Microwave assisted synthesis of a small library of substituted N, N ′-Bis((8-hydroxy-7-quinolinyl)methyl)-1,10-diaza-18-crown-6 ethers

Article abstract of DOI:10.1021/jo101173t

Figure presented. N,N′-Bis-((8-hydroxy-7-quinolinyl)methyl)-1,10- diaza-18-crown-6 ether 1a and its analogue 1c are known as fluorescent sensors of magnesium in living cells. With the aim to investigate the effects of the substitution pattern on the photophysical properties of ligands 1 and their metal complexes, we developed an efficient microwaves enhanced one-pot Mannich reaction to double-armed diaza-crown ligands 1 carrying a variety of substituents. This new protocol is characterized by shorter reaction times, enhanced yields, and improved product purities with respect to the use of conventional conductive heating.

Full text of DOI:10.1021/jo101173t

pubs.acs.org/joc
SCHEME 1. Thermal One-Pot Mannich Synthesis of Ligands 1
Microwave Assisted Synthesis of a Small Library
of Substituted N,N0-Bis((8-hydroxy-
7-quinolinyl)methyl)-1,10-diaza-18-crown-6 Ethers
Giovanna Farruggia,† Stefano Iotti,‡,§ Marco Lombardo,*,
Chiara Marraccini,‡ Diego Petruzziello, Luca Prodi,
Massimo Sgarzi, Claudio Trombini,*, and
Nelsi Zaccheroni
†Universitaꢀ di Bologna, Dipartimento di Biochimica
‡
“G. Moruzzi”, Bologna, Italy, Universita di Bologna,
ꢀ
Dipartimento di Medicina Interna, dell’Invecchiamento e
ꢀ
Malattie Nefrologiche, Bologna, Italy, and Universita di
Bologna, Dipartimento di Chimica “G. Ciamician”, Bologna,
Italy. §Stefano Iotti is a member of the National Institute of
Biostructures and Biosystems.
marco.lombardo@unibo.it; claudio.trombini@unibo.it
Received June 16, 2010
in living cells,1 cadmium(II),2 and mercury(II).3 While un-
dertaking a structural study on the effects of substituents on
2, 4, and 5 position of the quinoline ring on the complexing
power and selectivity of 1, the task of synthesizing good
amounts of 1 of good purity proved to be somewhat trouble-
some. Indeed, the most practical synthetic approach to 1 was
a thermal three-component classic Mannich reaction of 1,10-
diaza-18-crown-6 ether 2 (0.02-0.045 M in toluene) with
a slight excess of paraformaldehyde and the appropriate
8-hydroxyquinoline 3 (1.2 equiv). After refluxing in toluene
for 15-20 h, the desired products were obtained in moderate
to good yields (Scheme 1).4 However, in our hands, complex
crude reaction mixtures were obtained, from which pure 1
was recovered in a low amount after a sluggish purification
by crystallization.
N,N0-Bis-((8-hydroxy-7-quinolinyl)methyl)-1,10-diaza-
18-crown-6 ether 1a and its analogue 1c are known as
fluorescent sensors of magnesium in living cells. With the
aim to investigate the effects of the substitution pattern on
the photophysical properties of ligands 1 and their metal
complexes, we developed an efficient microwaves en-
hanced one-pot Mannich reaction to double-armed dia-
za-crown ligands 1 carrying a variety of substituents. This
new protocol is characterized by shorter reaction times,
enhanced yields, and improved product purities with
respect to the use of conventional conductive heating.
Since the pioneering works of Gedye, Giguere, and
Majetich,5 microwave-assisted organic synthesis (MAOS)
has received an ever increasing attention and has now
become a well-established and reproducible technique.6
(2) Prodi, L.; Montalti, M.; Zaccheroni, N.; Bradshaw, J. S.; Izatt, R. M.;
Savage, P. B. Tetrahedron Lett. 2001, 42, 2941.
(3) Prodi, L.; Bargossi, C.; Montalti, M.; Zaccheroni, N.; Su, N.; Bradshaw,
J. S.; Izatt, R. M.; Savage, P. B. J. Am. Chem. Soc. 2000, 122, 6769.
(4) Su, N.; Bradshaw, J. S.; Zhang, X. X.; Song, H.; Savage, P. B.; Xue,
G.; Krakowiak, K. E.; Izatt, R. M. J. Org. Chem. 1999, 64, 8855. For
modified approaches to 1, see: Bordunov, A. V.; Bradshaw, J. S.; Zhang,
X. X.; Dalley, N. K.; Kou, X.; Izatt, R. M. Inorg. Chem. 1996, 35, 7229.
(5) (a) Gedye, R.; Smith, F.; Westaway, K.; Ali, H.; Baldisera, L.;
Laberge, L.; Rousell, J. Tetrahedron Lett. 1986, 27, 279. (b) Giguere, R. J.;
Bray, T. L.; Duncan, S. M.; Majetich, G. Tetrahedron Lett. 1986, 27, 4945.
(6) For leading reviews on microwave heating in organic synthesis, see:
(a) Kappe, C. O.; Dallinger, D.; Murphree, S. S. Practical Microwave
Synthesis for Organic Chemists; Wiley-VCH: Weinheim, Germany, 2009.
(b) Caddick, S.; Fitzmaurice, R. Tetrahedron 2009, 65, 3325. (c) Kappe,
C. O.; Dallinger, D. Mol. Diversity 2009, 13, 71. (d) Polshettiwar, V.; Varma,
R. S. Chem. Soc. Rev. 2008, 37, 1546. (e) Kappe, C. O. Chem. Soc. Rev. 2008,
37, 1127. (f) Dallinger, D.; Kappe, C. O. Chem. Rev. 2007, 127, 2563. (g)
Microwaves in Organic Synthesis; Loupy, A., Ed.; Wiley-VCH: Weinheim,
1,10-Diaza-18-crown-6 ethers of general structure 1
(Scheme 1), bearing two 8-hydroxy quinoline side arms, have
been recently proposed and applied as fluorescent chemo-
sensors for a variety of metal ions, including magnesium(II)
(1) (a) Farruggia, G.; Iotti, S.; Prodi, L.; Zaccheroni, N.; Montalti, M.;
Savage, P. B.; Andreani, G.; Trapani, V.; Wolf, F. I. J. Fluoresc. 2009, 19, 11.
(b) Farruggia, G.; Iotti, S.; Prodi, L.; Montalti, M.; Zaccheroni, N.; Savage,
P. B.; Trapani, V.; Sale, P.; Wolf, F. I. J. Am. Chem. Soc. 2006, 128, 344. (c)
Bronson, R. T.; Montalti, M.; Prodi, L.; Zaccheroni, N.; Lamb, R. D.;
Dalley, N. K.; Izatt, R. M.; Bradshaw, J. S.; Savage, P. B. Tetrahedron 2004,
60, 11139.
€
Germany, 2006. (h) Nuchter, M.; Ondruschka, B.; Bonrath, W.; Gum, A.
Green Chem. 2004, 6, 128. (i) Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43,
€
6250. (j) Lindstrom, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron
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DOI: 10.1021/jo101173t
r
Published on Web 08/17/2010
J. Org. Chem. 2010, 75, 6275–6278 6275
2010 American Chemical Society

Farruggia et al.
JOCNote
Even if the so-called “specific” or “non-thermal” microwave
effects have not been definitely established up to date, a large
number of publications demonstrate that microwave heating
often results in enhanced reaction rates, as well as reduced
formation of byproduct, with respect to conventional con-
ductive heating. Microwave heating has now been success-
fully applied to a number of one-pot multicomponent
procedures,7 including Mannich and related reactions.8
Here we report how the one-pot three-component Man-
nich synthesis of ligands 1 can be greatly improved by the use
of microwave heating, with a reduction of the reaction time,
an increase of the yield, and the minimization of side reac-
tions, allowing much easier purification operations.
FIGURE 1. Differently substituted ligands 1 synthesized.
TABLE 2. Microwave-Assisted Synthesis of Ligands 1e-ka
We started optimizing the reported procedure4 for ligands
1a-d using microwave heating in different solvents. Using
the same reagent ratios and concentrations adopted in
thermal reactions (2 0.045 M in toluene or 1,4-dioxane as
the solvent), an encouraging improvement of the efficiency
was observed, as shown in entries 1 and 2 of Table 1. The
procedure was further optimized using a 5-fold increase in
the concentration of 2 (0.25 M), a slight increased excess of
paraformaldehyde (1.5 equiv) and an equimolar amount of
hydroxyquinolines 3. Using a power of 600 W for 2 h at
atmospheric pressure, products 1a-d were obtained in ex-
cellent yieldsin allthe casesexamined (entries 3-10, Table 1).
entry
t (h)
1, Y b (%)
1
2
3
4
5
6
7
2
4
3
3
4
3
3
1e, 95
1f, 92
1g, 98
1h, 95
1i, 86
1j, 91
1k, 90
aReactions were run on 0.5 mmol of diazacrown 2, 1.5 mmol of
paraformaldehyde, and 1.0 mmol of 8-hydroxy-quinoline in 2 mL of 1,4-
dioxane, using a power of 600 W for the reported time. bIsolated yields.
In terms of product quality, the use of stoichiometric
amounts of 3 had a significant impact on the crude reaction
mixture purity that in all cases was >95% as judged by its 1H
NMR analysis. Thus, solvent removal under vacuum deliv-
ered ligands 1a-d as solid compounds that were purified
simply by washing with diethyl ether. If necessary, 1a-d
can be recrystallized from dichloromethane/diethyl ether
mixtures.
With this simple and efficient procedure in our hands, we
synthesized a new group of ligands (1e-k, Figure 1), to
investigate their capability to bind magnesium(II) cation in
water and living cells or to modulate their solubility and
distribution among the cellular compartments.
The results obtained using the microwave enhanced pro-
tocol in 1,4-dioxane as solvent are reported in Table 2. All the
new compounds 1e-k were obtained in very good yields and
purity according to the microwave-assisted protocol.
Concerning the synthesis of new ligands, it is worth under-
lining that, while 2-methyl-8-hydroxy-quinoline 3e is a com-
mercial product, quinolines 3f-k were prepared following
reported literature procedures. 5-Phenyl-8-hydroxy-quino-
line (3f) was prepared by a Suzuki cross-coupling, starting
from commercial 5-chloro-8-hydroxy-quinoline 3c, as re-
ported by Hormi.9
TABLE 1. Microwave-Assisted Synthesis of Ligands 1a-d
entry
solvent
1, Y a (%)
1b
2b
3c
4c
5c
6c
7c
8c
9c
10c
toluene
1,4-dioxane
toluene
1,4-dioxane
toluene
1,4-dioxane
toluene
1,4-dioxane
toluene
1,4-dioxane
1a, 83
1a, 80
1a, 90
1a, 88
1b, 98
1b, 95
1c, 98
1c, 99
1d, 98
1d, 96
aIsolated yields. bReactions were run on 0.5 mmol of diazacrown 2,
1.2 mmol of paraformaldehyde, and 1.2 mmol of 8-hydroxy-quinoline in
11 mL of solvent, using a power of 600 W for 2 h. cReactions were run
on 0.5 mmol of diazacrown 2, 1.5 mmol of paraformaldehyde, and
1.0 mmol of 8-hydroxy-quinoline in 2 mL of solvent, using a power of
600 W for 2 h.
(7) For recent examples, see: (a) Presset, M.; Coquerel, Y.; Rodriguez, J.
Org. Lett. 2009, 11, 5706. (b) Zhu, S.-L.; Ji, S.-J.; Zhao, K.; Liu, Y.
Tetrahedron Lett. 2008, 49, 2578. (c) Polshettiwar, V.; Varma, R. S. Pure
Appl. Chem. 2008, 80, 777. (d) Chebanov, V. A.; Sakhno, Y. I.; Desenko,
S. M.; Chernenko, V. N.; Musatov, V. I.; Shishkina, S. V.; Shishkina, O. V.;
Kappe, C. O. Tetrahedron 2007, 63, 1229. (e) Santra, S.; Andreana, P. R. Org.
Lett. 2007, 9, 5035. (f) Chebanov, V. A.; Saraev, V. E.; Desenko, S. M.;
Chernenko, V. N.; Shishkina, S. V.; Shishkin, O. V.; Kobzar, K. M.; Kappe,
C. O. Org. Lett. 2007, 9, 1691. (g) Tu, S.; Jiang, B.; Zhang, Y.; Jia, R.; Zhang,
J.; Yao, C.; Shi, F. Org. Biomol. Chem. 2007, 5, 355. (h) DiMauro, E. F.;
Kennedy, J. M. J. Org. Chem. 2007, 72, 1013. (i) Vugts, D. J.; Koningstein,
M. M.; Schmitz, R. F.; de Kanter, F. J. J.; Groen, M. B.; Orru, R. V. A.
Chem.;Eur. J. 2006, 12, 7178.
5-Alkyloxymethyl-8-hydroxy-quinolines (3g-j) were syn-
thesized with a slighlty modified procedure with respect
to the one reporteb by Burckhalter and Leib (Scheme 2,
see Supporting Information).10
Finally, 4-methoxy-8-hydroxy-quinoline 3k was pre-
pared from the commercially available xanthurenic acid, as
recently reported by Hormi.11
ꢁ
€ €
(8) (a) Szatmari, I.; Fulop, F. Synthesis 2009, 775. (b) Hao, W.-J.; Jiang,
B.; Tu, S.-J.; Cao, X.-D.; Wu, S.-S.; Yan, S.; Zhang, X.-H.; Hana, Z.-G.; Shi,
F. Org. Biomol. Chem. 2009, 7, 1410. (c) Ohta, Y.; Chiba, H.; Oishi, S.; Fujii,
N.; Ohno, H. Org. Lett. 2008, 10, 3535. (d) Rodrı
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Chem. 2006, 71, 2888. (e) Mosse, S.; Alexakis, A. Org. Lett. 2006, 8, 3577. (f)
guez, B.; Bolm, C. J. Org.
ꢁ
A complete set of results on the complexing and sensing
abilities of the new ligands for divalent cations will be
€
Follmann, M.; Graul, F.; Schafer, T.; Kopec, S.; Hamley, P. Synlett 2005,
1009. (g) Westermann, B.; Neuhaus, C. Angew. Chem., Int. Ed. 2005, 44,
4077. (h) McLean, N. J.; Tye, H.; Whittaker, M. Tetrahedron Lett. 2004, 45,
993. (i) Pemberton, N.; Aberg, V.; Almstedt, H.; Westermark, A.; Almqvist,
F. J. Org. Chem. 2004, 69, 7830. (j) Lehmann, F.; Pilotti, A.; Luthman, K.
Mol. Diversity 2003, 7, 145. (k) Leadbeater, N. E.; Torenius, H. M.; Tye, H.
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6276 J. Org. Chem. Vol. 75, No. 18, 2010

Farruggia et al.
JOCNote
FIGURE 2. (a) SaOs-2 cells grown on a glass coverslip were stained
for 5 min with 1a dye (25 μM) dissolved in Dulbecco’s phosphate-
buffered saline (DPBS) and observed exciting the sample at 390 nm
and collecting the emitted fluorescence around 500 nm. (b) The
same field observed by phase contrast.
SCHEME 2. Synthesis of Quinolines 3g-j
FIGURE 3. Fluorescence spectra (λex = 350 nm) of probe 1a (25 μM)
in Dulbecco’s phosphate-buffered saline (DPBS) at room temperature
upon addition of increasing amounts of Mg2þ ions.
TABLE 3. Intracellular Magnesium Content (nmol/106 Cells)
Evaluated by Atomic Absoption Spectroscopy (AAS) and Fluorescence
Spectroscopy (FS) Using 1a
cell line
AAS
FS
undifferentiated HL60
DMSO-differentiated HL60
Jurkat-6
11.2 ( 4.9
6.6 ( 3.5
26.5 ( 8.9
39.4 ( 5.9
11.9 ( 1.5a
7.2 ( 1.8a
25.4 ( 8.2b
37.2 ( 2.7b
A 2780
a1a synthesized according to ref 4. b1a synthesized by the herein
proposed method.
In conclusion, N,N0-bis-((8-hydroxy-7-quinolinyl)methyl)-
1,10-diaza-18-crown-6 ether (1a) and 10 analogues carrying
one more substituent on position 2, 4, or 5 of the quinoline ring
system (1b-k) have been efficiently synthesized via a simple
catalyst-free three-component bidirectional Mannich reaction
under microwave heating, using toluene or 1,4-dioxane as
solvent. The new reaction protocol benefited from a remark-
able improvement with respect to the analogous thermal
reaction, in terms of reaction time, yield, and suppression of
side reactions, resulting in an increased purity of the crude
products. Previous results on the use of 1a and 1c as fluorescent
chemosensors for magnesium ions in water and living cells
were confirmed by a preliminary assessment of the photophy-
sical properties of the new ligands. The results obtained clearly
indicate that the higher purity ensured by this new synthetic
methodology has a beneficial effect also on the analytical
performance of the probes.
reported in due course. Here we wish to report the photo-
physical properties of 1a synthesized by the present methods,
which allowed to reach a higher grade of purity compared to
the method previously published.4 We will particularly point
out how this higher purity affects its sensing performance.
The probe 1a was used to stain SaOs-2 cells as shown in
Figure 2. It is clear, especially in the flattened attached cells,
the ubiquitous distribution of the probe with a more intense
fluorescence in the nuclear and perinuclear regions, as
expected for the magnesium cellular distribution.
For a quantitative evaluation of the complexing ability of
this species, the probe was dissolved in buffered water
and titrated with Mg2þ. The titration was followed by
spectrofluorimetric measurements and the fluorescence
spectra are reported in Figure 3.
It has to be noted that the observed fluorescence enhance-
ment of the dye 1a obtained with this new synthetic method is
remarkably higher than the one previously observed.1a This
is due to the higher purity of the probe that results in a 5-fold
lower initial signal in the absence of magnesium ions, allow-
ing a lower detection limit.
The analytical capability of 1a is thus maintained if not
increased, as shown in Table 3, where the fluorescence data
obtained with the probes obtained with the two synthetic
methodologies are compared with atomic absorption deter-
minations for different cell lines.
Experimental Section
Microwave Assisted Synthesis of 1,10-Bis((2-methyl-8-hydroxy-
7-quinolinyl)methyl)-1,10-diaza-18-crown-6 Ether (1e, Table 2,
Entry 1). A mixture of 2-methyl-8-hydroxy-quinoline 3e (0.16 g,
1.0 mmol), 1,10-diaza-18-crown-6 ether 2 (0.131 g, 0.5 mmol),
and paraformaldehyde (0.045 g, 1.5 mmol) in 1,4-dioxane (2 mL)
was irradiated under inert atmosphere at 600 W for 2 h in a
MILESTONE MicroSYNTH Labstation microwave apparatus.
The internal temperature was monitored using a fiber-optic
temperature sensor. The solution was allowed to reach room
temperature, the solvent was evaporated at reduced pressure
and the crude residue was purified by washing with anhydrous
diethyl ether. The title compound was obtained as a faint yellow
solid after solvent removal under vacuum (0.288 g, 0.48 mmol,
Finally, the more sensitive response to magnesium allowed to
carry out the same applications at a lower concentration (20 μM
compared to concentration ranging between 25 and 50 μM).
J. Org. Chem. Vol. 75, No. 18, 2010 6277

Farruggia et al.
JOCNote
95%): mp = 163-164 °C; 1H NMR (200 MHz, CD3OD) δ 7.97
(d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 7.20-7.11 (m, 4H),
4.00 (s, 4H), 3.72 (t, J = 5.3 Hz, 8H), 3.62 (s, 8H), 2.95 (t, J = 5.4
Hz, 8H), 2.72 (s, 6H); 13C NMR (50 MHz, CD3OD) δ 158.0,
152.5, 138.6, 136.5, 127.1, 127.1, 122.5, 119.5, 117.6, 70.9, 69.4,
58.1, 53.8, 24.9. Anal. Calcd for C34H44N4O6 (604.74): C, 67.53;
H, 7.33; N, 9.26. Found: C, 67.16; H, 7.20; N, 9.15.
fluorescence microscope (Zeiss, Germany) exciting the sample
around 390 nm and collecting the emitted fluorescence around
500 nm.
Cellular Magnesium Determination. Concentrations of cell
magnesium contents were calculated by fluorescence spectros-
copy and by atomic absorption spectroscopy as previously
reported on sonicated cells samples.1
Cell Culture and Staining. The cells were grown in RPMI-123
medium supplemented with 2 mM glutamine and 10% foetal
calf serum, at 37 °C and in presence of 5% CO2. For microscope
examination, SaOs-2 cells were seeded on glass coverslip and
allowed to growth for 24 h, then rinsed in Dulbecco’s phosphate
buffered saline without calcium and magnesium (DPBS) and
stained in the dark for 5 min with 1a dye 25 μM dissolved in
DPBS. The sampled were gently blotted to remove the excess of
liquid, put on a microscope slide and observed with a Axioplan
Acknowledgment. The University of Bologna and MIUR
(Rome) are acknowledged for financial support.
Supporting Information Available: Characterization data,
experimental details, and copies of 1H and 13C NMR spectra of
compounds synthesized. This material is available free of charge
via the Internet at http://pubs.acs.org.
6278 J. Org. Chem. Vol. 75, No. 18, 2010