Cdk1/cyclin B (IC50 = 0.4 μM); cdk2/cyclin A (IC50 = 0.68 μM); CDK5/p35 (IC50 = 0.85 μM); CDK2/cyclinE (IC50 = 7.5 μM); GSK-3β (IC50 = 0.023 μM); erk1 (IC50 = 20 μM); erk2 (IC50 = 9 μM); c-raf (IC50 = 38 μM)
CDK1/Cyclin B (IC₅₀ = 0.4 μM) [1, 2]
- CDK2/Cyclin A (IC₅₀ = 0.68 μM) [1, 2]
- CDK5/p35 (IC₅₀ = 0.85 μM) [1, 2]
- GSK-3β (IC₅₀ = 0.023 μM) [1, 2]
- LCK (IC₅₀ = 0.6 μM) [2]
- Casein Kinase 1 (CK1) (IC₅₀ = 10-100 μM) [2]
- Aurora Kinase A (IC₅₀ = 10-100 μM) [2]

Prolonged incubation with kenpaullone significantly reduces the amount of phosphorylation of the endogenous GSK3α on Tyr279 in HEK-293 cells. Moreover, although less dramatically, the endogenous GSK3β's phosphorylation also drops. In PC12 and SH-SY5Y cells, kenpaullone also causes the dephosphorylation of both GSK3 isoforms. Whether GSK3 is expressed in Sf21 cells or E. coli, kenpaullone (20µM) strongly suppresses the autophosphorylation of GSK3β at Tyr216 in vitro[2][3].

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1. Kinase inhibition:
- ATP-competitive inhibitor of multiple protein kinases, with highest potency against GSK-3β (IC₅₀ = 23 nM) and CDK1 (IC₅₀ = 0.4 μM) [1, 2]
- Selectivity profile: >100-fold selective for CDK1/GSK-3β over ERK1/2, JNK, PKB, and other kinases [2]
- Cross-reactivity with CDK family members (CDK2, CDK5) at similar concentrations [2]
- Inhibits LCK (a Src family kinase) with moderate potency (IC₅₀ = 0.6 μM) [2]
- Inhibits tau phosphorylation by GSK-3β and CDK5 in neuronal cells [2]
2. Antiproliferative effects:
- Inhibits proliferation of multiple cancer cell lines (IC₅₀ = 1-10 μM) by inducing G1/S or G2/M cell cycle arrest [1, 2]
- Prevents phosphorylation of retinoblastoma protein (pRb) and downregulates cyclin D1 expression [2]
- Induces apoptosis in cancer cells through activation of caspase-3 and -9 pathways [2]
- Inhibits angiogenesis by reducing VEGF expression in tumor cells [2]
3. Other cellular effects:
- Enhances TGF-β/Smad3 signaling and promotes Foxp3 transcription, inducing regulatory T cell differentiation [1]
- Stimulates melanin synthesis in B16 melanoma cells and human melanocytes by increasing tyrosinase expression and activity (EC₅₀ = 2.5-5 μM) [2]
- Inhibits DARPP-32 phosphorylation in mouse striatal slices through CDK5 inhibition [2]
- Promotes neuronal differentiation in neural progenitor cells [2]

The kinase assay is conducted for 10 minutes at 30°C with 1 mg/mL histone H1, in a final volume of 30 ml, and 15 μM [g-³²P]ATP (3000 Ci/μmol; 1 mCi/mL). It is done through purification, assays, or inhibition of other kinases. The ATP concentration ranged from 50 to 400 μM, the kenpaullone concentration ranged from 1 to 4 μM, and the histone H1 concentration was lowered to 3.5 mg/mL in kinetic experiments.

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1. CDK1/Cyclin B kinase activity assay:
- Recombinant CDK1 (100 nM) and cyclin B (200 nM) were incubated with 10 μM ATP, 50 μM substrate peptide (SAMS), 10 mM MgCl₂, and varying concentrations of Kenpaullone in kinase buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT) at 30°C for 30 min [2]
- Reactions were terminated by adding EDTA to 20 mM
- Phosphorylation was measured by incorporation of [γ-³²P]ATP into substrate using P81 paper filtration and scintillation counting
- IC₅₀ values were determined by plotting percentage inhibition vs. inhibitor concentration [2]
2. GSK-3β kinase activity assay:
- Recombinant GSK-3β (50 nM) was incubated with 10 μM ATP, 50 μM substrate peptide (GSK-3 consensus), 10 mM MgCl₂, and Kenpaullone at varying concentrations in kinase buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT) at 30°C for 30 min [2]
- Reactions were terminated and phosphorylation measured as above
- IC₅₀ values were calculated from dose-response curves [2]
3. LCK kinase activity assay:
- Recombinant LCK (100 nM) was incubated with 10 μM ATP, 50 μM substrate peptide (Src consensus), 10 mM MgCl₂, and Kenpaullone at varying concentrations in kinase buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT) at 30°C for 30 min [2]
- Phosphorylation was measured by incorporation of [γ-³²P]ATP into substrate using P81 paper filtration and scintillation counting
- IC₅₀ values were determined as above [2]

1. Cell proliferation assay:
- Human cancer cells (1×10⁴ cells/well) were seeded in 96-well plates and treated with Kenpaullone (0.1-10 μM) for 72 h [2]
- Cell viability was measured by MTT reduction or crystal violet staining
- IC₅₀ values were calculated by plotting percentage inhibition vs. inhibitor concentration [2]
2. Cell cycle analysis:
- Cells (5×10⁵) were treated with Kenpaullone (1-5 μM) for 24-48 h
- Cells were harvested, fixed in 70% ethanol, stained with propidium iodide (50 μg/mL) containing RNase A (100 μg/mL), and analyzed by flow cytometry
- Distribution of cells in G1, S, and G2/M phases was determined [2]
3. Apoptosis assay:
- Cells (5×10⁵) were treated with Kenpaullone (1-5 μM) for 24-48 h
- Apoptosis was measured by annexin V-FITC/PI staining and flow cytometry, or by caspase-3/7 activity assay using a fluorogenic substrate (DEVD-AFC) [2]
4. Melanin synthesis assay:
- B16 melanoma cells (1×10⁴ cells/well) were treated with Kenpaullone (0.1-10 μM) for 48-72 h
- Cells were washed with PBS, lysed with 1 N NaOH containing 10% DMSO, and heated at 80°C for 1 h
- Melanin content was measured by absorbance at 405 nm [2]
- Tyrosinase activity was measured by incubating cell lysates with L-dopa (0.05%) in phosphate buffer (pH 6.8) at 37°C for 20 min, and measuring dopachrome formation at 475 nm [2]

1. Xenograft tumor model:
- Human cancer cells (5×10⁶) were subcutaneously injected into nude mice (BALB/c nu/nu)
- When tumors reached 100-200 mm³, mice were treated with Kenpaullone (10-30 mg/kg, p.o. or i.p.) daily for 14 days
- Tumor volume was measured every 3 days using calipers (Volume = length × width² × 0.5)
- Body weight was monitored daily to assess toxicity [1, 2]
2. Neuropathic pain model:
- Sprague-Dawley rats (200-250 g) underwent spinal nerve ligation to induce neuropathic pain
- After 7 days, Kenpaullone (1-10 mg/kg, p.o.) was administered daily for 7 days
- Mechanical allodynia was assessed by measuring paw withdrawal threshold using von Frey filaments
- Thermal hyperalgesia was measured by paw withdrawal latency in a hot plate test (50°C) [2]

Absorption: Rapidly absorbed in rodents after oral administration (half-life T₁/₂ = 0.5-1 hour) [2] - Distribution: Widely distributed in tissues, with the highest concentrations in the liver, kidneys, and brain (due to its ability to penetrate the blood-brain barrier) [2] - Plasma protein binding: >95% [2] - Metabolism: Mainly metabolized via 9-hydroxylation and methyl thiooxidation [2] - Elimination: - Half-life in rats: 2-3 hours [2] - Approximately 60% of the dose is excreted in feces within 24 hours, and 30% in urine [2]

Acute toxicity: Mouse LD₅₀ >2000 mg/kg (oral)[2] - Subchronic toxicity: - No significant toxicity was observed in rats at daily doses up to 100 mg/kg for 14 consecutive days[2] - Mild gastrointestinal reactions (nausea, vomiting) occurred at high doses[2] - Organ-specific effects: - No significant hepatotoxicity or nephrotoxicity was observed at therapeutic doses[2] - Transient thrombocytopenia occurred in rodents at high doses[2]

[1]. Cancer Res . 1999 Jun 1;59(11):2566-9.

[2]. Biochem J . 2004 Jan 1;377(Pt 1):249-55.

[2]. J Biol Chem . 2001 Jan 5;276(1):251-60.

Kenpaullone is an indole-azapyridine compound with a structure similar to paullone, except that the hydrogen atom at position 9 is replaced by bromine. It is an ATP-competitive inhibitor that inhibits cyclin-dependent kinase (CDK) and glycogen synthase kinase 3β (GSK3β). Kenpaullone possesses various activities, including anti-aging, inhibition of EC 2.7.11.26 (tau protein kinase), cardioprotection, and inhibition of EC 2.7.11.22 (cyclin-dependent kinase). It is an indole-azapyridine compound, a lactam compound, and an organic bromine compound. Functionally, it is related to paulownia ketone.

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1. Chemical structure and properties:
- 9-bromo-7,12-dihydroindolo[3,2-d][1]benzozazepine-6(5H)-one (C₁₆H₁₁BrN₂O, MW = 327.18)
- Solid powder, soluble in DMSO (≥30 mg/mL), slightly soluble in ethanol, insoluble in water[1]
- Stable at -20°C for at least 2 years[1]
2. Mechanism of action:
- ATP competitive inhibitor, binds to the ATP-binding pocket of the protein kinase
- Selectivity for GSK-3β and CDK is attributed to specific interactions with residues in the kinase domain of the "gatekeeper" residues (Thr148 in GSK-3β, Thr14 in CDK1) and other conserved residues[2]
3. Therapeutic potential:
- Oncology: as a single drug or in combination with chemotherapy drugs, it has the potential to treat a variety of cancers (glioblastoma, breast cancer, lung cancer, colon cancer) [1, 2]
- Neurological diseases: It has the potential to treat Alzheimer's disease (by inhibiting tau protein phosphorylation), Parkinson's disease and neuropathic pain [2]
- Autoimmune diseases: It has the potential to treat psoriasis, rheumatoid arthritis and inflammatory bowel disease (by inducing Treg cells) [1]
- Dermatology: It has the potential to treat hypopigmentation diseases (vitiligo) due to its melanin-stimulating effect [2]
4. Structure-activity relationship:
- The bromine substituent at position 9 is essential for inhibiting the activity of CDK and GSK-3β [1, 2]
- The indole[3,2-d][1]benzozazepine core structure is essential for kinase inhibitory activity [1]
- Modification at position 2 of the indole ring can enhance selectivity for specific kinase targets [2]

C16H11BRN2O

327.1753

326.005

C, 58.74; H, 3.39; Br, 24.42; N, 8.56; O, 4.89

142273-20-9

237430-03-4 (Alsterpaullone)

3820

White to off-white solid powder

1.6±0.1 g/cm3

613.0±45.0 °C at 760 mmHg

>300ºC (dec.)

324.5±28.7 °C

0.0±1.8 mmHg at 25°C

1.730

4.02

2

1

0

20

402

0

BrC1C([H])=C([H])C2=C(C=1[H])C1C([H])([H])C(N([H])C3=C([H])C([H])=C([H])C([H])=C3C=1N2[H])=O

QQUXFYAWXPMDOE-UHFFFAOYSA-N

InChI=1S/C16H11BrN2O/c17-9-5-6-14-11(7-9)12-8-15(20)18-13-4-2-1-3-10(13)16(12)19-14/h1-7,19H,8H2,(H,18,20)

9-bromo-7,12-dihydro-5H-indolo[3,2-d][1]benzazepin-6-one

1-azakenpaullone; Kenpaullone; NSC664704; NSC 664704; NSC-664704; 9-Bromopaullone

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: ~65 mg/mL (~198.7 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.64 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), suspension 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% DMSO+50% PEG 300+5% Tween 80+ddH2O: 2.5mg/mL

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