Terephthalamide derivatives as mimetics of the helical region of Bak peptide target Bcl-xL protein

Article abstract of DOI:10.1016/j.bmcl.2003.09.096

A group of novel Bcl-xL/Bak antagonists, based on a terephthalamide scaffold, were designed to mimic the α-helical region of the Bak peptide. Good in vitro inhibition potencies in disrupting the Bak/Bcl-xL complex have been observed (terephthalamide 4, K<

Full text of DOI:10.1016/j.bmcl.2003.09.096

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1375–1379
Terephthalamide derivatives as mimetics of the helical region of
Bak peptide target Bcl-xL protein
Hang Yin and Andrew D. Hamilton*
Department of Chemistry, Yale University, PO Box 20810, New Haven, CT06511, USA
Received 12 August 2003; accepted 15 September 2003
Abstract—A group of novel Bcl-xL/Bak antagonists, based on a terephthalamide scaffold, were designed to mimic the a-helical
region of the Bak peptide. Good in vitro inhibition potencies in disrupting the Bak/Bcl-xL complex have been observed
(terephthalamide 4, Ki=0.78ꢀ0.07 mM).
# 2004 Elsevier Ltd. All rights reserved.
Proteins in the Bcl-2 family play a critical role in deter-
mining the fate of a cell through the process of apopto-
sis.1 Many oncogenic mutations, particularly those to
p53, result in defects in DNA damage-induced apopto-
sis through a Bcl-2 dependent mechanism.2 In addition,
overexpression of Bcl-2 can inhibit the potency of many
currently available anticancer drugs by blocking the
apoptotic pathway.3 Therefore, agents that directly
mimic the death-promoting region BH3 domain of the
pro-apoptotic subfamily of Bcl-2 proteins4 are of
potential therapeutic value.
Bcl-xL/Bak antagonists based on a terephthalamide
scaffold, designed to mimic the a-helical region of the
Bak peptide. Using a fluorescence polarization assay,
we have observed high in vitro inhibition potencies in
disrupting the Bcl-xL/Bak complex and a significant
improvement in water solubility relative to the terphenyl
derivatives.
The goal of our design was to maintain the similarity
between the arrangement of the i, i+4, i+7 side chains
of an a-helix and the substituents on the 3, 20, 20-posi-
tions on terphenyl 1,7 while minimizing the structural
complexity and increasing the solubility of the inhibi-
tors. This strategy of simplifying a proven proteomi-
metic was accomplished by using terephthalamide 2 as
the scaffold. The flanking phenyl rings in 1 were
replaced by two functionalized carboxamide groups,
which also retain a planar geometry due to the restricted
rotation of the amide bonds. Figures 1A and B show,
respectively, superimpositions of energy-minimized 2 on
1 (R1=R2=R3=Me) with an RMS deviation of 0.34 A
and on the i, i+4, i+7 side chains of an a-helix with an
RMS deviation of 1.03 A, suggesting good stereo-
chemical similarity between both pairs.8
The NMR-derived structure of the Bcl-xL/Bak BH3
domain complex indicates that the Bak peptide is an
amphipathic a-helix that interacts with Bcl-xL by pro-
jecting hydrophobic side chains (Val74, Leu78, Ile81 and
Ile85) on one face of the a helix, into a hydrophobic cleft
of Bcl-xL.5 Several low molecular weight inhibitors of
Bcl-2/Bcl-xL have been reported with the majority
showing potency in the low mM range.6 An alternative
approach for identifying inhibitors is to design synthetic
scaffolds that reproduce structural features of the BH3
helix region. We have previously reported function-
alized terphenyls based on 1 as mimetics of the dis-
continuous binding epitopes of BH3.7 However, the
hydrophobicity of the terphenyls and their challenging
syntheses prompted us to search for simpler scaffolds
that could similarly mimic the side chain presentation
on an a-helix. Herein, we report a group of novel
A modular synthesis of terephthalamide derivative 4 is
shown in Scheme 1. The 2-alkoxy group was introduced
by O-alkylation and the 1-carboxylic acid was installed
through Sandmeyer reaction and Stille coupling fol-
lowed by Lemieux-Johnson and PDC oxidation. The
amide bond formation steps were accomplished by
using standard coupling conditions.
* Corresponding author. Tel.: +1-203-432-5570; fax: +1-203-432-
3221; e-mail: andrew.hamilton@yale.edu
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.096

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H. Yin, A. D. Hamilton / Bioorg. Med. Chem. Lett. 14 (2004) 1375–1379
CDCl3, 298 K) and temperature (Ád=5.5 ppb/K)
dependence of the aniline proton, suggesting inter-
rather than intramolecular hydrogen bonding.
Two strategies were used to orient the interacting N-
alkyl group of the lower tertiary amide into the desired
Z-conformation. In 3, the problem was avoided by
using identical substituents on the tertiary amide nitro-
gen. In a second series of derivatives (5, Scheme 2),
steric differentiation of the substituents favored the pla-
cement of the isobutyl group in the Z-position (Fig.
3A). The conformation of 5 in solution was probed by
ROESY and NOESY 1H spectroscopy.11 NOE cross
peaks between Hb and the ortho-aryl protons were
detected, while no significant NOE effect could be seen
between Ha and the ortho-protons. However, corre-
lations corresponding to the chemical exchange of Ha
and Hb were observed in the ROESY experiment (Fig.
3B), which indicated there were two conformations of 5
existing in DMSO solution at 298 K. Furthermore, the
signals of both Ha and Hb, which are split at room
temperature, coalesced at 353 K. These combined
experimental results suggest that both Z- and E-amide
conformations are present with the Z-conformation
being favored by 72% (from NMR integration), and by
8.01 kJ/mole in water solution (from MM2 energy
minimization using Macromodel). The consequence of
these constraints is that in low energy, accessible con-
formations of 5, the three substituents project from one
face of the terephthalamide scaffold in a manner analo-
gous to the terphenyl helix mimetics (e.g., Fig. 1A).7
Figure 1. (A) The superimposition of terphenyl 1 on terephthalamide
2. (B) Stereo view of the superimposition of 2 on the i, i+4, i+7
positions of an a-helix.
Another conformational constraint in the molecule was
imposed by an intramolecular hydrogen bond between
the amide –NH and the alkoxy oxygen atom, to influ-
ence the position of the upper alkyl group (Fig. 2).9 The
intramolecular hydrogen bond was confirmed by variable
temperature NMR, which showed very little change in
the amide-NH resonance (d=8.54 ppm) on heating
(Ád=1.54 ppb/K) or changing concentration.10 As
The binding affinity of the terephthalamide molecules
for Bcl-xL was assessed by a fluorescence polarization
assay using a fluorescently labeled 16-mer Bak-peptide
(Fl-GQVGRQLAIIGDDINR-CONH2).5 Displacement
of this probe through competitive binding of the
a
comparison, 2-isopropylaminoterephthalamide 17
showed both concentration (7.36 ppm, 0.5 M in CDCl3;
6.58 ppm, 0.05 M in CDCl3; 6.46 ppm, 0.005 M in
Scheme 1. Modular synthesis of terephthalamide derivatives 3 and 4.

H. Yin, A. D. Hamilton / Bioorg. Med. Chem. Lett. 14 (2004) 1375–1379
1377
Figure 2. 1H NMR spectra (low field region) for 4 at different concentrations.
Scheme 2. Synthesis of terephthalamide derivative 5.
Figure 3. (A) Energy minimized Z- and E-isomers of 5. (B) ROESY 1H experiments showed cross peaks corresponding to chemical exchange of Ha.

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H. Yin, A. D. Hamilton / Bioorg. Med. Chem. Lett. 14 (2004) 1375–1379
terephthalamide into the hydrophobic cleft of Bcl-xL
would lead to a decrease in its fluorescence polarization,
which in turn could be related to the known affinity of
the 16-mer Bak peptide.12 A series of terephthalamides
with varied side chains was prepared. All the assays
were carried out with 10ꢁ8 to 10ꢁ4 M terephthalamide
solution in 10 mM PBS buffer (pH 7.4, 298 K) with less
than 0.1% DMSO, indicating good solubility of ter-
ephthalamides in water. Figure 4 shows that ter-
ephthalamide 3 has good affinity for Bcl-xL with a Ki
value of 0.78ꢀ0.07 mM. By screening compounds with a
range of side chains on the upper carboxamide, we
found that the isobutyl group as the upper substituent
provided the best inhibition results (3, 10, 11, 12). The
newly introduced stereogenic center in the terephthalamide
did not affect the affinity, as seen by comparing 6 and 7.
The optimal alkoxy group in the 2-position of ter-
ephthalamide was found to be isopropoxy (3, 4), which
closely mimics the size of Leu78 of the Bak peptide; both
larger (8, 9) and smaller (6, 7) substituents gave
decreased affinities. The N,N-alkyl substituents on the
lower carboxamide were shown to be crucial in the
interaction since most of the affinity was lost when the
symmetrical isopropyl groups on the amide nitrogen
were replaced by other substituents (5, 13, 14, 15). The
importance of hydrophobic side chains was further
confirmed by the weak binding of 16, which lacks the key
substituents. The 2-isopropylaminoterephthalamide 17
showed affinity 4-fold less than its 2-isopropoxy analo-
gue 4, suggesting the intramolecular hydrogen bond in 4
helps to orient the side chain and in turn to enhance
binding. These assay results confirmed that the ter-
ephthalamide derivatives retained the high in vitro affi-
nity of the original terphenyl scaffold while reducing its
complexity.
A computational docking study using AutodockTM lent
support that the binding cleft for the BH3 domain of the
Bak peptide on the surface of Bcl-xL is the target area
for the synthetic inhibitors. Over 90% of the con-
formational search results showed the terephthalamide
docked to this region. Figure 5 shows the overlay of the
top-ranked docking result with the BH3 domain of the
Bak peptide in the Bcl-xL/Bak complex, suggesting that
the side chains of the terephthalamide scaffold have an
analogous spatial arrangement to the three key alkyl
side chains of the Bak peptide.
In conclusion, a novel family of Bcl-xL antagonists,
based on a terephthalamide scaffold has been successfully
Figure 4. Results of the fluorescence polarization competition assay of
terephthalamide derivatives as antagonists of Bcl-xL/Bak complex
formation. Yellow, non-labeled Bak BH3 peptide; purple, 3; light blue,
6; red, 8; brown, 9; green, 10; dark blue, 16; black, 17.
Figure 5. Results of docking studies of 6 and Bcl-xL. (A) Full view.
(B) Close up of an overlay of the highest-ranked binding mode and the
Bak peptide. The key hydrophobic side chains of Bak are shown as
stick representations.

H. Yin, A. D. Hamilton / Bioorg. Med. Chem. Lett. 14 (2004) 1375–1379
1379
developed. Systematic synthesis, conformational studies
and fluorescence polarization binding assays were com-
pleted and sub-mM level in vitro affinities were observed.
6. (a) Wang, J. L.; Liu, D. X.; Zhang, Z. J.; Shan, S. M.;
Han, X. B.; Srinivasula, S. M.; Croce, C. M.; Alnemri,
E. S.; Huang, Z. W. Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 7124. (b) Degterev, A.; Lugovskoy, A.; Cardone, M.;
Mulley, B.; Wagner, G.; Mitchison, T.; Yuan, J. Y. Nat.
Cell Biol. 2001, 3, 173. (c) Tzung, S. P.; Kim, K. M.;
Basanez, G.; Giedt, C. D.; Simon, J.; Zimmerberg, J.;
Zhang, K. Y. J.; Hockenbery, D. M. Nat. Cell Biol. 2001,
3, 183. (d) Enyedy, I. J.; Ling, Y.; Nacro, K.; Tomita, Y.;
Wu, X. H.; Cao, Y. Y.; Guo, R. B.; Li, B. H.; Zhu, X. F.;
Huang, Y.; Long, Y. Q.; Roller, P. P.; Yang, D. J.; Wang,
S. M. J. Med. Chem. 2001, 44, 4313. (e) Lugovskoy, A. A.;
Degterev, A. I.; Fahmy, A. F.; Zhou, P.; Gross, J. D.; Yuan,
J. Y.; Wagner, G. J. Am. Chem. Soc. 2002, 124, 1234.
7. Kutzki, O.; Park, H. S.; Ernst, J. T.; Orner, B. P.; Yin, H.;
Hamilton, A. D. J. Am. Chem. Soc. 2002, 124, 11838.
8. Energy minimization and compound superimpositions
were carried out using the MM2 forcefield within the
Macromodel program.
Acknowledgements
We thank the National Institutes of Health for financial
support of this work.
References and notes
1. (a) Adams, J. M.; Cory, S. Science 1998, 281, 1322. (b)
Reed, J. C. Nature 1997, 387, 773.
2. (a) Graeber, T. G.; Osmanian, C.; Jacks, T.; Housman,
D. E.; Koch, C. J.; Lowe, S. W.; Giaccia, A. J. Nature
1996, 379, 88. (b) Fearon, E. R.; Vogelstein, B. Cell 1990,
61, 759.
9. Ernst, J. T.; Becerril, J.; Park, H. S.; Yin, H.; Hamilton,
A. D. Angew. Chem., Int. Ed. 2003, 42, 535.
10. Crisp, G. T.; Jiang, Y. L. Arkivoc 2001, 2, U60.
11. 2-D NMR experiments were conducted with Bruker 500
Hz under following conditions: 0.1 mM 2 in DMSO at
298 K.
3. Strasser, A.; Huang, D. C. S.; Vaux, D. L. Biochim. Bio-
phys. Acta-Rev. on Cancer 1997, 1333, F151.
4. Adams, J. M.; Cory, S. Trends in Biochem. Sci. 2001, 26, 61.
5. Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.;
Harlan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S. B.;
Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S. W.
Science 1997, 275, 983.
12. Under this assay condition, the unlabeled 16-mer Bak
peptide gave a Kd of 120 nM.7