Discovery of N,4-Di(1H-pyrazol-4-yl)pyrimidin-2-amine-Derived CDK2 Inhibitors as Potential Anticancer Agents: Design, Synthesis, and Evaluation.

Cyclin-dependent kinase 2 (CDK2) has been garnering considerable interest as a target to develop new cancer treatments and to ameliorate resistance to CDK4/6 inhibitors. However, a selective CDK2 inhibitor has yet to be clinically approved. With the desire to discover novel, potent, and selective CDK2 inhibitors, the phenylsulfonamide moiety of our previous lead compound 1 was bioisosterically replaced with pyrazole derivatives, affording a novel series of N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amines that exhibited potent CDK2 inhibitory activity. Among them, 15 was the most potent CDK2 inhibitor (Ki = 0.005 µM) with a degree of selectivity over other CDKs tested. Meanwhile, this compound displayed sub-micromolar antiproliferative activity against a panel of 13 cancer cell lines (GI50 = 0.127-0.560 µM). Mechanistic studies in ovarian cancer cells revealed that 15 reduced the phosphorylation of retinoblastoma at Thr821, arrested cells at the S and G2/M phases, and induced apoptosis. These results accentuate the potential of the N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amine scaffold to be developed into potent and selective CDK2 inhibitors for the treatment of cancer.

2-Anilino-4-(1-methyl-1H-pyrazol-4-yl)pyrimidine-derived CDK2 inhibitors as anticancer agents: Design, synthesis & evaluation.

Deregulation of cyclin-dependent kinase 2 (CDK2) and its activating partners, cyclins A and E, is associated with the pathogenesis of a myriad of human cancers and with resistance to anticancer drugs including CDK4/6 inhibitors. Thus, CDK2 has become an attractive target for the development of new anticancer therapies and for the amelioration of the resistance to CDK4/6 inhibitors. Bioisosteric replacement of the thiazole moiety of CDKI-73, a clinically trialled CDK inhibitor, by a pyrazole group afforded 9 and 19 that displayed potent CDK2-cyclin E inhibition (Ki = 0.023 and 0.001 µM, respectively) with submicromolar antiproliferative activity against a panel of cancer cell lines (GI50 = 0.025-0.780 µM). Mechanistic studies on 19 with HCT-116 colorectal cancer cells revealed that the compound reduced the phosphorylation of retinoblastoma at Ser807/811, arrested the cells at the G2/M phase, and induced apoptosis. These results highlight the potential of the 2-anilino-4-(1-methyl-1H-pyrazol-4-yl)pyrimidine series in developing potent and selective CDK2 inhibitors to combat cancer.

Discovery of Potent Inhibitors of Cyclin-Dependent Kinases 7 and 9: Design, Synthesis, Structure-Activity Relationship Analysis and Biological Evaluation.

Cyclin-dependent kinases (CDKs) 7 and 9 are deregulated in various types of human cancer and are thus viewed as therapeutic targets. Accordingly, small-molecule inhibitors of both CDKs are highly sought-after. Capitalising on our previous discovery of CDKI-73, a potent CDK9 inhibitor, medicinal chemistry optimisation was pursued. A number of N-pyridinylpyrimidin-2-amines were rationally designed, chemically synthesised and biologically assessed. Among them, N-(6-(4-cyclopentylpiperazin-1-yl)pyridin-3-yl)-4-(imidazo[1,2-a]pyrimidin-3-yl)pyrimidin-2-amine was found to be one of the most potent inhibitors of CDKs 7 and 9 as well as the most effective anti-proliferative agent towards multiple human cancer cell lines. The cellular mode of action of this compound was investigated in MV4-11 acute myeloid leukaemia cells, revealing that the compound dampened the kinase activity of cellular CDKs 7 and 9, arrested the cell cycle at sub-G1 phase and induced apoptosis.

An Orally Bioavailable and Highly Efficacious Inhibitor of CDK9/FLT3 for the Treatment of Acute Myeloid Leukemia.

Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in approximately one-third of AML patients and are associated with a particularly poor prognosis. The most common mutation, FLT3-ITD, is a self-activating internal tandem duplication (ITD) in the FLT3 juxtamembrane domain. Many FLT3 inhibitors have shown encouraging results in clinical trials, but the rapid emergence of resistance has severely limited sustainable efficacy. Co-targeting of CDK9 and FLT3 is a promising two-pronged strategy to overcome resistance as the former plays a role in the transcription of cancer cell-survival genes. Most prominently, MCL-1 is known to be associated with AML tumorigenesis and drug resistance and can be down-regulated by CDK9 inhibition. We have developed CDDD11-8 as a potent CDK9 inhibitor co-targeting FLT3-ITD with Ki values of 8 and 13 nM, respectively. The kinome selectivity has been confirmed when the compound was tested in a panel of 369 human kinases. CDDD11-8 displayed antiproliferative activity against leukemia cell lines, and particularly potent effects were observed against MV4-11 and MOLM-13 cells, which are known to harbor the FLT3-ITD mutation and mixed lineage leukemia (MLL) fusion proteins. The mode of action was consistent with inhibition of CDK9 and FLT3-ITD. Most importantly, CDDD11-8 caused a robust tumor growth inhibition by oral administration in animal xenografts. At 125 mg/kg, CDDD11-8 induced tumor regression, and this was translated to an improved survival of animals. The study demonstrates the potential of CDDD11-8 towards the future development of a novel AML treatment.

Synthesis and evaluation of 2'H-spiro[cyclohexane-1,3'-imidazo[1,5-a]pyridine]-1',5'-dione derivatives as Mnk inhibitors.

Post-translational modulation of eIF4E through phosphorylation by Mnks is highly integral to the pathogenesis of different cancers. Therefore, inhibition of Mnks offers a strategy for cancer treatment. Herein, a series of 2'H-spiro[cyclohexane-1,3'-imidazo[1,5-a]pyridine]-1',5'-dione derivatives is presented as Mnk inhibitors. Some of them showed sub-micromolar to low nanomolar inhibitory activities against Mnk1/2 with a high level of selectivity for both kinases over CDKs. Biochemical assays revealed that compounds 4c and 4t are non-ATP-competitive inhibitors of Mnks. Lead compound 4t demonstrated a high selectivity for Mnk1/2 over a selection of 51 kinases, and displayed anti-proliferative activities against a panel of cancer cell lines. However, this compound in combination with our in-house CDK4/6 inhibitor 83 did not show a synergistic effect in A2780 ovarian cancer cells, suggesting that caution be exercised in the selection of an agent to be combined with an Mnk inhibitor.

Discovery of CDK5 Inhibitors through Structure-Guided Approach.

Specific abrogation of cyclin-dependent kinase 5 (CDK5) activity has been validated as a viable approach for the development of anticancer agents. However, no selective CDK5 inhibitor has been reported to date. Herein, a structure-based in silico screening was employed to identify novel scaffolds from a library of compounds to identify potential CDK5 inhibitors that would be relevant for drug discovery. Hits, representatives of three chemical classes, were identified as inhibitors of CDK5. Structural modification of hit-1 resulted in 29 and 30. Compound 29 is a dual inhibitor of CDK5 and CDK2, whereas 30 preferentially inhibits CDK5. Both leads exhibited anticancer activity against acute myeloid leukemia (AML) cells via a mechanism consistent with targeting cellular CDK5. This study provides an effective strategy for discovery of CDK5 inhibitors as potential antileukemic agents.

Discovery of N-Phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine Derivatives as Potent Mnk2 Inhibitors: Design, Synthesis, SAR Analysis, and Evaluation of in vitro Anti-leukaemic Activity.

BACKGROUND: Aberrant expression of eukaryotic translation initiation factor 4E (eIF4E) is common in many types of cancer including acute myeloid leukaemia (AML). Phosphorylation of eIF4E by MAPK-interacting kinases (Mnks) is essential for the eIF4E-mediated oncogenic activity. As such, the pharmacological inhibition of Mnks can be an effective strategy for the treatment of cancer. METHODS: A series of N-phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine derivatives was designed and synthesised. The Mnk inhibitory activity of these derivatives as well as their anti-proliferative activity against MV4-11 AML cells was determined. RESULTS: These compounds were identified as potent Mnk2 inhibitors. Most of them demonstrated potent anti-proliferative activity against MV4-11 AML cells. The cellular mechanistic studies of the representative inhibitors revealed that they reduced the level of phosphorylated eIF4E and induced apoptosis by down-regulating the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and by cleaving poly(ADP-ribose)polymerase (PARP). The lead compound 7k possessed desirable pharmacokinetic properties and oral bioavailability. CONCLUSION: This work proposes that exploration of the structural diversity in the context of Nphenyl- 4-(1H-pyrrol-3-yl)pyrimidin-2-amine would offer potent and selective Mnk inhibitors.

Dual Inhibition of Mnk2 and FLT3 for potential treatment of acute myeloid leukaemia.

The discovery of novel anti-AML therapeutic agents is urgently needed, but the complex heterogeneity of the disease has so far hampered the development of a curative treatment. FLT3 inhibitors have shown therapeutic potential in clinical trials; but a monotherapy regimen has been associated with resistance mediated by the activation of parallel signalling circuitry, including MAPK and mTOR. Therefore, inhibiting a nexus of the two signalling pathways along with inhibition of FLT3 might be advantageous. Herein, we propose that a dual inhibition of FLT3 and Mnk would provide a better clinical option for AML patients compared to targeting FLT3 alone. Thus, a series of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines and 4-(indol-3-yl)-N-phenylpyrimidin-2-amines were prepared. Potent Mnk2 inhibitors, FLT3 inhibitors, and dual inhibitors of Mnk2 and FLT3 were identified and their anti-proliferative activities assessed against MV4-11 AML cell lines. Dual inhibition of FLT3 and Mnk2 caused the increased apoptotic cell death of MV4-11 cells compared to inhibition of FLT3 or Mnk2 alone.

In Search of Novel CDK8 Inhibitors by Virtual Screening.

Aberrant activity of cyclin-dependent kinase (CDK) 8 is implicated in various cancers. While CDK8-targeting anticancer drugs are highly sought-after, no CDK8 inhibitor has yet reached clinical trials. Herein a large library of drug-like molecules was computationally screened using two complementary cascades to identify potential CDK8 inhibitors. Thirty-three hits were identified to inhibit CDK8 and seven of them were active against colorectal cancer cell lines. Finally, the primary target was confirmed using three promising hits.

Highly Potent, Selective, and Orally Bioavailable 4-Thiazol-N-(pyridin-2-yl)pyrimidin-2-amine Cyclin-Dependent Kinases 4 and 6 Inhibitors as Anticancer Drug Candidates: Design, Synthesis, and Evaluation.

Cyclin D dependent kinases (CDK4 and CDK6) regulate entry into S phase of the cell cycle and are validated targets for anticancer drug discovery. Herein we detail the discovery of a novel series of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as highly potent and selective inhibitors of CDK4 and CDK6. Medicinal chemistry optimization resulted in 83, an orally bioavailable inhibitor molecule with remarkable selectivity. Repeated oral administration of 83 caused marked inhibition of tumor growth in MV4-11 acute myeloid leukemia mouse xenografts without having a negative effect on body weight and showing any sign of clinical toxicity. The data merit 83 as a clinical development candidate.

Inhibition of Mnk enhances apoptotic activity of cytarabine in acute myeloid leukemia cells.

Cytarabine (Ara-C) is a first line clinical therapeutic agent for treatment of acute myeloid leukemia (AML). However, this therapy is limited due to high rate of resistance and relapse. Recent research has revealed that the poor prognosis and resistance to Ara-C in AML were associated with its abnormally activated MAPK pathways. In this study, we showed a strong synergistic effect of Ara-C with either our Mnk inhibitor (MNKI-8e) or short hairpin RNA (shRNA) mediated knockdown of Mnks in MV4-11 AML cells. We investigated the underlying mechanisms for this synergism. We showed that both MNKI-8e and Mnk shRNAs enhanced the ability of Ara-C to induce apoptosis. We found that Ara-C increased the phosphorylation of Erk1/2, p38 and eIF4E, which correlated with an enhanced level of anti-apoptotic Mcl-1 protein. Inhibition of Mnk activity suppressed the Ara-C-induced MAPK activity, and thus enhanced apoptosis in MV4-11 cells. Taken together, our study suggests that MAPK-Mnk-eIF4E pathway plays a critical role in Ara-C-treated MV4-11 cells and targeting Mnk may be a promising therapeutic strategy for sensitizing leukemic cells to Ara-C therapy.

Unveiling new chemical scaffolds as Mnk inhibitors.

The discovery of small molecules that selectively inhibit Mnks is considered of paramount importance towards deciphering the exact role of these proteins in carcinogenesis and to further validate them as anti-cancer drug targets. However, the dearth of structural information of Mnks is a major hurdle. This study unveils the 7H-pyrrolo[2,3-d]pyrimidine derivatives as potent inhibitors of Mnks. ATP and substrate competition assays showed that this scaffold interacts with the ATP binding site, but not with the substrate site. Screened against a panel of cancer cells, Mnk inhibitors were most potent against MV4-11 acute myeloid leukemia cells. The induction of apoptosis was shown to be mediated by downregulation of Mcl-1.

An integrated approach for discovery of highly potent and selective Mnk inhibitors: Screening, synthesis and SAR analysis.

Deregulation of protein synthesis is a common event in cancer. As MAPK-interacting kinases (Mnks) play critical roles in regulation of protein synthesis, they have emerged as novel anti-cancer targets. Mnks phosphorylate eukaryotic initiation factor 4E (eIF4E) and promote eIF4E-mediated oncogenic activity. Given that the kinase activity of Mnks is essential for oncogenesis but is dispensable for normal development, the discovery of potent and selective pharmacological Mnk inhibitors provides pharmacological target validation and offers a new strategy for cancer treatment. Herein, comprehensive in silico screening approaches were deployed, and three thieno[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives were identified as hit compounds. Further chemical modification of thieno[2,3-d]pyrimidine derivative 3 has given rise to a series of highly potent Mnk2 inhibitors that could be potential leads for the treatment of acute myeloid leukemia.

Identification of a Highly Conserved Allosteric Binding Site on Mnk1 and Mnk2.

Elevated levels of phosphorylated eukaryotic initiation factor 4E (eIF4E) have been implicated in many tumor types, and mitogen activated protein kinase-interacting kinases (Mnks) are the only known kinases that phosphorylate eIF4E at Ser209. The phosphorylation of eIF4E is essential for oncogenic transformation but is of no significance to normal growth and development. Pharmacological inhibition of Mnks therefore provides a nontoxic and effective strategy for cancer therapy. However, a lack of specific Mnk inhibitors has confounded pharmacological target validation and clinical development. Herein, we report the identification of a novel series of Mnk inhibitors and their binding modes. A systematic workflow has been established to distinguish between type III and type I/II inhibitors. A selection of 66 compounds was tested for Mnk1 and Mnk2 inhibition, and 9 out of 20 active compounds showed type III interaction with an allosteric site of the proteins. Most of the type III inhibitors exhibited dual Mnk1 and Mnk2 activities and demonstrated potent antiproliferative properties against the MV4-11 acute myeloid leukemia cell line. Interestingly, ATP-/substrate-competitive inhibitors were found to be highly selective for Mnk2, with little or no activity for Mnk1. Our study suggests that Mnk1 and Mnk2 share a common structure of the allosteric inhibitory binding site but possess different structural features of the ATP catalytic domain. The findings will assist in the future design and development of Mnk targeted anticancer therapeutics.

Pharmacologic Inhibition of MNKs in Acute Myeloid Leukemia.

The Ras/Raf/MAPK and PI3K/Akt/mTOR pathways are key signaling cascades involved in the regulation of cell proliferation and survival, and have been implicated in the pathogenesis of several types of cancers, including acute myeloid leukemia (AML). The oncogenic activity of eIF4E driven by the Mnk kinases is a convergent determinant of the two cascades, suggesting that targeting the Mnk/eIF4E axis may provide therapeutic opportunity for the treatment of cancer. Herein, a potent and selective Mnk2 inhibitor (MNKI-85) and a dual-specific Mnk1 and Mnk2 inhibitor (MNKI-19), both derived from a thienopyrimidinyl chemotype, were selected to explore their antileukemic properties. MNKI-19 and MNKI-85 are effective in inhibiting the growth of AML cells that possess an M5 subtype with FLT3-internal tandem duplication mutation. Further mechanistic studies show that the downstream effects with respect to the selective Mnk1/2 kinase inhibition in AML cells causes G1 cell cycle arrest followed by induction of apoptosis. MNKI-19 and MNKI-85 demonstrate similar Mnk2 kinase activity and cellular antiproliferative activity but exhibit different time-dependent effects on cell cycle progression and apoptosis. Collectively, this study shows that pharmacologic inhibition of both Mnk1 and Mnk2 can result in a more pronounced cellular response than targeting Mnk2 alone. However, MNKI-85, a first-in-class inhibitor of Mnk2, can be used as a powerful pharmacologic tool in studying the Mnk2/eIF4E-mediated tumorigenic mechanism. In conclusion, this study provides a better understanding of the mechanism underlying the inhibition of AML cell growth by Mnk inhibitors and suggests their potential utility as a therapeutic agent for AML.

Discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimidine derivatives as potent Mnk inhibitors: synthesis, structure-activity relationship analysis and biological evaluation.

Phosphorylation of the eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) is essential for oncogenesis but unnecessary for normal development. Thus, pharmacological inhibition of Mnks may offer an effective and non-toxic anti-cancer therapeutic strategy. Herein, we report the discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimidine derivatives as potent Mnk inhibitors. Docking study of 7a in Mnk2 suggests that the compound is stabilised in the ATP binding site through multiple hydrogen bonds and hydrophobic interaction. Cellular mechanistic studies on MV-4-11 cells with leads 7a, 8e and 8f reveal that they are able to down-regulate the phosphorylated eIF4E, Mcl-1 and cyclin D1, and induce apoptosis.

Discovery of 5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-one derivatives as potent Mnk2 inhibitors: synthesis, SAR analysis and biological evaluation.

Phosphorylation of eIF4E by human mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) is crucial for human tumourigenesis and development. Targeting Mnks may provide a novel anticancer therapeutic strategy. However, the lack of selective Mnk inhibitors has so far hampered pharmacological target validation and clinical drug development. Herein, we report, for the first time, the discovery of a series of 5-(2-(phenylamino)pyrimidin-4-yl)thiazole-2(3H)-one derivatives as Mnk inhibitors. Several derivatives demonstrate very potent Mnk2 inhibitory activity. The most active and selective compounds were tested against a panel of cancer cell lines, and the results confirm the cell-type-specific effect of these Mnk inhibitors. Detailed cellular mechanistic studies reveal that Mnk inhibitors are capable of reducing the expression level of anti-apoptotic protein Mcl-1, and of promoting apoptosis in MV4-11 acute myeloid leukaemia cells.