HIF-1α(hypoxia-inducible factor-1α)

For in vitro studies, BAY 87-2243 was prepared as a 10 mmol/L stock solution in dimethyl sulfoxide (DMSO) and diluted in the relevant assay media. Luciferase activity is inhibited by BAY 87-2243, with an IC50 value of around 0.7 nM. BAY 87-2243 inhibits hypoxic expression of the HIF target gene CA9 on the protein level in HCT116luc cells, with an IC50 value of around 2 nM. With an IC50 value of less than 10 nM, BAY 87- 2243 inhibits mitochondrial oxygen consumption as determined by the oxygen-sensitive fluorescent dye LUX-MitoXpress[1]. The nuclear HIF-1α protein expression is inhibited by BAY-87-2243. In contrast to relative vascular area (RVA) and perfused vessels (PF), administration of BAY-87-2243 for approximately 18 days significantly reduces HIF-1α protein expression, necrotic fraction (NF) (mean 9% vs. 35.6%, p=0.0002), and pimonidazole hypoxic fraction (pHF) (mean 2.4% (BAY-87-2243) vs. 17.6% (carrier), p<0.0001)[2].

H460 cells are subcutaneously injected into naked mice, and once tumors have developed, the mice are given daily oral gavage treatments with BAY 87-2243 (0.5, 1, 2, and 4 mg/kg) for three weeks. In accordance with a dose-dependent decrease in the mRNA expression levels of the HIF-1 target genes CA9, ANGPTL4, and EGLN3, BAY 87-2243 decreased tumor weight. However, the compound treatment in vivo had no effect on the mRNA expression levels of the hypoxia-insensitive EGLN2 gene or HIF-1α itself[1].

Prolyl hydroxylase activity assay[1]
The influence of the test compound on prolyl hydroxylase 2 (PHD2) activity was assayed as described previously15 with some modifications: Recombinant human PHD2 was purified from Sf9 cell lysates and used for hydroxylation of biotinylated HIF-1α 556-574 peptide coated on NeutrAvidin plates. Hydoxylated peptide was quantified after incubation with purified Von-Hippel-Lindau L-Elongin B-Elongin Complex complex labeled with europium and addition of enhancer solution by measuring time-resolved fluorescence with a Tecan infinite M200 plate reader.

---

Western blot analysis of HIF-1α[2]
Western blotting was performed one time according to the established protocol as described previously [24]. Protein samples were prepared using the NE-PER Nuclear and Cytoplasmic KIT according to the manufacturer’s instructions. Antibodies used were mouse monoclonal anti-human HIF-1α (1:250) and rabbit polyclonal anti-histone-H2B (1:500) and anti-Calpain 1 (1:100) served as the loading controls for nuclear or cytoplasmic cell compartments, respectively. Nuclear HIF-1α band intensities were normalized to histone-H2B levels.

Cellular assays[1]
For high-throughput screening of a small molecule library consisting of ∼830,000 compounds, HCT-116 cells were stably transfected with a vector containing a luciferase reporter system coupled four times to the HRE from human vascular endothelial growth factor (VEGF) promoter (HCT 116-4xVEGF-Luc). Cells were plated at 3 × 10E4 cells/well and incubated overnight before test compounds (5 mmol/L in DMSO) were added and plates were placed in a hypoxic chamber for 16 h at 1% pO2. Results are given as luminescence counts in arbitrary units after subtraction of baseline levels from normoxic, nontreated controls. For the measurement of cellular complex I activity, H1299 cells were cotransfected with a pcDNA3 vector encoding for Pyrearinus termitilluminans larval click beetle luciferase. Clones (H1299tluc) showing high luminescence and dose-dependent rotenone sensitivity were subcloned and then used for further in depth analysis of cellular complex I activity by luminescence measurements. In brief, H1299tluc cells (1500/well) were seeded into white 384 well plates. After 2 days in culturing in Dulbecco's modified eagle medium (DMEM) without glucose, but supplemented with 11 mmol/L galactose, 10 μL of a luciferin/inhibitor mixture (150 μmol/L d-luciferin, 0.4% DMSO final concentration in Tyrode) was added to each well and incubated for 1 h at 37°C. Luminescence measurements were performed with an in house developed plate reader. After this measurement 20 μL succinate (0.67 mol/L, pH 5.3 in Tyrode, final concentration 25 mmol/L) was added. The plate was then incubated for another 1 h at room temperature before the second measurement was performed. H1299tluc cells expressing NADH-Q-Oxidoreductase from Saccharomyces cerevisiae (NDI1) were generated by transfection with a pcDNA3 vector encoding for NDI1 under control of a cytomegaly virus (CMV) promoter and a C-terminal HA-tag using PiggyBac transposon-mediated gene transfer11. Selection for positive clones was performed by cultivation in the presence of 20 nmol/L rotenone in DMEM medium with 11.2 mmol/L glucose. Rotenone insensitive clones with high luminescence were used as described above. Luciferase activity is given in % of DMSO-treated cells. To evaluate the cytotoxicity of BAY 87-2243, 2.000 cells of the respective cell lines were seeded in 96-well plates and cultured in the appropriate growth medium containing 10% FCS. BAY 87-2243 at various concentrations was added at 24 h after seeding for additional 48 h and cell viability was determined using Cell Titer Glow Assay.

---

Quantification of CA9 protein[1]
HCT 116-4xVEGF-Luc cells were seeded at 3 × 10E4 cells/well in 96-well plates and incubated overnight at 37°C in a humidified incubator containing 5% CO2 under normal oxygen levels before shifting hypoxic conditions (1% pO2, 24 h) in the absence or presence of various concentrations of BAY 87-2243. Protein expression levels of the HIF target gene carbonic anhydrase 9 (CA9) in cell lysates was quantified using MN/CAIX enzyme linked immunosorbent assay.

In vivo tumor study [1]
For in vivo studies, BAY 87-2243 was formulated in a 1% (v/v) solution of ethanol/solutol/water (10/40/50%). Animals were given BAY 87-2243 (0.5, 1, 2, and 4 mg/kg) or vehicle control once daily by oral gavage. Tumor xenograft experiment was carried out on female immune-deficient, athymic NMRI nude mice, aged 7–9 weeks, weighing 20–25 g in full accordance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing and Education issued by the New York Academy of Sciences' Ad Hoc Committee on Animal Research. The lung carcinoma xenograft mouse model was established by subcutaneous injection into the right flank with 0.1 mL H460 tumor cells (1.5 × 10E6) mixed 1:1 with Matrigel. Mice were randomized into control and treatment groups when tumors reached a size of more than 40 mm2. Body weight was monitored as a measure for treatment-related, acute toxicity. Tumor area (measured by caliper) or tumor weight (measured when mice were sacrificed 21 days after cell injection) was calculated by the formula 100−100 × (tumor weight/area of treatment group)/(tumor weight/area of vehicle group).

---

Murine plasma pharmacokinetic analyses[1]
Plasma concentrations of unchanged BAY 87-2243 were determined by liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS). Briefly, murine plasma was centrifuged and subsequently precipitated by addition of acetonitrile and an internal standard. The supernatants were subjected to high-performance LC connected to a MS/MS (API 3000, Applied Biosystems, Darmstadt, Germany) via a Turbo Ion Spray interface.

---

Tumor sampling[2]
BAY-87-2243 was dissolved in carrier solution (10% ethanol, 40% Solutol® HS15, 50% sterile distilled water) and administered orally by gavage (9 mg/kg/body weight [b.w.]). When UT-SCC-5 hSCC xenografts in nude mice reached 6 mm in diameter BAY-87-2243 or carrier was administered before and/or during RT or radiochemotherapy with concomitant cisplatin (RCT). Local tumor control was evaluated 150 days after irradiation and the doses to control 50% of tumors (TCD50) were compared between treatment arms. Tumors were excised at different time points during BAY-87-2243 or carrier treatment for western blot and immunohistological investigations.

[1]. Ellinghaus P, et al. BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I. Cancer Med. 2013 Oct;2(5):611-24.
[2]. Helbig L, et al. BAY 87-2243, a novel inhibitor of hypoxia-induced gene activation, improves local tumor control after fractionated irradiation in a schedule-dependent manner in head and neck human xenografts. Radiat Oncol. 2014 Sep 19;9:207

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors[1].

C26H26F3N7O2

525.5256

525.2

C, 59.42; H, 4.99; F, 10.85; N, 18.66; O, 6.09

1227158-85-1

1227158-85-1

67377767

Typically exists as solids (or liquids in special cases) at room temperature

1.5±0.1 g/cm3

677.7±65.0 °C at 760 mmHg

363.7±34.3 °C

0.0±2.1 mmHg at 25°C

1.674

4.2

85.34

FC(OC1C([H])=C([H])C(=C([H])C=1[H])C1=NOC(C2C([H])=C(C([H])([H])[H])N(C([H])([H])C3C([H])=C([H])N=C(C=3[H])N3C([H])([H])C([H])([H])N(C([H])([H])C3([H])[H])C3([H])C([H])([H])C3([H])[H])N=2)=N1)(F)F

CDJNNOJINJAXPV-UHFFFAOYSA-N

InChI=1S/C26H26F3N7O2/c1-17-14-22(25-31-24(33-38-25)19-2-6-21(7-3-19)37-26(27,28)29)32-36(17)16-18-8-9-30-23(15-18)35-12-10-34(11-13-35)20-4-5-20/h2-3,6-9,14-15,20H,4-5,10-13,16H2,1H3

1-cyclopropyl-4-[4-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl]-1H-pyrazol-1-yl]methyl]-2-pyridinyl]-piperazine

BAY-872243; BAY 872243; BAY-872243; BAY-87-2243; BAY87-2243; BAY 87-2243

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO:<1 mg/mL Water:<1 mg/mL Ethanol: 8 mg/mL(15.22 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)