Aromatic oligoamide foldamers with a "wet edge" as inhibitors of the α-helix-mediated p53-hDM2 protein-protein interaction

Article abstract of DOI:10.1002/ejoc.201300069

This paper describes the design, synthesis and structural analysis of a 3-O-alkylated aromatic oligoamide that incorporates an additional hydrophilic 6-O-alkyl substituent in the central monomer. This oligomer exhibits low μM inhibitory potency against th

Full text of DOI:10.1002/ejoc.201300069

FULL PAPER
DOI: 10.1002/ejoc.201300069
Aromatic Oligoamide Foldamers with a “Wet Edge” as Inhibitors of the α-
Helix-Mediated p53–hDM2 Protein–Protein Interaction
Panchami Prabhakaran,[a,b] Anna Barnard,[a,b] Natasha S. Murphy,[a,b] Colin A. Kilner,[b,c]
Thomas A. Edwards,[b,c] and Andrew J. Wilson*[a,b]
Keywords: Proteins / Bioorganic chemistry / Amides / Foldamers
This paper describes the design, synthesis and structural
analysis of a 3-O-alkylated aromatic oligoamide that incorpo-
rates an additional hydrophilic 6-O-alkyl substituent in the
potency against the p53–hDM2 interaction compared with its
unfunctionalised counterpart and significantly improved sol-
ubility.
central monomer. This oligomer exhibits low μM inhibitory
aromatic oligoamides[37–40] have received considerable at-
tention for a number of reasons: 1) they exhibit predictable
conformations determined by the rotameric preference of
the amide and non-covalent interactions with donor or ac-
ceptor functional groups at the adjacent ortho positions,
2) they are (potentially) amenable to library synthesis (by
using solid-phase methodologies) and 3) when suitably
functionalised, side-chains may be projected in a similar
spatial orientation to key binding residues on the surfaces
of α helices.[41–44]
Our group has pursued a number of aromatic oligoamide
scaffolds as inhibitors of the model interaction p53–
hDM2.[44–47] p53 is the major tumour suppressor in humans
and its activity is regulated by binding to hDM2.[48,49] Over
expression of hDM2 in cancer cells prevents p53 from exert-
ing its normal pro-apoptotic function and, as such, inhibi-
tion of this PPI is a major target for chemotherapy.[50] The
binding interaction involves three key hydrophobic residues;
Phe19, Trp23 and Leu26 in an α-helical conformation (see
Figure 1) of p53 presented to a hydrophobic cleft on
hDM2.[49] Previously we have reported on the solid-phase
synthesis[47,51] and inhibition of the p53–hDM2 interaction
with N-alkylated p-benzamides[47] and the inhibition of the
p53–hDM2 interaction by using both 2- and 3-O-alkylated
p-benzamides[44,46] together with detailed structural stud-
ies[46,52,53] and, for the 3-O-alkylated series, MD simulations
on the hDM2 bound ligands.[45]
Introduction
Although many biological processes are controlled by en-
zyme–substrate interactions, protein–protein interactions
(PPIs) mediate a far greater number of essential processes
and are important in a number of therapeutic areas, notably
oncology.[1–5] The inhibition of PPIs represents a major
challenge due to the wide range of topographies and large
surface areas found at binding interfaces.[6,7] It may there-
fore be necessary for a small molecule to cover Ͼ1000 Å2
of a protein surface to achieve effective inhibition. In an
important early observation, Clackson and Wells noted
that, although PPIs involve large surface areas, the vast ma-
jority of the binding affinity can be attributed to a few key
residues, known as a “hot-spot”.[8] α-Helices are the most
abundant secondary structure found in proteins, and it is
perhaps therefore unsurprising that a large number of PPIs
are mediated by this motif.[9,10] Thus, α-helices have been
commonly used as a template for the design of inhibi-
tors.[10–15] Within the foldamer[16,17] family,[18–24] a number
of ligands, including β peptides,[25,26] mixed α/β pept-
ides,[27–29] peptoids[30] and constrained peptides,[31–34] have
been used to target α-helix-mediated PPIs. Proteomimetics
have emerged from this family as inhibitors of PPIs by
matching the special orientation of key binding residues on
the native α-helix.[35,36] Within the proteomimetic family,
[a] School of Chemistry, University of Leeds,
Woodhouse Lane, Leeds, LS2 9JT, UK
Fax: +44-113-3431409
In our initial screening of the 3-O-alkylated series[44] we
identified a trimer bearing benzyl, naphthyl and isopropyl
binding residues (Figure 1) to be an effective inhibitor of
the p53–hDM2 interaction. A significant drawback of this
class of inhibitor is its poor aqueous solubility, which has
limited further structural studies and could be problematic
for further biological studies. A strategy elaborated by sev-
eral other groups working on proteomimetics has been to
design scaffolds that possess a “wet-edge” along the non-
E-mail: A.J.Wilson@leeds.ac.uk
Homepage: http://www.chem.leeds.ac.uk/andrew-wilson/
wilson-group.html
[b] Astbury Centre for Structural and Molecular Biology,
University of Leeds,
Woodhouse Lane, Leeds, LS2 9JT, UK
[c] School of Molecular and Cellular Biology, University of Leeds,
Woodhouse Lane, Leeds, LS2 9JT, UK
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300069.
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Inhibitors of Protein–Protein Interactions
protein-binding face of the ligand.[54–56] In the strictest ated aromatic oligoamide inhibitor that incorporates an ad-
sense, the “wet-edge” approach has yet to be validated ditional and hydrophilic ethylene glycol chain at the 6-posi-
either because a suitable “dry-edge” comparison is absent tion (i.e., on the non-binding face) of the central monomer.
from the screening set or because inhibition studies have
not yet been reported for these scaffolds.
We describe detailed structural and conformational stud-
ies of this oligomer and show that it has the ability to mimic
p53 similarly to the unfunctionalised analogue but has im-
proved solubility.
Results and Discussion
Synthesis
The introduction of solubilising groups onto an existing
scaffold necessitated the design of a novel bifunctional
monomer unit. The strategy chosen was based on a scaffold
and synthetic methodology recently reported by Ahn and
co-workers who envisioned that use of such a monomer
could allow four side-chain functional groups at the i, i +
2, i + 5 and i + 7 positions of an α-helix to be mimicked.[57]
Use of this building block has the advantage of preserving
the O-alkylation procedure previously described by our
group,[53] the products of which have been demonstrated to
be effective PPI inhibitors.[44] The monomer was synthe-
sised as shown in Scheme 1. Initially, 2-hydroxy-4-nitro-
benzoic acid was hydroxylated selectively ortho to the nitro
group by using an established methodology.[57,58] This step
could also be performed starting from 3-hydroxy-4-nitro-
benzoic acid giving comparable yields. Dihydroxylated
nitrobenzoic acid 3 was then ketal-protected and sub-
sequently subjected to the standard O-alkylation procedure
developed in our laboratory.[53] The protecting group was
Figure 1. Schematic representation of the p53 helix with “hot-spot”
residues highlighted along with the chemical structure of an oli-
goamide inhibitor of the p53–hDM2 interaction (1) and the modi-
fied “wet-edge” inhibitor (2).
We envisioned that modification of our scaffolds along then readily removed with sodium methoxide to afford a
these lines would lead to more desirable properties. Herein second free hydroxy group for functionalisation with a
we describe the design and synthesis of a novel 3-O-alkyl- tetraethylene glycol chain by using standard Mitsunobu
Scheme 1. Synthesis of the bifunctional O-alkylated aromatic monomer 8.
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A. J. Wilson et al.
FULL PAPER
Scheme 2. Synthesis of the trimer 2.
conditions. The final monomer 8 was obtained by hydroly- studies on 2- and 3-O-alkylated trimers and model dimers
sis of the methyl ester.
by our group[46,52] revealed that intramolecular hydrogen
The procedure for the synthesis of the trimer is shown bonding between the NH and adjacent O-alkyl group gives
in Scheme 2 and follows our previously described methods pseudo-six- or five-membered rings with restricted rotation
involving iterative coupling followed by reduction of nitro- around the relevant aryl–amide bond. The other aryl–
masked monomers. The key steps involve monomer cou- amide bond remains free to rotate. Of concern to us here
pling between 8 and 9 with Ghosez’s reagent by in situ for- was the fact that intramolecular hydrogen bonding from the
mation of an acyl chloride intermediate. The nitro group of central amide NH could occur to the phenolic oxygen on
10 was converted into an amine group by reduction with both adjacent monomers. Such a “bifurcated” hydrogen-
sodium borohydride in the presence of cobalt(II) chlor- bonding interaction has been proposed and extensively
ide.[59] This dimer 11 then underwent coupling as above to studied by Gong and co-workers.[60,61] Although the simul-
afford 13 and then trimer 2 after deprotection of both the taneous formation of two non-covalent interactions
nitro and methyl ester groups. The ethylene glycol chain was through a single hydrogen might seem unlikely, the ability
found to be fully compatible with all the reaction conditions to form either interaction alongside the repulsive interac-
employed in the trimer synthesis demonstrating the versatil- tion between the phenolic oxygen and carbonyl group that
ity of this functionalisation strategy. Note, however, that is possible for alternative conformers may still render the
standard tin(II) chloride mediated nitro group reduction re- preferred conformation as one in which the C-terminal iso-
sulted in some cleavage of the ethylene glycol chain and that propyloxy and central PEG groups are located on the same
reduction in the absence of cobalt(II) chloride gave a mix- face. Such a conformation would be unproductive for bind-
ture of products resulting from partial reduction of the es- ing to hDM2. Against this, we postulated that intermo-
ter.
lecular interactions with hDM2 might bias the preference
towards the conformer presenting all three hDM2 binding
groups on one face (see the next section). In this regard it
is noteworthy that we previously found evidence that N-
Structural Studies
We performed detailed structural and conformational alkylated p-benzamides adopt the trans rotamer overriding
analysis on trimer 2 by using 1D and 2D NMR experiments the intrinsic cis preference[62] about the amide bond in the
alongside single-crystal diffraction studies on a number of presence of hDM2.[47] Furthermore, in silico studies have
intermediates (10 and 13) en route to trimer 2. Previous indicated that for model compounds even the S(6) hydrogen
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Inhibitors of Protein–Protein Interactions
bond in o-alkoxybenzamides can be broken, particularly in indicated similar conformational properties and confirmed
more polar solvent.[63,64] The crystal structures of 10 and the intramolecular nature of the NOEs (see the Supporting
13 (Figure 2, a,b) show the oligomers adopting undesired Information). These data therefore imply that the desired
conformations in which the central NH resides between the conformation is thermodynamically accessible even in the
phenolic oxygens appended to the C3 and C6 positions of absence of protein.
adjacent monomers forming pseudo-five- and six-mem-
bered rings with a longer and therefore weaker interaction
Binding Analysis
to the former. To our surprise, 1H–1H NOESY analysis
(Figure 2c) of 13 indicated that free rotation around both
the aryl–CO and aryl–NH axes occurs, as evidenced by the
presence of correlations between 3-NH and 3-C5 and be-
tween 3-NH and 2-C2. Similar unrestricted rotation around
the N-terminal amide is also observed. 1H–1H NOESY
analyses on trimer 2 at concentrations of 10, 5, and 2 mm
The ability of the bifunctionalised trimer 2 to inhibit the
p53–hDM2 interaction was measured by means of a fluo-
rescence anisotropy assay (Figure 3).[65] A fluorescently lab-
elled analogue of p53 can be displaced from the binding
site in hDM2 by an inhibitor leading to a characteristic de-
crease in anisotropy. Upon direct titration with unlabelled
peptide (positive control), an IC50 value of 1.35 μm was
measured (Table 1). Proteomimetic 1 (Figure 1) recorded an
IC50 of 4.15 μm. The binding of the new PEGylated ana-
logue gave a similar value of 7.54 μm. This clearly demon-
strates that functionalisation of the non-binding face of the
α-helix mimetic has little effect on its binding behaviour
towards the target protein. Furthermore, trimer 2 is signifi-
cantly more soluble in the assay buffer (see the Supporting
Information for qualitative solubility studies) than the orig-
inal unfunctionalised derivative, which demonstrates that
this is a viable strategy for conferring additional, advan-
tageous properties on these molecules.
Figure 3. Fluorescence anisotropy competition titration data
(40 mm sodium phosphate buffer pH 7.5, 54 nm p5315–31Flu, 42 nm
hDM2). Displacement by native peptide (black squares), trimer 1
(dark-grey circles) and PEGylated trimer 2 (light-grey triangles).
Table 1. Dissociation constants determined by the fluorescence an-
isotropy displacement assay.[a]
Compound
IC50 [μm]
p53
1
2
1.35 Ϯ 0.09
4.15 Ϯ 0.2
7.54 Ϯ 0.37
Figure 2. Structural characterisation of compounds 10 and 13.
(a) Solid-state structure of compound 10 (carbon in grey, hydrogen
in white, nitrogen in blue and oxygen in red) illustrating S(5) and
S(6) intramolecular hydrogen bonding to the central amide. (b) So-
lid-state structure of compound 13 illustrating S(5) intramolecular
[a] Conditions as indicated in Figure 3.
hydrogen bonding of the N-terminal amide and S(5) and S(6) intra- Conclusions
molecular hydrogen bonding of the C-terminal amide. (c) 1H–1H
NOESY spectra (aromatic region, 500 MHz, CDCl3) illustrating
We have described the design and synthesis of a novel
key NOEs and thus the free rotation around both ArCONH axes. bifunctional monomer that can be readily incorporated into
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A. J. Wilson et al.
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6-Hydroxy-2,2-dimethyl-7-nitro-4H-benzo[d][1,3]dioxin-4-one
(4):
2,5-Dihydroxy-4-nitrobenzoic acid (3; 1.57 g, 7.89 mmol) was sus-
pended in TFA (10 mL) and TFAA (7.7 mL, 55.2 mmol) was added
followed by acetone (4.1 mL, 55.2 mmol). The solution was heated
at reflux at 80 °C for 5 d. The solvent was then removed in vacuo
and the crude product purified by column chromatography (SiO2,
4:1 hexane/EtOAc) to afford the desired product as an orange solid
(354 mg, 1.48 mmol, 19%). Rf = 0.24 (4:1 hexane/EtOAc). 1H
NMR (500 MHz, MeOD): δ = 7.64 (s, 1 H, ArCH), 7.63 (s, 1 H,
ArCH), 1.73 (s, 6 H, CH3) ppm. 13C NMR (75 MHz, MeOD): δ
= 160.8, 149.0, 147.0, 121.9, 120.6, 114.7, 113.1, 108.9, 25.9 ppm.
HRMS: calcd. for C10H8NO6 238.0357 [M – H]–; found 238.0364.
an aromatic oligoamide trimer capable of acting as a prote-
omimetic. When tested in a fluorescence anisotropy compe-
tition assay, this novel α-helix mimetic was found to be cap-
able of inhibiting the p53–hDM2 PPI with an affinity com-
parable to that of the originally developed non-function-
alised analogue. The solubilising group was found to have
little impact on binding affinity, which indicates that this
type of orthogonal functionalisation is a promising tool for
improving the properties of α-helix mimetics.
Experimental Section
IR: νmax = 3244 (s, O–H), 3070 (m, Ar C–H), 1736 (s, C=O), 1589
˜
(s), 1446 (s, N–O), 1371 (m), 1294 (m), 1085 (m), 1020 (m) cm–1.
C10H9NO (159.19): calcd. C 50.22, H 3.79, N 5.86; found C 50.30,
H 3.75, N 5.60.
General: All chemicals and solvents were purchased and used with-
out further purification. 1H, 13C and 2D NMR Spectra were re-
corded with a Bruker DRX 500 MHz or DPX 300 MHz spectrom-
eter. 1H NMR spectra are referenced to tetramethylsilane (TMS)
and chemical shifts are given as parts per million downfield from
TMS. Coupling constants are reported to the nearest 0.1 Hz.
Microanalyses were obtained on a Carlo Erba Elemental Analyser
MOD 1106 instrument. IR spectra were recorded with a Perkin–
Elmer FTIR spectrometer and samples were analysed in the solid
phase. Mass spectra were obtained with a Bruker microTOF spec-
trometer using electrospray ionisation. Analytical TLC was per-
formed on 0.2 mm silica gel 60 F254 precoated aluminium sheets
(Merck) and visualised by using UV irradiation or, in the case of
amine intermediates, by staining with ninhydrin solution. Flash
chromatography was carried out on silica gel 60 (35–70 micron par-
ticles, FluoroChem). Expression of hDM2 and fluorescence
anisotropy assays were performed as described previously.[46] The
convention used to assign the spectroscopic data for this series of
aromatic oligoamides has been described previously.[52] Com-
pounds 9 and 12 were obtained as described previously.[44,53]
2,2-Dimethyl-6-(2-naphthylmethoxy)-7-nitro-4H-benzo[d][1,3]-
dioxin-4-one (5): 6-Hydroxy-2,2-dimethyl-7-nitro-4H-benzo[d][1,3]-
dioxin-4-one (4; 960 mg, 4.02 mmol) was dissolved in anhydrous
DMF (20 mL) and K2CO3 (2.77 g, 20.08 mmol) was added fol-
lowed by 2-(bromomethyl)naphthalene (1.24 g, 5.62 mmol) and the
reaction was stirred overnight at 50 °C under nitrogen. The solu-
tion was then poured into EtOAc (150 mL) and washed with H2O
(2ϫ 50 mL) and brine (50 mL). The organic phase was dried with
Na2SO4, filtered and the filtrate evaporated. The crude product was
purified by column chromatography (SiO2, 85:15 hexane/EtOAc)
to afford the desired product as a pale-yellow solid (1.5 g,
3.96 mmol, 98 %). Rf = 0.14 (85:15 hexane/EtOAc). 1H NMR
(500 MHz, CDCl3): δ = 7.90–7.84 (m, 4 H, CH naph), 7.77 (s, 1
H, ArCH), 7.54–7.49 (m, 3 H, CH naph), 7.39 (s, 1 H, ArCH),
5.38 (s, 2 H, CH2 naph), 1.74 (s, 6 H, CH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 159.4, 149.2, 146.3, 133.2, 133.2, 132.3,
128.7, 128.1, 127.7, 126.4, 126.4, 124.7, 117.0, 115.4, 114.3, 107.6,
2,5-Dihydroxy-4-nitrobenzoic Acid (3) from 2-Hydroxy-4-nitrobenz-
oic Acid: Compound 3 was synthesised following minor modifica-
tions to the published method.[57] 2-Hydroxy-4-nitrobenzoic acid
(17.5 g, 95 mmol) was dissolved in aqueous NaOH (2 m, 200 mL)
and K2S2O8 (19.25 g, 71 mmol) dissolved in H2O (400 mL) was
then added and the reaction mixture was stirred at room tempera-
ture for 4 d. The reaction mixture was then cooled in an ice bath
and acidified with conc. H2SO4. The solution was washed with
Et2O (3ϫ 400 mL) to recover unreacted starting material and the
aqueous layer was heated at reflux for 1 h. After cooling to room
temperature the solution was refrigerated for 2 d and the resulting
brown precipitate was isolated by filtration and dried in vacuo to
afford the desired product (8.77 g, 44 mmol, 46%). Rf = 0.12 (9:1
DCM/MeOH). 1H NMR (500 MHz, [D6]DMSO): δ = 7.51 (s, 1 H,
ArCH), 7.40 (s, 1 H, ArCH) ppm. 13C NMR (75 MHz, [D6]-
DMSO): δ = 169.8, 152.0, 142.8, 141.3, 119.3, 118.8, 112.2 ppm.
72.1, 25.7 ppm. IR: ν
= 3075 (br s, C–H), 1728 (s, C=O), 1632
˜
max
(s), 1591 (m), 1538 (s, N–O), 1487 (m), 1434 (s), 1350 (m), 1222 (m),
1126 (w), 1088 (s), 1026 (s) cm–1. HRMS: calcd. for C21H17NNaO6
402.0948 [M + Na]+; found 402.0955. C21H17NO6 (379.37): calcd.
C 66.49, H 4.52, N 3.69; found C 66.1, H 4.60, N 3.65.
Methyl 2-Hydroxy-5-(2-naphthylmethoxy)-4-nitrobenzoate (6): 2,2-
Dimethyl-6-(2-naphthylmethoxy)-7-nitro-4H-benzo[d][1,3]dioxin-4-
one (160 mg, 0.42 mmol) was suspended in MeOH (10 mL) and
sodium methoxide in methanol (0.5 mL, 25%) was added and the
reaction mixture stirred overnight at room temperature. The sol-
vent was removed in vacuo and the residue taken up in EtOAc
(50 mL) and washed with HCl (2ϫ 30 mL, 1 m) and H2O (30 mL).
The organic layer was dried with Na2SO4, filtered and the filtrate
evaporated to afford the desired product as a dark-yellow solid
(137.5 mg, 0.39 mmol, 93 %). Rf = 0.78 (9:1 DCM/MeOH). 1H
NMR (500 MHz, CDCl3): δ = 10.36 (s, 1 H, OH), 7.76–7.72 (m, 4
H, CH naph), 7.48 (s, 1 H, ArCH), 7.42–7.37 (m, 3 H, CH naph),
7.26 (s, 1 H, ArCH), 5.18 (s, 2 H, CH2 naph), 3.86 (s, 3 H,
CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 168.8, 155.4, 143.3,
133.1, 132.9, 128.6, 128.0, 127.7, 126.3, 126.3, 124.8, 116.5, 115.4,
IR: ν
= 3306s (O–H), 3071 (m, Ar C–H), 1695 (s, C=O), 1627
˜
max
(s), 1437 (s, N–O), 1311 (m), 1221 (m), 1036 (m) cm–1. HRMS:
calcd. for C7H5NO6 198.0044 [M – H]–; found 198.0048.
2,5-Dihydroxy-4-nitrobenzoic Acid (3) from 3-Hydroxy-4-nitrobenz-
oic Acid: 3-Hydroxy-4-nitrobenzoic acid (25 g, 137 mmol|) was dis-
solved in aqueous NaOH (2 m, 500 mL) and K2S2O8 (37 g,
137 mmol) dissolved in H2O (750 mL) was then added and the re-
action mixture stirred at room temperature for 4 d. The reaction
mixture was then cooled in an ice bath and strongly acidified with
conc. H2SO4. The resulting precipitate was removed by filtration.
The aqueous solution was heated at reflux for 1 h. After cooling
to room temperature the resulting precipitate was isolated by fil-
tration and dried in vacuo to afford the desired product (9.23 g,
46.4 mmol, 34%). For the characterisation data, see above.
114.1, 72.3, 53.0 ppm. IR: ν
= 3229 (s, O–H), 2859 (m, C–H),
˜
max
1701 (s, C=O), 1626 (m), 1527 (m, N–O), 1493 (m), 1437 (m), 1340
(s), 1263 (s), 1198 (s), 1102 (m), 1040 (m), 1003 (m) cm–1. HRMS:
calcd. for C19H15NNaO6 376.0792 [M + Na]+; found 376.0798.
C19H15NO6 (353.33): calcd. C 64.59, H 4.28, N 3.96; found C
64.65, H 4.55, N 3.65.
Methyl 2{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}-5-(2-naphthyl-
methyoxy)-4-nitrobenzoate (7): Methyl 2-hydroxy-5-(2-naphth-
ylmethoxy)-4-nitrobenzoate (6; 52.5 mg, 0.15 mmol) and triethyl-
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Inhibitors of Protein–Protein Interactions
ene glycol monomethyl ether (26 μL, 0.16 mmol) were dissolved in
anhydrous THF (10 mL) and cooled in an ice bath. Triphenylphos-
phane (58.4 mg, 0.22 mmol) and diisopropyl azodicarboxylate
(DIAD; 44 μL, 0.22 mmol) were then added and the reaction mix-
ture was warmed to room temperature and stirred overnight under
nitrogen. The solvent was then removed in vacuo and the crude
product purified by column chromatography (SiO2, 3:2 hexane/
EtOAc) to afford the desired product as a yellow solid (57.8 mg,
(m, 2 H, CH naph), 5.44 (s, 2 H, CH2 naph), 4.74 [sept, J = 6 Hz,
1 H, CH(CH3)2], 4.47 (t, J = 4.5 Hz, 2 H, CH2OAr), 3.91–3.89 [m,
5 H, CH3OC(O), CH2CH2OC], 3.59 (m, 2 H, CH2CH2OCH3), 3.49
(t, J = 4.5 Hz, 4 H, CH2O), 3.44–3.42 (m, 2 H, CH2OCH3), 3.31
(s, 3 H, CH3OCH2), 1.40 (d, J = 6 Hz, 6 H, CH3CH) ppm. 13C
NMR (125 MHz, CDCl3): δ = 166.7, 161.3, 150.0, 146.5, 146.3,
141.6, 133.2, 133.1, 133.0, 132.8, 128.5, 128.1, 127.8, 127.7, 126.3,
126.3, 126.2, 125.7, 124.9, 123.1, 120.2, 118.9, 113.7, 113.1, 71.9,
0.12 mmol, 77 %). Rf = 0.13 (1:1 hexane/EtOAc). 1H NMR 71.8, 71.8, 71.2, 70.9, 70.5, 70.4, 69.4, 58.9, 52.1, 22.2 ppm. IR:
(500 MHz, CDCl3): δ = 7.88–7.83 (m, 4 H, CH naph), 7.59 (s, 1 max = 3332 (s), 2923 (s, C–H), 1716 (s, C=O), 1674 (m), 1597 (m),
H, ArCH), 7.53–7.47 (m, 4 H, CH naph, ArCH), 5.33 (s, 2 H, CH2 1529 (m), 1486 (w), 1451 (w), 1420 (w), 1344 (m), 1291 (w), 1218
ν
˜
naph), 4.19–4.17 (m, 2 H, CH2OAr), 3.89 (s, 3 H, CH3), 3.87–3.85
(m), 1111 (m), 1032 (m) cm–1. HRMS: calcd. for C36H40N2NaO11
(m, 2 H, CH2CH2OAr), 3.74–3.72 (m, 2 H, CH2CH2OCH3), 3.66– 699.2524 [M + Na]+; found 699.2548.
3.62 (m, 4 H, CH2O), 3.54–3.52 (m, 2 H, CH2OCH3), 3.35 (s, 3 H,
Methyl 4-(4-Amino-2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-5-(2-
CH3OCH2) ppm. 13C NMR (125 MHz, CDCl3): δ = 164.9, 151.8,
145.2, 142.0, 133.1, 132.7, 128.0, 127.9, 127.6, 126.3, 126.2, 124.8,
118.7, 111.7, 72.1, 71.8, 70.9, 70.6, 70.4, 70.2, 69.5, 58.9, 52.5 ppm.
HRMS: calcd. for C26H29NNaO9 522.1735 [M + Na]+; found
naphthylmethoxy)benzamido)-3-isopropoxybenzoate (11): Methyl 3-
isopropoxy-4-(2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-5-(2-
naphthylmethoxy)-4-nitrobenzamido)benzoate (10; 50 mg,
0.07 mmol) was dissolved in MeOH/CHCl3 (2:1, 12 mL) and
CoCl2·6H2O (53 mg, 0.22 mmol, 3 equiv.) was added. NaBH4
(17 mg, 0.44 mmol, 6 equiv.) was then added slowly and the reac-
tion mixture was stirred at room temperature for 30 min. Further
CoCl2·6H2O (53 mg, 0.22 mmol, 3 equiv.) was then added followed
by NaBH4 (17 mg, 0.44 mmol, 6 equiv.) and the reaction mixture
522.1732. IR: ν
= 2878 (s, C–H), 1741 (s, C=O), 1529 (s, N–
˜
max
O), 1427 (m), 1350 (s), 1240 (s), 1210 (s), 1107 (s), 1085 (s), 1055
(m) cm–1.
2-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}-5-(2-naphthylmethoxy)-
4-nitrobenzoic Acid (8): Methyl 2-{2-[2-(2-methoxyethoxy)ethoxy]-
ethoxy}-5-(2-naphthylmethoxy)-4-nitrobenzoate (7; 929 mg, was stirred at room temperature for a further 30 min. The solution
1.86 mmol) was dissolved in MeOH/THF (1:1, 50 mL) and aque-
ous NaOH (2 m, 10 mL) was added and the reaction mixture stirred
at 65 °C for 5 h. The solution was then neutralised with HCl (1 m)
and the organic solvents removed under reduced pressure. The
product was extracted with EtOAc (3ϫ 20 mL). The organic phase
was dried with Na2SO4, filtered and the filtrate evaporated to af-
ford the product as a pale-yellow solid (900 mg, 1.85 mmol, 99%).
1H NMR (500 MHz, CDCl3): δ = 7.94 (s, 1 H, ArCH), 7.90–7.81
was filtered through Celite, diluted with DCM (30 mL) and washed
with HCl (1 m, 2ϫ 30 mL) and H2O (30 mL). The organic phase
was dried with Na2SO4, filtered and the filtrate removed in vacuo
to afford the product as an orange oil (48.5 mg, 0.07 mmol, 100%).
Rf = 0.2 (3:2 EtOAc/hexane). 1H NMR (500 MHz, CDCl3): δ =
10.48 (s, 1 H, NH), 8.70 (d, J = 8.5 Hz, 1 H, 5-H2), 7.88–7.83 (m,
5 H, CH naph, 5-H1), 7.71–7.68 (dd, J = 8.5, J = 1.5 Hz, 1 H, 6-
H2), 7.59 (d, J = 1.5 Hz, 1 H, 2-H2), 7.55–7.53 (dd, J = 7.8, J =
(m, 4 H, CH naph), 7.54–7.47 (m, 4 H, CH naph, ArCH), 5.38 (s, 1.25 Hz, 1 H, CH naph), 7.49–7.47 (m, 2 H, CH naph), 6.55 (s, 1
2 H, CH2 naph), 4.34–4.33 (m, 2 H, CH2OAr), 3.89–3.88 (m, 2 H, H, 5-H1), 5.28 (s, 2 H, CH2 naph), 5.27 (s, 2 H, NH2), 4.70 [sept,
CH2CH2OAr), 3.72–3.71 (m, 2 H, CH2CH2OCH3), 3.66–3.61 (m, J = 6 Hz, 1 H, CH(CH3)2], 4.36 (t, J = 5 Hz, 2 H, CH2OAr), 3.90
4 H, CH2O), 3.54–3.52 (m, 2 H, CH2OCH3), 3.36 (s, 3 H, [s, 3 H, CH3OC(O)], 3.85 (t, J = 5 Hz, 2 H, CH2CH2O), 3.60 (t, J
CH3OCH2) ppm. 13C NMR (125 MHz, CDCl3): δ = 164.1, 150.6,
146.3, 142.5, 133.2, 132.5, 128.6, 128.0, 127.7, 126.5, 126.3, 124.8,
119.8, 111.6, 72.0, 71.8, 70.7, 70.5, 70.4, 70.4, 68.5, 58.9 ppm. IR:
= 5 Hz, 2 H, CH2CH2OCH3), 3.52–3.49 (m, 4 H, CH2O), 3.46–
3.45 (m, 2 H, CH2OCH3), 3.32 (s, 3 H, CH3OCH2), 1.40 (d, J =
6 Hz, 6 H, CH3CH) ppm. 13C NMR (125 MHz, CDCl3): δ = 166.9,
152.8, 146.2, 141.7, 141.0, 134.5, 134.2, 133.2, 133.0, 128.3, 127.9,
ν
max
= 3218 (br, O–H), 2871 (s, C–H), 1725 (s, C=O), 1625 (m),
˜
1605 (w), 1584 (m), 1538 (m), 1495 (m), 1461 (m), 1416 (m), 1394 127.6, 126.7, 126.2, 126.1, 125.6, 124.2, 123.3, 119.8, 114.3, 113.9,
(w), 1378 (w), 1344 (m), 1286 (m), 1209 (m), 1128 (s), 1039
(m) cm–1. HRMS: calcd. for C25H27NNaO6 508.1578 [M + Na]+;
found 508.1600.
111.8, 101.4, 71.9, 71.7, 71.0, 70.7, 70.4, 70.4, 69.4, 58.9, 51.9,
22.2 ppm. IR: νmax = 3344 (s, N–H), 2919 (s, C–H), 1712 (s, C=O),
˜
1659 (m, C=O), 1593 (s), 1514 (s, Ar C=C), 1486 (s), 1436 (s), 1343
(m), 1289 (s), 1265 (s), 1209 (s), 1107 (s), 1035 (m), 1003 (m) cm–1.
HRMS: calcd. for C36H42N2NaO9 669.2783 [M + Na]+; found
669.2786.
Methyl 3-Isopropoxy-4-(2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-
5-(2-naphthylmethoxy)-4-nitrobenzamido)benzoate (10): 2-{2-[2-(2-
Methoxyethoxy)ethoxy]ethoxy}-5-(2-naphthylmethoxy)-4-nitro-
benzoic acid (8; 50 mg, 0.1 mmol) was dissolved in anhydrous
CHCl3 (5 mL) and preactivated with Ghosez’s reagent (204 μL,
Methyl 4-(4-[3-(Benzyloxy)-4-nitrobenzamido]-2-{2-[2-(2-methoxy-
ethoxy)ethoxy]ethoxy}-5-(2-naphthylmethoxy)benzamido)-3-iso-
0.3 mmol, 20% in CHCl3) for 3 h at room temperature. Methyl 4- propoxybenzoate (13): 3-(Benzyloxy)-4-nitrobenzoic acid (12;
amino-3-isopropoxybenzoate (73.2 mg, 0.35 mmol) was dissolved
in anhydrous CHCl3 (5 mL) and N,N-diisopropylethylamine (DI-
PEA) was added (125 μL, 0.72 mmol). The solution containing the
acid chloride was then added and the reaction mixture stirred over-
night at room temperature under nitrogen. The solvent was then
removed and the crude product purified by column chromatog-
raphy (SiO2, 1:1 EtOAc/hexane) to afford the desired product as a
yellow solid (45 mg, 0.067 mmol, 67%). Rf = 0.17 (1:1 EtOAc/hex-
ane). 1H NMR (500 MHz, CDCl3): δ = 10.46 (s, 1 H, NH), 8.64
64 mg, 0.23 mmol) was suspended in anhydrous CHCl3 (5 mL) and
Ghosez’s reagent (31 μL, 0.23 mmol) was then added. The solution
was stirred at room temperature for 10 min to allow the acid chlor-
ide to form. Methyl 4-(4-amino-2-{2-[2-(2-methoxyethoxy)ethoxy]-
ethoxy}-5-(2-naphthylmethoxy)benzamido)-3-isopropoxybenzoate
(11; 150 mg, 0.23 mmol) was dissolved in anhydrous CHCl3
(10 mL) and DIPEA (80 μL, 0.46 mmol) was added. This solution
was then added to the acid chloride and the reaction mixture stirred
overnight at room temperature under nitrogen. The solvent was
(d, J = 8.5 Hz, 1 H, 5-H2), 8.14 (s, 1 H, 5-H1), 7.94 (s, 1 H, CH removed in vacuo and the crude product purified by column
naph), 7.88–7.82 (m, 3 H, CH naph), 7.79 (s, 1 H, 2-H1), 7.70–7.68
(dd, J = 8.5, J = 1.5 Hz, 1 H, 6-H2), 7.59 (d, J = 1.5 Hz, 1 H, 2-
H2), 7.56–7.54 (dd, J = 8.5, J = 1.5 Hz, 1 H, CH naph), 7.47–7.45
chromatography (SiO2, 3:2 EtOAc/hexane) and crystallised by
diffusing hexane into a solution of the compound dissolved in
EtOAc to afford the pure product as a yellow solid (134 mg,
Eur. J. Org. Chem. 2013, 3504–3512
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3509

A. J. Wilson et al.
FULL PAPER
0.15 mmol, 64 %). Rf = 0.39 (3:2 EtOAc/hexane). 1H NMR Methyl Ester Deprotection: Amino ester trimer methyl 4-(4-[4-
(500 MHz, CDCl3): δ = 10.57 (s, 1 H, NH), 8.75 (s, 1 H, NH), 8.67 amino-3-(benzyloxy)benzamido]-2-{2-[2-(2-methoxyethoxy)-
(d, J = 8.5 Hz, 1 H, 5-H3), 8.46 (s, 1 H, 5-H2), 7.99 (s, 1 H, 2-H1),
7.86 (s, 1 H, 2-H2), 7.82 (d, J = 8 Hz, 1 H, 5-H1), 7.78–7.75 (m, 2
ethoxy]ethoxy}-5-(2-naphthylmethoxy)benzamido)-3-isopropoxy-
benzoate (113 mg, 0.13 mmol) was dissolved in MeOH/THF (1:1,
H, CH naph), 7.69–7.67 (m, 2 H, 6-H3, CH naph), 7.59 (s, 1 H, 2- 20 mL) and NaOH (2 mL, 2 m) was added. The reaction mixture
H3), 7.58 (s, 1 H, CH naph), 7.50 (d, J = 8 Hz, 1 H, CH naph),
7.47–7.45 (m, 2 H, CH naph), 7.36–7.29 (m, 5 H, CH benzyl), 7.18
was stirred at room temperature for 2 d and then neutralised with
HCl (1 m). The volatile solvent was then removed in vacuo and the
(d, J = 8 Hz, 1 H, 6-H1), 5.31 (s, 2 H, CH2 naph), 5.07 (s, 2 H, product extracted with EtOAc (2ϫ 40 mL). The organic phase was
CH2 benzyl), 4.71 [sept, J = 6 Hz, 1 H, CH(CH3)2], 4.51 (t, J =
dried with Na2SO4, filtered and the filtrate evaporated to afford
5 Hz, 2 H, CH2OC), 3.91 (t, J = 5 Hz, 2 H, CH2CH2OC), 3.88 [s, the desired product as an orange solid (90 mg, 0.1 mmol, 79%). Rf
3
1
3 H, CH3OC(O)], 3.59 (t, J = 5 Hz, 2 H, CH2CH2OCH3), 3.47– = 0.41 (EtOAc). H NMR (500 MHz, CDCl3): δ = 10.56 (s, 1 H,
3
3.46 (m, 4 H, CH2O), 3.41–3.39 (m, 2 H, CH2OCH3), 3.29 (s, 3
NH), 8.70 (s, 1 H, NH), 8.65 (d, J = 8 Hz, 1 H, 5-H3), 8.48 (s, 1
H, CH3OCH2), 1.43 (d, J = 6 Hz, 6 H, CH3CH) ppm. 13C NMR
H, 5-H2), 7.90 (s, 1 H, 2-H1), 7.83 (s, 1 H, 2-H2), 7.75–7.66 (m, 4
(125 MHz, CDCl3): δ = 166.8, 162.9, 162.9, 152.0, 151.9, 146.6, H, CH naph, 6-H3), 7.58 (s, 1 H, 2-H3), 7.47 (d, J = 8 Hz, 1 H,
142.3, 142.0, 139.1, 134.8, 133.9, 133.4, 133.2, 131.9, 128.8, 128.8,
128.4, 127.9, 127.8, 127.2, 127.2, 126.7, 126.7, 125.9, 125.3, 125.0,
123.2, 120.1, 118.2, 118.1, 115.0, 114.1, 114.0, 106.5, 72.4, 72.1,
CH naph), 7.39–7.19 (m, 8 H, CH naph, CH benzyl), 7.13 (d, J =
7.5 Hz, 1 H, 6-H1), 6.55 (d, J = 7.5 Hz, 1 H, 5-H1), 5.26 (s, 2 H,
CH2 naph), 4.85 (s, 2 H, CH2 benzyl), 4.64 [t, J = 4 Hz, 1 H,
CH(CH3)2], 4.46 (s, 2 H, CH2OC), 3.85 (s, 2 H, CH2CH2OC), 3.52
(s, 2 H, CH2CH2OCH3), 3.40 (br. s, 4 H, CH2O), 3.33 (br. s, 2
71.8, 71.2, 70.8, 70.5, 70.3, 69.1, 59.0, 22.3 ppm. IR: ν
= 3426
˜
max
(m), 3333 (m), 2982 (s, C–H), 2902 (s, C–H), 1706 (s, C=O), 1678
(m, C=O), 1659 (m, C=O), 1588 (s), 1515 (m, Ar C=C), 1480 (m, H, CH2OCH3), 3.22 (s, 3 H, CH3OCH2), 1.36 (d, J = 5 Hz, 6 H,
N–O), 1416 (w), 1379 (w), 1350 (m), 1281 (w), 1257 (w), 1218 (w),
CH3CH) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.7, 165.0,
1203 (w), 1103 (m) cm–1. HRMS: calcd. for C50H51N3NaO13 163.2, 151.8, 146.4, 145.8, 141.8, 136.2, 134.5, 133.6, 133.2, 133.1,
924.3314 [M + Na]+; found 924.3316. C50H51N3O13 (901.97): calcd.
C 66.58, H 5.70, N 4.66; found C 66.30, H 5.65, N 4.40.
128.6, 128.5, 128.1, 127.9, 127.7, 127.7, 126.8, 126.4, 126.3, 125.2,
124.0, 123.9, 123.4, 120.6, 120.0, 116.5, 114.2, 114.1, 113.6, 110.6,
105.7, 72.0, 71.7, 71.7, 70.7, 70.3, 69.9, 68.9, 58.8, 22.2 ppm. IR:
4-(4-[4-Amino-3-(benzyloxy)benzamido]-2-{2-[2-(2-methoxyethoxy)-
ethoxy]ethoxy}-5-(2-naphthylmethoxy)benzamido)-3-isopropoxy-
benzoic Acid (2)
ν
= 3346 (s, N–H, O–H), 2922 (s, C–H), 1665 (s, C=O), 1589
˜
max
(s), 1506 (s, Ar C=C), 1486 (m), 1347 (m), 1252 (w), 1109 (w), 1022
(s) cm–1. HRMS: calcd. for C49H51N3NaO11 880.3416 [M + Na]+;
found 880.3459.
Nitro Group Reduction: Methyl-4-(4-[3-(benzyloxy)-4-nitrobenz-
amido]-2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-5-(2-naphthyl-
methoxy)benzamido)-3-isopropoxybenzoate (13; 98 mg,
0.11 mmol) was dissolved in MeOH/CHCl3 (2:1, 15 mL) and
CoCl2·6H2O (78 mg, 0.33 mmol, 3 equiv.) was added. NaBH4
(25 mg, 0.66 mmol, 6 equiv.) was then added slowly and the reac-
tion mixture stirred at room temperature for 30 min. Further
CoCl2·6H2O (78 mg, 0.33 mmol, 3 equiv.) was then added followed
by NaBH4 (25 mg, 0.66 mmol, 6 equiv.) and the reaction mixture
stirred at room temperature for a further 30 min. The solution was
filtered through Celite, diluted with DCM (30 mL) and washed
with HCl (1 m, 2ϫ 30 mL) and H2O (30 mL). The organic phase
was dried with Na2SO4, filtered and the filtrate removed in vacuo
to afford the product as an orange oil (99 mg, 0.11 mmol, 100%).
Single Crystal X-ray Crystallographic Studies
Crystal Data for 10: Yellow crystals of 10 were obtained from a
solvent mixture of ethyl acetate and hexane. Measurements were
carried out at 150 K with a Bruker–Nonius Apex X8 diffractometer
equipped with an Apex II CCD detector and by using graphite-
monochromated Mo-Kα radiation from a FR591 rotating anode
generator. The structure was solved by direct methods and refined
by using SHELXL-97.[66] The COOMe (C18, O18, O19, C19)
group was modelled as disordered over two equally occupied posi-
tions. The naphthyl group was modelled as disordered over two
positions in the ratio 0.72:0.28.
A
crystal of size
0.37ϫ0.14ϫ0.04 mm3 was used for data collection, 2.34 Յ θ Յ
¯
27°, triclinic space group P1, C36H40N2O11, Mr = 676.7, a =
1
Rf = 0.36 (3:2 EtOAc/hexane). H NMR (500 MHz, CDCl3): δ =
7.4105(7), b = 14.7799(15), c = 17.7449(17) Å, α = 113.771(5), β =
96.518(4), γ = 92.914(5)°, V = 1757.2(3) Å3, Z = 2, D(calcd.) =
1.279 Mg/m3, μ = 0.095 mm–1, reflections collected 51762, indepen-
dent reflections 7491, observed reflections 5329 [IϾ2σ(I)], R value
= 0.0563, wR2 = 0.0816.
10.54 (s, 1 H, NH), 8.71 (s, 1 H, NH), 8.60 (d, J = 8.5 Hz, 1 H, 5-
H3), 8.47 (s, 1 H, 5-H2), 7.90 (s, 1 H, 2-H1), 7.82 (s, 1 H, 2-H2),
7.76–7.73 (m, 2 H, CH naph), 7.67 (d, J = 7.5 Hz, 1 H, CH naph),
7.63 (d, J = 8.5 Hz, 1 H, 6-H3), 7.54 (s, 1 H, 2-H3), 7.47 (d, J =
8 Hz, 1 H, CH naph), 7.41–7.33 (m, 3 H, CH naph), 7.28–7.21 (m,
5 H, CH benzyl), 7.13 (d, J = 8 Hz, 1 H, 6-H1), 6.56 (d, J = 8 Hz,
1 H, 5-H1), 5.26 (s, 2 H, CH2 naph), 4.86 (s, 2 H, CH2 benzyl),
4.64 [sept, J = 6 Hz, 1 H, CH(CH3)2], 4.44 (t, J = 5 Hz, 2 H,
CH2OC), 3.91 [s, 5 H, CH2CH2OC, CH3OC(O)], 3.50 (t, J = 5 Hz,
CCDC-919240 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
2 H, CH2CH2OCH3), 3.37 (br. s, 4 H, CH2O), 3.33–3.32 (m, 2 Crystal Data for 13: Yellow plate-like crystals of 13 were obtained
H, CH2OCH3), 3.21 (s, 3 H, CH3OCH2), 1.35 (d, J = 6 Hz, 6 H,
CH3CH) ppm. 13C NMR (125 MHz, CDCl3): δ = 166.8, 163.2,
151.9, 146.5, 146.0, 141.8, 136.2, 134.0, 133.6, 133.1, 133.1, 132.9,
128.6, 128.5, 128.1, 127.9, 127.8, 127.7, 126.8, 126.4, 126.3, 125.2,
124.7, 123.7, 123.2, 120.6, 120.1, 116.6, 114.1, 114.0, 113.9, 110.5,
105.7, 74.1, 72.1, 71.7, 71.7, 70.7, 70.3, 69.9, 68.9, 58.8, 51.9,
by diffusing methanol into a solution of trimer 13 in THF for 2–
3 d. Measurements were carried out at 150 K on a Bruker–Nonius
Apex X8 diffractometer equipped with an Apex II CCD detector
and using graphite-monochromated Mo-Kα radiation from a
FR591 rotating anode generator. The structure was solved by direct
methods and refined using SHELXL-97[66]. Despite long exposures
22.2 ppm. IR: νmax = 3355 (s, N–H), 2924 (s, C–H), 1713 (s, C=O), and high power, the crystal diffracted weakly and consequently the
˜
1667 (s, C=O), 1592 (s), 1505 (s, Ar C=C), 1347 (m), 1255 (w), 1194 complete structure of the molecule was not determined. The
(w), 1026 (s) cm–1. HRMS: calcd. for C50H53N3NaO11 894.3572 [M
+ Na]+; found 894.3590.
–O(CH2CH2O)3CH3 chain was disordered to such an extent that
several of the most remote atoms were not resolved from the elec-
3510
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 3504–3512

Inhibitors of Protein–Protein Interactions
tron density map. A crystal of size 0.30ϫ0.17ϫ0.03 mm3 was used
for data collection, 1.96 Յ θ Յ 26.47°, orthorhombic space group
P212121, C50H51N3O13, Mr = 901.94, a = 10.0889(10), b =
10.7013(12), c = 42.258(5) Å, β = 90°, V = 4562.4(9), Z = 4,
D(calcd.) = 1.313 Mg/m3, μ = 0.095 mm–1, reflections collected
42987, independent reflections 9335, observed reflections 5491
[IϾ2σ(I)], R value = 0.1074, wR2 = 0.2762.
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Acknowledgments
This work was supported by the European Research Council [ERC-
StG-240324]. N. S. M. acknowledges the University of Leeds for
the award of a Mary & Alice Smith Ph. D. Scholarship. Prof.
Michaele J. Hardie (University of Leeds) is thanked for additional
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Received: January 14, 2013
Published Online: May 8, 2013
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