A diaminocyclohexyl analog of SNS-032 with improved permeability and bioavailability properties
Abstract
The identification of a selective CDK2, 7, 9 inhibitor 4 with improved permeability is described. Com- pound 4 exhibits comparable CDK selectivity profile to SNS-032, but shows improved permeability and higher bioavailability in mice.
The CDKs (cyclin-dependent kinases) are a family of serine/ threonine protein kinases that, in conjunction with their cyclin (cyc) partners, play key roles in cell-cycle progression and tran- scriptional regulation.1–3 Cell-cycle regulatory CDKs include cycD/CDK4 and cycD/CDK6, as well as cycE/CDK2, which sequen- tially phosphorylate the retinoblastoma protein to facilitate G1 ? S transition. CDK2 and CDK1 paired with cycA, and cycB/ CDK1 are required for orderly progression through S-phase and the G2 ? M transition, respectively. Transcriptional CDKs include cycH/CDK7 and cycT/CDK9. The CDK7 subunit of transcription fac- tor II H (TFIIH) phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (pol II) on serine 5 at early stages of the tran- scriptional cycle.4 CDK9, a member of the elongation factor P-TEFb, phosphorylates serine 2 on the CTD to transition RNA pol II into productive elongation.5 Inhibition of these kinases is predicted to have the greatest effect on the expression of proteins with short half-lives, many of which are encoded by antiapoptotic and growth regulatory genes. Some of the CDKs are involved in both processes. For example, CDK7 is a component of the CDK-activating kinase (CAK). Full activation of CDKs 1, 2, 4, 5 and 6 requires phosphory- lation by CAK; therefore, CDK7 serves as a ‘master regulator’ of the cell cycle. Inappropriate activation of both cell-cycle and transcrip- tional-regulatory CDKs can lead to unregulated proliferation, avoidance of apoptosis, and the presence of genetic instability in cancer cells. These attributes, which are among the hallmarks of
cancer, suggest that CDKs may be important targets for cancer therapeutics. SNS-032 (Fig. 1, formerly BMS-387032) is such a dual-acting CDK inhibitor, with potency and selectivity against CDK2, 7 and 9 (Table 3). SNS-032 is currently in a phase 1 clinical trial for multiple myeloma and chronic lymphocytic leukemia as an intravenous agent.
Early reports showed that SNS-032 has oral bioavailability of about 31% in rats. Bioavailability was limited by absorption rather than extensive first-pass metabolism.6 Since SNS-032 is a substrate of P-glycoprotein, this efflux transporter may be responsible for limiting its absorption. With an interest in a potential oral CDK program, we sought to develop a backup inhibitor to SNS-032 with comparable CDK2, 7, and 9 inhibitory activities, but with improved permeability and lack of transporter-mediated efflux. We hypoth- esized that improving the permeability of SNS-032 would possibly provide an inhibitor with improved oral bioavailability. In order to conserve the specificity and potency of SNS-032, we chose to main- tain the general scaffold of SNS-032, but to replace the isonipecotic amide fragment and explore N-alkyl instead of N-acyl moieties.
Inhibitors 2–10 (Tables 1 and 2) were synthesized as summarized in Scheme 1. Intermediate 11 was prepared as reported previously.7 Treatment of 11 with NaNO2 in CH3CN provided 2-bro- mothiazole 12 in 40–60% yield. Exposure of 12 to various primary and secondary amines in dimethylacetamide at 110 °C afforded the final products, 2–10.
All the compounds were screened in a CDK2/cycA8 biochemical assay and a CDK9 high-content screen cellular assay9 and com- pared against SNS-032 (Compound 1). Inhibition of CDK2/cycA was readily achieved, consistent with CDK2/SNS-032 crystallogra- phy which shows that the piperidinyl ring is directed into sol- vent,10 and therefore replaceable. However, sub-micromolar CDK9 cellular activity was only observed for the 1,4-diam- inocyclohexyl substitution in 4 (Table 1).
Further exploration around 4 indicated a CDK9 preference for trans-1,4-diaminocyclohexyl fragment over the cis-isomer 5, as well as the 1,4-regioisomer vs the 1,3-isomer 7 (Table 1). Similar SAR was not determined for SNS-032 amide series.Compound 4 was further evaluated in in vitro studies, where it was shown to have comparable activities against CDKs 2, 7, and 9 (Table 2). More significantly, 4 exhibited over 5-fold improved per- meability in MDCK11 cells over 1 (Table 2). On the basis of the per- meability data and acceptable liver microsomal stability,12 4 was selected for pharmacokinetic studies.
Plasma concentration–time profiles after IV and PO administra- tion of 5 and 10 mg/kg, respectively, of compounds 1 and 4 are shown in Figure 2.13 Pharmacokinetic parameters are summarized in Table 3. Pharmacokinetics after intravenous administration are similar for 1 and 4. Compounds 1 and 4 show moderate to high clearance, a large volume of distribution, and terminal half-lives of 0.6 and 1.9 h, respectively. After oral administration of 10 mg/ kg, bioavailability of 1 was 14%, whereas 4 showed bioavailability of 62%. Administration of 30 mg/kg compound 4, led to a more than dose-linear increase in AUC (4.4-fold increase for a 3-fold in- crease in dose), resulting in a bioavailability of 92%. The high bio- availability was maintained at the next higher dose level of 45 mg/kg. Given the similar pharmacokinetics after intravenous administration, the increased bioavailability of 4 compared to 1 may be the result of the improved permeability.
In summary, the incomplete bioavailability of compound 1 (SNS-032) in mice that resulted from poor absorption may be rem- edied by improving the permeability of the compound. Through replacing the N-isonipecotic fragment with the N-1,4-trans-diam- inocyclohexyl fragment, we identified compound 4 which exhibits comparable CDK selectivity profile to SNS-032, but shows im- proved permeability and higher bioavailability in mice. Further evaluation of compound 4 is required to determine the effects of improved permeability on in vivo efficacy.