Linear and Branched Pyridyl-Oxazole Oligomers: Synthesis and Circular Dichroism Detectable Effect on c-Myc G-Quadruplex Helicity

Article abstract of DOI:10.1002/ejoc.201501269

Five unprecedented pyridyl-oxazole oligomers exhibiting either linear or branched connectivity of their subunits were developed as a family of potential G-quadruplex-interacting ligands. Our synthesis employed variations of a key Pd/Cu-mediated C-C cross-coupling/C-H activation reaction to gain access to the oligomer products from a small set of substituted pyridine building blocks. The effect of the compounds on the conformation of a c-myc oncogene promoter G-quadruplex was investigated by circular dichroism under various conditions. Some or all of the compounds induced detectable helicity enhancement in low-cation and Na⁺-rich Tris-HCl (pH 7.4) buffers, respectively, in which the helix was only partially prefolded.

Full text of DOI:10.1002/ejoc.201501269

DOI: 10.1002/ejoc.201501269
Full Paper
Pyridyl–Oxazole Oligomers
Linear and Branched Pyridyl–Oxazole Oligomers: Synthesis and
Circular Dichroism Detectable Effect on c-Myc G-Quadruplex
Helicity
Natalia Rizeq[a] and Savvas N. Georgiades*[a]
Abstract: Five unprecedented pyridyl–oxazole oligomers ex- pyridine building blocks. The effect of the compounds on the
hibiting either linear or branched connectivity of their subunits conformation of a c-myc oncogene promoter G-quadruplex was
were developed as a family of potential G-quadruplex-interact- investigated by circular dichroism under various conditions.
ing ligands. Our synthesis employed variations of a key Pd/Cu- Some or all of the compounds induced detectable helicity en-
mediated C–C cross-coupling/C–H activation reaction to gain hancement in low-cation and Na+-rich Tris-HCl (pH 7.4) buffers,
access to the oligomer products from a small set of substituted respectively, in which the helix was only partially prefolded.
Introduction
cancer research.[7] In more recent years, this field has taken gi-
ant leaps forward and it is now widely recognized that G-quad-
ruplexes play various roles in both healthy and cancer cells,
with impact on chromatin remodeling, genomic instability and
repair, telomerase dysfunction, gene expression, and cancer
progression.[3a,7,8] High-throughput methods for the detection
and mapping of G-quadruplex-forming sequences in the hu-
man genome[9] and in cellular transcripts[10] have been re-
ported. Moreover, evidence for the in vivo occurrence of G-
quadruplexes has been obtained by the use of G-quadruplex-
recognizing antibodies in fixed cells[11] and, very recently, by
the use of “smart” small-molecule bioimaging agents in live
cells.[12]
High-order biomolecular structures are critical in nearly all bio-
logical processes, and the complex mechanisms for the dy-
namic conversion of unfolded primary sequences into folded –
and functional – 3D entities have formed the object of numer-
ous investigations aimed at gaining insight into relations be-
tween structure and function.[1] Several non-canonical second-
ary structures have been identified for nucleic acid sequences,[2]
some of which are believed to be biologically relevant.
Guanine-rich sequences, in particular, have attracted enormous
attention,[3] owing to their inherent propensity to self-assemble
into tetrastranded helices, termed G-quadruplexes, in the pres-
ence of monovalent cations at near-physiological pH. Thermo-
dynamic and kinetic data suggest a relative stability of G-quad-
ruplexes under these conditions, as opposed to other non-B-
DNA structures.[4]
About a decade ago, early bioinformatic studies detected
that G-rich sequences, with potential to fold into G-quadru-
plexes, are prevalent in the human genome[5] and most notably
in the telomere as well as in promoter regions of several genes.
These included powerful oncogenes, such as c-myc, c-kit, and
k-ras. The abundance of G-rich tracts in gene promoters led
to hypotheses that in nature these sequences might serve as
regulatory elements of gene transcription.[6] Furthermore, the
observation that stabilization of G-quadruplexes within onco-
gene promoters causes suppression of the oncogene's expres-
sion established these sequences as promising targets for anti-
Progress in the G-quadruplex field has been chemistry
driven, and small molecules have been at the epicenter of many
of the aforementioned discoveries, as it was first realized that
one of the most effective ways to induce and/or stabilize a G-
quadruplex is through its direct interaction with appropriately
designed small molecules.[6a,7] Efforts toward the development
of improved G-quadruplex stabilizers remain very active, as they
are anticipated to yield novel anticancer pharmaceuticals or
specialized bioimaging probes for elucidating yet unknown bio-
logical functions of G-quadruplexes.
Terminal guanine tetrads, the planar arrangements of four
guanines exposed to either end of any G-quadruplex helix, have
been the most frequently exploited binding sites for small-mol-
ecule interaction. Owing to the planarity of G-tetrads, conven-
tional binder design has focused on polycyclic (hetero)aromatic
chromophores that offer the possibility for π–π stacking[13] (Fig-
ure 1, a). Whereas such systems may reach sufficiently high af-
finity in engaging with G-quadruplexes, especially if reinforced
with cationic/ionizable functionalities or metal cations, most
tend to lack specificity for one particular G-quadruplex. Unde-
sired affinity for duplex DNA, cell membranes, or other hydro-
phobic biomolecular surfaces may occasionally become limiting
[a] Laboratory of Synthetic Organic Chemistry and Chemical
Biology, Department of Chemistry, University of Cyprus,
1 Panepistimiou Ave., Aglandjia, Nicosia 2109, Cyprus
E-mail: georgiades.savvas@ucy.ac.cy
http://ucy.ac.cy/dir/el/component/comprofiler/userprofile/
savvasg
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201501269.
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for their progression to medicinal application. Quarfloxin is the Results and Discussion
first compound from this class to have progressed to phase II
clinical trials.[7,14]
Compound Design and Considerations
The emergence of TOxaPy as a target-specific telomeric G-
quadruplex binder, for which spectroscopic data and molecular
docking suggested a groove-interaction mode,[22] stressed the
marked absence of analogous neutral, modular binders for c-
myc and other oncogene promoter sequences. This encouraged
us to consider how small heteroaromatic moieties, such as the
pyridyl–oxazole fragment, could be employed in a new context
to assemble an extended family of oligomeric compounds with
variable architectural features (i.e., shape and size). We envis-
aged that an atom-economic synthesis employing a few com-
mon key intermediates to construct all members of this diverse
set of oligomers could be employed, in combination with a
small number of recurring reaction conditions. In this way a
“privileged” chemical moiety could be evaluated as a building
unit and incorporated in various architectures to identify conve-
nient ones for interaction with c-myc.
Figure 1. Alternative small-molecule binding modes on a G-quadruplex (gray
rectangles represent guanine bases): (a) end stacking (π–π stacking) to termi-
nal guanine tetrads, typical for rigid planar binders; (b) interaction with resi-
dues displayed in grooves and loops, believed to occur with conformationally
flexible, modular binders.
Employing combinations of pyridine and oxazole subunits as
building blocks, both of which have been previously validated
as part of other successful (including macrocyclic) DNA-binding
motifs,[22–24] seemed like a reasonable starting point. Both ring
systems are small-size heteroaromatics likely to fit well into DNA
cavities. They can serve as H-bond acceptors, which is a desired
property in guanine-rich groove environments, whereas they
do not exclude the possibility of π–π stacking. The aryl–aryl
connectivity of these heteroaromatics was chosen to provide
“ribbon-like” compounds that serve as mimics of protein helical
structures,[25] owing to the way they “exhibit” their heteroatoms
in a helical arrangement around their periphery. This type of
connectivity also allows for necessary rotational flexibility, so
that the compounds can “sample” potential binding modes be-
fore developing the most favorable multivalent interaction with
the DNA. Finally, their neutral character differentiates these
compounds from the vast majority of previous c-myc quadru-
plex-interacting molecules that are cationic, which might en-
hance selectivity.
We anticipated that the size and shape of the oligomers
would be two critical parameters for association to the target,
as G-quadruplexes have very specific spatial and conforma-
tional requisites. Thus, a range of linear oligomers from 5–7
constituent units (rings) was deemed ideal. G-quadruplex asym-
metry and twist helicity creates nonsymmetric environments in
the grooves and loops; therefore, to increase the probability of
binding, our synthetic scheme for the linear oligomers relied
on nonsymmetric, directional (“head-to-tail”) connectivity of the
subunits. Branched counterparts were also included in our com-
pound design to test the impact of nonlinear architectures on
c-myc interaction, although these were symmetric compounds
to simplify their synthesis. Their molecular weights were chosen
to be comparable to those of their linear relatives.
The realization that the most differentiating element of di-
verse G-quadruplexes is the shape of their grooves and loops,
which are also viewed as their most elusive domains because
of their dynamic nature,[15] has turned researchers toward con-
sidering longer modular molecules that offer conformational
flexibility (Figure 1, b). Studies have demonstrated that the
grooves of G-quadruplexes can accommodate diverse natural
products such as aminoglycosides[16] and nonplanar alka-
loids,[17] and analogous binding should be possible for syn-
thetic ligands. Following this line of thinking, deviations from
conventional binder design have delivered few groove/loop
binders for G-quadruplexes. For example, the cationic dye 3,3′-
diethyloxadicarbocyanine (DODC),[18] BINOL derivatives,[19] heli-
cally folded oligoamides,[20] and distamycin A with some of its
synthetic derivatives[21] have been reported as such. Prominent
work by Teulade-Fichou and co-workers has identified the neu-
tral, symmetric pyridine/oxazole-based oligoheteroaryl system
TOxaPy[22] and related cationic oxadiazole compound BOxAz-
aPy[23] as potential groove binders with the ability to discrimi-
nate between two distinct folds of human telomeric G-quadru-
plex DNA.
These last reports demonstrate that conformationally flexible
ligands for G-quadruplexes may offer potential for target-spe-
cific interactions. This prompted us to embark on a synthetic
effort to expand the available pool of conformationally versatile
ligands, the design of which would not pose any limitations as
to their possible modes of binding (i.e., not exclude a priori any
of the aforementioned binding modes) but would instead allow
adaptability to the G-quadruplex DNA target at hand. In partic-
ular, we became interested in developing a modular synthetic
method to gain access to heteroaryl oligomers with diverse ar-
chitectures and featuring the “privileged” pyridyl–oxazole unit.
Herein, we describe the synthesis of a small library of oligomers
and present preliminary circular dichroism data on compound
interaction with the c-myc oncogene promoter G-quadruplex
under various conditions to determine the ability of some of
Compound Synthesis
the compounds to induce favorable conformational changes to Our synthetic route for constructing a nonsymmetric pentaaryl
the helix.
oligomer in which alternating pyridines and oxazoles are con-
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nected in a linear “head-to-tail” fashion (i.e., Pyr-Oxa-Pyr-Oxa- based on a modification of a pre-existing procedure[29] involv-
Pyr) is outlined in Scheme 1. This synthesis relied on the use of ing extended heating with an excess amount of LiCl (10 equiv.)
two key substituted pyridine building blocks in its initial stages, in a mixed DMSO/H2O (1:1) solvent. In this case, satisfactory
from which all subsequent intermediates were derived. The conversion into the aldehyde (67 %) was observed, with no
first, 2-pyridinecarbaldehyde (1) was submitted to 1,3-dipolar starting material degradation. The TOSMIC reaction proved use-
cycloaddition with the carbenoid reagent p-toluenesulfonyl- ful once more in the conversion of aldehyde 6 into tetraaryl
methyl isocyanide (TOSMIC)[26] under deprotonating condi- intermediate 7 in 68 % yield. Next, another C–H activation–PdII/
tions, which led to the formation of oxazole heterocycle 2 in CuI co-catalyzed cross-coupling reaction was performed to pair
98 % yield. The second, 6-bromo-2-pyridinecarbaldehyde (3) compound 7 with a third key building block, 2-bromopyridine
was converted into dioxolane 4 under standard conditions[27] (8), to obtain pentaaryl product 9 (46 % yield). Compound 7
in 96 % yield. The availability of intermediates 2 and 4 enabled represents a critical synthetic intermediate in our overall
their direct cross-coupling under PdII/CuI co-catalysis to furnish scheme that was essential for assembling not only pentameric
extended intermediate 5. This step, which involves C–H activa- compound 9 but also all larger linear oligomers in this study.
tion of the 2-position of oxazole 2 and its C–C connection to
the 2-position of bromide 4 (with loss of HBr), is a key reaction
for the rapid assembly of oligomers of this type. It was demon-
strated by Hamon et al. in the synthesis of TOxaPy[22] and by
us in the construction of a “propeller-like” counterpart.[28] The
current work further extends the applicability of this reaction
and demonstrates its tolerance for certain chemical functionali-
ties (e.g., dioxolanes and esters). The moderate yield in this case
(53 %) was due to the competing formation of homocoupling
byproducts from the bromide, which appeared to be a general
limitation in all the examples we investigated. Compound 5 was
subsequently deprotected to regenerate the aldehyde moiety.
By employing a similar reaction set, the preparation of a
hexaaryl counterpart was possible (Scheme 2). In this case, we
made use of pre-existing intermediate 7, which was cross-
coupled directly to bromopyridine 4 to afford pentaaryl dioxol-
ane 10 (43 % yield). The dioxolane offers a handle for further
extension, and this took place after its deprotection to aldehyde
11 (80 % yield); submission of the aldehyde to TOSMIC cyclo-
addition affords hexaaryl product 12 (42 % yield). In contrast to
its 5-aryl and 7-aryl counterparts, the 6-aryl compound, which
“terminates” with an oxazole ring instead of a pyridine ring,
offers the possibility for further elongation by means of repeat-
ing the last three synthetic steps. This could potentially provide
access to larger even-numbered polyaryl counterparts (i.e., 8-
aryl, 10-aryl, etc.). Longer “head-to-tail” counterparts might be
useful in targeting the high-order G-quadruplex DNA structures
and will form the focus of a future investigation.
This step proved problematic if aqueous 2
M HCl was applied
under reflux conditions, in which case we observed conversion
of the dioxolane into a stable hydrate derivative of the alde-
hyde. Therefore, we devised an alternative set of conditions
Scheme 2. Synthesis of the linear 6-aryl oligomer. Reagents and conditions:
(a) 1,4-dioxane, Cs2CO3, Pd(OAc)2, CuI, Cy3P·HBF4, 130 °C, 24 h, 43 % yield;
(b) DMSO/H2O (1:1), LiCl, 150 °C, 48 h, 80 % yield; (c) MeOH, TOSMIC, K2CO3,
65 °C, 4 h, 42 % yield.
Scheme 1. Synthesis of the linear 5-aryl oligomer. Reagents and conditions:
(a) MeOH, TOSMIC, K2CO3, 65 °C, 4 h, 98 % yield; (b) benzene, ethylene glycol,
p-toluenesulfonic acid (TsOH, cat.), 100 °C, 24 h, 96 % yield; (c) 1,4-dioxane,
Cs2CO3, Pd(OAc)2, CuI, Cy3P·HBF4 (Cy = cyclohexyl), 130 °C, 24 h, 53 % yield;
(d) DMSO/H2O (1:1), LiCl, 150 °C, 48 h, 67 % yield; (e) MeOH, TOSMIC, K2CO3,
65 °C, 4 h, 68 % yield; (f) 1,4-dioxane, Cs2CO3, Pd(OAc)2, CuI, Cy3P·HBF4,
130 °C, 24 h, 46 % yield. Inset: structure of the TOSMIC reagent used in several
steps in this study.
To obtain a nonsymmetric “head-to-tail” 7-aryl compound, a
slightly modified route was applied (Scheme 3). A triaryl unit
with a bromine handle for cross-coupling was needed. This was
obtained in two steps: conversion of key building block 3 into
oxazole 13 (82 % yield), followed by cross-coupling of 13 to
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2-bromopyridine (8). The second step entailed the risk of 13 bromopyridine (an excess amount was needed) to afford final
undergoing “head-to-tail” homocoupling, as it contains both an C2-symmetric product 23 in 54 % yield.
aryl bromide and an oxazole functionality. Indeed, a competing
byproduct was formed in accord with a previous report,[22]
which was consistent with a 4-mer compound, and this thus
limited the yield of desired product 14 to 44 %. With compound
14 in hand, we proceeded to couple it to compound 7, the
result of which was desired 7-mer 15 (47 % yield).
Scheme 3. Synthesis of the linear 7-aryl oligomer. Reagents and conditions:
(a) MeOH, TOSMIC, K2CO3, 65 °C, 4 h, 82 % yield; (b) 1,4-dioxane, Cs2CO3,
Pd(OAc)2, CuI, Cy3P·HBF4, 130 °C, 24 h, 44 % yield; (c) 1,4-dioxane, Cs2CO3,
Pd(OAc)2, CuI, Cy3P·HBF4, 130 °C, 24 h, 47 % yield.
Scheme 4. Synthesis of the branched 5-aryl and 7-aryl oligomers. Reagents
and conditions: (a) EtOH, SOCl2, 0 °C, then r.t. for 18 h, then reflux for 2 h,
98 % yield; (b) PBr5 (neat), 95 °C, 3.5 h, then CHCl3, EtOH, 0 °C, 3 h, 92 %
To cover a broader range of pyridyl–oxazole architectures
with potentially different abilities to interact with c-myc, we
extended our synthetic efforts to include branched counter-
parts. A distinct synthetic scheme was developed (Scheme 4)
that was initiated from chelidamic acid (16), a pyridine-based
building block with three positions for chemical modification.
The 2- and 6-carboxylate positions were converted into ethyl
ester groups to obtain 17 in excellent yield (98 %) by using
thionyl chloride in EtOH.[30] The 4-OH group of 17 was subse-
yield; (c) 1,4-dioxane, Cs2CO3, Pd(OAc)2, CuI, Cy3P·HBF4, 130 °C, 24 h, 50 %
yield; (d) MeOH, NaBH4, 0 °C, then r.t. for 18 h, 76 % yield; (e) 1,4-dioxane,
SeO2, 100 °C, 5 h, 64 % yield; (f) MeOH, TOSMIC, K2CO3, 65 °C, 6 h, 77 % yield;
(g) 1,4-dioxane, Cs2CO3, Pd(OAc)2, CuI, Cy3P·HBF4, 130 °C, 24 h, 54 %.
Circular Dichroism Titrations
quently replaced by a bromine atom in high yield (92 %) upon Circular dichroism (CD) may be used to study G-quadruplex he-
treatment with PBr5 (neat) at 95 °C,[30] which afforded pyridyl licity and conformational polymorphism.[33] It can indicate con-
bromide 18. We next investigated this bromide as a substrate formational changes to the helix brought about by alteration
for PdII/CuI-mediated C–C cross-coupling to oxazole 2 to intro- of its environmental conditions or by small-molecule binding.
duce “branching” to the central pyridine ring. The obtained As part of this study, we performed preliminary CD titration
50 % conversion to compound 19 indicated that 4-bromo- experiments to determine whether final oligomeric compounds
pyridine 18 has reactivity comparable to that of the 2-bromo- 9, 12, 15, 22, and 23 had any effect on the helicity of prefolded
pyridines used for similar reactions in our previous schemes c-myc promoter G-quadruplex under various conditions. Any
and that the reaction tolerates ester functionalities as substitu- observed effect could serve as an initial indication of interac-
ents on the bromopyridine coupling partner. A stepwise reduc- tion. The CD experiments were conducted in 50 m
tion–reoxidation method was next applied on diethyl ester 19. buffer (pH 7.4), either in the absence of added salt or in the
Coordinating aluminum-based reducing agents [e.g., diisobutyl- presence of a monovalent cation (Na+ or K+, 100 m
concentra-
M Tris-HCl
M
aluminum hydride (DIBAL-H), sodium bis(2-methoxyethoxy)- tion) chloride. Specific cations may affect the topology assumed
aluminum hydride (Red-Al)] led only to partial reduction and by G-quadruplexes.[34] Under all three conditions used, the CD
loss of material; we, therefore, resorted to NaBH4 in anhydrous spectra exhibited a positive signal around 265 nm and a nega-
MeOH[31] for the full reduction of 19 to diol 20 (76 % yield). tive signal around 240 nm. This profile is typical of a unimolec-
Reoxidation of 20 to corresponding dialdehyde 21 was per- ular parallel-stranded G-quadruplex[35] (for a graphical repre-
formed with fresh SeO2 under reflux in 1,4-dioxane (64 % sentation see Figure 1) and is consistent with NMR structures
yield).[32] Treatment of dialdehyde 21 with TOSMIC for an ex- for c-myc 2345 (a variation of which is used in this study) under
tended time period ensured oxazole formation on both reactive K+ conditions.[15b,15c] The intensity of the CD signals correlates
positions to furnish branched pyridyl–oxazole 22 in 77 % yield. to helix thermal stability and followed the order K+ > Na+ > no
The synthesis was completed with C–C cross-coupling to 2- added salt.
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Control CD measurements (see the Supporting Information) differences in the three different buffer systems (see the Sup-
confirm that none of the five compounds exhibit intrinsic helic- porting Information). Nonetheless, these are likely to arise upon
ity on their own within their UV-absorbing region, even in helix binding.
100 m
M cation-containing buffer. This is consistent with their
The preliminary results of CD titrations are summarized in
Table 1, whereas a representative data set (for compound 15)
is shown in Figure 2. In the absence of added salt (partially
folded helix), significant helicity enhancement was observed
with the longest linear members of this compound family (i.e.,
compounds 12 and 15) and less so with compound 9, but no
change occurred with their branched counterparts. In Na+-rich
buffer, as would be expected given the higher G-quadruplex
stability under these conditions, the helicity enhancement upon
compound addition was moderate. Interestingly enough, the
observed signal intensity change was comparable for all five
compounds in this case, despite their different lengths and ar-
chitectures. This reinforces our earlier hypothesis that branched
pyridyl oxazoles can be potentially as effective as their linear
counterparts in inducing G-quadruplex conformational changes
under selected conditions.[28] In the CD titrations of the Na+-
induced quadruplex with the longest linear oligomers (i.e., com-
pounds 12 and 15), a small increase in ellipticity occurred just
below 300 nm, in addition to the two main signals. This could
be attributed to induced CD of the achiral ligand,[36] as a result
of its adapting to the DNA chiral environment during interac-
tion. Finally, none of the five compounds showed any effect on
the helicity of the robust K+-induced quadruplex, which indi-
cates that under these conditions, the interaction – if any –
does not lead to CD-detectable conformational change.
achiral and rotationally flexible nature. Besides, the UV data of
the free compounds is not suggestive of any conformational
Table 1. C-myc G-quadruplex helicity enhancement, as per CD
data.[a]
Ligand
Type[b]
Tris-HCl/low-salt
buffer[c]
Tris-HCl/Na+
buffer[c]
Tris-HCl/K+
buffer
9
linear-5
linear-6
linear-7
branched-5
branched-7
+
++
++
none
none
+
+
+
+
+
none
none
none
none
none
12
15
22
23
[a] For the full set of CD spectra, see the Supporting Information. [b] Number-
ing refers to aromatic rings present in each ligand structure. [c] Increase in
CD signal intensity: +: up to 25 %, ++: up to 60 %.
Conclusions
In the current work, we implemented a versatile and atom-eco-
nomic synthetic plan to deliver both nonsymmetric linear and
symmetric branched heteroaryl oligomers featuring alternating
pyridine and oxazole repetitive units as elements intended for
G-quadruplex DNA targeting. These new compounds with di-
verse lengths and architectures are neutral, modular, and rota-
tionally flexible and were designed with the aim to offer poten-
tial for favorable interaction with G-quadruplexes without im-
posing considerable constraints as to their mode of binding.
The synthesized compounds were evaluated by circular dichro-
ism (CD) spectroscopy for their ability to induce conformational
changes to a unimolecular parallel G-quadruplex formed from
a model c-myc oligonucleotide under three different buffered
conditions. CD led to initially identifying conditions under
which some or all of the compounds were able to assist in
increasing c-myc G-quadruplex helicity. Given that the identi-
fied conditions involved partially folded states of the c-myc
quadruplex, we are compelled to think of our oligomeric li-
gands as somewhat comparable to the molecular chaperones
employed by nature in the folding of biomolecules.[37] Investi-
gating the mechanisms by which members of this compound
family function on G-quadruplexes could prove critical for the
development of new improved generations of G-quadruplex
binders. Systematic spectroscopic and structural studies are
planned by our laboratory to elucidate the possible binding
modes for these compounds, and they will be reported in due
course.
Figure 2. CD titrations of prefolded c-myc promoter G-quadruplexes (3 μ
with increasing amounts of linear oligomer 15 (0–15 μ ) in 50 m Tris-HCl
buffer (pH 7.4) (a) without added salt, (b) with 100 m NaCl, and (c) with
100 m KCl. Arrows indicate signal intensity changes with increasing ligand.
M)
M
M
M
M
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1 H), 7.22 (t, J = 6.0 Hz, 1 H), 7.63 (d, J = 7.8 Hz, 1 H), 7.76 (t, J =
7.5 Hz, 1 H), 7.85–7.87 (m, 3 H, signals overlapping), 8.18 (d, J =
8.4 Hz, 1 H), 8.62 (d, J = 4.8 Hz, 1 H) ppm. 13C NMR (CDCl3): δ =
65.6, 103.5, 119.7, 121.7, 122.6, 123.1, 127.1, 136.8, 137.7, 145.4,
147.0, 149.9, 151.9, 157.9, 160.5 ppm. MS (MALDI-TOF): m/z (%) =
296.28 [M + H]+ (calcd. for C16H14N3O3: 296.10).
Experimental Section
General: All reactions were performed under an argon atmosphere
and anhydrous solvents were used, unless otherwise stated. In
chromatographic purifications, Merck silica gel 60, 0.06–0.2 mm,
was used. NMR spectra were obtained with a Bruker Avance III Ul-
1
trashield Plus spectrometer (at 500 MHz for H NMR and 125 MHz
for 13C NMR, at 25 °C, chemical shifts relative to tetramethylsilane).
MS data were collected with a Bruker Autoflex III Smartbeam
MALDI-TOF/TOF instrument. CD and UV spectra were obtained with
a Jasco J-815 spectrometer.
6-[5-(Pyridin-2-yl)oxazol-2-yl]picolinaldehyde (6): A round-bot-
tomed flask was charged with compound 5 (0.45 g, 1.5 mmol,
1 equiv.) and DMSO (9 mL). A solution of LiCl (0.635 g, 15 mmol,
10 equiv.) in water (9 mL) was added, and a vertical condenser was
fitted to the flask. The mixture was heated at reflux at 150 °C for
48 h and then cooled to room temperature, diluted with water, and
extracted with CH2Cl2 (3×). The organic extracts were combined
and dried with Na2SO4, and the solvent was removed under re-
duced pressure. The crude product was redissolved in CH2Cl2 and
applied to a silica gel column for flash chromatography (hexane/
EtOAc, gradient from 1:1 to 1:3). This led to the isolation of com-
pound 6 (0.25 g, 1 mmol, 67 % yield) as a white solid. 1H NMR
(CDCl3): δ = 7.28 (ddd, J1 = 7.6 Hz, J2 = 4.8 Hz, J3 = 1.2 Hz, 1 H),
5-(Pyridin-2-yl)oxazole (2): In a round-bottomed flask containing
dry MeOH (75 mL) were added 2-pyridinecarbaldehyde (1; 2.0 mL,
21 mmol, 1 equiv.), TOSMIC (4.51 g, 23.1 mmol, 1.1 equiv.) and
K2CO3 (6.39 g, 46.2 mmol, 2.2 equiv.), in this order. The flask was
fitted with a vertical condenser and the mixture was heated at re-
flux at 65 °C for 4 h. The solvent was entirely removed under re-
duced pressure and the residue was resuspended in EtOAc and
stirred for 30 min at room temperature Filtration led to removal of
insoluble inorganic materials and the solution was concentrated
under reduced pressure and applied to a silica column for flash
chromatography. Elution took place using a hexane/EtOAc step gra-
dient (from 2:1 to 1:2 to pure EtOAc) and led to isolation of com-
pound 2 (3.01 g, 20.6 mmol, 98 % yield) as a yellow oil. 1H NMR
(CDCl3): δ = 7.19 (ddd, J1 = 7.8 Hz, J2 = 4.7 Hz, J3 = 1.0 Hz, 1 H),
7.61 (d, J = 7.8 Hz, 1 H), 7.65 (s, 1 H), 7.71 (dt, J = 7.8 Hz, J2 = 1.8 Hz,
1 H), 7.93 (s, 1 H), 8.58 (d, J = 5.5 Hz, 1 H) ppm. 13C NMR (CDCl3):
δ = 119.0, 122.7, 124.5, 136.6, 146.7, 149.5, 150.7, 150.8 ppm. MS
(MALDI-TOF): m/z (%) = 146.22 [M] (calcd. for C8H6N2O: 146.05).
7.81 (dt, J1 = 7.6 Hz, J2 = 1.7 Hz, 1 H), 7.87 (td, J1 = 7.8 Hz, J2
=
1.0 Hz, 1 H), 7.90 (s, 1 H), 8.03 (m, 2 H, signals overlapping), 8.40
(dd, J1 = 5.7 Hz, J2 = 3.2 Hz, 1 H), 8.66 (dt, J1 = 4.9 Hz, J2 = 1.0 Hz,
1 H), 10.23 (s, 1 H) ppm. 13C NMR (CDCl3): δ = 119.9, 122.2,
123.5, 126.2, 127.4, 137.1, 138.1, 146.4, 146.8, 150.0, 152.3, 153.0,
159.7, 193.0 ppm. MS (MALDI-TOF): m/z (%)
=
251.18
[M]+ (calcd. for C14H9N3O2: 251.07).
2-[6-(Oxazol-5-yl)pyridin-2-yl]-5-(pyridin-2-yl)oxazole (7): Com-
pound 6 (0.21 g, 0.84 mmol, 1 equiv.), TOSMIC (0.18 g, 0.92 mmol,
1.1 equiv.), and K2CO3 (0.26 g, 1.88 mmol, 2.2 equiv.) were sequen-
tially added to a round-bottomed flask containing anhydrous MeOH
(10 mL). The flask was fitted with a vertical condenser, and the
mixture was heated at reflux at 65 °C for 4 h. The solvent was
entirely removed under reduced pressure, and the residue was re-
suspended in EtOAc and stirred for 30 min. Filtration led to removal
of the insoluble inorganic materials, and the solution was concen-
trated under reduced pressure and applied to a silica gel column
for flash chromatography (hexane/EtOAc, gradient from 1:2 to pure
EtOAc) and led to the isolation of compound 7 (0.165 g, 0.57 mmol,
2-Bromo-6-(1,3-dioxolan-2-yl)pyridine (4): In a round-bottomed
flask, 6-bromo-2-pyridinecarbaldehyde (3; 0.93 g, 5 mmol, 1 equiv.),
ethylene glycol (0.55 mL, 10 mmol, 2 equiv.), and p-toluenesulfonic
acid monohydrate (0.047 g, 0.25 mmol, 0.05 equiv.) were dissolved
in dry benzene (25 mL). The flask was fitted with a Dean–Stark
apparatus and reflux took place at 100 °C for 24 h. The mixture was
then cooled to room temperature and 1 % (w/w) aqueous Na2CO3
was added to quench. The mixture was transferred to a separatory
funnel and extracted with CH2Cl2 (3×). The combined organic ex-
tract was dried with Na2SO4, and the solvent was removed under
reduced pressure. The crude product was redissolved in CH2Cl2 and
applied to a silica column for flash chromatography (CH2Cl2/MeOH,
gradient from 95:5 to 90–10). This led to the isolation of 4 (1.10 g,
68 % yield) as a white solid. 1H NMR (CDCl3): δ = 7.28 (ddd, J1
=
7.6 Hz, J2 = 4.7 Hz, J3 = 1.0 Hz, 1 H), 7.76 (d, J = 7.8 Hz, 1 H), 7.83
(dt, J1 = 7.6 Hz, J2 = 1.5 Hz, 1 H), 7.89 (d, J = 8.1 Hz, 1 H), 7.92 (m,
3 H, signals overlapping), 8.02 (s, 1 H), 8.15 (d, J = 8.1 Hz, 1 H), 8.67
(d, J = 4.7 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 119.8, 120.3, 121.7,
123.3, 125.9, 127.3, 137.1, 138.0, 146.1, 146.9, 147.6, 149.9, 150.5,
151.4, 152.0, 160.2 ppm. MS (MALDI-TOF): m/z (%) = 290.17 [M]+
(calcd. for C16H10N4O2: 290.08).
1
4.8 mmol, 96 % yield) as a yellow oil. H NMR (CDCl3): δ = 4.07 (m,
2 H), 4.16 (m, 2 H), 5.81 (s, 1 H), 7.47 (d, J = 7.8 Hz, 1 H), 7.50 (d, J =
7.8 Hz, 1 H), 7.59 (t, J = 7.8 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 65.6,
102.8, 119.4, 128.5, 139.1, 141.7, 158.5 ppm. MS (MALDI-TOF): m/z
(%) = 228.95 [M]+ (calcd. for C8H8BrNO2: 228.97).
2-[6-(1,3-Dioxolan-2-yl)pyridin-2-yl]-5-(pyridin-2-yl)oxazole (5):
A round-bottomed flask was charged with compound 2 (0.15 g,
1 mmol, 1 equiv.), compound 4 (0.24 g, 1 mmol, 1 equiv.), Cs2CO3
(0.72 g, 2.2 mmol, 2.2 equiv.), Pd(OAc)2 (0.045 g, 0.2 mmol,
0.2 equiv.), CuI (0.21 g, 1.1 mmol, 1.1 equiv.), and Cy3P·HBF4 (0.035 g,
0.1 mmol, 0.1 equiv.). The flask was fitted with a vertical condenser
and was set under an argon atmosphere. Anhydrous 1,4-dioxane
(10 mL) was added by syringe, and the mixture was heated at reflux
at 130 °C for 24 h. The mixture was then cooled to room tempera-
2-(Pyridin-2-yl)-5-{6-[5-(pyridin-2-yl)oxazol-2-yl]pyridin-2-yl}-
oxazole (9): A round-bottomed flask was charged with compound
7 (0.070 g, 0.24 mmol, 1 equiv.), 2-bromopyridine (8; 0.070 mL,
0.73 mmol, 3 equiv.), Cs2CO3 (0.17 g, 0.52 mmol, 2.2 equiv.),
Pd(OAc)2 (0.011 g, 0.048 mmol, 0.2 equiv.), CuI (0.050 g, 0.26 mmol,
1.1 equiv.), and Cy3P·HBF4 (0.0085 g, 0.024 mmol, 0.1 equiv.). The
flask was fitted with a vertical condenser and was set under an
argon atmosphere. Anhydrous 1,4-dioxane (5 mL) was added by
syringe, and the mixture was heated at reflux at 130 °C for 24 h,
ture and filtered through a sintered Büchner funnel to remove insol- cooled to room temperature, and filtered through a sintered Büch-
uble inorganic material. The dioxane solution was dried under re- ner funnel to remove insoluble inorganic materials. The dioxane
duced pressure. The crude residue was redissolved in a small
amount of dichloromethane and then applied to a silica gel column
for flash chromatography (CH2Cl2/MeOH, 95:5), which afforded com-
pound 5 (0.156 g, 0.53 mmol, 53 % yield) as a white solid. H NMR
(CDCl3): δ = 4.10 (t, J = 7.0 Hz, 2 H), 4.20 (t, J = 7.0 Hz, 2 H), 5.97 (s,
solution was dried under reduced pressure, and the crude residue
was redissolved in a small amount of dichloromethane. It was ap-
plied to a silica gel column for flash chromatography (CH2Cl2/MeOH,
gradient from 99:1 to 90:10). This afforded compound 9 (0.040 g,
1
1
0.11 mmol, 46 % yield) as a pale yellow solid. H NMR (CDCl3): δ =
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7.29 (ddd, J1 = 7.9 Hz, J2 = 4.7 Hz, J3 = 1.0 Hz, 1 H), 7.42 (t, J =
6.0 Hz, 1 H), 7.84 (dt, J1 = 7.8 Hz, J2 = 1.6 Hz, 1 H), 7.87 (t, J = 7.7 Hz,
1 H), 7.92 (s, 1 H), 7.93 (d, J = 7.0 Hz, 1 H), 7.95 (t, J = 7.7 Hz, 1 H),
reduced pressure, and the residue was resuspended in dichloro-
methane and directly applied to a silica gel column for flash chro-
matography (CH2Cl2/MeOH, gradient from 99:1 to 90:10), which led
to the isolation of compound 12 (0.022 g, 0.05 mmol, 42 % yield)
7.99 (d, J = 7.7 Hz, 1 H), 8.11 (s, 1 H), 8.17 (dd, J1 = 7.7 Hz, J2
=
0.9 Hz, 1 H), 8.26 (d, J = 7.7 Hz, 1 H), 8.68 (d, J = 4.7 Hz, 1 H), 8.80
as a white solid. 1H NMR (CDCl3): δ = 7.32 (dd, J1 = 5.9 Hz, J2
=
(d, J = 3.4 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 119.8, 120.6, 121.7,
3.4 Hz, 1 H), 7.80 (d, J = 7.7 Hz, 1 H), 7.92 (t, J = 7.7 Hz, 1 H),
122.6, 123.3, 125.0, 127.2, 128.3, 136.9, 137.0, 137.9, 145.8, 146.1, 7.96–8.00 (m, 4 H, signals overlapping), 8.02–8.06 (m, 3 H, signals
147.0, 147.5, 150.0, 150.1, 151.4, 152.1, 160.2, 160.8 ppm. MS
overlapping), 8.11 (s, 1 H), 8.22 (d, J = 7.7 Hz, 1 H), 8.70 (d, J =
4.5 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 120.0, 120.5, 120.8, 121.8,
121.9, 123.4, 125.7, 126.3, 127.5, 128.8, 130.9, 132.4, 137.4, 137.9,
138.0, 146.0, 146.1, 146.8, 146.9, 147.4, 147.6, 149.7, 160.3,
160.8 ppm. MS (MALDI-TOF): m/z (%) = 435.55 [M + H]+ (calcd. for
(MALDI-TOF): m/z (%) = 366.99 [M]+ (calcd. for C21H13N5O2: 367.11).
UV (50 mM Tris-HCl aqueous buffer, pH 7.4): λmax = 306 nm.
2-[6-(1,3-Dioxolan-2-yl)pyridin-2-yl]-5-{6-[5-(pyridin-2-yl)ox-
azol-2-yl]pyridin-2-yl}oxazole (10): A round-bottomed flask was
charged with compound 7 (0.061 g, 0.21 mmol, 1 equiv.), com-
pound 4 (0.048 g, 0.21 mmol, 1 equiv.), Cs2CO3 (0.15 g, 0.46 mmol,
2.2 equiv.), Pd(OAc)2 (0.009 g, 0.042 mmol, 0.2 equiv.), CuI (0.044 g,
0.23 mmol, 1.1 equiv.), and Cy3P·HBF4 (0.007 g, 0.021 mmol,
0.1 equiv.). The flask was fitted with a vertical condenser and was
set under an argon atmosphere. Anhydrous 1,4-dioxane (5 mL) was
added by syringe, and the mixture was heated at reflux at 130 °C
for 24 h, then cooled to room temperature, and filtered through a
C24H15N6O3: 435.12). UV (50 m
M Tris-HCl aqueous buffer, pH 7.4):
λmax = 319 nm.
5-(6-Bromopyridin-2-yl)oxazole (13): 6-Bromo-2-pyridinecarbal-
dehyde (3; 0.615 g, 3.3 mmol, 1 equiv.), TOSMIC (0.71 g, 3.6 mmol,
1.1 equiv.), and K2CO3 (1.02 g, 7.4 mmol, 2.2 equiv.) were sequen-
tially added to a round-bottomed flask containing anhydrous MeOH
(15 mL). The flask was fitted with a vertical condenser, and the
mixture was heated at reflux at 65 °C for 4 h. The solvent was
sintered Büchner funnel to remove insoluble inorganic materials. entirely removed under reduced pressure, and the residue was re-
The dioxane solution was dried under reduced pressure, and the suspended in EtOAc and stirred for 30 min. Filtration led to removal
crude residue was redissolved in a small amount of dichlorometh- of the insoluble inorganic materials, and the solution was concen-
ane. It was applied to a silica gel column for flash chromatography trated under reduced pressure and applied to a silica gel column
(EtOAc/MeOH, gradient from 99:1 to 95:5). This afforded compound for flash chromatography (hexane/EtOAc, gradient from 3:1 to 1:1),
10 (0.040 g, 0.09 mmol, 43 % yield) as a white solid. 1H NMR (CDCl3):
δ = 4.14 (t, J = 6.2 Hz, 2 H), 4.24 (t, J = 6.2 Hz, 2 H), 6.02 (s, 1 H),
7.29 (t, J = 6.0 Hz, 1 H), 7.69 (d, J = 7.8 Hz, 1 H), 7.85 (t, J = 7.8 Hz,
1 H), 7.91–7.99 (m, 5 H, signals overlapping), 8.11 (s, 1 H), 8.18 (d,
J = 7.5 Hz, 1 H), 8.25 (d, J = 8.0 Hz, 1 H), 8.68 (d, J = 3.7 Hz, 1 H)
ppm. 13C NMR (CDCl3): δ = 65.8, 103.5, 119.8, 120.7, 121.7, 121.9,
122.9, 123.3, 127.2, 128.3, 137.0, 137.8, 137.9, 145.4, 146.1, 147.1,
147.5, 150.0, 151.4, 152.1, 157.9, 160.2, 160.6 ppm. MS (MALDI-TOF):
m/z (%) = 439.00 [M]+ (calcd. for C24H17N5O4: 439.13).
which led to the isolation of compound 13 (0.6 g, 2.7 mmol, 82 %
yield) as a white solid. 1H NMR (CDCl3): δ = 7.36 (dd, J1 = 7.0 Hz,
J2 = 1.9 Hz, 1 H), 7.56 (m, 2 H, signals overlapping), 7.69 (s, 1 H),
7.94 (s, 1 H) ppm. 13C NMR (CDCl3): δ = 117.9, 126.1, 127.4, 139.1,
142.2, 147.8, 149.7, 151.5 ppm. MS (MALDI-TOF): m/z (%) = 223.96
[M]+ (calcd. for C8H5BrN2O: 223.96).
5-(6-Bromopyridin-2-yl)-2-(pyridin-2-yl)oxazole (14): A round-
bottomed flask was charged with compound 13 (0.43 g, 1.9 mmol,
1 equiv.), 2-bromopyridine (8; 0.18 mL, 1.9 mmol, 1 equiv.), Cs2CO3
6-(5-{6-[5-(Pyridin-2-yl)oxazol-2-yl]pyridin-2-yl}oxazol-2-yl)- (1.36 g, 4.2 mmol, 2.2 equiv.), Pd(OAc)2 (0.085 g, 0.38 mmol,
picolinaldehyde (11): A round-bottomed flask was charged with
compound 10 (0.088 g, 0.20 mmol, 1 equiv.) and then DMSO
0.2 equiv.), CuI (0.40 g, 2.1 mmol, 1.1 equiv.), and Cy3P·HBF4 (0.067 g,
0.19 mmol, 0.1 equiv.). The flask was fitted with a vertical condenser
(1.5 mL) was added. A solution of LiCl (0.085 g, 2 mmol, 10 equiv.) and was set under an argon atmosphere. Anhydrous 1,4-dioxane
in water (1.5 mL) was added, and a vertical condenser was fit to
the flask. The mixture was heated at reflux at 150 °C for 48 h, then
cooled down to room temperature, diluted with water, and ex-
tracted with CH2Cl2 (3×). The organic extracts were combined and
(20 mL) was added by syringe, and the mixture was heated at reflux
at 130 °C for 24 h, then cooled to room temperature, and filtered
through a sintered Büchner funnel to remove insoluble inorganic
materials. The dioxane solution was dried under reduced pressure,
dried with Na2SO4, and the solvent was removed under reduced and the crude residue was redissolved in a small amount of di-
pressure. The crude product was redissolved in CH2Cl2 and applied chloromethane. It was applied to a silica gel column for flash chro-
to a silica gel column for flash chromatography (EtOAc/MeOH, gra- matography (hexane/EtOAc, gradient from 3:1 to 1:3). This afforded
dient from 99:1 to 95:5). This led to the isolation of 11 as a pale
yellow solid (0.063 g, 0.16 mmol, 80 % yield). H NMR (CDCl3): δ =
compound 14 (0.25 g, 0.83 mmol, 44 % yield) as a pale yellow solid.
1H NMR (CDCl3): δ = 7.37 (m, 2 H, signals overlapping), 7.58 (t, J =
7.8 Hz, 1 H), 7.81 (m, 2 H, signals overlapping), 7.86 (s, 1 H), 8.17 (d,
J = 8.1 Hz, 1 H), 8.74 (d, J = 4.7 Hz, 1 H) ppm. 13C NMR (CDCl3): δ =
118.2, 122.5, 125.1, 127.4, 128.2, 137.2, 139.1, 142.1, 145.3, 147.7,
149.9, 150.5, 160.6 ppm. MS (MALDI-TOF): m/z (%) = 300.99 [M]+
(calcd. for C13H8BrN3O: 300.99).
1
7.35 (t, J = 6.3 Hz, 1 H), 7.91 (t, J = 7.3 Hz, 1 H), 7.96–8.02 (m, 3 H,
signals overlapping), 8.03–8.09 (m, 3 H, signals overlapping), 8.13
(s, 1 H), 8.22 (dd, J1 = 5.5 Hz, J2 = 3.5 Hz, 1 H), 8.46 (t, J = 4.4 Hz, 1
H), 8.70 (d, J = 4.4 Hz, 1 H), 10.25 (s, 1 H) ppm. 13C NMR (CDCl3):
δ = 120.1, 120.8, 122.0, 122.1, 122.4, 123.5, 125.3, 126.3, 128.4, 138.1,
138.2, 138.3, 146.0, 146.3, 147.2, 149.1, 151.7, 153.0, 155.5, 159.9,
160.4, 192.9 ppm. MS (MALDI-TOF): m/z (%) = 395.12 [M]+ (calcd.
for C22H13N5O3: 395.10).
2-(Pyridin-2-yl)-5-[6-(5-{6-[5-(pyridin-2-yl)oxazol-2-yl]pyridin-2-
yl}oxazol-2-yl)pyridin-2-yl]oxazole (15): A round-bottomed flask
was charged with compound 14 (0.10 g, 0.34 mmol, 1 equiv.), com-
2-[6-(Oxazol-5-yl)pyridin-2-yl]-5-{6-[5-(pyridin-2-yl)oxazol-2-yl]- pound 7 (0.10 g, 0.34 mmol, 1 equiv.), Cs2CO3 (0.24 g, 0.74 mmol,
pyridine-2-yl}oxazole (12): Compound 11 (0.047 g, 0.12 mmol,
1 equiv.), TOSMIC (0.026 g, 0.13 mmol, 1.1 equiv.), and K2CO3
(0.036 g, 0.26 mmol, 2.2 equiv.) were sequentially added to a round-
bottomed flask containing anhydrous MeOH (2 mL). The flask was
fitted with a vertical condenser, and the mixture was heated at
2.2 equiv.), Pd(OAc)2 (0.015 g, 0.067 mmol, 0.2 equiv.), CuI (0.071 g,
0.37 mmol, 1.1 equiv.), and Cy3P·HBF4 (0.012 g, 0.034 mmol,
0.1 equiv.). The flask was fitted with a vertical condenser and was
set under an argon atmosphere. Anhydrous 1,4-dioxane (7 mL) was
added by syringe, and the mixture was heated at reflux at 130 °C
reflux at 65 °C for 4 h. The solvent was entirely removed under for 24 h, then cooled to room temperature, and filtered through a
Eur. J. Org. Chem. 2016, 122–131
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128 © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
sintered Büchner funnel to remove insoluble inorganic materials. romethane. It was applied to a silica gel column for flash chroma-
The dioxane solution was dried under reduced pressure, and the
tography (hexane/EtOAc, gradient from 2:1 to 1:2 to pure EtOAc) to
crude residue was redissolved in a small amount of dichlorometh- afford compound 19 (0.38 g, 1.0 mmol, 50 % yield) as a pale yellow
ane. It was applied to a silica gel column for flash chromatography
(CH2Cl2/MeOH, gradient from 99:1 to 90:10). This afforded com-
pound 15 (0.082 g, 0.16 mmol, 47 % yield) as a white solid. 1H NMR
(CDCl3): δ = 7.30 (dd, J1 = 7.8 Hz, J2 = 5.4 Hz, 1 H), 7.43 (br. s, 1 H),
7.89 (t, J = 7.6 Hz, 2 H), 7.94 (s, 1 H), 7.97–8.03 (m, 5 H, signals
overlapping), 8.13 (s, 1 H), 8.17 (br. s, 1 H), 8.21 (m, 2 H, signals
overlapping), 8.28 (d, J = 5.4 Hz, 1 H), 8.69 (d, J = 4.6 Hz, 1 H), 8.81
(br. s, 1 H) ppm. 13C NMR (CDCl3): δ = 119.6, 119.7, 120.2, 121.5,
122.3, 123.1, 123.2, 124.7, 125.9, 127.1, 127.2, 136.8, 136.9, 137.9,
145.9, 146.0, 146.9, 147.0, 147.4, 149.7, 149.8, 149.9, 150.0, 150.4,
151.3, 151.8, 152.0, 160.0, 160.5 ppm. MS (MALDI-TOF): m/z (%) =
solid. 1H NMR (CDCl3): δ = 1.47 (t, J = 7.2 Hz, 6 H), 4.51 (q, J = 7.2 Hz,
4 H), 7.30 (td, J1 = 5.5 Hz, J2 = 2.8 Hz, 1 H), 7.82 (br. s, 2 H, signals
overlapping), 7.91 (s, 1 H), 8.66 (dt, J1 = 4.7 Hz, J2 = 1.5 Hz, 1 H),
8.88 (s, 2 H) ppm. 13C NMR (CDCl3): δ = 14.3, 62.7, 119.9, 123.8,
123.9, 127.8, 136.7, 137.2, 146.4, 149.8, 150.1, 152.6, 158.2,
164.2 ppm. MS (MALDI-TOF): m/z (%) = 368.19 [M + H]+ (calcd. for
C19H18N3O5: 368.12).
{4-[5-(Pyridin-2-yl)oxazol-2-yl]pyridine-2,6-diyl}dimethanol
(20): A round-bottomed flask was charged with compound 19
(0.155 g, 0.42 mmol, 1 equiv.) and anhydrous MeOH (3 mL). The
mixture was cooled to 0 °C and NaBH4 (0.191 g, 5 mmol, 12 equiv.)
was added to the flask in three equal portions under continuous
stirring. The mixture was stirred at room temperature for 18 h and
then cooled down to 0 °C and quenched with water. The mixture
was extracted with CH2Cl2 (2×). The pH of the aqueous phase was
512.15 [M + H]+ (calcd. for C29H18N7O3: 512.14). UV (50 m
M Tris-HCl
aqueous buffer, pH 7.4): λmax = 306 nm.
Diethyl 4-Hydroxypyridine-2,6-dicarboxylate (17): Thionyl chlor-
ide (12.6 mL, 174 mmol, 7 equiv.) and chelidamic acid monohydrate
(16; 5.01 g, 24.9 mmol, 1 equiv.) were sequentially added to a
round-bottomed flask containing absolute EtOH (50 mL) at 0 °C.
The resulting mixture was warmed to room temperature, stirred at
this temperature for 18 h, and then heated at reflux at 80 °C for
then adjusted to 7 through the addition of aqueous 1
M HCl, and
two more extractions were performed with CH2Cl2. All organic ex-
tracts were combined together and dried with Na2SO4. The drying
agent was filtered, and the solvent was removed under reduced
2 h. Subsequently, the solvent was removed under reduced pres- pressure to afford compound 20 (0.091 g, 0.32 mmol, 76 % yield)
sure and distilled water was added to the crude product at 0 °C.
The mixture was neutralized with 10 % aqueous Na2CO3 (5 mL) and
50 % aqueous EtOH (5 mL). The precipitate was filtered, washed
with water, and dried under reduced pressure to afford compound
17 (5.84 g, 24.4 mmol, 98 % yield) as a white solid. 1H NMR (CDCl3):
δ = 1.41 (t, J = 7.1 Hz, 6 H), 4.46 (q, J = 7.1 Hz, 4 H), 7.35 (s, 2 H)
ppm. 13C NMR (CDCl3 with trace [D6]DMSO): δ = 13.6, 61.7, 115.7,
148.2, 163.9, 167.1 ppm. MS (MALDI-TOF): m/z (%) = 240.23 [M +
H]+ (calcd. for C11H14NO5: 240.09).
as a white solid. This was used in the next step without further
purification. H NMR ([D6]DMSO): δ = 4.63 (br. s, 4 H), 5.64 (br. s, 2
1
H), 7.44 (t, J = 5.8 Hz, 1 H), 7.96–7.98 (m, 4 H, signals overlapping),
8.07 (s, 1 H), 8.70 (d, J = 5.0 Hz, 1 H) ppm. 13C NMR ([D6]-
DMSO): δ = 64.1, 114.2, 119.9, 123.9, 127.4, 134.4, 137.6, 146.1,
150.1, 151.5, 159.7, 162.8 ppm. MS (MALDI-TOF): m/z (%) = 283.12
[M]+ (calcd. for C15H13N3O3: 283.10).
4-[5-(Pyridin-2-yl)oxazol-2-yl]pyridine-2,6-dicarbaldehyde (21):
A two-necked, round-bottomed flask was charged with fresh SeO2
(0.235 g, 2.1 mmol, 4 equiv.), and the flask was fitted with a reflux
condenser and was set under an argon atmosphere. Anhydrous 1,4-
dioxane (10 mL) was added by syringe, and this was followed by
the addition of a solution of compound 20 (0.150 g, 0.53 mmol,
1 equiv.) in 1,4-dioxane (5 mL). The mixture was heated at reflux at
Diethyl 4-Bromopyridine-2,6-dicarboxylate (18): A round-bot-
tomed flask was charged with compound 17 (1.48 g, 6.2 mmol,
1 equiv.) and phosphorus pentabromide (5.34 g, 12.4 mmol,
2 equiv.). The flask was fitted with a vertical condenser, and the
mixture was heated at 95 °C for 3.5 h, then cooled down to room
temperature, and CHCl3 (5 mL) was added. The temperature was 100 °C for 5 h, then cooled down to room temperature, and filtered
further reduced to 0 °C and EtOH (10 mL) was added dropwise. The
mixture was stirred at 0 °C for another 3 h. The solvent was then
removed under reduced pressure, and the crude mixture was redis-
solved in CH2Cl2. The organic phase was washed with 10 % aqueous
NaHCO3, aqueous NaCl (satd.), and water and was then dried with
Na2SO4. The drying agent was removed, and the solution was con-
centrated under reduced pressure. The sample was applied to a
short silica gel column (hexane/EtOAc, 2:1) to afford compound 18
to remove insoluble inorganic materials. The solvent was removed
under reduced pressure. The crude residue was redissolved in a
small amount of EtOAc and was applied to a silica gel column for
flash chromatography (hexane/EtOAc, gradient from 2:1 to 1:3) to
afford compound 21 (0.095 g, 0.34 mmol, 64 % yield) as a yellow
1
oil. H NMR (CDCl3): δ = 7.34 (dt, J1 = 5.6 Hz, J2 = 2.3 Hz, 1 H), 7.86
(m, 2 H, signals overlapping), 7.94 (s, 1 H), 8.70 (d, J = 4.7 Hz, 1 H),
8.80 (s, 2 H), 10.24 (s, 2 H) ppm. 13C NMR (CDCl3): δ = 120.0, 121.4,
123.9, 127.9, 136.9, 137.2, 146.5, 150.3, 153.0, 154.0, 157.9,
191.8 ppm. MS (MALDI-TOF): m/z (%) = 280.24 [M + H]+ (calcd. for
C15H10N3O3: 280.07).
1
(1.72 g, 5.7 mmol, 92 % yield) as a white solid. H NMR (CDCl3): δ =
1.45 (t, J = 7.2 Hz, 6 H), 4.49 (q, J = 7.2 Hz, 4 H), 8.42 (s, 2 H) ppm.
13C NMR (CDCl3): δ = 14.1, 62.7, 131.0, 134.9, 149.5, 163.5 ppm. MS
(MALDI-TOF): m/z (%) = 301.15 [M]+ (calcd. for C11H12BrNO4: 300.99).
5,5′-{4-[5-(Pyridin-2-yl)oxazol-2-yl]pyridine-2,6-diyl}bis-
(oxazole) (22): Compound 21 (0.084 g, 0.30 mmol, 1 equiv.), TOS-
MIC (0.125 g, 0.65 mmol, 2.2 equiv.), and K2CO3 (0.18 g, 1.3 mmol,
4.4 equiv.) were sequentially added to a round-bottomed flask con-
taining dry MeOH (5 mL). The flask was fitted with a vertical con-
denser, and the mixture was heated at reflux at 65 °C for 6 h. The
solvent was entirely removed under reduced pressure, and the resi-
due was resuspended in EtOAc and stirred for 30 min. Filtration led
to removal of the insoluble inorganic materials, and the solution
was concentrated under reduced pressure and applied to a silica
gel column (hexane/EtOAc, gradient from 1:1 to 1:2 to pure EtOAc;
then EtOAc/MeOH, gradient from 98:2 to 90:10). Compound 22
(0.082 g, 0.23 mmol, 77 % yield) was isolated as a white solid. 1H
Diethyl 4-[5-(Pyridin-2-yl)oxazol-2-yl]pyridine-2,6-dicarboxyl-
ate (19): A round-bottomed flask was charged with compound 18
(0.60 g, 2 mmol, 1 equiv.), compound 2 (0.29 g, 2 mmol, 1 equiv.),
Cs2CO3 (1.43 g, 4.4 mmol, 2.2 equiv.), Pd(OAc)2 (0.091 g, 0.40 mmol,
0.2 equiv.), CuI (0.42 g, 2.2 mmol, 1.1 equiv.), and Cy3P·HBF4 (0.070 g,
0.20 mmol, 0.1 equiv.). The flask was fitted with a vertical condenser
and was set under an argon atmosphere. Anhydrous 1,4-dioxane
(20 mL) was added by syringe, and the mixture was heated at reflux
at 130 °C for 24 h, then cooled to room temperature, and filtered
through a sintered Büchner funnel to remove insoluble inorganic
materials. The dioxane solution was dried under reduced pressure,
and the crude residue was redissolved in a small amount of dichlo-
Eur. J. Org. Chem. 2016, 122–131
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NMR (CDCl3): δ = 7.32 (dt, J1 = 5.2 Hz, J2 = 2.9 Hz, 1 H), 7.85 (m, 2
H, signals overlapping), 7.87 (s, 2 H), 7.93 (s, 1 H), 8.05 (s, 2 H), 8.27
(s, 2 H), 8.71 (d, J = 4.8 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 114.7,
119.8, 123.6, 126.4, 127.6, 136.1, 137.0, 146.7, 148.3, 150.2, 150.4,
151.6, 152.4, 158.9 ppm. MS (MALDI-TOF): m/z (%) = 358.28 [M +
MALDI-TOF/TOF, respectively. The University of Cyprus is
acknowledged for a New Faculty Startup Grant to S. N. G.
Keywords: G-Quadruplexes · DNA · N,O ligands · Nitrogen
heterocycles · C–H activation
H]+ (calcd. for C19H12N5O3: 358.09). UV (50 m
M Tris-HCl aqueous
buffer, pH 7.4): λmax = 326 nm.
5,5′-{4-[5-(Pyridin-2-yl)oxazol-2-yl]pyridine-2,6-diyl}bis[2-(pyr-
idin-2-yl)oxazole] (23): A round-bottomed flask was charged with
compound 22 (0.080 g, 0.22 mmol, 1 equiv.), 2-bromopyridine (8;
0.063 mL, 0.66 mmol, 3 equiv.), Cs2CO3 (0.316 g, 0.97 mmol,
4.4 equiv.), Pd(OAc)2 (0.020 g, 0.09 mmol, 0.4 equiv.), CuI (0.092 g,
0.48 mmol, 2.2 equiv.), and Cy3P·HBF4 (0.016 g, 0.045 mmol,
0.2 equiv.). The flask was fitted with a vertical condenser and was
set under an argon atmosphere. Anhydrous 1,4-dioxane (5 mL) was
added by syringe, and the mixture was heated at reflux at 130 °C
for 24 h, then cooled to room temperature, and filtered through a
sintered Büchner funnel to remove insoluble inorganic materials.
The dioxane solution was dried under reduced pressure, and the
crude residue was redissolved in a small amount of dichlorometh-
ane. It was applied to a silica gel column for flash chromatography
(CH2Cl2/MeOH, gradient from 98:2 to 90:10) to afford compound 23
(0.060 g, 0.12 mmol, 54 % yield) as a beige powder. 1H NMR (CDCl3):
δ = 7.33 (dd, J1 = 7.9 Hz, J2 = 4.9 Hz, 1 H), 7.45 (dd, J1 = 7.9 Hz, J2 =
4.9 Hz, 2 H), 7.85–7.93 (m, 3 H, signals overlapping), 7.95 (d, J =
6.5 Hz, 1 H), 7.96 (s, 1 H), 8.08 (s, 2 H), 8.30 (d, J = 7.9 Hz, 2 H), 8.49
(s, 2 H), 8.72 (d, J = 4.9 Hz, 1 H), 8.83 (d, J = 4.1 Hz, 2 H) ppm. 13C
NMR (CDCl3): δ = 115.1, 120.0, 122.7, 123.6, 125.1, 127.6, 128.8,
136.2, 137.0, 137.1, 145.8, 146.7, 148.3, 149.0, 150.2, 151.2, 152.5,
159.1, 161.2 ppm. MS (MALDI-TOF): m/z (%) = 512.41 [M + H]+
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Acknowledgments
The authors thank Prof. Nikos Chronakis for access to the CD
and MS-MALDI-TOF/TOF instruments, Prof. Panayiotis Koutentis
for very helpful discussions, and Dr. Elena Loizou and Ms. Maria
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Received: October 2, 2015
Published Online: November 24, 2015
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