Molecular recognition of biotin, barbital and tolbutamide with new synthetic receptors

Article abstract of DOI:10.1016/j.tet.2005.03.025

We have measured, by means of NMR titrations, the binding constants for the complexes between hosts N,N′-bis(6-methylpyridin-2-yl)-1,3- benzenedicarboxamide (7) and 4-chloro-N,N′-bis(6-methylpyridin-2-yl)-2,6- pyridinedicarboxamide (8, hydrated) with biot

Full text of DOI:10.1016/j.tet.2005.03.025

Tetrahedron 61 (2005) 5089–5100
Molecular recognition of biotin, barbital and tolbutamide
with new synthetic receptors
a
a
b
Rosa M. Claramunt,a, Fernando Herranz, M. Dolores Santa Marıa, Elena Pinilla,
*
´
b
M. Rosario Torres and Jose Elguero
c
´
a
´ ´ ´
Departamento de Quımica Organica y Bio-Organica, UNED, Facultad de Ciencias, Senda del Rey 9, E-28040 Madrid, Spain
´ ´ ´ ´
Laboratorio de Difraccion de Rayos X, Departamento de Quımica Inorganica, Facultad de Ciencias Quımicas, Universidad Complutense,
b
c
E-28040 Madrid, Spain
Instituto de Quımica Medica, Centro de Quımica Organica ‘Manuel Lora-Tamayo’, C.S.I.C., Juan de la Cierva 3, E-28006 Madrid, Spain
´
´
´
´
Received 14 January 2005; revised 4 March 2005; accepted 7 March 2005
Available online 19 April 2005
´
Dedicated to our friend Dr. Carmela Ochoa de Ocariz on her 65th anniversary
Abstract—We have measured, by means of NMR titrations, the0 binding constants for the complexes between hosts N,N0-bis(6-
methylpyridin-2-yl)-1,3-benzenedicarboxamide (7) and 4-chloro-N,N -bis(6-methylpyridin-2-yl)-2,6-pyridinedicarboxamide (8, hydrated)
with biotin methyl ester (1), N,N0-dimethylurea (2), 2-imidazolidone (3), N,N0-trimethylenurea (4), barbital (5) and tolbutamide (6) as guests.
Molecular Mechanics calculations (Monte Carlo Conformational Search, AMBER and OPLS force fields, MacroModel v.8.1) on the
complexes formed between the foregoing guests and hosts 7 and 8, comparatively with 4-oxo-N,N0-bis(6-methylpyridin-2-yl)-1,4-dihydro-
2,6-pyridinedicarboxamide (9a) have been carried out in order to determine the correlation between experimental and theoretical results and
to understand the behaviour of the designed new hosts. Finally we have performed single point DFT [B3LYP/6-31G(d,p)] calculations on the
optimised Molecular Mechanics geometries for the complexes between hosts 7–9 and water.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
In a preceding paper we have started the systematic study of
host–guest complexes using guests of biological interest,
with the final purpose to mimic the function of natural
receptors by means of an iterative optimisation approach.1
In that work we used two known hosts, those of Thummel2
and Goswami3 (Scheme 1) and five urea derivatives (the
first five ones of the present work, biotin methyl ester (1),
N,N0-dimethylurea (2), 2-imidazolidone (3), N,N0-trimethyl-
enurea (4) and barbital (5) (Scheme 2).
Now we present our results on the interaction of two new
hosts, N,N0-bis(6-methylpyridin-2-yl)-1,3-benzenedicarb-
oxamide (7) and 4-chloro-N,N0-bis(6-methylpyridin-2-yl)-
2,6-pyridinedicarboxamide (8), with six guests 1–6
(Scheme 2) adding to the previous list,1 a sulfonyl urea,
the anti-diabetic oral hypoglycaemic agent tolbutamide (6).4
Even if our attempts to prepare host 4-oxo-N,N0-bis-
Keywords: Molecular recognition; Binding constants; Molecular model-
ling; NMR titrations.
*
Corresponding author. Tel.: C34 91 3987322; fax: C34 91 3986697;
e-mail: rclaramunt@ccia.uned.es
Scheme 1. Thummel’s and Goswami’s hosts.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.025

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R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
analyse the binding constants (Kb) of all guests with host 7
by a direct method,1 using the Chemical Induced Shifts
(CIS) on the 2-CH benzenic proton and the NHs of the
1,3-dicarboxamide groups. The same method was employed
for complexes 8:water and 8:1, where the CIS on the NHs of
the 2,6-pyridinedicarboxamides were quantified. The com-
petitive method was needed to determine Kb in complexes
of 8 and the remaining guests 2–6 measuring the NH-CIS of
the urea moieties and the H2O-CIS.5
All complexes have been modelled at different theoretical
levels using the Monte Carlo conformational search with
both AMBER and OPLS Force Fields (MacroModel v.8.1).
We have carried out single point calculations at B3LYP/
6-31G(d,p) level on the optimised Molecular Mechanics
geometries (AMBER Force Field) for the complexes
between hosts 7, 8 and the two tautomers 9a and 9b
with water (Scheme 3). The pyridone/hydroxypyridine
tautomerism in 9 has also been approached with two models
10a and 10b at the B3LYP/ 6-31G(d,p) level but with
complete optimisation of the geometry.
Scheme 2. The six guests (N,N0-dimethylurea is represented in the E,E
conformation).
(6-methylpyridin-2-yl)-1,4-dihydro-2,6-pyridinedicarb-
oxamide (9a) have been unsuccessful, we have studied its
properties theoretically in comparison with hosts 7 and 8.
1H NMR titrations have been performed to measure and
2. Results and discussion
2.1. Chemistry
N,N0-Bis(6-methylpyridin-2-yl)-1,3-benzenedicarboxamide
(7) was prepared according to Scheme 4 by condensation
reaction of isophthaloyl chloride (11) with 2-amino-6-
methylpyridine (12).3
The various attempts to obtain host 9 resulted in the
synthesis of 8 (Scheme 5). 4-Oxo-1,4-dihydro-2,6-pyri-
dinedicarboxylic acid or chelidamic acid (13) was treated
with thionyl chloride to yield only 4-chloro-2,6-pyridine-
dicarbonyl dichloride (15),6 being unable to reproduce
the literature results where a mixture of 14 with 15 was
formed and used without isolation to obtain several
dicarboxamides.7
We also prepared the diethyl ester 16 from chelidamic acid
13 and ethyl orthoformate in acetic acid which would be
further reacted with 12, but the condensation between the
ester and the amine did not occur. Other assays were the
reaction of chelidamic acid 13 with 2-amino-6-methylpyri-
dine (12) in the presence of several dehydrating agents
(EDC/DMAP, DCC/DMAP) in different conditions but no
signals attributable to 9 were apparent in the 1H NMR
spectra.
Scheme 3. Hosts 7, 8 and 9 and model compound 10.
Scheme 4. Synthesis of host 7 from isophthaloyl chloride (11) and 2-amino-6-methylpyridine (12).

R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
5091
Scheme 5. Synthesis of host 8 from chelidamic acid (13) and 2-amino-6-methylpyridine (12).
Table 1. Experimental binding constants (MK1) for complexes of host 7
Guest
Kb (20–80%) 2-CH CIS
950G86
%10
1400G85
Kb (20–80%) NH CIS
Average Kb
DG (kJ molK1
)
1
2
3
4
5
6
1000G62
%10a
1500G90
2350G412
2308G294
600G125
975
%10
1450
2300
2375
600
K17.2
K4.0b
K18.1
K19.3
K19.4
K15.9
a
2250G353
2442G384
c
a No CIS were observed.
b Calculated from KbZ5.
c No CIS was observed on the 2-CH proton.
Table 2. Experimental binding constants (MK1) for complexes of host 8
2.2. Binding constants
Guest
Kb (20–80%)
Round Kb
DG (kJ molK1
)
The experimental binding constants (MK1) measured in
CDCl3 at 300 K for complexes of the six guests 1–6 with
host 7 are gathered in Table 1, and in Table 2 that with host 8
plus the measured binding constant 8:water. The interaction
energies of the process (KEmin in kJ molK1) evaluated by
Molecular Mechanics calculations for hosts 7, 8 and 9a with
AMBER force field are shown in Table 3 and with OPLS
force field in Table 4. As usual with this kind of studies,
entropy changes have been assumed to be the same or rather
close for all series.1
Water
93G10a
3600G640b
%10
95
3600
%10
140
100
275
K11.4
K20.4
K4.0c
K12.3
K11.5
K14.0
K16.5
1
2c,d
3
141G25d
100G18d
274G74d
735G207d
4
5
6
735
a Direct titration measuring the NH-CIS of the host 8.
b Direct titration measuring the NH-CIS of the host 8, the biotin NH
chemical shifts do not change on complexation.
c Calculated from KbZ5. No CIS was observed.
d Competitive titration measuring the NH-CIS of the urea derivative and
H2O protons.
Special mention deserves the case of N,N0-dimethylurea 2.
For both hosts 7 and 8 we have failed to measure CIS with
this guest. To determine values of Kb lower than 10 MK1 it
would be necessary to use very concentrated solutions,
0.2 M or larger, thus preventing to attain the 20–80%
saturation range in the titration procedure. Therefore, we
Table 3. Interaction energy values (KEmin in kJ molK1) obtained with
AMBER
Guest
7
8
8a
9a
Table 4. Interaction energy values (KEmin in kJ molK1) obtained with
OPLS
1
(E,E)-2
(Z,E)-2
(Z,Z)-2
3
4
5
6
60.7
29.0
47.8
47.8
51.7
53.0
65.0
63.4b
69.3
31.5
51.3
48.9
49.2
51.3
75.4
71.6
23.0
44.4
44.2
38.5
41.1
62.8
84.2
58.6
43.0
37.2
39.0
43.1
44.6
68.3
72.6
Guest
7
8
9a
1
(E,E)-2
(Z,E)-2
(Z,Z)-2
3
4
92.4
56.0
70.4
57.5
80.3
81.7
92.5
—
98.3
58.0
68.0
56.3
77.0
81.0
84.4
43.1
59.4
42.6
53.2
55.6
73.2
—
107.8
a With the GB/SA model for water.
b This value has been calculated taking into account the real interactions in
the 7/6 complex where only the SO2–NH intervenes, on the basis of
NOESY experiments. There is a minimum energy value for a theoretical
complex that considers both urea NHs with a KEmin in kJ molK1 of 73.4.
5
101.5
—
6a
a Lack of parameters in OPLS force field for this compound.

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R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
addition, these entities (8:H2O) are bonded with the
centrosymmetric ones through C3–H3/O2 (KxC1,
KyC2,KzC2) forming dimers, which are arranged in
chains via C21–H21/O1 (xK1,yK1,z). These hydrogen
˚
bonds give rise to a ribbon of 19.22 A width as depicted in
Fig. 2.
A comparison of the molecular structure determined by
X-ray crystallography with the structures obtained by
molecular mechanics for the 8:H2O complex is reported in
Table 5.
Table 5. Experimental and calculated geometries for 8:H2O
˚
D (A)/!(8)
X-Ray
AMBER
OPLS
N1/H17
N1/H8
N8–H8
2.26
2.20
1.00
2.13
0.95
2.15
1.09
2.04
1.20
1.76
144.1
154.9
2.18
2.24
1.02
1.87
1.02
1.88
0.97
2.12
0.97
2.08
152.1
154.3
143.6
137.7
2.14
2.13
1.02
1.86
1.02
1.85
0.97
2.13
0.97
2.14
152.2
152.5
138.8
138.2
O3/H8
N17–H17
O3/H17
O3–H3A
N10/H3A
O3–H3B
N19/H3B
N8–H8/O3
N17–H17/O3
O3–H3A/N10 129.2
O3–H3B/N19 140.5
Figure 1. The X-ray structure of host 8 including a water molecule as a
guest.
have indicated in Tables 1 and 2 a value of %10. We do not
know the actual value (that can be different for 7 and 8) of
Kb, somewhere between near 0 and 10, so we have decided
to adopt KbZ5 in both cases as a working hypothesis. Using
other values such as KbZ1 or KbZ0.1, the conclusions do
not change very much only the correlations slightly
worsened.
Taking into account the difficulty to reproduce hydrogen
bond interactions with molecular mechanics, the agreement
is best than acceptable and gives confidence in both
AMBER and OPLS optimised geometries.
We have represented in Fig. 1 the X-ray structure of 8:H2O.
The crystal consists of molecules bonded through hydrogen
bonds to water molecules. The 8:H2O entity is almost
coplanar, the maximum dihedral angle between the pyridine
rings on the 2,6-dicarboxamide nitrogens is 12.1 (1)8, and
2.3. Tautomerism pyridone/hydroxypyridine:
compounds 9a/9b and 10a/10b
˚
the oxygen atom O3 is located 0.543(3) A out the least
square plane formed by N10–N8–N17–N19 atoms. In
Before discussing the host–guest properties of the com-
pounds under study, we will examine the pyridone (a)/
hydroxypyridine (b) tautomerism of compounds 9 and 10
based on B3LYP/6-31G(d,p) calculations as well as the
influence of a water molecule on these equilibria. Some of
us have reported a theoretical study of the unsubstituted
pyridone/hydroxypyridine equilibrium [B3LYP/6-31G(d)].8
In the absence of any perturbation, 4-hydroxypyridine is
more stable then 4-pyridone by 6.3 kJ molK1; the presence
of two methyl ester substituents at positions 2,6
(the conformation of the arms was fixed to simulate the
beginning of a crown ether), produces an inversion of the
stability in favour of the pyridone by 6.5 kJ molK1. In
the case of model compound 10 (fully optimisation,
B3LYP/ 6-31G(d,p) and ZPE correction) the hydroxypyri-
dine derivative 10b is 30.2 kJ molK1 more stable than 10a
(Scheme 6). This effect can be interpreted as due to the
amide N–H groups that interact attractively with the lone
pair on NK1 in the minimum conformation of 10b.
When the complexes formed by 10a and 10b with water
are calculated, the energy difference becomes larger
in favour of the hydroxy tautomer: 10b Erel(CZPE)Z
K61.2 kJ molK1 and 10a, Erel(CZPE)Z0.0 kJ molK1, as
the conformation of the N,N0-dimethyl-2,6-dicarboxamides
Figure 2. Dimers of Host 8 including a water molecule as a guest forming a
ribbon. Dashed lines show hydrogen bonds.

R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
5093
111.3 kJ molK1 if a water molecule is placed in the cavity of
both 9a and 9b because the less stable tautomer 9a is a better
host for water than 9b.
According to the B3LYP6-31G(d,p)//Monte Carlo Confor-
mational Search, AMBER force field level calculations, the
interaction energies of the four hosts with water (H2O was
always situated in the concave part) defined as Ecomplex
K
(EhostCEwater), are: C19.9 kJ molK1 for 7 (destabilisation),
K51.4 kJ molK1 for 8 (stabilisation), K88.7 kJ molK1 for
9a (stabilisation), and K32.6 kJ molK1 for 9b (stabilis-
ation). Note that 8:H2O fits the experimental X-ray structure
(Fig. 1).
2.4. Experimental versus calculated binding constants
Scheme 6. The hydroxy/oxo tautomerism of pyridone derivatives taken
from Ref. 8 (diesters) and from this work (diamides).
The interactions with guest 6 cannot be calculated with
OPLS (Table 4), but it is possible to compare the AMBER
and OPLS results for the remaining compounds. There is
a rough proportionality between both series of values
(Eq. (1)):
changes on formation of the complex 10a:H2O to include
the water molecule into the cavity.
Although for compound 9 the calculations correspond
to B3LYP/6-31G(d,p)//MM ones, tautomer 9b is
167.3 kJ molK1 more stable than 9a, possibly due to the
increased acidity of the N–H protons at position 2 of the
pyridine ring. The 167.3 kJ molK1 difference decreases to
KEminðOPLSÞ ¼ ð1:38G0:04Þ!KEminðAMBERÞ;
(1)
n ¼ 21; r2 ¼ 0:983
Scheme 7. The different conformations of N,N0-dimethylurea 2.
Figure 3. A plot the experimental results (Table 1) vs. the calculated ones (Table 3) for host 7.

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R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
Comparison with the experimental results in the case of 7 is
not good enough to discriminate between both kinds of
calculations. Therefore, we will use the more complete
AMBER values (Table 3).
we have based our discussion on the AMBER calculations
of Table 3. Like in the preceding case, the best agreement in
the case of guest 2 is found for the E,E conformation with an
experimental Kb value of 5. Of the two columns for 8, the
results are better using the GB/SA model for water as shown
in Fig. 4.
When comparing the average Kb values for complexes of
host 7 in Table 1 (after transforming them into Ln Kb) with
KEmin AMBER in Table 3, the first problem that arises is
the isomerism of N,N0-dimethylurea 2 (Scheme 7). What of
the three conformations of this compound is the most
consistent with the experimental value?
The blue line corresponds to:
Ln Kb Z ð0:8G1:4Þ Cð0:083G0:024Þ
Since the CIS are too small we have assumed an
experimental value of KbZ5, and from the variation of
ln Kb against KEmin AMBER it appears that the (E,E)-2
isomer is the one interacting with the host. For (Z,E)-2 and
(Z,Z)-2 the residuals are much larger, so we have excluded
these points in Fig. 3.
(4)
!KEmin AMBER ðWater KGB=SAÞ;
n Z 6; r2 Z 0:76
The red line corresponds to:
Here again, tolbutamide (6) is the worse point. Removing it
one obtains as indicated in the black line:
Ln Kb ZKð2:1G2:6Þ Cð0:155G0:047Þ!KEminðAMBERÞ;
Ln Kb ZKð0:3G1:3Þ Cð0:112G0:025Þ
n Z 6; r2 Z 0:73
(2)
(5)
!KEmin AMBER ðWater KGB=SAÞ;
Removing tolbutamide (6) one obtains the black line:
n Z 5; r2 Z 0:87
Ln Kb ZKð2:9G2:5Þ Cð0:176G0:047Þ!KEminðAMBERÞ;
It is possible to treat together hosts 7 and 8 using AMBER
(CHCl3-GB/SA) for 7 and AMBER (Water-GB/SA) for 8:
n Z 5; r2 Z 0:82
(3)
Three regression lines can be calculated for the points of
Fig. 5:
We turn now to the data of host 8. For homogeneity reasons
Figure 4. A plot the experimental results (Table 2) vs. the calculated ones (Table 3) for host 8.

R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
5095
Figure 5. A plot the experimental results (Tables 1 and 2) vs. the calculated ones (Table 3) for hosts 7 and 8.
All points : Ln Kb Z ð0:4G1:3Þ Cð0:101G0:023Þ
!KEmin AMBER; n Z 12; r2 Z 0:65
Without 6 : Ln Kb ZKð1:1G1:2Þ Cð0:135G0:023Þ
!KEmin AMBER; n Z 10; r2 Z 0:81
(6)
(7)
Figure 6. Structures of complexes: a. 7:3; b. 7:1; c. 7:5; d. 7:6.

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R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
Figure 7. Structures of complexes: a. 8:3; b.8:1; c. 8:5; d. 8:6.

R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
5097
case of biotin methyl ester (1) where no CIS were observed
on the NH chemical shifts (Table 2) proving that there is no
interaction through the urea moiety. The binding takes place
involving the amide NHs of the host and the carbonyl group
of the biotin side chain. In the two views of the structure of
the complex 8:1 shown in Fig. 7b, that corresponds to the
energy minimum, the two internal hydrogen bonds remain
intact and there are no changes in the original conformations
nor in the host neither in the guest.
No intercept : Ln Kb Z ð0:115G0:007Þ!KEmin AMBER;
n Z 10; r2 Z 0:97
(8)
To explain why tolbutamide (6) deviates it is necessary to
examine the structure of the complexes as calculated by
AMBER for hosts 7 and 8. If we consider the complex
between 7 and 2-imidazolidone (3) (Fig. 6a) as representa-
tive of the studied complexes, we0 observe that those of
biotin methyl ester (1) (Fig. 6b), N,N -dimethylurea (2) in its
E,E-conformation and trimethyleneurea (4) are very similar.
That of barbital (5) uses only the carbonyl group (Fig. 6c)
and tolbutamide (6) only the SO2–NH moiety (Fig. 6d).
The binding modes were confirmed in the complex of
tolbutamide (6) with host 7 by means of NOESY NMR
experiments that proved the closeness of the p-tolyl protons
of 6 to the methyl group of the host (Fig. 8a) and the guest
aliphatic chain protons to host pyridine ones (Fig. 8b).
This information allowed us to calculate the new energy
minimum of K63.4 kJ molK1 depicted in Table 3, that fits
the experimental binding constant Kb.
Similar features are found for host 8 (Fig. 7a–c), save in the
Figure 8. Enlarged regions of the NMR NOESY spectra.

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R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
Moreover, no changes in the 13C NMR chemical shift of the
urea carbonyl group of tolbutamide (6) are induced when
complexes 7:6 and 8:6 are formed.
4.3. Synthesis of N,N0-bis(6-methylpyridin-2-yl)-1,3-
benzenedicarboxamide (7)
See Scheme 4. Isophthaloyl chloride (11, 1 g, 4.9 mmol)
was dissolved, under Ar, in 100 mL of dry CH2Cl2, then a
solution of 2-amino-6-methylpyridine (12, 1.1 g, 9.9 mmol)
and Et3N freshly distilled (4 mL) in 90 mL of dry CH2Cl2
was added slowly from a pressure-equalising addition
funnel. The resulting solution was stirred for 4 h and then
washed with saturated solution of NaHCO3 and water, dried
(Na2SO4) and concentrated to yield a yellow-pale solid
which is recrystallized from MeOH to obtain 0.74 g (44%)
of 7, mp 230 8C. 1H NMR (CDCl3): d (ppm) 8.81 (broad s,
2H, NH), 8.48 (dd, 1H, H-2, J2,4ZJ2,6Z1.8 Hz), 8.16 (d,
3. Conclusions
As a result of our studies, two different conformations in the
complexation mode of biotin methyl ester (1) with 7 and 8
have been found. The conformation with 7 is similar to the
normal one shown by the ureas, but with 8 is completely
different and takes place through the carbonyl group of the
biotin side chain. Concerning barbital (5), the hosts are only
able to accommodate one part of the molecule but the Kb
values are high (the highest with 7), a similar observation
was made with the hosts of our precedent paper.1
2H, H-30, J3’,4Z8.2 Hz), 8.10 (dd, 2H, H-4/H-6, J4,5/6,5
Z
7.8 Hz), 7.63 (dd, 2H, H-40, J4 ,5 Z7.7 Hz), 7.58 (dd, 1H,
H-5), 6.92 (d, 2H, H-50), 2.42 (s, 6H, CH3). 13C NMR
(CDCl3): d (ppm) 164.5 (CO), 156.9 (C60), 150.5 (C20, 3JZ
9.2 Hz), 138.9 (C40, 1JZ161.0 Hz), 135.0 (C1/C3, 3JZ
0
0
Tolbutamide (6) is rather different from the other five ureas
because the sulfonyl group increases considerably the
acidity of the contiguous NH11 but also modifies the
conformation. Based on its X-ray structure,12 we have
represented it in Scheme 2 with both NHs opposite to the
CZO (like a Z,Z-dimethylurea). Although this is the
conformation found in the complexes, tolbutamide is too
different from classical ureas to fit well in the same series of
calculations.
1
3
7.7 Hz), 130.8 (C4/C6, JZ161.8 Hz, JZ6.1 Hz), 129.4
(C5, 1JZ162.6 Hz), 125.9 (C2, 1JZ161.0 Hz, 3JZ6.1 Hz),
1
3
1
119.7 (C50, JZ162.6 Hz, JZ6.2 Hz), 111.0 (C30, JZ
171.8 Hz, 3JZ6.1 Hz), 23.9 (CH3, 1JZ127.3 Hz, 3JZ
3.1 Hz). 15N NMR (CDCl3): d (ppm) K242.9 (NH),
K98.9 (N10). Anal. Calcd for C20H18N4O2: C, 69.35; H,
5.24; N, 16.17%. Found: C, 69.09; H, 5.29; N, 16.09%.
4.4. Synthesis of 4-chloro-N,N0-bis(6-methylpyridin-2-
yl)-2,6-pyridindicarboxamide (8)
4. Experimental
See Scheme 5. Chelidamic acid (13, 0.5 g, 2.73 mmol) is
dissolved in thionyl chloride (10 mL, 137.4 mmol) with the
minimum quantity of DMF and the solution is heated at
110 8C for 3 h. After that, DMF and thionyl chloride are
evaporated at reduce pressure until a white solid of
4-chlorochelidamic acid dichloride (15) is obtained, 0.45 g
4.1. General
The six guests are commercially available:0 biotin methyl
ester (1) (O99%, dried under vacuum), N,N -dimethylurea
(2) (99%, recrystallized from ethyl acetate), 2-imidazo-
lidone (3) (96%, recrystallized from ethyl acetate),
N,N0-trimethyleneurea (4) (O98%, recrystallized from
ethyl acetate), barbital (5) (O99%) and tolbutamide (6)
(O99%). Melting points were determined in a Thermo-
Galen hot stage microscope and are uncorrected. Elemental
analyses for carbon, hydrogen, and nitrogen were carried
out by the Microanalytical Service of the Complutense
University on a Perkin-Elmer 240 analyser.
1
(74%), mp 200 8C. H NMR (DMSO-d6): d (ppm) 8.46 (s,
2H, H3/H5).
4-Chlorochelidamic acid dichloride (15, 0.5 g, 2.10 mmol)
was dissolved, under Ar, in 20 mL of dry CH2Cl2, then
a solution of 2-amino-6-methylpyridine (12, 0.68 g,
4.55 mmol) and Et3N freshly distilled (3 mL) in 30 mL of
dry CH2Cl2 was added slowly from a pressure-equalising
addition funnel. The resulting solution was stirred for 4 h
and then washed with saturated solution of NaHCO3 and
water, dried (Na2SO4) and concentrated to yield a white
solid which is recrystallized from MeOH to obtain 0.35 g
(40%) of 8, mp 244 8C. 1H NMR (CDCl3): d (ppm)
4.2. NMR spectroscopy
NMR spectra were recorded on a Bruker DRX 400 (9.4 T,
400.13 MHz for H, 100.62 MHz for 13C and 40.56 MHz
1
for 15N) spectrometer at 300 K. Chemical shifts (d in ppm)
10.23 (broad s, 2H, NH), 8.46 (s, 2H, H-3/H-5), 8.20
0
(d, 2H, H-30, J3 ,4 Z8.2 Hz), 7.67 (dd, 2H, H-4 , J4 ,5
Z
are given from internal solvent CDCl3 7.26 for 1H and 77.0
0
0
0
7.5 Hz), 6.98 (d, 2H, H-50), 2.52 (s, 6H, CH3). 13C NMR
(CDCl3): d (ppm) 160.5 (CO), 157.2 (C60), 150.2 (C2/C6),
150.0 (C20, 3JZ9.2 Hz), 148.3 (C4, 2JZ3.1 Hz), 138.8
(C40, 1JZ161.0 Hz), 126.1 (C3/C5, 1JZ174.6 Hz, 3JZ
4.5 Hz), 119.9 (C50, 1JZ162.9 Hz, 3JZ6.1 Hz), 111.3
(C30, 1JZ171.6 Hz, 3JZ6.1 Hz), 24.0 (CH3, 1JZ
127.3 Hz). 15N NMR (CDCl3): d (ppm) K247.1 (NH),
K97.9 (N10), K97.2 (N1). Anal. Calcd for C19H16ClN5O2.
H2O: C, 57.07; H, 4.54; N, 17.52%. Found: C, 57.17; H,
4.67; N, 17.49%.
for 13C, DMSO-d6 2.49 for 1H and 39.5 for 13C and for 15
NMR nitromethane was used as external standard. Coupling
N
1
constants (J in Hz) are accurate to JZG0.2 Hz for H and
13C and JZG0.6 Hz for 15N. 2D-Inverse proton detected
homonuclear shift correlation spectra gs-COSY (1H–1H),
NOESY and 2D inverse proton detected heteronuclear shift
correlation spectra, gs-HMQC (1H–13C), gs-HMBC
(1H–13C) and gs-HMBC (1H–15N), were carried out with
the standard pulse sequences to assign the H, 13C and 15N
signals.
1

R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
5099
4.5. NMR titrations
Titrations between host 8 and biotin methyl ester (1) are
carried out by the same method but keeping the concen-
tration of water under 1 mM in order to avoid a competitive
behaviour of the water.5
Each NMR titration was carried out at least three times at
300 K in CDCl3 as a solvent (Merck S33657, deuterium
content O99.8%, water content !0.01%). The syringes are
from Hamilton–Bonaduz, 5 mL (divisions 0.05 mL), 10 mL
(divisions 0.1 mL), 250 mL (divisions 5 mL) and the balance
for weighting the host and the guest a Metler AE260-Delta
For guests 3, 4, 5 and 6 a competitive titration is used, the
CIS of the two guests are measured while aliquots of the
host are added. The fitting of the data to the (Eq. (10))
allows to obtain a relative Kb for the guest we are studying,
as the Kb for the complex 8:water was previously measured
we can calculate the value for the complex 8:guest.
1
Range (error G0.00005 g). H NMR titrations are used in
order to quantify Kb values, these titrations are carried out
following the Chemical Induced Shift (CIS) in one or
several protons of host or guest while the concentration of
the complex formed is changed by the addition of one of the
components. For host 7 we performed a double independent
quantification following the CIS for amide protons and the
H-2 in the central benzene ring, while guest solution
aliquots are added. There are a large number of ways to fit
the data from a titration,9 but that consisting in non-linear
curve fitting is generally accepted as the method with the
lowest error in the determination of Kb values, in
comparison to others that employ approximations to reach
a linear relationship between d and Kb. To fit the
experimental data the Sigmaplot 8.1 program from SPSS
Science Software Gmbh was employed. The basic equation
used in this kind of titrations is represented by [Eq. (9)],
showing the relationship between chemical shifts (d),
concentrations of host H, guest G and complex C, and the
binding constant KbZ[C]/([H][G]), this equation is valid
only for 1:1 stoichiometry as is our case.10
K
bðwaterÞ=KbðguestÞ Z ½ð1=FguestÞ K1ꢀ=½ð1=FwaterÞ K1ꢀ
(10)
Fwater and Fguest are the molar fractions of water and guest
that are bound to the host, if no another equilibria arise
(which it has been proved with titrations of the guest versus
water), then FiZ(di,FreeKdiObserved)/(di,FreeKdi,Complexed).
4.6. MM calculations
MacroModel v.8.1, with the GB/SA model for chloroform13
was used in order to perform the molecular simulations
of the complexes in all cases, save as indicated in Table 3.
All calculations were achieved with Monte Carlo (MC)
conformational analyses.14 Minimisation is carried out
using Polak-Ribiere conjugate gradient optimiser.15 In a
typical MC run a MCMM is performed with never less than
8000 steps, to carry out the search both torsional rotations in
˚
host and guest and translation/rotation (10 A/3608) of the
guest is performed, for all the MC a cutoff is applied to van
der Waals, electrostatic and H-bond interactions with 7, 12
˚
with two different force fields, AMBER*,16 and OPLS*,17
as implemented in the version of the program.
dOBS Z ðdC KdHÞðfð1 C½Gꢀ=½Hꢀ C1=Kb½HꢀÞ=2g
and 4 A, respectively. These calculations were carried out
2
1=2
Kfð1 C½Gꢀ=½Hꢀ C1=Kb½HꢀÞ =4 K½Gꢀ=½Hꢀg Þ CdH
(9)
4.7. DFT calculations
Single point calculations were carried out at the B3LYP/
6-31G(d,p) level of theory18,19 with the Gaussian ’03
program on the optimised Molecular Mechanics geome-
tries.20 At this level we calculated the energies for the
complexes between hosts 7, 8 and 9a/9b with water,
analysing the energy differences between the two possible
tautomers in host 9 (a, 4-pyridone/b, 4-hydroxypyridine).
BSSE were determined for all the complexes computed by
DFT with the counterpoise correction.21
In order to obtain Kb values with the lowest error the
titrations are carried out in the 20–80% saturation range for
the compound which CIS is being followed. This condition
determines the concentrations to be used in the titrations for
both host and guest and a calculation has to be done to find
those concentrations that best cover the whole range of p in
order to get the maximum information from the titration
curve. The accuracy in the concentration range to be used in
titrations is usually disregarded in most publications of the
host–guest field, affording Kb values totally different from
those obtained following this procedure. The error deter-
mined by this magnitude is intrinsic to the measurement
method and it is not reflected by the standard deviation (Sd)
which is a measure of the fit goodness of the data employed.
Model compound 10a/10b and its water complexes were
fully optimised (no imaginary frequencies).
4.8. Crystallographic data collection and structure
determination of 8a:H2O
The titrations for the complex between host 8 and water are
carried out on the same way (20–80% saturation range),
water concentration is determined by the integration of its
NMR signal. The host sample for the titration is prepared
Suitable crystal for X-ray diffraction experiments was
obtained by crystallization from acetone/hexane. Data
collection was carried out at room temperature on a Bruker
Smart CCD diffractometer using graphite-monochromated
˚
with freshly distilled CDCl3 and molecular sieve 4 A is
˚
added to keep water at the minimum concentration, on this
way all the samples had an initial saturation range between
20–30%, so the titration was carried out in the right
saturation range. The results were reported in Table 2.
Mo K( radiation (lZ0.71073 A) operating at 50 Kv and
30 mA. Data were collected over a hemisphere of the
reciprocal space by combination of three exposure sets.
Each exposure of 30 s covered 0.3 in u. Structure was

5100
R. M. Claramunt et al. / Tetrahedron 61 (2005) 5089–5100
5. Adrian, J. C.; Wilcox, C. S. J. Am. Chem. Soc. 1991, 113,
678–680.
Table 6. Crystal data and structure refinement for 8:H2O
Empirical formula
Formula weight
Temperature
Crystal system
Space group
Unit cell dimensions
C19H18ClN5O3
399.83
6. Hirao, T.; Moriuchi, T.; Ishikawa, T.; Nishimura, K.; Mikami,
S.; Ohshiro, Y.; Ikeda, I. J. Mol. Catal. A 1996, 113, 117–130.
7. Jouaiti, A.; Hosseini, M. W.; Kyritsakas, N. Chem. Commun.
2003, 1898–1899.
293(2) K
Triclinic
P-1
˚
aZ8.274(2) A
aZ114.003(5)8
bZ91.997(5)8
gZ94.132(6)8
8. Alkorta, I.; Elguero J. Heterocycl. Chem. 2001, 38,
1387–1391.
˚
bZ10.560(3) A
˚
cZ11.942(3) A
948.4(4) A
3
˚
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
9. Fielding, L. Tetrahedron 2000, 56, 6151–6170.
10. Hirose, K. J. Incl. Phenom. Macrocycl. Chem. 2001, 39,
193–209.
2
1.400 Mg/m3
0.233 mmK1
416
11. NH-acidity (pKa): tolbutamide: 5.3, barbital: 7.7: www.
medchem.ku.edu/MDCM625.
q (8)
Index ranges
1.87–25.008
K9%h%9,
K10%k%12,
K14%l%13
5030
12. Nirmala, K. A.; Gowda, D. S. S. Acta Crystallogr. Sect. B
1981, 37B, 1597–1599. Donaldson, J. D.; Leary, J. R.; Ross,
S. D.; Thomas, M. J. K.; Smith, C. H. Acta Crystallogr. Sect. B
1981, 37B, 2245–2248.
Reflections collected
Independent reflections
Data/restraints/
parameters
3315 [R(int)Z0.0494]
3315/0/255
¨
13. MacroModel, Schrodinger LLC, 2004. http://www.schrodinger.
G.o.f. (F2)
0.815
0.0489 (ref. obs. 1342)
0.1228
com/Products/macromodel.html.
R1 [IO2sigma(I)]a
R2 (all data)b
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111, 4379–4385.
a S[jFojKjFcj] /SjFoj.
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London, 1977; Chapter III. 1.7.
b {S[w(Fo2KFc2)2] /S[w(Fo2)2]}1/2
.
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All non-hydrogen atoms were refined anisotropically. All
hydrogen atoms were located in a difference Fourier map,
included and fixed. Crystal data and other structure
determination details are presented in Table 6.
18. Becke, A. D. Phys. Rev. A 1988, 38, 3098–3100. Becke, A. D.
J. Chem. Phys. 1993, 98, 5648–5652. Lee, C.; Yang, W.; Parr,
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54, 724–728.
CCDC-256026 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data centre, 12 union Road,
Cambridge CB2 1EZ, UK; fax: (C44) 1223-336033; or
www.deposit@ccdc.cam.uk.
20. Gaussian 03, Revision B.04, Frisch, M. J.; Trucks, G. W.;
Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J.
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C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
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G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda,
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Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
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Challacombe, M.; Gill, P.M. W.; Johnson, B.; Chen, W.;
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Acknowledgements
Thanks are given to MCyT for financial support (project
BQU2003-00976). One of us (F.H.) is grateful to the MCyT
for a FPI fellowship.
References and notes
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