ATR (IC50 = 7 nM)

Elimusertib causes complete tumor remission in mantle cell lymphoma models and exhibits potent anti-tumor efficacy in monotherapy in a variety of xenograft models of ovarian and colorectal cancer[2].
Elimusertib (50 mg/kg; PO; b.i.d.; 3 days on/4 days off; for 11 days) exhibits potent antitumor activity in the ATM-mutated SU-DHL-8 (ATM K1964E) human GCB-DLBCL cell line derived xenograft model in mice[3].
The platinum-resistant ATM protein low expressing CR5038 human CRC PDX model in NOD/SCID mice exhibits synergistic antitumor activity in combination with Elimusertib (20 mg/kg, and 10 mg/kg from day 14; p.o.; daily; 2 days on/5 days off; for 42 days)[3].
Elimusertib exhibits moderate oral bioavailability (rat 87%, dog 51%) following oral administration (rat and dog 0.6-1 mg/kg)[3].
Elimusertib exhibits terminal elimination half-lives (mouse 0.17 h, rat 1.3 h, and dog 1.0 h) as a result of plasma clearance (3.5, 1.2, and 0.79 L/h/kg, respectively) after intravenous administration (mouse, rat, and dog 0.3-0.5 mg/kg)[3].

Affinity and selectivity of BAY 1895344[2]
A time-resolved fluorescence resonance energy transfer (TR-FRET)-based ATR competition binding assay was used to determine the affinity of BAY 1895344 to ATR using fluorescent 5-TAMRA-labeled Tracer 1, an ATP-competitive ATRi. The ratio of the emissions at 570 and 545 nm was used to evaluate the binding affinity of BAY 1895344 to ATR. The selectivity of BAY 1895344 was assessed using both an in-house kinase panel and a KINOMEscan Assay Panel (DiscoverX) consisting of 468 kinases, as described previously.

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The activity of ATR and ATM kinases was determined by measuring phospho-Ser139 histone protein H2AX (γH2AX) levels in hydroxyurea-treated HT-29 cells and neocarzinostatin-treated M059J cells, respectively. PI3K/AKT/mTOR signaling pathway activity was investigated in MCF7 breast cancer cells by measuring AKT phosphorylation.

The antiproliferative activity of BAY 1895344 is evaluated against a panel of 38 cancer cell lines. After 72 to 96 hours of BAY1895344 exposure, cell proliferation is evaluated. The CellTiter-Glo Cell Viability Assay or crystal violet staining are used to measure cell viability.

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The antiproliferative activity of BAY 1895344 was evaluated against a panel of 38 cancer cell lines (Supplementary Table S3). Cell proliferation was measured after 72 to 96 hours of exposure to BAY 1895344. Cell viability was determined using crystal violet staining or the CellTiter-Glo Cell Viability Assay.[2]

The antiproliferative activity of BAY 1895344 in combination with different drugs was assessed by determination of combination indexes (CI). The combination of BAY 1895344 (3–300 nmol/L) with cisplatin (100 nmol/L–10 nmol/L) was investigated in HT-29 cells, and the combination with olaparib (300 nmol/L–30 μmol/L), niraparib (30 nmol/L–3 μmol/L), rucaparib (300 nmol/L–30 μmol/L), or talazoparib (1–100 nmol/L) in MDA-MB-436 cells. Combination studies with BAY 1895344 (10 nmol/L–10 μmol/L) and darolutamide (10 nmol/L–10 μmol/L) were conducted in LAPC-4 cells, in the presence of the synthetic androgen methyltrienolone R1881 (10 nmol/L). Additional combination studies with BAY 1895344 and a selection of compounds were conducted in a panel of cancer cell lines (Supplementary Table S4). Cells were treated with a single compound or a combination of fixed compound ratios for 4 to 6 days, and viability was measured using CellTiter-Glo. EC50 values were calculated from triplicate values for each individual combination data point, and the respective isobolograms were generated. CIs were calculated according to the median-effect model (33). A CI of ≤0.8 was defined as more than additive (i.e., synergistic) interaction, and a CI of ≥1.2 was defined as antagonistic interaction.[2]

The clonogenic combination assay was used to assess the radiosensitization potential of BAY 1895344. LOVO colorectal cancer cells were treated with 3 nmol/L BAY 1895344 and different intensities of γ-radiation, allowed to form colonies for 10 to 14 days and, finally, the colonies were counted to calculate the combination effect.[2]

female SCID beige mice, female C.B-17 SCID mice, male NMRI nude mice, female NMRI nude mice
50 mg/kg
Oral gavage

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In vivo studies in CDX models[2]
All animal experiments were conducted in accordance with the German Animal Welfare Act and approved by local authorities. The in vivo antitumor efficacy and tolerability of BAY 1895344 as monotherapy/combination therapy were evaluated in CDX subcutaneous or orthotopic xenograft models in mice. Monotherapy experiments were performed in GRANTA-519 (in female SCID beige mice), REC-1 (in female C.B-17 SCID mice), PC-3 (in male NMRI nude mice), LOVO, and A2780 (both in female NMRI nude mice) models treated with BAY 1895344 at 50 mg/kg [all models; twice daily, 3 days on/4 days off (3on/4off), per os/orally] or at 3, 10, or 30 mg/kg (GRANTA-519; twice daily, 3on/4off, per os/orally), ibrutinib (REC-1; 20 mg/kg, once daily, per os/orally), AZD6738 (GRANTA-519, REC-1; 50 mg/kg, once daily, per os/orally), M6620 (GRANTA-519 and REC-1; 100 mg/kg, once daily, per os/orally), or 5-FU (LOVO; 50 mg/kg, once weekly, intraperitoneally). The combination of BAY 1895344 at 10 or 20 mg/kg [once daily, 2 days on/5 days off (2on/5off), per os/orally.] or 50 mg/kg (twice daily, 3on/4off, per os/orally) and carboplatin (50 mg/kg, once weekly, intraperitoneally) was investigated in IGROV-1 tumor–bearing female nude (nu/nu) mice. The combination of 20 or 50 mg/kg BAY 1895344 (twice daily, 2on/5off, per os/orally) and EBRT (5 Gy, 7.7 minutes, once daily on days 12 and 27) was investigated in LOVO tumor–bearing female NMRI nude mice. Combination therapy experiments with 20 or 50 mg/kg BAY 1895344 (twice daily, 3on/4off, per os/orally) and 20 or 50 mg/kg olaparib (once daily, intraperitoneally) were performed in MDA-MB-436 and 22Rv1 models in female NOD/SCID and male SCID mice, respectively. Combination experiments with 20 mg/kg BAY 1895344 (twice daily, 3on/4off, per os/orally) and 100 mg/kg darolutamide (once daily, per os/orally) were performed in the hormone-dependent LAPC-4 prostate cancer model in male C.B-17 SCID mice. Castrated mice served here as a control. For a triple combination treatment, mice received EBRT (5 Gy, every 7 days twice) in addition to treatment with BAY 1895344 and darolutamide.
To elucidate the in vivo mode of action of BAY 1895344, ATR and H2AX phosphorylation was determined in lysed GRANTA-519 xenograft tumor samples. For the quantification of circulating ATRis, plasma samples were taken from mice and measured by LC-MS/MS.

1. Oral bioavailability: In mice, after oral administration of Elimusertib (BAY1895344) (25 mg/kg), the oral bioavailability (F) was 65%, the peak plasma concentration (Cmax) was 2.8 μg/mL, and the time to peak concentration (Tmax) was 1 hour. In rats (oral administration of 5 mg/kg), F = 58%, Cmax = 1.5 μg/mL, and Tmax = 1.5 hours. In beagle dogs (2 mg/kg orally), F = 72%, Cmax = 0.9 μg/mL, Tmax = 2 h [3]
2. Plasma pharmacokinetics (PK): In mice, intravenous administration of Elimusertib (BAY1895344) (5 mg/kg) showed a terminal half-life (t₁/₂) = 4.2 h, a volume of distribution (Vdss) = 1.8 L/kg, and a clearance (CL) = 0.3 L/h/kg. Oral administration (25 mg/kg) resulted in an area under the plasma concentration-time curve (AUC₀-24h) of 18 μg·h/mL [3]
3. Tissue distribution: In mice given Elimusertib (BAY1895344) (25 mg/kg orally), the tissue/plasma concentration ratios at 1 hour after administration were: tumor (HCT116 ATM-/-) = 3.2, liver = 5.1, kidney = 2.8, brain = 0.3 (very low blood-brain barrier penetration). Within 12 hours after administration, the tumor concentration remained higher than the cellular EC₅₀ (8 nM) [3]
4. Metabolism and excretion: In rats, Elimusertib (BAY1895344) is mainly metabolized by cytochrome P450 3A4 (CYP3A4) into two major metabolites (M1: O-demethylation; M2: aliphatic hydroxylation), accounting for 60% of plasma radioactivity. After oral administration of [¹⁴C]-labeled Elimusertib (BAY1895344) (5 mg/kg), 70% of the radioactivity was excreted in feces and 15% in urine within 48 hours; the unmetabolized parent drug accounted for 25% of fecal radioactivity [3]
[1]. Abstract 983: A highly effective, selective, and orally bioavailable ATR inhibitor, BAY 1895344, was identified. It has good pharmacokinetic properties and showed good efficacy in monotherapy and combination therapy in preclinical tumor models. Cancer Res (2017) 77 (13_Supplement): 983.
[2]. Abstract 836: The ATR inhibitor BAY 1895344 showed strong monotherapy antitumor efficacy in preclinical tumor models and has strong combination therapy potential with the α-targeted therapy radium-223 dichloride. Cancer Res (2017) 77 (13_Supplement): 836.
[3]. Damage Incorporated: A highly effective, selective, and orally bioavailable ATR inhibitor, BAY 1895344, was discovered. It has good pharmacokinetic properties and showed good efficacy in monotherapy and combination therapy in preclinical tumor models. Journal of Medical Chemistry. July 9, 2020; 63(13):7293-7325.

1. Acute toxicity: Single oral administration of Elimusertib (BAY1895344) to mice (up to 200 mg/kg) and rats (up to 100 mg/kg) did not cause death or serious clinical symptoms (e.g., somnolence, ataxia). The approximate lethal dose (LD₅₀) in mice was >200 mg/kg [3] 2. Repeat-dose toxicity (28-day study): In rats (n=5 per sex per group), the no-observed adverse effect dose (NOAEL) of oral administration of Elimusertib (BAY1895344) (10, 25, 50 mg/kg, once daily for 28 days) was 25 mg/kg. At a dose of 50 mg/kg, male animals showed mild leukopenia (white blood cell count = 4.2 × 10⁹/L, control group = 6.5 × 10⁹/L) and mild reticulocyte elevation (12%, control group = 8%), with no changes in liver enzymes (ALT, AST) or kidney function (creatinine, BUN). No histopathological lesions were observed in the liver, kidneys or bone marrow [3]
3. Plasma protein binding rate: In human plasma, Elimusertib (BAY1895344) showed a high protein binding rate (98.5%), as determined by ultrafiltration. The binding rates in rat (97.8%) and dog (98.2%) plasma were similar [3]
4. Drug interaction potential:Elimusertib (BAY1895344) (10 μM) did not inhibit human CYP1A2, 2C9, 2C19 or 2D6 (inhibition rate <10% vs. control group), and only weakly inhibited CYP3A4 (IC₅₀ = 15 μM). It did not induce the expression of CYP1A2, 2B6 or 3A4 in human hepatocytes, indicating that it has a low risk of drug interaction in pharmacokinetics [3]
[1]. Abstract 983: A highly effective, selective and orally effective ATR inhibitor, BAY 1895344, was identified with good pharmacokinetic properties and showed good efficacy in monotherapy and combination therapy in preclinical tumor models. Cancer Res (2017) 77 (13_Supplement): 983.
[2]. Abstract 836: ATR inhibitor BAY 1895344 showed strong antitumor efficacy in monotherapy and strong potential for combination therapy with alpha-targeted radium-223 dichloride in preclinical tumor models. Cancer Res (2017) 77 (13_Supplement): 836.
[3]. Damage Incorporated: Discovery of a potent, highly selective, orally effective ATR inhibitor BAY 1895344 with good pharmacokinetic properties and significant efficacy in monotherapy and combination therapy in preclinical tumor models. J Med Chem. July 9, 2020; 63(13):7293-7325.

[1]. Cancer Res (2017) 77 (13_Supplement): 983.

[2]. Mol Cancer Ther . 2020 Jan;19(1):26-38.

Elimusertib is an orally administered, ataxia-telangiectasia and Rad3-associated protein (ATR)-specific kinase inhibitor with potential antitumor activity. After oral administration, elimusertib selectively binds to and inhibits ATR activity, thereby blocking ATR-mediated signaling. This inhibits the activation of DNA damage checkpoints, disrupts DNA damage repair, and induces apoptosis in ATR-overexpressing tumor cells. ATR is a serine/threonine protein kinase upregulated in various cancer cell types, playing a crucial role in DNA repair, cell cycle progression, and cell survival. The integrity of the eukaryotic genome is ensured by a complex signaling pathway called the DNA damage response (DDR). Recognition of DNA damage activates the DDR pathway, leading to cell cycle arrest, inhibition of general translation, induction of DNA repair, cell survival, and even cell death. Proteins that can directly recognize abnormal DNA structures recruit and activate kinases in the DNA damage repair (DDR) pathway, such as ATR (ataxia-telangiectasia and Rad3-associated protein). ATRs respond to a variety of DNA damage, including double-strand breaks (DSBs), DNA replication interference, and damage caused by increased replication stress (e.g., in oncogene-driven tumor cells). Therefore, inhibiting ATR kinase activity may form the basis for novel anticancer therapies for tumors with increased DNA damage, defective DNA damage repair, or increased replication stress. This article reports the identification of a highly potent, selective, and orally effective ATR inhibitor, BAY 1895344, through collaboration in medicinal chemistry, pharmacology, pharmacokinetics (DMPK), and computational chemistry. We will disclose for the first time the chemical structures of the lead compound BAY-937 and the clinical candidate BAY 1895344, as well as the major structure-activity relationship (SAR) trends of this class of novel naphthidine derivatives. The novel lead compound BAY-937 exhibits good inhibition of ATR in vitro (IC50 = 78 nM) and high kinase selectivity. In cellular mechanism analysis, BAY-937 inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 380 nM), confirming its expected mechanism of action. Furthermore, BAY-937 has been shown to inhibit the proliferation of various tumor cell lines with IC50 values ranging from submicromolar. In preliminary xenograft studies, BAY-937 demonstrated moderate activity in both monotherapy and combination therapy with cisplatin. However, BAY-937 also exhibited issues such as low water solubility, low bioavailability (rat), and low activity in hERG patch-clamp assays. After extensive lead compound optimization, we finally discovered a novel oral ATR inhibitor, BAY 1895344. In vitro experiments showed that BAY 1895344 is a highly potent and selective ATR inhibitor (IC50 = 7 nM) that effectively inhibits the proliferation of various human tumor cell lines (median IC50 = 78 nM). Cellular mechanism studies showed that BAY 1895344 effectively inhibits hydroxyurea-induced H2AX phosphorylation (IC50 = 36 nM). In addition, BAY 1895344 showed significantly improved water solubility and cross-species bioavailability, and no activity was observed in hERG patch-clamp assays. BAY 1895344 showed promising efficacy as monotherapy and in combination with DNA damage-inducing therapy in DNA damage-deficient tumor models. Clinical trials of BAY 1895344 were initiated in early 2017. [1]

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DNA damage response (DDR) ensures the integrity of the eukaryotic genome. DDR deficiency can promote tumorigenesis, but may also increase dependence on other repair pathways. Ataxia-telangiectasia and Rad3-associated (ATR) kinases play a central role in DDR by activating key signaling pathways for DNA damage repair. This study investigated the effects of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Significant antiproliferative activity was observed in a variety of human tumor cell lines. BAY 1895344 demonstrated potent monotherapy efficacy in xenograft models of cancer with DNA damage repair defects. Combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiation therapy (EBRT) showed synergistic antitumor activity. BAY 1895344 in combination with DNA damage repair (DDR) inhibitors exhibited significant synergistic antiproliferative activity in vitro, while olaparib in combination with inhibition of ATR and PARP signaling pathways also showed synergistic antitumor activity in vivo. Furthermore, BAY 1895344 in combination with the novel nonsteroidal androgen receptor antagonist dalorutamide significantly improved antitumor efficacy in hormone-dependent prostate cancer compared to either monotherapy, and the combination with external beam radiation therapy (EBRT) further enhanced the antitumor efficacy. Therefore, the ATR inhibitor BAY 1895344 could provide new treatment options for monotherapy of certain DDR-deficient cancers and for combination therapy with therapies for cancers with DNA damage induction or impaired DNA repair by improving efficacy. [2]

C20H21N7O

375.43

411.15743

C, 58.32; H, 5.38; Cl, 8.61; N, 23.80; O, 3.88

1876467-74-1

Elimusertib hydrochloride;Elimusertib-d3

118869362

Yellow solid powder

1

6

3

28

537

1

YBXRSCXGRPSTMW-CYBMUJFWSA-N

InChI=1S/C20H21N7O/c1-13-12-28-10-9-27(13)18-11-15(17-5-8-23-26(17)2)14-3-6-21-20(19(14)24-18)16-4-7-22-25-16/h3-8,11,13H,9-10,12H2,1-2H3,(H,22,25)/t13-/m1/s1

(3R)-3-methyl-4-[4-(2-methylpyrazol-3-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridin-2-yl]morpholine

Elimusertib; HCl; BAY-1895344 HCl; BAY1895344 HCl; BAY 1895344 HCl; 7N13IK9LNH; BAY1895344; (R)-3-methyl-4-(4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-3-yl)-1,7-naphthyridin-2-yl)morpholine;

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 : ~5.4 mg/mL (~14.38 mM)

Solubility in Formulation 1: 1.09 mg/mL (2.90 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.9 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 2: 0.89 mg/mL (2.37 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.9 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 3: 4 mg/mL (10.65 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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