We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.

We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion protein in KM12 colorectal carcinoma cells and inhibited downstream PLCγ1 signaling at sub-nanomolar concentrations. Both degraders also degraded human wild-type TRKA with similar potency. Interestingly, both degraders, especially 6, showed selectivity for the degradation of endogenous TPM3-TRKA over ectopically expressed ATP/GTP binding protein-like 4 (AGBL4)-TRKB or ETS variant transcription factor 6 (ETV6)-TRKC fusion proteins in KM12 cells. Global proteomic profiling assays demonstrated that 5 is highly selective for the intended target. TPM3-TRKA protein degradation induced by 5 and 6 was further confirmed to be mediated through cereblon and the ubiquitin-proteasome system. Compared with the parental TRK kinase inhibitor, both degraders exhibited higher potency for inhibiting growth of KM12 cells. Moreover, both 5 and 6 showed good plasma exposure levels in mice. Therefore, 5 and 6 are valuable chemical tool compounds for investigating the in vivo function of TRK fusion during tumorigenesis. Our study also paves the way for pharmacological degradation of TRK.

BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.

Abstract Small molecule-mediated targeted protein degradation offers a systemic approach to deplete disease-causing proteins. The tropomyosin receptor kinase (TRK) family kinases are receptors for neurotrophic factors and primarily function in the central nervous system. As results of chromosomal rearrangement events, TRK kinases are re-expressed as fusion proteins and implicated in a wide diversity of human malignancies. These fusion proteins universally retain the kinase domain and function as key oncodrivers. Two TRK kinase inhibitors, entrectinib and larotrectinib, have been approved to treat TRK fusion-expressing cancers with compelling clinical evidence, highlighting the significance of TRK fusion kinases as oncology targets. In the current study, we report the development of selective TRK degraders. Lead compounds induced rapid degradation of different TRK fusion variants identified in patients at subnanomolar concentrations. Degradation of the fusion kinases significantly compromised TRK-dependent cancer cell growth with low nanomolar IC50. The lead compound exhibited approximately 16% oral bioavailability in mouse and effectively controlled TRK-driven xenograft tumor growth. Pharmacodynamic analysis demonstrated sustained degradation of the TRK fusion in xenograft tumors. Collectively, these findings provide the framework to develop a novel approach that modulates oncogenic TRK fusion kinases for cancer treatment. Citation Format: Xiaoran Han, Liqun Chen, Chengwei Zhang, Bingyang Jiao, Yanke Chen, Jing Liu, Michael Plewe, Jialiang Wang. Targeted protein degradation for the treatment of TRK fusion-driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5331.

We identified and explored the structure-activity-relationship (SAR) of an adamantane carboxamide chemical series of Ebola virus (EBOV) inhibitors. Selected analogs exhibited half-maximal inhibitory concentrations (EC50 values) of ∼10-15 nM in vesicular stomatitis virus (VSV) pseudotyped EBOV (pEBOV) infectivity assays, low hundred nanomolar EC50 activity against wild type EBOV, aqueous solubility >20 mg/mL, and attractive metabolic stability in human and nonhuman liver microsomes. X-ray cocrystallographic characterizations of a lead compound with the EBOV glycoprotein (GP) established the EBOV GP as a target for direct compound inhibitory activity and further provided relevant structural models that may assist in identifying optimized therapeutic candidates.

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.

Corrigendum Corrigendum to ‘‘Design and synthesis of a novel pyrrolidinyl pyrido pyrimidinone derivative as a potent inhibitor of PI3Ka and mTOR’’ [Bioorg. Med. Chem. Lett. 22 (2012) 5098–5103] Phuong T. Le a,⇑, Hengmiao Cheng , Sacha Ninkovic , Michael Plewe , Xiaojun Huang , Hai Wang , Shubha Bagrodia , Shaoxian Sun , Daniel R. Knighton , Caroline M. LaFleur Rogers , Andrew Pannifer , Samantha Greasley , Deepak Dalvie , Eric Zhang c Cancer Chemistry, Pfizer Worldwide Research & Development, La Jolla Laboratories, Pfizer Inc., La Jolla, CA 92121, United States Oncology Research Unit, Pfizer Worldwide Research & Development, La Jolla Laboratories, Pfizer Inc., La Jolla, CA 92121, United States PDM, Pfizer Worldwide Research & Development, La Jolla Laboratories, Pfizer Inc., La Jolla, CA 92121, United States d Pfizer Worldwide Research & Development, Sandwich Laboratories, Pfizer Inc., UK

Lead optimization efforts that employed structure base drug design and physicochemical property based optimization leading to the discovery of a novel series of 4-methylpyrido pyrimidinone (MPP) are discussed. Synthesis and profile of 1, a PI3Kα/mTOR dual inhibitor, is highlighted.

HIV-1 integrase (IN) is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the synthesis of orally bioavailable azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. Here we disclose the design and synthesis of novel tricyclic N-hydroxy-dihydronaphthyridinones as potent, orally bioavailable HIV-1 integrase inhibitors displaying excellent ligand and lipophilic efficiencies.

Abstract The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss of function of tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Hence, inhibition of the key targets in the pathway, e.g. PI3K, AKT, mTOR, offers great potential for the treatment of cancer. In an effort to discover compounds that inhibit PI3Kα, a high throughput screen was carried out, and 4-methyl-pyrido-pyrimidine (MPP) derivatives were identified as potent and selective inhibitors of PI3Kα. For example, PF-00271897, 8-cyclopentyl-6-[3-(hydroxymethyl)phenyl]-4-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one demonstrated PI3Kα Ki of 2.2 nM. Multiple crystal structures of inhibitors bound to PI3K gamma were determined to inform design and optimization of the ADMET properties of this lead series. Crystallographic studies with PI3K gamma protein indicated that the aminopyrimidine moiety forms two hydrogen bonds to the kinase backbone, and the aromatic moiety at the 6 position binds in a hydrophobic pocket. The X-ray structure suggested that the 4-methyl group on the MPP core structure conferred the excellent overall kinase selectivity to the series. The structure and SAR suggested optimization could come from keeping N-R group at 2 position very small and maintaining aromatic moiety at 6 position for hydrophobic interaction. Introduction of polar groups to the 8N side chains that are located in the ribose binding pocket increased both metabolic stability and solubility. Based on the overall properties, PF-04691502, 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one, was selected as a clinical candidate. PF-04691502 demonstrated Ki's of 1.2-2.2 nM against PI3K α, β, γ and δ isoforms, and Ki of 9.1 nM against recombinant mTOR. PF-04691502 inhibited AKT phosphorylation at S473 in BT20 breast cancer line with IC50 of 12 nM. PF-04691502 is highly selective for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the class III PI3K hVps34. In the in vivo rat PK studies, PF-04691502 demonstrated the following properties: Clearance = 5.2 ml/min/kg, Vdss = 1.4 L/kg, T1/2 = 3.1 h, F% = 63%. The design, synthesis, in vitro potency SAR, selectivity, ADMET of the MPP derivatives will be discussed. The crystal structure of PF-04691502 in PI3K gamma will also be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5779.

HIV-1 integrase is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the discovery of azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. N-Methyl hydroxamic acids were stable against oxidative metabolism, however were cleared rapidly through phase 2 glucuronidation pathways. We were able to introduce polar groups at the β-position of the azaindole core thereby altering physical properties by lowering calculated logD values (cLogD) which resulted in attenuated clearance rates in human hepatocytes. Pharmacokinetic data in dog for representative compounds demonstrated moderate oral bioavailability and reasonable half-lives. These ends were accomplished without a large negative impact on enzymatic and antiviral activity, thus suggesting opportunities to alter clearance parameters in future series.

HIV-1 integrase (IN) is one of three enzymes encoded by the HIV genome and is essential for viral replication. Recently, HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Herein, we report the discovery of azaindole carboxylic acids and azaindole hydroxamic acids as potent inhibitors of the HIV-1 IN enzyme and their structure-activity relationships. Several 4-fluorobenzyl substituted azaindole hydroxamic acids showed potent antiviral activities in cell-based assays and offered a structurally simple scaffold for the development of novel HIV-1 IN inhibitors.

rac-Benzyl 3-oxohexa-hydro-1H-pyrrolo[3,4-c]pyridine-5(6H)-carboxyl-ate was separated by chiral chromatography, and one of the enanti-omers ([α](22) (D) = +10°) was hydrogenated in the presence of Pd/C in methanol, producing octa-hydro-3H-pyrrolo[3,4-c]pyridin-3-one. The latter was reacted with (2R)-3,3,3-trifluoro-2-meth-oxy-2-phenyl-propanoyl chloride [(R)-(-)-Mosher acid chloride], giving rise to the title compound, C(17)H(19)F(3)N(2)O(3)·H(2)O. The present structure established the absolute configuration of the pyrrolopiperidine fragment based on the known configuration of the (R)-Mosher acid chloride. The piperidine ring has a somewhat distorted chair conformation and is cis-fused with the five-membered envelope-shaped ring; the plane of the exocyclic amide bond is approximately orthogonal to the plane of the phenyl ring, making a dihedral angle of 82.31 (3)°. The water mol-ecule acts as an acceptor to the proton of the amino group in an N-H⋯O inter-action, and as a double proton donor in O-H⋯O hydrogen bonds, generating infinite bands along the a axis.

A weak human glucagon receptor antagonist with an IC50 of 7 microM was initially found by screening of libraries originally targeted to mimic the binding of the glucagon-like peptide (GLP-1) hormone to its receptor. Optimization of this hit for binding affinity for the glucagon receptor led to ligands with affinity in the nanomolar range. In addition to receptor binding, optimization efforts were made to stabilize the molecules against fast metabolic turnover. A potent antagonist of the human human glucagon receptor was obtained that had 17% oral availability in rats with a plasma half-life of 90 min. The major metabolites of this lead were identified and used to further optimize this series with respect to pharmacokinetic properties. This final optimization led to a potent glucagon antagonist that was orally available in rats and dogs and was efficacious in lowering blood glucose levels in a diabetic animal model.