Y. Hu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6067–6072
6071
Table 8. In vitro (lM) and in vivo (%) inhibition of TNFa production
with selected inhibitors
Drs. Allan Wissner, Hwei-Ru Tsou, Bernard Johnson,
and Middleton Floyd for the intermediates toward the
synthesis of C7-substituted compounds; Dr. Alex Gont-
charov for the large-scale preparation of compound 9;
Dr. Nelson Huang and Ms. Ning Pan for LC–MS mea-
surements; Dr. Walter Massefski for NMR measure-
ments; Mrs. Julie Liu for selectivity assays in Table 7;
and Mrs. Qiuna Bi for LPS-TNF assay in Table 8.
Compound
Tpl2
Monocytes
HWB
LPS-TNF
29
31
32
37
38
41
0.031
0.019
0.011
0.003
0.003
0.014
0.6
0.46
1.7
0.4
0.4
0.7
>20
3.3
7.8
NT
70%b
54%a
84%a
NT
7.9
15.8
13.2
NT
Monocytes: TNF inhibition in LPS-treated human monocytes.
HWB: TNF inhibition in LPS-treated human whole blood.
References and notes
a
LPS-TNF: LPS-induced TNF inhibition in mice (IP dosing): 50 mg/
kg; b25 mg/kg; NT, not tested.
1. (a) Choy, E. H. S.; Panayi, G. S. P. N. Eng. J. Med. 2001,
344, 907; (b) Feldmann, M.; Mani, R. Ann. Rev. Immunol.
2001, 19, 163; (c) Feldmann, M.; Brennan, F. M.; Foxwell,
B. M.; Maini, R. N. Curr. Dir. Autoimmun. 2001, 3, 188.
2. Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. (Drug Discov.)
2003, 2, 717.
3. (a) Miyoshi, J.; Higashi, T.; Mukai, H.; Ohuchi, T.;
Kakunaga, T. Mol. Cell. Biol. 1991, 11, 4008; (b) Dumitru,
C. D.; Ceci, J. D.; Tsatsanis, C.; Konoyiannis, D.;
Stamatakis, K.; Lin, J. H.; Patriotis, C.; Jenkins, N. A.;
Copeland, N. G.; Kollias, G.; Tsichlis, P. N. Cell 2000,
103, 1071; (c) Schlesinger, T. K.; Franger, G. R.; Yujiri,
T.; Johnson, G. L. Front. Biosci. 1998, 3, D1181.
or OH (48)9) positions (Table 6). Both R1 and R2 substi-
tutions are detrimental to activity.
Compound 3110 was screened for selectivity over a panel
of kinases both in in vitro assays and in cellular assays
(Table 7).11 This compound is >120-fold selective
against MEK and >300-fold selective against PKA
while much more selective against other kinases such
as p38, Src, CAMKII, MK2, PKC, and S6. In human
monocytes, compound 31 selectively inhibits the phos-
phorylation of the downstream MEK kinase (P-MEK:
0.21 lM) over other MAP kinase signaling pathways,
for example, P-p38, P-MK2, P-cJun, and P-lkBa. How-
ever, in A431 cells, compound 31 appears to also inhibit
EGFR kinase (30 nM).
4. Chiarello, M.; Marinissen, M. J.; Gutkind, J. S. Mol. Cell.
Biol. 2000, 20, 1747.
5. Gavrin, L. K.; Green, N.; Hu, Y.; Janz, K.; Kaila, N.; Li,
H.-Q.; Tam, S. Y.; Thomason, J. R.; Gopalsamy, A.;
Ciszewski, G.; Cuozzo, J. W.; Hall, J. P.; Hsu, S.; Telliez,
J.-B.; Lin, L.-L. Bioorg. Med. Chem. Lett. 2005, 15, 5288.
6. A typical preparation of target compound 2 through
reductive amination is illustrated by the following proce-
dure for 31: in a 50 mL round-bottomed flask 6-amino-4-
(3-chloro-4-fluoro-phenylamino)-quinoline-3-carbonitrile
(200 mg, 0.64 mmol) and 4(5)-imidazole carboxaldehyde
(147 mg, 1.53 mmol) were taken up in ethanol (10 mL).
Then, acetic acid was added to bring the pH of the
solution to 4, and the mixture was stirred for 15 min.
NaCNBH3 (48 mg, 0.77 mmol) was then added and the
reaction mixture warmed to 30 ꢂC for 2.5 h or until
complete by TLC. The reaction mixture was stripped to
dryness and the residue was purified via preparative
HPLC and lyophilized to give the product as a yellow
solid (166 mg, 66%): 1H NMR (400 MHz, DMSO-d6) d
ppm 4.26 (d, J = 4.80 Hz, 2H) 6.53 (t, J = 5.43 Hz, 1H)
7.05 (s, 1H) 7.20 (d, J = 2.53 Hz, 1H) 7.22–7.28 (m, 1H)
7.38 (dd, J = 8.97, 2.40 Hz, 1H) 7.43 (t, J = 9.09 Hz, 1H)
7.48 (dd, J = 6.57, 2.78 Hz, 1H) 7.62–7.70 (m, 2H) 8.15 (s,
2H) 9.36 (s, 1H); HRMS (ESI+) calcd for C20H14ClFN6
(MH+) 393.10252, found 393.1019.
7. (a) Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.;
Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani,
C.; Davis, R.; Shi, Xi.; Rabindran, S. K.; Cruber, B. C.;
Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang,
Y.-F.; Greenberger, L. M.; Tsou, H.-R. J. Med. Chem.
2003, 46, 49; (b) Tsou, H.-R.; Overbeek-Klumpers, E. G.;
Hallett, W. A.; Reich, M. F.; Floyd, M. B.; Johnson, B.
D.; Michalak, R. S.; Nilakantan, R.; Discafani, C.; Golas,
J.; Rabindran, S. K.; Shen, R.; Shi, X.; Wang, Y.-F.;
Upeslacis, J.; Wissner, A. J. Med. Chem. 2005, 48, 1107.
8. (a) Smaill, J. B.; Showalter, H. D. H.; Zhou, H.; Bridges,
A. J.; McNamara, D. J.; Fry, D. W.; Nelson, J. M.;
Sherwood, V.; Vincent, P. W.; Roberts, B. J.; Elliott, W.
L.; Denny, W. A. J. Med. Chem. 2001, 44, 429; (b)
Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.;
Selected compounds were evaluated for TNFa inhibito-
ry activities in cells, blood, and in vivo (Table 8).11 Com-
pounds 29, 31, 32, 37, 38, and 41 are all potent in LPS-
treated human monocytes with compound 32 being the
least potent, possibly due to low permeability of the
charged N-oxide moiety in cells. In human whole blood,
compound 31 is most potent with an IC50 of 3.3 lM.
Compounds 32, 31, and 37 were tested intraperitoneally
in a mouse model of LPS-stimulated production of
TNFa.12 As indicated in Table 8, all three compounds
demonstrated in vivo efficacy on LPS/D-Gal induced
TNFa release. The fact that compound 31 appears to
be more potent than compounds 32 and 37 in the in vivo
model (70% inhibition at 25 mg/kg) correlates with their
respective whole blood activities.
In summary, we have successfully identified potent and
selective Tpl2 inhibitors with a quinoline-3-carbonitrile
scaffold. Compound 31 is selective against a panel of ki-
nases in enzymatic assays, and it also selectively inhibits
phospho-MEK (P-MEK) formation in LPS-treated hu-
man monocytes. Selected compounds with cellular and
whole blood activity also effectively inhibited LPS/D-
Gal-induced TNFa release when administered intraper-
itoneally in mice. Further studies to improve EGFR
selectivity for this series will be reported in due course.
Acknowledgments
The authors thank Drs. Tarek Mansour and Dennis
Powell for their support; Dr. Ariamala Gopalsamy
and Mr. Greg Ciszewski for tailpiece SAR studies;
´
Johnstone, C.; Ple, P. A.; Ogilvie, D. J.; Dukes, M.;
Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem.