New macrocyclic compounds with naphthyridine units for molecular recognition studies of biotin and urea derivatives

Abstract

Two macrocyclic hosts containing benzenedi-carboxamide or pyridinedicarboxamide moieties and two 1,8-naphthyridine units linked by a crown ether like chain, have been synthesized and fully characterized by multi-nuclear NMR spectroscopy. X-ray diffraction analysis is provided for one of the macrocycles including a DMSO guest molecule. Binding constant determination of both hosts with four ureido derivatives, amongst them (?)-bio-tin methyl ester, was achieved by means of 1 H NMR titrations.

Full text

ORIGINAL ARTICLE
New macrocyclic compounds with naphthyridine units
for molecular recognition studies of biotin and urea derivatives
M. A
´ngeles Farra
´n•Dolores Santa Marı
´a•
M. A
´ngeles Garcı
´a•Rosa M. Claramunt •
Guy J. Clarkson
Received: 28 March 2014 / Accepted: 26 June 2014
ÓSpringer Science+Business Media Dordrecht 2014
Abstract Two macrocyclic hosts containing benzenedi-
carboxamide or pyridinedicarboxamide moieties and two
1,8-naphthyridine units linked by a crown ether like chain,
have been synthesized and fully characterized by multi-
nuclear NMR spectroscopy. X-ray diffraction analysis is
provided for one of the macrocycles including a DMSO
guest molecule. Binding constant determination of both
hosts with four ureido derivatives, amongst them (?)-bio-
tin methyl ester, was achieved by means of
1
HNMR
titrations.
Keywords Bisnaphthyridineamides 
Benzenedicarboxamides Pyridinedicarboxamides Host–
guest Binding constants MM calculations
Introduction
Macrocycles research has continually increased since Pe-
dersen reported the synthesis of crown ethers in 1967. The
interest in these compounds range from methodological
aspects of their synthesis to a large number of applications
in important areas such as catalysis, membrane transport,
switches, sensors and biochemical systems, as demon-
strated by the large number of contributions devoted to the
subject. Molecular recognition studies are usually per-
formed to understand features like complementarity in size,
shape, and functional groups responsible for the macrocy-
cles properties [1].
In the particular area of design of receptors for bioactive
substances, ureas and biotin stand out for their significance.
Biotin is a cofactor found in many enzymes that have diverse
metabolic functions and is also used as a tag in different bio-
logical assays due to its capability of forming strong complexes
via hydrogen bond (HB) with avidin (K
b
=2.5 910
15
M
-1
)
or streptavidin (K
b
=1.7 910
15
M
-1
). The avidin–biotin
binding is the strongest protein–ligand interaction reported and
is a consequence of a combination of two factors: (i) a perfect fit
of biotin through van der Waals interactions in a hydrophobic
pocket formed by tryptophan and phenylalanine residues and
(ii) the binding of biotin by the formation of 10 additional
hydrogen bonds [2,3].
Synthetic receptors that try to mimic this system tuning
these two main non-covalent interactions have been
reported. Wilcox and coworkers [4] described a Troger’s
base that binds biotin methyl ester exclusively by HB
interactions. Goswami and Dey [5] reported two neutral
isophthaloyl pyridine bisamide receptors with one and two
methyl amido pyridine pendant arms, designed for tandem
binding of biotin. We studied similar receptors for biotin
methyl ester and urea derivatives fully soluble in chloro-
form, a solvent having comparable polarity to that of the
interior cavity of an enzyme [6], and Ghosh and Sen [7]a
benzothiazole-based receptor for recognition of biotin ester
and urea.
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-014-0433-3) contains supplementary
material, which is available to authorized users.
M. A
´. Farra
´n(&)D. Santa Marı
´aM. A
´. Garcı
´a
R. M. Claramunt (&)
Departamento de Quı
´mica Orga
´nica y Bio-Orga
´nica, Facultad de
Ciencias, UNED, Senda del Rey 9, 28040 Madrid, Spain
e-mail: afarran@ccia.uned.es
R. M. Claramunt
e-mail: rclaramunt@ccia.uned.es
G. J. Clarkson
Department of Chemistry, University of Warwick, University
Library Rd., Coventry CV4 7AL, UK
e-mail: guy.clarkson@warwick.ac.uk
123
J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-014-0433-3

On the other hand, the introduction of the 1,8-naph-
thyridine moiety provides extra HB acceptor sites for
complexation. Zimmerman and coworkers [8,9] and
Hamilton and Pant [10] were among the first groups to use
7-amido-1,8-naphthyridines to bind guanine derivatives.
Our group [11–13], as well as Ghosh and Sen [14], have
also contributed to this field using isophthaloyl or pyridine
naphthyridyl bisamide systems to build receptors for
methyl biotin ester and various ureas. Others have used the
same motif to construct macrocycles [15–17], hydrogen
bonded polymers [18,19] or monosaccharide and disac-
charide receptors [20,21].
The aim of the present work has been to synthesize two
new symmetrical macrocycles, Ia and Ib, with benzen-
edicarboxamide or pyridinedicarboxamide moieties and
two 1,8-naphthyridine units linked by a chain similar to
that found in crown ethers of 17 atoms with pendant nosyl
groups (Fig. 1). These systems appear to be suitable for
molecular recognition studies of ureas in low polarity
solvents such as chloroform and dichloromethane, and they
potentially offer recognition sites, HB and p-stacking and
van der Waals interactions, for the ureido functionality and
for the side chain of (?)-biotin methyl ester.
Bearing this in mind, the binding properties of the
artificial receptors Ia and Ib have been studied with four
guests: (?)-biotin methyl ester (1), 2-imidazolidinone (2),
N,N’-trimethyleneurea (3), and barbital (4) (Fig. 2).
Experimental section
Materials
Guests are commercially available: biotin methyl ester
(methylbiotin, 1)([99 %, dried under vacuum), 2-imi-
dazolidinone (2) (96 %, recrystallized from ethyl acetate),
N,N’- trimethyleneurea (3)([98 %, recrystallized from
ethyl acetate), and barbital (4)([
99 %). All starting
reagents were obtained from commercial suppliers and
used as received without further purification. Solvents were
purified and dried with use of standard procedures. Com-
pounds 5[22], 6[23] and 7[23] were prepared according to
literature. Melting points were determined with a Thermo-
Galen hot stage microscope and are given uncorrected.
Elemental analyses for carbon, hydrogen and nitrogen were
performed by the Microanalytical Service of the Univers-
idad Complutense of Madrid, using a Perkin-Elmer 240
analyzer. Mass spectrometry experiments were carried out
on a Finnigan Surveyor (Thermo Electron, San Jose
´, CA,
USA) pump coupled with a Finnigan LCQ Deca (Thermo
Electron, San Jose
´, CA, USA) ion trap mass spectrometer
using an electrospray ionization (ESI) interface. Metha-
nolic solutions of the compounds (1.3 mg L
-1
) were
infused into the mass spectrometer at a flow rate of
5lLmin
-1
using the syringe pump included in the LCQ
instrument and mixing it with 100 lL min
-1
of metha-
nol:acetic acid 0.2 % (50:50, v/v) by means of a zero-dead
volume T–piece. Mass spectra were acquired in positive
mode, scanning from m/z 700 to m/z 2000. Spray voltage
was set at 4.5 kV, heated capillary temperature at 200 °C,
sheath gas at 0.6 L min
-1
and, auxiliary gas at 6 L min
-1
.
Synthesis
N,N’-(7,7’-(2,16-bis(4-nitrophenylsulfonyl)-6,9,12-trioxo-
2,16-diazaheptadecano-1,17-diyl)bis(1,8-naphthyridin-2,7-
diyl))diacetamide (9)
To a solution of N-(7-formyl-1,8-naphthyridin-2-yl)acet-
amide (7), 1.90 g (8.82 mmol), in 120 mL of methanol,
0.973 g (4.41 mmol) of 4,7,10-trioxatridecane-1,13-dia-
mine were added. After 12 h at room temperature an ali-
quot of the reaction mixture was analyzed to verify that the
starting material was consumed and the diimine formed.
Then, slow addition of 0.7 g (17.64 mmol) of sodium
borohydride was achieved. The reaction was maintained at
room temperature with continuous stirring for 24 h and
afterwards water was added to quench the excess NaBH
4
.
XOO
N N
N N
N N
NN
OOO
4
3
2
5
6
1
H H 2' 3'
4'
4a'
5'
6'
7'
1'
8'
8a'
9'
11'
12'
13'
14'
15'
Ia X=C Y= p-nosyl
Ib X=N Y= p-nosyl
Y = NO2
O2S
10'
ortho meta
para
ipso
Y
Y
Fig. 1 Macrocyclic receptors Ia and Ib
J Incl Phenom Macrocycl Chem
123

The solvents were removed under vacuum and the crude
residue extracted twice with dichloromethane/water. The
organic fractions were combined, dried over anhydrous
sodium sulfate and the solvent evaporated off to yield 8as
a brown hygroscopic oil (1.78 g) that was used without
further purification.
Crude compound 8was dissolved in 150 mL of dry
dichoromethane (amylene stabilized) with 1.603 g
(15.85 mmol) of triethylamine and was maintained at 0 °C
using an ice bath. After addition of a solution of 1.75 g
(8 mmol) of 4-nitrobenzenosulfonyl chloride in 50 mL of
dichloromethane the reaction mixture was stirred at room
temperature for 8 h and then extracted with water and 1 M
hydrochloric acid. Organic layers were combined and the
solvent evaporated off to yield a brown solid (2.3 g, 81 %),
the diacetyl derivative 9, which was purified by column
chromatography over silica gel 60 mesh with dichlorome-
tane/methanol (98:2 in volume). During these operations
partial deacetylation occurred (0.2 g of 10) and only 0.7 g
of 9were obtained: m.p. 142–145 °C;
1
H NMR (CDCl
3
)d
(ppm) 8.94 (br s, 2H, CONH), 8.52 (d, 2 H,
3
J=8.8 Hz,
30-H), 8.32 (d, 4 H,
3
J=8.9 Hz, meta-H), 8.17 (d, 2 H,
3
J=8.8 Hz, 40-H), 8.14 (d, 2 H,
3
J=8.2 Hz, 50-H), 8.05
(d, 4 H, J=8.8 Hz, ortho-H), 7.63 (d, 2 H,
3
J=8.2 Hz,
60-H), 4.70 (s, 4 H, Ar-CH
2
NNs), 3.40 (t, 4H,
3
J=7.5 Hz,
OCH
2
CH
2
CH
2
N), 3.35 (m, 8H, OCH
2
CH
2
O), 3.29 (t, 4 H,
3
J=5.9 Hz, OCH
2
CH
2
CH
2
N), 2.27 (s, 6 H, CH
3
-CONH),
1.87-1.49 (m, 4H, OCH
2
CH
2
CH
2
N);
13
C NMR (CDCl
3
)d
(ppm) 169.7 (CO), 160.7 (C70), 154.2 (C20or C8a’), 154.1
(C8a’ or C20), 150.1 (C
para
), 145.5 (C
ipso
), 139.4 (C40),
137.6 (C50), 128.6 (C
ortho
), 124.5 (C
meta
), 119.8 (C4a’),
119.7 (C60), 115.5 (C30), 70.5 and 70.3 (OCH
2
CH
2
O), 67.9
(OCH
2
CH
2
CH
2
N), 54.4 (Ar-CH
2
NNs), 46.8 (OCH
2
CH
2-
CH
2
N), 28.5 (OCH
2
CH
2
CH
2
N), 25.1 (CH
3
CO). Anal.
Calcd for C
44
H
48
N
10
O
13
S
2
.H
2
O: C, 53.43; H, 4.89; N,
14.16, S, 6.48 Found: C, 53.10; H, 5.25; N, 14.10; S, 6.35.
N,N’-(3,30-(2,20-oxybis(ethane-1,2-
diyl)bis(oxy))bis(propane-1,3-diyl))bis(N-((7-amino-1,8-
naphthyridin-2-yl)methyl))-4-nitrobenzenesulfonamide
(10)
To a solution of 0.53 g (0.95 mmol) of 9in 10 mL of
dioxane and 2 mL of water, 1 mL of 4 M HCl in dioxane
was added and the mixture heated for 30 min at 110 °C.
The reaction mixture was cooled down and neutralized
with 2.5 M sodium hydroxide to pH 8–9. The dioxane was
evaporated under vacuum and the oily residue extracted
with dichloromethane to yield 10 (0.45 g, 93 %): m.p.
102–105 °C;
1
H NMR (CDCl
3
)d(ppm) 8.26 (d, 4 H,
3
J=8.8 Hz, meta-H), 7.98 (d, 4 H,
3
J=8.8 Hz, ortho-H),
7.92 (d, 2 H,
3
J=8.2, 50-H), 7.82 (d, 2 H,
3
J=8.8 Hz, 40-
H,), 7.37 (d, 2 H,
3
J=8.2 Hz, 60-H,), 6.75 (d, 2 H,
3
J=8.8 Hz, 30-H), 5.20 (s, 4 H, NH
2
), 4.63 (s, 4 H, Ar-
CH
2
NNs), 3.54 (t, 4 H,
3
J=7.5 Hz, OCH
2
CH
2
CH
2
N),
3.42 (m, 8 H, OCH
2
CH
2
O), 3.32 (4 H, t,
3
J=5.9 Hz,
OCH
2
CH
2
CH
2
N), 1.87-1.49 (m, 4 H, OCH
2
CH
2
CH
2
N);
13
C NMR (CDCl
3
)d(ppm) 160.0 (C70), 159.3 (C20), 155.8
(C8a’), 150.1 (C
para
), 145.8 (C
ipso
), 138.3 (C40), 137.4
(C50), 128.6 (C
ortho
), 124.2 (C
meta
), 117.4 (C60), 116.5
(C4a’), 112.6 (C30), 70.6 and 70.2 (OCH
2
CH
2
O), 68.1
(OCH
2
CH
2
CH
2
N), 54.4 (Ar-CH
2
NNs), 47.1 (OCH
2
CH
2-
CH
2
N), 28.6 (OCH
2
CH
2
CH
2
N). Anal. Calcd for C
40
H
44-
N
10
O
11
S
2
: C, 53.09; H, 4.90; N, 14.48; S,7.09 Found: C,
52.94; H, 4.82; N, 14.38; S, 7.01.
Macrocycle Ia
0.83 g (0.92 mmol) of 10 and 0.376 g (3.67 mmol) of tri-
ethylamine were dissolved in 50 mL of dry chloroform.
0.187 g (0.92 mmol) of isophthaloyl dichloride was also
dissolved in 50 mL of dry chloroform. In an oven dried 1 L
three-necked round bottomed flask, maintained at 0
°
C and
under Argon atmosphere, containing 200 mL of dry chlo-
roform, the two already prepared solutions were simulta-
neously added using independent pressure controlled
addition funnels during a 2 h period. Then the reaction
mixture was stirred for 48 h more at room temperature, the
solvent was evaporated off and the crude purified by column
chromatography over silica gel 60 mesh with dichloro-
methane-methanol (95.5 in volume) to afford Ia (200 mg,
21 %): m.p. [250 °C (dec.);
1
H NMR (CDCl
3
)d(ppm)
9.57 (br. s, 2 H, CONH), 8.71 (d, 2 H,
3
J
4
0
-H
=8.8 Hz, 30-H),
8.62 (s, 1 H, 1-H), 8.39 (d, 4 H,
3
J
ortho-H
=8.8 Hz, meta-H),
8.37 (d, 2H,
3
J
4-H
=7.8 Hz, 3-H/5-H), 8.26 (d, 2 H, 40-H),
8.16 (d, 2 H,
3
J
6
0
-H
=8.2 Hz, 50-H), 8.04 (d, 4 H, ortho-H),
7.77 (t, 1 H, 4-H), 7.58 (d, 2 H, 60-H), 4.78 (s, 4 H, 90-H), 3.54
(t, 4 H,
3
J
12
0
-H
=7.5 Hz, 110-H), 3.45 (m, 8 H, 140-H, 150-H),
NN
O
NN
O
H H
HH NN
O
O
O
H5C2C2H5
H H
S
NN
O
(CH2)4CO2CH3
H H
234
1
Hx
Hy
H4
Fig. 2 Selected guests 1–4
J Incl Phenom Macrocycl Chem
123

3.37 (t, 4 H,
3
J
12
0
-H
=5.9 Hz, 130-H), 1.75 (m, 4 H, 120-H);
13
C NMR (CDCl
3
)d(ppm) 164.8 (CO), 160.6 (C70), 154.0
(C20, C8a’), 150.1 (C
para
), 145.4 (C
ipso
), 139.6 (C40), 137.5
(C50), 134.0 (C2/C6), 133.2 (C3/C5), 130.4 (C4), 128.5
(C
ortho
), 124.6 (C
meta
), 124.3 (v. br. s, C1), 119.8 (C4a’),
119.7 (C60), 115.2 (C30), 70.6 (C140or C150), 70.2 (C150or
C140), 68.1 (C130), 53.8 (C90), 47.0 (C110), 28.9 (C120);
15
N
NMR (CDCl
3
)d(ppm) -79.5 (N80), -109.5 (N10); Anal.
Calcd for C
48
H
46
N
10
O
13
S
2
H
2
O: C, 54.75; H, 4.48; N, 13.30,
S, 6.09; Found: C, 54.27; H, 4.50; N, 13.14; S, 6.0; ESI–MS
[M ?H] 1035.2.
Macrocycle Ib
0.83 g (0.92 mmol) of 10 and 0.376 g (3.67 mmol) of tri-
ethylamine were dissolved in 50 mL of dry chloroform.
0.188 g (0.92 mmol) of 2,6-pyridinedicarbonyl dichloride
were also dissolved in 50 mL of dry chloroform. In an oven
dried 1 L three-necked round bottomed flask, maintained at
0
°
C and under Argon atmosphere, containing 200 mL of
dry chloroform the two already prepared solutions were
simultaneously added using independent pressure con-
trolled addition funnels during a 2 h period. Then the
reaction mixture was stirred for 48 h more at room tem-
perature, the solvent was evaporated off and the crude
purified by column chromatography over silica gel 60 mesh
with dichloromethane-methanol (95:5) to afford Ib
(100 mg, 10.5 %): m.p. C250 °C (dec);
1
H NMR (CDCl
3
)
d(ppm) 11.24 (br. s, 2 H, CONH), 8.88 (d, 2 H,
3
J
4
0
-
H
=8.8 Hz, 30-H), 8.64 (d, 2 H,
3
J
4-H
=7.7 Hz, 3-H/5-H),
8.43 (m, 4 H, meta-H), 8.28 (d, 2 H, 40-H), 8.25 (t, 1 H,
4-H), 8.17 (d, 2 H,
3
J
6
0
-H
=8.2 Hz, 50-H), 8.16 (m, 4 H,
ortho-H), 7.57 (d, 2 H, 60-H), 4.93 (s, 4 H, 90-H), 3.65 (t, 4
H,
3
J
12
0
-H
=7.7 Hz, 110-H), 3.46 (m, 8 H, 140-H, 150-H),
3.42 (t, 4 H,
3
J
12
0
-H
=6.1 Hz, 130-H), 1.80 (m, 4 H, 120-H);
13
C NMR (CDCl
3
)d(ppm) 162.4 (CO), 160.7 (C70), 153.9
(C20or C8a’), 153.8 (C8a’ or C20), 150.1 (C
para
), 148.4
(C2/C6), 145.2 (C
ipso
), 139.7 (C40), 139.6 (C4), 128.8
(C
ortho
), 124.7 (C
meta
), 126.6 (C3/C5), 119.7 (C4a’), 119.4
(C60), 115.4 (C30), 70.9 (C140or C150), 70.3 (C150or C140),
68.3 (C130), 53.9 (C90), 47.7 (C110), 29.6 (C120);
15
NNMR
(CDCl
3
)d(ppm) -78.8 (N80), -246.5 (
1
J
NH
=91.8 Hz,
HNCO); Anal. Calcd for C
47
H
45
N
11
O
13
S
2
3H
2
O: C, 51.78;
H, 4.36; N, 14.13; S, 5.88; Found: C, 51.43; H, 4.36; N,
13.97; S, 5.80; ESI–MS [M ?H] 1036.2.
Nuclear magnetic resonance studies
NMR spectra were recorded at 300 K (9.4 Tesla,
400.13 MHz for
1
H, 100.62 MHz for
13
C and 40.56 MHz for
15
N) with a 5-mm inverse-detection H–X probe equipped
with a z-gradient coil. Chemical shifts (din ppm) are given
from internal solvent CDCl
3
7.26 for
1
H and 77.0 for
13
C and
for
15
N NMR nitromethane was used as external standard.
2D experiments, gs-HMQC (
1
H-
13
C), gs-HMBC (
1
H-
13
C),
gs-HMQC (
1
H-
15
N) and gs-HMBC (
1
H-
15
N), were carried
out with the standard pulse sequences [24] to assign the
1
H,
13
C and
15
N signals. 2D NOESY spectra were acquired in the
phase sensitive mode using mixing time of 850 ms (Ia) and
650 ms (Ia:1).
Determination of the stoichiometry by Job plot titrations
The stoichiometry of the host–guest complexes was
determined by the continuous variation method [25–27].
Both stock solutions of the guests 1–4(3 mM) and
receptors Ia and Ib (3 mM) were prepared in a n/m (% v/v)
mixture of CDCl
3
. To obtain the desired host–guest ratios,
which varied from 0 to 1 (molar fraction of Ia), appropriate
volumes of stock solutions were mixed. In a final volume
equal to 1,000 ll, the sum of guest and host concentrations
is constant (3 mM). From the value of the maximum,
which can be obtained by means of equation X =m/
(m ?n), the stoichiometry of the complex is determined.
A representative curve for complex Ia:1 is shown in Fig. 3.
Quantification of the binding constants K
b
Each NMR titration was carried out at least three times at
300 K in CDCl
3
(Merck S33657, deuterium content
[99.8 %, water content \0.01 %). eVol
Ò
XR hand-held
automated analytical syringes (500 lL, 50 lL) from SGE
Analytical Sciences previously calibrated for CDCl
3
, were
used to perform additions. Host and guest were weighted in
a Metler AE260-Delta Range scale (error ±0.00005 g).
1
H
NMR titrations were used to quantify K
b
values, These
titrations were carried out monitoring the chemical induced
shift (CIS) in one or more host or guest protons as the
Molar fraction Χ (Ia)
0,0 0,2 0,4 0,6 0,8 1,0 1,2
Χ* Δδ
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Fig. 3 Job plot titration for Ia:1 (2 mM)
J Incl Phenom Macrocycl Chem
123

concentration of the formed complex varies upon addition
of one of the components. The CIS for amide protons while
guest solution aliquots were added was monitored. There is
a large number of ways to fit data from a titration [28]but
that consisting in non-linear curve fitting is generally
accepted as the method with the lowest error in the deter-
mination of K
b
values, in comparison to others that employ
approximations to reach a linear relationship between and
K
b
. Binding curves were fitted using Wineqnmr soft-
ware [29]. The quality of the fit was estimated using the
merit-function shown in Eq. 1where w
i
is the weight
attributed to observation i (normally data points were
assigned equal weights):
R¼100 Xwidobs dcalc
ðÞ
2
hi
=Xwidobs
ðÞ
2
no
1=2
ð1Þ
the basic equation for this kind of titrations is represented
in Eq. 2, showing the relationship between chemical shifts
(d), concentrations of host H, guest G and complex C, and
the binding constant K
b
=[HG]/([H][G]). This equation is
valid only for 1:1 stoichiometry.
Dd ¼dHG HG½=H½
o
 ð2Þ
Crystal structure determination
Single crystals of C
49
H
51
N
11
O
14
S
3
(Ib) were grown from
dimethylsulfoxide-water. A suitable crystal was selected
and mounted on a glass fibre with Fromblin oil on a
Xcalibur Gemini diffractometer with a Ruby CCD area
detector. The crystal was kept at 100.15 K during data
collection. Using Olex2 [30], the structure was solved with
the ShelXS [31] structure solution program using direct
methods and refined with the ShelXL [31] refinement
package using least squares minimization.
Crystal data for C
49
H
51
N
11
O
14
S
3
(M =1114.18): triclinic,
space group P-1 (No. 2), a =11.0495(3) A
˚,b =12.5732(4) A
˚,
c=18.6966(5) A
˚,a=85.817(2)°,b=79.788(2)°,
c=84.750(2)°,V =2541.34(12) A
˚
3
,Z =2, T =100.15 K,
l(Cu Ka)=2.007 mm
-1
,Dcalc =1.456 mg/mm
3
, 45532
reflections measured (7.072 B2HB124.114), 7955 unique
(R
int
=0.0271) which were used in all calculations. The final
R
1
was 0.0347 (I [2r(I)) and wR
2
was 0.0991 (all data).
CCDC reference number is CCDC-951726.
Molecular modeling
MacroModel v.8.1, with the GB/SA model for chloroform
was used to perform the molecular simulations of hosts,
guests and complexes [32]. All calculations were achieved
with Montecarlo (MC) conformational analyses [33].
Minimization is carried out using Polak-Ribiere Conjugate
Gradient (PRCG) optimizer [34,35] as implemented in the
program version, the energy gradient was chosen as the
convergence criteria with a value of 0.05, and at least 2,000
iterations. All MC calculations were performed with
Montecarlo multiple minimum (MCMM) method, and the
variables were torsion angles, molecule coordinates or
both. The minimization method was PRCG with the same
characteristics described above. In a typical MC run a
MCMM is never performed with less than 8,000 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 and
4A
˚respectively. These calculations were carried out with
the AMBER* force field [36] as implemented in the ver-
sion of the program.
Results and discussion
Receptors studies
Synthesis
Macrocycles Ia and Ib were synthesized according to
Scheme 1, starting from 2-amino-7-methyl-1,8-naphthyridine
(5)[22], which was first acetylated to yield 6and later its
methyl group oxidized to afford the corresponding aldehyde 7
[23]. Condensation of two equivalents of 7with 4,7,10-tri-
oxatridecane-1,13-diamine in methanol and subsequent
reduction of the imine groups with sodium borohydride gave
N,N’-(7,70-(6,9,12-trioxa-2,16-diazaheptadecane-1,17-diyl)-
bis(1,8-naphthyridine-2,7-diyl))diacetamide (8). The crude
product was treated first with 4-nitrobenzenesulfonyl chloride
(p-nosyl chloride, NsCl) and after with hydrochloric acid in
dioxane-water to yield (10). Reaction of 10 with isophthaloyl
dichloride or 2,6-pyridinedicarbonyl dichloride using trieth-
ylamine and chloroform in high dilution conditions gave the
macrocyclic receptors Ia and Ib, respectively.
Nuclear magnetic resonance studies
A complete characterization of the macrocycles was car-
ried out by
1
H,
13
C and
15
N NMR spectroscopy in CDCl
3
as solvent. For symmetry reasons, besides the isochronous
atoms in the central bisamido ring, benzene and pyridine
(3-H/5-H, C2/C6 and C3/C5), these molecules show
equivalent naphthyridinyl and methylene groups. Full
assignment of protons and carbons was achieved by ana-
lysis of the chemical shift values, multiplicity of the signals
and the coupling constants magnitude, as well as gs-COSY,
gs-HMQC and gs-HMBC bidimensional experiments. The
15
N-NMR chemical shifts were assigned using gs-HMQC
J Incl Phenom Macrocycl Chem
123

and gs-HMBC (
1
H-
15
N) correlation experiments, but only
N10at -109.5 ppm and N8‘at -79.5 ppm in receptor Ia,
and N80at -78.8 ppm and the NHCO at -246.5 ppm
(
1
J
NH
=91.8 Hz) in receptor Ib, were detected (See
Electronic supplementary material).
The NH amide protons appear at 9.57 ppm in Ia and
11.24 ppm in Ib, the more deshielded value encountered in
the macrocycle containing the 2,6-bisamidopyridine central
core confirming that these protons are involved in an
intramolecular hydrogen bond with the pyridine nitrogen
atom, just as in the open receptors previously studied in our
group [13]. Figures 4and 5show the
1
H NMR spectra of
both macrocycles. The protons of the benzene or pyridine
rings are easy to identify by integration and signal multi-
plicity. In Ia, the 1-H proton appears as a broad singlet at
8.62 ppm and the 4-H as a triplet, due to coupling with the
N N
N N
NN
OOO
HH
NH2
N
N
H3C
5
Ac2O
110oCN
NH3C
6
SeO2
50oC
N
H
N
NC
O
H
H2NNH2
OOO
1) MeOH/ RT
2) NaBH47
N
H
O
CH3
O
CH3
NH HN
OH3C
O
CH3
p-NsCl/ Et3N
N
N
N
N
N
N
O
O
O
Ns
Ns
NH2
NH2
+X
OO
Cl Cl Ia
8
X= C
Ib
Ia
X=N
10
Et3N/CHCl3
High dilution
N
N
N
N
N
N
O
O
O
Ns
Ns
N
H
H
N
O
CH3
O
CH3
1,4-dioxane/HCl/H2O
90 °C9
Scheme 1 Synthetic procedure used to obtain macrocycles Ia and Ib
J Incl Phenom Macrocycl Chem
123

C O
HN
N
N
N S
O
O
NO
2
OO
7.77
8.37
8.62
3.45 3.45 3.37 1.75
3.54
4.78
8.04 8.39
8.71
8.26
8.16
7.58
9.57
Fig. 4
1
H NMR spectrum of macrocycle Ia in CDCl
3
(15 mM)
N C O
HN
N
N
N S
O
O
NO
2
OO
8.25
8.64
3.46 3.46 3.42 1.80
3.65
4.93
8.16 8.43
8.88
8.28
8.17
7.57
11.24
Fig. 5
1
H NMR spectrum of macrocycle Ib in CDCl
3
(15 mM)
J Incl Phenom Macrocycl Chem
123

magnetically equivalent 3-H and 5-H with a coupling
constant of 7.8 Hz. The doublet signal at 8.37 ppm corre-
sponds to the 3-H and 5-H protons. In Ib, the signals of
equivalent pyridine protons 3-H and 5-H appear as a
doublet with a coupling constant of 7.7 Hz with 4-H at
8.25 ppm. In both receptors,
1
H-NMR chemical shifts of
the naphthyridine unit follow the order: 30-H [40-H [50-
H[60-H and appear as doublet signals in the range of
7.57–8.88 ppm with
3
J(30-H, 40-H) of 8.8. Hz and
3
J(50-H,
60-H) of 8.2 Hz.
For the methylene groups of the ether chain, the most
deshielded signals correspond to 90-H (singlet) and 110-H
(triplet) protons because their proximity to the electron-
withdrawing sulfonamide group. The values of chemical
shifts follow the order: 90-H [110-H [140-H =150-
H[130-H [120-H.
The 2D NOESY experiment carried out using a 15 mM
solution of host Ia in CDCl
3
shed some light on the spatial
arrangement of the p-nosyl groups around the cavity
(Fig. 6). On the basis of the following correlations, ortho-
(a)
(b)
Fig. 6 2D NOESY spectrum of
Ia in CDCl
3
:aortho-H with 60-
H, 90-H and 110-H correlations
benlarged region showing the
ortho-H/60-H correlation
J Incl Phenom Macrocycl Chem
123

H/90-H, ortho- H/60-H, and ortho-H/11
0-H, we concluded
that the p-nosyl groups are pointing upwards towards the
HB (benzenedicarboxamide/naphthyridines) recognition
site of the macrocycle.
Molecular modeling
The minimum energy conformations of macrocycles Ia and
Ib were obtained from Monte Carlo conformational sear-
ches with AMBER force field (Fig. 7). In Ia, the nitro
group of the two p-nosyl pendant arms is hydrogen bonded
to each NH of the isophthaloyl amides forming a small
aromatic box, in agreement with the experimental evidence
found in the NOESY experiment discussed above (Fig. 6).
In Ib these hydrogen bonds are also shared with the central
pyridine nitrogen. The intramolecular HB network main-
tains the p-pstacking between the p-nosyl groups and the
naphthyridine units.
X ray crystal structural analysis
We were able to obtain crystals of macrocycle 1b of suitable
quality for single crystal Xray diffraction from dimethylsulf-
oxide-water and it crystallizes in the triclinic space group P-1
and the asymmetric unit contains the macrocycle (C
47
H
45
N
11
O
13
S
2
) with a molecule of DMSO (Fig. 8).
Fig. 7 Minimum energy
conformations of macrocycles
Ia and Ib
Fig. 8 X-ray crystal structure
of Ib with heteroatom labeling,
showing the H bonding
interactions
J Incl Phenom Macrocycl Chem
123

The DMSO molecule is chelated by Hunter style [37]
bifurcated hydrogen bonds NHO to the biscarboxamides
and by several longer contacts between the DMSO methyl
groups C61 and C63 and ether chain oxygens and naph-
thyridine nitrogens of the macrocycle (Fig. 8).
These and other close contacts are tabulated in
Table 1. Internal p-pstacking between one of the nosyl
groups and a naphthyridine unit [closest atomic contact
N38-C48 of 3.08 A
˚], and the 2,6-pyridinebiscarboxamide
and a naphthyridine [closets atomic contact C4-C18 of
Table 1 Hydrogen bonding (A
˚
and °) with esds (except fixed
and riding H)
D-HA d(D-H) d(HA) d(DA) \(DHA)
Contacts from NHs of the pyridine carboxamides
N1-H1O6 0.81(2) 2.07(3) 2.852(2) 164(2)
N9-H9O62 0.81(2) 2.01(2) 2.803(2) 164(2)
Intra-contacts from NHs of the pyridine carboxamides to the pyridine N
N1-H1N3 0.81(2) 2.36(2) 2.704(2) 107(2)
N9-H9N3 0.81(2) 2.30(2) 2.695(2) 110.6(19)
Contacts from methyls C61 and C63 to the ether chain
C63-H63BO25 0.98 3.10 3.561(2) 110.4
C63-H63BO31A_b 0.98 3.00 3.824(4) 142.3
C61-H61CO28_a 0.98 2.39 3.296(10) 152.8
C61-H61CO28A_b 0.98 2.84 3.722(6) 149.5
C63-H63BO28_a 0.98 2.59 3.452(13) 146.2
C63-H63BO28A_b 0.98 2.78 3.671(8) 151.6
Contacts from methyls C61 and C63 to naphthyridine Ns
C63-H63AN11 0.98 2.92 3.626(2) 129.7
C63-H63AN13 0.98 2.71 3.683(2) 172.2
C63-H63CN38 0.98 2.87 3.821(2) 164.0
C63-H63CN40 0.98 2.77 3.641(2) 147.7
Fig. 9
1
H NMR spectra of the macrocycle Ia (10
-3
M in CDCl
3
) at the starting point of the titration (bottom) and when 1.2 equiv of guest 1have
been added (top)
J Incl Phenom Macrocycl Chem
123

3.33 A
˚] are also observed in the solid state structure of
macrocycle Ib.
Host–guest properties
Binding constant quantification
1
H NMR titrations in CDCl
3
at 300 K have been performed
to quantify the interactions between receptors Ia and Ib
and guests 1–4. Before the quantification of the binding
constants, the stoichiometry of the complexes was deter-
mined by using the method of continuous variation to
generate Job plots and 1:1 stoichiometries for all com-
plexes were obtained.
The host–guest binding constants (K
b
) have been mea-
sured using the CIS on the NH signal of the amide groups
of the receptors. In Figs. 9and 10 we present the
1
HNMR
spectra in the titrations of complexes Ia:1and Ia:2. The
complete set of titrations for all complexes can be found in
the Electronic supplementary material.
In macrocycle Ia, the aromatic protons 3-H and 5-H of
the isophthaloyl ring are shielded about 0.1 ppm upon
complexation of 1, whereas such effect is not observed
with the other guests (c. f. Ia:2in Fig. 10), indicating a
conformational change of Ia. Another interesting feature is
the fact that 90-H proton signals broaden and appear at
around 4.7 ppm in the region of 3a-H and 6a-H of biotin
methyl ester 1. Aliphatic protons of Ia and also the C5
chain protons of biotin show some deshielding due to their
exposure to the rich penvironment of the inner cavity of
the macrocycle. In the 2D NOESY spectra of the Ia:1
complex, the 90-H protons are correlated with Hx and 4-H
protons of the biotin ring, suggesting that the ureido part of
the latter sits in the central cavity of the macrocycle, and
ortho-H of the p-nosyl groups show correlation with 60-H
of the naphthyridine.
The experimental binding constants K
b
(M
-1
)of
receptors Ia and Ib with the four guests 1–4are gathered in
Table 2. Examination of the data indicates that the stability
constants are moderate ranging over 3 orders of magnitude
from Ia:1(1.8 910
4
)toIb:4(7.0 910). For all guests, Ia
Fig. 10
1
H NMR spectra of the macrocycle Ia (7 910
-4
M in CDCl
3
) at the starting point of the titration (bottom) and when 5 equiv of guest 2
have been added (top)
Table 2 Experimental binding constants K
b
(M
-1
) at 300 K for the
complexes
Guest Ia Ib
118,000 ±1,600 230 ±30
26,000 ±300 162 ±40
38,000 ±600 120 ±20
4800 ±70 70 ±7
J Incl Phenom Macrocycl Chem
123

has proved to be a better receptor than Ib by 2 orders of
magnitude, and no significant improvement has been found
with respect to known open receptors [11–13].
Molecular modeling
In the same manner as we did for the macrocyclic receptors,
all complexes were modeled using Monte Carlo conforma-
tional search with the AMBER force field. The calculated
enthalpies [E
interaction
=E
min
(complex)–E
min
(receptor)–
E
min
(guest), kJ mol
-1
] pointed out the following relative
stability order, Ia:1(-57.7) [Ia:3-41.6) [Ia:2-36.6)
and Ib:1(-49.7) [Ib:3(-40.0) *Ib:2(-39.8), which
agree with the experimental K
b
values (free energies DG)of
Table 2only in a qualitative way, most probably because
entropy variation is not the same for all cases.
The calculated enthalpies obtained for complexes
involving barbital Ia:4(-58.3) and Ib:4(-82.6) cannot be
compared with the previous ones. Its complexation mode is
quite different, involving the carbonyl amide group close to
the ethyl substituent and not the ureido motif.
We reproduce in Fig. 11 the geometries obtained for
complexes Ia:1and Ib:1, showing the hydrogen bonds that
held guest 1into the macrocycles and the two aromatic p-
nosyl arms maintaining the p-pstacking with the naph-
thyridine rings. Interesting to note that in Ia:1complex,
guest 1presents a folded side chain, the carboxyl group of
the ester forming an additional intramolecular HB with one
of the ureido NHs. The increased preorganization via
intramolecular HB in host Ib decreases the flexibility
needed to accommodate the guests inside the cavity, and
would account for the lower binding constants obtained for
the complexes involving this macrocycle.
Conclusions
We report here the design and synthesis of two new mac-
rocyclic receptors Ia and Ib and their molecular recogni-
tion studies for methyl biotin ester, 2-imidazolidinone,
trimethylene urea and barbital. The binding constant values
determined by
1
H NMR titration and the best results are
obtained for methyl biotin ester and the isophthaloyl bi-
samido naphthyridine Ia. Molecular mechanics calcula-
tions account for the experimental binding constants only
in a qualitative way due to the fact that these modeling
studies do not take into account entropic factors or solva-
tion-desolvation effects [38,39]. In our previous papers,
the entropy changes were the same or rather close for all
series of complexes and good results were obtained in
discussing the geometries of the energetic minima.
Acknowledgments This work is supported by Grant CTQ2010-
16122 (Ministerio de Ciencia e Innovacio
´n, MICINN, Spain).
References
1. Monographs in Supramolecular Chemistry. 11 vols. Royal Soci-
ety of Chemistry, London (2006–2013)
2. Livnah, O., Bayer, E.A., Wilchek, M., Sussman, J.L.: Three-
dimensional structures of avidin and the avidin-biotin complex.
Proc. Natl. Acad. Sci. USA. 90, 5076–5080 (1993)
3. Young, T., Abel, R., Kim, B., Berne, B.J., Friesner, R.A.: Motifs
for molecular recognition exploiting hydrophobic enclosure in
protein-ligand binding. Proc. Natl. Acad. Sci. USA. 104, 808–813
(2007)
4. Adrian Jr, J.C., Wilcox, C.S.: Chemistry of synthetic receptors
and functional group arrays. 10. Orderly functional group dyads.
Fig. 11 Minimum energy conformations of complexes between Ia and Ib with (?)-biotin methyl ester (1)
J Incl Phenom Macrocycl Chem
123

Recognition of biotin and adenine derivatives by a new synthetic
host. J. Am. Chem. Soc. 111, 8055–8057 (1989)
5. Goswami, S., Dey, S.: Directed molecular recognition: design
and synthesis of neutral receptors for biotin to bind both its
functional groups. J. Org. Chem. 71, 7280–7287 (2006)
6. Claramunt, R.M., Herranz, F., Santa Marı
´a, M.D., Pinilla, E.,
Torres, M.R., Elguero, J.: Molecular recognition of biotin, bar-
bital and tolbutamide with new synthetic receptors. Tetrahedron
61, 5089–5100 (2005)
7. Ghosh, K., Sen, T.: A benzothiazole-based simple receptor in
fluorescence sensing of biotin ester and urea. Tetrahedron Lett.
50, 4096–4100 (2009)
8. Murray, T.J., Zimmerman, S.C.: 7- Amido-1,8-naphthyridines as
hydrogen bonding units for the complexation of guanine deriv-
atives: the role of 2-alkoxyl groups in decreasing binding affinity.
Tetrahedron Lett. 42, 7627–7630 (1995)
9. Mayer, M.F., Nakashima, S., Zimmerman, S.C.: Synthesis of a
soluble ureido-naphthyridine oligomer that self-associates via
eight contiguous hydrogen bonds. Org. Lett. 7, 3005–3008 (2005)
10. Hamilton, A.D., Pant, N.: Nucleotide base recognition: ditopic
binding of guanine to a macrocyclic receptor containing naph-
thyridine and naphthalene units. Chem. Commun. 12, 765–766
(1988)
11. Claramunt, R.M., Herranz, F., Santa Marı
´a, M.D., Jaime, C., de
Federico, M., Elguero, J.: Towards the design of host–guest
complexes: biotin and urea derivatives versus artificial receptors.
Biosens. Bioelectron. 20, 1242–1249 (2004)
12. Herranz, F., Santa Marı
´a, M.D., Claramunt, R.M.: Molecular
recognition: improved binding of biotin derivatives with synthetic
receptors. J. Org. Chem. 71, 2944–2951 (2006)
13. Santa Marı
´a, D., Farra
´n, M.A., Garcı
´a, M.A., Pinilla, E., Torres,
M.R., Elguero, J., Claramunt, R.M.: Synthetic hosts for molecular
recognition of ureas. J. Org. Chem. 76, 6780–6788 (2011)
14. Ghosh, K., Sen, T.: Naphthyridine-based receptors for flurometric
detection of urea and biotin. J. Incl. Phenom. Macrocycl. Chem.
67, 271–280 (2010)
15. Petitjean, A., Cuccia, L.A., Lehn, J.M., Nierengarten, H., Sch-
mutz, M.: Cation-promoted hierarchical formation of supramo-
lecular assemblies of self-organized helical molecular
components. Angew. Chem. Int. Ed. 41, 1195–1198 (2002)
16. Katz, J.L., Geller, B.J., Foster, P.D.: Oxacalixarenes and oxa-
cyclophanes containing 1,8-naphthyridines: a new class of
molecular tweezers with concave-surface functionality. Chem.
Comm. 10, 1026–1028 (2007)
17. Petitjean, A., Cuccia, L.A., Schmutz, M., Lehn, J.M.: Naph-
thyridine-based helical foldamers and macrocycles: synthesis,
cation binding, and supramolecular assemblies. J. Org. Chem. 73,
2481–2495 (2008)
18. Sijbesma, R.P., Beijer, F.H., Brunsveld, L., Folmer, B.J.B., Hir-
schberg, J.H.K.K., Lange, R.F.M., Lowe, J.K.L., Meijer, E.W.:
Reversible polymers formed from self-complementary monomers
using quadruple hydrogen bonding. Science 278, 1601–1604
(1997)
19. Park, T., Zimmerman, S.C.: Formation of a miscible supramo-
lecular polymer blend through self-assembly mediated by a
quadruply hydrogen-bonded heterocomplex. J. Am. Chem. Soc.
128, 11582–11590 (2006)
20. Mazik, M., Sicking, W.: Molecular recognition of carbohydrates
by artificial receptors: systematic studies towards recognition
motifs for carbohydrates. Chem. Eur. J. 7, 664–670 (2001)
21. Mazik, M., Cavga, H.: An acyclic aminonaphthyridine-based
receptor for carbohydrate recognition: binding studies in com-
petitive solvents. Eur. J. Org. Chem. 3633–3638 (2007)
22. Brown, E.V.: 1,8-Naphthyridines. I. Derivatives of 2-and
4-methyl-1,8-naphthyridines. J. Org. Chem. 30, 1607–1610 (1965)
23. Goswami, S., Mukherjee, R., Mukherjee, R., Jana, S., Maity,
A.C., Adak, A.K.: Simple and efficient synthesis of 2,7-difunc-
tionalized-1,8-naphthyridines. Molecules 10, 929–936 (2005)
24. Berger, S., Braun, S.: 200 and More NMR Experiments. Wiley,
Weinheim (2004)
25. Job, P.: Formation and stability of inorganic complexes in solu-
tion. Ann. Chim. App. 9, 113–203 (1928)
26. MacCarthy, P., Hill, Z.D.: Novel approach to Job’s method.
J. Chem. Ed. 63, 162–167 (1986)
27. Renny, J.S., Tomasevich, L.L., Tallmadge, E.H., Collum, D.B.:
Method of continuous variations: Applications of Job plots to the
study of molecular associations in organometallic chemistry.
Angew. Chem. Int. Ed. 52, 2–18 (2013)
28. Thordarson, P.: Determining association constants from titration
experiments in supramolecular chemistry. Chem. Soc. Rev. 40,
1305–1323 (2011)
29. Hynes, M.J.: EQNMR: a computer program for the calculation of
stability constants from nuclear magnetic resonance chemical
shift data. J. Chem. Soc. Dalton Trans. 311–312 (1993)
30. Dolomanov, O.V., Bourhis, L.J., Gildea, R.J., Howard, J.A.K.,
Puschmann, H.: OLEX2: a complete structure solution, refine-
ment and analysis program. J. Appl. Cryst. 42, 339–341 (2009)
31. Sheldrick, G.M.: A short history of SHELX. Acta Cryst. A64,
112–122 (2008)
32. Mohamadi, F., Richards, N.G.J., Guida, W.C., Liskamp, R.,
Lipton, M., Caufield, C., Chang, G., Hendrickson, T., Still, W.C.:
MacroModel-an integrated software system for modeling organic
and bioorganic molecules using molecular mechanics. J. Comp.
Chem. 11, 440–467 (1990)
33. Chang, G., Guida, W.C., Still, W.C.: An internal-coordinate
Monte Carlo method for searching conformational space. J. Am.
Chem. Soc. 111, 4379–4386 (1989)
34. Polak, E.: Computational methods in optimization. Academic
Press, New York (1971)
35. Brodlie, K.W. In: The state of the art in numerical analysis.
Jacobs, D.A.H. (ed.). Academic Press, London (1977)
36. Weiner, P.K., Kollmann, P.A.: AMBER: assisted model building
with energy refinement. A general program for modeling mole-
cules and their interactions. J. Comput. Chem. 2, 287–303 (1981)
37. Carver, F.J., Hunter, C.A., Shannon, R.J.: Directed macrocycli-
zation reactions. J. Chem. Soc. Chem. Comm. 10, 1277–1280
(1994)
38. Cohen, N.C. (ed.): Guidebook on molecular modeling in drug
design. Academic Press Ltd, London (1996)
39. Lewars, E.G.: Computational chemistry. Introduction to the the-
ory and applications of molecular and quantum mechanics.
Springer, New York (2011)
J Incl Phenom Macrocycl Chem
123