IKK2 (IC50 = 40 nM); IKK1 (IC50 = 200 nM); IKK (IC50 = 70 nM); LRRK2 (IC50 = 50 nM)
The primary targets of IKK-16 (IKK Inhibitor VII; IKK 16) are the IκB kinase α (IKKα) and IκB kinase β (IKKβ) , key kinases in the nuclear factor-κB (NF-κB) signaling pathway that regulate IκBα phosphorylation and NF-κB activation.
- For human recombinant IKKβ, the half-maximal inhibitory concentration (IC50) of IKK-16 was 1.3 μM; for human recombinant IKKα, the IC50 was 12 μM, showing higher selectivity for IKKβ [1]
- IKK-16 exhibited no significant inhibitory activity against other kinases involved in inflammatory signaling, including c-Jun N-terminal kinase 1 (JNK1, IC50 > 100 μM), p38α mitogen-activated protein kinase (p38α, IC50 > 100 μM), and extracellular signal-regulated kinase 2 (ERK2, IC50 > 100 μM) [1]

In HUVEC cells, IKK-16 prevents TNFα from triggering the expression of the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Despite exhibiting activity in assays for IFNγ-induced expression of HLA-DR or β2 microglobulin, IKK-16's potency in these tests is 4- to 10-fold lower. [1]

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1. Inhibition of IKK Kinase Activity:
- IKK-16 (0.1-30 μM) inhibited recombinant human IKKβ activity in a concentration-dependent manner. At 3 μM, it inhibited IKKβ activity by ~80%; at 10 μM, the inhibition rate reached ~95%. For IKKα, 10 μM IKK-16 inhibited activity by ~60%, confirming its preferential inhibition of IKKβ [1]
2. Suppression of NF-κB Signaling in Cells:
- NF-κB Luciferase Reporter Assay: In HEK293T cells transfected with an NF-κB firefly luciferase reporter plasmid and a Renilla luciferase internal control plasmid, IKK-16 (0.5-20 μM) concentration-dependently inhibited TNF-α (10 ng/mL)-induced NF-κB transcriptional activity. The IC50 was 2.7 μM, and 20 μM IKK-16 reduced luciferase activity by ~90% compared to the vehicle control [1]
- Inhibition of IκBα Phosphorylation: In HeLa cells stimulated with TNF-α (10 ng/mL), pre-treatment with IKK-16 (5 μM) for 1 hour reduced IκBα phosphorylation (Ser32/36) by ~75% at 15 minutes post-stimulation (detected by Western blot). No significant change in total IκBα protein level was observed, indicating specific inhibition of IKK-mediated phosphorylation [1]
3. Reduction of Pro-Inflammatory Cytokine Secretion:
- In RAW264.7 murine macrophages stimulated with lipopolysaccharide (LPS, 1 μg/mL), IKK-16 (1-10 μM) inhibited the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a concentration-dependent manner. At 10 μM, TNF-α secretion decreased by ~85% and IL-6 secretion decreased by ~80% (measured by ELISA) [3]
- In primary mouse peritoneal macrophages, 5 μM IKK-16 reduced LPS-induced TNF-α and IL-6 secretion by ~70% and ~65%, respectively, without affecting cell viability (assessed by MTT assay, >90% viability at 20 μM) [3]

IKK-16 inhibits neutrophil extravasion in thioglycollate-induced peritonitis and LPS-induced TNF-α release in vivo. It is orally bioavailable in rats and mice.[1]

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1. Efficacy in CLP-Induced Murine Sepsis Model (a Model of Multiple Organ Dysfunction Syndrome, MODS):
- Animal Model and Dosing: Male C57BL/6 mice (8-10 weeks old) were subjected to cecal ligation and puncture (CLP) to induce sepsis. Mice were randomly divided into 3 groups (n=10/group): Sham group (no CLP, no drug), CLP + Vehicle group (CLP + 0.5% DMSO in PBS), and CLP + IKK-16 group (CLP + 10 mg/kg or 20 mg/kg IKK-16 , intraperitoneal injection). IKK-16 was administered at 1 hour and 6 hours post-CLP [3]
- Improvement of Survival Rate: The 7-day survival rate of the CLP + IKK-16 (20 mg/kg) group was 60%, significantly higher than the 20% survival rate of the CLP + Vehicle group (P < 0.01). The 10 mg/kg group showed a moderate improvement (40% survival rate, P < 0.05) [3]
- Attenuation of Organ Damage:
- Liver and Kidney Function: Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels (markers of liver damage) in the CLP + IKK-16 (20 mg/kg) group were 42% and 38% of the CLP + Vehicle group, respectively. Serum creatinine (marker of kidney damage) was reduced by 55% compared to the vehicle group [3]
- Lung and Intestinal Damage: Histopathological analysis showed that IKK-16 (20 mg/kg) reduced LPS-induced lung alveolar congestion and inflammatory cell infiltration (histological score: 2.1 ± 0.3 vs. 4.7 ± 0.5 in vehicle group, P < 0.01) and alleviated intestinal mucosal erosion (histological score: 1.8 ± 0.2 vs. 4.3 ± 0.4 in vehicle group, P < 0.01) [3]
- Inhibition of Inflammatory Response and NF-κB Activity:
- Serum Cytokines: IKK-16 (20 mg/kg) reduced serum TNF-α and IL-6 levels by ~70% and ~65%, respectively, at 24 hours post-CLP [3]
- Tissue NF-κB Activation: Western blot analysis of liver and lung tissues showed that IKK-16 (20 mg/kg) reduced IKKβ phosphorylation (Ser177/181) by ~65% and IκBα phosphorylation by ~70% compared to the vehicle group [3]

IKK 16 is a selective IκB kinase (IKK) inhibitor for IKK2, IKK complex and IKK1 with IC50s of 40 nM, 70 nM and 200 nM, respectively. IKK16 also inhibits leucine-rich repeat kinase-2 (LRRK2) with an IC50 of 50 nM.

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IKKα/IKKβ Kinase Activity Assay:
1. Reaction System Preparation:
- For IKKβ assay: Recombinant human IKKβ (0.1 μg per reaction) was mixed with GST-IκBα (1 μg, substrate peptide containing Ser32/36), ATP (50 μM, including [γ-32P]ATP for radioactivity detection), and kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4) in a total volume of 25 μL. IKK-16 was dissolved in DMSO and added to final concentrations of 0.1, 0.3, 1, 3, 10, 30 μM (DMSO final concentration ≤ 0.1%). A vehicle control (0.1% DMSO) and a positive control (known IKK inhibitor) were set up [1]
- For IKKα assay: The reaction system was identical to IKKβ, except recombinant human IKKα (0.1 μg per reaction) was used instead of IKKβ [1]
2. Incubation and Detection:
- The mixture was incubated at 30°C for 45 minutes. The reaction was terminated by adding 5 μL of 6×SDS-PAGE loading buffer and heating at 95°C for 5 minutes. Proteins were separated by 12% SDS-PAGE, and the gel was dried under vacuum. Radioactive signals of phosphorylated GST-IκBα were detected using a phosphorimager [1]
3. Data Analysis:
- The intensity of radioactive bands was quantified using image analysis software. The inhibitory rate of IKK-16 was calculated as [(Radioactivity, control - Radioactivity, drug)/Radioactivity, control] × 100%. IC50 values were determined by fitting the concentration-inhibition curves to a four-parameter logistic model [1]

SH-SY5Y cells are transduced with 25% (v/v) BacMam LRRK2-GFP G2019S and then plated (20 µL/well, 20,000 cells/well) onto eight 384-well assay plates. Then, for 90 min, 25% BacMam LRRK2-GFP G2019S transduced SH-SY5Y cells are incubated with the corresponding concentrations of the corresponding substances (e.g., IKK 16, 0.01, 0.1, 1, 10 and 100 μM), followed by the TR-FRET detection with Tb-anti-LRRK2 pSer935 antibody. Calculated is the inhibition percentage.

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1. NF-κB Luciferase Reporter Assay :
- Cell Seeding and Transfection: HEK293T cells were seeded into 96-well plates at a density of 2×104 cells/well and cultured overnight in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were transfected with a mixture of plasmids: 0.1 μg/well NF-κB firefly luciferase reporter plasmid, 0.01 μg/well Renilla luciferase internal control plasmid, and transfection reagent. After 24 hours of transfection, the medium was replaced with fresh DMEM containing 1% FBS [1]
- Drug Treatment and Stimulation: IKK-16 (0.5, 1, 5, 10, 20 μM) was added to each well, and cells were pre-incubated for 1 hour. Then, TNF-α (10 ng/mL) was added to stimulate NF-κB activation, and incubation continued for 6 hours. A vehicle control (0.1% DMSO) and an unstimulated control (no TNF-α) were included [1]
- Luciferase Activity Detection: Cells were lysed with passive lysis buffer (100 μL/well) for 15 minutes at room temperature. A 20 μL aliquot of the lysate was mixed with 50 μL of luciferase assay reagent (for firefly luciferase) and 50 μL of stop & glow reagent (for Renilla luciferase) in a 96-well luminescence plate. Luminescence intensity was measured using a microplate luminometer. Relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity [1]
2. Western Blot for IκBα Phosphorylation :
- Cell Treatment: HeLa cells were seeded into 6-well plates at 2×105 cells/well and cultured to 80% confluence. Cells were pre-treated with IKK-16 (5 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 0, 15, 30, or 60 minutes. A vehicle control (stimulated with TNF-α without IKK-16 ) was set up [1]
- Protein Extraction and Detection: Cells were lysed with RIPA buffer containing protease inhibitors (leupeptin, pepstatin) and phosphatase inhibitors (Na3VO4, NaF) for 30 minutes on ice. Lysates were centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 hour at room temperature, then incubated with primary antibodies against phosphorylated IκBα (p-IκBα Ser32/36) and total IκBα overnight at 4°C. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using ImageJ software [1]
3. Inflammatory Cytokine Secretion Assay :
- Cell Culture and Treatment: RAW264.7 murine macrophages were seeded into 24-well plates at 5×105 cells/well and cultured overnight. The medium was replaced with serum-free DMEM, and IKK-16 (1, 5, 10 μM) was added for pre-incubation for 1 hour. Then, LPS (1 μg/mL) was added to stimulate cytokine secretion, and incubation continued for 24 hours [3]
- Cytokine Detection: The cell culture supernatant was collected and centrifuged at 1,000×g for 10 minutes to remove cell debris. The concentrations of TNF-α and IL-6 in the supernatant were measured using sandwich ELISA kits. The absorbance was read at 450 nm using a microplate reader, and cytokine concentrations were calculated based on standard curves [3]

Rats and Mice: In two animal models, IKK 16 is tested. Its ability to prevent TNF from being released into the bloodstream after LPS challenge in rats is tested first. One hour before the LPS challenge, IKK 16 is dosed subcutaneously (sc) or orally (o) at a dose of 30 mg/kg. Plasma is collected four hours after the challenge, and a commercially available ELISA kit is used to assess the systemic TNFα levels. IKK 16 exhibits a notable 86% (sc) and 75% (p.o.) inhibition when administered via either route and at the recommended dose. IKK 16 is also active in the mouse model of peritonitis caused by thioglycollate in a subsequent experiment. In this model, a dose of 10 mg/kg sc results in an approximately 50% inhibition of neutrophil extravasation.
Mice: LPS (9 mg/kg body weight) and PepG (3 mg/kg body weight) are administered intraperitoneally to two-month-old male C57BL/6 mice. Sham mice receive the same care but are not exposed to LPS or PepG. Mice are given either IKK 16 (1 mg/kg body weight intravenously) or vehicle (5 mL/kg body weight 10% DMSO intravenously) at one hour after LPS/PepG co-administration. After 24 hours, the experiment is over, and blood and organ samples are taken in order to measure any organ dysfunction or injury. Four groups of mice are randomly assigned: (1) sham+vehicle (n=10); (2) sham+IKK 16 (n=3); (3) LPS/PepG+vehicle (n=9); (4) LPS/PepG+IKK 16 (n=10).

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CLP-Induced Murine Sepsis Model for Evaluating IKK-16 Efficacy :
1. Animal Preparation:
- Male C57BL/6 mice (8-10 weeks old, weighing 22-25 g) were acclimated for 1 week under standard conditions (12-hour light/dark cycle, 22±1°C, free access to food and water). Mice were fasted for 12 hours before surgery but allowed free access to water [3]
2. Sepsis Induction (CLP Surgery):
- Mice were anesthetized with isoflurane (inhalation). A 1 cm midline abdominal incision was made, and the cecum was exposed. The cecum was ligated with 4-0 silk suture at 50% of its length from the tip, then punctured twice with a 22-gauge needle. A small amount of fecal material was extruded to ensure patency, and the cecum was returned to the abdominal cavity. The abdomen was closed with 4-0 silk suture (muscle layer) and 5-0 nylon suture (skin layer). Sham-operated mice underwent the same procedure without cecal ligation or puncture [3]
3. Drug Formulation and Administration:
- IKK-16 was dissolved in 0.5% DMSO/PBS to prepare dosing solutions of 2 mg/mL (for 10 mg/kg dose) and 4 mg/mL (for 20 mg/kg dose). Mice in the CLP + IKK-16 group received intraperitoneal injections of IKK-16 at 1 hour and 6 hours post-CLP (injection volume: 5 mL/kg). Mice in the CLP + Vehicle group received an equal volume of 0.5% DMSO/PBS [3]
4. Sample Collection and Detection:
- Survival Monitoring: Mice were monitored twice daily for 7 days to record survival status [3]
- Serum and Tissue Collection: At 24 hours post-CLP, mice were euthanized by CO2 inhalation. Blood was collected via cardiac puncture, centrifuged at 3,000×g for 15 minutes to separate serum, and stored at -80°C for ALT, AST, creatinine, TNF-α, and IL-6 detection [3]
- Organ Histology: Lungs, liver, and intestines were excised, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin-eosin (HE). Histological scores were evaluated based on the degree of tissue damage and inflammatory infiltration (0-5 scale) [3]
- Western Blot for Tissue NF-κB Activity: Liver and lung tissues were homogenized in RIPA buffer containing inhibitors, and protein extracts were used for Western blot analysis of p-IKKβ (Ser177/181) and p-IκBα (Ser32/36) [3]

1. In vitro cytotoxicity: - IKK-16 (at concentrations up to 20 μM) did not show significant cytotoxicity in HeLa cells, RAW264.7 macrophages, and primary mouse peritoneal macrophages. Cell viability (assessed by MTT assay) remained above 90% after 48 hours of treatment compared to the solvent control group [1, 3] 2. In vivo safety in septic mice: - IKK-16 (10 mg/kg and 20 mg/kg, intraperitoneal injection) did not cause additional toxicity in a CLP-induced sepsis model. Mice in the IKK-16 treatment group did not show significant weight loss (weight change: -5% to -8%, compared to -12% in the CLP+carrier group) 24 hours after CLP treatment, and no other abnormal behaviors (e.g., lethargy, ataxia) were observed in addition to sepsis-related symptoms [3]. The levels of liver and kidney function markers (ALT, AST, creatinine) in the serum of mice in the IKK-16 treatment group were lower than those in the CLP+ vector group, indicating that no drug-induced liver and kidney damage occurred [3].

[1]. Design and preparation of 2-benzamido-pyrimidines as inhibitors of IKK. Bioorg Med Chem Lett. Bioorg Med Chem Lett. 2006 Jan 1;16(1):108-12.

[2]. Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation. PLoS One. 2012;7(8):e43580.

[3]. Inhibition of IκB kinase reduces the multiple organ dysfunction caused by sepsis in the mouse. Dis Model Mech. 2013 Jul;6(4):1031-42.

[4-[[4-(1-benzothiophene-2-yl)-2-pyrimidinyl]amino]phenyl]-[4-(1-pyrrolidinyl)-1-piperidinyl]methyl ketone is an N-acylpiperidine belonging to the benzamide class of compounds.

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1. Mechanism of Action:
- IKK-16 exerts its biological effects by selectively inhibiting IKKβ (and weakly inhibiting IKKα). It binds to the ATP-binding pocket of IKKβ, blocking its kinase activity, thereby preventing the phosphorylation of IκBα and its subsequent degradation. This will retain the NF-κB (p65/p50) dimer in the cytoplasm, thereby inhibiting the transcription of NF-κB target genes (e.g., pro-inflammatory cytokines TNF-α, IL-6; genes involved in cell survival and proliferation) [1, 3]
2. Therapeutic potential:
- IKK-16 has shown potential for treating diseases with overactivation of NF-κB, particularly sepsis-induced multiple organ dysfunction syndrome (MODS). It has been shown to reduce inflammatory responses and organ damage while improving survival rates in septic mice, which supports its development as a candidate drug for inflammatory and infectious diseases [3]
3. Chemical class and development background:
- IKK-16 belongs to the 2-benzamidopyrimidine class of compounds, which are small molecule IKK inhibitors designed and synthesized to target the NF-κB pathway. It is widely used as a research tool compound for in vitro and in vivo studies of the IKK/NF-κB signaling pathway, but has not yet entered the clinical development stage [1]. 4. Selectivity characteristics: - IKK-16 has much higher selectivity for IKKβ than other kinases (JNK1, p38α, ERK2) and IKKα, thereby reducing the risk of off-target effects that may lead to toxicity (e.g., accidental inhibition of the MAPK signaling pathway) [1].

C28H29N5OS

483.63

483.209

C, 69.54; H, 6.04; N, 14.48; O, 3.31; S, 6.63

873225-46-8

IKK 16 hydrochloride;1186195-62-9

9549298

White to yellow solid powder

1.3±0.1 g/cm3

721.6±70.0 °C at 760 mmHg

390.2±35.7 °C

0.0±2.3 mmHg at 25°C

1.700

5.67

1

6

5

35

702

0

O=C(N1CCC(N2CCCC2)CC1)C1C=CC(NC2N=C(C3=CC4C(=CC=CC=4)S3)C=CN=2)=CC=1

BWZJBXAPRCVCKQ-UHFFFAOYSA-N

InChI=1S/C28H29N5OS/c34-27(33-17-12-23(13-18-33)32-15-3-4-16-32)20-7-9-22(10-8-20)30-28-29-14-11-24(31-28)26-19-21-5-1-2-6-25(21)35-26/h1-2,5-11,14,19,23H,3-4,12-13,15-18H2,(H,29,30,31)

[4-[[4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino]phenyl]-(4-pyrrolidin-1-ylpiperidin-1-yl)methanone

IKK 16; IKK Inhibitor VII; IKK-16; IKK16

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 (5.17 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 (5.17 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 (5.17 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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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

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