2-anilino-4-aryl-8H-purine derivatives as inhibitors of PDK1.

A series of 2-anilino substituted 4-aryl-8H-purines were prepared as potent inhibitors of PDK1, a serine-threonine kinase thought to play a role in the PI3K/Akt signaling pathway, a key mediator of cancer cell growth, survival and tumorigenesis. The synthesis, SAR and ADME properties of this series of compounds are discussed culminating in the discovery of compound 6 which possessed sub-micromolar cell proliferation activity and 65% oral bioavailability in mice.

Preclinical metabolism and disposition of SB939 (Pracinostat), an orally active histone deacetylase inhibitor, and prediction of human pharmacokinetics.

The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino) ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human pharmacokinetics (PK) was predicted using Simcyp and allometric scaling. SB939 showed high aqueous solubility with high Caco-2 permeability. Metabolic stability was relatively higher in dog and human liver microsomes than in mouse and rat. The major metabolites formed in human liver microsomes were also observed in preclinical species. Human cytochrome P450 (P450) phenotyping showed that SB939 was primarily metabolized by CYP3A4 and CYP1A2. SB939 did not significantly inhibit human CYP3A4, 1A2, 2D6, and 2C9 (>25 µM) but inhibited 2C19 (IC(50) = 5.8 µM). No significant induction of human CYP3A4 and 1A2 was observed in hepatocytes. Plasma protein binding in mouse, rat, dog, and human ranged between ∼84 and 94%. The blood-to-plasma ratio was ∼1.0 in human blood. SB939 showed high systemic clearance (relative to liver blood flow) of 9.2, 4.5, and 1.5 l · h(-1) · kg(-1) and high volume of distribution at steady state (>0.6 l/kg) of 3.5, 1.7, and 4.2 l/kg in mouse, rat, and dog, respectively. The oral bioavailability was 34, 65, and ∼3% in mice, dogs, and rats, respectively. The predicted oral PK profile and parameters of SB939, using Simcyp and allometric scaling, were in good agreement with observed data in humans. Simcyp predictions showed lack of CYP3A4 and 2C19 drug-drug interaction potential for SB939. In summary, the preclinical ADME of SB939 supported its preclinical and clinical development as an oral drug candidate.

Association of IFN-γ : IL-10 Cytokine Ratio with Nonsegmental Vitiligo Pathogenesis.

Background and Objectives. Cytokines regulate immune response and inflammation and play a crucial role in depigmentation process of vitiligo. The present study aimed to estimate the serum levels of pro- and anti-inflammatory cytokines, IFN-γ and IL-10, and their ratios in nonsegmental vitiligo patients and healthy individuals from India. Methods. Blood samples were collected from 280 subjects and serum IFN-γ and IL-10 levels were measured using standard ELISA. Results. Nonsegmental vitiligo patients showed increased levels of IFN-γ (12.4 ± 3.2 versus 9.9 ± 4.4 pg/mL) and decreased levels of IL-10 (9.3 ± 1.7 versus 11.5 ± 5 pg/mL) compared to controls. Ratio of IFN-γ : IL-10 differed significantly from patients to controls (p < 0.05). IFN-γ concentrations and IFN-γ : IL-10 ratio varied significantly with respect to clinical variants, disease stability, and social habits (smoking and alcohol consumption) and showed a positive correlation with disease duration. Family history of vitiligo was significantly associated with IFN-γ : IL-10 ratio but not with their individual levels. Conclusion. The ratio of IFN-γ : IL-10 serum levels may be considered as one of the promising immunological markers in nonsegmental vitiligo. This is the first study considering multiple aspects in relation to ratio of cytokine levels. Similar studies with large samples are warranted to confirm our observations.

Metabolism and Disposition of Pacritinib (SB1518), an Orally Active Janus Kinase 2 Inhibitor in Preclinical Species and Humans.

The ADME of Pacritinib (SB1518), an orally active JAK 2 inhibitor, was investigated in vitro and in vivo in preclinical species and humans. Pacritinib showed ~5 fold higher affinity to human plasma proteins relative to mouse in vitro. It was metabolized by human CYP3A4 in vitro, and did not significantly induce CYP3A and 1A2 in human hepatocytes. In vitro metabolism studies with mouse and human liver microsomes showed the presence of four major metabolites of Pacritinib -M1 (oxidation), M2 (dealkylation), M3 (oxidation), M4 (reduction). The in vitro and in vivo metabolic patterns observed in mice and humans were in good agreement. Qualitatively and quantitatively, none of the metabolites formed in vivo was >10% of Pacritinib in mouse, dog and humans. Pacritinib showed systemic clearance of 8.0, 1.6, 1.6 l/h/kg, volume of distribution of 14.2, 7.9, 8.5 l/kg, t1/2 of 5.6, 6.0, 4.6 h, and oral bioavailability of 39, 10, and 24% in mouse, rat and dog, respectively. In radiolabeled mass balance and QWBA studies in mice, ~91% of the dose was recovered in feces, suggesting biliary clearance, and maximum radioactivity was seen in the gastrointestinal tract followed by the kidney, heart and low activity in the brain. The relatively high exposures of Pacritinib in humans might be attributed to its very high plasma protein binding, low metabolic and/or biliary clearance.

Preclinical metabolism and pharmacokinetics of SB1317 (TG02), a potent CDK/JAK2/FLT3 inhibitor.

SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed > 99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 µM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans. In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution ( > 0.6 L/kg), oral bioavailability of 24%, ∼ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate.

Discovery of kinase spectrum selective macrocycle (16E)-14-methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a potent inhibitor of cyclin dependent kinases (CDKs), Janus kinase 2 (JAK2), and fms-like tyrosine kinase-3 (FLT3) for the treatment of cancer.

Herein, we describe the design, synthesis, and SAR of a series of unique small molecule macrocycles that show spectrum selective kinase inhibition of CDKs, JAK2, and FLT3. The most promising leads were assessed in vitro for their inhibition of cancer cell proliferation, solubility, CYP450 inhibition, and microsomal stability. This screening cascade revealed 26 h as a preferred compound with target IC(50) of 13, 73, and 56 nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies of 26 h in preclinical species showed good oral exposures. Oral efficacy was observed in colon (HCT-116) and lymphoma (Ramos) xenograft studies, in line with the observed PK/PD correlation. 26h (SB1317/TG02) was progressed into development in 2010 and is currently undergoing phase 1 clinical trials in advanced leukemias and multiple myeloma.

Pharmacodynamic evaluation of the target efficacy of SB939, an oral HDAC inhibitor with selectivity for tumor tissue.

SB939 is an oral histone deacetylase (HDAC) inhibitor currently in phase II clinical trials potently inhibiting class I, II, and IV HDACs with favorable pharmacokinetic properties, resulting in tumor tissue accumulation. To show target efficacy, a Western blot assay measuring histone H3 acetylation (acH3) relative to a loading control was developed, validated on cancer cell lines, peripheral blood mononuclear cells (PBMC), and in animal tumor models. Exposure of cells to 60 nmol/L (22 ng/mL) SB939 for 24 hours was sufficient to detect an acH3 signal in 25 µg of protein lysate. AcH3 levels of liver, spleen, PBMCs, bone marrow and tumor were measured in BALB/c mice, HCT-116 xenografted BALB/c nude mice, or in SCID mice orthotopically engrafted with AML (HL-60) after oral treatment with SB939. AcH3 could only be detected after treatment. In all tissues, the highest signal detected was at the 3-hour time point on day 1. On day 15, the signal decreased in normal tissues but increased in cancerous tissues and became detectable in the bone marrow of leukemic mice. In all tissues, acH3 correlated with SB939 dose levels (r(2)=0.76-0.94). When applied to PBMCs from 30 patients with advanced solid malignancies in a phase I clinical trial, a dose-dependent (10-80 mg) increase in relative acH3 was observed 3-hour postdose on day 1, correlating with C(max) and AUC of SB939 concentrations in plasma (r=0.97, P=0.014). Our data show that the favorable pharmacokinetic and pharmacodynamic properties of SB939 are translated from preclinical models to patients.

Discovery of the macrocycle 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (SB1518), a potent Janus kinase 2/fms-like tyrosine kinase-3 (JAK2/FLT3) inhibitor for the treatment of myelofibrosis and lymphoma.

Discovery of the activating mutation V617F in Janus Kinase 2 (JAK2(V617F)), a tyrosine kinase critically involved in receptor signaling, recently ignited interest in JAK2 inhibitor therapy as a treatment for myelofibrosis (MF). Herein, we describe the design and synthesis of a series of small molecule 4-aryl-2-aminopyrimidine macrocycles and their biological evaluation against the JAK family of kinase enzymes and FLT3. The most promising leads were assessed for their in vitro ADME properties culminating in the discovery of 21c, a potent JAK2 (IC(50) = 23 and 19 nM for JAK2(WT) and JAK2(V617F), respectively) and FLT3 (IC(50) = 22 nM) inhibitor with selectivity against JAK1 and JAK3 (IC(50) = 1280 and 520 nM, respectively). Further profiling of 21c in preclinical species and mouse xenograft and allograft models is described. Compound 21c (SB1518) was selected as a development candidate and progressed into clinical trials where it is currently in phase 2 for MF and lymphoma.

In vitro phase I cytochrome P450 metabolism, permeability and pharmacokinetics of SB639, a novel histone deacetylase inhibitor in preclinical species.

In vitro liver microsomal stability, permeability, pharmacokinetics (PK) and oral bioavailability of SB639, a novel HDACi (Histone Deacetylase inhibitor), were determined. The in vitro metabolism was examined in mouse, rat, dog and human liver microsomes. The permeability and efflux potential of SB639 were determined using Caco-2 cell monolayers. To determine pharmacokinetics and oral bioavailability, blood samples were drawn at pre-determined intervals up to 24 h post-dose after single intravenous (i.v.) or oral (p.o.) administration of SB639 to mouse or rat. The concentrations of SB639 in plasma samples were determined by a validated LC-MS/MS method. In vitro liver microsomal stability data revealed that SB639 was stable in human and dog liver microsomes, unstable in mouse and rat liver microsomes. The Caco-2 data has shown that SB639 is highly permeable with an apparent permeability of 3.01.10(-6) cm/s at 10 microM. After oral administration, maximum concentrations of SB639 were achieved within 0.5 h of post dose. Following i.v. administration, the concentration of SB639 declined in a bi-exponential fashion with terminal elimination half-life of 1.67 h for mice and 1.12 h for rats. The systemic clearance and volume of distribution of SB639 in mice were 15.8 l/h/kg and 38 l/kg, respectively, while the respective values in rats were 3.84 l/h/kg and 3.67 l/kg. Elimination half-life in rats ranged between 1.12-2.26 h. Absolute oral bioavailability of SB639 in mouse and rat was 13% and 10%, respectively. In conclusion, the superior potency, physicochemical and PK properties of SB639 compared to the recently FDA approved drug Zolinza (Suberoylanilide hydroxamic acid or Vorinostat) in the preclinical setting makes it a potential clinical candidate.