Discovery of triazole-based uracil derivatives bearing amide moieties as novel dipeptidyl peptidase-IV inhibitors

Dipeptidyl peptidase-IV (DPP-4) is a validated target for T2DM treatment. We previously reported a novel series of triazole-based uracil derivatives bearing aliphatic carboxylic acids with potent DPP-4 inhibitory activities in vitro, but these compounds showed poor hypoglycemic effects in vivo. Herein we further optimized the triazole moiety by amidation of the carboxylic acid to improve in vivo activities. Two series of compounds 3a-f and 4a-g were designed and synthesized. By screening in DPP-4, compound 4c was identified as a potent DPP-4 inhibitor with the IC₅₀ value of 28.62 nM. Docking study revealed compound 4c has a favorable binding mode and interpreted the SAR of these analogs. DPP-8 and DPP-9 tests indicated compound 4c had excellent selectivity over DPP-8 and DPP-9. Further in vivo evaluations revealed that compound 4c showed more potent hypoglycemic activity than its corresponding carboxylic acid in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice. The overall results have shown that compound 4c could be a promising lead for further development of novel DPP-4 agents treating T2DM.     

Discovery of conformationally rigid 3-azabicyclo[3.1.0]hexane-derived dipeptidyl peptidase-IV inhibitors

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P₂ region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure–activity relationship, and selectivity against related proteases are delineated.     

Isolation of rugosin A,B and related compounds as dipeptidyl peptidase-IV inhibitors from rose bud extract powder

Dipeptidyl peptidase-IV (DPP-IV) is a protease responsible for the degradation of the incretin hormone. A number of DPP-IV inhibitors have been approved for use in the treatment of type 2 diabetes. While these inhibitors are effective for this treatment, methods for the prevention of this disease are also required as diabetes patient numbers are currently increasing rapidly worldwide. We screened the DPP-IV inhibitory activities of edible plant extracts with the intention of using these extracts in a functional food supplement for the prevention of diabetes. Rose (Rosa gallica) bud extract powder was a promising material with high inhibitory activity. In this study, seven ellagitannins were isolated as active compounds through activity-guided fractionations, and their DPP-IV inhibitory activities were measured. Among them, rugosin A and B showed the highest inhibitory activities and rugosin B was shown as the major contributing compound in rose bud extract powder.     

Structural basis of proline-specific exopeptidase activity as observed in human dipeptidyl peptidase-IV

Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.     

Inhibition of dipeptidyl peptidase-IV (DPP-IV) by atorvastatin

Dipeptidyl peptidase-IV (DPP-IV) is an enzyme responsible for the inactivation of the glucoregulatory incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In this report, we show that the hypolipidemic agent atorvastatin is a competitive inhibitor of porcine DPP-IV in vitro, with K(i)=57.8+/-2.3 microM. These results may have implications in the development of novel DPP-IV inhibitors based on the use of atorvastatin as a lead compound for the treatment of type 2 diabetes.     

Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments

Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (P<0.05) reduced serum DPP-IV activity and glycosylated hemoglobin. Glyburide did not lower DPP-IV activity or glycosylated hemoglobin. Regression analysis showed serum DPP-IV activity correlated with glycosylated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.     

Fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors

Amides derived from fluorinated pyrrolidines and 4-substituted cyclohexylglycine analogues have been prepared and evaluated as inhibitors of dipeptidyl dipeptidase IV (DP-IV). Analogues which incorporated (S)-3-fluoropyrrolidine showed good selectivity for DP-IV over quiescent cell proline dipeptidase (QPP). Compound 48 had good pharmacokinetic properties and was orally active in an oral glucose tolerance test in lean mice.     

Guanidine-substituted imidazoles as inhibitors of nitric oxide synthase

Guanidine-substituted imidazoles were prepared and evaluated as inhibitors of the three isoforms of nitric oxide synthase. These results identify a new 2-substituted imidazole as an isoform selective inhibitor and illustrate the possible importance of the L-arginine side chain in selective isoform recognition.     

Lead discovery of quinoxalinediones as an inhibitor of dipeptidyl peptidase-IV (DPP-IV) by high-throughput screening

N-Ureido-quinoxalinedione derivatives have been discovered as leads for a novel series of dipeptidyl peptidase-IV (DPP-IV) inhibitors through high-throughput screening of our chemical library. A brief structure-activity relationship of the compounds was investigated. Among them, entry 5 showed the most potent inhibitory activity. The nitro group in quinoxaline moiety and the aromatic sulfonyl substituted ureido functional group seem to be important to increase the potency dramatically.     

Dipeptidyl nitriles as human dipeptidyl peptidase I inhibitors

Using a dipeptide nitrile scaffold we have identified a potent and selective inhibitor of human dipeptidyl peptidase I.     

Bicyclic cyanothiazolidines as novel dipeptidyl peptidase 4 inhibitors

The synthesis and biochemical evaluation of novel cyanothiazolidine inhibitors of dipeptidyl peptidase 4 (DPP4) is described. Their main structural feature is a constrained bicyclic core that prevents the intramolecular formation of inactive cyclic species. The inhibitors show good to moderate biochemical potency against DPP4 and display distinct selectivity profiles towards DPP7, DPP8 and DPP9 depending on their substitution.     

Diprolyl nitriles as potent dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP4) is a multifunctional type II transmembrane serine peptidase which regulates various physiological processes, most notably plasma glucose homeostasis by cleaving peptide hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. Inhibition of DPP4 is a potentially valuable therapy for type 2 diabetes. Synthesis and structure-activity relationships of a series of substituted diprolyl nitriles are described, leading to the identification of compound 1 with a measured DPP4 K(i) of 3.6 nM.     

Substituted piperazines as novel dipeptidyl peptidase IV inhibitors

Incorporation of a fluorophenyl beta-amino amide moiety into piperazine screening lead 2 has resulted in the discovery of a structurally novel series of potent and selective DP-IV inhibitors. Simplification of the molecule and incorporation of multiple fluorine atoms on the phenyl ring has provided low molecular weight analogs such as compound 32, which is a 19nM DP-IV inhibitor with >4000-fold selectivity over QPP.     

Xanthine mimetics as potent dipeptidyl peptidase IV inhibitors

A series of xanthine mimetics containing 5,5 and 5,6 heterocycle fused imidazoles were synthesized as dipeptidyl peptidase IV inhibitors. Compound 7 is potent (h-DPPIV K_i = 2 nM) and exhibits excellent selectivity and no species specificity against rat and human enzymes. The X-ray structure confirms that the binding mode of 7 to rat DPPIV is similar to the parent xanthines.     

Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors

Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.     

Structure-based design of pyridopyrimidinediones as dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP-4) inhibitors have been shown to enhance GLP-1 levels and thereby improve hyperglycemia in type II diabetes. From a small fragment hit, using structure-based design, we have discovered a new class of non-covalent, potent and selective DPP-4 inhibitors.     

Probing prime substrate binding sites of human dipeptidyl peptidase-IV using competitive substrate approach

Dipeptidyl peptidase-IV is a cell surface protease which plays an important role in glucose homeostasis through proteolytic inactivation of incretin hormones, primarily glucagon like peptide-1 (GLP-1). Substrate N-terminal amino acid (S2-S1) specificity is rather clearly defined, while no substantial information is available on the significance of amino acid interactions towards the C-terminus after the scissile bond (so called prime S1'-S4' or distant S5'-S28' sites). In the present study the increasing length of the peptide towards prime sites (S1'-S4') resulted in approximately 7-fold decrease in Km. Moreover, the Km for GLP-1 cleavage was comparable to that of an S2-S4' peptide, suggesting that few, if any, important enzyme-substrate interactions occur beyond the active site. Effect of substrate length on kcat was less obvious, but kcat/Km showed an increasing trend when His-Ala-pNA (representing the natural two N-terminal residues) was compared to GLP-1. To probe the impact of increasing substrate length on the free energy of activation (as has been suggested for elastase and chymotrypsin) we performed temperature studies. To adequately interpret thermodynamic data we sought to understand what steps limit the kcat expression. Steady-state parameters of the reactions catalyzed by serine proteases are composed of microscopic constants describing binding, acylation, and deacylation steps. Viscosity and pre-steady-state studies suggested that His-Ala-pNA cleavage is limited in the deacylation half-reaction, most likely the product release step. Thus, the free energy of activation, as calculated from the Eyring equation, is underestimated (at least for His-Ala-pNA) and the effect of substrate length on the acylation step (and transition-state stabilization) could not be unambiguously assessed.     

Fluoroolefins as amide bond mimics in dipeptidyl peptidase IV inhibitors

The synthesis, selectivity, rat pharmacokinetic profile, and drug metabolism profiles of a series of potent fluoroolefin-derived DPP-4 inhibitors (4) are reported. A radiolabeled fluoroolefin 33 was shown to possess a high propensity to form reactive metabolites, thus revealing a potential liability for this class of DPP-4 inhibitors.     

4-Amino cyclohexylglycine analogues as potent dipeptidyl peptidase IV inhibitors

Substituted 4-amino cyclohexylglycine analogues were evaluated for DP-IV inhibitory properties. Bis-sulfonamide 15e was an extremely potent 2.6 nM inhibitor of the enzyme with excellent selectivity over all counterscreens. 2,4-difluorobenzenesulfonamide 15b and 1-naphthyl amide 16b, however, combined an acceptable in vitro profile with good pharmacokinetic properties in the rat, and 15b was orally efficacious at 3 mpk in an OGTT in lean mice.     

Quantitative structure-activity relationship study of benzylsulfanyl imidazoles as cytokine release inhibitors

Benzylsulfanyl imidazole derivatives (Figure 1) have shown their ability to inhibit the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from peripheral blood mononuclear cells or human whole blood. Such anticytokine actions of these congeners are quantitatively studied using Fujita-Ban and Hansch type analyses. The Fujita-Ban study resulted in the contributions of different substituents and the parent moiety for the inhibitions of cytokines TNF-alpha and IL-1beta. The substituents that have a higher positive contribution to the given activity, relative to substituents of the parent moiety at different positions were then used to obtain a trend for the active analogues. None of the substituents present at X, Y, 2-R and 3-R, appears to be advantageous over the substituents of the parent moiety for inhibition of both the cytokines. However, the substituents at 4-R, 5-R and 6-R help to improve the inhibitory actions of the compounds for both cytokines. The optimal activities seem to be manifested by compounds in which 4-R, 5-R and 6-R are substituted respectively by OH (or SOCH3 and SO2CH3), Cl and OH for inhibition of TNF-alpha, whereas by SOCH3 (or SO2CH3 and OH), H and OH for inhibition of IL-1beta. The Hansch type analysis, on the other hand, revealed that the F-substituents of the X-position and a less bulky structural moiety such as--S(CH2)2--at the Y-incision are advantageous in improving the inhibitory action towards TNF-alpha. Similarly, a less bulky/polar substituent present at 2-R and not having a hydrogen-bond donor property, while a more hydrophobic substituent at 3-R and hydrogen-bond acceptor substituent at 4-R are helpful in augmenting inhibitory activity of a compound. However, for inhibition of cytokine IL-1beta, it emerged that the X-substituents that transmits a higher negative resonance effect, the Y-substituent that offers less molecular bulk are beneficial. The R-substituents, being more electron donors at the meta-position, less hydrophobic at the para-position and offering smaller refractivity (less bulky and or polar) at the ortho-position are likewise helpful in improving the activity of a compound.