GSK-3β (IC50 = 0.71 μM)
Glycogen Synthase Kinase-3β (GSK-3β) and GSK-3α (both isoforms inhibited due to high homology) [1]

Glycogen Synthase Kinase-3α and GSK-3β [2]

9-ING-41, a clinically relevant small molecule GSK-3β inhibitor with broad spectrum pre-clinical antitumor activity, suppresses the growth of neuroblastoma cells.[1] 9-ING-41 induces apoptosis and cell cycle arrest at prophase by targeting centrosomes and microtubule-bound GSK3β.[2]

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In neuroblastoma cells (SK-N-DZ and SK-N-BE(2)), 9-ING-41 (0.1-1 μM) inhibited GSK-3 activity as shown by downregulation of phospho-Glycogen Synthase (direct downstream target of GSK-3), decreased expression of the anti-apoptotic protein XIAP (NF-κB target), and induced significant apoptosis as shown by PARP cleavage. GI50 of 9-ING-41 for inhibiting growth of SK-N-DZ and SK-N-BE(2) cells was 50-100 nM, which was 10-60 times lower than that of AR-A014418 and TDZD-8. [1]

In neuroblastoma cells, using a clinically relevant pulsed in vitro treatment (5 hours) to mimic transient drug exposure in vivo, 9-ING-41 (30-500 nM) was combined with 0.5 μM CPT-11 (based on pediatric plasma Cmax). In SK-N-BE(2) cells, monotherapy with 0.5 μM 9-ING-41 for 5 hours caused 75% growth inhibition, while CPT-11 alone caused 20% inhibition. 9-ING-41 at 120 nM and 250 nM potentiated the antitumor effect of 0.5 μM CPT-11 (p < 0.05). [1]

In lymphoma cell lines (TCL, MCL, DLBCL), 9-ING-41 induced apoptosis after 48 hours treatment. Normal unstimulated T lymphocytes or PBMCs showed no significant apoptosis even at 10 μM. IC50 values for cell survival: OCI-LY1 3.05 μM, OCI-LY19 2.21 μM, OCI-Ly3 3.76 μM, SU-DHL6 1.58 μM, Granta-519 0.77 μM, Jeko 1.28 μM, Mino 0.55 μM, Karpas-299 3.34 μM, MyLa 0.69 μM, SeAx 1.60 μM. IC50 for cell proliferation: OCI-LY1 0.69 μM, OCI-LY19 1.96 μM, OCI-Ly3 1.03 μM, SU-DHL6 0.84 μM, Granta-519 0.38 μM, Jeko 0.94 μM, Mino 0.72 μM, Karpas-299 0.26 μM, MyLa 0.19 μM, SeAx 0.38 μM. [2]

In lymphoma cells, 9-ING-41 treatment (1.0-2.0 μM for 24 hours) caused cell cycle arrest at G2/M (prophase stage). In Jeko cells treated with 1.0 μM 9-ING-41 for 24 hours, only prophase (M1) mitotic cells were observed, with no cells in prometaphase (M2), metaphase (M3), anaphase (M4), or telophase (M5). 9-ING-41 did not alter the localization of GSK3β to centrosomes and mitotic spindles. [2]

In primary lymphoma patient cells (MCL, high-grade B-cell lymphoma, follicular lymphoma grade 3B, DLBCL, angioimmunoblastic TCL), 9-ING-41 inhibited proliferation. [2]

In a mouse model of MCL, 9-ING-41 has antitumor activity as a single agent.[2] The combination of clinically relevant doses of CPT-11 and 9-ING-41 results in a greater antitumor effect than either agent alone, according to mouse xenograft studies. [1]

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In SK-N-BE(2) and SK-N-DZ neuroblastoma xenograft models, mice were treated with 9-ING-41 (70 mg/kg, intraperitoneal) alone or combined with CPT-11 (5 mg/kg). 9-ING-41 monotherapy inhibited SK-N-BE(2) tumor growth; combination with CPT-11 led to tumor regression. In SK-N-DZ tumors, 9-ING-41 monotherapy did not significantly inhibit growth, but combined CPT-11+9-ING-41 led to tumor regression and significantly increased apoptosis measured by TUNEL staining (p < 0.05). [1]

In a Jeko (MCL) xenograft mouse model (NSG mice with subcutaneous Fluc-expressing Jeko cells), 9-ING-41 (40 mg/kg, intraperitoneal, every other day) significantly reduced tumor growth compared to untreated controls as measured by bioluminescence imaging. [2]

Measurement of cell viability. Relative number of viable cancer cells is determined by measuring the optical density using CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit according to the manufacturer’s instructions. GraphPad Prism 6.0 software is used to determine a GI50 value for each compound using a non-linear regression model with standard slope.

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Cell viability was measured using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. Cells were treated with compounds for indicated times, then MTS reagent added, and optical density measured. GI50 was calculated using non-linear regression model. [1]

For pulsed in vitro treatment to mimic transient in vivo exposure, cells were treated with 9-ING-41 and/or CPT-11 for 5 hours, then washed and replaced with compound-free fresh media, and allowed to grow for 72 hours before cell viability measurement. [1]

Immunoblot analysis: Cells were lysed, protein concentration determined by Bradford assay, equal amounts (30 μg) of protein loaded on SDS-polyacrylamide gel, separated by 10% SDS-PAGE, transferred to PVDF membrane, and probed with antibodies for phospho-Glycogen Synthase, GAPDH, PARP, XIAP. [1]

Apoptosis detection: TUNEL staining was performed on xenograft tumor sections according to manufacturer’s protocol. Percentage of apoptosis was calculated as number of TUNEL positive cells per 1000 cells counted in three randomly selected microscopic fields. [1]

CRISPR/CAS9 knockout: Guide RNAs targeting first coding exons of GSK3α and GSK3β genes were cloned into px458 vector (co-expressing GFP). Lymphoma cells were nucleofected, GFP-positive single cells sorted into 96-well plates (1 cell/well). After 2 weeks expansion, subclones were genotyped by PCR and Sanger sequencing. [2]

Cell cycle analysis: Cells were treated with 9-ING-41, then fixed and stained for DNA content analysis by flow cytometry. [2]

Immunofluorescence staining: Cells were stained for GSK3β, pericentrin (centrosome marker), α-tubulin (microtubule marker), and DNA. Colocalization was observed. [2]

Morphological analysis of mitotic stages: Wright’s stained cells were examined. Untreated cells showed all stages (prophase M1, prometaphase M2, metaphase M3, anaphase M4, telophase M5). 9-ING-41-treated cells showed only prophase (M1) cells. [2]

6–8 week-old female athymic nude mice
70 mg/kg
IP

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Female athymic nude mice (6-8 weeks old) were inoculated subcutaneously with 1×10^6 SK-N-BE(2) or SK-N-DZ neuroblastoma cells mixed with Matrigel. Tumors were staged prior to treatment: SK-N-BE(2) tumors ~500 mm³, SK-N-DZ tumors ~200 mm³. Mice were randomized into 4 groups: control (DMSO), 9-ING-41 (70 mg/kg), CPT-11 (5 mg/kg), and CPT-11+9-ING-41. Drugs were injected intraperitoneally. At study end, tumors were fixed in formalin and paraffin-embedded. [1]

NSG mice (8 weeks old, 8-10 mice per group) were subcutaneously injected with 5×10^6 Fluc-expressing Jeko cells in right flanks. Tumor engraftment verified by imaging 4 days after inoculation. Mice were randomized into control (untreated) and treatment groups. 9-ING-41 (40 mg/kg) was injected intraperitoneally every other day. Tumor volumes measured with IVIS Imager 20 minutes after intraperitoneal injection of D-Luciferin (15 mg/mL, 200 μL) and anesthesia with 2.5% isoflurane. Experiment terminated when largest tumor met size limit. [2]

9-ING-41 showed good tolerability in rodents. [1]

9-ING-41 did not induce significant apoptosis in normal unstimulated T lymphocytes or peripheral blood mononuclear cells even at concentration of 10.0 μM. [2]

[1]. Anticancer Drugs . 2018 Sep;29(8):717-724.

[2]. Blood . 2019 Jul 25;134(4):363-373.

Elraglusib is being investigated in the clinical trial NCT04218071 (Actuate 1901: 9-ING-41 for myelofibrosis). Elraglusib is a maleimide-based small molecule glycogen synthase kinase-3 (GSK-3; serine/threonine protein kinase GSK3) inhibitor with potential antitumor activity. After intravenous injection, Elraglusib binds to and competitively inhibits GSK-3, which may lead to downregulation of nuclear factor κB (NF-κB) and reduced expression of NF-κB target genes, including cyclin D1, B-cell lymphoma 2 (Bcl-2), the anti-apoptotic protein XIAP, and B-cell lymphoma superlarge (Bcl-XL) . This may inhibit NF-κB-mediated cell survival and chemotherapy resistance in certain tumor types. GSK-3 is a constitutively active serine/threonine kinase that plays a role in multiple pathways, including protein synthesis, cell proliferation, differentiation, and metabolism. It is abnormally overexpressed in certain tumor types, which may promote tumor cell survival and lead to resistance to chemotherapy and radiotherapy.

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9-ING-41 is a clinically relevant small molecule GSK-3β inhibitor with broad spectrum pre-clinical antitumor activity. It has been shown to be more selective for GSK-3 than for 320 other related kinases. FDA has granted 9-ING-41 orphan drug status for the treatment of neuroblastoma. An IND for 9-ING-41 has been approved by FDA for first in human Phase I/II clinical trials. [1]

9-ING-41 targets GSK3β localized to centrosomes and mitotic spindles, causing mitotic prophase arrest. A Phase I trial of 9-ING-41 (NCT03678883) has begun. [2]

C22H13FN2O5

404.347429037094

404.081

C, 65.35; H, 3.24; F, 4.70; N, 6.93; O, 19.78

1034895-42-5

1034895-42-5

44582816

Pink to red solid powder

3.688

1

6

2

30

802

0

FC1C=CC2=C(C=1)C(=CO2)C1C(NC(C=1C1=CN(C)C2C=C3C(=CC1=2)OCO3)=O)=O

FARXPFGGGGLENU-UHFFFAOYSA-N

InChI=1S/C22H13FN2O5/c1-25-7-13(11-5-17-18(6-15(11)25)30-9-29-17)19-20(22(27)24-21(19)26)14-8-28-16-3-2-10(23)4-12(14)16/h2-8H,9H2,1H3,(H,24,26,27)

3-(5-fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)-1H-pyrrole-2,5-dione

9-ING-41; 9 ING 41; 9ING41

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: 50~81 mg/mL (123.7~200.3 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.18 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)