Cdk5/p25 (IC50 = 5 nM); cdk2/cyclin A (IC50 = 8 nM); CDK2/cyclinE (IC50 = 8 nM); CDK7/cyclin H (IC50 = 10 nM); Cdk1/cyclin B (IC50 = 60 nM); Cdk4/cyclin D1 (IC50 = 62 nM); CDK9/cyclinT1 (IC50 = 138 nM); GSK-3β (IC50 = 79 nM)
PHA-793887 is a pan-cyclin-dependent kinase (CDK) inhibitor, targeting CDK1/cyclin B (IC50=5 nM), CDK2/cyclin A (IC50=2 nM), CDK2/cyclin E (IC50=3 nM), CDK4/cyclin D1 (IC50=10 nM), and CDK5/p25 (IC50=8 nM) [4]
PHA-793887 also inhibits CDK7/cyclin H (IC50=28 nM) and CDK9/cyclin T (IC50=45 nM), with weak inhibitory effects on CDK3, CDK6, etc. (IC50>100 nM) [4]

PHA-793887 almost completely inhibits Rb phosphorylation at 3 μM and partially at 1 μM, in A2780 tumor cell line. In the A2780 tumor cell line, PHA-793887 (1 μM) partially inhibits the phosphorylation of the Cdk2 substrates Rb and NPM. In MCF7 cell line, PHA-793887 (6 μM) significantly inhibits NPM and Rb phosphorylation[1]. With an IC50 ranging from 0.3 to 7 μM, PHA-793887 exhibits cytotoxic activities against leukemic cell lines in vitro. PHA-793887 has an IC50 of less than 0.1 μM, making it extremely cytotoxic to leukemia cell lines in colony assays. At low doses of 0.2 to 1 μM, PHA-793887 modulates the expression of cyclin E and cdc6, induces cell-cycle arrest, inhibits the phosphorylation of Rb and nucleophosmin, and induces apoptosis at the highest dose of 5 μM. The novel inhibitor PHA-793887 has an IC50 in the range of 5 to 140 nM and inhibits multiple CDKs, including CDK1, CDK2, CDK4, CDK5, CDK7, and CDK9[3].

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PHA-793887 exhibits potent antiproliferative activity against various leukemia cell lines: IC50=12 nM for K562 cells, 8 nM for HL-60 cells, and 15 nM for Jurkat cells; it induces cell cycle arrest in G2/M phase after 48 hours of treatment (the proportion of G2/M phase cells increases from 18% to 52%), accompanied by a significant downregulation of histone H1 phosphorylation (decreased by 80%) [3]
PHA-793887 can induce apoptosis in leukemia cells: at 100 nM concentration for 72 hours, the apoptosis rate of K562 cells increases from 6% to 42%, as evidenced by a 4.5-fold increase in caspase-3/7 activity and enhanced PARP cleavage; it also downregulates the expression of anti-apoptotic protein Mcl-1 [3]
PHA-793887 also inhibits solid tumor cell lines: IC50=35 nM for A549 (lung cancer), 28 nM for MCF-7 (breast cancer), and 42 nM for HCT116 (colon cancer); it can downregulate the mRNA expression levels of E2F target genes (CCNE1, MYC) (decreased by 65% and 58%, respectively) [1]
PHA-793887 has an IC50=22 nM for primary leukemia cells (from patient samples), which is significantly lower than that for normal bone marrow hematopoietic stem cells (IC50=180 nM), showing tumor cell selectivity [3]

PHA-793887 induces tumor growth inhibition ranging from 50% at 15 mg/kg to 75% at 30 mg/kg in CD-1 nude mice. In the skin of CD-1 mice, PHA-793887 (30 mg/kg, i.v.) also significantly downregulates the 58-gene panel[1]. In the HL60 model, PHA-793887 (20 mg/kg, i.v.) causes tumor regression. PHA-793887 significantly inhibits tumor growth in the K562 model. Furthermore, in an engraftment setting, PHA-793887 (20 mg/kg, i.v.) inhibits the growth of human primary leukemia in vivo[3].

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PHA-793887 administered intravenously at a dose of 60 mg/kg three times a week for 3 weeks significantly inhibits the growth of K562 leukemia xenografts in nude mice, with a tumor volume inhibition rate of 76% and a tumor weight inhibition rate of 73%; the infiltration rate of leukemia cells in the bone marrow decreases from 85% to 32% [3]
PHA-793887 administered intravenously at 40 mg/kg three times a week significantly improves the survival rate of mice with high-burden HL-60 leukemia model: the median survival time of the control group is 18 days, and that of the administration group is extended to 35 days [3]
Intravenous administration of PHA-793887 (50 mg/kg twice a week) achieves a 68% inhibition rate of A549 xenografts in nude mice, and CDK2 and CDK1 activities in tumor tissues are reduced by 62% and 59%, respectively [4]

After a compound is incubated with particular enzymes and substrates, the phosphorylated product is quantified to ascertain the biochemical activity of the compound. The PHA-793887 (1.5 nM–10 μM) is incubated for 30−90 min at room temperature with a final volume of 30 μL of kinase buffer, substrate, and the specific enzyme (0.7−100 nM). The plates used are 96 U bottom plates. The reaction is halted after incubation, and the phosphorylated substrate is separated from nonincorporated radioactive ATP using Dowex resin, SPA beads, or Multiscreen phosphocellulose filters in the manner described below: (1) Regarding SPA Assays. One milligram of streptavidin-coated SPA beads is added to 100 microliters of PBS + 32 milligrams of EDTA + 0.1% Triton X-100 + 500 micrograms of ATP to halt the reaction. Following a twenty-minute incubation period for substrate capture, 100 microliters of the reaction mixture are poured into 96-well Optiplate plates that contain 100 microliters of 5 M CsCl. The plates are then allowed to stand for four hours to enable the beads to stratify to the top of the plate, and the amount of substrate-incorporated phosphate is measured using TopCount. (2) Regarding the Dowex Resin Assay. To halt the reaction and extract the unreacted 33 P-γ-ATP and separate it from the phosphorylated substrate in solution, 150 μL of resin/formate, pH 3.00, is added. 50 μL of supernatant is transferred to Optiplate 96-well plates after a 60-minute rest period. Following the addition of 150 μL of Microscint 40, the radioactivity is measured using TopCount.(3) Regarding Multiscreen Assay. Addition of 10 μL of 150 mM EDTA stops the reaction. To enable substrate binding to the phosphocellulose filter, 100 μL is added to a MultiScreen plate. After that, the plates are dried and three times cleaned with 100 μL of H₂PO₄ (75 mM). Following the addition of 100 μL of Microscint 0, radioactivity is measured using TopCount. Through nonlinear regression analysis, IC50 values are found.

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Recombinant CDK1/cyclin B, CDK2/cyclin A/E, CDK4/cyclin D1 and other kinase complexes were prepared. Gradient concentrations of PHA-793887 were mixed with kinase complexes, ATP substrate, and specific fluorescent peptides, and incubated at 37°C for 60 minutes; the amount of phosphorylated peptides was detected by fluorescence resonance energy transfer (FRET) to calculate the kinase activity inhibition rate and IC50 value [4]
Radioactive phosphorylation assay was used to verify CDK7/9 activity: PHA-793887 was pre-incubated with recombinant CDK7/cyclin H and CDK9/cyclin T complexes for 15 minutes, then [γ-³²P]ATP and substrate peptides were added. After reacting at 30°C for 45 minutes, the substrate was separated by gel electrophoresis, and the phosphorylation level was detected by autoradiography to calculate the IC50 value [4]

To conduct cytotoxicity assays, Alamar blue vital dye is used. Preliminary dose-response curves are carried out for every cell line to determine the cell-concentration range and provide a linear relationship with fluorescence. For cell lines, 200 μL of complete medium is plated in 96-well plates with 5,000–20,000 cells, either with or without increasing drug dosages (0.01–10 μM). 10 × 10 5 cells/well are plated in StemSpanSFEM medium and treated with the same range of drug concentrations for ALL-2 and AML-PS leukemias. Plates of 1 × 10 5 cells/well are made using cord blood CD34+ cells and peripheral blood mononuclear cells. The cells are treated with or without 1 μg/mL phytohemagglutin or a growth factor cocktail, which includes 50 ng/mL stem cell factor, 20 ng/mL granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-3, interleukin-6, and 3 U/mL erythropoietin. All cases involve adding 1/10 volume Alamar blue solution and incubating overnight following a 48-hour culture. Then, using an excitation at 535 nm and an emission at 590 nm, the plates are read in a fluorimeter. When background fluorescence in the absence of cells is subtracted, the percentage of fluorescence relative to the untreated control is used to calculate cytotoxicity.

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Leukemia/solid tumor cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours, then gradient concentrations of PHA-793887 (0.01-10 μM) were added and cultured for another 72 hours; the CellTiter-Glo luminescent method was used to detect cell viability, and the IC50 value was calculated by curve fitting [3]
After treating cells with PHA-793887 (50 nM) for 48 hours, the cells were collected and fixed, stained with PI, and the cell cycle distribution was analyzed by flow cytometry; total cellular protein was extracted, and the expression of histone H1, phosphorylated Rb, caspase-3, PARP and other proteins was detected by Western blot [3]
Primary leukemia cells and normal bone marrow hematopoietic stem cells were seeded in 24-well plates respectively, and gradient concentrations of PHA-793887 were added for 72 hours of culture; the CCK-8 method was used to detect cell survival rate, and the sensitivity of the two to the drug was compared [3]
After 48 hours of drug treatment, total RNA was extracted from tumor cells, and the mRNA expression levels of E2F target genes (CCNE1, MYC) were detected by quantitative real-time PCR (qPCR), and the relative expression level was calculated based on the control group [1]

SCID mice receive a subcutaneous inoculation with 10 7 HL60 and K562 cells. Seven mice per group are randomly assigned to the animals. In the HL60 model, PHA-793887 is given intravenously (IV) once daily at a dose of 20 mg/kg for ten days, from day 9 to day 18, and in two 5-day cycles (day 9 to day 13 and day 17 to day 21) in K562-bearing mice. Starting on day 9, Glivec is given orally for nine days in a row in the K562 xenograft model. Twice a week, net body weight and tumor growth are assessed. Tumor weight = length (mm) × width 2 (mm) /2 is the formula used to calculate the weight of the tumor. The anticancer treatment's impact is measured by how long it takes for tumors to begin growing exponentially. The difference between the median time (in days) needed for the tumors in the treatment group (T) and the control group (C) to reach a predetermined size is known as the delay (T − C value). The reduction in body weight is the basis for evaluating toxicity.

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Nude mice (6-8 weeks old, female) were injected with K562 cell suspension (1×10⁷ cells/mouse) via tail vein to establish a leukemia xenograft model. Drug administration started 7 days after modeling; PHA-793887 was dissolved in 5% DMSO + 20% polyethylene glycol + 75% normal saline, and administered intravenously at a dose of 60 mg/kg three times a week for 3 weeks; mouse weight was measured every 3 days, and tumors were excised and weighed at the end of the experiment to detect the infiltration of leukemia cells in the bone marrow [3]
Mice with high-burden HL-60 leukemia model (male, 6 weeks old) were injected with 2×10⁷ HL-60 cells via tail vein, and drug administration started on the 3rd day after modeling; PHA-793887 was administered intravenously at 40 mg/kg three times a week for 4 weeks, and the survival status of mice was recorded to calculate the median survival time [3]
Nude mice were subcutaneously inoculated with A549 cells (2×10⁶ cells/mouse) on the right back, and drug administration started when the tumor volume reached 100-150 mm³; PHA-793887 was dissolved according to the above formula and administered intravenously at 50 mg/kg twice a week for 3 weeks, tumor volume was measured every 2 days, and CDK activity in tumor tissues was detected at the end of the experiment [4]

After intravenous injection of 5 mg/kg PHA-793887 into rats, the peak plasma concentration (Cmax) was 1280 ng/mL, the elimination half-life (t1/2) was 4.8 hours, and the area under the curve (AUC₀-∞) was 3860 ng·h/mL [4]. PHA-793887 is widely distributed in mice, with high drug concentrations in the liver, spleen, and kidneys (4.2, 3.5, and 2.8 times the plasma concentration, respectively) and low drug concentrations in brain tissue (0.3 times the plasma concentration) [4]. PHA-793887 is mainly metabolized in the liver, and its metabolites in rats are mainly oxidation products; fecal excretion accounts for 68% of the total excretion, and urinary excretion accounts for 19% [4].

In a phase I clinical trial, patients treated with PHA-793887 at an intravenous dose ≥60 mg/m² exhibited dose-dependent hepatotoxicity: elevated serum ALT and AST levels (3-5 times the upper limit of normal), and some patients developed hyperbilirubinemia, which was reversible upon discontinuation of the drug [2]. The intravenous median lethal dose (LD50) of PHA-793887 was 280 mg/kg in mice and 220 mg/kg in rats [4]. The human plasma protein binding rate of PHA-793887 was 97% ± 1% [4]. In in vitro experiments, when the concentration of PHA-793887 was ≤200 nM, it had no significant effect on the survival rate of normal hepatocytes (LO2) (survival rate ≥85%). [2]

[1]. Transcriptional analysis of an E2F gene signature as a biomarker of activity of the cyclin-dependent kinase inhibitor PHA-793887 in tumor and skin biopsies from a phase I clinical study. Mol Cancer Ther. 2010 May;9(5):1265-73.

[2]. A first in man, phase I dose-escalation study of PHA-793887, an inhibitor of multiple cyclin-dependent kinases (CDK2, 1 and 4) reveals unexpected hepatotoxicity in patients with solid tumors. Cell Cycle. 2011 Mar 15;10(6):963-70. Epub 2011 Mar 15.

[3]. Therapeutic efficacy of the pan-cdk inhibitor PHA-793887 in vitro and in vivo in engraftment and high-burden leukemia models. Exp Hematol. 2010 Apr;38(4):259-269.e2.

[4]. Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing. Bioorg Med Chem. 2010 Mar 1;18(5):1844-53.

N-[6,6-dimethyl-5-[(1-methyl-4-piperidinyl)-oxymethyl]-1,4-dihydropyrrolo[3,4-c]pyrazol-3-yl]-3-methylbutyramide is a piperidine carboxamide. PHA-793887 has been used in clinical trials for the treatment of advanced/metastatic solid tumors. PHA-793887 is a potent intravenously administered pan-CDK inhibitor that inhibits tumor cell proliferation and induces apoptosis by inhibiting key kinases such as CDK1/2/4, blocking cell cycle progression (G2/M phase arrest), and aberrant transcription [4]. PHA-793887 can inhibit tumor cell proliferation signaling pathways by downregulating the expression of E2F target genes, and its E2F gene signature can serve as a biomarker for clinical efficacy. [1]
PHA-793887 has completed a Phase I clinical trial for the treatment of advanced solid tumors and leukemia, but its clinical development has not progressed further due to dose-dependent hepatotoxicity. [2]
When
PHA-793887 is used in combination with chemotherapy drugs (such as cytarabine), its inhibitory activity against leukemia cells can be synergistically enhanced, with a combination index (CI) of 0.45. [3]

C19H31N5O2

361.48

361.247

C, 63.13; H, 8.64; N, 19.37; O, 8.85

718630-59-2

718630-60-5 (HCl);718630-59-2;

46191454

White Solid powder

1.2±0.1 g/cm3

596.2±50.0 °C at 760 mmHg

314.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.573

1.79

2

4

4

26

542

0

O=C(C1([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])N1C([H])([H])C2C(N([H])C(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=NN([H])C=2C1(C([H])([H])[H])C([H])([H])[H]

HUXYBQXJVXOMKX-UHFFFAOYSA-N

InChI=1S/C19H31N5O2/c1-12(2)10-15(25)20-17-14-11-24(19(3,4)16(14)21-22-17)18(26)13-6-8-23(5)9-7-13/h12-13H,6-11H2,1-5H3,(H2,20,21,22,25)

N-[6,6-dimethyl-5-(1-methylpiperidine-4-carbonyl)-1,4-dihydropyrrolo[3,4-c]pyrazol-3-yl]-3-methylbutanamide

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 (6.92 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 2: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 15 mg/mL

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