Structure-Activity Relationships of N-Cinnamoyl and Hydroxycinnamoyl Amides on α-Glucosidase Inhibition

1Faculty ofMathematics andNatural Sciences, South-WestUniversity “Neofit Rilski”, 66 IvanMihailov Str., 2700Blagoevgrad, Bulgaria 2REQUIMTE-UCIBIO, Departamento de Quı́mica e Bioquı́mica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal 3Faculty of Science, Section of Chemistry, Charles University, Hlavova 2030/8, 12843 Prague 2, Czech Republic 4Faculty of Education, Department of Chemistry, Janos Selye University, Bratislavská Cesta 3322, 94501 Komárno, Slovakia


Introduction
Universally it is accepted that overproduction of radicals in biological systems, termed as oxidative and nitrosative stress [1][2][3][4], has been implicated in the pathophysiology of a variety of diseases, including cancer, ischemia/reperfusion injury, inflammatory diseases, neurodegenerative disorders, ageing, and diabetes mellitus [5].
Nowadays, diabetes mellitus is known to affect nearly 30 million people worldwide.Being the most serious chronic metabolic disorder, diabetes is associated with high concentration of blood glucose [6,7].Glucosidases (EC 3.2.1.20)[8,9] belong to glycoside hydrolase enzymes which convert complex sugars into their monomeric forms.Therefore, the possible way for reduction of the rate of carbohydrate digestion and hence decrease of the after-meal glucose levels is an inhibition of the activity of -glucosidase.
Therefore, the search for potential therapeutic glucosidase inhibitors from both synthetic and natural origin is of great demand.
In this context, the aim of the current study is the evaluation of -glucosidase inhibitory potential of cinnamic and hydroxycinnamic acid amides with amino acid.
Thin-layer chromatography (TLC) was conducted on precoated Kieselgel 60F254 plates (Merck, Germany).Separation of the compounds was accomplished by using preparative thin-layer chromatography with silica gel 60 GF245 (Merck, Bulgaria), and the solvents were used without further purification.
The NMR experiments were recorded on Bruker Avance III 600 or Bruker Avance III 400 spectrometer, operating at 600.13 and 400.15 MHz for protons, respectively.The measurements in CDCl 3 solutions were carried out at ambient temperature (300 K) and tetramethylsilane (TMS) was used as an internal standard.The UV spectra of the compounds were measured with an "Agilent 8453" UV-vis spectrophotometer.Electrospray Ionisation (ESI) mass spectra were recorded on an Esquire 3000.

Synthesis of N-Cinnamoyl and
Hydroxycinnamoyl Derivatives
Briefly, loading of the Fmoc-Gly-OH to Rink amide resin and all acylation reactions were carried out for 1 h using a 3-fold excess of Fmoc-amino acids activated with DIC (3.5 mmol) in the presence of HOBt (3.5 mmol).The efficiency of coupling was checked by ninhydrin or isatin tests.The sinapoyl-peptide was removed from the resin with acidolysis with 95% TFA/H 2 O for 45 min.The crude products were purified by preparative TLC (CHCl 3 /CH 3 OH = 2 : 1).
SA-Tyr-Pro-Leu-Gly- 2 (16) -Glucosidase Inhibitory Assay.The -glucosidase inhibition activity of tested compounds was determined according to the method reported by Mayur et al. [36] with slight modifications.Briefly, the reaction mixture contained 50 l of 0.1 M phosphate buffer (pH 7.0), 25 l of 0.5 mM 4nitrophenyl--D-glucopyranoside, 20 l of test sample at various concentrations, and 25 l of -glucosidase solution (0.8 Unit/ml).The mixtures were then incubated at 37 ∘ C for 10 min, and then 100 l of 0.2 M Na 2 CO 3 was added to terminate the reaction.Absorbance of p-nitrophenol released during the enzymatic hydrolysis of substrate 4nitrophenyl -D-glucopyranoside was monitored at 405 nm using microplate reader Stat Fax 303 Plus.Acarbose was used as standard glucosidase inhibitor.
All experiments were carried out in triplicate.The inhibition percentage of -glucosidase was estimated by the following formula: where IC 50 values were calculated by the graphic method.

Results and Discussion
Following the assumption that hyperglycemia causes oxidative stress, herein we investigated if the antioxidant active hydroxycinnamoyl amides might serve as potential glucosidase inhibitors.
In order to investigate the structure--glucosidase inhibitory relationships of hydroxycinnamoyl amides, some of the previous evaluated cinnamoyl compounds were used for comparison (Table 1).These results were previously obtained by us [32] and were used for comparison.

Inhibition of a-Glucosidase Activity by N-Hydroxycinnamoyl
Amides and Cinnamic Acids.The inhibitory effects of synthesized N-hydroxycinnamoyl amides and substituted cinnamic acids on yeast a-glucosidase were estimated in vitro.For comparison of their inhibitory potencies, the IC 50 values of the tested compounds and acarbose (a clinically used a-glucosidase inhibitor) were determined (IC 50 = 2.5 ± 0.04 g/ml).
However, our findings revealed that N-hydroxycinnamoyl amides with one tryptophan residue (3, 4) were inactive (IC 50 > 200 g/ml), whereas the introduction of two tryptophan moieties in caffeic and sinapic acid amides (1, 2) pronouncedly exerted a higher glucosidase inhibition.Interestingly, the observed significant differences are due to the binding affinity of hydroxycinnamoyl derivatives with four catalytic regions of the family I -glucosidases [37].Our ongoing experiment should be focused on the clarification of molecular mechanisms of hydroxycinnamic acid derivatives against -glucosidase.
Considering the cinnamoyl and hydroxycinnamoyl amides with the same phenylalanine moiety (5-8) it was found that the presence of hydroxyl or methoxyl groups at cinnamoyl moiety resulted in an enhancement of glucosidase inhibitory activity.Likewise, amongst the hydroxycinnamic acids, the cinnamic acid displayed the lowest potency, whereas sinapic acid showed the highest inhibition (the IC 50 value of 1.17 ± 0.1 g/ml) on yeast a-glucosidase.

Conclusions
In conclusion, sixteen N-cinnamoyl and hydroxycinnamoyl amides were synthesized in satisfactory yields using solution or solid phase carbodiimide-mediated couplings.
A preliminary study of synthetically obtained amides on a-glucosidase inhibition indicated that N-hydroxycinnamoyl amides 1, 2, 5, 6, 9, 14, and 15 exhibited extremely high in vitro inhibitory potency against -glucosidase and can serve as very promising therapeutic glucosidase inhibitors.

Table 1 :
Glucosidase inhibitory activity of N-hydroxycinnamoyl amides.The given values are the means ± SD of triplicate samples.