VEGFR-2; c-Kit; FLT3; RET (IC50 = 170 nM)
FLT3 (including FLT3ITD mutants, FLT3D835Y mutant, and wild-type FLT3) – IC50 for growth inhibition in FLT3ITD-NPOS-transformed Ba/F3 cells: 175 nM; IC50 for growth inhibition in FLT3D835Y-transformed Ba/F3 cells: 250 nM; IC50 for growth inhibition in MV4-11 cells (FLT3ITD): 150 nM (MTT assay); IC50 for growth inhibition in MM6 cells: 450 nM; IC50 for growth inhibition in MM1 cells: 600 nM; IC50 for dephosphorylation of FLT3 in MM6 cells: 150 nM; IC50 for apoptosis induction in MM6 cells: 650 nM [1]
KIT – IC50 = 200 nM (biological assay in Kasumi-1 cells); IC50 = 30 nM (kinase assay) [1]
VEGFR-2 – IC50 = 70 nM (biological assay); IC50 = 460 nM (kinase assay) [1]
PDGFRβ – IC50 = 500 nM (biological assay); IC50 = 360 nM (kinase assay) [1]
FMS – IC50 = 13 μM (kinase assay) [1]
EGFR, FGFR-1 – IC50 > 100 μM (kinase and biological assays) [1]
TEL-ABL, BCR-ABL, TEL-JAK2 – IC50 > 10 μM (biological assays) [1]

In Vitro: SU5614 (1 μM) induced growth inhibition of Ba/F3 cells transformed by FLT3ITD-NPOS, FLT3ITD-W51, and FLT3D835Y mutants, but not by TEL-ABL. This growth inhibitory activity was completely abolished when cells were grown in the presence of IL-3. [1]
SU5614 induced growth arrest in Ba/F3 cells transformed by FLT3ITD and FLT3D835Y mutants with IC50 values of 175 nM and 250 nM, respectively, after 72 hours of incubation. [1]
SU5614 induced rapid apoptotic cell death in FLT3ITD-transformed Ba/F3 cells as measured by annexin V/7-AAD staining and DNA fragmentation (DNA laddering). The addition of IL-3 rescued FLT3-transformed cells from apoptosis induction at concentrations up to 10 μM. [1]
SU5614 induced dose-dependent cell cycle arrest in FLT3ITD-transformed Ba/F3 cells in the absence of IL-3, increasing the percentage of cells in G₀/G₁ phase from 65.4% ± 0.8% (0 μM) to 79.9% ± 3.3% (2.5 μM), with a parallel reduction in S phase and G₂/M phase. [1]
SU5614 (0.1-5 μM) dose-dependently inhibited the hyperphosphorylation of FLT3ITD receptor and FLT3D835Y receptor, and down-regulated STAT5 and MAPK activity in Ba/F3 cells in a time- and dose-dependent manner. [1]
SU5614 selectively inhibited growth of AML cell lines expressing constitutively activated FLT3 (MM1, MM6, MV4-11) with IC50 values of 300-600 nM, while cell lines expressing nonactivated FLT3 (THP-1, OCI-AML5) or no FLT3 (NB4, U937, K562, HL60, PLB985) were completely resistant (IC50 > 10 μM). [1]
SU5614 (5 μM, 48 hours) induced apoptosis in 90-98% of MM1, MM6, and MV4-11 cells as determined by annexin V/7-AAD staining. [1]
SU5614 (0.1-5 μM) dose-dependently inhibited FLT3 phosphorylation, STAT3, STAT5, and MAPK activity in MM6 cells, and down-regulated the STAT5 target genes BCL-XL and p21 in FLT3ITD-transformed Ba/F3 cells. [1]
SU5614 (1-5 μM) completely inhibited the anti-apoptotic and pro-proliferative activity of FL (100 ng/mL) in serum-starved OCI-AML5 cells and inhibited FL-induced FLT3 tyrosine phosphorylation. [1]
SU5614 did not inhibit the growth of Ba/F3 cells transformed by TEL-ABL, BCR-ABL, or TEL-JAK2 at concentrations up to 10 μM. [1]

Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are adverse prognostic features in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDSs). VEGF is a soluble circulating angiogenic molecule that stimulates signaling via receptor tyrosine kinases (RTKs), including VEGF receptor 2 (VEGFR-2). AML blasts may express VEGFR-2, c-kit, and FLT3. SU5416 is a small molecule RTK inhibitor (RTKI) of VEGFR-2, c-kit, and both wild-type and mutant FLT3. A multicenter phase 2 study of SU5416 was conducted in patients with refractory AML or MDS. For a median of 9 weeks (range, 1-55 weeks), 55 patients (33 AML: 10 [30%] primary refractory, 23 [70%] relapsed; 22 MDS: 15 [68%] relapsed) received 145 mg/m2 SU5416 twice weekly intravenously. Grade 3 or 4 drug-related toxicities included headaches (14%), infusion-related reactions (11%), dyspnea (14%), fatigue (7%), thrombotic episodes (7%), bone pain (5%), and gastrointestinal disturbance (4%). There were 11 patients (20%) who did not complete 4 weeks of therapy (10 progressive disease, 1 adverse event); 3 patients (5%) who achieved partial responses; and 1 (2%) who achieved hematologic improvement. Single agent SU5416 had biologic and modest clinical activity in refractory AML/MDS. Overall median survival was 12 weeks in AML patients (range, 4-41 weeks) and not reached in MDS patients. Most observed toxicities were attributable to drug formulation (polyoxyl 35 castor oil or hyperosmolarity of the SU5416 preparation). Studies of other RTKI and/or other antiangiogenic approaches, with correlative studies to examine biologic effects, may be warranted in patients with AML/MDS.[1]

Enzyme Assay: Kinase assays for various protein tyrosine kinases (KIT, VEGFR-2, PDGFR, FMS, EGFR, FGFR) were performed as previously described (references cited in paper). The IC50 values were determined. [1]
For FLT3 dephosphorylation assay, MM6 cells were incubated with SU5614 at indicated concentrations for 3 hours. Protein lysates were immunoprecipitated with FLT3 antibody and precipitates were analyzed by Western blot using anti-phosphotyrosine antibody. The IC50 for dephosphorylation was calculated by densitometry. [1]

---

Thyroid neoplasia is frequently associated with rearranged during transfection (RET) proto-oncogene mutations that cause hyperactivation of RET kinase activity. Selective inhibition of RET-mediated signaling should lead to an efficacious therapy. SU5416 is a potent inhibitor of vascular endothelial cell growth factor receptor, c-Kit, and FLT-3 receptor tyrosine kinases presently used in clinical trials. We found that SU5416 inhibits RET with similar potency, both in cell-free assays and in cells, thus causing proliferation arrest in oncogenic RET-transfected cells and in papillary thyroid carcinoma (PTC) cells expressing the RET/PTC1 oncogene, but not in RET-negative control cells. SU5416 inhibited RET-mediated signaling through the extracellular signal regulated kinase (ERK) and JNK pathways. In addition, we show that a naturally occurring MEN2 mutation at codon 804 confers resistance to SU5416, but not to the related compound SU4984. We provide a possible explanation to these results by using molecular docking. Finally, SU5416 was also assessed against an array of 52 tyrosine and serine/threonine kinases.[2]

Cell Assay: Ba/F3 cell proliferation assay: Cells were seeded at 4 × 10⁴ cells/mL in RPMI 1640 containing 10% FBS with or without IL-3 (10 ng/mL) and SU5614 as indicated. Viable cells were counted in a Neubauer chamber after trypan blue staining for up to 3 days. [1]
AML cell line proliferation assay: Cells were washed with PBS and cultured in 1 mL RPMI 1640 medium containing 10% FBS (20% FBS for MV4-11). Cells were seeded at 1 × 10⁵ cells/mL in 24-well plates for 72 hours with or without PTK inhibitors. Viable cells were counted by trypan blue exclusion. MTT assays were performed in 96-well plates in triplicate. Cells were seeded at 2 × 10⁵ cells/mL in RPMI 1640 containing 10% FBS with different concentrations of SU5614. After 84 hours, absorbance at 570 nm (reference 690 nm) was measured. [1]
Apoptosis assay: Cells were stained with annexin V-PE and 7-AAD and analyzed by flow cytometry. DNA fragmentation was assessed by DNA laddering on 2% agarose gel electrophoresis. Hypodiploid DNA content was analyzed after staining cell nuclei with PI by flow cytometry. [1]
Cell cycle analysis: Cells were stained with PI and cell nuclei were analyzed by flow cytometry. [1]
OCI-AML5 serum starvation assay: OCI-AML5 cells were starved in RPMI 1640 with 0.25% FBS for 16 hours, washed, and seeded at 2 × 10⁵ cells/mL in RPMI without FBS. FL (100 ng/mL) and SU5614 (5 μM) were added as indicated. Viable cells were counted for 4 days. [1]
Immunoprecipitation and Western blot: Protein lysates were immunoprecipitated with FLT3 antibody and precipitates were analyzed by Western blot with anti-phosphotyrosine antibody. Membranes were probed with phospho-specific antibodies against STAT3 (Tyr705), STAT5 (Tyr694), and MAPK (Thr202/Tyr204), followed by reblotting with specific STAT3, STAT5, and MAPK antibodies. BCL-XL, p21, and β-actin were detected by Western blot. [1]
Flow cytometry for CD135 and CD117 expression: Cells were incubated for 30 minutes on ice with PE-labeled isotype-matched control antibody, CD117-PE, or CD135-PE antibody. Viable cells were analyzed using a flow cytometer. [1]
Transient transfection and transduction: BOSC23 cells were transfected using calcium-phosphate coprecipitation. Ba/F3 cells were transduced with retroviral supernatant in the presence of polybrene (8 μg/mL). EGFP/EYFP+ cells were purified by FACS 48 hours after transduction. [1]

[1]. Blood. 2003 Aug 1;102(3):795-801.

[2]. J Mol Endocrinol. 2006 Oct;37(2):199-212.

SU5614 is a small-molecule protein tyrosine kinase inhibitor that inhibits FLT3 (including FLT3ITD and FLT3D835Y mutants), KIT, VEGFR-2, and PDGFRβ. It selectively induces growth arrest, apoptosis, and cell cycle arrest in Ba/F3 cells transformed by FLT3 mutants and in AML-derived cell lines expressing constitutively activated FLT3 (MM1, MM6, MV4-11). SU5614 reverts the anti-apoptotic and pro-proliferative activity of FLT3 ligand in FL-dependent cells. The compound has no cytotoxic activity in leukemic cell lines that express nonactivated FLT3 or no FLT3 protein. SU5614 inhibits FLT3 hyperphosphorylation and downstream STAT3, STAT5, and MAPK pathways, and down-regulates STAT5 target genes BCL-XL and p21. SU5614 is dissolved in DMSO at stock concentrations of 10 mM or 50 mM, aliquoted, and stored at -20°C. Final DMSO concentration in growth medium does not exceed 0.1%. [1]

---

SU5614 belongs to the oxindole class of compounds, specifically 5-chlorooxindole, in which the two hydrogen atoms at the 3-position are replaced by (3,5-dimethylpyrrole-2-yl)methylene groups. It is a vascular endothelial growth factor receptor antagonist. SU5614 belongs to the pyrrole class, oxindole class, and organochlorine class of compounds.

C15H13CLN2O

272.73

272.072

C, 66.06; H, 4.80; Cl, 13.00; N, 10.27; O, 5.87

1055412-47-9

Semaxanib HCl;204005-46-9;1055412-47-9 (Chloro-SU5416);

6536806

Brown to black solid powder

3.862

2

1

1

19

409

0

ClC1C([H])=C([H])C2=C(C=1[H])C(C(N2[H])=O)=C([H])C1=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])N1[H]

XLBQNZICMYZIQT-GHXNOFRVSA-N

InChI=1S/C15H13ClN2O/c1-8-5-9(2)17-14(8)7-12-11-6-10(16)3-4-13(11)18-15(12)19/h3-7,17H,1-2H3,(H,18,19)/b12-7-

(3Z)-5-chloro-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1H-indol-2-one

SU-5614; SU 5614; (Z)-SU5614; (Z)-SU-5614; CID:6536806; CHEBI:87159; SU5614.

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: 10~16.7 mg/mL (61.1~36.7 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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