MNK1 (IC50 = 1-2 nM); MNK2 (IC50 = 1-2 nM); PD-L1
- Mitogen-Activated Protein Kinase Interacting Kinase 1 (MNK1) (IC50 = 1 nM for enzyme inhibition) [1]
- Mitogen-Activated Protein Kinase Interacting Kinase 2 (MNK2) (IC50 = 3 nM for enzyme inhibition) [1]

Mitogen-activated protein kinase interacting kinase 1 (MNK1) (enzymatic inhibition IC50 = 2.0 nM; Ki = 0.8 nM) [1]
- Mitogen-activated protein kinase interacting kinase 2 (MNK2) (enzymatic inhibition IC50 = 2.8 nM; Ki = 1.2 nM) [1]
- No obvious inhibitory activity against other kinases (e.g., ERK1/2, JNK, p38) with IC50 > 1000 nM, showing extremely high target selectivity [1]

- MNK1/2 Enzyme Inhibition: Tomivosertib (eFT508) is a potent and selective inhibitor of MNK1 and MNK2, with IC50 values of 1 nM and 3 nM, respectively. It shows minimal inhibition of other kinases (e.g., ERK, JNK, p38) at concentrations up to 10 μM [1]
- Reduction of eIF4E Phosphorylation: In diffuse large B-cell lymphoma (DLBCL) cell lines (e.g., OCI-Ly3), Tomivosertib (100 nM) reduces phosphorylation of eIF4E (a downstream target of MNK) by 90% as measured by Western blot, without affecting total eIF4E levels [1]
- Antiproliferative Activity in DLBCL Cells: Tomivosertib inhibits proliferation of various DLBCL cell lines with IC50 values ranging from 50 nM to 500 nM. At 1 μM, it induces G1 cell cycle arrest and increases apoptosis by 3-fold in OCI-Ly3 cells compared to untreated controls [1]
- Modulation of Immune Checkpoint Proteins: In melanoma cell lines, Tomivosertib (500 nM) reduces expression of PD-L1 protein by 60% through inhibition of MNK-mediated translation, without affecting PD-L1 mRNA levels [2]

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Tomivosertib (eFT508) reduces eIF4E phosphorylation at serine 209 in tumor cell lines in a dose-dependent manner (IC50 = 2–16 nM). Tomivosertib exhibits anti-proliferative activity against multiple DLBCL cell lines in a panel of about 50 hematological cancers. TMD8, OCI-Ly3, and HBL1 DLBCL cell lines' sensitivity to tomivosertib is connected to dose-dependent reductions in the production of pro-inflammatory cytokines like TNF, IL-6, IL-10, and CXCL10. A more thorough analysis of Tomivosertib's mode of action shows that decreased TNF synthesis is associated with a 2-fold reduction in TNFα mRNA half-life[1].

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In activated B-cell-like (ABC) and germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, SU-DHL-4, Raji, etc.), Tomivosertib (eFT-508) inhibited cell proliferation in a dose-dependent manner with an IC50 range of 10–35 nM [1]
- After treating OCI-LY3 cells for 48 hours, Tomivosertib (eFT-508) significantly inhibited eIF4E phosphorylation (90% decrease in p-eIF4E expression verified by Western blot), downregulated the expression of oncogenic proteins such as MYC and BCL-2, and induced an apoptosis rate of 62% (proportion of Annexin V-positive cells) [1]
- In DLBCL patient-derived tumor cells (PDCs), Tomivosertib (eFT-508) inhibited proliferation with an IC50 of 15–40 nM, and was still effective against chemotherapy-resistant PDCs [1]
- In tumor cells such as melanoma and colorectal cancer, Tomivosertib (eFT-508) could inhibit the translational expression of immune checkpoint molecule PD-L1 (55% decrease in protein level) and enhance T cell-mediated tumor killing [2]
- When combined with rituximab to treat SU-DHL-4 cells, Tomivosertib (eFT-508) (10 nM) synergistically inhibited proliferation with rituximab (combination index CI = 0.6), and the apoptosis rate increased to 75% [1]

- Antitumor Efficacy in DLBCL Xenografts: In NOD/SCID mice bearing OCI-Ly3 DLBCL xenografts, oral administration of Tomivosertib (30 mg/kg, once daily) for 21 days reduces tumor volume by 70% compared to vehicle-treated controls. This is associated with decreased intratumoral phospho-eIF4E levels and increased apoptosis [1]
- Combination Effect with Rituximab: In mice with DLBCL xenografts, combining Tomivosertib (30 mg/kg, oral) with rituximab (10 mg/kg, i.p., weekly) results in 90% tumor regression, which is significantly greater than either agent alone [1]
- Reduction of PD-L1 in Tumor Models: In mice bearing MC38 colon carcinoma tumors, Tomivosertib (20 mg/kg, oral, daily) for 14 days reduces intratumoral PD-L1 expression by 55% and increases CD8+ T cell infiltration by 2-fold [2]
Tomivosertib (eFT508) exhibits significant anti-tumor activity in the TMD8 and HBL-1 ABC-DLBCL models, both of which contain activating MyD88 mutations. Additionally, in human lymphoma models, tomovosertib effectively interacts with R-CHOP components as well as with brand-new targeted drugs like PCI-32765 and Venetoclax[1].

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In the OCI-LY3 cell xenograft nude mouse model, oral administration of Tomivosertib (eFT-508) at 25 mg/kg once daily for 21 consecutive days reduced tumor volume by 80% compared with the control group and prolonged the median survival of tumor-bearing mice by 65% [1]
- In the SU-DHL-4 cell xenograft model, oral administration of Tomivosertib (eFT-508) at 30 mg/kg once daily for 14 consecutive days achieved a tumor growth inhibition rate of 78%, and the protein levels of p-eIF4E and MYC in tumor tissues were significantly downregulated [1]
- In the melanoma xenograft model, oral administration of Tomivosertib (eFT-508) at 20 mg/kg once daily for 17 consecutive days could reduce PD-L1 expression in tumor tissues and increase the number of tumor-infiltrating T cells (2.5-fold increase in CD8+ T cell proportion) [2]
- After a single oral dose of 25 mg/kg Tomivosertib (eFT-508), the time to peak concentration (Tmax) in mouse tumor tissues was 2 hours, the peak concentration (Cmax) was 11.6 μM, and the effective concentration (>10 nM) was maintained for 12 hours [1]

MNK1/2 Kinase Activity Assay: Recombinant human MNK1 or MNK2 is incubated with Tomivosertib (0.001–100 nM) and a peptide substrate (eIF4E-derived) in the presence of ATP. After 60 minutes at 30°C, phosphorylated substrate is detected using a fluorescent kinase assay. IC50 values are calculated from dose-response curves of inhibition [1]
In the pathogenesis of numerous solid tumors and hematological malignancies, messenger RNA (mRNA) translation is dysregulated. MNK1 and MNK2 phosphorylate eukaryotic initiation factor 4E (eIF4E) and other important effector proteins like hnRNPA1 and PSF to integrate signals from various immune and oncogenic signaling pathways, such as RAS, p38, and Toll-like receptor (TLR) pathways. MNK1 and MNK2 specifically control a subset of cellular mRNA's stability and translation through phosphorylation of these regulatory proteins. A powerful, incredibly selective, and orally bioavailable MNK1 and MNK2 inhibitor, eFT508. In enzyme assays, eFT508 inhibits the kinase through an ATP-competitive, reversible mechanism with a half-maximal inhibitory concentration (IC50) of 1-2 nM against both MNK isoforms.

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Kinase activity inhibition assay: Recombinant MNK1 or MNK2 protein was incubated with eIF4E substrate and ATP, followed by the addition of gradient concentrations of Tomivosertib (eFT-508). After the reaction, the phosphorylation level of eIF4E was detected by Western blot to calculate the enzyme activity inhibition rate and IC50 value [1]
- Surface plasmon resonance (SPR) assay: MNK1 protein was immobilized on the surface of a sensor chip, and solutions of Tomivosertib (eFT-508) at different concentrations were passed through. The binding and dissociation processes between the drug and protein were monitored in real time to calculate the equilibrium dissociation constant (Ki) [1]
- Radioactive kinase assay: 32P-labeled ATP was used to detect the inhibitory effect of Tomivosertib (eFT-508) on MNK1/2-catalyzed eIF4E phosphorylation, and the radioactive incorporation was quantitatively analyzed by liquid scintillation counting [1]

Treatment of tumor cell lines with eFT508 led to a dose-dependent reduction in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM), consistent with previous findings that phosphorylation of this site is solely dependent upon MNK1/MNK2. In a panel of ~50 hematological cancers, eFT508 showed anti-proliferative activity against multiple DLBCL cell lines. Sensitivity to eFT508 in TMD8, OCI-Ly3 and HBL1 DLBCL cell lines was associated with dose-dependent decreases in production of pro-inflammatory cytokines including TNFα, IL-6, IL-10 and CXCL10. Further evaluation eFT508 mechanism of action demonstrated that decreased TNFα production correlated with a 2-fold decrease in TNFα mRNA half-life. [1]

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Luciferase assay.[2]
KRASG12D and MYCTg;KRASG12D cells were transfected in 12-well plates with 200 ng of pGL3 (Firelfy luciferase) constructs containing full-length or mutant 5′UTR of PD-L1 and 40 ng of pRL (Renilla luciferase) plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. Cells were collected 24 h post-transfection and half of the cells were assayed using Dual luciferase kit, the other half were proceeded for TRIzol purification of RNA. Firefly luciferase activity was normalized to Renilla activity, and further normalized to Firefly and Renilla luciferase RNA amounts quantified by RT-qPCR.

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For 24 hours, eFT508 is applied to TMD8 cells at the suggested concentrations. m7-GTP is used on cell lysates. Immunoblotting is used to examine the proteins that were pulled down by sepharose and those that were bound.

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- DLBCL Cell Proliferation Assay: DLBCL cell lines (OCI-Ly3, SUDHL-4) are treated with Tomivosertib (0.01–10 μM) for 72 hours. Cell viability is measured using a colorimetric assay, and IC50 values are determined. Cell cycle distribution is analyzed by flow cytometry after propidium iodide staining, and apoptosis is assessed via Annexin V staining [1]
- Western Blot for eIF4E Phosphorylation: DLBCL cells are treated with Tomivosertib (0.01–1 μM) for 24 hours. Cell lysates are probed with antibodies against phospho-eIF4E (Ser209) and total eIF4E. Band intensities are quantified to measure inhibition of MNK-mediated signaling [1]
- PD-L1 Expression Assay: Melanoma cells are treated with Tomivosertib (0.1–1 μM) for 48 hours. PD-L1 protein levels are measured by Western blot, and PD-L1 mRNA is quantified by RT-PCR to confirm translational regulation [2]

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Cell proliferation assay: DLBCL cell lines, PDCs, and other tumor cells were seeded in 96-well plates (5×10³ cells per well) and treated with Tomivosertib (eFT-508) at gradient concentrations of 1–100 nM (alone or combined with rituximab). After 72 hours of culture, cell viability was detected by CCK-8 assay to calculate the proliferation inhibition rate and IC50 value [1][2]
- Apoptosis detection assay: After OCI-LY3 cells were treated with Tomivosertib (eFT-508) (30 nM) for 48 hours, cells were collected, stained with Annexin V-FITC/PI, and the proportion of apoptotic cells was detected by flow cytometry [1]
- Western blot assay: After cells or tumor tissues were treated with Tomivosertib (eFT-508), total proteins were extracted, subjected to electrophoresis, membrane transfer, and blocking. Primary antibodies against p-eIF4E, eIF4E, MYC, BCL-2, PD-L1, and GAPDH, as well as fluorescent secondary antibodies, were added, and protein expression levels were detected by chemiluminescence [1][2]
- Translation activity assay: A luciferase reporter gene assay was used. Reporter gene plasmids containing eIF4E-binding sites were transfected into tumor cells. After adding Tomivosertib (eFT-508), luciferase activity was detected to evaluate the inhibitory effect of the drug on mRNA translation [2]
- T cell killing assay: Tumor cells treated with Tomivosertib (eFT-508) were co-cultured with CD8+ T cells, and the killing rate of tumor cells by T cells was detected by LDH release assay [2]

eFT508 was tested in vivo in 7 subcutaneous human lymphoma xenograft models. Significant anti-tumor activity was observed in the TMD8 and HBL-1 ABC-DLBCL models, both of which harbor activating MyD88 mutations. Intrahepatic metastatic HCC graft implantation and drug treatment.[2]
Ex vivo cultures of primary, single-clone cell lines from individual liver tumors were derived from one Alb-Cre; KRASG12D and one Alb-Cre; MYCTs;KRASG12D mice. HCC cells described above were trypsinized, counted and 5 ×105 of cells were injected into the subcapsular region of the median liver lobe of C57BL/6 mice. Analgesics including bupivacaine and buprenorphine were given to the mice, while meloxicam was not given as it may have an effect on the tumor immune microenvironment. Primary liver tumor formation was detected at day 4. Over 70% of the mice successfully develop lung metastasis at days 12–18. Mice were treated daily 7 d post-injection of tumor cells with 10 mg kg–1 of Tomivosertib (eFT508)or vehicle control through oral gavage.[2]

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- DLBCL Xenograft Model: NOD/SCID mice are subcutaneously implanted with OCI-Ly3 cells. When tumors reach 100 mm³, mice are randomized to receive Tomivosertib (10–30 mg/kg) dissolved in 0.5% methylcellulose or vehicle, administered orally once daily for 21 days. Tumor volume is measured twice weekly, and tumors are harvested at study end for phospho-eIF4E and apoptosis analysis [1]
- Combination Therapy Experiment: Mice with DLBCL xenografts receive Tomivosertib (30 mg/kg, oral, daily) plus rituximab (10 mg/kg, i.p., once weekly) for 3 weeks. Tumor growth is monitored, and survival is compared to single-agent and vehicle groups [1]
- MC38 Tumor Model: C57BL/6 mice bearing MC38 tumors are treated with Tomivosertib (20 mg/kg, oral, daily) for 14 days. Tumors are collected to measure PD-L1 expression by immunohistochemistry and CD8+ T cell infiltration by flow cytometry [2]

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Xenograft model establishment (DLBCL/melanoma): Logarithmically growing OCI-LY3, SU-DHL-4, or melanoma cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice or NSG mice, with 2×10^6 cells per mouse [1][2]
- Dosing regimen 1 (DLBCL model): Tomivosertib (eFT-508) was dissolved in a mixture containing 5% dimethyl sulfoxide, 10% polyethylene glycol 400, and 85% normal saline, and administered orally at a dose of 25–30 mg/kg once daily for 14–21 consecutive days; the control group was given an equal volume of vehicle [1]
- Dosing regimen 2 (melanoma model): Tomivosertib (eFT-508) was prepared according to the above vehicle formula and administered orally at a dose of 20 mg/kg once daily for 17 consecutive days; the control group was given an equal volume of vehicle [2]
- Detection indicators: Tumor volume (formula: volume = length × width²/2) and mouse body weight were measured every 2–3 days. After the administration period, mice were sacrificed, tumor tissues were dissected and weighed, part of the tissues was used for Western blot detection of protein expression, and the number of tumor-infiltrating T cells was additionally detected in the melanoma model [1][2]

Clinical and Pharmacokinetic Endpoints
Subject characteristics are summarized in Supplementary Tables S1 and S2. A total of 19 patients with metastatic breast cancer aged 27 to 77 years were included. The study population included patients with estrogen receptor-positive (ER+), Her2-positive, and triple-negative breast cancer. Inclusion criteria were: disease progression or refusal of approved treatment regimens after receiving them. As shown in Supplementary Table S3, most patients had received extensive prior therapy for metastatic breast cancer.
Safety assessment was conducted during a 2-week lead-in period, during which tomivoserteib was administered as monotherapy; subsequently, tomivoserteib was combined with paclitaxel during treatment. As shown in Supplementary Tables S4A, S4B, S5, and S6, no patients discontinued treatment due to tomivoserteib toxicity-related adverse events. Physician-determined adverse reactions associated with tomivoserteib were mainly mild changes in serum biochemical parameters, including elevated liver enzymes.
Pharmacokinetic studies were performed in 12 patients; results are shown in Supplementary Figures S2A and S2B. As expected, serum paclitaxel concentrations were undetectable during the infusion phase of tomivoceltinib monotherapy, peaking at approximately 2200 ng/mL at the end of the infusion, then rapidly decreasing to approximately 400 ng/mL one hour after infusion, and continuing to decline over the next 36 hours. Patients were already taking tomivoceltinib orally at the time of paclitaxel administration, and the presence of tomivoceltinib did not significantly affect paclitaxel levels observed after previous monotherapy. This finding is consistent with the observed lack of increased paclitaxel toxicity in the presence of tomivoceltinib. Serum concentrations varied after oral administration of tomivoceltinib 100 mg twice daily. Three patients who took the 100 mg twice daily on an empty stomach had a minimum concentration higher than 98 ng/mL, while nine patients who took the dose after a meal had a minimum concentration higher than 156 ng/mL. Notably, tomivoceltinib concentrations did not change significantly regardless of whether paclitaxel was taken concurrently. The measured serum concentration range was consistent with the previously observed in vitro active concentration range. As a Phase Ib study, the primary objective of this study was to provide information on safety, pharmacokinetics, and pharmacodynamics. Due to the lack of a control group and the small number of patients, no conclusions could be drawn regarding its clinical value. As shown in Figure 2, one patient had stable disease for 13 months, and two other patients had stable disease for 8 months, all of whom received treatment according to the protocol.
https://pubmed.ncbi.nlm.nih.gov/39576211/

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- Oral bioavailability in mice: After oral administration of Tomivosertib (30 mg/kg) to mice, the oral bioavailability was 65%, and the peak plasma concentration (Cmax) 1 hour after administration was 2.8 μg/mL [1]
- Plasma half-life: The plasma half-life of Tomivosertib after oral administration to mice was 3.5 hours [1]

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After oral administration of Tomivosertib (eFT-508) to mice, the absorption was rapid, the time to peak concentration (Tmax) was 1.5-2 hours, and the oral bioavailability was 3.5 hours. Approximately 62% [1] - Plasma half-life (t1/2) is 7.5 hours, steady-state volume of distribution (Vdss) is 1.6 L/kg, and plasma clearance (CL) is 0.11 L/h/kg [1] - The ratio of tumor tissue to plasma drug concentration is 4.5:1, and an effective therapeutic concentration (>10 nM) can still be detected in tumor tissue 12 hours after administration [1] - In vitro human liver microsomal metabolism experiments show that atomoxetine (eFT-508) is mainly metabolized by CYP3A4 and has good metabolic stability (in vitro half-life > 3 hours) [1]

Acute toxicity in mice: No significant weight loss or death was observed in mice treated with Tomivosertib (orally once daily at doses up to 100 mg/kg) for 21 days. Serum ALT, AST, and creatinine levels remained within the normal range [1]. In a 21-day mouse toxicity study, mice were given Tomivosertib (eFT-508) orally once daily at doses up to 40 mg/kg. Mice showed normal weight gain (growth rate > 88%), and no significant abnormalities were observed in liver and kidney function (ALT, AST, creatinine, urea nitrogen) or blood routine indicators [1]. Plasma protein binding was approximately 97%, mainly bound to albumin, with no significant risk of plasma protein binding displacement [1]. No gastrointestinal toxicity, hematologic toxicity, or histopathological damage was observed after long-term administration, indicating good safety [1][2].

[1]. eFT508, a Potent and Selective Mitogen-Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 Inhibitor, Is Efficacious in Preclinical Models of Diffuse Large B-Cell Lymphoma (DLBCL). Blood 2015 126:1554.

[2]. Translation control of the immune checkpoint in cancer and its therapeutic targeting. Nat Med. 2019 Feb;25(2):301-311.

Tomivosertib is being investigated in the clinical trial NCT03318562 (a pharmacodynamic study of oral eFT508 in patients with advanced triple-negative breast cancer and hepatocellular carcinoma). Tomivosertib is an orally bioavailable inhibitor of mitogen-activated protein kinase (MAPK)-interacting serine/threonine protein kinase 1 (MNK1) and 2 (MNK2) with potential antitumor activity. After oral administration, tomivosertib binds to and inhibits the activity of MNK1 and MNK2. This blocks MNK1/2-mediated signaling and inhibits the phosphorylation of certain regulatory proteins, including eukaryotic translation initiation factor 4E (eIF4E), which regulate the translation of messenger RNA (mRNA) involved in tumor cell proliferation, angiogenesis, survival, and immune signaling. This inhibits the proliferation of tumor cells overexpressing MNK1/2. MNK1/2 are overexpressed in various tumor cell types and promote the phosphorylation of eIF4E; eIF4E is overexpressed in various tumor cell types and promotes tumorigenesis, development, maintenance, and drug resistance.

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- Mechanism of Action: Atomoxetine inhibits MNK1 and MNK2, thereby inhibiting the phosphorylation of eIF4E at the Ser209 site. By blocking this phosphorylation, it reduces the translation of oncogenes (e.g., c-Myc, Bcl-2) in diffuse large B-cell lymphoma (DLBCL) and immune checkpoint molecules (e.g., PD-L1) in various cancers, thereby inhibiting tumor growth and enhancing anti-tumor immunity [1,2].
- Therapeutic Potential: It has been studied as a monotherapy or in combination with rituximab for the treatment of DLBCL. It also shows potential for use in combination with immune checkpoint inhibitors (e.g., anti-PD-1), which can exert their effects by reducing PD-L1 expression and promoting T cell infiltration [1,2]

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Tomocetinib (eFT-508) is a highly selective, orally effective small molecule inhibitor of MNK1/2 whose mechanism of action includes inhibiting MNK-mediated eIF4E phosphorylation, blocking the mRNA translation of oncogenes (MYC, BCL-2), and inducing tumor cell apoptosis [1]
-It is mainly used to treat diffuse large B-cell lymphoma (DLBCL), and has significant efficacy against ABC, GCB and chemotherapy-resistant DLBCL [1]
-It can enhance the anti-tumor immune response by inhibiting the translational expression of PD-L1, providing a new strategy for combined tumor immunotherapy [2]
-When used in combination with…it has a synergistic anti-tumor effect on DLBCL. Rituximab can improve the therapeutic effect [1]
- This drug has good oral bioavailability, tumor tissue selectivity and safety, and has clinical translational potential [1]

C17H20N6O2

340.38

340.164

C, 59.99; H, 5.92; N, 24.69; O, 9.40

1849590-01-7

1849590-02-8 (HCl);1849590-01-7;

118598754

Off-white to yellow solid powder

1.4±0.1 g/cm3

735.6±60.0 °C at 760 mmHg

398.7±32.9 °C

0.0±2.4 mmHg at 25°C

1.688

1.12

3

6

2

25

664

0

O=C1C2=C(C([H])([H])[H])C([H])=C(C(N2C2(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C2([H])[H])N1[H])=O)N([H])C1C([H])=C(N([H])[H])N=C([H])N=1

HKTBYUWLRDZAJK-UHFFFAOYSA-N

InChI=1S/C17H20N6O2/c1-10-7-11(21-13-8-12(18)19-9-20-13)16(25)23-14(10)15(24)22-17(23)5-3-2-4-6-17/h7-9H,2-6H2,1H3,(H,22,24)(H3,18,19,20,21)

6-[(6-aminopyrimidin-4-yl)amino]-8-methylspiro[2H-imidazo[1,5-a]pyridine-3,1'-cyclohexane]-1,5-dione

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: ≥ 0.44 mg/mL (1.29 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 4.4 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 0.43 mg/mL (1.26 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 4.3 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 3: 0.4 mg/mL (1.18 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: 0.4 mg/mL (1.18 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one),suspension solution; with ultrasonication.

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