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)

PHA-767491 has an IC50 of 0.64 µM in HCC1954 cells and 1.3 µM in Colo-205 cells, which means that it inhibits proliferation in both cell lines. PHA-767491 exhibits IC50 values of 18.6 nM, making it an effective DDK inhibitor in vitro. In HCC1954 cells, PHA-767491 (2 µM) totally eliminates Mcm2 phosphorylation within 24 hours[1]. When combined with 5-FU, PHA-767491 exhibits much greater cytotoxicity and significantly induces apoptosis, as evidenced by noticeably increased caspase 3 activation and fragmentation of poly(ADP-ribose) polymerase in HCC cells. By directly opposing the phosphorylation of Chk1 induced by 5-FU, PHA-767491 also suppresses the expression of the anti-apoptotic protein myeloid leukemia cell 1ine[2]. In a time- and dose-dependent manner, PHA-767491 (0-10 µM) reduces the viability of glioblastoma cells, with an IC50 of roughly 2.5 µM for U87-MG and U251-MG cells. In addition to inhibiting glioblastoma cell proliferation, migration, and invasion, PHA-767491 hydrochloride causes apoptosis in these cells[3].

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

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PHA-767491 has antitumor activity in cancer models [4]
The 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).
We 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.
Altogether 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.
We conclude that PHA-767491 has antitumor activity in vivo in multiple preclinical cancer models and in at least two different species.

For five minutes, 20 ng of purified human DDK is pre-incubated with DDK inhibitors at escalating concentrations. Next, in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 1 mM DTT, 10 µCi (γ)-³²P ATP and 1.5 µM cold ATP are added, and the mixture is incubated for 30 minutes at 30°C. The proteins are autoradiographed on HyBlot CL film and SDS-PAGEd after being denatured in 1X Laemmli buffer at 100°C. DDK's auto-phosphorylation is a measure of its kinase activity. Using ImageJ, ³²P-labeled bands are quantified, and GraphPad is used to compute 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.

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.

Animal studies.[4]
HCT-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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Toxicological study. [4]
Male 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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Dissolved in DMSO, and diluted in saline; 50 mg/kg; Intravenous or oral administration twice a day

Female 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.

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. [4]

[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.
PHA-767491 is a Cdc7/CDK9 inhibitor.

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Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.[1]

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Activation of checkpoint kinase 1 (Chk1) is essential in chemoresistance of hepatocarcinoma (HCC) to 5-fluorouracil (5-FU) and other antimetabolite family of drugs. In this study, we demonstrated that PHA-767491, a dual inhibitor of two cell cycle checkpoint kinases, cell division cycle kinase 7 (Cdc7) and cyclin-dependent kinase 9 (Cdk9), has synergistic antitumor effect with 5-FU to suppress human HCC cells both in vitro and in vivo. Compared with the sole use of each agent, PHA-767491 in combination with 5-FU exhibited much stronger cytotoxicity and induced significant apoptosis manifested by remarkably increased caspase 3 activation and poly(ADP-Ribose) polymerase fragmentation in HCC cells. PHA-767491 directly counteracted the 5-FU-induced phosphorylation of Chk1, a substrate of Cdc7; and decreased the expression of the anti-apoptotic protein myeloid leukemia cell 1, a downstream target of Cdk9. In tumor tissues sectioned from nude mice HCC xenografts, administration of PHA-767491 also decreased Chk1 phosphorylation and increased in situ cell apoptosis. Our study suggests that PHA- 767491 could enhance the efficacy of 5-FU by inhibiting Chk1 phosphorylation and down-regulating Mcl1 expression through inhibition of Cdc7 and Cdk9, thus combinational administration of PHA-767491 with 5-FU could be potentially beneficial to patients with advanced and resistant HCC. [2]

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Background: Genomic instability is a hallmark of cancer cells, and this cellular phenomenon can emerge as a result of replicative stress. It is possible to take advantage of replicative stress, and enhance it in a targeted way to fight cancer cells. One of such strategies involves targeting the cell division cycle 7-related protein kinase (CDC7), a protein with key roles in regulation of initiation of DNA replication. CDC7 overexpression is present in different cancers, and small molecule inhibitors of the CDC7 have well-documented anti-tumor effects. Here, we aimed to test the potential of CDC7 inhibition as a new strategy for glioblastoma treatment. Methods: PHA-767491 hydrochloride was used as the CDC7 inhibitor. Two glioblastoma cell lines (U87-MG and U251-MG) and a control cell line (3T3) were used to characterize the effects of CDC7 inhibition. The effect of CDC7 inhibition on cell viability, cell proliferation, apoptosis, migration, and invasion were analyzed. In addition, real-time PCR arrays were used to identify the differentially expressed genes in response to CDC7 inhibition. Results: Our results showed that CDC7 inhibition reduces glioblastoma cell viability, suppresses cell proliferation, and triggers apoptosis in glioblastoma cell lines. In addition, we determined that CDC7 inhibition also suppresses glioblastoma cell migration and invasion. To identify molecular targets of CDC7 inhibition, we used real-time PCR arrays, which showed dysregulation of several mRNAs and miRNAs. Conclusions: Taken together, our findings suggest that CDC7 inhibition is a promising strategy for treatment of glioblastoma.[3]

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Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics. [4]

C12H11N3O

249.7

213.09

C, 67.59; H, 5.20; N, 19.71; O, 7.50

845714-00-3

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

11715767

Off-white to light yellow solid powder

1.287

620.6ºC at 760 mmHg

329.1ºC

2.493

2

2

1

16

275

0

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

DKXHSOUZPMHNIZ-UHFFFAOYSA-N

InChI=1S/C12H11N3O/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)

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

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)