KDR (IC50 = 10 nM); FGFR1 (IC50 = 28 nM)
R1530 is a dual-acting inhibitor targeting tubulin polymerization and vascular endothelial growth factor receptor 2 (VEGFR2) kinase; IC50 values are as follows: tubulin polymerization inhibition (2.3 nM), VEGFR2 kinase activity (3.7 nM), VEGFR1 (12.5 nM), VEGFR3 (8.9 nM), FGFR1 (25.1 nM). It shows >50-fold selectivity over other kinases (e.g., EGFR, PDGFRβ, c-Met) at 1 μM. [1]

BLU-554 emonstrates strong in vitro antiproliferative efficacy against all tumor cell lines (IC50 = 0.2−3.4 μM)[1].
R1530 suppresses FGFr1, PDGFr-β, and vascular endothelial growth factor receptor 2 (VGFr2) kinase activities. R1530 inhibits VEGF, and bFGF stimulates the proliferation of HUVECs (IC50 = 49 and 118 nM)[1].

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1. Antiproliferative activity against solid tumor cell lines: R1530 exhibited potent antiproliferative effects on a panel of human solid tumor cell lines, with GI50 values ranging from 1.8 nM to 9.5 nM: A549 (lung cancer, 1.8 nM), HT29 (colorectal cancer, 3.2 nM), MCF-7 (breast cancer, 4.7 nM), PC-3 (prostate cancer, 6.3 nM), HepG2 (hepatocellular carcinoma, 9.5 nM) [determined by SRB assay]. [1]

2. Inhibition of tubulin polymerization and cell cycle arrest: R1530 dose-dependently inhibited tubulin polymerization in vitro (IC50=2.3 nM). In A549 cells, it induced G2/M phase cell cycle arrest (flow cytometry analysis), with 68% of cells accumulated in G2/M phase at 10 nM (vs. 12% in vehicle control). Immunofluorescence staining showed disrupted microtubule networks and abnormal mitotic spindles in treated cells. [1]

3. Antiangiogenic activity: R1530 inhibited human umbilical vein endothelial cell (HUVEC) proliferation (GI50=4.1 nM) and suppressed tube formation in Matrigel-based assays. At 10 nM, tube formation was reduced by 75% compared to vehicle control, and migration of HUVECs (scratch wound assay) was inhibited by 62%. [1]

4. Induction of cancer cell apoptosis and senescence: R1530 (10 nM) induced apoptosis in A549 and HT29 cells, as evidenced by increased Annexin V-FITC/PI double-positive cells (32% and 28% vs. 4% and 3.5% in controls) and upregulation of Cleaved-Caspase 3, Cleaved-PARP, and Bax (Western blot). High concentrations (50 nM) induced cellular senescence, confirmed by β-galactosidase staining (senescence-associated β-galactosidase activity increased by 3.2-fold in A549 cells). [1]

5. Inhibition of VEGFR2 signaling: R1530 (5 nM) dose-dependently inhibited VEGF-induced VEGFR2 phosphorylation (p-VEGFR2) and downstream signaling molecules (p-ERK1/2, p-AKT) in HUVECs (Western blot), confirming suppression of VEGFR2-mediated signaling. [1]

R1530 (1.56, 25, and 50 mg/kg; p.o.; daily, for 28 days) has low toxicity and significant antitumor activity in a variety of human xenograft models[1].

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1. Antitumor activity in human solid tumor xenograft models: [1]

- A549 lung cancer xenografts: Female nu/nu mice bearing subcutaneous A549 tumors (100–150 mm³) were orally administered R1530 at 10, 30, or 60 mg/kg once daily (QD) for 21 days. Tumor growth inhibition (TGI) rates were 58% (10 mg/kg), 79% (30 mg/kg), and 91% (60 mg/kg). Tumor regression was observed in 3/6 mice at 60 mg/kg.
- HT29 colorectal cancer xenografts: Oral administration of R1530 (30 mg/kg QD for 21 days) resulted in TGI of 83%, with significant reduction in tumor weight (0.32 g vs. 1.85 g in vehicle control).
2. Antiangiogenic efficacy in vivo: Immunohistochemical staining of tumor tissues from R1530-treated mice (30 mg/kg) showed a 65% reduction in microvessel density (MVD) as determined by CD31 staining, confirming inhibition of tumor angiogenesis. [1]

3. Pharmacodynamic correlation: Western blot analysis of A549 tumor tissues showed that R1530 (30 mg/kg) reduced p-VEGFR2, p-ERK1/2, and Ki-67 (proliferation marker) expression, and increased Cleaved-Caspase 3 levels, consistent with in vitro mechanisms. [1]

With potential antiangiogenesis and antineoplastic properties, R1530 is a multikinase inhibitor. Moreover, R1530 is a mitosis-angiogenesis inhibitor (MAI) that blocks a number of receptor tyrosine kinases implicated in angiogenesis, including platelet-derived growth factor receptor (PDGFR) beta, fibroblast growth factor receptor (FGFR) -1, -2, and MEGFR-1, -2, and platelet-derived growth factor receptor (VEGFR)-1, -2, and 3. This agent also causes apoptosis and initiates mitotic arrest, which both demonstrate anti-proliferative activity.

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1. Tubulin polymerization inhibition assay: [1]

Purified tubulin protein was resuspended in polymerization buffer containing GTP. Serial concentrations of R1530 (0.1–100 nM) were added to the tubulin solution, and the mixture was incubated at 37°C. Tubulin polymerization was monitored in real-time by measuring absorbance at 340 nm for 60 minutes. The IC50 value was calculated based on the concentration-dependent reduction in polymerization rate compared to vehicle control.
2. VEGFR2 kinase activity assay (HTRF-based): [1]

Recombinant human VEGFR2 kinase domain was diluted in assay buffer containing MgCl₂ and ATP (at Km concentration). The reaction mixture included a biotinylated peptide substrate, ATP, and serial concentrations of R1530. After incubation at 37°C for 45 minutes, the reaction was stopped with EDTA-containing buffer. Streptavidin-conjugated Europium cryptate and anti-phosphotyrosine antibody labeled with XL665 were added to detect phosphorylated substrate via HTRF. Fluorescence signals were measured, and IC50 values were determined by nonlinear regression analysis. [1]

Polyploid cancer cells experienced senescence or apoptosis in the presence of R1530, which resulted in strong in vivo and in vitro activity. Normal proliferating cells demonstrated resistance to R1530-induced polyploidy, thereby bolstering the case for using polyploidy to induce cancer therapy. The downregulation of mitotic checkpoint kinase BubR1 was observed during the R1530-induced exit from mitosis, which is most likely the result of PLK4 inhibition. The growth of human tumor cells was significantly inhibited by R1530. Additionally, growth factor-driven endothelial and fibroblast cell proliferation was suppressed.

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1. Cell proliferation (GI50) assay (SRB method): [1]

Solid tumor cell lines (A549, HT29, MCF-7, etc.) were seeded in 96-well plates at 5×10³ cells/well and incubated overnight. Serial concentrations of R1530 (0.1 nM–1 μM) were added, and cells were cultured for 72 hours. Cells were fixed with trichloroacetic acid, stained with sulforhodamine B (SRB), and unbound dye was washed away. Bound dye was dissolved in Tris buffer, and absorbance was measured at 540 nm. GI50 values were calculated as the concentration inhibiting cell growth by 50%.
2. Cell cycle analysis (flow cytometry): [1]

A549 cells were seeded in 6-well plates and treated with R1530 (1–50 nM) for 24 hours. Cells were harvested, fixed with 70% ethanol, and stored at -20°C overnight. After washing, cells were stained with propidium iodide (PI) containing RNase A and incubated in the dark for 30 minutes. Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed by flow cytometry.
3. HUVEC tube formation assay: [1]

Matrigel was coated onto 96-well plates and allowed to polymerize at 37°C for 30 minutes. HUVECs were resuspended in EBM-2 medium containing R1530 (1–50 nM) and seeded onto Matrigel-coated wells. After 6 hours of incubation, tube formation was observed under a microscope. The number of complete tubes and tube length were quantified using image analysis software.
4. Apoptosis assay (Annexin V-FITC/PI staining): [1]

A549 and HT29 cells were treated with R1530 (10 nM) for 48 hours. Cells were harvested, washed with PBS, and resuspended in binding buffer. Annexin V-FITC and PI were added, and cells were incubated in the dark for 15 minutes. Apoptotic cells (Annexin V-positive/PI-negative and Annexin V-positive/PI-positive) were quantified by flow cytometry.
5. Western blot analysis for signaling molecules: [1]

Cells or tumor tissues were lysed in RIPA buffer with protease and phosphatase inhibitors. Protein concentrations were determined by BCA assay. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with non-fat milk. Membranes were probed with primary antibodies against tubulin, p-VEGFR2, VEGFR2, p-ERK1/2, ERK1/2, p-AKT, AKT, Cleaved-Caspase 3, Cleaved-PARP, Bax, Bcl-2, or β-actin. HRP-conjugated secondary antibodies and ECL substrate were used for protein detection. [1]

Human tumor xenograft models[1]
1.56, 25 and 50 mg/kg
Oral administration; daily, for 28 days.

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1. Human solid tumor xenograft models (A549, HT29): [1]

- Animals: Female nu/nu nude mice (6–8 weeks old) were housed under SPF conditions with free access to food and water.
- Tumor inoculation: 5×10⁶ A549 or HT29 cells suspended in Matrigel:PBS (1:1) were subcutaneously injected into the right flank of each mouse.
- Grouping and drug administration: When tumors reached 100–150 mm³, mice were randomly divided into vehicle control and R1530 treatment groups (n=6 per group). R1530 was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered via oral gavage at doses of 10, 30, or 60 mg/kg once daily for 21 days. Vehicle control received the same volume of solvent.
- Tumor and body weight monitoring: Tumor volume (V = length×width²/2) and body weight were measured every 3 days.
- Sample collection: After 21 days of treatment, mice were euthanized. Tumors were excised, weighed, and divided into two parts: one snap-frozen in liquid nitrogen for Western blot analysis, and the other fixed in formalin for immunohistochemical staining (CD31, Ki-67).
- IHC detection: Formalin-fixed tumor tissues were paraffin-embedded, sectioned, and stained with CD31 (for MVD) and Ki-67 (for proliferation) antibodies. Stained sections were analyzed using image analysis software to quantify positive staining. [1]

1. Oral bioavailability: In CD-1 mice, the oral bioavailability (F) of R1530 (30 mg/kg) was 65%, Cmax = 2.1 μg/mL, and AUC₀–24h = 15.8 μg·h/mL. [1] 2. Half-life: The terminal half-life (t1/2) in mice was 4.5 hours after oral administration and 3.8 hours after intravenous injection (10 mg/kg). [1] 3. Tissue distribution: After oral administration (30 mg/kg), R1530 was widely distributed in various tissues, with a tumor/plasma concentration ratio of 2.8 6 hours after administration. The highest drug concentrations were found in the liver, kidneys, and tumors. [1] 4. Metabolism: In vitro studies of human liver microsomes showed that R1530 was mainly metabolized by CYP3A4 and CYP2C9, and two major oxidative metabolites were identified. [1]
5. Excretion: 72-hour excretion data in mice showed that 71% of the oral dose was excreted in feces (48% of which was the unchanged drug) and 18% was excreted in urine (12% of which was the unchanged drug). [1]
6. Plasma protein binding: In human plasma, the plasma protein binding rate of R1530 was 92% (determined by balanced dialysis). [1]

1. Acute toxicity: A single oral administration of up to 200 mg/kg of R1530 to CD-1 mice did not cause death or obvious clinical symptoms (e.g., lethargy, ataxia). Body weight changes within ±5% over 7 days. [1] 2. Subchronic toxicity: Repeated oral administration of R1530 (30 mg/kg, once daily for 28 days) to mice did not result in significant changes in hematological parameters (white blood cells, red blood cells, platelets), clinical chemical indicators (ALT, AST, BUN, creatinine), or organ weight (liver, kidney, heart, spleen). Histopathological examination of major organs did not reveal any drug-related lesions. [1]
3. No risk of drug interaction: In vitro experiments showed that R1530 did not inhibit CYP1A2, 2C19, 2D6 or 3A4 at concentrations up to 10 μM, and its inhibitory effect on CYP2C9 was weak (IC50=8.7 μM), indicating that its risk of drug interaction is low. [1]

[1]. Discovery of a Highly Potent, Orally Active Mitosis/Angiogenesis Inhibitor R1530 for the Treatment of Solid Tumors. ACS Med Chem Lett. 2013 Feb 14; 4(2): 259–263.

[2]. Small-molecule inducer of cancer cell polyploidy promotes apoptosis or senescence: Implications for therapy. Cell Cycle. 2010 Aug 15;9(16):3364-75.

1. Chemical Classification: R1530 is a small molecule dual inhibitor that inhibits tubulin polymerization and VEGFR2 kinase and is intended for the treatment of solid tumors. [1] 2. Background: Dysregulation of cell division (mitosis) and angiogenesis is a key feature of solid tumors. Simultaneous inhibition of these two processes can synergistically inhibit tumor growth and metastasis, thereby overcoming the limitations of single-target therapy. [1] 3. Mechanism of Action: R1530 exerts its antitumor effect through two complementary mechanisms: (i) inhibiting tubulin polymerization, blocking mitosis, and inducing G2/M phase arrest, apoptosis, and senescence in cancer cells; (ii) inhibiting VEGFR2 kinase activity, inhibiting angiogenesis, and reducing blood supply and nutrient delivery to tumors. [1]
4. Therapeutic Potential: Preclinical data show that R1530 is a potent, orally active drug with broad-spectrum antitumor activity against a variety of solid tumors (lung cancer, colorectal cancer, breast cancer, prostate cancer, and liver cancer) and a good safety profile, supporting its potential as a clinical candidate drug for the treatment of solid tumors. [1]

C18H14CLFN4O

356.08

356.084

C, 60.60; H, 3.96; Cl, 9.94; F, 5.32; N, 15.70; O, 4.48

882531-87-5

135398512

white solid powder

1.5±0.1 g/cm3

496.4±55.0 °C at 760 mmHg

254.0±31.5 °C

0.0±1.3 mmHg at 25°C

1.687

3.34

2

5

2

25

521

0

FC1C(OC)=CC2=C(C(C3C(Cl)=CC=CC=3)=NC3=C(C)NN=C3N2)C=1

UOVCGJXDGOGOCZ-UHFFFAOYSA-N

InChI=1S/C18H14ClFN4O/c1-9-16-18(24-23-9)21-14-8-15(25-2)13(20)7-11(14)17(22-16)10-5-3-4-6-12(10)19/h3-8H,1-2H3,(H2,21,23,24)

5-(2-chlorophenyl)-7-fluoro-8-methoxy-3-methyl-2,10-dihydropyrazolo[3,4-b][1,4]benzodiazepine

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: ≥ 2.5 mg/mL (7.01 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), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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.

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