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.

Phase I dose escalation study of NMS-1286937, an orally available Polo-Like Kinase 1 inhibitor, in patients with advanced or metastatic solid tumors.

Background Pharmacological inhibition of polo-like kinase 1 (PLK1) represents a new approach for the treatment of solid tumors. This study was aimed at determining the first cycle dose-limiting toxicities (DLTs) and related maximum tolerated dose (MTD) of NMS-1286937, a selective ATP-competitive PLK1-specific inhibitor. Secondary objectives included evaluation of its safety and pharmacokinetic (PK) profile in plasma, its antitumor activity, and its ability to modulate intracellular targets in biopsied tissue. Methods This was a Phase I, open-label, dose-escalation trial in patients with advanced/metastatic solid tumors. A treatment cycle comprised 5 days of oral administration followed by 16 days of rest, for a total of 21 days (3-week cycle). Results Nineteen of 21 enrolled patients with confirmed metastatic disease received study medication. No DLTs occurred at the first 3 dose levels (6, 12, and 24 mg/m2/day). At the subsequent dose level (48 mg/m2/day), 2 of 3 patients developed DLTs. An intermediate level of 36 mg/m2/day was therefore investigated. Four patients were treated and two DLTs were observed. After further cohort expansion, the MTD and recommended phase II dose (RP2D) were determined to be 24 mg/m2/day. Disease stabilization, observed in several patients, was the best treatment response observed. Hematological toxicity (mostly thrombocytopenia and neutropenia) was the major DLT. Systemic exposure to NMS-1286937 increased with dose and was comparable between two cycles of treatment following oral administration of the drug. Conclusions This study successfully identified the MTD and DLTs for NMS-1286937 and characterized its safety profile.

Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury.

Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRß, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRß inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative IC50s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models in vivo In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. Mol Cancer Ther; 17(3); 603-13. ©2017 AACR.

Establishment and genomic characterization of the new chordoma cell line Chor-IN-1.

Chordomas are rare, slowly growing tumors with high medical need, arising in the axial skeleton from notochord remnants. The transcription factor "brachyury" represents a distinctive molecular marker and a key oncogenic driver of chordomas. Tyrosine kinase receptors are also expressed, but so far kinase inhibitors have not shown clear clinical efficacy in chordoma patients. The need for effective therapies is extremely high, but the paucity of established chordoma cell lines has limited preclinical research. Here we describe the isolation of the new Chor-IN-1 cell line from a recurrent sacral chordoma and its characterization as compared to other chordoma cell lines. Chor-IN-1 displays genomic identity to the tumor of origin and has morphological features, growth characteristics and chromosomal abnormalities typical of chordoma, with expression of brachyury and other relevant biomarkers. Chor-IN-1 gene variants, copy number alterations and kinome gene expression were analyzed in comparison to other four chordoma cell lines, generating large scale DNA and mRNA genomic data that can be exploited for the identification of novel pharmacological targets and candidate predictive biomarkers of drug sensitivity in chordoma. The establishment of this new, well characterized chordoma cell line provides a useful tool for the identification of drugs active in chordoma.

Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.

Discovery of Entrectinib: A New 3-Aminoindazole As a Potent Anaplastic Lymphoma Kinase (ALK), c-ros Oncogene 1 Kinase (ROS1), and Pan-Tropomyosin Receptor Kinases (Pan-TRKs) inhibitor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

Sensitivity to Entrectinib Associated With a Novel LMNA-NTRK1 Gene Fusion in Metastatic Colorectal Cancer.

In metastatic colorectal cancer (CRC), actionable genetic lesions represent potential clinical opportunities. NTRK1, 2, and 3 gene rearrangements encode oncogenic fusions of the tropomyosin-receptor kinase (TRK) family of receptor tyrosine kinases in different tumor types. The TPM3-NTRK1 rearrangement is a recurring event in CRC that renders tumors sensitive to TRKA kinase inhibitors in preclinical models. We identified abnormal expression of the TRKA protein in tumor and liver metastases of a CRC patient refractory to standard therapy. Molecular characterization unveiled a novel LMNA-NTRK1 rearrangement within chromosome 1 with oncogenic potential, and the patient was treated with the pan-TRK inhibitor entrectinib, achieving partial response with decrease in hepatic target lesions from 6.8 and 8.2cm in longest diameter to 4.7 and 4.3cm, respectively. To our knowledge, this is the first clinical evidence of efficacy for therapeutic inhibition of TRKA in a solid tumor, illuminating a genomic-driven strategy to identify CRCs reliant on this oncogene to be clinically targeted with entrectinib.

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer.

BACKGROUND: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. METHODS: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. RESULTS: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1-35 of CAD with exons 20-29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. CONCLUSIONS: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.

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.

Novel pyrrole carboxamide inhibitors of JAK2 as potential treatment of myeloproliferative disorders.

Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.

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.

Mcl-1 antagonism is a potential therapeutic strategy in a subset of solid cancers.

Cancer cell survival is frequently dependent on the elevated levels of members of the Bcl-2 family of prosurvival proteins that bind to and inactivate BH3-domain pro-apoptotic cellular proteins. Small molecules that inhibit the protein-protein interactions between prosurvival and proapoptotic Bcl-2 family members (so-called "BH3 mimetics") have a potential therapeutic value, as indicated by clinical findings obtained with ABT-263 (navitoclax), a Bcl-2/Bcl-xL antagonist, and more recently with GDC-0199/ABT-199, a more selective antagonist of Bcl-2. Here, we report study results of the functional role of the prosurvival protein Mcl-1 against a panel of solid cancer cell lines representative of different tumor types. We observed silencing of Mcl-1 expression by small interfering RNAs (siRNAs) significantly reduced viability and induced apoptosis in almost 30% of cell lines tested, including lung and breast adenocarcinoma, as well as glioblastoma derived lines. Most importantly, we provide a mechanistic basis for this sensitivity by showing antagonism of Mcl-1 function with specific BH3 peptides against isolated mitochondria induces Bak oligomerization and cytochrome c release, therefore demonstrating that mitochondria from Mcl-1-sensitive cells depend on Mcl-1 for their integrity and that antagonizing Mcl-1 function is sufficient to induce apoptosis. Thus, our results lend further support for considering Mcl-1 as a therapeutic target in a number of solid cancers and support the rationale for development of small molecule BH3-mimetics antagonists of this protein.

Discovery of 2-(cyclohexylmethylamino)pyrimidines as a new class of reversible valosine containing protein inhibitors.

Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity.

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.

Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors.

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.