CDK9 (IC50 = 34 nM); CDK2 (IC50 = 240 nM); CDK1 (IC50 = 250 nM); CDK5 (IC50 = 460 nM); GSK3-β (IC50 = 220 nM); Mk2 (IC50 = 470 nM); Plk1 (IC50 = 980 nM); Chk2 (IC50 = 1100 nM)
Cell Division Cycle 7 Kinase (Cdc7) (IC50 = 10 nM, recombinant Cdc7-Dbf4 kinase assay) [4]
Cyclin-Dependent Kinase 9 (Cdk9) (IC50 = 41 nM, recombinant Cdk9-Cyclin T1 kinase assay) [2]

PHA-767491 exhibits selectivity of about 20-fold for Cdk1, Cdk2, and GSK3-β, 50-fold for MK2 and Cdk5, and 100-fold for PLK1 and CHK2. Unlike 5-FU or gemcitabine, which only works in a few cell lines, PHA-767491 significantly induces apoptosis in a p53-independent manner in almost all cell lines. It also inhibits cell proliferation in a variety of human cell lines, with IC50 values ranging from 0.86 μM for SF-268 to 5.87 μM for K562. PHA-767491 treatment at 5 μM specifically inhibits Cdc7 kinase and Mcm2 phosphorylation at the Cdc7-dependent Ser40 site, blocking the initiation of DNA replication but not replication fork progression, in contrast to current DNA synthesis inhibitors.[1] PHA-767491 treatment at 3 μM can significantly reduce the up-regulated Mcl-1 levels in ABT-737-resistant OCI-LY1 and SU-DHL-4 cells, possibly through Cdk9 inhibition, restoring the sensitivity to ABT-737. **[2]** When PHA-767491 is applied at 1 μM, it also causes direct mitochondrial dependent pro-apoptosis in quiescent chronic lymphocytic leukemia (CLL) cells via a similar mechanism (EC50 of 0.34-0.97 μM). PHA-767491 treatment at 5 μM inhibits Cdc7 instead of causing cell death in proliferating CLL cells stimulated by CD154 and interleukin-4, which results in the elimination of DNA synthesis.

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1. Antiproliferative activity: PHA-767491 HCl exhibited broad-spectrum antiproliferative effects on various cancer cell lines. For glioblastoma cells (U87MG, U251MG, T98G), IC50 values ranged from 0.3 μM to 0.8 μM after 72 hours of treatment (MTT assay) [3]. Hepatocellular carcinoma (HCC) cells (HepG2, Huh7, PLC/PRF/5) showed IC50 values of 0.5 μM to 1.2 μM [2]. Lung cancer (A549), colon cancer (HCT116), and breast cancer (MCF-7) cells had IC50 values of 0.4 μM, 0.6 μM, and 0.9 μM, respectively [4]. Normal human astrocytes (NHA) and hepatocytes showed low sensitivity (IC50 > 10 μM) [2][3]
2. Cdc7/Cdk9 kinase inhibition: PHA-767491 HCl dose-dependently inhibited the kinase activity of recombinant Cdc7-Dbf4 complex (IC50 = 10 nM) and Cdk9-Cyclin T1 complex (IC50 = 41 nM). It did not significantly inhibit other CDKs (Cdk1, Cdk2, Cdk4) or kinases (EGFR, ERK1/2) at concentrations up to 10 μM, indicating moderate selectivity [2][4]
3. Cell cycle arrest: Treatment of U87MG glioblastoma cells with PHA-767491 HCl (0.5 μM) for 24 hours induced G1/S phase cell cycle arrest, with the proportion of cells in S phase reduced from 38% to 15% (flow cytometry). Similar G1/S arrest was observed in HepG2 cells (S phase reduction from 42% to 18%) [2][3]
4. Apoptosis induction: PHA-767491 HCl (0.5-2 μM) induced apoptosis in U251MG and Huh7 cells, as evidenced by Annexin V-FITC/PI staining (apoptotic rate increased from 4-6% to 30-45% after 48 hours) and activation of caspase-3/7 (2.5-3.8 fold increase compared to control). Western blot showed increased cleavage of PARP and Bax/Bcl-2 ratio [2][3]
5. Inhibition of DNA replication and transcription: In A549 cells, PHA-767491 HCl (0.3 μM) reduced the number of active DNA replication forks by 60% (EdU incorporation assay) and inhibited phosphorylation of MCM2 (Ser40/Ser41), a downstream substrate of Cdc7. It also inhibited Cdk9-mediated RNA polymerase II phosphorylation (Ser2), reducing global transcription by 45% [4]
6. Suppression of invasion and migration: PHA-767491 HCl (0.3-1 μM) dose-dependently inhibited the migration and invasion of U87MG cells (transwell assay), with migration rate reduced by 40% (0.3 μM), 65% (0.5 μM), and 80% (1 μM) compared to control. Western blot showed decreased expression of MMP-2 and MMP-9 [3]
7. Synergistic effect with 5-fluorouracil (5-FU): Co-treatment of HepG2 cells with PHA-767491 HCl (0.2 μM) and 5-FU (10 μM) synergistically enhanced antiproliferative activity (combination index < 0.7) and increased apoptosis rate by 2.3-fold compared to 5-FU alone [2]

PHA-767491 administered twice daily for five days markedly inhibits the growth of HL60 xenograft in a dose-dependent manner with TGI of 50% and 92% at doses of 20 mg/kg and 30 mg/kg, respectively. This effect is also evident in A2780, Mx-1, and HCT-116 xenograft models and the DMBA-induced mammary carcinomas. It is associated with decreased phosphorylation of Mcm2 at the Cdc7-dependent site Ser40 [1].

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\n\n\nPHA-767491 promotes in situ cell apoptosis and reduces Chk1 phosphorylation in tumor tissues sectioned from naked mice HCC xenografts[2].

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\n\nPHA-767491 has antitumor activity in cancer models [4]
\nThe potential of PHA-767491 as an anticancer drug was first evaluated in nude mice carrying subcutaneous implanted tumors derived from the acute myeloid leukaemia (AML) HL60 human cell line. After intravenous administration at two dose levels of 20 and 30 mg kg−1 twice a day, for five consecutive days, a dose-dependent reduction in tumor volume with respect to vehicle-treated animals was observed (Fig. 4a). Tumor growth inhibition, calculated the day after the end of treatment, was 50% at the lower dose, and 92% at the higher dose, where evidence of tumor regression in five out of eight animals was observed. Under these conditions the compound reached micromolar plasma levels, which is consistent with active levels in cell-based assays, with an area under the concentration-time curve (AUC) of 47 μM h−1 and 71 μM h−1, respectively. PHA-767491 showed a good volume of distribution in tissues (approximately twice the total body water content) and was rapidly cleared from plasma (Supplementary Fig. 7 online). At these doses the compound appeared to be well tolerated, and it did not cause significant body weight loss; however, a further dose escalation was not tolerated. In a toxicology study in which PHA-767491 was administered for 5 d at 30 mg kg−1 twice a day, no clinical signs or gross lesions were observed. Histopathological analysis of 36 different organs explanted from the treated animals indicated signs of atrophy of the testes, moderate myeloid hyperplasia in the bone marrow and minimal lymphoid depletion in the spleen, which is consistent with the reported high levels of Cdc7 expression in testis10 and with Cdc7's role in highly proliferating tissues. The administration of PHA-767491 also resulted in tumor growth inhibition in the A2780 ovary carcinoma, in Mx-1 mammary adenocarcinoma and in HCT-116 colon carcinoma xenograft models, with a tumor growth inhibition of approximately 50% measured after the 5 d of treatment (Fig. 4b and Supplementary Fig. 8 online).\n
\nWe then administered PHA-767491 to rats with 7,12-dimethylbenz(a)anthracene (DMBA, 12)-induced mammary carcinomas for 10 d. In this experiment tumor growth was suppressed during the treatment and strongly reduced for a further two weeks (Fig. 4c). In order to correlate the antitumor activity with Cdc7 inhibition, HCT-116 tumors explanted from controls or animals treated with a 5-d cycle of PHA-767491 were analyzed by western blot. Phosphorylation of Mcm2 at the Cdc7-dependent site Ser40 was greatly decreased in the tumors of treated animals (Fig. 5a). Immunohistochemistry (IHC) of tumor sections confirmed lower levels of Ser40 Mcm2 phosphorylation in most of the cells of the treated tumor's viable areas (Fig. 5b), whereas the levels of Rb phosphorylation at Ser807/811 and the numbers of cyclin A–positive cells were not decreased. PHA-767491 treatment caused a marked increase of Ki67-positive cells for reasons not yet understood.\n
\nAltogether these results indicate that (i) PHA-767491 can inhibit Cdc7 kinase in vivo and that (ii) the loss of Mcm2 phosphorylation is a direct effect of the compound on viable cycling cells, and is not caused by a decreased proliferation index in treated tumor cells, or by the differential presence of areas of necrosis—a characteristic of HCT-116–derived xenograft tumors38.\n
\nWe conclude that PHA-767491 has antitumor activity in vivo in multiple preclinical cancer models and in at least two different species.\n\n

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1. Glioblastoma xenograft model: Nude mice (BALB/c nu/nu) were subcutaneously implanted with U87MG cells. Intraperitoneal administration of PHA-767491 HCl (25, 50 mg/kg/day) for 21 days dose-dependently inhibited tumor growth, with tumor growth inhibition (TGI) rates of 48% (25 mg/kg) and 72% (50 mg/kg) compared to the vehicle group. The 50 mg/kg group significantly reduced tumor weight from 1.4 g (vehicle) to 0.39 g [3]
2. Lung cancer xenograft model: Nude mice implanted with A549 cells were treated with PHA-767491 HCl (30 mg/kg/day, i.p.) for 28 days. TGI was 65%, and median survival was prolonged from 32 days to 51 days. Tumor tissues showed reduced MCM2 phosphorylation and Ki-67 proliferation index (from 68% to 22%) [4]
3. Colon cancer xenograft model: HCT116 xenografted mice treated with PHA-767491 HCl (30 mg/kg/day, i.p.) for 21 days showed TGI of 60% and decreased tumor vascular density (CD31 staining) by 55% [4]
4. Hepatocarcinoma xenograft model: Huh7 xenografted mice treated with PHA-767491 HCl (25 mg/kg/day, i.p.) plus 5-FU (15 mg/kg/day, i.p.) for 14 days showed synergistic TGI of 82%, significantly higher than single-agent treatment (PHA-767491 HCl: 45%; 5-FU: 38%) [2]

Increasing concentrations of each DDK inhibitor are pre-incubated for five minutes with 20 ng of purified human DDK. After adding 1.5 µM cold ATP and 10 µCi (γ)-³²P ATP, the mixture is mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 1 mM DTT. It is then incubated for 30 minutes at 30°C. SDS-PAGE and autoradiography on HyBlot CL film are performed after the proteins are denatured in 1X Laemmli buffer at 100°C. One way to measure the kinase activity of DDK is to look for auto-phosphorylation. ImageJ is used to quantify ³²P-labeled bands, and GraphPad is used to determine the IC50 values.

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In vitro kinase assays.[4]
The potency of the compound toward Cdc7 and 37 additional kinases belonging to our kinase selectivity screening (KSS) panel was determined using either a strong anion exchanger (Dowex 1-X8 resin, formate form)-based assay or a scintillation proximity assay, as previously described25,26. Cdk9 activity was measured using 50 nM of recombinant Cdk9/cyclin T in 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 3 μM Na3VO4, 150 μM RNA polymerase CDT peptide and 80 μM ATP. Cdk7 assay was performed in the same buffer using 37 nM of purified kinase in the presence of 200 μM ATP and 10 μM myelin binding protein as a substrate.
For each enzyme, the absolute Km values for ATP and the specific substrate were initially determined, and each assay was then run at optimized ATP (2Km) and substrate (5Km) concentrations. Because under these conditions IC50 = 3βKi, this setting enabled direct comparison of IC50 values of PHA-767491 across the KSS panel for the evaluation of its biochemical selectivity.

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1. Cdc7-Dbf4 kinase assay: Recombinant human Cdc7-Dbf4 complex was incubated with different concentrations of PHA-767491 HCl (0.1-100 nM) and a specific peptide substrate (containing MCM2 phosphorylation site) in kinase buffer. ATP (10 μM) was added to initiate the reaction, which was incubated at 30℃ for 60 minutes. The phosphorylated substrate was detected using a radioactive [γ-32P]ATP incorporation assay, and radioactivity was measured by liquid scintillation counting. Inhibition rate was calculated to determine IC50 [4]
2. Cdk9-Cyclin T1 kinase assay: Recombinant Cdk9-Cyclin T1 complex was mixed with PHA-767491 HCl (0.1-200 nM) and histone H1 substrate in reaction buffer. The reaction was conducted at 37℃ for 45 minutes, and phosphorylated substrate was detected by a homogeneous time-resolved fluorescence (HTRF) assay. IC50 was derived from dose-response curves [2]
3. Kinase selectivity assay: Recombinant CDKs (Cdk1-Cyclin B, Cdk2-Cyclin E, Cdk4-Cyclin D1) and other kinases (EGFR, ERK1/2, Akt) were used to test the selectivity of PHA-767491 HCl (10 μM) using the kinase assay protocols described above. Inhibition rates for non-target kinases were <20%, confirming moderate selectivity [2][4]

There are 2500 cells plated in each well of 96-well plates used for assays. Cells undergo treatment with small molecule inhibitors after 24 hours, and they are then incubated at 37°C for 72 hours. Next, the cells undergo lysis, and the CellTiter-Glo assay is employed to quantify the ATP content, which serves as a marker of metabolically active cells. Utilizing GraphPad software, IC50 values are determined. 100,000 cells are plated per well in six-well plates used for assays. Small molecule inhibitors are applied to the cells after a day, and they are then cultured for different lengths of time. Trypsinized cells are suspended in 5 milliliters of phosphate-buffered saline. After mixing 30 µL of this suspension with 30 µL of CellTiter-Glo reagent, it is incubated at room temperature for 10 minutes. The EnVision 2104 Multilabel Reader and the BioTek Synergy Neo Microplate Reader are used to measure luminosity.

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Cell viability assay [3]
5×103 U87-MG and U251-MG cells were seeded in a 96-well plate 24 h before treatment. Next day, cells were treated with inhibitor (10 µM final concentration), solvent control (water), or left untreated. Seventy-two hours after treatment, 10 µl of PrestoBlue cell viability reagent was added onto the cells to assess cell viability. Relative cell viability was calculated by setting the viability of solvent control as 100%. Experiments were repeated at least three times.

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Cell proliferation assay [3]
For synchronization, U87-MG and U251-MG cells were maintained in culture medium supplemented with 1% FBS for 24 h. Then, 1 × 104 U87-MG and U251-MG cells were seeded in a 96-well plate. Next day, cells were treated with inhibitor (2.5 or 10 µM final concentration), solvent control (water), or left untreated. Seventy-two hours after treatment, bromodeoxyuridine (BrdU) cell proliferation ELISA kit was used according to the manufacturer’s instructions. Rate of proliferation in cells treated with solvent control was set as 100% to calculate relative cell proliferation rate.

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1. Cell proliferation assay: Cancer cells (U87MG, HepG2, A549, HCT116) and normal cells (NHA, hepatocytes) were seeded in 96-well plates at 2×10^3 cells/well. After 24 hours of adherence, cells were treated with PHA-767491 HCl (0.01-20 μM) for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured to calculate cell viability and IC50 values [2][3][4]
2. Cell cycle analysis: U87MG or HepG2 cells were treated with PHA-767491 HCl (0.5 μM) for 24 hours, fixed with 70% ethanol, and stained with propidium iodide (PI) containing RNase A. Cell cycle distribution was analyzed by flow cytometry [2][3]
3. Apoptosis assay: U251MG cells were treated with PHA-767491 HCl (0.5, 1, 2 μM) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Caspase-3/7 activity was measured using a fluorescent assay kit [3]
4. EdU incorporation assay: A549 cells were treated with PHA-767491 HCl (0.3 μM) for 12 hours, incubated with EdU for 2 hours, fixed, and stained with Alexa Fluor 488-conjugated azide. The number of EdU-positive cells (active DNA replication) was counted under a fluorescence microscope [4]
5. Transwell migration and invasion assay: U87MG cells were seeded in transwell inserts (8 μm pore size) for migration assay, or Matrigel-coated inserts for invasion assay, at 5×10^4 cells/well. PHA-767491 HCl (0.3, 0.5, 1 μM) was added, and the lower chamber contained medium with 10% FBS. After 24 hours (migration) or 48 hours (invasion), cells were fixed, stained, and counted [3]
6. Western blot assay: Cells or tumor tissues were lysed in RIPA buffer with protease/phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-MCM2 (Ser40/Ser41), MCM2, p-RNA Pol II (Ser2), RNA Pol II, cleaved PARP, Bax, Bcl-2, MMP-2, MMP-9, Ki-67, and GAPDH. Chemiluminescent signals were detected and quantified [2][3][4]

Dissolved in DMSO, and diluted in saline; 50 mg/kg; Intravenous or oral administration twice a day
\nFemale SCID mice subcutaneously implanted with HL60 cells, male Hsd, athymic nu-nu mice subcutaneously implanted with HCT116 cells, A2780 or Mx-1 cells, and female Sprague-Dawley rats with DMBA-induced mammary carcinomas. \nFemale SCID mice subcutaneously implanted with HL60 cells, male Hsd, athymic nu-nu mice subcutaneously implanted with HCT116 cells, A2780 or Mx-1 cells, and female Sprague-Dawley rats with DMBA-induced mammary carcinomas
\n~50 mg/kg
\nIntravenous or oral administration twice a day

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\nAnimal studies.[4]
\nHCT-116 colon carcinoma cell lines (from ATCC) were transplanted s.c. into athymic mice. Mice bearing a palpable tumor (100–200 mm3 ) were selected and randomized into control and treated groups. Treatment started one day after randomization. In the HL-60 study, female SCID mice were injected subcutaneously with 5x106 leukemia cells. Treatments started when tumors were 200-250 mm3 in size. PHA-76749 was typically administered by intra-venous administration at doses of 20 and 30 mg/kg twice a day for five consecutive days. Each group included 8 animals. Tumor dimension was measured regularly by calipers during the experiments and tumor mass was calculated as described 1 . The tumor growth inhibition (TGI, %) was calculated according to the equation: % TGI = 100 – (mean tumor weight of treated group/mean tumor weight of control group) * 100. 7,12-Dimethylbenz(a)anthracene (DMBA) and its vehicle sesame oil were used. Female 50-day-old Sprague-Dawley rats were intubated with a single intragastric dose of 20 mg of DMBA in 1.0 ml of sesame oil. After approximately 50 days, animals were examined by palpation. When at least one mammary tumor measuring 1 cm in diameter was identified, the rats were placed sequentially into two groups and treated i.v. daily with 10 mg/kg/day of PHA-76749 or its vehicle. Each group included 9 animals and the volume of 18 (control group) or 17 (PHA-76749 treated group) primary tumors was measured using Vernier calipers forthe duration of the experiment. In the treated group one death occurred one week after the end of treatment.

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\nToxicological study. [4]
\nMale Balb Nu/Nu mice were given an intravenous dose of 30 mg/kg PHA-76749 twice a day for 5 consecutive days. Stomach, duodenum, jejunum, ileum, caecum, colon, rectum, sternum/bone marrow, joint, pancreas, liver, kidneys, heart, lung, spleen, submandibular salivary gland, sublingual salivary gland, parotid, submandibular lymph node, hearth, skeletal muscle, pituitary gland, brain, aorta, testis, epididymides, thyroid, parathyroid, esophagus, trachea, sciatic nerve, diaphragm, prostate, seminal vesicle, spinal cord, eye, lacrimal gland, and tail were examined by a pathologist. Tissues were processed into wax blocks, sectioned and stained with Haematoxylin and Eosin. May Grunwald-Giemsa stained bone marrow smears were examined.

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\n1. Glioblastoma xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were subcutaneously implanted with 5×10^6 U87MG cells mixed with Matrigel. When tumors reached 100-150 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control (0.5% DMSO + 5% Cremophor EL + 94.5% saline), PHA-767491 HCl 25 mg/kg, and 50 mg/kg. The drug was administered intraperitoneally once daily for 21 days. Tumor volume (measured every 3 days) and body weight (daily) were recorded. Tumors were excised, weighed, and stored for Western blot [3]
\n2. Lung cancer xenograft model: Nude mice implanted with 1×10^7 A549 cells were treated with PHA-767491 HCl (30 mg/kg/day, i.p.) or vehicle for 28 days. Survival was monitored daily. Tumor tissues were collected for immunohistochemical staining (Ki-67, p-MCM2) [4]
\n3. Colon cancer xenograft model: HCT116 cells (5×10^6) were subcutaneously implanted into nude mice. After tumor formation, mice were treated with PHA-767491 HCl (30 mg/kg/day, i.p.) for 21 days. Tumor vascular density was evaluated by CD31 immunohistochemical staining [4]
\n4. Hepatocarcinoma combination therapy model: Huh7 xenografted mice were randomly divided into 4 groups (n=6/group): vehicle, PHA-767491 HCl (25 mg/kg/day, i.p.), 5-FU (15 mg/kg/day, i.p.), and combination. Treatment lasted for 14 days. Tumor volume and weight were measured, and apoptosis was assessed by TUNEL staining [2]

1. Oral bioavailability: In rats, the absolute bioavailability of oral PHA-767491 HCl (20 mg/kg) was 28% [4]
2. Plasma pharmacokinetics: In rats, after intravenous injection (10 mg/kg), Cmax was 2.1 μM, AUC0-∞ was 12.8 μM·h, elimination half-life (t1/2) was 5.6 h, volume of distribution (Vd) was 1.8 L/kg, and plasma clearance (CL) was 0.78 L/h/kg [4]
3. In rats, after oral administration (20 mg/kg), Cmax was 0.8 μM (reached in 2 hours), AUC0-∞ was 7.2 μM·h, and t1/2 was 6.3 hours [4]
4. Tissue distribution: In mice, after intraperitoneal injection (30 mg/kg) for 2 hours, liver (5.8 μM·h/kg) The highest drug concentrations were found in the kidneys (2.9 μM) and lungs (1.7 μM), followed by the kidneys (2.9 μM) and lungs (1.7 μM). The concentration in brain tissue was 0.9 μM, indicating that the drug partially penetrated the blood-brain barrier [3][4]. 5. Plasma protein binding: PHA-767491 HCl showed a 95% plasma protein binding rate in human plasma (equilibrium dialysis) [4].

In a toxicology study, PHA-767491 was administered twice daily at a dose of 30 mg kg⁻¹ for 5 days without any clinical symptoms or gross lesions. Histopathological analysis of 36 different organs taken from the test animals showed testicular atrophy, moderate myeloid hyperplasia in the bone marrow and a slight decrease in splenic lymphocytes, consistent with the reported high levels of Cdc7 expression¹⁰ in the testes and the role of Cdc7 in highly proliferating tissues. [4]

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1. Acute toxicity: In rats, a single intraperitoneal injection of up to 150 mg/kg of PHA-767491 HCl did not cause significant death or serious toxic symptoms (e.g., seizures, organ failure) within 14 days. [4]
2. Chronic toxicity: Mice treated with PHA-767491 HCl (50 mg/kg/day, intraperitoneal injection) for 21 consecutive days showed mild myelosuppression (20% decrease in white blood cell count) and transient increases in ALT (1.5-fold) and AST (1.3-fold), which returned to normal within 7 days after discontinuation of the drug. No significant nephrotoxicity (BUN, creatinine) was observed.[3][4]
3. Combination drug toxicity: In Huh7 xenograft mice treated with PHA-767491 HCl in combination with 5-FU, no significant increase in toxicity was observed compared with monotherapy, and histopathological analysis did not reveal serious organ damage.[2]

[1]. The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds. PLoS One. 2014 Nov 20;9(11):e113300.

[2]. Dual Inhibition of Cdc7 and Cdk9 by PHA-767491 Suppresses Hepatocarcinoma Synergistically with 5-Fluorouracil. Curr Cancer Drug Targets. 2015;15(3):196-204.

[3]. Cell division cycle 7-kinase inhibitor PHA-767491 hydrochloride suppresses glioblastoma growth and invasiveness. Cancer Cell Int. 2016 Nov 18;16:88.

[4]. A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nat Chem Biol. 2008 Jun;4(6):357-65.

2-Pyridin-4-yl-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one is a pyrrolopyridine compound. PHA-767491 is a Cdc7/CDK9 inhibitor. Cdc7-Dbf4 kinase, or DDK (Dbf4-dependent kinase), initiates DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells, and because DDK inhibition induces apoptosis in various cancer cell types but not in normal cells, DDK has become an emerging chemotherapeutic target. PHA-767491 and XL413 are two of several potent DDK inhibitors with low nanomolar IC50 values for purified kinases. Although XL413 exhibits high selectivity for DDK, its activity in cell lines has not been fully characterized. We determined the antiproliferative and pro-apoptotic effects of XL413 against a range of tumor cell lines and compared it with that of PHA-767491, whose activity has been well characterized. Both compounds are potent DDK biochemical inhibitors, but surprisingly, their activities varied considerably across different cell lines. Unlike PHA-767491, XL413 showed significant antiproliferative activity against only one of the ten cell lines tested. Since XL413 failed to effectively inhibit DDK in multiple cell lines, its bioavailability may be limited. To identify other potential DDK inhibitors, we also tested the cross-reactivity of approximately 400 known kinase inhibitors with DDK using DDK thermostability variation analysis (TSA). We identified 11 compounds that significantly stabilized DDK. Some of these compounds exhibited DDK inhibition potency comparable to PHA-767491, including Chk1 and PKR kinase inhibitors, but their chemical skeletons are distinctly different from known DDK inhibitors. In summary, these data indicate that several known kinase inhibitors cross-react with DDK, highlighting the opportunity to design more specific and bioactive DDK inhibitors as chemotherapeutic drugs. [1]

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Activation of checkpoint kinase 1 (Chk1) is a key factor in the development of chemotherapy resistance in hepatocellular carcinoma (HCC) to 5-fluorouracil (5-FU) and other antimetabolites. In this study, we demonstrated that PHA-767491 is a dual inhibitor that inhibits two cell cycle checkpoint kinases—cell division cycle kinase 7 (Cdc7) and cyclin-dependent kinase 9 (Cdk9)—and has a synergistic antitumor effect with 5-FU, inhibiting human HCC cells in vitro and in vivo. Compared with the use of each drug alone, the combination of PHA-767491 and 5-FU showed stronger cytotoxicity and significantly induced apoptosis in liver cancer cells, as evidenced by a significant increase in caspase 3 activation and poly(ADP-ribose) polymerase (PARP) fragmentation. PHA-767491 directly antagonizes 5-FU-induced phosphorylation of Chk1 (a substrate of Cdc7) and reduces the expression of the anti-apoptotic protein myeloid leukemia cell 1 (a downstream target of Cdk9). In nude mouse hepatocellular carcinoma xenograft tissue sections, administration of PHA-767491 also reduced the phosphorylation level of Chk1 and increased in situ apoptosis. Our study suggests that PHA-767491 can enhance the efficacy of 5-FU by inhibiting Chk1 phosphorylation and downregulating Mcl1 expression (by inhibiting Cdc7 and Cdk9), and therefore, the combination of PHA-767491 and 5-FU may be beneficial for patients with advanced and drug-resistant hepatocellular carcinoma (HCC). [2]

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Background: Genomic instability is a hallmark of cancer cells, and this cellular phenomenon may be caused by replication stress. Replication stress can be utilized and its effects enhanced in a targeted manner to combat cancer cells. One strategy is to target cell cycle 7-related protein kinase (CDC7), which plays a crucial role in the regulation of DNA replication initiation. CDC7 is overexpressed in various cancers, and small molecule inhibitors of CDC7 have been shown to have anti-tumor effects. This study aimed to explore the potential of CDC7 inhibitors as a novel therapeutic strategy for glioblastoma. Methods: PHA-767491 hydrochloride was used as a CDC7 inhibitor. The effects of CDC7 inhibitors were characterized using two glioblastoma cell lines (U87-MG and U251-MG) and a control cell line (3T3). The effects of CDC7 inhibitors on cell viability, proliferation, apoptosis, migration, and invasion were analyzed. In addition, differentially expressed genes after CDC7 inhibitor treatment were identified using real-time quantitative PCR. Results: The results showed that CDC7 inhibitors reduced glioblastoma cell viability, inhibited cell proliferation, and induced glioblastoma cell apoptosis. Furthermore, we found that CDC7 inhibitors inhibited glioblastoma cell migration and invasion. To identify the molecular targets of CDC7 inhibition, we used real-time PCR arrays, which showed dysregulation of expression of multiple mRNAs and miRNAs. Conclusion: In summary, our results suggest that CDC7 inhibition is a promising strategy for the treatment of glioblastoma. [3]

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Cdc7 is an important kinase that promotes DNA replication by activating the origin of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) by biochemical and cellular experiments and tested its antitumor activity in rodents. We found that the compound blocked DNA synthesis and affected the phosphorylation of replicating DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevented activation of the origin of replication but did not hinder the extension of the replication fork or induce a sustained DNA damage response. PHA-767491 treatment induced apoptosis in multiple cancer cell types and inhibited tumor growth in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule to directly affect DNA replication initiation rather than elongation mechanisms, and its activity suggests that Cdc7 kinase inhibition may be a novel strategy for developing anticancer therapies. [4]

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1. PHA-767491 HCl is a dual inhibitor of Cdc7 and Cdk9 kinases. It inhibits Cdc7-mediated DNA replication initiation by blocking MCM2 phosphorylation and Cdk9-dependent RNA transcription by inhibiting RNA polymerase II phosphorylation. This dual mechanism leads to cell cycle arrest and apoptosis in the G1/S phase of cancer cells. [2][4]
2. This drug has broad-spectrum antitumor activity against solid tumors (glioblastoma, liver cancer, lung cancer, colon cancer, breast cancer) and exhibits synergistic effects with chemotherapy drugs (5-fluorouracil), making it a potential candidate drug for combination therapy [2][3][4]
3. PHA-767491 HCl has partial blood-brain barrier penetration, which is beneficial for the treatment of brain tumors such as glioblastoma. Its moderate oral bioavailability and good tissue distribution support its clinical development, but mild myelosuppression and transient hepatotoxicity need to be monitored [3][4]
4. This drug is more toxic to cancer cells than to normal cells, which may be due to the higher dependence of cancer cells on the proliferation and survival of Cdc7/Cdk9. It can also inhibit the invasion and migration of cancer cells by downregulating MMP-2 and MMP-9, suggesting its potential to prevent metastasis [3]

C12H11N3O.HCL

249.7

249.067

C, 57.72; H, 4.84; Cl, 14.20; N, 16.83; O, 6.41.

942425-68-5

PHA-767491;845714-00-3; PHA-767491 hydrochloride;942425-68-5; 845538-12-7 (2HCl)

11715766

Light yellow to yellow solid powder

2.493

3

2

1

17

275

0

Cl.O=C1C2=C(NC(C3C=CN=CC=3)=C2)CCN1

IMVNFURYBZMFDZ-UHFFFAOYSA-N

InChI=1S/C12H11N3O.ClH/c16-12-9-7-11(8-1-4-13-5-2-8)15-10(9)3-6-14-12;/h1-2,4-5,7,15H,3,6H2,(H,14,16);1H

2-pyridin-4-yl-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one;hydrochloride

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)

Solubility in Formulation 1: ≥ 1 mg/mL (4.00 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (4.00 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 10.0 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: 5% DMSO+30% PEG 300+2% Tween 80+ddH2O: 1mg/mL

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Solubility in Formulation 4: 50 mg/mL (200.24 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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