Rapid identification of a novel phosphodiesterase 7B tracer for receptor occupancy studies using LC─MS/MS.

Phosphodiesterase 7B (PDE7B) inhibition has been considered as a therapeutic target for the treatment of several neurological disorders. Currently, there are no radio-labeled tracers available to determine receptor occupancy (RO) of this target. Developing such a tracer could greatly facilitate the identification of viable PDE7B inhibitors. In the current study, a liquid chromatography tandem mass spectrometry (LC─MS/MS) method was utilized to evaluate the brain distribution of unlabeled tracer candidates following intravenous micro-dosing. This novel approach resulted in an accelerated identification of a potential novel RO tracer for PDE7B. The identified molecule, Compound 30, showed reasonable target-tissue specificity (striatum/cerebellum ratio of 2.2) and suitable uptake (0.25% of the injected dose/g brain tissue) as demonstrated in rats dosed with the unlabeled compound. Compound 30 was subsequently labeled with tritium (3H). In vitro characterization of 3H-Compound 30 demonstrated that this compound possessed a high target affinity with a subnanomolar Kd (0.8 nM) and a Bmax of 58 fmol/mg of protein using rat brain homogenate. Intravenous microdosing of 3H-Compound 30 showed preferential binding in the rat striatum, consistent with the mRNA distribution of PDE7B. In vitro displacement study with other structurally distinct PDE7B target-specific inhibitors using rat brain homogenate indicated that 3H-Compound 30 is an ideal tracer for Ki analysis. This is the first report of a preclinical tracer for PDE7B. With further characterization, Compound 30 may ultimately show the appropriate properties required to be further developed as a PDE7B PET ligand for clinical studies.

Identification of 5-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-Carboxamides as Novel and Selective Monoamine Oxidase B Inhibitors Used to Improve Memory and Cognition.

Initial work in Drosophila and mice demonstrated that the transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is a master control gene for memory formation. The relationship between CREB and memory has also been found to be true in other species, including aplysia and rats. It is thus well-established that CREB activation plays a central role in memory enhancement and that CREB is activated during memory formation. On the basis of these findings, a phenotypic high-throughput screening campaign utilizing a CRE-luciferase (CRE-Luci) SK-N-MC cell line was performed to identify compounds that enhance transcriptional activation of the CRE promoter with a suboptimal dose of forskolin. A number of small-molecule hits of unknown mechanisms of action were identified in the screening campaign, including HT-0411. Follow-up studies suggested that the CREB activation by HT-0411 is attributed to its specific and selective inhibition of monoamine oxidase B (MAO-B). Further, HT-0411 was shown to improve 24 h memory in rodents in a contextual fear conditioning model. This report describes the lead optimization of a series of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl) thiophene-2-carboxamides that were identified as novel, potent, and selective inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral evaluations of this and other related analogue series identified a number of potential clinical development candidates; ultimately, compound 8f was identified as a candidate molecule with high selectivity toward MAO-B (29-56 nM) over MAO-A (19% inhibition at a screening concentration of 50 µM), an excellent profile against a panel of other enzymes and receptors, good pharmacokinetic properties in rodents and dogs, and efficacy in multiple rodent memory models.

Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone-targeted lead compounds, successfully achieving in vivo proof-of-concept and providing the framework for the next-generation molecules that have further advanced, in terms of preclinical studies, for the treatment of osteoporosis and related bone diseases, including osteolytic bone metastases.

Regulation of gene expression by synthetic dimerizers with novel specificity.

New synthetic chemical inducers of dimerization, comprising homodimeric FKBP ligands with engineered specificity for the designed point mutant F36V, have been evaluated for inducing targeted gene expression in mammalian cells. Structure-activity studies indicated that high-affinity dimerizers such as AP1903 are ineffective, perhaps due to kinetic trapping of non-productive dimers, whereas lower-affinity molecules, exemplified by AP1889 and AP1966, potently activate transcription.

Bone-targeted Src kinase inhibitors: novel pyrrolo- and pyrazolopyrimidine analogues.

Src tyrosine kinase is a therapeutic target for bone diseases that has been validated by gene knockout studies. Furthermore, in vitro cellular studies implicate that Src has a positive regulatory role in osteoclasts and a negative regulatory role in osteoblasts. The potential use of Src inhibitors for osteoporosis therapy has been previously shown by novel bone-targeted ligands of the Src SH2 (e.g., AP22408) and non-bone-targeted, ATP-based inhibitors of Src kinase. Significant to this study, compounds 2-12 exemplify novel analogues of known pyrrolopyrimidine and pyrazolopyrimidine template-based Src kinase inhibitors that incorporate bone-targeting group modifications designed to provide tissue (bone) selectivity and diminished side effects. Accordingly, we report here the structure-based design, synthetic chemistry and biological testing of these compounds and proof-of-concept studies thereof.

Bone-targeted 2,6,9-trisubstituted purines: novel inhibitors of Src tyrosine kinase for the treatment of bone diseases.

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.

Bone-targeted pyrido[2,3-d]pyrimidin-7-ones: potent inhibitors of Src tyrosine kinase as novel antiresorptive agents.

The design of bone-targeted pyrido[2,3-d]pyrimidin-7-ones as Src tyrosine kinase inhibitors is described. Leveraging SAR from known compounds and using structure-based methods, we were able to rapidly incorporate bone binding components, which maintained, and even increased potency against the target enzyme. Compound 4 displayed a high affinity for hydroxyapatite, a major constituent of bone, and demonstrated antiresoprtive activity in our cell-based assay.