WHI-P258

Bcl-W (Ki = 1 nM); Bcl-xL (Ki = 1 nM); Bcl-2 (Ki = 1 nM)

WHI-P258 is a potent and selective Janus kinase 3 (JAK3) inhibitor discovered from homology modeling. Acute lymphoblastic leukemia, the most prevalent type of childhood cancer, may be the target of novel therapeutic approaches using potent and targeted JAK3 inhibitors like WHI-P131. In order to create dimethoxyquinazoline compounds with potent and precise inhibitory activity against JAK3, a novel homology model of the kinase domain of Janus kinase (JAK) 3 was used. This model led to the identification of WHI-P258. With a volume of about 530 A3 available for inhibitor binding, the active site of JAK3 in this homology model has dimensions of roughly 8 A x 11 A x 20 A. Modeling studies showed that WHI-258 would probably fit into the catalytic site of JAK3 and that derivatives of this substance that contain an OH group at the 4' position of the phenyl ring would more strongly bind to JAK3 because of added interactions with Asp-967, a crucial residue in the catalytic site of JAK3.

Using a methylcellulose colony assay system, the antileukemic activity of WHI-P131 was evaluated against clonogenic tumor cells. A variety of concentrations of WHI-P131 were applied to cells (105 cells/ml in RPMI-10% fetal bovine serum) overnight at 37°C. Following treatment, cells were twice washed, plated at 104 or 105 cells/ml in RPMI, 10% fetal bovine serum, and 0.9% methylcellulose in Petri dishes, and cultured for 7 days at 37°C in a humidified 5% CO2 incubator. Then, a phase-contrast inverted microscope was used to count the number of leukemic cell (or tumor cell) colonies. This formula was used to determine the percentage inhibition of colony formation.

Sf21 (IPLB-SF21-AE) cells were purchased from Invitrogen (Carlsbad, CA) and were maintained at 26–28°C in Grace's insect cell medium supplemented with 10% fetal bovine serum and 1.0% antibiotic/antimycotic (Life Technologies, Inc.). These cells were derived from the ovarian tissue of the fall armyworm Spodotera frugiperda. Stock cells were kept in suspension in 1-liter Bellco spinner flasks at 60-90 rpm with a total culture volume of 600 ml at 0.2 × 106–1.6 × 106 cells/ml. Trypan blue dye exclusion was used to measure the cell viability, which was kept between 95 and 100 percent. According to a previous report, a baculovirus expression vector for BTK, SYK, JAK1, JAK2, or JAK3 was introduced into Sf21 cells. Cells were harvested and lysed [10 mm Tris (pH 7.6), 100 mm NaCl, 1% NP40, 10% glycerol, 50 mmNaF, 100 μm Na3VO4, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotonin, and 10 μg/ml leupeptin], the kinases were immunoprecipitated from the lysates, and their enzymatic activity was assayed, as reported previously. As previously mentioned, Western blot analysis was performed on the immunoprecipitates.
HepG2 human hepatoma cells were grown to an approximate 80% confluency before being washed once with serum-free DMEM and starved for 3 hours at 37°C in a CO2 incubator for IRK assays. After that, cells were stimulated with insulin (Eli Lilly and Co., Indianapolis, IN; 10 units/ml, 10 × 106 cells) for 10 min at room temperature. After this IRK activation step, cells were washed once with serum-free medium, lysed in NP40 buffer, and then IRK was immunoprecipitated from the lysates using an anti-IRβ antibody (Santa Cruz Biotechnology, Santa Cruz, CA; polyclonal IgG). We adjusted the beads with the kinase buffer [30 mm HEPES (pH 7.4), 30 mm NaCl, 8 mm MgCl2, and 4 mm MnCl2] prior to running the immune complex kinase assays. NALM-6 human leukemia cells' whole cell lysates were used to produce LYN, as previously reported.
In JAK3 immune complex kinase assays, KL-2 EBV-transformed human lymphoblastoid B cells (native JAK3 kinase assays) or insect ovary cells (recombinant JAK3 kinase assays) were lysed with NP40 lysis buffer [50 mm Tris (pH 8), 150 mm NaCl, 5 mm EDTA, 1% NP40, 100 μmsodium orthovanadate, 100 μm sodium molybdate, 8 μg/ml aprotinin, 5 μg/ml leupeptin, and 500 μmphenylmethylsulfonyl fluoride] and centrifuged 10 min at 13,000 × g to remove insoluble material. Samples were immunoprecipitated using JAK3-specific antibodies. Immune complexes were obtained by incubating with 15 μl of protein A-Sepharose after the antisera had been diluted. The protein A-Sepharose beads were washed four times with NP40 lysis buffer before being resuspended in the same buffer after being washed once with kinase buffer (20 mm MOPS (pH 7)-10 mm MgCl2). 25 μCi of [γ-32P]ATP (5000 Ci/mmol) and unlabeled ATP were added to a final concentration of 5 μm to start the reactions. Boiling for 4 minutes in SDS sample buffer was used to stop the reactions. Labeled proteins were found using autoradiography after samples were run on 9.5% SDS polyacrylamide gels. Kinase gels were electrophoresed, dried onto Whatman 3M filter paper, and then phosphorimaged on a Molecular Imager (Bio-Rad, Hercules, CA) and autoradiographed on film. The kinase activity in phosphorimager units was compared to that of the control sample to produce a kinase activity index for each drug concentration. Cold kinase assays were carried out in some experiments, as previously mentioned.

The following cell lines were used in various biological assays: BT-20 (breast cancer), M24-MET (melanoma), SQ20B (squamous cell carcinoma), NALM-6 (pre-B-ALL), LC1;19 (pre-B-ALL), DAUDI (B-ALL), RAMOS (B-ALL), MOLT-3 (T-cell ALL), and PC3 (prostate cancer). As was previously reported, these cell lines were maintained in culture. In a treatment medium containing varying concentrations of WHI-P131, cells were seeded in six-well tissue culture plates at a density of 50 × 104 cells/well and incubated for 24-48 h at 37°C in a humidified 5% CO2 atmosphere.

[1]. Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin Cancer Res. 1999 Jun;5(6):1569-82.

[2]. Role of a JAK3-dependent biochemical signaling pathway in platelet activation and aggregation. J Biol Chem. 2001 May 25;276(21):17815-22.

C16H15N3O2

281.32

281.12

C, 68.31; H, 5.37; N, 14.94; O, 11.37

21561-09-1

Solid powder

COC1=C(C=C2C(=C1)C(=NC=N2)NC3=CC=CC=C3)OC

MJKCGAHOCZLYDG-UHFFFAOYSA-N

InChI=1S/C16H15N3O2/c1-20-14-8-12-13(9-15(14)21-2)17-10-18-16(12)19-11-6-4-3-5-7-11/h3-10H,1-2H3,(H,17,18,19)

6,7-dimethoxy-N-phenylquinazolin-4-amine

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)

Solubility in Formulation 1: ≥ 2.25 mg/mL (8.00 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 22.5 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.

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Solubility in Formulation 2: ≥ 2.25 mg/mL (8.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 22.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.25 mg/mL (8.00 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 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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