Mps1/monopolar spindle 1 (IC50 = 0.63 nM)

BAY 1217389 has an IC50 value of 0.63±0.27 nmol/L in biochemical assays. The protein exhibits a high degree of selectivity towards other kinases and can bind to PDGFRβ (<10 nmol/L), Kit (between 10 and 100 nmol/L), CLK1, CLK2, CLK4, JNK1, JNK2, JNK3, LATS1, MAK, MAPKAP2, MERTK, p38β, PDGFRα, PIP5K1C, PRKD1, and RPS6KA5 (between 100 and 1,000 nmol/L). By causing an early exit from mitosis (referred to as "mitotic breakthrough") and thereby abrogating nocodazole-induced SAC activity, BAY1217389 induces multinuclearity and tumor cell death in cellular mechanistic assays. It has been discovered that it inhibits cell proliferation, with a median IC50 of 6.7 nmol/L (range: 3 to >300 nmol/L).

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BAY 1161909 and BAY1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis (“mitotic breakthrough”), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range).[1]

BAY 1217389 exhibits a moderate level of efficacy when used in monotherapy in tumor xenograft studies. In the tested species, its blood clearance is found to be low. The terminal half-lives were long and the Vss was high. Male Wistar rats (0.5 mg/kg) and female NMRI mice (1 mg/kg) are given oral BAY 1217389. Peak plasma concentrations are measured in the 1.5–7 hour time range. In rats, oral bioavailability is high, whereas in mice, it is moderate[1].

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In vivo, Empesertib (BAY1161909) and BAY1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance [1].

Through the phosphorylation of a biotinylated peptide (Biotin-Ahx-PWDPDDADITEILG-NH2), TRFRET-based in vitro kinase assays evaluate the inhibition of recombinant human Mps1 by BAY 1161909 or BAY 1217389. Standard assay conditions call for a 15-minute preincubation period between the kinase and test compound, followed by the addition of substrate and ATP at 10 μM to initiate the enzyme reaction.

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Kinase assay [1]
The inhibition of recombinant human Mps1 by Empesertib (BAY1161909) or BAY1217389 was assessed in TR-FRET–based in vitro kinase assays via phosphorylation of a biotinylated peptide (biotin-Ahx-PWDPDDADITEILG-NH2). Kinase and test compound were preincubated for 15 minutes before enzyme reaction was started by the addition of substrate and ATP upon 10 μmol/L. For further details, see Supplementary Materials and Methods.

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Kinase selectivity profiling [1]
Empesertib (BAY1161909) and BAY1217389 were counterscreened against a panel of kinases using the Millipore Kinase or DiscoveRx profiler screen. Empesertib (BAY1161909) was initially tested at 1 μmol/L in the DiscoveRx kinase panel, followed by KD determination for 11 kinases (Supplementary Table S1). BAY 1161909 was tested at 10 μmol/L in the Millipore kinase panel, followed by retesting at 1 and 0.1 μmol/L and IC50 determination for JNK1alpha, JNK2alpha, and JNK3 (Supplementary Table S1). BAY 1217389 was initially tested at 1, 0.1, and 0.01 μmol/L in the DiscoveRx kinase panel (Supplementary Table S2).

Cells are seeded in the appropriate medium supplemented with 10% FCS into 96-well plates at densities ranging from 1,000 to 5,000 cells per well. Cells are treated with compound dilutions in quadruplicates after a 24-hour period. Adherent cells are stained with crystal violet and fixed with glutaraldehyde after an additional 96 hours. The company's software is used to calculate IC50 values through a 4-parameter fit.

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Multinuclearity assay. [2]
U-2 OS (osteosarcoma ATCC: HTB-96) cells were plated at a density of 2,500 cells per well in a 384-well microtiter plate in 20 μL cell culture medium and incubated overnight at 37°C. Empesertib (BAY1161909) or BAY1217389 was added at a final concentration of 100 nmol/L in triplicates. Cells were treated for 0, 24, 48, and 72 hours at 37°C in the presence of test compounds. Thereafter, cells were fixed with 4% (v/v) PFA, permeabilized with 0.5% (v/v) Triton X-100, and blocked with 0.5% (v/v) BSA in PBS. α-Tubulin structures were detected by antibody labeling. After blocking with goat IgG, secondary antibodies were applied in the blocking solution. Cells were washed with PBS, and nuclei were marked with Hoechst 33342. Finally, cells were washed and covered with PBS and stored at 4°C until image acquisition. Images were acquired with a PerkinElmer Opera High-Content Analysis reader.

Mice: Female athymic NMRI nu/nu mice, aged 50 days, with an average body weight of 20-22 g, are utilized for tumor xenograft studies. Animals are randomized to treatment and control groups (8–10 mice / group) when tumors reach a size of 20–40 mm², depending on the growth of the tumor model. They are then treated p.o. with vehicle (70% polyethylene glycol 400, 5% ethanol, 25% solutol), BAY 1161909, BAY1217389, and/or paclitaxel. A2780cis tumor-bearing female NMRI nude mice are treated with paclitaxel (i.v., once at a dose of 24 mg/kg), BAY 1161909 (p.o., twice daily for two days at a dose of 2.5 mg/kg), and in combination with paclitaxel (i.v., once at a dose of 24 mg/kg) and BAY 1161909 (p.o., twice daily for two days at a dose of 1 mg/kg). This allows for the analysis of polyploidy and multinuclearity induction in vivo.\n

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\n\nPharmacokinetic investigations [2]
\nPharmacokinetic studies were performed in male Wistar rats and female CD1 or NMRI nu/nu mice. For i.v. studies in rats and mice, Empesertib (BAY1161909) was solubilized in 1% DMSO, 99% plasma; for p.o. studies in rats in 50% polyethylene glycol (PEG) 400, 10% ethanol, 40% water, and for p.o. studies in mice in 75% PEG 400, 5% ethanol, and 25% solutol. BAY1217389 was solubilized in 50% PEG 400, 10% ethanol, and 40% water for intravenous and p.o. dosing in rats and mice. In pharmacokinetic studies, plasma samples were collected after 2, 5, 15, 30, 45 minutes, 1, 2, 4, 6, 8, and 24 hours after intravenous application and after 8, 15, 30, 45 minutes, 1, 2, 4, 6, 8, and 24 hours after p.o. administration and precipitated with ice-cold acetonitrile (1:5). Supernatants were analyzed via LC/MS-MS. Pharmacokinetic parameters were estimated from the plasma concentration data, e.g., using the log-linear trapezoidal rule for AUC estimation. Maximal plasma concentrations (Cmax) and time thereof (Tmax) were taken directly from the concentration time profiles.

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\n\n\nAnimal efficacy studies [2]
\nFor tumor xenograft studies, female athymic NMRI nu/nu mice (Taconic), 50 days old, average body weight 20 to 22 g, were used after an acclimatization period of 14 days. Feeding and drinking was ad libitum 24 hours per day. Human tumor cells derived from exponentially growing cell cultures were resuspended for A2780cis, NCI-H1299, and SUM-149 models in 100% Matrigel to a final concentration of 2 × 107, 3 × 107, or 5 × 107 cells/mL, respectively. Subcutaneous implants of 0.1 mL of 2 × 106 A2780cis, 3 × 106 NCI-H1299, or 5 × 106 SUM-149 cells were inoculated into the inguinal region of athymic mice. Tumor fragments of patient tumor explants MAXF 1384 or LU384, obtained from serial passage in nude mice, were cut into fragments of 4 to 5 mm diameter and transplanted subcutaneously into the flank of athymic mice. Tumor area (product of the longest diameter and its perpendicular), measured with a caliper, and body weight were determined two to three times a week. Tumor growth inhibition is presented as treatment/control ratio (T/C) calculated with tumor areas at the end of the study. Animal body weight was used as a measure for treatment-related toxicity. Body weight loss > 20% was dedicated as toxic. When tumors reached a size of approximately 20 to 40 mm², depending on growth of the tumor model, animals were randomized to treatment and control groups (8–10 mice/group) and treated p.o. with vehicle (70% PEG 400, 5 % ethanol, and 25% Solutol), Empesertib (BAY1161909), BAY1217389, and/or paclitaxel, as indicated in Tables and Figure legends. In combination treatment groups, Mps1 inhibitor and paclitaxel were applied at the same day within a time frame of 1 hour. The treatment of each animal was based on individual body weight.

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\n\n\nIn vivo mode of action studies [2]
\nFor analysis of polyploidy and multinuclearity induction in vivo, A2780cis tumor–bearing female NMRI nude mice (see above) were treated with paclitaxel (intravenously once with 24 mg/kg), Empesertib (BAY1161909) (p.o. twice daily for 2 days with 2.5 mg/kg), and in combination with paclitaxel (i.v. once 24 mg/kg) and Empesertib (BAY1161909) (p.o. twice daily for 2 days 1 mg/kg). Treatment for all groups started at a tumor size of 60 mm² at day 14 after tumor cell inoculation. Tumor samples were prepared 4 and 8 hours after first Empesertib (BAY1161909) application at treatment day 1, as well as 4, 8, and 24 hours after first application of BAY 1161909 on treatment day 2. At each time point, 3 animals per treatment group were analyzed. Tumors were used for histologic examination after paraffin embedding and hematoxylin and eosin staining.

Pharmacokinetic parameters in vivo [1]
Pharmacokinetic parameters were determined in mice and rats. The plasma clearance of BAY 1161909 was low after intravenous injection of 0.5 mg/kg into male CD1 mice and male Wistar rats. The steady-state volume of distribution (Vss) was high in both animals, and the terminal half-life was long. Peak plasma concentrations were reached 4 hours after oral administration of 1 mg/kg and 0.5 mg/kg to female NMRI mice and male Wistar rats, respectively. Oral bioavailability in mice and rats was moderate (Table 1). BAY1217389 was administered intravenously at 1.0 mg/kg and 0.5 mg/kg to female CD1 mice and male Wistar rats, respectively. The plasma clearance was low in both tested animals. The steady-state volume of distribution (Vss) was high, and the terminal half-life was long. BAY1217389 was administered orally to female NMRI mice (1 mg/kg) and male Wistar rats (0.5 mg/kg), respectively. Peak plasma concentrations occurred between 1.5 and 7 hours. Oral bioavailability was high in rats and moderate in mice (Table 1). Our data demonstrate that we have identified two novel Mps1 kinase inhibitors with favorable pharmacokinetic profiles, supporting their further development for clinical application.

Objective: The monopolar spindle 1 (MPS1) kinase inhibitor BAY 1217389 (BAY) has a synergistic effect with paclitaxel. This phase I study used a novel randomized continuous reassessment (rCRM) method to evaluate the efficacy of BAY in combination with paclitaxel in order to improve dose determination. [2]
Patients and Methods: Patients with solid tumors were randomly assigned to receive oral BAY (twice daily for 2 days on and 5 days off) in combination with weekly paclitaxel (90 mg/m2) or paclitaxel monotherapy (first cycle). Dose escalation was guided by a CRM model. The primary objectives were to assess safety, determine the maximum tolerated dose (MTD) of BAY, and evaluate the pharmacokinetic characteristics of the two compounds. Simulations were performed to determine the contribution of rCRM to dose determination. [2]
Results: A total of 75 patients were included. The main dose-limiting toxicity was hematologic toxicity (55.6%). The maximum tolerated dose (MTD) for the combination of BAY and paclitaxel was determined to be 64 mg twice daily. After including the control group, we confirmed that ≥ grade 3 neutropenia was caused by higher BAY exposure [AUC0-12 (P< 0.001)]. After determining the MTD, we included 19 breast cancer patients treated at this dose for a dose extension study. Other common toxicities included nausea (45.3%), fatigue (41.3%), and diarrhea (40.0%). In evaluable patients, a confirmed overall response was observed in 31.6%. The simulation results showed that rCRM was superior to conventional designs in determining the true MTD. [2] Conclusion: The combination of BAY and paclitaxel has considerable toxicity and no therapeutic window. However, the rCRM design allowed us to determine the exposure-toxicity relationship of BAY. Therefore, we believe that rCRM can improve dose determination when drugs with overlapping toxicities are used in combination in phase I trials.

[1]. Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity. Mol Cancer Ther. 2016 Apr;15(4):583-92.

[2]. A Phase I Study of an MPS1 Inhibitor (BAY 1217389) in Combination with Paclitaxel Using a Novel Randomized Continual Reassessment Method for Dose Escalation. Clin Cancer Res . 2021 Dec 1;27(23):6366-6375.

Monopolar spindle 1 (Mps1) has been identified as a key kinase activating the spindle assembly checkpoint (SAC) to ensure the proper distribution of chromosomes to daughter cells. This article reports the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, belonging to different chemical classes. Both BAY 1161909 and BAY 1217389 exhibit excellent selectivity and IC50 values for inhibiting Mps1 kinase activity, both below 10 nmol/L. In cellular mechanism experiments, both Mps1 inhibitors inhibited nocodazole-induced SAC activity and induced premature cell exit from mitosis (“mitotic breakthrough”), ultimately leading to multinucleated cell formation and tumor cell death. Both compounds effectively inhibited tumor cell proliferation in vitro (IC50 values in the nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy as monotherapy in tumor xenograft studies. However, consistent with its unique mechanism of action, low-dose Mps1 inhibitors, when used in combination with paclitaxel, can reduce paclitaxel-induced mitotic arrest by attenuating SAC activity. Therefore, in various xenograft models, including those exhibiting acquired or inherent paclitaxel resistance, combination therapy significantly improved efficacy at their respective maximum tolerated doses (MTDs), superior to paclitaxel or Mps1 inhibitor monotherapy. Both Mps1 inhibitors demonstrated good tolerability and did not increase the toxicity of paclitaxel monotherapy. These preclinical results validate the innovative concept of SAC inhibition in cancer treatment and provide a basis for clinical proof-of-concept studies to evaluate the combination of Mps1 inhibitors BAY 1161909 and BAY 1217389 with antimitotic anticancer drugs to improve efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92.

C27H24F5N5O3

561.5032

561.179

C, 57.75; H, 4.31; F, 16.92; N, 12.47; O, 8.55

1554458-53-5

1554458-53-5

78320750

White to off-white solid powder

1.5±0.1 g/cm3

1.617

4.64

2

11

9

40

867

0

FC(C([H])([H])C([H])([H])N([H])C1=C([H])C(=NN2C1=NC([H])=C2C1C([H])=C([H])C(=C(C([H])([H])[H])C=1[H])C(N([H])C1([H])C([H])([H])C1([H])[H])=O)OC1C([H])=C([H])C(=C(C=1F)F)OC([H])([H])[H])(F)F

WNEILUNVMHVMPH-UHFFFAOYSA-N

InChI=1S/C27H24F5N5O3/c1-14-11-15(3-6-17(14)26(38)35-16-4-5-16)19-13-34-25-18(33-10-9-27(30,31)32)12-22(36-37(19)25)40-21-8-7-20(39-2)23(28)24(21)29/h3,6-8,11-13,16,33H,4-5,9-10H2,1-2H3,(H,35,38)

N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-(3,3,3-trifluoropropylamino)imidazo[1,2-b]pyridazin-3-yl]-2-methylbenzamide

BAY-1217389; BAY 1217389; 1554458-53-5; BAY1217,389; BAY-1217,389; Bay 1217,389; Benzamide, N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-[(3,3,3-trifluoropropyl)amino]imidazo[1,2-b]pyridazin-3-yl]-2-methyl-; M964LB1114; N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxyphenoxy)-8-(3,3,3-trifluoropropylamino)imidazo[1,2-b]pyridazin-3-yl]-2-methylbenzamide; UNII-M964LB1114; BAY1217389

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: ~100 mg/mL (~178.1 mM)
Ethanol: ~8 mg/mL (~14.3 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.45 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (4.45 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 25.0 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.)