Sequences in the HSP90 promoter form G-quadruplex structures with selectivity for disubstituted phenyl bis-oxazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.07.065

The HSP90 protein is an important target in cancer. We report here that stable quadruplex DNAs can be formed from a promoter sequence in the HSP90 gene, on the basis of melting, circular and NMR studies, and show that these can be selectively targeted by

Full text of DOI:10.1016/j.bmcl.2012.07.065

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5930–5935
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sequences in the HSP90 promoter form G-quadruplex structures
with selectivity for disubstituted phenyl bis-oxazole derivatives
Stephan A. Ohnmacht, Marialuisa Micco, Vanessa Petrucci, Alan K. Todd, Anthony P. Reszka,
⇑
Mekala Gunaratnam, Marta A. Carvalho, Mire Zloh, Stephen Neidle
CRUK Biomolecular Structure Group, UCL School of Pharmacy, 29-39 Brunswick Square, WC1N 1AX London, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The HSP90 protein is an important target in cancer. We report here that stable quadruplex DNAs can be
formed from a promoter sequence in the HSP90 gene, on the basis of melting, circular and NMR studies,
and show that these can be selectively targeted by non-macrocyclic quadruplex-stabilizing phenyl bis-
oxazole derivatives. These do not bind significantly to duplex DNA and show low stabilization of the
human telomeric quadruplex. These results suggest an approach to targeting HSP90 at the DNA level.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 24 June 2012
Revised 11 July 2012
Accepted 16 July 2012
Available online 23 July 2012
Keywords:
HSP90
Quadruplex
Promoter
Small molecule
Guanine-rich sequences of nucleic acids can form a wide variety
of inter- or intramolecular higher-order structures containing re-
peats of four guanines hydrogen-bonded together, the G-quartet
motif.1 G-tract repetitive telomeric DNA and RNA sequences at
the end of eukaryotic chromosomes can form quadruplexes under
the influence of small molecules, mostly based on polycyclic het-
eroaromatic motifs.2 These telomeric quadruplex-small molecule
complexes can inhibit the action of the telomerase enzyme, which
is involved in telomere maintenance in many cancer cell types.
Since telomerase is a key driver of tumorigenesis and cellular
immortalization, this can result in anticancer effects.3 Putative
quadruplex sequences in the human genome4 occur within the
promoter regions of many genes5 and in 50-untranslated regions.6
These quadruplexes have been studied as selective targets for
down-regulating individual genes in cancer and other diseases,
using quadruplex-binding organic7 and metallo-containing8 small
molecules to stabilize the target quadruplex and hence to elicit a
biological response. Down-regulation of transcription has been
demonstrated for a number of promoter quadruplexes, including
the c-myc,9a c-kit9b and src genes.9c
dressed in order to ensure that off-target effects are minimised
in any therapeutic application of quadruplex targeting. Structural
data on human genomic quadruplexes is available for telomeric
G4s10 and those from the c-myc,11 c-kit,12 bcl-2,13 hTERT14 and
RET promoters and attempts at structure-based design are cur-
rently15 restricted to using those quadruplexes. We report here
on results from an alternative library-based strategy to generate
quadruplex-selective ligands, in which we have screened a small
panel of quadruplexes using a high-throughput quadruplexes-
melting assay.
HSP90 is a well-established anticancer target at the protein le-
vel. It is involved in ensuring correct folding of many cellular pro-
teins, and plays this role in rapidly-proliferating cells, especially in
many cancers. Many small molecules have been developed as
inhibitors of the HSP90 protein and are in clinical trial.16 Resistance
to such inhibitors occurs via mutations in the ATP binding site.17
Targeting the HSP90 gene rather than the gene product offers the
possibility of a distinct profile of activity and reduced susceptibility
to resistance. HSP90 also plays a role in telomere maintenance and
telomerase regulation so HSP90 down-regulation may have a con-
sequence for these pathways.18
A number of small molecules have been identified with >103 or
more selectivity for quadruplexes over genomic duplex DNA.2 The
basis for selectivity has not generally been explored but may arise
as a result of differences in steric requirements between duplex
and quadruplexes. However selectivity for one quadruplex over an-
other remains a largely unsolved challenge that needs to be ad-
A search of the human genome using the Ensemble database
and in-house software shows that the sequence occurs 77
nucleotides upstream of the transcription start-site of the HSP90
gene and contains the sp1 promoter consensus sequence. This sug-
gests a functional role for the sequence. The presence of five short
guanine tracts (highlighted in red) in the sequence also suggest
⇑
Corresponding author. Tel.: +44 207 753 5969.
E-mail address: s.neidle@ucl.ac.uk (S. Neidle).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.065

S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5930–5935
5931
that either it is polymorphic, capable of forming several inter-re-
lated quadruplexes, or that it corresponds to a single structurally
complex quadruplex.
UV & CD spectra of annealed HSp90A/B/C at 23 oC
a
in 200mM K+ Phosphate buffer, 10mm cell pathlength
1.5
Analogy with the c-myc promoter sequence,7,11 which similarly
contains >4 short G-tracts, suggests that the former is more likely.
We have examined the full-length sequence and two shorter, over-
lapping ones, each with four G-tracts (see below). Spectroscopic
studies demonstrate that these are quadruplex-forming sequences
in ionic conditions analogous to those in cells.
HSp90A
HSp90B
HSp90C
1.0
0.5
Quadruplexes in solution have distinct circular dichroism (CD)
spectra depending on their topologies. Parallel-folded quadruplex-
es show a maximum positive signal at 260 nm with a negative sig-
nal at 240 nm. Anti-parallel topologies produce a positive signal at
295 nm with a negative peak at 260 nm. Two 21-mer G-rich sub-
sections (HSP90A and B)
0.0
40
35
30
25
20
15
10
5
and the full sequence (HSP90C) were analyzed in K+ and Na+ phos-
phate-buffer at 10 mM and 200 mM concentrations. All three se-
quences have absorption properties consist with quadruplex
topologies.
Both sub-sequences and the complete 27-mer sequence
(HSP90C) form quadruplex structures in high K+ concentration,
with CD spectra indicative of mixed topology: a positive signal in
the CD spectrum at 260 nm, a negative signal at 240 nm and some
features around 295 nm (Fig. 1). The HSP90B sequence showed a
single positive absorbance maximum in 200 mM Na+ buffer (pH
7.0), suggesting an anti-parallel conformation (Fig. 1b). The
HSP90 and the HSP90A sequences remain in a predominantly par-
allel arrangement under these conditions.
0
-5
-10
-15
200
250
300
Wavelength (nm)
1-D NMR experiments on all three sequences show imino re-
gion of the spectra having the characteristic signals arising from
G-quartets with patterns of imino resonances in the 11–12 ppm re-
gion, consistent with a quadruplex core comprising three stacked
G-quartets (Fig. 2) .10–12 The integration of this region indicates
that multiple conformations are probably present.
In view of the indications that the HSP90 promoter sequences
form stable quadruplex arrangements, these were included in the
panel of quadruplexes used for small-molecule screening, with a
human telomeric and c-kit12 and k-ras19 promoter quadruplex
sequences.
UV & CD spectra annealed HSp90A/B/C at 23 oC
b
in 200mM Na+ Phosphate buffer,10mm cell pathlength
1.5
HSp90A
HSp90A repeat
HSP90B annealed
HSp90C
1.0
0.5
BRACO-19 and telomestatin are well-studied and effective
quadruplex stabilizing compounds (Fig. 3) BRACO-19 has a polyh-
eteroaromatic acridine platform together with two cationic ‘han-
dles’ (pyrrolidines), which are significant contributors to
quadruplex binding.20 The hepta-oxazole telomestatin has high
quadruplex affinity and quadruplex:duplex selectivity.21 Telomest-
atin has a largely planar structure. BRACO-19 contains the acridine
planar structural motive but is not macrocylic and all three substit-
uents interact strongly with quadruplex grooves and loops.20
Telomestatin can interact only with the planar G-quartet at the
terminus of a quadruplex and thus has a stringent surface area
requirement. We have generated a focused library of phenyl
bis-oxazoles with terminal tertiary amines of the type found in
BRACO-19 and other quadruplex-binding ligands.2 The hypothesis
was that these acyclic compounds would bind to a range of quad-
ruplex structural types, analogous to acyclic ‘click’ chemistry
compounds.22
0.0
20
15
10
5
0
-5
-10
200
250
300
The side-arms were also envisioned to improve the solubility,
Wavelength (nm)
and overall drug-likeness compared to
quadruplex ligand with either triazole22 or oxazole groups. We
have utilized regioselective and atom-economic transition
a purely macrocyclic
Figure 1. CD spectra of the three HSP90 sequences, (a) in 200 mM K+ and (b) in
200 mM Na+ solution.
a

5932
S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5930–5935
Figure 2. 1-D NMR spectrum of the HSP90A sequence, taken on a Bruker Avance 500 MHz instrument. Experiments in H2O used excitation suppression of the water signal.
Samples were made up in 10 mM KCl solution with 10 mM potassium phosphate pH 7.0 buffer. Samples were annealed by heating to 90 °C followed by slow cooling over a
period of 15 h; 540
lL of sample and 60
lL of D2O were then mixed to give a strand concentration of 200
l
M with a 10% D2O content.
O
O
N
N
S
N
N
H
N
N
O
NH
O
N
O
O
Me
N
O
N
O
O
N
N
H
N
N
H
N
Me
BRACO-19
Telomestatin
Figure 3. Structures of BRACO-19 and the hepta-oxazole macrocycle telomestatin.
metal-catalyzed direct arylation approach (Fig. 4) for the synthesis
of a small library based on the phenyl bis-oxazole framework
instead of the traditional sp2–sp2 cross-coupling reactions nor-
mally used for oxazoles.23 Prior to the arylation step, 5-substituted
phenyl bis-oxazole precursor 1 was synthesized from cheap and
commercially available isophathalaldehyde using the tosylmethy-
lisocyanate-procedure.24 The meta-substituted phenyl bis-oxazole
building block 1 was then arylated twice, either with para- or
meta-substituted arylbromides or aryliodides.25 Most of the
arylhalides were freshly prepared or commercially available as
the iodides and C2-regioselective and very mild ‘on-water’ oxazole
arylation conditions were utilized.26 The synthetic procedure and
analytical data for compound 2f is given below.27 That for the other
compounds is provided in the Supplementary data.
Concentration-dependent FRET (Fluorescence Resonance
Energy Transfer) melting assays28 using doubly labelled (Fam-
Tamra) short G-rich DNA sequences revealed (Fig. 4) an
unexpected selectivity profile for the compounds in K+ buffer.
Compounds 2a–i showed significant stabilization of the two
HSP90 quadruplexes A and B, yet no stabilization of a human
quence (G4 h-tel DNA) (
of 14 °C and 23 °C at 1 and 2
plex DNA was observed for 2a–i. Even at ligand concentrations as
high as 3 M the Tm value for duplex DNA are <1 °C, showing that
compound 2f discriminates between quadruplexes. Selectivity
over duplex DNA was demonstrated using a 300-fold excess of du-
plex by a competition assay in which increasing excess of calf thy-
mus duplex DNA was titrated into the HSP90A quadruplex FRET
assay (Fig. 5).
D
Tm values for the HSP90A quadruplex
lM). No significant stabilization of du-
l
D
The selectivity of compound 2f was examined further with
other promoter quadruplex (Table 2), and showed modest stabi-
lisation of the K-ras promoter quadruplex but none with the
c-kit2 quadruplex. Compound 2f also shows low-micromolar cell
growth inhibitory activity in cancer cells,29 yet only modest
selectivity compared to its effect on normal WI38 fibroblast cells
(Table 2). Only a modest change in HSP90 expression was ob-
served in these cell lines (data not shown), suggesting that either
the HSP90 promoter quadruplexes are not readily formed, are
inaccessible to compound 2f, or possibly that the affinity for
these complexes is not sufficient for effective transcription fac-
tor/polymerase inhibition. This compound is also not an inhibitor
of human telomerase activity (unpublished observations). We
have previously reported that a structurally unrelated naphtha-
lene diimide compound has exceptionally high affinity for
telomeric quadruplex DNA sequence (Table 1). The
x-N-methyl
piperazine bis-oxazole analogue 2f, which at physiological pH is
likely to be doubly-charged, is the best compound in this series
with high selectivity for the HSP90A over HSP90B quadruplexes
and especially over the human telomeric quadruplex DNA se-
HSP90 (and other) quadruplexes,30 with a
DTm at 1.0 lM ligand

S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5930–5935
5933
Br
R
Van Leusen
TosMIC
O
O
N
N
O
O
Conditions A
H
H
1
N
N
O
O
Isophthalaldehyde
2.20£ / g
I
R
R
R
Conditions B
2a-k
N
N
N
N
N
N
N
O
N
N
O
O
O
O
O
2a
2b
2c
N
N
N
N
N
O
O
N
N
N
N
N
O
O
O
O
O
O
2d
2e: n=1
2f: n=2
2g
n
n
N
N
N
N
N
N
N
N
N
O
O
Reagents and Conditions
(a) TosMIC (2.2 eq.), K2CO3 (3 eq.), MeOH, reflux, 3 hrs
Conditions A
2h: n=1
2i: n=2
2k: n=3
(b)
: sealed tube, K2CO3 (2.5 eq.), PivOH (20 % mol),
Pd(OAc)2 (10 % mol), Ru-Phos (20 % mol), toluene, 110 °C, 36 hrs
(c) Conditions B: sealed tube, Ag2CO3 (2.2 eq.), PPh3 (20 % mol),
Pd(dppf)Cl2xDCM (10 % mol), H2O, 60 °C, 36 hrs
Note: 2d-2f and 2h were synthesized using conditions A
O
O
n
n
N
N
Figure 4. Structures (2a–k) of the final compounds generated via the double direct arylation of the C2-position of phenyl bis-oxazole 1.
Table 1
Melting data for compounds 2a–k with the human telomeric and HSP90 quadruplexes. Average esds are 0.1 °C from triplicate experiments
Compd
G4 h-tel DNA
mol
HSP-90A
HSP-90B
ds DNA
1
2
<2
3
<2
<2
<2
<2
<2
<2
2
l
2
l
mol
1
lmol
2
l
mol
1
lmol
2
l
mol
1
lmol
2
lmol
2a
2b
2c
2d
2e
2f
2g
2h
2i
6
2
7
2
2
2
<2
<2
<2
15
7
14
6
2
5
9
8
3
2
3
2
2
2
4
6
11
9
12
7
6
6
6
5
0
0
1
1
0
0
1
0
0
0
0
0
1
1
0
0
1
0
0
0
6
8
8
7
14
5
7
7
5
9
8
16
23
8
14
14
19
2k
Note: All values are
DTm in degree Celsius (rounded to the nearest degree based on triplicate runs in 60 mmol potassium cacodylate buffer at pH-7.4).
concentration, of 36.3 °C for the HSP90A quadruplex and 32.0 °C
for the HSP90B quadruplex. This particular compound also pro-
duces down-regulation of HSP90 expression in the Mia-Pa-Ca-2
pancreatic cancer cell line and in treated Mia-Pa-Ca-2 xenograft
tumours, consistent with the hypothesis that a threshold level of
quadruplex binding is needed in order to produce a significant
cellular effect (although at this stage we cannot rule out other
mechanisms of down-regulating HSP90 expression).

5934
S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5930–5935
moter quadruplex could circumvent resistance induction,
although the current lead compound 2f will require optimization
so that its affinity for the HSP90 quadruplexes is significantly
higher in order for this concept to be fully validated. Structural
studies of small-molecule-HSP90 quadruplex complexes, analogous
to those reported for BRACO-1920 and naphthalene diimide dervi-
atives with human telomeric quadruplexes32 and quindoline with
a c-myc promoter quadruplex,33 will undoubtedly aid optimiza-
tion. None of these structures provide obvious insight into the
structural basis of the selectivity shown by the present com-
pounds, in particular compound 2f, binding to HSP90 quadruplex-
es. One can speculate, in the absence of structural data, that the
sequence CCAAA present in the HSP90 quadruplexes, may form a
long loop that folds into a binding cleft, analogous to that found
in the c-kit1 quadruplex.12 More extensive studies on the complete
and partial HSP90 quadruplex sequences will also clarify which (or
other variants) are the more biologically relevant.
Figure 5. FRET competition plot for compound 2f. Points 1–5 represent HSP90A:-
calf thymus DNA molar ratios of 1:0, 1:1, 1:10, 1:100 and 1:300 respectively. With
this duplex DNA no significant decrease in
DTm values for the HSP90A quadruplex
were observed. Experiments were performed in triplicate.
Acknowledgments
This work was supported by CRUK Programme Grant No. C129/
A4489 to S. N. M. M. thanks the University of Bologna for Fellow-
ship support.
Table 2
Results of studies on compound 2f. Top: FRET melting studies using a range of
quadruplex sequences and a duplex (ds) DNA probe, with two concentrations for
compound 2f (DTm values in °C 0.1 °C.). F21T is the 21-mer human telomeric
quadruplex. c-kit2 and k-ras are sequences from the promoter of the c-kit and k-ras
oncogenes. Bottom: data on inhibition of cell growth proliferation following 96 h
exposure using a panel of cancer cell lines and a normal fibroblast line (WI38). All
experiments were performed in triplicate
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.07.
065.
Sequence
1
l
mol
2
lmol
F21T
c-kit2
k-ras
HSP90A
HSP90B
ds DNA
<2
<2
7
14
2
2
2
17
23
7
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0
0
Cell line
IC50, in lmol
A549
MCF7
RCC4
786-o
Mia-Pa-Ca2
WI38
1.02 0.13
1.32 0.29
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20. Capbell, N. H.; Parkinson, G. N.; Reszka, A. P.; Neidle, S. J. Am. Chem. Soc. 2008,
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21. See also Nielsen (a) Kim, M.-Y.; Vankayalapati, H.; Shin-ya, K.; Wierzba, K.;
Hurley, L. H. J. Am. Chem. Soc. 2002, 124, 2098; (b) Rzuczek, S. G.; Pilch, D. S.;
Liu, A.; Liu, L.; LaVoie, E. J.; Rice, J. E. J. Med. Chem. 2010, 53, 3632; (c) Ulven, M.
C. T. Curr. Med. Chem. 2010, 17, 3438. for a recent review of macrocyclic
quadruplex ligands..
28. FRET DNA melting assays on compounds 2a–k were performed as described
previously (Guyen, B.; Schultes, C. M.; Hazel, P.; Mann, J.; Neidle, S. Org. Biomol.
Chem. 2004, 2, 981) using a fluorescence resonance energy transfer (FRET)
assay modified to be used as a high-throughput screen in a 96-well format.
The labelled oligonucleotides had attached the donor fluorophore FAM:
6-carboxyfluorescein and the acceptor fluorophore TAMRA: 6-carboxytetram-
ethylrhodamine. The FRET probe sequences were diluted from stock to the
correct concentration (400 nM) in a 60 mM potassium cacodylate buffer (pH
7.4) and then annealed by heating to 95 °C for 10 min, followed by cooling to
room temperature in the heating block (3 –3.5 h). The compounds were stored
as a 1 mM stock solution in 10% DMSO / 90% 1 mmol HCl; final solutions (at
2 ꢁ concentration) were prepared using 60 mM potassium cacodylate buffer
(pH 7.4). Relevant controls using BRACO-19 (in addition to blank runs) were
also performed to check for quality of DNA samples (eg. F21T). 96-Well plates
(MJ Research, Waltham, MA) were prepared by aliquoting 50
ll of the annealed
DNA into each well, followed by 50 of the compound solutions.
ll
22. (a) Moorhouse, A. D.; Santos, A. M.; Gunaratnam, M.; Moore, M.; Neidle, S.;
Moses, J. E. J. Am. Chem. Soc. 2006, 128, 15972; (b) Drewe, W. C.; Neidle, S.
Chem. Commun. 2008, 5295.
23. Reeder, M. R.; Gleaves, H. E.; Hoover, S. A.; Imbordino, H. R.; Pangborn, J. J. Org.
Process Res. Dev. 2003, 7, 696.
24. van Leusen, A. M.; Hoogenboom, B. E.; Siderius, H. Tetrahedron Lett. 1972, 23,
2369.
25. Strotman, N. A.; Chobanian, H. R.; Gou, Y.; He, J.; Wilson, J. E. Org. Lett. 2010, 16,
3578.
Measurements were made on a DNA Engine Opticon (MJ Research) with
excitation at 450–495 nm and detection at 515–545 nm. Fluorescence readings
were taken at intervals of 0.5 °C in the range 30–100 °C, with a constant
temperature being maintained for 30 s prior to each reading to ensure a stable
value. Final analysis of the data was carried out using a script written in the
program Origin 7.0 (OriginLab Corp., Northampton, MA). The advanced curve-
fitting function in Origin 7.0 was used for calculation of
DTm are 0.1 °C.
DTm values. Esds in
29. Sulforhodamine B assay (SRB). Cells were counted and diluted to the required
concentration in 20 mL medium. For cell lines MCF7, A549, MIA-Pa-Ca-2, RCC4
26. Ohnmacht, S. A.; Mamone, P.; Culshaw, A. J.; Greaney, M. F. Chem. Commun.
2008, 10, 1241.
and 786-0, 1000–4000 cells with 160
into each well of a 96 well plate (Nunc, Denmark). After incubation for 24 h, the
compound to be tested was dissolved in 40 L of medium and was added in a
lL media (WI38: 6000/well) were seeded
27. Synthesis and analytical data for compound 2f, 1,3-bis(2-(4-(2-(4-
methylpiperazine-1-yl)ethyl)phenyl)-oxazol-5-yl)benzene. A 5 mL microwave
vial was charged with phenyl bis-oxazole (40 mg, 0.19 mmol, 1 equiv), 4-(4-
iodobenzyl)-4-methylpiperazine (120 mg, 0.4 mmol, 2.1 equiv), Pd(dppf)Cl2ꢀ-
CH2Cl2 (16 mg, 0.019 mmol, 10 mol%), Ag2CO3 (105 mg, 0.38 mmol, 2 equiv)
and PPh3 (10 mg, 0.038 mmol, 20 mol %). A magnetic stirrer bar was added and
the mixture of solids was gently shaken for a few seconds to ensure all solids
were well mixed. Distilled water (3 mL) was added and the vial was covered
with a serum cap. The vial and its contents were then heated and stirred in a
pre-heated oil bath at 60 °C for 36 h. After this time the reaction mixture was
cooled down to room temperature. CH2Cl2 (4 ml) was added and the contents
of the vial were filtered through a short pad of celite (in a Pasteur pipette with
cotton wool at bottom). The vial was rinsed once with an additional 1 ml of
CH2Cl2. The organic layer was separated, and the aqueous phase extracted once
with CH2Cl2 (1 ml). The organic layers were combined and concentrated in
l
range of concentrations, and the cells incubated for 96 h. The medium was then
removed and the cells fixed by incubation with TCA (10%, Sigma-Aldrich, UK)
for 30 min at 4 °C. After removal of the TCA, the cells were washed with
deionised water 5 times and dried at 60 °C for 1 h. The cells were then
incubated with sulforhodamine
Organics, UK) for 15 min at RT. The SRB was removed, the wells washed with
1% acetic acid (200 L), and dried at 60 °C for 1 h. Tris-base (100 L, 10 mM,
B (80 lL, 0.4 % in 1% acetic acid, Acros
l
l
Acros Organics, UK) solution was added to each well, and the plates were
gently shaken for 5 min. The absorbance at 540 nm was measured with a plate
reader (Spectrostar Omega, BMG Labtech, Germany). The data were
normalized to the value of 100 for the control experiment (untreated cells),
and the IC50 values were obtained by interpolation from a plot with Origin
(Version 7.0, OriginLab Corp.), as the concentration leading to an absorbance
intensity of 50%.
vacuo. The residue was purified by flash chromatography (silica; CH2Cl2
/
MeOH 9:1) to provide the title compound as an off-white solid in 78% yield.
Best purification results were obtained when a gradient from CH2Cl2 (100%) to
CH2Cl2 : MeOH (7:3) was applied. 1H NMR (400 MHz, CDCl3) d 8.06 (4H, d,
J = 8 Hz, Ar-H), 8.00 (1H, s, Ar-H), 7.68 (2H, dd, J = 8 Hz, 8 Hz, Ar-H), 7.54–7.50
(3H, m, Ar-H), 7.35 (4H, d, J = 8 Hz, Ar-H), 2.91–2.87 (4H, m, 2 ꢁ CH2), 2.68–
2.50 (20H, m, 10 ꢁ CH2), 2.31 (6H, s, 2 ꢁ CH3). 13C NMR (100 MHz, CDCl3) d
161.7 (quat), 150.5 (quat), 143.4 (quat), 129.6 (quat), 129.3 (CH), 128.9 (CH),
126.5 (CH), 125.3 (CH), 124.2 (CH), 124.0 (CH), 119.6 (quat), 60.0 (CH2), 55.2
30. Gunaratnam, M.; de la Fuente, M.; Hampel, S. M.; Todd, A. K.; Reszka, A. P.;
Schatzlein, A.; Neidle, S. Bioorg. Med. Chem. 2011, 19, 7151.
31. Hamon, F.; Large, E.; Guédin-Beaurepaire, A.; Rouchon-Dagois, M.; Sidibe, A.;
Monchaud, D.; Mergny, J.-L.; Riou, J.-F.; Nyugen, C.-H.; Teulade-Fichou, M.-P.
Angew. Chem., Int. Ed. 2011, 50, 8745.
32. Collie, G. W.; Promontorio, R.; Hampel, S. M.; Micco, M.; Neidle, S.; Parkinson,
G. N. J. Am. Chem. Soc. 2012, 134, 2723.
33. Dai, J.; Carver, M.; Hurley, L. H.; Yang, D. J. Am. Chem. Soc. 2012, 134, 17673.