AA-115 (Alrizomadlin; APG115)

MDM2 (IC50 = 3.8 nM; Ki = 1 nM)

With concentration-dependent effects, alrizomadlin (0.001-100 μM; 72 hours) suppresses cell proliferation in AGS and MKN45 cells, with IC50s of 18.9 ± 15.6 nM and 103.5 ± 18.3 nM, respectively [3]. To improve the effects of irradiation at various radiation doses, alrizomadlin (0.02 μM, 0.2 μM; Alrizomadlin (0.02 μM, 0.2 μM; 48) was induced by induction with wild-type p53 [3] at 48 h in G0/G1 phase in AGS and MKN45 cells. impact against ischemia[3]. In AGS and MKN45 cells, alrizomadlin (0.02 μM, 0.2 μM; 24 hours) activates p53 to improve radiation direction; stable p53 knockdown removes MDM2, p53, p21, PUMA, BAX, and Cleaved-caspase3 cells to influence progression. In p53 wide cell lines (TPC-1, KTC-1), alrizomadlin (0.3 μM, 1 μM, 3 μM, 10 μM; 24 hours) induces the production of the G2/M phase focused cell cycle marker γH2AX [3].

In vivo radioresistance of adenocarcinoma tumors is improved by alrizomadlin (oral formulation; 100 mg/kg; once daily; 10 days) [3].

Fluorescence Prolarization (FP)-Based Protein Binding Assay [5]
The binding affinity of MDM2 inhibitors was determined by an optimized, sensitive, and quantitative FP-based binding assay, using a recombinant human His-tagged MDM2 protein (residues 1–118) and a FAM tagged p53-based peptide as the fluorescent probe. The design of the fluorescent probe was based upon a previously reported high affinity p53-based peptidomimetic compound (5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2).31 This tagged peptide was named as PMDM6-F. The equilibrium dissociation constant (Kd) of PMDM6-F to the MDM2 protein was determined to be 1.4 ± 0.3 nM by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the MDM2 protein with increasing concentrations up to full saturation. Fluorescence polarization values were measured using the Infinite M-1000 plate reader in Microfluor 1 96-well, black, round-bottom plates. In the saturation experiments, 1 nM of PMDM6-F and increasing concentrations of proteins were added to each well to a final volume of 125 μL in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide (Invitrogen), with 0.01% Triton X-100 and 4% DMSO). Plates were mixed and incubated at room temperature for 30 min with gentle shaking to ensure equilibrium. The polarization values in millipolarization units (mP) were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The Kd value was then calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software. IC50 and Ki values of tested compounds were determined in a dose-dependent competitive binding experiment. Mixtures of 5 μL of the tested compound with different concentrations in DMSO and 120 μL of pre-incubated protein/fluorescent probe complex with fixed concentrations in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide, with 0.01% Triton X-100) were added into assay plates and incubated at room temperature for 30 min with gentle shaking. Final concentrations of the protein and fluorescent probe in the competitive assays were 10 and 1 nM, respectively, and final DMSO concentration was 4%. Negative controls containing protein/fluorescent probe complex only (equivalent to 0% inhibition), and positive controls containing free fluorescent probe only (equivalent to 100% inhibition), were included in each assay plate. FP values were measured as described above. IC50 values were determined by nonlinear regression fitting of the sigmoidal dose-dependent FP decreases as a function of total compound concentrations using Graphpad Prism 6.0 software. Ki values of competitive inhibitors were obtained directly by nonlinear regression fitting as well, based upon the Kd values of the probe to different proteins and concentrations of the proteins and probes in the competitive assays.

Cell proliferation assay [3]
Cell Types: AGS and MKN45 cells
Tested Concentrations: 0.0001μM, 0.001μM, 0.01μM, 0.1μM, 1μM, 10μM, 100μM
Incubation Duration: 72 hrs (hours)
Experimental Results: and S phase reduction [4]. Inhibits cell proliferation in a concentration-dependent manner.

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RT-PCR[3]
Cell Types: AGS and MKN45 Cell
Tested Concentrations: 0.02 μM, 0.2 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: MDM2, p21, PUMA and BAX mRNA expression increased.

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Cell cycle analysis[3]
Cell Types: AGS and MKN45 Cell
Tested Concentrations: 0.02 μM, 0.2 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Cell arrest in G0/G1 phase.

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Apoptosis analysis [4]
Cell Types: DePTC p53 wide
Cell Types: TPC-1 cells, KTC-1 cells
Tested Concentrations: 0.3μM, 1μM, 3μM, 10μM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished number of cells in S phase, while cells accumulate in the G2/M phase.

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Western Blot Analysis[3]
Cell Types: AGS and MKN45 Cell
Tested Concentrations: 0.2 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: MDM2 and p53 expression were enhanced, and stable knockdown of p53 eliminated them.

Animal/Disease Models: 4weeks old male BALB/c athymic nude mice, MKN45 cells [3]
Doses: 100 mg/kg
Route of Administration: po (po (oral gavage)) one time/day; 10-day
Experimental Results: Xenograft tumor growth diminished.

[1]. Angelo Aguilar, et al. 4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-fluorophenyl)-1′-ethyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development. J Med Chem. 2017 Apr 13; 60(7): 2819–2839.
[2]. A W Tolcher et al, A phase Ib/II study of APG-115 in combination with MK-3475 in patients with unresectable or metastatic melanomas or advanced solid tumors, Ann Oncol. 2019 Feb 1; 30(Supplement_1). pii: mdz027.
[3]. Hanjie Yi ea al, A novel small molecule inhibitor of MDM2-p53 (APG-115) enhances radiosensitivity of gastric adenocarcinoma, J Exp Clin Cancer Res. 2018 May 2;37(1):97.
[4]. Chen H, et al. Restoration of p53 using the novel MDM2-p53 antagonist APG115 suppresses dedifferentiated papillary thyroid cancer cells. Oncotarget. 2017 Jun 27;8(26):43008-43022.
[5]. Design of Chemically Stable, Potent, and Efficacious MDM2 Inhibitors That Exploit the Retro-Mannich Ring-Opening-Cyclization Reaction Mechanism in Spiro-oxindoles. J Med Chem. 2014 Dec 26; 57(24): 10486–10498.

C34H38CL2FN3O4

642.5934

641.2223

C, 63.55; H, 5.96; Cl, 11.03; F, 2.96; N, 6.54; O, 9.96

1818393-16-6

1818393-16-6

White to off-white solid

7.63

O=C1NC2=CC(Cl)=CC=C2[C@]13C4(CCCCC4)N(CC)[C@@H](C(NC5(CC6)CCC6(C(O)=O)CC5)=O)[C@@H]3C7=C(F)C(Cl)=CC=C7

YJCZPJQGFSSFOL-MNZPCBJKSA-N

InChI=1S/C34H38Cl2FN3O4/c1-2-40-27(28(41)39-32-16-13-31(14-17-32,15-18-32)30(43)44)25(21-7-6-8-23(36)26(21)37)34(33(40)11-4-3-5-12-33)22-10-9-20(35)19-24(22)38-29(34)42/h6-10,19,25,27H,2-5,11-18H2,1H3,(H,38,42)(H,39,41)(H,43,44)/t25-,27+,31?,32?,34+/m0/s1

4-((3'R,4'S,5'R)-6''-chloro-4'-(3-chloro-2-fluorophenyl)-1'-ethyl-2''-oxodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indoline]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic acid

AA-115; APG115; AA 115; APG115; AA115; APG-115

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 (~155.62 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (7.78 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 50.0 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: ≥ 5 mg/mL (7.78 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 50.0 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: ≥ 2.5 mg/mL (3.89 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.)