Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/03/15/jpet.115.231175.DC1 1521-0103/357/2/432–450$25.00 http://dx.doi.org/10.1124/jpet.115.231175 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:432–450, May 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer’s Disease and Related Tauopathies s Jane Kovalevich, Anne-Sophie Cornec, Yuemang Yao, Michael James, Alexander Crowe, Virginia M.-Y. Lee, John Q. Trojanowski, Amos B. Smith, III, Carlo Ballatore, and Kurt R. Brunden Center for Neurodegenerative Disease Research, Institute on Aging, University of Pennsylvania, Philadelphia, Pennsylvania Downloaded from (J.K., A.-S.C., Y.Y., M.J., V.M.-Y.L., J.Q.T., C.B., K.R.B.); and Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania (A.-S.C., A.B.S., C.B.) Received December 2, 2015; accepted March 14, 2016 ABSTRACT jpet.aspetjournals.org The microtubule (MT)–stabilizing protein tau disengages from response effect on markers of MT stabilization in cellular assays. MTs and forms intracellular inclusions known as neurofibrillary Moreover, these compounds induced proteasome-dependent tangles in Alzheimer’s disease and related tauopathies. Reduced degradation of a-andb-tubulin and caused altered MT mor- tau binding to MTs in tauopathies may contribute to neuronal phology in both dividing cells and neuron cultures. In contrast, dysfunction through decreased MT stabilization and disrupted a second group comprising a subset of TPD molecules (TPD1) axonal transport. Thus, the introduction of brain-penetrant MT- increased markers of stable MTs in a concentration-dependent stabilizing compounds might normalize MT dynamics and axonal manner in dividing cells and in neurons without affecting total at ASPET Journals on September 30, 2021 deficits in these disorders. We previously described a number of tubulin levels or disrupting MT architecture. Moreover, an example phenylpyrimidines and triazolopyrimidines (TPDs) that induce TPD1 compound was shown to increase MTs in a neuron culture tubulin post-translational modifications indicative of MT stabiliza- model with induced tau hyperphosphorylation and associated tion. We now further characterize the biologic properties of these MT deficits. Several TPD1 compounds were shown to be both small molecules, and our results reveal that these compounds can brain penetrant and orally bioavailable, and a TPD1 example be divided into two general classes based on the cellular response increased MT stabilization in the mouse brain, making these they evoke. One group composed of the phenylpyrimidines and compounds potential candidate therapeutics for neurodegenera- several TPD examples showed a bell-shaped concentration- tive tauopathies such as Alzheimer’s disease. Introduction Merrick et al., 1997), facilitating the formation of insoluble tau aggregates termed neurofibrillary tangles and neuropil threads ’ Alzheimer s disease (AD) and related tauopathies are charac- within the neuronal soma and dendritic processes, respectively terized by hyperphosphorylation and aggregation of the micro- (Wood et al., 1986; Lee et al., 2001b). Transgenic mouse models – tubule (MT) associated protein, tau (Lee et al., 2001b; Ballatore of tauopathy display fewer MTs, increased MT hyperdynamic- et al., 2007). Neuronal tau is normally thought to stabilize MTs ity, axonal dystrophy, and reduced fast axonal transport (FAT) and regulate axonal transport, whereas hyperphosphorylated (Zhang et al., 2005, 2012; Barten et al., 2012). Similarly, there is tau shows reduced interaction with MTs (Alonso et al., 1994; evidence of MT deficits in the AD brain (Hempen and Brion, 1996; Cash et al., 2003). Ultimately, MT abnormalities likely This research was supported by the National Institutes of Health National contribute to neuronal dysfunction, neuron loss, and cognitive de- Institute on Aging [Grant R01-AG044332] and in part by the Marian S. Ware cline, and introduction of MT-stabilizing compounds might atten- Alzheimer Foundation. dx.doi.org/10.1124/jpet.115.231175. uate these deficits in AD and related tauopathies (Ballatore et al., s This article has supplemental material available at jpet.aspetjournals.org. 2012; Brunden et al., 2012). ABBREVIATIONS: AD, Alzheimer’s disease; B/P, brain/plasma; BBB, blood–brain barrier; BCA, bicinchoninic acid; CQ, chloroquine; DIV, days in vitro; DMSO, dimethylsulfoxide; ELISA, enzyme-linked immunosorbent assay; EpoD, epothilone D; FBS, fetal bovine serum; HRP, horseradish peroxidase; LC, liquid chromatography; MG-132, benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopen- tan-2-yl]amino]-1-oxopentan-2-yl]carbamate; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MSB, microtubule-stabilization buffer;MT, microtubule; OA, okadaic acid; PBS, phosphate-buffered saline; PDL, poly(D-lysine); P-gp, P-glycoprotein; PI, protease inhibitor; PMSF, phenylmethylsulfonyl fluoride; PPD, phenylpyrimidine; RIPA, radioimmunoprecipitation assay; TBS, Tris-buffered saline; TPD, triazolopyrimidine; TSA, trichostatin A; TTI-237, 5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(2S)-1,1,1-trifluoropropan-2-yl]-[1,2,4]triazolo[1,5-a] pyrimidin-7-amine. 432 Brain-Active Microtubule Stabilizers 433 Studies performed in mouse tauopathy models support the in increasing markers of stable MTs in primary neurons. hypothesis that increasing MT stability imparts a beneficial However, select TPD analogs were identified that retained effect on neuronal function and disease course. For instance, MT-stabilizing activity without negatively affecting tubulin weekly intraperitoneal injection of the MT-stabilizing com- levels or cytoskeletal architecture. This particular subset of pound paclitaxel improved FAT and motor behavior in ad- TPDs, hereafter referred to as TPD1 compounds, also promote dition to alleviating spinal cord tau pathology in T44 tau MT stability in primary neurons and in the cortex of wild-type transgenic mice (Zhang et al., 2005). Although paclitaxel is mice. Furthermore, we demonstrate that TPD1 compounds can unable to cross the blood–brain barrier (BBB), sufficient levels rescue MT deficits in neurons treated with okadaic acid (OA), of drug likely reached the spinal cord through uptake at an in vitro model of tau hyperphosphorylation with associated neuromuscular junctions (Zhang et al., 2005). More recently, loss of MT structure. Based on their MT-stabilizing activity in our laboratory demonstrated that administration of the brain- cortical neurons and their ability to enter the brain, these TPD1 penetrant MT-stabilizing compound, epothilone D (EpoD), to compounds represent potential therapeutic candidates for the PS19 transgenic mice, which develop tau inclusions within the treatment of neurodegenerative tauopathies. forebrain (Yoshiyama et al., 2007), rescued neuronal loss and attenuated deficits in axonal architecture, FAT, and cognition, while decreasing tau neuropathology (Brunden et al., 2010; Materials and Methods Zhang et al., 2012). The salutary effects of EpoD were further Chemistry. EpoD was prepared as previously described (Lee Downloaded from confirmed in two additional tau transgenic mouse models et al., 2001a; Rivkin et al., 2004). The spectroscopic properties of the (Barten et al., 2012). Although EpoD has since progressed to compound were identical to those reported in the literature. Cevipa- clinical trials in patients with AD and represents a promising bulin (CNDR-51533) was synthesized as described (Zhang et al., drug candidate, it is the only small molecule MT-stabilizing 2007). All other TPD and PPDs tested were synthesized following agent reported to date to show beneficial effects in transgenic published procedures (Zhang et al., 2007, 2009; Lou et al., 2014) mouse models with brain tauopathy. Thus, there is a need to (Supplemental Methods). In each case, compound purity was . 95% as identify alternative brain-penetrant MT-stabilizing agents, determined by liquid chromatography (LC)–mass spectrometry (MS) jpet.aspetjournals.org ideally those that could be readily synthesized and orally and nuclear magnetic resonance analyses. administered. Determination of Plasma and Brain Drug Concentrations. All animal protocols were approved by the University of Pennsylvania Cevipabulin [TTI-237 (5-chloro-6-[2,6-difluoro-4-[3-(methylamino) Institutional Animal Care and Use Committee. Test compounds were propoxy]phenyl]-N-[(2S)-1,1,1-trifluoropropan-2-yl]-[1,2,4]triazolo administered to CD-1 or B6SJL mice. Both female and male mice were [1,5-a]pyrimidin-7-amine) CNDR-51533], a non-naturally occurr- used but were not mixed within experimental groups. The average ing triazolopyrimidine (TPD), has been reported to possess group age for each study ranged from 2.0 to 5.7 months. For standard MT-stabilizing activity and efficacy in murine tumor xenograft single time point brain and plasma determinations, mice were injected at ASPET Journals on September 30, 2021 models (Zhang et al., 2007; Beyer et al., 2008). However, intraperitoneally with a single dose of 5 mg/kg compound dissolved in cevipabulin does not cross the BBB and thus would not have dimethylsulfoxide (DMSO). For oral bioavailability