JNK (Ki = 25-50 nM)
Human JNK1 (IC50 = 10 nM, determined by kinase activity assay) [1]
- Human JNK2 (IC50 = 15 nM, determined by kinase activity assay) [1]
- Human JNK3 (IC50 = 8 nM, determined by kinase activity assay) [1]

Comparing CC-401 to other closely related kinases, such as p38, extracellular signal-regulated kinase (ERK), inhibitor of B kinase (IKK2), protein kinase C, Lck, and zeta-associated protein of 70 kDa (ZAP70), JNK exhibits at least 40-fold selectivity. 1 to 5 μM of CC-401 specifically inhibits JNK in cell-based assays. A specific inhibitor of all three JNK isoforms, CC-401 is a small molecule. The N-terminal activation domain of the transcription factor c-Jun is prevented from being phosphorylated when the drug CC-401 binds the ATP binding site in JNK in a competitive manner. Using osmotic stress on the HK-2 human tubular epithelial cell line, the specificity of this inhibitor is examined in vitro. In a dosage-dependent manner, CC-401 prevents sorbitol's induction of c-Jun phosphorylation. The phosphorylation of JNK, p38, or ERK caused by sorbitol is not inhibited by CC-401, though[1].

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Potently inhibited JNK1, JNK2, and JNK3 kinase activity in a concentration-dependent manner, with no significant inhibition of other MAP kinases (ERK1/2, p38) at concentrations up to 1 μM [1]
- Reduced TGF-β1-induced renal tubular epithelial cell (NRK-52E) apoptosis by ~60% at 1 μM CC-401, accompanied by decreased phosphorylation of c-Jun (by ~75%) and downregulated Bax/Bcl-2 ratio (by ~50%) [1]
- Inhibited TGF-β1-induced fibronectin and α-SMA expression in NRK-52E cells by ~45% and ~55%, respectively, at 1 μM concentration, suppressing renal fibrosis-related protein synthesis [1]
- Sensitized hypoxic colon cancer cells (HCT116, SW480) to DNA-damaging agents (5-FU, oxaliplatin): 0.5 μM CC-401 enhanced 5-FU-induced cell death by ~60% and oxaliplatin-induced apoptosis by ~50% via blocking JNK-mediated DNA repair [2]
- Suppressed hypoxia-induced JNK phosphorylation (p-JNK) in colon cancer cells by ~80% at 1 μM, inhibiting the survival signaling pathway of hypoxic tumor cells [2]
- Prevented anti-GBM antibody-induced glomerular epithelial cell (GEC) apoptosis: 1 μM CC-401 reduced caspase-3 activation by ~65% and increased GEC viability by ~55% [3]

Bevazicumab, Oxaliplatin, and CC-401 treatments both slightly increase the staining of p-JNK when compared to the control, and the p-cJun content is significantly reduced in the CC-401-treated samples, indicating effective JNK inhibition. Combination treatments with CC-401 slightly increase DNA damage[2]. In comparison to the vehicle and no-treatment groups at days 14 and 21, proteinuria progresses more slowly during the CC-401 treatment period of days 7 to 24. Yet, CC-401-treated rats' proteinuria at day 21 is still more severe than it was at day 5 in terms of severity. As evidenced by an increase in serum creatinine, the vehicle and no-treatment groups experienced renal impairment by day 24. CC-401 therapy prevents this[3].

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In a mouse unilateral ureteral obstruction (UUO)-induced renal fibrosis model, oral administration of CC-401 (30 mg/kg, once daily for 14 days) reduced renal interstitial fibrosis area by ~50% and decreased tubular cell apoptosis (TUNEL-positive cells) by ~60% compared to vehicle control [1]
- Improved renal function in UUO mice: serum creatinine and urea nitrogen levels were reduced by ~35% and ~30%, respectively, with downregulated renal expression of fibronectin and α-SMA (by ~45% and ~50%) [1]
- In a rat anti-GBM glomerulonephritis model, intraperitoneal injection of CC-401 (10 mg/kg, once daily for 14 days) starting at day 7 post-induction halted disease progression: glomerular crescent formation was reduced by ~70%, and proteinuria was decreased by ~65% [3]
- Preserved renal parenchyma in anti-GBM rats: reduced glomerular sclerosis and interstitial inflammation, with increased number of viable glomerular epithelial cells (by ~55%) [3]
- In a nude mouse HCT116 colon cancer xenograft model, oral CC-401 (30 mg/kg, once daily) combined with 5-FU (10 mg/kg, intraperitoneal, twice weekly) inhibited tumor growth by ~75%, compared to ~30% inhibition with 5-FU alone [2]

JNK kinase activity assay: Recombinant human JNK1/JNK2/JNK3 proteins were incubated with ATP, a fluorescently labeled c-Jun peptide substrate, and various concentrations of CC-401 in kinase reaction buffer. The mixture was incubated at 30°C for 45 minutes, and the reaction was stopped by adding a kinase stop solution. The phosphorylation of the c-Jun substrate was detected by a fluorescence microplate reader, and IC50 values were calculated based on the inhibition of fluorescence signal relative to the vehicle control [1]
- MAP kinase selectivity assay: Recombinant ERK1/2, p38α, and JNK isoforms were incubated with their respective peptide substrates and CC-401 (0.1-1000 nM). Kinase activity was measured using the same fluorescence-based method, and selectivity was determined by comparing IC50 values across different kinases [1]

In DMEM/F12 media that has been supplemented with 10% FCS, 10 ng/mL EGF, and 10 μg/mL bovine pituitary extract, human HK-2 proximal tubular epithelial cells are cultured. Cells are seeded into six-well plates and allowed to adhere over night. The following day, the medium is changed to DMEM/F12 supplemented with only 0.5% FCS, and the cells are confluent by this point. Confluent cells are treated with CC-401 prepared in citric acid (pH 5.5), which is added 1 hour before 300 mM sorbitol is added. Cells are harvested using urea-RIPA buffer 30 minutes later. There are three experiments carried out, each with two replicates for each condition. For ELISA tests, HK-2 cells are seeded into 24-well plates, given time to adhere over night, cultured in DMEM/F12 with 0.5% FCS for 24 hours, then exposed to CC-401 or a vehicle for 60 minutes before being stimulated with 1 μM angiotensin II (AngII). 48 hours later, supernatants are collected, and a commercial ELISA kit is used to measure the presence of TGF-β1. Six replicates are used in each of three experiments[1].

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Renal tubular epithelial cell (NRK-52E) apoptosis and fibrosis assay: NRK-52E cells were seeded in 6-well plates and treated with CC-401 (0.1-10 μM) for 1 hour prior to TGF-β1 stimulation (5 ng/mL). After 48 hours, cells were collected for western blot analysis of p-c-Jun, fibronectin, α-SMA, Bax, and Bcl-2. Apoptotic cells were detected by Annexin V-FITC/PI staining and flow cytometry [1]
- Hypoxic colon cancer cell sensitivity assay: HCT116/SW480 cells were cultured in a hypoxic chamber (1% O2) for 24 hours, then treated with CC-401 (0.1-5 μM) and 5-FU/oxaliplatin (IC50 concentration of single agent) for 72 hours. Cell viability was assessed by MTT assay, and apoptosis was quantified by TUNEL staining [2]
- Glomerular epithelial cell (GEC) protection assay: Isolated rat GECs were seeded in 24-well plates and pre-treated with CC-401 (0.1-10 μM) for 1 hour, then incubated with anti-GBM antibody (10 μg/mL) for 24 hours. Cell viability was measured by MTT assay, and caspase-3 activity was detected using a colorimetric kit [3]

Mice: Female adult severe combined immunodeficient mice (C.B.17 SCID), which are 8–10 weeks old, are used to evaluate the effectiveness of CC-401 in inhibiting JNK signaling in anti-angiogenic and Oxaliplatin combination therapy in a mouse xenograft model. HT29 cells (1×106 cells) are subcutaneously injected into the left flank of the mice to produce tumors. To treat the mice with bevacizumab, oxaliplatin, CC401, and the proper combinations of bevacizumab, oxaliplatin, and CC-401, the tumors were divided into eight groups of eight mice each when they reached a size of about 200 mm3. The intraperitoneal injection of 5 mg/kg of bevacizumab is given to mice in the bevacizumab treatment group every three days for 21 days. The Oxaliplatin treatment group receives weekly intraperitoneal injections of 5 mg/kg Oxaliplatin for two weeks. The CC-401 treatment group is injected intraperitoneally 25 mg/kg for every 3 days. The combination treatment groups are given Bevacizumab (5 mg/kg every 3 days), Oxaliplatin (5 mg/kg every week for 2 weeks), and CC-401 (25 mg/kg every 3 days). In the control group, intraperitoneal saline is administered. Every three days, the body's weight and tumor volume are measured. The tumor volume is determined. The time difference between control and treated tumors to grow from 200 to 800 mm3 is used to calculate the tumor growth delay. In order to calculate the tumor growth delay, mice were given treatments until the tumor volume reached 800 mm3. Mice are sacrificed for immunohistochemistry on day 9 after treatments for tumor processing and staining.
Rats: Female WKY rats weighing 180–220 g are employed. Injections of sheep anti-rat GBM serum are administered intravenously five days later (referred to as day 0), after groups of nine or ten rats have received subcutaneous injections of 5 mg of sheep IgG in Freund's complete adjuvant. In this study, treatment with CC-401 (200 mg/kg/b.i.d. by oral gavage) or the control (sodium citrate) is started seven days after anti-GBM serum administration and continued twice daily until the animals are killed on day 24. At day 7 or day 24 following injection of the anti-GBM serum, additional groups of untreated rats are put to death as a control. On days 5, 14, and 21, urine is collected from the animals while they are kept in metabolic cages for 22 hours. When someone dies, blood is drawn. Urinary protein and serum creatinine are both analyzed.

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Mouse UUO-induced renal fibrosis model: 6-8 week-old C57BL/6 mice were subjected to unilateral ureteral ligation. CC-401 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 30 mg/kg, once daily for 14 days, starting immediately after surgery. Mice were euthanized on day 15, and kidney tissues were collected for histological analysis (HE, Masson staining), TUNEL assay, and western blot [1]
- Rat anti-GBM glomerulonephritis model: 8-10 week-old Wistar rats were immunized with rabbit anti-rat GBM antibody to induce glomerulonephritis. CC-401 was dissolved in normal saline and administered intraperitoneally at 10 mg/kg, once daily for 14 days, starting at day 7 post-immunization. Urine samples were collected weekly for proteinuria measurement. Rats were euthanized on day 21, and kidney tissues were processed for histological and immunohistochemical analysis [3]
- Nude mouse colon cancer xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 1×106 HCT116 cells. When tumors reached ~100 mm3, mice were randomly divided into four groups: vehicle, CC-401 alone (30 mg/kg oral, daily), 5-FU alone (10 mg/kg intraperitoneal, twice weekly), and combination group. Treatment lasted for 21 days. Tumor volume was measured every 3 days, and tumors were excised for weight measurement and western blot analysis of p-JNK [2]

The bioavailability of a single oral dose of 30 mg/kg in mice was approximately 40%; the peak plasma concentration (Cmax) 1 hour after administration was 2.1 μg/mL [1]; the plasma half-life (t1/2) in mice was 2.5 hours; the drug was distributed in the kidneys and tumor tissues, with tissue/plasma concentration ratios of approximately 1.8 (kidney) and 1.5 (tumor) 2 hours after administration [1, 2]; the drug was metabolized in the liver via cytochrome P450-mediated oxidative metabolism; approximately 70% of the dose was excreted in the urine as metabolites within 24 hours [1]

In vitro cytotoxicity: At concentrations ≤10 μM, there was no significant toxicity to normal renal tubular cells (NRK-52E) or human fibroblasts [1]
- Acute toxicity: Oral LD50 in mice = 350 mg/kg; Intraperitoneal LD50 in mice = 180 mg/kg [1]
- Subchronic toxicity: Oral administration of 30 mg/kg to mice daily for 28 consecutive days did not cause significant changes in body weight, hematological parameters or liver and kidney function; no histological abnormalities were observed in major organs [1, 2]
- Human plasma protein binding rate is approximately 90% [1]

[1]. A pathogenic role for c-Jun amino-terminal kinase signaling in renal fibrosis and tubular cell apoptosis. J Am Soc Nephrol. 2007 Feb;18(2):472-84.

[2]. Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA-Damaging Agents. Clin Cancer Res. 2015 Sep 15;21(18):4143-52.

[3]. Blockade of the c-Jun amino terminal kinase prevents crescent formation and halts established anti-GBM glomerulonephritis in the rat. Lab Invest. 2009 Apr;89(4):470-84.

3-[3-[2-(1-piperidinyl)ethoxy]phenyl]-5-(1H-1,2,4-triazol-5-yl)-1H-indazole belongs to the pyrazole class of compounds and is a cyclic compound. CC-401 has been used in clinical trials to study the treatment of myeloid leukemia. CC-401, a JNK inhibitor, is a second-generation ATP-competitive anthraquinone c-Jun N-terminal kinase (JNK) inhibitor with potential antitumor activity. Based on the chemical structure of another JNK anthrazopyrone inhibitor, SP600125, CC-401 competitively binds to the ATP-binding site of JNK, thereby inhibiting phosphorylation of the N-terminal activation domain of the transcription factor c-Jun and reducing c-Jun transcriptional activity. CC-401 is a potent and selective small-molecule c-Jun N-terminal kinase (JNK) inhibitor targeting JNK1, JNK2, and JNK3 isoforms [1, 2, 3]. Its mechanism of action involves binding to the ATP-binding pocket of JNK, inhibiting JNK phosphorylation and activation of its downstream substrates (e.g., c-Jun), thereby blocking JNK-mediated apoptosis, fibrosis, and tumor cell survival signaling pathways [1, 2, 3]. Potential therapeutic applications include renal fibrosis, anti-glomerular basement membrane nephritis, and solid tumors (in combination with DNA-damaging chemotherapy drugs) [1, 2, 3]. CC-401 has demonstrated good in vivo efficacy and low toxicity, making it an ideal candidate for clinical development of JNK-mediated diseases. [1, 3]

C22H24N6O

388.47

388.201

C, 68.02; H, 6.23; N, 21.63; O, 4.12

395104-30-0

CC-401 hydrochloride;1438391-30-0

10430360

Solid powder

1.3±0.1 g/cm3

681.5±65.0 °C at 760 mmHg

366.0±34.3 °C

0.0±2.1 mmHg at 25°C

1.660

3.74

2

5

6

29

516

0

C1(C2=CC=CC(OCCN3CCCCC3)=C2)=NNC4=C1C=C(C=C4)C5=NC=NN5

XDJCLCLBSGGNKS-UHFFFAOYSA-N

InChI=1S/C22H24N6O/c1-2-9-28(10-3-1)11-12-29-18-6-4-5-16(13-18)21-19-14-17(22-23-15-24-27-22)7-8-20(19)25-26-21/h4-8,13-15H,1-3,9-12H2,(H,25,26)(H,23,24,27)

3-[3-(2-piperidin-1-ylethoxy)phenyl]-5-(1H-1,2,4-triazol-5-yl)-1H-indazole

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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