5868
S. Lin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5864–5868
Dodd, J.; Barrish, J. C.; Schieven, G. L.; Leftheris, K. Bioorg. Med. Chem. Lett. 2008,
18, 2739.
shorter than the sum of the van der Waals radii of nitrogen and sul-
fur atoms (3.35 Å) indicative of a stabilizing nonbonding interac-
tion. Furthermore, the less potent pyrimidine isomer 43 also
crystallized in a conformation having a short nitrogen-to-sulfur
contact distance of 2.94 Å indicating a similar nonbonding interac-
tion. However, in the case of 43, this conformation is not the pre-
ferred conformation for binding to p38, and as such, provides an
explanation for the decreased potency observed for this isomer.
In conclusion, we have designed and synthesized 2-amin-
othiazol-5-yl-pyrimidines as a novel series of potent and selective
4. (a) Burling, F. T.; Goldstein, B. M. Acta Crystallogr., Sect. B 1993, 49, 738; (b)
Haginoya, N.; Kobayashi, S.; Komoriya, S.; Yoshino, T.; Suzuki, M.; Shimada, T.;
Watanabe, K.; Hirokawa, Y.; Furugori, T.; Nagahara, T. Journal Med. Chem 2004,
47, 5167; (c) Hayashi, K.; Ogawa, S.; Sano, S.; Shiro, M.; Yamaguchi, K.; Sei, Y.;
Nagao, Y. Chem. Pharm. Bull. 2008, 56, 802.
5. General procedure for borylation: To a solution of tert-butyl 5-bromothiazol-2-
yl(isopropyl)carbamate (1.87 g, 5.79 mmol) in THF at ꢀ78 °C was added n-BuLi
(2.5 M in hexane, 6.37 mL, 6.36 mmol) dropwise. After stirring at ꢀ78 °C for
30 min, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was added and
stirred at ꢀ78 °C for 1 h and rt for 2 h. The reaction mixture was quenched with
addition of satd aq NH4Cl. The THF was removed under vacuum and the aqueous
portion was extracted with EtOAc (40 mL ꢁ 3). The combined organics were
washed with brine, dried over Na2SO4, filtered and concentrated to afford 2.06 g
(96%) as a light yellow solid. 1H NMR (400 MHz, CDCl3) d ppm 7.86 (1H, s), 5.23
ꢀ5.49 (1H, m), 1.59 (9H, s), 1.44 (6H, d, J = 6.61 Hz), 1.32 (12H, s).
p38
a
inhibitors. These SAR efforts were performed with the aim of
inhibitors having a suitable
identifying structurally unique p38
a
balance of potency and physicochemical properties and were
guided by structure-based design principles leading to the discov-
ery of 41 as a promising analog for further investigation. Highlights
of these efforts also include the use of a novel 2-aminothiazole-5-
boronate ester intermediate to enable a highly modular synthesis
of the targeted compounds and the use of a unique intramolecular
nitrogen–sulfur nonbonding interaction to stabilize the conforma-
6. Experimental procedure for p38
bottomed 96-well plates. The final assay volume was 60
three 20 L additions of enzyme, substrates (MBP and ATP) and test compounds
in assay buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 50 mM NaCl and 1 mM DTT).
Bacterially expressed, activated p38 was pre-incubated with test compounds
for 10 min prior to initiation of reaction with substrates. The reaction was
incubated at 25 °C for 45 min and terminated by adding 5 L of 0.5 M EDTA to
a
assay: The assays were performed in V-
lL prepared from
l
a
l
each sample. The reaction mixture was aspirated onto a pre-wet filtermat using
a Skatron Micro96 Cell Harvester (Skatron, Inc.), then washed with PBS. The
filtermat was then dried in a microwave oven for 1 min, treated with MeltilLex A
scintillation wax (Wallac), and counted on a Microbeta scintillation counter
Model 1450 (Wallac). Inhibition data were analyzed by nonlinear least-squares
regression using Prism (GraphPadSoftware). The final concentration of reagents
tion required for binding to p38
graphic data.
a as supported by X-ray crystallo-
Acknowledgement
in the assays are ATP, 1
p38, 10 nM; and DMSO, 0.3%.
lM; [c- lg/well;
33P]ATP, 3 nM; MBP (Sigma, #M1891), 2
The authors thank our colleague Mr. Gerry Everlof for the deter-
mination of aqueous solubility measurements reported herein.
7. Experimental procedure for PBMC assay: Heparinized human whole blood was
obtained from healthy volunteers. Peripheral blood mononuclear cells (PBMCs)
were purified from human whole blood by Ficoll-Hypaque density gradient
centrifugation and resuspended at
medium (RPMI medium containing 10% fetal bovine serum). Fifty microliters of
a
concentration of 5 ꢁ 106/mL in assay
References and notes
cell suspension was incubated with 50
lL of test compound (4ꢁ concentration
1. (a) Adams, J. L.; Badger, A. M.; Kumar, S.; Lee, J. C. Prog. Med. Chem. 2001, 38, 1;
(b) Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.; Kumar, S.; Green, D.;
McNulty, D.; Blumenthal, M. J.; Heyes, J. R. Nature 1994, 372, 739.
in assay medium containing 0.2% DMSO) in 96-well tissue culture plates for
5 min at rt. Hundred microliters of LPS (200 ng/mL stock) was then added to the
cell suspension and the plate was incubated for 6 h at 37 °C. Following
2. For reviews of this area see: (a) Hynes, J., Jr.; Leftheris, K. Curr. Top. Med. Chem.
2005, 5, 967; (b) Goldstein, D. M.; Gabriel, T. Curr. Top. Med. Chem. 2005, 5, 1017;
(c) Diller, D. J.; Lin, T. H.; Metzger, A. Curr. Top. Med. Chem. 2005, 5, 953; (d)
Dominguez, C.; Powers, D. A.; Tamayo, N. Curr. Opin. Drug Discovery Dev. 2005, 8,
421.
incubation, the culture medium was collected and stored at ꢀ20 °C. TNF-
concentration in the medium was quantified using standard ELISA kit
(Pharmingen-San Diego, CA). Concentrations of TNF- and IC50 values for test
a
a
a
compounds (concentration of compound that inhibited LPS-stimulated TNF-
production by 50%) were calculated by linear regression analysis.
a
3. (a) Hynes, J., Jr.; Wu, H.; Pitt, S.; Shen, D. R.; Zhang, R.; Schieven, G. L.; Gillooly, K.
M.; Shuster, D. J.; Taylor, T. L.; Yang, X.; McIntyre, K. W.; McKinnon, M.; Zhang,
H.; Marathe, P. H.; Doweyko, A. M.; Kish, K.; Kiefer, S. E.; Sack, J. S.; Newitt, J. A.;
Barrish, J. C.; Dodd, J.; Schieven, G. L.; Leftheris, K. Bioorg. Med. Chem. Lett. 2008,
18, 1762; (b) Wrobleski, S. T.; Lin, S.; Hynes, J., Jr.; Wu, H.; Pitt, S.; Shen, D. R.;
Zhang, R.; Gillooly, K. M.; Shuster, D. J.; Taylor, T. L.; McIntyre, K. W.; Doweyko,
A. M.; Kish, K.; Kiefer, S. E.; Tredup, J. A.; Duke, G. J.; Sack, J. S.; McKinnon, M.;
8. Kinase selectivity data determined for 41 based on Ambit screening at 10 lM:
no activity observed (100% of control) for the following kinases: ABL1, AMPKA1,
AURA, AURC, CAMK1A, CAMK1D, CDK2, CK1E, CLK1, CSK, DAPK2, EGFR, EPHA2,
EPHB4, FGF1R, FLT3, GAK, IGF1R, INSR, KDR, MAP3K4, MARK2, MNK2, NEK2,
PAK1, PAK4, PAK5, PHKG1, PIM1, SKMLCK, SRC, STK16, SYK, TIE2, TRKA. Activity
observed as% of control: JNK1 (24.9%), JNK3 (0.2%), KIT (14.5%), PDGRB (11.3%),
p38a (0%).