Sunday, October 4, 2015

Entrectinib | Multi-Kinase Inhibitor | pan-TRK Inhibitor | ALK Inhibitor | ROS-1 Inhibitor | Orphan Drug

Entrectinib [N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)benzamide] is an oral small molecule inhibitor of TrkA (IC50 = 1 nM), TrkB (IC50 = 2, 3 nM) and TrkC (IC50 = 5 nM), as well as ROS1 (IC50 = 7 nM) and ALK (IC50 = 12 nM), with high potency and selectivity. Entrectinib has demonstrated potent pharmacological activity in preclinical studies and has the potential to be first-in-class against the Trk family of kinases [1, 5].
Entrectinib: 2D and 3D Structure

Entrectinib was being developed as Anaplastic Lymphoma Kinase (ALK) inhibitor where studies showed that it induces tumor regression in mouse models of NPM-ALK–driven lymphoma and EML4-ALK-driven NSCLC; it also has activity against the Crizotinib-resistant ALK mutants L1196M and C1156Y. Entrectinib also crosses the blood-brain barrier, inhibits tumor growth, and prolongs survival in mice with intracranially injected NCI-H2228 EML4-ALK cells [2]. 

Due to the structural similarity between ALK and ROS1 kinases, the activity of Entrectinib on ROS1 was evaluated in biochemical and cellular assays. The compound was found to be a potent, ATP competitive inhibitor of ROS1 (IC50 = 7 nM). This biochemical potency translates into inhibition of the proliferation of HCC-78, a NSCLC cell line characterized by ROS1-driven activated signaling, due to the presence of the SCL34A2-ROS1 fusion gene. To further characterize the compound in cells, an activated form of ROS1 was expressed in Ba/F3 cells generating an IL3-independent, ROS1-driven tumor model. NMS-E628 strongly inhibits the proliferation of Ba/F3-ROS1 cells in vitro with confirmed dose-dependent decrease of ROS1 phosphorylation. When tested in vivo, the compound was found to induce complete tumor regression after 10-day continuous treatment in nude mice bearing established Ba/F3-ROS1 tumors [3].
Important timelines in the history of Entrectinib:
a: In Nov 2009, Nerviano Medical Sciences revealed NMS-E628, a small molecule inhibitor of anaplastic lymphoma kinase with antitumor efficacy in ALK-dependent lymphoma and non-small cell lung cancer models at AACR-NCI-EORTC International Conference. 
b: In Nov 2011, Nerviano Medical Sciences revealed NMS-E628 also potently inhibits ROS1 and induces tumor regression in ROS-driven models at American Association Cancer Society Annual Meeting.
c: In Nov 2013 Nerviano Medical Sciences signed a license agreement with Ignyta Inc granting the American biotech company exclusive global development and marketing rights to RXDX-101 and RXDX-102. 
d: In 2014 at ASCO Annual Meeting Nerviano Medical Sciences/Ignyta Inc added pan-TRK activity to the pre-existing ALK and ROS1 activities.
Dosages and Approvals:

On December 29, 2014, Ignyta, Incorporated announced that the FDA had granted Orphan Drug status and a Rare Pediatric Disease designation for Entrectinib for the treatment of neuroblastoma.
On Feburary 05, 2015 Ignyta announced that the Food and Drug Administation (FDA) has granted Orphan Drug designation to Entrectinib for the treatment of TrkA-positive, TrkB-positive, TrkC-positive, ROS1-positive and ALK-positive non-small cell lung cancer (NSCLC).
Entrectinib is currently in Phase I/II clinical trials with activity against TrkA as well as TrkB, TrkC, ALK and ROS1 proteins (encoded by NTRK1, NTRK2, NTRK3, ALK and ROS1 genes, respectively). Entrectinib is indicated for patients with advanced, refractory, relapsed or metastatic solid tumors who have exhausted standard treatment options and who exhibit molecular alterations in any of the target genes. 
The activity of Entrectinib is as follows:
IC50 (TRKA enzyme assay) = 1 nM
IC50 (TRKB enzyme assay) = 2 nM; 3 nM [5]
IC50 (TRKC enzyme assay) = 5 nM
IC50 (ROS1 enzyme assay) = 7 nM
IC50 (ALK enzyme assay) = 12 nM; 55 nM [4]
IC50 (IGF1R enzyme assay) = 263 nM
IC50 (AURKA enzyme assay) = 338 nM

Common Name: Entrectinib
Synonyms: RXDX-101; RXDX 101; RXDX101; NMS-E628; NMSE628; NMS E628
IUPAC Name: N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)benzamide
CAS Number: 1108743-60-7
Mechanism of Action: Kinase Inhibitor; Multi-Kinase Inhibitor; pan-TRK Kinase Inhibitor; ALK Kinase Inhibitor; ROS1 Kinase Inhibitor
Indication: Various Cancers; Anti-tumor Agents
Development Stage: Phase II
Company: Nerviano Medical Sciences/Ignyta Inc

The Trk family of kinases, which include TrkA, TrkB and TrkC, are high affinity receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activities of Trk family members due to chromosome rearrangements, gene mutations, splicing variants and overexpression have been shown to be associated with tumorigenesis and poor prognosis in a number of cancer types. Particularly, several chromosomal rearrangements involving TrkA have been reported in lung, colorectal, papillary thyroid, glioblastoma, melanoma and other cancers, and are believed to be the key oncogenic driver in these tumors. Therefore oncogenic Trk may represent a promising therapeutic target in Trk-driven tumors.
In KM-12, a human colorectal cancer cell line driven by constitutively active TrkA fusion TPM3-NTRK1, Entrectinib exhibited in vitro anti-proliferative activity with an IC50 of 17 nM, accompanied by inhibition of TrkA phosphorylation and concomitant inactivation of downstream effectors, PLCgamma1, AKT and ERK, as well as cell cycle arrest and apoptosis. In mice bearing KM- 12 xenografts, treatment with Entrectinib resulted in tumor regression and durable stasis under either intermittent or continuous dosing regimens, accompanied by sustained intratumoral inhibition of phospho-TrkA and PLCgamma1. In these studies, Entrectinib was well tolerated during the course of treatment [1].

Entrectinib Synthesis

J Med Chem 201659(7), 3392-3408: The article reports synthesis of Entrectinib and its analogues. Pre-clinical data is also reported.


Final Synthesis:


1H NMR (Estimated) for Entrectinib

Experimental: 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.29-1.41 (m, 2H) 1.89-1.97 (m, 2H) 2.24 (s, 3 H) 2.41-2.48 (m, 4H) 3.23-3.29 (m, 4 H) 3.49 (ddd, J = 11.7, 10.2, 2.3 Hz, 2 H) 3.62-3.72 (m, 1 H) 3.80(ddd, J = 11.7, 3.8, 3.8 Hz, 2 H) 4.04 (s, 2 H) 6.13 (d, J = 2.1 Hz, 1 H) 6.23 (dd, J = 9.0, 2.2 Hz, 1 H) 6.93-7.04 (m, 3 H) 7.25 (dd, J = 8.7, 1.5 Hz, 1 H) 7.40 (d, J = 8.7 Hz, 1 H) 7.48 (s, 1 H) 7.80 (d, J = 9.0 Hz, 1 H) 8.29 (d, J = 7.6 Hz, 1 H) 10.07 (s, 1 H) 12.62 (s, 1 H).
1. Anderson, D.; et. al. Inhibition of Trk-driven tumors by the pan-Trk inhibitor RXDX-101. Euro J Cancer 201450(supp 6),101. (FMO only)
2. Ardini, E.; et. al. Abstract A243: Characterization of NMS-E628, a small molecule inhibitor of anaplastic lymphoma kinase with antitumor efficacy in ALK-dependent lymphoma and non-small cell lung cancer models Mol Cancer Ther 20098(12 suppl), A244. (FMO only)
3. Ardini, E.; et. al. Abstract 2092: The ALK inhibitor NMS-E628 also potently inhibits ROS1 and induces tumor regression in ROS-driven models . Cancer Res 201373(8 suppl), 2092. (free copy)
4. Lombardi, B. A.; et. al. Substituted indazole derivatives active as kinase inhibitors. WO2009013126A1
5. Menichincheri, M.; et. al. 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. J Med Chem 2016, 59(7), 3392-3408. (FMO only)