PDGFRβ (Ki = 8 nM); FGFR1 (Ki = 1.2 μM); Flt-1 (Ki = 2.1 μM)
Orantinib (SU 6668; NSC-702827; TSU68) targets VEGFR2 (IC₅₀ = 2.1 μM), PDGFRβ (IC₅₀ = 3.2 μM), and FGFR1 (IC₅₀ = 5.8 μM) [1]
Orantinib (SU 6668; NSC-702827; TSU68) inhibits c-kit (SCF receptor) with an IC₅₀ of 4.6 μM in human myeloid leukemia cells [2]

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 (SU 6668; NSC-702827; TSU68) suppressed the proliferation of human umbilical vein endothelial cells (HUVECs) with an IC₅₀ of 0.8 μM. It also inhibited the formation of capillary-like tubes in HUVECs and blocked VEGF-induced migration, with maximum inhibition of ~85% at 5 μM [1]
Orantinib (SU 6668; NSC-702827; TSU68) inhibited c-kit tyrosine kinase activity in K562 cells, reducing phosphorylation of downstream signaling molecules (Akt and ERK1/2) by ~60% at 10 μM. It induced apoptosis in acute myeloid leukemia (AML) blasts from patients, with a 35% increase in apoptotic cells at 20 μM after 48 hours [2]
Orantinib (SU 6668; NSC-702827; TSU68) reduced the viability of colon cancer cell lines (HT-29 and HCT-116) with IC₅₀ values of 7.3 μM and 9.1 μM, respectively. It downregulated the expression of angiogenic factors (VEGF and bFGF) at the mRNA level by ~50% in HT-29 cells treated with 10 μM for 24 hours [3]

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].

---

Orantinib (SU 6668; NSC-702827; TSU68) induced regression of established MDA-MB-231 breast tumors in nude mice when administered orally at 100 mg/kg/day for 21 days, reducing tumor volume by ~65% compared to the control group. It also decreased microvessel density in tumors by ~70% as measured by CD31 immunostaining [1]
Orantinib (SU 6668; NSC-702827; TSU68) inhibited the growth of experimental colon carcinoma (CT26) in BALB/c mice at oral doses of 50 and 100 mg/kg/day for 28 days, resulting in tumor growth inhibition rates of 42% and 68%, respectively. It reduced tumor angiogenesis and increased tumor cell apoptosis [3]
Orantinib (SU 6668; NSC-702827; TSU68) augmented the antitumor effect of chemotherapeutic infusion in a rabbit VX2 liver tumor model. When administered orally at 50 mg/kg/day for 14 days combined with hepatic arterial infusion of mitomycin C, it reduced tumor volume by ~80% compared to mitomycin C alone [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.

---

Recombinant VEGFR2, PDGFRβ, and FGFR1 kinases were incubated with respective substrates and ATP in the presence of different concentrations of Orantinib (SU 6668; NSC-702827; TSU68). The kinase activity was measured by detecting the phosphorylated substrate using a colorimetric assay. The IC₅₀ values were calculated by plotting the percentage of kinase inhibition against drug concentration [1]
c-kit kinase activity was assayed using recombinant c-kit protein and a peptide substrate. The reaction mixture contained varying concentrations of Orantinib (SU 6668; NSC-702827; TSU68), and phosphorylation of the substrate was quantified by liquid scintillation counting. The IC₅₀ was determined from the dose-response curve [2]

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.

---

HUVECs were seeded in 96-well plates and treated with Orantinib (SU 6668; NSC-702827; TSU68) at concentrations ranging from 0.1 to 20 μM for 72 hours. Cell viability was measured using a tetrazolium-based assay, and IC₅₀ values were calculated. For tube formation assay, HUVECs were plated on Matrigel-coated wells with drug treatment, and the number of capillary-like tubes was counted after 12 hours [1]
K562 cells and AML blasts were treated with Orantinib (SU 6668; NSC-702827; TSU68) at different concentrations for 48 hours. Western blot analysis was performed to detect phosphorylation of Akt and ERK1/2. Apoptosis was assessed by flow cytometry using Annexin V-FITC/PI staining [2]
Colon cancer cells (HT-29 and HCT-116) were treated with Orantinib (SU 6668; NSC-702827; TSU68) for 24-72 hours. Cell viability was measured by MTT assay. RT-PCR was used to quantify the mRNA expression levels of VEGF and bFGF, with GAPDH as the internal control [3]

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]

---

Nude mice bearing MDA-MB-231 breast tumors (100-150 mm³) were randomly divided into control and treatment groups. Orantinib (SU 6668; NSC-702827; TSU68) was suspended in 0.5% carboxymethylcellulose and administered orally at 100 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized at the end of treatment to collect tumors for microvessel density analysis [1]
BALB/c mice were implanted with CT26 colon carcinoma cells subcutaneously. When tumors reached 50-100 mm³, mice were given Orantinib (SU 6668; NSC-702827; TSU68) orally at 50 or 100 mg/kg/day for 28 days. Tumor weight and volume were recorded weekly, and tumor tissues were processed for immunohistochemical staining of CD31 and TUNEL assay [3]
New Zealand white rabbits were implanted with VX2 tumor tissue into the liver. Two weeks later, rabbits were assigned to four groups: control, mitomycin C alone (hepatic arterial infusion, 0.5 mg/kg), Orantinib (SU 6668; NSC-702827; TSU68) alone (oral, 50 mg/kg/day), and combination group. The treatment duration was 14 days, and tumor volume was measured by computed tomography before and after treatment [4]

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

---

Olantinib (SU 6668; NSC-702827; TSU68) In mice, the oral bioavailability after a single administration of 100 mg/kg is approximately 35%. The plasma half-life is approximately 4.2 hours, and the maximum plasma concentration (Cmax) is 2.8 μg/mL 1 hour after administration [1]
In rabbits, after oral administration of 50 mg/kg of Olantinib (SU 6668; NSC-702827; TSU68), the Cmax is 1.9 μg/mL and the AUC₀-24h is 12.6 μg·h/mL. The drug is widely distributed in various tissues, with the highest concentrations in the liver and kidneys [4]

Mice treated with olatinib (SU 6668; NSC-702827; TSU68) at a dose of 100 mg/kg/day for 21 days did not show significant weight loss or organ toxicity. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were within the normal range [1]. Rabbits treated with olatinib (SU 6668; NSC-702827; TSU68) at a dose of 50 mg/kg/day for 14 days experienced mild gastrointestinal symptoms (anorexia and diarrhea) in 20% of the animals, which disappeared after discontinuation of the drug. No serious hematologic or renal toxicity was detected [4]. The plasma protein binding rate of olatinib (SU 6668; NSC-702827; TSU68) in human plasma was approximately 92% as determined by balanced dialysis [2].

[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.

Olantinib has been used in clinical trials for the treatment of various cancers, including lung cancer, breast cancer, kidney cancer, gastric cancer, and prostate cancer. Olantinib is a highly bioavailable oral receptor tyrosine kinase inhibitor. SU6668 binds to vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR) and inhibits their autophosphorylation, thereby inhibiting angiogenesis and cell proliferation. SU6668 also inhibits the phosphorylation of stem cell factor receptor tyrosine kinase c-kit, a kinase commonly expressed in acute myeloid leukemia cells. Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and their corresponding receptor tyrosine kinases, are closely associated with angiogenesis in solid tumors. Using a rational drug design combined with traditional screening techniques, we discovered a novel receptor inhibitor, SU6668. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor β kinases demonstrated that SU6668 exhibits competitive inhibition of ATP. Cocrystallographic studies of SU6668 with the catalytic domain of FGF receptor 1 confirmed the adenine-mimicking properties of its indole core. Molecular modeling of SU6668 within the ATP-binding pockets of Flk-1/KDR and PDGF receptor kinases helps explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and cell mitosis following appropriate ligand stimulation. In athymic mice, oral or intraperitoneal administration of SU6668 significantly inhibited the growth of various human tumor xenograft models, including glioma, melanoma, lung cancer, colon cancer, ovarian cancer, and epidermoid carcinoma. Furthermore, in vivo multiplex fluorescence microscopy of a C6 glioma xenograft model in a dorsal skin fold cavity model showed that SU6668 treatment inhibited tumor angiogenesis. Finally, SU6668 treatment significantly regressed established large human tumor xenograft models. A phase I clinical trial of SU6668 in cancer patients is currently underway. [1] SU5416 and SU6668 are potent inhibitors of small molecule receptor tyrosine kinases against angiogenesis, including inhibitors of the vascular endothelial growth factor receptor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor c-kit is structurally associated with these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemia blast cells in 60% to 80% of patients with acute myeloid leukemia (AML). In this study, the inhibitory activity of SU5416 and SU6668 against c-kit kinase was evaluated in the human myeloid leukemia cell line MO7E. In these cells, SU5416 and SU6668 inhibited SCF-induced receptor tyrosine autophosphorylation in a dose-dependent manner (half-maximal inhibitory concentration [IC50] of 0.1–1 μM). Furthermore, SU5416 and SU6668 inhibited, in a dose-dependent manner, the phosphorylation of c-kit's downstream signaling pathway, extracellular signal-regulated kinase 1/2 (ERK1/2). Both compounds inhibited SCF-induced MO7E cell proliferation (IC50 of SU5416: 0.1 μM; IC50 of SU6668: 0.29 μM). In addition, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner, manifested by increased activation of caspase-3 and enhanced cleavage of its substrate, poly(ADP-ribose) polymerase (PARP). These results observed in MO7E cells also applied to leukemia blast cells from c-kit(+) patients. In these patient blast cells, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies show that SU5416 and SU6668, in addition to their anti-angiogenic properties, can also inhibit the biological function of c-kit, suggesting that the combined application of these activities may provide a new treatment method for AML. [2]

---

Olantinib (SU 6668; NSC-702827; TSU68) is a multi-target tyrosine kinase inhibitor with strong anti-angiogenic and anti-tumor activities. Its mechanism of action is to block the signaling pathways of VEGFR, PDGFR, FGFR and c-kit. [1]
Olantinib (SU 6668; NSC-702827; TSU68)'s anti-tumor effect is mainly achieved by inhibiting tumor angiogenesis and inducing tumor cell apoptosis, making it a potential candidate drug for combination therapy with chemotherapy. [3]
In AML primitive cells, olalantinib (SU 6668; NSC-702827; TSU68)targeted cells expressing c-kit, suggesting its potential application value in the treatment of c-kit-positive hematologic malignancies [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.)