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Based on the structure of an early lead identified in Deciphera's proprietary compound collection of switch control kinase inhibitors and using a combination of medicinal chemistry guided structure activity relationships and structure-based drug design, a novel series of potent acyl urea-based CSF1R inhibitors was identified displaying high selectivity for CSF1R versus the other members of the Type III receptor tyrosine kinase (RTK) family members (KIT, PDGFR-α, PDGFR-β, and FLT3), VEGFR2 and MET. Based on in vitro biology, in vitro ADME and in vivo PK/PD studies, compound 10 was selected as an advanced lead for Deciphera's CSF1R research program.

Background: Mutations in the RAS/MAPK pathway are a frequent driver of cancer, with oncogenic RAS or RAF mutations occurring in >30% of all cancers. First generation BRAF inhibitors are approved for use for tumors with Class I BRAF mutations (V600X). However, these drugs are not efficacious in RAF dimer mutant and RAS mutant cancers due to paradoxical activation of RAF dimers. Herein, we describe DCC-3084, a potent and selective investigational Switch Control inhibitor of BRAF and CRAF kinase dimers that targets Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers. DCC-3084 combines with inhibitors of additional nodes in the MAPK pathway to potentially target a large unmet medical need in RAS and RAF mutant cancers. Methods: Inhibition of RAF kinases, including off-rate analysis, was measured using recombinant enzymes. X-ray crystallography was used for structure-based drug design. Cellular proliferation was measured using resazurin to monitor cell viability. Synergy in cells was measured using BLISS scores and curve shift analysis. Inhibition of ERK or RSK phosphorylation was measured by AlphaLISA or ELISA. Pharmacokinetics (PK) in the plasma, brain and CSF compartments were measured following oral dosing in Wistar rats. RAF and RAS mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 is a potent and selective Switch Control inhibitor of RAF dimers that was designed to target Class I, II, III BRAF mutants, BRAF fusions, and BRAF/CRAF heterodimers. DCC-3084 inhibits BRAF and CRAF, exhibiting slow off-rates (t1/2 >20 hr). Potent single-agent inhibition of MAPK pathway signaling and cellular proliferation was observed in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. Synergy was observed in combination with inhibitors of other nodes in the RAS/MAPK pathway in RAS mutant cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the RAS/MAPK pathway in PK/PD models. DCC-3084 accumulated in tumor tissue relative to plasma, further demonstrating a favorable pharmaceutical profile. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in BRAF mutant and KRAS Q61K mutant mouse xenograft models and tumor growth inhibition in KRAS G12C/D mutant models. Additionally, DCC-3084 in combination with a MEKi resulted in tumor regression in KRAS mutant models. Conclusions: The Switch Control inhibitor DCC-3084 broadly inhibits Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers leading to tumor regression in preclinical models. The overall preclinical profile of DCC-3084 supports IND-enabling activities towards clinical development in a key area of unmet medical need in RAS and RAF mutant cancers. Citation Format: Stacie L. Bulfer, Bertrand Le Bourdonnec, Jeffery D. Zwicker, Yu Mi Ahn, Gada Al-Ani, Hikmat Al-Hashimi, Chase Crawley, Kristin M. Elliott, Saqib Faisal, Andrew M. Harned, Cale L. Heiniger, Molly M. Hood, Salim Javed, Michael Kennedy, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, Max D. Petty, Hunter R. Picard, Justin T. Proto, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Hanumaiah Telikepalli, Mary J. Timson, Lakshminarayana Vogeti, Subha Vogeti, Sihyung Yang, Lexy H. Zhong, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a RAF dimer inhibitor, broadly inhibits BRAF class I, II, III, BRAF fusions, and RAS-driven solid tumors leading to tumor regression in preclinical models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4045.

Background: The Integrated Stress Response (ISR) is a major adaptive stress response pathway in cancer cell maintenance. The ISR kinase family member PERK controls one of the three arms of the Unfolded Protein Response (UPR). The UPR is considered an Achilles’ heel in B-cell cancers. Multiple myeloma (MM) and B-cell lymphomas are dependent on a well-balanced UPR pathway to cope with the high demand for protein folding and their secretory nature. Given the double-edge sword nature of the UPR, the activation of PERK and downstream pathway can have cytoprotective or cytotoxic effects. In B-cell cancers the UPR is at close to maximum cytoprotective capacity, such that further pharmacological stimulation of PERK can potentially be leveraged to cause a cancer cell cytotoxic response and induce antitumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular assays of PERK activation assessed ATF4 by ELISA. Cellular assays of GCN2 modulation assessed phospho-GCN2 and ATF4 by Western blot or ELISA (under basal or low amino acid conditions). DP-9024-induced upregulation of components of the ISR/UPR pathway (ATF4, CHOP) or the apoptosis pathway (PARP and Caspase 3/7) was measured by Western blot or ELISA assays. Compound-mediated PERK activation was investigated mechanistically using a cellular nanoBRET dimerization assay. In vivo upregulation of tumoral ATF4 was determined in a MM PK/PD xenograft model. In vivo inhibition of tumor growth was determined in MM and B-cell lymphoma xenografts. Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to upregulate the ISR/UPR pathway (ATF4, CHOP). The mechanism by which DP-9024 treatment induced the UPR pathway was found to be through the dimerization and activation of PERK. Upregulation of the UPR pathway downstream of PERK led to induction of apoptosis (PARP and Caspase 3/7) in MM and B-cell lymphoma lines in vitro. DP-9024 mediated activation of the UPR pathway in cell lines with high basal level of endoplasmic reticulum (ER) stress led to growth arrest in combination with FDA approved therapies. Oral dosing of DP-9024 in MM xenograft models induced ATF4, and combination efficacy was observed in MM and B-cell lymphoma xenografts in combination with FDA approved agents in vivo. Conclusions: The ISR/UPR is a targetable vulnerability in cancers with high basal levels of ER stress. DP-9024 increases UPR signaling via activating PERK dimerization. This novel mechanism leads to antitumoral effects in B-cell cancers in vitro and in vivo likely through the induction of unresolved ER stress, which may potentially provide an alternative mechanism to current UPR targeting therapies. Citation Format: Gada Al-Ani, Qi Groer, Aaron J. Rudeen, Kristin M. Elliott, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javed, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9024, an investigational small molecule modulator of the Integrated Stress Response kinase PERK, causes B-cell cancer growth inhibition as single agent and in combination with standard-of-care agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1640.

Introduction: Gastrointestinal stromal tumors (GISTs) are typically driven by primary mutations in KIT exons 9 or 11. Heterogeneous drug-resistant secondary mutations arise in patients treated with FDA approved KIT inhibitors, including imatinib and sunitinib. Drug resistant secondary mutations are found at multiple regions in the ATP pocket (encoded by exons 13 and 14) or activation switch (encoded by exons 17 and 18) of KIT kinase. In addition, multiple drug-resistant clones can arise within a tumor or in metastatic tumor sites. An inhibitor that can broadly and potently inhibit the spectrum of KIT mutations is highly sought. Ripretinib has been FDA approved as a 4th line treatment for GIST and has broad activity against KIT mutations, including clinical potency in patients with mutations in KIT exons 11, 17, or 18. DCC-3009 was designed as a next generation KIT inhibitor that broadly and potently inhibits primary KIT mutations in exons 9 and 11 and secondary drug-resistant mutations across exons 13, 14, 17, and 18. DCC-3009 is a potent and selective inhibitor in enzyme and cell-based assays, and has demonstrated efficacy in xenograft models driven by drug resistant KIT mutations. Methods: DCC-3009 was tested for inhibition of KIT mutants using standard enzyme and cell-based assays. Levels of phosphorylated KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. KIT mutant xenograft or patient-derived xenograft models were performed at Crown Biosciences or Labcorp, AAALAC accredited facilities, with the approval of Animal Care and Use Committees. Results: In BaF3 cells transfected with KIT mutants, DCC-3009 was shown to potently inhibit the spectrum of known primary and secondary drug-resistant mutations in GIST. The pan-mutant KIT profile of DCC-3009 was shown in vitro to be superior to 2nd and 3rd line standard of care therapies sunitinib and regorafenib. DCC-3009 was selective for KIT when screened against a large panel of kinases. DCC-3009 has optimized pharmaceutical properties for oral administration. In pharmacokinetic/pharmacodynamic studies DCC-3009 achieved sufficient free drug levels to significantly inhibit drug-resistant KIT mutants for 12 hr post dose. In xenograft studies, treatment with DCC-3009 twice daily led to tumor regression in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Conclusions: DCC-3009 is a pan-exon mutant KIT inhibitor exhibiting high potency in KIT mutants in pre-clinical models spanning exons 9, 11, 13, 14, 17 and 18. In vivo, DCC-3009 exhibited efficacy in drug-resistant models with KIT exon 9/13, 11/13 or 11/17 mutations. Based on this profile, DCC-3009 has entered formal preclinical development. Citation Format: Bryan D. Smith, Subha Vogeti, Timothy M. Caldwell, Hanumaiah Telikepalli, Yu Mi Ahn, Gada Al-Ani, Stacie L. Bulfer, Andrew Greenwood, Cale L. Heiniger, Joshua W. Large, Cynthia B. Leary, Wei-Ping Lu, Kylie Luther, William C. Patt, Max D. Petty, Yeni K. Romero, Forrest A. Stanley, Kristen L. Stoltz, Daniel C. Tanner, Sihyung Yang, Yu Zhan, Bertrand Le Bourdonnec, Daniel L. Flynn. Pan-exon mutant KIT inhibitor DCC-3009 demonstrates tumor regressions in preclinical gastrointestinal stromal tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4033.

Background: The integrated stress response (ISR) is a major adaptive pathway stress response pathway and plays an important role in cell fate determination in response to stress. Oncogene addicted tumors are under high levels of stress, both extrinsic as well as intrinsic, and are dependent on a well-balanced ISR to cope with the high metabolic demands for accelerated growth. The ISR is a double edge sword of survival and cell death, and depending on context, activation of the ISR kinase GCN2 can have either cytoprotective or cytotoxic effects. Activation of GCN2 was identified as a resistance mechanism to asparaginase (ASNase) in asparagine synthetase (ASNS)-low leukemic cells and MAPK-driven solid tumors.1-3 The inhibition of GCN2 in the context of ASNase-resistant leukemic cells can be pharmacologically leveraged to induce anti-tumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. ISR pathway modulation was assessed using cellular assays of phospho-GCN2 and ATF4 by Western blot or ELISA under basal, ASNase-treated, or amino acid starvation conditions. Re-sensitization of leukemic cells to ASNase was tested in cell proliferation assays in vitro. In vivo compound-mediated reversal of ASNase-induced upregulation of tumoral ATF4 was determined in a leukemia PK/PD xenograft model. Inhibition of tumor growth was determined in leukemia xenograft models in vivo. Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to reverse ASNase-mediated upregulation of ISR readouts (phospho-GCN2 and ATF4) in leukemic cells in vitro. Additionally, DP-9024-mediated inhibition of GCN2 was found to re-sensitize leukemic cells to ASNase in vitro. In vivo asparagine depletion by Leunase (L-asparaginase) led to upregulation of ATF4 in leukemia tumors in a PK/PD xenograft model and oral dosing of DP-9024 reversed Leunase-mediated upregulation of tumoral ATF4 down to basal levels. Importantly, we found that DP-9024-mediated inhibition of GCN2 strongly synergized with ASNase in vivo, leading to potent inhibition of tumor growth in leukemic xenografts and causing a reversal of ASNase resistance in leukemic tumors in vivo. Conclusions: The ISR kinase GCN2 was identified as a resistance mechanism to ASNase in ASNS-low leukemic cells. Inhibition of the ISR pathway through the potent and selective small molecule modulator of GCN2, DP-9024, synergized with ASNase and re-sensitized leukemic cells to amino acid withdrawal in vitro as well as in leukemic xenograft models in vivo. Re*****ferences: Nakamura et al. 2018. PNAS 115(33):E7776-E7785 Apfel et al. 2021. ACS Pharamacology and Trans Sci 4(1):327-337 Gwin et al. 2018. Cancer Cell 33(1):91-107 Citation Format: Gada Al-Ani, Kristin M. Elliott, Qi Groer, Aaron J. Rudeen, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javed, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9024, an investigational small molecule modulator of the integrated stress response kinase GCN2, synergizes with asparaginase therapy in leukemic tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4938.

Background: The Integrated Stress Response (ISR) is a major adaptive stress response pathway in cancers. The ISR kinase family member PERK controls one of the three arms of the Unfolded Protein Response (UPR). The UPR is considered an Achilles’ heel in B-cell cancers. Myelomas and B-cell lymphomas are dependent on a well-balanced UPR pathway to cope with the high demand for protein folding and their secretory nature. Given the double-edge sword nature of the UPR, the activation of PERK and downstream pathway can have cytoprotective or cytotoxic effects. In B-cell cancers the UPR is at close to maximum cytoprotective capacity, such that further pharmacological stimulation of PERK drives a cytotoxic outcome leveraged to induce antitumoral effects. Methods: Recombinant WT and mutant PERK constructs were assayed in the presence of DP-9024. Structures of compound-bound PERK were determined by X-ray crystallography. Kinome profiling was determined using enzymatic and cellular assays. Cellular modulation of the ISR/UPR pathway (phospho-GCN2, PERK, ATF4, CHOP) or the apoptosis pathway (cleaved-PARP, cleaved-Caspase 3/7) was measured by Western blot or ELISA. The level of DP-9024-induced PERK activation was determined using a cellular nanoBRET dimerization assay utilizing WT and mutant PERK constructs. Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to upregulate the ISR/UPR pathway (ATF4, CHOP). The mechanism by which DP-9024 induced the UPR pathway was found to be through dimerization-dependent activation of PERK. Utilizing recombinant biophysical and cellular assays of WT and mutant PERK constructs, we found that DP-9024 directly binds to a switch control site in the kinase domain of PERK that governs dimerization and that the binding of the compound to one monomer was sufficient to induce dimerization-mediated activation of the unoccupied monomer. This paradoxical stimulation of the unbound PERK monomer is reminiscent of the phenomenon observed with some BRAF inhibitors.1 X-ray crystallography studies revealed that PERK crystalizes as a dimer with both monomers bound to compound, due to the high concentration of compound used during crystallization. DP-9024-mediated PERK dimerization and transactivation led to the activation of downstream pathways (ATF4, CHOP), apoptotic pathway (Caspase 3/7, PARP1), and growth arrest in cell lines with high levels of endoplasmic reticulum (ER) stress such as multiple myeloma and B-cell lymphoma. Conclusions: Paradoxical stimulation of the ISR family member kinase PERK, through direct binding and dimerization by DP-9024, led to unresolved ER stress that can potentially be leveraged as a novel mechanism to induce growth arrest in UPR vulnerable cancers, including myelomas and B-cell lymphomas. References: 1. Poulikakos et al. 2010. Nature 464:427-30 Citation Format: Gada Al-Ani, Aaron J. Rudeen, Qi Groer, Kristin M. Elliott, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javid, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. Dimerization-induced activation of the integrated stress response kinase PERK by an investigational small molecule modulator, DP-9024 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1613.

Background: The Integrated Stress Response (ISR) is one of the major adaptive stress response pathways in cancer and plays an important role in cell fate determination. Oncogene addicted solid tumors are under high stress levels, both extrinsic as well as intrinsic, and are dependent on a well-balanced ISR pathway activity to cope with the high demand for accelerated growth. The ISR is well known to be a double edge sword of survival and cell death and depending on context, the activation of the ISR kinase, GCN2, and downstream pathway can have either cytoprotective or cytotoxic effects. Given the context-dependent nature of the ISR pathway, the inhibition or stimulation of GCN2 in solid tumors can be pharmacologically leveraged to induce anti-tumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular modulation of the ISR pathway (phospho-GCN2, ATF4, CHOP) or the apoptosis pathway (PARP and Caspase3/7) was assessed via Western blot or ELISA. In vivo upregulation of tumoral ATF4 was determined in a fibrosarcoma PK/PD xenograft model. In vivo inhibition of tumor growth was determined in solid tumor xenografts. Results: Selective and potent modulators of GCN2 kinase with favorable drug-like properties were designed. These compounds were found to upregulate components of the ISR pathway (phospho-GCN2, ATF4, CHOP). The mechanism by which GCN2 modulator DP-9149 treatment induced the ISR pathway was found to be through the direct binding and activation of GCN2. Upregulation of the ISR pathway downstream of GCN2 led to induction of a programmed cell death pathway in oncogene-driven solid tumor cell lines in vitro. DP-9149-mediated activation of the ISR pathway led to cell growth arrest both as a single agent and in combination with standard-of-care (SOC) agents. Furthermore, oral dosing of DP-9149 in RAS mutant and other oncogene-driven xenograft models in vivo induced ATF4, and significantly inhibited tumor growth as a single agent and in combination with SOC agents. Additionally, therapeutic agents targeting the tumor microenvironment, including anti-angiogenic agents, synergized with DP-9149 to induce tumor regressions in vivo. Conclusions: The ISR is a targetable vulnerability in oncogene addicted solid tumors. Upregulating the ISR by paradoxical activation of the ISR family member kinase, GCN2, by DP-9149 can be leveraged as a novel mechanism to cause anti-tumoral effects in solid tumors in vitro and in vivo, likely through the induction of an unresolved stress response. In particular, DP-9149 exhibited robust activity in RAS mutant cancers and in VHL-mutant renal cancers as a single agent and in combination with SOC agents in vivo. Citation Format: Gada Al-Ani, Qi Groer, Kristin M. Elliott, Aaron J. Rudeen, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javid, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9149, an investigational small molecule modulator of the Integrated Stress Response kinase GCN2, pre-clinically causes solid tumor growth inhibition as a single agent and regression in combination with standard of care agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1639.

Summary Stearoyl-CoA desaturase (SCD) is a potential therapeutic target for Parkinson’s and related neurodegenerative diseases. SCD inhibition ameliorates neuronal toxicity caused by aberrant α-synuclein, a lipid-binding protein implicated in Parkinson’s disease. Its inhibition depletes monounsaturated fatty acids, which may modulate α-synuclein conformations and membrane interactions. Herein, we characterize the pharmacokinetic and pharmacodynamic properties of YTX-7739, a clinical-stage SCD inhibitor. Administration of YTX-7739 to rats and monkeys for 15 days caused a dose-dependent increase in YTX-7739 concentrations that were well-tolerated and associated with concentration-dependent reductions in the fatty acid desaturation index (FADI), the ratio of monounsaturated to saturated fatty acids. An approximate 50% maximal reduction in the carbon-16 desaturation index was observed in the brain, with comparable responses in the plasma and skin. A study with a diet supplemented in SCD products indicates that changes in brain C16 desaturation were due to local SCD inhibition, rather than to changes in systemic fatty acids that reach the brain. Assessment of pharmacodynamic response onset and reversibility kinetics indicated that approximately 7 days of dosing were required to achieve maximal responses, which persisted for at least 2 days after cessation of dosing. YTX-7739 thus achieved sufficient concentrations in the brain to inhibit SCD and produce pharmacodynamic responses that were well-tolerated in rats and monkeys. These results provide a framework for evaluating YTX-7739 pharmacology clinically as a disease-modifying therapy to treat synucleinopathies.

Based on knowledge of kinase switch-control inhibition and using a combination of structure-based drug design and standard medicinal chemistry principles, we identified a novel series of dihydropyrimidone-based CSF1R kinase inhibitors displaying exquisite selectivity for CSF1R versus a large panel of kinases and non-kinase protein targets. Starting with lead compound 3, an SAR optimization campaign led to the discovery of vimseltinib (DCC-3014; compound 20) currently undergoing clinical evaluation for the treatment of Tenosynovial Giant Cell Tumor (TGCT), a locally aggressive benign tumor associated with substantial morbidity. 2021 Elsevier ltd. All rights reserved.

N,N-diethyl-4-(5-hydroxyspiro[chromene-2,4′-piperidine]-4-yl) benzamide (ADL5859) and N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4′-piperidine]-4-yl)benzamide (ADL5747) are novel δ-opioid agonists that show good oral bioavailability and analgesic and antidepressive effects in the rat and represent potential drugs for chronic pain treatment. Here, we used genetic approaches to investigate molecular mechanisms underlying their analgesic effects in the mouse. We tested analgesic effects of ADL5859 and ADL5747 in mice by using mechanical sensitivity measures in both complete Freund's adjuvant and sciatic nerve ligation pain models. We examined their analgesic effects in δ-opioid receptor constitutive knockout (KO) mice and mice with a conditional deletion of δ-receptor in peripheral voltage-gated sodium channel (Nav)1.8-expressing neurons (cKO mice). Both ADL5859 and ADL5747, and the prototypical δ agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethyl-piperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethyl-benzamide (SNC80) as a control, significantly reduced inflammatory and neuropathic pain. The antiallodynic effects of all three δ-opioid agonists were abolished in constitutive δ-receptor KO mice and strongly diminished in δ-receptor cKO mice. We also measured two other well described effects of δ agonists, increase in locomotor activity and agonist-induced receptor internalization by using knock-in mice expressing enhanced green fluorescence protein-tagged δ receptors. In contrast to SNC80, ADL5859 and ADL5747 did not induce either hyperlocomotion or receptor internalization in vivo. In conclusion, both ADL5859 and ADL5747 showed efficient pain-reducing properties in the two models of chronic pain. Their effects were mediated by δ-opioid receptors, with a main contribution of receptors expressed on peripheral Nav1.8-positive neurons. The lack of in vivo receptor internalization and locomotor activation, typically induced by SNC80, suggests agonist-biased activity at the receptor for the two drugs.

In Parkinson's disease (PD), dyskinesia develops following long-term treatment with 3,4-dihydroxyphenylalanine (L-dopa). Given the prominent role of the opioid system in basal ganglia function, nonselective opioid receptor antagonists have been tested for antidyskinetic efficacy in the clinic (naltrexone and naloxone), although without success. In the current study, ADL5510, a novel, orally active opioid antagonist with mu opioid receptor selectivity, was examined in L-dopa-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) macaques. Antidyskinetic effects were compared with those of naltrexone. Parkinsonian monkeys with established L-dopa-induced dyskinesia (LID) received acute challenges with L-dopa (subcutaneously) in combination with either vehicle, ADL5510 (0.1, 1, 3 or 10 mg/kg by mouth), or naltrexone (1, 3, or 10 mg/kg subcutaneously). Following treatments, behavior was monitored for 6 hours. Parameters assessed were total activity, parkinsonism, and dyskinesia. ADL5510 (1, 3, and 10 mg/kg) reduced activity and LID (chorea and dystonia) without affecting the antiparkinsonian benefits of L-dopa. The antidyskinetic effect of ADL5510 showed a U-shaped dose-response. It was inactive at 0.1 mg/kg, efficacious at 1 and 3 mg/kg (72% and 40% reductions, respectively), and then less effective at 10 mg/kg. The quality of ON time produced by L-dopa was improved, as indicated by a reduction in the percentage of ON time spent experiencing disabling dyskinesia (70% and 61% reductions with 1 and 3 mg/kg, respectively, compared with L-dopa). Naltrexone, in contrast, did not alleviate LID or affect the antiparkinsonian actions of L-dopa. Mu-selective opioid antagonists have the potential to form the basis of novel antidyskinetic therapies for PD.

A lead optimization campaign in our previously reported sulfamoyl benzamide class of CB2 agonists was conducted to improve the in vitro metabolic stability profile in this series while retaining high potency and selectivity for the CB2 receptor. From this study, compound 14, N-(3,4-dimethyl-5-(morpholinosulfonyl)phenyl)-2,2-dimethylbutanamide, was identified as a potent and selective CB2 agonist exhibiting moderate in vitro metabolic stability and oral bioavailability. Compound 14 demonstrated in vivo efficacy in a rat model of post-surgical pain.

A series of imidazopyrimidine derivatives with the general formula I was synthesized and identified as potent inhibitors of iNOS dimer formation, a prerequisite for proper functioning of the enzyme. Stille and Negishi coupling reactions were used as key steps to form the carbon-carbon bond connecting the imidazopyrimidine core to the central cycloalkenyl, cycloalkyl and phenyl ring templates.

A general and efficient synthetic approach to tricyclic spiroketones of interest as useful scaffolds in drug discovery was developed. Starting from commercially available benzyl 4-oxo-1-piperidinecarboxylate (5), spirocyclic tetralone 4, spirocyclic indanone 14, and spirocyclic benzocycloheptanone 15 were synthesized via six reaction steps in excellent overall yield.

Previous research within our laboratories identified sulfamoyl benzamides as novel cannabinoid receptor ligands. Optimization of the amide linkage led to the reverse amide 40. The compound exhibited robust antiallodynic activity in a rodent pain model when administered intraperitoneally. Efficacy after oral administration was observed only when ABT, a cytochrome P450 suicide inhibitor, was coadministered.

Selective delta opioid receptor agonists are promising potential therapeutic agents for the treatment of various types of pain conditions. A spirocyclic derivative was identified as a promising hit through screening. Subsequent lead optimization identified compound 20 (ADL5859) as a potent, selective, and orally bioavailable delta agonist. Compound 20 was selected as a clinical candidate for the treatment of pain.

A series of N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines, mu opioid receptor antagonists, analogs of alvimopan, were prepared using solid phase methodology. This study led to the identification of a highly selective mu opioid receptor antagonist, which interacts selectively with mu peripheral receptors.

Incorporation of a naphthalene-dialdehyde moiety into the delta antagonist, 6'-aminonaltrindole afforded a potent, selective, irreversible delta-agonist 1. However, flow cytometry studies revealed no time-dependent specific fluorescence, suggesting that both Lys214 and Cys216 at the recognition site are not involved in covalent binding. Molecular simulation studies suggest that compound 1 may form a Schiff base with the epsilon-amino group of Lys214, which could explain its irreversibility and transformation into a delta-agonist through a conformational change of TM5.

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC5010 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

N-alkyl pyrazolidine-3,5-diones and tetrahydropyridazine-3,6-diones have been synthesized by a new method in a three-step sequence from dialkyl malonates or succinates respectively.

In Parkinson's disease (PD), dyskinesia develops following long-term treatment with 3,4-dihydroxyphenylalanine (L-dopa). Given the prominent role of the opioid system in basal ganglia function, nonselective opioid receptor antagonists have been tested for antidyskinetic efficacy in the clinic (naltrexone and naloxone), although without success. In the current study, ADL5510, a novel, orally active opioid antagonist with mu opioid receptor selectivity, was examined in L-dopa-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) macaques. Antidyskinetic effects were compared with those of naltrexone. Parkinsonian monkeys with established L-dopa-induced dyskinesia (LID) received acute challenges with L-dopa (subcutaneously) in combination with either vehicle, ADL5510 (0.1, 1, 3 or 10 mg/kg by mouth), or naltrexone (1, 3, or 10 mg/kg subcutaneously). Following treatments, behavior was monitored for 6 hours. Parameters assessed were total activity, parkinsonism, and dyskinesia. ADL5510 (1, 3, and 10 mg/kg) reduced activity and LID (chorea and dystonia) without affecting the antiparkinsonian benefits of L-dopa. The antidyskinetic effect of ADL5510 showed a U-shaped dose-response. It was inactive at 0.1 mg/kg, efficacious at 1 and 3 mg/kg (72% and 40% reductions, respectively), and then less effective at 10 mg/kg. The quality of ON time produced by L-dopa was improved, as indicated by a reduction in the percentage of ON time spent experiencing disabling dyskinesia (70% and 61% reductions with 1 and 3 mg/kg, respectively, compared with L-dopa). Naltrexone, in contrast, did not alleviate LID or affect the antiparkinsonian actions of L-dopa. Mu-selective opioid antagonists have the potential to form the basis of novel antidyskinetic therapies for PD.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

The crystal structures of three angiotensin-II receptor antagonists involving different spacer groups (CO, CONH and NHCO) between the aryl rings are presented, namely 2-{4-[(3-butyl-1,4-dioxo-2,3-di­aza­spiro­[4.4]­non-2-yl)­methyl]­benzoyl}benzoic acid, C26H28N2O5, (I), 2-{4-[(3-butyl-1,4-dioxo-2,3-di­aza­spiro­[4.4]­non-2-yl)­methyl]­benz­amido}benzoic acid, C26H29N3O5, (II), and 2-{4-[(3-butyl-1,4-dioxo-2,3-di­azaspiro­[4.4]­non-2-yl)­methyl]­anilino­carbonyl}benzoic acid monohydrate, C26H29N3O5·H2O, (III). The aryl rings of (II) are almost coplanar, in contrast with compounds (I) and (III). The conformation of (II) is induced by an intramolecular N—H⋯O hydrogen bond between the amide and carboxyl­ic acid groups.

Adolor Corporation, 700 PennsylVania DriVe, Exton, PennsylVania 19341, Semafore Pharmaceuticals, Inc., 8496 Georgetown Road, Indianapolis, Indiana 46268, NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center DriVe, RockVille, Maryland 20850, and Department of Chemistry, UniVersity of Massachusetts Boston, 100 Morrissey BouleVard, Boston, Massachusetts 02125

Selective, nonpeptidic delta opioid receptor agonists have been the subject of great interest as potential novel analgesic agents. The discoveries of BW373U86 (1) and SNC80 (2) contributed to the rapid expansion of research in this field. However, poor drug-like properties and low therapeutic indices have prevented clinical evaluation of these agents. Doses of 1 and 2 similar to those required for analgesic activity produce convulsions in rodents and nonhuman primates. Recently, we described a novel series of potent, selective, and orally bioavailable delta opioid receptor agonists. The lead derivative, ADL5859 (4), is currently in phase II proof-of-concept studies for the management of pain. Further structure activity relationship exploration has led to the discovery of ADL5747 (36), which is approximately 50-fold more potent than 4 in an animal model of inflammatory pain. On the basis of its favorable efficacy, safety, and pharmacokinetic profile, 36 was selected as a clinical candidate for the treatment of pain.

Among the various human cytochrome P450s (CYP450s) that catalyze the biotransformation of xenobiotics, CYP450 2D6 (CYP2D6) is one of the most important based on the number and wide variety of its drug substrates. CYP2D6 shows a high degree of interindividual variability, which is primarily due to the extensive genetic polymorphism that influences its expression and function. A number of drugs have been clinically implicated in major drug-drug interactions (DDI) via CYP2D6 inhibition. In order to avoid or minimize issues related to CYP2D6-mediated DDIs, pharmaceutical companies routinely screen for potential CYP2D6 liability of lead candidates in the early stage of the drug discovery process. This review summarizes the medicinal chemistry tactics employed to mitigate inhibitory activity at CYP2D6, identified through an extensive literature survey covering the 1998-2008 period.

Replacement of the phenyl ring in our previous (morpholinomethyl)aniline carboxamide cannabinoid receptor ligands with a pyridine ring led to the discovery of a novel chemical series of CB2 ligands. Compound 3, that is, 2,2-dimethyl-N-(5-methyl-4-(morpholinomethyl)pyridin-2-yl)butanamide was identified as a potent and selective CB2 agonist exhibiting in vivo efficacy after oral administration in a rat model of neuropathic pain.

For thousands of years mu opioid agonists such as morphine have been utilized for their analgesic properties. Today, morphine and related compounds are still used as a first line therapy in the treatment of moderate to severe pain. However, despite the clear benefits of mu agonists in pain management, severe side effects such as dependence and respiratory depression are associated with use of these drugs. To date, there are only two approved mu opioid antagonists for use in the treatment of these adverse effects, that is, naloxone and naltrexone. However, many other clinical and therapeutic areas have been linked to mu opioid receptor antagonism. These include treatment of opioid induced pruritis of the skin, obesity, and Parkinson-induced tardive dyskinesia. Currently there are two compounds, N-methylnaltrexone and alvimopan, under FDA review as possible treatments for opioid induced bowel dysfunction and postoperative ileus. These compounds are of special interest as they are peripherally restricted. This attribute enables treatment of peripheral side effects induced by opioid agonists without reversal of the centrally mediated analgesia of the agonist. In this article we discuss the structural classes of mu opioid antagonists, their potential clinical applications, and review the relevant patents of the last ten years.

Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes of nitric oxide synthase (NOS). Potential candidate clinical drugs should be devoid of inhibitory activity against endothelial NOS (eNOS), since eNOS plays an important role in maintaining normal blood pressure and flow. A new series of aminopiperidines as potent inhibitors of iNOS were identified from a HTS lead. From this study, we identified compound 33 as a potent iNOS inhibitor, with >25-fold selectivity over eNOS and 16-fold selectivity over nNOS.

The ninth annual review provides listings of chemical libraries, solid-phase reagents and scavengers, linkers, and polymer-supported chiral ligands reported during the year 2005.

To better understand structural requirements for a mu ligand of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class to interact with the mu opioid receptor, we have described in the previous article (Le Bourdonnec, B. et al. J. Med. Chem. 2006, 25, 7278-7289) new, constrained analogues of the N-phenethyl derivative 3. One of the active constrained analogues, compound 4, exhibited subnanomolar mu-opioid receptor affinity (K(i) = 0.62 nM) and potent mu-opioid antagonist activity (IC(50) = 0.54 nM). On the basis of structure 4, a new series of mu-opioid receptor antagonists were designed. In these compounds the octahydroquinolizine template of 4 was replaced by an octahydro-1H-pyrido[1,2-a]pyrazine scaffold. The new derivatives were tested for their binding affinities and in vitro functional activity against the cloned human mu-, delta-, and kappa-opioid receptors. From this study, we identified compound 36, which displays high affinity toward the mu-opioid receptor (K(i) = 0.47 nM), potent mu in vitro antagonist activity (IC(50) = 1.8 nM) and improved binding selectivity profile mu/kappa and mu/delta, when compared to 4.

Structure–activity relationships at the 2α-position of the piperidine ring of the trans-4,5-dimethyl-4-(3-hydroxyphenyl)piperidine μ-opioid antagonist series were investigated. This study showed that only small linear alkyl groups (methyl, propyl) are tolerated at the 2α-position of the piperidine ring of this series.

trans-3,4-Dimethyl-4-(3-carboxamidophenyl)piperidines constitute a novel class of micro opioid receptor antagonists. The CONH(2) group was found to be an effective isostere of the phenolic OH moiety. Structure-activity relationships at the piperidine nitrogen position led to the identification of several ligands displaying high affinity toward the cloned human micro opioid receptors, good selectivity micro/delta, micro/kappa, and potent in vitro antagonist activity.

A new series of nonpeptide AT(1) receptor antagonists were recently developed, based on the structure of irbesartan (Le Bourdonnec et al. J. Med. Chem. 2000, 43, 2685-2697). The lead compound 1 displayed high selectivity for the AT(1) receptor subtype but lower binding affinity than irbesartan. As expected from molecular modeling studies, extension of the pyrazolidine-3,5-dione scaffold to the six-membered heterocycle tetrahydropyridazine-3,6-dione led to an enhancement of the binding affinity toward the AT(1) receptor.

To develop ligands with fluorogenic properties amenable for following the kinetics of cross-linking to receptors, a naphthalene dicarboxaldehyde moiety has been attached to an opiate pharmacophore 2 and evaluated in mu-opioid receptors. The fluorescence of the benzo[f]isoindole formed upon cross-linking of mu-opioid receptors by 2 permitted the time-course of covalent bonding to be followed. This demonstrated proof-of-concept suggests the usefulness of naphthalene dicarboxaldehyde-containing affinity labels as kinetic probes.

The crystal structures of three angiotensin-II receptor antagonists involving different spacer groups (CO, CONH and NHCO) between the aryl rings are presented, namely 2-[4-[(3-butyl-1,4-dioxo-2,3-diazaspiro[4.4]non-2-yl)methyl]benzoyl]benzoic acid, C(26)H(28)N(2)O(5), (I), 2-[4-[(3-butyl-1,4-dioxo-2,3-diazaspiro[4.4]non-2-yl)methyl]benzamido]benzoic acid, C(26)H(29)N(3)O(5), (II), and 2-[4-[(3-butyl-1,4-dioxo-2,3-diazaspiro[4.4]non-2-yl)methyl]anilinocarbonyl]benzoic acid monohydrate, C(26)H(29)N(3)O(5) x H(2)O, (III). The aryl rings of (II) are almost coplanar, in contrast with compounds (I) and (III). The conformation of (II) is induced by an intramolecular N-H.O hydrogen bond between the amide and carboxylic acid groups.

N-alkyl pyrazolidine-3,5-diones and tetrahydropyridazine-3,6-diones have been synthesized by a new method in a three-step sequence from dialkyl malonates or succinates respectively.