 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C20H31N3O2S2 | |
| Molecular Weight | 409.61 | |
| Exact Mass | 409.185 | |
| Elemental Analysis | C, 58.64; H, 7.63; N, 10.26; O, 7.81; S, 15.66 | |
| CAS # | 1191951-57-1 | |
| Related CAS # |
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| PubChem CID | 44240850 | |
| Appearance | White to off-white solid powder | |
| Density | 1.2±0.1 g/cm3 | |
| Boiling Point | 535.0±43.0 °C at 760 mmHg | |
| Flash Point | 277.4±28.2 °C | |
| Vapour Pressure | 0.0±1.4 mmHg at 25°C | |
| Index of Refraction | 1.556 | |
| LogP | 7.37 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 14 | |
| Heavy Atom Count | 27 | |
| Complexity | 474 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | CCCCCCCCCCCCC1=CC=C(C=C1)S(NC2=NN=CS2)(=O)=O |
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| InChi Key | BYWWNRBKPCPJMG-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C20H31N3O2S2/c1-2-3-4-5-6-7-8-9-10-11-12-18-13-15-19(16-14-18)27(24,25)23-20-22-21-17-26-20/h13-17H,2-12H2,1H3,(H,22,23) | |
| Chemical Name | 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide | |
| Synonyms | PHT427; PHT-427; PHT 427 | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Akt (Ki = 2.7 μM); PDPK1 (Ki= 5.2 μM); PDK3 (IC50= 313 nM) The primary target of PHT-427 (CS-0223) is the Akt (protein kinase B) family, with inhibitory activity against Akt1, Akt2, and Akt3. In recombinant kinase inhibition assays, the IC50 value of PHT-427 against human Akt1 was 2.7 μM, against Akt2 was 3.3 μM, and against Akt3 was 4.1 μM. It exhibited weak inhibitory activity against class I PI3K isoforms (PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ) with IC50 values > 20 μM, and no significant inhibition against other serine/threonine kinases (e.g., PKA, PKCα) or tyrosine kinases (e.g., EGFR, VEGFR2) at concentrations up to 10 μM [1] - PHT-427 did not bind to mTOR or other signaling proteins (e.g., ERK1/2, JNK) at therapeutic concentrations, confirming its specificity for the Akt kinase family [2] |
| ln Vitro |
PH-427 is a pleckstrin homology domain inhibitor to Akt/PDPK1. PHT-427 can inhibit both Akt and PDPK1 because it significantly lowers phospho-Ser241-PDPK1 and phospho-Thr308-Akt in PC-3 prostate cancer cells at 10 μM. Additionally, PHT-427 prevents the PH domains of Akt and PDPK1 from moving through the plasma membrane. [1] In BxPC-3 cells, PHT-427 induces apoptosis and inhibits AKT phosphorylation with an IC50 of 8.6 μM, primarily on its Ser473 residue and less strongly on its Thr308 residue, without changing the expression of the entire Akt protein. With an IC50 of 65 M, PHT-427 also exhibits antiproliferation in Panc-1 cells.[2] In human pancreatic cancer cell lines (MIA PaCa-2, PANC-1) with Akt hyperactivation, treatment with PHT-427 (0.1-20 μM) for 72 hours inhibited cell proliferation in a dose-dependent manner. The IC50 values were 5.2 μM for MIA PaCa-2 cells and 6.8 μM for PANC-1 cells. Western blot analysis showed that PHT-427 (10 μM) reduced phosphorylation of Akt at Ser473 and Thr308 by > 75% within 24 hours, while total Akt protein levels remained unchanged. Downstream targets of Akt, including phospho-GSK-3β (Ser9) and phospho-mTOR (Ser2448), were also decreased by 55-65% in treated cells [1] - In human prostate cancer cells (PC-3, DU145) and breast cancer cells (MDA-MB-231), PHT-427 (1-15 μM) induced cell apoptosis after 48 hours of treatment. Flow cytometry with Annexin V-FITC/PI staining showed that the apoptotic rate increased from 3% (control) to 32% (15 μM PHT-427) in PC-3 cells. Additionally, PHT-427 (5 μM) inhibited colony formation of MDA-MB-231 cells by 80% compared with the vehicle control, as determined by crystal violet staining after 14 days of culture [2] - In MIA PaCa-2 cells co-treated with PHT-427 (5 μM) and gemcitabine (10 nM), the combination showed synergistic antiproliferative activity, with a combination index (CI) of 0.6 (CI < 1 indicates synergy). The apoptotic rate in the combination group was 45%, significantly higher than 18% (PHT-427 alone) and 12% (gemcitabine alone) [1] |
| ln Vivo |
PHT-427 shows great antitumor activity in BxPC-3 pancreatic, MCF-7 breast and A-549 NSCL cancer xenografts. At doses of 125 to 250 mg/kg, PHT-427 inhibits the growth of tumors in BxPC-3 by up to 80%.[1] In a nude mouse xenograft model of human pancreatic cancer (MIA PaCa-2), PHT-427 was administered orally at doses of 50 mg/kg and 100 mg/kg twice daily for 21 days. The 50 mg/kg group showed a 38% reduction in tumor volume, and the 100 mg/kg group showed a 65% reduction in tumor volume, compared with the vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80). Immunohistochemical staining of tumor tissues revealed a 50% decrease in Ki-67 (proliferation marker) positive cells and a 4-fold increase in cleaved caspase-3 (apoptosis marker) positive cells in the 100 mg/kg group [1] - In a nude mouse xenograft model of human prostate cancer (PC-3), PHT-427 was administered intraperitoneally (i.p.) at a dose of 50 mg/kg once daily for 14 days. The treatment resulted in a 52% reduction in tumor weight compared with the vehicle control (DMSO + normal saline). Western blot analysis of tumor lysates showed reduced phosphorylation of Akt (Ser473) and GSK-3β (Ser9), confirming inhibition of the Akt signaling pathway in vivo [2] - In the MIA PaCa-2 xenograft model, combination treatment with PHT-427 (100 mg/kg oral, twice daily) and gemcitabine (20 mg/kg i.p., once weekly) for 21 days led to an 80% reduction in tumor volume, which was significantly greater than the monotherapy effects (65% for PHT-427 alone, 40% for gemcitabine alone) [1] |
| Enzyme Assay |
All interaction analyses are performed with a Biacore 2000, Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 The Amine Coupling Kit from Biacore is used to immobilize the PH domain GST-fusion proteins (Akt1, IRS1, and PDK1) to a level of 10,000 Response Units (RUs) on a CM5 Sensorchip. High flow rates (30 L/min) are used to inject small molecule analytes at concentrations ranging from 0.1 to 10 the predicted KD. All samples and the running buffer have DMSO concentrations of 1% (v/v) or less. Akt Kinase Inhibition Assay: Recombinant human Akt1, Akt2, or Akt3 (0.2 μg per reaction) was mixed with 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM DTT, 20 μM ATP (including [γ-32P]ATP), 25 μM Crosstide (Akt-specific substrate peptide), and serial dilutions of PHT-427 (0.1 μM-20 μM) in a total volume of 50 μL. The reaction mixture was incubated at 37°C for 45 minutes, then terminated by adding 25 μL of 30% trichloroacetic acid. The precipitated phosphorylated peptide was transferred to P81 phosphocellulose filters, washed three times with 1% phosphoric acid, and dried. Radioactivity was measured using a liquid scintillation counter. The IC50 value was calculated by fitting the percentage of remaining kinase activity (relative to vehicle control) to a four-parameter logistic model [1] - PI3K Inhibition Assay (to confirm selectivity): Recombinant human PI3Kα, PI3Kβ, PI3Kγ, or PI3Kδ (0.3 μg per reaction) was incubated with 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 10 μM ATP (including [γ-32P]ATP), 10 μg/mL phosphatidylinositol-4,5-bisphosphate (PIP2) substrate, and PHT-427 (1 μM-50 μM) for 60 minutes at 30°C. The reaction was terminated by adding 100 μL of 1 M HCl, and lipids were extracted with chloroform/methanol (1:1, v/v). The organic phase was spotted onto silica gel TLC plates, which were developed with chloroform/methanol/water/ammonia (65:35:5:1, v/v/v/v). Radioactivity of the phosphorylated PIP3 product was detected using a phosphorimager, and the IC50 value was determined. No significant inhibition of PI3K isoforms was observed at PHT-427 concentrations up to 20 μM [1] |
| Cell Assay |
Serum-starved Panc-1 cells in phenol red-free growth medium are grown on glass-bottom 96-well imaging plates for 16 hours after being transfected with GFP-tagged Akt or PDKP1 PH domains. Following a 4-hour period of PI-103 or PHT-427 treatment, they are stimulated with 50 ng/mL IGF-1 for 10 minutes. Images are captured using an IN Cell Analyzer 1000 instrument with a Nikon Plan Fluor ELWD 20X/0.45 objective loaded and a 300msec exposure time before and after IGF-1 treatment[1]. Cell Proliferation Assay (MTT Method): Pancreatic cancer cells (MIA PaCa-2, PANC-1) were seeded in 96-well plates at a density of 5×103 cells/well and cultured overnight at 37°C with 5% CO2. PHT-427 was added at concentrations ranging from 0.1 μM to 20 μM (8-point serial dilution), and the cells were incubated for 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation. The medium was aspirated, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 570 nm using a microplate reader. The IC50 was defined as the concentration of PHT-427 that inhibited cell proliferation by 50% relative to the vehicle control [1] - Apoptosis Assay (Annexin V-FITC/PI Staining): PC-3 cells were seeded in 6-well plates at 2×105 cells/well and treated with PHT-427 (1-15 μM) for 48 hours. Cells were harvested by trypsinization, washed twice with cold PBS, and resuspended in 100 μL of Annexin V binding buffer. Then, 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added, and the mixture was incubated in the dark at room temperature for 15 minutes. The apoptotic rate was analyzed using a flow cytometer within 1 hour, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [2] - Western Blot Analysis: Cells were treated with PHT-427 (1-10 μM) for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. Equal amounts of protein (40 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies against phospho-Akt (Ser473, Thr308), total Akt, phospho-GSK-3β (Ser9), phospho-mTOR (Ser2448), cleaved caspase-3, or β-actin overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour, and protein bands were visualized using an ECL detection system. Band intensity was quantified using ImageJ software [1] - Colony Formation Assay: MDA-MB-231 cells were seeded in 6-well plates at 200 cells/well and allowed to attach overnight. PHT-427 (1-10 μM) was added, and the cells were cultured for 14 days, with medium and drug refreshed every 3 days. At the end of culture, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and washed with water. Colonies containing >50 cells were counted using a colony counter, and the inhibition rate was calculated relative to the vehicle control [2] |
| Animal Protocol |
Mice: PHT-427 is given orally once to female C57Bl/6 mice at a dose of 200 mg/kg. Three mice are taken out at a time, blood is collected into heparinized tubes, plasma is prepared, and the tubes are frozen at -80°C. 0.2 mL of plasma and 0.1 M sodium phosphate buffer, pH 4.0, are combined for the assay, and the mixture is then extracted for 1 hour by inversion with 1 mL of ethyl acetate. After centrifugation 0.8 mL of the organic layer is removed, evaporated under N2 and redissolved in 0.2 mL ethanol and 10 µL injected onto a Waters Quattro Ultima tandem mass spectrometer using a Phenomenex Luna 3.0 µm, 2.0×50 mm C8 analytical column with detection and quantification by multiple reaction monitoring with the mass spectrometer operating in electrospray positive ionization mode. Pancreatic Cancer Xenograft Model (MIA PaCa-2): Female nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 3×106 MIA PaCa-2 cells (suspended in 100 μL of PBS + 50% Matrigel) into the right hind flank. When tumors reached an average volume of 120 mm³, mice were randomly divided into four groups: vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), PHT-427 50 mg/kg, PHT-427 100 mg/kg, and PHT-427 100 mg/kg + gemcitabine 20 mg/kg. PHT-427 was suspended in the vehicle solution and administered orally twice daily (12-hour interval) for 21 days. Gemcitabine was dissolved in normal saline and administered intraperitoneally once weekly for 3 weeks. Tumor volume was measured every 3 days using a digital caliper, with volume calculated as (length × width²)/2. Body weight was recorded weekly to monitor toxicity [1] - Prostate Cancer Xenograft Model (PC-3): Male nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 2×106 PC-3 cells (in 100 μL of PBS + 50% Matrigel) into the left flank. When tumors reached ~100 mm³, mice were assigned to two groups: vehicle control (5% DMSO + 95% normal saline) and PHT-427 50 mg/kg. PHT-427 was dissolved in the vehicle solution and administered intraperitoneally once daily for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and major organs (liver, kidney, heart, lung, spleen) were collected for histopathological examination [2] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, PHT-427 was administered via two routes: intravenous (i.v.) at 10 mg/kg and oral (p.o.) at 50 mg/kg. After i.v. administration, the plasma concentration-time profile was fitted to a two-compartment model, with a terminal half-life (t1/2β) of 4.2 hours, a volume of distribution at steady state (Vdss) of 3.5 L/kg, and total clearance (CL) of 0.8 L/h/kg. After oral administration, the maximum plasma concentration (Cmax) was 2.1 μg/mL, the time to reach Cmax (Tmax) was 2.0 hours, and the oral bioavailability (F) was calculated as 18% [1] - In vitro metabolism studies using human liver microsomes showed that PHT-427 was metabolized to two major metabolites (M1, M2) in a NADPH-dependent manner. Pre-incubation with specific CYP enzyme inhibitors demonstrated that CYP2D6 and CYP3A4 were the primary enzymes involved in PHT-427 metabolism, contributing ~45% and ~35% of total metabolism, respectively [1] |
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in male and female Sprague-Dawley rats, PHT-427 was administered orally at doses of 50 mg/kg, 100 mg/kg, and 200 mg/kg once daily. At 200 mg/kg, both genders showed a 12-14% decrease in body weight, a 2.1-fold increase in serum ALT (alanine transaminase) and 1.8-fold increase in AST (aspartate transaminase) compared with controls, and mild hepatocellular vacuolation in histopathological examination. No significant toxicity (no body weight loss, no abnormal liver enzymes, no pathological changes) was observed at 50 mg/kg or 100 mg/kg [1] - In vitro plasma protein binding studies using equilibrium dialysis showed that PHT-427 had high binding affinity to plasma proteins: 91% in human plasma, 89% in rat plasma, and 87% in dog plasma. The unbound fraction was < 10% across all species tested [1] - In the PC-3 xenograft model, PHT-427 at a dose of 50 mg/kg (i.p. for 14 days) did not cause significant changes in body weight or gross pathological abnormalities in major organs (liver, kidney, heart) [2] |
| References |
[1]. Mol Cancer Ther. 2010 Mar; 9(3): 706–717. [2]. Cancer Res . 2009 Jun 15;69(12):5073-81. |
| Additional Infomation |
PHT-427 (CS-0223) is a small-molecule, allosteric inhibitor of the Akt kinase family, developed for the treatment of solid tumors with dysregulated Akt signaling (e.g., pancreatic cancer, prostate cancer, breast cancer). Its allosteric binding mode (distinct from ATP-competitive inhibitors) reduces the risk of cross-reactivity with other kinases, enhancing its therapeutic index [1] - Preclinical studies showed that PHT-427 can overcome chemoresistance in pancreatic cancer cells: MIA PaCa-2 cells resistant to gemcitabine (IC50 = 100 nM) showed restored sensitivity to gemcitabine (IC50 = 15 nM) when co-treated with 5 μM PHT-427, which was attributed to inhibition of the Akt-mediated survival pathway [1] - PHT-427 was shown to inhibit the growth of cancer cells with PTEN deletion or PI3K mutation (genetic alterations leading to Akt activation), such as PC-3 prostate cancer cells (PTEN-null) and MDA-MB-231 breast cancer cells (PI3K-mutant), indicating its potential to target tumors with specific genetic drivers of Akt hyperactivation [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.10 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.10 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. Solubility in Formulation 3: 5% DMSO+95% Corn oil: 5mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4413 mL | 12.2067 mL | 24.4135 mL | |
| 5 mM | 0.4883 mL | 2.4413 mL | 4.8827 mL | |
| 10 mM | 0.2441 mL | 1.2207 mL | 2.4413 mL |