Synthesis, structure-activity relationship and biological evaluation of 2,4,5-trisubstituted pyrimidine CDK inhibitors as potential anti-tumour agents.

A series of 2,4,5-trisubstituted pyrimidines have been synthesised and characterised, which exhibited potent CDK inhibition and anti-proliferative activities. The structure-activity relationship is analysed and a rational for CDK9 selectivity is discussed. Compound 9s, possessing appreciable selectivity for CDK9 over other CDKs, is capable of activating caspase 3, reducing the level of Mcl-1 anti-apoptotic protein, and inducing cancer cell apoptosis.

Substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidines are highly active CDK9 inhibitors: synthesis, X-ray crystal structures, structure-activity relationship, and anticancer activities.

Cancer cells often have a high demand for antiapoptotic proteins in order to resist programmed cell death. CDK9 inhibition selectively targets survival proteins and reinstates apoptosis in cancer cells. We designed a series of 4-thiazol-2-anilinopyrimidine derivatives with functional groups attached to the C5-position of the pyrimidine or to the C4-thiazol moiety and investigated their effects on CDK9 potency and selectivity. One of the most selective compounds, 12u inhibits CDK9 with IC(50) = 7 nM and shows over 80-fold selectivity for CDK9 versus CDK2. X-ray crystal structures of 12u bound to CDK9 and CDK2 provide insights into the binding modes. This work, together with crystal structures of selected inhibitors in complex with both enzymes described in a companion paper, (34) provides a rationale for the observed SAR. 12u demonstrates potent anticancer activity against primary chronic lymphocytic leukemia cells with a therapeutic window 31- and 107-fold over those of normal B- and T-cells.

Comparative structural and functional studies of 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile CDK9 inhibitors suggest the basis for isotype selectivity.

Cyclin-dependent kinase 9/cyclin T, the protein kinase heterodimer that constitutes positive transcription elongation factor b, is a well-validated target for treatment of several diseases, including cancer and cardiac hypertrophy. In order to aid inhibitor design and rationalize the basis for CDK9 selectivity, we have studied the CDK-binding properties of six different members of a 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile series that bind to both CDK9/cyclin T and CDK2/cyclin A. We find that for a given CDK, the melting temperature of a CDK/cyclin/inhibitor complex correlates well with inhibitor potency, suggesting that differential scanning fluorimetry (DSF) is a useful orthogonal measure of inhibitory activity for this series. We have used DSF to demonstrate that the binding of these compounds is independent of the presence or absence of the C-terminal tail region of CDK9, unlike the binding of the CDK9-selective inhibitor 5,6-dichlorobenzimidazone-1-ß-d-ribofuranoside (DRB). Finally, on the basis of 11 cocrystal structures bound to CDK9/cyclin T or CDK2/cyclin A, we conclude that selective inhibition of CDK9/cyclin T by members of the 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile series results from the relative malleability of the CDK9 active site rather than from the formation of specific polar contacts.

In vitro antitumor mechanism of a novel cyclin-dependent kinase inhibitor CDKI-83.

Cancer is regarded as a proliferative disorder. Inhibition of cyclin-dependent kinases (CDKs), which are the key regulators of the cell-cycle and RNA transcription, represents an attractive strategy for cancer therapy. In this study, we report the cellular mechanistic investigation of CDKI-83, a K (i)-nanomolar CDK9 inhibitor. The compound shows effective anti-proliferative activity in human tumour cell lines with GI(50) <1 µM, and is capable of inducing apoptosis in A2780 human ovarian cancer cells as determined by the activated caspase-3, Annexin V/PI double staining and accumulated cells at the sub-G1 phase of cell-cycle. While A2780 cells were arrested in G(2)/M phase with CDKI-83 treatment, phosphorylation at Thr(320) of PP1α was significantly reduced, indicating CDK1 inhibition. Importantly, this compound reduced phosphorylation at Ser-2 of RNA polymerase II (RNAPII) by inhibiting cellular CDK9 activity, and down-regulated Mcl-1 and Bcl-2. These results suggest that combined inhibition of CDK9 and CDK1 may result in the effective induction of apoptosis and CDKI-83 has the potential to be developed as an anti-cancer agent.

CDKI-71, a novel CDK9 inhibitor, is preferentially cytotoxic to cancer cells compared to flavopiridol.

Cancer cells appear to depend heavily on antiapoptotic proteins for survival and so targeted inhibition of these proteins has therapeutic potential. One innovative strategy is to inhibit the cyclin-dependent kinases (CDKs) responsible for the regulation of RNA polymerase II (RNAPII). In our study, we investigated the detailed cellular mechanism of a novel small-molecule CDK inhibitor (CDKI-71) in cancer cell lines, primary leukemia cells, normal B - & T- cells, and embryonic lung fibroblasts and compared the cellular and molecular responses to the clinical CDK inhibitor, flavopiridol. Like flavopiridol, CDKI-71 displayed potent cytotoxicity and caspase-dependent apoptosis induction that were closely associated with the inhibition of RNAPII phosphorylation at serine-2. This was caused by effective targeting of cyclinT-CDK9 and resulted in the downstream inhibition of Mcl-1. No correlation between apoptosis and inhibition of cell-cycle CDKs 1 and 2 was observed. CDKI-71 showed a 10-fold increase in potency in tumor cell lines when compared to MRC-5 human fibroblast cells. Significantly, CDKI-71 also demonstrated potent anti-chronic lymphocytic leukemia activity with minimal toxicity in normal B- and T-cells. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Here, we provide the first cell-based evidence that flavopiridol induces DNA double-strand breaks: a fact which may explain why flavopiridol has such a narrow therapeutic window in preclinical and clinical settings. Taken together, our data provide a rationale for the development of selective CDK inhibitors as therapeutic agents and CDKI-71 represents a promising lead in this context.

Online concentration of aristolochic acid I and II in Chinese medicine preparations by micellar electrokinetic chromatography.

In this study, an online concentration method in micellar electrokinetic chromatography (MEKC) applying field-enhanced sample injection (FESI) mode was developed for the detection of aristolochic acids (AAs) in Chinese medicine preparations. AA-I and AA-II were baseline separated with high separation efficiency, and 100-fold enhancement of the detection sensitivity was achieved compared with those obtained from normal capillary zone electrophoresis (CZE) or simple MEKC method. The proposed method was successfully applied for the determination of AAs in Chinese medicine preparations.

Fragmentation study of hexanitrostilbene by ion trap multiple mass spectrometry and analysis by liquid chromatography/mass spectrometry.

The fragmentation pathways of three explosive compounds with similar structures, hexanitrostilbene (HNS), cyclotrimethylene trinitramine (RDX), and 2,4,6-trinitrotoluene (TNT), have been investigated by multiple mass spectrometry (MSn, n = 1, 2, 3) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The electron capture mechanism for these compounds in negative ion APCI and ESI mode differs from the usual negative ion mechanism, deprotonation or addition of other species. This was shown for HNS and TNT, which both gave a [M]- anion but not a [M-H]- ion in APCI, and the [M]- anion of HNS was observed in ESI. The quantitative analysis of HNS was performed by liquid chromatography (LC)/ESI-MS, and the results obtained by the internal standard (ISTD) method were compared with those from the external standard (ESTD) method, demonstrating that both quantitation approaches are useful, with good sensitivity, reproducibility and linearity, and ESTD is preferable in routine applications.