PDGFRβ (Ki = 8 nM); FGFR1 (Ki = 1.2 μM); Flt-1 (Ki = 2.1 μM)

Orantinib (SU6668; 0.03-10 μM) inhibits the tyrosine phosphorylation of KDR in VEGF-stimulated HUVECs and prevents PDGF-stimulated PDGFRβ tyrosine phosphorylation in NIH-3T3 cells that overexpress PDGFRβ. The phosphorylation of the FGFR1 substrate 2 is inhibited by orantinib (≥10 μM) when it is exposed to acidic FGF. Nonetheless, in NIH-3T3 cells overexpressing EGFR, orantinib (up to 100 μM) has no effect on EGF-stimulated EGFR tyrosine phosphorylation. Moreover, orantinib blocks HUVECs' ability to proliferate when stimulated by VEGF and FGF, with mean IC50 values of 0.34 μM and 9.6 μM, respectively[1]. Orantinib (SU6668) inhibits both ERK1/2 phosphorylation and the tyrosine autophosphorylation of the stem cell factor (SCF) receptor, c-kit, in human myeloid leukemia MO7E cells, with an IC50 of 0.1-1 μM. Furthermore, orantinib induces apoptosis and, at an IC50 of 0.29 μM, suppresses the proliferation of MO7E cells induced by SCF[2].

Orantinib (SU6668; 75-200 mg/kg) inhibits the growth of various tumor types, including A375, Colo205, H460, Calu-6, C6, SF763T, and SKOV3TP5 cells, in xenograft models in athymic mice. C6 glioma xenograft tumor angiogenesis is likewise inhibited by orantinib (75 mg/kg)[1]. Orantinib (200 mg/kg) reduces average vessel permeability and average fractional plasma volume in the tumor rim and core of an HT29 human colon carcinoma tumor model. Around the edges of carcinomas, orantinib promotes aberrant stromal development[3]. In addition, a rabbit VX2 liver tumor model shows that Orantinib (TSU-68; 200 mg/kg) enhances the effects of chemotherapy infusion[4].

Tyrosine kinase assays are used in 96-well microtiter plates that have been precoated (20 μg/well) in PBS and incubated overnight at 4 °C with the peptide substrate poly-Glu,Tyr (4:1). The purpose of these assays is to quantify the trans-phosphorylation activity of Flk-1 and FGFR1. One to five percent (w/v) BSA in PBS is used to block excess protein binding sites. The microtiter wells are then filled with purified GST-FGFR1 (kinase domain) or GST-Flk-1 (cytoplasmic domain) fusion proteins in a 2 × concentration kinase dilution buffer that contains 40 μM NaVO₄, 50 mM NaCl, 100 mM HEPES, and 0.02% (w/v) BSA. For GST-Flk-1 and GST-FGFR1, the final enzyme concentration is 50 ng/mL. SU6668 is diluted 1:25 in H₂O after being dissolved in DMSO at 100× the final required concentration. Each reaction well is then filled with 25 μL of diluted SU6668. Different concentrations of ATP are added to a solution of MnCl2 to start the kinase reaction. The final concentration of MnCl₂ is 10 mM, and the final ATP concentrations span the Km for the enzyme. Before adding EDTA to stop the reaction, the plates are incubated for five to fifteen minutes at room temperature. TBST is then used to wash the plates three times. In TBST containing 0.5% (w/v) BSA, 0.025% (w/v) nonfat dry milk, and 100 μM NaVO₄, rabbit polyclonal antiphosphotyrosine antisera are diluted 1: 10,000 and added to the wells. The incubation process lasts for one hour at 37 °C. Next, goat anti-rabbit antisera conjugated with HRP is added to the plates after three TBST washes. Three TBST washes are performed after the plates are incubated for an hour at 37 °C. After adding 2,2 to each well, the amount of phosphotyrosine is measured.

In DMEM containing 10% (v/v) FBS, cells (HUVECs and NIH-3T3 cells overexpressing PDGFRβ or EGFR) are seeded (3 × 10₅ cells/35-mm well), grown to confluence, and then quiesced in DMEM containing 0.1% serum for two hours prior to drug treatment. After being seeded at a density of 2 × 10₆ cells/10-cm plate, HUVECs are grown to confluence in endothelial cell growth media and subsequently quiesce for 24 hours in endothelial cell basal media containing 0.5% FBS prior to drug treatment. Before being stimulated with ligand (100 ng/mL) for 10 minutes, all cell lines are incubated with SU6668 for 1 hour.

Mouse (Female, BALB/c, nu/nu) xenograft models of A375, Colo205, H460, Calu-6, C6, SF763T, and SKOV3TP5 tumor cells
75-200 mg/kg
Via i.p. injection or oral gavage once daily.

---

A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. DCE-MRI with a macromolecular contrast agent was used to measure transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. CD31 immunohistochemical staining was used for assessing microvessels density and vessels area. Experiments were performed after 24 h, and 3, 7, and 14 days of treatment. Results: DCE-MRI clearly detected the early effect (after 24 h of treatment) of SU6668 on tumor vasculature as a 51% and 26% decrease in the average vessel permeability measured in the tumor rim and core (respectively). A substantial decrease was also observed in average fractional plasma volume in the rim (59%) and core (35%) of the tumor. Histological results confirmed magnetic resonance imaging findings. After 3, 7, and 14 days of treatment, postcontrast magnetic resonant images presented a thin strip of strongly enhanced tissue at the tumor periphery; histology examination showed that this hyperenhanced ring corresponded to strongly vascularized tissue adjacent but external to the tumor. Histology also revealed a strong decrease in the thickness of peripheral viable tissue, with a greatly reduced vessel count. SU6668 greatly inhibited tumor growth, with 60% inhibition at 14 days of treatment.[3]

Metabolism / Metabolites
TSU-68 has known human metabolites that include TSU-68 metabolite 1, TSU-68 metabolite 2, and TSU-68 metabolite 3.

[1]. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res, 2000, 60(15), 4152-4160.

[2]. The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts. Blood, 2001, 97(5), 1413-1421.

[3]. In vivo assessment of antiangiogenic activity of SU6668 in an experimental colon carcinoma model. Clin Cancer Res, 2004, 10(2), 739-750.

[4]. Augmentation of chemotherapeutic infusion effect by TSU-68, an oral targeted antiangiogenic agent, in a rabbit VX2 liver tumor model. Cardiovasc Intervent Radiol. 2012 Feb;35(1):168-75.

Orantinib has been used in trials studying the treatment of Lung Cancer, Breast Cancer, Kidney Cancer, Gastric Cancer, and Prostate Cancer, among others.
Orantinib is an orally bioavailable receptor tyrosine kinase inhibitor. SU6668 binds to and inhibits the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), thereby inhibiting angiogenesis and cell proliferation. SU6668 also inhibits the phosphorylation of the stem cell factor receptor tyrosine kinase c-kit, often expressed in acute myelogenous leukemia cells.

---

Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.[1]

---

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC(50)] 0.1-1 microM). Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC(50) 0.1 microM for SU5416; 0.29 microM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit(+) patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.[2]

C18H18N2O3

310.35

310.131

C, 69.66; H, 5.85; N, 9.03; O, 15.47

252916-29-3

(Z)-Orantinib;210644-62-5

5329099

Yellow to orange solid powder

1.3±0.1 g/cm3

590.5±50.0 °C at 760 mmHg

310.9±30.1 °C

0.0±1.7 mmHg at 25°C

1.675

2.49

3

3

4

23

516

0

CC1=C(/C=C2C3=CC=CC=C3NC/2=O)NC(C)=C1CCC(O)=O

NHFDRBXTEDBWCZ-ZROIWOOFSA-N

InChI=1S/C18H18N2O3/c1-10-12(7-8-17(21)22)11(2)19-16(10)9-14-13-5-3-4-6-15(13)20-18(14)23/h3-6,9,19H,7-8H2,1-2H3,(H,20,23)(H,21,22)/b14-9-

3-[2,4-dimethyl-5-[(Z)-(2-oxo-1H-indol-3-ylidene)methyl]-1H-pyrrol-3-yl]propanoic acid

Orantinib; NSC 702827; NSC-702827; NSC702827; TSU68; TSU-68; SU-6668; SU 6668; SU6668; NSC702827; TSU 68

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: ≥ 10 mg/mL (32.22 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 100.0 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.

---

Solubility in Formulation 2: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

---

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