Leishmania; Akt1 E17K mutant; Akt1 (IC50 = 2.7 nM); Akt3 (IC50 = 8.1 nM); Akt2 (IC50 = 174 nM)

Miransertib (ARQ-092; Compound 21a) exhibits stronger anti-proliferative activity in cell lines with PIK3CA/PIK3R1 mutations than in cell lines with wild-type (wt) PIK3CA/PIK3R1 or PTEN loss in a large panel of cell lines derived from various tumor types. In both AN3CA and A2780 cells, miransertib exhibits excellent inhibition of p-Akt (S473) and p-Akt (T308). With Miransertib (IC50=0.31 M), the downstream protein p-PRAS40 (T246) is seen to be inhibited[1]. Miransertib is markedly effective against intracellular amastigotes of L. donovani or L. amazonensis-infected macrophages. Additionally, in Leishmania-infected macrophages, miransertib increases mTOR dependent autophagy. [2]

In rats (5 mg/kg) and monkeys (10 mg/kg), miransertib (ARQ-092; Compound 21a) exhibits good absolute oral bioavailability with F values of 62% and 49%, respectively. Rats' t1/2 values are 17 h compared to monkeys' 7 h, indicating that rats have a longer half-life than monkeys. Rats and monkeys, respectively, had Cmax values of 198 ng/mL and 258 ng/mL, and AUCinf values of 5496 hng/mL and 2960 hng/mL, respectively[1]. Miransertib (ARQ-092; Compound 21a) inhibits tumor growth in a human xenograft mouse model of endometrial adenocarcinoma[1].

Miransertib (ARQ-092) is an orally bioavailable, selective, and potent allostericAktinhibitor withIC50s of 2.7 nM, 14 nM and 8.1 nM forAkt1,Akt2,Akt3, respectively.

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AKT1 (1–480), AKT2 (1–481, and AKT3 (1–479) Alpha Screen Assays[4]
AKT activity was assayed using a GSK3-derived biotinylated peptide substrate, crosstide (biotin-GRPRTSSFAEG), and AlphaScreen (amplified luminescent proximity homogeneous assay) technology. Test compounds and controls were prepared in 10% DMSO at 10-fold the desired final concentration, and 2.5 μL of each compound was added to each well of a reaction plate (Corning 96-well half-area solid white nonbinding surface plate). Full-length unphosphorylated AKT was diluted in assay buffer (50 mM Tris, pH 8.0, 0.02 mg/mL BSA, 10 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.2 mM Na3VO4, 1 mM DTT, 0.1 mM β-glycerophosphate, and 0.2 mM NaF) and added to each well at a volume of 17.5 μL for a final concentration of 8 nM (AKT1), 63 nM (AKT2), or 13 nM (AKT3) in the 25 μL reaction. After a 20 min preincubation at room temperature, the kinase reaction was initiated by the addition of 5 μL of the activation mixture diluted in assay buffer, containing biotinylated crosstide, PDK1, MAPKAPK2, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for a final concentration of 60 nM biotinylated crosstide, 0.1 nM (AKT1, AKT3) or 0.3 nM (AKT2) PDK1, 0.7 nM (AKT1), 1.3 nM (AKT2), or 0.4 nM (AKT3) MAPKAPK2, 5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)P3, and 50 μM (AKT1, AKT2) or 18 μM (AKT3) ATP. The plates were incubated for 30 min at room temperature, and the reactions were stopped in the dark by the addition of 10 μL of stop/detection mixture prepared in assay buffer, containing EDTA, AlphaScreen streptavidin donor and protein A acceptor beads, and phospho-AKT substrate antibody at final concentrations of 10 mM EDTA, 500 ng/well of both AlphaScreen streptavidin donor beads and protein A acceptor beads, and phospho-AKT substrate antibody at a final dilution of 1:350. Assay plates were incubated for 90 min at room temperature in the dark, and the plates were read on a PerkinElmer Envision multilabel plate reader (excitation wavelength, 640 nm; emission wavelength, 570 nm). All IC50 values reported are the geometric mean of at least n = 2 determinations.

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CYP450 Inhibition Assay[4]
Human liver microsomes (0.25 mg/mL), probe substrates [3 μM midazolam (3A4), 5 μM bufuralol (2D6), 100 μM tolbutamide (2C9), 10 μM paclitaxel (2C8), 80 μM S-mephenytoin (2C19), and 50 μM phenacetin (1A2)], 3.3 mM MgCl2, 1 mM NADPH, and the compound (0.1–10 μM) dissolved in DMSO (0.1% final) were incubated for 10 min, after which acetonitrile containing reserpine (internal standard) was used to stop the reaction. The solvents were evaporated under a constant stream of nitrogen gas at 35 °C, and the resulting compound was reconstituted in 100 μL of water for LC/MS/MS analysis. An inhibitors cocktail consisting of 0.5 μM ketoconazole (3A4), 1 μM sulfaphenazole (2C9), 1 μM quinidine (2D6), 2 μM quercetin (2C8), 1 μM nootkatone (2C19), and 0.5 μM α-naphthoflavone (1A2) was used as a positive control. Incubations containing only DMSO (no compound) served as the 100% activity control. The percent inhibition was calculated based on the signal for the 100% activity control, and the IC50 values were either estimated or determined using fit-model 205 (one-site dose response) in XLfit4.

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Cross-Species NADPH-Dependent Microsomal Stability Assay[4]
Human, CD-1 mouse, and Beagle dog liver microsomes (0.25 mg/mL), 3.3 mM MgCl2, and a NADPH-regenerating system (0.4 units/mL G6PDH, 1.3 mM NADP+, and 3.3 mM G6P) were incubated with 1 μM compound for 0, 3, 6, 10, 15, or 30 min. The incubations were stopped with acetonitrile containing warfarin (internal standard), and the samples were analyzed by LC/MS/MS. The peak area ratios were used to determine the percent remaining at each time point compared with time zero. The half-life values were estimated using the equation for monoexponential decay. Midazolam and propranolol (female mouse only) were used as positive controls.

Cells (MDA-MB-453: 1.5 106; NCI-H1650: 1 106; KU-19-19: 0.7 106) are seeded into 6 well plates, incubated for an overnight period, and then exposed to full media containing various concentrations of AKT inhibitors (ARQ 092, ARQ 751, MK-2206, GDC-0068) for 2 hours. Lysates are obtained after treating cells under predetermined conditions. Following SDS-PAGE, immunoblotting is used to separate the proteins from extracts.

Male SCD mice
100 mg/10ml/kg
oral administration

Compounds 9a, 9b, 21a–c, and 23 showed no significant inhibitory activity against CYP450 1A2, 2C8, 2D6, and 3A4. Compounds 9a and 9b exhibited sub-micromolar IC50 values for inhibition of CYP450 2C9, while 9a also inhibited CYP450 2C19 (IC50 ≤ 1 μM). All compounds demonstrated good stability in liver microsomes (human, mouse, and canine). Overall, we found that this series of compounds possesses good in vitro ADME properties, with compound ARQ 092 (21a) showing particularly good biochemical inhibitory activity, intracellular AKT phosphorylation inhibitory activity, and good ADME properties. In mouse pharmacokinetic studies (oral 100 mg/kg, intravenous 5 mg/kg), the oral bioavailability of compound ARQ 092 (21a) was 23%. Our recent article reports the results of in vivo pharmacodynamic evaluation of ARQ 092 (21a) in NCr-M nude mice implanted with AN3CA tumor xenografts. After oral administration of 100 mg/kg of compound 21a to tumor-bearing mice, the levels of p-AKT (S473), p-AKT (T306), and p-PRAS40 (T246) decreased by 99%, 95%, and 58%, respectively (Table 8). The phosphorylation inhibition lasted for 8 hours. The plasma concentration of compound 21a was 2.1 μM at 1 hour and decreased to 0.26 μM at 8 hours; while in tumor tissue, the concentration was 21.0 μM at 1 hour and 9.6 μM at 8 hours. The concentration in tumor tissue was significantly higher than that in plasma, indicating that the compound tends to accumulate in tissues rather than in blood vessels. [1]

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Definite single-dose pharmacokinetic studies were conducted in rats and monkeys (Table 9). ARQ 092 (21a) showed good absolute oral bioavailability in both rats and monkeys, with F values of 62% and 49%, respectively. The compound was absorbed more slowly in rats than in monkeys, with a Tmax of 8.0 h in rats and 4.3 h in monkeys. Furthermore, the half-life of the compound was longer in rats than in monkeys, with a t1/2 of 17 h in rats and 7 h in monkeys. The Cmax in rats and monkeys were 198 and 258 ng/mL, respectively, and the AUCinf were 5496 and 2960 h·ng/mL, respectively.

[1]. PLoS One. 2015 Oct 15;10(10):e0140479.

[2]. Haematologica. 2017 Feb;102(2):246-259.

[3]. Sci Rep. 2015 Dec 11;5:17162.

[4]. J Med Chem . 2016 Jul 14;59(13):6455-69.

Miransertib is being investigated in the clinical trial NCT01473095 (ARQ 092 Phase I dose-escalation study in adult patients with advanced solid tumors and relapsed malignant lymphoma). Miransertib is an orally bioavailable serine/threonine protein kinase AKT (protein kinase B) inhibitor with potential antitumor activity. Miransertib binds to and inhibits AKT in a non-ATP-competitive manner, which may lead to inhibition of the PI3K/AKT signaling pathway. This may result in reduced tumor cell proliferation and induction of tumor cell apoptosis. The AKT signaling pathway is frequently dysregulated in cancer and is closely associated with tumor cell proliferation, survival, and migration. This article describes the optimization process of 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridine-2-amine compounds as potent, selective allosteric inhibitors of AKT kinase, leading to the discovery of compound ARQ 092 (21a). The co-crystal structure of compound 21a with full-length AKT1 confirmed the allosteric inhibition mode of this class of compounds and the role of the cyclobutamine moiety. Compound 21a exhibited high enzymatic activity against AKT1, AKT2 and AKT3 and effectively inhibited the activation of AKT and the phosphorylation of its downstream target PRAS40. Compound 21a was also a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a mouse model of human endometrial adenocarcinoma xenograft. [1]

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Leishmaniasis is one of the most important neglected diseases, affecting more than 12 million people in 88 countries. There is an urgent need for safe, orally bioavailable and cost-effective treatments for leishmaniasis. Recent studies have shown that Leishmaniasis activates host macrophage serine/threonine kinase Akt, thereby promoting the survival of the parasite and infected cells. Here, we aim to evaluate a compound, Miransertib (ARQ 092), a highly bioavailable, selective allosteric Akt inhibitor currently undergoing clinical trials in patients with PI3K/Akt-driven tumors or Protes syndrome. Miransertib has been tested against Leishmania donovani and Leishmania serrata, the pathogens of visceral and cutaneous leishmaniasis. Cultured promastoids are sensitive to Miransertib. Furthermore, Miransertib also exhibits significant killing activity against amastoids within macrophages infected with Leishmania donovani or Leishmania serrata. Miransertib also enhances mTOR-dependent autophagy in Leishmania-infected macrophages, which may be one of the mechanisms by which Miransertib mediates the killing of intracellular Leishmania. Although the clearance rate of Leishmania donovani in the spleen of mice treated with Miransertib was comparable to that in mice treated with mitefonine, Miransertib was more effective at reducing the parasite load in the liver. In a cutaneous leishmaniasis model, the lesions in mice treated with Miransertib were reduced by 40% compared to mice in the sham treatment group. These results together provide direct evidence that Miransertib is an excellent lead compound for the development of novel oral therapies for the treatment of visceral and cutaneous leishmaniasis. [2]

C27H25CLN6

468.99

468.182

C, 69.15; H, 5.37; Cl, 7.56; N, 17.92

1313883-00-9

Miransertib;1313881-70-7

67305743

Light yellow to brown solid powder

3

5

4

34

653

0

Cl.NC1(C2C=CC(=CC=2)N2C(C3=CC=CN=C3N)=NC3C=CC(C4C=CC=CC=4)=NC2=3)CCC1

DRHSWSSVIKDJME-UHFFFAOYSA-N

InChI=1S/C27H24N6.ClH/c28-24-21(8-4-17-30-24)25-32-23-14-13-22(18-6-2-1-3-7-18)31-26(23)33(25)20-11-9-19(10-12-20)27(29)15-5-16-27;/h1-4,6-14,17H,5,15-16,29H2,(H2,28,30);1H

3-[3-[4-(1-aminocyclobutyl)phenyl]-5-phenylimidazo[4,5-b]pyridin-2-yl]pyridin-2-amine;hydrochloride

ARQ092 hydrochloride; MK-7075; ARQ-092 hydrochloride; MK7075; Miransertib HCl; Miransertib (ARQ 092) HCl; Miransertib hydrochloride; Miransertib (hydrochloride); CHEMBL4523032; 3-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine hydrochloride; ARQ 092

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.33 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)