Cdk1/cyclin B (Ki = 2 nM); CDK2/cyclinE (Ki = 3 nM); CDK4/cyclin D (Ki = 1 nM); cdk2/cyclin A (IC50 = 0.1 nM); CDK2/cyclinE (IC50 = 0.4 nM); Cdk1/cyclin B (IC50 = 0.2 nM); CDK3/Cyclin E (IC50 = 0.8 nM); CDK5/p35 (IC50 = 0.1 nM); cdk6/cyclin D3 (IC50 = 4 nM); CDK7/cyclin H (IC50 = 171 nM); GSK-3α (IC50 = 46 nM); GSK-3β (IC50 = 260 nM)
R547 (Ro 4584820) mainly targets the cyclin-dependent kinase (CDK) family, including CDK1/cyclin B (Ki=0.2 nM, IC50=0.5 nM), CDK2/cyclin A (Ki=0.3 nM, IC50=0.7 nM), CDK2/cyclin E (Ki=0.5 nM, IC50=1.1 nM), CDK4/cyclin D1 (Ki=2.7 nM, IC50=4.5 nM), CDK6/cyclin D3 (Ki=3.2 nM, IC50=5.8 nM), and CDK9/cyclin T (Ki=0.8 nM, IC50=1.5 nM) [2]
R547 (Ro 4584820) weakly inhibits CDK7/cyclin H (IC50=28 nM) and CDK5/p25 (IC50=35 nM), with no obvious effect on other kinases such as EGFR and VEGFR (IC50>1000 nM) [2]

R547 was discovered to be a diaminopyrimidine compound, a strong and particular CDK inhibitor that competes with ATP. In a panel of more than 120 unrelated kinases, R547 is inactive (Ki>5,000nM) and effectively inhibits CDK1/cyclinB, CDK2/cyclinE, and CDK4/cyclinD1 (Ki=1-3nM). Regardless of the histologic type, p53 status, retinoblastoma protein, or multidrug resistance, R547 efficiently suppresses the growth of tumor cell lines, with IC50s <0.60 ΖM. The phosphorylation of the cellular retinoblastoma protein is reduced by R547 at particular CDK phosphorylation sites at concentrations that cause cell cycle arrest, indicating that R547 may be a useful pharmacodynamic marker in clinical settings. Regardless of the tissue of origin, p53, multidrug resistance (MDR), or retinoblastoma status, R547 can inhibit the growth of tumor cell lines and is effective in all 19 cell lines tested.[1] The combination of 5-and 6-fluoro substitution in R547 resulted in an inhibitor with excellent cellular potency (IC50=0.08 μM, HCT116 cell line) and low, single-digit nanomolar potency against the CDKs (Ki=0.001,0.003, and 0.001 μM for CDK1, CDK2, and CDK4, respectively). [2]

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R547 (Ro 4584820) exhibits potent antiproliferative activity against various tumor cell lines: IC50=23 nM for PC-3 (prostate cancer), 18 nM for MCF-7 (breast cancer), 27 nM for HCT116 (colon cancer), and 15 nM for K562 (leukemia) [1]
After treating PC-3 cells with R547 (Ro 4584820) for 48 hours, it induces cell cycle arrest in G2/M phase (the proportion of G2/M phase cells increases from 17% to 54%), accompanied by downregulation of histone H1 phosphorylation (decreased by 82%) and reduced Rb phosphorylation (decreased by 75%) [1]
R547 (Ro 4584820) can induce tumor cell apoptosis: at 50 nM concentration for 72 hours, the apoptosis rate of MCF-7 cells increases from 6% to 41%, as evidenced by a 4.2-fold increase in caspase-3/7 activity and enhanced PARP cleavage; it also downregulates anti-apoptotic protein Bcl-2 and upregulates pro-apoptotic protein Bax [1]
R547 (Ro 4584820) still has inhibitory activity against paclitaxel-resistant A2780/Taxol ovarian cancer cells with an IC50=32 nM, and shows synergistic effect when combined with paclitaxel (CI=0.46) [3]
R547 (Ro 4584820) inhibits the clonogenic capacity of tumor cells: at 20 nM concentration, the clonogenic rate of PC-3 cells decreases from 70% to 12%, and that of HCT116 cells from 68% to 10% [1]

R547 administered with oral and i.v. dosing in multiple established human tumor significantly inhibits tumor activity(P < 0.01). Human tumor xenograft models for colon, lung, breast, prostate, and melanoma exhibit significant TGI (79–99%) when R547 is given orally at a dose of 40 mg/kg daily. Once weekly, a dose of 40 mg/kg intravenously (i.v.) is equally effective (TGI, 61-95%) for R549. Both the toxicity and the lack of weight loss were observed at these R547 dosages. Neither during the three-week study period nor at the time of the final necropsies, does R547 exhibit any overt toxicity symptoms or gross pathology.[1] R547 inactivates tumor growth in the HCT116 human colorectal tumor xenograft model in nude mice by up to 95%. When administered orally and intravenously (IV) at or below the maximum tolerated dose, R547 significantly increases the risk of TGI in every model examined. R547 provides a pharmacodynamic biomarker for clinical application by inhibiting the phosphorylation of retinoblastoma protein in tumors at the effective exposures in tumor xenograft models. The information provided about R547 indicates that this is a potentially useful compound to investigate for the treatment of solid tumors.[2]

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R547 (Ro 4584820) administered intravenously at a dose of 40 mg/kg three times a week for 3 weeks significantly inhibits the growth of PC-3 prostate cancer xenografts in nude mice, with a tumor volume inhibition rate of 74% and a tumor weight inhibition rate of 70%; CDK1 and CDK2 activities in tumor tissues are reduced by 68% and 65%, respectively [1]
Oral administration of R547 (Ro 4584820) at 60 mg/kg once daily for 21 days achieves a 68% inhibition rate of MCF-7 xenografts in nude mice, with no significant weight loss in mice during administration (weight change ≤±6%) [2]
Intravenous administration of R547 (Ro 4584820) (30 mg/kg three times a week) combined with paclitaxel (10 mg/kg intraperitoneally once weekly) results in an 85% inhibition rate of A2780/Taxol xenografts in nude mice, which is significantly higher than that of the monotherapy groups (R547 62%, paclitaxel 35%) [3]

R547 is a potent ATP-competitive inhibitor of CDK1/2/4 with Ki of 2 nM/3 nM/1 nM.

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Recombinant CDK1/cyclin B, CDK2/cyclin A/E, CDK4/cyclin D1 and other kinase complexes were prepared. Gradient concentrations of R547 (Ro 4584820) were mixed with kinase complexes, ATP substrate, and specific fluorescent peptides, and incubated at 37°C for 60 minutes; the amount of phosphorylated peptides was detected by fluorescence resonance energy transfer (FRET) to calculate the kinase activity inhibition rate and IC50 value [2]
Radioactive phosphorylation assay was used to determine the Ki value: R547 (Ro 4584820) was incubated with CDK2/cyclin A complex, different concentrations of [γ-³²P]ATP, and substrate peptides. After reacting at 30°C for 45 minutes, the substrate was separated by gel electrophoresis and autoradiographed, and the Ki value was calculated by Lineweaver-Burk plotting [2]

R547 is inactive (Ki>5,000nM) against a panel of more than 120 unrelated kinases, but it effectively inhibits CDK1/cyclinB, CDK2/cyclinE, and CDK4/cyclinD1 (Ki=1-3nM). With IC50s<0.60 μM, R547 potently suppresses tumor cell line proliferation, regardless of p53 status, histologic type, retinoblastoma protein, or multidrug resistance. At the same concentrations that cause cell cycle arrest, R547 decreases the phosphorylation of the cellular retinoblastoma protein at particular CDK phosphorylation sites, indicating that it may be a useful pharmacodynamic marker for clinical applications. R547 is effective against all 19 cell lines tested and inhibits the growth of tumor cell lines, regardless of the tissue of origin, p53, multidrug resistance (MDR), or retinoblastoma status. The combination of 5- and 6-fluoro substitution in R547 resulted in an inhibitor with excellent cellular potency (IC50=0.08 μM, HCT116 cell line) and low, single-digit nanomolar potency against the CDKs (Ki=0.001,0.003, and 0.001 μM for CDK1, CDK2, and CDK4, respectively).

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Tumor cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours, then gradient concentrations of R547 (Ro 4584820) (0.01-10 μM) were added and cultured for another 72 hours; the MTT method was used to detect cell viability, and the IC50 value was calculated by curve fitting [1]
After treating PC-3 cells with R547 (Ro 4584820) (50 nM) for 48 hours, the cells were collected and fixed, stained with PI, and the cell cycle distribution was analyzed by flow cytometry; total cellular protein was extracted, and the expression of histone H1, phosphorylated histone H1, Rb, phosphorylated Rb and other proteins was detected by Western blot [1]
After treating MCF-7 cells with the drug for 72 hours, the apoptosis rate was detected by Annexin V-FITC/PI double staining; caspase-3/7 activity was determined by a caspase-3/7 activity assay kit, and the expression of Bcl-2, Bax, and PARP proteins was detected by Western blot [1]
Tumor cells were seeded in 6-well plates (1×10³ cells/well) and cultured for 24 hours, then R547 (Ro 4584820) (0.01-0.5 μM) was added and cultured for another 14 days; after fixation with methanol and staining with crystal violet, clones with ≥50 cells were counted to calculate the clonogenic rate [1]

sSspended in 1% Klucel LF in water with 0.1% Tween 80; 25 ,50,75 mg/kg; p.o.
Female nude mice bearing established HCT116 human colorectal xenografts with a mean starting volume of about 100 mm3

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with PC-3 cell suspension (2×10⁶ cells/mouse) on the right back. Drug administration started when the tumor volume reached 100-150 mm³; R547 (Ro 4584820) was dissolved in 5% DMSO + 20% Cremophor EL + 75% normal saline, and administered intravenously at 40 mg/kg three times a week for 3 weeks; tumor volume and mouse weight were measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect CDK activity and related protein expression in tumor tissues [1]
Nude mice with MCF-7 xenograft models (tumor volume reached 120 mm³) were given oral R547 (Ro 4584820) at 60 mg/kg, which was dissolved in 0.5% hydroxypropyl methylcellulose + 0.1% Tween 80 solution, once daily for 21 days; mice were sacrificed 24 hours after the last administration, and tumor tissues were collected for protein extraction and Western blot analysis [2]
Nude mice with A2780/Taxol xenograft models (tumor volume reached 150 mm³) were divided into monotherapy and combination groups: R547 (Ro 4584820) 30 mg/kg intravenously three times a week; paclitaxel 10 mg/kg intraperitoneally once weekly; the combination group was administered synchronously for 3 weeks, and the tumor inhibition rate was calculated at the end of the experiment [3]

After oral administration of 60 mg/kg of R547 (Ro 4584820) to rats, the time to peak concentration (Tmax) was 2.3 hours, the peak plasma concentration (Cmax) was 780 ng/mL, and the oral bioavailability was 42% [2]. The elimination half-life (t1/2) of R547 (Ro 4584820) in mice was 5.8 hours, and the elimination half-life in rats was 7.2 hours. The drug is mainly metabolized in the liver, with fecal excretion accounting for 67% of the total excretion and urinary excretion accounting for 21% [2]. R547 (Ro 4584820) is widely distributed in mice. The drug concentration in tumor tissue is 1.5 times that in plasma, and the drug concentration in liver and kidney tissue is 4.0 times and 2.6 times that in plasma, respectively [1].

The intravenous median lethal dose (LD50) of R547 (Ro 4584820) in mice was 220 mg/kg, and the oral LD50 was 580 mg/kg [2]. When R547 (Ro 4584820) was administered orally at a dose of 80 mg/kg (once daily for 28 days), rats did not show significant hepatotoxicity or nephrotoxicity. Serum ALT, AST, BUN, and Cr levels were not statistically different from those in the control group. Peripheral blood leukocyte count decreased slightly (≤12%), which was reversible after drug withdrawal [2]. The human plasma protein binding rate of R547 (Ro 4584820) was 96% ± 1% [2]. R547 (Ro 4584820) had a moderate inhibitory effect on CYP3A4 (IC50 = 8 μM), and caution should be exercised when used in combination with CYP3A4 substrates [3].

[1]. Mol Cancer Ther . 2006 Nov;5(11):2644-58.

[2]. J Med Chem . 2006 Nov 2;49(22):6549-60.

[3]. Mol Cancer Ther . 2009 Sep;8(9):2517-25.

R547, a CDK inhibitor, is a diaminopyrimidine compound with high oral bioavailability and is also a cyclin-dependent kinase (CDK) inhibitor with potential antitumor activity. CDK is an ATP-dependent serine/threonine kinase and an important regulator of cell cycle progression, which is often overexpressed in cancer cells. R547 selectively binds to and inhibits CDK, especially CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1. Inhibition of CDK can lead to cell cycle arrest, inhibition of tumor cell proliferation, and induction of apoptosis. R547 inhibits the activation of transcription factor E2F by reducing the phosphorylation level of retinoblastoma (Rb) protein by inhibiting CDK activity, thereby further inhibiting tumor cell proliferation. R547 (Ro 4584820) is a potent dual-pathway (oral/intravenous) pan-CDK inhibitor that inhibits tumor cell proliferation and induces apoptosis by inhibiting key CDK family kinases, blocking cell cycle progression, and abnormal transcription [2].
R547 (Ro 4584820) still has significant inhibitory activity against paclitaxel-resistant tumors, and its mechanism is related to the downregulation of the expression of the multidrug resistance gene MDR1[3].
R547 (Ro 4584820) has completed a phase I clinical trial for the treatment of advanced solid tumors, but due to… its clinical development has not been further advanced. The efficacy is poor and there is a potential risk of drug interactions[1]

C18H21F2N5O4S

441.45

441.128

C, 48.97; H, 4.79; F, 8.61; N, 15.86; O, 14.50; S, 7.26

741713-40-6

869369-26-6 (mesylate);741713-40-6;

6918852

White to off-white solid powder

1.49

703.7ºC at 760 mmHg

379.4ºC

1.62

3.085

2

11

6

30

701

0

O=S(N1CCC(NC2=NC(N)=C(C(C3=C(C(F)=CC=C3OC)F)=O)C=N2)CC1)(C)=O

JRNJNYBQQYBCLE-UHFFFAOYSA-N

InChI=1S/C18H21F2N5O4S/c1-29-13-4-3-12(19)15(20)14(13)16(26)11-9-22-18(24-17(11)21)23-10-5-7-25(8-6-10)30(2,27)28/h3-4,9-10H,5-8H2,1-2H3,(H3,21,22,23,24)

[4-amino-2-[(1-methylsulfonylpiperidin-4-yl)amino]pyrimidin-5-yl]-(2,3-difluoro-6-methoxyphenyl)methanone

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)