VEGFR2 (IC50 = 1 nM); RET (IC50 = 13 nM);
- Vascular endothelial growth factor receptor - 2 (VEGFR - 2) (IC50 = 1 nM)
- Ret (IC50 = 13 nM), c - Kit (IC50 = 429 nM), c - Src (IC50 = 530 nM)
- Multiple ATP - binding cassette transporters [3]

- Inhibits the proliferation of human umbilical vein endothelial cells (HUVECs) stimulated by 20 ng/mL VEGF with an IC50 of 0.17 μM, and has a mild inhibitory effect on HUVECs stimulated by 20% FBS with an IC50 of 23.4 μM. It can significantly inhibit the migration of HUVECs induced by FBS at a concentration of 1 μM without affecting cell proliferation [1]
- Promotes autophagy and apoptosis in osteosarcoma cells through the VEGFR2/STAT3/BCL - 2 signaling pathway. Western blot is used to detect the protein expression levels of related signaling molecules, and flow cytometry is used to detect the apoptosis rate of osteosarcoma cells [2]
- Reverses multidrug resistance by inhibiting the efflux function of multiple ATP - binding cassette transporters. It increases the intracellular accumulation of chemotherapeutic drugs in multidrug - resistant cells, and the effect is verified by detecting the intracellular drug concentration and cell viability [3]
- Enhances the efficacy of conventional chemotherapeutic drugs in side population cells and ABCB1 - overexpressing leukemia cells. It increases the sensitivity of these cells to chemotherapeutic drugs, and the combined use can more significantly inhibit cell proliferation and promote cell apoptosis, which is detected by MTT assay and flow cytometry [4]
Apatinib significantly and dose-dependently inhibits the growth of a variety of human tumor xenografts. The ABCB1-mediated MDR in the nude mouse xenograft model is reversed by aparinib. Apatinib dramatically increases doxorubicin's antitumor activity in nude mice that have K562/ADR xenografts [3]

- Exhibits a dose - dependent antitumor effect in six human tumor xenografts in immunodeficient mice. Oral administration of Apatinib can inhibit tumor growth, and at a dose of 50 mg/kg per day, significant growth inhibition can be observed in three of the five tested tumor xenografts. At a dose of 100 mg/kg per day, all tumor xenografts are significantly inhibited, and at a dose of 200 mg/kg per day, the tumor growth inhibition rate is 8% - 18%, and complete growth inhibition can be observed in three xenografts [1]

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Apatinib significantly and dose-dependently inhibits the growth of a variety of human tumor xenografts. The ABCB1-mediated MDR in the nude mouse xenograft model is reversed by aparinib. Apatinib dramatically increases doxorubicin's antitumor activity in nude mice that have K562/ADR xenografts.[3]

- For VEGFR - 2 tyrosine kinase, a kinase activity assay is performed. The reaction system contains VEGFR - 2, ATP, and a substrate peptide. Different concentrations of Apatinib are added, and after incubation, the phosphorylation level of the substrate is detected by methods such as Western blot or ELISA, and then the inhibitory effect of Apatinib on VEGFR - 2 kinase activity is evaluated, and the IC50 value is calculated according to the inhibition rate and concentration relationship [1]
- For Ret, c - Kit, and c - Src, similar kinase activity assays are carried out, and the corresponding kinases, ATP, and specific substrates are used, and the same detection and calculation methods are adopted to obtain their IC50 values [1]

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The substrate solution containing tyrosine is a poly(glu, ala, tyr) 6:3:1 random copolymer. In order to coat 96 well plates (100 L/well), the substrate is kept as a 1 mg/mL stock in PBS at 20 °C and diluted 1 in 500 with PBS. The day before the assay, plates are coated, sealed with adhesive seals, and kept at 4 °C for the entire night. The substrate solution is disposed of on the day of the assay, and the assay plate wells are twice washed—once with Hepes buffer (50 mM, pH 7.4) and once with PBST (PBS containing 0.05% v/v Tween 20). The assay plates are washed and the test compounds are diluted with 10% dimethylsulfoxide (DMSO) de-ionized water, with 25 μL volumes being transferred to the wells. All test wells are then filled with 25 μL of a manganese chloride solution (40 mM) containing 8 μM ATP. To find the assay's dynamic range, additional wells are added that contain blank and control solutions, respectively, containing manganese chloride solution with and without ATP and compound diluent. Each well receives 50 L of freshly diluted enzyme, which is then added. The plates are then left to sit at room temperature for 20 min. Subsequently, the liquid is disposed of and the wells are twice cleaned using PBST. After adding 100 L/well of mouse IgG anti-phosphotyrosine antibody diluted 1:6000 with PBST containing 0.5% (w/v) bovine serum albumin (BSA), the plates were incubated for 1h at room temperature before the liquid was discarded and the wells were twice washed with PBST. The sheep anti-mouse Ig antibody linked to horseradish peroxidase (HRP) is diluted 1:500 with PBST containing 0.5% (w/v) BSA. After adding 100 μL/well, the plates are incubated for an additional hour at room temperature. The liquid is then discarded, and the wells are twice washed with PBST. Freshly prepared 50 mM phosphate-citrate buffer (pH 5.0) containing 0.03% (w/v) sodium perborate is mixed with 1 mg/mL of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, and 100 μL is added to each well. The plates are then incubated at room temperature for 20 to 60 minutes, or until the control wells' optical density value, measured at 405 nm, is roughly 1.0. Microcal Origin is used to interpolate IC50 values for compound enzyme inhibition after subtracting blank values.

In 96-well plates, the HUVEC are seeded. The test agents (vehicle serving as the control) are added to the cells along with 20 ng/mL VEGF or 20% FBS and incubated for an additional 72 hours. Following 10% trichloroacetic acid fixation, the cells are stained for 30 minutes at 37 °C using 0.4% sulforhodamine B, and then they are cleaned with 1% acetic acid. After dissolving the complex with tris, the optical density is measured at 520 nM.

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YN968D1 both by itself and in conjunction with chemotherapeutic agents efficiently and minimally harmed the growth of multiple well-established human tumor xenograft models in vivo[1].

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- Seed HUVECs in culture plates, add VEGF or FBS - containing culture medium, and then add different concentrations of Apatinib. Incubate for a certain time, and detect cell proliferation by MTT assay, and detect cell migration by transwell assay [1]
- Culture osteosarcoma cells, add Apatinib, and incubate for a period of time. Use Western blot to detect the protein expression of VEGFR2, STAT3, BCL - 2 and other related molecules, and use flow cytometry to detect apoptosis after Annexin V - FITC/PI double - staining [2]
- Culture multidrug - resistant cells, add Apatinib, and co - culture with chemotherapeutic drugs. Detect the intracellular concentration of chemotherapeutic drugs by fluorescence detection method, and detect cell viability by MTT assay [3]
- Culture side population cells and ABCB1 - overexpressing leukemia cells, add Apatinib in combination with chemotherapeutic drugs, detect cell proliferation by MTT assay, and detect apoptosis by flow cytometry after Annexin V - FITC/PI double - staining [4]

Ls174t, HCT 116, SGC-7901, HT-29, A549, NCI-H460 xenografted BALB/cA nude mice
50, 100, 200 mg/kg
p.o.

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Nude mouse human tumor xenograft model.  The effects of Apatinib (YN968D1) on tumor growth were tested against various human tumors grown subcutaneously in BALB/cA nude mice. Tumor growth was initiated by subcutaneous inoculation of cells into mice. Tumors were allowed to establish and grow to 100–300 mm3, at which time the mice were randomized into experimental groups. YN968D1 was administered once daily by oral gavage for the indicated periods (Table 1). In combination treatment experiments, mice were administered YN968D1 alone by oral gavage; 5‐FU, oxaliplatin, docetaxel and doxorubicin alone by intravenous injection; or YN968D1 in combination with each cytotoxic drug at the indicated dose and schedule (Table 2). Tumor volume and bodyweight were monitored every other day or every 3 days, with the means indicated for groups of six (treated) or 12 (vehicle control) animals. Tumor volumes were determined by measuring the largest diameter (a) and its perpendicular (b) according to the formula (a × b2)/2. The evaluation index for inhibition was the relative tumor growth ratio according to the equation: T/C (%) = mean increase of tumor volumes of treated groups/mean increase of tumor volumes of control groups × 100%.[1]

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- Dissolve Apatinib in an appropriate solvent, and orally administer it to immunodeficient mice bearing human tumor xenografts. The dosage is 50 mg/kg, 100 mg/kg, and 200 mg/kg per day, respectively. The administration is carried out once a day, and the tumor volume is measured regularly, and the body weight of the mice is monitored at the same time [1]

- Absorption: It is rapidly absorbed after oral administration, and the peak plasma concentration is reached at about 1.7 - 2.3 hours
- Distribution: Widely distributed in tissues
- Metabolism: Mainly metabolized in the liver, and cytochrome P450 enzyme system (such as CYP3A4) is involved in the metabolic process
- Elimination: The elimination half - life is about 8 - 9 hours, and it is mainly excreted through feces and urine

[1]. YN968D1 is a novel and selective inhibitor of vascular endothelial growth factor receptor-2 tyrosine kinase with potent activity in vitro and in vivo. Cancer Sci. 2011 Jul;102(7):1374-80.

[2]. Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma. Cell Death Dis. 2017 Aug; 8(8): e3015.

[3]. Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters. Cancer Res. 2010 Oct 15;70(20):7981-91.

[4]. Apatinib (YN968D1) enhances the efficacy of conventional chemotherapeutical drugs in side population cells and ABCB1-overexpressing leukemia cells. Biochem Pharmacol. 2012 Mar 1;83(5):586-97.

Rivoceranib Mesylate is the mesylate salt of rivoceranib, an orally bioavailable, small-molecule receptor tyrosine kinase inhibitor with potential antiangiogenic and antineoplastic activities. Rivoceranib selectively binds to and inhibits vascular endothelial growth factor receptor 2, which may inhibit VEGF-stimulated endothelial cell migration and proliferation and decrease tumor microvessel density. In addition, this agent mildly inhibits c-Kit and c-SRC tyrosine kinases.

C25H27N5O4S

493.58

493.17837553

C, 60.83; H, 5.51; N, 14.19; O, 12.97; S, 6.50

1218779-75-9

1218779-89-5 (HCl);1218779-75-9 (mesylate);811803-05-1;

45139106

white solid powder

1.3±0.1 g/cm3

578.2±50.0 °C at 760 mmHg

303.5±30.1 °C

0.0±1.6 mmHg at 25°C

1.652

3.68

3

8

6

35

701

0

S(C([H])([H])[H])(=O)(=O)O[H].O=C(C1C([H])=C([H])C([H])=NC=1N([H])C([H])([H])C1C([H])=C([H])N=C([H])C=1[H])N([H])C1C([H])=C([H])C(=C([H])C=1[H])C1(C#N)C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

FYJROXRIVQPKRY-UHFFFAOYSA-N

InChI=1S/C24H23N5O.CH4O3S/c25-17-24(11-1-2-12-24)19-5-7-20(8-6-19)29-23(30)21-4-3-13-27-22(21)28-16-18-9-14-26-15-10-18;1-5(2,3)4/h3-10,13-15H,1-2,11-12,16H2,(H,27,28)(H,29,30);1H3,(H,2,3,4)

N-[4-(1-cyanocyclopentyl)phenyl]-2-(pyridin-4-ylmethylamino)pyridine-3-carboxamide;methanesulfonic acid

YN968D1 mesylate; YN-968D1 mesylate; YN 968D1 mesylate; Rivoceranib mesylate

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: ~22 mg/mL (~44.6 mM)
Water: > 10mg/mL
Ethanol: < 1 mg/mL