Nigel Brooks

Small-molecule androgen receptor downregulators as an approach to treatment of advanced prostate cancer

Chemical starting points were investigated for downregulation of the androgen receptor as an approach to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a designed for intramuscular administration showed insufficient cellular potency, a medicinal chemistry program derived from a novel androgen receptor ligand 8a led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), for which high plasma levels following oral administration in a preclinical model compensate for moderate cellular potency.

Inhibitors of the tyrosine kinase EphB4. Part 3: identification of non-benzodioxole-based kinase inhibitors.

Starting from the initial bis-anilinopyrimidine 1, good potency against EphB4 was retained when benzodioxole at C-4 was replaced by an indazole. The key interactions of the indazole with the protein were characterised by crystallographic studies. Further optimisation led to compound 20, a potent inhibitor of the EphB4 and Src kinases with good pharmacokinetics in various preclinical species and high fraction unbound in plasma. Compound 20 may be used as a tool for evaluating the potential of EphB4 kinase inhibitors in vivo.

Inhibitors of the tyrosine kinase EphB4. Part 4: Discovery and optimization of a benzylic alcohol series

Optimization of our bis-anilino-pyrimidine series of EphB4 kinase inhibitors led to the discovery of compound 12 which incorporates a key m-hydroxymethylene group on the C4 aniline. 12 displays a good kinase selectivity profile, good physical properties and pharmacokinetic parameters, suggesting it is a suitable candidate to investigate the therapeutic potential of EphB4 kinase inhibitors.

Abstract 3568: Characterization of AZD4547: An orally bioavailable, potent and selective inhibitor of FGFR tyrosine kinases 1, 2 and 3

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signalling axis plays an important role in normal organ, vascular and skeletal development. Deregulation of FGFR signalling through genetic modification or over-expression of the receptors (or their ligands) has been observed in numerous tumour settings, whilst the FGF/FGFR axis also plays a key role in driving tumor angiogenesis. A growing body of data demonstrates that inhibition of FGFR signalling can result in anti-proliferative and/or pro-apoptotic effects, both in vitro and in vivo, thus confirming the validity of FGFR as a therapeutic target. AZD4547 is a small molecule inhibitor which competes with ATP for binding to FGF receptors 1, 2 and 3, thus inhibiting autophosphorylation and downstream signalling. Using in vitro kinase assays, we demonstrate that AZD4547 is a potent inhibitor of FGFR9s 1,2 and 3 with IC50 values of 0.2, 2.5 and 1.8nM respectively. AZD4547 is also highly selective vs a panel of over 70 other kinases (no activity at 10μM) and importantly vs Kinase Insert Domain Receptor (KDR) (>120-fold window in cellular assays vs. FGFR2). In vitro treatment of a large panel of cell lines (>100) with AZD4547 resulted in varying anti-proliferative responses, the most potent of which were almost exclusively confined to those cell-lines in which FGFR signalling was de-regulated. Treatment of these sensitive cell lines with AZD4547 for 1hr, demonstrated potent inhibition of phosphorylation of FGF receptors and downstream substrates including p44/42 MAPK. The anti-proliferative effects of AZD4547 appear to be cell line dependent and vary between potent G1-phase cell cycle arrest and apoptotic induction. Chronic in vivo dosing of AZD4547 up to 12.5mg/kg qd was well tolerated and resulted in dose-dependent growth inhibition in a KMS11 tumour xenograft model. This in vivo anti-tumor activity is correlated with exposure to AZD4547 and associated pharmacodynamic modulation of phospho-FGFR. These data confirm the novelty of AZD4547 as a potent and selective agent for the therapeutic treatment of tumors with deregulated FGF/FGFR signalling. 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 3568. doi:10.1158/1538-7445.AM2011-3568

Abstract 3848: Preclinical profile of AZD3514: A small molecule-targeting androgen receptor function with a novel mechanism of action and the potential to treat castration-resistant prostate cancer

The androgen receptor (AR), an important molecular target in the aetiology and progression of prostate cancer, has been found recently to drive key signalling responses in castration resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed. Here we describe the biological characterisation of AZD3514 an oral drug that targets AR function, with a novel mechanism of action that can result in down-regulation of AR protein. AZD3514 binds to the AR ligand binding domain and has selectivity for binding to AR over other nuclear hormone receptors. In vitro AZD3514 inhibits cell growth in prostate cancer cells expressing wild-type (VCaP) and mutated (T877A) AR (LNCaP), but is inactive in AR-negative prostate cancer cells, indicating a dependency on AR for efficacy. In vivo, we assessed activity initially in the Hershberger castrated rat assay in which oral dosing of AZD3514 (100mg/kg once-daily for 7 days) significantly inhibited testosterone-induced growth of sexual accessory organs. Studies to investigate the mode of action of AZD3514 revealed a number of cellular events associated with loss of AR function. Consistent with an inhibition of AR signalling, AZD3514 caused a rapid reduction in PSA synthesis in vitro; with a significant decrease in PSA mRNA being evident in LNCaP cells within 2 - 3 h of compound treatment. Additional experiments in LNCaP cells and U2OS AR-transfected cells, demonstrated that AZD3514 inhibits an androgen-induced translocation of AR from the cytoplasm to the nucleus within a comparable time-frame. In addition to the effects on AR localisation and AR-dependent transcription, AZD3514 treatment also reduced AR protein in LNCaP cells maintained in steroid-depleted conditions; an effect which was evident within 6 - 8h, and maximal at 18 - 24h. The ability to down-regulate AR under such conditions differentiates AZD3514 from the AR antagonists bicalutamide and MDV3100, which do not reduce AR protein levels. Administration of AZD3514 (100 mg/kg/day orally) for 3 days to Copenhagen rats bearing R3327H Dunning prostate tumours, indicates that AZD3514 treatment also reduces tumour AR in vivo. AZD3514 has also been shown to reduce AR protein expression, PSA synthesis and cell growth in vitro in a subclone of cells serially maintained in the presence of bicalutamide (LNCaP-CR) and an androgen-independent subclone of cells that was serially maintained in steroid-depleted medium (LNCaP-AI), suggesting that this novel mechanism of AR inhibition can deliver activity in CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3848. doi:1538-7445.AM2012-3848

Abstract 1575: Novel small molecule inhibitors of p300/CBP down-regulate AR and c-Myc for the treatment of castrate resistant prostate cancer

Background: E1A binding protein (p300) and CREB binding protein (CBP) are two closely related, paralogue histone acetyl transferase proteins that act as transcriptional co-activators of a variety of cancer related genes. We have developed potent, selective and orally active small molecule inhibitors of the bromodomain of p300/CBP and investigated their role in regulating androgen receptor expression and function. We have also examined their role in driving synthetic lethality in tumours. Loss of function mutations in either p300 or CBP (including in significant proportions of lung and bladder tumours), can lead to a dependency on the corresponding paralogue protein. Methods: Binding affinity to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity was demonstrated in a panel of prostate cells lines representing hormone responsive (LNCaP), hormone independent (DU145, PC3) and castrate resistant disease (22Rv1, C4-2, VCaP, LNCaP-AR) as well as wildtype (A549) and CBP deficient (H520, H1703, LK2) lung cancer cells. Combination effects of p300/CBP inhibitors with a PARP or CDK4/6 inhibitor were determined in LNCaP and C4-2 cells. Effects of p300/CBP inhibitors (and by comparison, the BET inhibitor, JQ1), on AR, AR-V7 splice variant and c-Myc protein, as well as c-Myc, KLK3 and TMPRSS2 gene expression, were assessed in 22Rv1 cells in vitro. In vivo effects on biomarkers were measured in a 22Rv1 xenograft model. Results: CCS1357, an in vitro probe compound, binds to p300 and CBP with high affinity (Kd=4nM) and selectivity (Kd=245nM; BRD4). It is a potent inhibitor of cell proliferation in castrate resistant cell lines (IC50=100nM in LnCaP-AR; 350nM in 22Rv1) with minimal effects in hormone independent lines. CCS1357 combined with palbociclib (CDK4/6) or olaparib (PARP) in LNCaP or C4-2 cells, showed reduced cell viability compared with any of these drugs given alone. In 22RV1 cells, CCS1357 significantly down-regulated AR-FL, AR-V7 and c-Myc protein by Western, an effect not seen with JQ1 at equivalent proliferation IC50s. CCS1357 effects were reversed by the proteasome inhibitor, MG132. CCS1357 also caused a profound inhibition of c-Myc, KLK3 and TMPRSS2 genes measured by qPCR in 22Rv1 cells in vitro. A preclinical candidate (CCS1477) given as a single oral dose (30mg/kg) inhibited plasma PSA and tumour AR, AR-V7 and c-Myc in a 22Rv1 xenograft model. In the lung cancer cell lines, we observed differential sensitivity to CCS1357; CBP deficient lines were more sensitive (cell proliferation) compared with normal. Conclusions: Taken together these data support the clinical testing of p300/CBP inhibition in patients in two settings; firstly, castrate resistant prostate cancer by down-regulating of AR, AR-SV and c-MYC expression and function; and secondly in patients with loss of function mutations in p300 or CBP by driving synthetic lethality. Citation Format: Nigel Brooks, Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser, Jordan Lane, David Taddei, Matthew Schiewer, Renee deLeeuw, Jennifer McCann, Karen Knusden. Novel small molecule inhibitors of p300/CBP down-regulate AR and c-Myc for the treatment of castrate resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1575. doi:10.1158/1538-7445.AM2017-1575

Abstract 3912: The discovery of AZD4547: An orally bioavailable, potent and selective N-(5-Pyrazolyl)benzamide FGFR1-3 inhibitor

There is increasing evidence that FGFR signaling plays an important role within human cancer, with members of FGFR family acting as driving oncogenes in a significant number of human tumors. Deregulation of FGFR-signaling has been documented within clinical samples of breast multiple myeloma, bladder, endometrial, gastric, squamous NSCLC and prostate cancers. This dysregulation most frequently occurs through gene amplification, or through genetically altered forms of FGFR proteins. This increasing body of evidence implicating FGFR signaling in cancer has provided rationale for the identification and testing of selective inhibitors of FGFR signaling in the clinic. In this presentation, we describe the progress of our FGFR tyrosine kinase inhibitor programme and report the discovery of N-(5-pyrazolyl)benzamide FGFR inhibitors. Early compounds in this series suffered from poor in vivo pharmacokinetic (PK) properties. The key site of metabolism was identified to be at a basic N-methyl group. This group was shown to be located in the solvent channel of the ATP binding site on binding to FGFR1, and modification could be made without causing major changes to intrinsic binding affinity. However, the first compounds identified with low metabolic clearance also showed a significant reduction in oral bioavailability, due to apparent low permeability and increased efflux potential. The characterization of these PK issues and the discovery of compounds which overcame them, through modulation of pKa, lipophilicity and masking of the polar groups, will be described. Leading compounds showed significant anti-tumor activity in xenograft tumors grown in mice. Detailed characterization of these compounds led to the identification of AZD4547, a potent and selective FGFR tyrosine kinase inhibitor currently in Phase I clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3912. doi:1538-7445.AM2012-3912

Abstract B107: Characterization of a small molecule selective androgen receptor downregulator: A novel approach for the treatment of castration-resistant prostate cancer.

The androgen receptor (AR), an important molecular target in the aetiology and progression of prostate cancer, has been found recently to drive key signalling responses in castration resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed. Here we describe the biological characterisation of a novel and selective small molecule AR downregulator and propose this mechanism as a potential new approach for the treatment of CRPC. The compound is a derivative of a novel AR binding core with selectivity over other nuclear hormone receptors, and causes a reduction of AR protein in human LNCaP prostate cancer cells in vitro. The reduction in AR protein is observed in steroid depleted serum (CSS) conditions and demonstrates a differential mode of action from a classical AR antagonist (bicalutamide) which has no effect on AR protein expression. This AR downregulation translates into functional activity in vitro and in vivo. In cell viability assays in vitro the compound has activity in cells expressing wild-type (VCaP) and mutated (T877A) AR, but is inactive in AR-negative PC3 and DU145 prostate cancer cells, indicating a dependency on AR for efficacy. We have also observed a reduction in PSA synthesis in vitro, consistent with the inhibition of AR signalling. The compound also reduced AR protein expression, PSA synthesis and cell viability in LNCaP-Casodex™-Resistant and LNCaP-Androgen-Independent cells in vitro. We investigated in vivo activity using the Hershberger castrated rat assay where oral dosing (100mg/kg twice-daily for 7 days) resulted in a significant inhibition of testosterone-induced growth of sexual accessory organs. In summary we describe data supporting our hypothesis that a selective AR downregulator offers a novel approach for delivering therapeutic benefit in CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B107.

Abstract DDT01-01: Preclinical profile of AZD4547: A selective inhibitor of FGF-receptors 1, 2, and 3

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling axis comprises 18 ligands which exert their actions via 4 transmembrane tyrosine kinase receptors. FGFs signaling though their cognate receptors play an important role in normal organ, vascular and skeletal development. Deregulation of FGFR signaling through genetic modification or overexpression of the receptors (or their ligands) has been observed in numerous tumor settings, whilst the FGF/FGFR axis also plays an important role in driving tumor angiogenesis. A growing body of data demonstrates that inhibition of FGFR signaling can result in antiproliferative and/or proapoptotic effects, both in vitro and in vivo, thus confirming the validity of FGFR as a cancer therapeutic target. AZD4547 is a small molecule inhibitor which competes with ATP for binding to FGF receptors 1, 2 and 3, thus inhibiting autophosphorylation and downstream signaling. Using in vitro kinase assays, we demonstrate that AZD4547 is a potent inhibitor of FGFR9s 1,2 and 3 with IC 50 values of 0.2, 2.5 and 1.8 nM respectively. AZD4547 is also highly selective vs. a panel of over 70 other kinases (no activity at 10 μM) and importantly vs. kinase insert domain receptor (KDR) (>120-fold window in cellular assays vs. FGFR2). In vitro treatment of a large panel of cell lines (>100, representing several tumor types) with AZD4547 resulted in varying antiproliferative responses, the most potent of which were almost exclusively confined to those cell-lines in which FGFR signaling was deregulated by either gene amplification, mutation or translocation. Treatment of these sensitive cell lines with AZD4547 for 1 hr, demonstrated potent inhibition of phosphorylation of FGF receptors and downstream substrates including pFRS2α and p44/42 MAPK. The antiproliferative effects of AZD4547 appear to be cell line dependent and vary between potent G1-phase cell cycle arrest and apoptotic induction. Chronic in vivo dosing of AZD4547 up to 12.5 mg/kg qd was well tolerated and resulted in dose-dependent growth inhibition in KMS11 (FGFR3 translocated, multiple myeloma) and Snu16 (FGFR2 amplified, gastric) tumour xenograft models. This in vivo antitumor activity is correlated with exposure to AZD4547 and associated pharmacodynamic modulation of phospho-FGFR and downstream substrates. In addition to the single-agent efficacy, combining of AZD4547 with cytotoxic agents, currently used for the treatment of gastric cancer patients, revealed enhanced growth inhibition of Snu-16 xenografts. Collectively, these data confirm the suitability of AZD4547 as a potent and selective agent for the therapeutic treatment of tumors with deregulated FGF/FGFR signaling. 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 DDT01-01. doi:10.1158/1538-7445.AM2011-DDT01-01