Phosphodiesterase 7B (PDE7B) inhibition has been considered as a therapeutic target for the treatment of several neurological disorders. Currently, there are no radio-labeled tracers available to determine receptor occupancy (RO) of this target. Developing such a tracer could greatly facilitate the identification of viable PDE7B inhibitors. In the current study, a liquid chromatography tandem mass spectrometry (LC─MS/MS) method was utilized to evaluate the brain distribution of unlabeled tracer candidates following intravenous micro-dosing. This novel approach resulted in an accelerated identification of a potential novel RO tracer for PDE7B. The identified molecule, Compound 30, showed reasonable target-tissue specificity (striatum/cerebellum ratio of 2.2) and suitable uptake (0.25% of the injected dose/g brain tissue) as demonstrated in rats dosed with the unlabeled compound. Compound 30 was subsequently labeled with tritium (3H). In vitro characterization of 3H-Compound 30 demonstrated that this compound possessed a high target affinity with a subnanomolar Kd (0.8 nM) and a Bmax of 58 fmol/mg of protein using rat brain homogenate. Intravenous microdosing of 3H-Compound 30 showed preferential binding in the rat striatum, consistent with the mRNA distribution of PDE7B. In vitro displacement study with other structurally distinct PDE7B target-specific inhibitors using rat brain homogenate indicated that 3H-Compound 30 is an ideal tracer for Ki analysis. This is the first report of a preclinical tracer for PDE7B. With further characterization, Compound 30 may ultimately show the appropriate properties required to be further developed as a PDE7B PET ligand for clinical studies.

吲哚酮结构是很多药物的重要活性基团,含这类结构的化合物通常具有消炎、杀菌、抗病毒、抗高血压和抗肿瘤等生物活性.吲哚酮类化合物因具有广谱的生物活性和潜在的药效研究价值,是药物研究的热点.按照结构分类,分别为亚胺类、硫脲类、酰腙类、亚甲基类以及联氨类3-取代吲哚酮衍生物,及其在抑菌和抗肿瘤等方面的生物活性进行概述,以期为该类化合物的药物化学方面的研究提供参考价值.

Phosphodiesterase (PDE) inhibition has been broadly investigated as a target for a wide variety of indications including central nervous system (CNS) disorders. Cyclic nucleotide (cNT) changes within associated tissues may serve as a biomarker of PDE inhibition. We recently developed robust sample harvesting and bioanalytical methods to quantify cNT levels in rodent brain and cerebrospinal fluid (CSF). Herein, we report on the application of those methods to study rodent species-specific and rodent brain region-specific cNT changes following individual or concomitant PDE inhibitor administration. Male Sprague Dawley (Crl:CD (R) [SD]) rats were dosed subcutaneously (sc) with a PDE1B inhibitor (DNS-0056), a PDE2A inhibitor (PF-05180999), a PDE9A inhibitor (PF-4447943), and a PDE10A inhibitor (MP10), each at a single dose of 10 or 30 mg/kg, or concomitantly with all 4 inhibitors at 10 mg/kg each. Male Carworth Farms (Crl:CF1 (R)[CF-1]) mice were dosed intraperitoneally (ip) with the four individual inhibitors at a single dose of 10 mg/kg or concomitantly with all 4 inhibitors at 10 mg/kg each. The doses studied are generally adequate for affecting measurable cNT levels in the tissues of interest and were thereby chosen for this investigation. Measured 3',5'-cyclic adenosine monophosphate (cAMP) changes were generally statistically insignificant in the brain, striatum and CSF after administration of the aforementioned PDE inhibitors. However, the levels of 3',5'-cyclic guanosine monophosphate (cGMP) increased in both rat and mouse striatum (2.2-, 2.1 and 1.7-fold and 6.4-, 2.8- and 1.7-fold, respectively) after PDE2A, 9A, and 10A inhibitor dosing. In all cases, the cNT changes followed the same trend in the brain, striatum and CSF after PDE inhibitor dosing and dose response was observed in rats. Concomitant treatment with PDE1B, PDE2A, PDE9A and PDE10A inhibitors resulted in a 4.4- and 36.7-fold increase of cGMP in rat and mouse striatum. The drug exposures after concomitant treatment were also higher than in the individual inhibitor-treated animals. cGMP enhancement observed could be due to synergistic effects, though an additive effect of the combined inhibitor concentrations may also contribute.

Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC). By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents. Citation Format: Gaofeng Xiong, Rachel Stewart, Jie Chen, Tianyan Gao, Timothy Scott, Luis Samayoa, Kathleen O’Connor, Andrew Lane, Ren Xu. Collagen hydroxylation promotes TNBC chemoresistance through stabilizing HIF1á [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3035.

A robust high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3 ',5 '-cyclic adenosine -C-13(5) monophosphate (C-13(5)-cAMP) and 3 ',5 '-cyclic guanosine-C-13 N-15(2) monophosphate ((C5N2)-C-13-cGMP) were used as surrogate analytes to measure endogenous 3 ',5 '-cyclic adenosine monophosphate (cAMP) and 3 ',5 '-cyclic guanosine monophosphate (cGMP). Pre weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC-MS/MS. The calibration curves for both analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r(2) > 0.996). Relative surrogate analyte-to-analyte LC-MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for C-13(5)-CAMP and (C15N2)-C-13-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for (13)C(5)cAMP and (13)C(15)N(2-)cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies. (C) 2017 The Authors. Published by Elsevier B.V.

The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.

The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.

The glucokinase-glucokinase regulatory protein (GK-GKRP) complex plays an important role in controlling glucose homeostasis in the liver. We have recently disclosed a series of arylpiperazines as in vitro and in vivo disruptors of the GK-GKRP complex with efficacy in rodent models of type 2 diabetes mellitus (T2DM). Herein, we describe a new class of aryl sulfones as disruptors of the GK-GKRP complex, where the central piperazine scaffold has been replaced by an aromatic group. Conformational analysis and exploration of the structure-activity relationships of this new class of compounds led to the identification of potent GK-GKRP disruptors. Further optimization of this novel series delivered thiazole sulfone 93, which was able to disrupt the GK-GKRP interaction in vitro and in vivo and, by doing so, increases cytoplasmic levels of unbound GK.

The HTS-based discovery and structure-guided optimization of a novel series of GKRP-selective GK-GKRP disrupters are revealed. Diarylmethanesulfonamide hit 6 (hGK-hGKRP IC50 = 1.2 μM) was optimized to lead compound 32 (AMG-0696; hGK-hGKRP IC50 = 0.0038 μM). A stabilizing interaction between a nitrogen atom lone pair and an aromatic sulfur system (nN → σ*S-X) in 32 was exploited to conformationally constrain a biaryl linkage and allow contact with key residues in GKRP. Lead compound 32 was shown to induce GK translocation from the nucleus to the cytoplasm in rats (IHC score = 0; 10 mg/kg po, 6 h) and blood glucose reduction in mice (POC = -45%; 100 mg/kg po, 3 h). X-ray analyses of 32 and several precursors bound to GKRP were also obtained. This novel disrupter of GK-GKRP binding enables further exploration of GKRP as a potential therapeutic target for type II diabetes and highlights the value of exploiting unconventional nonbonded interactions in drug design.

Small molecule activators of glucokinase have shown robust efficacy in both preclinical models and humans. However, overactivation of glucokinase (GK) can cause excessive glucose turnover, leading to hypoglycemia. To circumvent this adverse side effect, we chose to modulate GK activity by targeting the endogenous inhibitor of GK, glucokinase regulatory protein (GKRP). Disrupting the GK-GKRP complex results in an increase in the amount of unbound cytosolic GK without altering the inherent kinetics of the enzyme. Herein we report the identification of compounds that efficiently disrupt the GK-GKRP interaction via a previously unknown binding pocket. Using a structure-based approach, the potency of the initial hit was improved to provide 25 (AMG-1694). When dosed in ZDF rats, 25 showed both a robust pharmacodynamic effect as well as a statistically significant reduction in glucose. Additionally, hypoglycemia was not observed in either the hyperglycemic or normal rats.

We have recently reported a novel approach to increase cytosolic glucokinase (GK) levels through the binding of a small molecule to its endogenous inhibitor, glucokinase regulatory protein (GKRP). These initial investigations culminated in the identification of 2-(4-((2S)-4-((6-amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (1, AMG-3969), a compound that effectively enhanced GK translocation and reduced blood glucose levels in diabetic animals. Herein we report the results of our expanded SAR investigations that focused on modifications to the aryl carbinol group of this series. Guided by the X-ray cocrystal structure of compound 1 bound to hGKRP, we identified several potent GK-GKRP disruptors bearing a diverse set of functionalities in the aryl carbinol region. Among them, sulfoximine and pyridinyl derivatives 24 and 29 possessed excellent potency as well as favorable PK properties. When dosed orally in db/db mice, both compounds significantly lowered fed blood glucose levels (up to 58%).

Structure-activity relationship investigations conducted at the 5-position of the N-pyridine ring of a series of N-arylsulfonyl-N'-2-pyridinyl-piperazines led to the identification of a novel bis-pyridinyl piperazine sulfonamide (51) that was a potent disruptor of the glucokinase-glucokinase regulatory protein (GK-GKRP) interaction. Analysis of the X-ray cocrystal of compound 51 bound to hGKRP revealed that the 3-pyridine ring moiety occupied a previously unexplored binding pocket within the protein. Key features of this new binding mode included forming favorable contacts with the top face of the Ala27-Val28-Pro29 ("shelf region") as well as an edge-to-face interaction with the Tyr24 side chain. Compound 51 was potent in both biochemical and cellular assays (IC50=0.005 μM and EC50=0.205 μM, respectively) and exhibited acceptable pharmacokinetic properties for in vivo evaluation. When administered to db/db mice (100 mg/kg, po), compound 51 demonstrated a robust pharmacodynamic effect and significantly reduced blood glucose levels up to 6 h postdose.

In the previous report, we described the discovery and optimization of novel small molecule disruptors of the GK-GKRP interaction culminating in the identification of 1 (AMG-1694). Although this analogue possessed excellent in vitro potency and was a useful tool compound in initial proof-of-concept experiments, high metabolic turnover limited its advancement. Guided by a combination of metabolite identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor (27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.