Stereoselective synthesis of 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-one derivatives as PIM kinase inhibitors inspired from marine alkaloids.

We previously demonstrated that natural product-inspired 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-ones derivatives delivered potent and selective PIM kinases inhibitors however with non-optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure-based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure-activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose-dependently promoted c-Myc degradation and appear to be promising lead compounds for further development.

Identification of unprecedented ATP-competitive choline kinase inhibitors.

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.

Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-P515.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 µM) and cellular (IC50: 0.027 µM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.

The target landscape of clinical kinase drugs.

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118): A Potent, Orally Available, and Highly Selective PARP-1 Inhibitor for Cancer Therapy.

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.

Optimization of diarylthiazole B-raf inhibitors: identification of a compound endowed with high oral antitumor activity, mitigated hERG inhibition, and low paradoxical effect.

Aberrant activation of the mitogen-activated protein kinase (MAPK)-mediated pathway components, RAF-MEK-ERK, is frequently observed in human cancers and clearly contributes to oncogenesis. As part of a project aimed at finding inhibitors of B-Raf, a key player in the MAPK cascade, we originally identified a thiazole derivative endowed with high potency and selectivity, optimal in vitro ADME properties, and good pharmacokinetic profiles in rodents, but that suffers from elevated hERG inhibitory activity. An optimization program was thus undertaken, focused mainly on the elaboration of the R(1) and R(2) groups of the scaffold. This effort ultimately led to N-(4-{2-(1-cyclopropylpiperidin-4-yl)-4-[3-(2,5-difluorobenzenesulfonylamino)-2-fluorophenyl]thiazol-5-yl}-pyridin-2-yl)acetamide (20), which maintains favorable in vitro and in vivo properties, but lacks hERG liability. Besides exhibiting potent antiproliferative activity against only cell lines bearing B-Raf V600E or V600D mutations, compound 20 also intriguingly shows a weaker "paradoxical" activation of MEK in non-mutant B-Raf cells than other known B-Raf inhibitors. It also demonstrates very good efficacy in vivo against the A375 xenograft melanoma model (tumor volume inhibition >90% at 10 mg kg(-1) ); it is therefore a suitable candidate for preclinical development.

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors.

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.

Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

Discovery and optimization of pyrrolo[1,2-a]pyrazinones leads to novel and selective inhibitors of PIM kinases.

A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors of PIM isoforms with IC50 values in the low micromolar range. The application of a rational optimization strategy, guided by the determination of the crystal structure of the complex in the kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases, representative of each family. The synthesis, structure-activity relationship studies, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore, the cellular activities including inhibition of cell growth and modulation of downstream targets are also described.

Structural insight into maternal embryonic leucine zipper kinase (MELK) conformation and inhibition toward structure-based drug design.

Maternal embryonic leucine zipper kinase (MELK) is upregulated in several types of tumor, including breast, prostate, and brain tumors. Its expression is generally associated with cell survival, cell proliferation, and resistance to apoptosis. Therefore, the potential of MELK inhibitors as therapeutic agents is recently attracting considerable interest. Here we report the first structures of MELK in complex with AMP-PNP and with nanomolar inhibitors. Our studies shed light on the role of the MELK UBA domain, provide a characterization of the kinase active site, and identify key residues for achieving high potency, laying the groundwork for structure-based drug design efforts.

Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

NMS-E973, a novel synthetic inhibitor of Hsp90 with activity against multiple models of drug resistance to targeted agents, including intracranial metastases.

PURPOSE: Recent developments of second generation Hsp90 inhibitors suggested a potential for development of this class of molecules also in tumors that have become resistant to molecular targeted agents. Disease progression is often due to brain metastases, sometimes related to insufficient drug concentrations within the brain. Our objective was to identify and characterize a novel inhibitor of Hsp90 able to cross the blood-brain barrier (BBB). EXPERIMENTAL DESIGN: Here is described a detailed biochemical and crystallographic characterization of NMS-E973. Mechanism-based anticancer activity was described in cell models, including models of resistance to kinase inhibitors. Pharmacokinetics properties were followed in plasma, tumor, liver, and brain. In vivo activity and pharmacodynamics, as well as the pharmacokinetic/pharmacodynamic relationships, were evaluated in xenografts, including an intracranially implanted melanoma model. RESULTS: NMS-E973, representative of a novel isoxazole-derived class of Hsp90 inhibitors, binds Hsp90α with subnanomolar affinity and high selectivity towards kinases, as well as other ATPases. It possesses potent antiproliferative activity against tumor cell lines and a favorable pharmacokinetic profile, with selective retention in tumor tissue and ability to cross the BBB. NMS-E973 induces tumor shrinkage in different human tumor xenografts, and is highly active in models of resistance to kinase inhibitors. Moreover, consistent with its brain penetration, NMS-E973 is active also in an intracranially implanted melanoma model. CONCLUSIONS: Overall, the efficacy profile of NMS-E973 suggests a potential for development in different clinical settings, including tumors that have become resistant to molecular targeted agents, particularly in cases of tumors which reside beyond the BBB.

PARP inhibitors in cancer therapy: an update.

INTRODUCTION: Inhibitors of the poly(ADP-ribose) polymerases (PARPs) family of proteins are currently being evaluated as potential anticancer medicines at both preclinical and clinical levels. They have the peculiarity to increase the efficacy of DNA-damaging agents and to selectively target tumor cells with specific DNA repair defects. This later development of these drugs should make it possible, in principle, to selectively target neoplastic vs healthy cells, thus realizing the Ehrlich's magic bullet concept of a personalized and tailored cure of diseases. AREAS COVERED: This review is designed to provide the readers with a brief summary and an update on PARP inhibitors in the oncology field, by covering the recent patent literature (2010 - 2012: and Questel Intellectual Property Portal [QPat] database search). EXPERT OPINION: Presently, along with a number of preclinical candidates, there are eight PARP inhibitors in the clinic as either single agents or in combination with various chemotherapy and radiotherapy regimens. The tremendous efforts underneath those results testify the high interest on the target. The investigation and understanding of the cross-reactivity among members of the PARPs family as well as a deeper knowledge of their biological functions may lead to a more profound characterization of the PARP inhibitor's profile. This, in turn, will cast additional light on this exciting approach in treating cancer.

Alkylsulfanyl-1,2,4-triazoles, a new class of allosteric valosine containing protein inhibitors. Synthesis and structure-activity relationships.

Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.

Structure-based optimization of potent PDK1 inhibitors.

In this Letter is described the structure-based design of potent dihydro-pyrazoloquinazolines as PDK1 inhibitors. Starting from low potency HTS hits with the aid of X-ray crystallography and modeling, a medicinal chemistry activity was carried out to improve potency versus PDK1 and selectivity versus CDK2 protein kinase.

Identification of potent pyrazolo[4,3-h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitors.

Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents to complement the existing therapies. An expansion of pyrazolo[4,3-h]quinazoline chemical class oriented to the development of three points of variability was undertaken leading to a series of compounds able to inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound 1, we succeeded in obtaining several compounds showing enhanced inhibitory activity both on CDKs and on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in vivo render this compound very interesting as potential antineoplastic agent.

Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing.

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Identification of N,1,4,4-tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a potent, orally available cyclin dependent kinase inhibitor.

The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a panel of serine-threonine and tyrosine kinases, new analogues were synthesized. Enhancement in selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of the physical properties and pharmacokinetic profile led to the identification of highly potent and orally available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments, was identified as a drug candidate for further development. Compound 28 is currently undergoing phase I and phase II clinical trials.

Crystal structure of the T315I Abl mutant in complex with the aurora kinases inhibitor PHA-739358.

Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine.