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. 2022 Feb 24;65(4):3420-3433.
doi: 10.1021/acs.jmedchem.1c01951. Epub 2022 Feb 3.

A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics

Li Zhang  1 , Chen Cheng  1 , Jing Li  1 , Lili Wang  1 , Alexander A Chumanevich  1 , Donald C Porter  2 , Aleksei Mindich  3   4 , Svetlana Gorbunova  3 , Igor B Roninson  1   2 , Mengqian Chen  1   2 , Campbell McInnes  1
Affiliations

A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics

Li Zhang et al. J Med Chem. .

Abstract

Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and Senexin B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship (SAR). An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.

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Conflict of interest statement

The authors declare the following competing financial interest(s):

D.C.P, M.C. and I.B.R. are or have been employees or officers, and J.L. is a consultant of Senex Biotechnology, Inc. A.M. and S.G. are or have been employees or officers of CSC BIOCAD.

Figures

Figure 1.
Figure 1.
The chemical structures of Senexin A (1) and Senexin B (2).
Figure 2.
Figure 2.
Comparison of the predicted binding modes of Senexin A (1) (quinazoline) and 8a (quinoline) docked to CDK8/cyclin C protein structure 4F7S.
Figure 3.
Figure 3.
The predicted binding modes of Senexin B (2) and 20a docked to CDK8/cyclin C protein structure 4F7S.
Figure 4.
Figure 4.
Kinome profiling dendrogram (Discover X) of Senexin C at a concentration of 2μM. Kinases with >65% inhibition are labeled.
Figure 5.
Figure 5.
Cell-based IC50 assays of Senexin C (SnxC) and Senexin B (SnxB). (A) 293-WT and dKO cells expressing Luciferase reporter under CDK8/19-dependent NFκB promoter treated with 10ng/mL TNF-α and SnxC at different concentrations for 3 hours. (B) Effects of SnxB and SnxC (3 hours treatment) on MYC mRNA expression in 293 cells. (C) Effects of SnxB and SnxC on CXCL8 mRNA expression in 293 cells treated with 10ng/mL TNF-α.
Figure 6.
Figure 6.
Effects of Senexin B and Senexin C on durability of the inhibition of CDK8/19-dependent gene expression in the wash-off study in 293 cells. 293 cells were pre-treated with 1 μM Senexin B or Senexin C or solvent control DMSO (0.1%) for 3 h before removal of the drug-containing media and then incubated with drug-free media for indicated period of time. Percentage of inhibition was calculated by normalization to the expression levels in control samples and presented as mean ± SEM (n = 3).
Figure 7.
Figure 7.
Metabolic stability of Senexin B and Senexin C in human hepatocytes.
Figure 8.
Figure 8.
PK/PD analysis of Senexin C in the CT26 tumor model in Balb/c mice. 8-week-old female Balb/c mice were inoculated with 1×106 CT26 murine colon cancer cells subcutaneously. When average tumor size reached 200~300 mm3, mice were dosed with Senexin C i.v. (2.5 mg/kg) in 5% Dextrose (A) or orally (100mg/kg) in 30% propylene glycol, 70% PEG-400 with one dose (B-C) or daily for 7 days (D-F). A. Senexin C accumulation in the plasma and tumor tissues 0.5, 1, 2, 4, 8, 12 and 24 hrs after receiving a single i.v. dose at 2.5 mg/kg. B. The same after a single oral dose at 100 mg/kg. C. Expression of CDK8/19 dependent genes in CT26 tumors from the mice in (B). D. Changes in body weights of animals orally dosed by Senexin C for 7 days (100 mg/kg, q.d.). E. Expression of PD marker genes in endpoint CT26 tumors collected at 12 hours post oral dosing on day 7. F. Endpoint PK of Senexin C in blood and tissues from the mice in (E).
Figure 9.
Figure 9.
In vivo efficacy study of Senexin C (40mg/kg, p.o., BID) in the MV4-11 AML model in female NSG mice. Treatment started 7 days post inoculation (1×106 MV4-11-luc cells via tail vein) and tumor growth was monitored through weekly bioluminescence imaging (BLI).
Scheme 1.
Scheme 1.
Synthesis of 6-substituted-N-phenethylquinolin-4-amine derivatives. Reagents and conditions: (a) triethoxymethane, 100°C, 94%; (b) p-substituted aniline, DCM, r.t., 20–64%; (c) phenoxybenzene, 220°C, 30–91%; (d) POCl3, 1,4-dioxane, 90°C, 27–91%; (e) 2-phenylethan-1-amine, DIEA, DMSO, 100°C, 6–40%; (f) HNO3, H2SO4, 0°C to r.t., 32%; (g) KOH, t-BuOH, 90°C, 40%; (h) NH4Cl, Fe, EtOH, H2O, reflux, 94%; (i) acetic anhydride, pyridine, r.t., 84%.
Scheme 2.
Scheme 2.
Synthesis of 4-((2-(6-substituted-naphthalen-2-yl)ethyl)amino)quinoline-6-carbonitrile derivatives. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, toluene, water, 80oC, 85%; (b) NBS, AIBN, CCl4, reflux, 83% for 12; NCS, AIBN, DCE, 80oC, 23% for 13; (c) KCN, MeOH, reflux, 40%; (d) LiOH, THF, water, 59% for 15, 94% for 16; (e) SOCl2, 1-methylpiperazine, DCM, 60oC to r.t., 42%; (f) KCN, ethanol, water, 60oC, 48%; (g) HATU, DIEA, DCM/DMF, r.t., 44–91%; (h) raney Ni, MeOH/H2O/NH4OH, H2, r.t., 42–98%; (i) 4-chloroquinoline-6-carbonitrile, DIEA, DMSO, 120oC, 17–77%; (j) NaIO4, MeOH, H2O, r.t., 73%; (k) TFA, DCM, r.t.,62–96%; (l) rhodium acetate dimer, iodobenzene diacetate, 2,2,2-trifluoroacetamide, MgO, DCM, r.t, 60%; (m) acetyl chloride, DIEA, DCM, 0oC to r.t., 91%
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References

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