Heterocyclic α-helix mimetics for targeting protein-protein interactions

Article abstract of DOI:10.1016/j.bmcl.2007.05.075

The design and synthesis of α-helix peptidomimetics using inverse electron demand Diels-Alder reactions is described. The potency of the resulting pyridazine-based library to disrupt the Bak/Bcl-X_L interaction was tested using an in vitro fluorescence polarization assay.

Full text of DOI:10.1016/j.bmcl.2007.05.075

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4641–4645
Heterocyclic a-helix mimetics for targeting
protein–protein interactions
Shannon M. Biros,a, Lionel Moisan,a, Enrique Mann,a Alexandre Carella,a
Dayong Zhai,b John C. Reedb and Julius Rebek, Jr.a,*
aThe Skaggs Institute for Chemical Biology, The Scripps Research Institute, Mail MB-26, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
bBurnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 17 April 2007; revised 22 May 2007; accepted 24 May 2007
Available online 27 May 2007
Abstract—The design and synthesis of a-helix peptidomimetics using inverse electron demand Diels–Alder reactions is described.
The potency of the resulting pyridazine-based library to disrupt the Bak/Bcl-XL interaction was tested using an in vitro fluorescence
polarization assay.
Ó 2007 Elsevier Ltd. All rights reserved.
Protein–protein interactions are involved in the regula-
tion of a wide variety of biological processes. Since the
sequencing of the human genome, some research groups
have put forward the challenge of developing a small
molecule inhibitor for every protein–protein interac-
tion.1 This is unlikely for many protein–protein com-
The helices present their i, i + 3/i + 4, and i + 7 residues
to make crucial contacts with the target protein that
constitute the majority of binding energy (Fig. 1a).5
These residues project from one face of the helix with
well-known distance and angular relationships as shown
in Figure 1a.
2
˚
plexes: large surface areas with ꢀ1600 A or 170 atoms
are involved,2,3 and their relatively flat shapes do not of-
fer purchase for small molecules. Two exceptions are
possible. Allosteric sites, particularly those deep within
the core of a protein, can modify protein–protein inter-
actions such as those well known in the association of
hemoglobin subunits.4a A second favorable situation
arises when one of the interacting surfaces features a
deep, narrow invagination. An appropriate small mole-
cule can be more or less surrounded in this environment,
with the consequence of high binding affinity through
the molecular recognition elements on offer. The natural
complement for the cleft may be a strand or loop struc-
ture,4b but a-helices are often involved.4c Moreover,
only a few side chains of the helices typically occupy
the binding site on complexation, and we are concerned
with these cases here.
The development of non-peptide based scaffolds capable
of displaying functionality in a fashion imitating the rel-
evant binding residues of an a-helix was pioneered by
Hamilton.6–9 Many of these compounds have shown rel-
atively high affinity for a-helix binding sites, as well as
in vitro and in vivo activity. Terphenyl derivatives func-
tionalized at the 3, 20, and 200 positions such as 1 can
achieve a staggered conformation where the substituents
are displayed in a way that closely resembles the i, i + 3,
and i + 7 residues of an a-helix (Scheme 1). A series of
these molecules were synthesized in a modular fashion
by Hamilton and coworkers, allowing for the incorpora-
tion of multiple components.10
Inspired by the success of the Hamilton terphenyl scaf-
folds, we set out to devise synthetic methods for access
to structurally similar molecules that feature more
hydrophilic components via an easier synthetic route.
Our major alteration was the incorporation of a pyrid-
azine heterocycle as the center ring of our scaffolds.
These heterocycles are readily accessible through an
inverse electron demand Diels–Alder reaction between
an electron deficient 1,2,4,5-tetrazine and an electron
Keywords: a-helix mimetic; Pyridzaine; Bak/Bcl-XL; Protein–protein
interaction; Diels–Alder.
*
Corresponding author. Tel.: +1 858 784 2250; fax: +1 858 784
2876; e-mail: jrebek@scripps.edu
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.075

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S. M. Biros et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4641–4645
Figure 1. (a) Overlay of energy minimized 4 with a generic a-helix (minimization carried out using Macromodel (AMBER)); (b) general
retrosynthetic approach of the target molecule 4.
the final oxazole–pyridazine–piperazine compound 4 are
made at the amide bonds to give a pyridazine diester (7),
an amino alcohol (6), and a piperazine (8). This synthe-
sis is modular, as each piece can be synthesized sepa-
rately and attached in sequence. Many amino-alcohols
are commercially available bearing either natural amino
acid side chains or some of the common homologs. The
central pyridazine ring is readily available from the in-
verse electron demand Diels–Alder reaction of known
dimethyl-1,2,4,5-tetrazine-dicarboxylate 913 and a suit-
able dienophile. Any piperazine (bearing a variety of
protecting groups) can be easily prepared from the cor-
responding dipeptide. This synthesis also allows for the
attachment of the piperazine module (or amino-alcohol)
to either position of the pyridazine ring.
Scheme 1. Examples of a-helix mimetics: terphenyl 1,10 oligoamide
A small variety of mono-protected 2-substituted pipera-
zines are commercially available. These compounds
present standard, hydrophobic side chains (i.e., benzyl,
foldamer 2,11 and terephtalamide 3.12
rich dienophile. This type of chemistry has been exten-
sively studied over the past two decades by the Boger
and Snyder groups.13–16
isobutyl, etc.) and are available as the 2-Boc or 4-benzyl
derivatives. As an alternative, these structures can be
prepared containing any of the desired amino acid resi-
dues following the procedure of Hartman et al.17
An overlay of an a-helix with a general depiction of
one of our target molecules (4, Fig. 1b) is shown in
Figure 1a. The i, i + 3, and i + 7 residues of the a-helix
as well as their counterparts on the synthetic scaffold
are highlighted as small spheres. It is clear that the pre-
sentation of functionality by scaffold 4 is close to that
of a natural a-helix. The new scaffolds are intended to
present both a hydrophobic surface for recognition and
a ‘wet edge’ that is rich in hydrogen bond donors and
acceptors. This was intended to enhance solubility and
to ensure that during complexation with its target, the
wet edge remains directed toward the solvent; in other
words, no entropic penalty (or advantage) should
occur by solvation at the wet edge as it is not altered
during docking of the helix mimetic. We also hoped
that these molecules would exhibit an increased
solubility in water through protonation of the basic
piperazine ring at physiological pH. These scaffolds
may be thought of as synthetic counterparts of
amphiphilic a-helices.
Since some of our desired piperazines were not commer-
cially available, we synthesized a series of 2-N-methyl pip-
erazines containing a variety of substituents.18
A
representative example is shown in Scheme 2, (S)-2-sec-
butyl-1-methylpiperazine. Coupling of amino acid deriv-
ative 10 and Boc-protected glycine using standard
coupling reagents provided the dipeptide 11 in good yield.
Removal of the Boc group with TFA followed by thermal
cyclization in the presence of a hydrogen bonding sol-
vent19 gave the crystalline diketopiperazine 12. Reduction
The general retrosynthetic approach to these molecules
is laid out in Figure 1b. The major disconnections from
Scheme 2. Synthesis of N-methyl-piperazine derivative 13.

S. M. Biros et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4641–4645
4643
with lithium aluminum hydride proceeded smoothly with
mild heating to give the enantiomerically pure piperazine
13.
of the amide–aldehyde to PPh3/I2/DMAP25 was not
effective in the conversion of this function to an oxazole.
A slight modification of the reaction conditions to PPh3,
2,6-di-tert-butyl pyridine, dibromo-tetrachloroethane,
and DBU26 resulted in the transformation of amide-
aldehyde 20 to the oxazole 21 in good yield.
For construction of the central pyridazine ring, the elec-
tron deficient 3,6-dimethyl-1,2,4,5-tetrazine dicarboxy-
late 9 was synthesized following the procedure of
Boger and coworkers.13 Tetrazine 9 was reacted with
4-methylpentyne 14 to give pyridazine 15 in good yield
(Scheme 3). The two heterocycles can then be connected
by preparation of the aluminum amide20–22 of pipera-
zine 13. Exposure of this intermediate to a solution of
pyridazine diester 15 with gentle heating (41 °C) results
in smooth conversion to the mono-amide 16.23 It is clear
from the 1H NMR spectrum of product 16 that only one
methyl ester is present, as the two esters in the starting
material are not magnetically equivalent. An HMBC
NMR spectrum of the mono-ester 16 shows coupling
between the protons of the piperazine ring and the pyr-
idazine aryl proton to the same carbonyl carbon; the
carbonyl at the less hindered 6-position.
We were also interested in preparing substituted pyrida-
zines where the groups at the 3- and 6-positions are
equivalent. As discussed above, while AlMe3 is a very
reactive reagent, the yields obtained for aminolysis at
the 3-position of substituted pyridazine rings are quite
low. Magnesium chloride is another effective Lewis Acid
capable of carrying out the aminolysis of pyridazine
methyl esters.27 Exposure of iso-butyl substituted diester
15 to an excess of MgCl2 and (S)-valinol proceeds
smoothly to give the diamide 22 in 63% yield (Scheme
4). Oxidation to the dialdehyde followed by oxazole for-
mation as described above gives the dioxazole–pyrida-
zine compound 23. While these bis-oxazole compounds
do not display functionality in exactly the same orienta-
tion as the oxazole–pyridazine–piperazine scaffold 4,
their synthesis is straightforward and is a potential theme
for preparation of a small library for structure–activity
correlations.
Exposure of mono-ester 16 to protected-valinol in the
presence of AlMe3 also resulted in the aminolysis of
the remaining methyl ester function, however the yield
of this reaction was quite low (20%). To increase the
yield, the Curtius coupling procedure was used.24 The
methyl ester was transformed into the acyl hydrazide
17, diazotized to give the acyl-azide 18, and displaced
with (S)-valinol to give the di-amide 19 in 64% overall
yield.
The synthetic sequences described above were applied to
combine a series of piperazines, piperidines, pyridazines,
and amino alcohols bearing hydrophobic groups to
prepare a library of twenty-four compounds shown in
Figure 2b.
To increase the rigidity of the final a-helix mimetics mol-
ecule and decrease loss of entropy upon binding, we pur-
sued methods to close the b-hydroxy amide to an
oxazole. Our first attempt to form an oxazole ring from
the b-hydroxy amide moiety required oxidation of the
alcohol to the aldehyde 20, and this proceeded as ex-
pected using the Dess–Martin periodinane. Exposure
To evaluate the activity of our a-helix mimetics we tested
their ability to disrupt the Bak/Bcl-XL interaction. Bak
and Bcl-XL are two proteins involvedin apoptosis and thus
have become targets as new therapeutic approaches for the
treatment of cancer and other diseases.9 It is known that
when the domain of interaction of Bak (termed ‘BH3’)
interacts with Bcl-XL, it adopts an a-helix conformation
Scheme 3. Modular synthesis of the oxazole–pyridazine–piperazine scaffold 21 bearing hydrophobic substituents.

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S. M. Biros et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4641–4645
Scheme 4. Modular synthesis of the bis-oxazole scaffold.
Figure 2. (a) FPA for the disruption of the Bak-BH3 peptide and Bcl-XL interaction; pale yellow bars: 10 lM of assayed compound, pale blue bars:
50 lM of assayed compound. Ctrl-1 (dark blue bar): measured polarization for Bak-BH3 and Bcl-XL alone, Ctrl-2 (red bar): terphenyl 1 positive
control, Ctrl-3 (green bar): measured polarization for Bak-BH3 peptide alone; (b) library tested in the FPA.
thatliesinahydrophobiccleftatthesurfaceofBcl-XL. Key
contacts are made between the hydrophobic residues
Val74, Leu78, Ile81, and Ile85 of Bakand hydrophobic res-
idues lining the pocket of Bcl-XL.5b
control (red bar, far right). Values from ca. 100–
140 mP represent a ‘target area’ for polarization values.
Since this was a preliminary screening assay, we sought
compounds having high enough affinity for Bcl-XL to
generate a polarization value within this range.
The binding affinity of our molecules for Bcl-XL was
determined by
a
fluorescence polarization assay
The left part of Figure 2a lists the results from com-
pounds shown in Figure 2b that appear to have little to
no affinity for Bcl-XL. This group of compounds is com-
posed of molecules containing b-hydroxyamides (com-
pounds 22 and 24–33) and/or unsubstituted piperazines
(compounds 34–37). It is not surprising that this series
of molecules have low affinity for Bcl-XL. Those
compounds presenting three R-groups also contain b-hy-
droxy amide functions that possess six freely rotating
bonds each. The entropic cost for ‘freezing’ out these
rotations on receptor binding is high. However, the com-
pounds containing the more rigid oxazole function in
(FPA)28 using fluorescently labeled Bak-BH3 peptide.29
The polarization amount (mP) is plotted on the y-axis in
the graph in Figure 2. It provides a rough estimate of the
affinity for the small molecules 22–44 for Bcl-XL. The
dark blue bar in the column on the far left of the graph
represents the amount of polarization observed when
the fluorescein-labeled Bak peptide is bound to Bcl-
XL. The green bar in the column on the far right is the
fluorescein-labeled Bak peptide alone in solution. Ter-
phenyl 130 has been previously shown to bind to
Bcl-XL with nanomolar affinity10 and acts as a positive

S. M. Biros et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4641–4645
4645
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place of the b-hydroxy amide (three freely rotating
bonds) only present two R-groups. It is likely these struc-
tures are not involved in enough interactions with the
receptor to induce binding. Also, most of the compounds
in this set contain basic piperazine functions that are pro-
tonated under the assay conditions. While we included
this feature in these molecules for ease of synthesis and
to increase water-solubility, the cationic ammonium
group may be causing unfavorable interactions on bind-
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set of data shown in Figure 2a contains compounds
belonging to the oxazole–pyridazine–piperazine and bis-
oxazole scaffold classes. Of this group, four compounds
approximate our desired mP values: 23, 41, 43, and 44.
Three of these compounds (23, 43, and 44) are neutral,
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These compounds represent the strongest binders for Bcl-
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Some insight can be gleaned from these results that
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see that neutral compounds have a higher affinity for
Bcl-XL than those containing a positive charge, except
in the case of 41. The cationic ammonium may cause
unfavorable electrostatic interactions while in the hydro-
phobic binding pocket and/or the penalty for desolvation
of this ionic center is too high. Compound 1 possesses
two carboxylate functions thought to be involved in
favorable electrostatic interactions with Bcl-XL along
the upper ridge of the binding site.10 The extra favorable
interaction(s) experienced by compound 1 may be enough
to bring its binding affinity into the nanomolar range.
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Acknowledgments
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25. Ceide, S. C.; Trembleau, L.; Haberhauer, G.; Somogyi, L.;
Lu, X.; Bartfai, T.; Rebek, J., Jr. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 16727.
We are grateful to the Skaggs Institute for Research, the
ARCS Foundation for a Fellowship to S.M.B., and the
National Institutes of Health (CA 113318) for support.
Both Kemia, Inc. and Novartis provided support. We
thank Prof. A.D. Hamilton for a gift of terphenyl 1,
and Prof. D.L. Boger and Prof. E. Roberts for advice
and helpful discussions. We are pleased to acknowledge
Drs. D.-H. Huang and L. Pasternack for assistance with
NMR experiments.
26. Wipf, P.; Lim, S. J. Am. Chem. Soc. 1995, 117, 558.
´
´
27. Gonzalez-Gomez, J. C.; Santana, L.; Uriarte, E. Tetrahe-
dron 2003, 59, 8171.
28. Zhang, H.; Nimmer, P.; Rosenberg, S. H.; Ng, S. C.;
Joseph, M. Anal. Biochem. 2002, 307, 70.
29. Assays for Bcl-XL were performed using as the binding partner
the 5-carboxyfluorescein-labeled 25-mer peptide tracer Flu-
Bak-BH3 (FITC-Ahx-PSSTMGQVGRQLAIIGDDINR-
RYDS) derived from the Bak BH3 domain. The assays were
performed in a 96-well format, in 20 mM potassium phosphate
buffer, pH 7.4, containing 150 mM NaCl. The final concen-
tration of DMSO in all assays was 1%. The reaction was
carried out in a 100 lL volume and the resulting polarization
signal was measured at kex = 485 nm/kem = 535 nm using an
Analyst TM AD Assay Detection System (LJL Biosystem,
Sunnyvale, CA) after 20-min incubation of the reaction
mixture at room temperature.
References and notes
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30. Compound 1 was generously provided by Prof. A.D.
Hamilton.