Akt1 (IC50 = 5 nM); Akt3 (IC50 = 8 nM); Akt2 (IC50 = 18 nM); PKA (IC50 = 3100 nM)

Over 600-fold and over 100-fold, respectively, more selective for Akt1 at IC50 than for the closely related PKA and p70S6K, is exhibited by imatasertib diHClide. Ipatasertib diHClide inhibited only three protein conjugates (PRKG1α, PRKG1β, and p70S6K) by more than 70% at 1 μM in a panel of 230 protein conjugates, including 36 members of the human AGC family. ..For these three species, the IC50 values that were periodically measured were 98, 69, and 860 nM, in that order. Hence, in comparison to the next most potent non-Akt inhibitor, p70S6K Multiples panel above, ipatasertib dihydrochloride is 100 times more selective for Akt1 in the screen, with the exception of PKG1 (where ipatasertib dihydrochloride has >10-fold greater Akt1 selectivity). Three xenograft models demonstrating dose-dependent responses to drug treatment were used to study the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD) of ipatasertib dihydrochloride: MCF7-neo. The average cell viability IC50 of /HER2, TOV-21G.x1, and LNCaP Ipatasertib diHClide in these three cell lines were 2.56, 0.44, and 0.11 μM, respectively [2].

In transplantation models where Akt is triggered by genetic changes such as PTEN loss, PIK3CA mutations or mutations, or HER2 overexpression, imatinib diHClide is often successful. The tumors in these models developed slowly, were generated, or reverted at or below 100 mg/kg, the maximum well-tolerated dose observed in immunocompromised mice. When examined in vivo, the daily combination of RP-56976 with Ipatasertib diHClide produced analgesia and tumor regression in PC-3 and MCF7-neo/HER2 xenograft mice, but the toxin by itself was either ineffective or just caused the tumor to develop. gradually increasing dosages. Similarly, when Ipatasertib diHClide and NSC 241240 were coupled, a rise in TGI was seen in an OVCAR3 ovarian cancer xenograft model. Compared to treatment with extra chemotherapy, Ipatasertib diHClide in combination with RP-56976 or NSC 241240 alone resulted in less than 5% weight loss [2].

Enzymatic Assays[1]
The assay for the determination of Akt1/2/3 and PKA kinase activity employs the IMAP fluorescence polarization (FP) phosphorylation detection reagent to detect fluorescently labeled peptide substrates that have been phosphorylated by the respective kinases. The Akt enzymes employed in these studies consisted of recombinant baculovirus expressed, amino-terminal, polyhistidine-tagged, full-length, wild-type human forms and were obtained commercially. The PKA enzyme employed in these studies consisted of the recombinant untagged human isolated catalytic subunit of PKA expressed in Escherichia coli obtained commercially. Inhibitor, enzyme (9 nM Akt1 or 100 pM PKA), and substrate (100 nM Crosstide) were incubated with 5 μM ATP in assay buffer (10 mM Tris–HCl (pH 7.2), 10 mM MgCl2, 0.1% BSA (w/v), final DMSO 2% (v/v)) for 60 min at ambient temperature in a 5 μL reaction volume. Reactions were initiated by addition of enzyme + peptide substrate to ATP solutions. IMAP binding reagent (15 μL) was added to terminate the reaction, and the stopped reactions were incubated for a minimum of 30 min at room temperature (rt).

The 384-well plates are seeded with 2,000 cells per well in a volume of 54 L, and then incubated overnight (roughly 16 hours) at 37°C with 5% CO2. To create the desired stock concentrations, compounds (like Ipatasertib) are diluted in DMSO before being added in a volume of 6 L per well. Each treatment is tested four times. After four days of incubation, total luminescence is measured on a Wallac Multilabel Reader, and relative viability is estimated using CellTiter-Glo. The 4-parameter curve analysis (XLfit) is used to calculate the drug concentration that produces an IC50 and is based on the results of a minimum of three experiments. The highest concentration tested (10 M) is listed for cell lines that failed to reach an IC50[2].

Mice：Numerous patient-derived xenograft models and tumor cell line models are used to evaluate in vivo efficacy. Immunocompromised mice have cells or tumor fragments implanted subcutaneously into their flanks. Mice that are severely combined immunodeficient (SCID) or beige, or both, are used. Male mice are castrated prior to the implantation of tumor fragments, and the LuCaP35V patient-derived primary tumors are obtained. When tumor cells or fragments are implanted into mice, the tumors are watched until they reach mean tumor volumes of 180 to 350 mm3 and are then divided into groups of 8 to 10 animals each. Ipatasertib is administered every day (QD) via oral (per os; PO) gavage and is formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT). Every week (QW), 2.5 or 7.5 mg/kg of RP-56976 is administered intravenously (IV) in a solution of 3% EtOH/97% saline. Saline-based NSC 241240 is administered intraperitoneally (IP) once a week at a dose of 50 mg/kg.

[1]. Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors. J Med Chem. 2012 Sep 27;55(18):8110-27.

[2]. Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models. Clin Cancer Res. 2013 Apr 1;19(7):1760-72.

(2S)-2-(4-chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]-1-piperazinyl]-3-(propan-2-ylamino)-1-propanone is a N-arylpiperazine.
Ipatasertib has been used in trials studying the treatment of Cancer, Neoplasms, Solid Cancers, Breast Cancer, and Gastric Cancer, among others.

Ipatasertib is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Ipatasertib 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 and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.
Drug Indication: Treatment of breast cancer , Treatment of prostate cancer.

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The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.[1]

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Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling.[2]

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Colon cancer is one of the three common malignant tumors, with a lower survival rate. Ipatasertib, a novel highly selective ATP-competitive pan-Akt inhibitor, shows a strong antitumor effect in a variety of carcinoma, including colon cancer. However, there is a lack of knowledge about the precise underlying mechanism of clinical therapy for colon cancer. We conducted this study to determine that ipatasertib prevented colon cancer growth through PUMA-dependent apoptosis. Ipatasertib led to p53-independent PUMA activation by inhibiting Akt, thereby activating both FoxO3a and NF-κB synchronously that will directly bind to PUMA promoter, up-regulating PUMA transcription and Bax-mediated intrinsic mitochondrial apoptosis. Remarkably, Akt/FoxO3a/PUMA is the major pathway while Akt/NF-κB/PUMA is the secondary pathway of PUMA activation induced by ipatasertib in colon cancer. Knocking out PUMA eliminated ipatasertib-induced apoptosis both in vitro and in vivo (xenografts). Furthermore, PUMA is also indispensable in combinational therapies of ipatasertib with some conventional or novel drugs. Collectively, our study demonstrated that PUMA induction by FoxO3a and NF-κB is a critical step to suppress the growth of colon cancer under the therapy with ipatasertib, which provides some theoretical basis for clinical assessment.[3]

C₂₄H₃₄CL₃N₅O₂

530.9181

529.178

C, 54.30; H, 6.46; Cl, 20.03; N, 13.19; O, 6.03

1396257-94-5

Ipatasertib;1001264-89-6; 1489263-16-2 (HCl); 1491138-24-9; 1396257-94-5 (2HCl); 1491138-23-8 (besylate)

62707512

Light yellow to yellow solid powder

5.098

4

6

6

34

622

3

ClC1C([H])=C([H])C(=C([H])C=1[H])[C@@]([H])(C([H])([H])N([H])C([H])(C([H])([H])[H])C([H])([H])[H])C(N1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C1C2=C([C@@]([H])(C([H])([H])[C@@]2([H])C([H])([H])[H])O[H])N=C([H])N=1)=O.Cl[H].Cl[H]

SRKVNRNRVFDUTG-VISIQVHMSA-N

InChI=1S/C24H32ClN5O2.2ClH/c1-15(2)26-13-19(17-4-6-18(25)7-5-17)24(32)30-10-8-29(9-11-30)23-21-16(3)12-20(31)22(21)27-14-28-23/h4-7,14-16,19-20,26,31H,8-13H2,1-3H32*1H/t16-,19-,20-/m1../s1

(2S)-2-(4-Chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]piperazin-1-yl]-3-(propan-2-ylamino)propan-1-onedihydrochloride

Ipatasertib dihydrochloride; Ipatasertib; GDC 0068; GDC-0068; GDC0068; RG7440; 1396257-94-5; GDC-0068 (dihydrochloride); UNII-M2JCB5A8EV; M2JCB5A8EV; Ipatasertib (dihydrochloride); (2S)-2-(4-Chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]piperazin-1-yl]-3-(propan-2-ylamino)propan-1-one;dihydrochloride; RG 7440; RG-7440

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (188.35 mM; Need ultrasonic)
H2O: ≥41 mg/mL (77.22 mM)

Solubility in Formulation 1: ≥ 3.88 mg/mL (7.31 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 3.88 mg/mL (7.31 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (3.92 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 20.8 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 4: ≥ 2.08 mg/mL (3.92 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 20.8 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 5: ≥ 2.08 mg/mL (3.92 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 16.67 mg/mL (31.40 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)