CDK9- Cyclin T1 (IC50 = 0.020 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 = 2.500 μM); cdk6-cyclin D3 (IC50 = 0.396 μM); CDK9-cyclin H (IC50 = 2.900 μM)
Cyclin-dependent kinases (Cdks)
Potent inhibitor of Cdk4-D1 and Cdk1-B. The study does not provide specific IC50 values for these kinases but references an earlier publication. [2]

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Riviciclib HCl (P276-00) demonstrated potent antiproliferative activity against a panel of 16 human tumor cell lines in a propidium iodide (PI) assay. The mean IC₇₀ value was 0.50 µg/mL (1.1 µmol/L), making it approximately 30-fold more potent than cisplatin (mean IC₇₀ = 11.9 µg/mL, 40 µmol/L). The most sensitive cell lines included colon (DLD1, HCT-116, CXF 94L), lung (LXFA 629L), breast (MCF-7, MAXF 401NL), and melanoma (MEXF 462NL). It was also active against cisplatin-resistant colon (DLD1, CXF 94L) and breast (MCF-7) cancer cell lines.
In a clonogenic assay using 22 human tumor xenograft-derived cell lines, Riviciclib HCl (P276-00) significantly inhibited colony formation in a dose-dependent manner. At a concentration of 1 µmol/L, it was active in 41% (9/22) of the models; at 10 µmol/L, it was active in 91% (20/22). In comparison, cisplatin showed no activity at 1 µmol/L and activity in only 9% (2/22) of models at 10 µmol/L. P276-00 was particularly active against tumors of the CNS (CNXF SF268), NSCLC (LXFE 397), breast (MAXF 401), melanoma (MEXF 276, MEXF 462), prostate (PRXF 22RV1), renal (RXF 393, RXF 944LX), and sarcoma (SXF 1301).
In cell cycle analysis using asynchronous populations, Riviciclib HCl (P276-00) (1.5 µmol/L) induced a G2-M arrest in slow-growing PC-3 prostate cancer cells, with minimal apoptosis up to 72 hours. In contrast, in fast-growing HL-60 leukemia cells, it induced significant apoptosis from 6 hours onwards, with >75% apoptosis at 24 hours.
In synchronized H-460 NSCLC cells released from G0 arrest, Riviciclib HCl (P276-00) (1.5 µmol/L) caused a G1 arrest, with increasing apoptosis over time (27.4% at 48h, 34.8% at 72h). In synchronized WI-38 normal lung fibroblasts, the compound also caused a G1 arrest, but apoptosis remained negligible (3-4% at 72h).
In recovery experiments, after 48h treatment with 1.5 µmol/L P276-00 followed by drug-free recovery, H-460 cancer cells showed significant apoptosis (27.7% at 0h recovery), while WI-38 normal cells did not (5.5% at 0h recovery), confirming selectivity for cancer cells. [2]

P276-00 exhibits selectivity toward Cdk4-D1 that is 40 times greater than that of Cdk2-E[1]. It is found to be highly selective for cancer cells as compared with normal fibroblast cells[1]. It exhibits potent antiproliferative effects against a variety of human cancer cell lines, including HCT-116, U2OS, H-460, HL-60, HT-29, SiHa, MCF-7, Colo-205, SW-480, PC-3, Caco2, and T-24. The IC50 ranges from 300 to 800 nmol/L. In an ATP-competitive manner, P276-00 can down-regulate Cdk4 and cyclin D1, as well as lower phosphorylation of pRb Ser780 that is specific to Cdk4. Through the activation of cellular caspase-3 activity and the creation of DNA ladders, P276-00 also causes apoptosis[1].

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Murine Colon Carcinoma (CA-51) Model: BALB/c mice bearing CA-51 tumors were treated with Riviciclib HCl (P276-00) at 50 mg/kg i.p. daily for 20 days. This resulted in a significant 81% tumor growth inhibition on day 33, with a T/C ratio of 19%.
Murine Lewis Lung Carcinoma Model: Mice with established Lewis lung tumors were treated with two regimens: 35 mg/kg i.p. daily for 14 days, or 30 mg/kg i.p. twice daily (60 mg/kg/day) every other day for 7 treatments. The twice-daily regimen was more efficacious, achieving 70% growth inhibition on day 17 compared to 43% with the daily regimen.
Human Colon Carcinoma (HCT-116) Xenograft Model: SCID mice bearing HCT-116 xenografts were treated with Riviciclib HCl (P276-00) at 35 mg/kg i.p. daily for 10 days. This resulted in significant (P < 0.05) tumor growth inhibition of >70% on day 21, with average tumor weights of 517.2 ± 70.3 mg in treated vs. 1,349 ± 130.9 mg in control.
Human Non-Small Cell Lung Carcinoma (H-460) Xenograft Model: SCID mice bearing H-460 xenografts were treated with Riviciclib HCl (P276-00) at 50 mg/kg i.p. once daily for 20 days, or 30 mg/kg i.p. twice daily for 20 days. Both regimens were similarly efficacious, achieving 75.7% and 68.4% tumor growth inhibition, respectively, on day 40. [2]

P276-00 can significantly inhibit the growth of murine colon cancer (CA-51) when given intraperitoneally (i.p.) at a dose of 50 mg/kg per day for 20 treatments. However, an increased dose of 60 mg/kg (30 mg/kg twice daily) given every other day intraperitoneally for seven treatments, in the murine lung carcinoma model (Lewis lung), exhibits a notable inhibition in the growth[2]. Additionally, it inhibits the growth of human non-small cell lung carcinoma H-460 and human colon carcinoma HCT-116 xenografts[2]. Its maximum tolerated dose, according to efficacy studies, is 78 mg/kg/d[2].

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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Cdk4-D1 and Cdk2-E Assays: Human Cdk4 or Cdk2 was co-expressed with cyclin D1 or E in a baculovirus system and purified. GST-retinoblastoma fusion protein (amino acids 776-928) was used as substrate. The assay was performed in 96-well filter plates. GST-Rb bound to GSH-Sepharose beads was added to wells, followed by a reaction mix containing [γ-³²P]ATP and test compound. The reaction was initiated by adding the enzyme, incubated for 30 minutes at 30°C, and then filtered and washed. Incorporated radioactivity was quantified by scintillation counting. Flavopiridol was used as a standard.
Kinase Selectivity Profiling: In vitro kinase assays for a panel of other Cdks and non-Cdk kinases (c-Src, GSK3β, LCK, MAPK1, MAPK2, PKCα, PKA, total PKC) were performed in the presence of increasing concentrations of P276-00 and appropriate substrates using a commercial kinase profiler service. [1]

In a 96-well plate, the cells are seeded at a density of 3,000–5,000 per well, depending on the type of cell, in 180 μL of culture medium. The cells are then allowed to adhere over night in the incubator. Compounds in varying concentrations are added to the wells, and they are then incubated at 37°C for 48 hours. Each well receives an addition of 3H-thymidine (0.25 μCi), and the radiolabel is allowed to be incorporated for a duration of 5 to 7 hours. After the incubation period, cells are harvested onto GF/B unifilter plates using a Packard Filtermate Universal harvester, and the 96-well liquid scintillation counter Packard Top Count is used to count the plates.

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Propidium Iodide (PI) Assay for Cell Proliferation: Cells were seeded in 96-well plates and allowed to recover for 24h. They were then exposed to five concentrations (0.003-30 µg/mL) of Riviciclib HCl (P276-00) or cisplatin for 4 days. After treatment, the medium was replaced with a PI solution (7 µg/mL). Plates were frozen to permeabilize cells, and fluorescence was measured. Growth inhibition was expressed as T/C%. IC₅₀ and IC₇₀ values were determined by plotting concentration vs. cell viability.
Clonogenic Assay: Solid human tumor xenografts were mechanically disaggregated and incubated with an enzyme cocktail. The resulting cell suspension was used in a clonogenic assay. Riviciclib HCl (P276-00) and cisplatin were tested at concentrations from 0.001 to 100 µmol/L with continuous exposure. Antitumor activity was defined as inhibition of colony formation by >70% compared to untreated control.
Cell Cycle Analysis by Flow Cytometry: Cells (H-460, PC-3, HL-60, WI-38) were seeded and treated with Riviciclib HCl (P276-00) (1.5 or 5 µmol/L) for various times (0-72h). For recovery studies, cells were treated for 48h and then allowed to grow in drug-free medium for 6-48h. Both detached and adherent cells were harvested, fixed in 70% ethanol, and stained with PI and RNase A. Cell cycle distribution was analyzed by flow cytometry. [2]

H-460 xenograft
50 mg/kg once daily or 30 mg/kg twice daily
i.p.

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Murine Tumor Models: For the CA-51 colon model, BALB/c mice were injected s.c. with tumor cells. 24h later, they were randomized and treated i.p. with Riviciclib HCl (P276-00) at 50 mg/kg in water daily for 20 days. For the Lewis lung model, when tumors reached ~5 mm, mice were randomized and treated i.p. with 35 mg/kg daily for 14 days or 30 mg/kg twice daily every other day for 7 treatments. Tumor diameters were measured periodically, and tumor weight was calculated. Growth inhibition and T/C% were determined.
Human Tumor Xenograft Models: SCID mice were injected s.c. with HCT-116 or H-460 cells. When tumors reached ~5 mm, mice were randomized. For HCT-116, they received Riviciclib HCl (P276-00) at 35 mg/kg i.p. daily for 10 days. For H-460, they received 50 mg/kg i.p. once daily for 20 days or 30 mg/kg i.p. twice daily for 20 days. Tumor growth was monitored, and tumor weight and growth inhibition were calculated as in murine models. Animal weight and signs of health deterioration were monitored. [2]

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Murine Tumor Models: For the CA-51 colon model, BALB/c mice were injected s.c. with tumor cells. 24h later, they were randomized and treated i.p. with Riviciclib HCl (P276-00) at 50 mg/kg in water daily for 20 days. For the Lewis lung model, when tumors reached ~5 mm, mice were randomized and treated i.p. with 35 mg/kg daily for 14 days or 30 mg/kg twice daily every other day for 7 treatments. Tumor diameters were measured periodically, and tumor weight was calculated. Growth inhibition and T/C% were determined.
Human Tumor Xenograft Models: SCID mice were injected s.c. with HCT-116 or H-460 cells. When tumors reached ~5 mm, mice were randomized. For HCT-116, they received Riviciclib HCl (P276-00) at 35 mg/kg i.p. daily for 10 days. For H-460, they received 50 mg/kg i.p. once daily for 20 days or 30 mg/kg i.p. twice daily for 20 days. Tumor growth was monitored, and tumor weight and growth inhibition were calculated as in murine models. Animal weight and signs of health deterioration were monitored. [2]

The study provides some information on tolerability from in vivo experiments.
Maximum Tolerated Dose (MTD): Dose-finding studies identified a dose of 78 mg/kg/day administered i.p. once daily for 5 days as the maximum tolerated dose in mice.
In Vivo Toxicity Observations: In murine tumor models, no significant reduction in body weight loss was seen. In human xenograft models, after chronic treatment, animals had an average weight loss of 10-15%. No other specific toxicities (e.g., hepatic, renal) were reported. The study also notes that Riviciclib HCl (P276-00) was less effective against normal cells (WI-38 fibroblasts) in vitro, causing G1 arrest without significant apoptosis, suggesting a degree of selectivity for cancer cells. [2]

[1]. Mol Cancer Ther . 2007 Mar;6(3):918-25.

[2]. Mol Cancer Ther . 2007 Mar;6(3):926-34.

Riviciclib hydrochloride is the hydrochloride form of Riviciclib, 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 crucial roles in the regulation of cell cycle and cell proliferation. Inhibition of these kinases leads to cell cycle arrest in the G1/S phase, thereby inducing apoptosis and inhibiting tumor cell proliferation.

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Riviciclib HCl (P276-00) is a flavone that inhibits cyclin-dependent kinases, identified as a novel antineoplastic agent. Its mechanism of action involves disrupting cell cycle progression by causing G1 or G2-M arrest, which leads to apoptosis, particularly in fast-proliferating cancer cells. It shows significant antitumor activity against a broad range of human tumor cell lines and xenografts, including those resistant to cisplatin. The compound also demonstrates selectivity for cancer cells over normal cells in vitro. Based on its potent in vitro cellular activity and promising in vivo efficacy in murine and human xenograft models, Riviciclib HCl (P276-00) is considered a candidate for further preclinical and clinical development. [2]

C21H21CL2NO5

438.301

437.08

C, 57.55; H, 4.83; Cl, 16.18; N, 3.20; O, 18.25

920113-03-7

Riviciclib;920113-02-6

23643975

Off-white to light yellow solid powder

4.044

4

6

3

29

628

2

OC1C=C(O)C2C(C=C(C3C=CC=CC=3Cl)OC=2C=1[C@@H]1CCN(C)[C@H]1CO)=O.Cl

OOVTUOCTLAERQD-OJMBIDBESA-N

InChI=1S/C21H20ClNO5.ClH/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;1H/t12-,14+;/m1./s1

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

P-27600; P 27600; P27600; P276-00; P-276-00; P 276-00; Riviciclib HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50~88 mg/mL (114.1~200.8 mM)
Ethanol: ~7 mg/mL (~16 mM)
Water: ~88 mg/mL (~200.8 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.75 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.75 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), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.75 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30 mg/mL

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