Novel isoindoline compounds for potent and selective inhibition of prolyl dipeptidase DPP8

DPP8 is a prolyl dipeptidase homologous to DPP-IV, which is a drug target for Type II diabetes. The biological function of DPP8 is not known. To identify potent and selective chemical compounds against DPP8, we have synthesized a series of isoquinoline and isoindoline derivatives and have tested their inhibitory activity against DPP8, DPP-IV and DPP-II. Isoindoline derivatives were found to be more potent DPP8 inhibitors than isoquinoline derivatives. Isoindoline with a 1-(4,4'-difluor-benzhydryl)-piperazine group at the P2 site was observed to be a very potent DPP8 inhibitor, having an IC(50) value of 14nM with at least a 2500-fold selectivity over either DPP-IV or DPP-II. From SAR results, we speculate that the S1 site of DPP8 may be larger than that of DPP-IV, which would allow the accommodation of larger C-terminal residues, such as isoquinoline or isoindoline.     

Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors

Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.     

Glutamic acid analogues as potent dipeptidyl peptidase IV and 8 inhibitors

To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that incorporated a bulky substituent at the first carbon position of benzylamine or isoquinoline showed over 30-fold selectivity for DPP-IV over both DPP8 and DPP-II. From structure-activity relationship studies, we speculate that the S2 site of DPP8 might be similar to that of DPP-IV, while DPP-IV inhibitor with N-substituted glycine in the P2 site and/or with a moiety involving in hydrophobic interaction with the side chain of Phe357 might provide a better selectivity for DPP-IV over DPP8.     

Synthesis and activity of a potent, specific azabicyclo[3.3.0]-octane-based DPP II inhibitor

A cell permeable DPP II [also known as DPP2, DPP7, and quiescent cell proline dipeptidase (QPP)] inhibitor has been synthesized. The azabicyclo[3.3.0]octane-based inhibitor is potent and selective and elicits very similar quiescent lymphocyte death to previously characterized inhibitors that are not as selective.     

New homocamptothecins: synthesis, antitumor activity, and molecular modeling

Homocamptothecins (hCPTs) represent a class of new emerging antitumor agents, which contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring (E ring) of camptothecins. Some novel 7-substituted hCPTs were designed and synthesized based on a newly developed synthetic route which couples ring A with ring C, E and D. Most of the synthesized compounds exhibit very high cytotoxic activity on tumor cell line A549. Some compounds, such as 9b, 9l, and 9y, show broad in vitro antitumor spectrum and are more potent than topotecan. Three-dimensional quantitative structure-activity relationship (3D-QSAR) methods, CoMFA and CoMSIA, were applied to explain the structure-activity relationship (SAR) of the synthesized compounds. Furthermore, molecular docking was used to clarify the binding mode of the synthesized compounds to human DNA topoisomerase I. The important hydrophobic, base-pair stacking, and hydrogen-bonding interactions were observed between the hCPT derivatives and their receptor. The results from molecular modeling will guide the design of novel hCPTs with higher antitumor activity.     

DA-1229, a novel and potent DPP4 inhibitor, improves insulin resistance and delays the onset of diabetes

AimTo characterize the pharmacodynamic profile of DA-1229, a novel dipeptidyl peptidase (DPP) 4 inhibitor.Main methodsEnzyme inhibition assays against DPP4, DPP8 and DPP9. Antidiabetic effects of DA-1229 in HF-DIO mice and young db/db mice.Key findingsDA-1229 was shown to potently inhibit the DPP4 enzyme in human and murine soluble forms and the human membrane-bound form with IC₅₀ values of 0.98, 3.59 and 1.26 nM, respectively. As a reversible and competitive inhibitor, DA-1229 was more selective to human DPP4 (6000-fold) than to human DPP8 and DPP9. DA-1229 (0.1–3 mg/kg) dose-dependently inhibited plasma DPP4 activity, leading to increased levels of plasma GLP-1 and insulin, and thereby lowering blood glucose levels in mice. In high fat diet-fed (HF) mice, a single oral dose of 100 mg/kg of DA-1229 reduced plasma DPP4 activity by over 80% during a 24 h period. Long-term treatment with DA-1229 for 8 weeks revealed significant improvements in glucose intolerance and insulin resistance, accompanied by significant body weight reduction. However, it remains unclear whether there is a direct causal relationship between DPP4 inhibition and body weight reduction. In young db/db mice, the DA-1229 treatment significantly reduced blood glucose excursions for the first 2 weeks, resulting in significantly lower levels of HbA1c at the end of the study. Furthermore, the pancreatic insulin content of the treatment group was significantly higher than that of the db/db control.SignificanceDA-1229 as a novel and selective DPP4 inhibitor improves the insulin sensitivity in HF mice and delays the onset of diabetes in young db/db mice.     

Design, syntheses and 3D-QSAR studies of novel N-phenyl pyrrolidin-2-ones and N-phenyl-1H-pyrrol-2-ones as protoporphyrinogen oxidase inhibitors

The characteristics of low application rates, good crop selectivity, low residue and environmental safety exhibited by Protoporphyrinogen oxidase (PPO, EC 1.3.3.4)-inhibiting herbicides have attracted a world-wide research interests. As continuation of our research work on the development of new PPO inhibitors, a series of mono-carbonyl analogues of cyclic imides, N-phenyl pyrrolidin-2-ones and N-phenyl-1H-pyrrol-2-ones, were designed and synthesized based on previously established DFT-QSAR results. The PPO inhibition activities of 29 newly synthesized compounds were tested and a predictive comparative molecular field analysis (CoMFA) model was established with the conventional correlation coefficient r(2)=0.980 and the cross-validated coefficient q(2)=0.518. According to the CoMFA model, the substituent effects on the PPO inhibition activity were explained reasonably. Further greenhouse assay showed that 2-(4-chloro-2-fluoro-5-propoxy-phenyl)-2,3,4,5,6,7-hexahydro-isoindol-1-one (C(6), k(i)=0.095μM) and 2-(5-allyloxy-4-chloro-2-fluorophenyl)-2,3,4,5,6,7-hexahydro-isoindol-1-one (C(7), k(i)=0.12μM) displayed excellent post-emergency herbicidal activity at the concentration of 150g.ai/ha against seven tested weeds. Due to their high PPO inhibition effect and broad spectrum herbicidal activity, these two compounds have the potential for further study on crop selectivity and field trial. These results confirmed once again that only one of the carbonyl groups of cyclic imides is essential to the PPO inhibition activity.     

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.     

Structures of Human DPP7 Reveal the Molecular Basis of Specific Inhibition and the Architectural Diversity of Proline-Specific Peptidases

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.     

Synthesis and SAR of azolopyrimidines as potent and selective dipeptidyl peptidase-4 (DPP4) inhibitors for type 2 diabetes

Several pyrazolo-, triazolo-, and imidazolopyrimidines were synthesized and evaluated as inhibitors of DPP4. Of these three classes of compounds, the imidazolopyrimidines displayed the greatest potency and demonstrated excellent selectivity over the other dipeptidyl peptidases. SAR evaluation for these scaffolds was described as they may represent potential treatments for type 2 diabetes.     

7-Oxopyrrolopyridine-derived DPP4 inhibitors-mitigation of CYP and hERG liabilities via introduction of polar functionalities in the active site

Design, synthesis, and SAR of 7-oxopyrrolopyridine-derived DPP4 inhibitors are described. The preferred stereochemistry of these atropisomeric biaryl analogs has been identified as Sa. Compound (+)-3t, with a K(i) against DPP4, DPP8, and DPP9 of 0.37 nM, 2.2, and 5.7 μM, respectively, showed a significant improvement in insulin response after single doses of 3 and 10 μmol/kg in ob/ob mice.     

Molecular docking and 3D-QSAR studies of Yersinia protein tyrosine phosphatase YopH inhibitors

Three-dimensional quantitative structure-activity relationship (QSAR) studies were conducted on two classes of recently explored compounds with known YopH inhibitory activities. Docking studies were employed to position the inhibitors into the YopH active site to determine the probable binding conformation. Good correlations between the predicated binding free energies and the inhibitory activities were found for two subsets of phosphate mimetics: alpha-ketocarboxylic acid and squaric acid (R2=0.70 and 0.68, respectively). The docking results also provided a reliable conformational alignment scheme for 3D-QSAR modeling. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed based on the docking conformations, giving q2 of 0.734 and 0.754 for CoMFA and CoMSIA models, respectively. The 3D-QSAR models were significantly improved after removal of an outlier (q2=0.829 for CoMFA and q2=0.837 for CoMSIA). The predictive ability of the models was validated using a set of compounds that were not included in the training set. Mapping the 3D-QSAR models to the active site of YopH provides new insight into the protein-inhibitor interactions for this enzyme. These results should be applicable to the prediction of the activities of new YopH inhibitors, as well as providing structural implications for designing potent and selective YopH inhibitors as antiplague agents.     

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.     

Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity

A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating structure-activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation protein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic structures for future FAP-selective-inhibitor discovery.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure–activity-relationship data obtained in this way led to the preparation of a series of α-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.     

Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine (23)

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate.     

Design, synthesis, and biological evaluation of substituted hydrazone and pyrazole derivatives as selective COX-2 inhibitors: Molecular docking study

New arylhydrazone derivatives and a series of 1,5-diphenyl pyrazoles were designed and synthesized from 1-(4-chlorophenyl)-4,4,4-trifuorobutane-1,3-dione 1. The newly synthesized compounds were investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw oedema model. Moreover, they were tested for their inhibitory activity against ovine COX-1 and COX-2 using an in vitro cyclooxygenase (COX) inhibition assay. Some of the new compounds (2f, 6a and 6d) showed a reasonable in vitro COX-2 inhibitory activity, with IC?? value of 0.45 μM and selectivity index of 111.1. A virtual screening was carried out through docking the designed compounds into the COX-2 binding site to predict if these compounds have analogous binding mode to the COX-2 inhibitors. Docking study of the synthesized compounds 2f, 6a and 6d into the active site of COX-2 revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead-like screening hit

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.     

Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.