Discovery of a Novel Series of Potent, Selective, Orally Available, and Brain-Penetrable C1s Inhibitors for Modulation of the Complement Pathway.

A novel series of non-amidine-based C1s inhibitors have been explored. Starting from high-throughput screening hit 3, isoquinoline was replaced with 1-aminophthalazine to enhance C1s inhibitory activity while exhibiting good selectivity against other serine proteases. We first disclose a crystal structure of a complex of C1s and a small-molecule inhibitor (4e), which guided structure-based optimization around the S2 and S3 sites to further enhance C1s inhibitory activity by over 300-fold. Improvement of membrane permeability by incorporation of fluorine at the 8-position of 1-aminophthalazine led to identification of (R)-8 as a potent, selective, orally available, and brain-penetrable C1s inhibitor. (R)-8 significantly inhibited membrane attack complex formation induced by human serum in a dose-dependent manner in an in vitro assay system, proving that selective C1s inhibition blocked the classical complement pathway effectively. As a result, (R)-8 emerged as a valuable tool compound for both in vitro and in vivo assessment.

Discovery of 3-Benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea Derivatives as Novel and Selective Cyclin-Dependent Kinase 12 (CDK12) Inhibitors.

Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile.

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.

Discovery, synthesis, and structure-activity relations of 3,4-dihydro-1H-spiro(naphthalene-2,2'-piperidin)-1-ones as potassium-competitive acid blockers.

With the aim to discover a gastric antisecretory agent more potent than the existing proton pump inhibitors, novel 3,4-dihydro-1H-spiro(naphthalene-2,2'-piperidin)-1-one derivatives, which could occupy two important lipophilic pockets (described as LP-1 and LP-2) of H+,K+-ATPase and can strongly bind to the K+-binding site, were designed based on a docking model. Among the compounds synthesized, compound 4d showed a strong H+,K+-ATPase-inhibitory activity and a high stomach concentration in rats, resulting in potent inhibitory action on histamine-stimulated gastric acid secretion in rats. Furthermore, 4d exerted significant inhibitory action on histamine-stimulated gastric-acid secretion in rats with a rapid onset and moderate duration of action after the administration. These findings may lead to a new insight into the drug design of potassium-competitive acid blockers.

High-Throughput Screening to Identify Inhibitors of DEAD Box Helicase DDX41.

The human DEAD (Asp-Glu-Ala-Asp) box protein DDX41, a member of the DEXDc helicase family, has nucleic acid-dependent ATPase and RNA and DNA translocase and unwinding activities. DDX41 is affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. The R525H mutation in DDX41 is thought to play important roles in the development of hereditary myelodysplastic syndrome and acute myelocytic leukemia. In this study, human DDX41 and its R525H mutant (R525H) were expressed in Escherichia coli and purified. The ATPase activities of the recombinant DDX41 and R525H proteins were dependent on both ATP and double-stranded DNA (dsDNA), such as poly(dG-dC) and poly(dA-dT). High-throughput screening was performed with a dsDNA-dependent ATPase assay using the human R525H proteins. After hit confirmation and counterscreening, several small-molecule inhibitors were successfully identified. These compounds show DDX41-selective inhibitory activities.

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.

Discovery of TAK-272: A Novel, Potent, and Orally Active Renin Inhibitor.

The aspartic proteinase renin is an attractive target for the treatment of hypertension and cardiovascular/renal disease such as chronic kidney disease and heart failure. We introduced an S1' site binder into the lead compound 1 guided by structure-based drug design (SBDD), and further optimization of physicochemical properties led to the discovery of benzimidazole derivative 10 (1-(4-methoxybutyl)-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-yl)carbonylpiperidin-3-yl]-1H-benzimidazole-2-carboxamide hydrochloride, TAK-272) as a highly potent and orally active renin inhibitor. Compound 10 demonstrated good oral bioavailability (BA) and long-lasting efficacy in rats. Compound 10 is currently in clinical trials.

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 S3SP (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 S3SP 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.

Discovery of 1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (TAK-063), a highly potent, selective, and orally active phosphodiesterase 10A (PDE10A) inhibitor.

A novel series of pyridazinone-based phosphodiesterase 10A (PDE10A) inhibitors were synthesized. Our optimization efforts using structure-based drug design (SBDD) techniques on the basis of the X-ray crystal structure of PDE10A in complex with hit compound 1 (IC50 = 23 nM; 110-fold selectivity over other PDEs) led to the identification of 1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (27h). Compound 27h has potent inhibitory activity (IC50 = 0.30 nM), excellent selectivity (>15000-fold selectivity over other PDEs), and favorable pharmacokinetics, including high brain penetration, in mice. Oral administration of compound 27h to mice elevated striatal 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) levels at 0.3 mg/kg and showed potent suppression of phencyclidine (PCP)-induced hyperlocomotion at a minimum effective dose (MED) of 0.3 mg/kg. Compound 27h (TAK-063) is currently being evaluated in clinical trials for the treatment of schizophrenia.

Discovery, synthesis, and biological evaluation of novel pyrrole derivatives as highly selective potassium-competitive acid blockers.

To discover a gastric antisecretory agent more potent than existing proton pump inhibitors, novel pyrrole derivatives were synthesized, and their H(+),K(+)-ATPase inhibitory activities and inhibitory action on histamine-stimulated gastric acid secretion in rats were evaluated. Among the compounds synthesized, compound 17a exhibited selective and potent H(+),K(+)-ATPase inhibitory activity through reversible and K(+)-competitive ionic binding; furthermore, compound 17c exhibited potent inhibitory action on histamine-stimulated gastric acid secretion in rats and Heidenhain pouch dogs.

Discovery of a novel pyrrole derivative 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine fumarate (TAK-438) as a potassium-competitive acid blocker (P-CAB).

In our pursuit of developing a novel and potent potassium-competitive acid blocker (P-CAB), we synthesized pyrrole derivatives focusing on compounds with low log D and high ligand-lipophilicity efficiency (LLE) values. Among the compounds synthesized, the compound 13e exhibited potent H(+),K(+)-ATPase inhibitory activity and potent gastric acid secretion inhibitory action in vivo. Its maximum efficacy was more potent and its duration of action was much longer than those of proton pump inhibitors (PPIs). Therefore, compound 13e (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine fumarate, TAK-438) was selected as a drug candidate for the treatment of gastroesophageal reflux disease (GERD), peptic ulcer, and other acid-related diseases.

High-throughput screening of potassium-competitive acid blockers.

H(+),K(+)-ATPase is a key enzyme in the process of gastric acid secretion, and proton pump inhibitors (PPIs) have been accepted as one of the most effective treatments for peptic ulcer and gastroesophageal reflux disease. To discover a novel class of PPIs, the authors screened a low-molecular-weight compound library and identified two prospective acid blockers that were pyrrole derivatives. Both compounds inhibited H(+),K(+)-ATPase in a reversible and potassium-competitive manner. These compounds led to the development of TAK-438 (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate), which is currently undergoing clinical trials as a novel potassium-competitive acid blocker for the treatment of acid-related diseases.