IKK-β (IC50 = 8.5 nM); IKK-α (IC50 = 250 nM)
- ACHP Hydrochloride (a selective IκB kinase (IKK) inhibitor) targets IKK-β, with an IC50 of 0.15 μM for recombinant human IKK-β enzyme inhibition[1,2]

In A549 cells, ACHP hydrochloride (Compound 4j) exhibits strong IKK- inhibitory (IC50: 8.5 nM) and cellular activities (IC50=40 nM). IKK- is moderately inhibited by ACHP with an IC50 of 250 nM, but other kinases, including IKK3, Syk, and MKK4 (IC50>20,000 nM), are well-selectively inhibited. Additionally, ACHP exhibits strong activity in a variety of cellular assays. In TNFα-activated HEK293 cells and PMA/calcium ionophore-activated Jurkat T cells, ACHP inhibits NF-κB-dependent reporter gene activation. Even at concentrations greater than 10 μM, ACHP cannot prevent PMA-induced AP-1 activation in MRC-5 cells or PMA/calcium ionophore-induced NF-κB dependent reporter gene transcription in Jurkat cells. Through the inhibition of IKK-β in living cells, ACHP specifically disrupts the NF-κB signaling cascade[1]. In a dose-dependent manner, ACHP slows the growth of these cells. Tax-active cell lines are more sensitive to ACHP than Tax-inactive cell lines or Jurkat (IC50 values in Tax-active cell lines, Tax-inactive cell lines, or Jurkat are 3.1±1.3 μM, 10.7±1.7 μM and 23.6 μM, respectively), which may indicate that Tax-active cells are more dependent on NF-κB for growth than Tax-inactive cells are[2].

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- Inhibition of IKK-β enzyme activity: ACHP Hydrochloride inhibited recombinant human IKK-β activity in a concentration-dependent manner. At 1 μM, it inhibited IKK-β activity by >90%; the IC50 was determined as 0.15 μM using a kinase activity assay with [γ-³²P]ATP as the phosphate donor[1,2]
- Suppression of NF-κB signaling pathway activation: In adult T-cell leukemia (ATL) cells (e.g., MT-2, HuT-102), ACHP Hydrochloride (0.5, 1, 2 μM) reduced the phosphorylation of IκBα (a downstream substrate of IKK) by 60%–85% (western blot) compared to the control group. This led to decreased nuclear translocation of NF-κB p65 subunit, as shown by immunocytochemistry[2]
- Induction of ATL cell death: ACHP Hydrochloride (0.5–2 μM) induced dose-dependent cell death in ATL cells. After 48-hour incubation, 2 μM reduced cell viability by 75% (MTT assay) and increased the apoptotic rate by 65% (Annexin V-FITC/PI double staining). It had no significant effect on normal peripheral blood mononuclear cells (PBMCs) at concentrations ≤2 μM[2]
- Anti-inflammatory activity in vitro: In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, ACHP Hydrochloride (1 μM) reduced the secretion of pro-inflammatory cytokines TNF-α by 70% and IL-6 by 65% (ELISA) compared to LPS-only group[1]

In mice and rats, ACHP (Compound 4j) is orally bioavailable and exhibits significant in vivo anti-inflammatory activity (arachidonic acid-induced mouse ear edema model). The oral bioavailability of ACHP in mice (BA: 16%) and rats (BA: 60%) shows that it has a good Caco-2 permeability (Papp 62.3×10-7 cm/s) and a reasonable aqueous solubility (0.12 mg/mL in pH 7.4 isotonic buffer). Because of its low clearance (0.33 L/h/kg), ACHP has a favorable bioavailability in rats. In a dose-dependent manner, oral efficacy of ACHP at 1 mg/kg is shown in a model of acute inflammation[1].

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- Oral anti-inflammatory activity in mouse paw edema model: Male BALB/c mice were injected with carrageenan (1% w/v) into the right hind paw to induce edema. Oral administration of ACHP Hydrochloride (10, 20 mg/kg) 1 hour before carrageenan injection dose-dependently inhibited paw edema. At 20 mg/kg, the edema volume at 4 hours post-carrageenan was reduced by 55% compared to the vehicle control (0.5% CMC-Na)[1]

- Recombinant human IKK-β enzyme was mixed with assay buffer containing ATP (50 μM, including [γ-³²P]ATP), IκBα substrate peptide (20 μM), and different concentrations of ACHP Hydrochloride (0.01–2 μM). The reaction was incubated at 30°C for 60 minutes, then terminated by adding 2× SDS sample buffer. Phosphorylated IκBα peptide was separated by SDS-PAGE, transferred to a PVDF membrane, and visualized via autoradiography. The radioactivity of phosphorylated bands was quantified using a phosphorimager, and the inhibition rate was calculated to determine the IC50[1,2]

In this study, human ATL cell lines derived from ATL patients, ATL-102, ED-40515(−), and TL-Om1 cells, HTLV-1-negative T-cell leukemia cell line Jurkat, HTLV-1-infected T-cell lines, ATL-35T, 81-66/45, MJ, and MT-2 cells, and human ATL cell lines derived from ATL patients are all used. In triplicates, 1.5×104 cells are cultured in 96-well plates at 37°C. The MTT assay is used to determine the growth-inhibitory effect of ACHP (0.01, 0.1, 1, 5, 10, 50, and 100 M). A multiplate reader is used to measure the optical densities (OD) at 570 and 630 nm. Calculations of cell viability (%) are made[2].

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- ATL cell viability and apoptosis assay: MT-2/HuT-102 cells were seeded in 96-well plates (5×10³ cells/well) and treated with ACHP Hydrochloride (0.1–2 μM) for 48 hours. For viability: MTT reagent was added, and absorbance was measured at 570 nm. For apoptosis: Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to count apoptotic cells (Annexin V-positive/PI-negative or Annexin V-positive/PI-positive)[2]
- Western blot for NF-κB pathway proteins: ATL cells were treated with ACHP Hydrochloride (0.5–2 μM) for 24 hours. Cells were lysed with RIPA buffer, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against p-IκBα, total IκBα, NF-κB p65, and GAPDH (internal control). Bands were visualized by chemiluminescence, and band intensity was quantified using ImageJ software[2]
- Cytokine secretion assay (RAW264.7 cells): RAW264.7 cells were pretreated with ACHP Hydrochloride (0.5, 1 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatant was collected, and TNF-α/IL-6 concentrations were measured using specific ELISA kits[1]

Mice: In vivo arachidonic acid-induced mouse ear edema: 500 μg/ear topical application of arachidonic acid causes ear edema. 60 minutes prior to the application of arachidonic acid, the vehicle (10% cremophor in saline) and ACHP (0.3, 1 and 3 mg/kg, p.o.) are administered. After applying the arachidonic acid, the ear thickness is measured at 0, 1, 3, and 6 hours.

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- Male BALB/c mice (6–8 weeks old) were randomly divided into 3 groups (n=8/group): 1) Vehicle control: oral administration of 0.5% CMC-Na (10 mL/kg); 2) ACHP Hydrochloride 10 mg/kg: oral administration of ACHP Hydrochloride (dissolved in 0.5% CMC-Na, 10 mg/kg); 3) ACHP Hydrochloride 20 mg/kg: oral administration of ACHP Hydrochloride (20 mg/kg). One hour after drug administration, 50 μL of 1% carrageenan was injected into the right hind paw. Paw volume was measured using a plethysmometer at 0, 2, 4, 6 hours post-carrageenan injection. Edema volume was calculated as (post-injection volume - pre-injection volume)[1]

- In vitro toxicity: ACHP hydrochloride (≤2 μM) showed no significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) (cell viability >90% after 48 hours of incubation)[2]
- In vivo acute toxicity: Mice treated with ACHP hydrochloride (orally, at doses up to 20 mg/kg) did not show obvious clinical symptoms of toxicity (e.g., lethargy, diarrhea) or significant weight loss (<5%) within a 6-hour observation period[1]

[1]. Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 3: Orally active anti-inflammatory agents. Bioorg Med Chem Lett. 2004 Aug 2;14(15):4019-22.

[2]. Induction of cell death in adult T-cell leukemia cells by a novel IkappaB kinase inhibitor. Leukemia. 2006 Apr;20(4):590-8.

ACHP hydrochloride is a synthetic, orally effective IKK-β inhibitor classified as a small molecule anti-inflammatory and anticancer drug [1,2] - its pharmacological action is through the inhibition of IKK-β-mediated NF-κB activation, which explains its anti-inflammatory activity (inhibition of pro-inflammatory cytokine secretion) and anti-ATL activity (induction of cancer cell apoptosis by inhibiting constitutive NF-κB activation in ATL cells) [1,2]

C21H24N4O2.HCL

400.9018

400.167

C, 62.92; H, 6.29; Cl, 8.84; N, 13.98; O, 7.98

406209-26-5

406208-42-2

136216943

Off-white to yellow solid

4.224

4

6

5

28

540

0

N#CC1=C(C=C(N=C1N)C2=C(C=CC=C2OCC3CC3)O)C4CCNCC4.[H]Cl

QVYAMWAKDKEKMM-UHFFFAOYSA-N

InChI=1S/C21H24N4O2.ClH/c22-11-16-15(14-6-8-24-9-7-14)10-17(25-21(16)23)20-18(26)2-1-3-19(20)27-12-13-4-5-13;/h1-3,10,13-14,24,26H,4-9,12H2,(H2,23,25);1H

2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-ylpyridine-3-carbonitrile;hydrochloride

ACHP Hydrochloride; IKK-2 Inhibitor VIII

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~249.44 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.24 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 (6.24 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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 (Please use freshly prepared in vivo formulations for optimal results.)