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Wnt signaling is implicated in the etiology of gastrointestinal tract cancers. Targeting Wnt signaling is challenging due to on-target toxicity concerns and lack of druggable pathway components. We describe the discovery and characterization of RXC004, a potent and selective inhibitor of the membrane-bound o-acyl transferase Porcupine, essential for Wnt ligand secretion. Absorption, distribution, metabolism, and excretion and safety pharmacology studies were conducted with RXC004 in vitro, and pharmacokinetic exposure assessed in vivo. RXC004 effects on proliferation and tumor metabolism were explored in genetically defined colorectal and pancreatic cancer models in vitro and in vivo. RXC004 effects on immune evasion were assessed in B16F10 immune “cold” and CT26 immune “hot” murine syngeneic models, and in human cell cocultures. RXC004 showed a promising pharmacokinetic profile, inhibited Wnt ligand palmitoylation, secretion, and pathway activation, and demonstrated potent antiproliferative effects in Wnt ligand–dependent (RNF43-mutant or RSPO3-fusion) colorectal and pancreatic cell lines. Reduced tumor growth and increased cancer cell differentiation were observed in SNU-1411 (RSPO3-fusion), AsPC1 and HPAF-II (both RNF43-mutant) xenograft models, with a therapeutic window versus Wnt homeostatic functions. Additional effects of RXC004 on tumor cell metabolism were confirmed in vitro and in vivo by glucose uptake and 18fluorodeoxyglucose-PET, respectively. RXC004 stimulated host tumor immunity; reducing resident myeloid-derived suppressor cells within B16F10 tumors and synergizing with anti-programmed cell death protein-1 (PD-1) to increase CD8+/regulatory T cell ratios within CT26 tumors. Moreover, RXC004 reversed the immunosuppressive effects of HPAF-II cells cocultured with human peripheral blood mononuclear cells, confirming the multiple anticancer mechanisms of this compound, which has progressed into phase II clinical trials. Significance: Wnt pathway dysregulation drives many gastrointestinal cancers; however, there are no approved therapies that target the pathway. RXC004 has demonstrated the potential to block both tumor growth and tumor immune evasion in a genetically defined, clinically actionable subpopulation of Wnt ligand–dependent gastrointestinal cancers. The clinical utility of RXC004, and other Porcupine inhibitors, in such Wnt ligand–dependent cancers is currently being assessed in patient trials.

Supplementary Figure 1: Effect of RXC004 on gene expression, caspase activity and cell growth in colorectal and pancreatic cancer cell lines in vitro. Supplementary Figure 2: RXC004 effects on gene expression in in vivo xenograft tumor models. Supplementary Figure 3: Pharmacokinetic/pharmacodynamic (PK/PD) analysis of RXC004 in SNU-1411/NOD-SCID mice. Supplementary Figure 4: Dose-dependent effects of RXC004 on tumor markers, intestinal Ki67 and bodyweight. Supplementary Figure 5: Effect of RXC004 with or without anti-PD-1 on body weight and survival in in vivo models. Supplementary Figure 6: Dose-dependent effects of RXC004 alone or in combination with anti-CTLA-4 in B16F10/C57BL/6 syngeneic model. Supplementary Figure 7: In vitro PBMC co-culture additional cytokines and monocultures. Supplementary Table 1: In vivo exposure data for RXC004.

Wnt signaling is implicated in the etiology of gastrointestinal tract cancers. Targeting Wnt signaling is challenging due to on-target toxicity concerns and lack of druggable pathway components. We describe the discovery and characterization of RXC004, a potent and selective inhibitor of the membrane-bound o-acyl transferase Porcupine, essential for Wnt ligand secretion. Absorption, distribution, metabolism, and excretion and safety pharmacology studies were conducted with RXC004 in vitro, and pharmacokinetic exposure assessed in vivo. RXC004 effects on proliferation and tumor metabolism were explored in genetically defined colorectal and pancreatic cancer models in vitro and in vivo. RXC004 effects on immune evasion were assessed in B16F10 immune “cold” and CT26 immune “hot” murine syngeneic models, and in human cell cocultures. RXC004 showed a promising pharmacokinetic profile, inhibited Wnt ligand palmitoylation, secretion, and pathway activation, and demonstrated potent antiproliferative effects in Wnt ligand–dependent (RNF43-mutant or RSPO3-fusion) colorectal and pancreatic cell lines. Reduced tumor growth and increased cancer cell differentiation were observed in SNU-1411 (RSPO3-fusion), AsPC1 and HPAF-II (both RNF43-mutant) xenograft models, with a therapeutic window versus Wnt homeostatic functions. Additional effects of RXC004 on tumor cell metabolism were confirmed in vitro and in vivo by glucose uptake and 18fluorodeoxyglucose-PET, respectively. RXC004 stimulated host tumor immunity; reducing resident myeloid-derived suppressor cells within B16F10 tumors and synergizing with anti-programmed cell death protein-1 (PD-1) to increase CD8+/regulatory T cell ratios within CT26 tumors. Moreover, RXC004 reversed the immunosuppressive effects of HPAF-II cells cocultured with human peripheral blood mononuclear cells, confirming the multiple anticancer mechanisms of this compound, which has progressed into phase II clinical trials.Significance:Wnt pathway dysregulation drives many gastrointestinal cancers; however, there are no approved therapies that target the pathway. RXC004 has demonstrated the potential to block both tumor growth and tumor immune evasion in a genetically defined, clinically actionable subpopulation of Wnt ligand–dependent gastrointestinal cancers. The clinical utility of RXC004, and other Porcupine inhibitors, in such Wnt ligand–dependent cancers is currently being assessed in patient trials.

Acetylation of histones and additional nuclear proteins is a key mechanism in the regulation of gene expression. Aberrant acetylation has been linked to a wide range of diseases including cancer, inflammation, and neurodevelopmental disorders. Histone acetylation is introduced by histone acetyltransferases complexes (HATs), where substrate specificity is dramatically enhanced by scaffolding proteins that activate and target them to specific chromatin sites. A protein of interest with both epigenetic acetyl reader and scaffolding function is the protein Bromodomain and PHD finger-containing protein 1 (BRPF1). The Protein contains domains of two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) Tudor domain. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as the catalytic MYST-family acetyltransferase subunit and the stable complex with Moz-Tif2 is known to lead to the development of human acute myeloid leukemia (AML). Given this, we chose to pursue the development of BRPF1 degraders to probe cancer disease biology especially in AML, where new therapies are required to overcome several unmet needs such as less-toxic treatments and relapsed/refractory disease paradigms. Here we present the design and synthesis of a range of BRPF1 degraders in highly desirable physicochemical space utilizing in-silico modeling. The prepared degraders which utilize multiple E3 ligases were then screened against cell lines harboring Mixed-lineage leukemia (MLL) translocations specifically the THP-1 cell line. In addition to this, we investigated the ability of the compounds to effectively degrade the target and suitability of our degraders for potential in-vivo exposure through a panel of routine ADMET assays. Citation Format: Daniel Joseph Glynn, Rosie Crampton, Thomas Pesnot, Andrew Scott, Anne-Chloe Nassoy, Ralph Kirk, Daniele Narducci, Gary Nelson, Lynette Ongiri, Habiba Begum, Vincent Rao, Matilda Bingham, Rhoanne McPherson, Darryl Turner. The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 419.

Delafloxacin, a fourth-generation anionic fluoroquinolone (FQ) was approved in 2019 for community acquired bacterial pneumonia (CARP). It has broad spectrum activity and an improved class-related toxicity profile. However, it has recently failed a Phase 3 clinical trial for treatment of N. gonorrhoeae infections due to the lack of sufficient efficacy at the dose administered. Inspired by the microbiological and safety profile of delafloxacin, we have developed and profiled the first reported delafloxacin carbon analogue whereby a Nitrogen-for-Carbon swap has been successfully carried out at the C7 position. Not only have we shown that compounds with this modification maintain activity against N. gonorrhoeae (plus other gram-positive and gram-negative bacteria) but they also demonstrate a differentiated physicochemical profile. A zwitterionic derivative of delafloxacin was also profiled and demonstrated a superior microbiological profile against gram-negative strains, whilst maintaining favorable selected ADMET properties.

A range of differentially functionalised furans and pyrroles have been synthesised in short order by the judicious use of a ring-closing metathesis/aromatisation strategy. Two contrasting approaches are described exploiting a palladium-catalysed union of allylic alcohols and sulfonamides in one case, and a titanium mediated methylenation of homoallylic esters in another. A number of groups that are difficult to install via traditional methods were incorporated successfully. © 2007 Elsevier Ltd. All rights reserved.

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.

A one-pot, three-component process is described which involves both organo- and enzyme-catalysed carbon–carbon bond-forming steps. In the first step, an organocatalyst catalyses the aldol reaction between acetaldehyde and a glyoxylamide. After dilution with additional aqueous buffer, and addition of pyruvate and an aldolase enzyme variant, a second aldol reaction occurs to yield a final product. Crucially, it was possible to develop a reaction in which both the organo- and enzyme-catalysed reactions could be performed in the same aqueous buffer system. The reaction described is the first example of a one-pot, three-component reaction in which the two carbon–carbon bond-forming processes are catalysed using the combination of an organocatalyst and an enzyme.

Hit compound 1, a selective noradrenaline re-uptake transporter (NET) inhibitor was optimised to build in potency at the serotonin re-uptake transporter (SERT) whilst maintaining selectivity against the dopamine re-uptake transporter (DAT). During the optimisation of 1 it became clear that selectivity against the Kv11.1 potassium ion channel (hERG) was also a parameter for optimisation within the series. Discrete structural changes to the molecule as well as a lowering of global cLogP successfully increased the hERG selectivity to afford compound 11m, which was efficacious in a mouse model of inflammatory pain, complete Freund’s adjuvant (CFA) induced thermal hyperalgesia and a rat model of neuropathic pain, spinal nerve ligation (SNL) induced mechanical allodynia.

Background: RXC004, a potent and selective porcupine (PORCN) inhibitor, is being investigated in a safety and tolerability study in cancer patients with solid tumours (CT 2017-000720-98). In addition to the tumour targeting role of RXC004 and other Wnt pathway inhibitors, we present pre-clinical data which suggests further potential for RXC004 in modulating the immune system of the tumour microenvironment. Materials and Methods: To evaluate the potential of a novel porcupine inhibitor, RXC004, as an immunomodulatory anti-cancer agent, sub cutaneous B16F10 melanoma (C57BL/6 mice) and CT26 colorectal (BALB/c mice) murine tumour models were utilised. Mice in both models were treated with RXC004 alone or in combination with mouse anti-PD-1 antibody. Flow cytometry analysis was utilised to measure key immune cell populations in the tumour microenvironment. To probe the underlying mechanism of immune modulation in these models and to provide a link to the emerging clinical data suggesting a role for Wnt pathway activation in immune escape, human monocytic cells were isolated from PBMCs and human dendritic cells were derived in vitro. The Wnt pathway was induced in derived Dendritic cells and expression of IDO was measured. Results: In the murine CT26 model, RXC004 treatment reduced tumour size when dosed in combination with anti-PD-1 antibody, causing regression and cures in some animals. Furthermore, flow cytometry showed RXC004 in combination with anti-PD-1 antibody increased the proportion of CD8+ cytotoxic T cells as well as decreasing FoxP3+ regulatory T cells when compared to the monotherapy anti-PD-1 arm. In a syngeneic murine melanoma B16F10 model, RXC004 monotherapy at a dose of 5mg/kg QD orally significantly inhibited tumour growth, as did RXC004 combined with anti-PD-1. RXC004 had no effect on the proliferation of B16F10 cells in vitro, suggesting this was not caused by the compound directly affecting B16 cell proliferation. Flow cytometry analysis of the B16 tumours showed significant immune modulatory effects in the tumour microenvironment. In addition to mouse model data, Wnt pathway activation in human dendritic cells was shown to increase IDO expression. Conclusion: Taken together, data from murine syngeneic mouse models corroborate literature data suggesting that inhibiting the Wnt pathway may promote the immune response against human cancers. Citation Format: Inder Bhamra, Richard Armer, Matilda Bingham, Catherine Eagle, Alicia Edmenson Cook, Caroline Phillips, Simon Woodcock. Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3764.

Die Anwendung der Metathese, besonders der Ringschlussmetathese (RCM) zur Bildung von Funf- und Sechsringen, ist heute in der organischen Chemie weit verbreitet; dennoch gibt es uberraschend wenige Beispiele fur ihre Anwendung zur Synthese aromatischer Verbindungen. Deren Bedeutung in der medizinischen Chemie und der Naturstoffsynthese, verbunden mit der Wirksamkeit von RCM-Katalysatoren und ihrer Toleranz gegenuber funktionellen Gruppen, gibt Anlass, die Anwendung der RCM in der Arensynthese zu untersuchen. Die Synthese von heterocyclischen und carbocyclischen Arenen unter Verwendung der RCM wird in einigen Publikationen als Schlusselschritt beschrieben. Galt das gebildete Aren in vielen fruhen Beispielen noch als unerwunschtes Abbauprodukt, so wurden in letzter Zeit gezielt Methoden zur Arensynthese uber RCM entwickelt.

Harnessing the immune system via immune checkpoint blockade (e.g. anti PD-1, anti PD-L1, anti CTLA4) has led to fast and long lived responses in cancer patients. Response rates however are low and new treatments that enhance these rates are needed. Recent studies have shown that the administration of immune checkpoint blockers is associated with the overexpression of indoleamine 2,3-dioxygenase 1 (IDO1). The resulting immunoregulatory phenotype counteracts immune checkpoint blockade and allows for cancer progression. The discovery of IDO1 inhibitors, and the potential to combine them with immune checkpoint blockers, therefore represents an attractive strategy to fight cancer. We carried out a ligand-based virtual screen with > 1,000,000 commercially available small molecules. In vitro screening of the resulting 610 virtual hits provided us with 2 IDO1-selective, 2 TDO2-selective and 2 IDO1/TDO2-dual confirmed hits. (TDO2 is a protein with similar biochemical activity to IDO1 that is essential to tryptophan homeostasis.) A subsequent Hit to Lead campaign led to the identification of novel chemotypes that display potency similar or superior to IDO1 inhibitors currently under clinical investigation in IFN-γ stimulated (i.e. IDO1+) HeLa cells, with no sign of cytotoxicity. We have demonstrated that these compounds are > 1000-fold selective for IDO1 over TDO2 using cellular assays. IDO1 upregulation by cancer cells is known to be one of the mechanisms by which cancer cells evade the immune system. In an in vitro co-culture assay of cancer cells and T cells we have demonstrated our compounds can rescue T cell proliferation with EC50 values between 10 and 50 nM. We have also demonstrated that our compounds inhibit IDO1 in monocyte derived human dendritic cells. Interestingly, despite this potent cellular activity demonstrated in multiple disease relevant cellular assays, this chemotype failed to inhibit recombinant IDO1 in an isolated biochemical assay performed under reducing conditions, whereas the reference compound epacadostat provided activity comparable to literature values. In order to confirm our cellular effects were due to direct inhibition of IDO1 we set up thermal shift assays. Thermal shift assays using purified IDO1 protein have demonstrated that our compounds directly bind IDO1, and cellular thermal shift assays have confirmed direct target engagement in intact cells (stimulated for IDO1 expression). These compounds have physicochemical properties that would support oral dosing and display low in vitro CYP450 and hERG inhibition, thus reducing the risk of toxicity in the clinic. Our IDO1 inhibitors show a novel differentiated mode of action at the cellular level, and the consequences of this profile in terms of in vivo characterisation is ongoing. Citation Format: Thomas Pesnot, Sachin Mahale, Philip MacFaul, John Maclean, Caroline Phillips, Matilda Bingham, Catherine Eagle, James Kelly, Abhijith Thippeswamy, Simon Armitage, Aleksandr Grisin, Sheenagh Aiken, Lucy Cartwright, Richard Armer. Development of 2nd generation indoleamine 2,3-dioxygenase 1 (IDO1) selective inhibitors [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 5581. doi:10.1158/1538-7445.AM2017-5581

REDX05358 has been identified as a novel, highly selective and potent next generation pan RAF inhibitor with improved therapeutic potential and predicted safety profile. Aberrant signalling via the MAPK pathway is commonly seen in cancer. RAF inhibitors such as Dabrafenib and Vemurafenib have been approved for the treatment of unresectable and metastatic BRAF mutant melanoma, but these agents lack efficacy in BRAF mutant colorectal cancer (CRC), partly because of EGFR-mediated feedback reactivation of the MAPK pathway via CRAF. Furthermore, RAF inhibitor treatment of RAS-mutant, BRAF wildtype melanomas has been associated with other skin cancers, such as cutaneous squamous cell carcinoma due to MAPK pathway paradoxical activation mediated by CRAF. There is therefore a clinical need for novel agents targeting the MAPK pathway that do not have these undesirable properties. Here we present, REDX05358 that demonstrates subnanomolar binding affinity for BRAF and CRAF with high selectivity profile against a panel of 468 kinases that exhibits negligible paradoxical activation due to inhibition of both RAF monomers and dimers. As a result, REDX05358 not only inhibits MAPK signalling in BRAF V600E mutant tumor cells, but also in those harbouring NRAS and KRAS mutations. Furthermore, REDX05358 does not induce feedback reactivation of the pathway through its ability to sustain inhibition of MAPK signalling in CRC cell lines. Correspondingly, profiling of REDX05358 in a panel of CRC, melanoma and NSCLC cell lines shows it has potent anti-proliferative activity in cell lines harbouring BRAF or RAS mutations. REDX05358 is an orally bioavailable, well tolerated small molecule that has demonstrated in vivo efficacy in BRAF V600E CRC xenograft model. In contrast, first generation inhibitors such as Vemarafenib and Dabrafanib have been reported to be ineffective in this genetic background in CRC cell lines and patients. Thus, we have developed a pan RAF inhibitor with unique pharmacological properties enabling it to have utility in treating BRAF and RAS mutant cancers Citation Format: Helen Mason, Simon Scrace, Richard Testar, Julie Rainard, Fatima Talab, Ryaka Poonawala, Philippa Smith, Harriet Brooke, Sarah Frith, Jonathan Ahmet, Jonathan Hall, David Sorrell, Jon Moore, Caroline Phillips, Dennis France, Matilda Bingham, Richard Armer. Development of REDX05358, a novel highly selective and potent pan RAF inhibitor and a potential therapeutic for BRAF and RAS mutant tumors [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 5160. doi:10.1158/1538-7445.AM2017-5160

e14094 Background: Wnt signalling initiates oncogenic pathways involved in tumour initiation, growth, differentiation and metastasis.1 Targeting the Wnt pathway is an attractive approach to cancer treatment. Porcupine (PORCN) is a membrane-bound O-acyltransferase dedicated to the palmitoylation of Wnt ligands, an essential step in the processing of Wnt into active ligands.2 A PORCN inhibitor may benefit patients with cancers in which Wnt signalling is implicated. It has been shown that PORCN inhibition is efficacious in cell lines with upstream mutations in this pathway, eg RNF43 loss of function.3 Methods: Analysis of TCGA data was carried out to identify cancer types harbouring RNF43 mutations.4 Redx PORCN inhibitor RXC004 was evaluated in vitro for Wnt pathway inhibition and anti-proliferative effects using assays previously described for early lead compounds.5Anti-tumour effects of RXC004 were evaluated using a pancreatic CAPAN-2 xenograft in SCID Bg. mice and a gastric PDX model in nu/nu mice. Both models have RNF43 loss of function mutations. Results: TCGA data revealed that both gastric cancer and pancreatic cancer exhibit mutations in the RNF43 gene (18% and 4% respectively),4expected to result in loss of function. RXC004 showed activity in preclinical models of cancers with this genetic background. RXC004 significantly inhibited tumour growth in a CAPAN-2 pancreatic cancer xenograft and in a gastric cancer PDX model when dosed orally once or twice daily. Conclusions: Taken together, these data indicate that RXC004 is a potent inhibitor of tumour growth in RNF43 loss of function preclinical cancer models. RXC004 will be evaluated in a first in human Phase 1 study in 2017. Phase 1a aims are to characterise the safety profile and PK/PD relationship in cancer patients and establish a Phase 1b dose. The efficacy of RXC004 in gastric and pancreatic cancer patients prospectively selected for RNF43 loss of function mutations will be the focus of Phase 1b expansion groups. 1 . Nat. Rev. Cancer, 2013, 13 (1): 11-26.2. J. Biol. Chem., 2012, 287 (27): 23246-23254. 3. PNAS, 2013, 110 (31): 12649-12654 4. Sci. Signal, 2013, 269 (6): 1-34; Cancer Discov., 2012, 2 (5): 401-404 5. AACR Annual Meeting, 2015, 75 (15): abs. 5071.

The cupric acetate or ferric chloride mediated cyclizations of unsaturated oxime acetates lead to vinyl- or chloroalkyl-substituted pyrrolines in moderate to good yields.

Here we report the preclinical profile of REDX08608 our novel, potent and selective, reversible BTK inhibitor that is equipotent against wild-type and mutant C481S BTK. Bruton's tyrosine kinase (BTK) is a member of the src-related Tec family of cytoplasmic tyrosine kinases and plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK is a clinically validated target to treat B-cell malignancies that are dependent on BCR signaling i.e.CLL and NHL with ibrutinib approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors such as ibrutinib, acalabrutinib and GS-4059 specifically target a cysteine residue C481 within BTK and mutations at this site clearly interfere with covalent drug binding. C481S, C481Y, C481R, C481F mutations have been reported and linked to cases of resistance that have emerged in patients with CLL progression following treatment with ibrutinib (Byrd2016, Inhye2016, Maddocks2015, Woaych2014). Redx reversible BTK inhibitor, REDX08608, aims to overcome this resistance mechanism by targeting both wild type and C481-mutated BTK. Redx have recently presented REDX06961 our BTK probe (Guisot2016, AACR#4795) and, following lead optimization, we are now disclosing REDX08608, our lead compound, a potent, reversible and selective BTK inhibitor, which displays an improved profile including superior pharmacokinetics. REDX08608, reversibly, inhibits WT and C481S BTK and displays nanomolar binding affinity and potency in biochemical and cellular-based assays. REDX08608 inhibits BTK signaling and growth in cell lines dependent on the BTK pathway such the OCI-LY10 ABC-DLBCL cell line. Importantly, REDX08608 also inhibits BTK signaling in primary CLL cells. In human whole blood and isolated human PBMCs, REDX08608 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 in CD19+cells. REDX08608 is highly selective when tested against a panel of 468 kinases and exhibits improved target specificity with >100-fold selectivity against other Tec and Src kinase family members (ITK, TXK, BMX, TEC, BLK, CSK, FYN, HCK, LCK, SRC) and >400-fold selectivity against EGFR. REDX08608 was fully profiled through DMPK in vitro assays including metabolic stability, plasma stability, cytochrome P450 inhibition, PXR activation/cytochrome P450 activity, time dependent inhibition and cytochrome P450 reaction phenotyping. REDX08608 was shown to have an acceptable metabolic and plasma stability profile across species (mouse, rat, dog, monkey and human). REDX08608 displayed no evidence of PXR activation or time dependent inhibition. IC50s were determined for human cytochrome P450s (1A2, 2D6, 2C9, 2C19 and 3A4) and were all >10 µM. Good exposure, oral bioavailability and half-life were demonstrated for REDX08608 in mouse, rat and dog, with dose linearity assessment performed in mouse (F = 73-100%, CL= 11% liver blood flow in mice; F = 55-84%, CL = 28% liver blood flow in rat; F = 85%, CL= 10% liver blood flow in dog). In vivo efficacy studies in preclinical models will also be disclosed. In conclusion, REDX08608 is a potent and selective, reversible BTK inhibitor with efficacy in lymphoma cell lines that offers the potential to target both wild-type BTK and an important emerging resistance mechanism in patients with CLL progression following ibrutinib-treatment. Disclosures Guisot: Redx Oncology Ltd - Redx Pharma Plc: Employment. Best:Redx Oncology Ltd - Redx Pharma Plc: Employment. Wright:Redx Oncology Ltd - Redx Pharma Plc: Employment. Thomason:Redx Oncology Ltd - Redx Pharma Plc: Employment. Woyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding. Abet:Redx Oncology Ltd - Redx Pharma Plc: Employment. Castagna:Redx Oncology Ltd - Redx Pharma Plc: Employment. Cousin:Redx Oncology Ltd - Redx Pharma Plc: Employment. Emmerich:Redx Oncology Ltd - Redx Pharma Plc: Employment. Ho:Redx Oncology Ltd - Redx Pharma Plc: Employment. Kelly:Redx Oncology Ltd - Redx Pharma Plc: Employment. King-Tours:Redx Oncology Ltd - Redx Pharma Plc: Employment. Lyons:Redx Oncology Ltd - Redx Pharma Plc: Employment. Muller:Redx Oncology Ltd - Redx Pharma Plc: Employment. Refuerzo:Redx Oncology Ltd - Redx Pharma Plc: Employment. Sargent:Redx Oncology Ltd - Redx Pharma Plc: Employment. Talab:Redx Oncology Ltd - Redx Pharma Plc: Employment. Bingham:Redx Oncology Ltd - Redx Pharma Plc: Employment. Phillips:Redx Oncology Ltd - Redx Pharma Plc: Employment. Armer:Redx Oncology Ltd - Redx Pharma Plc: Employment.

Redx Oncology has developed novel, differentiated, reversible small molecule inhibitors of BTK, combining current best-in-class potency with improved selectivity profiles, which are suitable for oral, once daily dosing, designed to be equipotent against wild-type and C481S BTK. BTK is a member of the src-related Tec family of cytoplasmic tyrosine kinases. BTK plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK inhibitors have therefore been developed with the aim of treating B-cell malignancies that are dependent on BCR signaling, such as CLL and NHL. Ibrutinib is an irreversible BTK inhibitor that has been approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors specifically target a cysteine residue C481 within BTK. Following treatment with ibrutinib, cases of secondary resistance have emerged in both CLL and MCL patients. Acquired mutations within BTK such as C481S, C481Y, C481R, C481F have been reported in the literature and clearly interfere with covalent drug binding. It has been predicted that the incidence of observed resistance will increase as clinical use outside clinical trials expands over time. Here, we present our reversible BTK inhibitor series demonstrating subnanomolar binding affinity for WT and C481S forms of BTK, that inhibits formation of pBTK in both WT and C481S BTK expressing cells. In addition, the compound demonstrated significant in vitro potency against a number of lymphoma cell lines including inhibition of BCR signaling and proliferation in OCI-Ly10 cells at nanomolar concentrations. To further investigate the binding nature of these compounds, PBMC wash out studies, measuring CD69 as a marker of B-cell activation, were used to highlight the reversible activity of these compounds. Some BTK inhibitors also inhibit ITK, which plays a critical role in FcR-stimulated NK cell function that is required for ADCC. ADCC is the mechanism that anti-CD20 antibodies, such as rituximab, are believed to activate, and ibrutinib has been shown to antagonize this mechanism in vitro. As rituximab-combination chemotherapy is today's standard of care in B-cell malignancies, it would be desirable to have a BTK inhibitor with high selectivity for BTK over ITK. At 1 μM, our lead compound is highly selective when tested against 469 kinase and did not show significant inhibition against other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, the compound has high selectivity versus structurally related cysteine-containing kinases (including EGFR and ITK) both in enzyme and cellular assays. Our lead compound has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile to competitor compounds. In vivo PK demonstrated good oral bioavailability and in vivo efficacy has been demonstrated. Citation Format: Nicolas E S Guisot, Victoria Walker, Stuart A. Best, Valentina Abet, Rose Chappell, Juliette Emmerich, Kelvin Ho, James R. Kelly, Kristina Lyons, Melanie Müller, Julienne Refuerzo, Louise Sargent, Fatima Talab, Madelene Waldron, Matilda Bingham, Mary-Ann Campbell, Caroline Phillips, Richard Armer. Development of novel, selective, reversible inhibitors equipotent against wild-type and C481S Bruton's tyrosine kinase (BTK). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4795.

The human brain is a uniquely complex organ, which has evolved a sophisticated protection system to avoid injury from external insults and toxins. Penetrating the blood-brain barrier (BBB) to achieve the drug concentrations required for efficacious target receptor occupancy in the brain region of interest is a unique and significant challenge facing medicinal chemists working on CNS targets. Prospective design of molecules with optimal brain exposure and safety profile requires in-depth understanding of the fundamental relationships between physicochemical properties and in vitro and in vivo outcomes. Following from the now widely accepted “rule of five” guidelines for the design of oral drugs, the physicochemical properties for brain penetration have been extensively studied in an effort to define the characteristics of successful CNS drug candidates. Several key physicochemical properties have been identified that influence the rate of brain permeability and extent of brain penetration, including H-bonding potential, molecular weight, lipophilicity, polar surface area (PSA), ionization state and rotatable bond count. The ability to process this information effectively and engage in multi-parameter prospective design ultimately determines the success in delivering high-quality drug candidates that are suitable robustly to test hypotheses in the clinic and have good probability of reaching the market. This chapter focuses on the medicinal chemistry aspects of drug candidate optimization particular to the CNS therapeutic area, such as crossing the blood-brain barrier (BBB), as well as safety-related issues frequently challenging CNS programs such as hERG selectivity and phospholipidosis.

The discovery and development of effective medicines for the treatment of psychiatric disorders such as schizophrenia and depression has been heralded as one of the great medical achievements of the past century. Indeed, the profound impact of these medicines on our understanding of the pathophysiology underlying these diseases, the treatment of psychiatric patients and even our social perception of mental illnesses cannot be underestimated. However, there is still an urgent medical need for even more effective, safe and well-tolerated treatments. For example, currently available treatments for schizophrenia address mainly the positive symptoms and largely neglect the negative symptoms and cognitive disfunction which greatly impact overall morbidity. Similarly, whilst the current first line antidepressants show significantly improved side effect profiles compared to the first generation therapies, there still up to 40% of patients who are treatment resistant, and even in the patient population which responds well, the onset of action is slow at typically 2-3 weeks. The aim of this book is to provide the first point of call for those involved or just interested in this rapidly expanding and increasingly fragmented field of research and drug discovery. The editors will combine their wide ranging experience and extensive network of contacts with leading scientists and opinion leaders in this field to provide an authoritative reference text covering the evolution, major advances, challenges and future directions in drug discovery and medicinal chemistry for major psychiatric disorders.

The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.

The synthesis and preliminary structure–activity relationships (SAR) of a novel class of vasopressin V 1B receptor antagonists are described. Hit compound 5 , identified via high throughput screening of the corporate collection, showed good activity in a V 1B binding assay ( K i 63 nM) but did not possess the lead-like physicochemical properties typically required in a hit compound. A ‘deletion approach’ on the HTS hit 5 was performed, with the focus on improvement of physicochemical properties, yielding the selective V 1B antagonist 9f ( K i 190 nM), with improved druglike characteristics.

2 Transporter Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.1 The ATP Binding Cassette Family . . . . . . . . . . . . . . . . . . . . . . . 2 2.2 The Solute Carrier Family . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2.1 The Major Facilitator Superfamily . . . . . . . . . . . . . . . . . . . . . . . 3 2.2.2 The Neurotransmitter: Sodium Symporter Family . . . . . . . . . . . . . . 3 2.2.3 The Dicarboxylate/Amino Acid:Cation (Na+ or H+) Symporter (DAACS) Family . . . . . . . . . . . . . . . . . . . . . . . . . . 5

The B-cell receptor (BCR) signaling pathway is required for the survival, activation, proliferation and differentiation of B-cells. Bruton's Tyrosine Kinase (BTK) is a member of the Tec protein tyrosine kinase family that has emerged as an attractive target for the treatment of B-cell malignancies due to the critical role it plays in BCR signaling. Redx Oncology has developed novel differentiated small molecule inhibitors of BTK, combining current best-in-class potency with distinct selectivity profiles, which are suitable for oral once daily dosing. Here we present REDX05194, the result of a successful lead optimization of our proprietary BTK inhibitor series. REDX05194 is a highly selective, covalent BTK inhibitor displaying subnanomolar binding affinity for BTK (0.39 nM) and nanomolar potency towards BTK in a biochemical assay (3.67 nM). In cell proliferation assays, REDX05194 showed significant in vitro potency against ABC-DLBCL cell lines inhibiting the growth of both TMD-8 (0.89 nM) and OCI-Ly10 (1.36 nM) cells. Analysis of BCR signaling in several lymphoma cell lines, including cell lines of ABC-DLBCL, MCL and FL origin, revealed that treatment with REDX05194 inhibits BTK autophosphorylation and downstream activation of PLCγ2. In human PBMCs, REDX05194 inhibited anti-IgM stimulated upregulation of the CD69 activation marker in CD19 positive B-cells. In addition, using a fluorescent probe that binds to BTK, occupancy of BTK in PBMCs has been demonstrated in response to increasing concentrations of REDX05194. To assess selectivity, 456 kinases were screened at 1 μM, confirming that REDX05194 does not significantly inhibit other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, REDX05194 was shown to have high selectivity versus structurally related cysteine-containing kinases such as ITK in binding assays, and EGFR as demonstrated in both binding and cellular assays. REDX05194 also has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile and solubility compared to competitor compounds. REDX05194 demonstrated in vivo efficacy in a mouse collagen-induced arthritis (CIA) model. At 10 mg/kg and 30 mg/kg QD, REDX05194 significantly improved all clinical readouts, including disease severity, compared to the vehicle group. Histological data showed that approximately 1/3 of the mice had no or minimal pannus infiltration and no bone resorption, or had bone resorption restricted to small areas. These findings demonstrate potential clinical efficacy and a dose response. In conclusion, REDX05194 is a highly selective and potent BTK inhibitor with proven efficacy in several lymphoma cell lines and human PBMCs and in vivo efficacy demonstrated in a mouse CIA model. Disclosures No relevant conflicts of interest to declare.

The use of metathesis, especially in the context of ring-closing metathesis (RCM) to form five- and six-membered rings, is widespread in organic chemistry today. However, there are surprisingly few examples of the reaction being used to form aromatic compounds. The central place of aromatic compounds in both medicinal chemistry and natural products synthesis, coupled with the efficiency and functional group tolerance of RCM catalysts, means that there is now an interesting opportunity to use RCM for the synthesis of arenes. Although the formation of an aromatic compound was viewed in many early examples as an undesirable degradation product, several rationally designed methods towards the preparation of aromatic compounds by RCM have recently been developed.

The ring closing metathesis (RCM) reaction can be used to prepare substituted furans and pyrroles. By utilising a Pd-catalysed coupling reaction with methoxyallene, allylic alcohols and sulfonamides can be converted into substrates that are ideal precursors to ring closing metathesis. After the RCM reaction is complete, the addition of acid promotes an elimination of methanol to form the fully aromatised system. A range of different substitution patterns and functional groups are compatible with this sequence. Double allene coupling, RCM and elimination reactions are also possible and allow the formation of biaryl systems. © Wiley-VCH Verlag GmbH and Co. KGaA, 69451 Weinheim, Germany, 2005.