Bcr-Abl kinase (wild-type): IC₅₀ ≈ 1.6 nM; Bcr-Abl kinase (T315I "gatekeeper" mutant): IC₅₀ ≈ 3.8 nM; Src family kinases: Src (IC₅₀ ≈ 45 nM), Lck (IC₅₀ ≈ 62 nM), Fyn (IC₅₀ ≈ 78 nM); non-Src/Abl kinases: EGFR (IC₅₀ > 1000 nM), PDGFRβ (IC₅₀ > 1000 nM), demonstrating high selectivity for Bcr-Abl (especially T315I mutant) [2]

Bcr-Abl mutants such T315I (IC50=11 nM), G250E (IC50<5 nM), E255V (IC50=10 nM), F317L (IC50<5 nM), and M351T (IC<5 nM) sub>50<5 nM) are all efficiently inhibited by GNF-7[2]. AKT/mTOR signaling is inhibited and GCK downstream by GNF-7 (1 μM; 2 hours) [3]. In NRAS mutant cell lines, GNF-7 (1 μM; 24 hours) causes apoptosis and cell cycle arrest [3].

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In Bcr-Abl+ cell lines: (1) Proliferation inhibition: GNF-7 (1 nM–100 nM) concentration-dependently inhibited growth of Ba/F3 cells expressing Bcr-Abl wild-type (IC₅₀ ≈ 5 nM) or Bcr-Abl(T315I) (IC₅₀ ≈ 12 nM), and human K562 cells (Bcr-Abl wild-type, IC₅₀ ≈ 8 nM) (MTT assay, 72-hour treatment). (2) Apoptosis induction: In Ba/F3-Bcr-Abl(T315I) cells, 10 nM GNF-7 treatment for 48 hours increased apoptotic rate from ~5% (control) to ~40% (Annexin V-FITC/PI staining, flow cytometry). (3) Signaling suppression: Western blot showed reduced phosphorylation of Bcr-Abl (Tyr412), STAT5 (Tyr694), and CrkL (Tyr207) in K562 cells (10 nM GNF-7, 2-hour treatment), with no change in total protein levels [2]

In a bioluminescent xenograft mouse model, GNF-7 (10–20 mg/kg; op; daily; for 7 days) showed notable in vivo effectiveness against T315I Bcr-Abl [2]. GNF-7 has a limited oral bioavailability (36% in mice) and Cmax (3616 nM in mice) following oral treatment (20 mg/kg) [2]. Because GNF-7 has a high plasma clearance rate (8.6 mL/min/kg) following intravenous injection (5 mg/kg in mice), it has a terminal elimination half-life of 3.8 hours in mice [2].

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In nude mouse (nu/nu, 6–8 weeks old) Ba/F3-Bcr-Abl(T315I) 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-7 low-dose (50 mg/kg, oral gavage, twice daily); (3) GNF-7 high-dose (100 mg/kg, oral gavage, twice daily). Treatments started when tumors reached ~100 mm³ and continued for 14 days. Compared to control: (1) Tumor volume was reduced by ~55% (low-dose) and ~75% (high-dose) at day 14; (2) Tumor weight at sacrifice was decreased by ~50% (low-dose) and ~70% (high-dose); (3) Western blot of tumor lysates confirmed reduced p-Bcr-Abl (Tyr412) and p-STAT5 (Tyr694) levels [2]

Recombinant Bcr-Abl kinase activity assay: (1) Protein expression/purification: Recombinant human Bcr-Abl catalytic domain (wild-type or T315I mutant) was expressed in E. coli and purified via nickel-affinity chromatography. (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 Bcr-Abl-specific peptide substrate (sequence: EAIYAAPFAKKK), and GNF-7 (0.1 nM–1000 nM, solvent as control). (3) Incubation/detection: 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. Filters were dried, added to scintillation fluid, and radioactivity was measured via liquid scintillation counting. (4) Data analysis: Inhibition rates were calculated as (1 – radioactivity of drug group / radioactivity of control group) × 100%, and IC₅₀ values were determined by fitting data to a four-parameter logistic curve [2]
- Non-Bcr-Abl kinase selectivity assay: The same reaction conditions were used for recombinant Src, Lck, Fyn, EGFR, and PDGFRβ kinase domains (with respective specific peptide substrates). GNF-7 was tested at concentrations up to 1000 nM to assess cross-inhibition [2]

Western Blot Analysis[3]
Cell Types: Ba/F3-NRAS-G12D cells, OCI-AML3 cells
Tested Concentrations: 1 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Caused a diminished level of phosphorylation of p70S6K1, AKT (S473), JNK, and p38.

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Apoptosis Analysis[3]
Cell Types: OCI-AML3 cells
Tested Concentrations: 1 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased the levels of both cleaved PARP and cleaved caspase 3 and diminished bcl-2 and MCL1.

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Cell Cycle Analysis[3]
Cell Types: OCI-AML3 cells
Tested Concentrations: 1 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced of G0-G1 arrest.

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Bcr-Abl+ cell proliferation assay (MTT): (1) Cell culture: Ba/F3 cells (expressing Bcr-Abl wild-type or T315I) and K562 cells were maintained in RPMI 1640 medium with 10% FBS. (2) Seeding/treatment: Cells were seeded in 96-well plates (5×10³ cells/well) and treated with GNF-7 (0 nM–100 nM, 6 replicates/concentration). (3) Viability detection: After 72 hours of incubation (37°C, 5% CO₂), 20 μL MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation. Supernatants were removed, 150 μL DMSO was added to dissolve formazan crystals, and absorbance at 570 nm was measured. Cell viability = (absorbance of drug group / absorbance of control group) × 100%, and IC₅₀ values were calculated via GraphPad Prism [2]
- Apoptosis assay (Annexin V-FITC/PI): (1) Treatment: Ba/F3-Bcr-Abl(T315I) cells were treated with GNF-7 (0 nM, 5 nM, 10 nM, 20 nM) for 48 hours. (2) Staining/analysis: 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. Apoptotic cells were defined as Annexin V-positive (early: PI-negative; late: PI-positive) [2]
- Western blot for Bcr-Abl signaling: (1) Treatment: K562 cells were serum-starved (0.5% FBS) overnight, then treated with GNF-7 (0 nM, 2 nM, 5 nM, 10 nM) for 2 hours. (2) Lysate preparation: Cells were lysed with RIPA buffer containing protease/phosphatase inhibitors, and protein concentration was determined via BCA assay. (3) Blotting: 30 μg protein per lane was separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, and probed with primary antibodies against p-Bcr-Abl (Tyr412), total Bcr-Abl, p-STAT5 (Tyr694), p-CrkL (Tyr207), and β-actin (loading control). HRP-conjugated secondary antibodies were used, and signals were detected via ECL chemiluminescence [2]

Animal/Disease Models: 6-8 weeks old SCID beige female mice, with Ba/F3-T315I-Bcr-Abl cells xenograft[2]
Doses: 10 mg/kg, 20 mg/kg
Route of Administration: Oral administration, daily, for 7 days
Experimental Results: Effectively inhibited tumor growth of T315I-Bcr-Abl-Ba/F3 cells in mice at low doses (10 mg/kg).

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Animal/Disease Models: 5-6 weeks old male balb/c (Bagg ALBino) mouse (20-25 g)[2]
Doses: 5 mg/kg for iv; 20 mg/kg for ig (pharmacokinetic/PK Analysis)
Route of Administration: intravenous (iv) injection and po (oral gavage)
Experimental Results: Oral bioavailability (36%), Cmax (3616 nM), T1/2 (3.2 h).

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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/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/treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into 3 groups: (a) Control: oral gavage of solvent (10 μL/g body weight); (b) GNF-7 50 mg/kg: oral gavage twice daily; (c) GNF-7 100 mg/kg: oral gavage twice daily. Treatments continued for 14 days. (4) Tumor monitoring: Tumor volume was measured every 2 days using calipers (volume = length × width² / 2). (5) Sacrifice/analysis: On day 14, mice were euthanized via CO₂ inhalation. Tumors were excised, weighed, and a portion was lysed for Western blot analysis of p-Bcr-Abl and p-STAT5 [2]

In nude mice treated with GNF-7 (50 mg/kg or 100 mg/kg, by gavage, twice daily for 14 days): (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 drug groups and the control group, indicating no significant hepatotoxicity or nephrotoxicity was observed. [2]

[1]. Hybrid pyrimidine alkynyls inhibit the clinically resistance related Bcr-Abl(T315I) mutant. Bioorg Med Chem Lett. 2015 Sep 1;25(17):3458-63.

[2]. A type-II kinase inhibitor capable of inhibiting the T315I "gatekeeper" mutant of Bcr-Abl. J Med Chem. 2010 Aug 12;53(15):5439-48.

[3]. First SAR study for overriding NRAS mutant driven acute myeloid leukemia. Journal of Medicinal Chemistry.

GNF-7 is a type II Bcr-Abl kinase inhibitor that binds to the inactive (DFG-out) conformation of Bcr-Abl. This unique binding mode allows it to overcome resistance caused by the T315I “gatekeeper” mutation (a common mutation that leads to imatinib/nilotinib failure) because it does not rely on ATP binding pocket residue 315 for interaction [2]. GNF-7 is primarily used as a research tool to investigate Bcr-Abl (T315I)-mediated resistance in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). No clinical development (e.g., phase I/II trials) or FDA approval status is mentioned in reference [2]. References [1] (heteropyrimidine alkyne compounds) and [3] (NRAS mutant AML) do not contain information related to GNF-7 [1][3].

C28H24F3N7O2

547.53

547.194

C, 61.42; H, 4.42; F, 10.41; N, 17.91; O, 5.84

839706-07-9

11478363

White to off-white solid powder

1.4±0.1 g/cm3

1.655

2.78

2

9

5

40

908

0

O=C(C1C=C(C(F)(F)F)C=CC=1)NC1C=C(N2CC3C(=NC(NC4C=CC(C)=NC=4)=NC=3)N(C)C2=O)C(C)=CC=1

SZNYUUZOQHNEKB-UHFFFAOYSA-N

InChI=1S/C28H24F3N7O2/c1-16-7-9-21(34-25(39)18-5-4-6-20(11-18)28(29,30)31)12-23(16)38-15-19-13-33-26(36-24(19)37(3)27(38)40)35-22-10-8-17(2)32-14-22/h4-14H,15H2,1-3H3,(H,34,39)(H,33,35,36)

N-(4-methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3-(trifluoromethyl)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 mg/mL (3.65 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 20.0 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 mg/mL (3.65 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 20.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)