Synthesis , characterization and evaluation of antidiabetic activity of novel indoline derivatives

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Introduction
Diabetic is characterized by high blood glucose level.When a person has diabetes, the body either does not produce sufficient insulin or is unable to use its own insulin effectively.Inhibition of saccharide hydrolysing enzyme (α-amylase) have been useful as oral hypoglycemic drugs for the control of hyperglycemia mainly in patients with type-2 diabetes mellitus.Inhibition of this enzyme hold-up carbohydrate digestion and extend overall carbohydrate digestion time, causing a decrease in the rate of glucose absorption and, therefore, reducing the postprandial plasma glucose rise (Keri et al., 2015).
Heterocyclic compounds are the basis in antidiabetic treatment for many years.The hyperglycemia in diabetes mellitus is reduced by drugs like sulfonylurea and biguanides.However, there is a continuous search for alternative drugs for management of diabetes is still a challenge to the medicinal chemist.
In addition, the indoline nucleus is incorporated in various natural products such as alkaloids (Zhang et al., 2011).Encouraged by the above observations and considering the interesting pharmacological profile of indoline, N-(4-aminophenyl)indoline-1-carbothioamide scaffold based compounds were synthesized as antidiabetic agents.The biological activity and structureactivity relationship (SARs) of the newly synthesized indoline derivatives are also discussed.

Materials and Methods
The starting materials such as reagents and solvents used for the synthesis are of analytical grade and they are purchased from Sigma-Aldrich Chemical Co., Spectrochem Chemical Co, and Merck Chemical Co.Melting points were recorded by labtronics digital melting point apparatus. 1H-NMR and 13 C-NMR spectra were recorded in CDCl3 or DMSO solvent on Bruker 300 MHz spectrophotometer using TMS as an internal standard.Infrared spectroscopy was recorded in the frequency range of 400-4000 cm -1 by Perkin Elmer.The Mass spectra were recorded on a Waters Synapt G2 High detection Mass spectrometry.The thin layer chromatography (TLC) analysis was carried out using 5 × 20 cm plate coated with silica gel GF254.

In vitro antidiabetic activity of indoline derivatives
The α-amylase inhibitory activities were performed by (Nickavar and Amin, 2011), which was originally proposed by (Patil et al., 2013).The solution of compounds was prepared in DMSO to give the various concentrations (50,100,150,200 and 250 µg/mL).500 µg/mL αamylase solutions prepared in 0.02M sodium phosphate buffer (pH 6.9) was added to different concentrations of the compounds and incubated for 15 min at 25ºC.After 10 min, 500 µg/mL of 1% starch solution in 0.02M of sodium phosphate buffer was added to each tube.The mixture was further incubated at 25ºC for 10 min.Then the reaction mixture was terminated by adding 0.5 mL of DNS reagent (12.0 g of sodium potassium tartrate tetrahydrate in 8 mL of 2 M NaOH and 96 mM 3,5dinitrosalicylic acid solution) and the contents were heated in a boiling water bath for 5 min.The absorption of resulting reaction mixture was measured at 540 nm.Acarbose was used as positive control/standard.The antidiabetic activity of the compounds was determined by the inhibition of α-amylase.The percentage of inhibition was calculated by the equation: Where, AC and AS are the absorbance of the control and sample respectively To calculate the IC50 (the concentration of a sample required to inhibit the activity of a given enzyme by 50%) values for each sample, the %inhibition was plotted against the sample concentration and a logarithmic regression curve was established.

Results
The intermediate 1 was prepared (Sidoova et al., 1998) by using indoline with the reagent 4-nitrophenyl isothiocyanate.The FT-IR spectrum of intermediate 1 had strong absorption about 3300 cm -1 , 1270 cm -1 corresponds to NH (thiocarboxamide) and C=S, respectively.The 1 H-NMR spectrum of intermediate 1 exhibited characteristic thiocarbamide NH at δ 10.4 ppm and in the case of 13 C-NMR, characteristic thiocarbamide carbon (C=S) observed at δ 177.5 ppm.The nitro group in intermediate 1 was reduced to equivalents of SnCl2.2H2O in 12N HCl.
FT-IR spectrum of intermediate 2 showed the characteristics absorption at 3398 cm -1 for amine group.In 1 H-NMR the nitro group reduction was confirmed by characteristics broad at δ 5.05 ppm.For the synthesis of new series of indoline derivatives the intermediate -2 was used as a common scaffold.The synthetic path way for intermediate 2 is outlined below:

Scheme 1
The amide derivatives 5a, 5b and 5c were prepared by coupling reagent EDCI (Jing et al., 2009).For all amide derivatives NH proton were observed between δ 9.7 and 10.1 ppm as a broad singlet in 1 H-NMR.In the 13 C-NMR of all new carboxamide carbons observed between δ 163.9 and 178.4 ppm.
The compound 3a and 3b were obtained by reacting intermediate 2 with 4-fluoro phenyl isothiocynate and 3-methoxy-phenyl isocyanate.In 13 C-NMR, characteristics the new thiourea carbon (C=S) and urea carbon (C=O) seen at δ 179.9 and 159.6 respectively.
The sulfonamide derivates 4a and 4b were prepared (Macıas et al., 2002) by reaction between corresponding sulfonyl chloride and key intermediate-2 and corresponding sulfonyl chloride.The 1 H-NMR of synthesised sulfonamide derivatives showed new singlet peak at δ 9.6 ppm and 9.6 ppm.The synthetic scheme for the preparation of thiourea, urea, sulfonaminde and carboxamide derivatives are shown in Scheme 2.
The antidiabetic activities of all the above indoline derivatives were examined by standard α-amylase inhibition assay.The inhibition efficiency of all synthesized compounds was tested at a concentration ranging from 50 to 250 µg/mL.The %inhibition and IC50 values of all indoline derivatives are listed in Table I.
The above result showed that 4a and 4b inhibit αamylase due to presence of sulfonamide substitution.Furthermore, all moderate active of indoline based compounds have amide group substitution.

Conclusion
Compounds 4a and 4b reported have potent in vitro αamylase inhibition activity, similarly the compounds 3a and 3b were found to be moderate α-amylase inhibition activity.