The following are the supplementary data related to

The following are the supplementary data related to this article.
Introduction Target therapies have achieved significant gains in the fight against cancer, however, they are still a long way from providing generally curative treatments for the majority of cancers. Targeted agents can be used with each other or with traditional chemo- or radiotherapy [1]. However more research is required to develop new combinations of targeted agents by finding pathways which can be inhibited together and lead to better and more durable responses in patients [2]. Targeting the JAK-STAT pathway with JAK kinase inhibitors, such as ruxolitinib [3], has provided value for patients with myeloproliferative neoplasms, conditions which can lead to leukemia [4]. Likewise, inhibitors of the histone deacetylase (HDAC) pathway have found value in T-cell lymphomas [5]. In an exciting new way forward, we propose to combine the targeting of JAK kinases and HDACs with a single, dual inhibitor. Clinical trials of ruxolitinib with panobinostat support the notion that blockade of these pathways may be useful in treating solid tumors or hematological malignancies [6]. Recently we have reported new dual JAK-HDAC inhibitors, such as 1 and 2 (Fig. 1), based on the JAK inhibitor templates pacritinib and ruxolitinib, respectively [7,8]. In this latest study we report the discovery of dual JAK-HDAC agents based on XL019 (3). Key discoveries with this series are the very potent JAK and HDAC1/6 activities which have led to very potent cellular activites. Compound 3 is a potent and selective JAK2 inhibitor with IC50 of 2.2 nM, exhibiting >50-fold selectivity over JAK1, JAK3 and TYK2. It has been studied in a phase 1 clinical trial but was forced to terminate due to CNS side effects [9]. In our study, CNS penetration is not expected due to the higher molecular weight and polarity of the compounds. Importantly, Neuregulin/Heregulin-1β (NRG-1β/HRG-1β), human recombinant protein 3 has inherent selectivity for JAK2 over the other JAK family enzymes, unlike ruxolitinib which has a JAK1/2 profile, possibly a factor in its safety profile where patients with low platelet counts cannot be treated [10]. In this report we demonstrate the design and synthesis of novel JAK-HDAC dual inhibitors based on 3 and the marketed HDAC inhibitor vorinostat (4).
Results and discussion
Experimental section All reagents purchased from commercial sources (Sigma-Aldrich, Combi-blocks, Merck and Alpha chemical) were of the highest purity grade available and were used without further purification. Commercially available AR grade solvents or anhydrous solvents packed in resealable bottles were used as received. Only ethyl acetate was distilled before use. All reaction temperatures stated in the procedures are external bath temperatures. Non-aqueous reactions were performed under a positive pressure of nitrogen in oven-dried glassware. Yields refer to chromatographically and spectroscopically homogeneous materials, unless otherwise stated. Reaction progress was monitored by analytical thin layer chromatography (TLC) with 0.25 mm Merck pre-coated silica gel plates (60F-254) using UV light (254 nm) as visualizing agent, or potassium permanganate solutions as a developing stain. All products were purified by flash chromatography using silica gel (Merck 60–200 mesh, purchased from SiliCycle or Merck) as the stationary phase or purified by crystallization. The structures of synthesized compounds were verified by 1H NMR, 13C NMR, and mass spectrometry. 1H (400 MHz) and 13C (101 MHz) NMR spectra were recorded in deuterated solvents (purchased from Cambridge Isotopes and used without further purification) on a Bruker Avance III 400 (Ultrashield Plus) spectrometer. Chemical shifts are assigned and reported in ppm (δ); corresponding to reference standard solvents CDCl3 (7.26 ppm), DMSO‑d6 (2.50 ppm) and CD3OD (3.31 ppm). Multiplicities are reported as singlet (s), doublet (d), triplet (t), multiplet (m), and broad singlet (bs). Coupling constants (J) are reported in Hz. Mass spectra were recorded on a Liquid Chromatography-Mass Spectrometry (LCMS) system (Agilent 1260 Infinity quaternary LC system with 150 mm column and Agilent 6130 quadrupole mass spectrometer). The unit of molecular weight reported is g/mol. Analytical HPLC purity was determined using an Agilent technologies 1200 series instrument with Zorbax SB-C18 4.6 × 250 mm 5 μm column at 254 nM, using gradient MeCN/H2O 5:95 to 95:5 over 20 min. Preparative HPLC was performed using a Gilson GX281 with 21.2 × 250 mm 7 μm, Hypersil™ BDS C18 column to purify polar compounds. All purity profiles were recorded with gradient MeCN/H2O 5:95 to 95:5 at 0.5 mL per minute over 20 min. All the structures and their IUPAC names have been generated using ChemBioDraw Ultra 12.0.