1H, 13C NMR studies of new 3-aminophenol isomers linked to pyridinium salts

Article abstract of DOI:10.1002/mrc.4025

¹H and ¹³C NMR spectroscopic data of 20 new non-symmetrical compounds were assigned by a combination of 1D and 2D NMR experiments (DEPT, HSQC, and HMBC). These compounds contain a 4-(N,N-dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium moiety and a 3-nitro-, 3-amino-, or 3-hydroxyphenyl ring, linked by p-xylene, 4,4-dimethylbiphenyl, 1,2-bis(p-tolyl)ethane, or 1,4-bis(p-tolyl)butane.

Full text of DOI:10.1002/mrc.4025

MRC letters
Received: 3 September 2013
Revised: 30 September 2013
Accepted: 5 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4025
1
13
H, C NMR studies of new 3-aminophenol
isomers linked to pyridinium salts
Santiago Schiaffino-Ortega, Antonio Espinosa, Miguel A. Gallo,
L. Carlota López-Cara* and Antonio Entrena*
1H and 13C NMR spectroscopic data of 20 new non-symmetrical compounds were assigned by a combination of 1D and 2D
NMR experiments (DEPT, HSQC, and HMBC). These compounds contain a 4-(N,N-dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium
moiety and a 3-nitro-, 3-amino-, or 3-hydroxyphenyl ring, linked by p-xylene, 4,4′-dimethylbiphenyl, 1,2-bis(p-tolyl)ethane,
or 1,4-bis(p-tolyl)butane. Copyright © 2013 John Wiley & Sons, Ltd.
1
Keywords: 1H–13C 1D NMR; H–13C 2D NMR (HSQC; HMBC); choline kinase inhibitors; antiproliferative compounds
constants (J) are given in Hz. The following parameters were used
in DEPT experiments: PW (Pulse Width: 135°), 9.0ms; recycle time,
Introduction
Evidence for the requirement of choline kinase-α activity in
cancer has been obtained from observations that the expression
of this enzyme is really high in many tumor cells.[1]
Theoretical studies initially performed on a human choline
kinase homology model[2] and further on the X-ray crystal struc-
ture of the enzyme indicated that non-symmetrical inhibitors
could insert the cationic head into the choline binding site and
the adenine moiety into the ATP binding site.[3]
1 s; 1/2J (CH)= 4 ms; 65.536 data points acquired and transformed
from 1024 scans; spectral width, 15 KHz; and line broadening,
1.3 Hz. HMBC spectra were measured with a pulse sequence
gc2hmbc (Standard sequence, Agilent Vnmrj_3.2A software)
optimized for 8 Hz (inter-pulse delay for the evolution of long-
range couplings: 62.5 ms). The HSQC spectra were measured
with a pulse sequence gc2hsqcse (Standard sequence Agilent
Vnmrj_3.2A software).
In a recent paper, the synthesis and the biological evaluation of
a novel family of compounds bearing a 3-aminophenol fragment
and a pyridinium salt connected by means of different linkers,
which were designed and evaluated as ChoK inhibitors and as
antiproliferative agents, have been described.[4]
Results and Discussion
Scheme 1 represents the previously reported synthetic pathway
followed in the preparation of two families of compounds 4a–h
and 5a–h.[4]
Although the structures of these derivatives have been
determined by means of standard spectroscopic techniques (1H
and 13C NMR, and MS), a detailed NMR study has been performed
in some of them, in order to unequivocally corroborate their
structures.
Compounds with general structure 2 were prepared using
linkers 1a–d, which were synthetized following the reported
route.[4,5] Reaction of compounds 1a–d with a half equivalent
of 4-(N,N-dimethylamino)pyridine or 4-pyrrolidinopyridine, in
butanone during 3 days at room temperature, yields the corre-
sponding derivatives 2a–h. Compounds 3e–h were obtained
by treatment of compounds 2a–h with 3-nitrophenol in the
presence K2CO3, with general good yields. Preparation of
compounds 5a–h needs an initial protection of the OH group
in 3-aminophenol and a further reaction with intermediates 2a–h
in the presence of K2CO3, being the OH group liberated during
this last reaction. Finally, compounds 4a–h were obtained using
two routes depending on the starting intermediate. In this sense,
compounds 5a–d were obtained by reaction of 3-aminophenol
and intermediates 2a–d in the presence of NaH, while reduction
1
This paper reports the H and 13C NMR unambiguous signal
assignments corresponding to the pyridinium salts, the linkers,
and the 3-aminophenol moiety (4a–h and 5a–h) using 1D and
2D NMR techniques. The spectra of nitro derivatives 3e–h,
precursors in the synthetic pathway for the preparation of
compounds 4a–h, are also included.
Experimental
NMR spectra were recorded on 500 MHz 1H and 125 MHz 13C
NMR Agilent Direct-Drive (Santa Clara, CA, USA), 400 MHz
1H NMR Agilent Direct-Drive, or 300 MHz 1H and 75 MHz 13C
NMR Agilent Inova-Unity spectrometers at 298 K. Chemical shifts
(δ) are quoted in parts per million (ppm) and are referenced to
the residual solvent peak: CD3OD, δ = 3.31 ppm (1H), δ = 49.05
ppm (13C); DMSO-d6, δ = 2.50 ppm (1H), δ = 39.5 ppm (13C). Spin
multiplicities are given as s (singlet), bs (broad singlet), d (doublet),
dd (doublet doublet), ddd (doublet, doublet doublet), t (triplet), dt
(doublet triplet), q (quadruplet), and m (multiplet). Coupling
*
Correspondence to: L. C. López-Cara and Antonio Entrena, Departamento de
Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de
Granada, Campus de Cartuja, 18071 Granada, Spain. E-mail: lcarlotalopez@
ugr.es; aentrena@ugr.es
Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia,
Universidad de Granada, Campus de Cartuja, 18071, Granada, Spain
Magn. Reson. Chem. (2013)
Copyright © 2013 John Wiley & Sons, Ltd.

S. Schiaffino-Ortega et al.
Scheme 1. General synthetic pathway followed in the preparation of final compounds 4a–h and 5a–h. (a) 4-(N,N-dimethylamino)pyridine (DMAP) or
4-pyrrolidinopyridine, butanone, room temperature, 72 h; (b) 3-aminophenol, NaH, dimethylformamide (DMF), reflux, 20 h; (c) 3-nitrophenol, NaH,
DMF, reflux, 22 h; (d) Fe/FeSO4/H2O, reflux, 3 h; (e) tert-butyldimethylsilyl chloride, triethylamine, DMAP, dichloromethane, room temperature,
6 h; (f) K2CO3, DMF, 110 °C, 20–24 h.
of the NO2 group in intermediates 3e–h yields the final com-
pounds 5e–h.
carbon atoms of R1 and R2, C-2ph, C-5ph, C-2,6pyr, and C-3,5pyr
.
These carbon atoms show signals at 41.59, 111.71, 132.67,
144.34, and 110.37 ppm, respectively, in compound 3f; 49.69/
26.11, 103.34, 130.80, 142.90, and 109.63 ppm, in compound
4c; and 48.46/24.69, 99.55, 129.29, 141.38, and 108.22 ppm in
compound 5c. Chemical shifts of the hydrogen atoms of the
pyridine ring (H-2,6pyr and H-3,5pyr) coincide with those previ-
ously described.3b
For the remaining atoms, this information must be
coordinated with HMBC experiments simultaneously. The HMBC
spectra performed on 3f, 4c, and 5c reveal a correlation be-
tween H-2,6pyr and the carbon atom of the –CH2N+– methylene
group in the three HMBC spectra (Table S2 and Figs S2–S4 in the
Supporting information). This information is the key to identify
all other proton and carbon atoms.
Structural elucidation of compounds 3, 4, and 5 has been
made by routine 1H and 13C NMR techniques. Nevertheless, a
definitive assignment of all signals needs the use of several
NMR techniques as follows: (i) DEPT experiments to determine
the number of protons attached to each carbon atom; (ii)
gs-HSQC spectra to determine the 13C resonances of the tertiary,
secondary, and primary carbons; and (iii) gs-HMBC sequences to
assign the signals of quaternary carbons via two-bond and
three-bond interactions.
Tables 1–3 show the 1H NMR signals of compounds 3e–h,
4a–h, and 5a–h, respectively, while Tables 4–6 show the
corresponding 13C NMR chemical shifts for the same molecules.
NMR spectra of all compounds were run in CD3OD solution,
except for compounds 4b, 4e, 4h, and 5f, which were registered
in DMSO-d6. For this reason, some significant variations are
observed in the chemical shifts depending on the solvent. Hydro-
For the final compounds, when the terminal benzene ring is
connected to the linker through the O atom (compounds
1
4a–h), the H and 13C NMR signals of benzene are in ranges of
gen atoms of the 3-substituted phenyl ring (H-2ph, H-5ph, H-4ph
,
6.14–7.35 and 99.99–161.31 ppm, respectively, while when is
connected through the NH group (compounds 5a–h), the
1H and 13C NMR signals ranges are 6.02–6.87 and 99.21–
159.16 ppm. Apparently, no big differences in the chemical shifts
are observed, but a more in detail study of the chemical shifts
clearly shows the differences between both families of isomeric
compounds (4a–h and 5a–h).
H-2ph appears in a range of 6.22–6.36 ppm in the first family
(4a–h), while in the second family (5a–h), the chemical shifts
are in a range of 5.99–6.09 ppm. This could be because the
addition of the electronic effects in the first family is greater than
in the second one. Thus, the H-2ph protons of 3e–h compounds
appear less shielded than 4a–h and these ones more than
5a–h because of the mesomeric effects (+M and ÀM) of the
3-position substituent upon these protons. NO2 group exert a
ÀM effect (deshielding effect) upon overall phenyl ring, and
NH2 and OH groups have a +M effect (shielding effect) being
and H-6ph) can be identified in accordance with the estimated
values calculated by means of the additive rules for
polysubstituted benzenes.[6]
1H NMR and 13C NMR signals of intermediate nitroderivatives
(3e–h) are similar to the signals of the final compounds (4a–h
and 5a–h) being the main differences in the terminal benzene
ring, which is influenced by the substituent in 3-position (nitro,
amino, or hydroxy group).
HSQC and HMBC experiments were performed on some
compounds of each series, and the results of these experi-
ments have been extrapolated to the other compounds.
Tables S1 and S2 in the Supporting information show the
HSQC and HMBC correlations, respectively, for compounds
3f, 4c, and 5c.
HSQC experiments performed on compounds 3f, 4c, and 5c
allowed the unequivocal chemical shifts assignment to the
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Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)

NMR studies of new 3-aminophenol isomers
Magn. Reson. Chem. (2013)
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

S. Schiaffino-Ortega et al.
wileyonlinelibrary.com/journal/mrc
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)

NMR studies of new 3-aminophenol isomers
Magn. Reson. Chem. (2013)
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

S. Schiaffino-Ortega et al.
wileyonlinelibrary.com/journal/mrc
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)

NMR studies of new 3-aminophenol isomers
higher in 4a–h than in 5a–h compounds. H-4ph and H-6ph suffers
and the ‘Ministerio de Ciencia e Innovación’ (SAF2009-11955). The
award of grant from the ‘Consejería de Innovación, Ciencia y
Empresa, Junta de Andalucía’ to S. S. O. is gratefully acknowledged.
the same effect for the same reason.
The same behavior is observed in 13C NMR signals of C-2ph
,
C-4ph, and C-6ph, which appear more shielded in 5a–h than in
4a–h derivatives and their direct precursors (3e–h).
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Finally, the methylene group connected to the ether bond in
1
4a–h compounds appears less shielded as in H NMR as in 13C
NMR signals, than when this methylene is connected to amino
group in the 5a–h compounds.
In the rest of the molecule, no significant differences have been
found. The 1H and 13C NMR chemical shifts of the linker and the cat-
ionic head are very similar in isomeric compounds of families 4 and 5.
In conclusion, two families of structural isomers bearing
3-aminophenol moiety (4a–h and 5a–h) and their direct precur-
sors (3e–h) have been unequivocally identified by 1D and 2D
NMR techniques. The only differences between both isomers are
the chemical shift of H-2ph, H-4ph, and H-6ph and of C-2ph, C-4ph
,
and C-6ph respectively in the 3-aminophenol moiety, which
appears more deshielding for 4a–h compounds than for 5a–h.
Thus, only one difference between two isomers is enough to
unequivocally identify them.
Supporting Information
Acknowledgements
This work was supported by a project from the ‘Consejería de
Innovación, Ciencia y Empresa, Junta de Andalucía’ (P07-CTS-03210)
Additional supporting information may be found in the online
version of this article at the publisher’s web site.
Magn. Reson. Chem. (2013)
Copyright © 2013 John Wiley & Sons, Ltd.
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