Evaluation of (S)- and (R)-misonidazole as GPX inhibitors: Synthesis, characterization including circular dichroism and in vitro testing on bovine GPx-1

Article abstract of DOI:10.1002/ardp.201300285

Racemic misonidazole, a radiosensitizer formally used in radiation therapy of cancer and to date still applied, was once reported to exhibit strong inhibitory effects on mouse glutathione peroxidases (GPX). This appeared to qualify misonidazole as a lead structure for the development of novel GPX inhibitors to cause oxidative stress in chemotherapy-resistant tumors. A unique feature of misonidazole as an inhibitor of GPX is the absence of a thiol functionality. Therefore, it was expected to selectively target inhibition devoid of promiscuous interactions with cations and sulfhydryl groups. We synthesized the isomers of misonidazole and analyzed the ability of chiroptical high-performance liquid chromatography (HPLC) to identify the particular enantiomers. Due to the chiral pool synthesis, the assignment of the correct configuration could be verified. Finally, we evaluated both isomers for their inhibitory activities on bovine erythrocyte GPx-1, which is 87% homologous to the human enzyme. Despite the previously reported inhibition of racemic misonidazole on the less homologous mouse GPx-1, we did not find any significant inhibitory activity on the bovine enzyme for either isomer. Though misonidazole appears unlikely to be an inhibitor of human GPx-1 activity, we still spotlight misonidazole as a promising fragment-like lead structure in general. The (S)- and (R)-isomers of misonidazole were synthesized and analyzed for their inhibitory activities on bovine erythrocyte GPx-1, which is 87% homologous to the human enzyme. Although misonidazole was found to be not likely to be an inhibitor of human GPx-1 activity, misonidazole is still spotlighted as a promising fragment-like lead structure in general.

Full text of DOI:10.1002/ardp.201300285

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
1
Full Paper
Evaluation of (S)- and (R)-Misonidazole as GPX Inhibitors:
Synthesis, Characterization Including Circular Dichroism
and In Vitro Testing on Bovine GPx-1
Felix Wilde, Chamseddin Chamseddin, Heidi Lemmerhirt, Patrick J. Bednarski, Thomas Jira, and
Andreas Link
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Ernst-Moritz-Arndt University
Greifswald, Greifswald, Germany
Racemic misonidazole, a radiosensitizer formally used in radiation therapy of cancer and to date still
applied, was once reported to exhibit strong inhibitory effects on mouse glutathione peroxidases (GPX).
This appeared to qualify misonidazole as a lead structure for the development of novel GPX inhibitors
to cause oxidative stress in chemotherapy-resistant tumors. A unique feature of misonidazole as an
inhibitor of GPX is the absence of a thiol functionality. Therefore, it was expected to selectively target
inhibition devoid of promiscuous interactions with cations and sulfhydryl groups. We synthesized the
isomers of misonidazole and analyzed the ability of chiroptical high-performance liquid
chromatography (HPLC) to identify the particular enantiomers. Due to the chiral pool synthesis,
the assignment of the correct configuration could be verified. Finally, we evaluated both isomers for
their inhibitory activities on bovine erythrocyte GPx-1, which is 87% homologous to the human
enzyme. Despite the previously reported inhibition of racemic misonidazole on the less homologous
mouse GPx-1, we did not find any significant inhibitory activity on the bovine enzyme for either isomer.
Though misonidazole appears unlikely to be an inhibitor of human GPx-1 activity, we still spotlight
misonidazole as a promising fragment-like lead structure in general.
Keywords: Circular dichroism / Glutathione peroxidase / In vitro inhibition / Lead structure / Misonidazole
Received: August 1, 2013; Revised: September 24, 2013; Accepted: September 24, 2013
DOI 10.1002/ardp.201300285
Introduction
nodules [5]. Kumar and Weiss [6] reported that misonidazole
also has potent inhibitory activity against glutathione
peroxidase (GPX) from mouse liver. GPX, the first selenocys-
Racemic misonidazole was developed as one of the first
nitroimidazoles used as a radiosensitizer [1], but it showed
dose-limiting neurotoxicity and overall poor results in clinical
trials [2]. The idea of generating nitro radicals in hypoxic
tissue to bring about covalent binding to the DNA was
confirmed [3]. The absence of oxygen also leads to an
accumulation of the drug, making it one of the most
extensively explored radiotracers for hypoxia in tissues [4].
Recently it was even reported to be a promising tool for the
detection of hypoxia in micrometastases, which might help to
delimit malign tumors from noncancerous diseases, like lung
2 2
teine enzyme described [7], metabolizes H O and organic
peroxides to water and alcohols, respectively. To survive redox
stress incurring from permanent proliferation, cancer cells
often raise their levels of GPX [8]. Thus, GPX inhibitors might
offer a novel therapy to treat cancer, especially after they have
become resistant to chemotherapy [9]. Most of the known
inhibitors of GPX, however, contain a thiol functionality,
which is easily oxidized and complexes readily with
ubiquitous metal ions, thus making such compounds poor
drug candidates. Non-thiol substances such as misonidazole
would appear interesting starting points for the development
of novel GPX inhibitors. Thus, we were interested in re-
evaluating the GPX inhibitory activity of misonidazole on
bovine GPx-1, which has a closer homology to the human
enzyme than mouse GPx-1. Here we present the synthesis and
Correspondence: Prof. Andreas Link, Department of Pharmaceutical and
Medicinal Chemistry, Institute of Pharmacy, Ernst-Moritz-Arndt University
Greifswald, 17487 Greifswald, Germany.
E-mail: link@pharmazie.uni-greifswald.de
Fax: þ49 3834 864802
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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F. Wilde et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
characterization of the misonidazole enantiomers as well as
their ability to inhibit bovine GPx-1.
crude product had to be purified by column chromatography;
product-containing fractions were evaporated in vacuo and
freeze-dried to give (S)-misonidazole as an orange oil. The (R)-
enantiomer was prepared in a similar fashion by using (2R)-2-
Results
(methoxymethyl)oxirane (R)-5, but the crude product (R)-6 had
Synthesis of misonidazole enantiomers
to be purified by a second column chromatography with
various solvents since the first run revealed impurities and a
subsequent bulb-to-bulb distillation revealed no success.
We reported a straightforward approach to synthesize the
misonidazole enantiomers independently in a reasonable and
practicable manner. By the combination of steps from three
discrete syntheses [10–12], we obtained the chiral products
from readily available chiral intermediates with overall
slightly lower yields than reported. A key step was the
synthesis of 2-nitroimidazole (4), which turned out to be
accessible only in very poor yields in our hands (4%) in
contrast to the reported outcome (43%) [11]. However, since
the commercially available 2-nitroimidazole is very expensive,
it still proved more reasonable to synthesize this intermediate
from larger quantities of cheap starting material. The
characterization of misonidazole enantiomers has only been
The synthesis of 2-aminoimidazole (3) as hemisulfate salt was
performed in analogy to a previously reported procedure [10].
Accordingly, 2-aminoacetaldehyde dimethyl acetal 2 was
added to O-methylisourea sulfate 1 under an argon atmo-
sphere. The reaction mixture was adjusted to pH of 2–3 by
means of concentrated sulfuric acid, giving a brownish
mixture that further darkened upon stirring at elevated
temperature. After work-up, the hemisulfate salt 3 was
obtained as light brown crystals in good yields (Scheme 1).
The synthesis of 2-nitroimidazole (4) was based on the
procedure reported by Yang and Goldberg [11]. Dilute sulfuric
acid was cooled to 0°C; then copper sulfate and hemisulfate 3
were added slowly to prevent the reaction temperature from
rising above 20°C. The resulting green solution was cooled to
ꢀ
20°C under vigorous stirring. During the careful addition of
1
an aqueous solution of sodium nitrite, brown gas evolved and
the color turned dark blue. The reaction mixture was allowed
to stand at room temperature for 1 week to give a green
solution with some white precipitate. While keeping the
incompletely described in the literature, by H NMR and IR
spectra as well as by optical rotation [12]. We hereby report on
the characterization of the enantiomers of misonidazole by
1
3
C, DEPT, as well as the HSQC NMR for the (R)-misonidazole
temperature below ꢀ8°C, NH
3
gas was slowly bubbled
salt. Furthermore, high-resolution mass spectra (HRMS) and
circular dichroism spectra (CDS) completed the identification
and characterization of the target products. The optical
rotation could not be determined by polarimetry, due to
insufficient amounts of enantiomeric products, and thus had
to be determined by chiral LC-UV and LC-CD.
through the solution until it was adjusted to pH 1. Afterwards
the reaction mixture was stirred for another 3 h at 0°C. The
yield of the yellow precipitate of 4 was insufficient. Therefore,
the product containing reaction mixture was worked up by
extraction. The combined precipitated and extracted product
fractions together gave a small but sufficient amount of
yellow 2-nitroimidazole (4) for the subsequent alkylations
with enantiomerically pure 2-(methoxymethyl)oxiranes 5
Identification of (þ)- and (ꢀ)-enantiomers of
misonidazole using chiral LC-UV and LC-CD
(
Scheme 2).
Since enantiomers have identical chemical and physical
properties, the only way to discriminate and quantify them is
by interaction with polarized light or maybe one day by
nonlinear, resonant, phase-sensitive microwave spectrosco-
py [13]. Therefore, chiroptical high-performance liquid
chromatography (HPLC) detectors to date are the logical
The synthesis of enantiomeric misonidazole was performed
according to the procedure developed by Jin et al. [12]. For the
preparation of (S)-misonidazole (S)-6, 2-nitroimidazole (4) was
added to commercially available (2S)-2-(methoxymethyl)oxir-
ane (S)-5 and sodium carbonate in dry ethanol and heated. The
Scheme 1. Synthesis of 2-aminoimidazole hemisulfate. (I) Four hours at 50°C, then 20°C; addition of concentrated sulfuric acid. (II) Two
hours stirring at 100°C.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Evaluation of (S)- and (R)-Misonidazole as GPx-Inhibitors
3
Scheme 2. Synthesis of misonidazole enantiomers (I) dilute sulfuric acid, 0°C; CuSO
gaseous NH . (II) Na CO , EtOH, 5 h at 60°C.
4
· 5H
2
O in water 20°C. Sodium nitrite, ꢀ10°C, ꢀ20°C
3
2
3
complement to any chiral separation. Circular dichroism
spectroscopy is the technique of choice for determining the
stereochemistry of chiral drugs and may well be used with
chiral columns to verify elution order and to measure the
spectra of the compounds. Selectivity and short response time
also make CD an excellent detector for HPLC [14]. The
influence of working wavelength, isoenantioselective temper-
ature, and mobile phase on the output signal was recently
reviewed [15]. A chiral separation method was necessary to
control the enantioselective synthesis of misonidazole
enantiomers. The chiral LC-UV and LC-CD separation of
misonidazole enantiomers is demonstrated in Fig. 1.
S-(þ)-misonidazole, which could be assigned, since we had
both analytes with known configuration in hand.
In vitro inhibition assay on bovine GPx-1
In order to determine in vitro inhibition of (S)-6, and (R)-6 on
bovine GPx-1, an enzyme assay was used. The method has been
briefly described by Kumar et al. but some details such as the
exact GPX concentration were not reported. We thus
optimized the method to improve the reproducibility of
the literature method [9]. The assay is based on the GPX-
dependent oxidation of glutathione to the disulfide (GSSG),
which is recycled to GSH by glutathione reductase (GR). This
step is coupled to the consumption of NADPH and the
resulting decrease at l ¼ 340 nm is monitored photometrical-
ly. We used 96-well UV-transparent microtiter plates for a
Figure 2 shows the CD spectra of misonidazole enantiomers
as mirror images of each other, indicating their enantiomeric
nature. The CD spectra in Figs. 1 and 2 indicate R-(ꢀ)- and
Figure 1. Identification of misonidazole enantiomers elution order by (a) HPLC-UV chromatogram at 295 nm for the chiral separation of the
racemic mixture as well as for each enantiomer (shown in the small box). (b) HPLC-CD at 352 nm with flow cell for the LC-coupling. Chiralcel
1
OD-H column (5 mm, 250 mm ꢁ 4.6 mm). Mobile phase: hexane/isopropanol 93:7 v/v at 0.6 mL/min flow rate and 40°C column temperature.
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F. Wilde et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Figure 2. CD spectra of (R)- and (S)-misonidazole as mirror images of each other, indicating their enantiomeric nature, recorded for each
enantiomer as a solution in acetonitrile (1 mg/mL) at room temperature using quartz cells (path length of 5 mm).
threefold determination of the remaining GPx-1 activity by
addition of 180 mL of tripotassium phosphate buffer (pH 7.4,
inhibition at 40 mM, neither (R)- nor (S)-misonidazole, nor the
racemic mixture showed significant inhibitory activity at
500 mM (Fig. 3). (S)-Misonidazole free base showed extensive
absorption at 340 nm, which limited the concentration that
could be used in the enzyme assay. The salt formation of the
(R)-enantiomer led to a shift of the spectral maxima, resulting
in lower levels of absorption.
50 mM, 1.1 mM EDTA), 20 mL of bovine erythrocyte GPx
solution (0.75 IU/mL), 30 mL of GR/NADPH mixture (baker’s
yeast GR solution: 2 IU/mL, NADPH solution: 2 mM), 30 mL of
inhibitor stock solution (5 mM), 30 mL of GSH solution
(
2.5 mM). The addition of 30 mL H
2 2
O (1 mM) started the
reaction, which was measured for 12 min at intervals of 10 s at
room temperature. The total volume in the microtiter plate Discussion
was 300 mL and contained 0.1% Triton X-100 to avoid
precipitation of the tested compounds. The final concen-
Analysis of misonidazole enantiomers
trations of the used reagents were as follows: GPx 0.05 U/mL,
GR 0.20 U/mL, GSH 250 mM, NADPH 200 mM, H O 500 mM,
2 2
The analysis of the final products revealed (S)-misonidazole as
a free base, but the (R)-misonidazole salt, which was proved by
in-depth analysis and led to slightly differing purification
processes required for both enantiomers. The reason for this
different behavior of the two enantiomeric products might be
attributed to different qualities of the commercial samples of
the two enantiomeric oxiranes (S)-5 and (R)-5. Due to the
detection cutoff of the used IT-TOF mass spectrometer, the
nature of the low molecular mass counter ion could not be
determined. In order to ensure the identity of (R)-misonidazole
a small aliquot was prepared as free base and the NMR signals
were identical to those of the sample of (S)-misonidazole. The
enantiomeric impurity of both products proved to be
negligibly small (Fig. 1, small box).
misonidazole 500 mM, and mercaptosuccinic acid 40 mM.
Mercaptosuccinic acid and misonidazole were dissolved in
DMSO and hydrogen peroxide in water, respectively. All
other reagents were dissolved in tripotassium phosphate
buffer. The GPx-independent NADPH oxidation was con-
trolled by the substitution of GPx solution with buffer. By
replacing the inhibitor with just DMSO we determined the
full enzymatic activity. The known inhibitor, mercaptosuc-
cinic acid, served as positive control. Due to the calculation of
the quotient of slopes with and without addition of the
inhibitor, the remaining GPX activity was obtained.
Biological activity
Kumar et al. reported that racemic misonidazole inhibits
mouse GPx-1 both in vivo as well as in vitro [6]. Our findings
with bovine GPx-1 show very contrary results. While the
known GPX inhibitor mercaptosuccinic acid caused 83%
Suitability of misonidazole as a lead structure
In general, a good starting point for the lead optimization
study is characterized by fragment-like structures with three-
3
dimensional shape. This comprises sp -rich backbones with
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Evaluation of (S)- and (R)-Misonidazole as GPx-Inhibitors
5
Figure 3. Results of the bovine GPx-1 inhibition assay: NADPH decrease over a 8-min period. Significant inhibition (lower NADPH
vc ao lnu se us m ap r tei o pn r) o wp ao sr t ido en t ae lcl yt e hd i go hn .l y for mercaptosuccinic acid. Due to the strong absorption of (S)-misonidazole free base at l ¼ 340 nm the SD
defined stereochemistry and planarity disrupting structural
elements, frequently present in compounds found in nature.
The advantage that often comes along with such properties is
a rigorous drop in crystallization tendency, which is
advantageous during screening and of special importance
in the later phases of galenics and pharmacokinetics. This also
positively affects the water solubility, as described [16] by the
general solubility equation (GSE), and thus is commonly
beneficial for bioanalytical approaches. We were particularly
interested in the enantiomers of misonidazole, as it meets
many characteristics of a promising lead structure with
tongue in cheek but still quite seriously termed as “slim and
shapely” [17]. It proved to exhibit a reduced affinity for
crystallization, since the melting point of racemic misonida-
zole was reported to be 110–111°C [18] and both enantiomers
of misonidazole were found to be semi-solid, like indicated
before [12]. These low-melting properties illustrate the fact
that crystal packaging of single enantiomers may differ from
those of racemates considerably. In the case of the isolated
tical formulations due to the inherent inability to crystallize
during storage of drug formulations and in the GI tract.
Though our approach accordingly focused on a virtually ideal
and highly promising fragment-like lead structure with “slim
and shapely” design and easily exploitable synthetic diversity,
the biological results restricted further efforts on the specified
target enzyme.
Evaluation of biological results
In contrast to Kumar and coworkers, we were not able to
detect a significant inhibition for bovine GPx-1 at the same
concentration (500 mM). Since a racemate can show activity,
while each enantiomer separately appears inactive [19], we
independently screened the pure enantiomers as well as the
racemic mixture on bovine GPx-1 inhibition. No effect was
detected for both enantiomers, whereas the racemate showed
an average inhibition of 5.3 ꢂ 3.7% at 500 mM by means of
threefold determination, but was not considered an effective
inhibitor as well, since this activity was so low that it remains
unclear whether there exists a synergistic effect between the
two enantiomers. A notable difference between the two
enzyme systems is the use of isolated GPx-1 from bovine
(R)-misonidazole salt one could even speak of an ionic liquid,
since the product represents a semi-solid salt under ambient
conditions. Such ionic liquids are a hot topic in pharmaceu-
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6
F. Wilde et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Figure 4. Alignment of mouse, human, and bovine GPx-1; highlighted in gray: active site, which are all residues containing at least one atom
ꢄ
within 4.5 A of the catalytic tetrad atoms; highlighted in gray and underlined: non-conserved active site amino acids; framed in black: catalytic
ꢅ
tetrad; , identical; :, conserved; ., semiconserved; data compiled on www.uniprot.com.
erythrocytes in our work and the use of mouse liver cytosol
from post-mitochondrial supernatant fractions by Kumar
et al. Still the enzyme activity measured by Kumar and
coworkers should mainly come from the GPx-1 activity in the
mouse liver cytosol, since GPx-2 [20] and GPx-3 [21] have only
minor significance in mouse liver and GPx-4 does not utilize
polarimeter P1000 from A.KRÜSS Optronic. Nuclear magnetic
resonance (NMR) spectra were recorded using an Avance III
1
13
instrument with Ultrashield 400 ( H: 400.2 MHz, C: 100.6 MHz)
from Bruker at 25°C with tetramethylsilane (TMS) as internal
standard, using ppm scale. HRMS were obtained after HPLC with a
mass spectrometer (LC-IT-TOF) from Shimadzu based on a
deviance tolerance limit ꢃ5 ppm. Infrared (IR) spectra were
recorded on a Nicolet IR200 FT-IR from Thermo Electron
Corporation with diamond ATR accessory. Column chromatogra-
phy for the purification of (S)- and (R)-6 was done with silica gel
2 2
H O as a substrate [22]. GPx-5 to GPx-8 are oxidoreductases
not found in the liver and can be ignored in this case. The
homologies between the mouse, bovine, and human proteins
were examined. The overall protein consensus between
bovine and mouse GPx-1 was found to be 84% and at the
active site still only 86%. In contrast, the bovine and human
GPx-1 show overall homology of 87%, but 93% at the active
site. The catalytic tetrad is completely preserved among all
enzymes (Fig. 4). Especially the differences in the protein
primary structure in the active site might suffice for
discriminative three-dimensional orientation of mouse and
bovine proteins and thus account for the presented results.
Since the X-ray crystal structure of the mouse GPx-1 is not yet
available, this remains speculative.
6
2
0 H. O-Methylisourea sulfate 1 was donated by AlzChem AG,
-aminoacetaldehyde dimethyl acetal 2 was purchased from
Acros Organics, and chiral 2-(methoxymethyl)oxiranes 5 were
purchased from ABCR GmbH and Co. KG. All chemicals were used
without further purification. Glutathione, GR, and GPX from
bovine erythrocytes were purchased from Sigma–Aldrich. NADPH
was bought from Carl Roth GmbH.
2
-Aminoimidazole hemisulfate (3)
The synthesis of 2-aminoimidazole was performed according to a
procedure developed by Weinmann et al. [10]. Thus, 0.20 mol
2-aminoacetaldehyde dimethyl acetal 2 was added under inert
argon atmosphere to 0.24 mol O-methylisourea sulfate 1. The
reaction mixture was stirred for 4 h at 50°C, then allowed to cool
at 20°C, and 2.8 mL of concentrated sulfuric acid was added to
reach a pH of 2–3, upon which the reaction mixture turned
brownish. The solution further darkened while it was stirred
for another 2 h at 100°C. Afterwards it was cooled with ice
before slowly introduced over a 12 min period into 600 mL of
ice-cold ethanol. The formed suspension was stirred for an
additional hour at 0°C, then filtered off, washed twice with 20 mL
of ice-cold ethanol, and dried to give 3 as light brown crystals
(14.3 g). Yield: 14.3 g (0.11 mol, 54%), light brown crystals,
purity: 100% (at 220 nm), m.p.: 212.1–229.7°C (decomp.);
Experimental
Synthesis
General
Medium-pressure liquid chromatography was done on silica gel
from Macherey-Nagel (particle size 50–100 mm, 140–270 mesh
ASTM) with Buechi devices C-630, C-601, and C-660 (column
length 40 cm, column diameter 3.5 cm). Melting points were
determined on a hot stage microscope by Kofler PHMK 81/3035
1
13
“Boëtius” (VEB Wägetechnik Rapido) with 16-fold amplification
H NMR (DMSO-d þ TFA-d ) d ¼ 6.85, 7.45, 11.01;
C NMR
6
1
and are uncorrected. The specified purities were determined by
the 100% method of the DAD chromatogram at wavelengths
as indicated. Optical rotation was determined on a manual
(DMSO-d þ TFA-d ) d ¼ 113.0, 113.1, 147.1, 147.2; HRMS [m/z]:
6
1
þ
found 265.0717 (2 ꢁ M þ H SO þ H) ; IR (n): 600, 1036, 1068,
2
4
ꢀ
1
1085, 1666, 3149 cm
.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Evaluation of (S)- and (R)-Misonidazole as GPx-Inhibitors
7
ꢀ
2
-Nitroimidazole (4)
159.8; HRMS [m/z]: found 200.0678 (MꢀH) ; IR (n): 1108, 1269,
ꢀ
1
The synthesis of 2-nitroimidazole was based on the procedure by
Yang and Goldberg [11] and started with 2.0 mol sulfuric acid in
1569, 1674 cm
6
2.5 mL of water, which was stirred and cooled to 0°C. During the
slow addition of 25 mmol CuSO 5H O in 62.5 mL of water and
5 mmol 3 (4.1 g) in 37.5 mL of water the temperature was kept
Chiral LC-UV and LC-CD separation of misonidazole
enantiomers
4
2
2
Circular dichroism CD (Jasco J-710 spectropolarimeter), with flow
cell for the LC-coupling (LCCD-311) connected to Jasco HPLC
system consisting of DG-980-50 degasser and PU-980 intelligent
HPLC-pump, was employed to develop a chiral liquid chro-
matographic method to control the enantioselective synthesis. By
using both UV and CD detectors a good selectivity between
misonidazole enantiomers was achieved. CD spectra of each
enantiomer were recorded as a solution in acetonitrile (1 mg/mL)
at room temperature using quartz cells with path length of 5 mm,
under the following conditions: scan rate (speed) 20 nm/min,
bandwidth 1 nm, response 1 s, step resolution 0.5 nm, accumula-
tion 3, and wavelength range 200–400 nm. The spectra are
average computed over three instrumental scans, and the
intensities are presented in terms of ellipticity values (mdeg). A
Daicel 250 mm ꢁ 4.6 mm chromatographic column packed
below 20°C. The resulting green solution was cooled to ꢀ20°C
and stirred vigorously. During the very careful addition of 0.5 mol
sodium nitrite in 112.5 mL water the temperature was kept below
ꢀ
10°C; brown gas evolved and the color turned into a dark blue.
The reaction mixture was allowed to stand at room temperature
for 1 week to give a green solution with some white precipitate.
The reaction mixture was cooled to ꢀ20°C before NH
3
gas was
initiated under the surface to titrate the solution slowly to pH 1,
while the temperature was kept below ꢀ8°C. Afterwards the
reaction mixture was stirred for another 3 h at 0°C. The yellow
precipitate of 4 was washed twice with 10 mL of water and dried in
vacuo. The aqueous solution was extracted six times with 100 mL
of ethyl acetate and evaporated in vacuo. The combined product
gave 2.40 g of yellow 2-nitroimidazole. Yield: 2.4 g (21.2 mmol,
4
%), yellow crystals, purity: 97% (at 254 nm), m.p.: 245.1–269.7°C
1
1
13
with Chiralcel OD-H [cellulose tris(3,5-dimethyl-phenyl-carba-
(
(
decomp.); H NMR (DMSO-d
DMSO-d
6
) d ¼ 7.41, 7.44, 14.49; C NMR
mate)] coated on 5 mm silica gel spherical particles (Daicel, Japan)
was used for the chiral chromatography of misonidazole
enantiomers. The following mobile phase for the chiral separa-
tion was used: hexane/isopropanol 93:7 v/v at 0.6 mL/min flow
rate and 40°C column temperature. UV detection was performed
in single wavelength mode (295 nm), and circular dichroic
detection was performed at 352 nm.
6
) d ¼ 126.5, 146.1 (the two methine carbon atoms give
only one signal, as confirmed by HSQC and HMBC spectra); HRMS
ꢀ
ꢀ1
[
m/z]: found 112.0155 (MꢀH) ; IR (n): 791, 1100, 1360, 1490 cm
.
(
S)-Misonidazole ((S)-6)
The synthesis of (S)-misonidazole was performed according to the
procedure by Jin et al. [12]. Thus, 5.5 mmol 4 was added to
1
0 mmol of the (2S)-2-(methoxymethyl)oxirane (S)-5, 2.7 mmol
Na CO , and 17 mL of dry ethanol. The reaction mixture was
heated for 5 h at 60°C, followed by further addition of 3.3 mmol of
The authors have declared no conflict of interest.
2
3
(
S)-5 and heated for another 2 h. After addition of 50 mL of water, References
the product was extracted with 30 mL of dichloromethane, dried
over Na SO , und evaporated in vacuo. The crude product was
2
4
[1] C. N. Coleman, Oncologist 1996, 1, 227–231.
further purified by column chromatography; product-containing
fractions were evaporated in vacuo and freeze-dried to give an
orange oil. Mobile phase for MPLC run: petroleum ether/ethyl
acetate (1:3); yield: 230 mg (1.14 mmol, 43%), orange oil, purity:
[
[
[
2] M. M. Poggi, C. N. Coleman, J. B. Mitchell, Curr. Probl. Cancer
001, 25, 334–411.
2
3] G. V. Martin, J. H. Caldwell, J. S. Rasey, Z. Grunbaum,
M. Cerqueira, K. A. Krohn, J. Nucl. Med. 1989, 30, 194–201.
20
1
9
7% (at 254 nm); ½aꢆ ꢀ16.7 (c 0.3, EtOH); H NMR (CDCl
3
) d ¼ 3.40,
D
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2
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D
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3
6
.65, 6.66; C NMR (CDCl
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