Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors

Dipeptidyl Peptidase-IV (DPP-4) is a validated therapeutic target for type 2 diabetes. Aiming to interact with both residues Try629 and Lys554 in S₂′ site, a series of novel uracil derivatives 1a–l and 2a–i incorporating benzoic acid moieties at the N₃ position were designed and evaluated for their DPP-4 inhibitory activity. Structure-activity relationships (SAR) study led to the identification of the optimal compound 2b as a potent and selective DPP-4 inhibitor (IC₅₀?=?1.7?nM). Docking study revealed the additional salt bridge formed between the carboxylic acid and primary amine of Lys554 has a key role in the enhancement of the activity. Furthermore, compound 2b exhibited no cytotoxicity in human hepatocyte LO2 cells up to 50?μM. Subsequent in vivo evaluations revealed that the ester of 2b robustly improves the glucose tolerance in normal mice. The overall results have shown that compound 2b has the potential to a safe and efficacious treatment for T2DM.     

Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors

In recent years, dipeptidyl peptidase IV inhibitors have been noted as valuable agents for treatment of type 2 diabetes. Herein, we report the discovery of a novel potent DPP-4 inhibitor with 3H-imidazo[4,5-c]quinolin-4(5H)-one as skeleton. After efficient optimization of the lead compound 2a at the 7- and 8-positions using a docking study, we found 28 as a novel DPP-4 inhibitor with excellent selectivity against various DPP-4 homologues. Compound 28 showed strong DPP-4 inhibitory activity compared to marketed DPP-4 inhibitors. We also found that a carboxyl group at the 7-position could interact with the residue of Lys554 to form a salt bridge. Additionally, introduction of a carboxyl group to 7-position led to both activity enhancement and reduced risk for hERG channel inhibition and induced phospholipidosis. In our synthesis of compounds with 7-carboxyl group, we achieved efficient regioselective synthesis using bulky ester in the intramolecular palladium coupling reaction.     

Design of potent dipeptidyl peptidase IV (DPP-4) inhibitors by employing a strategy to form a salt bridge with Lys554

We report a design strategy to obtain potent DPP-4 inhibitors by incorporating salt bridge formation with Lys554 in the S1′ pocket. By applying the strategy to the previously identified templates, quinoline 4 and pyridines 16a, 16b, and 17 have been identified as subnanomolar or nanomolar inhibitors of human DPP-4. Docking studies suggested that a hydrophobic interaction with Tyr547 as well as the salt bridge interaction is important for the extremely high potency. The design strategy would be useful to explore a novel design for DPP-4 inhibitors having a distinct structure with a unique binding mode.     

New dipeptidyl peptidase 4 inhibitors among adamantane derivatives

Fifteen adamantane derivatives were synthesized. Preliminary evaluation of their potential as dipeptidyl peptidase 4 (DPP-4) inhibitors was performed in silico by the Microcosm informational technology, PASS system, and docking in AutoDock Vina. The DPP-4 inhibition was studied in vitro. The selectivity of action of the most active compounds was studied by the direct inhibition of human plasma DPP-4 and recombinant human DPP-8. The highest activity was found for the compounds containing a nitrogen atom in the β-position of the side chain, namely, derivatives of adamantane carboxylic acid and N-(3-adamantyl-allyl) thiourea. We demonstrated that the most active compound of the series, 3,5-dimethyladamantane 1-carboxamide, was a selective DPP-4 inhibitor with IC₅₀ 53.94 μM.     

(R)-3-Amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives as potent inhibitors of dipeptidyl peptidase-4: Design,synthesis, biological evaluation,and molecular modeling

A series of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives was designed, synthesized, and evaluated as novel inhibitors of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes. Most of the synthesized compounds demonstrated good inhibition activities against DPP-4. Among these, compounds 3e, 4c, 4l, and 4n exhibited prominent inhibition activities against DPP-4, with IC₅₀s of 0.07, 0.07, 0.14, and 0.17 μM, respectively. The possible binding modes of compounds 3e and 4n with dipeptidyl peptidase-4 were also explored by molecular docking simulation. These potent DPP-4 inhibitors were optimized for the absorption, distribution, metabolism, and excretion (ADME) properties, and compound 4n displayed an attractive pharmacokinetic profile (F = 96.3%, t_1/2 = 10.5 h).     

Structure-activity relationship study between Ornithyl-Proline and Lysyl-Proline based tripeptidomimics as angiotensin-converting enzyme inhibitors

A designed library of tripeptidomimics of Ornithyl-Proline (Orn-Pro) and Lysyl-Proline (Lys-Pro) conjugated with various unnatural amino acids and carboxylic acid derived heterocyclics was synthesized and screened for possible inhibitors of angiotensin-converting enzyme (ACE). Among the tripeptidomimics 10[MTP-Orn-Pro], 11[HTP-Orn-Pro], 14[TA-Orn-Pro] and 20[BPA-Orn-Pro] showed prominent inhibition with IC50 values in micromolar concentrations. Structure-activity relationship study indicated that C3 side chain of Orn as compared to C4 side chain of Lys at P1' position was better suited to inhibit ACE, with propionic acid (C3) derived heterocyclics and unnatural amino acids.     

DPP-4 inhibition increases GIP and decreases GLP-1 incretin effects during intravenous glucose tolerance test in Wistar rats

GIP metabolite [GIP (3-42)] and GLP-1 metabolite [GLP-1 (9-36) amide] have been reported to differ with regard to biological actions. Systemic DPP-4 inhibition can therefore reveal different actions of GIP and GLP-1. In catheter wearing Wistar rats, insulinotropic effects of equipotent doses of GIP (2.0 nmol/kg) and GLP-1 (7-36) amide (4.0 nmol/kg) and vehicle were tested in the absence/presence of DPP-4 inhibition. Blood glucose and insulin were frequently sampled. DPP-4 inhibitor was given at -20 min, the incretin at -5 min and the intravenous glucose tolerance test (0.4 g glucose/kg) commenced at 0 min. G-AUC and I-AUC, insulinogenic index and glucose efflux, were calculated from glucose and insulin curves. Systemic DPP-4 inhibition potentiated the acute GIP incretin effects: I-AUC (115±34 vs. 153±39 ng·min/ml), increased the insulinogenic index (0.74±0.24 vs. 0.99±0.26 ng/mmol), and improved glucose efflux (19.8±3.1 vs. 20.5±5.0 min?1). The GLP-1 incretin effects were diminished: I-AUC (124±18 vs. 106±38 ng·min/ml), the insulinogenic index was decreased (0.70±0.18 vs. 0.50±0.19 ng/mmol), and glucose efflux declined (14.9±3.1 vs. 11.1±3.7 min?1). GLP-1 and GIP differ remarkably in their glucoregulatory actions in healthy rats when DPP-4 is inhibited. These previously unrecognized actions of DPP-4 inhibitors could have implications for future use in humans.     

Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.     

A DPP-4 Inhibitor Suppresses Fibrosis and Inflammation on Experimental Autoimmune Myocarditis in Mice

Myocarditis is a critical inflammatory disorder which causes life-threatening conditions. No specific or effective treatment has been established. DPP-4 inhibitors have salutary effects not only on type 2 diabetes but also on certain cardiovascular diseases. However, the role of a DPP-4 inhibitor on myocarditis has not been investigated. To clarify the effects of a DPP-4 inhibitor on myocarditis, we used an experimental autoimmune myocarditis (EAM) model in Balb/c mice. EAM mice were assigned to the following groups: EAM mice group treated with a DPP-4 inhibitor (linagliptin) (n = 19) and those untreated (n = 22). Pathological analysis revealed that the myocardial fibrosis area ratio in the treated group was significantly lower than in the untreated group. RT-PCR analysis demonstrated that the levels of mRNA expression of IL-2, TNF-α, IL-1β and IL-6 were significantly lower in the treated group than in the untreated group. Lymphocyte proliferation assay showed that treatment with the DPP-4 inhibitor had no effect on antigen-induced spleen cell proliferation. Administration of the DPP-4 inhibitor remarkably suppressed cardiac fibrosis and reduced inflammatory cytokine gene expression in EAM mice. Thus, the agents present in DPP-4 inhibitors may be useful to treat and/or prevent clinical myocarditis.     

Structure-based design and synthesis of pyrazinones containing novel P1 'side pocket' moieties as inhibitors of TF/VIIa

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P(1) amidine designed to explore additional interactions with the VIIa residues in the so-called 'S(1) side pocket'. A crystal structure of the designed inhibitors demonstrates the ability of the P(1) side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.     

Discovery of DA-1229: a potent, long acting dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes

A series of β-amino amide containing substituted piperazine-2-one derivatives was synthesized and evaluated as inhibitors of dipeptidyl pepdidase-4 (DPP-4) for the treatment of type 2 diabetes. As results of intensive SAR study of the series, (R)-4-[(R)-3-amino-4-(2,4,5-trifluorophenyl)-butanoyl]-3-(t-butoxymethyl)-piperazin-2-one (DA-1229) displayed potent DPP-4 inhibition pattern in several animal models, was selected for clinical development.     

Macrocarpal C isolated from Eucalyptus globulus inhibits dipeptidyl peptidase 4 in an aggregated form

Dipeptidyl peptidase 4 (DPP-4) inhibitors are used for the treatment of type-2 diabetes mellitus. Various synthetic inhibitors have been developed to date, and plants containing natural DPP-4 inhibitors have also been identified. Here, 13 plant samples were tested for their DPP-4 inhibitory activity. Macrocarpals A–C were isolated from Eucalyptus globulus through activity-guided fractionation and shown to be DPP-4 inhibitors. Of these, macrocarpal C showed the highest inhibitory activity, demonstrating an inhibition curve characterised by a pronounced increase in activity within a narrow concentration range. Evaluation of macrocarpal C solution by turbidity, nuclear magnetic resonance spectroscopy and mass spectrometry indicated its aggregation, which may explain the characteristics of the inhibition curve. These findings will be valuable for further study of potential small molecule DPP-4 inhibitors.     

Diacylglycerol Acyltransferase-1 (DGAT1) Inhibition Perturbs Postprandial Gut Hormone Release

Diacylglycerol acyltransferase-1 (DGAT1) is a potential therapeutic target for treatment of obesity and related metabolic diseases. However, the degree of DGAT1 inhibition required for metabolic benefits is unclear. Here we show that partial DGAT1 deficiency in mice suppressed postprandial triglyceridemia, led to elevations in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) only following meals with very high lipid content, and did not protect from diet-induced obesity. Maximal DGAT1 inhibition led to enhanced GLP-1 and PYY secretion following meals with physiologically relevant lipid content. Finally, combination of DGAT1 inhibition with dipeptidyl-peptidase-4 (DPP-4) inhibition led to further enhancements in active GLP-1 in mice and dogs. The current study suggests that targeting DGAT1 to enhance postprandial gut hormone secretion requires maximal inhibition, and suggests combination with DPP-4i as a potential strategy to develop DGAT1 inhibitors for treatment of metabolic diseases.     

Structure-based design, synthesis, evaluation, and crystal structures of transition state analogue inhibitors of inosine monophosphate cyclohydrolase

The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATIC in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.     

Peptidases of the rumen bacterium, Prevotella ruminicola

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.     

Novel mimics of sialyl Lewis X: design, synthesis and biological activity of a series of 2- and 3-malonate substituted galactoconjugates.

A series of potent inhibitors of P-selectin as potential anti-inflammatory agents is reported. These compounds are derivatives of galactocerebrosides bearing a malonate side chain in positions 2 and 3 of the galactose moiety. Based on the binding mode of sialyl Lewis X, the two acidic groups of the malonate are designed to form ionic interactions with two important lysines in the active site of P-selectin, Lys113 and Lys111. On the other hand, the 4- and 6-hydroxy groups on the galactose ring are arranged to chelate the calcium ion in the P-selectin active site. The synthesis and the biological activity of this series of compounds are described. Lead compounds having a greater potency than sialyl Lewis X are identified.     

17Alpha-alkan (or alkyn) amide derivatives of estradiol as inhibitors of steroid-sulfatase activity

To develop inhibitors of steroid sulfatase without residual estrogenic activity, we have designed a series of estradiol (E2) derivatives bearing an alkan (or alkyn) amide side chain at position 17alpha. A hydrophobic alkyl group was selected from our previous study where 17alpha-octyl-E2 was found to inhibit strongly the steroid-sulfatase activity. Furthermore, it is known that an alkylamide side chain blocks the estrogen-receptor activation. Starting from ethynylestradiol, the chemical synthesis of target compounds was short and efficient with overall yields of 22-42% (3 or 4 steps). Among these compounds, N-octyl,N-methyl-3-(3',17'beta-dihydroxy-1',3',5'(10')-estratrien- 17'alpha-yl)-propanamide (15) was the most potent inhibitor, with an IC50 value of 0.08 microM for the transformation of estrone sulfate (E1S) to estrone (E1) by homogenated JEG-3 cells. N-butyl, N-hexyl, and N,N-dioctyl propanamide derivatives of E2 (IC50 values of 6.4, 2.8, and >20 microM, respectively) were less potent inhibitors than N-octyl analog 15. Furthermore, the unsaturated propynamide analog of 15 gave lower inhibition (four times) than the saturated compound. Compound 15 is also about 100-fold more effective in interacting with the enzyme than substrate E1S itself. The ability of target compounds to bind the estrogen receptor, to stimulate the proliferation of estrogen-sensitive ZR-75-1 cells, or to inhibit the E2-stimulation of ZR-75-1 cells was also evaluated. Although a mixed estrogenic/anti-estrogenic activity was obtained for tested compounds at 1 microM, no estrogenic activity was observed at 0.03 microM for 15. In conclusion, a promising inhibitor of steroid-sulfatase activity was obtained by introducing a hydrophobic octyl group in a 17alpha-propanamide side chain of E2, but further structure-activity relationships (SAR) studies are necessary to minimize the residual estrogenic activity.     

Peptide conformations--49(1): synthesis and structure-activity relationships of side chain modified peptides of cyclo(-D-Pro-Phe-Thr-Lys-Trp-Phe.)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.     

Design and synthesis of 3-pyridylacetamide derivatives as dipeptidyl peptidase IV (DPP-4) inhibitors targeting a bidentate interaction with Arg125

We have previously discovered nicotinic acid derivative 1 as a structurally novel dipeptidyl peptidase IV (DPP-4) inhibitor. In this study, we obtained the X-ray co-crystal structure between nicotinic acid derivative 1 and DPP-4. From these X-ray co-crystallography results, to achieve more potent inhibitory activity, we targeted Arg125 as a potential amino acid residue because it was located near the pyridine core, and some known DPP-4 inhibitors were reported to interact with this residue. We hypothesized that the guanidino group of Arg125 could interact with two hydrogen-bond acceptors in a bidentate manner. Therefore, we designed a series of 3-pyridylacetamide derivatives possessing an additional hydrogen-bond acceptor that could have the desired bidentate interaction with Arg125. We discovered the dihydrochloride of 1-{[5-(aminomethyl)-2-methyl-4-(4-methylphenyl)-6-(2-methylpropyl)pyridin-3-yl]acetyl}-l-prolinamide (13j) to be a potent and selective DPP-4 inhibitor that could interact with the guanidino group of Arg125 in a unique bidentate manner.     

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.