Design, synthesis and conformational analyses of bifacial benzamide based foldamers

Article abstract of DOI:10.1039/c5ra20451h

The design, synthesis and conformational analyses of novel backbones represents a key focus of research that underpins efforts to exploit foldamers (i) in a biological setting e.g. as inhibitors of protein-protein interactions (PPIs) and (ii) for the purp

Full text of DOI:10.1039/c5ra20451h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Design, synthesis and conformational analyses of
bifacial benzamide based foldamers†
Cite this: RSC Adv., 2015, 5, 104187
Silvia Rodriguez-Marin,ab Natasha S. Murphy,ab Helena J. Shepherdc
ab
*
and Andrew J. Wilson
The design, synthesis and conformational analyses of novel backbones represents a key focus of research
that underpins efforts to exploit foldamers (i) in a biological setting e.g. as inhibitors of protein–protein
interactions (PPIs) and (ii) for the purposes of constructing functional architectures that adopt defined
tertiary and quaternary folds. The current manuscript addresses a need to develop aromatic oligoamide
backbones that are regioisomeric in terms of backbone connectivity and/or functionalized on more than
one face. We describe the design, synthesis and comparative conformational analyses of foldamers
derived from 2-, 3- and 2,5-O-alkylated derivatives of para-aminobenzoic acid, and, derived from 2-,3-
and 2,5-O-alkylated derivatives of 1,4-diaminobenzene/terephthalic acid monomers. Analysis of the
accessible conformational space for these oligomers indicates that despite different connectivity they
can adopt conformations that position side chains in a manner that mimic the i, i + 3, i + 4 of an a-helix.
Received 2nd October 2015
Accepted 20th November 2015
DOI: 10.1039/c5ra20451h
www.rsc.org/advances
face of the a-helix; however, there are also examples of these
scaffolds mimicking more than one face.25–27 In the context of
Introduction
foldamer synthesis and structure, the construction of back-
bones functionalised with different side-chains on multiple
faces of the scaffold represents an as yet unrealised approach to
achieve control over secondary conformation and higher order
tertiary/quaternary organisation. Similarly, there is an obvious
need for PPI inhibiting helix mimetics that target more than
one face of an interaction, such as the case of the estrogen
receptor (ER), a ligand-activated transcription factor that plays
a key role in the development of certain cancers.28,29 In response
to binding with its natural ligand, ER undergoes a conforma-
tional change to promote recruitment of co-regulators, thereby
up- or down-regulating the expression of specic genes.30 The
Protein–protein interactions (PPIs) are considered difficult
targets for drug discovery due to their large and moderately
convex surfaces, and the availability of fewer “obvious” well-
dened binding sites when compared to classical enzymes
and receptors.1 Among the wide range of PPI recognition motifs,
the a-helix is the most common secondary structure in nature
and thus represents a good generic template for inhibitor
design.2,3 The proteomimetic4 approach utilises suitably func-
tionalized non-peptidic foldamers5,6 (oligomers that adopt well
dened conformations) to topographically mimic the spatial
orientation of the key recognition residues on the native a-helix
surface (Fig. 1a). Most of these scaffolds mimic the i, i + 4 and i +
7 residues on a single face.4,7–12 In particular oligobenzamides
have been described as effective proteomimetics by our group
and others;9,13–17 they may be accessed through robust modular
solution18 and solid phase syntheses,19,20 and, as is typical for
aromatic oligoamide foldamers21–24 adopt reasonably predict-
able conformations. Most published studies focus on the design
of oligobenzamides mimicking the key residues located on one
aSchool of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. E-mail:
J.Wilson@leeds.ac.uk
bAstbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse
Lane, Leeds, LS2 9JT, UK
cSchool of Physical Sciences, University of Kent, Ingram Building, Canterbury, Kent,
CT2 7NH, UK
Fig. 1 (a) Bifacial helix mimetics as inhibitors of PPIs (steroid ligand in
orange, ER in purple, a-helix containing co-activator in red and key
side chain residues represented as coloured circles). (b) Crystal
structure of the ERa (in purple) bound to an LXXLL co-activator motif
(in red) (PDB ID: 3ERD) (charge clamp shown in green).40
† Electronic supplementary information (ESI) available: Synthetic procedures,
characterisation, modelling and docking studies. CCDC 1426299. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c5ra20451h
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 104187–104192 | 104187

View Article Online
RSC Advances
Paper
nuclear receptor box is an alpha helical LXXLL motif (where L is scaffold (4) was also synthesised to explore the role of a 4th side
leucine and X any amino acid), which acts as a recognition chain in helix mimicry.
element between co-activators and their receptors (Fig. 1b).31
A novel second generation scaffold was designed based on
Direct inhibition of the receptor/co-activator protein–protein a N-(4-aminophenyl)terephthalamidic acid backbone, where the
interaction,32–37 notably using helix mimetics35,36,38 is of poten- central amide bond is inverted in comparison with the bis-
tial therapeutic interest as an alternative to the use of compet- benzamide scaffold. The dimer is formed from a para-phenyl-
itive inhibitors for the ligand binding site.39 Herein, we enediamine monomer linked to a terephthalate monomer
introduce two bifacial proteomimetic scaffolds; bis-benzamide through an amide bond. The backbone can be functionalized at
and N-(4-aminophenyl)terephthalamidic as novel foldamers different positions using a variety of O-alkylated monomers. A
designed as tools to (a) enhance our understanding of aromatic convergent synthetic methodology was developed to provide the
oligoamide foldamer conformation and (b) ligands that could monomeric building blocks (Scheme 1). Noteworthy features of
mimic the key side chains at i, i + 3, i + 4 positions of a-helices the monomer syntheses include: (i) the use of a Curtius rear-
that participate in PPIs mediated by such a side chain constel- rangement to convert monomer 6 to 7 rendering this syntheti-
lation. A comprehensive analysis of the different scaffolds cally efficient for both building blocks (ii) the use of a common
reveals that different combinations of monomers lead to starting material 8 to access monomers 9 and 10 for construc-
a plethora of side chain spatial relationships which effectively tion of different regioisomers (see below). Note also that no N-
mimic the intended a-helix side-chains.
alkylation was observed on transformation of 8 to 9. For mon-
osubstituted alkoxy derivatives of terephthalic acid, it was
necessary to perform
manipulations.
a sequence of protecting group
Results and discussion
To effect amide bond formation, the acyl chloride of the
diacid monomer 6 was obtained using thionyl chloride before
coupling to its amino-monomer partners 9 or 10 (Scheme 2). By
using an excess of the diacid 6 it was possible to bias the product
distribution towards the monoamide. The nal products 14ba
and 14bb were obtained by hydrogenation of the nitro group or
hydrolysis of the Fmoc group respectively. Due to oxidation upon
exposure to air, the diamine derivative of compound 7 was ob-
tained through in situ Boc deprotection and direct reaction with
A rst generation of scaffold 1–4 (Fig. 2) was designed using the
modular oligobenzamide synthetic methodology previously re-
ported by our group (Schemes S2 and S3†).15,41,42 Combinations
of 3-O-alkylated, 2-O-alkylated and 2,5-O-dialkylated monomers
were used to obtain a regioisomeric set of compounds for
conformational analyses. The regioisomer of compound 2 could
not be obtained due to unsuccessful coupling between methyl 4-
amino-2,5-diisobutoxybenzoate
and
4-nitro-2-isobutoxy-
benzoic acid under multiple conditions. The tetrasubstituted
Fig. 2 Minimal benzamide foldamers comprising 3-O, 2-O, 2,5-O Scheme 1 Synthesis of monomers for N-(4-aminophenyl)tereph-
alkylated p-aminobenzoic acid monomers.
thalamidic derived foldamers.
104188 | RSC Adv., 2015, 5, 104187–104192
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Scheme 2 Synthesis of minimal N-(4-aminophenyl)terephthalamidic foldamers.
the acid chloride derivative of 12 or 13; these were obtained by in ve- and six-membered hydrogen-bonded rings being popu-
situ activation using Ghosez's reagent. Again, the monoamide lated in solution.
product was biased by using the starting diamine in excess. The
An X-ray crystal structure of compound 1 reinforces this
˚
nal compounds 14aa and 14ab were obtained aer appropriate result with NH to O distances of 2.007 and 2.223 A respectively
deprotection sequences (Scheme 2) specically acid mediated for the S(6) and S(5) H-bonded rings (Fig. 3c). Intriguingly,
Boc deprotection and ethyl ester hydrolysis respectively. Despite similar NMR analyses for compound 4 were indicative only of
numerous efforts we were unable to isolate a tetrasubstituted pseudo-six-membered hydrogen bonding in solution.
foldamer derived from 6 and 7.
Compounds 14aa, 14bb form pseudo-ve- or six-membered
Previous studies on 2- and 3-O-alkylated trimers and model hydrogen-bonded rings as expected whereas 14ab (Fig. 3b)
dimers on the oligobenzamide scaffold revealed intramolecular and 14ba showed evidence of only pseudo-six-membered
pseudo-six- or ve-membered hydrogen bonding between the intramolecular hydrogen bonded ring formation in solution.
NH and adjacent O-alkyl group.15,43 This results in restricted These results are supported by H/D exchange experiments
rotation around one of the Ar–CO or Ar–NH bonds leaving the performed on compounds 1, 2 and 14ba as models of the three
other free to rotate. The conformation of such scaffolds can be types of intramolecular hydrogen bonding interaction (Fig. 3d,
further restricted by introduction of a second alkoxy group S1† and Table 1). The rate of exchange is entirely consistent with
leading to a “bifurcated” hydrogen bonding interaction, where that which is observed for S(6) type hydrogen bonded rings for
the NH is located between two phenolic oxygens from adjacent the regioisomeric oligomers derived from 2-O-alkoxy-4-
monomers forming pseudo-six- and ve-membered rings.42,44,45 aminobenzoic acid and suggests the different electronic struc-
In principle, the set of compounds discussed here can display ture of the 2,5-dialkoxyterephthalamide monomer does not
a similar array of conformations, resulting in different projec- dramatically affect the strength of hydrogen-bonding.
tions of the alkoxy side chains and a distribution of 3D struc-
Molecular modelling was also performed on all the
tures some of which effectively mimic an i, i + 3, i + 4 helical compounds (Fig. 3a). The lowest energy conformations all adopt
pharmacophore. Consequently, structural and conformational an extended structure, where the amide bond is trans. The low
analyses were performed on each compound (summarised in energy conformations for each compound are consistent with
Fig. 3a–d). Compounds 2 and 3 form pseudo-ve- or six- those that are accessible in solution phase according to the
membered hydrogen-bonded rings whereas the 1H–1H 2D NOESY data. The nature of the structure permits the superim-
NOESY spectra for compound 1 were indicative of both pseudo- position in both parallel and antiparallel N-to-C orientation
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 104187–104192 | 104189

View Article Online
RSC Advances
Paper
Fig. 3 (a) Preferred conformation and intramolecular hydrogen-bonding interactions of the compounds 1–3 and 14 supported by molecular
modelling, 2D NMR studies and H/D exchange experiments. Distances and angles between side chains (green and black respectively), H-bonds
(dashed red line) and free rotation axes (red arrow) are shown. (b) 1H–1H NOESY (10 mM, 500 MHz CDCl3) of 14ba (c) X-ray structure of 1 (H-
bonding distances (A˚) are shown in red) (d) H/D exchange data for 1, 2 and 14ba.
104190 | RSC Adv., 2015, 5, 104187–104192
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Table 1 Kinetic constants and t1/2 based on H/D exchange in 10%
CD3OD/CDCl3
kH/D (minÀ1
)
t1/2 (min)
H bonding
1
2
6.7857 Â 10À4 Æ 0.0000093
0.01485 Æ 0.00017
1021.5 Æ 14
46.7 Æ 0.5
228 Æ 3
S(5)/S(6)
S(5)
S(6)
14ba
0.00305 Æ 0.00005
with respect to an a-helical peptide.19 Accordingly, both align-
ments were analysed using an ERa co-activator sequence. The
match was assessed on the basis of the RMSD between a-
carbons on the helix and oxygen atoms on the foldamer
together with an evaluation on the quality of orientation with
respect to the helical axis of the peptide (Table 2 and ESI† for
details). For compounds 1–3, 14aa and 14bb, in the poses pre-
senting the best overlay, the three side chains overlap reason-
ably well with the leucine residues at positions i, i + 3 and i + 4 of
the co-activator helix (Fig. 3a for 14aa and Fig. S5–S11† for other
dimers) and the distances between the oxygens of the dimers
match the distance between the a-CH of those residues.
Compounds 14ab and 14ba, matched less well in terms of
alignment with the helical backbone.
Docking studies using the lowest energy conformation of each
foldamer were also performed to ascertain the extent to which they
might act as ERa/co-activator inhibitors (see ESI†). The results
from the docking analyses reveal binding poses that display
favourable interaction of the foldamers 1–3 and 14 with the co-
activator binding groove. Electrostatic interactions are observed
for both termini of the foldamer. However, in all cases only one of
these involves the precise “charge clamp” residues from ER
exploited by co-activator ligands. Shown in Fig. 4b is a good pose
for 14aa; the three hydrophobic side chains of the foldamer
occupy the hydrophobic space normally occupied by the co-
activator peptide. The terminal carboxamide and aniline groups
of the dimer are suitably positioned to form electrostatic interac-
tions with glutamic acid (542) and glutamine 372 (rather than
lysine 362) in the region of the “charge clamp”. This behaviour is
reproduced for the other compounds (e.g. Fig. S12† for 1).
To perform a preliminary assessment of the ability of these
compounds to act as PPI inhibitors, we carried out uorescence
polarisation competition assays for three nuclear receptor/co-
Fig. 4 (a) Overlay of compound 14aa with a co-activator peptide. Co-
activator residues are in dark colours and helix mimetic residues are in
light colours (side and top views are given). (b) Potential binding mode
of compound 14aa in the ER co-activator-binding groove with the
native helix in transparent red.
activator interactions (ERa/SrcBox2, ERb/Src1B2 and RXRa/
D22), however the compounds were not sufficiently potent to
show a signicant effect in these assays. Future studies will
focus on the synthesis of libraries bearing different side-chains
and terminal groups together with a broader array of biophys-
ical and cellular assays to explain this observation and identify
potent inhibitors.
Conclusions
In conclusion, we have described the design, synthesis and
comparative structural/conformational analyses of two
minimal bifacial foldamer scaffolds, using for rst time a N-(4-
aminophenyl)terephthalamidic acid. The synthetic route to the
N-(4-aminophenyl)terephthalamidic acid scaffold has been
readily adapted to obtain a multitude of analogues displaying
diverse side chain spacing. Furthermore, the ability of these
scaffolds to mimic more than one face of an a-helix has been
assessed using molecular modelling. These new foldamer
backbones should be entirely compatible with the plethora of
aromatic oligoamide backbones and could readily be utilised
to construct well dened secondary and tertiary structures by
exploiting side-chain/side-chain interactions as appropriate. In
future, our own efforts will focus on exploiting these aromatic
benzamide scaffolds as components of proteomimetic inhibi-
tors of PPIs.
Table 2 Summary of modelling
Alignmenta
RMSD
1
2
3
Antiparallel
Parallel
1.322
1.625
2.084
1.038
1.622
1.027
2.046
Acknowledgements
Antiparallel
Antiparallel
No good alignment
No good alignment
Parallel
14aa
14ab
14ba
14bb
This work was supported by the European Research Council
[ERC-StG-240324] and [ERC-PoC-632207]. We thank Prof Luc
Brunsveld and Marcel Scheepstra (Technische Universiteit
Eindhoven) for assistance in performing preliminary ER/co-
activator assays. We also thank Dr Chris Pask (University of
Leeds) for assistance with Crystallographic data deposition.
a
Where N and C termini of the benzamide and helix match, they are
dened as being parallel and where they oppose, they are dened as
being antiparallel.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 104187–104192 | 104191

View Article Online
RSC Advances
Paper
21 I. Huc, Eur. J. Org. Chem., 2004, 2004, 7.
22 A. Roy, P. Prabhakaran, P. K. Baruah and G. J. Sanjayan,
Chem. Commun., 2011, 47, 11593–11611.
23 B. Gong, Chem. Eur. J., 2001, 7, 4336–4342.
24 D.-W. Zhang, X. Zhao, J.-L. Hou and Z.-T. Li, Chem. Rev.,
2012, 112, 5271–5316.
25 S. Marimganti, M. N. Cheemala and J. M. Ahn, Org. Lett.,
2009, 11, 4418–4421.
26 S. Thompson, R. Vallinayagam, M. J. Adler, R. T. W. Scott
and A. D. Hamilton, Tetrahedron, 2012, 68, 4501–4505.
27 I. C. Kim and A. D. Hamilton, Org. Lett., 2006, 8, 1751–1754.
28 E. P. Gelmann, J. Clin. Oncol., 2002, 20, 3001–3015.
29 M. H. Herynk and S. A. W. Fuqua, Endocr. Rev., 2004, 25, 869–
898.
References
1 S. Surade and T. L. Blundell, Chem. Biol., 2012, 19, 42–50.
2 B. N. Bullock, A. L. Jochim and P. S. Arora, J. Am. Chem. Soc.,
2011, 133, 14220–14223.
3 V. Azzarito, K. Long, N. S. Murphy and A. J. Wilson, Nature
Chem., 2013, 5, 161–173.
4 B. P. Orner, J. T. Ernst and A. D. Hamilton, J. Am. Chem. Soc.,
2001, 123, 5382–5383.
5 S. H. Gellman, Acc. Chem. Res., 1998, 31, 173–180.
6 S. Fahs, Y. Patil-Sen and T. J. Snape, ChemBioChem, 2015, 16,
1840–1853.
7 D. Xin, L. M. Perez, T. R. Ioerger and K. Burgess, Angew.
Chem. Int. Ed., 2014, 53, 3594–3598.
¨
´
¨
30 R. Evans, Science, 1988, 240, 889–895.
31 K. A. Green and J. S. Carroll, Nat. Rev. Cancer, 2007, 7, 713–
722.
32 J. R. Gunther, T. W. Moore, M. L. Collins and
J. A. Katzenellenbogen, ACS Chem. Biol., 2008, 3, 282–286.
33 A. A. Parent, J. R. Gunther and J. A. Katzenellenbogen, J. Med.
Chem., 2008, 51, 6512–6530.
¨
8 G. Schafer, J. Milic, A. Eldahshan, F. Gotz, K. Zuhlke,
C. Schillinger, A. Kreuchwig, J. M. Elkins, K. R. Abdul
Azeez, A. Oder, M. C. Moutty, N. Masada, M. Beerbaum,
B. Schlegel, S. Niquet, P. Schmieder, G. Krause, J. P. von
Kries, D. M. F. Cooper, S. Knapp, J. Rademann,
W. Rosenthal and E. Klussmann, Angew. Chem. Int. Ed.,
2013, 52, 12187–12191.
34 J. R. Gunther, A. A. Parent and J. A. Katzenellenbogen, ACS
Chem. Biol., 2009, 4, 435–440.
35 J. Becerril and A. D. Hamilton, Angew. Chem. Int. Ed., 2007,
46, 4471–4473.
9 J. L. Yap, X. Cao, K. Vanommeslaeghe, K.-Y. Jung,
C. Peddaboina, P. T. Wilder, A. Nan, A. D. MacKerell,
W. R. Smythe and S. Fletcher, Org. Biomol. Chem., 2012, 10,
2928–2933.
36 M. Scheepstra, L. Nieto, A. K. H. Hirsch, S. Fuchs, S. Leysen,
C. V. Lam, L. in het Panhuis, C. A. A. van Boeckel, H. Wienk,
R. Boelens, C. Ottmann, L.-G. Milroy and L. Brunsveld,
Angew. Chem. Int. Ed., 2014, 53, 6443–6448.
37 P. Ravindranathan, T. K. Lee, L. Yang, M. M. Centenera,
L. Butler, W. D. Tilley, J. T. Hsieh, J. M. Ahn and G. V. Raj,
Nat. Commun., 2013, 4, e1923.
38 A. B. Williams, P. T. Weiser, R. N. Hanson, J. R. Gunther and
J. A. Katzenellenbogen, Org. Lett., 2009, 11, 5370–5373.
39 M.-E. Taplin, Nat Clin Prac Oncol, 2007, 4, 236–244.
40 A. K. Shiau, D. Barstad, P. M. Loria, L. Cheng, P. J. Kushner,
D. A. Agard and G. L. Greene, Cell, 1998, 95, 927–937.
41 J. Plante, F. Campbell, B. Malkova, C. Kilner, S. L. Warriner
and A. J. Wilson, Org. Biomol. Chem., 2008, 6, 138–146.
42 P. Prabhakaran, A. Barnard, N. S. Murphy, C. A. Kilner,
T. A. Edwards and A. J. Wilson, Eur. J. Org. Chem., 2013,
3504–3512.
10 J.-M. Ahn and S.-Y. Han, Tetrahedron Lett., 2007, 48, 3543–
3547.
11 W. E. Martucci, J. M. Rodriguez, M. A. Vargo, M. Marr,
A. D. Hamilton and K. S. Anderson, MedChemComm, 2013,
4, 1247–1256.
ˇ
´
12 P. Tosovska and P. S. Arora, Org. Lett., 2010, 12, 1588–1591.
13 F. Campbell, J. P. Plante, T. A. Edwards, S. L. Warriner and
A. J. Wilson, Org. Biomol. Chem., 2010, 8, 2344–2351.
14 J. P. Plante, T. Burnley, B. Malkova, M. E. Webb,
S. L. Warriner, T. A. Edwards and A. J. Wilson, Chem.
Commun., 2009, 5091–5093.
15 V. Azzarito, P. Prabhakaran, A. I. Bartlett, N. S. Murphy,
M. J. Hardie, C. A. Kilner, T. A. Edwards, S. L. Warriner
and A. J. Wilson, Org. Biomol. Chem., 2012, 10, 6469–6472.
16 A. Barnard, K. Long, H. L. Martin, J. A. Miles, T. A. Edwards,
D. C. Tomlinson, A. Macdonald and A. J. Wilson, Angew.
Chem. Int. Ed., 2015, 54, 2960–2965.
43 P. Prabhakaran, V. Azzarito, T. Jacobs, M. J. Hardie,
C. A. Kilner, T. A. Edwards, S. L. Warriner and A. J. Wilson,
Tetrahedron, 2012, 68, 4485–4491.
44 K. Yamato, L. Yuan, W. Feng, A. J. Helsel, A. R. Sanford,
J. Zhu, J. Deng, X. C. Zeng and B. Gong, Org. Biomol.
Chem., 2009, 7, 3643–3647.
17 V. Azzarito, J. A. Miles, J. Fisher, T. A. Edwards, S. L. Warriner
and A. J. Wilson, Chem. Sci., 2015, 6, 2434–2443.
18 G. M. Burslem and A. J. Wilson, Synlett, 2014, 25, 324–335.
19 N. S. Murphy, P. Prabhakaran, V. Azzarito, J. P. Plante,
M. J. Hardie, C. A. Kilner, S. L. Warriner and A. J. Wilson,
Chem. Eur. J., 2013, 19, 5546–5550.
45 J. Zhu, R. D. Parra, H. Zeng, E. Skrzypczak-Jankun, X. C. Zeng
and B. Gong, J. Am. Chem. Soc., 2000, 122, 4219–4220.
20 K. Long, T. A. Edwards and A. J. Wilson, Bioorg. Med. Chem.,
2013, 21, 4034–4040.
104192 | RSC Adv., 2015, 5, 104187–104192
This journal is © The Royal Society of Chemistry 2015