MAP4K2 (IC50 = 21.7 nM); TAK1 (IC50 = 149 nM); LYN (IC50 = 12.9 nM); GSK (IC50 = 56.4 nM); ABL,ARG (IC50 = 75.2 nM); FER (IC50 = 82.3 nM); SRC (IC50 = 113 nM); Eph B2 (IC50 = 672 nM); ZAK (IC50 = 698 nM); Eph A2 (IC50 = 773 nM); Eph B4 (IC50 = 999 nM); ZC1/HGK (IC50 = 3250 nM); RAF1 (IC50 = 7590 nM)
NG25 is a potent dual TAK1 and MAP4K2 inhibitor, with IC50s of 149 nM and 21.7 nM, respectively. Additionally, NG25 effectively inhibits a number of kinases, including LYN, CSK, FER, p38α, ABL, ARG, and SRC, with respective IC50 values of 12.9, 56.4, 82.3, 102, 75.2, and 113 nM[1]. NG25 completely inhibits the secretion of IFNα and IFNβ when stimulated by CL097 or CpG B or A, respectively[2]. All tested breast cancer cell lines experience a dose-dependent reduction in cell viability after NG25 treatment. The cytotoxic effect of Dox on breast cancer cells is enhanced by NG25 (2 μM)[3].
NG-25 (also referred to as compound 1) is a type II kinase inhibitor. Its primary targets are Transforming Growth Factor β-Activated Kinase 1 (TAK1, MAP3K7) with an IC₅₀ of 149 nM and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2, GCK) with an IC₅₀ of 21.7 nM.
It also inhibits p38α (MAPK14) and ABL kinase.
Other kinases showing inhibition in profiling include ZAK, LYN, SRC, FER, FES, and EPH-family kinases. [1]

NG25 is a potent dual TAK1 and MAP4K2 inhibitor, with IC50s of 149 nM and 21.7 nM, respectively. Additionally, NG25 effectively inhibits a number of kinases, including LYN, CSK, FER, p38α, ABL, ARG, and SRC, with respective IC50 values of 12.9, 56.4, 82.3, 102, 75.2, and 113 nM[1]. NG25 completely inhibits the secretion of IFNα and IFNβ when stimulated by CL097 or CpG B or A, respectively[2]. All tested breast cancer cell lines experience a dose-dependent reduction in cell viability after NG25 treatment. The cytotoxic effect of Dox on breast cancer cells is enhanced by NG25 (2 μM)[3].

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NG-25 (1) at 100 nM effectively reduced TNFα-induced phosphorylation of IKKα/β and inhibited IκB-α degradation in mouse L929 cells, comparable to the irreversible TAK1 inhibitor 37 (5Z-7-oxozeaenol). [1]
In 293 IL-1R cells and mouse embryonic fibroblasts (MEFs) stimulated with IL-1α, NG-25 (1) dose-dependently inhibited phosphorylation of downstream p105, p38, and JNK1/2. [1]
In mouse RAW cells stimulated with LPS, NG-25 (1) inhibited phosphorylation of p105, p38, and JNK1/2. [1]
In human Gen2.2 cells stimulated with the TLR7 agonist CL097, NG-25 (1) at 1 µM blocked signaling. [1]
NG-25 (1) exhibited antiproliferative activity against BCR-ABL-dependent Ba/F3 cells. [1]

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NG-25 (1) at 100 nM effectively reduced TNFα-induced phosphorylation of IKKα/β and inhibited IκB-α degradation in mouse L929 cells, comparable to the irreversible TAK1 inhibitor 37 (5Z-7-oxozeaenol). [1]
In 293 IL-1R cells and mouse embryonic fibroblasts (MEFs) stimulated with IL-1α, NG-25 (1) dose-dependently inhibited phosphorylation of downstream p105, p38, and JNK1/2. [1]
In mouse RAW cells stimulated with LPS, NG-25 (1) inhibited phosphorylation of p105, p38, and JNK1/2. [1]
In human Gen2.2 cells stimulated with the TLR7 agonist CL097, NG-25 (1) at 1 µM blocked signaling. [1]
NG-25 (1) exhibited antiproliferative activity against BCR-ABL-dependent Ba/F3 cells. [1]

IRF7 is produced in Escherichia coli as a fusion protein with the enzyme glutathione S-transferase (GST), which is broken down by the enzyme PreScission proteinase. IRF7 is released from GST and glutathione-Sepharose by PreScission proteinase digestion, and GST-IRF7 is captured on glutathione-Sepharose. Insect Sf21 cells express His6-tagged forms of IKKβ and TBK1 that have been phosphorylated to become active, and these purified forms are then used in nickel nitrilotriacetate-agarose affinity chromatography. The Millipore company sells active GST-IKKα, which is then tested.

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The inhibitory activity of NG-25 against various kinases was determined using biochemical enzyme assays. For TAK1 and MAP4K2, IC₅₀ values were determined using a commercial kinase profiling service (SelectScreen). The assay measures compound inhibition of kinase enzymatic activity. [1]
Additionally, kinome-wide selectivity profiling was performed using an in vitro ATP-site competition binding assay (KinomeScan) against a panel of over 420 kinases at concentrations of 10 µM or 1 µM. [1]

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The inhibitory activity of NG-25 against various kinases was determined using biochemical enzyme assays. For TAK1 and MAP4K2, IC₅₀ values were determined using a commercial kinase profiling service (SelectScreen). The assay measures compound inhibition of kinase enzymatic activity. [1]
Additionally, kinome-wide selectivity profiling was performed using an in vitro ATP-site competition binding assay (KinomeScan) against a panel of over 420 kinases at concentrations of 10 µM or 1 µM. [1]

In 96-well plates, 3.5×105 Gen 2.2 cells or Flt3-DCs are incubated for 1 hour without or with the indicated concentrations of the inhibitor. They are then stimulated with 1 M CpG (type A or B), 1 μg/mL of CL097, or 1 μg/mL of R848. The cell culture supernatants are collected after 5 or 12 hours, clarified by centrifugation, and stored at 80°C pending cytokine level analysis. Unstimulated cells are incubated for 12 hours, either in the absence or presence of inhibitors, for cell viability assays. Following cell fixation, flow cytometry is used to determine the percentage of living cells.

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or cell-based kinase inhibition profiling, A375 melanoma cells were treated with NG-25 at 5 µM and 0.5 µM, lysed, and probe-labeled using KINativ technology. This approach measures the compound's ability to protect kinases in cell lysates from labeling with a lysine-reactive ATP or ADP-biotin probe, indicating target engagement in a cellular context. [1]
To assess inhibition of downstream signaling, L929 cells were pretreated with compounds (e.g., 100 nM) for 30 min, then stimulated with TNFα for 5 min. Cells were lysed, and phosphorylation of IKKα/β and levels of IκB-α were analyzed by Western blot. [1]
Similarly, 293 IL-1R cells, MEFs, and RAW cells were pretreated with NG-25 at various concentrations for 1 h, then stimulated with IL-1α or LPS for 30 min. Phosphorylation of p105, p38, and JNK1/2 was analyzed by Western blot. [1]
Gen2.2 cells were pretreated with compounds (1 µM) for 1 h, then stimulated with CL097 for 30 min, followed by Western blot analysis. [1]
Antiproliferative activity was assessed in Ba/F3 cells (BCR-ABL-dependent and parental). Cells were co-cultured with serially diluted compounds for 48 h, and viability was measured using a luminescent cell viability assay. [1]

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or cell-based kinase inhibition profiling, A375 melanoma cells were treated with NG-25 at 5 µM and 0.5 µM, lysed, and probe-labeled using KINativ technology. This approach measures the compound's ability to protect kinases in cell lysates from labeling with a lysine-reactive ATP or ADP-biotin probe, indicating target engagement in a cellular context. [1]
To assess inhibition of downstream signaling, L929 cells were pretreated with compounds (e.g., 100 nM) for 30 min, then stimulated with TNFα for 5 min. Cells were lysed, and phosphorylation of IKKα/β and levels of IκB-α were analyzed by Western blot. [1]
Similarly, 293 IL-1R cells, MEFs, and RAW cells were pretreated with NG-25 at various concentrations for 1 h, then stimulated with IL-1α or LPS for 30 min. Phosphorylation of p105, p38, and JNK1/2 was analyzed by Western blot. [1]
Gen2.2 cells were pretreated with compounds (1 µM) for 1 h, then stimulated with CL097 for 30 min, followed by Western blot analysis. [1]
Antiproliferative activity was assessed in Ba/F3 cells (BCR-ABL-dependent and parental). Cells were co-cultured with serially diluted compounds for 48 h, and viability was measured using a luminescent cell viability assay. [1]

Male Swiss albino mice
1 mg/kg
i.v. or o.g.

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Pharmacokinetic studies were performed in male Swiss albino mice. For intravenous (IV) administration, NG-25 was formulated as a solution in 20% (v/v) hydroxypropyl β-cyclodextrin in 25 mM sodium phosphate buffer and administered via tail vein at a dose of 1 mg/kg.
For oral (PO) administration, NG-25 was formulated as a suspension in 0.5% (w/v) sodium carboxymethyl cellulose (Na CMC) with 0.1% (v/v) Tween-80 in water and administered via oral gavage at a dose of 10 mg/kg.
Blood samples were collected at various time points post-dose (e.g., 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h). Plasma was separated and stored for bioanalysis by LC/MS/MS. [1]

After intravenous injection of 1 mg/kg NG-25 into mice, its half-life (T₁/₂) was 2.03 h, its clearance (CL) was 80.8 mL min⁻¹ kg⁻¹, and its steady-state volume of distribution (Vₛₛ) was 11.9 L/kg. After oral administration of 10 mg/kg NG-25, the maximum plasma concentration (Cmax) was 316 ng/mL, the time to peak concentration (Tmax) was 0.5 h, and the area under the concentration-time curve (AUC) was 1369 h·ng/mL. The oral bioavailability (F) was 70.2%. [1]

At the concentrations used in cell experiments (the highest concentration in the cytokine secretion experiment was 400 nM, and the highest concentration in the Western blot experiment was 2 μM), NG-25 had no effect on the viability of Gen2.2 cells, as confirmed in control experiments. [2]

[1]. Discovery of type II inhibitors of TGFβ-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2). J Med Chem. 2015 Jan 8;58(1):183-96.

[2]. Essential role for IKKβ in production of type 1 interferons by plasmacytoid dendritic cells. J Biol Chem. 2012 Jun 1;287(23):19216-28.

[3]. TAK1 inhibitor NG25 enhances doxorubicin-mediated apoptosis in breast cancer cells. Sci Rep. 2016 Sep 7;6:32737.

NG-25 was discovered using a pharmacophore model and whole kinase assay of type II inhibitors.
It binds to the DFG-out conformation of TAK1, as confirmed by the 2.4 Å co-crystal structure (PDB: 4O91). Its binding involves hydrogen bonds with the hinge region (Ala107), the linker amide with the Glu77 and Asp175 backbone, and the piperazine tails with the carbonyl groups of Ile153 and His154 on the activation ring.
It can serve as a pharmacological probe for TAK1 due to its reversible inhibitory mode and unique off-target effects compared to the covalent inhibitor 5Z-7-oxozeaenol.
Structural modifications yielded analogs with higher selectivity for MAP4K2 (e.g., compound 17) or p38α (e.g., compound 11). [1]

C29H30F3N5O2

537.5760

537.235

C, 64.79; H, 5.63; F, 10.60; N, 13.03; O, 5.95

1315355-93-1

NG25 trihydrochloride;2108554-00-1

53340664

White to off-white solid powder

6.332

2

8

7

39

808

0

FC(C1C([H])=C(C([H])=C([H])C=1C([H])([H])N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C1([H])[H])N([H])C(C1C([H])=C([H])C(C([H])([H])[H])=C(C=1[H])OC1C([H])=C([H])N=C2C=1C([H])=C([H])N2[H])=O)(F)F

SMPGEBOIKULBCT-UHFFFAOYSA-N

InChI=1S/C29H30F3N5O2/c1-3-36-12-14-37(15-13-36)18-21-6-7-22(17-24(21)29(30,31)32)35-28(38)20-5-4-19(2)26(16-20)39-25-9-11-34-27-23(25)8-10-33-27/h4-11,16-17H,3,12-15,18H2,1-2H3,(H,33,34)(H,35,38)

N-[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-4-methyl-3-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)benzamide

NG25; NG-25; NG 25

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)

DMSO: 16.7~25 mg/mL (31.0~46.1 mM)
Ethanol: ~3 mg/mL (~5.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.65 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 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 (4.65 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.65 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.)