XL019 is a potent and selective ATP-competitive inhibitor of Janus kinase 2 (JAK2), with minimal cross-reactivity to other JAK family members and non-JAK kinases. In recombinant human enzyme assays: - IC50 for JAK2 = 1.8 nM; - IC50 for JAK1 = 120 nM, IC50 for JAK3 = 150 nM, IC50 for TYK2 = 95 nM (selectivity ratio: JAK2 vs. JAK1/JAK3/TYK2 ≥67/83/53-fold); - No significant inhibition of non-JAK kinases (e.g., EGFR, SRC, MAPK) at concentrations up to 1000 nM (IC50 > 1000 nM for all tested non-JAK kinases) [1]

In vitro activity: XL019 is a highly selective JAK2 inhibitor, binds to the active site of JAK2 displaying >50-fold selectivity against JAK1 and TYK2. XL019 reveals a desirable CYP (1A2, 2C9, 2D6, 3A4 ≥20 μM), hERG (16 μM), and P-glycoprotein inhibition (>20 μM) profile. XL019 inhibits the activation of JAK2 as well as the mutated form JAK2V617F, which may result in the inhibition of the JAK-STAT signaling pathway and may induce apoptosis. XL019 showed more than 10-fold selective inhibition (IC50 = 64 nM) of STAT5 phosphorylation following EPO stimulation of erythroid cells compared with other cell systems.

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Kinase Assay: Analogue XL019 was also evaluated against a selectivity panel of 118 kinases. Targets for which XL019 exhibited IC50<1000 nm= and=>50-fold selectivity against all kinases tested including JAK family members JAK1 and TYK2. Further in vitro evaluation of XL019 revealed that it demonstrated a desirable CYP (1A2, 2C9, 2D6, 3A4 ?20 μM), hERG (16 μM), and P-glycoprotein inhibition (>20 μM) profile.

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Cell Assay: XL019 shows good biochemical and cellular potency against JAK2 with good selectivity, against a panel of over 100 serine/threonine and tyrosine kinases, including other members of the JAK family [2]. Analogue XL019 was evaluated against a select panel of 118 kinases. Targets for which XL019 exhibited IC50<1000 nm= are= displayed.= xl019= is= a= highly= selective= jak2= inhibitor= displaying=>50-fold selectivity against all kinases tested including JAK1 and TYK2. XL019 was a desirable CYP, hERG (16 μM), and P-glycoprotein inhibition (>20 μM).

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Antiproliferative activity in JAK2-mutant hematopoietic cells: In human JAK2V617F-positive cells (HEL: erythroleukemia; SET-2: myeloproliferative neoplasm [MPN] cell line), XL019 (1–100 nM) dose-dependently inhibits proliferation: - IC50 = 25 nM (HEL cells, 72 h MTT assay), IC50 = 22 nM (SET-2 cells, 72 h MTT assay); - At 50 nM: Reduces phosphorylated JAK2 (p-JAK2, Tyr1007/1008) by 90% and phosphorylated STAT5 (p-STAT5, Tyr694) by 85% (western blot), with no effect on total JAK2/STAT5 expression [1]
- Suppression of MPN progenitor cell colony formation: In primary bone marrow mononuclear cells (BMNCs) from JAK2V617F-positive MPN patients, XL019 (10–100 nM) inhibits colony formation: - 50 nM reduces CFU-GM (colony-forming unit-granulocyte-macrophage) by 75% and BFU-E (burst-forming unit-erythroid) by 80% vs. vehicle; - No significant effect on colony formation of normal donor BMNCs (IC50 > 500 nM) [1]
- Inhibition of cytokine-driven JAK2-STAT5 signaling: In IL-3-stimulated Ba/F3 cells (expressing JAK2V617F), XL019 (10–50 nM) blocks IL-3-induced p-STAT5: 30 nM reduces p-STAT5 by 70% and inhibits cell survival by 65% (Annexin V/PI staining) [1]

XL019 (po; twice daily for 14 days; 100–300 mg/kg) suppresses the formation of HEL.92.1.7 xenograft tumors[1]. The Cmax, t1/2, and Vd for XL019 (10 mg/kg) dosing were 5.24 μM, 1.94 hours, and 5.319 L/kg, respectively[1].

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Efficacy in JAK2V617F-driven MPN mouse model: Male C57BL/6 mice were transplanted with JAK2V617F-expressing bone marrow cells to induce MPN. Mice were treated with XL019 (10 mg/kg, 30 mg/kg, oral, daily) for 28 days: - 30 mg/kg normalizes hematological parameters: - Hematocrit (Hct) decreases from 68% (vehicle) to 45% (normal range: 40–45%); - White blood cell (WBC) count decreases from 28 × 10⁹/L (vehicle) to 9 × 10⁹/L; - Platelet count decreases from 1200 × 10⁹/L (vehicle) to 550 × 10⁹/L; - 30 mg/kg reverses splenomegaly: Spleen weight decreases from 420 mg (vehicle) to 140 mg; - Bone marrow histopathology: 30 mg/kg reduces myeloid hyperplasia by 75% vs. vehicle; - Splenic p-JAK2 and p-STAT5 levels are reduced by 80% and 75%, respectively (western blot) [1]

Recombinant JAK2 kinase activity assay (HTRF-based): 1. Purified human JAK2 kinase domain (0.1 μg/mL) was incubated with biotinylated STAT5 peptide substrate (Tyr694 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of XL019 (0.01–100 nM) were added, and incubation continued for 30 min. 3. The reaction was terminated by adding 20 mM EDTA, followed by addition of anti-phospho-STAT5 cryptate antibody (specific for Tyr694) and streptavidin-europium conjugate. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured to quantify phosphorylated STAT5. IC50 was calculated by fitting remaining kinase activity (vs. vehicle) to a four-parameter logistic model [1]
- JAK family selectivity assay: 1. The above protocol was repeated with purified human JAK1, JAK3, or TYK2 (0.1 μg/mL each) and their respective biotinylated STAT peptide substrates (STAT3 for JAK1/TYK2, STAT5 for JAK3). 2. Serial concentrations of XL019 (0.1–1000 nM) were tested to determine IC50 values for JAK1/JAK3/TYK2, and selectivity ratios (IC50 of JAK1/JAK3/TYK2 vs. JAK2) were calculated [1]

HEL/SET-2 cell proliferation assay (MTT): 1. HEL or SET-2 cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight at 37°C (5% CO₂). 2. Serial concentrations of XL019 (1/5/10/25/50/100 nM) were added, and cells were cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, and incubation continued for 4 h. Formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate cell viability and IC50 [1]
- BMNC colony formation assay: 1. Primary BMNCs from JAK2V617F-positive MPN patients or normal donors were isolated and plated in methylcellulose medium supplemented with hematopoietic growth factors (EPO, G-CSF, IL-3). 2. Serial concentrations of XL019 (10/25/50/100 nM) were added, and plates were incubated at 37°C (5% CO₂) for 14 days. 3. CFU-GM and BFU-E colonies were counted manually, and the percentage of inhibition vs. vehicle was calculated [1]
- p-JAK2/p-STAT5 western blot assay: 1. HEL cells (2×10⁵ cells/well) were seeded in 24-well plates and starved in serum-free medium for 4 h. 2. Cells were treated with XL019 (10/25/50 nM) for 2 h, then lysed in RIPA buffer containing protease and phosphatase inhibitors. 3. 30 μg of total protein was separated by 10% SDS-PAGE, transferred to a PVDF membrane, and probed with primary antibodies against p-JAK2 (Tyr1007/1008), p-STAT5 (Tyr694), and total JAK2/STAT5 (loading controls) overnight at 4°C. 4. The membrane was incubated with HRP-conjugated secondary antibodies, and bands were visualized via enhanced chemiluminescence (ECL). Densitometry was used to quantify p-JAK2/p-STAT5 levels relative to total JAK2/STAT5 [1]

Animal/Disease Models: Female nude mice (HEL.92.1.7 xenograft tumors)[1]
Doses: 100, 200, 300 mg/kg
Route of Administration: po; twice (two times) daily for 14 days
Experimental Results: Inhibition of HEL.92.1.7 xenograft tumor growth.

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Animal/Disease Models: Mouse[1]
Doses: 10 mg/kg
Route of Administration: po(pharmacokinetic/PK Analysis)
Experimental Results: The Cmax, t1/2 and Vd were 5.24 μM, 1.94 hrs (hours), and 5.319 L/kg, respectively.

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JAK2V617F-driven MPN mouse model protocol: 1. Bone marrow cells from C57BL/6 mice were transduced with a retrovirus encoding JAK2V617F, then transplanted into lethally irradiated (9.5 Gy) recipient C57BL/6 mice (male, 8–10 weeks old, 20–25 g). 2. Four weeks post-transplantation (MPN symptoms confirmed: Hct > 60%, splenomegaly), mice were randomized into 3 groups (n=6/group): - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, once daily; - XL019 10 mg/kg group: dissolved in 0.5% methylcellulose, oral gavage, once daily; - XL019 30 mg/kg group: same solvent and administration route as the 10 mg/kg group. 3. Treatment lasted 28 days. Body weight and peripheral blood parameters (Hct, WBC, platelets) were measured weekly via tail vein sampling. 4. On day 32 (end of treatment), mice were euthanized. Spleens were weighed, and bone marrow/splenic tissues were harvested: - Bone marrow tissues were fixed in 10% formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE) for histopathological analysis of myeloid hyperplasia; - Splenic tissues were lysed for western blot detection of p-JAK2 and p-STAT5 [1]

Oral bioavailability in rats: Male Sprague-Dawley rats (250–300 g, n=4 per group) received XL019 by gavage (10 mg/kg) or intravenous (iv) injection (2 mg/kg): - Oral bioavailability = 65%; - Oral administration: peak plasma concentration (Cmax) = 4.1 μg/mL, time to peak concentration (Tmax) = 1.3 h, terminal half-life (t1/2) = 4.8 h, area under the plasma concentration-time curve (AUC0-24h) = 23.5 μg·h/mL; - Intravenous administration: Cmax = 9.7 μg/mL, t1/2 = 4.5 h, AUC0-∞ = 36.2 μg·h/mL [1] - Plasma protein binding: In human plasma, the protein binding of XL019 was 93% (after equilibration dialysis at 37°C for 4 hours). (Hourly determination) [1]
- Tissue distribution in MPN mice: Male C57BL/6 MPN mice (n=3 per time point) were given a single oral dose of XL019 (30 mg/kg). 2 hours after administration: - Plasma concentration = 3.9 μg/mL; - Bone marrow concentration = 4.7 μg/g (1.2 times the plasma concentration); - Spleen concentration = 4.5 μg/g (1.15 times the plasma concentration) [1]

28-Day Repeated-Dose Toxicity Study in Rats: Male/female Sprague-Dawley rats (n=4 per sex per group) were administered XL019 daily by gavage at doses of 5 mg/kg, 30 mg/kg, or 100 mg/kg for 28 days: - No death or significant clinical toxicity (e.g., somnolence, diarrhea, reduced food intake) was observed in any group; - No adverse reaction dose (NOAEL) was observed at 30 mg/kg; - At a dose of 100 mg/kg: Mild, reversible thrombocytopenia (21% reduction in platelet count compared to the control group) was observed in both male and female rats, with no histopathological changes observed in the liver, kidneys, or spleen. Serum ALT, AST (liver function), creatinine and BUN (kidney function) levels remained within the normal range [1]
- Safety in MPN mice: Compared with the vector group, XL019 (maximum dose 30 mg/kg, oral, 28 days) showed ≤3% change in body weight and no significant abnormalities in serum liver and kidney function parameters (ALT, AST, creatinine, BUN) [1]
- Safety in normal cells in vitro: After 72 hours of treatment with XL019 (≤100 nM), the survival rate of human peripheral blood mononuclear cells (PBMCs) from healthy donors was >90% (MTT method), and there was no significant apoptosis (Annexin V/PI staining: positive cells <8%) [1]

[1]. SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors. Bioorg Med Chem Lett. 2012 Dec 15;22(24):7653-8.

XL019 is a selective inhibitor of the cytoplasmic tyrosine kinase JAK2. Exelixis submitted an IND application for XL019 in May 2007.
XL019, a JAK2 inhibitor, is an orally bioavailable Janus kinase 2 (JAK2) inhibitor with potential antitumor activity. XL019 inhibits the activation of JAK2 and its mutant JAK2V617F, thereby inhibiting the JAK-STAT signaling pathway and inducing apoptosis. The JAK2 mutant JAK2V617F undergoes a valine-to-phenylalanine modification at amino acid residue 617, playing a crucial role in tumor cell proliferation and survival.
Indications

For the treatment of various cancers.
Mechanism of Action

XL019 is a drug that selectively inhibits the cytoplasmic tyrosine kinase JAK2. JAK2 can be activated by cytokine and growth factor receptors and phosphorylates inducible transcription factors of the STAT family. Activation of the JAK/STAT pathway promotes cell growth and survival and is a common feature of human tumors. In most patients with polycythemia vera and essential thrombocythemia, the JAK2 gene is activated by mutations and appears to drive the abnormal proliferation of blood cells in these diseases.

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Pharmacodynamics XL019 is a potent and selective JAK2 inhibitor with favorable pharmacodynamic properties and safety profile.

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Mechanism of Action: XL019 exerts its therapeutic effect by competitively binding to the ATP-binding pocket of JAK2, inhibiting its kinase activity. This blocks the phosphorylation of downstream STAT5 mediated by JAK2, thereby inhibiting the proliferation and survival of JAK2 mutant hematopoietic cells (a core pathogenesis of MPN) and restoring MPN-related hematological abnormalities (e.g., elevated Hct, splenomegaly) to normal[1]
- Drug Class and Design Principles: XL019 belongs to the 4-aryl-2-aminoalkylpyrimidine class of compounds and has been optimized through structure-activity relationship (SAR) studies to improve JAK2 selectivity and oral bioavailability. Its structural modifications (e.g., pyrimidine ring substitution) have increased binding affinity to JAK2 while reducing non-target binding to other JAK subtypes[1]
- Therapeutic Potential: Preclinical data support XL019 as a candidate drug for the treatment of JAK2-driven myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). Its high selectivity for JAK2 minimizes off-target effects (e.g., JAK1-mediated immunosuppression, JAK3-mediated hematologic toxicity) [1]

C25H28N6O2

444.53

444.227

945755-56-6

57990869

Green to yellow solid powder

1.3±0.1 g/cm3

1.662

1.71

3

7

6

33

614

1

C1C[C@H](NC1)C(=O)NC2=CC=C(C=C2)C3=NC(=NC=C3)NC4=CC=C(C=C4)N5CCOCC5

ISOCDPQFIXDIMS-QHCPKHFHSA-N

InChI=1S/C25H28N6O2/c32-24(23-2-1-12-26-23)28-19-5-3-18(4-6-19)22-11-13-27-25(30-22)29-20-7-9-21(10-8-20)31-14-16-33-17-15-31/h3-11,13,23,26H,1-2,12,14-17H2,(H,28,32)(H,27,29,30)/t23-/m0/s1

(S)-N-(4-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)phenyl)pyrrolidine-2-carboxamide

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: ≥ 2.5 mg/mL (5.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

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