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M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4257–4261
Table 2. IL-2, Hck kinase, and Src kinase inhibition data for select
compounds.
References and notes
a
b,13a
b,13b
1. (a) Molina, T. J.; Kishihara, K. I.; Siderovski, D. P.;
van Ewijk, W.; Narendran, A.; Timms, E.; Wakeham,
A.; Paige, C. J.; Hartmann, K. U.; Veillette, A.;
Davidson, D.; Mak, T. W. Nature 1992, 357, 161; (b)
Levin, D. S.; Anderson, S. J.; Forbush, K. A.;
Perlmutter, R. M. EMBO J. 1993, 12, 1671; (c) August,
A. J. Biol. Chem. 1996, 271, 10054; (d) Bolen, J. B.;
Brugge, J. S. Annu. Rev. Immunol. 1997, 15, 371; (e)
Kamens, J. S.; Ratnofsky, S. E.; Hirst, G. C. Curr.
Opin. Invest. Drugs 2001, 2, 1213.
Compound
IL-2 IC50
(lM)
Hck IC50
(nM)
Src IC50
(nM)
12l
2.00
3.21
0.546
2.10
160
48
104
3
12m
12p
12q
546
456
273
764
a IL-2 synthesis inhibition measured from Jurkat cells.
b IC50s were determined with a commercial Proflour assay (Promega
Corp., Cat. #1271). See also Ref. 13.
2. Zhu, X.; Kim, J. L.; Newcomb, J. R.; Rose, P. E.; Stover,
D. R.; Toledo, L. M.; Zhao, H.; Morgenstern, K. A.
Structure 1999, 7, 651 (1QPC pdb, Research Collabora-
tory for Structural Bioinformatics).
3. (a) Kimura, Y.; Miyamoto, T.; Matsumoto, J.; Minami, S.
Chem. Pharm. Bull. 1976, 24, 2637; (b) Minami, S.;
Kimura, Y.; Miyamoto, T.; Matsumoto, J. Tetrahedron
Lett. 1974, 44, 3893.
4. Youssef, M. M.; Youssef, A. M. S. Phosphorus Sulfur
Silicon Relat. Elem. 2003, 178, 67.
5. Coffen, D. L.; Schaer, B.; Bizzarro, F. T.; Cheung, J. B. J.
Org. Chem. 1984, 49, 296.
6. Collins, I.; Moyes, C.; Davey, W. B.; Rowley, M.;
Bromidge, F. A.; Quirk, K.; Atack, J. R.; McKernan, R.
M.; Thompson, S.; Wafford, K.; Dawson, G. R.; Pike, A.;
Sohal, B.; Tsou, N. N.; Ball, R. G.; Castro, J. L. J. Med.
Chem. 2002, 45, 1887.
7. Oxidation with a single equivalent of m-CPBA at 0 ꢁC
favored formation of sulfoxide. However, small amounts
of the sulfone were formed. The ratio favored sulfone
when excess oxidant was present or the temperature was
raised. Both intermediates underwent nucleophilic
displacement.
8. Perri, S. T.; Slater, S. C.; Toske, S. G.; White, J. D. J.
Org.Chem. 1990, 55, 6037.
Figure 3. Key hydrogen bonds between 12m and Lck.
9. TsOH acid was found to be the optimal reagent for this
cyclization. A more detailed account of the methodology
to these ring systems will be disclosed in a separate future
publication. See also Ref. 11.
including a hydrogen bond with Lys273, as well as a
possible salt bridge with Asp326 (Fig. 3).
10. The ability of compounds to inhibit human Lck enzyme
(IC50) was determined using the commercially available
ProFlour Src-family Kinase Assay (Promega Corporation,
Madison, WI; cat. #1271). The assay was performed
according to manufacturer’s instructions, with 2 nM
recombinant human active Lck (Upstate Cell Signaling
Solutions, Charlottesville, VA; cat. #14-442) at an ATP
concentration of 10 mM.
11. A more detailed account of the methodology used to
synthesize the various pyridazine analogs including com-
pounds 12n-q is in preparation and will be disclosed in the
literature.
Analog 12m (Lck IC50 = 2 nM) also displayed better
aqueous solubility (33.8 lg/ml) and was subsequently
screened for in vitro metabolism using rat hepatocytes.14
This compound suffered low metabolic loss with >84%
of the parent molecule remaining after 1 h. This mole-
cule was subsequently evaluated in a rat PK study to
determine bioavailability and half-life. Pyridazine 12m
displayed modest bioavailability (F = 14%) and a short
half-life (T1/2 = 1.5 h).
In summary, we have described the design, synthesis,
biological activity, and proposed binding mode in Lck
kinase of a novel class of tri-cyclic derivatives of
1,2-dihydro-pyrimido[4,5-c]pyridazines. SAR studies
identified several low nanomolar inhibitors with the
most advanced analog 12m being progressed into
pharmacokinetic evaluation.
12. IL-2 release was measured by stimulating the Jurkat E6-1
T cell line (human acute T cell leukemia, ATCC, Manas-
sas, VA) with monoclonal anti-human CD3e antibody and
Phorbol Myristate Acetate (PMA) (Sigma, St. Louis,
MO). Flat-bottomed 96-well plates were pre-coated with
400 ng/well of anti-human CD3e mouse antibody UCHT1
(R&D systems, Minneapolis, MN) and incubated for 2 h
at 37 ꢁC. Jurkat cells maintained in RPMI-1640 contain-
ing 10% fetal bovine serum and 1% antibiotics in the log
growth phase (2 · 105–1 · 106 cells/ml) were harvested and
incubated in triplicate in 96-well plates for 30 min at 37 ꢁ C
in the presence or absence of various concentrations of
Lck inhibitors. The cell-inhibitor mixture was then trans-
ferred into the wells of the anti-CD3e-coated 96-well
plates, and PMA was added to the wells at a final
concentration of 10 ng/ml (1 ng/well). The plates were
Acknowledgments
We are grateful to A. L. Roe, C. A. Cruze, W. E.
Schwecke, and C. R. Dietsch for pharmacokinetic stud-
ies, M. Buchalova for chemical stability and solubility
studies.