Yuan Yuan Ling

Synthesis, Biological Evaluation, X-ray Structure, and Pharmacokinetics of Aminopyrimidine c-jun-N-terminal Kinase (JNK) Inhibitors

Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors with good cell potency and good brain penetration properties. The structure-activity relationships (SAR) around a series of aminopyrimidines were evaluated utilizing biochemical and cell-based assays to measure JNK inhibition and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC(50) values < 100 nM. Moreover, compound 9l showed an IC(50) = 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rats along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.

Structure-activity relationships and X-ray structures describing the selectivity of aminopyrazole inhibitors for c-Jun N-terminal kinase 3 (JNK3) over p38.

c-Jun N-terminal kinase 3α1 (JNK3α1) is a mitogen-activated protein kinase family member expressed primarily in the brain that phosphorylates protein transcription factors, including c-Jun and activating transcription factor-2 (ATF-2) upon activation by a variety of stress-based stimuli. In this study, we set out to design JNK3-selective inhibitors that had >1000-fold selectivity over p38, another closely related mitogen-activated protein kinase family member. To do this we employed traditional medicinal chemistry principles coupled with structure-based drug design. Inhibitors from the aminopyrazole class, such as SR-3576, were found to be very potent JNK3 inhibitors (IC50 = 7 nm) with >2800-fold selectivity over p38 (p38 IC50 > 20 μm) and had cell-based potency of ∼1 μm. In contrast, indazole-based inhibitors exemplified by SR-3737 were potent inhibitors of both JNK3 (IC50 = 12 nm) and p38 (IC50 = 3 nm). These selectivity differences between the indazole class and the aminopyrazole class came despite nearly identical binding (root mean square deviation = 0.33 Å) of these two compound classes to JNK3. The structural features within the compounds giving rise to the selectivity in the aminopyrazole class include the highly planar nature of the pyrazole, N-linked phenyl structures, which better occupied the smaller active site of JNK3 compared with the larger active site of p38.

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors.

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) Inhibitors

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC(50)=40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.

Synthesis and SAR of 4-(pyrazol-3-yl)-pyridines as novel c-jun N-terminal kinase inhibitors.

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK) inhibitors is described. The development of the 4-(pyrazol-3-yl)-pyridine series was discovered from an earlier pyrimidine series of JNK inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.

Synthesis and SAR of novel isoxazoles as potent c-jun N-terminal kinase (JNK) inhibitors

The design and synthesis of isoxazole 3 is described, a potent JNK inhibitor with two fold selectivity over p38. Optimization of this scaffold led to compounds 27 and 28 which showed greatly improved selectivity over p38 by maintaining the JNK3 potency of compound 3. Extensive SAR studies will be described as well as preliminary in vivo data of the two lead compounds.

Synthesis and SAR of 2,4-diaminopyrimidines as potent c-jun N-terminal kinase inhibitors

The design and synthesis of a potent series of c-jun N-terminal kinase (JNK2) inhibitors is described. The development and optimization of the 2,4-diaminopyrimidines series was carried out from an earlier in-house kinase inhibitor program. Through the optimization of the scaffold 2, several cell potent compounds with good in vivo profiles were discovered.

Identification of an EGFRvIII-JNK2-HGF/c-Met-Signaling Axis Required for Intercellular Crosstalk and Glioblastoma Multiforme Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor–c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.

Abstract 1810: Analysis of EGFRvIII mediated responses driven by JNK2 in Glioblastoma Multiforme.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The most malignant central nervous system cancer, Glioblastoma Mulitforme (GBM), accounts for the majority of primary brain cancer-related deaths (Porter, McCarthy et al. 2010) and has an average post-diagnostic survival rate of just over 14 months (Behin, Hoang-Xuan et al. 2003; Stupp, Mason et al. 2005; Louis, Ohgaki et al. 2007). The epidermal growth factor receptor (EGFR) (Wong, Ruppert et al. 1992; Mukasa, Wykosky et al. 2010) has been shown to play central roles in GBM tumorigenesis and therapeutic resistance and reports have indicated that ∼50% of all GBMs display aberrant activation of EGFR and of these ∼50% also express a ligand-independent, constitutively active mutant, EGFRvIII (Nishikawa, Ji et al. 1994) that is associated with increased tumorigenecity and mortality (Heimberger, Hlatky et al. 2005; Heimberger, Suki et al. 2005). In addition, several reports have indicated that the c-Jun N-terminal kinase 2 (JNK2) is activated in the majority of GBM tumors when compared to normal brain tissue (Antonyak, Kenyon et al. 2002; Tsuiki, Tnani et al. 2003; Cui, Han et al. 2006) and JNK2 activation strongly correlates with both GBM histological grade and expression of EGFR (Li, Wang et al. 2008). Current therapies specifically targeting the EGFR have limited success and the universal resurgence of tumors post-treatment occurs due to infiltrating tumor cells that escape initial surgery and exhibit profound resistance to irradiation and current chemotherapy treatments (Claes, Idema et al. 2007). Therefore, the identification of novel tractable targets for improved therapeutics and the molecular and genetic profiling of GBMs continues to be a necessity. To this end we have compared the gene expression profiles of U87 GBM cells with cells expressing EGFRvIII (U87EGFRvIII) and specifically studied genes modulated by JNK2 using microarray analysis comparing U87EGFRvIII cells expressing either short hairpin RNA directed against JNK2 (U87EGFRvIIIshJNK2) or a non-silencing control (U87EGFRvIIIshNS). Gene expression profiles revealed 390 EGFRvIII-induced genes that were significantly associated with targeted loss of JNK2 expression by a minimum of 2 fold on comparison to non-silencing controls. JNK2 modulated gene clusters related to cell movement were revealed with Ingenuity Pathway Analysis (IPA) and included interesting genes such as HGF, MMP7, FGF10, PDGFRA and IGFBP5. Ultimately, these studies, allowed us to further understand the molecular mechanisms driving EGFRvIII-induced tumorigenesis and such knowledge will ultimately enhance our ability to develop an efficacious GBM therapy that can increase the effectiveness of clinically approved drugs. Citation Format: Vanessa Saunders, Mohammad Fallahi-Sichani, Yuan Yuan Ling, Derek Duckett. Analysis of EGFRvIII mediated responses driven by JNK2 in Glioblastoma Multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2013-1810

Abstract 1119: Interplay between EGFRvIII and c-Met in glioblastoma multiforme

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastoma multiforme (GBM) is a highly invasive and lethal brain tumor which is associated with a high recurrence rate after surgery and an increased resistance to irradiation and chemotherapies. Consequently, patients suffering from GBM have a poor prognosis and a high mortality rate with an average life expectancy of less than 15 months. Key signaling components, especially receptor tyrosine kinases (RTKs), are aberrantly activated in GBM. The most frequent is the epidermal growth factor receptor (EGFR) and here the most commonly activated form, EGFRvIII, augments GBM invasiveness, promotes therapy resistance and connotes poor prognosis. There are five EGFR approved inhibitors and interestingly clinical studies have indicated that targeting the activity of the EGFR in association with conventional GBM therapies does little to inhibit tumor growth. The underlying mechanistic reasoning for this poor response are not understood and may reflect EGFR-induced trans-activation of other RTKs such as the hepatocyte growth factor receptor (c-Met), or uncoupling of EGFR signaling from its downstream mediators. Several studies have indicated that EGFR can trans-activate c-Met and consequently the dual targeting of both EGFR and c-Met significantly decreases the growth of EGFRvIII expressing tumor xenografts, indicating that inhibiting both EGFR and c-Met activity may prove beneficial in GBM therapy. Currently, the only known ligand for c-Met is the hepatocyte growth factor (HGF). Thus we hypothesize that EGFR induced HGF production is critical for the increased tumorgenicity observed in EGFRvIII expressing GBM cells. Using GBM cell lines we show that c-Met activation is increased in GBM cells that both stably and transiently express EGFRvIII. Furthermore, conditioned media taken from GBM cells expressing EGFRvIII can activate c-Met signaling in GBM cells that lack EGFRvIII. In both instances we see that the kinase activity of the EGFRvIII partially contributes to the production of HGF and the activation of c-Met in GBM cells. Aberrant c-Jun terminal kinase signaling (JNK) can be detected in the majority of patient GBMs. Using GBM cells stably lacking JNK2α2 we identify JNK2α2 signaling as a potential mechanism behind EGFRvIII activation of c-Met via the production of HGF in EGFRvIII GBM expressing cells. Taken together our data describes a novel role for JNK2α2 in mediating GBM tumorgenicity through the regulation of HGF production and the subsequent activation of c-Met signaling in GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1119. doi:10.1158/1538-7445.AM2011-1119