ATR kinase
Camonsertib (RP-3500) is a potent and selective inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. Biochemical IC50 for ATR/ATRIP is 1.0 ± 0.40 nmol/L, and Ki is 0.022 ± 0.002 nmol/L. It exhibits 30-fold selectivity over mTOR (IC50 = 120 nmol/L), >2,000-fold selectivity over ATM and DNA-PK (IC50 >10,000 nmol/L and >10,000 nmol/L, respectively), and >2,000-fold selectivity over PI3Kα (IC50 = 780 nmol/L) in cell-based assays [1].

Camonsertib (RP-3500; 1 μM; 1-24 hours) inhibits CHK1 (Ser345) phosphorylation for 1 to 3 hours [1]. Camonsertib inhibits gemcitabine-stimulated ATR phosphorylation of its substrate pCHK1 (Ser345), based on Western Blot analysis of LoVo cells [1]

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In cell-based assays, Camonsertib inhibits gemcitabine-stimulated ATR phosphorylation of CHK1 (Ser345) with an IC50 of 0.33 nmol/L in LoVo cells. It shows potent antiproliferative activity in a panel of cell lines with defective ATM or BRCA1/2 pathways, with IC50 values ranging from 8 to 64 nmol/L in 5-day CellTiter-Glo assays [1].
Western blot analysis in LoVo and CW-2 cells treated with 1 μmol/L Camonsertib shows inhibition of pCHK1 (Ser345) within 1-3 hours, followed by rephosphorylation after 4 hours and activation of DNA-PKcs, KAP1, and H2AX, indicating induction of DNA double-strand breaks [1].
In RPE1-hTERT TP53KO ATM-WT and ATM-KO cells, exposure to 30 nmol/L Camonsertib for 48-72 hours increases γH2AX staining similarly in both lines; after washout, ATM-WT cells recover within 24 hours, while ATM-KO cells sustain DNA damage for at least 48 hours [1].
Synergy studies in SUM149PT (BRCA1-mutant) cells show that concomitant administration of Camonsertib and olaparib for 3 days produces greater synergy (ZIP synergy score >10) and cell killing compared to sequential treatment [1].

Camonsertib (RP-3500; 3, 7, 15 mg/kg; lateral; once daily for 18 days) produces dose-dependent tumor growth inhibition in LoVo xenografts with a minimum effective dose (MED) of 7 mg/kg [Camonsertib (5, 10 mg/kg; vascular; once daily) produces statistically significant tumor growth inhibition in the CW-2 floating xenograft model [1]. Camonsertib (7 mg/kg; for 7 days) is claimed to elicit 8.1-fold and 2.7-fold stimulation of KAP1 and DNA-PKcs phosphorylation in mice with LoVo tumors [1]. High-dose Camonsertib tumors, 3 days on/4 days off (30 mg/kg) and 5 days on/2 days off (25 mg/kg), showed greater incidental anti-tumor effects compared with continuous dosing, daily A lower dose formulation (10 mg/kg) for 14 days [1]. /kg) in conjunction with PARPi Olaparib (80mg/kg; both medications on days 1-3 on/4 days off) or sequential (PARPi on for 3 days, then RP-3500 on 3 days, then 1 day) with sequential In contrast, schedules produce greater anti-tumor effects when affecting intolerance [1].

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In LoVo colorectal xenograft-bearing mice, oral administration of Camonsertib once daily for 17 days produces dose-dependent tumor growth inhibition, with a minimum effective dose (MED) of 7 mg/kg. At 15 mg/kg, one mouse was sacrificed due to body weight loss, establishing the MTD as 10 mg/kg once daily on a continuous schedule [1].
In CW-2 colon xenografts, Camonsertib at 5 and 10 mg/kg once daily significantly inhibits tumor growth [1].
In a gastric cancer patient-derived xenograft (PDX) with biallelic ATM loss, Camonsertib at 5 and 10 mg/kg once daily for 28 days induces complete tumor regressions without body weight loss [1].
Pharmacodynamic analysis in LoVo tumors shows dose-dependent inhibition of pCHK1, with an estimated in vivo tumor IC80 of 18.6 nmol/L (85% CI: 7.23–145 nmol/L) for free plasma concentration. At the MED (5–7 mg/kg), free plasma levels above the IC80 are maintained for 10–12 hours, sufficient for efficacy [1].
Intermittent dosing schedules (e.g., 3 days on/4 days off) allow higher doses (up to 30 mg/kg) with improved efficacy and reduced anemia compared to continuous daily dosing. In Granta-519 xenografts, 30 mg/kg on a 3 days on/4 days off schedule produces superior tumor growth inhibition and less red blood cell depletion than continuous lower doses [1].
Combination studies with PARP inhibitors (olaparib or niraparib) show that concomitant intermittent administration (3 days on/4 days off) of low-dose Camonsertib (e.g., 15 mg/kg) plus PARPi is more efficacious and better tolerated than sequential administration, with no significant drug-drug interactions and preserved reticulocyte regeneration during drug holidays [1].

ATR biochemical kinase assays are performed using human recombinant tagged ATR/ATRIP proteins purified from mammalian cells. The assay uses a GST-tagged p53 substrate and 3 μmol/L ATP in buffer containing HEPES, Brij, EGTA, and glycerol. Phosphorylated substrate is detected by incubation with anti-phospho-p53 (Ser15) antibody, donor beads, and acceptor beads for 4-5 hours in EDTA/Tris/BSA buffer. Phosphorylation is quantified by fluorescence measurement (excitation 680 nm, emission 520-620 nm) using a plate reader. IC50 values are determined from dose-response curves [1].

Western Blot analysis
Tested Concentrations: 1 μM
Incubation Duration: 1, 2, 4, 6, 8, 16, 24, IC50 was 0.33 nM[1]. Hourly
Experimental Results: CHK1(Ser345) phosphorylation is inhibited for 1 to 3 hrs (hours). Beginning at 4 hrs (hours), CHK1(Ser345) is re-phosphorylated as DNA-PKcs and its substrates KAP1 and H2AX are activated in treated cells.

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Cell viability is measured using CellTiter-Glo assay. Cells are plated in 96-well plates at optimal densities and treated with compound for 120 hours. Luminescence is read on a plate reader, and viability is normalized to untreated controls. Synergy with PARP inhibitors is analyzed using SynergyFinder with the Zero Interaction Potency (ZIP) model [1].
Immunoblotting: Cells or tumor fragments are lysed in buffer with protease and phosphatase inhibitors. Lysates are separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against pCHK1 (Ser345), pKAP1 (Ser824), KAP1, ATM, pDNA-PKcs, CHK1, γH2AX, and loading controls. Signals are detected with HRP-conjugated secondary antibodies and chemiluminescence [1].
γH2AX immunofluorescence: Treated cells are fixed with 4% paraformaldehyde, permeabilized with Triton X-100, blocked, and incubated with anti-γH2AX antibody followed by Alexa Fluor 488-conjugated secondary antibody and DAPI. Images are acquired on an Operetta high-content microscope and analyzed with Harmony software to quantify nuclear γH2AX intensity [1].

Animal/Disease Models: Female mice (6-8 weeks old) bearing LoVo xenografts [1]
Doses: 3, 7, 15 mg/kg (0.5% methylcellulose/0.02% SDS vehicle)
Route of Administration: Oral; one time/day for 18 days
Experimental Results: Produced dose-dependent tumor growth inhibition with a minimum effective dose (MED) of 7 mg/kg. The maximum tolerated dose (MTD) is 10 mg/kg, one time/day, continuously.

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For xenograft studies, female mice (6-8 weeks old; CB17 SCID, NOD/SCID, or BALB/c nude) are subcutaneously implanted with 5×10⁶ cells (or tumor fragments for PDX) in 1:1 media:Matrigel. When tumors reach 100-200 mm³, mice are randomized and treated orally with Camonsertib formulated in 0.5% methylcellulose/0.02% SDS. Dosing schedules include daily continuous or intermittent (e.g., 2, 3, or 5 days on/off per week). Tumor volume is measured three times weekly using calipers and calculated as 0.52 × length × width². Body weight and clinical signs are monitored for tolerability [1].
For pharmacokinetic/pharmacodynamic studies, whole blood is collected at 0.5, 1, 3, 8, and 24 hours post-dose, diluted with citrate buffer, and plasma is separated. Tumor samples are flash-frozen and analyzed by immunoblotting for pCHK1, pKAP1, and pDNA-PKcs. Plasma concentrations of Camonsertib are determined by LC-MS/MS [1].
Hematology: At study endpoints, whole blood is collected by cardiac puncture into EDTA tubes and analyzed with a hematology analyzer for red blood cell (RBC) and reticulocyte counts [1].

In mice carrying LoVo tumors, oral administration of carmonertib resulted in dose-dependent plasma free drug concentrations. At the lowest effective dose (5-7 mg/kg once daily), plasma free drug concentrations remained above the tumor IC80 (18.6 nmol/L) for approximately 10-12 hours, sufficient to achieve therapeutic efficacy. The compound exhibits good oral bioavailability, and its pharmacokinetic profile is consistent with once-daily dosing regimens. Specific half-life or percentage of bioavailability has not yet been reported [1].

In mice, continuous daily oral administration of carmonertib at doses ≥15 mg/kg resulted in weight loss and anemia (reduced red blood cell count and reticulocyte depletion). The maximum tolerated dose (MTD) for continuous dosing was 10 mg/kg once daily. Intermittent dosing regimens (e.g., 3 days on/4 days off) allowed for higher doses (up to 30 mg/kg) with better tolerability; reticulocyte counts recovered during the 4-day withdrawal period, indicating that erythroid toxicity was reversible. In studies of combination therapy with PARP inhibitors (olaparib or niraparib), continuous combination therapy was intolerable, but intermittent combination therapy (3 days on/4 days off) with reduced doses (e.g., 15 mg/kg carmonertib + low-dose PARP inhibitors) was well tolerated without significant weight loss or severe anemia, and no drug interactions were observed based on pharmacokinetic analysis [1].

[1]. RP-3500: A Novel, Potent and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors. Mol Cancer Ther. 2022 Feb;21(2):245-256.

Camonsertib is a pyrazolopyridine compound with the structure 1H-pyrazolo[3,4-b]pyridine, substituted at positions 1, 4, and 6 with 1H-pyrazol-3-yl, (3-endo)-3-hydroxy-8-oxabicyclo[3.2.1]octane-3-yl, and (3R)-3-methylmorpholin-4-yl, respectively. It is a highly potent and selective ATR kinase inhibitor (IC50 = 1 nM). It can be used as an antitumor drug and an EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitor. It belongs to the morpholino, imidazole, pyrazolopyridine, tertiary alcohol, and oxabicycloalkyl groups. Camonsertib is an oral ataxia-telangiectasia and Rad3-associated kinase (ATR) inhibitor with potential antitumor activity. After oral administration, carmonesertib selectively targets and inhibits ATR activity, blocking phosphorylation of downstream serine/threonine protein kinase checkpoint kinase 1 (CHK1). This blocks ATR-mediated signaling, thereby inhibiting activation of DNA damage checkpoints, disrupting DNA damage repair, and inducing tumor cell apoptosis. ATR is a serine/threonine protein kinase upregulated in multiple cancer cell types, playing a crucial role in DNA repair, cell cycle progression, and cell survival. It is activated by DNA damage induced by DNA replication-related stress. Camonsertib (RP-3500) is a novel, potent, selective, orally bioavailable ATR kinase inhibitor currently undergoing clinical evaluation in patients with advanced solid tumors (NCT04497116). It is designed to utilize synthetic lethal effects to treat tumors with defective DNA damage responses, such as ATM deletion or BRCA1/2 mutations. Preclinical studies have shown that intermittent dosing (e.g., 3 days on/4 days off) maximizes efficacy while minimizing targeted hematologic toxicity (anemia). When used in combination with PARP inhibitors (olaparib, niraparib) in an intermittent dosing regimen, the compound showed a significant synergistic effect, resulting in a superior antitumor response compared to sequential dosing. These findings support clinical trials of intermittent Camonsertib monotherapy and combination regimens with PARP inhibitors to improve the therapeutic index [1].

C21H26N6O3

410.47

410.206

C, 61.45; H, 6.38; N, 20.47; O, 11.69

2417489-10-0

156487652

White to off-white solid powder

1.6

2

7

3

30

628

3

OC1(CC2CCC(O2)C1)C1C=C(N2CCOCC2C)N=C2N(C3=NNC=C3)N=CC=12

YIHHYCIYAIVQKX-YNOVCBQDSA-N

InChI=1S/C21H26N6O3/c1-13-12-29-7-6-26(13)19-8-17(21(28)9-14-2-3-15(10-21)30-14)16-11-23-27(20(16)24-19)18-4-5-22-25-18/h4-5,8,11,13-15,28H,2-3,6-7,9-10,12H2,1H3,(H,22,25)/t13-,14-,15+,21?/m1/s1

(1R,5S)-3-[6-[(3R)-3-methylmorpholin-4-yl]-1-(1H-pyrazol-5-yl)pyrazolo[3,4-b]pyridin-4-yl]-8-oxabicyclo[3.2.1]octan-3-ol

Camonsertib; 2417489-10-0; RP-3500; RP3500; Rp 3500;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~50 mg/mL (~121.81 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.05 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 12.5 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: ≥ 1.25 mg/mL (3.05 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 12.5 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: ≥ 1.25 mg/mL (3.05 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 12.5 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.)