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Purity: ≥98%
B02 (also called RAD51-IN-02)is a potent and specific inhibitor of human RAD51 with an IC50 of 27.4 μM. In many types of cancer cells, RAD51 is overexpressed. Chemo- or radio-sensitive cancer cells are enhanced when RAD51 is downregulated by siRNA. B02 induces a heightened sensitivity to various agents that damage DNA and inhibits HR repair in human embryonic kidney (HEK) and breast cancer cells. B02 also makes decitabine-induced apoptosis and DNA damage in MM cells more pronounced”. B02 exhibits a high degree of specificity for RAD51 and does not substantially inhibit RAD54 across a concentration range of 0 to 200 μM.
| Targets |
hRAD51 ( IC50 = 27.4 μM )
RAD51 (Ki = 1.5 μM); RAD51-mediated DNA strand exchange (IC50 = 2.0 μM) [2] RAD51 (IC50 = 1.8 μM for DNA binding inhibition) [3] |
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| ln Vitro |
In vitro activity: B02 is a particular inhibitor of human RAD51 recombinase that prevents HR repair in HEK and breast cancer cells and makes them more vulnerable to various agents that damage DNA. Additionally, B02 increases the apoptosis and DNA damage that decitabine causes in MM cells[1]. In the range of concentrations from 0 to 200 μM, B02 does not significantly inhibit RAD54 and exhibits high specificity for RAD51[2]. B02 exhibits physiological effects in both mouse and human cells. B02 suppresses the formation of RAD51 foci in response to DNA damage in human embryonic kidney (HEK) cells, which in turn inhibits DSB repair and DSB-dependent HR. Additionally, B02 may make cancer cells more susceptible to DNA-damaging chemotherapeutic agents[3]. B02 specifically binds to RAD51 with a Ki of 1.5 μM, inhibiting RAD51-mediated DNA strand exchange activity with an IC50 of 2.0 μM [2] It suppresses homologous recombination (HR) repair in U2OS cells transfected with DR-GFP reporter, reducing HR efficiency by 75% at 10 μM [3] The compound enhances the cytotoxicity of cisplatin and camptothecin in BRCA-proficient cancer cell lines: A549 (cisplatin IC50 reduced from 5.2 μM to 1.8 μM at 5 μM B02), MCF-7 (camptothecin IC50 reduced from 4.7 μM to 1.5 μM at 5 μM B02) [1][3] It induces G2/M cell cycle arrest in A549 cells at 8 μM, accompanied by increased γ-H2AX foci (3.6-fold) indicating DNA damage accumulation [1] Western blot analysis shows that B02 (10 μM) does not affect RAD51 protein expression but blocks its nuclear localization in HCC1937 cells [3] It inhibits colony formation of HeLa cells by 68% at 10 μM and induces Annexin V-positive apoptosis in A549 cells (2.8-fold increase at 15 μM) [1] |
| ln Vivo |
B02 increases cisplatin's anti-tumor activity in vivo by a considerable amount. In the liver and kidneys, the primary organs involved in detoxification, no observable morphological alterations brought on by B02 have been established[3].
Intraperitoneal administration of B02 at 50 mg/kg twice weekly, combined with cisplatin (3 mg/kg weekly), inhibits tumor growth in A549 xenograft mice by 82% after 21 days of treatment, compared to 45% inhibition with cisplatin alone [1] In HeLa xenografts, B02 (60 mg/kg, i.p., twice weekly) combined with camptothecin (2 mg/kg twice weekly) reduces tumor volume by 76%, with increased cleaved caspase-3 expression in tumor tissues [1] Pharmacodynamic analysis shows that combined treatment increases tumor γ-H2AX levels by 2.5-fold, confirming enhanced DNA damage [1] |
| Enzyme Assay |
RAD51 DNA strand exchange assay: Recombinant RAD51 protein was incubated with circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in reaction buffer. Serial dilutions of B02 were added, and the mixture was incubated at 37°C for 60 minutes. The reaction was terminated, and DNA products were separated by agarose gel electrophoresis. The intensity of the strand exchange product band was quantified to calculate IC50 [2]
RAD51-DNA binding assay: Fluorescein-labeled dsDNA was mixed with RAD51 protein and serial dilutions of B02 in binding buffer. After incubation at 25°C for 30 minutes, fluorescence polarization was measured to assess binding affinity, with IC50 calculated from dose-response curves [3] |
| Cell Assay |
The WST-1 colorimetric cell count assay is used to track the proliferation of MM-cell lines. Approximately 8000 cells/well are seeded in 96-well plates with MM cell lines. After one hour of treatment with or without B02 (10 μM), the cells are treated for a further 72 hours with either vehicle (DMSO) or DOX (20–160 nM). Each well's culture medium receives a 1:10 addition of WST-1 reagent, and the incubation process lasts for four hours at 37°C. With a spectrophotometer, the absorbance at 450 nm is used to estimate the relative cell number.
HR repair efficiency assay: U2OS cells stably expressing DR-GFP reporter were transfected with I-SceI expression plasmid to induce DNA double-strand breaks. Cells were treated with B02 (0.5–20 μM) for 48 hours, and GFP-positive cells were counted by flow cytometry to determine HR efficiency [3] Chemosensitization assay: A549 and MCF-7 cells were seeded in 96-well plates, pretreated with B02 (0–15 μM) for 1 hour, then incubated with cisplatin or camptothecin for 72 hours. Cell viability was measured by colorimetric assay to determine IC50 shifts [1][3] Cell cycle and apoptosis assay: A549 cells were treated with B02 (0–20 μM) for 24 hours (cell cycle) or 48 hours (apoptosis). For cell cycle, cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. For apoptosis, cells were stained with Annexin V-FITC/PI and detected by flow cytometry [1] Colony formation assay: HeLa cells were seeded in 6-well plates at 500 cells/well, treated with B02 (0–15 μM), and incubated for 14 days. Colonies were stained with crystal violet and counted [1] |
| Animal Protocol |
Mice: The solutions of cisplatin and B02 are made up of NS and cremophor/DMSO/NS (1:1:3), respectively, right before injection. B02 (50 mg/kg or as indicated) and cisplatin (4 mg/kg or as indicated) are injected into mice in a combination treatment group. Mice are injected with B02 and NS in the B02 group and cisplatin and B02 vehicle in the cisplatin group. Three hours after the B02 (or its vehicle) injection, cisplatin (or NS) is administered. On days 11, 13, 15, and 17 following tumor cell inoculations, intraperitoneal injections (I.P.) are used to administer all treatments.
A549 xenograft model: Female nude mice were subcutaneously implanted with 5×106 A549 cells. When tumors reached 150–200 mm3, mice were randomized into four groups: vehicle, cisplatin alone, B02 alone, and combination. B02 was formulated in 10% DMSO + 90% corn oil and administered intraperitoneally at 50 mg/kg twice weekly. Cisplatin was given intraperitoneally at 3 mg/kg weekly. Tumor volume and body weight were measured twice weekly for 21 days [1] HeLa xenograft model: Female nude mice were subcutaneously inoculated with 1×107 HeLa cells. Treatment was initiated at tumor volume 200 mm3: B02 (60 mg/kg, i.p., twice weekly) combined with camptothecin (2 mg/kg, i.p., twice weekly) for 21 days. Tumor samples were collected for cleaved caspase-3 immunohistochemical analysis [1] |
| Toxicity/Toxicokinetics |
In a 21-day repeated-dose study in nude mice, intraperitoneal injection of up to 60 mg/kg of B02 twice weekly did not cause significant weight loss or abnormal hematological parameters [1]. No significant hepatotoxicity or nephrotoxicity was observed, and serum AST, ALT, creatinine, and BUN levels were normal in the treated mice [1]. B02 has a plasma protein binding rate of 85% in human plasma and 82% in mouse plasma [2].
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| References | |
| Additional Infomation |
3-(phenylmethyl)-2-[2-(3-pyridyl)vinyl]-4-quinazolinone belongs to the quinazoline class of compounds.
B02 is a small molecule RAD51 inhibitor, RAD51 being a key protein in homologous recombination (HR) DNA repair [2][3]. Its mechanism of action involves binding to RAD51, blocking its DNA strand exchange activity and nuclear localization, thereby inhibiting HR repair and enhancing the cytotoxicity of DNA damage chemotherapeutic agents [1][2][3]. This compound has shown potential as a chemosensitizer for cancers with normal BRCA function, overcoming resistance to platinum-based and topoisomerase inhibitor chemotherapy [1][3]. It has been widely used as a research tool to study HR repair and DNA damage response pathways in cancer cells [2]. |
| Molecular Formula |
C22H17N3O
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| Molecular Weight |
339.39
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| Exact Mass |
339.137
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| Elemental Analysis |
C, 77.86; H, 5.05; N, 12.38; O, 4.71
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| CAS # |
1290541-46-6
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| Related CAS # |
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| PubChem CID |
5738263
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.01
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
550
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1C2=C([H])C([H])=C([H])C([H])=C2N=C(/C(/[H])=C(\[H])/C2=C([H])N=C([H])C([H])=C2[H])N1C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]
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| InChi Key |
GEKDQXSPTHHANP-OUKQBFOZSA-N
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| InChi Code |
InChI=1S/C22H17N3O/c26-22-19-10-4-5-11-20(19)24-21(13-12-17-9-6-14-23-15-17)25(22)16-18-7-2-1-3-8-18/h1-15H,16H2/b13-12+
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| Chemical Name |
3-benzyl-2-[(E)-2-pyridin-3-ylethenyl]quinazolin-4-one
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 10 mg/mL (29.46 mM) in 20% DMSO 20% Cremophor EL + 60% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2 mg/mL (5.89 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 20.0 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. View More
Solubility in Formulation 3: ≥ 2 mg/mL (5.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9465 mL | 14.7323 mL | 29.4646 mL | |
| 5 mM | 0.5893 mL | 2.9465 mL | 5.8929 mL | |
| 10 mM | 0.2946 mL | 1.4732 mL | 2.9465 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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