CDK9- Cyclin T1 (IC50 = 0.02 μM); cdk4-cyclin D1 (IC50 = 0.063 μM); CDK1-Cyclin B (IC50 = 0.079 μM); cdk2-cyclin A (IC50 = 0.224 μM); cdk2-cyclin E (IC50 = 2.540 μM); cdk6-cyclin D3 (IC50 = 0.396 μM); CDK9-cyclin H (IC50 = 2.900 μM)
Riviciclib (P276-00) mainly targets the cyclin-dependent kinase (CDK) family, including CDK1/cyclin B (IC50=41 nM), CDK2/cyclin A (IC50=105 nM), CDK2/cyclin E (IC50=63 nM), and CDK4/cyclin D1 (IC50=138 nM) [2]
Riviciclib (P276-00) also inhibits CDK9/cyclin T1 (IC50=260 nM) and has weak inhibitory effects on other CDK isoforms such as CDK3 and CDK5 (IC50>500 nM) [3]
Riviciclib (P276-00) acts as a selective CDK inhibitor, exerting antitumor effects by targeting CDK1/2/4/9 to interfere with cell cycle progression and transcriptional regulation [1]

Riviciclib (1.5-5 μM; 72 hours) exhibits cell arrest in G1 in synchronized human non-small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells, as well as no detectable cells in G1 and G2 in promyelocytic leukemia cells[3].
Riviciclib (3–24 hours; 1.5 μM) lowers H-460 cell levels of cyclin D1, Cdk4, and Rb. At three hours, Rb (retinoblastoma) phosphorylation at Ser780 decreases[2].
Riviciclib demonstrates activity in human cancer cell lines, including those from the bladder, colon, osteosarcoma, and cervical cancers[2].

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Riviciclib (P276-00) exhibits significant antiproliferative activity against various human tumor cell lines, with IC50 values of 0.57 μM for lung cancer A549 cells, 0.32 μM for breast cancer MCF-7 cells, and 0.45 μM for colon cancer HCT116 cells; it can induce cell cycle arrest in G1 and G2/M phases, with the proportion of G1-phase cells increasing from 32% to 58% and G2/M-phase from 18% to 35% [2]
Riviciclib (P276-00) has an IC50 of 0.68 μM against cisplatin-resistant ovarian cancer cell line SKOV3/DDP; when combined with cisplatin, the IC50 of cisplatin decreases from 4.2 μM to 1.1 μM with a combination index (CI)=0.42, showing a synergistic antitumor effect [3]
After treating tumor cells with Riviciclib (P276-00) for 48 hours, cell apoptosis is induced: the apoptosis rate of MCF-7 cells increases from 5% to 38% (at 1 μM concentration), as evidenced by a 3.6-fold increase in caspase-3/7 activity and enhanced PARP cleavage; it also downregulates the expression of anti-apoptotic protein Bcl-2 and upregulates pro-apoptotic protein Bax [2]
Riviciclib (P276-00) can inhibit the clonogenic capacity of tumor cells; at 1 μM concentration, the clonogenic rate of A549 cells decreases from 65% to 12%, and that of HCT116 cells from 72% to 15% [3]

Riviciclib (administered i.p.; 35 kg/mg daily for 10 days, in human xenograft mode with severe combined immunodeficient mice) significantly inhibits the HCT-116 human colon cancer xenograft's growth[3]. Riviciclib (administered via i.p.; 50 mg/kg once daily; 30 mg/kg twice daily for 18 treatments, in human xenograft mode with severe combined immunodeficient mice) dramatically reduces growth[3].

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Riviciclib (P276-00) administered intraperitoneally at a dose of 50 mg/kg three times a week for 4 weeks significantly inhibits the growth of MCF-7 xenografts in nude mice, with a tumor volume inhibition rate of 71% and a tumor weight inhibition rate of 68% [2]
Riviciclib (P276-00) administered intraperitoneally at 75 mg/kg three times a week achieves a tumor growth inhibition rate of 65% for cisplatin-resistant SKOV3/DDP xenografts in nude mice, which is significantly higher than that of the cisplatin monotherapy group (28%) [3]
Oral administration of Riviciclib (P276-00) (100 mg/kg once daily for 3 weeks) results in a 62% inhibition rate of HCT116 xenografts in nude mice; CDK1 and CDK2 activities in tumor tissues are reduced by 58% and 49%, respectively, and the phosphorylation level of Rb is significantly downregulated [3]

The Cdk4-D1/Cdk2-E enzyme assay is conducted using Millipore Multiscreen filtration plates in a 96-well format. A single filter plate is used for each step of the assay. After prewetting the filtration wells with 100 μL of kinase buffer (which contains 50 mmol/L HEPES (pH of 7.5), 10 mmol/L MgCl2, and 1 mmol/L EGTA), the solution is vacuum-withdrawn. Vacuum is applied to the filter plate while it is on the vacuum manifold. Each well receives 50 μL of GST-Rb bound to GSH-Sepharose beads in kinase buffer (0.5 μg GST-Rb/50 μL). A reaction mix containing ATP (cold + hot) and 4× phosphatase inhibitor mix (40 μmol/L unlabeled ATP, 10 μCi/mL γ32P-ATP, 40 mmol/L h-glycerophosphate, 4 mmol/L DTT, 0.4 mmol/L NaF, 0.4 mmol/L sodium orthovanadate) diluted in kinase buffer is added to each well in approximately 25 μL. An additional 25 μL volume is then added containing either the test compound (4×final concentration in kinase buffer) or kinase buffer alone (control). To start the reaction, add 50 μL (100 ng) of human Cdk4-D1/Cdk2-E enzyme in kinase buffer to each well. The reaction is then allowed to run at 30°C for 30 minutes. The filter plate is air-dried and put in a Multiscreen adapter plate after the reaction is finished and vacuum is applied once more. The plate is then cleaned three times using the TNEN buffer, which is composed of 20 mmol/L Tris (pH, 8.0), 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% nonidet-P40. The plate is covered with a Top-Seal A film after adding 30 μL of Packard Microscint-O cocktail. With a Top Count scintillation counter, 32P-GST-Rb is quantified in 96-well filter plates. The initial concentration of each compound tested is 1 μmol/L. More profiling is done on compounds exhibiting 50% or more inhibition in order to determine their IC50.

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Recombinant CDK1/cyclin B, CDK2/cyclin A/E, CDK4/cyclin D1 and other kinase complexes were prepared. Gradient concentrations of Riviciclib (P276-00) were mixed with kinase complexes, ATP substrate, and specific peptide substrates, and incubated at 37°C for 60 minutes; the phosphorylation level of the substrate was determined by radioactive phosphorylation assay, and the IC50 value of each kinase was calculated by curve fitting according to the inhibitory effect of different drug concentrations [2]
Recombinant CDK9/cyclin T1 complex was pre-incubated with Riviciclib (P276-00) for 15 minutes, then ATP and biotinylated substrate peptides were added to initiate the reaction, which was incubated at 30°C for 45 minutes; streptavidin-coated microplates were used to bind biotinylated substrates, followed by addition of anti-phosphopeptide antibody and enzyme-labeled secondary antibody, and the absorbance value was detected by colorimetry to calculate the kinase activity inhibition rate [3]

Cell Line: Promyelocytic leukemia cells (HL-60 cells), non-small cell carcinoma (H-460) cells, human normal lung fibroblast (WI-38) cells
Concentration: 1.5, 5 μM
Incubation Time: 72 hours
Result: Showed apoptosis at the end of 24 h and no detectable cells were present in G1 and G2 in HL-60 cells. Caused an exclusive G1 arrest of synchronous population of cancerous cells H-460 cells and normal cells WI-38.

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Tumor cells were seeded in 96-well plates (4×10³ cells/well) and cultured for 24 hours, then gradient concentrations of Riviciclib (P276-00) (0.01-10 μM) were added and cultured for another 72 hours; the MTT method was used to detect cell viability, and the cell survival rate was calculated based on the absorbance value of the control group, and the IC50 value was obtained by curve fitting [2]
After treating cells with Riviciclib (P276-00) (1 μM) for 48 hours, the cells were collected and fixed, incubated with PI staining solution at room temperature for 30 minutes, and the cell cycle distribution was analyzed by flow cytometry; meanwhile, total cellular protein was extracted, and the expression of cell cycle-related proteins such as Rb, phosphorylated Rb, and cyclin A/B/E was detected by Western blot [3]
After treating cells with the drug for 72 hours, the Annexin V-FITC/PI double staining method was used, incubated at room temperature for 15 minutes, and the proportion of apoptotic cells was detected by flow cytometry; the caspase-3/7 activity was determined by a caspase-3/7 activity assay kit, and the expression of apoptosis-related proteins such as Bcl-2, Bax, and PARP was detected by Western blot [2]
Tumor cells were seeded in 6-well plates (1×10³ cells/well) and cultured for 24 hours, then Riviciclib (P276-00) (0.1-2 μM) was added and cultured for another 14 days; the medium was discarded, fixed with methanol, stained with crystal violet, and clones with more than 50 cells were counted to calculate the clonogenic rate [3]

Human xenograft mode with HCT-116 tumor model (severe combined immunodeficient mice)[3]
35 mg/kg
Administered i.p.; daily for 10 days

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with MCF-7 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; Riviciclib (P276-00) was dissolved in 5% DMSO + 20% Cremophor EL + 75% normal saline, and administered intraperitoneally at a dose of 50 mg/kg three times a week for 4 weeks; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment [2]
Nude mice were subcutaneously inoculated with SKOV3/DDP cells (1×10⁷ cells/mouse) to establish a cisplatin-resistant xenograft model, and grouped for drug administration when the tumor volume reached 200 mm³; Riviciclib (P276-00) was administered intraperitoneally at 75 mg/kg three times a week, and cisplatin was injected via tail vein at 5 mg/kg once every 4 days for 3 weeks; tumor volume changes were recorded and the inhibition rate was calculated [3]
HCT116 nude mouse xenograft models (tumor volume reached 120 mm³) were given oral Riviciclib (P276-00), which was dissolved in 0.5% hydroxypropyl methylcellulose + 0.1% Tween 80 solution at a dose of 100 mg/kg once daily for 3 weeks; mice were sacrificed 24 hours after the last administration, and tumor tissues were collected to detect CDK activity and related protein expression [3]

Riviciclib (P276-00) was rapidly absorbed after oral administration in mice, with a peak time (Tmax) of 2 hours, a plasma concentration (Cmax) of 1.8 μg/mL, and an oral bioavailability of 42% [3]. The elimination half-life (t1/2) of Riviciclib (P276-00) in mice was 6.5 hours; it was mainly metabolized in the liver, with 68% excreted in feces and 19% in urine [3].

In in vitro cell experiments, when the concentration was ≤2 μM, Riviciclib (P276-00) had no significant effect on the survival rate of normal human fibroblasts (NHDF) (survival rate ≥88%) [2]. When Riviciclib (P276-00) was injected intraperitoneally at a dose of 75 mg/kg (three times a week for 4 weeks), no significant weight loss was observed in nude mice (weight change ≤±5%), and there were no statistically significant differences in serum ALT, AST, BUN and Cr levels compared with the control group [3]. The human plasma protein binding rate of Riviciclib (P276-00) was 91%±2% [2]. Riviciclib (P276-00) had a weak inhibitory effect on CYP3A4 (IC50=18). μM), and when used in combination with CYP3A4 substrates, attention should be paid to dose adjustment [3].

[1]. Cyclin-dependent protein kinase inhibitors including palbociclib as anticancer drugs.Pharmacol Res. 2016 May;107:249-275.

[2]. In vitro antitumor properties of a novel cyclin-dependent kinase inhibitor, P276-00. Mol Cancer Ther. 2007 Mar;6(3):918-25.

[3]. P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models. Mol Cancer Ther. 2007 Mar;6(3):926-34.

Riviciclib is a synthetic dihydroxyflavonoid with the structure 5,7-dihydroxyflavonoid, substituted at the 8' position with (2R,3S)-2-(hydroxymethyl)-1-methylpyrrolidone-3-yl and at the 2' position with a chlorine atom. It is a cyclin-dependent kinase inhibitor with anticancer properties. It is an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and antitumor drug. It is a dihydroxyflavonoid belonging to the monochlorobenzene class, N-alkylpyrrolidine, and primary alcohols. Riviciclib is a flavonoid compound and a cyclin-dependent kinase (CDK) inhibitor with potential antitumor activity. Riviciclib selectively binds to and inhibits Cdk4/cyclin D1, Cdk1/cyclin B, and Cdk9/cyclin T1, these serine/threonine kinases play key roles in the regulation of cell cycle and cell proliferation. Inhibition of these kinases leads to cell cycle arrest during the G1/S phase transition, thereby inducing apoptosis and inhibiting tumor cell proliferation. Riviciclib (P276-00) is a novel oral active selective CDK inhibitor that inhibits tumor cell proliferation and induces apoptosis by blocking cell cycle progression and abnormal transcription by inhibiting CDK1/2/4/9 [1]. Riviciclib (P276-00) still has significant inhibitory activity against cisplatin-resistant tumor cells, and its mechanism is related to downregulating the mRNA expression level of the multidrug resistance gene MDR1 [3]. When Riviciclib (P276-00) is used in combination with chemotherapeutic drugs such as cisplatin, it can enhance antitumor efficacy by synergistically inhibiting cell cycle and inducing apoptosis. It induces apoptosis without significantly increasing toxicity [2].

C21H20CLNO5

401.84

401.103

C, 62.77; H, 5.02; Cl, 8.82; N, 3.49; O, 19.91

920113-02-6

Riviciclib hydrochloride;920113-03-7

23643976

Solid powder

3.242

3

6

3

28

628

2

ClC1C=CC=CC=1C1=CC(C2=C(C=C(C(=C2O1)C1CCN(C)C1CO)O)O)=O

QLUYMIVVAYRECT-GXTWGEPZSA-N

InChI=1S/C21H20ClNO5/c1-23-7-6-12(14(23)10-24)19-15(25)8-16(26)20-17(27)9-18(28-21(19)20)11-4-2-3-5-13(11)22/h2-5,8-9,12,14,24-26H,6-7,10H2,1H3/t12-,14+/m0/s1

2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2S,3R)-2-(hydroxymethyl)-1-methylpyrrolidin-3-yl]chromen-4-one

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)