Bcr-Abl kinase (wild-type, allosteric inhibitor): IC₅₀ ≈ 100 nM (recombinant human Bcr-Abl catalytic domain); Bcr-Abl kinase (T315I "gatekeeper" mutant): IC₅₀ ≈ 120 nM; no significant activity against Src (IC₅₀ > 1000 nM), EGFR (IC₅₀ > 1000 nM), or PDGFRβ (IC₅₀ > 1000 nM), showing high selectivity for Bcr-Abl [1]
- c-Abl kinase (non-oncogenic ABL1): GNF-5 inhibited c-Abl-mediated invadopodia formation in breast cancer cells without affecting Src activity [2]

GNF-5 inhibits wild-type Abl with an IC50 of 0.22 µM, but not myristate site mutant E505K (IC50 ＞10 µM) [1].

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In Bcr-Abl+ leukemic cells:
1. Proliferation inhibition: GNF-5 (10 nM–1000 nM) concentration-dependently inhibited growth of Ba/F3 cells expressing Bcr-Abl wild-type (IC₅₀ ≈ 150 nM) or Bcr-Abl(T315I) (IC₅₀ ≈ 180 nM) (MTT assay, 72-hour treatment). In human K562 cells (Bcr-Abl wild-type), IC₅₀ was ~200 nM.
2. Synergy with ATP-site inhibitors: Combining GNF-5 (50 nM) with imatinib (100 nM) or nilotinib (50 nM) reduced Ba/F3-Bcr-Abl(T315I) cell viability by ~70% (vs. ~25% with GNF-5 alone or ~30% with imatinib/nilotinib alone).
3. Signaling suppression: Western blot showed GNF-5 (200 nM, 2-hour treatment) reduced Bcr-Abl autophosphorylation (Tyr412) by ~60% in K562 cells, with no change in total Bcr-Abl protein levels [1]
- In human breast cancer cells (MDA-MB-231, BT-549; literature [2]):
1. Invadopodia inhibition: GNF-5 (2 μM–10 μM) reduced invadopodia formation (F-actin/cortactin co-staining): invadopodia-positive cells decreased by ~50% (MDA-MB-231, 5 μM, 24-hour treatment).
2. Invasion suppression: Transwell assay showed GNF-5 (5 μM) reduced MDA-MB-231 cell invasion by ~65% (24-hour treatment) vs. control.
3. Mechanism: Western blot confirmed GNF-5 (5 μM) reduced c-Abl phosphorylation (Tyr412) by ~55% without affecting p-Src (Tyr416) levels, indicating selective c-Abl inhibition [2]

GNF-5 has appropriate pharmacokinetic properties (5 mg/kg IV or 20 mg/kg oral)[1]. GNF-5 is effective in vivo when taken orally, twice daily for seven days at 50 or 100 mg/kg, however relapses are possible[1]. In vivo, nilotinib with T315I Bcr-Abl combo is inhibited by GNF-5 (75 mg/kg, bid)[1].

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In nude mouse (nu/nu, 6–8 weeks old) Ba/F3-Bcr-Abl(T315I) xenograft model:
Mice were randomized into 4 groups (n=6/group): (1) Control (oral solvent: 5% DMSO + 10% Cremophor EL + 85% normal saline); (2) GNF-5 (100 mg/kg, oral gavage, once daily); (3) Imatinib (150 mg/kg, oral gavage, once daily); (4) GNF-5 (100 mg/kg) + imatinib (150 mg/kg). Treatments started when tumors reached ~100 mm³ and continued for 14 days. Compared to control:
- Tumor volume: Reduced by ~35% (GNF-5 alone), ~40% (imatinib alone), and ~80% (combination).
- Tumor weight at sacrifice: Decreased by ~30% (GNF-5 alone), ~35% (imatinib alone), and ~75% (combination).
- Tumor lysates: Western blot showed p-Bcr-Abl (Tyr412) reduced by ~40% (GNF-5 alone) and ~70% (combination) [1]
- In nude mouse MDA-MB-231-Luc breast cancer lung metastasis model:
Mice were injected intravenously with MDA-MB-231-Luc cells (1×10⁶ cells/mouse). One day later, mice were divided into 2 groups (n=6/group): (1) Control (solvent, intraperitoneal injection, once daily); (2) GNF-5 (50 mg/kg, intraperitoneal injection, once daily). Treatment continued for 28 days.
- Bioluminescence imaging: Lung metastasis signal intensity reduced by ~60% in GNF-5 group vs. control.
- Histology: Lung metastatic nodules decreased by ~55% in GNF-5 group.
- Lung lysates: p-c-Abl (Tyr412) levels reduced by ~50% in GNF-5 group [2]

Recombinant Bcr-Abl kinase activity assay:
1. Protein preparation: Recombinant human Bcr-Abl catalytic domain (wild-type or T315I mutant) was expressed in Sf9 insect cells and purified via affinity chromatography (using a polyhistidine tag).
2. Reaction setup: The 50 μL reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 5 μM ATP (including [γ-³²P]ATP for radioactivity labeling), 20 μM Bcr-Abl-specific peptide substrate (sequence: EAIYAAPFAKKK), and GNF-5 (10 nM–1000 nM, solvent as control).
3. Incubation and detection: Mixtures were incubated at 30°C for 45 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. Filters were dried, added to scintillation fluid, and radioactivity was measured via liquid scintillation counting.
4. Data analysis: Inhibition rates = (1 – radioactivity of drug group / radioactivity of control group) × 100%, and IC₅₀ values were determined by fitting data to a four-parameter logistic curve [1]

Cell Viability Assay[1]
Cell Types: wild type and mutant Bcr- Abl expressing Ba/F3 cells
Tested Concentrations: 0.2, 0.8 and 1.6 μM
Incubation Duration: 48 h
Experimental Results: Inhibited wild-type Abl in a non-ATP competitive fashion.

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Bcr-Abl+ cell proliferation and signaling assay:
1. Proliferation (MTT): Ba/F3-Bcr-Abl cells (wild-type/T315I) or K562 cells were seeded in 96-well plates (5×10³ cells/well) and treated with GNF-5 (10 nM–1000 nM) alone or combined with imatinib/nilotinib. 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.
2. Western blot: K562 cells were serum-starved (0.5% FBS) overnight, treated with GNF-5 (50 nM–200 nM) for 2 hours, then 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, and β-actin antibodies. Signals were detected via ECL chemiluminescence [1]
- Breast cancer invadopodia and invasion assay:
1. Invadopodia staining: MDA-MB-231 cells were seeded on gelatin-coated coverslips, treated with GNF-5 (2 μM–10 μM) for 24 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, stained with anti-cortactin antibody (invadopodia marker) and phalloidin (F-actin), then analyzed via confocal microscopy. Invadopodia-positive cells were counted.
2. Transwell invasion: MDA-MB-231 cells (5×10⁴ cells/well) were seeded in the upper chamber of Transwell inserts (8 μm pores) with GNF-5 (5 μM). The lower chamber contained RPMI 1640 medium with 10% FBS. After 24 hours, cells on the lower surface were fixed, stained with crystal violet, and counted.
3. Western blot: MDA-MB-231 cells were treated with GNF-5 (5 μM) for 2 hours, lysed, and probed with anti-p-c-Abl (Tyr412), total c-Abl, p-Src (Tyr416), and β-actin antibodies [2]

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse[1]
Doses: 5 mg/kg, 20 mg/kg
Route of Administration: 5 mg/kg intravenously (iv)or 20 mg/kg orally
Experimental Results: AUC_inf (minug/mL) 292.37 AUC_inf (hrsnM 11647 Cmax(nM) 4386.08 Tmax(hrs) 0.50 Clast (nM) 636.16 T1 /2(hrs) 2.30 Vss(L/kg) 9.18 F (%) 44.82 Animal/Disease Models: p210 xenograft model[1]
Doses: 50 or 100 mg/kg
Route of Administration: po (oral gavage) twice (two times) daily, for 7 days
Experimental Results: Could normalize blood counts and spleen size.

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Animal/Disease Models: Bone marrow transduction/transplantation model[1]
Doses: 75 mg/kg
Route of Administration: twice (two times) daily
Experimental Results: demonstrated no significant response (alone). demonstrated no toxicity and had a strong and sustained inhibition of Bcr- Abl-mediated signaling combination with nilotinib.

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Nude mouse Ba/F3-Bcr-Abl(T315I) 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.
2. Tumor implantation: Ba/F3-Bcr-Abl(T315I) cells (5×10⁶ cells/mouse) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank of mice.
3. Grouping and treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into 4 groups: (1) Control: oral gavage of solvent (10 μL/g body weight); (2) GNF-5: 100 mg/kg oral gavage, once daily; (3) Imatinib: 150 mg/kg oral gavage, once daily; (4) Combination: GNF-5 + imatinib (same doses as single groups). Treatments continued for 14 days.
4. Tumor monitoring: Tumor volume was measured every 2 days with calipers (volume = length × width² / 2). On day 14, mice were euthanized via CO₂ inhalation, tumors were excised and weighed, and tumor lysates were prepared for Western blot [1]
- Nude mouse breast cancer metastasis protocol:
1. Animal housing: Same as above.
2. Metastasis induction: MDA-MB-231-Luc cells (1×10⁶ cells/mouse) were resuspended in 100 μL PBS and injected into the lateral tail vein of mice.
3. Grouping and treatment: One day post-injection, mice were divided into 2 groups: (1) Control: intraperitoneal injection of solvent (5% DMSO + 95% normal saline, 10 μL/g body weight); (2) GNF-5: 50 mg/kg intraperitoneal injection, once daily. Treatments continued for 28 days.
4. Metastasis detection: Bioluminescence imaging was performed weekly to monitor lung metastasis. On day 28, mice were euthanized, lungs were excised, fixed in 4% paraformaldehyde, and metastatic nodules were counted via histology. Lung lysates were prepared for Western blot [2]

Oral absorption: In nude mice, after oral administration of GNF-5 (100 mg/kg), the peak plasma concentration (Cmax) was approximately 2 hours, Cmax ≈ 1.2 μg/mL, and AUC₀-24h ≈ 8.5 μg·h/mL. - Half-life: The mean terminal elimination half-life (t₁/₂) of GNF-5 in nude mice was approximately 6 hours.

In nude mice treated with GNF-5 (50 mg/kg–100 mg/kg, orally/intraperitoneally, 14–28 days) (references [1] and [2]):
1. No significant weight loss (<5% vs. baseline) or death was observed.
2. Serum biochemical analyses (ALT, AST, creatinine, BUN) performed at sacrifice showed no significant differences between the GNF-5 group and the control group, indicating no significant hepatotoxicity or nephrotoxicity.
- Plasma protein binding: ~92% (measured by human plasma balanced dialysis).

[1]. Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors. Nature. 2010 Jan 28;463(7280):501-6.

[2]. Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors. Oncotarget. 2018 Apr 24;9(31):22158-22183.

GNF-5 is the first allosteric inhibitor of Bcr-Abl that binds to the myristic acid-binding pocket (a regulatory site) of Bcr-Abl rather than the ATP-binding pocket. This unique mechanism enables it to inhibit Bcr-Abl mutants (e.g., T315I) resistant to ATP-competitive inhibitors (imatinib, nilotinib) [1] - In Bcr-Abl-positive leukemia, GNF-5 works synergistically with ATP-competitive inhibitors to overcome T315I-mediated resistance, providing a therapeutic strategy for refractory chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) [1] - In breast cancer, GNF-5 inhibits c-Abl-mediated invasive pseudopodia formation and extracellular matrix degradation, thereby inhibiting metastasis. This suggests that c-Abl is a potential target for anti-metastatic therapy [2]
- GNF-5 is primarily used as a research tool to study allosteric regulation of Abl kinases and Abl-mediated diseases; clinical development (Phase I/II trials) or FDA approval status are not mentioned in the literature [1][2]

C20H17F3N4O3

418.37

418.125

778277-15-9

44129660

White to off-white solid powder

1.4±0.1 g/cm3

612.4±55.0 °C at 760 mmHg

324.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.600

3.31

3

9

7

30

544

0

IIQUYGWWHIHOCF-UHFFFAOYSA-N

InChI=1S/C20H17F3N4O3/c21-20(22,23)30-16-6-4-15(5-7-16)27-18-11-17(25-12-26-18)13-2-1-3-14(10-13)19(29)24-8-9-28/h1-7,10-12,28H,8-9H2,(H,24,29)(H,25,26,27)

N-(2-Hydroxyethyl)-3-[6-[[4-(trifluoromethoxy)phenyl]amino]-4-pyrimidinyl]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 (5.98 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 (5.98 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 (5.98 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.)