Novel generation of pH indicators for proton magnetic resonance spectroscopic imaging

Article abstract of DOI:10.1021/jm070044j

We describe the synthesis of 1,ω-di-1H-imidazoles 2 and 3, derived from L-threitol and D-mannitol, respectively, showing suitable magnetic and toxicological properties, as novel extracellular pH indicators for ¹H spectroscopic imaging by magnet

Full text of DOI:10.1021/jm070044j

J. Med. Chem. 2007, 50, 4539-4542
4539
Novel Generation of pH Indicators for Proton Magnetic Resonance Spectroscopic Imaging
†
†
‡
§
§
Jordi Soler-Padr o´ s, Elena P e´ rez-Mayoral, Laura Dom ´ı nguez, Pilar L o´ pez-Larrubia, Elena Soriano,
‡
§
,†
Jos e´ Luis Marco-Contelles, Sebasti a´ n Cerd a´ n, and Paloma Ballesteros*
Laboratorio de S ´ı ntesis Org a´ nica e Imagen Molecular por Resonancia Magn e´ tica, Instituto UniVersitario de InVestigaci o´ n,
UNED, Paseo Senda del Rey 9, 28040 Madrid, Spain, Laboratorio de Radicales Libres, Instituto de Qu ´ı mica Org a´ nica General (CSIC),
c/Juan de la CierVa 3, 28006 Madrid, Spain, and Laboratorio de Imagen y Espectroscop ´ı a por Resonancia Magn e´ tica (LIERM),
Instituto de InVestigaciones Biom e´ dicas “Alberto Sols” (CSIC), c/Arturo Duperier 4, 28029 Madrid, Spain
ReceiVed January 12, 2007
We describe the synthesis of 1,ω-di-1H-imidazoles 2 and 3, derived from L-threitol and D-mannitol,
respectively, showing suitable magnetic and toxicological properties, as novel extracellular pH indicators
1
for H spectroscopic imaging by magnetic resonance methods.
1
Introduction
Chart 1. H MRSI pH Indicators Containing an Imidazole Ring
1
The imidazole ring is a major biochemical contributor found
in many natural products such as histidine, histamine, and
guanine, as well as a crucial component of nucleic acids DNA
and RNA. Additionally, a number of pyrrole-imidazole com-
2
pounds, such as those derived from oroidin, and indole-
3
imidazole alkaloids with interesting biological properties have
Chart 2. Polyhydroxylated 1,ω-Diimidazoles 2 and 3
been isolated from marine sponges and terrestial plants. At the
same time, synthetic fluoro-2-nitroimidazoles have been exten-
sively investigated and considered as ideal markers for the
4
measurement of tumor hypoxia by noninvasive methods. These
1
8
19
F (or F) labeled bioreductive drugs bind to hypoxic cells in
tumors, a process that can be detected by positron emission
6
tumors. These results provided the first pH map in vivo from
e
1
9
tomography (PET) or F magnetic resonance spectroscopy
a model of C6 glioblastoma multiforme implanted in the brain
of adult rats. Although IEPA was an excellent reporter molecule
for the in vitro measurements, the presence of only one proton
(H-2 of imidazole ring) demanded the use of relatively high
concentrations of the probe, often resulting in unfavorable
1
9
(
F MRS). As part of our current work on molecular imaging
5
and diagnosis, we reported the synthesis and in vivo evaluation
of 3-(ethoxycarbonyl)-2-imidazol-1-ylpropionic acid (IEPA)
6
(
Chart 1), the first probe made available for the noninvasive
1
1
measurement of extracellular pH (pHe) in tumors by H NMR
signal-to-noise ratios in the corresponding in vivo H MRSI
1
a
spectroscopic imaging ( H MRSI ). The pHe in tumors is usually
acid compared to the surrounding normal tissue. This feature
has been confirmed with numerous microelectrode measure-
ments and confirmed by P magnetic resonance spectroscopy
(
spectra. Moreover, IEPA became rapidly removed from the
circulation by renal clearance, a circumstance that imposed very
high delivery doses to compensate its elimination. To overcome
some of these drawbacks, we recently described a new pH probe
7
31
8
10
MRS) as a less invasive method. Moreover, pHe in tumors is
derived from L-histidine 1 to increase the polar nature of the
known to provide valuable information concerning the most
appropriate chemotherapic agent, as well as in the characteriza-
tion of the tumoral microenvironment, a crucial variable
molecule and increase its retention time within the circulation
(Chart 1). However, its apparent pK ′ ) 5.58 remained far from
a
the physiological range, a circumstance that precluded further
9
determining tumoral invasion and metastatic behavior. On these
developments as a pH indicator.
e
grounds, the development of optimal probes for the measurement
of extracellular pH in tumors is of considerable clinical interest.
The selection of an imidazole ring in the design of IEPA is
based on two reasons: (i) imidazole pKa (∼7.0) is very close
to physiological pH, and (ii) its H-2, H-4 and H-5 proton
resonances are easily resolved and observed in the aromatic
To further improve the performance of these pHe probes, we
herein report the design and synthesis of 2 and 3 containing
two imidazole rings linked to a polyhydroxylated skeleton (Chart
2
). The hydroxy groups of the polyalcohol moiety should
provide higher polarity to the molecule, while the presence of
two magnetically equivalent imidazole rings would increase the
intensity of the H2 MRSI resonance, improving both the
unfavorable dose and elimination rates of the previous probes.
Diimidazoles 2 and 3 can easily be prepared from readily
available carbohydrate templates, being very useful in the
preparation of novel families of pHe probes with optimized
magnetic and pharmacological properties.
1
region of H NMR spectra, far away enough from the water
resonance and the more crowded aliphatic regions of the
spectrum. In vivo pHe measurements using this probe were
obtained from chemical shifts of its H-2 proton, as the indicator
is distributed within the different pHe environments of the
*
To whom correspondence should be addressed. Phone: 34 913987320.
Fax: 34 913986697. E-mail: pballesteros @ccia.uned.es.
Results and Discussion
†
Instituto Universitario de Investigaci o´ n.
Instituto de Qu ´ı mica Org a´ nica General (CSIC).
‡
In order to setup the experimental conditions for the synthesis
of 2 and 3, we first pursued the preparation of 4 from the
previously known acetate 5 as a model compound (Scheme
§
Instituto de Investigaciones Biom e´ dicas “Alberto Sols” (CSIC).
11
a
1
Abbreviations: H MRSI, proton magnetic resonance spectroscopic
imaging.
1
2
1
0.1021/jm070044j CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/11/2007

4
540 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Brief Articles
Scheme 1. Synthesis of 1,3-Diimidazol-1-yldeoxyglycerol (4)
Scheme 2. Synthesis of Compound 2 from Commercial
Tosylate 6
Figure 1. Proton NMR of (A) 25 mM IEPA in cultures of C6 glioma
cells, (B) 25 mM 3 in cultures of C6 glioma cells, (C) 12.5 mM IEPA
and 12.5 mM 3 erythrocytes, and (D) 12.5 mM IEPA and 12.5 mM 3
in cultures of C6 glioma cells, at 500.13 MHz. Numbers in the left
Scheme 3. Synthesis of Diimidazole 3 from the Known
Mesylates 8 and 9
e
indicate the final pH value of the suspension.
Table 1. Apparent pKa (pKa′) Values of Compounds 2-4 Determined
1
by H NMR (500.13 MHz, 37 °C)
compd
pKa′ (D2O)a
pKa′ (FBS)b
pKa′ (calcd)c
2
3
4
6.56
7.00
6.45
6.67
6.63
6.65
7.12
6.38
a
Model solution of 25 mM 2, 3 or 4 in D2O. b Model solution of 25
c
mM 2, 3, or 4 in FBS (20% D2O). Calculated values are those described
by Soriano et al.15
1
). Reaction of 5 with imidazole in the presence of sodium
resonance intensity and by 1.43 the signal-to-noise ratio for the
same number of scans and acquisition conditions.
amide in dry DMF afforded product 4 in 17% yield (not
optimized) (Scheme 1). Considering this, we tried the synthesis
of 2 from commercial 1,4-di-O-p-toluenesulfonyl-2,3-O-iso-
propylidene-L-threitol (6) under the same experimental condi-
tions. In this case, 7 was obtained in high chemical yield (88%)
and was submitted to acid hydrolysis to give 2 (Scheme 2),
characterized as its dihydrochloride salt, in almost quantitative
We then performed in vitro NMR experiments using eryth-
rocyte preparations (from healthy rats) and cultures of C6 glioma
cells (a well-characterized tumor astrocytoma cell line) in order
to compare the chemical shifts of the H2 protons from the probes
in different environments (parts C and D of Figure 1). Although
the imidazole ring resonances of 3 and IEPA appeared partially
overlapping, the double intensity of the H2 resonance from the
diimidazolic probe was well observed. The assessments of
chemical shift of H2 proton of imidazole rings in 3 were
achieved in the presence and absence of IEPA as a reference
compound. It is important to remark here that the pKa′ value of
yield. In the same way, 3 was synthesized from a mixture of
mesylates 8 and 9,1
3,14
leading to diimidazoles 10 and 11 in
7
7% chemical yield that afforded the dihydrochloride salt of 3
after acid hydrolysis without separation (Scheme 3).
With these compounds in hand, we pursued the determination
of the corresponding apparent pKa′ of N-3 of diimidazoles 2, 3,
and 4. Values of pKa were determined by H NMR (500.13
3
was not affected by the presence of IEPA, a circumstance
1
confirmed by the combined titration of 3 and IEPA (see Figure
MHz, 37 °C) from pH titrations of the solutions (25 mM) of
the corresponding compounds in deuterated water solutions and
by simulating biological conditions through the use of fetal
bovine serum (FBS). Table 1 shows the calculated and
experimental pKa′ values for 2-4. The experimental values were
determined from the titration behavior of H2 resonance of the
imidazole ring in the range 3 < pH < 11. As indicated in Table
1
in Supporting Information).
Figure 1 shows only the aromatic resonances of the tested
compounds, confirming the uncrowded environment of the
aromatic region of H NMR spectra from cells and tissues, and
1
the absence of the overlap of the H2 resonance from our probes
with other endogenous resonances from the cells. As shown in
parts A and B of Figure 1, the in vitro spectra (cultures of C6
cells) depict only the resonances from added IEPA and 3, but
very similar results were obtained with 2. These resonances
correspond to the extracellular space, since they reveal a pH
very similar to that measured by potentiometric methods with
a pH electrode in the same suspension. It is important to
remember that methylimidazol-1-yl acetate, considered as an
indicator of intra- and extracellular pH (pHi), has two distinct
H2 resonances, derived from the intra- and extracellular spaces,
respectively. This is due to the fact that the uncharged form of
1
7
, the pKa′ of these indicators remained in the range 6.45-
.00, in good agreement with the theoretical values determined
15
by computational calculations. Noteworthy is the fact that pKa′
values determined in D2O become higher for longer chain
lengths, from three to six carbons. The pKa′ values of 2 and 3
in FBS are 6.67 and 6.63, respectively. Under these conditions
the pKa′ of 3 is slightly lower than the calculated and
experimental pKa′ in D2O, although it remains in the range of
physiological pH.
5
As mentioned above, the presence of only one H2 proton
per molecule in IEPA limited the sensitivity of the in vivo
this probe freely crosses the cellular membranes distributing
in the intracellular and extracellular spaces and consequently
originating two resolved resonances as previously reported. This
6
5
determination because of signal-to-noise considerations. The
compounds reported herein are ideally functionalized to over-
come this inconvenience because they contain two magnetically
equivalent imidazole rings per molecule, improving by 2 the
was not observed in the present study. In contrast, our results
reveal the presence of only one resonance, corresponding to a
pH virtually identical to that measured with a potentiometric

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4541
Figure 2. T
2
-weighted spin-echo anatomical image acquired with a RARE sequence in axial orientation (TR ) 3300 ms, TE ) 60 ms, RARE
2
factor ) 8, av ) 3, FOV ) 38 × 38 mm, acquisition matrix ) 256 × 256, in-plane resolution of 148 × 148 µm , slice thickness ) 1.00 mm, and
number of slices ) 24) of a representative rat. In vivo spectroscopy protocol using a point-resolved spectroscopy sequence (PRESS) combined with
VAPOR water suppression, located in the injection zone of the white matter in the right ventricle (TR ) 3000 ms, TE ) 35 ms, av ) 128, dummy
scans ) 8, volume ) 3 mm ): (a) 60 min after injection of 3 (125 mM, 25 µL); (b) 25 min after injection of IEPA (125 mM, 25 µL); (c) 35 min
after injection of IEPA (125 mM, 25 µL). Note the faster elimination of IEPA compared to 3.
3
glass electrode. This finding allows us to conclude that the new
probes monitor primarily the extracellular pH compartment.
Furthermore, we initiated the in vivo spectroscopic imaging
of 3 by intravenous injection of this probe in rats bearing an
implanted C6 glioma in their brains. Earlier studies using IEPA
required the iv infusion of a 0.6 M solution of this probe (4-5
mL total infusion volume). However, the maximal concentration
of 3 was limited by its solubility at physiological pH to 0.175
M, making it very difficult to detect the corresponding
added. The mixture was left to react at the temperature and time
stated in each case. The solvent was evaporated, and the residue
was purified by column chromatography using mixtures of CH
Cl /MeOH as eluent.
3-[(2S,3S)-(2,3-Dihydroxybutane-1,4-diyl)]}-di-1H-imidazo-
2
-
2
{
lium Dihydrocholoride (2). Following the general method for the
synthesis of diimidazole derivatives above, imidazole (0.441 g, 6.48
2
mmol), NaNH (0.253 g, 6.48 mmol) in dry DMF (9 mL), and
1,4-di-O-p-toluenesulfonyl-2,3-O-isopropylidene-L-threitol (6) (0.764
g, 1.62 mmol) were mixed and stirred at room temperature for 24
h. Purification by column chromatography (CH Cl /MeOH, 98:2)
1
resonances by in vivo H NMR. To overcome this limitation, 3
2
2
was administered by direct injection into the brains of healthy
Wistar rats, and in vivo PRESS spectra were acquired at 7 T as
indicated in the Experimental Section (Figure 2). In this case,
the resonances of the imidazole ring could easily be detected
and adequately resolved even 60 min after the intracranial
injection of 3 (Figure 2a). However, the resonances from IEPA
injected intracraneally under the same conditions became
undetectable 5 min after the injection, revealing a much faster
elimination rate (Figure 2b and Figure 2c, respectively). These
experiments confirmed that the retention time of 3 in cerebral
tissue was higher than that of IEPA, overcoming one of the
main limitations of the parental compound.
gave (-)-1,4-deoxy-1,4-diimidazol-1-yl-2,3-O-isopropylidene-L-
threitol (7) (0.373 g, 88%). Compound 7 (0.180 g, 0.69 mmol) was
dissolved in 1.2 M HCl (3 mL) at 50 °C for 3 h. Then the solvent
was evaporated, and the product was purified by recrystallization
from methanol to give 2 (0.149 g, 97%) as a colorless solid: mp
25
2
1
13-5 °C; [R] -25 (c 0.67, MeOH); IR (KBr) ν 3232, 1576,
D
1 1
-
2
544, 1436, 1283, 1087 cm ; H NMR (D O, 200 MHz) δ 8.61
(s, 2 H, H-2 Im), 7.38 (s, 2 H, H-5 Im), 7.32 (s, 2 H, H-4 Im),
13
4
.32-4.16 (m, 4 H, 2 × CH
NMR (D
O, 50 MHz) δ 135.2 (2 × C-2 Im), 122.3 (2 × C-5 Im),
19.7 (2 × C-4 Im), 69.7 (2 × CH), 52.0 (2 × CH ); MS (ES) m/z
223 [M + 1] , 445 [2M + 1] . Anal. (C H N O ‚2HCl‚H O) C,
2
), 3.98-3.91 (m, 2 H, 2 × CH);
C
2
1
2
+
+
10
14
4
2
2
H. N calcd, 38.35, 5.79, 17.89; found, 38.41, 5.80, 18.01.
Finally, in vitro toxicity of 2 and 3 was separately assayed
in cultures of C6 glioma cells by monitoring the intracellular
release of lactic dehydrogenase (LDH) to the incubation
medium. The amount of LDH released was measured after 1-6
h of incubation of the C6 cells with the concentration of the
probes ranging between 20 and 150 mM. No apparent signs of
toxicity were detected, with LDH release values being smaller
than 10% for 2 or 3, even at the highest concentrations.
In summary, the polyhydroxylated diimidazoles 2 and 3 were
easily obtained from available L-threitol and D-mannitol deriva-
tives, exhibited low toxicity, have pKa′ values in the range of
physiological extracellular pH (6.45-7), double the intensity
of H-2 imidazole resonance, and have higher retention times in
cerebral tissue than earlier monoimidazolic pHe probes. Taken
together, these favorable circumstances indicate that the new
{3-[(2R,3R,4R,5R)-(2,3,4,5-Tetrahydroxyhexane-1,6-diyl)]}di-
H-imidazolium Dihydrocholoride (3). Following the general
1
method for the synthesis of diimidazole derivatives, imidazole
0.462 g, 6.80 mmol), NaNH (0.265 g, 6.80 mmol), dry DMF (10
mL), and 1,6-dimethanesulfonyl-2,4;3,5-di-O-isopropylidene-D-
(
2
14
mannitol (8) /1,6-dimethanesulfonyl-2,3;4,5-di-O-isopropylidene-
14
D-mannitol (9) (0.710 g, 1.70 mmol) were mixed, stirred at
5
0 °C for 24 h, and purified and separated by chromatography (CH
2
-
Cl /MeOH, 97:3), affording (-)-1,6-deoxy-1,6-diimidazol-1-yl-2,4:
2
3,5-di-O-isopropylidene-D-mannitol (10) (0.312 g, 51%) and (-)-
1,6-deoxy-1,6-diimidazol-1-yl-2,3:4,5-di-O-isopropylidene-D-
mannitol (11) (0.159 g, 26%). Total yield was 77%. A mixture of
the isomers 10 and 11 (0.470 g, 1.29 mmol) was dissolved in 1.2
M HCl (10 mL) at 50 °C for 2 h. Then the solvent was evaporated,
and the residue was washed several times with absolute ethanol.
The product was recrystallized from methanol to give 3 (0.215 g,
2
5
5
1%) as a colorless solid: mp 210-2 °C; [R] +10 (c 0.43,
D
1
1
probes are good and promising pHe indicators for H MRSI.
MeOH); H NMR (D
2
O, 200 MHz) δ 8.57 (s, 2 H, H-2), 7.36 (s,
), 3.86
O, 50 MHz)
2
(
H, H-5), 7.31 (s, 2 H, H-4), 4.46-4.13 (m, 4 H, 2 × CH
2
Experimental Section
13
m, 2H, 2 × CH), 3.47 (d, 2H, 2 × CH); C NMR (D
2
General Methods. Melting points were determined on a digital
δ 135.4 (2 × C-2), 122.7 (2 × C-5), 119.7 (2 × C-4), 69.6 (2 ×
1
+
melting-point apparatus (Electrothermal) and are uncorrected. H
CH), 68.8 (2 × CH), 52.6 (2 × CH ); MS (ES) m/z 283 [M + 1] ,
2
and 13C NMR spectra were recorded in CDCl
at 200 and at 50
+
3
565 [2M + 1] . Anal. (C H N O ‚2HCl‚2H O) C, H. N calcd,
12
18
4
4
2
MHz. TLC was performed on silica F254 (Merck), and detection
was by UV light at 254 nm or by charring with phosphomolybdic
H SO reagent. Column chromatography was carried out on silica
2 4
gel 60 (Merck, 230 mesh).
General Method for the Synthesis of the Diimidazole Deriva-
36.84, 6.18, 14.32; found, 37.01, 6.21, 14.45.
Acknowledgment. This work was supported in part by MEC
Grants SAF2003-01810 and CTQ2006-06505/BQU to P.B.),
Grants SAF 2004-02145, NAN2004-09125-C07-03, FIS C03/
(
(
tives. To a solution of imidazole (4 equiv) and NaNH
in dry DMF (0.8 M), the corresponding ditosylate (1 equiv) was
2
(4 equiv)
10, FIS C03/08, and FIS G03/155 to S.C.), European Com-
munity (MEDITRANS 2006, Integrated EU Project FP6-2004-

4
542 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Brief Articles
Extracellular pH in Rat Brain Gliomas in Vivo by 1H Magnetic
Resonance Spectroscopy: Comparison with Maps of Other Metabo-
lites. Cancer Res. 2001, 61, 6524-6531. Bhujwala, Z.; Artemov,
D.; Ballesteros, P.; Cerd a´ n, S.; Gillies, R.; Solaiyappan, M. Combined
Vascular and Extracellular pH Imaging of Solid Tumors. NMR
Biomed. 2002, 15, 114-119.
NMP-NI-4/IP 026668-2), Grant S-BIO/0179/2006 from the
Community of Madrid to P.B. and S.C., and Grant BQU2003-
00813 to J.L.M.-C.
Supporting Information Available: Experimental procedures
and characterization of 2-4 and figure of combined titration of
IEPA and 3. This material is available free of charge via the Internet
at http://pubs.acs.org.
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8) Gillies, R. J.; Liu, Z.; Bhujwalla, Z. 31P MRS Measurement of
Extracellular pH of Tumor Using 3-Aminopropylphosphonate. Am.
J. Physiol. 1994, 267, C195-C203.
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