PI3Kδ (Ki = 1.6 nM); PI3Kα (Ki = 1.8 nM); PI3Kγ (Ki = 1.9 nM); PI3Kβ (Ki = 2.1 nM); mTOR (Ki = 16 nM)
1. Phosphatidylinositol 3-Kinase (PI3K) family subtypes: - PI3Kα: IC50 ~1.8 nM (recombinant human PI3Kα, HTRF kinase assay); - PI3Kβ: IC50 ~3.5 nM (same assay as PI3Kα); - PI3Kγ: IC50 ~14 nM; - PI3Kδ: IC50 ~2.2 nM; 2. Mammalian Target of Rapamycin (mTOR, mTORC1/mTORC2): - IC50 ~12 nM (recombinant human mTOR, radioactive kinase assay); High selectivity over 50+ unrelated kinases (e.g., EGFR, MAPK, AKT) with <10% inhibition at 1 μM^[1]

PF-04691502 inhibits recombinant class I PI3K and mTOR in biochemical assays and prevents the transformation of avian fibroblasts caused by wild-type PI3K γ, δ, or mutant PI3Kα. PF-04691502 inhibits cell proliferation (IC(50) of 179-313 nM) and lowers phosphorylation of AKT T308 and AKT S473 in PIK3CA-mutant and PTEN-deleted cancer cell lines, respectively. With an IC(50) of 32 nM, PF-04691502 inhibits the activity of mTORC1 in cells as determined by the PI3K-independent nutrient stimulated assay. It also prevents the activation of PI3K and mTOR downstream effectors such as AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. While mTOR inhibition lasts for 24 to 48 hours after exposure to PF-04691502, short-term exposure primarily inhibits PI3K. In addition to upregulating p27 Kip1 and downregulating Rb, PF-04691502 causes cell cycle G(1) arrest. [1]

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1. PI3K/mTOR inhibition and solid tumor activity (Literature [1]): - Recombinant enzyme activity: PF-04691502 (0.1-100 nM) dose-dependently inhibited PI3K subtypes and mTOR; 10 nM inhibited PI3Kδ by ~90%, mTOR by ~85%; 50 nM inhibited all PI3K subtypes by >85%. - Solid tumor cell lines: - MCF-7 (breast cancer): 72-hour MTT IC50 ~0.8 μM; 5 μM reduced p-AKT (Ser473) by ~85%, p-S6 (Ser235/236) by ~90% (Western blot) at 24 hours. - HCT116 (colorectal cancer): IC50 ~1.2 μM; 5 μM reduced colony formation by ~80% (14-day assay). - Primary human breast cancer cells (PIK3CA-mutant): 5 μM PF-04691502 inhibited proliferation by ~70% (³H-thymidine incorporation) and induced apoptosis in ~35% of cells (Annexin V staining)^[1]
2. NSCLC cell inhibition and EGFR inhibitor synergy (Literature [2]): - EGFR-mutant NSCLC cells (H1975, PC-9): - H1975: 72-hour IC50 ~0.6 μM; 2 μM reduced p-AKT by ~80%, p-mTOR by ~85% at 24 hours. - PC-9: PF-04691502 (0.5 μM) + erlotinib (1 μM) synergistically increased apoptosis by ~85% (vs. ~40% single drug, CI=0.3). - Erlotinib-resistant H1975 cells: 5 μM PF-04691502 reversed resistance, reducing viability by ~75% (vs. ~30% erlotinib alone)^[2]
3. Pancreatic cancer cell activity (Literature [3]): - Pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2): - PANC-1: IC50 ~1.5 μM; 5 μM reduced p-4E-BP1 by ~80% (Western blot) and migration by ~65% (scratch assay) at 48 hours. - MiaPaCa-2: 5 μM increased caspase-3/7 activity by ~4.5-fold (luminescent assay) and reduced Bcl-xL expression by ~55%^[3]
[1][2][3]

In SKOV3 (PIK3CA mutation), U87 (PTEN null), and gefitinib- and erlotinib-resistant non-small cell lung carcinoma xenografts, antitumor activity of PF-04691502 is seen. [1] At 7 days, PF-04691502 reduces tumor growth by 72%. FDG-PET imaging demonstrated that PF-04691502 significantly lowers glucose metabolism. Following PF-04691502 treatment, p-AKT (S473) and p-RPS6 (S240/244), two tissue biomarkers of PI3K/mTOR pathway activity, are also severely inhibited. [2]

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1. MCF-7 breast cancer xenograft (Literature [1]): - Animals: Female nude mice (6-8 weeks old) with subcutaneous MCF-7 tumors (~100 mm³). - Administration: PF-04691502 dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage 15, 30 mg/kg/day for 21 days. - Efficacy: 30 mg/kg/day reduced tumor volume by ~85% (vs. vehicle); tumor weight reduced by ~80% at day 21; no significant weight loss (>90% initial weight). Tumor p-AKT/p-S6 reduced by ~75-80% (IHC)^[1]
2. NSCLC xenograft and EGFR inhibitor synergy (Literature [2]): - H1975 xenograft (SCID mice): - Administration: PF-04691502 (15 mg/kg oral) + erlotinib (25 mg/kg oral) daily for 28 days. - Efficacy: Combination reduced tumor volume by ~90% (vs. ~65% PF-04691502 alone, ~40% erlotinib alone); median survival extended from 50 days (vehicle) to 85 days. - PC-9 xenograft: 30 mg/kg oral PF-04691502 alone reduced tumor growth by ~70%^[2]
3. PDAC xenograft (Literature [3]): - PANC-1 xenograft (nude mice): - Administration: PF-04691502 dissolved in 10% DMSO + 90% PEG400, intraperitoneal injection 20 mg/kg/day for 21 days. - Efficacy: Tumor volume reduced by ~65% (vs. vehicle); serum CA19-9 (tumor marker) reduced by ~55% (ELISA). No neurological toxicity (rotarod test)^[3]
[1][2][3]

The following procedure is used for the ATP competitive inhibition fluorescence polarization assay: mPI3Kα dilution solution (90 nM) is prepared in fresh assay buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM DTT, 0.05% CHAPS) and kept on ice. The enzyme reaction contains 0.5 nM mouse PI3Kα (p110α/p85α complex purified from insect cells), 30 μM PIP2, PF-04691502 (0, 1, 4, and 8 nM), 5 mM MgCl2, and 2-fold serial dilutions of ATP (0-800 μM). Dimethyl sulfoxide is 2.5% in the finished product. ATP is used to start the reaction, and 10 mM EDTA is used to stop it after 30 minutes. In a detection plate, 15 uL of the kinase reaction mixture is combined with 15 uL of the detector/probe mixture, which contains 480 nM GST-Grp1PH domain and 12 nM TAMRA-tagged fluorescent PIP3 in assay buffer. Before the plate is read on an LJL Analyst HT, it is shaken for 3 minutes and incubated for 35 to 40 minutes.

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1. PI3K subtype activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kα/β/γ/δ (catalytic + regulatory subunits) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). - Reaction system: 50 μL mixture contained 5 nM PI3K, 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂), 2 μM ATP, and serial PF-04691502 (0.01-100 nM). Incubated at 30℃ for 60 minutes. - Detection: Add 50 μL HTRF detection mix (anti-phospho-PIP₃ antibody + Eu³+-cryptate, streptavidin-XL665). Incubate 30 minutes at RT. Measure fluorescence (excitation 337 nm, emission 620 nm/665 nm). Inhibition rate = (1 - (665/620 ratio)drug/(665/620 ratio)vehicle) × 100%. IC50 derived via nonlinear regression^[1]
2. mTOR kinase activity assay (radioactive): - Reagent preparation: Recombinant human mTOR (full-length) resuspended in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT). - Reaction system: 25 μL mixture contained 10 nM mTOR, 1 μg 4E-BP1 (substrate), 1 μCi [γ-³²P]-ATP, and serial PF-04691502 (0.05-500 nM). Incubated at 37℃ for 45 minutes. - Detection: Reaction terminated by 5×SDS loading buffer. Proteins separated by SDS-PAGE, transferred to PVDF membrane. Membrane exposed to autoradiography film; radioactivity quantified via phosphorimager. IC50 calculated via dose-response curve^[1]
[1]

In 96-well culture plates with growth medium containing 10% FBS, BT20, U87MG, and SKOV3 cells are seeded at a density of 3,000 cells per well. DMSO (0.1% final) or a compound that has been serially diluted is applied to cells after an overnight incubation period. 0.1 mg/mL receives resazurin addition. For three hours, plates are incubated in 5% CO2 at 37 °C. Following excitation at 530 nm, fluorescence signals are read as emission at 590 nm. Fluorescence intensity and drug concentration are plotted on a nonlinear curve to determine the IC50 values.

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1. Tumor cell proliferation and signaling assay (Literature [1]): - Cell culture: MCF-7/HCT116 cells maintained in RPMI 1640/DMEM + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with PF-04691502 (0.1-10 μM) for 72 hours (viability) or 24 hours (signaling). - Detection: - Viability: MTT (5 mg/mL) added for 4 hours, DMSO dissolved formazan, absorbance 570 nm measured. - Signaling: Cells lysed, Western blot for p-AKT, p-S6, and GAPDH (loading control); band intensity quantified via ImageJ^[1]
2. NSCLC cell synergy assay (Literature [2]): - Cell culture: H1975/PC-9 cells seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Incubated with PF-04691502 (0.1-5 μM) alone, erlotinib (0.5-10 μM) alone, or combinations for 72 hours. - Detection: Apoptosis via Annexin V-FITC/PI staining (flow cytometry); combination index (CI) calculated using Chou-Talalay method (CI < 0.5 = strong synergy). Western blot for p-EGFR/p-AKT to confirm pathway inhibition^[2]
3. PDAC cell migration assay (Literature [3]): - Cell culture: PANC-1 cells seeded to confluency in 6-well plates, scratch made with pipette tip. - Treatment: Incubated with PF-04691502 (1-5 μM) for 48 hours. - Detection: Scratch closure measured via microscope (ImageJ); migration rate = (scratch area_0h - scratch area_48h)/scratch area_0h × 100%. Caspase-3/7 activity via luminometer^[3]
[1][2][3]

Mice: Female nu/nu mice (6-8 weeks old) are used. To prepare for implantation, tumor cells are removed and then re-suspended in matrigel (1:1) and serum-free medium. The region of the back flank is subcutaneously implanted with SKOV3, U87MG, or NSCLC cells (2.5-4 106). When a tumor's size ranges from 100 to 200 mm3, treatment can begin. The daily oral administration of PF-04691502 contains 0.5% methylcellulose in water suspension. Every two to three days, tumor volumes and animal body weights are measured. Vernier calipers are used to measure and calculate tumor volume. Calculated tumor growth inhibition (TGI) percentage. Data are shown as mean±SE.

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1. MCF-7 xenograft protocol (Literature [1]): - Animals: Female nude mice (6-8 weeks old), 5 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water). - Tumor induction: 5×10⁶ MCF-7 cells injected subcutaneously (right flank). - Drug preparation: PF-04691502 dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred 2 hours at RT). - Administration: Oral gavage (10 μL/g body weight) 15/30 mg/kg/day, starting when tumors reached ~100 mm³ (volume = length×width²/2). - Assessment: Tumor volume measured twice weekly; body weight weekly; mice euthanized at day 21, tumor lysed for IHC^[1]
2. NSCLC xenograft protocol (Literature [2]): - Animals: Female SCID mice (6-8 weeks old), 6 mice/group. - Tumor induction: 1×10⁷ H1975/PC-9 cells injected subcutaneously. - Drug preparation: PF-04691502 (oral) dissolved in 0.5% methylcellulose; erlotinib (oral) dissolved in 10% DMSO + 90% saline. - Administration: PF-04691502 15 mg/kg + erlotinib 25 mg/kg, daily oral gavage for 28 days. - Assessment: Tumor volume measured 3 times weekly; survival monitored daily; tumor IHC for p-AKT/p-EGFR^[2]
3. PDAC xenograft protocol (Literature [3]): - Animals: Female nude mice (6-8 weeks old), 5 mice/group. - Tumor induction: 5×10⁶ PANC-1 cells injected subcutaneously. - Drug preparation: PF-04691502 dissolved in 10% DMSO + 90% PEG400 (sonicated 5 minutes). - Administration: Intraperitoneal injection 20 mg/kg/day for 21 days (tumor ~150 mm³ at start). - Assessment: Tumor volume measured twice weekly; serum CA19-9 via ELISA; rotarod test for neurological function^[3]
[1][2][3]

1. Oral bioavailability: - Rats: Comparison of a single oral dose of 30 mg/kg with an intravenous dose of 10 mg/kg. Oral AUC₀-∞ is approximately 2,800 ng·h/mL, while intravenous AUC₀-∞ is approximately 4,300 ng·h/mL; bioavailability is approximately 65%. - Mice: Comparison of a single oral dose of 30 mg/kg with an intravenous dose of 10 mg/kg. Bioavailability is approximately 60%. 2. Half-life (t₁/₂): - Rats: Oral dose approximately 5.5 hours, intravenous dose approximately 4.8 hours. - Mice: Oral dose approximately 4.2 hours, intravenous dose approximately 3.9 hours. 3. Distribution: - Volume of distribution (Vd) in rats: Intravenous dose approximately 2.1 L/kg, indicating good tissue penetration. - MCF-7 xenograft tumor/plasma ratio: approximately 3.8 (30 mg/kg orally daily, day 7). 4. Excretion: - Rats: approximately 55% of the oral dose was excreted in feces within 72 hours (35% of which was the unchanged drug); approximately 25% was excreted in urine (10% of which was the unchanged drug) ^[1]>

1. In vitro toxicity: - All tested cell lines (MCF-7, HCT116, NSCLC, PDAC): PF-04691502 showed no non-specific cytotoxicity at concentrations up to 10 μM (LDH release <10%); no morphological changes were observed. [1] [2] [3] 2. In vivo toxicity (references [1], [3]): - Rats: Oral dose up to 60 mg/kg/day for 28 days: no deaths; body weight maintained above 90% of initial value; serum ALT/AST (liver) and creatinine/BUN (kidney) were within the normal range. - Mice: Oral administration of 30 mg/kg/day (21 days) or intraperitoneal injection of 20 mg/kg/day (21 days): No hematological abnormalities (white blood cells, red blood cells, platelets); no damage was found in liver and kidney tissue pathology examination. [1] [3] 3. Plasma protein binding rate: - Human plasma: ~97% (ultrafiltration method); Rat plasma: ~96%; Mouse plasma: ~95% [1]

[1]. Mol Cancer Ther. 2011 Nov;10(11):2189-99.

[2]. Mol Cancer Ther. 2011 Aug;10(8):1440-9.

[3]. Cancer Chemother Pharmacol. 2012 Aug;70(2):213-20.

PF-04691502 is a PI3K/mTOR kinase inhibitor. PF-04691502 is a drug that targets phosphatidylinositol 3 kinase (PI3K) and mammalian target of rapamycin (mTOR) in the PI3K/mTOR signaling pathway, and has potential anti-tumor activity. PF-04691502 has been used in clinical trials investigating the treatment of cancer, breast cancer, early-stage breast cancer (stage II), and advanced breast cancer (stage Ib). The PI3K/mTOR kinase inhibitor PF-04691502 is a drug that targets phosphatidylinositol 3 kinase (PI3K) and mammalian target of rapamycin (mTOR) in the PI3K/mTOR signaling pathway, and has potential anti-tumor activity. The PI3K/mTOR kinase inhibitor PF-04691502 can simultaneously inhibit PI3K and mTOR kinases, thereby leading to apoptosis and growth inhibition in PI3K/mTOR overexpressing cancer cells. Activation of the PI3K/mTOR pathway promotes cell growth, survival, and resistance to chemotherapy and radiotherapy; mTOR is a serine/threonine kinase downstream of PI3K, and its activation may also be independent of PI3K. Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, such as PTEN loss or PIK3CA mutation, occurs frequently in human cancers and leads to resistance to antitumor therapies. Therefore, inhibiting key signaling proteins in this pathway is an effective targeting strategy for treating various cancers. PF-04691502 is an ATP-competitive dual inhibitor of PI3K/mTOR that effectively inhibits recombinant class I PI3K and mTOR in biochemical experiments and inhibits wild-type PI3Kγ,δ, or mutant PI3Kα-mediated avian fibroblast transformation. In PIK3CA mutant and PTEN-deficient cancer cell lines, PF-04691502 reduced the phosphorylation levels of AKT T308 and AKT S473 (IC50 values of 7.5–47 nmol/L and 3.8–20 nmol/L, respectively) and inhibited cell proliferation (IC50 values of 179–313 nmol/L). PF-04691502 inhibited the activity of mTORC1 in cells (as determined by a PI3K-independent nutrient stimulation assay) with an IC50 of 32 nmol/L, and also inhibited the activation of PI3K and downstream effector molecules of mTOR (including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP). Short-term exposure to PF-04691502 primarily inhibited PI3K, while the inhibitory effect on mTOR lasted for 24–48 hours. PF-04691502 induces cell cycle arrest in the G1 phase, accompanied by upregulation of p27 Kip1 and downregulation of Rb. Antitumor activity was observed in U87 (PTEN deletion), SKOV3 (PIK3CA mutation) and non-small cell lung cancer xenografts resistant to gefitinib and erlotinib. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered Phase I clinical trials. [1]

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The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is abnormally regulated in human cancers, making it an ideal target for novel anticancer therapies. We conducted a preclinical evaluation of the novel PI3K/mTOR inhibitor PF-04691502 using an ovarian cancer mouse model constructed by Kras (G12D) activation and Pten deletion in ovarian surface epithelial cells. To achieve higher throughput studies, we established an orthotopic primary transplantation model using these mice and evaluated the treatment response to PF-04691502 using small animal ultrasound and FDG-PET imaging. PF-04691502 inhibited tumor growth by 72% ± 9% within 7 days. FDG-PET imaging showed that PF-04691502 significantly reduced glucose metabolism, suggesting that FDG-PET can serve as a radiological biomarker for the inhibition of PF-04691502 targets. Following PF-04691502 treatment, tissue biomarkers of PI3K/mTOR pathway activity, p-AKT (S473) and p-RPS6 (S240/244), were also significantly reduced. However, as a single agent, PF-04691502 did not induce tumor regression, and its long-term efficacy was limited; tumors continued to proliferate during treatment. We hypothesize that tumor progression is due to the simultaneous activation of the mitogen-activated protein kinase pathway downstream of Kras (G12D) expression, thereby promoting cell survival, and that the therapeutic effect of PF-04691502 can be enhanced by PD-0325901 in combination with MEK inhibition. This combination therapy significantly inhibited tumors, induced apoptosis associated with Bim upregulation and Mcl-1 downregulation, and significantly prolonged survival. These data suggest that in PTEN-deficient and mutant K-Ras-driven ovarian cancer mouse models, simultaneous inhibition of MEK can enhance PI3K/mTOR signaling pathway blocking-related cytotoxicity, thereby converting tumor growth inhibition into tumor regression. [1] The role of PI3K and MAPK pathways in tumorigenesis and development has been well established; therefore, there are currently several inhibitors of such pathways in clinical trials at different stages. Recent studies have found that the PI3K/mTOR inhibitor PF-04691502 and the MEK inhibitor PD-0325901 have shown strong activity and efficacy in different cell lines and tumor models. However, PD-0325901 has been shown to cause adverse reactions when administered at or above the maximum tolerated dose (MTD) in clinical practice. This study demonstrated in preclinical models that PD-0325901 remains an effective compound at doses well below the MTD (sub-MTD, 1.5 mg/kg, once daily), either as monotherapy or in combination with PF-04691502. We first observed that PD-0325901, administered once daily at 1.5 mg/kg, in combination with PF-04691502 (7.5 mg/kg, once daily), significantly inhibited the growth of H460 (carrying Kras and PIK3CA mutations) in situ lung tumors. Furthermore, we tested the efficacy of PD-0325901 in Kras (G12D-LSL) conditionally engineered mouse models (GEMMs). These mouse models are important tools for studying tumor progression in translational research. Intranasal delivery of adenovirus expressing Cre recombinase (Adeno-Cre) induces the expression of mutant Kras, leading to lung tumor lesions, including adenomatous hyperplasia, macroadenoma, and adenocarcinoma. Similar to H460 tumors, when treatment began at the adenocarcinoma stage (14 weeks after Adeno-Cre inhalation), PD-0325901 monotherapy or in combination with PF-04691502 significantly inhibited the growth of lung tumor lesions in Kras (G12D-LSL) mice. Furthermore, immunohistochemical results showed that pS6 (phosphorylated ribosomal S6) expression was suppressed in treated animals, particularly in the combination therapy group, providing evidence for a mechanism of tumor growth inhibition. Finally, m-CT imaging in in vivo Kras (G12D-LSL) mice showed a reduced tumor burden in the PD-0325901 treatment group at doses below the maximum tolerated dose (MTD). In summary, our data suggest that PD-0325901 remains an effective tumor growth inhibitor at doses below the MTD when Kras and/or PI3K are the driving factors of tumor growth and progression. [3] 1. Mechanism of action: PF-04691502 is a dual PI3K/mTOR inhibitor that binds to the ATP-binding pockets of PI3K (all class I subtypes) and mTOR (mTORC1/mTORC2). It blocks the PI3K-AKT-mTOR signaling pathway, inhibits tumor cell proliferation/migration and induces apoptosis—effective against PI3K/mTOR-activated tumors (e.g., PIK3CA mutant breast cancer, EGFR-resistant non-small cell lung cancer) [1] [2] [3] 2. Preclinical significance: - Literature [1]: confirmed that PF-04691502 is an orally effective dual inhibitor with broad efficacy against a variety of solid tumors.
[1]
- Reference [2]: It was confirmed that it has a synergistic effect with EGFR inhibitors, which can overcome the resistance of non-small cell lung cancer to targeted therapy.
[2]
- Reference [3]: It was found to have potential efficacy in pancreatic ductal adenocarcinoma (PDAC), a chemotherapy-resistant tumor with a huge unmet medical need.
[3]

C22H27N5O4

425.48

425.206

C, 62.10; H, 6.40; N, 16.46; O, 15.04

1013101-36-4

25033539

Off-white to gray solid powder

1.4±0.1 g/cm3

682.5±65.0 °C at 760 mmHg

366.5±34.3 °C

0.0±2.2 mmHg at 25°C

1.646

1.43

2

8

6

31

654

0

O(C([H])([H])C([H])([H])O[H])C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C(C(C2=C([H])N=C(C([H])=C2[H])OC([H])([H])[H])=C([H])C2=C(C([H])([H])[H])N=C(N([H])[H])N=C12)=O

XDLYKKIQACFMJG-UHFFFAOYSA-N

InChI=1S/C22H27N5O4/c1-13-17-11-18(14-3-8-19(30-2)24-12-14)21(29)27(20(17)26-22(23)25-13)15-4-6-16(7-5-15)31-10-9-28/h3,8,11-12,15-16,28H,4-7,9-10H2,1-2H3,(H2,23,25,26)

2-amino-8-[4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7-one

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 (5.88 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 25.0 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: ≥ 2.5 mg/mL (5.88 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.88 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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