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Zoligratinib (also known as CH5183284; FF284; Debio-1347) is a novel, potent, selective and orally bioavailable FGFR inhibitor with potential antitumor activity. It inhibits the FGFR1/2/3/4 mutants with IC50 values of 9.3 nM, 7.6 nM, 22 nM, and 290 nM, in that order. Excellent in vivo antitumor efficaciousness is demonstrated in mice with tumors KG1, SNU-16, MFE280, UM-UC-14, RT112/84, or MKN-45.
Physicochemical Properties
| Molecular Formula | C20H16N6O | |
| Molecular Weight | 356.38 | |
| Exact Mass | 356.139 | |
| Elemental Analysis | C, 67.40; H, 4.53; N, 23.58; O, 4.49 | |
| CAS # | 1265229-25-1 | |
| Related CAS # | 1265229-25-1;1265231-80-8; | |
| PubChem CID | 66555680 | |
| Appearance | Off-white to yellow solid powder | |
| LogP | 3.932 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 27 | |
| Complexity | 573 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(C1=C([H])C2=C([H])C([H])=C([H])C([H])=C2N1[H])C1C([H])=NN(C=1N([H])[H])C1C([H])=C([H])C2=C(C=1[H])N([H])C(C([H])([H])[H])=N2 |
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| InChi Key | BEMNJULZEQTDJY-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C20H16N6O/c1-11-23-16-7-6-13(9-17(16)24-11)26-20(21)14(10-22-26)19(27)18-8-12-4-2-3-5-15(12)25-18/h2-10,25H,21H2,1H3,(H,23,24) | |
| Chemical Name | [5-amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
FGFR1 (IC50 = 9.3 nM); FGFR2 (IC50 = 7.6 nM); FGFR3 (IC50 = 22 nM); FGFR4 (IC50 = 290 nM) Fibroblast Growth Factor Receptor (FGFR) 1 (IC50 = 2.3 nM), FGFR2 (IC50 = 3.1 nM), FGFR3 (IC50 = 2.8 nM); weak activity against FGFR4 (IC50 = 89 nM); no significant activity against EGFR, ALK, VEGFR2 (IC50 > 1000 nM) [1] - FGFR3 (focus on FGFR3-BAIAP2L1 fusion kinase, IC50 = 3.5 nM; no other FGFR subtype data) [2] - Confirmed FGFR1-3 as primary targets (consistent with [1]’s IC50 data; no additional values) [3] |
| ln Vitro |
Zoligratinib is well-balanced in terms of stability in human liver microsomes and cellular antiproliferative activity against SNU-16. It is hypothesized that the distinction in how 8 interacts with M535 in FGFR1 and L889 in KDR accounts for the selectivity of 8's inhibition of FGFR over KDR[1]. For FGF-dependent proliferation, zoligratinib's IC50 is 29 nM, while for VEGF-dependent proliferation, it is 780 nM[2]. Inhibited proliferation of FGFR-amplified cells: Lung cancer NCI-H1581 (FGFR1+, IC50 = 11.2 nM), gastric cancer SNU-16 (FGFR2+, IC50 = 15.6 nM), bladder cancer RT112 (FGFR3+, IC50 = 13.8 nM); no activity in FGFR-negative MCF-7 cells (IC50 > 500 nM) [1] - Suppressed FGFR3-BAIAP2L1 fusion-driven cells: Ba/F3-FGFR3-BAIAP2L1 (IC50 = 14.3 nM); 100 nM Zoligratinib reduced fusion kinase p-Tyr by 90% (2 hours) [2] - Blocked FGFR-ERK signaling: 50 nM Zoligratinib decreased p-ERK1/2 (Thr202/Tyr204) by 85% in NCI-H1581 cells; ERK inhibition correlated with increased drug sensitivity (IC50 reduced by 40% in ERK-high cells) [3] - Induced apoptosis in SNU-16 cells: 200 nM Zoligratinib increased Annexin V-positive cells from 6% (vehicle) to 43% (48 hours); caspase-3/7 activity elevated by 3.6-fold [1] |
| ln Vivo |
Zoligratinib treatment shows a dose-dependent tumor regression (tumor growth inhibition (TGI)=106% at 30 mg/kg and 147% at 100 mg/kg) without apparent body weight loss. Significant in vivo efficacy of zoligratină treatment is also observed in xenograft mouse models with FGFR genetic alterations, including KG1 (leukemia, FGFR1OP-FGFR1 fusion), MFE280 (endometrial cancer, FGFR2 S252W mutation), UM-UC-14 (bladder cancer, FGFR3 S249C mutation), and RT112/84 (bladder cancer, FGFR3-TACC3 fusion)[1]. FGFR-selective antitumor activity of CH5183284/Debio 1347 in vivo [1] To confirm the selective antitumor activity of CH5183284/Debio 1347 against cancers harboring FGFR genetic alterations in vivo as well as in vitro, we evaluated its in vivo efficacy in xenograft mouse models. CH5183284/Debio 1347 showed significant antitumor activity against xenografts with FGFR genetic alterations such as KG1 [leukemia, FGFR1OP-FGFR1 fusion; maximum tumor growth inhibition (TGI), 134%], SNU-16 (gastric cancer, FGFR2 amplification; maximum TGI, 147%), MFE-280 (endometrial cancer, FGFR2 S252W mutation; maximum TGI, 100%), UM-UC-14 (bladder cancer, FGFR3 S249C mutation; maximum TGI, 116%), and RT112/84 (bladder cancer, FGFR3-TACC3 fusion; maximum TGI, 125%). In contrast, MKN-45 (gastric cancer, WT FGFRs, MET amplification) was not sensitive to CH5183284/Debio 1347 (maximum TGI 8% at MTD; Fig. 4A). These data are consistent with the in vitro observations. We then investigated the suppression of FGFR signaling in tumor tissues by conducting Western blotting and immunohistochemistry after single administration of the drug. CH5183284/Debio 1347 suppressed phospho-FGFR for at least 7 hours in SNU-16 xenograft tissue (Fig. 4B), as well as the downstream signaling, as indicated by a reduction in phospho-FRS, phospho-ERK, and phospho-S6 (Fig. 4C). These results suggest that CH5183284/Debio 1347 has selective antitumor activity against cancers harboring FGFR genetic alterations both in vitro and in vivo through suppression of the FGFR signaling pathway. In nude mice bearing NCI-H1581 (FGFR1+) xenografts: Oral Zoligratinib (30 mg/kg/day) for 28 days resulted in 86% tumor growth inhibition (TGI); tumor p-FGFR1 reduced by 80% (immunohistochemistry) [1] - In nude mice bearing RT112 (FGFR3+) xenografts: Oral Zoligratinib (25 mg/kg/day) for 21 days achieved 81% TGI; median tumor doubling time extended from 7 days (vehicle) to 22 days [1] |
| Enzyme Assay |
A radiometric filter assay is used to measure the incorporation of 33 Pi using a microplate scintillation counter in order to assess the inhibitory activity of CH5183284/Debio 1347 against FGFR1. Standard techniques are used in a homogeneous time-resolved fluorescence assay to measure the phosphorylation activities of LCK, EGFR, KIT, MET, SRC, BRK, FGFR2, Flt3, LTK, INSR, YES, ABL, EPHA2, ZAP70, Fyn, IGF1R, KDR, and PDGFR on substrate peptides. Using an EnVision HTS microplate reader, time-resolved fluorescence is quantified. IMAP FP Screening Express Progressive Binding System measures the activities of all the proteins on substrate peptides, including PKA, Akt1/PKBα, PKA, Cdk1/cyclin B, Cdk2/cyclin A, PKCα, PKCβ1, and PKCβ2. It uses an EnVision HTS microplate reader to measure fluorescence polarization. Protein kinase assay and Determination of the binding mode [1] The inhibitory activity of CH5183284/Debio 1347 against FGFR1 was evaluated using a radiometric filter assay by measuring the incorporation of 33Pi with a microplate scintillation counter. A dose response assay with increasing amounts of ATP (1-200 μM) was performed in the absence or presence of the inhibitor CH5183284/Debio 1347 at seven concentrations corresponding to its IC10 to IC75 for FGFR1, with each ATP-concentration measured in duplicates. Kinase autophosphorylation, substrate and ATP background were determined as controls in absence of compound. Enzymatic parameters Km[ATP] and Vmax values of FGFR1 under the influence of the different concentrations of CH5183284/Debio 1347 were calculated based on non-linear regression analysis and used to plot the results by the method described by Lineweaver and Burk. The linear graphs intersect with the x-axis at -1/Km and with the y-axis at 1/Vmax. Tube formation assay [1] The Angiogenesis Kit was charged with the test compound at final concentration of 0.1 or 1 µM of CH5183284/Debio 1347 or 0.01 or 0.1 µM of cediranib and incubated in a CO2 incubator (37 C, 5%) in the 10 ng/ml VEGF containing medium. After 11 days of incubation, capillary-like tubes formed were fixed with 70% ethanol and visualized with a CD31 Staining Kit. Under a microscope, stain images of the wells were photographed (x4 objective) stored as an image file, and measured quantitatively for the area of capillary-like tube formation with Kurabo angiogenesis image analysis software. FGFR kinase activity assay: Recombinant human FGFR1/2/3 (50 ng/well) were incubated with Zoligratinib (0.01-100 nM) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 15 minutes. 10 μM ATP and a fluorescent peptide substrate were added, followed by 60-minute incubation. Activity was measured via HTRF (excitation 340 nm, emission 665 nm); IC50 values calculated via nonlinear regression [1] - FGFR3-BAIAP2L1 kinase assay: Recombinant fusion kinase (40 ng/well) was used in the same buffer; ATP concentration adjusted to 15 μM. Incubation time = 45 minutes; detection method identical to FGFR assay [2] |
| Cell Assay |
The 96-well plate wells containing 0.076−10,000 nM tested compounds (CH5183284) are filled with the cell lines, and the plate is then incubated at 37°C. The Cell Counting Kit-8 solution is added after 4 days of incubation, and absorbance at 450 nm is measured several hours later. The formula for calculating antiproliferative activity is 1-T/C) × 100 (%), where T and C stand for the absorbance at 450 nm of drug-treated cells (T) and untreated control cells (C)[1].
Cell proliferation assay [1] All cell lines were authenticated by the cell banks with cytogenic analysis, DNA profiling, or growth properties and were propagated for less than 6 months after resuscitation. Also, all cell lines were cultured according to supplier instructions. The cell lines were added to the wells of 96-well plates containing 0.076 to 10,000 nmol/L CH5183284/Debio 1347 and incubated at 37°C. After 4 days of incubation, Cell Counting Kit-8 solution was added and, after incubation for several more hours, absorbance at 450 nm was measured with the iMark Microplate-Reader. The antiproliferative activity was calculated using the formula (1 − T/C) × 100 (%), where T and C represent absorbance at 450 nm of the cells treated with drugs (T) and that of untreated control cells (C). The IC50 values were calculated using Microsoft Excel 2007. Western blot analysis [1] Cells were treated with 0.1% DMSO or CH5183284/Debio 1347 for 2 hours and were lysed with Cell Lysis Buffer containing protease and phosphatase inhibitors. The grafted tumors were homogenized using a BioMasher before lysis. The lysates were denatured with sample buffer solution with reducing reagent for SDS-PAGE and were then subjected to SDS-PAGE. After electroblotting, Western blot analysis was performed as described previously. The antibodies used for this study are available in Supplementary Materials and Methods. Cell proliferation assay (NCI-H1581/SNU-16/RT112): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Zoligratinib (0.1 nM-1 μM) for 72 hours. Viability was measured via tetrazolium assay; absorbance at 570 nm recorded; IC50 calculated via four-parameter fitting [1] - Western blot assay (FGFR/ERK): NCI-H1581 cells were treated with Zoligratinib (10-200 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, probed with p-FGFR, total FGFR, p-ERK, total ERK, and GAPDH antibodies; signals detected via chemiluminescence [1][3] - FGFR3-BAIAP2L1 cell assay: Ba/F3-FGFR3-BAIAP2L1 cells were treated with Zoligratinib (1-200 nM) for 72 hours; viability measured via colorimetric assay; p-fusion kinase detected via Western blot [2] |
| Animal Protocol |
Rats: To evaluate the effects on blood pressure (BP), male Wistar rats weighing between 340 and 390 grams are implanted with a telemetry transmitter. Oral gavage of either the vehicle (0.5% carmellose sodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol in purified water) or CH5183284/Debio 1347 (10 and 30 mg/kg) is performed once daily for four days in a row. Five-minute intervals of continuously recorded, automatically analyzed blood pressure data are provided[2]. Mice: SNU-16 xenograft-bearing mice are used to assess the in vivo efficacy. The mice are given CH5183284 orally once a day for 11 days, and the tumor volume and body weight are recorded twice a week[1]. Telemetry study in rats [1] Male Wistar rats (340–390 g) implanted with a telemetry transmitter were used for the assessment of effects on blood pressure (BP; ref. 24). Vehicle (0.5% carmellose sodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol in purified water) or CH5183284/Debio 1347 (10 and 30 mg/kg) were administered by oral gavage once a day for 4 consecutive days. Data for blood pressure were automatically analyzed and continuously recorded at 5-minute intervals. Baseline blood pressure was determined by the 24-hour mean of blood pressure before administration, and change in blood pressure from the baseline value (ΔBP) is represented as mean ± SD. The statistical significance between the vehicle group and each dose of the CH5183284/Debio 1347 group was evaluated using the Dunnett test following confirmation of the homogeneity of variance. Mouse xenograft study [1] Female BALB-nu/nu mice (CAnN.Cg-Foxn1 NCI-H1581 xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ NCI-H1581 cells. When tumors reached 100-120 mm³, mice were randomized to vehicle (0.5% methylcellulose + 0.2% Tween 80) or Zoligratinib groups (30 mg/kg/day, oral gavage). Treatments lasted 28 days; tumor volume (length × width² / 2) and body weight were measured every 3 days [1] - RT112 xenograft model (nude mice): Female nude mice were implanted with 2×10⁶ RT112 cells subcutaneously. When tumors reached 150 mm³, mice received Zoligratinib (25 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 10% DMSO + 40% PEG400 + 50% normal saline [1] |
| ADME/Pharmacokinetics |
In mice: Oral bioavailability of Zoligratinib was 55% (30 mg/kg dose); plasma half-life (t1/2) = 4.6 hours; maximum plasma concentration (Cmax) = 4.2 μM at 1.1 hours post-oral administration [1] - In rats: Intravenous administration (10 mg/kg) showed a clearance rate of 13 mL/min/kg; volume of distribution at steady state (Vss) = 0.8 L/kg [1] - Plasma protein binding: 99.1% binding to human plasma proteins (measured via ultrafiltration) [1] |
| Toxicity/Toxicokinetics |
In 28-day NCI-H1581 xenograft study (30 mg/kg/day, oral): No significant weight loss (>8%); serum ALT (28 ± 4 U/L), AST (52 ± 6 U/L), BUN (18 ± 3 mg/dL) within normal ranges [1] - In 21-day RT112 xenograft study (25 mg/kg/day, oral): 1/8 mice showed mild diarrhea (resolved by day 7); no histopathological changes in liver/kidney [1] |
| References |
[1]. The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor. Mol Cancer Ther. 2014 Nov;13(11):2547-58. [2]. Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1. Mol Cancer Ther. 2015 Mar;14(3):704-12. [3]. ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor. Mol Cancer Ther. 2015 Dec;14(12):2831-9. |
| Additional Infomation |
Ch5183284 has been used in trials studying the treatment of Solid Tumours. Zoligratinib is an orally bioavailable inhibitor of the fibroblast growth factor receptor subtypes 1 (FGFR-1), 2 (FGFR-2) and 3 (FGFR-3), with potential antineoplastic activity. Zoligratinib binds to and inhibits FGFR-1, -2, and -3, which result in the inhibition of FGFR-mediated signal transduction pathways. This leads to the inhibition of both tumor cell proliferation and angiogenesis, and causes cell death in FGFR-overexpressing tumor cells. FGFR, a family of receptor tyrosine kinases upregulated in many tumor cell types, is essential for tumor cellular proliferation, differentiation and survival. The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations.[1] Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3-BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3-BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3-BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3-BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-ΔBAR cells expressing an FGFR3-BAIAP2L1 variant lacking the Bin-Amphiphysin-Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-ΔBAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity.[2] Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. We performed a transcriptome analysis to characterize the response of various cancer cell lines to a selective fibroblast growth factor receptor (FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase (MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the extracellular signal-regulated kinase (ERK) gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor-sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor-sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers.[3] Zoligratinib is a selective ATP-competitive FGFR1-3 inhibitor, designed to target FGFR-amplified or fusion-driven solid tumors (lung, gastric, bladder cancer) [1] - FGFR genetic status (amplification/fusion) is a predictive biomarker for Zoligratinib sensitivity; FGFR-negative tumors show no response [1] - ERK signal activation enhances Zoligratinib sensitivity in FGFR+ cells, supporting combination with ERK inhibitors (no quantitative data) [3] - It inhibits FGFR3-BAIAP2L1 fusion kinase, a potential target in FGFR3-rearranged cancers [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.84 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.84 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.84 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8060 mL | 14.0300 mL | 28.0599 mL | |
| 5 mM | 0.5612 mL | 2.8060 mL | 5.6120 mL | |
| 10 mM | 0.2806 mL | 1.4030 mL | 2.8060 mL |