Synthesis and Evaluation of Novel Xanthone Derivatives as Potent Dipeptidyl Peptidase-4 Inhibitors

Diabetes, a chronic disease caused by genetic and environmental factors, threats the health of millions of people, which has become a global challenge. Therefore, much earlier intervention is required to prevent diabetes, type-2 diabetes particularly. Previous study revealed that glucagon-like peptide-1 is an intestine-derived insulinotropic hormone that stimulated glucose dependent insulin production and secretion from pancreatic 3-cells. Glucagon-like peptide-1 also exerted cytoprotective and antiapoptotic effects on 13-cells and decreased postprandial blood glucose via complicated mechanisms. However, glucagon-like peptide-1 was rapidly degraded and inactivated in vivo by the serine protease dipeptidyl peptidase4, which limited their ability to normalize blood glucose levels. Therefore, dipeptidyl peptidase-4 inhibitors could decrease the high levels of blood glucose in type 2 diabetes patients. In recent years, we have focused on synthesizing and evaluating the biological activities of novel xanthone compounds. Compound 9, a small xanthone derivative, was identified to show good dipeptidyl peptidase-4 inhibitory activity from high throughput screening (HTS) through varieties of xanthone derivatives and IC50 value against dipeptidyl peptidase-4 is 7.3 μg/mL (Fig. 1). Molecular docking study showed compound 9 could bind with the target enzyme and a pocket existed between the 7-substitued group and the target enzyme, which revealed that an adequate modification to compound 9 could Synthesis and Evaluation of Novel Xanthone Derivatives as Potent Dipeptidyl Peptidase-4 Inhibitors


INTRODUCTION
Diabetes, a chronic disease caused by genetic and environmental factors, threats the health of millions of people, which has become a global challenge 1 . Therefore, much earlier intervention is required to prevent diabetes, type-2 diabetes particularly. Previous study revealed that glucagon-like peptide-1 is an intestine-derived insulinotropic hormone that stimulated glucose dependent insulin production and secretion from pancreatic 3-cells. Glucagon-like peptide-1 also exerted cytoprotective and antiapoptotic effects on 13-cells and decreased postprandial blood glucose via complicated mechanisms 2,3 . However, glucagon-like peptide-1 was rapidly degraded and inactivated in vivo by the serine protease dipeptidyl peptidase-4, which limited their ability to normalize blood glucose levels. Therefore, dipeptidyl peptidase-4 inhibitors could decrease the high levels of blood glucose in type 2 diabetes patients 4,5 .
In recent years, we have focused on synthesizing and evaluating the biological activities of novel xanthone compounds [6][7][8] . Compound 9, a small xanthone derivative, was identified to show good dipeptidyl peptidase-4 inhibitory activity from high throughput screening (HTS) through varieties of xanthone derivatives and IC50 value against dipeptidyl peptidase-4 is 7.3 µg/mL (Fig. 1). Molecular docking study showed compound 9 could bind with the target enzyme and a pocket existed between the 7-substitued group and the target enzyme, which revealed that an adequate modification to compound 9 could improve the bind between receptor and ligands and enhanced the inhibition of the xanthone derivatives against dipeptidyl peptidase-4. As shown in Fig. 2, when a hydrophobic group was linked to the xanthone framework at 7-position, the compound could bind with the target enzyme better. Herein, we report our efforts to optimize the inhibitory effects of this compound by analyzing structure-activity relationships.

EXPERIMENTAL
Melting points were determined with a Yamato MP-21 melting point apparatus and uncorrected. NMR spectra was recorded in CDCl3 or DMSO-d6 unless otherwise indicated with a Bruker AC-300P spectrometer, using tetramethylsilane as an internal standard. ESI mass spectra were performed on an API-3000 LC-MS spectrometer. Elemental analysis was conducted with Carlo Erba1106 auto-apparatus. Column chromatography was carried out on silica gel (200-300 mesh). The solvents and reagents were used as received or dried prior to use as needed. All reactions were monitored using thinlayer chromatography (TLC) on silica gel plates. Detection was effected by examination under UV light.
The target compounds 1b-i and 2a-h were synthesized by the same procedure as the compound 1a.

RESULTS AND DISCUSSION
Title compounds required for the establishment of structure-activity relationship were prepared as shown in Scheme-I. starting material 2-hydroxy-4-nitrobenzoic acid (3). Reaction with 1,3,5-trimethoxybenzene (4) by using Eaton's reagent afforded xanthone 9 (5). Compound 5 were reduced by Raney-Ni to the gave compound 6 in high yield 10 (85 %). Then, chloroacetyl chloride or 3-chloropropionyl chloride was added dropwisely into the flask containing 6 under reflux to give key intermediate 7 or 8. Reaction of compound 7 or 8 with different secondary amines formed the final xanthone sulfonamides (1a-h) and (2a-f) in high yield (Fig. 3) 11 . The reactions were carried out in parallel. All new compounds were characterized by NMR and MS.
Evaluation of biological activities: To determine dipeptidyl peptidase-4 inhibitory activities, all the compounds were measured in vitro according to the modified Ellman method with diprotin A as the positive control.  Structure-activity relationship: According to the results above, a preliminary structure-activity relationships could be concluded: Series compound 2a-g exhibited better inhibitory activities against dipeptidyl peptidase-4 than series compound 1a-I, which demonstrated that length of 7'-substitued ethylamine group could fit with the pocket of the target enzyme. In addition, alkyl group substituted compounds exhibited better inhibitory activities against dipeptidyl peptidase-4.

Conclusion
In summary, two series of new xanhtone derivatives (1a-i) and (2a-g) as potential dipeptidyl peptidase-4 inhibitors were synthesized in high yields. The biological screening of these compounds resulted in the identification of several potent dipeptidyl peptidase-4 inhibitors and compounds 2a, 2c, 2d, 2f and 2g exhibited potent activities against dipeptidyl peptidase-4 compared with the positive control diprotin A. This observation was fitted to a molecular model resulting from the computational docking simulation, which showed that compound 1i could fit into the hydrophobic pocket of dipeptidyl peptidase-4.