Design and synthesis of novel pyrazole-phenyl semicarbazone derivatives as potential α-glucosidase inhibitor: Kinetics and molecular dynamics simulation study

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Abstract

A series of novel pyrazole-phenyl semicarbazone derivatives were designed, synthesized, and screened for in vitro α-glucosidase inhibitory activity. Given the importance of hydrogen bonding in promoting the α-glucosidase inhibitory activity, pharmacophore modification was established. The docking results rationalized the idea of the design. All newly synthesized compounds exhibited excellent in vitro yeast α-glucosidase inhibition (IC50 values in the range of 65.1–695.0 μM) even much more potent than standard drug acarbose (IC50 = 750.0 μM). Among them, compounds 8o displayed the most potent α-glucosidase inhibitory activity (IC50 = 65.1 ± 0.3 μM). Kinetic study of compound 8o revealed that it inhibited α-glucosidase in a competitive mode (Ki = 87.0 μM). Limited SAR suggested that electronic properties of substitutions have little effect on inhibitory potential of compounds. Cytotoxic studies demonstrated that the active compounds (8o, 8k, 8p, 8l, 8i, and 8a) compounds are also non-cytotoxic. The binding modes of the most potent compounds 8o, 8k, 8p, 8l and 8i was studied through in silico docking studies. Molecular dynamic simulations have been performed in order to explain the dynamic behavior and structural changes of the systems by the calculation of the root mean square deviation (RMSD) and root mean square fluctuation (RMSF).

Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder resulting from defects in insulin action (type 2 diabetes), insulin secretion (type 1 diabetes), or both, which affects the metabolic functions of the endocrine system. Diabetes is undoubtedly one of the major public health problems worldwide [1,2]. According to WHO reports, more than 420 million people are suffering from diabetes and this number is expected to be more than 642 million by 2040 [2,3]. Type-2 diabetes is the most common type and accounts for approximately 80–90% of all diabetic cases, which is responsible for around 5% death globally [[4], [5], [6]]. This dysfunction leads to high blood glucose concentration or hyperglycemia. Prolonged hyperglycemia causes a wide variety of critical complications, including diabetic neuropathy [7,8], cancer [9,10], retinopathy [11], stroke [12,13], amputations [14,15], microangiopathy, and cardiovascular diseases [[16], [17], [18]].

One of the critical strategies to control diabetes complications is by managing the blood glucose level [19]. α-Glucosidase inhibitors prevent the hydrolysis α-glucosidic bond of complex carbohydrates and delay monosaccharide (α-d-glucose) absorption, which is mainly responsible to cause hyperglycemia [20]. Thus, α-glucosidase inhibitors have been considered as a most attractive therapeutic target for drug discovery in the treatment of type 2 diabetes [[21], [22], [23]]. Beside, α-glucosidase is biocatalyst involved in a series of relevant processes like glycolipid and glycoprotein metabolic routes, cell-to-cell signaling, and virus recognition [24,25]. Therefore, glucosidase inhibitors have attracted plenty of interest by the pharmaceutical industry as novel agents to treat other carbohydrate mediated diseases including viral infections, hepatitis, cancer, pompe disease, and other degenerative diseases such as nojirimycin and castanospermine [[25], [26], [27], [28], [29], [30]]. Currently, glycosidic based α-glucosidase inhibitors such as acarbose (Glucobay), miglitol (Glyset), and voglibose (Volix, Basen) are clinically used in the treatment of type-2 diabetes mellitus [31]. Although these medications have a certain therapeutic effect on diabetes, they are associated with serious adverse. As the result, they have to be used in combination with other anti-diabetic agents to improve the effectiveness [32]. On the other hand, the synthesis of other similar derivatives of mentioned α- glucosidase inhibitors is tedious and need complicated multi-step procedures due to the presence of sugar moieties in their structures [33]. So, it is still an appealing and interesting area of medicinal chemistry research to discover and develop efficient small molecules capable of possessing potent α-glucosidase inhibitory activities.

The heterocyclic scaffolds have attracted considerable attention of medicinal chemists due to their exceptional chemical and versatile biological profiles. In this category, pyrazole and their derivatives represent one of the key structural units in numerous medicinally important compounds with various pharmacological effects including anti-inflammatory, analgesic, antimicrobial, neuroprotective, antihypercholestrolemia, antimalarial, anti-Alzheimer, antihypertensive, antitubercular, antiviral, and antihyperglycaemic activities [[34], [35], [36]]. Pyrazole scaffold has also reported to show selective and potent inhibitory activity against lung, colon, renal, prostate, and breast cancer cells [37,38]. Furthermore, pyrazole derivatives possess the LRRK2 inhibitory activity to identify new drugs to treat Parkinson's disease [39]. Several pyrazole containing drug molecules with diverse therapeutic activities such as Celecoxib, sildenafil, Crizotinib, Surinabant, Rimonabant, and Pyrazofurin are now available in the market [34]. Historically, different substituted pyrazole had been reported as in vivo anti hypoglycemic agent [40]. Also, recent studies have confirmed that several entities of the pyrazole have been possessed antidiabetic [[41], [42], [43]] and hypoglycemic [[44], [45], [46]] activity (Fig. 1). In addition to the mentioned successful studies, significant metabolic stability and pharmacological efficiency of pyrazole derivatives as antidiabetic agents and approved the “Teneligliptin”, a pyrazole motif containing antidiabetic drug, encouraged us to further study on pyrazole scaffolds [47].

Benzohydrazides are important classes of compounds due to its wide range of activities, including antiglycation, antioxidant, antileishmanial, antitumor, antibacterial, and anticonvulsant [[48], [49], [50], [51], [52], [53]]. In recent years, several compounds possessing benzohydrazide scaffolds with high α-glucosidase inhibitory activity have been reported [25,54]. For instance, benzothiazole hybrids having benzohydrazide moiety (Fig. 2 compound C) were reported by Taha et al. as a potent α- glucosidase inhibitor (IC50 = 5.31 ± 0.03 μM comparing with acarbose, 906 ± 6.3 μM) [54].

Molecular hybridization has been a strong tool to design and develop new biologically active compounds with improved potency. In this work, in continuation of our efforts for developing new α-glucosidase inhibitors using molecular hybridization and endeavoring to extend the chemical space, we designed a new scaffold in order to identify lead candidates for more advanced research in future [[55], [56], [57]]. Steps for the rational design of new hybrid are depicted in Fig. 2 Enormous importance of hydrogen bonding in promoting the α-glucosidase inhibitory activity has inspired the modification of benzohydrazide to the phenyl semicarbazide moiety, which introduced extra nitrogen atom into the structure as hydrogen bond donor (Fig. 2 compound D). Modifications were implemented in the designed hybrid in order to enhance the key factors required to promote the glucosidase inhibitory such as p-stacking, hydrogen bonding, and hydrophobicity [58,59]. Hydrazone linker was included in the design of the new hybrid as part of the initial design. It was hypothesized that the addition of the hydrazine linkage could substantially decrease the entropic penalty for the formation of the enzyme-inhibitor complex (Fig. 2 compound B) [25]. Accordingly, our research team designed and synthesized a new hybrid moiety that incorporated pyrazole, phenyl semicarbazide, and hydrazone in a single structural entity and evaluated them against α-glucosidase enzyme for the first time. The docking results rationalized the idea of conjugation of pyrazole with arylsemicarbazone moiety. The designed hybrid packed in the catalytic hydrophobic pocket of active site whereas nitrogen and oxygen atoms of the semicarbazide formed hydrogen bonds with the enzyme (Fig. 2E).

Apart from in vitro assessment of target compounds, in silico study and mechanism underlying enzymatic inhibition were also investigated to have a better understanding of the interactions of the title compounds with α-glucosidase. Moreover, molecular dynamic simulation was performed to explain of the dynamic behavior and structural changes of the system.

Section snippets

Chemistry

The pathway for the synthesis of pyrazole-phenyl semicarbazone hybrids 8a-p has been depicted in Scheme 1. Different substituted acetophenones 1 reacted with phenyl hydrazine or 4-methyl phenyl hydrazine 2 in the presence of sulfuric acid in absolute ethanol to afford the hydrazone intermediates 3a-p. The synthesized hydrazone intermediates 3a-p then undergo a Vilsmeier–Hack reaction in the presence of DMF and POCl3 to form pyrazole carbaldehydes 4a-p. On the other hand, 4-phenyl semicarbazide

Conclusion

In conclusion, a novel series of pyrazole-phenyl semicarbazone hybrids were designed and synthesized as potential α-glucosidase inhibitors. The docking results rationalized the idea of hybridization. As a result, designed hybrid packed in the catalytic hydrophobic pocket of the active site while nitrogen and oxygen atoms of the semicarbazide formed hydrogen bonds with the enzyme. It is worth mentioning that inhibitory activity of all new designed compounds in the range of 1–12 fold were better

Experimental

All reagents and solvents used in this study were purchased from Sigma Aldrich (USA) and Merck (Germany) and were used without further purification. All the reactions were monitored on pre-coated silica gel aluminum plates (Merck silica gel 60, F254) and visualized using UV lamp at 254 nm (UVGL-58; Upland, USA). Melting points of the target compounds 8a-p were determined with a Kofler hot stage apparatus and are uncorrected. The IR spectra recoded with Nicolet FT-IR Magna 550 spectrographs

CRediT authorship contribution statement

Fateme Azimi: Investigation, Conceptualization, Methodology, Validation,Writing - Original Draft.

Jahan B. Ghasemi: Software.

Homa Azizian: Software, Methodology.

Mohammad Najafi: Software, Writing - Review & Editing.

Mohammad Ali Faramarzi: Resources.

Lotfollah Saghaei: Conceptualization, Project administration.

Hojjat Sadeghi-aliabadi: Resources.

Bagher Larijani: Funding acquisition,

Farshid Hassanzadeh: Project administration, Conceptualization.

Mohammad Mahdavi: Conceptualization, Project

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