Advances in synthesis and biological evaluation of CDK2 inhibitors for cancer therapy.

One of the leading causes of mortality in the world is cancer. This disease occurs when responsible genes that regulate the cell cycle become inactive due to internal or external factors. Specifically, the G1/S and S/G2 transitions in the cell cycle are controlled by a protein called cyclin-dependent kinase 2 (CDK2). CDKs, which play a crucial role in managing the cell cycle, have been a wide area of research in cancer treatment. Over the past 11 years, significant research has been made in identifying potent, targeted, and efficient inhibitors of CDK2. In this summary, we have summarized recent developments in the synthesis and biological evaluation of CDK2 inhibitors.

Design, synthesis, and biological evaluation of N-(pyridin-3-yl)pyrimidin-4-amine analogues as novel cyclin-dependent kinase 2 inhibitors for cancer therapy.

The discovery and development of CDK2 inhibitors has currently been validated as a hot topic in cancer therapy. Herein, a series of novel N-(pyridin-3-yl)pyrimidin-4-amine derivatives were designed and synthesized as potent CDK2 inhibitors. Among them, the most promising compound 7l presented a broad antiproliferative efficacy toward diverse cancer cells MV4-11, HT-29, MCF-7, and HeLa with IC50 values of 0.83, 2.12, 3.12, and 8.61 µM, respectively, which were comparable to that of Palbociclib and AZD5438. Interestingly, these compounds were less toxic on normal embryonic kidney cells HEK293 with high selectivity index. Further mechanistic studies indicated 7l caused cell cycle arrest and apoptosis on HeLa cells in a concentration-dependent manner. Moreover, 7l manifested potent and similar CDK2/cyclin A2 nhibitory activity to AZD5438 with an IC50 of 64.42 nM. These findings revealed that 7l could serve as ahighly promisingscaffoldfor CDK2 inhibitors as potential anticancer agents and functional probes.

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 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.

Development of pyrazolo[3,4-d]pyrimidin-4-one scaffold as novel CDK2 inhibitors: Design, synthesis, and biological evaluation.

A series of pyrazolo[3,4-d]pyrimidin-4-one scaffold were designed and synthesized as novel CDK2 inhibitors. By analyzing the common motifs of various known inhibitors, the designed compounds 1 were virtually screen for their inhibitory activity by docking into the active pocket of CDK2. The influence of different substitutes on the docking results was investigated. A total of 15 pyrazolo[3,4-d]pyrimidin-4-ones 1 were synthesized by Paal-Knorr reaction, pyrimidine ring closure, bromination, Suzuki coupling reaction, amide formation and Knoevenagel condensation. The Cell Counting Kit-8 (CCK-8) was used to evaluate the inhibitory activity of pyrazolo[3,4-d]pyrimidin-4-ones 1 in the breast cancer cell line MCF-7 in vitro using Etoposide as a reference control substance. The screening results demonstrated that the designed compounds have significant antiproliferative activity, and compounds 1e and 1j were the most active compounds with IC50 values of 10.79 µM and 10.88 µM, respectively, being better than that of Etoposide (IC50 = 18.75 µM). The enzyme inhibition assay was carried out against CDK2, the results indicated that the compounds 1e and 1j significantly inhibited CDK2 with IC50 values of 1.71 µM and 1.60 µM.

Design, synthesis and molecular docking of new fused 1H-pyrroles, pyrrolo[3,2-d]pyrimidines and pyrrolo[3,2-e][1, 4]diazepine derivatives as potent EGFR/CDK2 inhibitors.

A new series of 1H-pyrrole (6a-c, 8a-c), pyrrolo[3,2-d]pyrimidines (9a-c) and pyrrolo[3,2-e][1, 4]diazepines (11a-c) were designed and synthesised. These compounds were designed to have the essential pharmacophoric features of EGFR Inhibitors, they have shown anticancer activities against HCT116, MCF-7 and Hep3B cancer cells with IC50 values ranging from 0.009 to 2.195 µM. IC50 value of doxorubicin is 0.008 µM, compounds 9a and 9c showed IC50 values of 0.011 and 0.009 µM respectively against HCT-116 cells. Compound 8b exerted broad-spectrum activity against all tested cell lines with an IC50 value less than 0.05 µM. Compound 8b was evaluated against a panel of kinases. This compound potently inhibited CDK2/Cyclin A1, DYRK3 and GSK3 alpha kinases with 10-23% compared to imatinib (1-10%). It has also arrested the cell cycle of MCF-7 cells at the S phase. Its antiproliferative activity was further augmented by molecular docking into the active sites of EGFR and CDK2 cyclin A1.

Eco-friendly sequential one-pot synthesis, molecular docking, and anticancer evaluation of arylidene-hydrazinyl-thiazole derivatives as CDK2 inhibitors.

One current approach in the treatment of cancer is the inhibition of cyclin dependent kinase (CDK) enzymes with small molecules. CDK are a class of enzymes, which catalyze the transfer of the terminal phosphate of a molecule of ATP to a protein that acts as a substrate. Among CDK enzymes, CDK2 has been implicated in a variety of cancers, supporting its potential as a novel target for cancer therapy across many tumor types. Here the discovery and development of arylidene-hydrazinyl-thiazole as a potentially CDK2 inhibitors is described, including details of the design and successful synthesis of the series analogs (27a-r) using one-pot approach under eco-friendly ultrasound and microwave conditions. Most of the newly synthesized compounds showed good growth inhibition when assayed for their in-vitro anti-proliferative activity against three cancer cell lines (HepG2, MCF-7 and HCT-116) compared to the reference drug roscovitine, with little toxicity on the normal fibroblast cell lines (WI-38). Furthermore, the compounds exhibiting the highest anti-proliferative activities were tested against a panel of kinase enzymes. These derivatives displayed an outstanding CDK2 inhibitory potential with varying degree of inhibition in the range of IC50 0.35-1.49 µM when compared with the standard inhibitor roscovitine having an IC50 value 0.71 µM. The most promising CDK2 inhibitor (27f) was selected for further studies to determine its effect on the cell cycle progression and apoptosis in HepG2 cell line. The results indicated that this compound implied inhibition in the G2/M phase of the cell cycle, and it is a good apoptotic agent. Finally, Molecular docking study was performed to identify the structural elements which involved in the inhibitory activity with the prospective target, CDK2, and to rationalize the structure-activity relationship (SAR).

Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles arrest cell cycle and induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological evaluation and in silico studies.

As a continuation for our previous work, a novel set of N-alkylindole-isatin conjugates (7, 8a-c, 9 and 10a-e) is here designed and synthesised with the prime aim to develop more efficient isatin-based antitumor candidates. Utilising the SAR outputs from the previous study, our design here is based on appending four alkyl groups with different length (ethyl and n-propyl), bulkiness (iso-propyl) and unsaturation (allyl) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the growth inhibitory profile for the target conjugates in comparison to the reported lead VI. The best results were obtained with N-propylindole -5-methylisatin hybrid 8a which displayed broad spectrum anti-proliferative action with efficient sub-panel GI50 (MG-MID) range from 1.33 to 4.23 µM, and promising full-panel GI50 (MG-MID) equals 3.10 µM, at the NCI five-dose assay. Also, hybrid 8a was able to provoke cell cycle disturbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/PI assays. Furthermore, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2 protein (IC50= 0.85 ± 0.03 and 0.46 ± 0.02 µM, respectively). Interestingly, molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising lead for further development and optimisation as an efficient antitumor drug.

A Series of Isatin-Hydrazones with Cytotoxic Activity and CDK2 Kinase Inhibitory Activity: A Potential Type II ATP Competitive Inhibitor.

Isatin derivatives potentially act on various biological targets. In this article, a series of novel isatin-hydrazones were synthesized in excellent yields. Their cytotoxicity was tested against human breast adenocarcinoma (MCF7) and human ovary adenocarcinoma (A2780) cell lines using MTT assay. Compounds 4j (IC50 = 1.51 ± 0.09 µM) and 4k (IC50 = 3.56 ± 0.31) showed excellent activity against MCF7, whereas compound 4e showed considerable cytotoxicity against both tested cell lines, MCF7 (IC50 = 5.46 ± 0.71 µM) and A2780 (IC50 = 18.96± 2.52 µM), respectively. Structure-activity relationships (SARs) revealed that, halogen substituents at 2,6-position of the C-ring of isatin-hydrazones are the most potent derivatives. In-silico absorption, distribution, metabolism and excretion (ADME) results demonstrated recommended drug likeness properties. Compounds 4j (IC50 = 0.245 µM) and 4k (IC50 = 0.300 µM) exhibited good inhibitory activity against the cell cycle regulator CDK2 protein kinase compared to imatinib (IC50 = 0.131 µM). A molecular docking study of 4j and 4k confirmed both compounds as type II ATP competitive inhibitors that made interactions with ATP binding pocket residues, as well as lacking interactions with active state DFG motif residues.

Design, synthesis and biological evaluation of pyrimidine derivatives as novel CDK2 inhibitors that induce apoptosis and cell cycle arrest in breast cancer cells.

Cyclin-dependent kinase 2 (CDK2) plays a key role in eukaryotic cell cycle progression which could facilitate the transition from G1 to S phase. The dysregulation of CDK2 is closely related to many cancers. CDK2 is utilized as one of the most studied kinase targets in oncology. In this article, 24 benzamide derivatives were designed, synthesized and investigated for the inhibition activity against CDK2. Our results revealed that the compound 25 is a potent CDK2 inhibitor exhibiting a broad spectrum anti-proliferative activity against several human breast cancer cells. Additionally, compound 25 could block cell cycle at G0 or G1 and induce significant apoptosis in MDA-MB-468 cells. These findings highlight a rationale for further development of CDK2 inhibitors to treat human breast cancer.

Design, synthesis, and molecular docking study of 3H-imidazole[4,5-c]pyridine derivatives as CDK2 inhibitors.

A novel series of imidazo[4,5-c]pyridine-based CDK2 inhibitors were designed from the structure of CYC202 via scaffold hopping strategy. These compounds were synthesized and biologically evaluated for their CDK2 inhibitory and in vitro anti-proliferation potential against cancer cell lines. Several compounds exhibited potent CDK2 inhibition with IC50 values of less than 1 µM. The most potent compound 5b showed excellent CDK2 inhibitory (IC50 = 21 nM) and in vitro anti-proliferation activity against three different cell lines (HL60, A549, and HCT116). The molecular docking and dynamic studies portrayed the potential binding mechanism between 5b and CDK2, and several key interactions between them were observed, which would be the reason for its potent CDK2 inhibitory and anti-proliferation activities. Therefore, the pyridin-3-ylmethyl moiety would serve as an excellent pharmacophore for the development of novel CDK2 inhibitors for targeted anti-cancer therapy.

Novel tetrahydroisoquinolines as DHFR and CDK2 inhibitors: synthesis, characterization, anticancer activity and antioxidant properties.

In this study, we synthesized new 5,6,7,8-tetrahydroisoquinolines and 6,7,8,9-tetrahydrothieno[2,3-c]isoquinolines based on 4-(N,N-dimethylamino)phenyl moiety as expected anticancer and/or antioxidant agents. The structure of all synthesized compounds were confirmed by spectral date (FT-IR, 1H NMR, 13C NMR) and elemental analysis. We evaluated the anticancer activity of these compounds toward two cell lines: A459 cell line (lung cancer cells) and MCF7 cell line (breast cancer cells). All tested compounds showed moderate to strong anti-cancer activity towards the two cell lines. Compound 7e exhibited the most potent cytotoxic activity against A549 cell line (IC50: 0.155 µM) while compound 8d showed the most potent one against MCF7 cell line (IC50: 0.170 µM) in comparison with doxorubicin. In addition, we examined the effect of compounds 7e and 8d regarding the growth of A549 and MCF7 cell lines, employing flow cytometry and Annexin V-FITC apoptotic assay. Our results showed that compound 7e caused cell cycle arrest at the G2/M phase with a 79-fold increase in apoptosis of A459 cell line. Moreover, compound 8d caused cell cycle arrest at the S phase with a 69-fold increase in apoptosis of MCF7 cell line. Furthermore, we studied the activity of these compounds as enzyme inhibitors against several enzymes. Our findings by docking and experimental studies that compound 7e is a potent CDK2 inhibitor with IC50 of 0.149 µM, compared to the Roscovitine control drug with IC50 of 0.380 µM. We also found that compound 8d is a significant DHFR inhibitor with an IC50 of 0.199 µM, compared to Methotrexate control drug with IC50 of 0.131 µM. Evaluation of the antioxidant properties of ten compounds was also studied in comparison with Vitamin C. Compounds 1, 3, 6, 7c and 8e have higher antioxidant activity than Vitamin C which mean that these compounds can used as potent antioxidant drugs.

Structure-Based Design of 2-Aminopurine Derivatives as CDK2 Inhibitors for Triple-Negative Breast Cancer.

Cyclin-dependent kinase 2 (CDK2) regulates the progression of the cell cycle and is critically associated with tumor growth. Selective CDK2 inhibition provides a potential therapeutic benefit against certain tumors. Purines and related heterocycle (e.g., R-Roscovitine) are important scaffolds in the development of CDK inhibitors. Herein, we designed a new series of 2-aminopurine derivatives based on the fragment-centric pocket mapping analysis of CDK2 crystal structure. Our results indicated that the introduction of polar substitution at the C-6 position of purine would be beneficial for CDK2 inhibition. Among them, compound 11l showed good CDK2 inhibitory activity (IC50 = 19 nM) and possessed good selectivity against other CDKs. Further in vitro tests indicated that compound 11l possesses anti-proliferation activity in triple-negative breast cancer (TNBC) cells. Moreover, molecular dynamics simulation suggested the favorable binding mode of compound 11l, which may serve as a new lead compound for the future development of CDK2 selective inhibitors.

Design, synthesis, and biological evaluation of 4-benzoylamino-1H-pyrazole-3-carboxamide derivatives as potent CDK2 inhibitors.

Cyclin-dependent kinases play significant roles in cell cycle progression and are promising targets for cancer therapy. However, most potent CDK inhibitors lack the balance between efficacy and safety because of poor selectivity. Given the roles of CDK2 in tumorigenesis, selective CDK2 inhibition may provide therapeutic benefits against certain cancer. In this study, a series of 4-benzoylamino-1H-pyrazole-3-carboxamide derivatives were designed, synthesized, and evaluated. The most selective compound DC-K2in212 in this series exhibited high potency towards CDK2 and had effective anti-proliferative activity against A2058 melanoma cell line and MV4-11 leukemia cell line while exhibiting low toxic effect on human normal cell lines MRC5 and LX2. The molecular modeling illustrated that compound DC-K2in212 had the similar binding mode with CDK2 as C-73, the most selective CDK2 inhibitor reported so far, which might account for selectivity against CDK2 over CDK1. Further biological studies revealed that compound DC-K2in212 suppressed CDK2-associated downstream signaling pathway, blocked cell cycle progression, and induced cellular apoptosis. Therefore, compound DC-K2in212 could serve as a potential CDK2 inhibitor for further development.

Actin-like protein 6A/MYC/CDK2 axis confers high proliferative activity in triple-negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high proliferative activity. TNBC tumors exhibit elevated MYC expression and altered expression of MYC regulatory genes, which are associated with tumor progression and poor prognosis; however, the underlying mechanisms by which MYC retains its high expression and mediates TNBC tumorigenesis require further exploration. METHODS: ACTL6A regulation of MYC and its target gene, CDK2, was defined using Co-IP, mass spectrometry and ChIP assays. To study the role of ACTL6A in TNBC, we performed soft-agar, colony formation, flow cytometry and tumor formation in nude mice. CDK2 inhibitor and paclitaxel were used in testing combination therapy in vitro and in vivo. RESULTS: ACTL6A bound MYC to suppress glycogen synthase kinase 3 beta (GSK3ß)-induced phosphorylation on MYC T58, which inhibited ubiquitination of MYC and stabilized it. Moreover, ACTL6A promoted the recruitment of MYC and histone acetyltransferase KAT5 on CDK2 promoters, leading to hyperactivation of CDK2 transcription. ACTL6A overexpression promoted, while silencing ACTL6A suppressed cell proliferation and tumor growth in TNBC cells in vitro and in vivo, which was dependent on MYC signaling. Furthermore, co-therapy with paclitaxel and CDK2 inhibitor showed synergistic effects in tumor suppression. Notably, ACTL6A/MYC/CDK2 axis was specifically up-regulated in TNBC and high expression of ACTL6A was correlated to shorter survival in patients with TNBC. CONCLUSIONS: These findings reveal a novel mechanism by which ACTL6A prolongs the retention of MYC in TNBC and suggest that pharmacological targeting ACTL6A/MYC/CDK2 axis might have therapeutic potential in patients with TNBC.

Discovery of novel pyrimidine-based benzothiazole derivatives as potent cyclin-dependent kinase 2 inhibitors with anticancer activity.

To develop novel CDK2 inhibitors as anticancer agents, a series of novel pyrimidine-based benzothiazole derivatives were designed and synthesized. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against five cancer cell lines. Especially, the analogue 10s exhibited approximately potency with AZD5438 toward four cells including HeLa, HCT116, PC-3, and MDA-MB-231 with IC50 values of 0.45, 0.70, 0.92, 1.80 µM, respectively. More interestingly, the most highly active compound 10s in this study also possessed promising CDK2/cyclin A2 inhibitory activities with IC50 values of 15.4 nM, which was almost 3-fold potent than positive control AZD5438, and molecular docking studies revealed that the analogue bound efficiently with the CDK2 binding site. Further studies indicated that compound 10s could induce cell cycle arrest and apoptosis in a concentration-dependent manner. These observations suggest that pyrimidine-benzothiazole hybrids represent a new class of CDK2 inhibitors and well worth further investigation aiming to generate potential anticancer agents.

Novel pyrazolopyrimidines: Synthesis, in vitro cytotoxic activity and mechanistic investigation.

A series of novel pyrazolo[3,4-d]pyrimidines bearing benzenesulfonamide moiety 5a-f, 6 and 7 were synthesized. Cytotoxic screening was conducted against MCF-7 and HepG2. 6-(4-Methoxyphenyl)-4-oxopyrazolopyrimidine derivative 5e and 4-imino-6-oxopyrazolopyrimidine derivative 6 revealed potent cytotoxic activity with IC50 1.4 µM (MCF-7) and 0.4 µM (HepG2), respectively compared to that of doxorubicin, (IC50 = 1.02 µM and 0.9 µM, respectively). Compounds 5e and 6 were subjected to cell cycle analysis and apoptosis assay after 24 h and 48 h treatment. Compound 5e arrested cell at G1 phase, while 6 arrested cell at S and G2/M phases, respectively. The apoptotic effect of both compounds were evidenced by pre G1 apoptosis as its percentage increased by time (7.38%, 11.61%) and (13.92%, 16.71%), respectively. Apoptosis induction capability was confirmed by the effect on early and late apoptosis and augmentation of caspase-3 level. Furthermore, compound 6 inhibited CDK2 enzyme with IC50 = 0.19 µM and increased levels of its regulators, P21 and P27 by 10.06% and 8.5%, respectively. Moreover, a molecular docking study of compound 6 on CDK2 enzyme was adopted to explore binding interaction with amino acid residues of its active site.