Chinese Chemical Letters  2016, Vol. 27 Issue (5): 627-630   PDF    
Metal complexes of anthranilic acid derivatives: A new class of noncompetitive α-glucosidase inhibitors
Jing-Wei Zheng, Lin Ma     
School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China
Abstract: Metal complexes of anthranilic acid derivatives that constitute a novel class of non-sugar-type α-glucosidase inhibitors were synthesized and assessed in vitro for inhibitory activity. All of the Ag(Ⅰ) complexes (9-16) inhibited α-glucosidase at the nanomolar scale, while 3,5-dichloroanthranilic acid silver(Ⅰ) (9) was the most potent (IC50=3.21 nmol/L). Analysis of the kinetics of enzyme inhibition indicated that the mechanism of the newly prepared silver complexes was noncompetitive. The structure-activity relationships were also analyzed, and they are discussed in this report.
Key words: α-Glucosidase     Anthranilic acid derivatives     Metal complexes     Noncompetitive inhibitors    
1. Introduction

α-Glucosidases are crucially important in the processing of glycoproteins and glycolipids,in the digestion of carbohydrates in the intestinal tract,and in other vital metabolic processes [1, 2, 3]. Glucosidase activity is also essential for hydrolyzing polysaccharides to monosaccharide units,catabolizing lysosomal glycoconjugates,and biosynthesizing oligosaccharide units [4]. α-glucosidase inhibitors have been shown to have therapeutic potential in the treatment of diabetes mellitus,obesity,human immunodeficiency virus infections,and metastatic cancer via the control or regulation of enzyme activity [5, 6, 7, 8]. Currently,the main α-glucosidase inhibitors are disaccharides,iminosugars,carbasugars,thiosugars,and sugar mimetics configurationally derived from glucose to yield high anti-glucosidase activity. Docking simulations of α-glucosidase inhibitors suggest that they possess ammonium cation moieties and carboxylic groups,both of which exhibit strong electrostatic interactions with amino acid residues in the binding pocket of the enzyme. The amino acid residues may have π-stacking and hydrophobic effects with the -NH and -OH groups. The formation of hydrogen bonds with amino and carboxyl groups could play a crucial role in specific molecular recognition and affinity. Hence,amino acid derivatives have been evaluated for their ability to inhibit enzyme activity [9]. Metal ions,such as Cu and Fe,are physiologically important ions,and they have broad application in biochemistry as well as in medicine. One previous report suggested that transition metal ions could specifically inhibit α-glucosidase and had strong affinity towards the enzyme [10]. Moreover,zinc,nickel,and vanadium have been shown to be beneficial in diabetes therapy [11].

The biological activity of anticancer [12],catalytic [13],and antibacterial [14] amino acid metal complexes has been extensively studied. Metal complexes of amino-acid ligands with both N and O donor atoms are considered suitable for efficient interactions between metal ions and proteins. However,due to the zwitterion effect,the pH must be tightly regulated to stabilize the ligands during the reaction. Moreover,their instability and poor solubility make structural analysis difficult. Anthranilic acid is a viable alternative to amino acids as a ligand; it is an amino acid analogue,containing both carboxyl and amino groups as well as O and N atoms with high electronegativity capable of coordinating transition metals. Anthranilic acid is also the biochemical precursor to tryptophan,and its chemistry is vital to medicinal and biological sciences. Anthranilic acid derivatives are reported to possess a wide variety of biological activities,including antibacterial [15],and anti-inflammatory [16]. In addition,N-phenylanthranilic acid is used as an important intermediate in the synthesis of pharmaceutically active molecules,such as antimalarial,antiinflammatory,and antineoplastic [17]. Recently,there has been increasing interest in studying pharmaceutical ligands that have halogens introduced into their structures [18]. In the areas of pharmaceutical chemistry and drug discovery,the presence of strong C-halogen bonds has been shown to increase lipophilicity and catabolic stability [19, 20, 21]. One of the principle findings in protein database surveys was that aromatically bound halogens generally have stronger electron-withdrawing properties,which means that halogens bound to aromatic groups may have a greater capacity for coordinating with amino acid residues in enzymes [22, 23].

Thus,complexes of anthranilic acid,with halogen (F/Cl/Br/I) and nitro substitutions,and metals (Ag/Zn/Fe/Cu/Mn/Sn/Mg/Co/Ni) should be evaluated for their inhibitory effect on α-glucosidase as well as their structure-activity relationships. To the best of our knowledge,no such studies have been reported to date.

2. Experimental

All commercially available chemicals used were of analytical grade and used as procured. Saccharomyces cerevisiae α-glucosidase and 4-nitrophenyl-β-D-glucopyranoside (PNPG) were purchased from Sigma-Aldrich Chemical Co.,(St. Louis,Mo); Absolute ethyl alcohol and dimethylsulfoxide were domestic analytical reagents. Water used was re-distilled and ion-free. All 1H NMR spectra were elucidated using Mercury-Plus 300 MHz 1H NMR spectrometers (VARIAN,American) and tetramethylsilane was used as an internal standard. The solvent used was DMSO. All chemical shifts (δ) are quoted in ppm and coupling constants (J) in hertz. Bio-rad 680 microplate reader.

2.1. Synthesis of metal complexes of anthranilic acid derivatives (1-16)

The preparation of the metal complexes was carried out solidstate grinding in the following general procedure: 2 mmol anthranilic acid derivatives,1 mmol metal salts were grounnded until finely blended in an agate mortar. Microwave (800 W) for 3 min (grind 30 s per min),recrystallized with 30%-50% aqueous ethanolic,yielding 80%-90%. All the complexes are coloured solids,air stable for an extended period of time. All the complexes are soluble in DMSO at room temperature. Full details on the synthesis,purification,and analysis of the compounds are deposited in supporting information.

2.2. Bioassay procedures

Assay for α-glucosidase inhibitory activity [24, 25]: The enzyme and the substrate solution was prepared by dissolving S. cerevisiae α-glucosidase in 0.01 mol/L potassium phosphate buffer (pH 7). Diluted enzyme solution (10 μL),test samples (0.016-2 μL,in DMSO) and buffer solution (90 μL) were mixed in each well of a 96- well microtiter plate. After pre-incubated for 20 min at 37 ℃,PNPG (10 μL,1.5 mg/mL) was added to start the enzymatic reaction measured by a microtiter plate reader immediately. The increment of absorption at 405 nm is based on the hydrolysis of PNPG. Controls without enzyme or without substrate were included. The resveratrol was used as reference and averages of three replicates were presented. The inhibition percentage (%) was calculated by the equation:

Inhibition percentage(%)=[Abssample=Absblank]×100%

where,Absblank represents the absorbance of the blank with the same volume DMSO.

Kinetic assay: The enzyme solution (10 μL),0.01 mol/L potassium phosphate buffer and test samples (2 μL in DMSO) were mixed in a 96-well microtiter plate. After incubation at 37 ℃ for 20 min,PNPG (0.45-1.50 mg/mL) was added and measured by a microtiter plate reader at 405 nm straight away. The X-axis of Lineweaver-Burk plot is the reciprocal of the concentration of PNPG; the Y-axis is the reciprocal of the rate of enzyme reaction. All the experiments were carried out in triplicate.

3. Results and discussion 3.1. Inhibitory activity of α-glucosidase

A concise synthesis of anthranilic acid-derivative metal complexes is reported in this paper by applying mechanochemistry under solvent-free conditions assisted by microwave irradiation,which in addition to experimental simplicity offers significant environmental advantages [17]. A generalized synthetic approach for the proposed compounds 1-16 is shown in Scheme 1. Refer to the coordination mode of azo-linked Schiff base Cu(Ⅱ) [26],we hypothesized the common structure of complexes is shown in the Scheme 1. The structures of the target compounds were verified by 1H NMR,IR,and mass spectrometry.

Scheme. 1. Synthetic route for compounds 1–16.

The activity of the synthesized compounds was evaluated in vitro against S. cerevisiae α-glucosidase using a Bio-Rad 680 microplate reader at 405 nm. The results are expressed as the inhibitor concentrations required to achieve 50% inhibition of α-glucosidase activity and are shown in Table 1.

Table 1
The IC50 values of the synthesized compounds and the reference inhibitors (IC50 nmol/L).

According to the IC50 data,bivalent metallic ion complexes of 3,5-dichloroanthranilic acid showed little or no detectable inhibitory activity. In contrast,Ag(Ⅰ) complexes of anthranilic acid derivatives demonstrated potent inhibition,with IC50 values below 4 nmol/L,suggesting that the Ag(Ⅰ) significantly contributed to the inhibitory effects. Ag(Ⅰ) complexes have long been known to have metabolically inhibitory and bactericidal effects [27],while few studies have demonstrated their ability to inhibit α-glucosidase.

Among the Ag(Ⅰ) complexes,3,5-dichloroanthranilic acid 9 exhibited the strongest inhibitory activity (IC50 = 3.21 nmol/L). The introduction of halogen and nitro substituents generally produced remarkably inhibitory activities,with IC50 values of 3.21 and 11.8 nmol/L,respectively.

On comparing two compounds simultaneously (10 and 11,12 and 13,15 and 16),it is obvious that the 4-position substituent had a negative impact on the inhibitory activity,indicating that the 4- substituent may be unfavorable for binding with α-glucosidase. With compounds 9,10,11,the increase in chlorine atoms may have played an important role in promoting the enzymatic activity. Nevertheless,the fluorine atoms did not show the same pattern (i.e.,by comparing compounds 12 and 13). It is plausible that the introduction of a 4-fluorine substituent sharply decreased the activity. Generally,complexes of anthranilic acid,with halogen and nitro substitutions,and silver exhibited conspicuous inhibitory activity. Halogen atoms are typically found on the periphery of molecules,which favors their participation in intermolecular interactions. In addition,halogens and nitro groups favorably interact with oxygen and nitrogen atoms with a lone electron pair,a scenario that exists in most enzymes and that could be involved in hydrogen-bond interactions with hydrogen-bond donors [18]. It is worth mentioning that the anthranilic acids with halogen (F/Cl/Br/I) and nitro substitutions were also screened for their inhibitory activity. These ligands did not show potent inhibition,which implies that the interaction between the metal ions and the ligands may play the most profound role in decreasing the activity of the enzyme.

The reactant silver acetate 18 displayed inhibitory activity with an IC50 value of 27.3 nmol/L,while all of the complexes 9-16 exhibited better inhibitory activities than that of the central metal ion Ag(Ⅰ). We speculated that inhibition of α-glucosidase resulted not from the metal ions or ligands themselves. The -NH2,-COO,and -NO2,halogen groups and the π electrons of benzene in these compounds may have π-stacking,hydrophobic effects,hydrogenbonded salt bridges,electrostatic interactions,and other noncovalent bonds with the amino acid residues of the enzyme. Metal complexes have many advantages; for example,metal chelates reduce the polarity of the metal by sharing the positive charge with the ligands over the entire chelating ring and increase the lipophilic nature of the central metal atom [28]. On the other hand,anthranilic acids with halogen and nitro substitutions are excellent electron donors that are capable of stabilizing a large number of organometallic compounds.

3.2. Kinetics and the mechanism of α-glucosidase inhibition

Compounds 9,12,and 13 were selected for further investigation of the kinetics of enzyme inhibition (Fig. 1). As mentioned above,9 showed the strongest inhibitory activity and 12 and 13,with different numbers of fluorine atoms,were used as a reference. The kinetics show that the mechanism of α-glucosidase inhibition by this series of compounds was noncompetitive,indicating that these inhibitors possibly do not bind to the active site of the enzyme,and instead reduce the catalytic activity by modulating the enzyme-substrate transition state.

Fig. 1. Lineweaver–Burk plot analyses of the inhibition kinetics of α-glucosidase by compounds 9 (a), 12 (b), and 13 (c).

4. Conclusion

In conclusion,a series of complexes of anthranilic acid,with halogen- (F/Cl/Br/I) and nitro substitutions,and metals (Ag/Zn/Fe/ Cu/Mn/Sn/Mg/Co/Ni) were designed,prepared via a simple method,and evaluated for their potential as α-glucosidase inhibitors. The in vitro biological studies showed that the ligand complexes have more potent activities than the corresponding free ligands and metal ions. Among them,3,5-dichloroanthranilic acid Ag(Ⅰ) complex 9 was identified as the most potent α-glucosidase inhibitor,showing 4000 times higher activity than the reference inhibitors. The other Ag(Ⅰ) complexes 10-16 also exhibited good inhibitory activities,with IC50 values less than 12 nmol/L. Studies on the inhibition kinetics of compounds 9,12,and 13 indicated that this class of Ag(Ⅰ) complexes uses a noncompetitive mechanism. To the best of our knowledge,the effect of Ag(Ⅰ) complexes on α-glucosidase have thus far been largely ignored. Hence,due to their potent inhibitory activity,they may provide a new direction for controlling or regulating α-glucosidase activity to treat or prevent diseases caused by metabolic disorders,immune responses,tumor metastases,and viral infections.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

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