A novel microtubule destabilizing entity from orthogonal synthesis of triazine library and zebrafish embryo screening

Article abstract of DOI:10.1021/ja026720i

The first orthogonal combinatorial synthesis of a high-purity triazine library was demonstrated. Novel triazine-based microtubule inhibitors were discovered by an efficient zebrafish embryo screening and in vitro microtubule polymerization assay. Copyrigh

Full text of DOI:10.1021/ja026720i

Published on Web 09/10/2002
A Novel Microtubule Destabilizing Entity from Orthogonal Synthesis of
Triazine Library and Zebrafish Embryo Screening
Ho-Sang Moon,† Eric M. Jacobson,‡ Sonya M. Khersonsky,† Michael R. Luzung,† Daniel P. Walsh,†
Wennan Xiong,† Jae Wook Lee,† Puja B. Parikh,† Jennifer C. Lam,† Tae-Wook Kang,†
Gustavo R. Rosania,*,§ Alexander F. Schier,*,‡ and Young-Tae Chang*,†
Department of Chemistry, New York UniVersity, New York, New York 10003, Skirball Institute of Biomolecular
Medicine, Department of Cell Biology, New York UniVersity School of Medicine, New York, New York 10016, and
Department of Pharmaceutics, College of Pharmacy, UniVersity of Michigan, Ann Arbor, Michigan 48109
Received May 2, 2002
To overcome the nonspecific toxicities of current chemothera-
peutic and radiation therapies, influencing microtubule functionality
has become a widespread strategy in the design of anticancer drugs.1
The recently characterized myoseverin, a tubulin binder with a novel
purine structure, has demonstrated promising ability in surmounting
the major problems of currently known tubulin binding drugs.2 The
moderate activity of myoseverin (low µM IC50) remains to be
Figure 1. Structure of myoseverin and triazine library structures.
improved through a thorough structure-activity relationships (SAR)
Scheme 1a
study.
To accelerate the development of an optimized myoseverin-like
anti-tubulin drug, we designed a novel triazine library utilizing
computer aided modeling and known SAR data from myoseverin
derivatives (Figure 1). The triazine scaffold has three-fold sym-
metry, and therefore positional modification is much more flexible
than in purine.
Triazine has elicited considerable interest as an ideal combina-
torial library scaffold due to its ease of manipulation and the low
price of the starting material, resulting in the publication of several
triazine libraries in the literature.3 All of the reported library
synthesis procedures utilize the reactivity differences of the three
reaction sites. This stepwise amination approach, however, was
difficult to generalize for a variety of nucleophiles with various
reactivities; thus, each reaction step may accumulate byproducts
yielding impure library compounds at the last stage. Herein, we
report a new straightforward orthogonal synthetic pathway, which
can be used in the general preparation of a trisubstituted triazine
library (Scheme 1).
In this new route, three types of building blocks were prepared
separately and assembled by orthogonal reactions. The building
blocks are (i) PAL-resin bound primary amine, (ii) amine, alcohol,
or thiol attached (at 2 position) 4,6-dichloro-[1,3,5]triazine, and (iii)
a series of primary and secondary amines. For Building block I,
primary amines including a variety of benzylamine were attached
to PAL aldehyde resins using the reductive amination reaction.
Building block II especially accommodated alkyl/aryl alcohols,
bulky amines, and anilines with low nucleophilic reactivity, utilizing
the high reactivity of the first substitution reaction on triazine
trichloride. Building block III is composed of many different
primary and secondary amines, but not anilines due to their low
reactivity.
a (a) R1NH2 (5 equiv), 2% acetic acid in THF, room temperature, 1 h,
followed by NaB(OAc)3H (7 equiv), room temperature, 12 h. (b) R2R2′NH2,
R2OH, or R2SH (1 equiv) in THF, 0 °C, 1 h. (c) Building block II (4 equiv),
DIEA (4 equiv) in THF, 60 °C, 3 h. (d) R3R3′NH (4 equiv), DIEA (4 equiv)
in NMP:n-BuOH ) 1:1, 120 °C, 3 h. (e) 5% TFA in DCM, 10 min.
compounds that share a structural motif similar to that of myo-
severin. The purity and identity of all of the products were
monitored by LC-MS, and more than 90% of the compounds
demonstrated >98% purity.
The synthesized triazine library compounds were first screened
using zebrafish embryos.4 A series of compounds induced very
similar morphological changes as the known microtubule disrupters
myoseverin and nocodazole at 10 µM concentration. 1K cell stage
embryos (3 h post fertilization: 3hpf), over the course of 5 h of
compound treatment, were characterized by delays in epiboly, as
well as larger blastoderm cells. Arrows mark the extent of migration
of the blastoderm over the yolk (Figure 2, upper row). Moreover,
treatment of somite stage embryos induced a characteristic devel-
opmental arrest (Figure 2, lower row).
Using this straightforward synthetic procedure with readily
available building blocks, we synthesized more than 100 triazine
To confirm that the selected compounds acted on tubulin directly,
the compounds were subjected to an in vitro polymerization assay
using purified bovine brain tubulin.5 The representative active
* To whom correspondence should be addressed. E-mail: yt.chang@nyu.edu.
† New York University.
‡ New York University School of Medicine.
§ University of Michigan.
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J. AM. CHEM. SOC. 2002, 124, 11608-11609
10.1021/ja026720i CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Morphological changes of zebrafish embryos upon treatment with different compounds. (a) upper: 1K cell stage embryo (3 hpf); lower: 17-
somite stage embryo (17 hpf). (b)-(f) After 5 h treatment of the compounds: (b) 1% DMSO, (c) nocodazole (10 µM), (d) myoseverin (10 µM), (e) 1 (active
triazine, 10 µM), (f) S79 (inactive triazine, 10 µM) in 1% DMSO/Hanks-derived buffer (scale bar, 100 µm).
Acknowledgment. We thank Dr. Victor Livengood and Dr.
Yeoun Jin Kim at the Laboratory of Bioorganic Chemistry, NIDDK,
NIH for the high resolution mass spectrometry measurements. We
also thank Dr. Natalia Glickman for her expert help with microscopy
and Young-Soo Kim for graphics. This project was supported by
the New York University Research Challenge Fund Program, and
E.M.J. was supported by an American Cancer Society postdoctoral
fellowship.
Supporting Information Available: Full experimental procedures
and characterization data (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 3. Structures of the representative active compounds (1-3) and
inactive compound (S79).
References
(1) (a) von Angerer, E. Curr. Opin. Drug DiscoVery DeV. 2000, 3, 575-584.
Table 1. In Vitro Microtubule Disassembly and Growth Inhibition
of U937 Cells by New Triazine Compounds and Known Inhibitors
(b) Li, Q.; Sham, H. L.; Rosenberg, S. H. Annu. Rep. Med. Chem. 1999,
34, 139-148. (c) Altmann, K.-H. Curr. Opin. Chem. Biol. 2001, 5, 424-
431.
MIC of tubulin polymerization (µM)a
GI50 in U937 (µM)
(2) (a) Rosania, G. R.; Chang, Y. T.; Perez, O.; Sutherlin, D.; Dong, H.;
Lockhart, D. J.; Schultz, P. G. Nat. Biotechnol. 2000, 18, 304-308. (b)
Chang, Y. T.; Wignall, S. M.; Rosania, G. R.; Gray, N. S.; Hanson, S.
R.; Su, A. I.; Merlie, J., Jr.; Moon, H. S.; Sangankar, S. B.; Perez, O.;
Heald, R.; Schultz, P. G. J. Med. Chem. 2001, 44, 4497-4500.
nocodazole
myoseverin
1
2
3
10
20
10
20
5
0.05
10
9
5
1
(3) (a) Gustafson, G. R.; Baldino, C. M.; O’Donnel, M. E.; Sheldon, A.; Tarsa,
R. J.; Verni, C. J.; Coffen, D. L. Tetrahedron 1998, 54, 4051-4065. (b)
Scharn, D.; Wenschuh, H.; Reineke, U.; Schneider-Mergener, J.; Germ-
eroth, L. J. Comb. Chem. 2000, 2, 361-369. (c) Johnson, C. R.; Zhang,
B.; Fantauzzi, P.; Hocker, M.; Yager, K. M. Tetrahedron 1998, 54, 4097-
4106. (d) Stankova, M.; Lebl, M. Mol. DiVersity 1996, 2, 75-80. (e) Silen,
J. L.; Lu, A. T.; Solas, D. W.; Gore, M. A.; MacLean, D.; Shah, N. H.;
Coffin, J. M.; Bhinderwala, N. S.; Wang, Y.; Tsutsui, K. T.; Look, G. C.;
Campbell, D. A.; Hale, R. L.; Navre, M.; DeLuca-Flaherty, C. R.
Antimicrob. Agents Chemother. 1998, 42, 1447-1453. (f) Teng, S. F.;
Sproule, K.; Hussain, A.; Lowe, C. R. J. Mol. Recognit. 1999, 12, 67-
75. (g) Filippusson, H.; Erlendsson, L. S.; Lowe, C. R. J. Mol. Recognit.
2000, 13, 370-381. (h) Masquelin, T.; Meunier, N.; Gerber, F.; Rosse,
G. Heterocycles 1998, 48, 2489-2505.
(4) (a) Peterson, R. T.; Link, B. A.; Dowling, J. E.; Schreiber, S. L. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 12965-12969. (b) Jesuthasan, S. J. Cell
Sci. 1998, 111, 3695-3703. (c) Ikegami, R.; Zhang, J.; Rivera-Bennetts,
A. K.; Yager, T. D. Zygote 1997, 5, 329-350. (d) Jesuthasan, S.; Stahle,
U. Curr. Biol. 1997, 7, 31-42. (e) Solnica-Krezel, L.; Driever, W.
DeVelopment 1994, 120, 2443-2455.
a MIC: minimum inhibitory concentration.
compounds (shown in Figure 3) were also tested in U937 human
leukemia cells to measure their cell growth inhibition (Table 1).6
While compound 1 and 2 showed activities comparable to that of
myoseverin, the best compound, 3, demonstrated at least a 4-10-
fold improvement in tubulin assays and U937 cell growth inhibition.
In conclusion, we have developed a straightforward solid phase
synthetic pathway for a highly pure trisubstituted triazine library,
and through zebrafish embryo screening we have identified
members of this library that have novel anti-tubulin activities in
vitro and in vivo. This high-quality triazine library will ultimately
provide a rich source of biologically active synthetic molecules,
just as its relative purine libraries have. Diversity extension of the
triazine library and its application to the development of better
derivatives and other biological screening are currently being
studied.
(5) Belmont, L. D.; Mitchison, T. J. Cell 1996, 84, 623-631.
(6) Rosania, G. R.; Merlie, J., Jr.; Gray, N.; Chang, Y. T.; Schultz, P. G.;
Heald, R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4797-4802.
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