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,b, 21a–c, and 23 did not significantly inhibit CYP450 1A2, 2C8, 2D6, and 3A4. Compounds 9a and 9b inhibited CYP450 2C9 with a submicromolar IC50 value, while 9a also inhibited CYP450 2C19 (IC50 ≤ 1 μM). All compounds were adequately stable in liver microsomes (human, mouse, and dog). In general, we found that compounds from this chemical series possess adequate in vitro ADME properties, and compound ARQ 092 (21a) in particular showed good biochemical inhibition, cellular knockdown of AKT phosphorylation, and ADME properties. In a mouse pharmacokinetic study (po at 100 mg/kg, iv at 5 mg/kg), compound ARQ 092 (21a) showed an oral bioavailability of 23%. An in vivo pharmacodynamic assessment of ARQ 092 (21a) using NCr-M nude mice implanted with AN3CA tumor xenografts was reported in our recent publication. Compound 21a resulted in 99%, 95%, and 58% reductions in p-AKT (S473), p-AKT (T306), and p-PRAS40 (T246), respectively, after tumor-bearing mice were treated with 100 mg/kg po (Table 8). The inhibition of phosphorylation was sustained at 8 h. The plasma concentration of compound 21a at 1 h was 2.1 μM and decreased to 0.26 μM at 8 h, while in the tumor, the concentration was 21.0 μM at 1 h and 9.6 μM at 8 h. The concentrations in the tumor tissues were significantly higher than in the plasma, indicating a marked preference for tissue accumulation compared with the vasculature compartment.[1]

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Definitive single dose pharmacokinetic studies were conducted in rats and monkeys (Table 9). ARQ 092 (21a) showed good absolute oral bioavailability in rats and monkeys with F values of 62% and 49%, respectively. The compound was more slowly absorbed in rats compared to monkeys with Tmax values of 8.0 h for rats versus 4.3 h for monkeys. The half-life was also longer in rats compared to monkeys with t1/2 values of 17 h in rats versus 7 h in monkeys. The Cmax was 198 and 258 ng/mL and the AUCinf was 5496 and 2960 h·ng/mL in rats and monkeys, 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 under investigation in clinical trial NCT01473095 (Phase 1 Dose Escalation Study of ARQ 092 in Adult Subjects With Advanced Solid Tumors and Recurrent Malignant Lymphoma).
Miransertib is an orally bioavailable inhibitor of the serine/threonine protein kinase AKT (protein kinase B) with potential antineoplastic activity. Miransertib binds to and inhibits the activity of AKT in a non-ATP competitive manner, which may result in the inhibition of the PI3K/AKT signaling pathway. This may lead to the reduction in tumor cell proliferation and the induction of tumor cell apoptosis. The AKT signaling pathway is often deregulated in cancer and is associated with tumor cell proliferation, survival and migration.

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The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma.[1]

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Leishmaniasis is amongst the most important neglected diseases, afflicting more than 12 million people in 88 countries. There is an urgent need for safe orally bioavailable and cost-effective drugs for the treatment of leishmaniasis. It has recently been shown that Leishmania activates host macrophage serine/threonine kinase Akt, to promote survival of both parasites and infected cells. Here, we sought to evaluate a compound, Miransertib (ARQ 092), an orally bioavailable and selective allosteric Akt inhibitor currently in clinical trials for patients with PI3K/Akt-driven tumors or Proteus syndrome. Miransertib was tested against Leishmania donovani and Leishmania amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Cultured promastigotes were susceptible to Miransertib. In addition, Miransertib was markedly effective against intracellular amastigotes of L. donovani or L. amazonensis-infected macrophages. Miransertib also enhanced mTOR dependent autophagy in Leishmania-infected macrophages, which may represent one mechanism of Miransertib-mediated killing of intracellular Leishmania. Whereas parasite clearance in the spleen of mice infected with L. donovani and treated with Miransertib was comparable to that when treated with miltefosine, Miransertib caused a greater reduction in the parasite load in the liver. In the cutaneous leishmaniasis infection model, lesions were reduced by 40% as compared to mock treated mice. Together, these results provide direct evidence to support the conclusion that Miransertib is an excellent lead compound for the development of a new oral drug therapy for 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.)