Design, synthesis and binding studies of a novel quadruple ADDA hydrogen-bond array

Article abstract of DOI:10.1039/c2ob25333j

The design and synthesis of a novel ADDA hydrogen-bond array is described. The ureidodiimidazole motif (UDIM) 2 engages in interactions with complementary diamidonaphthyridine (DAN) 3 motifs with an association constant K_a = 825 ± 16 M^-1 in chloroform. ¹H NMR and molecular modelling studies were carried out in order to explain the unexpected behaviour of this new supramolecular motif. These revealed that a combination of effects including; an energetic bias for the folded conformer, subtle differences in shape complementarity between the two components and the potential for self-association of UDIM 2 disfavour higher affinity interactions between the two components.

Full text of DOI:10.1039/c2ob25333j

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ARTICLE TYPE
Design, synthesis and binding studies of a novel quadruple ADDA
hydrogen-bond array
a
a
a
a,b
Maria L. Pellizzaro, Simon A. Barrett, Julie Fisher and Andrew J. Wilson*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
The design and synthesis of a novel ADDA hydrogen-bond array is described. The ureidodiimidazole
motif (UDIM) 2 engages in interactions with complementary diamidonaphthyridine (DAN) 3 motifs with
-1
1
a
an association constant K = 825 ± 16 M in chloroform. H NMR and molecular modelling studies were
carried out in order to explain the unexpected behaviour of this new supramolecular motif. These revealed
that a combination of effects including; an energetic bias for the folded conformer, subtle differences in
shape complementarity between the two components and the potential for self-association of UDIM 2
disfavour higher affinity interactions between the two components.
1
0
39, 40
has been extensively studied
as a DAAD array, was selected
Introduction
as a complementary partner to study the molecular recognition
properties of this new supramolecular building block (in this
work we used the novel derivative 3 with n-butyl chains). Our
results reveal weaker than anticipated heterodimerisation
resulting from a complex interplay of effects, including a variety
of stable conformational states, subtle differences in shape
complementarity, steric interactions and the potential for
homodimerisation of UDIM 2.
The design and synthesis of linear arrays possessing hydrogen
bond donor and acceptor moieties represents an ongoing
endeavour in supramolecular chemistry. Such motifs enhance
5
0
1
2
2
3
3
4
4
5
0
5
0
5
0
5
1
our fundamental understanding of co-operative non-covalent
interactions and form the bases of higher order supramolecular
2, 3
assemblies. The strength with which linear arrays participate in
molecular recognition with complementary partners is determined
by a complex interplay of different factors that is not wholly
understood. These factors include: the number and arrangement
of donor and acceptor interactions, the tautomeric and
conformational properties of the array and remote substituent
5
5
1, 4, 5,6
effects.
Additional subtle effects occur as a consequence of
proximal functional groups capable of weak interactions (e.g.
CH···O hydrogen-bonds) and small differences in shape
complementarity that arise as a result of small differences in bond
length within the covalent framework of the array (e.g. the C-O,
C-N and C-C bond length all differ). Although arrays employing
large numbers (≥4) of hydrogen bond donor and acceptor
moieties have been described,
hydrogen bonds have received considerable attention as targets
for design and synthesis,
7
7
8-15
linear arrays employing four
1
6-33
presumably because they represent
Figure 1 Structures of compounds 1-3 under study in this work together
with an illustration depicting the degenerate conformational/ tautomeric
properties of the N-acyl-2-aminoimidazole motif.
an ideal balance between synthetic accessibility and predictable
or controllable recognition properties. Our group previously
described a series of conformer independent linear arrays that
participate in molecular recognition through three hydrogen
Results and Discussion
15, 34-38
bonds.
In the current manuscript we describe the logical
6
0
Synthesis
extension of our work with N-alkyl/ arylureidoimidazoles (e.g.
34, 36, 38
UIM 1)
to the development of a quadruply hydrogen-
retains the
UDIM 2 was synthesised as illustrated in Scheme 1b by closely
34
bonded motif. Ureidodiimidazole (UDIM)
conformer independent features of the N-acyl-2-aminoimidazole
core (Figure 1) and presents an acceptor-donor-donor-acceptor
2
following the synthesis of UIM 1. Mono-protection of guanine
hydrochloride 4 with a Boc-group to give 5, followed by
cyclisation using 1-bromopinacolone gave the protected
(ADDA) arrangement of functional groups in the fully extended 65 aminoimidazole 6. Deprotection using hydrochloric acid in
conformation. The diamidonaphthyridine (DAN) motif, which
ethanol gave the hydrochloride salt 7, which was reacted with
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1
2
,1-carbonyldiimidazole (CDI) to give the final product, UDIM
. Because of the potential for UDIM 2 to adopt a range of
temperatures (333K) the NH resonances begin to coalesce;
indicating that inter-conversion between the different tautomers
and conformers becomes more rapid on the NMR timescale. On
conformational and tautomeric states, we attempted to synthesise
more symmetrical derivatives. The product of the reaction 25 cooling, the NMR spectrum is resolved further, however
5
between benzimidazole 8 and CDI gave the desired target 9
assignment of the individual conformers/tautomers was not
possible by 2D NOESY NMR because of the presence of
exchange peaks in the spectrum (see ESI). Two of the extended
conformers are symmetrical whilst all the folded conformers are
however this was insoluble in most solvents and we were only
1
able to obtain a H NMR spectrum. Aminoimidazole 10 and
dimethlyaminoimidazole 11 both failed to yield the desired
products 12 and 13 noting again that the reaction produced only 30 not; it is therefore tempting to suggest that a folded conformer
1
0
insoluble precipitates. DAN 3 was synthesized following a
predominates as there are four signals of comparable intensity in
the low temperature spectrum, although this might equally
suggest that two symmetrical extended conformers (e.g. 2-ex-i
and 2-ex-iii) are present in comparable proportions.
slightly modified literature procedure as illustrated in Scheme
41
1
b.
3
5
Figure 2 Plausible conformational states of UDIM 2
Scheme 1 Synthesis of (a) UDIM 2 and (b) DAN 3.
1
5
Conformational Analysis
UDIM 2 may exist in two different conformations as illustrated in
Figure 2 which we refer to as extended (ex) or folded (fo).
Similarly, two tautomers of each imidazole ring in UDIM 2 are
1
Figure 3 VT H NMR of 2 (500 MHz, 10 mM, CDCl
3
)
1
possible, leading to seven plausible states. The H NMR spectrum
Binding Studies
2
0
of UDIM 2 suggests that there is more than one conformer
1
present in solution (Figure 3). VT H NMR shows that at high
In order to study the interaction between UDIM 2 and DAN 3 in
2
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1
-1
deuterochloroform, a H NMR spectrum of a 1:1 mixture was
was found to have an association constant of about 834 M , in
1
-1
compared to that of the individual components. Significant shifts 60 agreement with the H NMR titrations (825 M ).
1
of the NH resonances of DAN 3 were observed, indicating that
intermolecular hydrogen bonding occurs. In contrast resonances
for UDIM 2, exhibited only minimal complexation induced shifts
Figure 4 H NMR based binding studies of UIM 1 and UDIM 2 towards
5
0
5
0
5
0
5
0
5
0
5
(
CIS), however an additional set of resonances were observed –
this is most clearly seen for the imidazole CH resonance and will
be discussed further below. It should be noted that there was no
change in the NMR spectrum of this sample over a period of 24
hours. The result indicates that weak binding (see below) is not
limited by slow interconversion between tautomeric and
conformational states of UDIM 2. The origin of this effect likely
derives from either geometrical/steric incompatibility of 2-ex-i-iii
with DAN 3 or because 2 exhibits a strong preference for a
conformational/ tautomeric state (i.e. 2-fo-i-iv)) that is
unproductive for binding to 3. On the basis of the CIS for DAN 3,
1
1
2
2
3
3
4
4
5
5
1
H NMR titrations were used to determine the association
constant for the interaction. Aliquots of UDIM 2, containing a
small amount of DAN 3, were added to a solution of DAN 3.
After each addition of UDIM 2 a H NMR spectrum was
recorded (Figure 4a) and the change in shift of the NH of DAN 3
was plotted against the concentration of UDIM 2. Fitting the data
to a 1:1 association model in HypNMR gave a binding affinity
of 825 ± 16 M (Figure 4b). It should be noted that no attempt
was made to account for self-association of either component in
the analytical treatment of the data. Such an assumption is
appropriate for DAN 3 which is known to only weakly self-
associate, however it is not clear if the assumption is appropriate
for UDIM 2.
The additional set of resonances observed for UDIM 2 in the
presence of DAN 3 is indicative of slow exchange between two
or more species. In addition to a simple equilibrium between
bound and unbound UDIM 2, these might include two different
conformations of UDIM 2 bound to DAN 3 and/or higher
1
42
-1
1
1
aggregates such as homodimers of UDIM 2. A H- H NOESY
experiment was performed to provide additional structural
information on the nature of interactions taking place, however it
was difficult to assign correlations as through-space or exchange
1
DAN 3 (a) H NMR spectra for the titration of UDIM 2 into DAN 3
300 MHz, CDCl , new UDIM CH resonance shown in light blue) (b)
proposed structure of the complex and the binding isotherm for the
(
3
1
1
correlations (see ESI).
A
H- H ROESY was therefore
1
performed; the spectrum (Figure 5) exhibits intramolecular 65 titration shown in (a), (c) H NMR spectra for the titration of UIM 1 into
3
DAN 3 (300 MHz, CDCl ) (d) proposed structure of the complex and the
correlations only for the additional set of resonances that appear
for UDIM 2 in the presence of DAN 3. Such a result is consistent
with the extended conformation of UDIM 2-ex-iii being bound to
DAN 3
Formation of a complex in solution was also observed by
diffusion ordered NMR spectroscopy (DOSY). DOSY is capable
of measuring the size of assemblies that are present in solution
and changes in the diffusion coefficients reflect changes in the
binding isotherm for the titration shown in (c).
The comparatively low binding constant determined and
observation of a distinct set of resonances attributable to
UDIM:DAN 2:3 prompted further study of the self-association of
UDIM 2. In particular, the major resonances for the NH protons
are observed at low field (11-15 ppm) which is indicative of
hydrogen-bonding however of the plausible conformations, only
NH’s can be hydrogen-bonded at any one time, suggesting the
possibility of intermolecular self-association. Dilution studies in
chloroform resulted in no change in the chemical shifts of UDIM
7
7
8
0
5
0
43
size of these species. All diffusion coefficients were measured
on 3.6 mM samples at 20 °C in CDCl . The diffusion coefficients
for DAN 3 and UDIM 2 in isolation were 10.61 and 9.33 m s ×
2
3
2
-1
-10
1
0
respectively. In the 1:1 mixture they both decreased (10.06
2
. Similarly for the VT experiment illustrated in Figure 3, larger
2
-1
-10
and 8.67 m s × 10 ), indicating that they are within larger
assemblies in solution, however the absence of coincident
diffusion coefficients for both components indicates weak
association between 2 and 3. It is possible to calculate the
association constant of the complex from the change in the
diffusion coefficients (see ESI). The UDIM:DAN 2:3 complex
chemical shift changes than those observed would be anticipated
for intermolecular hydrogen-bonds indicating either no
interaction or a very strong interaction. We therefore also
performed a DMSO titration to disrupt intermolecular hydrogen
bonding; as the concentration of DMSO-d
solution of UDIM 2 in CDCl , the NH resonances broaden and
6
increases in a 10 mM
3
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then converge to two peaks which may support an intermolecular 30 fact that formation of a heterodimer maximizes the non-covalent
mode of association but could also arise due to a change in the
position of conformational/ tautomeric equilibrium. We also
interactions of the system as a whole, however this is not always
the case; DAN derivatives have been shown to form weak
3
-1 19, 21
performed a dilution study at 5% DMSO-d
no change in the chemical shift of the key resonances was
observed. Overall, these data are ambiguous in defining the 35 self-associate weakly. It is noteworthy that multiple resonances
6
in CDCl
3
, however
complexes (K
a
~ 10 M )
with dipyridylurea (DPU)
5
derivatives which preferentially adopt a folded conformation and
10
extent of UDIM 2 self-association. However, in tandem with the
diffusion co-efficient determined by DOSY, we tentatively
suggest that UDIM 2 is largely monomeric under the conditions
of the experiment and that its self-association behaviour is not a
for the UDIM 2 imidazole protons are still observed in the
presence of DAN 3 consistent with there being no change in the
distribution of conformers at equilibrium. It is possible that this
derives from an excessive barrier to interconversion between
1
0
major characteristic that defines the intermolecular association 40 tautomers and conformers; as stated previously, we believe this to
1
1
behaviour with DAN 3.
be unlikely as our H- H NOESY data revealed correlations
16
indicative of exchange, whilst for UPy
ureidodiguanidinium DDDD motifs
kinetically rapid on the NMR timescale.
and
+
31
such transitions are
4
5
5
6
5
0
5
0
The Zimmerman group have shown that motifs capable of
interaction through only three hydrogen bonds can form stronger
interactions in comparison to arrays presenting four hydrogen
46
bonding groups, although it should be noted that this is
47
1
functional group dependent. We therefore also performed a H
NMR titration on the UIM:DAN, 1:3 complex (Figure 4c). CIS
were observed and these were in fact significantly larger than
those observed for UDIM:DAN 2:3. The binding affinity for the
-1
UIM:DAN 1:3 interaction – 2140 ± 42 M (Figure 4d) – is
stronger than the UDIM:DAN 2:3 complex and is presumably
38
weaker than triply hydrogen-bonded complexes (made by 1)
because the remaining H-bond donor on DAN 3 contributes an
1
additional unfavourable secondary interaction. Taken with the H
NMR observations this suggests that both the extend conformer
2-ex-i-iii and folded conformer 2-fo-i-iv of UDIM should be
capable of interaction with DAN 3.
Molecular Modelling
We performed modelling studies to provide additional insight
48
on the experimental observations. Gaussian 03 was used to
perform density functional theory (DFT) calculations, which can
predict the geometry of complexes that interact via intermolecular
hydrogen bonds. Gas phase calculations were run using the
hybrid B3LYP functional. Due to the size of the molecules, it was
necessary to use a mixed basis set C, 6-31G; N/O, 6-31G*; H, 6-
6
5
38
31++G** as we did in earlier work. This approach allows
1
1
70 polarisation and diffusion functionals to be added to heteroatoms
involved in hydrogen bonding, whilst atoms not directly involved
in the formation of hydrogen bonds (i.e. carbon), can be
calculated using a smaller basis set, reducing the computational
requirements. The structures and minimum energies of the
Figure 5 Phase sensitive H- H ROESY spectrum of 1:1 mix of
components UDIM 2 and DAN 3 at 10 mM concentration (with respect
1
5
3
to each component) in CDCl , 500 MHz.
We found the weak nature of the interaction between UDIM 2
and DAN 3 surprising; although the VT NMR experiments
suggest UDIM 2 adopts a variety of conformations and
tautomers, we still anticipated that when presented with the
complementary DAN array 3 the conformer 2-ex-i-iii would be
adopted in order to maximize hydrogen bonding interactions
within the entire system. This property has been observed for
other systems e.g. the ureidopyrimidine motif (UPy) despite
forming a stable homodimer, reconfigures in the presence of
a
DAN derivatives to form stable heterodimers (K > 10 M ).
Similarly, the N, N’-di-4-triazolylurea motif recently described by
Hisamatsu and co-workers (DTU) also forms stable heterodimers
7
8
8
5
0
5
individual molecules were calculated first. For UDIM 2 we first
performed calculations on the version without the Bu groups
t
present (compound 12) to minimize computational time. The
folded conformer 12-fo is more stable than the extended
2
0
-1
conformer 12-ex by 3.6 kJ mol (Figure 6a). We also preformed
the calculations for all conformers and tautomers of 2 – the
-1
energies of which were all within 5 kJ mol (see ESI). We next
4
4
calculated the structure and energy of DAN 3 in the binding
conformation (again the butyl chains were shortened to methyl
groups (compound 18)); alternative amide rotamers were not
considered because, given our earlier observations on
5
-1 22, 45
2
5
5
-1 29
with DAN derivatives (K
a
> 10 M ). In both instances the high
affinity interaction may be (at least partially) reconciled by the
38
amidonaphthyridine derivatives, these are disfavoured due to
electrostatic repulsion with the nitrogen atoms in the aromatic
4
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Figure 6 Optimized geometries and calculated energies of monomers and complexes used in this work as calculated using DFT (B3LYP, C, 6-31G;
N/O, 6-31G*; H, 6-31++G**), (a) UDIM in folded and extended conformations, (b) UDIM 12 bound to DAN 18 in folded and extended conformations,
c) UIM 19 bound to DAN 18 in both conformations.
(
38
ring. Calculations on the dimers were run using counterpoise in
order to overcome the basis set superposition error, using the
necessary in order to form the complex. For triply hydrogen-
bonded complexes based upon UIM 19, these distortions are not
basis set outlined above. The calculations were limited to 12:18 40 observed. Thus in order to interact through the desired ADDA-
due to the size of 2. Energies are counterpoise corrected and
given for the global minimum. The large energies relative to
those obtained by experiment derive from the fact that the
calculations were performed in the gas phase and no solvation
DAAD array both DAN 3/18 and UDIM 2/12 must adopt
distorted and unfavorable geometries. A further consideration is
that the t-butyl groups in conformation 2-ex-i-ii might sterically
impede interaction with 3, however we do not attribute the weak
5
0
5
0
5
0
5
energies are incorporated. For this reason, caution must be 45 binding affinity solely to this feature as our data clearly indicate
exercised in interpreting the results however the qualitative
observations are still useful. The UDIM:DAN complex when
optimized using UDIM in the extended conformation 12-ex:18
interconversion between the different conformations and
tautomers is possible; if strong hydrogen bonds formed this
would drive the equilibrium in favour of conformations capable
of making these interactions.
1
1
2
2
3
3
-1
has a binding affinity of -104 kJ mol and in the folded
-1
conformation 12-fo:18 an energy of -76 kJ mol (Figure 6b). For
comparison the energies of both conformers of UIM (with the tBu 50 Conclusions
groups removed 19) bound to DAN 18 were also calculated and
-1
In conclusion, we have described the design and synthesis of a
novel hydrogen-bonding motif that has the potential to present a
quadruple ADDA array. Lower than anticipated binding affinities
towards a complementary partner were observed that may be
attributed to a number of factors. This may include an energetic
bias for the folded conformer alongside subtle differences in
shape complementarity and steric interactions that hinder the
desired interaction. The results highlight the fact that the
conformational and tautomeric states adopted by hydrogen-
bonding motifs are difficult to predict and control. In addition to
found to be -78 and -79 kJ mol respectively (Figure 6c). These
results suggest that (a) the difference in energy between the
folded and extended states of UDIM 2/12 is quite small and (b)
the extended conformation of UDIM 12 (and most likely 2)
should be favourably recognized by DAN derivatives with
reasonable affinity, which conflicts with the experimental
observations. There are a number of explanations for this
discrepancy – (i) solvation energies affect the solution stability of
the two conformations of UDIM 2/12 to the extent that the
difference in stability is much larger than calculated (ii) solvation
energies offset the difference in experimental binding energies
5
6
6
7
5
0
5
0
16
31
the UPy and ureidodiguanidinium examples referred to
previously, where maximizing non-covalent interactions seems to
drive formation of a heterocomplex, the ureidodiguanidinium
(
3:2-ex and 3:2-fo) such that they do not match with those
calculated (18:12-ex and 18:12-fo) (iii) the calculated energies do
not account for additional factors. In the latter case, one such
factor may be the bond lengths within the covalent framework.
Because aromatic C-N bonds are shorter than aromatic C-C
+
which is capable of presenting a DDDD and engages in high
affinity interactions with AAAA arrays (described recently by the
31
Leigh group) is a similar motif to UDIM 2. On the other hand,
our observations follow the trend observed with DPUs, where a
strong preference for the folded conformation dominates
molecular recognition behaviour. Although undesired states can
be designed out as in the case of ureidoguanosine (UG), where
the energy of the self-complementary conformer is much higher
than the conformer that presents the desired ADDA array
46
bonds, the DAN 18 motif is slightly curved, not linear.
Similarly, the intramolecular hydrogen bonds for UDIM 12-fo
undergo minimal change upon complexation with DAN 18 to
form three hydrogen-bonds, whereas in UDIM 12-ex they
become shorter upon complex formation with DAN 18, implying
significant distortion of the covalent framework of UDIM 12 is
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(
facilitating high affinity and fidelity interaction with DAN
3
CDCl ); 162.9, 162.8 (× 2), 162.7 (× 2), 149.9, 149.1 (× 2),
149.0, 148.1, 141.3 (× 2), 140.1 (× 2), 136.7, 134.7, 111.8, 110.4
49
derivatives), this remains difficult to do predictably. Our future
work in this area will focus on this objective.
(× 2), 103.2, 30.9 (× 3), 30.8, 30.2 (× 2), 29.8 (× 2), 29.7; ESI-
+
6
0
HRMS found m/z 305.2087 [M + H]
15 25 6
C H N O requires
Experimental
305.2084.
1,3-Di(1H-benzo[d]imidazol-2-yl)urea 9
5
General Considerations
2-Aminobenzimidazole (100 mg, 0.75 mmol) was added to a
All reagents were purchased from Aldrich or Alfa-Aesar and used
solution of triethylamine (262 μL, 1.88 mmol) in THF (10 mL).
without further purification unless otherwise stated. Where 65 The reaction mixture was then heated to reflux and 1,1-carbonyl
anhydrous solvents were required, THF was freshly distilled from
sodium benzophenone ketyl radical, CH Cl was freshly distilled
from calcium hydride and CHCl was freshly distilled from
calcium chloride under a nitrogen atmosphere. Anhydrous DMF
was obtained “sure-sealed” from Sigma-Aldrich. Triethylamine 70 colourless solid; m.p. > 350 °C; R
diimidazole (73 mg, 0.45 mmol) was added. The reaction mixture
was then heated at reflux for a further 20 hr, before being cooled
2
2
1
1
2
2
3
0
5
0
5
0
3
to 0 °C and filtered. The resultant solid was washed (Et
give 1,3-di(1H-benzo[d]imidazol-2-yl)urea 8 (51 mg, 45%) as a
0.00 (1:9 MeOH–EtOAc); δ
); 12.15 (2H, s, 2 × NH), 7.47-7.44 (4H, m,
4 × ArCH), 7.14-7.11 (4H, m, 4 × ArCH); Molecule was too
2
O) to
f
H
was distilled from calcium hydride and stored, under nitrogen,
over potassium hydroxide pellets. All non-aqueous reactions
were carried out under a nitrogen atmosphere. Analytical thin
layer chromatography (TLC) was conducted using Merck
(300 MHz, DMSO-d
6
-1
C
insoluble to obtain δ ; νmax/cm (neat); 3369-2681 (br), 1633,
1593, 1564, 1274, 752; ESI-HRMS found m/z 293.1142
+
13 6
Kieslegel 0.25 mm silica gel pre-coated aluminium plates with 75 [M + H] , C15H N O requires 293.1145.
fluorescent indicator active at UV254
.
Purification by column
7-Amino-1,8-naphthyridin-2-ol 15
chromatography was carried out using Merck Kieselgel 60 silica
gel. NMR spectra were obtained using Bruker DRX500 or
Concentrated sulphuric acid (10 mL) was added dropwise to a
ground mixture of 2,6-diaminopyridine 14 (2.2 g, 20.0 mmol) and
malic acid (3.0 g, 22 mmol) cooled to 0 °C. The reaction mixture
Bruker DPX300 spectrometers operating at 500.13 MHz or
1
3
00.13 MHz for H spectra and 125.76 MHz or 75.47 MHz for 80 was then heated to 110 °C for 3 hr before being cooled to 0 °C.
1
3
Ammonium hydroxide solution was then added dropwise to pH 9
and the reaction mixture was filtered and washed with water and
C spectra as stated. Proton spectra are referenced to TMS at
.00 ppm, and carbon spectra to CDCl at 77.4 ppm, unless
0
3
Et
an orange solid; m.p. decomposes > 350 °C [Lit. > 350 °C] ; R
(300 MHz, DMSO-d ); 11.91 (1H, s, OH), 7.66
(1H, d, J = 9.5 Hz, ArCH), 7.65 (1H, d, J = 8.4 Hz, ArCH), 7.03
(2H, s, NH ), 6.36 (1H, d, J = 8.4 Hz, ArCH), 6.12 (1H, d,
J = 9.5 Hz, ArCH); δ (75 MHz, DMSO-d ); 164.1, 160.9, 150.7,
140.1, 137.7, 115.2, 105.6, 105.4; ESI found m/z 162.1 [M + H] ,
8 8 3
C H N O requires 161.2.
2
O to give 7-amino-1,8-naphthyridin-2-ol 15 (3.04 g, 95%) as
otherwise stated. Melting points were determined using a Griffin
D5 variable temperature apparatus and are uncorrected. IR
50
f
spectra were obtained using Perkin-Elmer FTIR spectrometer. 85 0.00 (EtOAc); δ
H
6
Microanalysis was carried out on a Carlo Erba Elemental
Analyser MOD 1106 instrument. High Resolution Mass Spectra
(HRMS) were recorded on a Micromass GCT Premier using
electron impact ionisation (EI) or a Bruker Daltonics micrOTOF
using electro spray ionisation (ESI).
2
C
6
+
90
N-(7-Hydroxy-1,8-naphthyridin-2-yl)pentanamide 16
Synthetic Procedures
Valeroyl chloride (3.70 mL, 31.7 mmol) was added dropwise to a
solution of 7-amino-1,8-naphthyridin-2-ol 15 (3.00 g, 18.6 mmol)
in pyridine (20 mL) and heated to 110 °C for 20 hr before the
2
-tert-Butoxyamido-4-tert-butylimidazole and UIM 1, were
36
3
5
synthesized as described previously by our group.
1
2
,3-Bis(5-tert-butyl-1H-imidazol-2-yl)urea 2
-tert-Butoxyamido-4-tert-butylimidazole 6 (1.36 g, 5.68 mmol)
95 reaction mixture was allowed to cool to room temperature. The
pyridine was evaporated and then co-evaporated with toluene and
was dissolved in 1M HCl-EtOH (100 mL) before being stirred at
3
the resultant black residue was dissolved in hot CHCl (20 mL)
reflux for 17 hr. It was then evaporated to dryness in vacuo to
and hexane (200 mL) was added. The solid was filtered and
4
0
give the aminoimidazole hydrochloride as
a
yellow oil.
triturated (MeOH) to give N-(7-hydroxy-1,8-naphthyridin-2-
Triethylamine (1.98 mL, 14.20 mmol) was added to a solution of 100 yl)pentanamide 16 (3.47 g, 76%) as a pale yellow powder; m.p.
the aminoimidazole hydrochloride (1.00 g, 5.68 mmol) in THF
316–319 °C; R 0.39 (1:19 MeOH–CH Cl ); δ (300 MHz,
100 mL) and the reaction mixture was stirred at room
DMSO-d ); 11.84 (1H, s, OH), 10.48 (1H, s, NH), 8.04 (1H, d,
temperature for 2 hr. It was then heated to reflux and 1,1-
carbonyldiimidazole (0.55 g, 3.41 mmol) was added. The
f
2
2
H
(
6
J = 8.5 Hz, ArCH), 7.94 (1H, d, J = 8.5 Hz, ArCH), 7.84 (1H, d,
J = 9.4 Hz, ArCH), 6.42 (1H, d, J = 9.4 Hz, ArCH), 2.45 (2H, t,
4
5
5
5
0
5
reaction mixture was then heated at reflux for a further 20 hr 105 J = 7.4 Hz, CH
2
), 1.55 (2H, m, CH
(3H, t, J = 7.3 Hz, CH ); ESI-HRMS found m/z 246.1239
requires 246.1237.
2 2
), 1.32 (2H, m, CH ), 0.89
before being cooled to room temperature and filtered. The filtrate
was evaporated in vacuo and the resultant solid was purified by
column chromatography (gradient elution: 0:1-1:19 MeOH-
3
+
16 3 2
[M + H] , C13H N O
N-(7-Chloro-1,8-naphthyridin-2-yl)pentanamide 17
N-(7-Hydroxy-1,8-naphthyridin-2-yl)pentanamide 16 (500 mg,
2 2
CH Cl ) followed by trituration (MeOH) to give 1,3-bis(5-tert-
butyl-1H-imidazol-2-yl)urea 2 (150 mg, 17%) as a colourless
solid; m.p. decomposes > 280 °C (Found: C, 59.1; H, 7.95; N,
1
10 2.04 mmol) was dissolved in POCl
for 3 hr. The reaction mixture was then cooled and POCl
evaporated in vacuo. The residue was poured onto iced water
25 mL) and neutralised with concentrated ammonium hydroxide
solution under vigorous stirring. The reaction mixture was
15 extracted into CH Cl (3 × 20 mL) and then the organic phase
3
(10 mL) and heated to 95 °C
3
was
2
(
7.7; C15
1:9 MeOH-CH
12.40 (1H, s, NH), 11.16 (1H, s, NH), 10.96 (1H, s, NH), 6.30
H
24
N
6
O requires C, 59.2; H, 7.95; N, 27.6%); R
f
0.45
(
2
Cl ); δ (300 MHz, CDCl ); 15.20 (1H, s, NH),
2
H
3
t
1
2
2
and 6.26 (2H, 2 × s, 2 × ArCH), 1.33 (18H, s, Bu); δ
C
(125 MHz,
6
| Journal Name, [year], [vol], 00–00
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1
1
was washed with saturated aqueous sodium bicarbonate
3 × 20 mL), water (3 × 20 mL) and saturated aqueous sodium
chloride (20 mL). The organic phase was dried (Na SO ) and
evaporated in vacuo before being crystallised (1:1
MeOH/MeCN–H O) to give N-(7-chloro-1,8-naphthyridin-2- 60 of 500 MHz. A pulse sequence of 8.2 µs pulse, 2.0 s delay, 16.4
yl)pentanamide 17 (380 mg, 71%) as yellow plates; m.p. 159–
62 °C; R 0.60 (EtOAc); δ (300 MHz, CDCl ); 8.57 (1H, d,
H- H NOESY data (256 increments, 1024 data points) was
collected on the individual components and their 1:1 mixtures at
10 mmol concentrations in CDCl . Spectra were recorded on a
Bruker Avance500 instrument operating at 300 K at a frequency
(
2
4
3
5
2
µs pulse, 1.2 s delay was used (note: spectra have been
symmetrised for presentation purposes).
1
f
H
3
J = 8.7 Hz, ArCH), 8.35 (1H, s, NH), 8.19 (1H, d, J = 8.7 Hz,
ArCH), 8.07 (1H, d, J = 8.3 Hz, ArCH), 7.40 (1H, d, J = 8.3 Hz,
ROESY Data Aquisition
1
1
1
0
ArCH), 2.48 (2H, t, J = 7.7 Hz, CH
2H, m, CH ), 0.96 (3H, t, J = 7.4 Hz, CH
); 172.7, 154.5, 154.4, 139.1, 138.8, 122.0, 119.2, 115.5,
2
), 1.75 (2H, m, CH
); δ
(75 MHz, 65 1:1 mixture of components UDIM 2 and DAN 3 at 10 mM
concentration with respect to each component in CDCl solvent.
The sample was analysed immediately after preparation to avoid
uptake of residual atmospheric H O. Spectra were recorded (104
2
), 1.44
Phase sensitive H- H ROESY experiments were performed on a
(
2
3
C
CDCl
3
C
C
3
3
+
7.7, 27.3, 22.3, 13.8;; ESI-HRMS found m/z 264.0896 [M + H] ,
+
13
H
H
15ClN
14ClN
3
O requires 264.0898. Found m/z 286.0713 [M + Na] ,
2
1
5
13
3
NaO requires 286.0718.
increments, 572 data points) on a Bruker Avance DRX500
70 instrument at 253 K at a frequency of 500 MHz. A mixing time of
350 ms was applied using a constant wave spin lock mixing
method. A pulse sequence repetition of 2.2 secs was used (note:
spectra have been symmetrised for presentation purposes).
N,N'-(1,8-Naphthyridine-2,7-diyl)dipentanamide 3
2
0
-Pentanoylamino-7-chloro-1,8-naphthyridine
.38 mmol), valeramide (46 mg, 0.46 mmol), potassium
17
(100 mg,
carbonate (73 mg, 0.53 mmol), palladium (II) acetate (4 mg,
0.02 mmol) and xantphos (18 mg, 0.04 mmol) were suspended in
2
0
1
,4-dioxane (10 mL) in a schlenck tube. The reaction mixture was
Acknowledgments
heated to 100 °C for 23hr before being cooled to room
temperature and filtered through celite. The solvent was
evaporated in vacuo and the crude product was purified by
column chromatography (gradient elution: 0:1-1:19 MeOH–
7
5
This work was supported by the Leverhulme Trust
F/00122/AN].
[
2
3
3
5
0
5
Notes and references
2 2 2
CH Cl ) followed by crystallisation (MeOH–H O) to give N,N'-
a
(
1,8-naphthyridine-2,7-diyl)dipentanamide 3 (79 mg, 63%) as
School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2
10
colourless needles; m.p. 214–216 °C [Lit. 216–217 °C] (Found:
9JT, United Kingdom, Fax: +44 (0)113 3436565; Tel: +44 (0)113
3431409; E-mail: A.J.Wilson@leeds.ac.uk
8
0
C, 65.6; H, 7.45; N, 17.0; C18
H
24
N
4
O
2
requires C, 65.8; H, 7.37;
); δ (300 MHz, CDCl );
b
Astbury Centre for Structural Molecular Biology, University of Leeds,
N, 17.1%); R 0.66 (1:9 MeOH–CH
f
2
Cl
2
H
3
Woodhouse Lane, Leeds LS2 9JT, United Kingdom
8
(
1
.44 (2H, d, J = 8.8 Hz, 2 × ArCH), 8.35 (2H, s, 2 × NH), 8.12
2H, d, J = 8.8 Hz, 2 × ArCH), 2.46 (4H, t, J = 7.5 Hz, 2 × CH ),
), 0.95 (6H, t, 85 See DOI: 10.1039/b000000x/
); 172.3, 153.9, 153.7,
139.0, 118.4, 113.4, 37.8, 27.3, 22.3, 13.8; ESI-HRMS found m/z
†
Electronic Supplementary Information (ESI) available: experimental
2
characterisation, additional 2D NMR spectra and modelling geometries.
.73 (4H, m, 2 × CH
2
), 1.42 (4H, m, 2 × CH
(75 MHz, CDCl
2
J = 7.3 Hz, 2 × CH
3
); δ
C
3
1
.
A. J. Wilson, Soft Matter, 2007, 3, 409-425.
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25 4 2
29.1968 [M + H] , C18H N O requires 329.1972.
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4
0
CDCl
3
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51
pair simulated echo (BPPSTE) sequence was employed
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7
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318-1319.
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.
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This journal is © The Royal Society of Chemistry [year]