Cdk1/cyclin B (IC50 = 7 nM); CDK2/cyclinE (IC50 = 9 nM); CDK4/cyclin D (IC50 = 11 nM); CDK9/cyclinT1 (IC50 = 5 nM); CDK7/Cyclin H/MAT1 (IC50 = 25 nM)
Roniciclib (BAY-1000394; BAY1000394) inhibits the kinase activity of the cell-cycle CDKs CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclinDwith IC50 values of 7, 9, and 11 nM, respectively. The range of inhibition for the transcriptional CDKs CDK9/cyclin T1 and CDK7/cyclin H/MAT1 is comparable (5 and 25 nM)[1]. With a very balanced profile (mean IC50 on human tumor cells: 16 nM), riciclib potently inhibits the proliferation of several human and murine tumor cell lines[2].
Roniciclib (BAY 1000394) is a potent pan-cyclin-dependent kinase (CDK) inhibitor. It inhibits the activity of cell-cycle CDKs CDK1/cyclin B (IC50 = 7 nmol/L), CDK2/cyclin E (IC50 = 9 nmol/L), CDK4/cyclin D1 (IC50 = 11 nmol/L), and transcriptional CDKs CDK9/cyclin T1 (IC50 = 5 nmol/L) and CDK7/cyclin H/MAT1 (IC50 = 25 nmol/L). It shows no off-target inhibition of carbonic anhydrase II (CA-II, IC50 > 10,000 nmol/L) [1, 2].

Roniciclib (BAY-1000394; BAY1000394) inhibits the kinase activity of the cell-cycle CDKs CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclinDwith IC50 values of 7, 9, and 11 nM, respectively. The range of inhibition for the transcriptional CDKs CDK9/cyclin T1 and CDK7/cyclin H/MAT1 is comparable (5 and 25 nM)[1]. With a very balanced profile (mean IC50 on human tumor cells: 16 nM), riciclib potently inhibits the proliferation of several human and murine tumor cell lines[2].

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Roniciclib inhibits proliferation of a broad spectrum of human cancer cell lines with IC50 values between 6 and 84 nmol/L (mean 16 nmol/L on a panel of 25 human cancer cell lines). On a panel of 40 human lung tumor cell lines, IC50 values range from 9 to 79 nmol/L (mean 39 nmol/L). On a panel of 24 human breast tumor and immortalized cell lines, IC50 values range from 6 to 84 nmol/L (mean 37 nmol/L). No cell line with IC50 >100 nmol/L was identified [1].
In HeLa cells, Roniciclib induces concentration-dependent increases in caspase-3/7 activity and DNA fragmentation, indicating apoptotic cell death. In wash-out experiments, IC50 values decrease from >1000 nmol/L to 14 nmol/L with increasing exposure time, with approximately 16 hours of exposure required for maximal effect [1].
Roniciclib arrests cells at multiple CDK-dependent steps within the cell cycle. In asynchronously growing HeLa cells, treatment reduces G0-G1 phase fraction from 69% to 52% and increases sub-2N DNA content from 1% to 16% (apoptotic cells). In double-thymidine block release experiments, the compound arrests cells at G1-S boundary and at various other CDK-dependent transitions [1].
Roniciclib inhibits phosphorylation of CDK downstream targets: it completely suppresses retinoblastoma protein phosphorylation at Ser780 in MCF7 cells at 30 nmol/L (indicating CDK4/2 inhibition); reduces T199-phosphorylated nucleophosmin in nocodazole-arrested HeLa cells at 30 nmol/L and abolishes it at 100 nmol/L (indicating CDK1 inhibition); and inhibits Ser2 phosphorylation of RNA polymerase II and reduces MCL-1 protein levels in A549 cells at 100 nmol/L (indicating CDK9 inhibition) [1].

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BAY 1000394 exhibited broad antiproliferative activity against a panel of 25 human cancer cell lines, with IC₅₀ values ranging from 8 to 33 nM (mean 16 nM). Similar potency was observed across panels of 40 human lung tumor cell lines (mean IC₅₀ 39 nM) and 24 human breast tumor cell lines (mean IC₅₀ 37 nM), indicating uniform sensitivity regardless of genetic background (e.g., p53, pRB, K-Ras status). [1]
In HeLa cells, BAY 1000394 induced apoptotic cell death in a concentration-dependent manner, as evidenced by increased caspase-3/7 activity and DNA fragmentation after 17 hours of treatment. [1]
Cell cycle analysis in HeLa cells showed that BAY 1000394 (100 nM, 24h) arrested cells at various CDK-dependent steps, reducing the G0-G1 population and increasing the sub-2N (apoptotic) population. In synchronized cells released from a double-thymidine block, the compound arrested the majority of cells at the G1-S boundary. [1]
Mechanistically, BAY 1000394 (30-100 nM) suppressed phosphorylation of retinoblastoma protein (pRb, a CDK2/4 substrate), reduced T199-phosphorylated nucleophosmin (a CDK1 substrate), and inhibited Ser-2 phosphorylation of RNA polymerase II (a CDK9 substrate) in various cancer cell lines. [1]

The T/C values at the lower dose are 0.19, indicating strong dose-dependent inhibition of tumor growth, and at the higher dose, they are 0.02 (tumor regression). Roniciclib also significantly suppresses the growth of HeLa-MaTu tumors that have grown to a size of about 50 mm2 prior to the initiation of treatment (day 8 following inoculation). Tumor growth is slowed to T/C values of 0.15 and 0.62 (respectively) by treatment with Roniciclib at 1.5 and 1 mg/kg. T/C values of 0.01 (1.0 mg/kg Roniciclib) and -0.02 (1.5 mg/kg Roniciclib) indicate a significant inhibition of tumor growth when Roniciclib is added to cisplatin[1]. Low blood clearance rates of riciclib have been reported in mice, rats, and dogs (0.51, 0.78, and 0.50 Lh-1kg-1, respectively) [2].

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In athymic mice bearing HeLa-MaTu human cervical tumor xenografts, oral administration of Roniciclib once daily for 21 days produces dose-dependent tumor growth inhibition with T/C values of 0.61 at 0.5 mg/kg, 0.03 at 2 mg/kg, and signs of tumor regression at the 2 mg/kg dose. Treatment is well tolerated with no body weight loss below initial body weight [1].
On an intermittent dosing schedule (2 days on/5 days off for 3 cycles), Roniciclib at 1.5, 2.0, and 2.5 mg/kg twice daily shows dose-dependent efficacy with T/C values of 0.19, 0.02 (tumor regression), and similar results in larger tumors (~50 mm²) [1].
In the triple-negative MX-1 human breast cancer model, cisplatin-resistant A2780-cis ovarian cancer model, and multidrug-resistant OVCAR-8-ADR ovarian cancer model, Roniciclib is more efficacious than doxorubicin, cisplatin, or paclitaxel [1].
In cell line-derived and patient-derived SCLC xenograft models (NCI-H82, etc.), Roniciclib strongly inhibits tumor growth with T/C values between 0.12 and 0.19, comparable or superior to cisplatin [1].
In NCI-H82 SCLC xenografts, combination of Roniciclib (1.0-1.5 mg/kg) with cisplatin (6 mg/kg) or etoposide (12 mg/kg) produces more than additive efficacy (observed T/C values below Bliss-calculated additive values). Triple combination with cisplatin, etoposide, and Roniciclib (0.75 mg/kg) yields T/C of 0.09, significantly below expected additive T/C of 0.40, without worsening body weight loss compared to cisplatin/etoposide alone [1].
Pharmacodynamic analysis in HeLa-MaTu tumors shows that a single oral dose of Roniciclib (2 mg/kg) suppresses pRb phosphorylation for up to 7 hours, with recovery by 48 hours. Unbound drug concentrations exceed the in vitro IC50 (11 nmol/L) for approximately 24 hours [1].

For CDK1 and CDK2 kinase assays, recombinant human CDK1/cyclin B or CDK2/cyclin E GST-fusion proteins (purified from baculovirus-infected Sf9 insect cells) are incubated with test compounds for 10 minutes at 22°C in assay buffer containing Tris-HCl, MgCl2, dithiothreitol, Na3VO4, PEG, ATP, [γ-33P]ATP, and calf thymus histone type IIIS as substrate. The reaction is stopped with EDTA solution. Reaction mixtures are transferred to P30 filter sheets, washed with phosphorous acid, and subjected to scintillation counting. IC50 values are determined as the inhibitor concentration resulting in 50% substrate phosphorylation [2].
For VEGF-R2 kinase assay, GST-tagged recombinant human VEGF-R2 kinase domain purified from Sf9 cells is incubated with test compounds for 10 minutes at 22°C in Tris-HCl buffer containing MgCl2, MnCl2, dithiothreitol, PEG, ATP, [γ-33P]ATP, and poly(Glu,Tyr) substrate. Reaction processing and IC50 determination are as described above [2].
Carbonic anhydrase II inhibition is determined using the 4-nitrophenyl acetate hydrolysis technique in a 96-well spectrophotometer [2].

For proliferation assays, cells are seeded in 96-well plates at 1,000-5,000 cells/well in medium containing 10% FCS. After 24 hours, serial dilutions of Roniciclib are added and cells are incubated for 96 hours. Relative cell numbers are quantified by crystal violet staining. IC50 values are calculated using 4-parameter fit [1].
For apoptosis assays, caspase-3/7 activity is measured using ApoONE Caspase-3/7 Assay, and DNA fragmentation is measured using Cell Death Detection ELISA Plus. Results are normalized to vehicle controls and cell number [1].
For cell cycle analysis, HeLa cells are grown asynchronously or released from double-thymidine block, treated with vehicle or Roniciclib (100 nmol/L), harvested at indicated times, stained with propidium iodide, and analyzed by flow cytometry [1].
For downstream target analysis, MCF7 cells are stimulated to enter cell cycle from quiescence and treated with Roniciclib; pRb phosphorylation is analyzed by immunoblotting. Nocodazole-arrested HeLa cells are treated with Roniciclib and analyzed for phospho-nucleophosmin. A549 cells are treated with Roniciclib and analyzed for phospho-RNA polymerase II and MCL-1 levels by immunoblotting [1].

Mice: Athymic mice with established HeLa-MaTu xenograft tumors measuring approximately 25 mm² are given oral doses of Roniciclib (BAY 1000394) once a day for 21 days. There is no weight loss below the starting body weight, indicating that the treatment is well tolerated. A cyclic intermittent dosing schedule is used to treat additional mouse groups. Doses of 1.5, 2.0, and 2.5 mg/kg are administered twice daily for two days, after which the mice receive no treatment for five days (2 on/5 off). Three treatment cycles have been finished in total[1].

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For efficacy studies, female athymic nu/nu mice (50 days old, 20-22 g) are subcutaneously implanted with 1.5×10⁶ tumor cells (HeLa-MaTu, MX-1, A2780-cis, OVCAR-8-ADR, NCI-H82, or patient-derived SCLC models) suspended in 50% Matrigel/50% culture medium. When tumors reach approximately 21-25 mm², mice are randomized into treatment and control groups (8 mice/group). Roniciclib is dissolved in vehicle (40% polyethylene glycol, 60% water) and administered orally (p.o.) at indicated doses and schedules (once daily or intermittent 2 days on/5 days off). Tumor area (length × perpendicular) and body weight are measured twice weekly. Statistical analysis uses one-way ANOVA with pairwise comparison versus control [1].
For pharmacodynamic studies, mice bearing HeLa-MaTu tumors receive a single oral dose of Roniciclib (2 mg/kg). Tumors are harvested at 1, 7, 24, and 48 hours post-dose, homogenized, and analyzed for pRb phosphorylation using Meso Scale Discovery Phospho (Ser780)/Total Rb Whole Cell Lysate Kit. Unbound drug concentrations in serum are determined [1].
For combination studies, cisplatin (6 mg/kg i.p. on day 1) and/or etoposide (12 mg/kg i.p. on days 1-3 or 3-5) are administered in 14-day cycles with Roniciclib (p.o. on days 1-3, 3-5, or 8-10) at indicated doses [1].

Roniciclib has high water solubility: its thermodynamic solubility in water at pH 7.4 was 182 mg/L (22 times higher than ZK 304709) as determined by the balanced shake flask method; its solubility was even higher in low pH values and organic solvents. The melting point is 134°C [2]. In vitro metabolic stability: the parent compound was recovered from human, mouse and rat liver microsomes after 60 minutes of incubation at 94%, 100% and 100% respectively [1, 2]. Cytochrome P450 inhibition: the IC50 values of all tested CYP isoenzymes were >20 μmol/L [1]. Caco-2 cell permeability: the apparent permeability coefficient (Papp ap-bas) was 79 nm/s, indicating good intestinal permeability [1]. Plasma protein binding: 93-95% in mouse, rat, dog and human plasma [1].
Pharmacokinetics in vivo: After intravenous administration, clearance rates were low for all species (mice: 0.51 L·h⁻¹·kg⁻¹; rats: 0.78 L·h⁻¹·kg⁻¹; dogs: 0.50 L·h⁻¹·kg⁻¹). Volume of distribution was relatively large (1.8–2.5 L/kg). Half-lives ranged from 2.1 h (rat), 3.7 h (mice), and 4.3 h (dogs). After oral administration, bioavailability was moderate (48–56%) for all species [1, 2].

In efficacy studies, roniciclib was well tolerated at therapeutic doses (up to 2.5 mg/kg), and no significant weight loss was observed in any treatment group (once daily or intermittent dosing). In studies of its use in combination with cisplatin and etoposide, the addition of roniciclib did not exacerbate weight loss compared to cisplatin/etoposide alone [1]. The compound does not have off-target inhibition of carbonic anhydrase II (IC50 > 10,000 nmol/L), thus eliminating the erythrocyte accumulation problem previously observed in sulfonamide analogues [2].

[1]. BAY 1000394, a novel cyclin-dependent kinase inhibitor, with potent antitumor activity in mono- and in combination treatment upon oral application. Mol Cancer Ther. 2012 Oct;11(10):2265-73.

[2]. The lab oddity prevails: discovery of pan-CDK inhibitor (R)-S-cyclopropyl-S-(4-{[4-{[(1R,2R)-2-hydroxy-1-methylpropyl]oxy}-5-(trifluoromethyl)pyrimidin-2-yl]amino}phenyl)sulfoximide (BAY 1000394) for the treatment of cancer. ChemMedChem. 2013 Jul;8(7):1067-85.

Roniciclib has been investigated for the treatment of small cell lung cancer. Roniciclib is an orally bioavailable cyclin-dependent kinase (CDK) inhibitor with potential antitumor activity. Roniciclib selectively binds to and inhibits the activity of CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D1, and CDK9/cyclin T1, these serine/threonine kinases play key roles in the regulation of cell cycle progression 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. CDKs are often dysregulated in cancer cells. See also: Roniciclib (note moved to). Human tumor xenograft efficacy study: Female athymic nude mice with established subcutaneous HeLa-MaTu cervical cancer xenografts (tumor size approximately 21-25 mm²) were randomly divided into several groups (n=8). BAY 1000394 was administered orally once daily for 21 days at doses of 0.5, 1.0, 1.5, and 2.0 mg/kg. The compound was formulated in a solvent of 40% polyethylene glycol and 60% water. Tumor growth was measured twice weekly using calipers. [1] Intermittent dosing studies: In the same model, BAY 1000394 was administered orally twice daily at doses of 1.5, 2.0, and 2.5 mg/kg for 2 days followed by a 5-day break (2-day dosing/5-day break regimen) for a total of 3 cycles. [1] Pharmacokinetic/pharmacodynamic (PK/PD) studies: Tumor-bearing mice (HeLa-MaTu) were administered a single oral dose of 2 mg/kg of BAY 1000394 (formulation as described above). Tumor and plasma samples were collected at 1, 7, 24 and 48 hours after administration to analyze the concentration of free drug in tumor tissue and the phosphorylation level of retinoblastoma protein (P-pRb). [1] Roniciclib (BAY 1000394) is a novel oral pan-CDK inhibitor designed to overcome the limitations of the early clinical candidate ZK 304709. ZK 304709 failed in a phase I clinical trial due to dose-limiting absorption and large inter-patient variability (attributed to low solubility and off-target carbonic anhydrase inhibition). Roniciclib introduces a sulfoxide imine group, which eliminates carbonic anhydrase inhibitory activity and improves solubility while maintaining potent CDK inhibitory activity. The compound inhibits cell cycle CDKs (1, 2, 4) and transcription CDKs (7, 9), leading to cell cycle arrest at multiple checkpoints and inducing apoptosis. It has demonstrated broad antitumor activity in various xenograft models, including chemotherapy-resistant and patient-derived models, and showed greater than additive efficacy when used in combination with cisplatin and etoposide in a small cell lung cancer (SCLC) model. Roniciclib is effective with both once-daily continuous and intermittent dosing regimens, providing flexibility for clinical optimization. It is currently in a Phase I clinical trial for the treatment of advanced solid tumors (ClinicalTrials.gov registration number: NCT01188252) [1, 2].

C18H21F3N4O3S

430.44500

430.129

C, 50.23; H, 4.92; F, 13.24; N, 13.02; O, 11.15; S, 7.45

1223498-69-8

1223498-69-8

45380979

White to off-white solid powder

4.993

3

10

7

29

661

2

C[C@@H](O)[C@H](OC1=NC(NC2=CC=C([S@@](=N)(C3CC3)=O)C=C2)=NC=C1C(F)(F)F)C

UELYDGOOJPRWGF-MFOHZAOFSA-N

InChI=1S/C18H21F3N4O3S/c1-10(26)11(2)28-16-15(18(19,20)21)9-23-17(25-16)24-12-3-5-13(6-4-12)29(22,27)14-7-8-14/h3-6,9-11,14,22,26H,7-8H2,1-2H3,(H,23,24,25)/t10-,11-,29?/m1/s1

(2R,3R)-3-[2-[4-(cyclopropylsulfonimidoyl)anilino]-5-(trifluoromethyl)pyrimidin-4-yl]oxybutan-2-ol

Roniciclib; BAY1000394; roniciclib; 1223498-69-8; 0W9Q8U337A; BAY10-00394; BAY-1000,394; BAY-1000394; BAY 1000394

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)

DMSO: ≥ 250 mg/mL (~580.8 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.83 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 (4.83 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 (4.83 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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 (Please use freshly prepared in vivo formulations for optimal results.)