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

GDC-0068 only inhibits 3 kinases by >70% at 1 μM concentration when tested against a large panel of 230 kinases (PRKG1α, PRKG1β, and p70S6K, with IC50 values of 98 nM, 69 nM, and 860 nM, respectively). GDC-0068 has an IC50 of 3.1 μM and exhibits >100-fold selectivity for Akt over PKA. With IC50 values of 157 nM, 197 nM, and 208 nM, respectively, GDC-0068 treatment inhibits the phosphorylation of the Akt substrate, PRAS40, in LNCaP, PC3, and BT474M1 cells. Additionally, the Akt-signaling-driven cancer cell lines that have defects in the tumor suppressor PTEN, oncogenic mutations in PIK3CA, and HER2 amplification are all selectively inhibited by GDC-0068, with the strongest effects seen in the HER2+ and Luminal subtypes. [1-3]

GDC-0068 oral administration causes the down-regulation of p-PRAS40 in PC3 prostate tumor xenograft models. GDC-0068 treatment in BT474-Tr xenografts lowers pS6 and peIF4G levels, relocalizes FOXO3a to the nucleus, and causes feedback upregulation of HER3 and pERK. In numerous xenograft tumor models, including the PTEN-deficient prostate cancer models LNCaP and PC3, the PIK3CA H1047R mutant breast cancer model KPL-4, and the MCF7-neo/HER2 tumor model, administration of GDC-0068 demonstrates potent antitumor efficacy.[1-3]

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).

Inhibition of cellular viability was measured in LNCaP cells plated in black, clear-bottomed 96-well plates at a density of 5000 cells/well and subsequently treated with 0–10 μM 28 (ipatasertib) for 72 h at 37 °C and 5% CO2. The extent of cell proliferation was determined by measuring the reduction of resazurin to resorufin as described in the manufacturer’s protocol using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Dose–response curves were generated using the four-parameter logistic model, and 50% inhibitory concentration (IC50) values were determined from these curve fits.[1]

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Briefly, after ipatasertib treatment, HCT116 WT or p53−/− were fixed with 1% formaldehyde and lysed in warm SDS lysis buffer. The genomic DNA was obtained and sheared to 200–1000 bp by sonication on ice. Samples were precleared with Protein A-Agarose/Salmon Sperm DNA (50% Slurry) for 1 h at 4 °C with agitation. Then anti-FoxO3a antibody or anti-p65 antibody was added and incubated overnight on a shaker at 4 °C. Normal rabbit IgG was used as a negative control. The protein agarose/salmon sperm DNA (50% slurry) bead was then added to precipitate the antibody/protein/DNA complexes. After washed with serial wash buffers, DNA–protein immunocomplexes were eluted from the beads by elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min. Finally, the protein–DNA cross-links were reversed to release DNA by incubation with 0.2 M NaCl at 65 °C for 4 h[3].

Female nude mice bearing LNCaP, PC3, KPL-4, or MCF7 tumor xenografts
~100 mg/kg/day
Orally

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For in vivo tumor xenograft studies, female nu/nu (nude) mice were inoculated subcutaneously in the right hind flank with PC3 cells suspended in Hank’s balanced salt solution (HBSS). When tumors reached a mean volume of 150 mm3, the animals were size matched and distributed into treatment groups consisting of 10 animals/group. Tumor volume was calculated as follows: tumor size (mm3) = (longer measurement × (shorter measurement)2) × 0.5. Following data analysis, p values were determined using Dunnett’s t test with JMP statistical software, version 7.0 (SAS Institute). Mouse body weights were recorded twice weekly using an Adventura Pro AV812 scale (Ohaus Corp.). Mice were promptly euthanized when the tumor volume exceeded 2000 mm3 or if body weight loss was ≥20% of the starting weight per IACUC protocol guidelines.[1]

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For PK/PD studies, blood and tumor samples were collected at 1, 3, 8, and 24 h after a single dose of ipatasertib from PC3 tumor bearing mice. Blood samples (approximately 800 μL) were collected from each animal at the scheduled sample collection time by terminal cardiac puncture into tubes containing K2EDTA as an anticoagulant and centrifuged at 1500–2000g to isolate plasma. The concentration of ipatasertib in each plasma sample was determined by a nonvalidated LC/MS/MS assay in the DMPK Bioanalytical Department at Genentech. The assay lower limit of quantitation (LLOQ) was 0.005 μM. Tumor samples were dissociated in Tris lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100. Protein concentrations were determined using the BCA Protein Assay Kit. The human enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of total PRAS40 and PRAS40 phosphorylated at Thr246 (p-PRAS40). The assay quantifies protein levels on the basis of measurements of absorbance. The colored product is directly proportional to the concentration of p-PRAS40 and tPRAS40 present in the specimen. The Meso Scale Discovery Multi-Spot Biomarker Detection System was used to determine the levels of total S6RP and S6RP phosphorylated at Ser235/236 (pS6RP). These assays quantify protein levels on the basis of measurements of electrochemiluminescence intensity. Levels of phosphorylated protein were normalized to total protein levels in ipatasertib-treated tumors and compared to the vehicle control.[1]

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In vivo efficacy was evaluated in multiple tumor cell line- and patient-derived xenograft models. Cells or tumor fragments were implanted subcutaneously into the flank of immunocompromised mice. Female or male nude (nu/nu) or severe combined immunodeficient mice (SCID)/beige mice were used. For the MCF7-neo/HER2 model, 17β-estradiol pellets (0.36 mg/pellet, 60-day release, no. SE-121) were implanted into the dorsal shoulder before cell inoculation. Male mice were castrated before implantation of tumor fragments. After implantation of tumor cells or fragments into mice, tumors were monitored until they reached mean tumor volumes of 180 to 350 mm3 and distributed into groups of 8 to 10 animals/group. ipatasertib/GDC-0068 was formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT) and administered daily (QD), via oral (per os; PO) gavage.[2]

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HCT116 WT and PUMA−/− were harvested, and 1 × 10~6 cells in 0.2 ml of medium were implanted subcutaneously into the back of athymic nude female mice. Female 5-week-old nude mice were housed in a sterile environment with microisolator cages and allowed access to water and chow ad libitum. Mice were treated daily with ipatasertib/GDC-0068 at 40 mg/kg by oral gavage for 21 days treatment after 7 days. Calipers were used to monitor the tumor growth, volume was calculated by the formula: 0.5 × length × width2. Mice were euthanized when tumors reached ~1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin.[3]

[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

[3]. Ipatasertib, a novel Akt inhibitor, induces transcription factor FoxO3a and NF-κB directly regulates PUMA-dependent apoptosis. Cell Death Dis. 2018 Sep 5;9(9):911.

(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.

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Drug Indication
Treatment of breast cancer , Treatment of prostate cancer

C24H32CLN5O2

457.9962

457.224

C, 62.94; H, 7.04; Cl, 7.74; N, 15.29; O, 6.99

1001264-89-6

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

24788740

White to light yellow solid powder

1.3±0.1 g/cm3

669.4±55.0 °C at 760 mmHg

358.7±31.5 °C

0.0±2.1 mmHg at 25°C

1.603

1.71

2

6

6

32

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

GRZXWCHAXNAUHY-NSISKUIASA-N

InChI=1S/C24H32ClN5O2/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,8-13H2,1-3H3/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-one

GDC0068; GDC 0068; GDC-0068; RG-7440; 1001264-89-6; Ipatasertib (GDC-0068); RG7440; (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one; RG-7440; GDC0068; RG 7440; RG7440; Ipatasertib

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: ~92 mg/mL (~200.9 mM)
Water: <1 mg/mL
Ethanol: ~92 mg/mL (~200.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.54 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 2: ≥ 2.08 mg/mL (4.54 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 3: ≥ 2.08 mg/mL (4.54 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 4: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 92mg/ml

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Solubility in Formulation 5: 10 mg/mL (21.83 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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