Elsevier

Bioorganic Chemistry

Volume 97, April 2020, 103647
Bioorganic Chemistry

Cholinesterases, α-glycosidase, and carbonic anhydrase inhibition properties of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives: Synthetic analogues for the treatment of Alzheimer's disease and diabetes mellitus

https://doi.org/10.1016/j.bioorg.2020.103647Get rights and content

Highlights

  • In this study, using the Cu (OTf) 2 catalyst, 1H-pyrazolo [1,2-b] phthalazine-5,10-dione derivative molecules were carried out in one step and with high yield (86–91%).

  • As a kinetic study, inhibition of the synthesized molecules on CA I, CA II, AAChE, BChE and α- Gly were performed.

  • They showed nanomolar inhibition levels on metabolic enzymes.

Abstract

In this study, using the Cu(OTf)2 catalyst, 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivative molecules were carried out in one step and with high yield (86–91%). The previously synthesized 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives, carbonic anhydrase I and II isozymes (hCA I and II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and α-glycosidase (α-Gly) enzymes with Ki values in the range of 4.88–15.94 nM for hCA I, 7.04–20.83 nM for hCA II, 68.25–158.27 for AChE, 60.17–91.27 for BChE and 0.36–2.36 nM for α-Gly, respectively. In silico studies were performed on the molecules inhibiting hCA I, hCA II, AChE, BChE and α-Gly receptors. When we evaluated the data obtained in this work, we determined the inhibition type of the 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives at the receptors. Reference inhibitors were used for all enzymes.

Introduction

Nitrogen-containing heterocyclic compounds are common in nature and their application to pharmaceuticals, agrochemicals, and functional materials is becoming increasingly important. 1H-pyrazolo[1,2-b]phthalazine-5,10-diones are important nitrogen-containing heterocyclics known to have many pharmacological and biological activities such as anticancer [1], antimicrobial [2], antifungal [3], anti-inflammatory [4] and anticonvulsant [5]. Therefore, it is very important to develop a simple method for 1H-pyrazolo[1,2-b]phthalazine-5,10-diones. The multicomponent reactions (MCRs) are one of the most promising methods for the synthesis of heterocyclics. These reactions have attracted great interest by organic chemists as they provide basic products in a single step with the formation of several new bonds. MCRs are crucial in the field of organic and medical chemistry for the development of new methodology since they are carried out in one-step to avoid time-consuming and costly processes for intermediate product isolation, eliminate molecular complexity and increase impressive selectivity. Organic reactions under solvent-free conditions are not only interesting from an environmental point of view but also offer significant advantages in terms of efficiency, selectivity, and simplicity of the reaction procedures. Due to the high toxicity of the chemical industry, organic solvents are a source of chemical contamination and their removal or replacement is very helpful to the environment. In most cases, the reaction was carried out under solvent-free conditions better and faster than conventional methods.

Carbonic anhydrases (CAs, E.C. 4.2.1.1) as zinc metalloenzymes, activate the reversible reaction of carbon dioxide and bicarbonate in prokaryotes and eukaryotes, and this reaction has a noteworthy role in many cases such as transportation and respiration of CO2 and HCO3, pH balance, biosynthetic reactions, calcification, electrolyte secretion, and tumorigenicity [6] Carbonic anhydrase inhibitors or activators show numerous bioactivities including anticancer, antiinfectives, diuretics, antiobesity, antiepileptics, antiglaucoma and anti-AD [7]. In the class of CA isoforms, CA I, which located in erythrocytes, and gastrointestinal tract, on the other hand, CA II isoenzyme is located in erythrocytes, eye, gastrointestinal tract, kidney, bone osteoclasts, brain, lung, and testis. Both of isoenzymes are the most investigated ones [8] and have remarkable metabolic roles like gas-exchange and ion transportation. Inhibition of human CA II is an effective process for the treatment of diverse illness including glaucoma, gastric ulcers, and epilepsy. Even with significant developments on CAIs, crucial problems in CAIs design are a high amount of isoenzymes in many tissues, and the deficiency of enzyme selectivity of them [9], [10].

Acetylcholinesterases (EC.3.1.1.7, AChE) are an enzyme in the hydrolase class. Other names used in the world of science; true cholinesterase, cholinesterase I, and erythrocyte cholinesterase [11]. The pharmacological importance of AChE encoded by chromosome 7 is the target of drugs used for the treatment of many diseases, especially Alzheimer's disease (AD) [12]. In various in vivo and in vitro studies, AChE enzyme has been shown to be effective in morphometric processes, cell differentiation in the nervous system, synaptogenesis, cell adhesion and migration, apoptotic pathways [13]. The primary biological role of AChE is to rapidly terminate the neural impulse that occurs when AChE is released in the synaptic space [14], [15]. Both in vitro and in vivo studies have shown that AChE levels are increased in apoptotic cells. For example, rat kidney cells have shown a high rate of AChE during apoptosis.

Butrylcholinesterases (BChE) is synthesized in the liver and released into plasma. It is also found in the intestinal mucosa, spleen, pancreas, white matter and many other tissues except erythrocytes. The physiological role is exactly unknown. Both enzymes can be distinguished by different catalytic activities. BChE hydrolyzes many choline esters such as butyrylcholine, mivacurium, procaine, chlorprocaine, tetracaine, cocaine, heroin, succinylcholine, and benzoylcholine [16], [17], [18], [19], [20].

α-Glycosidase (α-Gly) enzymes are found on the brushy surface of the small intestine. And they are responsible for the breakdown of complex carbohydrates. These enzymes wash oligo and disaccharides into monosaccharides. In monosaccharides, it is easily absorbed from the intestinal wall and into the blood pass α-glycosidase enzyme inhibitors they inhibit. The effects of α-glycosidase enzyme inhibitors on these enzymes are different. The net result of enzyme inhibition is the delay in the absorption of carbohydrates [21]. This delay does not cause malabsorption. In addition to delaying carbohydrate absorption, α-glycosidase enzyme inhibitors may also alter gastrointestinal hormonal axes [22].

The aim of this study was to investigate the inhibition impacts of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivative molecules (4a–f) that we worked on carbonic anhydrases, cholinesterases and α-glycosidase enzymes in in vitro conditions. In addition, enzyme and inhibitor interaction was examined by molecular docking study.

Section snippets

General procedure for the one-pot synthesis of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives (4a–f)

A mixture of phthalhydrazide (1) (1 mmol), substituted benzaldehyde (2) (1 mmol), malononitrile (3) (1 mmol), Cu(OTf)2 (10 mol %) in ethanol (5 mL) was refluxed at 80 °C for the appropriate time. The progress of the reaction was monitored by TLC. After completion of the reaction, the crude product was filtered and then washed with water. The solid product was purified by recrystallization procedure in ethanol or column chromatography. All the products were characterized by spectroscopic data

Chemicals

All of the chemicals used in the experimental studies, AChE, BChE, α-Gly enzymes were obtained from Sigma-Aldrich Co. Column packing prepared to purify the CA I, CA II enzyme were also purchased from Sigma-Aldrich Co. (Sigma-Aldrich Chemie GmbH Export Department Eschenstrasse 5, 82024 Taufkirchen, Germany). Molecular docking studies of synthesized novel compounds were performed using the Small Drug Discovery Suites package (Schrödinger 2017-2, LLC, USA). The molecular docking studies were

Conclusion

Herein, substituted 1H-pyrazolo[1,2-b]phthalazine-5,10-diones having biological activity were obtained via one-pot multi-component cyclocondensation reaction of phthalhydrazide, aromatic aldehydes and malononitrile catalyzed in the presence of Cu(OTf)2 in very good yields and short times. Molecular docking studies have provided insight to inhibition mechanism of the most active compounds on the receptors. The dioxo moiety of all most active compounds plays a critical role for inhibition of hCA

Acknowledgements

The synthesis part of this study was supported by Yildiz Technical University Research Fund. Project Number: 2012-01-02-GEP01.

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