Akt2 6 nM (IC50) PKA 168 nM (IC50) p70S6K 120 nM (IC50) Autophagy Apoptosis

CCT128930 hydrochloride's growth inhibition GI50 values for PTEN-deficient human tumor cell lines are 6.3 μM for U87MG human glioblastoma cells, 0.35 μM for LNCaP human prostate cancer cells, and 1.9 μM for PC3 human prostate cancer cells[1]. AKT phosphorylation at serine 473 is first induced up to 20 μM in response to CCT128930 (0.1-60 μM; 1 hour; U87MG human glioblastoma cells) hydrochloride, and then decreases at higher concentrations[1]. With essentially constant levels of the corresponding total proteins and GAPDH, CCT128930 hydrochloride inhibits the downstream target, pSer235/236 S6RP, at ≥10 μM, and the direct substrates of AKT (Ser9 GSK3β, pThr246 PRAS40, and pT24 FOXO1/p32 FOXO3a) at ≥5 μM[1]. After half an hour, pSer473 AKT phosphorylation increases in response to CCT128930 (18.9 μM; U87MG human glioma cells) hydrochloride, and this effect lasts for 48 hours. Between 8 and 48 hours after therapy, the total AKT protein signal gradually drops[1]. In HepG2 and A549 cells, CCT128930 hydrochloride (0–10 μM; 24 hours) phosphorylates Akt more than it inhibits. By downregulating cyclin D1 and Cdc25A and upregulating p21, p27, and p53, CCT128930 (0–20 μM; 24 hours) hydrochloride prevents cell division. Hydrochloride of CCT128930 (20 μM) activates caspase-3, caspase-9, and PARP, which in turn causes cell death. HepG2 cells' phosphorylation of ERK and JNK is increased by CCT128930 hydrochloride (0–20 μM; 24 hours). HepG2 cells' DNA damage response is activated by CCT128930 (0–20 μM; 24-hour exposure), which is characterized by the phosphorylation of H2AX, ATM (ataxia-telangiectasia mutant), Chk1, and Chk2[2].

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CCT128930 has GI50 values of 6.3 M for human glioblastoma cells (U87MG), 0.35 M for human prostate cancer cells (LNCaP), and 1.9 M for human prostate cancer cells (PC3), all of which are PTEN-deficient human tumor cell lines[1]. The human glioblastoma cell line U87MG exhibits an initial induction of AKT phosphorylation at serine 473 up to 20 M, followed by a decrease in phosphorylation at higher concentrations[1] when exposed to the chemical compound CCT128930 (0.1-60 M; 1 hour). With relatively constant levels of the corresponding total proteins and GAPDH, CCT128930 inhibits the downstream target, pSer235/236 S6RP, at 10 M, and the direct substrates of AKT (Ser9 GSK3, pThr246 PRAS40, and pT24 FOXO1/p32 FOXO3a) at 5 M[1].

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CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. [1]

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PI3K/Akt/mTOR pathway plays an important role in tumor progression and anti-cancer drug resistance. The aim of the present study is to determine the antitumor effect of CCT128930, a novel small molecule inhibitor of Akt, in the HepG2 hepatoma cancer cells. Our results showed that at low concentrations, CCT128930 increased, but not inhibited, the phosphorylation of Akt in HepG2 and A549 cells. CCT128930 inhibited cell proliferation by inducing cell cycle arrest in G1 phase through downregulation of cyclinD1 and Cdc25A, and upregulation of p21, p27 and p53. A higher dose (20 μM) of CCT128930 triggered cell apoptosis with activation of caspase-3, caspase-9, and PARP. Treatment with CCT128930 increased phosphorylation of ERK and JNK in HepG2 cells. CCT128930 activated DNA damage response of HepG2 cell characterized by phosphorylation of H2AX, ATM (ataxia-telangiectasia mutated), Chk1 and Chk2. Upon exposure to CCT128930 at a higher concentration, HepG2 cells exhibited autophagy was accompanied by an increase the levels of LC3-II and Beclin-1. Blocking autophagy using chloroquine magnified CCT128930-induced apoptotic cell death and the phosphorylation of H2AX. The results in this study have advanced our current understandings of the anti-cancer mechanisms of CCT128930 in cancer cells[2].

In U87MG and BT474 human breast cancer xenografts, CCT128930 hydrochloride (25 or 40 mg/kg; intraperitoneal daily or twice daily for 5 days) exhibits antitumor activities[1]. The pharmacokinetic parameters of CCT128930 (25 mg/kg) in CrTacNCr- Fox1nu mice are summarized as follows: Tissue Route T1/2 (h) Tmax (h) Cmax (μM) Vss (L) Cl (L/h) AUC0-∞ (μMh) Bioavailability (%) Plasma iv 0.95 0.083 6.36 0.25 0.325 4.62 100 MCE has not independently confirmed the clearance. precision of these techniques. They are merely meant to be used as references.

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CCT128930 at 25 mg/kg i.p. shows a marked antitumor effect in established PTEN-null U87MG human glioblastoma xenografts with a treated:control (T/C) ratio of 48% on day 12. CCT128930 at 40 mg/kg also exerts a potent antitumor effect in HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, with complete growth arrest and a T/C ratio of 29% on day 22. When given intravenously, CCT128930 reaches a plasma peak concentration of 6.4 M before being rapidly cleared with a high volume of distribution, a short half-life, and an area under the curve (AUC0-) of 4.6 M h. I.p. administration of CCT128930 results in a peak plasma drug concentration of 1.3 M and an associated AUC0- of 1.3 Mh. When CCT128930 is taken orally, the peak plasma concentration is only 0.43 M, and the AUC0- is a low 0.4 Mh.[1]

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Pharmacodynamic activity of CCT128930 in vivo [1]
Having demonstrated concentration-dependent and time-dependent inhibition of numerous AKT biomarkers by CCT128930 in vitro and promising levels of tumor exposure in vivo, the pharmacodynamic effects of the compound were then evaluated in the same mice bearing U87MG human glioblastoma tumors, as used for the pharmacokinetic studies (Figure 4B). Figures 4C and D summarize the effects of CCT128930 treatment (50 mg/kg i.p. × 4 days) on several AKT biomarkers in U87MG xenografts harvested 2 and 6 hours following the last dose (see Figure 4B). CCT128930 caused a significant increase in Ser473 phosphorylation on AKT at both the 2 and 6 hour time points (P<0.001 and P<0.01, respectively), consistent with the biomarker results in vitro (compare Figure 3 to Figure 2). In addition, at both 2 and 6 hour time points, there were clear and significant decreases in the phosphorylation of Ser9 GSK3β (P<0.001 and P<0.01, respectively), Ser235/236 S6RP (P<0.05 and P<0.01, respectively) and Thr246 PRAS40 (P<0.05 and P<0.05, respectively), with the total forms of the protein remaining relatively constant (Figures 4C and D). These observations are consistent with inhibition of AKT activity by CCT128930 occurring in U87MG tumors in vivo.

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Antitumor activity of CCT128930 [1]
Next, the antitumor activity of CCT128930 was evaluated in two molecularly relevant human tumor xenograft models. Figure 5A demonstrates that there was a marked antitumor effect of CCT128930 at 25 mg/kg i.p. (× 5 of 7 days) in established PTEN-null U87MG human glioblastoma xenografts, giving a treated:control (T/C) ratio on day 12 of 48%. There was no weight loss associated with this regime. Treatment of HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts with CCT128930 (40 mg/kg bid × 5 of 7 days) also had a profound antitumor effect with complete growth arrest and a T/C ratio of 29% on day 22. This regimen was associated with minimal weight loss, with a nadir of only 94.8% of the initial body weight on day 15 of treatment. These results clearly demonstrate that CCT128930 has antitumor activity as a single agent in two human tumor xenograft models that are molecularly relevant with respect to PI3K pathway activation.

Profiling against 50 different human kinases is carried out using 10 μM CCT128930 at an ATP concentration equivalent to the Km for each enzyme.

Cells are seeded in 96-well plates and allowed to attach for 36 hours to ensure exponential growth prior to treatment. In vitro antiproliferative activity is determined using a 96-hour SRB assay. TCA-fixed cells are stained for 30 minutes with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. At the end of the staining period, SRB is removed and cultures are quickly rinsed four times with 1% acetic acid to remove unbound dye. The acetic acid is poured directly into the culture wells from a beaker. This procedure permits rinsing to be performed quickly so that desorption of protein-bound dye does not occur. Residual wash solution is removed by sharply flicking plates over a sink, which ensures the complete removal of rinsing solution. Because of the strong capillary action in 96-well plates, draining by gravity alone often fails to remove the rinse solution when plates are simply inverted. After being rinsed, the cultures are air dried until no standing moisture is visible. Bound dye is solubilized with 10 mM unbuffered Tris base (pH 10.5) for 5 minutes on a gyratory shaker. OD is read in either a UVmax microtiter plate reader or a Beckman DU-70 spectrophotometer. For maximum sensitivity, OD is measured at 564 nm. Because readings are linear with dye concentrations only below 1.8 OD units, however, suboptimal wavelengths are generally used, so that all samples in an experiment remains within the linear OD range. With most cell lines, wavelengths of approximately 490-530 nm works well for this purpose.

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Cell viability assay and cell colony formation assay [2]
MTT assay was used to detect cell viability, as reported elsewhere. Briefly, cells were plated in 96-well plates for 24 h. The medium was then removed, and cells were treated with various concentrations of CCT128930. An amount of 20 μL MTT (5 mg/mL) was added for 4 h. After removing supernatant, 150 μL DMSO was added to resolve formazan crystals, and the absorbance was detected at 490 nm. For cell colony formation assay, HepG2 cells were seeded at 1500 cells per well (6-well cell culture plate) and treated with different concentrations of CCT128930 for 2 weeks. Cells were fixed in 1% glutaraldehyde and stained with 0.5% crystal violet. Colonies with >30 cells were counted under an inverted microscope.

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Cell cycle analysis [2]
Cells were treated with CCT128930 for 24 h. At the end of the treatment, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS), and fixed overnight in cold 70% ethanol at 4 °C. After washing with PBS, the cells was digested with RNase A and stained with PI. Samples were analyzed for their DNA content by using a FACSCalibur flow cytometry with CellQuest software

Animal/Disease Models: 6-8 weeks old female CrTacNCr-Fox1nu (nude) mice[1]
Doses: 25 mg/kg (U87MG human glioblastoma xenografts) or 40 mg/kg (BT474 human breast cancer xenografts)
Route of Administration: ip daily for 5 days (U87MG human glioblastoma xenografts); ip twice (two times) daily for 5 days (BT474 human breast cancer xenografts)
Experimental Results: Giving a treated:control (T/C) ratio on day 12 of 48%. There was no weight loss associated with this regime in U87MG human glioblastoma xenografts. Had a profound antitumor effect with complete growth arrest and a T/C ratio of 29% on day 22. This regimen was associated with minimal weight loss, with a nadir of only 94.8% of the initial body weight on day 15 of treatment in BT474 human breast cancer xenografts.

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6-8 weeks old female CrTacNCr-Fox1nu mice [1]
25 mg/kg (U87MG human glioblastoma xenografts) or 40 mg/kg (BT474 human breast cancer xenografts)
i.p. daily for 5 days (U87MG human glioblastoma xenografts); i.p. twice daily for 5 days (BT474 human breast cancer xenografts)

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In vivo Human Tumor Xenograft Studies [1]
PTEN-null U87MG human glioblastoma cells (2×106) were injected subcutaneously (s.c.) in the right flank of 6-8 weeks old female CrTacNCr-Fox1nu mice. For HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, cells (5 × 106) were administered s.c. in medium supplemented with matrigel (1:1) into the mammary fat pads of female mice implanted s.c. with estradiol pellets (0.025 mg, 90 day release #NE-121) 3 days previously. Animals were randomized and treatment was started with vehicle or CCT128930 when established tumors were ~100 mm3 in mean volume. Control mice received vehicle only (10% DMSO, 5% Tween 20, 85% saline) and treated mice received 50 mg/kg CCT128930 intraperitoneally (i.p.) daily for 5 days (U87MG human glioblastoma xenografts) or 40 mg/kg CCT128930 i.p. twice daily for 5 days (BT474 human breast cancer xenografts). Tumor size and body weight were monitored three times a week. Tumor size was evaluated by measurement of 2 orthogonal diameters with calipers and volume was calculated from the formula: V= 4/3π[(d1+d2)/4]3. At the end of the study, tumors were excised and weighed.

To assess the pharmacokinetic and pharmacodynamic profiles of CCT128930, a single dose of compound (50 mg/kg i.p.) was administered to mice bearing U87MG human glioblastoma xenografts. Plasma and tumor samples were harvested at 2 and 6 hours following dosing. Mice were bled by cardiac puncture and plasma samples were collected and frozen at −20°C until analysis. Tumors were dissected, divided into two approximately equal pieces and snap frozen in liquid nitrogen until analysis. For pharmacodynamic studies, tumors were homogenized using a buffer containing 50 mmol/L Tris (pH 7.4), 1 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 1 mmol/L NaF, 1 mmol/L NaVO4, 5 μmol/L Fenvalerate, 5 μmol/L Vbphen, 10 mg/mL TLCK, 1× Complete inhibitor tablet per 10-mL buffer, protease inhibitor cocktail, and phosphatase inhibitor 1 and 2. Protein content was measured using Bradford reagent and samples were analyzed using immunoblotting as described above.

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Pharmacokinetic Analysis [1]
Concentrations of CCT128930 in biological samples were determined using LC-MS. Drug was extracted using methanol and chromatography carried out on a Synergi-Polar RP column (5.0 cm × 4.6 mm ID, 4 µm particle size) using a Waters 600MS pump and a 717 autosampler with a gradient mobile phase of 0.1% formic acid/methanol at 0.6ml/min over 12 minutes. Detection was by LC-MS using a TSQ700 triple quadrapole in which the analyte was ionized by electrospray interface in positive mode to monitor the transition [M+H]+ 342.8 to 146.6.4. The spray voltage was optimized to 5.5Kv and capillary temperature to 260ºC. The assay was linear over the range 10 – 10,000 nM CCT128930.

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PRAS40 Immunofluorescence Studies [1]
U87MG human glioblastoma cells were plated at 5 × 104 cells/well on cover slips in 24-well plates for 36 hours, before being treated with increasing concentrations of CCT128930 for 24 hours. Cells were fixed in 3.8% formaldehyde and permeabilized with 0.01% Triton X-100. BALB/c mice were treated with 50 mg/kg CCT128930 i.p. daily for 4 days, before whiskers were plucked and immediately fixed by immersing, root first, in 10% formal saline for 30 minutes before storing in 4% saline at 4°C. Hair follicles from healthy volunteers were collected and processed as described above. The whisker and hair follicles underwent antigen retrieval with a citrate buffer prior to analysis. The cells, whisker and hair follicles were stained with anti-phospho-Thr246 PRAS40 and anti-PRAS40 antibodies (1:200 for cells; 1:50 for whisker and hair follicles), then visualized with 1:1000 AlexaFluor® 488 goat anti-rabbit IgG antibody. Nuclei were counterstained with 1:10,000 TOPRO-3. The cells or follicles were mounted using Vectashield and images were visualized and captured using a Leica SP1 confocal laser scanning fluorescence microscope All images were taken with the same detector settings based on the respective vehicle-treated control sample. Optical magnification was 250, with an additional 2-fold software (digital) amplification, giving a total magnification of 500. For follicles, images were taken from the middle of the bulb along the z-axis and included the entire cross-section of the bulb. Fluorescence intensity in individual cells was quantified using the INCell Investigator Developer Toolbox v1.6 software. TOPRO-3 was used to identify all nuclei in the image. The areas around the boundary of the nuclei were then expanded to identify cytoplasmic regions. The nuclear and cytoplasmic segmentations were linked together, so that only cytoplasmic areas of the image within cells with nuclei were quantified. The mean fluorescence intensity per cell was then reported from both the pThr246 PRAS40 and total PRAS40 images, respectively.

Pharmacokinetics of CCT128930 [1]
The pharmacokinetics of CCT128930 were determined to establish if therapeutically active drug concentrations could be established in vivo. The pharmacokinetics of CCT128930 after a single dose of 25 mg/kg are illustrated in Figure 4A and summarized in Supplementary Table 1. Following i.v. administration, CCT128930 reached a peak concentration of 6.4 µM in plasma and was eliminated with a relatively short half-life, high volume of distribution and rapid clearance, giving an AUC0-∞ of 4.6 µMh. Following i.p. administration, the peak plasma drug concentration was 4-fold lower and the plasma clearance was similar to that observed i.v‥ The corresponding AUC0-∞ was 1.3 µMh, giving an i.p. bioavailability of 29%.

Oral CCT128930 administration gave a comparable pharmacokinetic profile to other routes, but the peak plasma concentration was only 0.43 µM, with a plasma clearance comparable to that observed iv suggesting no first pass metabolism (Supplementary Table 1). This resulted in a correspondingly low AUC and an oral bioavailability of only 8.5%. More importantly, following i.p. administration, peak tumor CCT128930 concentrations were 6-fold higher than the corresponding plasma value at 8 µM and there was evidence of drug retention, as shown by the 2-fold longer half-life and 6-fold slower apparent clearance. This resulted in much higher tumor drug exposure relative to plasma with an AUC0-∞ of 25.8 µMh. Tumor:plasma drug concentrations did not reach steady-state but varied from 4:1 at 30 minutes to 163:1 at 6 hours, confirming tissue drug retention. Assuming linear kinetics, these data supported the use of higher doses and repeat administration to achieve potentially therapeutic tumor drug concentrations in vivo.

Figure 4B shows that following CCT128930 administration at 50 mg/kg i.p. per day for 4 days, drug concentrations in U87MG human glioblastoma tumors were consistently much greater than the corresponding plasma concentrations with tumor:plasma ratios of 27:1 and 42:1 at 2 and 6 hours following the last dose, respectively. Moreover, U87MG tumor drug concentrations greatly exceeded the CCT128930 GI50 for at least 6 hours following the final dose and were consistently 5-fold higher than the concentrations required for in vitro biomarker modulation (Figure 2). Metabolism studies revealed that only 0.23% of an administered dose (25 mg/kg i.p.) was recovered unaltered in urine after 24 hours (data not shown). The above results showed that pharmacologically active concentrations of CCT128930 were achieved in tumor tissue at well-tolerated doses.

[1]. Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930. Mol Cancer Ther. 2011 Feb;10(2):360-71.

[2]. CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition. Biochimie. 2014;103:118-125.

AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. [1]

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In conclusion, we have evaluated the pharmacological and therapeutic properties of the interesting and novel AKT inhibitor CCT128930. This is a leading example of the pyrrolopyrimidines series, with which we obtained selectivity for AKT over PKA by targeting a single amino acid difference, which we were the first to show. We have demonstrated that CCT128930 suppresses the phosphorylation of downstream markers of AKT in vitro and in vivo and exhibits promising single agent antitumor activity in molecularly relevant human cancer models with appropriate pharmacokinetic and pharmacodynamic properties. We have also used this AKT inhibitor to develop a novel assay to quantify pharmacodynamic biomarker changes in a normal tissue following PI3K-AKT pathway blockade, and this assay may have broader applications in clinical trials of other related pathway inhibitors. [1]

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Recent reports suggest that autophagy plays an important role in determining cell fate although apoptosis has been widely studied as a cellular response to DNA damage. Autophagy is a ubiquitous highly conserved pathway in eukaryotic cells that takes place as a response to a variety of conditions, such as nutrient deprivation, growth factors withdrawal, and oxidative stress. Conversion of LC3 from cytosolic form (LC3-I) to the proteolytic and lipidated form (LC3-II) is a characteristic hallmark of autophagy. We found that CCT128930 could induce autophagy evidenced by increased levels of LC3-II in HepG2 cells. It will also be interesting to investigate whether inhibitors of autophagy can enhance the anticancer activity of CCT128930. We noted that combination of CCT128930 and CQ led to the desired effects on apoptosis in HepG2 cells. Targeting the autophagy pathway may be a promising therapeutic strategy to enhance CCT128930 lethality via apoptosis for HCC treatment. In conclusion, our results suggest that CCT128930 inhibits cancer cell growth. CCT128930 can induce cell cycle arresting, DNA damage and autophagy in a dose dependent manner. Treatment of the cells with CCT128930 significantly enhanced phosphorylation of ERK1/2 and JNK1/2. Inhibition of autophagy with a lysosomal protease inhibitor CQ potentiates CCT128930's apoptosis-inducing activity and anticancer activity in cancer cells. [2]

C18H21CL2N5

378.298841238022

377.117

2453324-32-6

CCT128930;885499-61-6

146013807

White to yellow solid powder

3

4

3

25

418

0

ClC1C=CC(=CC=1)CC1(CCN(C2C3C=CNC=3N=CN=2)CC1)N.Cl

OFLOSUNRPFWODP-UHFFFAOYSA-N

InChI=1S/C18H20ClN5.ClH/c19-14-3-1-13(2-4-14)11-18(20)6-9-24(10-7-18)17-15-5-8-21-16(15)22-12-23-17;/h1-5,8,12H,6-7,9-11,20H2,(H,21,22,23);1H

4-[(4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine;hydrochloride

2453324-32-6; CCT128930 hydrochloride; 4-(4-Chlorobenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine hydrochloride; CCT128930 (hydrochloride); 4-(4-Chlorobenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-aminehydrochloride;

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 : 20 mg/mL (52.87 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (5.29 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 20.0 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 mg/mL (5.29 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 20.0 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.)