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

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

Elimusertib is an orally available ataxia telangiectasia and Rad3-related (ATR)-specific kinase inhibitor, with potential antineoplastic activity. Upon oral administration, elimusertib selectively binds to and inhibits the activity of ATR, which prevents ATR-mediated signaling. This inhibits DNA damage checkpoint activation, disrupts DNA damage repair and induces apoptosis in ATR-overexpressing tumor cells. ATR, a serine/threonine protein kinase upregulated in a variety of cancer cell types, plays a key role in DNA repair, cell cycle progression and cell survival.

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The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress (e.g. in oncogene-driven tumor cells). Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Herein we report the identification of the potent, highly selective and orally available ATR inhibitor BAY 1895344 by a collaborative effort involving medicinal chemistry, pharmacology, DMPK and computational chemistry. The chemical structures of lead compound BAY-937 and clinical candidate BAY 1895344 as well as the main SAR trends within this novel class of naphthyridine derivatives will be disclosed for the first time. The novel lead compound BAY-937 revealed promising inhibition of ATR (IC50 = 78 nM) and high kinase selectivity in vitro. In cellular mechanistic assays BAY-937 inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 380 nM) demonstrating the anticipated mode of action. Moreover, BAY-937 was shown to inhibit proliferation of a variety of tumor cell lines with low- to sub-micromolar IC50 values. In initial xenograft studies, BAY-937 revealed moderate activity in monotherapy and in combination with cis-platin. However, BAY-937 also revealed low aqueous solubility, low bioavailability (rat) and activity in the hERG patch clamp assay. Extensive lead optimization efforts led to the identification of the novel, orally available ATR inhibitor BAY 1895344. In vitro, BAY 1895344 was shown to be a very potent and highly selective ATR inhibitor (IC50 = 7 nM), which potently inhibits proliferation of a broad spectrum of human tumor cell lines (median IC50 = 78 nM). In cellular mechanistic assays BAY 1895344 potently inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 36 nM). Moreover, BAY 1895344 revealed significantly improved aqueous solubility, bioavailability across species and no activity in the hERG patch-clamp assay. BAY 1895344 also demonstrated very promising efficacy in monotherapy in DNA damage deficient tumor models as well as combination treatment with DNA damage inducing therapies. The start of clinical investigation of BAY 1895344 is planned for early 2017. [1]

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The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage-inducing or DNA repair-compromising cancer therapies by improving their 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.)