Synthesis and biological evaluation of a 5-6-5 imidazole-phenyl-thiazole based α-helix mimetic

Article abstract of DOI:10.1021/ol8022962

The development of small molecules that disrupt protein-protein interactions is a key goal in addressing a number of disease states. The α-helix is commonly found at protein interaction interfaces and has been the focus of substantial small molecule mimet

Full text of DOI:10.1021/ol8022962

ORGANIC
LETTERS
2009
Vol. 11, No. 1
25-28
Synthesis and Biological Evaluation of a
5-6-5 Imidazole-Phenyl-Thiazole Based
r-Helix Mimetic
Christopher G. Cummings, Nathan T. Ross, William P. Katt,
and Andrew D. Hamilton*
Department of Chemistry, Yale UniVersity, P.O. Box 20810,
New HaVen, Connecticut 06520
andrew.hamilton@yale.edu
Received October 3, 2008
ABSTRACT
The development of small molecules that disrupt protein-protein interactions is a key goal in addressing a number of disease states. The
r-helix is commonly found at protein interaction interfaces and has been the focus of substantial small molecule mimetic efforts. One of the
primary drawbacks of many small molecule r-helix mimetics is their hydrophobic core structures. To address this problem we have developed
a novel scaffold based on a more water soluble 5-6-5 imidazole-phenyl-thiazole core. An inhibitor of this class has been shown to disrupt the
Cdc42/Dbs protein-protein interaction at micromolar concentrations and may be useful in overcoming Cdc42-induced tumor resistance to
anticancer therapies.
Protein-protein interactions have been implicated in
numerous disease states including HIV and cancer, making
them important targets for disruption by small molecules.1
R-Helices are common protein secondary structure elements
and are often responsible for recognition between proteins
via one helical face where interacting residues occupy
predominantly the i, i + 3 or i + 4, and i + 7 positions.
Accordingly, these surfaces are critical targets for small
molecule mimicry. In an effort to disrupt R-helix mediated
protein-protein interactions, we have reported several small
molecules that act as structural and functional mimetics of
R-helices.2 Often the binding hot spots targeted by R-helix
mimetics lie in hydrophobic clefts that are solvent-exposed
when the two interacting proteins are not associated.3 We
have previously focused on scaffolds that rely on six-
membered rings (either aromatic or hydrogen-bonded) to
maintain their proper orientation.1 Earlier R-helix mimetics,
such as those based on terpyridine2a and terphenyl2b scaffolds
are inherently disadvantaged because of poor solubility,
whereas others based on terephthalamide2c and trispyri-
dylamide2d scaffolds, rely on hydrogen-bonding networks
to maintain their correct orientation. Recent work by Rebek
and Ko¨nig has begun to address the demand for more water
soluble R-helix mimetics, by making one side of the scaffold
(1) For a review, see: Davis, J. M.; Tsou, L. K.; Hamilton, A. D. Chem.
Soc. ReV. 2007, 36, 326–334.
(2) (a) Davis, J. M.; Truong, A.; Hamilton, A. D. Org. Lett. 2005, 7,
5405–5408. (b) Yin, H.; Lee, G. I.; Sedey, K. A.; Kutzki, O.; Park, H. S.;
Omer, B. P.; Ernst, J. Y.; Wang, H. G.; Sebti, S. M.; Hamilton, A. D. J. Am.
Chem. Soc. 2005, 127, 10191–10196. (c) Yin, H.; Lee, G. I.; Sedey, K. A.;
Rodriguez, J. M.; Wang, H. G.; Sebti, S. M.; Hamilton, A. D. J. Am. Chem.
Soc. 2005, 127, 5463–5468. (d) Ernst, J. T.; Becerril, J.; Park, H. S.; Yin,
H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2003, 42, 535–539.
(3) Fletcher, S.; Hamilton, A. D. J. R. Soc. Interface 2006, 3, 215–233.
10.1021/ol8022962 CCC: $40.75
Published on Web 11/26/2008
2009 American Chemical Society

hydrophilic4a,b or via a 1,4-dipiperazino benzene scaffold.4c
Our design focuses on two variations of a novel 5-6-5
imidazole-phenyl-thiazole scaffold that replaces six-mem-
bered aromatic end units with water-soluble five-membered
heterocyclic groups.
Scheme 1. Synthesis of Model 5-6-5 Scaffold 4
Using this novel class of R-helix mimetics, we have chosen
to target a protein-protein interaction governed largely by
hydrophilic contacts, that between Cdc42 (cell division cycle
42) and Dbs (Dbl’s big sister).5 Cdc42 is a GTPase (guanine
nucleotide triphosphatase) shown to mediate cancer cell
resistance to both cytotoxic therapies and cytotoxic T-
lymphocyte induced tumor suppression.6 Cdc42 has also been
linked to diabetes and cardiovascular and neurodegenerative
diseases.7 However, Cdc42 is only capable of these aberrant
activites in its activated, GTP-bound, form.7 Cdc42 is
activated by interaction with the GEF (guanine nucleotide
exchange factor), Dbs. We chose to target the Cdc42-Dbs
interaction in an effort to block the most upstream event
involved in Cdc42-related disease. In addition, to the best
of our knowledge, no rationally designed small molecule
inhibitors of this protein-protein interaction currently exist.
The interaction between these proteins is mediated by the
Q770, K774, and L777 residues of Dbs, which correspond
to the i, i + 4, and i + 7 of a key Dbs R-helix, making it an
appropriate target for our mimetics (Figure 1c).5 Herein we
Scheme 1 depicts the synthetic route to a model derivative,
4-methyl-2-(2-methyl-4-(2-methyl-1H-imidazol-1-yl)phe-
Scheme 2. Synthesis of 12
Figure 1. (a) Energy minimized polyalanine R-helix displaying i,
i + 4, and i + 7 positions. (b) Energy minimized structure of 4.
(c) Overlay of energy minimized 4 onto targeted region of Dbs,
residues Q770, K774, and L777.
detail the design and synthesis of a 5-6-5 imidazole-phenyl-
thiazole based, water soluble R-helix mimetic of these key
residues.
(4) (a) Volonterio, A.; Moisan, L.; Rebek, J., Jr Org. Lett. 2007, 9, 3733–
3736. (b) Biros, S. M.; Moisan, L.; Mann, E.; Carella, A.; Zhai, D.; Reed,
J. C.; Rebek, J., Jr Bioorg. Med. Chem. Lett. 2007, 17, 4641–4645. (c)
Maity, P.; Ko¨nig, B. Org. Lett. 2008, 10, 1473–1476.
(5) Rossman, K. L.; Worthylake, D. K.; Snyder, J. T.; Siderovski, D. P.;
Campbell, S. L.; Sondek, J. EMBO J. 2002, 21, 1315–1326.
(6) Marques, C. A.; Hahnel, P. S.; Wolfel, C.; Thaler, S.; Huber, C.;
Theobald, M.; Schuler, M. Blood 2008, 111, 1413–1419.
(7) Sinha, S.; Yang, W. Cell. Signalling 2008, 20, 1927–1934.
(8) Kouwer, P. H. J.; Swager, T. M. J. Am. Chem. Soc. 2007, 129,
14042–14052.
(9) Rudolph, J.; Chen, L.; Majumdar, D.; Bullock, W. H.; Burns, M.;
Claus, T.; Dela Cruz, F. E.; Daly, M.; Ehrgott, F. J.; Johnson, J. S.;
Livingston, J. N.; Schoenleber, R. W.; Shapiro, J.; Yang, L.; Tsutsumi,
M.; Ma, X. J. Med. Chem. 2007, 50, 984–1000.
26
Org. Lett., Vol. 11, No. 1, 2009

nyl) thiazole (4), with methyl groups in the key substituent
positions. Treatment of commercially available 1 with
phosphorus pentasulfide gave thioamide 2, which after
heating with chloroacetone furnished thiazole 3.8 Ullman
coupling with 2-methylimidazole gave 4 in excellent yield.9
A crystal structure of 4 (Figure 2) confirmed the extended
key functional groups. A benzimidazole diacid replaced the
flexibly substituted imidazole, and a 2-substituted tolyl group
was used in place of the isopropyl ether. Although it has a
lower pKa, 3-substituted aniline was used to rigidify the alkyl
amine group in 12 with the expectation that the arylamine
would still participate in hydrogen bonding to Cdc42 active
site residues. The synthetic route (Scheme 3) involves
Scheme 3. Synthesis of 19
Figure 2. Crystal structure of 4 in stereoview.
shape of the molecule and the staggered projections in a
nonplanar orientation (5-6-5 dihedral angles, 43.0° and
38.2°). The log P (o/w) of the corresponding trimethyl-
substituted terphenyl,10 terpyridine2a and 4 were calculated
as 7.3, 3.4 and 2.3, respectively, suggesting a significant
improvement in aqueous solubility of the 5-6-5 heterocyclic
system, and this was seen with 12 and 19, which were soluble
to g500 µM in 2% DMSO/buffer.11
Functionalization of the core scaffold with amino acid like
side chains similar to those of Dbs involved modification of
the synthetic route. Nucleophilic aromatic substitution of 5
with tert-butyl 3-hydroxypropylcarbamate (KHMDS in THF)
gave 6, which was coupled under Ullman conditions with
methyl 3-(1H-imidazol-2-yl)propanoate to form 7 in excellent
yield.8 Oxidation with (NH4)2S(aq) gave thioamide 8, which
on treatment with isopropyl chloroactetate formed 9.9
Saponification of 9 followed by oxidation and deprotection
gave 12 in high overall yield. The synthetic approach to 12
is flexible, allowing for the ready introduction of diverse side
chains.
Compound 12 was assayed for its ability to disrupt the
Cdc42-Dbs interaction using a gel pull-down assay that has
identified inhibitors of other GTPase/GEF pairs.12 However,
12 showed no detectable inhibitory activity, possibly due to
the highly flexible side groups.
In an attempt to reduce the flexibility of the side chains
in 12, we introduced into a second generation inhibitor design
a series of substituents based on constrained analogs of the
nucleophilic aromatic substitution of 2-bromo-4-fluoroben-
zonitrile with 14 to give N-arylbenzimidazole 15. Hantzsch
conditions with 2-bromo-1-o-tolylethanone and thioamide 16
produced thiazole 17, which was subjected to Suzuki
coupling with 3-aminophenyl boronic acid to give, after
deprotection, target 19.9,13 Diacid 19, in a nonplanar
conformation, could match the positions of the i, i + 4, and
i + 7 side chains in the key Dbs R-helix.
(10) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc. 2001,
123, 5382–5383.
Mimetic 19 was assayed for its ability to disrupt the
Cdc42-Dbs interaction using a mant-GDP fluorescence
(11) MOE: Molecular Operating EnVironment; Chemical Computing
Group: Montreal, 2007.
(12) Gao, Y.; Dickerson, J. B.; Guo, F.; Zheng, J.; Zheng, Y. Proc.
Nat. Acad. Sci. U.S.A. 2004, 101, 7618–7623.
(13) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457–2483.
(14) Hemsath, L.; Ahmadian, M. R. Methods 2005, 37, 173–182.
Org. Lett., Vol. 11, No. 1, 2009
27

based activity assay, in which the uptake of this GDP analog
into Cdc42 is monitored.14 Compound 19 was screened at
varying concentrations with 100% inhibition being defined
as the extent of mant-GDP uptake when no Dbs or inhibitor
was present.
Dbs-accelerated uptake of mant-GDP by Cdc42 was
inhibited in a dose-response fashion by 19 (Figure 3). Fitting
In summary, we have generated a novel class of R-helix
mimetics based on a 5-6-5 imidazole-phenyl-thiazole scaf-
fold. X-ray crystallographic studies confirm the core struc-
ture, which has an R-carbon rmsd of 1.014 Å between 4
and the side chain positions on a polyalanine R-helix. In
addition, to demonstrate the potential of our 5-6-5 imidazole-
phenyl-thiazole scaffold to disrupt a biologically relevant
protein-protein interaction, we have generated a mimic with
rigidified side chains that is a 67 µM inhibitor of the
Cdc42-Dbs interaction. We are currently preparing a diverse
set of rigidified mimetics and testing their affinity and
specificity for Cdc42-Dbs, as well as other members of the
Rho family of GTPases.
Acknowledgment. The authors thank the National Insti-
tutes of Health for financial support of this work (CA67771)
and our collaborators Channing Der, Adrienne Cox, Jon
Sondek (UNC), and Said Sebti (H. Lee Moffitt Cancer Center
and Research Institute) for invaluable discussions. We would
also like to thank Dr. Steven Fletcher and Dr. Patrick
Gunning for initial discussions and Dr. Christopher Incarvito
for X-ray crystallographic analysis.
Figure 3. IC50 of 19 against the Cdc42-Dbs interaction.
this data to a one-site binding model yielded an IC50 of 67 µM.
To further validate that the binding site of 19 was in fact the
Cdc42-Dbs interface, the ability of 19 to disrupt non-Dbs-
assisted uptake of mant-GDP was monitored. At 50 µM 19 less
than 10% of mant-GDP uptake was blocked, indicating that
the mechanism of action of this compound is not simply directly
interfering with mant-GDP uptake by Cdc42.
Supporting Information Available: X-ray crystallo-
graphic data, experimental procedures, characterizations of
all compounds and assay protocols, This material is available
free of charge via the Internet at http://pubs.acs.org.
OL8022962
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Org. Lett., Vol. 11, No. 1, 2009