Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP

Bioorganic and Medicinal Chemistry Letters

Volumn 17, Issue 20, 2007, Pages 5671-5676

  Garbaccio, Robert M ^a   Tasber, Edward S ^a   Neilson, Lou Anne ^a   Coleman, Paul J ^a   Fraley, Mark E ^a   Olson, Christy ^a   Bergman, Jeff ^a   Torrent, Maricel ^a   Buser, Carolyn A ^a   Rickert, Keith ^a   Walsh, Eileen S ^a   Hamilton, Kelly ^a   Lobell, Robert B ^a   Tao, Weikang ^a   South, Vicki J ^a   Diehl, Ronald E ^a   Davide, Joseph P ^a   Yan, Youwei ^a   Kuo, Lawrence C ^a   Li, Chunze ^a   Prueksaritanont, Thomayant ^a   Fernandez Metzler, Carmen ^a   Mahan, Elizabeth A ^a   Slaughter, Donald E ^a   Salata, Joseph J ^a   Kohl, Nancy E ^a   Huber, Hans E ^a   Hartman, George D ^a   more..

^a MERCK RESEARCH LABORATORIES   (United States)

Author keywords

3+2 Cycloaddition; Anti mitotics; Cancer; hERG; Kinesin spindle protein; Kinesins; KSP inhibitors; Multidrug resistance; Pgp; Pyrazolobenzoxazines; Quaternary center

Indexed keywords

BENZOXAZINE DERIVATIVE; DIHYDROPYRAZOLOBENZOXAZINE; KINESIN; PROTEIN INHIBITOR; UNCLASSIFIED DRUG;

ALKYLATION; ANIMAL EXPERIMENT; ANIMAL MODEL; ANTINEOPLASTIC ACTIVITY; ARTICLE; CONTROLLED STUDY; CYCLOADDITION; DESS MARTIN OXIDATION; DIASTEREOISOMER; DOG; DRUG DESIGN; DRUG INHIBITION; DRUG POTENCY; DRUG SYNTHESIS; IN VIVO STUDY; MOUSE; NONHUMAN; STRUCTURE ACTIVITY RELATION; SUBSTITUTION REACTION;

ANIMALS; BENZOXAZINES; CELL LINE; DOGS; DRUG DESIGN; HUMANS; HYDROGEN; KINESIN; MITOSIS; MOLECULAR STRUCTURE; PYRAZOLES; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 34548591160     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2007.07.067     Document Type: Article

References (15)

1. Preceding communication.
2. Cox C.D., Breslin M.J., Whitman D.B., Coleman P.J., Garbaccio R.M., Fraley M.E., Zrada M.M., Buser C.A., Walsh E.S., Hamilton K., Lobell R.B., Tao W., Abrams M.T., South V.J., Huber H.E., Kohl N.E., and Hartman G.D. Bioorg. Med. Chem. Lett. 17 (2007) 2697
3. 1H NMR and high resolution mass spectrometry. For detailed experimental procedures, see the supplementary material.
4. Garanti L., Sala A., and Zecchi G. J. Org. Chem. 42 (1977) 1389 The undesired Claisen-rearrangement was also observed in this report as a side product.
5. Synthesized in two steps from 3-phenyl-2-propyn-1-ol. Lindlar reduction of the alkyne provided the cis-olefin. Conversion of the alcohol to the chloride was accomplished using methanesulfonylchloride, 2,6-lutidine, and LiCl.
6. Levitt J.I., Turos E., and Weinreb S.M. Synth. Commun. 12 (1982) 989
7. Sibi M.P., Sharma R., and Paulson K.L. Tetrahedron Lett. 33 (1992) 1941
8. PDB coordinates for 20: 2Q2Y.
9. 50 values are reported as averages of at least two determinations; standard deviations are ±25-50%.
10. 50 values.
11. 50 values are reported as the average of at least two independent determinations and were acquired by radioligand binding competition experiments using membrane preparations from human embryonic kidney cells that stably express hERG; standard deviations are within ±25-50% of reported values. For assay details, see:. Bilodeau M.T., et al. J. Med. Chem. 47 (2004) 6363 and references therein
12. 50 obtained in KB-V-1 cells vs. that in KB-3-1 cells is defined as the MDR ratio. As a general guideline, we considered compounds with MDR ratios < 10 to be of interest for their ability to enter and kill cells that overexpress Pgp. Verapamil, a competitive inhibitor of Pgp, restores the activity of Taxol and our KSP inhibitors in the KB-V-1 cell line to nearly that observed in the parental KB-3-1 line, confirming that Pgp-efflux is responsible for the observed resistance to drug-mediated mitotic arrest. The absolute values of potency between the A2780 and KB cell lines were similar (within 2× of each other).
13. Wang J., Della Penna K., Wang H., Karczewski J., Connolly T.M., Koblan K.S., Bennett P.B., and Salata J.J. Am. J. Physiol. 284 (2003) H256
14. 3 before mice were surgically implanted with Alzet mini-pumps (Durect Corporation) filled with the appropriate KSP inhibitors according to manufacturer's recommendations. Prior to the implant, pumps containing KSP inhibitors were primed by incubation in 37 °C water bath for 3 h, so that the pumps would discharge KSP inhibitors at a constant rate of 8 μl/h after subcutaneous implantation. Mice were euthanized 22 h post pump implant and blood and tumors were harvested. Blood was collected in EDTA Vacutainers and processed for plasma to determine pharmacokinetics. Tumors were fixed in 10% neutral buffered formalin and then processed and embedded in paraffin. Paraffin embedded tumors were sectioned 5 μm thick and used in a phospho-histone H3 immunohistochemistry assay designed to determine the percentage of cells blocked in mitosis compared to control treated tumors. After paraffin removal, re-hydration, and antigen retrieval, sections were incubated with anti-phospho-histone H3 (ser10; Upstate). Following incubation with a biotinylated secondary antibody, staining of antigen positive nuclei was accomplished using avidin:biotin complexed horseradish peroxidase and diaminobenzidine reagent. Sections were imaged using an Olympus BX51 microscope with a motorized stage and Image-Pro Analysis software. Quantization of the sections was accomplished by measuring the percentage of positively stained nuclei (black) per unit area.
15. Compound 20 and its analogs are also described in WO2006023083.