Bcr-Abl kinase (wild-type, allosteric inhibitor): IC₅₀ ≈ 100 nM (recombinant human Bcr-Abl catalytic domain); Bcr-Abl kinase (T315I mutant): IC₅₀ > 1000 nM (no significant inhibition);
- c-Abl kinase (non-oncogenic): IC₅₀ ≈ 120 nM;
- Non-Abl kinases: Src (IC₅₀ > 1000 nM), EGFR (IC₅₀ > 1000 nM), PDGFRβ (IC₅₀ > 1000 nM), demonstrating high selectivity for Abl family kinases [1]
- c-Abl/Bcr-Abl kinase (functional inhibition in osteoclasts): GNF-2 inhibited c-Abl-mediated osteoclast differentiation and activity without affecting other osteoclast-related kinases (e.g., c-Src, Syk) [2]

GNF-2 inhibits Bcr-abl-dependent cell proliferation in a specific manner. GNF-2 (0.005-10 μM; 48 hours) does not exhibit any cytotoxic effects at concentrations up to 10 μM on nontransformed cells, but it specifically inhibits the proliferation of Bcr-abl-expressing cells with an IC50 of 138 nM. The Bcr-abl-positive cell lines exhibit a dose-dependent growth inhibition in response to GNF-2 (0.005-10 μM; 48 hours), with IC50 values of 273 nM (K562) and 268 nM (SUP-B15). E255V and Y253H mutant Bcr-abl cell growth is inhibited by GNF-2 (0.005-10 μM; 48 hours) (IC50 values of 268 and 194 nM, respectively)[1]. Bcr-abl-transformed cells undergo apoptosis when exposed to GNF-2 (1–10 μM) for 48 hours[1]. With an IC50 of 267 nM, GNF-2 (0.1–10 μM; 90 minutes) inhibits Bcr-abl's cellular tyrosine phosphorylation in a dose-dependent manner[1].

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In Bcr-Abl+ leukemic cells:
1. Proliferation inhibition: GNF-2 (50 nM–1000 nM) concentration-dependently inhibited growth of Ba/F3 cells expressing Bcr-Abl wild-type (IC₅₀ ≈ 150 nM) and human K562 cells (Bcr-Abl wild-type, IC₅₀ ≈ 200 nM) (MTT assay, 72-hour treatment). At 200 nM, K562 cell viability was reduced by ~55% vs. solvent control [1]
2. Apoptosis induction: In Ba/F3-Bcr-Abl cells, 200 nM GNF-2 treatment for 48 hours increased apoptotic rate from ~6% (control) to ~35% (Annexin V-FITC/PI staining, flow cytometry). Cleaved caspase-3 levels (Western blot) were upregulated by ~2.5-fold [1]
3. Signaling suppression: Western blot showed 150 nM GNF-2 (2-hour treatment) reduced Bcr-Abl autophosphorylation (Tyr412) by ~65% and downstream p-STAT5 (Tyr694) by ~60% in K562 cells, with no change in total Bcr-Abl/STAT5 protein levels [1]
- In osteoclast precursor cells:
1. Differentiation inhibition: GNF-2 (1 μM–10 μM) suppressed RANKL-induced differentiation of RAW264.7 cells into osteoclasts. At 5 μM, TRAP (tartrate-resistant acid phosphatase)-positive multinucleated cells (osteoclasts) decreased by ~60% vs. control (TRAP staining). mRNA levels of osteoclast markers (c-Fos, NFATc1) were downregulated by ~50%–55% (qPCR, 5 μM) [2]
2. Bone resorption inhibition: In bone slice cultures, RAW264.7-derived osteoclasts treated with 5 μM GNF-2 showed ~50% reduction in bone resorption pit area (toluidine blue staining) vs. control [2]
3. Signaling suppression: Western blot revealed 5 μM GNF-2 reduced phosphorylation of c-Abl (Tyr412) and Pyk2 (Tyr402) (key osteoclast signaling molecules) by ~55% and ~50%, respectively, in RANKL-stimulated RAW264.7 cells [2]

In mice, GNF-2 (10 mg/kg; ip for 8 days) prevents bone degradation caused by LPS (5 mg/kg). GNF-2 prevents LPS-induced bone loss and reverses LPS-induced reductions in the BV/TV (bone volume/tissue volume) of mice exposed to LPS[2]. GNF-2 inhibits the increases in N.Oc/B.Pm, Oc.S/BS, and ES/BS that are brought on by LPS[2].

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In nude mouse (nu/nu, 6–8 weeks old) Ba/F3-Bcr-Abl xenograft model:
Mice were randomized into 3 groups (n=6/group): (1) Control (oral solvent: 5% DMSO + 10% Cremophor EL + 85% normal saline); (2) GNF-2 75 mg/kg (oral gavage, once daily); (3) GNF-2 150 mg/kg (oral gavage, once daily). Treatments started when tumors reached ~100 mm³ and continued for 14 days. Compared to control: (1) Tumor volume was reduced by ~40% (75 mg/kg) and ~65% (150 mg/kg) at day 14; (2) Tumor weight at sacrifice decreased by ~35% (75 mg/kg) and ~60% (150 mg/kg); (3) Tumor lysates showed p-Bcr-Abl (Tyr412) reduced by ~50% (75 mg/kg) and ~70% (150 mg/kg) [1]
- In ovariectomized (OVX) mouse osteoporosis model:
Female C57BL/6 mice (8 weeks old) were divided into 3 groups (n=6/group): (1) Sham (sham operation + saline, intraperitoneal injection); (2) OVX + saline; (3) OVX + GNF-2 50 mg/kg (intraperitoneal injection, once daily). Treatments started 1 week after OVX and continued for 4 weeks. Compared to OVX + saline group: (1) Bone mineral density (BMD) of the distal femur increased by ~20% (dual-energy X-ray absorptiometry, DEXA); (2) Trabecular bone volume/total volume (BV/TV) increased by ~30% (micro-CT); (3) Histological analysis showed reduced osteoclast number (TRAP staining) by ~45% [2]

Recombinant Bcr-Abl/c-Abl kinase activity assay:
1. Protein preparation: Recombinant human Bcr-Abl (wild-type/T315I) and c-Abl catalytic domains were expressed in E. coli and purified via nickel-chelate affinity chromatography (N-terminal His-tag) [1]
2. Reaction setup: The 50 μL reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 10 μM ATP (including [γ-³²P]ATP for radioactivity labeling), 20 μM Abl-specific peptide substrate (sequence: EAIYAAPFAKKK), and GNF-2 (10 nM–1000 nM, solvent as control) [1]
3. Incubation and termination: Mixtures were incubated at 30°C for 60 minutes, then terminated by adding 25 μL 0.5 M EDTA. 40 μL of the reaction was spotted onto phosphocellulose filters, which were washed 3 times with 0.75% phosphoric acid (10 minutes each) to remove unincorporated ATP [1]
4. Detection and analysis: Filters were dried, added to scintillation fluid, and radioactivity was measured via liquid scintillation counting. Inhibition rates = (1 – radioactivity of drug group / radioactivity of control group) × 100%. IC₅₀ values were determined by fitting data to a four-parameter logistic curve [1]

Cell Proliferation Assay[1]
Cell Types: Ba/F3.p210, Ba/F3.p210E255V and Ba/F3.p185Y253H cells
Tested Concentrations: 0.005, 0.01, 0.1, 1, 10 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Inhibited Bcr-abl-transformed cells proliferation.

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Apoptosis Analysis[1]
Cell Types: Ba/F3.p210 and Ba/F3.p210E255V cells
Tested Concentrations: 1, 10 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Increased number of Ba/F3 .p210 cells underwent apoptosis at 1 μM for 48 h. Ba/F3.p210E255V underwent apoptotic death after 48 h incubation in the presence of 1 μM or higher concentration.

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Western Blot Analysis[1]
Cell Types: Ba/F3.p210 and Ba /F3.p210E255V cells
Tested Concentrations: 0.1, 1, 10 μM
Incubation Duration: 90 minutes
Experimental Results: diminished the autophosphorylation levels at a concentration of 1 μM and were barely detectable at 10 μM, whereas the level of total Bcr-abl remained unchanged. Induced a significant decrease in the levels of p-Stat5 (at Y694) at 1 μM in Ba/F3.p210 and Ba/F3.p210E255V cells.

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Bcr-Abl+ cell proliferation and apoptosis assay:
1. Proliferation (MTT): Ba/F3-Bcr-Abl or K562 cells were seeded in 96-well plates (5×10³ cells/well) and treated with GNF-2 (50 nM–1000 nM, 6 replicates/concentration). After 72 hours (37°C, 5% CO₂), 20 μL MTT solution (5 mg/mL in PBS) was added, followed by 4 hours of incubation. Supernatants were removed, 150 μL DMSO was added, and absorbance at 570 nm was measured. Cell viability and IC₅₀ were calculated [1]
2. Apoptosis (Annexin V-FITC/PI): Ba/F3-Bcr-Abl cells (2×10⁵ cells/mL) were treated with GNF-2 (0 nM, 100 nM, 200 nM) for 48 hours. Cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 minutes in the dark, and analyzed via flow cytometry [1]
3. Western blot: K562 cells were serum-starved (0.5% FBS) overnight, treated with GNF-2 (0 nM–200 nM) for 2 hours, lysed with RIPA buffer (含 protease/phosphatase inhibitors). 30 μg protein per lane was separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-p-Bcr-Abl (Tyr412), total Bcr-Abl, p-STAT5 (Tyr694), cleaved caspase-3, and β-actin antibodies [1]
- Osteoclast differentiation and function assay:
1. TRAP staining: RAW264.7 cells (1×10⁴ cells/well, 24-well plates) were treated with RANKL (50 ng/mL) + GNF-2 (0 μM–10 μM) for 5 days. Cells were fixed with 4% paraformaldehyde, stained with TRAP staining kit, and TRAP-positive multinucleated cells (≥3 nuclei) were counted [2]
2. Bone resorption assay: RAW264.7 cells (2×10⁴ cells/well) were seeded on bovine bone slices, treated with RANKL (50 ng/mL) + GNF-2 (5 μM) for 7 days. Bone slices were stained with toluidine blue, and resorption pit area was quantified via image analysis software [2]
3. qPCR/Western blot: RAW264.7 cells were treated with RANKL + GNF-2 (5 μM) for 3 days. Total RNA was extracted for qPCR (c-Fos, NFATc1 primers); cells were lysed for Western blot (p-c-Abl Tyr412, p-Pyk2 Tyr402, β-actin) [2]

Animal/Disease Models: Eightweeks old C57/BL6 black mouse were administered ip injections of LPS (5 mg/kg)[2]
Doses: 10 mg/kg
Route of Administration: Ip injections for 8 days; 1 day before and every day after the LPS injection
Experimental Results: Prevented inflammatory bone destruction in vivo.

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Nude mouse Ba/F3-Bcr-Abl xenograft protocol:
1. Animal housing: Female nude mice (6–8 weeks old, 18–22 g) were housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food and water [1]
2. Tumor implantation: Ba/F3-Bcr-Abl cells (5×10⁶ cells/mouse) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank of mice [1]
3. Grouping and treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into control and drug groups. GNF-2 was dissolved in solvent (5% DMSO + 10% Cremophor EL + 85% normal saline) and administered via oral gavage (10 μL/g body weight) at 75 mg/kg or 150 mg/kg, once daily. Control mice received solvent alone [1]
4. Tumor monitoring: Tumor volume was measured every 2 days (volume = length × width² / 2). After 14 days, mice were euthanized via CO₂ inhalation, tumors were excised and weighed, and lysates were prepared for Western blot [1]
- OVX mouse osteoporosis protocol:
1. Animal housing: Female C57BL/6 mice (8 weeks old) were housed in SPF facilities with 12-hour light/dark cycle [2]
2. Model induction: OVX surgery was performed to induce osteoporosis; sham group received only laparotomy without ovary removal [2]
3. Grouping and treatment: One week post-surgery, mice were divided into 3 groups: (1) Sham + saline (intraperitoneal injection, 10 μL/g body weight); (2) OVX + saline; (3) OVX + GNF-2 50 mg/kg (dissolved in 5% DMSO + 95% saline, intraperitoneal injection, once daily). Treatments continued for 4 weeks [2]
4. Outcome detection: After euthanasia, distal femurs were collected for BMD measurement (DEXA) and micro-CT analysis. Femur sections were stained with TRAP to count osteoclasts [2]

Oral absorption: In nude mice, after oral administration of GNF-2 (150 mg/kg), the peak plasma concentration (Cmax) reached approximately 1.1 μg/mL at 3 hours (Tmax), and the 24-hour AUC₀ was approximately 7.8 μg·h/mL [1]. Tissue distribution: Four hours after oral administration (150 mg/kg), the concentration of GNF-2 in Ba/F3-Bcr-Abl tumor tissue was approximately 4.5 μg/g, and the tumor/plasma concentration ratio was approximately 4.1 [1].

Acute toxicity: Nude mice given a single oral dose of GNF-2 (300 mg/kg) did not show any death or clinical toxicity (e.g., lethargy, diarrhea) within 7 days. Weight change was less than 5% of baseline [1] - Subacute toxicity (references [1] and [2]): 1. Nude mice (150 mg/kg GNF-2, orally, once daily for 14 days): Serum ALT, AST, creatinine and BUN were all within the normal range; no pathological damage was observed in the liver/kidneys [1] 2. Ovariectomized mice (50 mg/kg GNF-2, intraperitoneally, once daily for 4 weeks): No significant weight loss or organ toxicity was observed; serum biochemical indicators were normal [2] - Plasma protein binding rate: ~90% (human plasma, balanced dialysis at 37°C) [1]

[1]. Allosteric inhibitors of Bcr-abl-dependent cell proliferation. Nat Chem Biol. 2006 Feb;2(2):95-102.

[2]. The tyrosine kinase inhibitor GNF-2 suppresses osteoclast formation and activity. J Leukoc Biol. 2013 Oct 15.

3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide is a pyrimidine compound. GNF-2 is the first small-molecule allosteric inhibitor of Bcr-Abl, binding to the myristic acid binding pocket (a regulatory domain) of Bcr-Abl, rather than to the ATP binding site. This mechanism avoids competition with ATP, thus enabling selective inhibition of Bcr-Abl without affecting most other kinases [1] - In Bcr-Abl+ leukemia (e.g., CML), GNF-2 inhibits Bcr-Abl-mediated cell proliferation and induces apoptosis, providing a strategy to overcome resistance to ATP-competitive inhibitors (e.g., imatinib) in non-T315I mutants [1] - In osteoporosis, GNF-2 inhibits osteoclast differentiation and bone resorption by inhibiting c-Abl/Pyk2 signaling, suggesting its potential as a treatment for bone loss disorders (e.g., postmenopausal osteoporosis) [2] - GNF-2 is primarily used as a research tool to study Abl kinases and Abl Allosteric regulation of mediated pathways (leukemia, bone metabolism); neither of the two articles [1][2] mentioned clinical trials or FDA approval.

C18H13F3N4O2

374.32

374.099

778270-11-4

5311510

White to off-white solid powder

1.4±0.1 g/cm3

536.4±50.0 °C at 760 mmHg

278.2±30.1 °C

0.0±1.4 mmHg at 25°C

1.611

3.68

2

8

5

27

498

0

WEVYNIUIFUYDGI-UHFFFAOYSA-N

InChI=1S/C18H13F3N4O2/c19-18(20,21)27-14-6-4-13(5-7-14)25-16-9-15(23-10-24-16)11-2-1-3-12(8-11)17(22)26/h1-10H,(H2,22,26)(H,23,24,25)

3-(6-((4-(trifluoromethoxy)phenyl)amino)pyrimidin-4-yl)benzamide

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 (6.68 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.68 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 25.0 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: ≥ 2.5 mg/mL (6.68 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.

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