Novel approach of fragment-based lead discovery applied to renin inhibitors

A novel approach was conducted for fragment-based lead discovery and applied to renin inhibitors. The biochemical screening of a fragment library against renin provided the hit fragment which showed a characteristic interaction pattern with the target protein. The hit fragment bound only to the S1, S3, and S3^SP (S3 subpocket) sites without any interactions with the catalytic aspartate residues (Asp32 and Asp215 (pepsin numbering)). Prior to making chemical modifications to the hit fragment, we first identified its essential binding sites by utilizing the hit fragment’s substructures. Second, we created a new and smaller scaffold, which better occupied the identified essential S3 and S3^SP sites, by utilizing library synthesis with high-throughput chemistry. We then revisited the S1 site and efficiently explored a good building block attaching to the scaffold with library synthesis. In the library syntheses, the binding modes of each pivotal compound were determined and confirmed by X-ray crystallography and the library was strategically designed by structure-based computational approach not only to obtain a more active compound but also to obtain informative Structure Activity Relationship (SAR). As a result, we obtained a lead compound offering synthetic accessibility as well as the improved in vitro ADMET profiles. The fragments and compounds possessing a characteristic interaction pattern provided new structural insights into renin’s active site and the potential to create a new generation of renin inhibitors. In addition, we demonstrated our FBDD strategy integrating highly sensitive biochemical assay, X-ray crystallography, and high-throughput synthesis and in silico library design aimed at fragment morphing at the initial stage was effective to elucidate a pocket profile and a promising lead compound.     

Structure-based optimization of potent 4- and 6-azaindole-3-carboxamides as renin inhibitors

The control of hypertension and associated cardiovascular risk factors is possible by selective inhibition of the aspartyl protease renin due to its unique position in the renin-angiotensin system. Starting from a previously disclosed series of potent and nonchiral indole-3-carboxamides, we further explored this motif by structure-based drug design guided by X-ray crystallography in combination with efficient parallel synthesis. This resulted in the discovery of 4- or 6-azaindole derivatives with remarkable potency for renin inhibition. The best compound from these series showed an IC(50) value of 1.3 nM.     

Structure-based design and synthesis of novel furan-diketopiperazine-type derivatives as potent microtubule inhibitors for treating cancer

Plinabulin, a synthetic analog of the marine natural product “diketopiperazine phenylahistin,” displayed depolymerization effects on microtubules and targeted the colchicine site, which has been moved into phase III clinical trials for the treatment of non-small cell lung cancer (NSCLC) and the prevention of chemotherapy-induced neutropenia (CIN). To develop more potent anti-microtubule and cytotoxic derivatives, the co-crystal complexes of plinabulin derivatives were summarized and analyzed. We performed further modifications of the tert-butyl moiety or C-ring of imidazole-type derivatives to build a library of molecules through the introduction of different groups for novel skeletons. Our structure–activity relationship study indicated that compounds 17o (IC₅₀ = 14.0 nM, NCI-H460) and 17p (IC₅₀ = 2.9 nM, NCI-H460) with furan groups exhibited potent cytotoxic activities at the nanomolar level against various human cancer cell lines. In particular, the 5-methyl or methoxymethyl substituent of furan group could replace the alkyl group of imidazole at the 5-position to maintain cytotoxic activity, contradicting previous reports that the tert-butyl moiety at the 5-position of imidazole was essential for the activity of such compounds. Immunofluorescence assay indicated that compounds 17o and 17p could efficiently inhibit microtubule polymerization. Overall, the novel furan-diketopiperazine-type derivatives could be considered as a potential scaffold for the development of anti-cancer drugs.     

Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors

We report synthesis and optimization of a series of (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of ${{\text{P}}_1}^{\prime }$, ${{\text{P}}_2}^{\prime }$ and P₃ portions led to a promising 3,5-disubstituted piperidine 32o showing high renin inhibitory activity and favorable oral exposure in both rats and cynomolgus monkeys with acceptable CYP and hERG current inhibition. Compound 32o exhibited a significant blood pressure lowering effect by oral administration in two hypertensive animal models, double transgenic rats and furosemide pretreated cynomolgus monkeys.     

Structure-based design of thienobenzoxepin inhibitors of PI3-kinase

Starting from thienobenzopyran HTS hit 1, co-crystallization, molecular modeling and metabolic analysis were used to design potent and metabolically stable inhibitors of PI3-kinase. Compound 15 demonstrated PI3K pathway suppression in a mouse MCF7 xenograft model.     

Structure-based design and synthesis of macrocyclic human rhinovirus 3C protease inhibitors

The design and synthesis of macrocyclic inhibitors of human rhinovirus 3C protease is described. A macrocyclic linkage of the P1 and P3 residues, and the subsequent structure-based optimization of the macrocycle conformation and size led to the identification of a potent biochemical inhibitor 10 with sub-micromolar antiviral activity.     

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.     

Structure-based optimization of non-peptidic Cathepsin D inhibitors

We discovered a novel series of non-peptidic acylguanidine inhibitors of Cathepsin D as target for osteoarthritis. The initial HTS-hits were optimized by structure-based design using CatD X-ray structures resulting in single digit nanomolar potency in the biochemical CatD assay. However, the most potent analogues showed only micromolar activities in an ex vivo glycosaminoglycan (GAG) release assay in bovine cartilage together with low cellular permeability and suboptimal microsomal stability. This new scaffold can serve as a starting point for further optimization towards in vivo efficacy.     

Structure-based design and optimization of 2-aminothiazole-4-carboxamide as a new class of CHK1 inhibitors

Drug design efforts in the emerging 2-aminothiazole-4-carboxamide class of CHK1 inhibitors have uncovered specific combinations of key substructures within the molecule; resulting in significant improvements in cell-based activity while retaining a greater than one hundred-fold selectivity against CDK2. The X-ray crystal structure of a complex between compound 39 and the CHK1 protein detailing a ‘U-shaped’ topology and key interactions with the protein surface at the ATP site is also reported.     

Structure-based design and SAR development of 5,6-dihydroimidazolo[1,5-f]pteridine derivatives as novel Polo-like kinase-1 inhibitors

Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors. Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor xenograft model.     

Discovery of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides as small molecule inhibitors of PCSK9

A series of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides were identified as small molecule PCSK9 mRNA translation inhibitors. Analogues from this new chemical series, such as 4d and 4g, exhibited improved PCSK9 potency, ADME properties, and in vitro safety profiles when compared to earlier lead structures.     

Structure-based drug design of 1,3,6-trisubstituted 1,4-diazepan-7-ones as selective human kallikrein 7 inhibitors

A novel series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were investigated as human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Based on the X-ray co-crystal structure of compound 1 bound to human KLK7, the derivatives of this scaffold were designed, synthesized, and evaluated. Through structure-activity relationship studies focused on the side chain located in the prime site region of the enzyme, representative compounds 15, 33a, and 35a were identified as highly potent and selective inhibitors of human KLK7.     

Structure-based design and optimization of potent renin inhibitors on 5- or 7-azaindole-scaffolds

The selective inhibition of the aspartyl protease renin is of high interest to control hypertension and associated cardiovascular risk factors. Following on preceding contributions, we report herein on the optimization of two series of azaindoles to arrive at potent and non-chiral renin inhibitors. The previously discovered azaindole scaffold was further explored by structure-based drug design in combination with parallel synthesis. This results in the identification of novel 5- or 7-azaindole derivatives with remarkable potency for renin inhibition. The best compounds on both series show IC(50) values between 3 and 8nM.     

Structure-based design and synthesis of tricyclic IAP (Inhibitors of Apoptosis Proteins) inhibitors

The design and synthesis of a series of novel tricyclic IAP inhibitors is reported. Rapid assembly of the core tricycle involved two key steps: Rh-catalyzed hydrogenation of an unsaturated bicyclic ring system and a Ru-catalyzed ring closing alkene metathesis reaction. The final Smac mimetics bind to cIAP1 and XIAP BIR3 domains and elicit the desired phenotype in cellular proliferation assays. Dimeric IAP inhibitors were found to possess nanomolar potency in a cellular proliferation assay and favourable in vitro drug-like properties.     

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (K_d = 0.52 nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC₅₀ = 0.098 μM, NCI-N87 IC₅₀ = 0.066 μM) and also displayed high oral bioavailability in mice (F = 44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition = 136%).     

Conformational control in structure-based drug design

In structure-based drug design, the basic goal is to design molecules that fit complementarily to a given binding pocket. Since such computationally modeled molecules may not adopt the intended bound conformation outside the binding pocket, one challenge is to ensure that the designed ligands adopt similar low energy conformations both inside and outside of the binding pocket. Computational chemistry methods and conformational preferences of small molecules from PDB and Cambridge Structural Database (CSD) can be used to predict the bound structures of the designed molecules. Herein, we review applications of conformational control in structure-based drug design using selected examples from the recent medicinal chemistry literature. The main purpose is to highlight some intriguing conformational features that can be applied to other drug discovery programs.     

Structure-based drug design of novel carborane-containing nicotinamide phosphoribosyltransferase inhibitors

A series of carborane-containing NAMPT inhibitors were designed and synthesized based on the structure of compounds 1 and the NAMPT inhibitory activity was evaluated using NAMPT Colorimetric Assay. Among the compounds synthesized, compounds 2b and 2c showed significant NAMPT inhibitory activity with IC₅₀ values of 0.098 ± 0.008 and 0.057 ± 0.001 µM, respectively. Docking simulation of compound 2 toward NAMPT using the crystal structure of the FK866-NAMPT complex (PDB code: 2GVJ) with replacing the boron atom type by the C3 atom type of carboranes predicted that the NAMPT inhibitory activity of 2c was improved by the hydrogen bond formation between the carborane amide and H191 of NAMPT. Although dicarborane compounds 38, 50, 51, and 55 were synthesize aiming to two hydrophobic pockets present in the binding pocket of NAMPT, their inhibitory activity was moderate.     

Structure-based design of novel HCV NS5B thumb pocket 2 allosteric inhibitors with submicromolar gt1 replicon potency: Discovery of a quinazolinone chemotype

We describe the structure-based design of a novel lead chemotype that binds to thumb pocket 2 of HCV NS5B polymerase and inhibits cell-based gt1 subgenomic reporter replicons at sub-micromolar concentrations (EC₅₀ <200 nM). This new class of potent thumb pocket 2 inhibitors features a 1H-quinazolin-4-one scaffold derived from hybridization of a previously reported, low affinity thiazolone chemotype with our recently described anthranilic acid series. Guided by X-ray structural information, a key NS5B–ligand interaction involving the carboxylate group of anthranilic acid based inhibitors was replaced by a neutral two-point hydrogen bonding interaction between the quinazolinone scaffold and the protein backbone. The in vitro ADME and in vivo rat PK profile of representative analogs are also presented and provide areas for future optimization of this new class of HCV polymerase inhibitors.     

Structure-based design of aliskiren,a novel orally effective renin inhibitor

Hypertension is a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, and heart failure, the leading causes of death in the Western world. Inhibitors of the renin-angiotensin system (RAS) have proven to be successful treatments for hypertension. As renin specifically catalyses the rate-limiting step of the RAS, it represents the optimal target for RAS inhibition. Several peptide-like renin inhibitors have been synthesized previously, but poor pharmacokinetic properties meant that these compounds were not clinically useful. We employed a combination of molecular modelling and crystallographic structure analysis to design renin inhibitors lacking the extended peptide-like backbone of earlier inhibitors, for improved pharmacokinetic properties. This led to the discovery of aliskiren, a highly potent and selective inhibitor of human renin in vitro, and in vivo; once-daily oral doses of aliskiren inhibit renin and lower blood pressure in sodium-depleted marmosets and hypertensive human patients. Aliskiren represents the first in a novel class of renin inhibitors with the potential for treatment of hypertension and related cardiovascular diseases.