-e) involved a sequence of six consecutive reactions. Initially, the preparation of aryl-amidoximes (3ae) was carried out through a classical reaction between aromatic nitriles and hydroxylamine, following the methodological procedures described by Mayer et al.,(2021). Subsequently, the synthesis of 1,2,4-oxadiazolin-5-ones (5a-e) was performed, adapting the methodology from the works of Soldatova et al., (2021) and Reddy et al., (2017). These oxadiazolinones were prepared in two steps. First, the synthesized arylamidoximes (3a-e) reacted with ethyl chloroformate in the first step, yielding the reactive intermediate (4a-e). In the second step, after reacting with sodium hydroxide, the desired product was obtained. Next, the 1,2,4-oxadiazolidin-5-ones (5a-e) underwent alkylation with propargyl bromide in dimethylformamide, resulting in the formation of 3-aryl-4- (propinyl)-1,2,4-oxadiazolin-5-ones (7a-e) (WHITE, 1995). Subsequently, the "click" reaction, involving alkyne and azide, adapted from Wu et al.'s work (2009), was carried out between the 3-aryl-4-(propinyl)-1,2,4-oxadiazolin-5-ones (7a-e) and 4-azidobenzoic acid (8), catalyzed by copper, to produce the substances (9a-e). Following this, amidification was conducted between the methyl ester of tryptophan and the triazolic derivatives of 1,2,4-oxadiazolin-5-ones (9a-e), resulting in 5 novel substances (11a-e). It's worth noting that only the substances that demonstrated the best results in molecular docking were synthesized.
Molecular Docking Simulations
The in silico study (molecular docking) was conducted to investigate the binding modes of the synthesized compounds on ACE. These studies also provided valuable insights that guided the development of new selective inhibitors. Initially, redocking simulations were carried out using the GOLD Suite software (V5.3) to validate the employed methodology. In this experiment, the structure with PDB code: 1O86 (selective for the cECA domain) was utilized since its co-crystallized ligand was a well-known inhibitor (lisinopril). Considering the structural complexity of ACE, certain constraints were applied to identify the most appropriate docking poses for further in silico study. In this case, the constraints were directed towards the nitrogen atoms (3826 and 2552) of the residues His353 and His513 (CABALLERO, 2020). Following the validation of the assay through redocking simulations, the study proceeded to perform molecular docking with a maximum of 100 runs for all designed structures. In this scenario, ligand structures were generated using ChemBioDraw Ultra (v13.0) (Cambridgesoft, Billerica, MA, USA). The interactions (ligand-receptor) were subsequently analyzed and visualized through the PyMOL and Ligplot programs.
RESULTS AND DISCUSSION
Molecular Docking
Molecular docking in silico was employed to guide the synthesis of new oxadiazolidin-5-one derivatives. The simulations began with the selection of the crystallographic structure from the PDB database, identified by the code 1O86 (selective for the cECA domain). After selecting the structure, redocking simulations were carried out to validate the chosen method for screening new inhibitor candidates. However, molecular docking simulations for ACE are quite complex due to the considerable size of the binding sites (nECA and cECA) and the enzyme's shape. To reproduce docking experiments with consistent anchoring solutions, it was necessary to apply certain restrictions that consider the major hydrogen bonds. These restrictions ensure that all hydrogen bonds considered essential for activity are found during the docking process (CABALLERO, 2020). In this case, two restrictions were applied to the nitrogen atoms (3826 and 2552) of residues His353 and His513. Thus, the software (GOLD Suite) could recognize that the interaction of a polar group with the zinc ion was crucial for successful docking poses.
After the successful completion of the redocking simulation under the aforementioned conditions, the Root Mean Square Deviation (RMSD) value was found to be 0.961 Å, which aligns with the desired parameters for method validation. Following the validation of the method through redocking simulations, the molecular docking (MD) study for the inhibitor candidates (11a-e) was conducted. Upon completion of the assay, two analysis parameters were initially evaluated: the score and the number of hydrogen bonds. The score obtained in the redocking for the co-crystallized ligand lisinopril was 117.52. In the case of the inhibitor candidates (11a-e), slightly lower values ranging from 94.25 to 102.70 were observed. Regarding the number of hydrogen bonds, a somewhat more significant variation was noted. While lisinopril forms approximately eight hydrogen interactions, the designed compounds establish up to five hydrogen bonds.
In this context, the tested compounds (11a-e) formed at least one hydrogen bond with four specific residues (His353, His383, Asn66, and Tyr520). Concerning chelation interactions with the metallic zinc, it was observed that all designed compounds (11a-e) engaged in interactions with the catalytic zinc (Zn2+) through the carboxyl group. This interaction is considered crucial since most ACE inhibitors bind to the catalytic region of active sites via chelation interactions with the metallic zinc.
Furthermore, it was observed that the zinc atom interacts with three amino acid residues (His383, Glu411, and His387). Although compound 11d did not exhibit any interaction with the catalytic zinc, the formation of three hydrogen bonds with conserved residues in the S2 subsite of the cECA domain was observed. These interactions involve Gln281, Lys511, Tyr520, and Ala356. These interactions occur as follows: the oxygen from the 1,2,4-oxadiazolidin-5-one ring forms bonds with the Gln281 and Lys511 residues, while the carbonyl oxygen, also linked to the heterocyclic ring, interacts with the mentioned residues. Compound 11e also engages in this interaction with Ala356.
Synthesis of Inhibitor Candidates
Synthesis of Aril-Amidoximes
The preparation of the series (11a-e) began with the synthesis of 5 aryl amidoximes. To achieve this, the methodology described by Mayer et al., 2021, was employed, which outlines the synthesis as a conventional reaction between aromatic nitriles and hydroxylamine hydrochloride under alkaline conditions. The resulting products (3a-e) from this reaction exhibited yields ranging from 58% to 90%, and their melting points were determined and compared with literature data.
Synthesis of 1,2,4-Oxadiazolidin-5-ones
The 1,2,4-oxadiazolidin-5-ones described in this study were synthesized following the method developed by the current research group at LASOM (Medicinal Organic Synthesis Laboratory), which proves to be highly promising and innovative. The synthesis of compounds 5a-e proceeded in two steps, employing ultrasound at 50°C, with a total duration of two hours. Consequently, five products (5a-e) were obtained with yields ranging from 67% to 89%, and their melting points were determined and compared with values previously reported in the literature.
Synthesis of 3-Aryl-4-(propinyl)-1,2,4-oxadiazolidin-5-ones
The series (7a-e) was obtained through the alkylation reaction of 1,2,4- oxadiazolidin-5-ones with propargyl bromide in an alkaline medium. This synthesis led to the formation of five novel products (7a-e) with yields ranging from 76% to 94%. Additionally, melting point analyses and NMR spectroscopy were conducted for the characterization of these compounds.
Alkyne-Azide "Click" Reaction between 3-Aryl-4-(propinyl)-1,2,4-oxadiazolidin-5- ones and 4-Azidobenzoic Acid
The alkyne-azide "click" reaction, also known as CuAAC, involves a 1,3-dipolar cycloaddition between a terminal alkyne and an organic azide catalyzed by copper, resulting in the regiospecific formation of 1,2,3-triazoles-1,4-disubstituted compounds. Using the principles of the click reaction and following the methodology of Silva et al. (2012), five novel compounds (9a-e) were synthesized, with yields ranging from 56% to 96%. Subsequently, characterization was performed via NMR (1H and 13C), and the melting points of the synthesized compounds were determined experimentally.
Amidification Reaction between the Methyl Ester of L-Tryptophan and the Triazolic Acid Derivatives of 1,2,4-oxadiazolidin-5-ones
The amidification reaction was conducted at room temperature, where the L- tryptophan methyl ester (10) reacted with the triazolic derivatives (9a-e). The reaction was mediated by triethylamine as a base, with dry and distilled dimethylformamide as the solvent, and 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as the carbonyl activating agent. This reaction resulted in the synthesis of five novel substances (11a-e), which were purified through silica gel column chromatography and isolated with yields ranging from 66% to 96%. Subsequently, the products were characterized using 1H and 13C NMR spectroscopy, and their melting points were determined experimentally.
PERSPECTIVES
Through the research conducted in this study, we aim to demonstrate that the newly synthesized derivatives of oxadiazole-5-ones exhibit a selective ability to inhibit angiotensin-converting enzyme (ACE) in vitro, with the potential to be incorporated into the pharmaceutical treatment of arterial hypertension, thus providing a less harmful option for patients. Additionally, we believe that the molecular docking simulations will provide valuable insights to guide future structural modifications of inhibitor candidates, making them more targeted and identifying which functional groups are most relevant for maintaining the activity of the synthesized substances.
CONCLUSION
Through the six consecutive steps of the synthetic route proposed in this study, 15 novel substances, namely (7a-e), (9a-e), and (11a-e), were obtained with chemical yields ranging from 50% to 99%, 56% to 96%, and 66% to 96%, respectively. All these substances were characterized using 1H and 13C NMR spectroscopy techniques. Furthermore, the molecular docking study not only guided the synthesis of the compounds but also provided a deeper understanding of the interactions at the molecular level between the synthesized substances (11a-e) and ACE. The simulations indicated that these substances establish at least one hydrogen bond with four specific residues, namely, His353, His383, Asn66, and Tyr520. Regarding interactions with the catalytic zinc (Zn2+), it was observed that the substances form complexes with the metal through the carboxyl group. Moreover, it is believed that structural modifications increasing hydrogen bonds may positively influence the inhibitory potential of the substances, allowing for stronger interactions with the active site of ACE.
ACKNOWLEDGMENTS
I would like to thank the Federal University of Pernambuco and CAPES for the scholarship received.
REFERENCES
PAN AMERICAN HEALTH ORGANIZATION (PAHO). Arterial hypertension. Available at: https://www.paho.org/pt/campanhas/dia-mundial-da-hipertensao-2022. Accessed on: 11.Sep.2023.
BARROSO, W. K. S. et al. Brazilian Guidelines on Arterial Hypertension-2020. Arq Bras Cardiol, 2021. v. 116, n. 3, p. 516–658.
WHELTON, Paul K et al., 2017, ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: executive summary. Hypertension, [S.L.], v. 71, n. 6, p. 1269-1324, jun. 2018. Ovid Technologies (Wolters Kluwer Health).
ZAMAN, Mohammad Amin; OPARIL, Suzanne; CALHOUN, David A Drugs targeting the renin–angiotensin–aldosterone system. Nature Reviews Drug Discovery, [S.L.], v. 1, n. 8, p. 621-636, ago. 2002. Springer Science and Business Media LLC.
THOMOPOULOS, Costas; PARATI, Gianfranco; ZANCHETTI, Alberto. Effects of blood pressure lowering on outcome incidence in hypertension. 1. Overview, meta-analyses, and meta-regression analyses of randomized trials. Journal Of Hypertension, [S.L.], v. 32, n. 12, p. 2285-2295, dez. 2014. Ovid Technologies (Wolters Kluwer Health).
HOFFMAN, B. B. Catecholamines, Sympathomimetic Drugs and Adrenergic Receptor Antagonists. In Goodman & Gilman's The Pharmacological Basis of Therapeutics: Hardman, J. G.; Limbird, L. E.; Eds. McGraw-Hill: New Yprk, 2001, 10th Ed., pp. 215- 268. HUISGEN, Rolf; SZEIMIES, Günter; MÖBIUS, Leander. 1.3?Dipolare Cycloadditionen, XXXII. Kinetik der Additionen organischer Azide an CC? Mehrfachbindungen. Chemische Berichte, [S.L.], v. 100, n. 8, p. 2494-2507, ago. 1967.
BAKRIS, G. L. Is blockade of the renin-angiotensin system appropriate for all patients with diabetes? Journal of the American Society of Hypertension, v. 3, n. 5, p. 288-290, Oct. 2009.
CABALLERO, Julio. Considerations for Docking of Selective Angiotensin-Converting Enzyme Inhibitors. Molecules, [S.L.], v. 25, n. 2, p. 295, 11 jan. 2020. MDPI AG. CAI, Jin et al. Discovery and preliminary evaluation of 2-aminobenzamide and hydroxamate derivatives containing 1,2,4-oxadiazole moiety as potent histone deacetylase inhibitors. European Journal Of Medicinal Chemistry, [S.L.], v. 96, p. 1-13, maio 2015. Elsevier BV.
FUCHS, S et al. Angiotensin-converting enzyme: The C-terminal catalytic domain is the primary site for in vivo cleavage of angiotensin I. Hypertension 2008 , 51 , 267-274.
COTTON, Joël et al. Selective Inhibition of the C-Domain of Angiotensin I Converting Enzyme by Bradykinin Potentiating Peptides. Biochemistry, [S.L.], v. 41, n. 19, p. 6065- 6071, 20 abr. 2002. American Chemical Society (ACS).
NATESH, Ramanathan et al. Structural Details on the Binding of Antihypertensive Drugs Captopril and Enalaprilat to Human Testicular Angiotensin I-Converting Enzyme. Biochemistry, Reino Unido, v. 43, n. 27, p. 8718-8724, 18 jun. 2004.
GEORGIADIS, Dimitris et al. Structural Determinants of RXPA380, a Potent and Highly Selective Inhibitor of the Angiotensin-Converting Enzyme C-Domain. Biochemistry, [S.L.], v. 43, n. 25, p. 8048-8054, 1 jun. 2004. American Chemical Society (ACS).
NAYER, J. C. P. et al. Synthesis, spectroscopic characterization and DNA/HSA bind study of (phenyl/naphthyl)ethenyl-substituted 1,3,4-oxadiazolyl-1,2,4-oxadiazoles. New Journal of Chemistry, 2021. v. 45, p. 471–484.
SOLDATOVA, Natalia S. et al. Copper?Catalyzed Selective N?Arylation of Oxadiazolones by Diaryliodonium Salts. Advanced Synthesis & Catalysis, [S.L.], v. 363, n. 14, p. 3566- 3576, 8 jun. 2021. Wiley EDDY, I. K. et al. Synthesis of 1,2,4-Oxadiazol-5(4H)-Ones and Evaluation of their AntiMycobacterial Activity. Organic Chemistry: An Indian Journal, Índia, v. 13, n. 1, p. 1- 13, maio 2017.
WHITE, Donald A. Alkylations with Potassium Carbonate in Dimethylformamide. Synthetic Communications, [S.L.], v. 7, n. 8, p. 559-568, jan. 1977. Informa UK Limited. Catalyzed ‘ Click Chemistry ’ from Azides and Acetylene Gas. Synlett, 2009. v. 9, p. 1453–1456.
Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
Complete tool for submission, evaluation and publication of proceedings for scientific events.
