Design and synthesis of potent and selective azaindole-based Rho kinase (ROCK) inhibitors

Rho kinase plays a pivotal role in several cellular processes such as vasoregulation, making it a suitable target for the treatment of hypertension and related disorders. We discovered a new compound class of Rho kinase (ROCK) inhibitors containing a 7-azaindole hinge-binding scaffold tethered to an aminopyrimidine core. Herein we describe the structure-activity relationships elucidated through biochemical and functional assays. The introduction of suitable substituents at the 3-position of the bicyclic moiety led to an increase in activity, which was required to design compounds with favorable pharmacokinetic profile. Azaindole 32 was identified as a highly selective and orally available ROCK inhibitor able to cause a sustained blood pressure reduction in vivo.     

Urolithin as a converging scaffold linking ellagic acid and coumarin analogues: design of potent protein kinase CK2 inhibitors

Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase; its abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other relevant diseases. Previously, using different in silico screening approaches, two potent and selective CK2 inhibitors were identified by our group: ellagic acid, a naturally occurring tannic acid derivative (K(i)=20 nM) and 3,8-dibromo-7-hydroxy-4-methylchromen-2-one (DBC, K(i)=60 nM). Comparing the crystallographic binding modes of both ellagic acid and DBC, an X-ray structure-driven merging approach was taken to design novel CK2 inhibitors with improved target affinity. A urolithin moiety is proposed as a possible bridging scaffold between the two known CK2 inhibitors, ellagic acid and DBC. Optimization of urolithin A as the bridging moiety led to the identification of 4-bromo-3,8-dihydroxy-benzo[c]chromen-6-one as a novel, potent and selective CK2 inhibitor, which shows a K(i) value of 7 nM against the protein kinase, representing a significant improvement in affinity for the target compared with the two parent fragments.     

Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.     

The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and conserved water molecules

CK2 is a highly pleiotropic Ser/Thr protein kinase that is able to promote cell survival and enhance the tumour phenotype under specific circumstances. We have determined the crystal structure of three new complexes with tetrabromobenzimidazole derivatives that display K(i) values between 0.15 and 0.30 microM. A comparative analysis of these data with those of four other inhibitors of the same family revealed the presence of some highly conserved water molecules in the ATP-binding site. These waters reside near Lys68, in an area with a positive electrostatic potential that is able to attract and orient negatively charged ligands. The presence of this positive region and two unique bulky residues that are typical of CK2, Ile66 and Ile174, play a critical role in determining the ligand orientation and binding selectivity.     

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth‐factor‐like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy‐resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4‐tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4‐substituted benzylphosphonic acid and 6‐substituted naphthalen‐2‐ylmethylphosphonic acid analogues block ATX activity with K_i values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed‐mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b , the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose‐dependent manner. The average terminal half‐life for compound 22 is 10±5.4 h and it causes a long‐lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16‐F10 syngeneic mouse melanoma in a post‐inoculation treatment paradigm. The 4‐substituted benzylphosphonic acids and 6‐substituted naphthalen‐2‐ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX–LPA–LPAR axis both in vitro and in vivo.     

Discovery of 3H-imidazo[4,5-b]pyridines as potent c-Met kinase inhibitors: design, synthesis, and biological evaluation

To identify novel c-Met inhibitors, sequences and crystal structures of the human kinome were analyzed to find interesting hinge binders that have been underexplored within the tyrosine kinase subfamily. Through this study, the imidazolopyridine ring was selected as a novel c-Met hinge-binding inhibitor scaffold. A series of derivatives was prepared, and the structure-activity relationships were studied. Among these, one compound in particular showed excellent activities in enzymatic and cellular assays, good in vitro metabolic stability, and favorable pharmacokinetic parameters. When administered orally, the compound inhibited tumor growth in an NIH-3T3/TPR-Met xenograft model and did not show adverse effects on body weight. The present work not only conceptually demonstrates a new route for designing novel kinase inhibitors by using known structural information of ligand-hinge interactions but also provides a series of imidazolopyridine derivatives as potent c-Met inhibitors.     

Structure-activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins)

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD(+)) as a cofactor for their catalytic mechanism(.) Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD(+)-dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two-digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X-ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.     

Discovery and structure-activity relationship studies of a unique class of HIV-1 integrase inhibitors

HIV-1 integrase (IN) is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. Currently there are no approved drugs that target IN. However, new IN inhibitors are under clinical investigation. As more IN inhibitors enter human drug trials, there is a growing need for the design of novel lead compounds with diverse structural scaffolds and promising pharmacokinetic properties to counteract the difficulties observed with first-generation IN inhibitors. We have identified a novel class of IN inhibitors through the systematic exploration of structure-activity relationships in a series of linomide analogues. The predicted bound conformation of the most active analogues inside the IN active site also supports the observed structure-activity correlation in this new compound class.     

Synthesis and evaluation of 3-phenylpyrazolo[3,4-d]pyrimidine-peptide conjugates as Src kinase inhibitors

3-Phenylpyrazolo[3,4-d]pyrimidine (PhPP) derivatives substituted with an alkyl or aryl carboxylic acid at the N1-endocyclic amine, such as PhPP-CH(2)COOH (IC(50)=250 microM), and peptides Ac-CIYKYY (IC(50)=400 microM) and Ac-YIYGSFK (IC(50)=570 microM) were weak inhibitors of polyE(4)Y phosphorylation by active c-Src. A series of PhPP-peptide conjugates were synthesized using PhPP as an ATP mimic and CIYKYY or YIYGSFK as a peptide substrate to improve the inhibitory potency against active c-Src kinase. PhPP derivatives were attached to the N terminus or the side chain of amino acids in the peptide template. Two N-terminal substituted conjugates, PhPP-CH(2)CO-CIYKYY (IC(50)=0.38 microM) and PhPP-CH(2)CO-YIYGSFK (IC(50)=2.7 microM), inhibited the polyE(4)Y phosphorylation by active c-Src significantly higher than that of the parent compounds. The conjugation of PhPP with the peptides produced a synergistic inhibition effect possibly through creation of favorable interactions between the conjugate and the kinase domain as shown by molecular modeling studies.     

6-Substituted pyrrolo[3,4-c]pyrazoles: an improved class of CDK2 inhibitors

We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer-assisted solution phase chemistry provided a way of generating compounds with improved biochemical and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma.     

Synthesis and structure-activity relationships of FAAH inhibitors: cyclohexylcarbamic acid biphenyl esters with chemical modulation at the proximal phenyl ring

Fatty acid amide hydrolase (FAAH) is a serine hydrolase that catalyzes the intracellular hydrolysis of fatty acid ethanolamides such as anandamide and oleoylethanolamide. Targeting this enzyme may have important therapeutic potentials owing to the multiple physiological roles of these amides. Cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) was one of the most promising FAAH inhibitors so far described. We report the modulation of the electronic and steric features of the proximal phenyl ring of this compound by introducing a series of substituents at the ortho and para positions. pIC50 values were found to correlate with molecular features thought to be involved in the recognition step such as steric hindrance and hydrogen-bonding ability. Derivatives with small polar groups at the para position of the proximal phenyl ring were slightly better FAAH inhibitors than the parent compound URB524.     

Design, synthesis, and biological evaluation of 1-[(biarylmethyl)methylamino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as potent antifungal agents: new insights into structure-activity relationships

We recently reported the design and synthesis of azole antifungal agents with a focus on modifications to the side chain appended to the propanol group. Herein we have identified a series of new 1-[(biarylmethyl)methylamino] derivatives with broad-spectrum antifungal activities against the most prevalent human pathogenic fungi (Candida spp. and Aspergillus fumigatus). Compounds containing a flexible benzylamine moiety were clearly shown to yield the best antifungal activities, without the need for a hydrogen-bond acceptor substituent directly attached to the para position. We were also able to determine that selected compounds are able to overcome gene overexpression and point mutations that lead to reduced susceptibility or resistance against current treatments, such as fluconazole. As the minor differences observed with small structural modifications cannot be explain with only a three-dimensional model of CYP51, adequate physicochemical parameters must be evaluated in terms of antifungal potency, bioavailability, and toxicity. Therefore, structure-activity relationship studies such as these reveal new insights for the development of future antifungal therapies.