CDK2 (IC50 = 1 nM); CDK5 (IC50 = 1 nM); CDK1 (IC50 = 3 nM); CDK9 (IC50 = 4 nM)

Dinaciclib is another strong inhibitor of DNA replication that, at an IC50 of 4 nM, prevents the thymidine (dThd) DNA from being incorporated into A2780 cells. At concentrations greater than 6.25 nM, dinaciclib significantly inhibits the phosphorylation of Rb on Ser 807/811, which is consistent with the finding that in the same cell model, 4 nM concentrations are necessary to inhibit dThd DNA incorporation 50% of the time. The appearance of the p85 PARP cleavage product in cells exposed to >6.25 nM Dinaciclib indicates a significant correlation between the onset of apoptosis and complete suppression of Rb phosphorylation. Dinaciclib exhibits efficacy against a wide range of tumor cell lines in humans. In a dose-dependent manner, the addition of Dinaciclib during hydroxyurea exposure also suppresses the accumulation of γ-H2AX. [2] Dinaciclib causes a large-scale apoptosis in melanoma cells by suppressing their ability to proliferate. [3] Several osteosarcoma cell lines, including those resistant to dasatinib and doxorubicin, undergo apoptosis when exposed to dinaciclib. The phosphorylation of CDK inhibitor p27Kip1 at threonine 187 and RNAP II at serine 2 are both attenuated by dinaciclib. At 12 to 40 nM Dinaciclib, phosphorylation activity decreases (4 to 16 hours after Dinaciclib addition). Moreover, dinaciclib lowers Rb's phosphorylation at serine 807/811. Similar levels of apoptosis are induced in mock- and p53-depleted U2OS cells by dinaciclib.[4]

Tumor inhibition occurs at 70%, 70%, 89%, and 96%, respectively, after administering dinaciclib intraperitoneally (i.p.) at 8, 16, 32, and 48 mg/kg daily for ten days. The minimal effective dose (MED) of dinaciclib seems to be less than 8 mg/kg. Dinaciclib is well tolerated; in the highest dosage group, there is a maximum 5% reduction in body weight. In vivo, dinaciclib exhibits dose-dependent antitumor activity, with nearly total inhibition of tumor growth occurring at a dose level below the maximum tolerated dose (MTD). Dinaciclib in mice has a brief half-life in the plasma.[1]

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SCH 727965 was selected as the optimal drug candidate for clinical development by screening individual, diversely substituted compounds against the A2780 ovarian carcinoma mouse xenograft model, using flavopiridol as a benchmark control agent. This system established the ratio of maximum tolerated dose (MTD) and minimum effective dose (MED) for each tested compound. MTD was determined following i.p. administration of each compound to nude mice at varying dose levels once daily for 7 days and defined as the dose associated with a body weight reduction of 20%. In parallel, MED was defined as the dose, given by the same schedule, associated with >50% tumor growth inhibition. Promising compounds were further profiled in rats and dogs (21). The screening data pertinent to SCH 727965 are outlined in Table 1. Thus, MTD and MED of SCH 727965 were >60 and 5 mg/kg, respectively; in contrast, MTD and MED of flavopiridol were <10 and 10 mg/kg, respectively. Therefore, a screening therapeutic index (MTD/MED ratio) of SCH 727965 was >10, whereas the index of flavopiridol was <1, indicating that minimal antitumor efficacy was not attained with flavopiridol before the onset of dose-limiting toxicity. These data indicated that SCH 727965 has an attractive in vivo profile that is superior to flavopiridol. [1]

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SCH 727965 efficacy and tolerability in vivo [1]
The previously described A2780 ovarian cancer mouse xenograft model developed for initial selection of active agents was used for further assessment of SCH 727965 efficacy and tolerability; paclitaxel was a positive control. Nude mice with ∼100 mm3 A2780 tumors were randomized into groups of 10 animals ∼7 days after initial s.c. cell inoculation and assigned to each SCH 727965 dosage group, paclitaxel, or vehicle control. SCH 727965 i.p. administration at 8, 16, 32, and 48 mg/kg daily for 10 days resulted in tumor inhibition by 70%, 70%, 89%, and 96%, respectively; paclitaxel i.p. administration at 20 mg/kg twice weekly inhibited tumor growth by 63% (Fig. 3A). Consistent with earlier in vivo screening data, SCH 727965 MED appears to be <8 mg/kg. SCH 727965 was well tolerated, and the maximum body weight loss in the highest dosage group was 5% (data not shown). This is well below the MTD defined as 20% loss of body weight over the course of this experiment. Taken together, the data show that SCH 727965 has dose-dependent antitumor activity in vivo, and that nearly complete inhibition of tumor growth occurs at a dose level below the MTD (Table 4; Fig. 3A). SCH 7279765 has antitumor activity on various intermittent schedules [1]
Pharmacokinetic studies showed that SCH 727965 has a short plasma half-life in mouse. Thus, a dose of 5 mg/kg SCH 727965 given i.p. in mice was associated with a plasma half-life of ∼0.25 hour (Supplementary Table S2), perhaps suggesting a need for frequent dosing. However, previous results imply that continuous SCH 727965 exposure may not be a prerequisite for antiproliferative activity because short treatments with the drug induce long-term effects in vitro and in vivo. To test that hypothesis, a total SCH 727965 dose of 260 mg/kg, equivalent to 20 mg/kg once daily for 13 days, was fractionated over several diverse schedules and administered to nude mice bearing established (>100 mm3) A549 tumor xenografts (Fig. 3B; dosing days are indicated by arrows). Primary end points for this study were tumor volume/mass and body weight. SCH 727965 dosing of 87 mg/kg given once weekly exceeded the MTD and was terminated early. Similar tumor regressions were observed for all schedules. These data agree with earlier observations and indicate that similar in vivo responses can be generated on a wide range of intermittent SCH 727965 dosing schedules.

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Mechanism-based systemic effects in vivo following exposure to SCH 727965 [1]
In this study, phospho-Rb 807/811 has been used as a surrogate marker of CDK engagement and mechanism-based SCH 727965 activity. To show that 20-mg/kg to 60-mg/kg doses of SCH 727965 previously exhibiting significant antitumor activity in a mouse xenograft model are associated with modulation of the CDK mechanism, the expression of phospho-Rb 807/811 was analyzed in skin samples taken from SCH 727965–treated, tumor-naïve nude mice. Skin and hair follicles are an excellent peripheral source of surrogate proliferating (nontumor) tissue. Skin punch biopsies were harvested at various time points following the administration of SCH 727965. Immunohistochemical staining of murine skin indicates that a single 40-mg/kg SCH 727965 dose induces rapid and sustained suppression of phospho-Rb 807/811 within the proliferating epithelial cells of the basal epithelium and hair follicles (Fig. 3C). These observations suggest that doses of SCH 727965 associated with regressions in the A549 xenograft model (Fig. 3B) are correlated with the modulation of a mechanism-based marker in proliferating surrogate tissues. These data are consistent with the hypothesis that inhibition of CDKs can induce inhibition of cell cycle within proliferating normal tissues and suggest that proliferating compartments will likely be sensitive to SCH 727965. To assess effects of SCH 727965 on hematologic parameters, BALB/c mice were i.p. dosed daily with 40 mg/kg for 5 days, with controls nontreated or dosed with a vehicle, 20% hydroxypropyl-β-cyclodextran. Blood samples were obtained 1 day after administration of the last dose (day 6) as well as 7 days later (day 13). Blood cells were counted (with differential) to examine nadir and rebound kinetics of several hematologic parameters. Neutrophils and reticulocytes were most sensitive to SCH 727965, and nadirs in their absolute counts were detected on day 6 (Fig. 3D). Significantly, absolute neutrophil counts (Fig. 3D, left) and reticulocyte counts (Fig. 3D, right) returned to normal levels by day 13. No detectable effects on platelets or RBC over this time course were observed (data not shown). These results are consistent with the mechanism-based activity of SCH 727965 within the examined dose range and suggest that cell cycle inhibition effects are transient and reversible in proliferating normal cell compartments.

Recombinant cyclin/CDK holoenzymes are isolated from Sf9 cells that have been modified to generate baculoviruses expressing a particular CDK or cyclin. Usually, cyclin/CDK complexes are diluted to a final concentration of 50 μg/mL in a kinase reaction buffer that has 0.1 mM sodium orthovanadate, 10 mM MgCl₂, 1 mM DTT, and 50 mM Tris-HCl (pH 8.0) in it. In every kinase reaction, 10 μL of diluted Dinaciclib (SCH 727965) is mixed with 1 μg of enzyme and 20 μL of a 2 μM substrate solution (a biotinylated peptide derived from histone H1). The addition of 0.1 μCi of ³³P-ATP and 50 μL of 2 μM ATP initiates the reaction. The addition of 0.1% Triton X-100, 1 mM ATP, 5 mM EDTA, and 5 mg/mL streptavidin-coated SPA beads stops the kinase reactions after an hour of room temperature incubation. A Filtermate universal harvester in combination with a 96-well GF/B filter plate is used to collect SPA beads. Two M NaCl and two M NaCl containing 1% phosphoric acid are used to wash the beads twice. A TopCount 96-well liquid scintillation counter is then used to measure the signal. Two sets of eight-point serial dilutions of inhibitory compounds are used to create dose-response curves. Nonlinear regression analysis is used to derive IC50 values.

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Cyclin/CDK kinase assay [1]
Recombinant cyclin/CDK holoenzymes were purified from Sf9 cells engineered to produce baculoviruses that express a specific cyclin or CDK. Cyclin/CDK complexes were typically diluted to a final concentration of 50 μg/mL in a kinase reaction buffer containing 50 mmol/L Tris-HCl (pH 8.0), 10 mmol/L MgCl2, 1 mmol/L DTT, and 0.1 mmol/L sodium orthovanadate. For each kinase reaction, 1 μg of enzyme and 20 μL of a 2-μmol/L substrate solution (a biotinylated peptide derived from histone H1) were mixed and combined with 10 μL of diluted SCH 727965. The reaction was started by the addition of 50 μL of 2 μmol/L ATP and 0.1 μCi of 33P-ATP. Kinase reactions were incubated for 1 hour at room temperature and were stopped by the addition of 0.1% Triton X-100, 1 mmol/L ATP, 5 mmol/L EDTA, and 5 mg/mL streptavidin-coated SPA beads (Amersham). SPA beads were captured using a 96-well GF/B filter plate and a Filtermate universal harvester. Beads were washed twice with 2 mol/L NaCl and twice with 2 mol/L NaCl containing 1% phosphoric acid. The signal was then assayed using a TopCount 96-well liquid scintillation counter. Dose-response curves were generated from duplicate, eight-point serial dilutions of inhibitory compounds. IC50 values were derived by nonlinear regression analysis.

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Kinase counter-screening [1]
SCH 727965 and flavopiridol were counter-screened using the Millipore Kinase Profiler service. Both compounds were tested at 1.0 and 10.0 μmol/L against a panel of diverse kinases, using a fixed (10 μmol/L) concentration of ATP.

Plated A2780 cells are grown using the proper growth media in tissue culture dishes. Growing cultures are subjected, usually for seven days, to varying concentrations of Dinaciclib (0.75, 1.5, 3.15, 6.25, 12.5, 25, and 500 nM) or a vehicle control. Once the medium is removed, cells are fixed for five minutes using a 50% methanol/50% acetone solution and then stained for five minutes using a 0.2% crystal violet solution in 2% ethanol. Water (5–10 mL) is used to wash the cells after staining them. 1% deoxycholic acid is used to solubilize stained cells, and a SOFTmax PRO 4.3 plate reader is used to measure the absorbance of the resultant solution at 600 nm. Plotting the absorbance of samples treated with Dinaciclib as a percentage of a vehicle-treated control is done, and the results are presented as an IC50 value in relation to these controls. The alamarBlue Cell Viability Assay kit is utilized to obtain cell viability assessments for suspension cell lines.

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dThd uptake growth inhibition assay [1]
A2780 cells were maintained in DMEM plus 10% fetal bovine serum and passaged twice weekly by detaching the monolayer with trypsin-EDTA. One hundred microliters of A2780 cells (5 × 103 cells) were added per well to a 96-well Cytostar-T plate and incubated for 16 to 24 hours at 37°C. Compounds were serially diluted in complete media plus 2% 14C-labeled dThd. Media were removed from the Cytostar T plate; 200 μL of various compound dilutions were added in quadruplicate; and the cells were incubated for 24 hours at 37°C. Accumulated incorporation of radiolabel was assayed using scintillation proximity and measured on a TopCount (Packard/Perkin-Elmer Life Sciences). The percentage of dThd uptake inhibition, relative to a vehicle control, was calculated and plotted on log-linear plots to allow derivation of IC50 values.

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Bromodeoxyuridine incorporation assay [1]
A2780 cells were plated into six-well tissue culture dishes and allowed to adhere. Cells were then exposed to differing concentrations of Dinaciclib (SCH727965; PS-095760) or a DMSO control vehicle for 24 hours, followed by a brief (30 min) pulsed exposure to bromodeoxyuridine (BrdUrd). Cells were then harvested, immunostained using FITC-conjugated antibodies specific for BrdUrd, counter-stained with propidium iodide/RNase A solution, and analyzed using flow cytometry. Fluorescence-activated cell sorting analyses were done on a FACSCalibur instrument. FITC-positive BrdUrd staining and propidium iodide signal allowed assessment of ongoing DNA replication and the cell cycle stage. Percentages of the cell population in each cell cycle stage were plotted for each test article concentration.

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Immunoblotting [1]
Asynchronously growing tumor cell lines were exposed to differing concentrations of Dinaciclib (SCH727965; PS-095760). Subsequently, cells were harvested and lysed in a 50 mmol/L Tris-HCl buffer containing 350 mmol/L NaCl, 0.1% NP40, 1 mmol/L DTT, and a cocktail of protease and phosphatase inhibitors. Following protein concentration determination, cell lysates were separated on reducing SDS-PAGE gels and immunoblotted with antisera specific for Rb phosphorylated on serines 807, 811, hypophosphorylated Rb, or the p85 poly ADP ribose polymerase (PARP) caspase cleavage product.

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Induction of apoptosis assessed by activated caspase [1]
Assays of caspase activation were done using the Beckman Coulter CellProbe HT Caspase-3/7 Whole Cell Assay system. Asynchronously growing cells were plated into 96-well plates and allowed to adhere. Cells were exposed to differing concentrations of Dinaciclib (SCH727965; PS-095760) or vehicle for 24 hours. Cells were subsequently incubated with a fluorescently labeled caspase substrate; uptake and fluorescence of the substrate within cells correlate with the level of activated caspases. The caspase activity was determined using an Analyst AD 96.384 fluorometer (485 nm excitation and 530 nm emission).

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Clonogenicity and alamarBlue viability assays [1]
Cells were plated onto tissue culture dishes and propagated with the appropriate growth media. Growing cultures were exposed to increasing concentrations of Dinaciclib (SCH727965; PS-095760) or a vehicle control, typically for 7 days. After removing the medium, cells were fixed with 50% methanol/50% acetone for 5 minutes and stained with 0.2% crystal violet in 2% ethanol for 5 minutes. Following staining, cells were washed with 5 to 10 mL of water. Stained cells were solubilized in 1% deoxycholic acid, and the absorbance of the resulting solution was measured at 600 nm using a SOFTmax PRO 4.3 plate reader. Absorbance of SCH 727965–treated samples was plotted as a percent of that of a vehicle-treated control, and data were reported as an IC50 value relative to these controls. For suspension cell lines, assessments of cell viability were obtained using the alamarBlue Cell Viability Assay kit, using the manufacturers' recommended protocol.

Mice: Certain cell lines are grown in vitro, once they have been washed with PBS, they are resuspended in 50% Matrigel in PBS until they reach a final concentration of 4×10⁷ to 5×10⁷ cells per milliliter for tumor implantation. 0.1 mL of this suspension is subcutaneously injected into the flanks of naked mice. Every mouse has its tumor measured twice a week using a caliper to determine its length (L), width (W), and height (H). The tumor volume is then determined by applying the formula (L×W×H)/2. The animals are randomized to treatment groups (10 mice/group) when the tumor volume reaches 100 mm³. They are then given individual chemotherapeutic agents or Dinaciclib (8, 16, 32, and 48 mg/kg daily, i.p.) in accordance with the dosing schedule shown in the table and figure legends. Body weights and tumor volumes are measured both during and after treatment.

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In vivo tumor growth assessments [1]
For tumor implantation, specific cell lines were grown in vitro, washed once with PBS, and resuspended in 50% Matrigel in PBS to a final concentration of 4 × 107 to 5 × 107 cells per milliliter. Nude mice were injected with 0.1 mL of this suspension s.c. in the flank region. Tumor length (L), width (W), and height (H) were measured by a caliper twice weekly on each mouse and then used to calculate tumor volume using the formula (L × W × H)/2. When the tumor volume reached ∼100 mm3, the animals were randomized to treatment groups (10 mice/group) and treated i.p. with either Dinaciclib (SCH727965; PS-095760) or individual chemotherapeutic agents according to the dosing schedule indicated in table and figure legends. Tumor volumes and body weights were measured during and after the treatment periods.

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Assessments of Dinaciclib (SCH727965; PS-095760) effects on hematologic parameters [1]
A daily dose of Dinaciclib (SCH727965; PS-095760) (40 mg/kg) was administered i.p. to BALB/c mice for 5 days. Blood was collected on day 6 and day 13 (1st and 7th day after the final dose, respectively), diluted 1:5 in PBS, and immediately analyzed on an Advia 120 hematology analyzer (with differential).

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Pharmacokinetic determinations [1]
Plasma samples from mice were collected at various times after i.p. administration of Dinaciclib (SCH727965; PS-095760). At each time point, blood samples from three animals were combined and analyzed for SCH 727965 by liquid chromatography-tandem mass spectrometry. Pharmacokinetic variables were estimated from the plasma concentration data. Maximum plasma concentration values were taken directly from the plasma concentration time profiles, and the area under the plasma concentration versus time curve (0–24 h) was calculated using the linear trapezoidal rule.

Pharmacokinetic studies showed that SCH 727965 has a short plasma half-life in mouse. Thus, a dose of 5 mg/kg SCH 727965 given i.p. in mice was associated with a plasma half-life of ∼0.25 hour (Supplementary Table S2), perhaps suggesting a need for frequent dosing. However, previous results imply that continuous SCH 727965 exposure may not be a prerequisite for antiproliferative activity because short treatments with the drug induce long-term effects in vitro and in vivo. [1]

[1]. Mol Cancer Ther . 2010 Aug;9(8):2344-53.

[2]. Mol Cancer Ther . 2011 Apr;10(4):591-602.

[3]. Cell Cycle . 2011 Mar 15;10(6):977-88.

[4]. Mol Cancer Ther . 2011 Jun;10(6):1018-27.

2-[(2S)-1-[3-ethyl-7-[(1-oxido-3-pyridin-1-iumyl)methylamino]-5-pyrazolo[1,5-a]pyrimidinyl]-2-piperidinyl]ethanol is a pyrazolopyrimidine.
Dinaciclib has been used in trials studying the treatment of rrMM, rrCLL, rrDLBCL, Solid Tumors, and Solid Neoplasm, among others.
Dinaciclib is a pyrazolo[1,5-a]pyrimidine with potential antineoplastic activity. Dinaciclib selectively inhibits cyclin dependent kinases CDK1, CDK2, CDK5, and CDK9; inhibition of CDK1 and CDK2 may result in cell cycle repression and tumor cell apoptosis.

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Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC(50) values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent.[1]

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SCH 727965 was selected for clinical development following a functional in vivo screen that integrated both efficacy and tolerability of tested compounds. This rapid and discriminatory approach identified a candidate for clinical development that was significantly more effective and better tolerated than flavopiridol. SCH 727965 has several distinct in vitro properties consistent with an improved in vivo therapeutic index. Notably, the compound exhibits strong selectivity for the CDK family. These data suggest the activated CDK conformation has unique structural aspects, not present in closely related serine/threonine kinases (such as the extracellular signal-regulated kinase and GSK3 families), thus providing a potential explanation for the observed excellent selectivity and tolerability profiles of SCH 727965.
In vitro and in vivo analyses presented in this study support the conclusion that SCH 727965 has the potential to inhibit the growth of a broad spectrum of human cancers. SCH 727965 induced mechanism-based apoptosis in the vast majority of tested human tumor cell lines of diverse origin, following a single exposure. In agreement, SCH 727965 was effective at doses below the MTD level in multiple in vivo models and induced regression in several xenografts using continuous or intermittent schedules. Under similar conditions, the observed xenograft efficacy profiles of SCH 727965 were consistently superior to those achieved using approved benchmark agents, such as taxanes. Moreover, in mechanism-based biomarker studies, effective doses of the drug were sufficient to suppress phosphorylated Rb levels in surrogate tissues, such as skin and hair follicles. Likewise, active dose levels in the mouse were also associated with reversible effects on hematologic parameters. Quantitative tracking of leukocyte cell counts may offer an additional approach for tracking mechanism-based pharmacodynamic effects of SCH 727965.
Interestingly, the in vivo activity of SCH 727965 observed in murine systems was readily detectable despite rapid clearance of the parent compound from mouse plasma, indicating that continual exposure to SCH 727965 was not necessary for activity in vivo. Consistent with this, short exposure to SCH 727965 can induce long-lasting pharmacodynamic effects in vitro. Thus, a 2-hour exposure to ≤500 nmol/L SCH 727965 was sufficient to suppress BrdUrd incorporation 24 hours later. Similarly, transient in vitro exposure to SCH 727965 induced suppression of Rb phosphorylation that was correlated with induction of apoptosis. Significantly, escalation of SCH 727965 exposure (≤30 μmol/L) did not augment apoptotic phenotypes, suggesting a relative lack of nonspecific or off-target cytotoxicity. Taken together, the available in vitro and in vivo data show that long-lasting therapeutic effects can be induced within sensitive cells following short exposures to SCH 727965. It is possible that selecting compounds for further development solely on the basis of pharmacokinetic parameters would not have facilitated selection of SCH 727965 for clinical development. In summary, the approach of in vivo screening in mice ultimately led to the selection of a compound with attractive biochemical and pharmacologic properties.
Inhibitors of the CDK family have been proposed as attractive drug targets and pursued for oncology indications for several years, and several candidate molecules have entered clinical studies. In the case of flavopiridol, a combination of suboptimal selectivity, poor drug-like qualities, and adverse side effects may ultimately obscure any potentially desirable mechanism-based activities of this agent. In this study, we have described the novel pharmacologic properties of SCH 727965, a highly potent and selective CDK inhibitor that is differentiated from first generation CDK inhibitor compounds, such as flavopiridol. SCH 727965 is currently undergoing clinical testing against a range of solid and hematologic malignancies. The overall excellent profile of SCH 727965 suggests this molecule has the necessary properties to allow further pharmacologic exploration of the cell cycle mechanism in oncology. [1]

C21H28N6O2

396.49

396.227

C, 63.62; H, 7.12; N, 21.20; O, 8.07

779353-01-4

46926350

white solid powder

1.3±0.1 g/cm3

1.677

0.97

2

6

7

29

512

1

O([H])C([H])([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N1C1C([H])=C(N([H])C([H])([H])C2C([H])=C([H])C([H])=[N+](C=2[H])[O-])N2C(=C(C([H])=N2)C([H])([H])C([H])([H])[H])N=1

PIMQWRZWLQKKBJ-SFHVURJKSA-N

InChI=1S/C21H28N6O2/c1-2-17-14-23-27-19(22-13-16-6-5-9-25(29)15-16)12-20(24-21(17)27)26-10-4-3-7-18(26)8-11-28/h5-6,9,12,14-15,18,22,28H,2-4,7-8,10-11,13H2,1H3/t18-/m0/s1

2-[(2S)-1-[3-ethyl-7-[(1-oxidopyridin-1-ium-3-yl)methylamino]pyrazolo[1,5-a]pyrimidin-5-yl]piperidin-2-yl]ethanol

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 (6.31 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 (6.31 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), suspension 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 (6.31 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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Solubility in Formulation 4: 2% DMSO +30% PEG 300 +ddH2O: 10 mg/mL

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Solubility in Formulation 5: 10 mg/mL (25.22 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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