Determination of binding constants of hydrogen-bonded complexes by ITC, NMR CIS, and NMR diffusion experiments

Article abstract of DOI:10.1002/ejoc.201001684

The host-guest complex formation between barbital and various acylaminopyridyl isophthalamides (Hamilton receptors) has been determined quantitatively. The syntheses of nine isophthalamides are described. Their structures differ in the substitution patter

Full text of DOI:10.1002/ejoc.201001684

FULL PAPER
DOI: 10.1002/ejoc.201001684
Determination of Binding Constants of Hydrogen-Bonded Complexes by ITC,
NMR CIS, and NMR Diffusion Experiments[‡]
Christiane Dethlefs,[a] Jens Eckelmann,[a] Hauke Kobarg,[a] Thomas Weyrich,[a]
Stefan Brammer,[a] Christian Näther,[b] and Ulrich Lüning*[a]
Keywords: Host–guest systems / Hydrogen bonds / Receptors / Supramolecular chemistry / Association constants
The host–guest complex formation between barbital and
various acylaminopyridyl isophthalamides (Hamilton recep-
tors) has been determined quantitatively. The syntheses of
nine isophthalamides are described. Their structures differ in
the substitution patterns on the central isophthalic unit, and
the natures of the acyl residues. Ethylhexanoyl derivatives
proved to be more soluble than pentanoyl amides. The asso-
ciation constants were determined by 1H NMR titrations
monitoring chemically induced shifts (CIS values), by 1H
NMR diffusion experiments, and by isothermal titration calo-
rimetry (ITC), giving Kass values in chloroform at 298 K be-
–1
tween 33ϫ103 and 100ϫ103
M .
acid derivatives (Figure 1, R = RЈ = H, with the two amide
groups in the 6-positions of the pyridine rings being con-
nected to give a macrocycle).[13] 1H NMR titrations of sev-
eral related receptors with different guests gave association
constants in the 105 m–1 range (in chloroform) for such bent
arrays of six hydrogen bonds.[14] The original macrocyclic
Hamilton receptors[13] were synthesized from isophthalic
acid as starting material. Molecular recognition between
open structures such as 1 and barbiturates, however, is also
strong and orthogonal to other hydrogen bond patterns.[7]
Consequently, nonmacrocyclic “Hamilton receptors” have
been used preferentially, and today quite a number of sup-
ramolecular systems use the “Hamilton receptor” and bar-
bituric acid derivatives; examples include self-assembled
dendrimers,[15,16] multifunctional block copolymers[17] or
double dynamers.[18]
Introduction
Noncovalent interactions are the fundamental forces in
supramolecular chemistry and molecular recognition.[1] In
order to design supramolecular structures based on mul-
tiple hydrogen bonds, it is necessary to use complementary
acceptor and donor patterns.[2,3] Various linear hydrogen-
bond acceptor/donor sequences have been synthesized and
their host–guest chemistry has been investigated during re-
cent years.
When four hydrogen bonds are situated next to one an-
other, the pattern determines whether homodimers, as de-
scribed by Meijer,[4,5] or heterodimers, such as the quadru-
ple hydrogen bond system first synthesized in our group,[6]
are formed. For controlled self-assembly of large supramo-
lecules through hydrogen bonds it is not enough to use just
one complementary pair: various orthogonal recognition
domains are necessary. For four neighboring hydrogen
bonds, several orthogonal pairs have already been synthe-
sized.[7–12] Another type of orthogonality can be achieved
if the recognition domain shows nonlinear geometry.
In 1988, Chang and Hamilton synthesized a macrocyclic
angulate receptor.[13] Containing six converging hydrogen
bonds, this phthalamide host was able to bind barbituric
[‡] Multiple Hydrogen Bonds, 8. Part 7: C. Glockner, U. Lüning,
J. Incl. Phenom. Macrocycl. Chem. 2011, DOI: 10.1007/s10847-
010-9903-4.
[a] Otto Diels-Institut für Organische Chemie, Christian-
Albrechts-Universität zu Kiel,
Olshausenstraße 40, 24098 Kiel, Germany
E-mail: luening@oc.uni-kiel.de
[b] Institut für Anorganische Chemie, Christian-Albrechts-
Universität zu Kiel,
Olshausenstraße 40, 24098 Kiel, Germany
E-mail: cnaether@ac.uni-kiel.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001684.
Figure 1. Host–guest systems 1·3 with n-butyl residues and 2·3 with
ethylpentyl residues at the amido groups at the 6-positions of the
pyridine rings.
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Binding Constants of Hydrogen-Bonded Complexes
A prerequisite for introduction of a Hamilton receptor
into a larger structure is a covalent connection to another
moiety, so syntheses of receptors functionalized in the
isophthalic component have been carried out. 5-Iodo,[7]
-amino,[15] or -hydroxy[16] substitution have been reported.
For practical applications, however, not every combination
of Hamilton hosts and corresponding guests can be used,
due to solubility problems. Firstly, the hosts and guests are
flat, so they can stack, and secondly, the heterocycles con-
tain many hetero elements that favor intermolecular inter-
actions through dipole–dipole interactions, including hy-
drogen bonds. All these forces have to be overcome if appli-
cations are to be carried out in solution. To enhance solu-
bility, we therefore synthesized a set of Hamilton receptors
2 (Figure 1), each bearing a branched alkyl residue rather
than the n-alkyl substituent of receptors 1.
Here we report on the syntheses of different Hamilton
receptors of both families 1 and 2 and on the determination
of their association constants when forming a complex with
barbital (3, Figure 1). Besides the two different residues at
the amido group in the 6-positions in the pyridine rings, the
receptors differ in the character and position of the func-
tional group(s) in the isophthalic acid moiety. The influence
of these changes on binding constants was studied. Three
techniques for the determination of the association con-
stants – 1H NMR titrations monitoring chemically induced
shifts (CIS values), 1H NMR diffusion experiments, and
isothermal titration calorimetry (ITC) – were selected and
the results were compared.
Scheme 1. Synthesis of the Hamilton receptors 1a–c and 2a and 2b.
a) Et3N, THF, room temp., 3 h; b) pentanoyl chloride, Et3N, THF,
room temp., 16 h; c) 2-ethylhexanoyl chloride, Et3N, THF, room
temp., 16 h.
Results and Discussion
Syntheses of the Hamilton Receptors
The syntheses of the Hamilton receptors were carried out ton receptors 1a–c and 2a and 2b with different solubilities
by two different reaction pathways. In both cases the parent were synthesized by use of pentanoyl chloride or 2-ethyl-
compounds were isophthalic acid derivatives with func- hexanoyl chloride. Two factors improve the solubility of the
tional groups either at their 5- or at their 4- and 6-positions. ethylhexanoyl derivatives: the branching itself and the for-
The next step was the formation of isophthalamides either mation of diastereomeric mixtures due to the fact that two
with 2,6-diaminopyridine (5, Scheme 1) or with the already chiral centers are introduced.
monoacylated diaminopyridines 7 or 8 (Scheme 2, below).
Since the first description of a synthesis of these iso-
The preparation of the iodine Hamilton receptor 1a was phthalamides, an alternative route to build up the angular
developed in our group in 2002 in analogy to the synthesis receptor has been reported in the literature.[23,24] According
described by Chang and Hamilton.[13] First of all, 5-amino- to these procedures, we synthesized four additional Hamil-
isophthalic acid was converted into 5-iodoisophthalic acid ton receptors (Scheme 2) by starting from 5-hydroxyiso-
by a literature procedure, with sodium nitrite, potassium phthalic acid, which was protected with a benzoyl group
iodide, and a catalytic amount of iodine.[19] 5-Iodoiso- and afterwards converted into the isophthaloyl chloride
phthaloyl chloride (4a, Scheme 1) could be obtained by 9.[25] Unlike in the syntheses described above, in this route
treatment with oxalyl dichloride in toluene.[19] 5-Nitro- the side chain was introduced prior to treatment with the
isophthaloyl chloride (4b) was synthesized by the same pro- acid chloride 9. 2,6-Diaminopyridine (5) was monoacylated
cedure from commercially available 5-nitroisophthalic acid. with an aliphatic acid chloride such as pentanoyl chloride
Furthermore, 4,6-dibromoisophthaloyl chloride (4c) was or 2-ethylhexanoyl chloride. Next, the amidoaminopyr-
prepared in three steps starting from m-xylene, which was idines 7 or 8 were linked to 5-(benzoyloxy)isophthaloyl
first brominated, then oxidized, and finally treated with chloride (9) to form the diamides 1d and 2d. Crystals were
thionyl chloride.[20–22] These stable isophthaloyl chlorides obtained from 5-benzoyloxy-N,NЈ-bis(6-pentanoylamino-
4a–4c were each treated with 2,6-diaminopyridine (5, ex- pyrid-2-yl)isophthalamide (1d) and the X-ray structure
cess) to afford the corresponding diamines 6a–6c in a uni- could be solved, although the crystals were non-mero-
versally applicable precursor. Finally, the different Hamil- hedrally twinned.
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Scheme 2. Synthesis of different 5-oxy-substituted Hamilton receptors: a) pentanoyl chloride, NEt3, THF, 0 °C, 3 h, room temp., 16 h;
b) 2-ethylhexanoyl chloride, NEt3, THF, 0 °C, 3 h, room temp., 16 h; c) and d) 5-(benzoyloxy)isophthaloyl chloride (9), NEt3, THF, room
temp., 30 min, reflux, 18 h; e) and f) EtOH, NaOH (1 n), room temp., 3 h, HCl (2 n).
The Hamilton receptor 1d crystallized in the triclinic twist is visible and the binding sites are still in-plane. Ac-
¯
space group P1. The almost planar orientation of the Ham- cording to the crystal structure, there is no steric hindrance
ilton receptor binding sites is shown in its X-ray structure to binding of a guest.
(Figure 2). The planarity is comparable to that in another
The last step in the syntheses of 1e and 2e was the depro-
structure published by Vögtle and De Cola.[15,26] Unlike tection of the hydroxy group. Ester cleavage was achieved
their receptor, which contained tertiary butyl groups, our in alkaline medium followed by acidification. The solubility
receptor 1d has longer unbranched chains; nevertheless, no of the Hamilton receptor 1e with unbranched pentanoyl
chains was limited, and it dissolved only in DMSO, DMF,
and THF. Through the introduction of the branched 2-eth-
ylhexanoyl substituents, the solubility was improved, and
less polar and aprotic solvents such as chloroform can be
used for further syntheses and measurements. Thanks to
these results, the two chloroform-soluble protected Hamil-
ton receptors 1d and 2d were used for the determination
and comparison of binding constants with barbital (3). The
hydroxy derivatives 1e and 2e were only synthesized to al-
low incorporation of Hamilton receptors into larger arrays,
so investigation of the esters 1d and 2d, rather than the
corresponding phenols 1e and 2e, seemed more relevant.
Investigation of the Host–Guest Systems
The determination of association constants is an essen-
tial element when dealing with supramolecular structures
or self-assembly. Various methods for the determination of
association constants for host–guest complexes are known.
In this work, H NMR CIS titrations, H NMR diffusion
experiments, and isothermal titration calorimetry (ITC)
Figure 2. X-ray structure of 5-benzoyloxy-N,NЈ-bis(6-pentanoyl-
aminopyrid-2-yl)isophthalamide (1d). Only one orientation of the
disordered benzoyl protecting group is shown.
1
1
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Binding Constants of Hydrogen-Bonded Complexes
were selected to investigate several Hamilton receptor/bar- barbital (3) in each mixture was measured, and finally the
bital systems (see Figure 1). All measurements were carried association constants (Kass) were calculated.[28] The signals
out in CDCl3 or CHCl3 at 25 °C.
of the guest 3 were chosen because of the larger difference
1
The determination of binding constants by H NMR ti- in molecular weight, and thus diffusion constant, between
trations, by recording of changes in the chemically induced barbital (3) and the complexes 1·3 or 2·3 (for an example
shifts (CISs), is an easily applicable and well established see Figure 4).
method.[27] The association constants for host–guest forma-
tion between the Hamilton receptors 1a, 1d, 2a, and 2d as
hosts and barbital (3) as guest were calculated from the CIS
of the N–H signal of barbital (3) upon addition of increas-
ing amounts of the Hamilton host to a solution of the guest
(see Table 1 and Figure 3 for an example). The reason for
the inverse titration was the parallel determination of the
diffusion parameters (see below). The results for the recep-
tors 1a, 1d, 2a, and 2d are compared in Table 1.
Table 1. Kass values of complexes 1·3 and 2·3 determined by 1H
1
NMR CIS titration and H NMR diffusion experiments in CDCl3
at 298 K with a concentration of 3 of 0.4 mm. All errors were
smaller than 20% except for values in parentheses. For exact errors
see the Supporting Information.
Figure 4. Titration curve of barbital (3, 5 mm) with the Hamilton
receptor 2d determined by 1H NMR diffusion spectroscopy in
CDCl3. The signal of the CH2 groups of barbital (3) was used to
determine the diffusion constant (Dobs), which is plotted against
the 2d/3 molar ratio.
Kass [103 m–1] (CIS)
Kass [103 m–1] (diffusion)
1a
2a
1d
2d
64
34
(76)
44
59
(33)
85
37
Table 1 summarizes the association constants (Kass) de-
termined by the diffusion method and compares them with
1
the constants (Kass) obtained from H NMR CIS titrations.
The latter analyze the chemically induced shifts of certain H
atoms, preferentially those participating in hydrogen bond
formation (see above). The Kass values determined this way
thus only register the complexes that are indeed bound by
hydrogen bonds. In contrast, the hydrodynamic radius of a
guest will always increase when it is coordinated to a host,
regardless of whether it is bound by hydrogen bonds or by
other intermolecular forces such as van der Waals interac-
tions or π–π stacking. If such other forces play a role, it is
obvious that Kass values determined from diffusion experi-
ments may be larger than those determined by CIS ti-
trations. Despite these differences, the binding constants
(Kass) for 1·3 or 2·3 determined by diffusion experiments
are in the expected range, and they even have a smaller cal-
culation error than the CIS titration data.
1
Figure 3. H NMR CIS titration curve of barbital (3, 5 mm) with
the Hamilton receptor 2d in CDCl3. The CIS of the NH signals of
3 is plotted against the 2d/3 molar ratio.
The Hamilton receptors 2a and 2d with the branched
All four host–guest systems 1a/2a·3 and 1d/2d·3 show as- acyl groups show weaker binding of barbital (3) than the
sociation constants (Kass) of the same order of magnitude. unbranched 1a and 1d. Furthermore, the substituent on the
Nevertheless, a difference between the Hamilton receptors isophthalic acid moiety appears to be irrelevant for the
with unbranched acyl residues (1a, 1d) and those with the binding; Kass is not influenced. These results are in accord-
branched chains (2a, 2d) is detectable. The smaller associa- ance with the measurements described in the NMR CIS
tion constants (Kass) for receptors 2a and 2d are probably titration section.
the result of greater steric hindrance during complexation.
In addition to the applied NMR methods, the associa-
With regard to the substituents at the isophthalic acid part, tion constants (Kass) of the receptors 1a, 2a, 1d, and 2d and
there is only a marginal difference in the association con- three other complexes 1b·3, 1c·3, and 2b·3 were determined
stants (Kass).
by isothermal titration calorimetry. ITC is a calorimetric
For the determination of association constants (Kass
)
method increasingly used to determine the thermodynamic
through 1H NMR diffusion experiments, the solutions from parameters of supramolecular interactions. The results for
the titrations of barbital (3) with increasing amounts of the the association constants (Kass), the enthalpy changes (ΔH),
Hamilton receptors 1 or 2 were used. By detection of spin and also the changes in entropy (ΔS) are summarized in
echoes with pulsed gradients the diffusion constant (D) of Table 2.
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Table 2. Association constants (Kass) and thermodynamic param- 1a/1d·3 and 2a/2d·3, measurements of three more Hamilton
eters ΔH and ΔS for titration of the Hamilton receptors 1 or 2 (ca.
receptors – 1b, 1c, and 2b – with barbital (3) were carried
0.4 mm) with barbital (3) in chloroform as determined by ITC.
out. The two Hamilton receptors with a nitro group at the
Note that kcal rather than kJ is used.
5-position in the isophthalic acid component (1b, 2b) did
not differ from other 5-substituted receptors. With regard
Kass [103 m–1]
ΔH [kcalmol–1] ΔS [calmol–1 K–1]
1a
1b
1c
1d
2a
2b
2d
98
100
40
91
59
67
58
–37
–36
–37
–39
–40
–37
–40
–103
–99
–104
–109
–115
–103
–113
to the only receptor with substituents in the ortho positions
(1c), there is a decrease in the association constant (Kass
)
comparable to that when the acyl groups are branched.
Table 2 also shows that the negative Gibbs free enthalpy
(ΔG) for the binding results from an enthalpic contribution.
Although there is a considerable entropic compensation of
ca. 30 kcalmol–1 (298 K times ca. 100 calmol–1 K–1), ΔH is
ca. 30% larger, resulting in negative Gibbs free enthalpies
and corresponding association constants of 40–
100ϫ103 m–1.
The ITC association constants had larger values than
those obtained in the NMR CIS experiments. This is not
too surprising, because the CIS changes observed during a
1H NMR titration only occur when the corresponding pro-
ton becomes part of a hydrogen bond – in contrast to
calorimetric measurements, which register any binding re-
gardless of whether this binding is caused by hydrogen
bonds or by other interactions.[9b] In this respect, ITC mea-
surements are similar to NMR diffusion experiments. The
measurements confirm the expected results: the differences
between the association constants (Kass) for the interaction
of the Hamilton receptors bearing unbranched (1a, 1d) and
branched acyl groups (2a, 2d) with barbital (3) became ob-
vious (for an example see Figure 5). Again, the substituents
at the central phthaloyl moiety had only a minor influence
on the association constants (Kass). In addition to the pairs
Conclusions
Nine different Hamilton receptors 1 and 2 were synthe-
sized from substituted isophthalic acids, 2,6-diaminopyr-
idine (5), and different acyl chlorides. The Hamilton recep-
tors 1 each bear two linear pentanoyl residues, whereas
branched substituents were introduced in class 2, resulting
in enhanced solubilities in low-polarity organic solvents
such as chloroform. The two amide bonds in the 2- and 6-
positions of the pyridine rings in the receptors 1 and 2
could be formed in either sequence. For 1d it was possible
to obtain an X-ray crystal structure showing the planar pre-
organization of the binding sites. The association constants
of the receptors 1 and 2 with barbital (3) were determined
by monitoring of the chemically induced shifts (CISs) in 1H
NMR titrations, by 1H NMR diffusion experiments, and by
isothermal titration calorimetry (ITC). The Kass values were
found to be between 35ϫ103 and 100ϫ103 m–1 by the three
methods, displaying differences of a factor of 3 at most.
Slight decreases in the Kass values were detected for Hamil-
ton receptors with branched acyl groups (2a, 2b, and 2d) or
for receptor 1c with two ortho substituents at the phthaloyl
unit, possibly due to greater steric hindrance.
Experimental Section
General Remarks: NMR spectra were recorded with Bruker
AC 200, DRX 500, or AV 600 instruments. Assignments are sup-
ported by COSY, HSQC, and HMBC. Even when obtained by
DEPT, the type of 13C signal is always listed as singlet, doublet,
etc. All chemical shifts are referenced to TMS or to the residual
proton or carbon signal of the solvent. All diffusion and titration
measurements were carried out with a Bruker AV 600 spectrometer
with a triple-resonance cryo probehead with a maximum z-gradient
strength of 5.67 Gmm–1. As pulse sequence a double stimulated
echo with bipolar gradient pulses was chosen. The diffusion time
in all these experiments was 100 ms and each gradient pair was
applied for 2500 μs. The maximum gradient strength was varied
between 5 and 95% of the gradient strength in 16 steps. EI/CI mass
spectra were recorded with a Finnigan MAT 8200 or MAT 8230.
MALDI mass spectra were recorded with a Bruker–Daltonics Bi-
flex III instrument and Cl-CCA (α-cyano-4-chlorocinnamic acid)
Figure 5. Isothermal titration calorimetry (ITC). Titration of bar-
bital (3, 0.5 mm) with the Hamilton receptor 2d (5 mm) in chloro-
form (energy in kcal and not in kJ.).
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Binding Constants of Hydrogen-Bonded Complexes
as matrix. ESI mass spectra were recorded with an Applied Biosys-
tems Mariner Spectrometry Workstation. IR spectra were recorded
with a Perkin–Elmer Paragon 1000, Perkin–Elmer Spectrum 100
fitted with an MKII Golden GateTM Single Reflection ATR unit.
Elemental analyses were carried out with a Euro EA 3000 Elemen-
tal Analyzer from Euro Vector. 5-(Benzoyloxy)isophthaloyl chlo-
ride (9) was synthesized by the literature procedure.[25] 5-Iodoiso-
phthaloyl acid and 5-iodoisophthaloyl dichloride (4a) were synthe-
sized by the literature procedure.[19] N,NЈ-Bis(6-aminopyrid-2-yl)-5-
nitroisophthalamide (6b) was synthesized analogously to Hamilton
et al.[29]
trations of each host guest pair is shown in the Supporting Infor-
mation.
5-Iodo-N,NЈ-bis[6-(pentanoylamino)pyrid-2-yl]isophthalamide (1a):
N,NЈ-Bis(6-aminopyrid-2-yl)-5-iodoisophthalamide (6a, 4.94 g,
10.4 mmol) and triethylamine (3.0 mL, 22 mmol) were dissolved in
anhydrous tetrahydrofuran (80 mL) under nitrogen. A solution of
pentanoyl chloride (2.7 mL, 22 mmol) in anhydrous tetra-
hydrofuran (10 mL) was added dropwise. After the system had been
stirred at room temp. for 3 h, the solvent was evaporated in vacuo
and the residue was dissolved in chloroform (80 mL) and water
(80 mL). The organic layer was washed with sodium hydrogen car-
bonate (50 mL, 5%) and dried with magnesium sulfate, and the
solvent was removed. The residue was purified by column
1H NMR CIS Titrations and Diffusion Experiments: A new sample
was prepared for each titration measurement. For each component
a stock solution in CDCl3 (dry, as purchased) with a concentration
of about 5 mmolL–1 was prepared. TMS (ca. 10 μL) was added as
internal reference to the stock solution of barbital (3). The samples
were prepared by injecting the stock solution of barbital (3, 50 μL)
into a 5 mm NMR tube with a standard microliter syringe. A corre-
sponding volume of a stock solution of a Hamilton receptor (1a,
2a, 1d, 2d) in CDCl3 was added and the NMR tube was filled up
to 600 μL with CDCl3.
chromatography (silica gel, ethyl acetate/cyclohexane 1:1, Rf
=
0.38); yield 5.90 g (88%); m.p. 170–173 °C. 1H NMR (600 MHz,
CDCl3): δ = 8.56 (br. s, 2 H, Ar-CONH), 8.33 (s, 2 H, Ar-H-4,6),
8.32 (s, 1 H, Ar-H-2), 7.98 (br. s, 2 H, H9C4CONH), 7.95–7.88 (m,
3
4 H, Pyr-H-3,5), 7.70 (t, JH,H = 8.1 Hz, 2 H, Py-4-H), 2.38 (t,
3
3JH,H = 7.5 Hz, 4 H, COCH2), 1.70 (quin, JH,H = 7.5 Hz, 4 H,
3
COCH2CH2), 1.40 (sext, JH,H = 7.5 Hz, 4 H, CH2CH3), 0.94 (t,
3JH,H = 7.5 Hz, 6 H, CH2CH3) ppm. 13C NMR (150 MHz, CDCl3):
δ = 171.9 (s, COC4H9), 163.0 (s, Ar-CONH), 149.8 (s, Py-C-2),
149.0 (s, Py-C-6), 141.0 (d, Py-C-4), 139.7 (d, Ar-C-4,6), 136.2 (s,
Ar-C-1,3), 125.0 (d, Ar-C-2), 110.4 (d, Py-C-3), 109.7 (d, Py-C-5),
94.8 (s, Ar-C-5), 37.5 (t, COCH2), 27.4 (t, COCH2CH2), 22.3 (t
The final concentrations of the Hamilton receptors 1 and 2 in the
mixtures were checked by integration of the CH2 group of barbital
(3) and the CO–CH or CO–CH2 signals of 1 or 2, respectively.
The barbital (3) concentration was assumed to be constant in all
measurements. The same signals were used for the diffusion analy-
sis. In cases of slight signal overlap, biexponential fits with respect
to two components were applied to the diffusion data to provide
more exact integrals for concentration calculations on the one hand
and more accurate diffusion constants on the other.
CH CH ), 13.8 (q, CH ) ppm. IR (KBr): ν = 3428, 3302 (N–H),
˜
2
3
3
2958, 2930, 2871 (aliph. C–H), 1688, 1586, 1514 (C=O), 1295 cm–1.
MS (EI, 70 eV): m/z (%) = 642 (24) [M]+·, 600 (100) [M – C3H6]+·,
585 (90) [M – C4H9]+, 516 (35) [M – I]+·. MS (CI, isobutane): m/z
(%) = 643 (26) [M + H]+, 517 (18) [M – I + H]+, 194 (100)
[H9C4CONHPyNH + H]+. HRMS (EI): calcd. C28H31IN6O4:
642.14514; found 642.14180, calcd. C2713CH31IN6O4: 643.14850;
found 643.14570. C28H31IN6O4 (642.49): calcd. C 52.34, H 4.86, N
13.08. C28H31IN6O4·0.5H2O (651.15): calcd. C 51.62, H 4.95, N
12.90; found C 51.60, H 4.96, N 12.90.
The diffusion analyses were carried out with the aid of Bruker’s
Topsin 2.1 software. In the case of biexponential fitting, Origin 7.5
including the ONMR plug-in was used for further analyses and
plotting/fitting options.[30]
5-Nitro-N,NЈ-bis[6-(pentanoylamino)pyrid-2-yl]isophthalamide (1b):
Compound 1b was synthesized by the same procedure as used for
receptor 1a, from N,NЈ-bis(6-aminopyrid-2-yl)-5-nitroisophthal-
amide (6b, 1.00 g, 2.54 mmol), triethylamine (1.65 mL, 7.50 mmol),
and pentanoyl chloride (920 μL, 7.50 mmol) in anhydrous tetra-
hydrofuran (10 mL). The crude product was purified by column
Statistical errors are usually calculated for curve fittings of NMR
titrations but in most cases they do not include all errors (weighing
error, temp., how many measurements have been carried out at
what ratios, systematic error when always adding the same aliquot
of a guest solution to a given host solution, etc.). In our experi-
ments, for every data point in the NMR measurements, new solu-
tions were prepared so that weighing errors etc. of each single ex-
periment should cancel out. In this regard, each titration curve is
a set of several independent experiments, and the calculated errors
include the other errors. The specific statistical error for each ti-
tration can be found in the Supporting Information
chromatography (silica gel, cyclohexane/ethyl acetate 1:1, Rf
=
0.30); yield 1.06 g (74%) as a slightly yellow solid; m.p. 193–195 °C.
1H NMR (500 MHz, [D6]DMSO): δ = 10.92 (br. s, 2 H, Ar-
4
CONH), 10.12 (br. s, 2 H, H9C4CONH), 8.92 (d, JH,H = 1.5 Hz,
4
2 H, Ar-H-4,6), 8.90 (d, JH,H = 1.5 Hz, 1 H, Ar-H-2), 7.88–7.78
3
(m, 6 H, Pyr-3,4,5-H), 2.41 (t, JH,H = 7.5 Hz, 4 H, COCH2), 1.58
3
3
(quin, JH,H = 7.5 Hz, 4 H, COCH2CH2), 1.32 (sext, JH,H
=
H,
Isothermal Titration Calorimetry: Experiments were carried out
with a VP-ITC microcalorimeter (MicroCal LLC, GE Healthcare)
in anhydrous chloroform. A Hamilton receptor 1 or 2 (ca. 1.4 mL,
0.3–0.5 mm) was placed in the calorimeter cell. The titration syringe
was loaded with barbital (3) at a 10 times higher concentration
than in the cell. The titrations were usually carried out with 50
injections of 6 μL each with time intervals of 5 min. The solution
was stirred at 300 rpm as suggested by the manufacturer. Titrations
were carried out at a cell temperature of 298 K (shield: 297 K) and
with a reference power of 10 μcals–1. ITC data analyses were car-
ried out in Origin 7 SR 2 (OriginLab Corp.) with the provided
microcal ITC routines. Please note that all energies are listed as
kcalmol–1 rather than kJmol–1 by this routine. Each experiment
was carried out at least twice. In cases of larger deviations (due to,
for instance, changes in room temperature stability), more titrations
were carried out. One representative titration curve for the ti-
3
7.5 Hz,
4
H, CH2CH3), 0.90 (t, JH,H
=
7.4 Hz,
6
CH2CH3) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 172.2 (s,
COC4H9), 163.4 (s, Ar-CONH), 150.6 (s, Py-C-2), 149.8 (s, Py-C-
6), 147.7 (s, Ar-C-5), 140.1 (d, Py-C-4), 135.8 (s, Ar-C-1,3), 133.6
(d, Ar-C-2), 125.7 (d, Ar-C-4,6), 110.5 (d, Py-C-3), 110.3 (d, Py-C-
5), 35.8 (t, COCH2), 27.1 (t, COCH2CH2), 21.7 (t CH2CH3), 13.7
(q, CH ) ppm. IR (ATR): ν = 3332, 3274 (N–H), 3087 (arom. C–
˜
3
H), 2959, 2931, 2872 (aliph. C–H), 1663 (C=O), 1583, 1443 (arom.
C=C), 1511 (C–NO2) cm–1. MS (EI, 70 eV): m/z (%) = 504 (5) [M –
C4H9]+·. MS (CI, isobutane): m/z (%) = 562 (3) [M + H]+. MS
(MALDI): m/z = 562 [M + H]+, 584 [M + Na]+. C28H31N7O6
(561.59): calcd. C 59.88, H 5.56, N 17.46; found C 59.60, H 5.55,
N 17.34.
4,6-Dibromo-N,NЈ-bis[6-(pentanoylamino)pyrid-2-yl]isophthalamide
(1c): Compound 1c was synthesized by the same procedure as used
Eur. J. Org. Chem. 2011, 2066–2074
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2071

U. Lüning et al.
FULL PAPER
3
for receptor 1a, from N,NЈ-bis(6-aminopyrid-2-yl)-4,6-dibromo-
isophthalamide (6c, 450 mg, 0.889 mmol), triethylamine (2.00 mL,
9.09 mmol), and pentanoyl chloride (433 μL, 3.56 mmol) in anhy-
drous tetrahydrofuran (10 mL). The residue was purified by column
chromatography (silica gel, ethyl acetate/cyclohexane 1:1, Rf =
COCH2CH2), 1.32 (sext, JH,H = 7.5 Hz, 4 H, COCH2CH2CH2),
0.86 (t, JH,H = 7.3 Hz, 6 H, CH3) ppm. 13C NMR (125 MHz,
3
CDCl3): δ = 172.1 (s, CH3CH2CH2CH2CO), 166.1 (s, NHCO),
150.5 (s, Ar-C-5), 150.3 (s, Py-C-2), 139.9 (Py-C-6, Py-C-4), 135.1
(s, Ar-C-1,3), 119.5 (Ar-C-4,6, Ar-C-2), 110.1 (d, Py-C-3), 109.4 (d,
Py-C-5), 35.8 (t, COCH2), 27.1 (t, COCH2CH2), 21.7 (t, CH2CH3),
1
0.39); yield 475 mg (70%); m.p. 199–201 °C. H NMR (200 MHz,
CDCl3): δ = 8.47 (br. s, 2 H, Ar-CONH), 8.05 (br. s, 1 H, Ar-H-
13.7 (q, CH ) ppm. IR (KBr): ν = 3376 (O–H, N–H), 1654 (C=O),
˜
3
5), 7.83 (s, 1 H, Ar-H-2), 8.20 (br. s, 2 H, H9C4CONH), 7.93 (d, 2 1585, 1530 (arom.), 1293 (N–H) cm–1. MS (ESI): m/z (%) = 533 [M
H, Py-5-H), 7.86 (br. d, 4 H, Py-3-H), 7.61 (t, 3JH,H = 8.2 Hz, 2 H,
+ H]+, 555 [M + Na]+. C28H32N6O5 (532.6): calcd. C 63.14, H 6.06,
3
3
Py-4-H), 2.38 (t, JH,H = 7.5 Hz, 4 H, COCH2), 1.68 (quin, JH,H N 15.78. C28H32N6O5·0.5H2O: calcd. C 62.05, H 6.14, N 15.52;
3
= 7.5 Hz, 4 H, COCH2CH2), 1.37 (sext, JH,H = 7.4 Hz, 4 H, found C 61.85, H 6.15, N 15.59.
CH2CH3), 0.91 (t, JH,H = 7.4 Hz, 6 H, CH2CH3) ppm. 13C NMR
3
N,NЈ-Bis[6-(2-ethylhexanoylamino)pyrid-2-yl]-5-iodoisophthalamide
(125 MHz, CDCl3): δ = 172.2 (s, COC4H9), 164.0 (s, Ar-CONH),
(2a): Compound 2a was synthesized by the same procedure as used
150.0 (s, Py-C-2), 148.5 (s, Py-C-6), 141.0 (d, Py-C-4), 137.9 (s, Ar-
for receptor 1a, from N,NЈ-bis(6-aminopyrid-2-yl)-5-iodoisophthal-
C-4,6), 136.2 (s, Ar-C-1,3), 130.3 (d, Ar-C-5), 122.1 (d, Ar-C-2),
amide (6a, 1.23 g, 2.60 mmol), triethylamine (750 μL, 5.50 mmol),
110.6 (d, Py-C-3), 109.8 (d, Py-C-5), 37.6 (t, COCH2), 27.4 (t,
and 2-ethylhexanoyl chloride (950 μL, 5.50 mmol). Column
COCH CH ), 22.3 (t CH CH ), 13.8 (q, CH ) ppm. IR (ATR): ν
˜
2
2
2
3
3
chromatography (silica gel, ethyl acetate/cyclohexane 1:1, Rf
=
= 3289 (N–H), 3025 (arom. C–H), 2958, 2930, 2871 (aliph. C–H),
1674 (C=O), 1582, 1505 (arom. C=C), 1445 (CH2, CH3) 1052
(arom. C–Br) cm–1. MS (EI, 70 eV): m/z (%) = 672 (5) [M]+·, 615
(8) [M – C4H9]+. MS (MALDI): m/z = 673 [M + H]+, 695 [M +
Na]+, 711 [M + K]+. C28H30Br2N6O4 (674.38): calcd. C 49.87, H
4.48, N 12.46; found C 49.67, H 4.53, N 12.25.
0.59); yield 1.51 g (80%); m.p. 130–132 °C. 1H NMR (500 MHz,
CDCl3): δ = 8.40 (br. s, 2 H, Ar-CONH), 8.40–8.35 (m, 3 H, Ar-
3
H-2,4,6), 8.03, 7.97 (2ϫd, JH,H = 8.0 Hz, 4 H, Pyr-3,5-H), 7.90
3
(br. s, 2 H, H15C7CONH), 7.73 (t, JH,H = 8.0 Hz, 2 H, Py-4-H),
2.19 (mc, 2 H, COCH), 1.80–1.50 (m, 8 H, COCHCH2), 1.32 (mc,
3
8 H, CH2CH2CH3), 0.97 (t, JH,H = 7.4 Hz, 6 H, CHCH2CH3)
5-Benzoyloxy-N,NЈ-bis[6-(pentanoylamino)pyrid-2-yl]isophthalamide
(1d): N-(6-Aminopyrid-2-yl)pentanamide (7, 1.12 g, 6.18 mmol)
0.88 (mc, 6 H, CHCH2CH2CH2CH3) ppm. 13C NMR (150 MHz,
CDCl3): δ = 174.9 (s, COC7H15), 162.8 (s, Ar-CONH), 149.8 (s,
and anhydrous triethylamine (1.12 mL, 8.03 mmol) were dissolved Py-C-2), 148.9 (s, Py-C-6), 141.0 (d, Py-C-4), 139.6 (d, Ar-C-2),
in anhydrous tetrahydrofuran (60 mL) under nitrogen. A solution 136.3 (s, Ar-C-1,3), 125.0 (d, Ar-C-4,6), 110.5 (d, Py-C-3), 109.7
of 5-(benzoyloxy)isophthaloyl chloride (9, 1.00 g, 2.37 mmol) in an- (d, Py-C-5), 94.8 (s, Ar-C-5), 50.7 (d, COCH), 32.4 (t,
hydrous tetrahydrofuran (15 mL) was then added dropwise. After
the system had been stirred at room temp. for 30 min it was heated
to reflux for 18 h. The resulting dispersion was filtered and the
solvent was evaporated in vacuo. The residue was purified by col-
umn chromatography (silica gel, ethyl acetate/cyclohexane 3:2, Rf
= 0.8); yield 911 mg (60%); m.p. 115 °C. 1H NMR (300 MHz,
CH2CH2CH2CH3), 29.8 (t, CH2CH2CH2CH3), 26.1 (t,
CHCH2CH3), 22.8 (t, CH2CH2CH2CH3), 14.0 (q,
CH CH CH CH ), 12.0 (q, CHCH CH ) ppm. IR (ATR): ν =
˜
2
2
2
3
2
3
3279 (N–H), 3071 (arom. C–H), 2958, 2929, 2859 (aliph. C–H),
1668 (C=O), 1580, 1504 (arom. C=C), 1441 (alkyl C–H) cm–1. MS
(EI, 70 eV): m/z (%) = 726 (15) [M]+·, 670 (72) [M – C4H8]+·, 627
(100) [M – C7H15]+, 600 (5) [M – I]+·. MS (CI, isobutane): m/z (%)
= 727 (100) [M + H]+, 601 (18) [M – I + H]+. MS (ESI): m/z (%)
= 727 [M + H]+. MS (MALDI): m/z (%) = 727 [M + H]+, 749 [M
+ Na]+, 765 [M + K]+. C34H43IN6O4 (726.24): calcd. C 56.20, H
5.96, N 11.57; found C 56.20, H 6.13, N 11.60.
3
CDCl3): δ = 8.22 (s, 1 H, Ar-H-2), 8.21 (d, JH,H = 8.1 Hz, 2 H,
Py-H-5), 8.05–7.94 (m, 6 H, Ar-H-4,6, Bz-H-2,6, Py-H-3), 7.81–
7.74 (m, 2 H, Bz-H-3,5), 7.70 (t, 3JH,H = 7.5 Hz, 1 H, Bz-H-4), 7.56
3
3
(t, JH,H = 8.1 Hz, 2 H, Py-H-4), 2.38 (t, JH,H = 7.5 Hz, 4 H,
3
COCH2), 1.73 (quint, JH,H = 7.5 Hz, 4 H, COCH2CH2), 1.42
(sext, JH,H = 7.5 Hz, 4 H, COCH2CH2CH2), 0.96 (t, JH,H
3
3
=
N,NЈ-Bis[6-(2-ethylhexanoylamino)pyrid-2-yl]-5-nitroisophthalamide
(2b): Compound 2b was synthesized by the same procedure as used
for receptor 1a, from N,NЈ-bis(6-aminopyrid-2-yl)-5-nitroiso-
phthalamide (6b, 661 mg, 1.68 mmol), triethylamine (785 μL,
3.56 mmol), and 2-ethylhexanoyl chloride (615 μL, 3.56 mmol).
The crude product was purified by column chromatography (silica
gel, cyclohexane/ethyl acetate 1:1, Rf = 0.60); yield 500 mg (46%)
as a slightly yellow solid; m.p. 119–121 °C. 1H NMR (500 MHz,
7.3 Hz, 6 H, CH3), ppm. 13C NMR (75.5 MHz, CDCl3): δ = 172.1
(s, NHCO), 165.1 (s, NHCO), 163.7 (s, OCO), 151.5 (s, Ar-C-5),
150.0 (s, Py-C-2), 149.1 (s, Py-C-6), 140.9 (d, Py-C-4), 136.1 (s, Ar-
C-1,3), 134.3 (d, Bz-C-4), 130.3 (s, Bz-C-1), 128.8 (d, Bz-C-2,6),
128.3 (d, Bz-C-3,5), 125.0 (d, Ar-C-4,6), 122.9 (d, Ar-C-2), 110.3
(d, Py-C-3), 109.4 (d, Py-C-5), 37.4 (t, CH2), 27.4 (t, CH2), 22.4 (t,
CH ), 13.8 (q, CH ) ppm. IR (KBr): ν = 3408 (N–H), 2962 (aliph.
˜
2
3
C–H), 1686 (C=O), 1584, 1523 (arom.), 1260 (N–H) cm–1. MS
(ESI): m/z (%) = 637 [M + H]+, 659 [M + Na]+. C35H36N6O6
(636.7): calcd. C 66.02, H 5.70, N 13.20. C41H48N6O6·2.5H2O:
calcd. C 61.66, H 6.06, N 12.33; found C 61.81, H 6.04, N 12.23.
4
CDCl3): δ = 8.87 (d, JH,H = 1.5 Hz, 2 H, Ar-H-4,6), 8.83 (t, 4JH,H
= 1.5 Hz, 1 H, Ar-H-2), 8.76 (br. s, 2 H, Ar-CONH), 8.02 (d, 3JH,H
= 8.0 Hz, 2 H, Pyr-5-H), 7.99 (br. s, 2 H, H9C4CONH), 7.95 (d,
3
3JH,H = 8.0 Hz, 2 H, Pyr-3-H), 7.74 (t, JH,H = 8.0 Hz, 2 H, Py-4-
5-Hydroxy-N,NЈ-bis[6-(pentanoylamino)pyrid-2-yl]isophthalamide
H), 2.21 (mc, 2 H, COCH), 1.80–1.50 (m, 8 H, COCHCH2), 1.32
3
(1e):
5-Benzoyloxy-N,NЈ-bis[6-(pentanoylamino)pyrid-2-yl]iso-
(mc, 8 H, CH2CH2CH3), 0.97 (t, JH,H = 7.5 Hz, 6 H, CHCH2-
phthalamide (1d, 570 mg, 896 μmol) was dissolved in ethanol
(5 mL). After addition of sodium hydroxide (1 n, 10 mL), the solu-
tion was stirred for 3 h and then acidified with hydrochloric acid
(2 n). The mixture was filtered, and the solid was washed with
tetrahydrofuran. The organic filtrate was dried with sodium sulfate
and the solvent was evaporated in vacuo; yield 449 mg (94%); m.p.
172 °C. 1H NMR (300 MHz, [D6]DMSO): δ = 10.4 (br. s, 2 H,
CH3), 0.88 (t, 3JH,H = 6.9 Hz, 6 H, CHCH2CH2CH2CH3) ppm. 13
C
NMR (125 MHz, CDCl3): δ = 175.1 (s, COC7H15), 162.1 (s, Ar-
CONH), 149.9 (s, Py-C-2), 148.8 (s, Py-C-6), 148.7 (s, Ar-C-5),
141.0 (d, Py-C-4), 141.0 (d, Ar-C-2), 136.5 (s, Ar-C-1,3), 125.3 (d,
Ar-C-4,6), 110.8 (d, Py-C-3), 109.8 (d, Py-C-5), 50.7 (d, COCH),
32.4 (t, CH2CH2CH2CH3), 29.8 (t, CH2CH2CH2CH3), 26.1 (t,
CHCH2CH3),
22.8
(t,
CH2CH2CH2CH3),
14.0
(q,
CONH), 10.2 (br. s, 2 H, CONH), 8.00 (s, 1 H, Ar-H-2), 7.52 (s, 2 CH CH CH CH ), 12.0 (q, CHCH CH ) ppm. IR (ATR): ν =
˜
2
2
2
3
2
3
3
H, Ar-H-4,6), 7.86–7.75 (m, 6 H, Py-H-3,4,5), 2.41 (t, JH,H
=
H,
3283 (N–H), 3050 (arom. C–H), 2959, 2931, 2872 (aliph. C–H),
3
7.4 Hz,
2
H, COCH2), 1.58 (quint, JH,H
=
7.4 Hz,
4
1669 (C=O), 1583, 1505 (arom. C=C), 1441 (alkyl C–H) cm–1. MS
2072
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 2066–2074

Binding Constants of Hydrogen-Bonded Complexes
(EI, 70 eV): m/z (%) = 645 (4) [M]+·, 616 (10) [M – C2H5]+·, 546
(100) [M – C4H9]+. MS (CI, isobutane): m/z (%) = 646 (6) [M +
H]+. MS (MALDI): m/z = 646 [M + H]+, 668 [M + Na]+, 684 [M
+ K]+. C34H43N7O6 (645.749): calcd. C 63.24, H 6.71, N 15.18.
C34H43N7O6·0.1CH3COOCH2CH3 (663.37): calcd. C 63.12, H
6.74, N 14.98; found C 63.28, H 6.94, N 14.82.
(50 mL) was added dropwise over a period of 3 h to a solution of
2,6-diaminopyridine (5, 8.39 g, 76.9 mmol) and triethylamine
(2.13 mL, 15.4 mmol) in anhydrous tetrahydrofuran (150 mL). Af-
ter the system had been stirred for 2 h, the solvent was removed in
vacuo and the residue was washed with water (1 L). Purification by
column chromatography (neutral alumina, tetrahydrofuran/dichlo-
romethane 3:1, Rf = 0.52) yielded a light yellow solid; yield 1.54 g
5-Benzoyloxy-N,NЈ-bis[6-(2-ethylhexanoyl)aminopyrid-2-yl]iso-
phthalamide (2d): N-(6-Aminopyrid-2-yl)-2-ethylhexanamide (8,
1.24 g, 5.27 mmol) and anhydrous triethylamine (1.24 mL,
8.96 mmol) were dissolved in anhydrous tetrahydrofuran (60 mL)
under nitrogen. A solution of 5-(benzoyloxy)isophthaloyl chloride
(9, 1.10 g, 3.40 mmol) in anhydrous tetrahydrofuran (15 mL) was
then added dropwise. After the system had been stirred at room
temp. for 30 min it was heated to reflux for 18 h. The resulting
dispersion was filtered, and the solvent was evaporated in vacuo.
The residue was purified by column chromatography (silica gel,
ethyl acetate/cyclohexane 3:1, Rf = 0.38); yield 850 mg (35%); m.p.
111 °C. 1H NMR (500 MHz, CDCl3): δ = 8.52 (br. s, 2 H, NH),
8.31 (br. s, 2 H, NH), 8.25 (s, 1 H, Ar-H-2), 8.08 (d, 3JH,H = 7.3 Hz,
2 H, Py-H-5), 7.98–7.87 (m, 6 H, Ar-H-4,6, Bz-H-2,6, Py-H-3),
1
(79%); m.p. 248 °C. H NMR (200 MHz, [D6]DMSO): δ = 10.35
4
(br. s, 2 H, NH), 8.49 (s, 1 H, Ar-2-H), 8.38 (d, JH,H = 1.3 Hz, 2
3
H, Ar-4,6-H), 7.45 (t, JH,H = 7.8 Hz, 2 H, Pyr-4-H), 7.36 (dd,
4
3
3JH,H = 7.7, JH,H = 0.9 Hz, 2 H, Pyr-3-H), 6.28 (dd, JH,H = 7.7,
4JH,H = 0.9 Hz, 2 H, Pyr-5-H), 5.84 (br. s, 4 H, NH2) ppm. 13C
NMR (125 MHz, [D6]DMSO): δ = 163.4 (s, Ar-CONH), 158.6 (s,
Py-C-2), 150.1 (s, Py-C-6), 139.3 (d, Py-C-4), 139.0 (d, Ar-C-4,6),
136.2 (s, Ar-C-1,3), 126.3 (d, Ar-C-2), 104.3 (d, Py-C-3), 101.9 (d,
Py-C-5), 94.6 (s, Ar-C-5) ppm. IR (ATR): ν = 3326 (CONH), 1673,
˜
1612 (CONH) cm–1. MS (EI, 70 eV): m/z (%) = 474 (100) [M]+·,
366 (9) [M – NHPyrNH2]+·. MS (CI, isobutane): m/z (%) = 475
(100) [M + H]+, 349 (18) [M – I + H]+. HRMS (EI): C18H15IN6O2
calcd. 474.03012; found 474.02408, C1713CH15IN6O2 calcd.
475.03348; found 475.02854.
3
7.65–7.59 (m, 3 H, Bz-H-3,4,5), 7.46 (t, JH,H = 7.8 Hz, 2 H, Py-
H-4), 2.23–2.17 (m, 2 H, CH), 1.75–1.68 (m, 4 H, CHCH2CH3),
1.60–1.48 (m, 4 H, CHCH2), 1.29–1.25 (m, 8 H, CHCH2CH2CH2),
0.94 (t, JH,H = 7.4 Hz, 6 H, CHCH2CH3), 0.86–0.83 (m, 6 H,
N,NЈ-Bis(6-aminopyrid-2-yl)-4,6-dibromoisophthalamide (6c): 2,6-
Diaminopyridine (5, 986 mg, 9 mmol) was dissolved in anhydrous
tetrahydrofuran (100 mL), the mixture was stirred for 3 h at room
temp., the solution was filtered, and triethylamine (2 mL) was
added. The solution was cooled in ice and 4,6-dibromoisophthaloyl
chloride (4c, 1.08 g, 3.00 mmol) in tetrahydrofuran (10 mL) was
added under nitrogen. The reaction mixture was stirred at room
temp. for 10 h, concentrated by evaporation, and poured into boil-
ing water (200 mL) to remove excess 2,6-diaminopyridine and tri-
ethylamine hydrochloride. The crude product was dried and puri-
fied by basic alumina filtration (tetrahydrofuran/methanol 25:1).
Recrystallization from tetrahydrofuran/n-hexane afforded 6a as
white crystals (1.15 g, 76%); m.p. 250–255 °C (decomp.). 1H NMR
(200 MHz, [D6]DMSO): δ = 10.33 (br. s, 2 H, Ar-CONH), 8.03 (s,
3
CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 175.2 (s, CO), 164.9
(s, OCO), 163.5 (s, NHCO), 151.5 (s, Ar-C-5), 149.9 (s, Py-C-2),
149.0 (s, Py-C-6), 140.8 (d, Py-C-4), 136.1 (s, Ar-C-1,3), 134.3 (d,
Bz-C-4), 130.3 (s, Bz-C-1), 128.7 (d, Bz-C-2,6), 128.3 (d, Bz-C-3,5),
124.7 (d, Ar-C-4,6), 122.9 (d, Ar-C-2), 110.5 (d, Py-C-3), 109.7
(d, Py-C-5), 50.6 (d, COCH), 32.4 (t, COCHCH2), 29.8
(t,
COCHCH2CH2CH2), 13.9 (q, COCHCH2CH2CH2CH3), 12.1 (q,
CHCH CH ) ppm. IR (ATR): ν = 3292 (N–H), 2959 (aliph. C–H),
COCHCH2),
26.9
(t,
COCHCH2CH2),
22.8
(t,
˜
2
3
1670 (C=O), 1582, 1507 (arom.) cm–1. MS (ESI): m/z = 721 [M +
H]+, 743 [M + Na]+. C41H48N6O6 (720.8): calcd. C 68.31, H 6.71,
N 11.66. C41H48N6O6·0.5H2O: calcd. C 67.47, H 6.77, N 11.51;
found C 67.85, H 6.75, N 11.65.
3
1 H, Ar-H-5), 7.68 (s, 1 H, Ar-H-2), 7.42 (t, JH,H = 7.8 Hz, 2 H,
3
3
Py-4-H), 7.31 (br. d, JH,H = 7.8 Hz, 2 H, Py-3-H), 6.26 (dd, JH,H
4
= 7.8, JH,H = 1.0 Hz, 2 H, Py-5-H), 5.80 (br. s, 4 H, NH2) ppm.
N,NЈ-Bis[6-(2-ethylhexanoyl)aminopyrid-2-yl]-5-hydroxyisophthal-
amide (2e): 5-Benzoyloxy-N,NЈ-bis[6-(2-ethylhexanoyl)aminopyrid-
2-yl]isophthalamide (2d, 570 mg, 791 μmol) was dissolved in eth-
anol (5 mL). After addition of aqueous sodium hydroxide (1 n,
10 mL), the solution was stirred for 3 h and then acidified to pH 3
with hydrochloric acid (2 n). The mixture was filtered, and the solid
was washed with tetrahydrofuran. The organic filtrate was dried
with sodium sulfate, and the solvent was evaporated in vacuo; yield
410 mg (84%); m.p. 134 °C. 1H NMR (200 MHz, [D6]DMSO): δ =
10.36 (br. s, 2 H, CONH), 10.14 (br. s, 2 H, CONH), 8.01 (s, 1 H,
Ar-H-2), 7.86–7.79 (m, 6 H, Py-H-3,4,5), 7.53 (s, 2 H, Ar-H-4,6),
2.51–2.50 (m, 2 H, CH), 1.57–1.55 (m, 4 H, CHCH2CH3), 1.46–
1.36 (m, 4 H, CHCH2), 1.27–1.23 (m, 8 H, CHCH2CH2CH2), 0.87–
0.83 (m, 12 H, CH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 175.1
(s, CO), 165.1 (s, NHCO), 157.7 (s, Ar-C-5), 150.4 (s, Py-C-2),
150.1 (s, Py-C-6), 140.1 (d, Py-C-4), 135.6 (s, Ar-C-1,3), 118.1 (d,
Ar-C-4,6), 117.6 (d, Ar-C-2), 110.3 (d, Py-C-3), 110.0 (d, Py-C-5),
47.6 (d, COCH), 31.9 (t, COCHCH2CH3), 29.2 (t, COCHCH2),
26.3 (t, COCHCH2CH2), 22.2 (t, COCHCH2CH2CH2), 13.9 (q,
13C NMR (125 MHz, [D6]DMSO): δ = 164.51 (s, CONH), 158.6
(s, Py-2-C), 149.9 (s, Py-6-C), 138.92 (d, Py-4-C), 137.0 (s, Ar-1,3-
C), 136.0 (d, Ar-5-C), 129.3 (d, Ar-2-C), 120.9 (s, Ar-C-4,6), 104.2
(d, Py-3-C), 101.4 (d, Py-5-C) ppm. IR (ATR): ν = 3463, 3300,
˜
3197 (N–H), 3056 (arom. C–H), 1668 (C=O), 1620, 1532 1448
(arom. C=C), 1052 (arom. C–Br) cm–1. MS (EI, 70 eV): m/z (%) =
504 (48) [M]+·, 425 (100) [M – Br]+, 345 [M – Br2 + H]+. MS (CI,
isobutane): m/z (%) = 505 (13) [M + H]+. MS (MALDI): m/z =
505 [M + H]+, 527 [M + Na]+.
N-(6-Aminopyrid-2-yl)pentanamide (7): 2,6-Diaminopyridine (5,
3.25 g, 29.8 mmol) and anhydrous triethylamine (4.35 mL,
29.8 mmol) were dissolved in anhydrous tetrahydrofuran (50 mL).
A solution of pentanoyl chloride (3.80 mL, 31.3 mmol) in anhy-
drous tetrahydrofuran (6 mL) was added dropwise at 0 °C over
30 min. Afterwards, the mixture was stirred at 0 °C for 3 h and then
for 16 h at room temp. After filtration, the solvent was evaporated
in vacuo and the residue was purified by column chromatography
(silica gel, ethyl acetate/cyclohexane 3:2, Rf = 0.5); yield 3.44 g
1
CH CH CH ), 11.7 (q, CHCH CH ) ppm. IR (ATR): ν = 3270
˜
2
2
3
2
3
(60%); m.p. 66 °C. H NMR (300 MHz, CDCl3): δ = 7.65 (br. s, 1
(O–H, N–H), 1668 (C=O), 1582, 1506 (arom.) cm–1. MS (ESI): m/z
= 617 [M + H]+, 639 [M + Na]+. C34H44N6O5 (616.7): calcd. C
66.21, H 7.19, N 13.63. C41H48N6O6·0.5H2O: calcd. C 65.26, H
7.25, N 13.43; found C 65.13, H 7.19, N 13.26.
3
3
H, N–H), 7.55 (d, JH,H = 7.9 Hz, 1 H, Py-H-3), 7.45 (t, JH,H
=
3
7.9 Hz, 1 H, Py-H-4), 6.24 (d, JH,H = 7.9 Hz, 1 H, Py-H-5), 4.29
(br. s, 2 H, NH2), 2.35 (t, JH,H = 7.6 Hz, 2 H, COCH2), 1.70
(quint, JH,H = 7.6 Hz, 2 H, COCH2CH2), 1.40 (sext, JH,H
3
3
3
=
N,NЈ-Bis(6-aminopyrid-2-yl)-5-iodoisophthalamide (6a): 5-Iodo-
isophthaloyl dichloride (4a, 2.52 g, 7.69 mmol) in tetrahydrofuran
7.6 Hz, 2 H, COCH2CH2CH2), 0.94 (t, JH,H = 7.4 Hz, 3 H,
3
CH3) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 171.9 (s, CO), 156.9
Eur. J. Org. Chem. 2011, 2066–2074
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2073

U. Lüning et al.
FULL PAPER
(s, Py-C-6), 149.8 (s, Py-C-2), 140.3 (d, Py-C-4), 104.1 (d, Py-C-5),
103.2 (d, Py-C-3), 37.3 (t, COCH2), 27.4 (t, COCH2CH2), 22.2 (t,
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra and titration curves.
COCH CH CH ), 13.7 (q, CH ) ppm. IR (KBr): ν = 3410 (N–
˜
2
2
2
3
H), 2961 (aliph. C–H), 1673 (C=O), 1617, 1542 (arom.), 1299 (N–
H) cm–1. MS (ESI): m/z = 194 [M + H]+, 216 [M + Na]+.
C10H15N3O (193.2): calcd. C 62.15, H 7.82, N 21.74; found C
62.09, H 7.97, N 21.62.
Acknowledgments
We thank F. D. Sönnichsen for the support in performing NMR
measurements.
N-(6-Aminopyrid-2-yl)-2-ethylhexanamide (8): 2,6-Diaminopyridine
(5, 3.25 g, 29.8 mmol) and anhydrous triethylamine (4.35 mL,
29.8 mmol) were dissolved in anhydrous tetrahydrofuran (50 mL).
A solution of 2-ethylhexanoyl chloride (5.36 mL, 31.3 mmol) in an-
hydrous tetrahydrofuran (6 mL) was added dropwise over 30 min.
Afterwards, the mixture was stirred at 0 °C for 3 h and then for
16 h at room temp. After filtration, the solvent was evaporated in
vacuo and the residue was purified by column chromatography (sil-
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ica gel, ethyl acetate/cyclohexane 1:2, Rf = 0.37); yield 3.77 g (54%);
3
m.p. 102 °C. 1H NMR (500 MHz, CDCl3): δ = 7.61 (br. d, JH,H
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3
7.8 Hz, 2 H, Py-H-3, N–H), 7.44 (t, JH,H = 7.9 Hz, 1 H, Py-H-4),
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3
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26.1 (t, COCHCH2CH2), 22.8 (t, COCHCH2CH2CH2), 13.9 (q,
CH CH CH ) 12.06 (q, CHCH CH ) ppm. IR (ATR): ν = 3201
˜
2
2
3
2
3
(N–H), 2922 (aliph. C–H), 1638 (C=O) cm–1. MS (EI, 70 eV): m/z
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CCDC-803919 (for 1d) contains the supplementary crystallo-
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Received: December 15, 2010
Published Online: March 1, 2011
2074
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 2066–2074