significant improvement in the potency, and the result indicated
the importance of this interaction in the hydrophobic region of
ATP binding pocket, since 2-amino-1,3,5-triazine was reported to
have 10 mM of Kd values using surface Plasmon resonance
(SPR)-based binding assay.14
of Hsp90 client proteins of Her2, Met and Akt, and upregulated
Hsp70 expression level. Computational modeling indicated that
2-amino-4-chloro-1,3,5-triazine moiety of 3b bound to the
hydrophilic pocket of Hsp90 through hydrogen bonding, and 2,6-
dimethylphenyl moiety of 3b reached the hydrophobic region of
Hsp90 for Van der Waals interactions. Further SAR efforts of 6-
phenoxy-1,3,5-triazines are currently underwent to improve the
efficacy and the pharmacokinetic properties and the result will be
reported in due course.
To predict the binding pose of 3b in the N-terminal domain of
Hsp90, the inhibitor 3b was docked to Hsp90 using Autodock
4.2. The crystal structure of Hsp90 (PDB code: 3QDD) in
complex with BIIB021 was selected as the receptor model for the
docking of 3b. After removing the ligand of BIIB021 and solvent
molecules, molecular docking of 3b was carried out in the ATP-
binding site of N-terminal Hsp90. The docking study revealed
that compound 3b fits the ATP binding pocket in a similar
fashion as BIIB021. 2-Amino-4-chloro-triazine ring and 2,6-
dimethylphenyl ring of 3b were positioned in the hydrophilic and
the hydrophobic pocket of Hsp90, respectively (Fig. 5a).
Acknowledgments
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (2011-0023605), and by College of Pharmacy-
specialized Research Fund (from institute for new drug
development) of Keimyung University in 2012
Supplementary data
References and notes
1.
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Figure 5. Comparative binding modes of 3b and BII021. (a) Docking model
of 3b in the N-terminal of human Hsp90. (b) X-ray co-crystal structure of
Hsp90 with BIIB021 (PDB code: 3QDD). The carbon atoms of 3b and
BIIB021 are shown in yellow and pink, respectively. The oxygen, nitrogen,
hydrogen, and chlorine atoms of 3b and BIIB021 are shown in red, blue,
gray, and green, respectively. The side chains of Hsp90’s binding site are
colored by atom types (carbon, gray; nitrogen, red; sulfur, yellow) and
labeled with their residue name.
As expected, the amino group at 2-position of 1,3,5-triazine
interacted with Asp93 by hydrogen bonding and the hydrogen
bonding distance between the hydrogen atom at amino group and
the oxygen atom of Asp93 was calculated in 1.9Å. Despite 2,6-
dimethylphenyl moiety of 3b was located in the hydrophobic
cavity of Hsp90 and formed proximal contacts with Phe138,
Leu107 and Val150, the interactions between 2,6-
dimethylphenyl group and Phe138 was modest. The angle and
the distance between 1,3,5-triazine and 2,6-dimethylphenyl ring
didn’t seem to be optimally shaped so as to negate the proximal
contact between 2,6-dimethylphenyl group and Phe138. That
may explain the modest anti-proliferative effect of 3b against
H1975. Collectively, the hydrogen bonding of triazine ring and
Van der Waals interactions of phenyl ring contributed to the
binding of 3b to ATP binding pocket of Hsp90 and the estimated
binding energy (ΔGb) and inhibition constants (Ki) of 3b using
the Lamarckian genetic algorithm result in -6.78 kcal/mol and
10.71 M, respectively.
12. Chiosis, G.; Timaul, M. N.; Lucas, B.; Munster, P. N.; Zheng, F.
F.; Sepp-Lorenzino, L.; Rosen, N. Chem Biol 2001, 8, 289.
13. Neckers, L.; Workman, P. Clin Cancer Res 2012, 18, 64.
14. Miura, T.; Fukami, T. A.; Hasegawa, K.; Ono, N.; Suda, A.;
Shindo, H.; Yoon, D. O.; Kim, S. J.; Na, Y. J.; Aoki, Y.; Shimma,
N.; Tsukuda, T.; Shiratori, Y. Bioorg Med Chem Lett 2011, 21,
5778.
15. Shi, J.; Van de Water, R.; Hong, K.; Lamer, R. B.; Weichert, K.
W.; Sandoval, C. M.; Kasibhatla, S. R.; Boehm, M. F.; Chao, J.;
Lundgren, K.; Timple, N.; Lough, R.; Ibanez, G.; Boykin, C.;
Burrows, F. J.; Kehry, M. R.; Yun, T. J.; Harning, E. K.;
Ambrose, C.; Thompson, J.; Bixler, S. A.; Dunah, A.; Snodgrass-
Belt, P.; Arndt, J.; Enyedy, I. J.; Li, P.; Hong, V. S.; McKenzie,
A.; Biamonte, M. A. J Med Chem 2012, 55, 7786.
16. Kim, J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.;
Chiosis, G. J Biomol Screen 2004, 9, 375.
17. Procedure for the synthesis of 3b: A mixture of cyanuric chloride
(4) (0.5 g, 4.1 mmol), 2,6-xylenol (0.75 g, 4.1 mmol), and 2,6-
lutidine (0.53 g, 4.9 mol) in acetone was stirred for 12 h at rt. The
solvent was removed under reduced pressure, and the remaining
residue was dissolved in ethyl acetate. The organic layer was
washed with water, dried over Na2SO4, and concentrated under
reduced pressure to give compound 10b, quantitatively.
Compound 10b was used in the following step without further
purification. A mixture of 10b (0.4 g, 1.5 mmol) and ammonium
hydroxide (0.68 g, 11.2 mmol) in acetone was stirred for 12 h at rt.
The solvent was removed under reduced pressure, and the
remaining residue was purified by silica gel column
chromatography using hexane-ethyl acetate (4:1) as eluent, to give
compound 3b in 89% yield. 1H NMR (400 MHz, DMSO-d6)
(ppm) 2.07 (s, 6H), 7.07-7.15 (m, 3H), 8.10 (br s, 1H), 8.13 (br s,
1H); 13C NMR (100 MHz, DMSO-d6) (ppm) 16.43, 126.30,
129.17, 130.26, 149.14, 168.74, 170.10, 170.83; ESI-MS (m/e) =
251 ([M+1]+)
In summary, we rationally designed and synthesized a series
of 1,3,5-triazine compounds. To create additional hydrophobic
interactions in ATP binding pocket of Hsp90, we expanded 2-
amino-4-chloro-1,3,5-triazine structure by adding substituents of
benzyloxy, phenylamino and phenoxy groups at 6-postion of 2-
amino-4-chloro-1,3,5-triazine.
The
biological
assays
demonstrated that the phenoxy-triazine was the most effective
substituent to inhibit Hsp90 chaperoning machinery among
others. The cell viability assay indicated that 3b has an anti-
proliferative activity against H1975. Western blot analyses
revealed that 3b dose-dependently downregulated the expression