Hindered phenolic aminothiazoles – Synthesis , α-glucosidase and α-amylase inhibitory and antioxidant activities

Base-catalysed heterocyclization of either N-aryl-N'-[imino(nitroamino)methyl]thioureas or N-aryl-N'-cyanothioureas by reaction with 2-bromo1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone afforded 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles, designed as molecular hybrids of hindered phenolic and 2-aminothiazole moieties. These compounds were screened for their inhibition activity on carbohydrate hydrolyzing enzymes. Thus, [4-amino-2-(phenylamino)-5-thiazolyl](3,5-di-t-butyl-4-hydroxyphenyl)methanone exhibited α-glucosidase inhibition activity with an IC50 value of 117 μM while the standard compound acarbose showed an IC50 value of 48.3 μM and {4-amino-2-[(4-methylphenyl)amino]-5-thiazolyl}(3,5-di-t-butyl-4-hydroxyphenyl)methanone showed good α-amylase inhibition activity with an IC50 value of 283 μM compared to acarbose (IC50 532 μM). The antioxidant activities of the hindered phenolic thiazoles were also investigated and the 2-[(4-methoxyphenyl)amino] derivative showed an antioxidant activity better than that of butylated hydroxyanisole in the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, better than that of either vitamin C or curcumin in the ferric ion-reducing antioxidant potential assay and comparable with that of butylated hydroxyanisole in the β-carotene bleaching assay.


INTRODUCTION
Diabetes mellitus is a chronic endocrine disease that affects the metabolism of carbohydrates.The goal of diabetes treatment is to maintain a nearly normal level of glycemic control subsequent to food intake so as to maintain the postprandial hyperglycaemia. 1 This could be achieved by inhibiting the carbo-1088 SATHEESH et al.
hydrate hydrolyzing enzymes involved in the breakdown of carbohydrates, such as α-glucosidase and α-amylase.Hence, carbohydrate digestive enzyme inhibitors are widely investigated in the identification of lead compounds for the treatment of diabetes. 2][5] Hindered phenols in which the phenolic hydroxyl group is juxtaposed with a sterically demanding group, such as a t-butyl group, have found wide application as antioxidants and permissible food preservatives. 6Typical examples of hindered phenols used in food preservation are 2-t-butyl-4-methylphenol (butylated hydroxytoluene, BHT), 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA) and t-butylhydroquinone (TBHQ).A recent report highlighted the importance of free phenolic groups in flavone, isoflavone and chalcone derivatives on their α-glucosidase inhibitory activity. 7The incorporation of a 2,6-di-t-butylphenolic unit to improve the bioactivities of flavonoids by designing hindered phenol-flavonoid hybrids and the antioxidant activity of hydrazones bearing a 2,6-di-t-butylphenolic unit have also been reported recently. 8The 2-aminothiazole moiety is isosteric with a phenolic unit and devoid of the acidity of the latter and hence, it finds much use in drug design. 9In connection with our interest in the anticancer 10,11 and neuroprotective 12 activities of 2,4-diaminothiazoles, it was noted that only a few reports exist on the antioxidant activity of aminothiazoles. 13,14With this background, it was hypothesized that 2,4-diaminothiazoles bearing a hindered phenol moiety could show promising antioxidant activities.Accordingly, the design and synthesis of hitherto unreported 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles as molecular hybrids incorporating di-t-butylphenol and 2,4-diaminothiazole moieties, along with their α--glucosidase and α-amylase inhibitory and antioxidant activities are reported herein.

Chemistry
Melting points are uncorrected and were determined by the open capillary method.The thin layer chromatographic analyses were performed using silica gel 60 F 254 TLC aluminium sheets purchased from Merck, Mumbai, India.The elemental analyses were performed on a Vario EL III elemental analyzer.The IR spectra were recorded on JASCO, Bomem MB and Shimadzu FTIR spectrophotometers.The NMR spectra were recorded on Bruker DPX-400 and 500 MHz spectrometers and FAB mass spectra were recorded on Jeol SX-102 FAB mass spectrometer.HRMS-ESI spectrum was performed at a resolution of 61800 using a Thermo Scientific Exactive mass spectrometer.All chemicals were from Sigma-Aldrich and Merck.The required N-aryl-N'-[imino(nitroamino)methyl]thioureas 1a-e were obtained from nitroguanidine and aryl isothiocyanates 2a-e as reported earlier. 15Reported procedures with slight modifications were used to prepare 1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone from 2,6-di-t--butylphenol 16 and its bromination 17 to obtain 2-bromo-1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone 4.

PHENOLIC AMINOTHIAZOLES 1089
Analytical and spectral data of the synthesized compounds are given in Supplementary material to this paper.
The crude products obtained by methods A and B were purified either by crystallization from ethanol or by dry column flash chromatography on thin layer chromatography grade silica gel eluted with hexane-ethyl acetate.

α-Glucosidase inhibition activity
The mode of α-glucosidase inhibition was studied according to Apostolidis et al. 18 Briefly, about 50 µL of homogenized sample solutions of varying concentrations (5-250 µM) and 100 µL of 0.1 M phosphate buffer (pH 6.9) containing α-glucosidase solution (1.0 U mL -1 ) was incubated in 96 well plates at 25 °C for 10 min.After pre-incubation, 50 μL of p-nitrophenyl α-D-glucopyranoside solution (7.5 mg in 5 ml; 5 mM) in 0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals.Before and after incubation at 25 °C for 5 min, the absorbance at 405 nm was measured using an Enspire multimode reader (Perkin Elmer).Acarbose was used as the positive control and the results are expressed as percent inhibition, calculated as: (1-A sample /A control )×100 (1 where A is the absorbance.

α-Amylase inhibition activity
The inhibitory activity of α-amylase enzymes (from Aspergillus oryzae) was performed using a reported procedure with a slight modification. 18Briefly, different concentrations of the stock solutions of the samples (100-600 µM) were incubated with an α-amylase solution (0.5 mg/ml) in 0.02 M phosphate buffer (pH 6.9 with 0.006 M NaCl, 500 μL) at 25 °C for 10 min.After pre-incubation, 500 μL of a 1 % starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to each tube at timed intervals.The reaction was stopped with 500 µL of 3,5-dinitrosalicylic acid (1 %) as a colour reagent.The tubes were then incubated in a boiling water bath for around 5 min, cooled to room temperature and 1090 SATHEESH et al.
diluted to 10 mL with distilled water.The absorbance was measured at 540 nm using acarbose as the standard.The percentage of inhibition was calculated using the formula (1).

Antioxidant capacity assays
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity.The DPPH radical scavenging efficacy of the compounds was evaluated based on a reported procedure. 19Briefly, aliquots of the test samples leading to a concentration range of 100-600 µM were mixed with a methanolic solution of DPPH (1.5 mL; 25 mg L -1 ), kept in the dark for 30 min and the absorbance was measured at 517 nm against the control.BHA and curcumin served as standards.The percentage radical scavenging activity, calculated as the scavenging effect (SE) from formula (1), was plotted against concentration to obtain the concentration values resulting in 50 % inhibition (IC 50 ).
Ferric ion reducing potential (FRAP) assay The FRAP activity was measured according to the method of Benzie and Strain. 20Acetate buffer (300 mM; pH 3.6), 2,4,6-tripyridyl-s--triazine (TPTZ; 10 mM in 40 mM aq. hydrochloric acid) and FeCl 3 •6H 2 O (20 mM) were mixed in the ratio of 10:1:1 to obtain the working FRAP reagent.Test samples (500 µM) in methanol (10 mL) were mixed with 3 mL of working FRAP reagent and absorbance was measured at 593 nm after vortexing.Methanolic solutions of FeSO 4 •7H 2 O ranging from 100 to 2000 μM were used for the preparation of the calibration curve of known Fe 2+ concentration.The parameter equivalent concentration (EC) was defined as the concentration of antioxidant having a ferric-TPTZ reducing ability equivalent to that of 1 mM FeSO 4 •7H 2 O. BHA and curcumin were used as standards.
β-Carotene bleaching assay.The inhibition of the oxidative bleaching of β-carotene in a β-carotene/linoleic acid emulsion is measured in the β-carotene bleaching assay.It was realised using the method of Hidalgo et al. 21by using an emulsion obtained by mixing β-carotene (0.2 mg), linoleic acid (20 mg) and Tween 20 (200 mg, 0.180 mL) in chloroform (0.5 mL), evaporating off the chloroform and suspending in distilled water (50 mL).The thus obtained emulsion (4 mL) was treated with the test samples in methanol (180 µL) at a concentration 10 -3 M. BHA was used as the standard together with a control without sample and the absorbance was measured at 470 nm.Antioxidant activity was expressed as the percentage inhibition relative to the control using the equation: where DR C is the degradation rate of the control (DR C = ln (a/b)/60, where a is initial absorbance of control and b is the final absorbance of control after 60 min) and DR S is the degradation rate of the thiazole sample (DR S = ln (a/b)/60, where a is the initial absorbance of the sample and b is the final absorbance of sample after 60 min). 22

Antioxidant activity
The antioxidant activity of 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles (5a-e) were assessed based on the DPPH radical scavenging assay.These were selected as representative examples of aminothiazoles ( 5 II).It appears that the presence of an electron donating substituent on the 2arylamino group of 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles promotes antioxidant activity.The free radical scavenging activity largely depends on the hydrogen donating ability of phenolic compounds and the phenoxyl radicals thus formed are stabilized by resonance or intramolecular hydrogen bonding. 24The free radical scavenging activity was suggested to be enhanced by the presence of electron donating groups in the aromatic substituents. 25In the present case, the presence of a hindered phenolic group and the aminothiazole unit together could be responsible for the observed antioxidant potential of the studied 4-amino-(2-arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles.

TABLE I .
α-Glucosidase and α-amylase enzyme inhibition activity of compounds 5a-e

TABLE II .
Antioxidant activity studies on compounds 5a-e