Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5–14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 (‘Leu-in’ to ‘Leu-out’) by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.     

N-(2-alkylaminoethyl)-4-(1,2,4-oxadiazol-5-yl)piperazine-1-carboxamides as highly potent smoothened antagonists

Smoothened (Smo) antagonists are emerging as new therapies for the treatment of neoplasias with aberrantly reactivated hedgehog (Hh) signaling pathway. A novel series of 4-[3-(quinolin-2-yl)-1,2,4-oxadiazol-5-yl]piperazinyl ureas as smoothened antagonists was recently described, herein the series has been further optimized through the incorporation of a basic amine into the urea. This development resulted in identification of some exceptionally potent smoothened antagonists with low serum shifts, however, reductive ring opening on the 1,2,4-oxadiazole in rats limits the applicability of these compounds in in vivo studies.     

Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.     

Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.     

Synthesis of N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides as new selective ligands for sigma receptors

Novel benzofuran-2-carboxamide ligands, which are selective for sigma receptors, have been synthesized via a microwave-assisted Perkin rearrangement reaction and a modified Finkelstein halogen-exchange used to facilitate N-alkylation. The ligands synthesized are the 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides (KSCM-1, KSCM-5 and KSCM-11). The benzofuran-2-carboxamide structure was N-arylated and N-alkylated to include both N-phenyl and N-(3-(piperidin-1-yl)propyl substituents, respectively. These new carboxamides exhibit high affinity at the sigma-1 receptor with K_i values ranging from 7.8 to 34 nM. Ligand KSCM-1 with two methoxy substituents at C-5 and C-6 of the benzofuran ring, and K_i = 27.5 nM at sigma-1 was found to be more selective for sigma-1 over sigma-2.     

Discovery of 5-(pyridin-3-yl)-1H-indole-4,7-diones as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Early studies demonstrated that over expression of indoleamine 2,3-dioxygenase (IDO1) in tumor microenvironment results in tumor immune escape. Herein, in order to simplify the structure of two kinds of IDO1 inhibitors from marine alkaloid, Exiguamine A and Tsitsikammamines, we designed, synthesized a series of 1H-indole-4,7-dione derivatives and evaluated their inhibitory activity in IDO1 enzyme and in IFN-γ stimulated Hela cells in vitro. The structure-activity relationship demonstrated that 5-(pyridin-3-yl)-1H-indole-4,7-dione is a promising scaffold for IDO1 inhibitors and most compounds with this core showed moderate inhibition potency at micromole level. Our further enzyme kinetics experiments reveal that these new developed compounds might act as reversible competitive inhibitors of IDO1.     

The discovery of N-(1,3-thiazol-2-yl)pyridin-2-amines as potent inhibitors of KDR kinase

An azo-dye lead was modified to a novel N-(1,3-thiazol-2-yl)pyridin-2-amine series of KDR kinase inhibitors through the use of rapid analog libraries. This new class has been found to be potent, selective, and of low molecular weight. Molecular modeling has postulated an interesting conformational preference and binding mode for these compounds in the active site of the enzyme.     

3-[2-(Aminomethyl)-5-[(pyridin-4-yl)carbamoyl]phenyl] benzoates as soft ROCK inhibitors

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC₅₀ = 2.5 nM) with rapid inactivation in plasma (t_1/2 <5′). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.     

N-(4-{[4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamide derivatives as small molecule heparanase inhibitors

A novel class of N-(4-{[4-(1H-benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamides are described as inhibitors of the endo-beta-glucuronidase heparanase. Among them are N-(4-{[4-(1H-benzoimidazol-2-yl)-phenylamino]-methyl}-phenyl)-3-bromo-4-methoxy-benzamide (15h), and N-(4-{[5-(1H-benzoimidazol-2-yl)-pyridin-2-ylamino]-methyl}- phenyl)-3-bromo-4-methoxy-benzamide (23) which displayed good heparanase inhibitory activity (IC(50) 0.23-0.29 microM), with the latter showing oral exposure in mice.     

Expression of PCSK1 (PC1/3), PCSK2 (PC2) and PCSK3 (furin) in mouse small intestine

The family of serine proteases known as the proprotein convertases subtilisin/kexin type (PCSK) is responsible for the cleavage and maturation of many precursor hormones. Over its three successive regions, the duodenum, the jejunum and the ileum, the small intestine (SI) expresses over 40 peptide hormones necessary for normal intestinal physiology. Most of these hormones derive from proteolytic cleavage of their cognate inactive polypeptide precursors. Members of the PCSK family of proteases have been implicated in this process, although details of enzyme-substrate interactions are largely lacking. As a first step towards elucidating these interactions, we have analyzed by immunohistochemistry the regional distribution of PCSK1, PCSK2 and PCSK3 in mouse SI as well as their cellular co-localization with substance P (SP), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and somatostatin (SS), 4 peptide hormones known to result from PCSK-mediated processing. Results indicate that PCSK1 is found in all three regions of the SI while PCSK2 and PCSK3 are primarily expressed in the upper two, the duodenum and the jejunum. In these proximal regions, PCSK1 was detectable in 100% of SP-positive (+) cells, 85% of CCK+ cells and 50% of GIP+ cells; PCSK2 was detectable in 40% of SS+ cells and 35% of SP+ cells; PCSK3 was detectable in 75% of GIP+ cells and 60% of SP+ cells. These histological data suggest that the 3 PCSKs may play differential and overlapping roles in prohormone processing in the three regions of the SI.     

Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein

PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1-3) in the secretion and function of PCSK9. Deletion of PD residues 31-40, 41-50, or 51-60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61-70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.     

tert-Butyl 1,5-bis(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)-1,5-dioxopentan-2-ylcarbamate urea/thiourea derivatives as potent H+/K+-ATPase inhibitors

Amino acids are known to possess variable efficacy against ulceration. Considering the good antiulcer activity of amino acids, a series of urea/thiourea derivatives of glutamic acid conjugated benzisothiazole analogue 3a–u with various substituents on aryl ring were synthesized, spectroscopically characterized and evaluated for in vitro H⁺/K⁺-ATPase inhibition. Majority of the compounds possessed potency compared to that of omeprazole, a reference drug. In particular, methoxy derivatives 3p–u were the most active compounds possessing a significant 15-fold increase for para substituent thus, contributing positively to gastric H⁺/K⁺-ATPase inhibition.     

Synthesis and biological evaluation of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides as antimitotic agents

A library of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides (7a–al) have been designed, synthesized and screened for their anti-proliferative activity against some selected human cancer cell lines namely DU-145, A-549, MCF-7 and HeLa. Most of them have shown promising cytotoxicity against lung cancer cell line (A549), amongst them 7f was found to be the most potent anti-proliferative congener. Furthermore, 7f exhibited comparable tubulin polymerization inhibition (IC₅₀ value 2.04 µM) to the standard E7010 (IC₅₀ value 2.15 µM). Moreover, flow cytometric analysis revealed that this compound induced apoptosis via cell cycle arrest at G₂/M phase in A549 cells. Induction of apoptosis was further observed by examining the mitochondrial membrane potential and was also confirmed by Hoechst staining as well as Annexin V-FITC assays. Furthermore, molecular docking studies indicated that compound 7f binds to the colchicine binding site of the β-tubulin. Thus, 7f exhibits anti-proliferative properties by inhibiting the tubulin polymerization through the binding at the colchicine active site and by induction of apoptosis.     

Discovery of 5-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrazin-2(1H)-one derivatives as new potent PB2 inhibitors

PB2 is an important subunit of influenza RNA-dependent RNA polymerase (RdRP) and has been recognized as a promising target for the treatment of influenza. We herein report the discovery of a new series of PB2 inhibitors containing the skeleton 5-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrazin-2(1H)-one. Compound 12b is the most potent one, which showed K_D values of 0.11 μM and 0.19 μM in surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays, respectively. In antiviral activity and cellular cytotoxicity assays, compound 12b showed an EC₅₀ value of 1.025 μM and a CC₅₀ value greater than 100 μM. Molecular docking was also used to predict the binding mode of 12b with PB2. Collectively, this study provides a promising lead compound for subsequent anti-influenza drug discovery targeting PB2.     

Design and synthesis of novel 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine derivatives as selective G-protein-coupled receptor kinase-2 and -5 inhibitors

G-protein-coupled receptor kinase (GRK)-2 and -5 are emerging therapeutic targets for the treatment of cardiovascular disease. In our efforts to discover novel small molecules to inhibit GRK-2 and -5, a class of compound based on 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine was identified as a novel hit by high throughput screening campaign. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK-2 and -5.     

Discovery and optimization of arylsulfonyl 3-(pyridin-2-yloxy)anilines as novel GPR119 agonists

We describe the discovery of a series of arylsulfonyl 3-(pyridin-2-yloxy)anilines as GPR119 agonists derived from compound 1. Replacement of the three methyl groups in 1 with metabolically stable moieties led to the identification of compound 34, a potent and efficacious GPR119 agonist with improved pharmacokinetic (PK) properties.     

Suppressor of Cytokine Signaling-3 (SOCS-3) Induces Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Expression in Hepatic HepG2 Cell Line

The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by proinflammatory cytokines, including the tumor necrosis factor-α (TNF-α). SOCS3 is also implicated in hypertriglyceridemia associated to insulin resistance. Proprotein convertase subtilisin kexin type 9 (PCSK9) levels are frequently found to be positively correlated to insulin resistance and plasma very low density lipoprotein (VLDL) triglycerides concentrations. The present study aimed to investigate the possible role of TNF-α and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. TNF-α induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2^SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein, with no effect on its promoter activity and mRNA stability. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels, whereas an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2^SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fattyacid synthase, stearoyl-CoA desaturase (SCD)-1, and apoB. These responses were associated with a significant increase of SCD-1 protein, activation of sterol regulatory element-binding protein-1c (SREBP-1), accumulation of cellular triglycerides, and secretion of apoB. HepG2^SOCS3 show lower phosphorylation levels of insulin receptor substrate 1 (IRS-1) Tyr⁸⁹⁶ and Akt Ser⁴⁷³ in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, fatty acid synthase, and apoB mRNA. In conclusion, our data candidate PCSK9 as a gene involved in lipid metabolism regulated by proinflammatory cytokine TNF-α in a SOCS3-dependent manner.     

Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives as T-type calcium channel (Cav3.2) inhibitors

Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca(v)3.2) inhibitor with in vivo antihypertensive effect in rats.     

Discovery and initial SAR of 3-(1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-ones as inhibitors of insulin-like growth factor 1-receptor (IGF-1R)

The discovery and synthesis of 3-(1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one inhibitors of insulin-like growth factor 1-receptor (IGF-1R) are presented. Installing amine containing side chains at the 4-position of pyridone ring significantly improved the enzyme potency. SAR and biological activity of these compounds is presented.     

Design, synthesis, and biological evaluation of substituted-N-(thieno[2,3-b]pyridin-3-yl)-guanidines, N-(1H-pyrrolo[2,3-b]pyridin-3-yl)-guanidines, and N-(1H-indol-3-yl)-guanidines

Sulfonylureas stimulate insulin secretion independent of the blood glucose concentration and therefore cause hypoglycemia in type 2 diabetic patients. Over the last years, a number of aryl-imidazoline derivatives have been identified that stimulate insulin secretion in a glucose-dependent manner. In the present study, we have developed three series of substituted N-(thieno[2,3-b]pyridin-3-yl)-guanidine (2a-l), N-(1H-pyrrolo[2,3-b]pyridin-3-yl)-guanidine (3a-l), and N-(1H-indol-3-yl)-guanidine (4a-l) as new class of antidiabetic agents. In vitro glucose-dependent insulinotropic activity of test compounds 2a-l, 3a-l, and 4a-l was evaluated using RIN5F (Rat Insulinoma cell) based assay. All the test compounds showed concentration-dependent insulin secretion, only in presence of glucose load (16.7mmol). Some of the test compounds (2c, 3c, and 4c) from each series were found to be equipotent to BL 11282 (standard aryl-imidazoline), which indicated that the guanidine group acts as a bioisostere of imidazoline ring system.