cIAP1 (Ki = 1.9 nM); cIAP2 (Ki = 5.1 nM); XIAP (Ki = 66.4 nM)

In mice, rats, non-human primates, and dogs, AT-406 has good oral bioavailability and pharmacokinetic (PK) characteristics. AT-406 effectively induces cIAP1 degradation, procaspase-8 processing, and PARP cleavage in tumor tissues at 100 mg/kg with good tolerance even at 200 mg/kg in the MDA-MB-231 xenograft. At 100 mg/kg, AT-406 significantly inhibits tumor growth, with a p value of 0.0012. [1]

FL-AT-406 (the fluorescently tagged AT-406) is employed to develop a set of new FP assays for determination of the binding affinities of Smac mimetics to XIAP, cIAP-1, and cIAP-2 BIR3 proteins. Titration experiments using a fixed concentration of FL-AT-406 and varying concentrations of the protein up to full saturation are used to calculate the Kd value of FL-AT-406 to each IAP protein. A Microfluor 2 96-well, black, round-bottom plate is used to measure the fluorescence polarization values using an Infinite M-1000 plate reader. For experiments with XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3, FL-AT-406 (2, 1, and 1 nM for each well, respectively) and various protein concentrations are added to a final volume of 125 μL in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 g/mL bovine -globulin, 0.02% sodium azide, with 4% DMSO). After being thoroughly combined, the plates are gently shaken for two to three hours at room temperature. At an excitation wavelength of 485 nm and an emission wavelength of 530 nm, the polarization values in millipolarization units (mP) are measured. Then, using Graphpad Prism 5.0 software, equilibrium dissociation constants (Kd) are calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations. In competitive binding tests for XIAP3 BIR3, AT-406 is incubated with 20 nM XIAP BIR3 protein and 2 nM FL-AT-406 in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine γ-globulin; 0.02% sodium azide). 3 nM protein and 1 nM FL-AT-406 are used in experiments to determine competitive binding for the cIAP1 BIR3 protein. 5 nM protein and 1 nM FL-AT-406 are used in competitive binding tests for cIAP2 BIR3. Using an Infinite M-1000 plate reader, polarization values are determined for each competitive binding experiment after two to three hours of incubation. Using nonlinear least-squares analysis, the IC50 value, or inhibitor concentration at which 50% of the bound tracer is displaced, is extracted from the plot. The PRISM program is used to fit curves.

At a density of (3-4) × 103 cells/well, cells are seeded in 96-well flat-bottom cell culture plates with AT-406 and incubated for 4 days. Three to four 103 cells/well of AT-406-seeded cells are placed in 96-well flat-bottom cell culture plates, and the cells are then incubated for four days. The rate of cell growth inhibition after treatment with different concentrations of AT-406 is determined by assaying with (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8). WST-8 is added to each well to a final concentration of 10%, and then the plates are incubated at 37 °C for 2−3 hours. Using a TECAN ULTRA reader, the samples' absorbance is calculated at 450 nm. By comparing absorbance in cells that were not treated and cells that were treated with AT-406, the concentration of AT-406 that inhibited cell growth by 50% (IC50) can be determined. A TECAN ULTRA reader is used to measure the samples' absorbance at 450 nm. By comparing the absorbance of treated and untreated cells, the concentration of AT-406 that 50% inhibited cell growth (IC50) can be determined.

MDA-MB-231 xenograft tumors in severe combined immune deficiency (SCID) mice
10 mg/kg (i.v.), 10 mg/kg (p.o.), 30 mg/kg (p.o.) and 100 mg/kg (p.o.)
Administered via intravenously (i.v.) or oral gavage (p.o.)

[1]. J Med Chem . 2011 Apr 28;54(8):2714-26.

C32H43N5O4

561.71

561.3315

C, 68.42; H, 7.72; N, 12.47; O, 11.39

1071992-99-8

Xevinapant hydrochloride;1071992-57-8

White to off-white solid powder

C[C@@H](C(=O)N[C@H]1CN(CC[C@H]2CC[C@H](N2C1=O)C(=O)NC(C3=CC=CC=C3)C4=CC=CC=C4)C(=O)CC(C)C)NC

LSXUTRRVVSPWDZ-MKKUMYSQSA-N

InChI=1S/C32H43N5O4/c1-21(2)19-28(38)36-18-17-25-15-16-27(37(25)32(41)26(20-36)34-30(39)22(3)33-4)31(40)35-29(23-11-7-5-8-12-23)24-13-9-6-10-14-24/h5-14,21-22,25-27,29,33H,15-20H2,1-4H3,(H,34,39)(H,35,40)/t22-,25+,26-,27-/m0/s1

(5S,8S,10aR)-N-benzhydryl-5-[[(2S)-2-(methylamino)propanoyl]amino]-3-(3-methylbutanoyl)-6-oxo-1,2,4,5,8,9,10,10a-octahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide

DeBio-1143; DeBio1143; DeBio 1143; Xevinapant; AT 406; AT-406; AT406; SM406; SM 406; ARRY-334543; ARRY 334543; ARRY334543

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)