2398
J. L. Buchanan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2394–2399
N
N
N
a
b
N
+
HN
O
N
O
NH
Cl
N
NH
Cl
N
Cl
H2N
3
28
N
N
O
NH2
N
NO2
O
c,d
e
f
HN
O
N
NH
NH
26
N
O
N
N
29
30
N
N
NH2
NO2
NO2
Br
HN
N
NH
g
h
i
8
O
O
O
28
N
N
O
O
N
O
N
O
31
32
33
Scheme 1. Synthesis of 2,4-bis-arylamino-1,3-pyrimidines 3, 26, and 8. Reagents and conditions: (a) i-Pr2NEt, i-PrOH, 100 °C, 12 h, 40%; (b) 3,4,5-trimethoxyaniline, DMSO,
100 °C, 65%; (c) 1-bromo-3-chloropropane, THF, rt, 67%; (d) p-nitrophenol, K2CO3, DMF; (e) H2, Pd/C, rt, 86% (two steps); (f) 28, DMSO, 100 °C, 33%; (g) morpholine, Pd2(dba)3,
NaOt-Bu, BINAP, PhCH3, 80 °C, 3 h, 66%; (h) H2, Pd/C, EtOH, rt; (i) 28, DMSO, 100 °C.
6. (a) Human tumor cell lines or a rat fibroblast cell line were plated out in flat-
Acknowledgments
well plates in complete medium and allowed to adhere overnight. The cells
were then starved in medium containing 0.1% bovine serum albumin (BSA)
The authors thank Nick Lydon, David Armistead, Rick Kendall,
Murray Robinson, Dave Lacey, Joe Kim and Vinod Patel for their
support of this research program. We thank Jean Bemis for provid-
ing compound 17, Lucian DiPietro for providing compounds 1 and
2, Pedro Beltran for help with statistics and Erin DiMauro and Mar-
garet Chu-Moyer for helpful discussions.
overnight, pre-incubated for 1 h with or without dilutions of compound, then
activated overnight with 50 ng/mL insulin-like growth factor (IGF-1).
Proliferation was determined by the level of 3H-thymidine incorporation into
DNA. IC50’s were determined by comparing the level of thymidine
incorporation found in the presence of compound compared to controls. (b)
Compounds were routinely assessed in an assay measuring IGF-1-induced
auto-phosphorylation of IGF-1Rb and displayed good correlation with the
proliferation results.
7. (a) Armistead, D. M.; Bemis, J. E.; DiPietro, L. V.; Geuns-Meyer, S. D.; Habgood,
G. J.; Kim, J. L.; Nunes, J. J.; Patel, V. F.; Toledo-Sherman, L. M. WO 01/60816.; (b)
Buchanan, J. L.; Chaffee, S.; Harmange, J.-C.; Novak, P. M.; Zhu, X. WO 03/
018022.; (c) Harmange, J.-C.; Booker, S.; Buchanan, J. L.; Chaffee, S.; Novak, P.
M.; Van Der Plas, S.; Zhu, X. WO 03/018021.
8. The kinase domain of human IGF-1R (res 988 to 1286) with an N-terminal GST
tag was expressed in insect cells and purified by immobilized glutathione
affinity, anion exchange, and size exclusion chromatographies. The coordinates
for the X-ray co-crystal structure of IGF-1R and 8 have been deposited in the
PDB. The RCSB ID code is RCSB063987 and the PDB ID code is 3QQU.
9. Incubation of 3 with rat or human liver microsomes in the presence of NADPH
identified O-demethylation as a major route of metabolism.
References and notes
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505. and references cited therein.
2. (a) Tolcher, A. W.; Sarantopoulos, J.; Patnaik, A.; Papadopoulos, K.; Lin, C.-C.;
Rodon, J.; Murphy, B.; Roth, B.; McCaffery, I.; Gorski, K. S.; Kaiser, B.; Zhu, M.;
Deng, H.; Friberg, G.; Puzanov, I. J. Clin. Oncol. 2009, 23, 5800; (b) Beltran, P. J.;
Mitchell, P.; Chung, Y.-A.; Cajulis, E.; Lu, J.; Belmontes, B.; Ho, J.; Tsai, M. M.;
Zhu, M.; Vonderfecht, S.; Baserga, R.; Kendall, R.; Radinsky, R.; Calzone, F. J. Mol.
Cancer Ther. 2009, 8, 1095.
3. (a) Wittman, M. D.; Velaparthi, U.; Vyas, D. M. Ann. Rep. Med. Chem. 2009, 44,
281; (b) Li, R.; Pourpak, A.; Morris, S. W. J. Med. Chem. 2009, 52, 4981; (c)
Hewish, M.; Chau, I.; Cunningham, D. Recent Patents Anti-Cancer Drug Discov.
2009, 4, 54; (d) Gualberto, A.; Pollak, M. Oncogene 2009, 28, 3009; (e) Ryan, P.
D.; Goss, P. E. Oncologist 2008, 13, 16; (f) Rodon, J.; DeSantos, V.; Ferry, R. J., Jr.;
Kurzrock, R. Mol. Cancer Ther. 2008, 7, 2575; (g) Chitnis, M. M.; Yuen, J. S. P.;
Protheroe, A. S.; Pollak, M.; Macaulay, V. M. Clin. Cancer Res. 2008, 14, 6363; (h)
Hubbard, S. R.; Miller, W. T. Curr. Opin. Cell Biol. 2007, 19, 117; (i) Imai, K.;
Takaoka, A. Nat. Rev. Cancer 2006, 6, 714; (j) Yuen, J. S. P.; Macaulay, V. M. Expert
Opin. Ther. Targets 2008, 12, 589; (k) Weroha, S. J.; Haluska, P. J. Mammary Gland
Biol. Neoplasia 2008, 13, 471; (l) Paz, K.; Hadari, Y. R. Comb. Chem. High
Throughput Screening 2008, 11, 62; (m) Wang, Y.; Ji, Q.-S.; Mulvihill, M.;
Pachter, J. A. Recent Results Cancer Res. 2007, 172, 59; (n) Hubbard, R. D.;
Wilsbacher, J. L. ChemMedChem 2007, 2, 41; (o) Hartog, H.; Wesseling, J.;
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10. This strategy to improve cellular potency has been previously reported, for
example: Martin, M. W.; Newcomb, J.; Junes, J. J.; Boucher, C.; Chai, L.; Epstein,
L. F.; Faust, T.; Flores, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Kim, J.
L.; Middleton, S.; Morgenstern, K.; Oliveira-dos-Santos, A.; Patel, V. F.; Powers,
D.; Rose, P.; Tudor, Y.; Turci, S. M.; Welcher, A. A.; Zack, D.; Zhao, H.; Zhu, L.;
Zhu, X.; Ghiron, C.; Ermann, M.; Johnston, D.; Saluste, C.-G. P. J. Med. Chem.
2008, 51, 1637.
11. (a) Male Sprague–Dawley rats were administered a solution of compound in
DMSO at the indicated doses iv For oral dosing, a suspension of the compound
in 1% Tween 80 and 1% HPMC in water was administered. Samples were
collected over a 12–24 h period and analyzed for parent compound by LC–MS.
(b) In vitro clearance (RLM and MLM) was not predictive of in vivo clearance,
thus compounds were chosen for PK studies based on their enzyme and
cellular potency profiles.
12. Calu-6 cell lines were obtained originally from ATCC and were maintained in
RPMI 1640 10% FBS, 1 Â NEAA, 2 mM
L-glutamine (Gibco/BRL, Grand Island,
NW). CD1 mice within approximately 4–8 weeks were challenged with
subcutaneous injections of Calu-6 (human NSCLC) cells (5 Â 106 cells per
mouse). After 1 day, animals began continuous daily treatment with
compound administered po, b.i.d at the indicated dose levels in 1% Tween 80
and 1% HPMC in water (3) or in 20% Captisol in PBS at pH 3.5 (26). Tumor
volumes as established by caliper measurements were recorded twice per
week, along with body weights as an index of toxicity. Blood glucose levels
were recorded before dosing and once per week. Data are expressed as mean
4. Armistead, D. M.; Bemis, J. E.; Buchanan, J. L.; DiPietro, L. V.; Elbaum, D.;
Habgood, G. J.; Kim, J. L.; Marshall, T. L.; Geuns-Meyer, S. D.; Novak, P. M.;
Nunes, J. J.; Patel, V. F.; Toledo-Sherman, L. M.; Zhu, X. WO 01/25220.
5. All compounds were screened in a homogeneous time-resolved fluorescence
(HTRF) kinase assay at the apparent Km of ATP with respect to 1 lM of peptide
substrate.
values plus or minus standard errors as
a function of time. Statistical
significance of observed differences was evaluated by repeated measures