Supplementary Data from JNJ-26481585, a Novel Second-Generation Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity

Abstract The fibroblast growth factor receptor (FGFR) tyrosine kinase family members, FGFR1, 2, 3 and 4, have roles in a variety of key cellular processes, including proliferation, migration, survival, and differentiation1. Aberrant activation of FGFRs through mutation, amplification, chromosomal translocation, and ligand up-regulation being strongly implicated in oncogenic signalling in many tumour types, has triggered efforts to identify selective FGFR inhibitors. As a result, several potent FGFR kinase inhibitors are currently being evaluated in clinical studies across many tumor types, including non-small cell lung, breast and bladder cancers. We have designed novel 1,5 and 1,7-naphthyridine derivatives that are potent kinase inhibitors of all FGFR family members in enzymatic and cellular systems. Initial hits were further optimized to increase potency and ADME properties leading to identification of a novel 1,5-naphthyridine-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. In vivo screening using an FGFR3-driven xenograft model revealed efficacious compounds that could be explored further as antitumoral agents. This report represents the first disclosure of the structure-activity relationship and synthesis pathway of novel naphthyridine chemical series displaying nanomolar affinity for FGFRs1, 2, 3 and 4. 1 Dieci M. V., Ardenos M., Andre F., Soria J.C. Cancer Discovery. 2013, 3(3) 264-279. Citation Format: Patrick R. Angibaud, Michel Obringer, Julien Marin, Matthieu Jeanty, Norbert Esser, Ron Gilissen, Peter King, Lieven Meerpoel, Olivier Querolle, David C. Rees, Bruno Roux, Gordon Saxty, Tinne Verhulst, Berthold Wroblowski, Christopher C. Murray, Jorge Vialard. Identification of naphthyridines as potent inhibitors of fibroblast growth factor receptor kinase family. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2015-3641

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Fibroblast growth factors (FGFs) and their receptors (FGFR1 through 4) regulate a variety of key cellular processes, including proliferation, migration, survival, and differentiationa. Aberrant activation of FGF/FGFR is strongly implicated in oncogenic signalling in many tumor types. This has stimulated the development of a number of FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We conducted a fragment screening campaign and this resulted in identification of a 6-aminoquinoxalinyl fragment with a binding affinity in the micromolar range. Structure-guided medicinal chemistry led to the identification of a novel quinoxaline-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. Further optimisation resulted in the generation of JNJ-42756493, a compound with favourable drug-like properties that demonstrated strong anti-tumoral activity in a FGFR2-dependent SNU-16 human gastric carcinoma xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathway of the JNJ-42756493 series and illustrates how a fragment-based drug discovery approach has been efficiently used to discover FGFR1-4 inhibitors with nanomolar affinity. aTurner, N. and Grose, R. Nat. Rev. Cancer, 2010, 10, 116-129. Citation Format: Patrick R. Angibaud, Laurence Mevellec, Gordon Saxty, Christophe Adelinet, Rhalid Akkari, Valerio Berdini, Pascal Bonnet, Marine Bourgeois, Xavier Bourdrez, Anne Cleasby, Helene Colombel, Imre Csoka, Werner Embrechts, Eddy Freyne, Ronaldus Gilissen, Eleonora Jovcheva, Peter King, Jean Lacrampe, Delphine Lardeau, Yannick Ligny, Steve Mcclue, Lieven Meerpoel, David R. Newell, Martin Page, Alexandra Papanikos, Elisabeth Pasquier, Isabelle Pilatte, Virginie Poncelet, Olivier Querolle, David C. Rees, Sharna Rich, Bruno Roux, Elodie Sement, Yvan Simonnet, Matthew Squires, Virginie Tronel, Tinne Verhulst, Jorge Vialard, Marc Willems, Steven J. Woodhead, Berthold Wroblowski, Christopher W. Murray, Timothy Perera. Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2014-4748

The trapping mechanism of hydrogen in niobium has been investigated within the molec­ ular dynamics approach. Simulations of the diffusion process of this impurity were performed which require the knowledge of the interatomic potential. N b N b interaction was described by an N body potential. The potential parameters were adjusted with respect to static and dynamic properties of Nb crystal. Nb H interaction was represented by a two body poten­ tial. The Arrhenius diagram of the H diffusion coefficient achieved by molecular dynamics in the single crystal case provides too small an activation energy in comparison with exper­ imental results. However, molecular dynamics simulations indicate a large increase of these values in the presence of defects. It is only at around 1000 K that the diffusion of hydrogen is not altered by defects. This conclusion confirms the experimental results concerning a good characteristic of superconducting cavities after thermal treatments. t Soumis a : Phys. Rev. C Molecular dynamics simulation of hydrogen diffusion in niobium. Influence of point defects \ ) B. Roux, H. Jafi"rezic, A. Chevarier, N. Chevarier InlJtitut de Physique Nucleaire de Lyon, IN2P3GNRS et Universite Glaude Bernard 43, Bd du 11 Novembre 1918, 69622 Villeurbanne Gedez, France PACS nurnber(s) : 61.72 Ss, 66.30 Jt

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Abstract Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a second-generation class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy. Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors. Results:In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L). Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):684151)

Based on the structure of R115777 (tipifarnib, Zarnestra), a series of farnesyltransferase inhibitors have been synthesized by modification of the 2-quinolinone motif and transposition of the 4-chlorophenyl ring to the imidazole or its replacement by 5-membered rings. This has yielded a novel series of potent farnesyltransferase inhibitors.

A264 Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of haematological malignancies, resulting in the recent approval of Vorinostat (SAHA) for the treatment of cutaneous T-cell lymphoma. We previously have identified R306465 as highly potent class-I selective HDAC inhibitor, showing oral anti-tumor activity in human xenograft-bearing athymic nude mice. Pharmacodynamic studies revealed that, like most HDAC inhibitors in clinical development, R306465 only transiently induces histone acetylation in tumor tissue. In order to identify novel HDAC with superior pharmacodynamic properties, we developed an animal model allowing non-invasive real-time evaluation of the response to HDAC inhibitors. Human A2780 ovarian carcinoma cells were engineered to express ZsGreen fluorescent protein under control of the p21waf1, cip1 promoter. Induction of fluorescence protein in vivo was found to accurately predict long-term anti-tumor activity of HDAC inhibitors. (Belien et al., Mol. Cancer Ther., 2006). In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585, a novel “second generation” oral pan-HDAC inhibitor with broad-spectrum preclinical anti-tumoral activity. Pharmacodynamic analysis showed that once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation in human HCT116 colon tumors, resulting in complete tumor growth inhibition. Similarly, JNJ-26481585 completely inhibited tumor growth in both human ER-/PR-/Her2-negative MDA-MB-231 breast carcinoma, and also human K-ras mutant A549 non small cell lung carcinoma (NSCLC) tumor models; with observed efficacy greater than Paclitaxel. JNJ-26481585, was shown to be a potent pan-HDAC inhibitor (HDAC1, IC50 0.16 nM) and demonstrated both acetylation of HDAC1 substrates (histones) and HDAC6 substrates (Tubulin) in A2780 cells. JNJ-26481585 inhibited tumor cell proliferation (IC50 values 2 to 144 nM) and induced massive apoptosis in all tumor cell lines tested, irrespective of p53 or Ras mutational status. Patient-derived, adriamycin-resistant, breast carcinoma cells and Paclitaxel-resistant NSCLC cells were also highly sensitive to JNJ-26481585. The potent anti-tumoral activity as a single agent in preclinical models combined with its favourable pharmacodynamic profile, makes JNJ-26481585 a promising “second generation” HDAC inhibitor with potential applicability in a broad spectrum of human solid and haematological malignancies.

Tandem conjugate addition of homochiral lithium N-benzyl-N-(alpha-methyl-p-methoxybenzyl)amide to tert-butyl cinnamate and enolate trapping with (TsSBu)-Bu-t proceeds with high diastercoselectivity to give a homochiral anti-alpha-tert-butylthio-beta-amino ester. Stepwise deprotection gives the corresponding free alpha-tert-butylthio-beta-amino acid without epimerisation. Tandem conjugate addition of homochiral lithium N-allyl-N-(alpha-methylbenzyl)amide to tert-butyl cinnamate and enolate trapping with TsS'Bu followed by conversion of the S-tert-butyl group to a disulphide, and reduction with Lalancette's reagent generates polysubstituted thiomorpholine derivatives. (c) 2006 Elsevier Ltd. All rights reserved.

A series of pyrimidyl-5-hydroxamic acids was prepared for evaluation as inhibitors of histone deacetylase (HDAC). Amino-2-pyrimidinyl can be used as a linker to provide HDAC inhibitors of good enzymatic potency.