A fragment based click chemistry approach towards hybrid G-quadruplex ligands: Design, synthesis and biophysical evaluation

Article abstract of DOI:10.1016/j.tet.2011.10.066

A library of hybrid oxazole-triazole based compounds containing contiguously linked aromatic units were synthesised as G-quadruplex binding ligands. The design of these ligands was based upon combining features of our first generation of G-quadruplex bis-

Full text of DOI:10.1016/j.tet.2011.10.066

Tetrahedron 68 (2012) 197e203
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A fragment based click chemistry approach towards hybrid G-quadruplex ligands:
design, synthesis and biophysical evaluation
*
Dougal J. Ritson, John E. Moses
School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 August 2011
Received in revised form 5 October 2011
Accepted 17 October 2011
Available online 23 October 2011
A library of hybrid oxazoleetriazole based compounds containing contiguously linked aromatic units
were synthesised as G-quadruplex binding ligands. The design of these ligands was based upon com-
bining features of our first generation of G-quadruplex bis-triazole ligands and the natural product te-
lomestatin. The syntheses and biophysical studies of these ligands are described.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Click chemistry
G-quadruplex
Telomerase inhibitor
Cancer
Telomestatin
1. Introduction
In recent years numerous G-quadruplex binding ligands have
been identified, the vast majority possessing a large aromatic/het-
G-Quadruplex structures are organised polymorphic tertiary
assemblies formed by oligonucleotides and nucleic acids containing
G-tracts.1,9 A core of hydrophobic G-quartets and a periphery of
negatively charged loops characterise these structures, which ex-
hibit higher stability over other possible conformations in the
presence of monovalent cations, such as Naþ or Kþ. Putative G-
quadruplex sequences have been identified in the genome,2 with
over-representation in guanine rich sequences, such as telomeres,3
in the promoter regions of a number of proto-oncogenes,4 such as
c-myc5a and c-kit,5b in 50 untranslated regions6a and in introns.6b
Unsurprisingly, several of these structures have become attractive
targets for cancer chemotherapy.
For example, inducing the single-stranded telomeric DNA
overhang to fold into G-quadruplexes has been shown to inhibit
telomerase activity7a and cancer cell growth.7b This is significant,
since in >80% of cancers telomerase is up-regulated and contrib-
utes to the malignant phenotype by maintaining cancer cell
immortalization.7c In addition to perturbing telomere elongation,
the induced G-quadruplex structures result in telomere uncapping,
which leads to rapid growth arrest or apoptosis.8 This creates
a window of opportunity for the design and synthesis of G-quad-
ruplex binding ligands with a view to employ them as therapeutic
agents.9,10
eroaromatic unit, usually locked into a planar disposition.9,10 These
aromatic moieties
p-stack onto the terminal tetraplex of the
quadruplex providing extra stabilisation, and can even induce
folding into the higher order structures.9,11 Side chains, which have
a positive charge or that can become protonated at physiological
pH, are another common feature, and provide points for electro-
static interactions with the negatively charged phosphate residues
in the grooves.10 However, ligands that selectively target specific G-
quadruplex structures are limited, presenting a significant chal-
lenge for further development.10b
We have previously reported the synthesis and biological eval-
uation of a family of triazole based quadruplex binding ‘click’ li-
gands (e.g., 1, Scheme 1), which showed strong G-quadruplex
stabilising effects, telomerase inhibition and anti-cancer activity.12
The design of these compounds was inspired by the non-fused
complex polycyclic aromatic structure of telomestatin (2), a very
potent telomerase inhibitor isolated from Streptomyces annulatus.13
We proposed that the 1,4-triazole moiety would serve as
a suitable mimic of the oxazole functionality of telomestatin (2),
and could be readily installed using the powerful CuAAC click re-
action, thus facilitating the synthesis of libraries of potential li-
gands. Indeed, the ‘click’ ligands (e.g., 1) displayed impressive
selectivity for G-quadruplex structures over duplex DNA, compar-
ative to that of telomestatin (2).12 Although 2 is an incredibly potent
and selective quadruplex binder, it is also a complex synthetic
target requiring lengthy syntheses,14 whereas our ligands were
readily accessible in significant quantities.
* Corresponding author. E-mail address: john.moses@nottingham.ac.uk (J.E.
Moses).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.066

198
D.J. Ritson, J.E. Moses / Tetrahedron 68 (2012) 197e203
Scheme 1. Rational design and retrosynthesis of new hybrid G-quadruplex ‘click’ ligands.
We have since developed a second generation of tristriazole li-
gands (3), which were designed in order to examine whether G-
quadruplexes could be stabilised by end-on hydrogen atome in-
teractions.15 However, whilst the loss of effective
binding in-
teractions by the inclusion of such tetrahedral centres was not
balanced by potential hydrogene bonding interactions, the
quadruplex/duplex selectivity of the ligands once again remained
high, suggesting that the rotational freedom of non-fused poly-
aromatic systems is important with regard to selectivity.
which a series of N,N-dialkyldiamines were added providing the
amides 21e26. Finally, the azide 5 and alkyne 6 coupling partners
were reacted together, under our previously developed microwave
conditions,19 to deliver the new library of hybrid tri-
azoleebisoxazole ligands 27e35 in good yield (69e87%, Scheme 2).
The ability of these compounds to stabilise G-quadruplex and
double helix DNA was investigated using a high-throughput FRET
(fluorescence resonance energy transfer) assay.20 Table 1 shows the
effect of different concentrations of compounds 27e35 on the
melting temperature (Tm) of two labelled oligomers in 60 mM
potassium cacodylate buffer at pH 7.4. The G-quadruplex forming
human telomeric (h-Tel) sequence (50-FAM-d(GGG[TTAGGG]3)-
TAMRA-30) and the F10T (ds) sequence (50-FAM-dTATAGCTATA-
HEG-TATAGCTATA-TAMRA-30)da hairpin double helix labelled
oligomer with an internal hexaethyleneglycol (HEG) linker.
p
pep
p
Taking these two factors into consideration, we have designed
a third generation of ligands, again based on a non-fused poly-
aromatic system but excluding excessive conformational flexibility.
Since p-stacking interactions had proven to be particularly impor-
tant, we opted to increase the number of heteroaromatic units in the
ligand cf. 1, in an attempt to maximise binding between electron-
rich ligand heterocycles and guanine residues. The general design
of these hybrid ligands was to incorporate the oxazole motifs of
telomestatin (2), and the 1,4-triazole and side chain functionalities
present in our first generation (e.g., 1) (Scheme 1). Keeping in mind
the proposed use of click chemistry, we opted to target a series of
triazole-centred ligands (e.g., 4) flanked by phenyloxazoles (Scheme
1). Synthetically, the ligands would be accessed through a CuAAC
reaction16 of the corresponding alkynes 5 and azides 6.
At a concentration of 4
mM several ligands, 31, 34 and 35,
showed moderate to good stabilisation of the G-quadruplex
structure with excellent selectivity over duplex DNA. Ligand 30 was
by far the best binder (
D
Tm¼15 ꢀC) but it did not discriminate be-
tween duplex and quadruplex DNA quite as effectively as 31, 34 and
35, which is essential for development as potential therapeutics.
The ligands with side chains containing three methylene units (n/
n0¼2) were uniformly stronger quadruplex binders than the anal-
ogous ligands with side chains containing two methylene units (n/
n0¼1) i.e., 27 cf. 30, 28 cf. 31, 29 cf. 32, 33 cf. 34. This may be due to
the fact that the longer arms allow more effective positioning of the
protonated amines for interaction with the phosphate backbone, or
simply due to providing more interactions with the guanine quar-
tet. It appears that the smaller ammonium ions interact more ef-
fectively than larger analogues, regardless of the length of the side
chain, i.e., 27 cf. 29 and 30 cf. 32, suggesting that there is a pocket
with some steric constraints where the protonated amines and
2. Results and discussion
The synthesis of our library began using the procedure of
Scanlan et al., with a Sonogashira reaction of 4-bromobenzamide 7
with ethynyltrimethylsilane to give 8 in 88% yield (cf. 53%, Scanlan,
Scheme 2).17 Typical BlumleineLewy oxazole synthesis conditions
€
(8, ethyl bromopyruvate, EtOH, reflux) provided oxazole 9 in low
yield (<30%). Hence, we opted for the two step procedure described
by Panek,18 which provided the oxazole 9 in good overall yield. The
TMS-protected alkyne was then unmasked and the ester hydro-
lysed in one step to afford the acid 10, which was converted to the
acid chloride 11 by treatment with (COCl)2/DMF. Compound 11 was
subsequently reacted with various N,N-dialkyldiamines to afford
the alkyne coupling partners 12e17 for the ensuing click reaction.
The complementary azide-coupling partners were accessed via
an analogous route. Hence, 4-aminobenzamide 18 was converted to
the corresponding azide, which was then subjected to the same
oxazole forming conditions as before to give 19 (Scheme 2). Hy-
drolysis of 19 was followed by conversion to the acid chloride 20, to
phosphate backbone interact. At a concentration of 1 mM the li-
gands were quite poor at binding G-quadruplex forming DNA, the
unsymmetrical ligand 35 being the only ligand to show any real
affinity for h-Tel. Although these ligands show comparable quad-
ruplex binding ability to some previously reported ligands,21 they
were rather disappointing bearing in mind our first generation of
ligands, i.e., 1 (Scheme 1), which exhibited
centration of 1
D
Tm¼18 ꢀC at a con-
m
M for the h-Tel G-quadruplex forming sequence.
However, one critical feature was prominent from these studies;
the selectivity of the ligands for quadruplex DNA over duplex DNA
remained consistently high, even at higher concentrations of the

D.J. Ritson, J.E. Moses / Tetrahedron 68 (2012) 197e203
199
Scheme 2. (a) Ethynyltrimethylsilane, 5 mol % PdCl2(PPh3)2, 2 mol % CuI, Et2NH, 88%; (b) BrCH2C(O)CO2Et, NaHCO3, THF; (c) TFAA, THF, 68% (9, two steps), 76% (19, three steps); (d)
LiOH, H2O/THF; (e) (COCl)2, DMF (cat.), CH2Cl2; (f) NH2(CH2)nNR2, CH2Cl2; (g) t-BuONO, concd HCl, NaN3, THF/H2O; (h) 5 mol % CuSO4$5H2O, sodium ascorbate, t-BuOH/H2O,
microwave, 100 ꢀC, 69e87%.
Table 1
polycyclic ligands comprised both triazole and oxazole function-
ality with pendant side chains and demonstrated modest, yet se-
lective, affinity for the intramolecular h-Tel G-quadruplex.
We undertook SAR studies, which further confirmed the ad-
vantageous nature of designing ligands with non-fused poly-
aromatic motifs with regard to selectivity of the ligand for
quadruplex DNA over duplex DNA. We believe that these results
provide important information that will aid in the design of new G-
quadruplex binding ligands that could potentially lead to novel
cancer therapeutics.
Thermal stabilisation for ligands 27e35 interacting with the human telomeric se-
quence (h-Tel) and a duplex DNA sequence (F10T)
Ligand
D
Tm (ꢀC)a
h-Tel
F10T
1
m
M
4
m
M
1
m
M
4
mM
27
28
29
30
31
32
33
34
35
1
1
1
1
1
2
1
2
3
4
2
1
1
1
0
0
0
1
0
1
0
1
1
0
3
0
1
0
1
0
15
9
4
3
7
8
4. Experimental section
4.1. General
a
The FRET method was used to obtain values, which are a mean of three runs.
High Resolution Mass Spectra were recorded on VG micron
Autospec or Bruker microTOF. Fourier Transform Infrared Spec-
troscopy (FT-IR) spectra were obtained on PerkineElmer 1600 se-
ries or Bruker Tensor 27. 1H and 13C NMR spectra were recorded on
a Bruker AV(III) 400, Bruker AV 400, Bruker DPX 400 (400 MHz (1H)
and 100 MHz (13C)) spectrometers. Coupling constant are given in
hertz (Hz) and the following notations indicate the multiplicity of
the signals: s (singlet), d (doublet), br s (broad signal), t (triplet), q
(quartet), quint (quintet), m (multiplet). Thin layer chromatography
was performed on Merck precoated silica gel aluminium plates (60
F254) and visualised using UV absorption and/or an appropriate
stain. Column chromatography was performed using Merck silica
gel 60 (230e400 mesh). Petrol ethers refer to petroleum ethers
40e60 ꢀC. Anhydrous THF was distilled from sodium wire/benzo-
phenone and anhydrous CH2Cl2 from CaH2 immediately prior to
use. MeCN and CHCl3 were distilled from CaH2 and stored over
ligands. Although non-specific binding of the ligands to quadruplex
DNA cannot be ruled out, it is noteworthy that this series of non-
fused polyaromatic ligands have displayed excellent selectivity for
quadruplex DNA over duplex DNA. In this context perhaps ligand 35
is most interesting, which displayed complete selectivity for
quadruplex DNA over duplex DNA, even at a concentration of 4
and showed a small amount of stabilisation of h-Tel at a concen-
tration of 1 M.
mM,
m
3. Conclusion
In summary, we have prepared a small library of hybrid G-
quadruplex binding ligands inspired by the natural product telo-
mestatin and our first generation ‘click’ ligands. These non-fused

200
D.J. Ritson, J.E. Moses / Tetrahedron 68 (2012) 197e203
ꢀ
activated 4 A MS. Toluene was dried by passing the solvent over an
activated alumina column, which was pressurised with dry N2.
Anhydrous DMF was purchased and used as received. Microwave
reactions were conducted on a CEM Discover Explorer microwave
reactor in sealed tubes with stirring at a constant temperature for
the indicated time. Preparative HPLC was performed with an Agi-
lent Technologies 1200 series system using a reverse phase column
(YMC Co., Ltd., YMC-Pack R&D ODS-A, 100ꢁ20 mm I.D., particle size
anhydrous CH2Cl2/THF (4:1, 125 mL) under Ar atmosphere with
anhydrous DMF (0.2 mL) and cooled to 0 ꢀC. (COCl)2 (3.70 mL,
44.0 mmol) was added slowly, and after 1 h the reaction was
allowed to come to room temperature slowly and was stirred
overnight. The solvents were removed in vacuo and the crude
brown solid was used directly in the next step without further
purification.
S-5
m
m, 12 nm). Analytical HPLC was carried out on using the same
4.1.4. Ethyl
2-(4-azidophenyl)oxazole-4-carboxylate
(19). 4-
instrument as for preparative HPLC, using a reverse phase column
Aminobenzamide 18 (10.0 g, 73.4 mmol) was suspended in THF
(600 mL) and cooled to 0 ꢀC then concd HCl (35 mL) was added
dropwise followed by t-BuONO (22.0 mL, 186 mmol). The reaction
was stirred for 2 h then NaN3 (14.2 g, 218 mmol) was added in one
portion. H2O (150 mL) was then added very carefully and after ½ h
the reaction was warmed to room temperature. After 2 ½ h a sat-
urated solution of NaHCO3 (80 mL) was added cautiously and the
reaction was neutralised by the careful addition of solid NaHCO3.
The THF was removed in vacuo and the product was extracted with
EtOAc (5ꢁ200 mL), which was dried (MgSO4), filtered and con-
centrated. The pale yellow solid was used directly in the next step
without further purification.
The crude azide was suspended in anhydrous THF (300 mL),
under an Ar atmosphere and NaHCO3 (21.8 g, 260 mmol) then ethyl
bromopyruvate (9.40 mL, 74.7 mmol) were added. The reaction was
heated to reflux overnight and a second charge of ethyl bromo-
pyruvate (9.40 mL, 74.7 mmol) was added in the morning. After
a further 6 h reflux the reaction was cooled and filtered through
Celite, washing with EtOAc. The filtrate was concentrated then
redissolved in anhydrous THF (250 mL) under N2 atmosphere. The
mixture was cooled to 0 ꢀC and trifluoroacetic anhydride (31.2 mL,
223 mmol) was added slowly. The reaction was allowed to warm to
room temperature and stirred overnight before the slow addition of
a saturated solution of NaHCO3 (150 mL) at 0 ꢀC. The reaction was
neutralised by the careful addition of solid NaHCO3 and the THF
was evaporated in vacuo. The crude oxazole was extracted with
EtOAc (3ꢁ250 mL), which was washed with brine (150 mL), dried
(MgSO4), filtered and concentrated. The oxazole was recrystallised
from EtOAc/petrol to give 19 as light orange needles (9.90 g,
38.4 mmol). The mother liquors were purified by flash chroma-
tography (9:1, petrol/EtOAc) to give the title compound 19 as a pale
yellow solid (2.30 g, 8.91 mmol, total 47.3 mmol, 64% over three
steps). Rf 0.16 (9:1, petrol ethers/EtOAc); nmax/cmꢂ1 3012, 2130,
2097, 1734, 1610, 1598; dH (400 MHz, CDCl3) 8.28 (1H, s), 8.12 (2H, d,
J 8.6), 7.14 (2H, d, J 8.6), 4.45 (2H, q, J 7.1), 1.43 (3H, t, J 7.1); dC
(101 MHz, CDCl3) 161.8, 161.3, 143.6, 143.0, 134.8, 128.6, 123.1, 119.4,
61.4, 14.4; HRMS ESIþ calculated for C12H10N4NaO3 [MNa]þ
281.0651, found 281.0651.
(YMC Co., Ltd., YMC-Pack R&D ODS-A, 100ꢁ4.6 mm I.D., particle
size S-5 mm, 12 nm). Solvents were HPLC grade purchased from
Fischer Scientific.
4.1.1. 4-(Trimethylsilylethynyl)benzamide (8)17. To a solution of 4-
bromobenzamide
7
(8.00 g, 40.2 mmol) and ethynyl-
trimethylsilane (16.8 mL, 121 mmol) in degassed (Ar bubbled for 3/4
h) Et2NH (240 mL) under an Ar atmosphere were added
PdCl2(PPh3)2 (1.42 g, 2.02 mmol, 5 mol %) and CuI (152 mg,
0.804 mmol, 2 mol %). After 2 days the solvent was evaporated and
the residue was suspended in EtOAc, which was then filtered
through Celite. The filtrate was then washed with H2O (100 mL) and
brine (80 mL) before being dried (MgSO4), filtered and concen-
trated. The product was purified by flash chromatography (1:1,
EtOAc/petrol ethers) to give the title compound 8 as an off-white
solid (6.63 g, 30.6 mmol, 76%). Rf 0.16 (1:1, petrol ethers/EtOAc);
dH (400 MHz, CDCl3) 7.78 (2H, d, J 8.6), 7.56 (2H, d, J 8.6), 6.10 (1H, br
s), 5.75 (1H, br s), 0.28 (9H, s); dC (101 MHz, CDCl3) 168.9, 132.8,
132.1, 127.2, 126.9, 103.9, 97.4, ꢂ0.2; HRMS ESIþ calculated for
C12H16NOSi [MH]þ 218.1001, found 218.0992.
4.1.2. Ethyl 2-(4-(trimethylsilylethynyl)phenyl)oxazole-4-carboxylate
(9). The amide 8 (6.63 g, 30.6 mmol) was dissolved in dry THF
(125 mL) under N2. NaHCO3 (8.98 g, 107 mmol) and ethyl bromo-
pyruvate (3.90 mL, 31.0 mmol) were added, after which the reaction
was heated to reflux overnight. After 16 h ethyl bromopyruvate
(3.90 mL, 31.0 mmol) was added and the reaction refluxed a further
7 h. The reaction was cooled to room temperature and filtered
through Celite washing with EtOAc. The filtrate was concentrated
then redissolved in dry THF (125 mL) under N2 and cooled to 0 ꢀC.
Trifluoroacetic anhydride (12.9 mL, 91.8 mmol) was added drop-
wise and the reaction was allowed to come to room temperature
slowly. The mixture was stirred overnight before being cooled to
0
ꢀC, then a saturated solution of NaHCO3 (100 mL) was added
carefully. The reaction was neutralised by the careful addition of
solid NaHCO3. The product was extracted with EtOAc (3ꢁ150 mL)
and the combined organics were washed with brine (100 mL), dried
(MgSO4), filtered and concentrated. The product recrystallised from
Et2O/petrol to give 9 as colourless plates (5.3 g, 16.9 mmol) and the
mother liquors were purified by flash chromatography (9:1, petrol
ethers/EtOAc) to give the title compound 9 as a colourless solid
(2.2 g, 7.03 mmol, total 23.9 mmol, 78% over two steps). Rf 0.37 (4:1,
petrol ethers/EtOAc); nmax/cmꢂ1 2986, 2157, 1736; dH (400 MHz,
CDCl3) 8.29 (1H, s), 8.09 (2H, d, J 8.4), 7.58 (2H, d, J 8.4), 4.46 (2H, q, J
7.1), 1.43 (3H, t, J 7.1), 0.29 (9H, s); dC (101 MHz, CDCl3) 161.9, 161.3,
143.8, 134.9, 132.4, 126.7, 126.03, 126.00, 104.1, 97.4, 61.4, 14.4, ꢂ0.1;
HRMS ESIþ calculated for C17H20NO3Si [MH]þ 314.1212, found
314.1206.
4.1.5. 2-(4-Azidophenyl)oxazole-4-carbonyl chloride (20). The azide
19 (9.87 g, 38.3 mmol) was dissolved in THF/H2O (4.3:1, 160 mL)
and cooled to 0 ꢀC. LiOH (1.39 g, 57.9 mmol) in H2O (30 mL) was
added slowly then the reaction was stirred ½ h before being
warmed to room temperature. After a further 1 ½ h H2O was
added until all the solids had dissolved then the pH was ad-
justed to w2 with 10% HCl. The mixture was extracted with
EtOAc repeatedly (150 mL portions) until all the solids had
dissolved. The organics were combined, dried (MgSO4), filtered
and concentrated. The crude material was used directly in the
next step.
4.1.3. 2-(4-Ethynylphenyl)oxazole-4-carbonyl chloride (11). The
oxazole 9 (6.94 g, 22.2 mmol) was dissolved in THF/H2O (2.7:1,
110 mL). LiOH (1.08 g, 44.9 mmol) in H2O (30 mL) was added slowly,
stirred 1 h then heated to 50 ꢀC for 2 h. The reaction was cooled to
room temperature and 10% HCl was added until the pH w2. The
product was extracted with EtOAc (4ꢁ110 mL), washed with brine
(80 mL) and dried (MgSO4). The crude material was suspended in
The crude acid was suspended in anhydrous CH2Cl2/THF (7:1,
175 mL) under an Ar atmosphere with anhydrous DMF (0.2 mL) and
cooled to 0 ꢀC. (COCl)2 (6.50 mL, 77.3 mmol) was added slowly, and
after 1 h the reaction was allowed to come to room temperature
and was stirred overnight. The solvents were removed in vacuo and
the crude brown solid was used directly in the next step without
further purification.

D.J. Ritson, J.E. Moses / Tetrahedron 68 (2012) 197e203
201
4.2. General procedure for the synthesis of amines: 12e17;
21e26
1651, 1598; dH (400 MHz, CDCl3) 8.60 (1H, br s), 8.24 (1H, s), 7.98
(2H, d, J 8.5), 7.58 (2H, d, J 8.5), 3.56 (2H, m), 3.24 (1H, s), 2.49 (2H, t,
J 6.1), 2.44 (4H, br s), 1.81e1.69 (6H, m), 1.50 (2H, br s); dC (101 MHz,
CDCl3) 160.54, 160.53, 141.0, 138.1, 132.5, 126.9, 126.3, 124.6, 82.9,
79.7, 58.8, 54.9, 39.7, 25.8, 25.1, 24.6; HRMS ESIþ calculated for
C20H24N3O2 338.1869 [MH]þ, found 338.1902.
The crude acid chloride (11/20) (0.35 M solution in anhydrous
CH2Cl2þhalf the volume CH2Cl2 wash) was added to the desired
primary amine (2 equiv, 0.7 M solution in CH2Cl2) under an Ar at-
mosphere at 0 ꢀC. After ½ h the reaction was warmed to room
temperature and stirred overnight. A saturated solution of NaHCO3
(½ the volume of reaction) was added and the mixture was stirred
vigorously for 10 min. The layers were then separated and the
aqueous layer was extracted with EtOAc (3ꢁ½ the volume of re-
action). The organics were combined, dried (Na2SO4), filtered and
concentrated, and the product was purified by flash chromatogra-
phy CH2Cl2/MeOH to afford the product.
4.2.7. N-(2-(Dimethylamino)ethyl)-2-(4-azidophenyl)oxazole-4-
carboxamide (21). Yellow solid (1.30 mmol, 83% over three steps). Rf
0.23 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3413, 3011, 2826, 2130, 2097,
1661, 1611, 1600; dH (400 MHz, CDCl3) 8.22 (1H, s), 8.05 (2H, d, J 8.5),
7.40 (1H, br s), 7.13 (2H, d, J 8.5), 3.55 (2H, app q, J 5.7), 2.55 (2H, t, J
6.1), 2.32 (6H, s); dC (101 MHz, CDCl3) 160.73, 160.70, 142.8, 140.7,
137.5, 128.3, 123.4, 119.5, 58.1, 45.4, 36.6; HRMS ESIþ calculated for
C14H17N6O2 301.1413 [MH]þ, found 301.1419.
4.2.1. N-(2-(Dimethylamino)ethyl)-2-(4-ethynylphenyl)oxazole-4-
carboxamide (12). Off-whitesolid (1.46 mmol, 84%over three steps).
Rf 0.22 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3413, 3301, 3010, 2826, 2109,
1927, 1661, 1599; dH (400 MHz, CDCl3) 8.23 (1H, s), 7.97 (2H, d, J 8.6),
7.55 (2H, d, J 8.6), 7.40 (1H, br s), 3.57 (2H, app q, J 5.9), 3.25 (1H, s),
2.51 (2H, t, J 6.1), 2.57 (6H, s); dC (101 MHz, CDCl3) 160.6,160.5,141.0,
137.7,132.5,126.7,126.5,124.7, 82.9, 79.8, 58.0, 45.4, 36.7; HRMS ESIþ
calculated for C16H18N3O2 284.1399 [MH]þ, found 284.1407.
4.2.8. N-(2-(Pyrrolidin-1-yl)ethyl)-2-(4-azidophenyl)oxazole-4-
carboxamide (22). Pale yellow solid (1.73 mmol, 85% over three
steps). Rf 0.23 (9:1, CH2Cl2/MeOH); nmax/cmꢂ13413, 3006, 2809, 2130,
2097,1661,1611,1600; dH (400 MHz, CDCl3) 8.24 (1H, s), 8.07 (2H, d, J
8.8), 7.49 (1H, br s), 7.15 (2H, d, J 8.8), 3.64 (2H, m), 2.81 (2H, br s), 2.70
(4H, br s), 1.88 (4H, br s); dC (101 MHz, CDCl3) 160.73, 160.68; 142.8,
140.7, 137.5, 128.3, 123.4, 119.5, 54.9, 54.1, 37.9, 23.6; HRMS ESIþ cal-
culated for C16H19N6O2 327.1569 [MH]þ, found 327.1597.
4.2.2. N-(2-(Pyrrolidin-1-yl)ethyl)-2-(4-ethynylphenyl)oxazole-4-
carboxamide (13). Off-white solid (438 mg, 82% over three steps). Rf
0.21 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3413, 3301, 3004, 2809, 2109,
1927, 1662,1599; dH (400 MHz, CDCl3) 8.27 (1H, s), 8.04 (2H, d, J 8.4),
7.62 (2H, d, J 8.4), 7.48 (1H, br s), 3.67 (2H, br s), 3.24(1H, s), 2.92e2.62
(6H, m), 1.90 (4H, br s); dC (75 MHz, CDCl3) 160.6, 160.5, 141.0, 137.7,
132.5,126.6,126.4,124.7, 82.8, 79.8, 54.9, 54.0, 38.0, 23.6; HRMS ESIþ
calculated for C18H20N3O2 310.1556 [MH]þ, found 310.1566.
4.2.9. N-(2-(Piperidin-1-yl)ethyl)-2-(4-azidophenyl)oxazole-4-
carboxamide (23). Pale yellow solid (1.63 mmol, 81% over three
steps). Rf 0.28 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3413, 2941, 2130, 2097,
1660, 1611, 1600; dH (400 MHz, CDCl3) 8.22 (1H, s), 8.03 (2H, d, J 8.8),
7.52 (1H, br s), 7.13 (2H, d, J 8.8), 3.55 (2H, m), 2.57 (2H, t, J 6.3), 2.47
(4H, br s),1.64 (4H, m),1.48 (2H, m); dC (101 MHz, CDCl3) 160.7,160.6,
142.7,140.6,137.6,128.2,123.4,119.5, 57.3, 54.4, 36.0, 26.1, 24.4;HRMS
ESIþ calculated for C17H21N6O2 341.1726 [MH]þ, found 341.1755.
4.2.3. N-(2-(Piperidin-1-yl)ethyl)-2-(4-ethynylphenyl)oxazole-4-
carboxamide (14). Off-white solid (1.27 mmol, 74% over three
steps). Rf 0.25 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3413, 3301, 3011,
2941, 2109, 1926, 1661, 1599; dH (400 MHz, CDCl3) 8.25 (1H, s), 8.02
(2H, d, J 8.7), 7.61 (2H, d, J 8.6), 7.52 (1H, br s), 3.56 (2H, app q, J 6.3),
3.26 (1H, s), 2.58 (2H, t, J 6.3), 2.48 (4H, br s), 1.64 (4H, m), 1.49 (2H,
m); dC (101 MHz, CDCl3) 160.6, 160.5, 140.9,137.7, 132.6, 126.7,126.4,
124.7, 82.9, 79.8, 57.3, 54.4, 36.0, 26.1, 24.4; HRMS ESIþ calculated
for C19H22N3O2 324.1712 [MH]þ, found 324.1751.
4.2.10. N-(3-(Dimethylamino)propyl)-2-(4-azidophenyl)oxazole-4-
carboxamide (24). Pale yellow solid (1.71 mmol, 85% over three steps).
Rf 0.28 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3415, 3004, 2950, 2130, 2097,
1662, 1611, 1600; dH (400 MHz, CDCl3) 8.25 (1H, s), 8.05 (2H, d, J 8.8),
8.00 (1H, br s), 7.15 (2H, d, J 8.8), 3.57 (2H, app q, J 6.2), 2.56 (2H, br s),
2.39 (6H, s), 1.88 (2H, app quint, J 6.4); dC (101 MHz, CDCl3) 160.59,
160.57, 142.7, 140.5, 137.6, 128.1, 123.4, 119.4, 57.9, 45.3, 38.3, 26.6;
HRMS ESIþ calculated for C15H19N6O2 315.1569 [MH]þ, found 315.1588.
4.2.4. N-(3-(Dimethylamino)propyl)-2-(4-ethynylphenyl)oxazole-4-
carboxamide (15). Light orange solid (1.40 mmol, 81% over three
steps). Rf 0.13 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3416, 3301, 3005,
2824, 2109, 1926, 1663, 1599; dH (400 MHz, CDCl3) 8.20 (1H, s), 8.09
(1H, br s), 7.90 (2H, d, J 8.5), 7.64 (2H, d, J 8.5), 3.60 (2H, app q, J 5.8),
3.26 (1H, s), 2.39 (2H, t, J 5.8), 2.22 (6H, s), 1.73 (2H, app quint, J 5.8);
dC (101 MHz, CDCl3) 160.5, 160.4, 140.8, 137.9, 132.5, 126.7, 126.3,
124.7, 82.8, 79.8, 58.1, 45.5 38.4, 26.6; HRMS ESIþ calculated for
C17H20N3O2 298.1556 [MH]þ, found 298.1564.
4.2.11. N-(3-(Pyrrolidin-1-yl)propyl)-2-(4-azidophenyl)oxazole-4-
carboxamide (25). Pale brown/yellow solid (1.30 mmol, 65% over
three steps). Rf 0.16 (9:1, CH2Cl2/MeOH); nmax/cmꢂ13415, 3012, 2807,
2130, 2097, 1661, 1611, 1600; dH (400 MHz, CDCl3) 8.22 (1H, s), 8.16
(1H, br s), 8.03 (2H, d, J 8.7), 7.14 (2H, d, J 8.7), 3.59 (2H, m), 2.77e2.56
(6H, m),1.89 (6H, br s); dC (101 MHz, CDCl3) 160.5,160.4,142.5,140.5,
137.8, 128.0123.4, 119.3, 55.1, 54.1, 38.9, 27.6, 23.5; HRMS ESIþ cal-
culated for C17H21N6O2 341.1726 [MH]þ, found 341.1755.
4.2.12. N-(3-(Piperidin-1-yl)propyl)-2-(4-azidophenyl)oxazole-4-
carboxamide (26). Light brown solid (1.59 mmol, 79% over three
steps). Rf 0.25 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3414, 3006, 2940,
2130, 2097, 1651, 1611, 1599; dH (400 MHz, CDCl3) 8.54 (1H, br s),
8.20 (1H, s), 8.00 (2H, d, J 8.8), 7.10 (2H, d, J 8.8), 3.57 (2H, m), 2.49
(2H, t, J 6.2), 2.44 (4H, br s), 1.81e1.68 (6H, m), 1.50 (2H, br s); dC
(101 MHz, CDCl3) 160.59, 160.50, 142.6, 140.7, 137.9, 128.1, 123.6,
119.4, 58.7, 54.9, 39.6, 25.8, 25.2, 24.6; HRMS ESIþ calculated for
C18H23N6O2 355.1882 [MH]þ, found 355.1921.
4.2.5. N-(3-(Pyrrolidin-1-yl)propyl)-2-(4-ethynylphenyl)oxazole-4-
carboxamide (16). Yellow solid (1.15 mmol, 66% over three steps). Rf
0.14 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3415, 3301, 3004, 2807, 2109,
1926, 1661, 1599; dH (400 MHz, CDCl3) 8.26 (1H, s), 8.21 (1H, br s),
7.93 (2H, d, J 8.6), 7.54 (2H, d, J 8.5), 3.50 (2H, app q, J 6.3), 3.24 (1H,
s), 2.78e2.55 (6H, m), 1.91 (6H, br s); dC (101 MHz, CDCl3) 160.5,
160.4, 140.9, 138.0, 132.5, 126.8, 126.3, 124.6, 82.8, 79.9, 55.2, 54.1,
36.0, 27.6, 23.5; HRMS ESIþ calculated for C19H22N3O2 324.1712
[MH]þ, found 324.1756.
4.3. General procedure for click reactions
4.2.6. N-(3-(Piperidin-1-yl)propyl)-2-(4-ethynylphenyl)oxazole-4-
carboxamide (17). Tan solid (1.39 mmol, 80% over three steps). Rf
0.18 (9:1, CH2Cl2/MeOH); nmax/cmꢂ1 3414, 3301, 2940, 2109, 1925,
The alkyne (1 equiv) and azide (1 equiv) were dissolved in t-
BuOH (0.3 M) then CuSO4$5H2O (5 mol %) was added followed by

202
D.J. Ritson, J.E. Moses / Tetrahedron 68 (2012) 197e203
sodium ascorbate (0.25 equiv) dissolved in H2O (equal volume to t-
BuOH). The reaction was heated to 100 ꢀC in the microwave for
25 min then it was cooled to room temperature and diluted with
H2O (4 volumes). The suspension was filtered and washed with
a small amount of methanol then EtOAc. The residue was dissolved
in CH2Cl2/MeOH (9:1), dried (Na2SO4), filtered and concentrated. A
sample was purified by prep HPLC for analysis and biological
evaluation (see SD for details).
(101 MHz, MeOD) 162.1, 162.0, 161.1, 160.3, 147.0, 141.8, 141.5, 138.1,
136.9, 136.7, 132.2, 127.6, 126.7, 126.2, 125.8, 125.6, 119.6, 118.8,
2ꢁ54.5, 2ꢁ53.0, 2ꢁ35.8, 2ꢁ24.1, 2ꢁ22.9, 2ꢁ21.3; HRMS ESIþ cal-
culated for C38H46N9O4 692.3673 [MH]þ, found 692.3665.
4.3.7. N-(2-(Dimethylamino)ethyl)-2-(4-(4-(4-(4-((2-(piperidin-1-
yl)ethyl)carbamoyl)oxazol-2-yl)phenyl)-1,2,3-triazol-1-yl)phenyl)ox-
azole-4-carboxamide (33). Dark green solid (0.226 mmol, 81%).
nmax/cmꢂ1 1671; dH (400 MHz, MeOD) 8.66 (1H, s), 8.34 (1H, s), 8.30
(1H, s), 7.87 (2H, d, J 8.6), 7.65e7.77 (6H, m), 3.62e3.80 (6H, m), 3.39
(2H, t, J 5.5), 3.33 (2H, t, J 5.6), 2.98 (8H, m), 1.92 (2H, m), 1.80 (3H,
m), 1.51 (1H, m); dC (101 MHz, MeOD) 162.5, 162.3, 161.0, 160.2,
146.9,142.1,141.8,137.9,136.6,136.4,132.0,127.5,126.6,126.0,125.7,
125.4, 119.4, 118.5, 2ꢁ57.1, 56.3, 53.3, 2ꢁ42.5, 2ꢁ34.2, 33.7, 22.8,
21.2; HRMS ESIþ calculated for C33H38N9O4 624.3047 [MH]þ, found
624.3037.
4.3.1. 1,4-Bis-{N-(2-(dimethylamino)ethyl)-2-(4-phenyl)oxazole-4-
carboxamide}-1,2,3-triazole
(27). Pale
yellow/orange
solid
(0.232 mmol, 87%). nmax/cmꢂ1 1674; dH (400 MHz, MeOD) 8.90 (1H,
s), 8.47 (1H, s), 8.44 (1H, s), 8.09 (2H, d, J 8.5), 7.87e8.00 (6H, m),
3.80 (4H, br s), 3.45 (4H, br s), 3.04 (12H, s); dC (101 MHz, MeOD)
162.6, 162.5, 161.2, 160.4, 147.1, 142.2, 141.9,138.2, 136.7,136.5, 132.3,
127.7, 126.7, 126.3, 125.9, 125.6, 119.8, 118.9, 2ꢁ57.1, 2ꢁ42.5, 2ꢁ34.2;
HRMS ESIþ calculated for C30H34N9O4 584.2734 [MH]þ, found
584.2730.
4.3.8. N-(3-(Dimethylamino)propyl)-2-(4-(4-(4-(4-((3-(piperidin-1-
yl)propyl)carbamoyl)oxazol-2-yl)phenyl)-1,2,3-triazol-1-yl)phenyl)
oxazole-4-carboxamide (34). Orange/red solid (0.227 mmol, 85%).
nmax/cmꢂ1 1673; dH (400 MHz, MeOD) 8.94 (1H, s), 8.43 (1H, s), 8.39
(1H, s), 8.14 (2H, m), 7.90e8.03 (6H, m), 3.47e3.63 (6H, m), 3.20 (4H,
m), 2.94 (8H, m), 2.08 (4H, m),1.96 (2H, m),1.80 (3H, m),1.54 (1H, m);
dC (101 MHz, MeOD) 162.2,162.1,161.2,160.4,147.1,141.9,141.5,138.3,
137.0,136.8,132.4,127.7,126.8,126.4,126.0,125.7,119.9,119.0, 2ꢁ55.3,
54.5, 53.0, 2ꢁ42.1, 35.7, 2ꢁ35.5, 2ꢁ24.8, 24.1, 22.9, 21.3; HRMS ESIþ
calculated for C35H42N9O4 652.3360 [MH]þ, found 652.3356.
4.3.2. 1,4-Bis-{N-(2-(pyrrolidin-1-ylamino)ethyl)-2-(4-phenyl)ox-
azole-4-carboxamide}-1,2,3-triazole (28). Light orange solid
(0.236 mmol, 81%). nmax/cmꢂ11670, 1510; dH (400 MHz, MeOD) 8.76
(1H, s), 8.39 (1H, s), 8.36 (1H, s), 7.96 (2H, d, J 8.5), 7.74e7.88 (6H,
m), 3.68e3.88 (8H, br s), 3.46 (4H, br s), 3.15 (4H, s), 2.16 (4H, br s),
2.03 (4H, br s); dC (101 MHz, MeOD) 162.5, 162.3, 161.1, 160.3, 147.0,
142.2, 141.8, 138.1, 136.7, 136.5, 132.2, 127.6, 126.7, 126.2, 125.8,
125.6, 119.6, 118.7, 2ꢁ54.3, 2ꢁ54.2, 2ꢁ35.3, 2ꢁ22.6; HRMS ESIþ
calculated for C34H38N9O4 636.3047 [MH]þ, found 636.3036.
4.3.9. N-(2-(Pyrrolidin-1-yl)ethyl)-2-(4-(4-(4-(4-((3-(pyrrolidin-1-
yl)propyl)carbamoyl)oxazol-2-yl)phenyl)-1,2,3-triazol-1-yl)phenyl)
oxazole-4-carboxamide (35). Pale orange solid (0.216 mmol, 87%).
nmax/cmꢂ1 1673; dH (400 MHz, MeOD) 8.70 (1H, s), 8.37 (1H, s), 8.29
(1H, s), 7.93 (2H, d, J 8.5), 7.71e7.83 (6H, m), 3.82 (2H, m), 3.70 (4H,
m), 3.46 (4H, m), 3.28 (2H, m), 3.10 (4H, br s), 2.15 (4H, br s), 2.04
(6H, br s); dC (101 MHz, MeOD) 162.2, 162.0, 161.0, 160.2, 146.9,
142.1,141.5,138.0,136.7,136.6,132.1,127.5,126.6,126.0,125.8,125.4,
119.5, 118.6, 2ꢁ54.4, 2ꢁ54.2, 53.8, 52.5, 35.7, 35.3, 26.0, 2ꢁ22.62,
2ꢁ22.60; HRMS ESIþ calculated for C35H40N9O4 650.3203 [MH]þ,
found 650.3191.
4.3.3. 1,4-Bis-{N-(2-(piperidin-1-ylamino)ethyl)-2-(4-phenyl)ox-
azole-4-carboxamide}-1,2,3-triazole
(29). Pale
yellow
solid
(0.214 mmol, 77%). nmax/cmꢂ1 1672; dH (400 MHz, MeOD) 9.04 (1H,
s), 8.53 (1H, s), 8.49 (1H, s), 8.22 (2H, d, J 8.8), 8.01e8.12 (6H, m),
3.84 (4H, m), 3.75 (4H, m), 3.41 (4H, app t, J 5.7), 3.05 (4H, m), 2.02
(4H, m), 1.77e1.92 (6H, m), 1.60 (2H, m); dC (101 MHz, MeOD) 162.7,
162.6, 161.2,160.4, 147.1,142.2, 141.9, 138.3, 136.8, 136.6, 132.4, 127.8,
126.8, 126.4, 126.0, 125.7, 119.9, 119.0, 2ꢁ56.5, 2ꢁ53.3, 2ꢁ33.8,
2ꢁ22.9, 2ꢁ21.3; HRMS ESIþ calculated for C36H42N9O4 664.3360
[MH]þ, found 664.3342.
4.3.4. 1,4-Bis-{N-(3-(dimethylamino)propyl)-2-(4-phenyl)oxazole-4-
carboxamide}-1,2,3-triazole (30). Brown solid (0.208 mmol, 69%).
nmax/cmꢂ1 1673; dH (400 MHz, MeOD) 8.87 (1H, s), 8.42 (1H, s), 8.33
(1H, s), 7.84e8.11 (8H, m), 3.50 (4H, br s), 3.25 (4H, br s), 2.96 (12H,
s), 1.10 (4H, br s); dC (101 MHz, MeOD) 162.1, 162.0, 161.1, 160.3,
147.0, 141.8, 141.5, 138.1, 136.9, 136.8, 132.3, 127.7, 126.7, 126.3, 125.9,
125.6, 119.7, 118.8, 2ꢁ55.3, 2ꢁ42.2, 2ꢁ35.5, 2ꢁ24.8; HRMS ESIþ
calculated for C32H38N9O4 612.3047 [MH]þ, found 612.3033.
Acknowledgements
The authors thank AICR, EPSRC and the University of Notting-
ham for generous financial support. Thanks to Prof. S. Neidle and Dr.
C. Lombardo for performing FRET analyses.
Supplementary data
Supplementary data pertaining to this article including NMR
spectra. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2011.10.066.
4.3.5. 1,4-Bis-{N-(3-(pyrrolidin-1-ylamino)propyl)-2-(4-phenyl)ox-
azole-4-carboxamide}-1,2,3-triazole (31). Light orange solid
(0.201 mmol, 72%). nmax/cmꢂ1 1669; dH (400 MHz, MeOD) 8.86 (1H,
s), 8.39 (1H, s), 8.35 (1H, s), 8.08 (2H, d, J 8.6), 7.97e7.85 (6H, m),
3.69 (4H, br s), 3.48 (4H, app t, J 6.0), 3.35 (4H, m), 3.10 (4H, m), 2.16
(4H, br s), 2.04 (8H, m); dC (101 MHz, MeOD) 160.5, 160.4, 159.6,
158.8, 145.5, 140.3, 139.9, 136.6, 135.4, 135.2, 130.8, 126.1, 125.2,
124.8, 124.4, 124.1, 118.2, 117.4, 2ꢁ52.3, 2ꢁ51.0, 2ꢁ34.1, 2ꢁ24.5,
2ꢁ21.1; HRMS ESIþ calculated for C36H42N9O4 664.3360 [MH]þ,
found 664.3347.
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