Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC₅₀ = 150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC₅₀ = 25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC₅₀ = 7.5 nM) and TNF-α production in mouse splenocytes (IC₅₀ = 9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID₅₀ = 18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand–enzyme complex.     

Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of K_d using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 μM–46 nM) to JNK3. Among them, compound 16f exhibited potent activities (K_d = 46 nM). Especially, 16f was also found to present a potent cell protective effect (IC₅₀ = 1.09 μM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.     

Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC₅₀ = 11 nM; PC-3 antiproliferative IC₅₀ = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC₅₀ = 10 nM; A2780 antiproliferative IC₅₀ = 7 nM) in complex with the NAMPT protein was also determined.     

Development of amino-pyrimidine inhibitors of c-Jun N-terminal kinase (JNK): Kinase profiling guided optimization of a 1,2,3-benzotriazole lead

A series of amino-pyrimidines was developed based upon an initial kinase cross-screening hit from a CDK2 program. Kinase profiling and structure-based drug design guided the optimization from the initial 1,2,3-benzotriazole hit to a potent and selective JNK inhibitor, compound 24f (JNK1 and 2 IC₅₀ = 16 and 66 nM, respectively), with bioavailability in rats and suitable for further in vivo pharmacological evaluation.     

Discovery of potent dipeptidyl peptidase IV inhibitors through pharmacophore hybridization and hit-to-lead optimization

A novel dipeptidyl peptidase IV inhibitor hit (5, IC₅₀ = 0.86 μM) was structurally derived from our recently disclosed preclinical candidate 4 by replacing the cyanobenzyl with a butynyl based on pharmacophore hybridization. A hit-to-lead optimization effort was then initiated to improve its potency. Most N-substituted analogs exhibited good in vitro activity, and compound 18o (IC₅₀ = 1.55 nM) was identified to be a potent dipeptidyl peptidase IV inhibitor with a significantly improved pharmacokinetic properties (bioavailablity: 41% vs 82.9%; T_1/2: 2 h vs 4.9 h).     

Highly potent tyrosinase inhibitor,neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC₅₀ of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC₅₀ = 500 nM), significantly. Another potent inhibitor 1 (IC₅₀ = 2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: K_i^app = 1.48 nM, k₃ = 0.0033 nM⁻¹ min⁻¹ and k₄ = 0.0049 min⁻¹. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC₅₀. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.     

A novel and potent VLA-4 antagonist based on trans-4-substituted cyclohexanecarboxylic acid

During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the diphenylurea structure. However, this modification resulted in a significant decrease (3, IC₅₀ = 19 nM) in VLA-4 inhibitory activity compared to 1 (IC₅₀ = 1.6 nM). To address this discrepancy, we worked on optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discovery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC₅₀ = 2.8 nM) as a novel and potent VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties (CL = 3.3 ml/min/kg, F = 51%) in rats.     

Synthesis and biological evaluation of 5-carbamoyl-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

5-Carbamoyl-2-phenylpyrimidine derivative 2 has been identified as a phosphodiesterase 4 (PDE4) inhibitor with moderate PDE4B inhibitory activity (IC₅₀ = 200 nM). Modification of the carboxylic acid moiety of 2 gave N-neopentylacetamide derivative 10f, which had high in vitro PDE4B inhibitory activity (IC₅₀ = 8.3 nM) and in vivo efficacy against lipopolysaccharide (LPS)-induced pulmonary neutrophilia in mice (ID₅₀ = 16 mg/kg, ip). Furthermore, based on the X-ray crystallography of 10f bound to the human PDE4B catalytic domain, we designed 7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one derivative 39 which has a fused bicyclic lactam scaffold. Compound 39 exhibited excellent inhibitory activity against LPS-induced tumor necrosis factor alpha (TNF-α) production in mouse splenocytes (IC₅₀ = 0.21 nM) and in vivo anti-inflammatory activity against LPS-induced pulmonary neutrophilia in mice (41% inhibition at a dose of 1.0 mg/kg, i.t.).     

Optimization of 5-vinylaryl-3-pyridinecarbonitriles as PKCθ inhibitors

Analog 8, a 3-pyridinecarbonitrile with an (E)-2-{6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl}vinyl group at C-5, had an IC₅₀ value of 1.1 nM for the inhibition of PKCθ and potently blocked the production of IL-2 in both stimulated murine T cells (IC₅₀ = 34 nM) and human whole blood (IC₅₀ = 500 nM).     

Synthesis,biological evaluation,and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.     

Pyrrolidinyl phenylurea derivatives as novel CCR3 antagonists

Optimization starting with our lead compound 1 (IC₅₀ = 4.9 nM) led to the identification of pyrrolidinyl phenylurea derivatives. Further modification toward improvement of the bioavailability provided (R)-1-(1-((6-fluoronaphthalen-2-yl)methyl)pyrrolidin-3-yl)-3-(2-(2-hydroxyethoxy)phenyl)urea 32 (IC₅₀ = 1.7 nM), a potent and orally active CCR3 antagonist.     

Aroylguanidine-based factor Xa inhibitors: The discovery of BMS-344577

We report the design and synthesis of a novel class of N,N′-disubstituted aroylguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The structure–activity relationships (SAR) investigation led to the discovery of the nicotinoyl guanidine 22 as a potent FXa inhibitor (FXa IC₅₀ = 4 nM, EC_2×PT = 7 μM). However, the potent CYP3A4 inhibition activity (IC₅₀ = 0.3 μM) of 22 precluded its further development. Detailed analysis of the X-ray crystal structure of compound 22 bound to FXa indicated that the substituent at the 6-position of the nicotinoyl group of 22 would be solvent-exposed, suggesting that efforts to attenuate the unwanted CYP activity could focus at this position without affecting FXa potency significantly. Further SAR studies on the 6-substituted nicotinoyl guanidines resulted in the discovery of 6-(dimethylcarbamoyl) nicotinoyl guanidine 36 (BMS-344577, IC₅₀ = 9 nM, EC_2×PT = 2.5 μM), which was found to be a selective, orally efficacious FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models.     

NAD-based inhibitors with anticancer potential

Three classes of novel inhibitors of inosine monophosphate dehydrogenase have been prepared and their anti-proliferative properties were evaluated against several cancer cell lines.(1) Mycophenolic adenine dinucleotide analogues (8–13) containing a substituent at the C2 of adenine ring were found to be potent inhibitors of IMPDH (K_i’s in range of 0.6–82 nM) and sub-μM inhibitors of leukemic K562 cell proliferation. (2) Mycophenolic adenosine (d and l) esters (20 and 21) showed a potent inhibition of IMPDH2 (K_i = 102 and K_i = 231 nM, respectively) and inhibition of K562 cell growth (IC₅₀ = 0.5 and IC₅₀ = 1.6 μM). These compounds serve both as inhibitors of the enzyme and as a depot form of mycophenolic acid. The corresponding amide analogue 22, also a potent inhibitor of IMPDH (K_i = 84 nM), did not inhibit cancer cell proliferation. (3) Mycophenolic-(l)- and (d)-valine adenine di-amide derivatives 25 (K_i = 9 nM) and 28 (K_i = 3 nM) were found to be very potent enzymatically, but did not inhibit proliferation of cancer cells.     

Isoquinoline derivatives as potent CRTH2 antagonists: Design,synthesis and SAR

In this study, we describe the synthesis and structure–activity relationship (SAR) of a series of isoquinoline chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2) antagonists. TASP0376377 (15-20), one of the most potent compounds, showed a potent binding affinity (IC₅₀ = 19 nM) in addition to the excellent functional antagonist activity (IC₅₀ = 13 nM). Moreover, the efficacy of this compound in a chemotaxis assay (IC₅₀ = 23 nM) was in good agreement with its potency as a CRTH2 antagonist. In addition, 15-20 exhibited greater selectivity in binding to CRTH2 than to the DP1 prostanoid receptor (IC₅₀ >1 μM) or the enzymes COX-1 and COX-2 (IC₅₀ >10 μM).     

New halogenated 3-phenylcoumarins as potent and selective MAO-B inhibitors

With the aim to find out the structural features for the MAO inhibitory activity and selectivity, in the present communication we report the synthesis and pharmacological evaluation of a new series of bromo-6-methyl-3-phenylcoumarin derivatives (with bromo atom in both different benzene rings of the skeleton) with and without different number of methoxy substituent at the 3-phenyl ring. The methoxy substituents were introduced, in this new scaffold, in the meta and/or para positions of the 3-phenyl ring. The synthesized compounds 3–7 were evaluated as MAO-A and B inhibitors using R-(?)-deprenyl (selegiline) and iproniazide as reference inhibitors, showing, most of them, MAO-B inhibitory activities in the low nanomolar range. Compounds 4 (IC₅₀ = 11.05 nM), 5 (IC₅₀ = 3.23 nM) and 6 (IC₅₀ = 7.12 nM) show higher activity than selegiline (IC₅₀ = 19.60 nM) and higher MAO-B selectivity, with more than 9050-fold, 30,960-fold and 14,045-fold inhibition levels, with respect to the MAO-A isoform.     

Potent BRAF kinase inhibitors based on 2,4,5-trisubstituted imidazole with naphthyl and benzothiophene 4-substituents

The RAS–RAF–MEK–ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine–threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC₅₀ = 190 nM and with cellular GI₅₀ = 2100 nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC₅₀ = 9 nM and GI₅₀ = 220 nM.     

Second generation 4-(4-methyl-1H-indol-5-ylamino)-2-phenylthieno[2,3-b]pyridine-5-carbonitrile PKCθ inhibitors

Thieno[2,3-b]pyridine-5-carbonitrile 16 with a 4-methyl-5-indolylamine at C-4 and a 5-methoxy-2-(dimethylamino)-methylphenyl group at C-2 had an IC₅₀ value of 16 nM for the inhibition of PKCθ. While moderate inhibition of PKCδ was also observed (IC₅₀ = 130 nM), 16 had IC₅₀ values of greater than 5 μM against Lyn and other members of the Src kinase family.     

5,6-Dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase

5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC₅₀ (1b) < 10 nM; IC₅₀ (1a) < 25 nM, EC₅₀ (1b) = 16 nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F = 24%).     

3-(Arylsulfonyl)-1-(azacyclyl)-1H-indoles are 5-HT6 receptor modulators

Novel 3-(arylsulfonyl)-1-(azacyclyl)-1H-indoles 6 were synthesized as potential 5-HT₆ receptor ligands, based on constraining a basic side chain as either a piperidine or a pyrrolidine. Many of these compounds had good 5-HT₆ binding affinity with K_i values <10 nM. Depending on substitution, both agonists (e.g., 6o: EC₅₀ = 60 nM, E_max = 70%) and antagonists (6y: IC₅₀ = 17 nM, I_max = 86%) were identified in a 5-HT₆ adenylyl cyclase assay.     

5-(1,3-Benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives as potent and selective transforming growth factor-β type I receptor inhibitors

A series of 5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazole derivatives was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and for their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. As a representative compound, 16i was a potent and selective ALK5 inhibitor, exhibiting a good enzyme inhibitory activity (IC₅₀ = 5.5 nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC₅₀ = 36 nM). Furthermore, the topical application of 3% 16i lotion significantly inhibited Smad2 phosphorylation in Mouse skin (90% inhibition compared with vehicle-treated animals).