1168
L. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1165–1168
Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.;
Table 5
Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.;
Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;
Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;
Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.
3. Pollock, P. M.; Harper, U. L.; Hansen, K. S.; Yudt, L. M.; Stark, M.; Robbins, C. M.;
Moses, T. Y.; Hostetter, G.; Wagner, U.; Kakereka, J.; Salem, G.; Pohida, T.;
Heenean, P.; Duray, P.; Kallioniemi, O.; Hayward, N. K.; Trent, J. M.; Meltzer, P.
S. Nat. Genet. 2003, 33, 19.
ADME properties of 17 and 21
Compd Clearancea Caco-2b
CYP3A4
Sol. @ pH 6.5 and 7.4d
inhibitionc
17
21
28
10
Medium >25
High >25
21,32
1,1
a
Mouse microsome clearance (ml/min/kg).
b
Caco-2-permeability classification: low (<2 Â 10À6 cm/s), medium (2–8 Â
10À6 cm/s), high (>8 Â 10À6 cm/s).
4. (a) Gorden, A.; Osman, I.; Gai, W.; He, D.; Huang, W.; Davidson, A.; Houghton, A.
N.; Busam, K.; Polsky, D. Cancer Res. 2003, 63, 3955; (b) Kuman, R.; Angelini, S.;
Czene, K.; Sauroja, I.; Hahka-Kemppinen, M.; Pyrhonen, S.; Hemminki, K. Clin.
Cancer Res. 2003, 9, 3362.
c
lM.
d
lg/mL.
5. Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004, 116,
855.
Table 6
6. (a) Samowitz, W. S.; Sweeney, C.; Herrick, J.; Albertsen, H.; Levin, T. R.;
Murtaugh, M. A.; Wolff, R. K.; Slattery, M. L. Cancer Res. 2005, 65, 6063; (b)
Riesco-Eizaguirre, G.; Gutiérrez-Martínez, P.; García-Cabezas, M. A.; Nistal, M.;
Santisteban, P. Endocr.-Relat Cancer 2006, 13, 257; (c) Houben, R.; Becker, J. C.;
Kappel, A.; Terheyden, P.; Bröcker, E. B.; Goetz, R.; Rapp, U. R. J. Carcinog. 2004,
3, 6.
Pharmacokinetic properties of 17 versus 1
Compd
AUCa
CLb
%F
Vdc
Sol. @ pH 1.2, 6.5, 7.4d
17
1
733
426
1.0
1.3
98
48
0.16
0.11
18,21,32
3,4,9
a
b
c
7. Sawyers, C. Nature 2004, 432, 294.
8. Puzanov, I.; Flaherty, K. T.; Sosman, J. A.; Grippo, J. F.; Su, F.; Nolop, K.; Lee, R. J.;
Bollag, G. Drugs Future 2011, 36, 191.
9. Exelixis has reported preliminary Phase 1 data on XL281: (a) Schwartz, G. L.;
Roberson, S.; Shen, A.; Wang, E.; Pace, L.; Dials, H.; Mendelson, D.; Shannon, P.;
Gordon, M. J. Clin. Oncol. 2009, 27, 3513. 15sAbstr; GSK has reported phase 1/2
data on dabrafenib, GSK2118436: (b) Kefford, R.; Arkenau, H.; Brown, M. P.;
Millward, M.; Infante, J. R.; Long, G. V.; Ouellet, D.; Curtis, M.; Lebowitz, P. F.;
Falchook, G. S. J. Clin. Oncol. 2010, 28, 8503. 15sAbstr.
Mouse PO PK at 30 mg/kg (lM h).
Mouse IV PK at 2.5 mg/kg (ml/min/kg).
L/kg.
d
lg/mL.
result of the negative impact of increased molecular weight and
lipophilicity (ClogP = 3.1).
Based on optimal combination of activity and physiochemical
properties, 17 was advanced into mouse pharmacokinetic studies
(Table 6). Similar to 1, 17 exhibited low clearance and low volume
of distribution. However, an oral dose of 30 mg/kg (dosed as a
solution in 40/10/50, by volume, in PEG400/EtOH/H2O) delivered
a ꢀ2-fold increase in exposure and oral bioavailability (%F) in com-
parison to 1.
Furthermore, while the oral exposure of 1 as a crystalline sus-
pension in 1% methylcellulose/0.5% Tween 80 in water is fourfold
lower compared to solution dosing (30 mg/kg in mice, AUC of
105 vs 426), the oral exposure of 17 was equivalent to solution
dosing when dosed as a crystalline suspension in the same suspen-
sion formula (30 mg/kg in mice, AUC of 844 vs 733). These
improvements can be attributed to the improved aqueous solubil-
ity of 17.
In summary, we have utilized a pyrazolo[1,5-a]pyrimidine core
to produce B-Raf inhibitors with excellent potency and selectivity
profiles. Optimization led to the identification of compound 17, a
potent, selective and orally available B-Raf inhibitor with favorable
physiochemical and pharmacokinetic properties. These improve-
ments made it feasible to use crystalline suspensions for efficacy
and safety evaluations without relying on enabling vehicles, such
as amorphous spray-dried dispersion. Further progress on these
inhibitors will be reported in due course.
10. (a) Takle, A. K.; Brown, M. J. B.; Davies, S.; Dean, D. K.; Francis, G.; Gaiba, A.;
Hird, A. W.; King, F. D.; Lovell, P. J.; Naylor, A.; Reith, A. D.; Steadman, J. G.;
Wilson, D. M. Biorg. Med. Chem. Lett. 2006, 16, 378; (b) Hansen, J. D.; Grina, J.;
Newhouse, B.; Welch, M.; Topalow, G.; Littman, M.; Callego, M.; Gloor, G.;
Martinson, M.; Laird, E.; Brandhuber, B. J.; Vigers, G.; Morales, T.; Woessner, R.;
Randolph, N.; Lyssikatos, J.; Olivero, A. Biorg. Med. Chem. Lett. 2008, 18, 4692;
(c) Tang, J.; Hamajima, T.; Nakano, M.; Sato, H.; Dickerson, S. H.; Lackey, K. E.
Biorg. Med. Chem. Lett. 2008, 18, 4610; (d) Smith, A. L.; DeMorin, F. F.; Paras, M.
A.; Huang, Q.; Petkus, J. K.; Doherty, E. M.; Nixey, T.; Kim, J. L.; Whittington, D.
A.; Epstein, L. F.; Lee, M. R.; Rose, M. J.; Babij, C.; Fernando, M.; Hess, K.; Le, Q.;
Beltran, P.; Carnahan, J. J. Med. Chem. 2009, 52, 6289.
11. (a) Wenglowsky, S.; Ren, L.; Ahrendt, K. A.; Laird, E. R.; Aliagas, I.; Alicke, B.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Feng, B.; Gloor, S. L.; Gould, S. E.; Gross,
S.; Gunzner-Toste, J.; Hansen, J. D.; Hatzivassiliou, G.; Liu, B.; Malesky, K.;
Mathieu, S.; Newhouse, B.; Raddatz, N. J.; Ran, Y.; Rana, S.; Randolph, N.; Risom,
T.; Rudolph, J.; Savage, S.; Selby, L. T.; Shrag, M.; Song, K.; Sturgis, H. L.; Voegtli,
W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.-I.; Young, W. B.; Grina, J.
ACS Med. Chem. Lett. 2011, 2, 342; (b) Wenglowsky, S.; Ahrendt, K. A.;
Buckmelter, A. J.; Feng, B.; Gloor, S. L.; Gradl, S.; Grina, J.; Hansen, J. D.; Laird, E.
R.; Lunghofer, P.; Mathieu, S.; Moreno, D.; Newhouse, B.; Ren, L.; Risom, T.;
Rudolph, J.; Seo, J.; Sturgis, H. L.; Voegtli, W. C.; Wen, Z. Biorg. Med. Chem. Lett.
2011, 21, 5533.
12. Vasconcelos, T.; Sarmento, B.; Costa, P. Drug Discovery Today 2007, 12, 1068.
13. Wenglowsky, S.; Moreno, D.; Rudolph, J.; Ran, Y.; Ahrendt, K. A.; Arrigo, A.;
Colsen, B.; Gloor, S. L.; Hasting, G. Biorg. Med. Chem. Lett. 2011. doi:10.1016/
14. Inhibitor enzyme activity was determined utilizing full-length B-RafV600E
.
Inhibition of basal ERK phosphorylation in Malme-3 M cells was used as the
mechanistic cellular assay.
15. The Maestro program was used to generate and manually position the
structure into the X-ray coordinates of B-Raf in complex with
a
pyrazolopyridine analog,11 followed by 500 steps of local minimization with
the OPLS potential function. Maestro Version 9.0, Schrodinger, LLC; Portland
OR.
Acknowledgments
16. The conformation constraining the lone pairs in a syn orientation resides
3.6 kcal/mol higher in energy than the anti, as computed using the 6-31G(d)
basis set. GAMESS version 24: Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.;
Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K.
A.; Su, S. J.; Windus, T. L.; Dupuis, M.; Montgomery, J. A. J. Comput. Chem. 1993,
14, 1347.
17. Hatzivassiliou, G.; Song, K.; Yen, I.; Brandhuber, B. J.; Anderson, D. J.; Alvarado,
R.; Ludlam, M. C.; Stokoe, D.; Gloor, S. L.; Vigers, G.; Morales, T.; Aliagas, I.; Liu,
B.; Siberis, S.; Hoeflich, K. P.; Jaiswal, B. S.; Seshagiri, S.; Koeppen, H.; Belvin, M.;
Friedman, L. S.; Malek, S. Nature 2010, 464, 431.
The authors thank Susan Rhodes, Jennifer Otten and Ben Colsen
for Caco-2, CYP P450 inhibition and solubility determinations. The
authors also thank Dr. Steve Wenglowsky for critical review of the
manuscript and helpful suggestions.
References and notes
18. Gradl, S.; Rudolph, J.; Ren, L. WO 2011/025951.
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