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

Article abstract of DOI:10.1007/s10847-014-0433-3

Two macrocyclic hosts containing benzenedicarboxamide or pyridinedicarboxamide moieties and two 1,8-naphthyridine units linked by a crown ether like chain, have been synthesized and fully characterized by multinuclear 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 (+)-biotin methyl ester, was achieved by means of ¹H NMR titrations.

Full text of DOI:10.1007/s10847-014-0433-3

J Incl Phenom Macrocycl Chem (2015) 81:57–69
DOI 10.1007/s10847-014-0433-3
ORIGINAL ARTICLE
New macrocyclic compounds with naphthyridine units
for molecular recognition studies of biotin and urea derivatives
´
•
M. Angeles Farran Dolores Santa Marıa
•
´
´
´
•
•
´
M. Angeles Garcıa Rosa M. Claramunt
Guy J. Clarkson
Received: 28 March 2014 / Accepted: 26 June 2014 / Published online: 11 July 2014
Ó Springer 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 1H NMR
titrations.
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
Keywords Bisnaphthyridineamides ꢀ
Benzenedicarboxamides ꢀ Pyridinedicarboxamides ꢀ Host–
guest ꢀ Binding constants ꢀ MM calculations
via hydrogen bond (HB) with avidin (Kb = 2.5 9 1015 M-1
)
or streptavidin (Kb = 1.7 9 1015 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].
Introduction
Macrocycles research has continually increased since Pe-
dersen reported the synthesis of crown ethers in 1967. The
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.
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.
´
´
´
´
´
M. A. Farran (&) ꢀ D. Santa Marıa ꢀ M. A. Garcıa ꢀ
R. M. Claramunt (&)
´
´
´
Departamento de Quımica Organica y Bio-Organica, 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

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J Incl Phenom Macrocycl Chem (2015) 81:57–69
4
On the other hand, the introduction of the 1,8-naph-
5
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].
3
2
6
O
O
X
1
N
N
3'
2'
H
H
4'
1'
N
N
4a'
8a'
5'
6'
8'
N
N
7'
10'
N
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.
9'
Y
Y
N
11'
12'
15'
O
O
O
13'
14'
Ia X=C Y= p-nosyl
Ib X=N Y= p-nosyl
Y =
O2S ipso
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).
para NO2
ortho
meta
Fig. 1 Macrocyclic receptors Ia and Ib
infused into the mass spectrometer at a flow rate of
5 lL min-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,
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
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 NaBH4.
´
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
123

J Incl Phenom Macrocycl Chem (2015) 81:57–69
59
Fig. 2 Selected guests 1–4
O
O
O
O
H
O
H
O
H
N
H
N
N
N
H
H
H
H
N
N
N
N
H4
(CH2)4CO2CH3
Hx
H5C2
C2H5
S
Hy
1
2
3
4
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 8 as
a brown hygroscopic oil (1.78 g) that was used without
further purification.
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; 1H NMR (CDCl3) d (ppm) 8.26 (d, 4 H,
3J = 8.8 Hz, meta-H), 7.98 (d, 4 H, 3J = 8.8 Hz, ortho-H),
7.92 (d, 2 H, 3J = 8.2, 50-H), 7.82 (d, 2 H, 3J = 8.8 Hz, 40-
Crude compound 8 was 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 9 were obtained: m.p. 142–145 °C; 1H NMR (CDCl3) d
3
H,), 7.37 (d, 2 H, J = 8.2 Hz, 60-H,), 6.75 (d, 2 H,
3J = 8.8 Hz, 30-H), 5.20 (s, 4 H, NH2), 4.63 (s, 4 H, Ar-
CH2NNs), 3.54 (t, 4 H, 3J = 7.5 Hz, OCH2CH2CH2N),
3
3.42 (m, 8 H, OCH2CH2O), 3.32 (4 H, t, J = 5.9 Hz,
OCH2CH2CH2N), 1.87-1.49 (m, 4 H, OCH2CH2CH2N);
13C NMR (CDCl3) d (ppm) 160.0 (C70), 159.3 (C20), 155.8
(C8a’), 150.1 (Cpara), 145.8 (Cipso), 138.3 (C40), 137.4
(C50), 128.6 (Cortho), 124.2 (Cmeta), 117.4 (C60), 116.5
(C4a’), 112.6 (C30), 70.6 and 70.2 (OCH2CH2O), 68.1
(OCH2CH2CH2N), 54.4 (Ar-CH2NNs), 47.1 (OCH2CH2-
CH2N), 28.6 (OCH2CH2CH2N). Anal. Calcd for C40H44-
N10O11S2 : 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.
3
(ppm) 8.94 (br s, 2H, CONH), 8.52 (d, 2 H, J = 8.8 Hz,
3
30-H), 8.32 (d, 4 H, J = 8.9 Hz, meta-H), 8.17 (d, 2 H,
3
3J = 8.8 Hz, 40-H), 8.14 (d, 2 H, J = 8.2 Hz, 50-H), 8.05
Macrocycle Ia
3
(d, 4 H, J = 8.8 Hz, ortho-H), 7.63 (d, 2 H, J = 8.2 Hz,
60-H), 4.70 (s, 4 H, Ar-CH2NNs), 3.40 (t, 4H, 3J = 7.5 Hz,
OCH2CH2CH2N), 3.35 (m, 8H, OCH2CH2O), 3.29 (t, 4 H,
3J = 5.9 Hz, OCH2CH2CH2N), 2.27 (s, 6 H, CH3-CONH),
1.87-1.49 (m, 4H, OCH2CH2CH2N); 13C NMR (CDCl3) d
(ppm) 169.7 (CO), 160.7 (C70), 154.2 (C20 or C8a’), 154.1
(C8a’ or C20), 150.1 (Cpara), 145.5 (Cipso), 139.4 (C40),
137.6 (C50), 128.6 (Cortho), 124.5 (Cmeta), 119.8 (C4a’),
119.7 (C60), 115.5 (C30), 70.5 and 70.3 (OCH2CH2O), 67.9
(OCH2CH2CH2N), 54.4 (Ar-CH2NNs), 46.8 (OCH2CH2-
CH2N), 28.5 (OCH2CH2CH2N), 25.1 (CH3CO). Anal.
Calcd for C44H48N10O13S2.H2O: 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.
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,
1
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)
21 %): m.p. [ 250 °C (dec.); H NMR (CDCl3) d (ppm)
9.57 (br. s, 2 H, CONH), 8.71 (d, 2 H, 3J4 -H = 8.8 Hz, 30-H),
0
8.62 (s, 1 H, 1-H), 8.39 (d, 4 H, 3Jortho-H = 8.8 Hz, meta-H),
8.37 (d, 2H, 3J4-H = 7.8 Hz, 3-H/5-H), 8.26 (d, 2 H, 40-H),
8.16 (d, 2 H, 3J6 -H = 8.2 Hz, 50-H), 8.04 (d, 4 H, ortho-H),
0
To a solution of 0.53 g (0.95 mmol) of 9 in 10 mL of
dioxane and 2 mL of water, 1 mL of 4 M HCl in dioxane
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, 3J12 -H = 7.5 Hz, 110-H), 3.45 (m, 8 H, 140-H, 150-H),
0
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
3.37 (t, 4 H, 3J12 -H = 5.9 Hz, 130-H), 1.75 (m, 4 H, 120-H);
13C NMR (CDCl3) d (ppm) 164.8 (CO), 160.6 (C70), 154.0
(C20, C8a’), 150.1 (Cpara), 145.4 (Cipso), 139.6 (C40), 137.5
(C50), 134.0 (C2/C6), 133.2 (C3/C5), 130.4 (C4), 128.5
(Cortho), 124.6 (Cmeta), 124.3 (v. br. s, C1), 119.8 (C4a’),
119.7 (C60), 115.2 (C30), 70.6 (C140 or C150), 70.2 (C150 or
C140), 68.1 (C130), 53.8 (C90), 47.0 (C110), 28.9 (C120); 15N
NMR (CDCl3) d (ppm) -79.5 (N80), -109.5 (N10); Anal.
Calcd for C48H46N10O13S2 H2O: 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.
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0
Macrocycle Ib
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Molar fraction Χ (Ia)
Fig. 3 Job plot titration for Ia:1 (2 mM)
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
for 15N NMR nitromethane was used as external standard.
2D experiments, gs-HMQC (1H-13C), gs-HMBC (1H-13C),
gs-HMQC (1H-15N) and gs-HMBC (1H-15N), were carried
out with the standard pulse sequences [24] to assign the 1H,
13C and 15N signals. 2D NOESY spectra were acquired in the
phase sensitive mode using mixing time of 850 ms (Ia) and
650 ms (Ia:1).
°
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
Determination of the stoichiometry by Job plot titrations
(100 mg, 10.5 %): m.p. C 250 °C (dec); 1H NMR (CDCl3)
3
0
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 CDCl3. 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.
d (ppm) 11.24 (br. s, 2 H, CONH), 8.88 (d, 2 H, J4 -
= 8.8 Hz, 30-H), 8.64 (d, 2 H, 3J4-H = 7.7 Hz, 3-H/5-H),
H
8.43 (m, 4 H, meta-H), 8.28 (d, 2 H, 40-H), 8.25 (t, 1 H,
3
4-H), 8.17 (d, 2 H, J6 -H = 8.2 Hz, 50-H), 8.16 (m, 4 H,
0
ortho-H), 7.57 (d, 2 H, 60-H), 4.93 (s, 4 H, 90-H), 3.65 (t, 4
3
H, J12 -H = 7.7 Hz, 110-H), 3.46 (m, 8 H, 140-H, 150-H),
0
3.42 (t, 4 H, 3J12 -H = 6.1 Hz, 130-H), 1.80 (m, 4 H, 120-H);
0
13C NMR (CDCl3) d (ppm) 162.4 (CO), 160.7 (C70), 153.9
(C20 or C8a’), 153.8 (C8a’ or C20), 150.1 (Cpara), 148.4
(C2/C6), 145.2 (Cipso), 139.7 (C40), 139.6 (C4), 128.8
(Cortho), 124.7 (Cmeta), 126.6 (C3/C5), 119.7 (C4a’), 119.4
(C60), 115.4 (C30), 70.9 (C140 or C150), 70.3 (C150 or C140),
68.3 (C130), 53.9 (C90), 47.7 (C110), 29.6 (C120); 15N NMR
(CDCl3) d (ppm) -78.8 (N80), -246.5 (1JNH = 91.8 Hz,
HNCO); Anal. Calcd for C47H45N11O13S2 3H2O: 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.
Quantification of the binding constants Kb
Each NMR titration was carried out at least three times at
300 K in CDCl3 (Merck S33657, deuterium content
[99.8 %, water content \0.01 %). eVolÒ XR hand-held
automated analytical syringes (500 lL, 50 lL) from SGE
Analytical Sciences previously calibrated for CDCl3, were
used to perform additions. Host and guest were weighted in
a Metler AE260-Delta Range scale (error 0.00005 g). 1H
NMR titrations were used to quantify Kb values, These
titrations were carried out monitoring the chemical induced
shift (CIS) in one or more host or guest protons as the
Nuclear magnetic resonance studies
NMR spectra were recorded at 300 K (9.4 Tesla,
400.13 MHz for 1H, 100.62 MHz for 13C and 40.56 MHz for
15N) with a 5-mm inverse-detection H–X probe equipped
with a z-gradient coil. Chemical shifts (d in ppm) are given
from internal solvent CDCl3 7.26 for 1H and 77.0 for 13C and
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
61
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 Kb values, in comparison to others that employ
approximations to reach a linear relationship between and
Kb. Binding curves were fitted using Wineqnmr soft-
ware [29]. The quality of the fit was estimated using the
merit-function shown in Eq. 1 where wi is the weight
attributed to observation i (normally data points were
assigned equal weights):
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
˚
4 A respectively. These calculations were carried out with
nh
i
o
X
X
1=2
2
2
R ¼ 100 ꢁ
wiðdobs ꢂ dcalc
Þ
=
wiðdobsÞ
the AMBER* force field [36] as implemented in the ver-
sion of the program.
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 Kb = [HG]/([H][G]). This equation is
valid only for 1:1 stoichiometry.
Results and discussion
Receptors studies
Synthesis
ꢀ
ꢁ
Dd ¼ dHG ½HGꢃ=½Hꢃo
Crystal structure determination
ð2Þ
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 6 and later its
methyl group oxidized to afford the corresponding aldehyde 7
[23]. Condensation of two equivalents of 7 with 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.
Single crystals of C49H51N11O14S3 (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 C49H51N11O14S3 (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)°,
3
˚
c = 84.750(2)°, V = 2541.34(12) A , Z = 2, T = 100.15 K,
l(Cu Ka) = 2.007 mm-1, Dcalc = 1.456 mg/mm3, 45532
reflections measured (7.072 B 2H B 124.114), 7955 unique
(Rint = 0.0271) which were used in all calculations. The final
R1 was 0.0347 (I [2r(I)) and wR2 was 0.0991 (all data).
CCDC reference number is CCDC-951726.
Nuclear magnetic resonance studies
A complete characterization of the macrocycles was car-
1
ried out by H, 13C and 15N NMR spectroscopy in CDCl3
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
15N-NMR chemical shifts were assigned using gs-HMQC
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
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
Ac2O
O
110oC
NH2
CH3
N
H3C
NH2
N
N
N
N
H3C
H
5
6
CH3
H3C
HN
O
SeO2
50oC
O
H2N
NH
O
O
N
N
O
O
N
N
O
1) MeOH/ RT
2) NaBH4
C
H
N
N
N
CH3
H
7
N
N
H
H
O
O
N
O
O
Ns
N
8
N
N
H
O
CH3
CH3
p-NsCl/ Et3N
O
H
N
N
N
O
Ns
O
N
1,4-dioxane/HCl/H2O
90 °C
9
N
Ns
N
O
N
NH2
High dilution
Et3N/CHCl3
X= C
X=N
Ia
Ib
Cl
Cl
+
O
X
O
O
NH2
N
O
N
Ns
N
10
Scheme 1 Synthetic procedure used to obtain macrocycles Ia and Ib
and gs-HMBC (1H-15N) correlation experiments, but only
N10 at -109.5 ppm and N8‘at -79.5 ppm in receptor Ia,
and N80 at -78.8 ppm and the NHCO at -246.5 ppm
(1JNH = 91.8 Hz) in receptor Ib, were detected (See
Electronic supplementary material).
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
1
group [13]. Figures 4 and 5 show the 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
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
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
63
7.77
8.62
8.37
C
O
HN
9.57
8.71
8.26
N
N
8.16
7.58
4.78
O
N
S
NO2
O
O
O
3.54
8.39
8.04
1.75
3.45 3.45 3.37
Fig. 4 1H NMR spectrum of macrocycle Ia in CDCl3 (15 mM)
8.25
N
8.64
C
O
HN
11.24
8.88
8.28
N
N
8.17
7.57
4.93
3.42
O
S
N
NO2
O
O
O
3.65
8.16 8.43
3.46 3.46
1.80
Fig. 5 1H NMR spectrum of macrocycle Ib in CDCl3 (15 mM)
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
Fig. 6 2D NOESY spectrum of
Ia in CDCl3: a ortho-H with 60-
H, 90-H and 110-H correlations
b enlarged region showing the
ortho-H/60-H correlation
(a)
(b)
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
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 CDCl3 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-
1
8.25 ppm. In both receptors, 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 3J (30-H, 40-H) of 8.8. Hz and 3J (50-H,
60-H) of 8.2 Hz.
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
65
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
H/90-H, ortho- H/60-H, and ortho- H/110-H, we concluded
that the p-nosyl groups are pointing upwards towards the
HB (benzenedicarboxamide/naphthyridines) recognition
site of the macrocycle.
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-p stacking between the p-nosyl groups and the
naphthyridine units.
Molecular modeling
X ray crystal structural analysis
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
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 (C47H45
N11O13S2) with a molecule of DMSO (Fig. 8).
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
Fig. 9 1H NMR spectra of the macrocycle Ia (10-3 M in CDCl3) at the starting point of the titration (bottom) and when 1.2 equiv of guest 1 have
been added (top)
˚
Table 1 Hydrogen bonding (A
and °) with esds (except fixed
and riding H)
D-HꢀꢀꢀA
d(D-H)
d(HꢀꢀꢀA)
d(DꢀꢀꢀA)
\(DHA)
Contacts from NHs of the pyridine carboxamides
N1-H1ꢀꢀꢀO6
0.81(2)
0.81(2)
2.07(3)
2.01(2)
2.852(2)
2.803(2)
164(2)
164(2)
N9-H9ꢀꢀꢀO62
Intra-contacts from NHs of the pyridine carboxamides to the pyridine N
N1-H1ꢀꢀꢀN3
N9-H9ꢀꢀꢀN3
Contacts from methyls C61 and C63 to the ether chain
0.81(2)
0.81(2)
2.36(2)
2.30(2)
2.704(2)
2.695(2)
107(2)
110.6(19)
C63-H63BꢀꢀꢀO25
0.98
0.98
0.98
0.98
0.98
0.98
3.10
3.00
2.39
2.84
2.59
2.78
3.561(2)
3.824(4)
3.296(10)
3.722(6)
3.452(13)
3.671(8)
110.4
142.3
152.8
149.5
146.2
151.6
C63-H63BꢀꢀꢀO31A_b
C61-H61CꢀꢀꢀO28_a
C61-H61CꢀꢀꢀO28A_b
C63-H63BꢀꢀꢀO28_a
C63-H63BꢀꢀꢀO28A_b
Contacts from methyls C61 and C63 to naphthyridine Ns
C63-H63AꢀꢀꢀN11
C63-H63AꢀꢀꢀN13
C63-H63CꢀꢀꢀN38
C63-H63CꢀꢀꢀN40
0.98
0.98
0.98
0.98
2.92
2.71
2.87
2.77
3.626(2)
3.683(2)
3.821(2)
3.641(2)
129.7
172.2
164.0
147.7
The DMSO molecule is chelated by Hunter style [37]
bifurcated hydrogen bonds NHꢀꢀꢀO 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-p stacking 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
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J Incl Phenom Macrocycl Chem (2015) 81:57–69
67
Fig. 10 1H NMR spectra of the macrocycle Ia (7 9 10-4 M in CDCl3) at the starting point of the titration (bottom) and when 5 equiv of guest 2
have been added (top)
˚
3.33 A] are also observed in the solid state structure of
Table 2 Experimental binding constants Kb (M-1) at 300 K for the
complexes
macrocycle Ib.
Guest
Ia
Ib
Host–guest properties
1
2
3
4
18,000 1,600
6,000 300
8,000 600
800 70
230 30
162 40
120 20
Binding constant quantification
1H NMR titrations in CDCl3 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.
70
7
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 p environment 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 Kb (M-1) of
receptors Ia and Ib with the four guests 1–4 are 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 9 104) to Ib:4 (7.0 9 10). For all guests, Ia
The host–guest binding constants (Kb) have been mea-
sured using the CIS on the NH signal of the amide groups
of the receptors. In Figs. 9 and 10 we present the 1H NMR
spectra in the titrations of complexes Ia:1 and 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: 2 in Fig. 10), indicating a
conformational change of Ia. Another interesting feature is
the fact that 90-H proton signals broaden and appear at
123

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J Incl Phenom Macrocycl Chem (2015) 81:57–69
Fig. 11 Minimum energy conformations of complexes between Ia and Ib with (?)-biotin methyl ester (1)
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].
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
Molecular modeling
1
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 [Einteraction = Emin (complex)–Emin (receptor)–
Emin (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 Kb values (free energies DG) of
Table 2 only in a qualitative way, most probably because
entropy variation is not the same for all cases.
determined by 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.
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.
Acknowledgments This work is supported by Grant CTQ2010-
´
16122 (Ministerio de Ciencia e Innovacion, MICINN, Spain).
We reproduce in Fig. 11 the geometries obtained for
complexes Ia:1 and Ib:1, showing the hydrogen bonds that
held guest 1 into the macrocycles and the two aromatic p-
nosyl arms maintaining the p-p stacking with the naph-
thyridine rings. Interesting to note that in Ia:1 complex,
guest 1 presents 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.
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