Synthesis of [2H]- and [13C]-labeled pyronaridine tetraphosphate - An antimalarial drug

Article abstract of DOI:10.1002/jlcr.1568

The increasing prevalence of strains of Plasmodium falciparum resistant to chloroquine and other antimalarial drugs, necessitates the need for developing novel antimalarial drugs with a potent pharmacological activity. Pyronaridine tetraphosphate (PNDP) is one such drug that is currently undergoing preclinical and clinical trials for use in a chemotherapy treatment of malaria. The present investigation was carried out with the objective of synthesizing carbon-13 [¹³C]- and deuterium [²H]-labeled PNDP for use in studying the ADME and pharmacokinetics of the drug. Here, we present a methodology to synthesize [¹³C]- and [²H]-PNDP using a microwave irradiation technique as this method was found to be more advantageous than the classical method. The labeled compounds thus synthesized had a chemical purity of >99% as determined by HPLC and were also found to be relatively stable up to 3 months when stored under standard conditions. Further they also revealed satisfactory instrumental analysis data. Copyright

Full text of DOI:10.1002/jlcr.1568

Research Article
Received 20 August 2008,
Revised 24 October 2008,
Accepted 3 November 2008
Published online 24 December 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1568
Synthesis of [2H]- and [13C]-labeled
pyronaridine tetraphosphate—an
antimalarial drug
Ã
Sang Hyun Park Yeon Jun Jeong and Kannampalli Pradeep
The increasing prevalence of strains of Plasmodium falciparum resistant to chloroquine and other antimalarial drugs,
necessitates the need for developing novel antimalarial drugs with a potent pharmacological activity. Pyronaridine
tetraphosphate (PNDP) is one such drug that is currently undergoing preclinical and clinical trials for use in a chemotherapy
treatment of malaria. The present investigation was carried out with the objective of synthesizing carbon-13 [13C]- and
deuterium [2H]-labeled PNDP for use in studying the ADME and pharmacokinetics of the drug. Here, we present a
methodology to synthesize [13C]- and [2H]-PNDP using a microwave irradiation technique as this method was found to be
more advantageous than the classical method. The labeled compounds thus synthesized had a chemical purity of 499% as
determined by HPLC and were also found to be relatively stable up to 3 months when stored under standard conditions.
Further they also revealed satisfactory instrumental analysis data.
Keywords: carbon-13; deuterium; pyronaridine tetraphosphate; microwave irradiation technique; antimalarial drug
involving several thousand cases in China, to have a promising
therapeutic value in the treatment of a malarial infection. In a
Introduction
Malaria, a life-threatening disease caused by the Plasmodium
parasites transmitted by female anopheles mosquito, is reported
to be responsible for about 1 to 2 million deaths each year
globally. It is endemic to 90 countries worldwide and affects
nearly 40% of the world’s population, making it one of the most
onerous global health problems.1 Several antimalarial drugs have
been developed over the years and they are being used for
treating malaria with varying degree of success. Chloroquine, a
4-aminoquinoline derivative, emerged during the first part of the
20th century as the drug of choice against malaria.2,3 It proved to
be the most successful antimalarial drug on a worldwide scale
owing to its wide deployment coinciding with the geographical
distribution of Plasmodium and also for its high intrinsic
antiparasitic efficacy and low toxicity.4 However, the malarial
parasite P. falciparum has started to develop a widespread
resistance to chloroquine and this has become a major cause for
concern. Presently, as the strains of P. falciparum have become
resistant to chloroquine and their resistance to other alternative
antimalarial drugs has also been reported,5–9 it has become
imperative to develop new antimalarial drugs and understand
their mechanism of action, an offspring of which is pyronaridine.
Pyronaridine, (2-methoxy-7-chloro-10 [3,5-bis(pyrrolidinyl-1-
methyl-) 4-hydroxy-phenyl] aminobenzyl-(b)1,5-naphthyridine),
is a relatively new antimalarial agent synthesized in the 1970s
at the Institute of Parasitic Disease, Chinese Academy of
Preventive Medicine.10–13 It is a benzonaphthyridine derivative
that has been in use in China for more than 20 years for the
treatment of malaria. The antimalarial activity of pyronaridine has
been attributed to both blood schizontocidal and gametocyto-
clinical trial, pyronaridine was found to be safe and well tolerated
by symptomatic children and it is highly efficacious in regions
where chloroquine resistance is well established. Pyronaridine is
available as a free base and also as pyronaridine tetraphosphate,
which is soluble in water.14 The World Health Organization
(WHO) plans to complete preclinical and clinical trials with the
aim of replacing chloroquine with pyronaridine as the first line of
treatment for malaria, particularly in Africa.
Preclinical studies are usually performed on experimental
animals to obtain information regarding the absorption,
distribution, metabolism and excretion of a drug in addition
to the pharmacological (effective dose) and toxicological (lethal
dose) properties of a drug under investigation. The use of a
radiolabeled drug is considered to be one of the most effective
methods of tracking a drug inside a biological system. It is highly
sensitive and is also effective in detecting the metabolites of a
drug at desired intervals of time after administering a drug
to experimental animals. The present investigation was carried
out with the objective of synthesizing carbon-13 [13C]- and
deuterium [2H]-labeled PNDP so as to use them in studying the
ADME and pharmacokinetics of the drug. Using PNDP-labeled
Radiation Research Division for Biotechnology, Advanced Radiation
Technology Research Institute, Korea Atomic Energy Research Institute, 1266
Shinjeong-dong, Jeongeup, Jeonbuk 580-185, Republic of Korea
*Correspondence to: Sang Hyun Park, Radiation Research Division for
Biotechnology, Advanced Radiation Technology Research Institute, Korea
Atomic Energy Research Institute, 1266 Shinjeong-dong, Jeongeup, Jeonbuk
580-185, Republic of Korea.
cidal activities. Pyronaridine was shown in several field studies E-mail: parksh@kaeri.ac.kr
J. Label Compd. Radiopharm 2009, 52 56–62
Copyright r 2008 John Wiley & Sons, Ltd.

S. H. Park et al.
with stable isotopes such as [13C] and [2H] is relatively safe when product of a drug undergoing a cellular metabolism. In this
compared with [14C]-, [3H]- and [131I]-labeled PNDP as it does not study, we present a methodology to synthesize [13C]- and [2H]-
pose the risk of a radiation exposure. Further, it will also facilitate labeled PNDP using a microwave irradiation technique. Figure 1
in an easy detection of its metabolites while performing ADME shows the expected metabolites of pyronaridine. The position at
and pharmacokinetic studies in in vivo and in vitro systems, due which the isotopes [13C] and [2H] were introduced to replace the
to the advancement in the field of mass spectroscopy (MS). An stable atoms is shown in Figures 2 and 3, respectively, and they
important point to be considered during a radiolabeling of any were determined by considering the structure of the several
drug is the position at which the radioisotope has to be expected primary metabolites of pyronaridine.
introduced into the chemical structure of a drug. The structure
of the expected metabolites of a drug has to be taken into Results and discussion
account before selecting the position of
introduction. Generally, the position of
a
radioisotope
a
The synthesis of pyronaridine tetraphosphate (3), [13C]- and
[2H]-labeled pyronaridine tetraphosphate starting from PNC (1)
is outlined in Schemes 1, 2 and 3, respectively.
radio-
labeling is selected such that the labeling is highly stable and
is not cleaved from the parent compound during the process of
a cellular metabolism when administered to a biological system.
This will result in the metabolites also retaining their radio-
activity, thus facilitating in a continuous tracking of a drug inside
the body. Further, this will enable one to detect not only the
metabolites of a drug but will also allow one to detect the end
Synthesis of pyronaridine tetraphosphate (3) by a classical
method
Paraformaldehyde (20 eq, 2.862 g), pyrrolidine (20 eq, 7.96 mL)
and anhydrous ethanol (10 eq, 16.7 mL) were mixed and refluxed
Glc
OH
O
O
O
O
N
N
N
N
N
N
N
N
OH
NH
N
NH
N
NH
N
NH
N
N
OH
N
OCH
N
OCH
N
OH
3
3
Cl
Cl
Cl
Cl
OH
OH
OH
O
OH
N
N
N
N
N
N
HO
NH
N
NH
N
NH
N
N
OH
N
OCH
N
OCH
3
3
Cl
Cl
Cl
Pyronaridine
OH
OH
OH
O
O
O
O
O
N
N
N
N
N
N
N
N
OH
OH
HO
NH
N
NH
N
NH
N
NH
N
N
OCH
N
OCH
N
OCH
N
OCH
3
3
3
3
Cl
Cl
Cl
Cl
Glc
Glc
O
O
O
O
N
N
N
N
OH
NH
N
NH
N
N
OCH
N
OCH
3
3
Cl
Cl
Figure 1. Expected primary metabolites of pyronaridine.
J. Label Compd. Radiopharm 2009, 52 56–62
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S. H. Park et al.
at 50–601C for 1 h. Then 1.676 g of PNC (2-2-methoxy-7-chloro-
10-(40-hydroxyanilino)-[b]-1,5-naphthyridine, (1) was added to
the mixture and it was stirred for 14–16 h at 45–501C. Upon a
completion of the reaction, distilled water (10 eq, 16.7 mL) was
added to the mixture and stirred at 15–251C for 1 h and then
filtered. The residue was dissolved in anhydrous methanol
(10 eq, 16.7 mL) and stirred at 5–101C for 2 h, then filtered and
washed with excessive methanol and finally dried to yield 40%
of PND (2-methoxy-7-chloro-10 [3.5-bis(pyrrolidinyl-1-methyl-)
4-hydroxyphenyl] aminobenzyl-(b)-1,5-naphthyridine, (2). PND
(2) thus obtained was mixed with distilled water (10 eq, 16.7 mL)
and ortho phosphoric acid (7.5 eq, 3.59 g) and stirred at room
temperature for 30 min. The mixture was filtered and washed
with distilled water. To the crude product, methanol (3 eq,
5.3 mL) was added and stirred at 5–101C for 1 h. Then acetone
was added to the mixture until the PNDP particles were
observed at the bottom, and filtered and washed with acetone
again. The solution of the crude product was mixed with 70%
ethanol (14 eq, 23.38 mL) and ortho phosphoric acid (2 eq,
0.93 g) and stirred at 60–651C for 1 h. The mixture was allowed
to cool to 10–201C, filtered and washed with acetone, then dried
H
13C
N
H
OH
N
H
HN
13C
H
N
OCH3
4H3PO4
Cl
N
5
Figure 2. Structure of [13C]-labeled pyronaridine tetraphosphate showing the
position of [13C]-labeling.
D
N
D
C
N
1
under a vacuum at 501C to yield 90% of 3. H-NMR, 13C-NMR,
OH
C
MS, Fourier transform infrared spectroscopy (FT-IR) and ele-
mental analysis (EA) were performed to identify the recovered
product. The chemical purity of PNDP (3) as determined by a
high performance liquid chromatography (HPLC) was found to
be 499% as the chromatogram revealed a single peak with a
retention time of 26.3 min detected by a UV–V is detector.
N
D
HN
D
OCH3
4H3PO4
Cl
N
Synthesis of pyronaridine tetraphosphate (3) by a micro-
wave irradiation technique
7
Figure 3. Structure of [2H]-labeled pyronaridine tetraphosphate showing the
Microwave has become a useful tool for use in an organic
synthesis, in analytical chemical laboratories and in many
position of [1H]-labeling.
N
N
H
OH
N
OH
OH
HN
H3PO4/Acetone
H2O/EtOH
N
N
N
OMe
HN
N
HN
N
(CH2O)n
N
OMe
N
OMe
Cl
N
4H3PO4
Cl
Cl
1
2
3
Scheme 1. Synthesis of PND (2) and PNDP (3) from PNC (1).
H
13C
OH
H
13C
N
H
N
N
H
H
OH
OH
HN
H3PO4/Acetone
N
OCH3
N
N
H
13CH2O
13C
HN
13C
H O/EtOH
2
HN
H
H
H
N
OCH3
N
OCH3
Cl
N
4H3PO4
Cl
N
Cl
N
1
4
5
Scheme 2. Synthesis of [13C]-PND (4) and [13C]-PNDP (5) from PNC (1).
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J. Label Compd. Radiopharm 2009, 52 56–62

S. H. Park et al.
OH
D
N
D
H
N
D
N
D
C
N
C
N
OH
C
OH
C
HN
H3PO4/Acetone
H2O/EtOH
N
OCH3
N
D
N
D
(CD2O)n
HN
HN
D
D
OCH3
OCH3
Cl
N
4H3PO4
Cl
N
Cl
N
1
6
7
Scheme 3. Synthesis of [2H]-PND (6) and [2H]-PNDP (7) from PNC (1).
[ a
13C]-PNDP (5) and [2H]-PNDP (7) by
chemical manipulations during the process of standardizing
procedures. It is becoming evident that rapid microwave
protocols can be developed for a large number of chemical
transformations requiring heat. The main benefits of performing
reactions under microwave irradiation conditions are the
significant rate enhancements and the higher product
yields that are otherwise not possible by conventional
methods. While different hypotheses have been proposed to
account for the observed rate enhancements under a micro-
wave irradiation, a generally accepted rationalization remains
Synthesis of
microwave irradiation technique
On the basis of the optimum condition obtained from the above
reactions, [13C]-labeled PNDP (5) was synthesized by using
formaldehyde-13C by using a microwave irradiation technique.
A mixture of formaldehyde-13C (2.5 eq, 0.36 g) and pyrrolidine
(5 eq, 1.99 mL) in anhydrous ethanol (2.5 eq, 4.175 mL) was
refluxed at 50–601C for 1 h, then 1 (0.25 eq, 0.419 g) was added
and the mixture was irradiated for 25 min so as to reach a
temperature of 1401C. The reaction yield 45% of 4. 4 (0.1 eq,
0.246 g) was mixed with distilled water (2.5 eq, 4.2 mL) and ortho
phosphoric acid (1.9 eq, 0.89 g) and stirred at room temperature
for 30 min. The mixture was filtered and washed with distilled
water. To the crude product 5, methanol (0.75 eq) was added
and stirred at 5–101C for 1 h. The mixture was filtered and
washed with acetone. The solution of crude product 5 was
mixed with 70% ethanol (3.5 eq, 5.84 mL) and ortho phosphoric
acid (0.5 eq, 0.223 g) and stirred at 60–651C for 1 h. The mixture
was allowed to cool at 10–201C, filtered and washed with
acetone, then dried under a vacuum at 501C to yield 96% of 5. In
order to investigate the structural change upon a proton,
carbon exchange due to a [13C]-labeling, 1H and 13C-NMR
analyses were performed and it was foundthat there was no
significant structural difference between the labeled and
unlabeled compounds. It can be seen from the 13C-NMR
spectrum of 5 that owing to a C–13C coupling, the signals of
the aromatic carbons occur as a multiplet with spans of
122–123 ppm. 13C-labelling causes the signals in the 13C-NMR
spectra of 5 and their corresponding one in 3 and allows for a
correct assignment of the chemical shifts to the carbon atoms.
The signal of the 13C-labeled atoms in 5 was observed at m/z
53.2 and for 3 the signals were found to be too high in the MS
elusive.15 Regardless of the origin/existence of
a special
microwave effect, microwave-enhanced chemistry can be
extremely efficient and is applicable to a broad range of
practical synthesis.16,17
In this paper, a microwave irradiation technique was used in
the synthesis of [13C]-labeled PNDP and [2H]-labeled PNDP for a
reduction of the chemical reaction time and a high product
yield. Purification of 2 and synthesis of 3 were performed
essentially in the same manner described for the classical
method. In both the classical and microwave irradiation
methods, after purification procedures, the products provided
1
satisfactory H-NMR, 13C-NMR, MS, IR data and element analysis
data.
Paraformaldehyde (5 eq, 0.716 g), pyrrolidine (5 eq, 1.99 mL)
and anhydrous ethanol (2.5 eq, 4.175 mL) were mixed and
refluxed at 50–601C for 1 h. Then 1 (0.25 eq, 0.419 g) was added
to the mixture and it was irradiated in a microwave reactor for
25 min at 1401C. On completion of the reaction, distilled water
(2.5 eq, 4.175 mL) was added to the mixture and stirred at
15–251C for 1 h, then filtered. The residue was dissolved in
anhydrous methanol (2.5 eq, 4.175 mL) and stirred at 5–101C for
2 h, then filtered and washed with excessive methanol and
dried. The residue was dissolved in a mixture of dimethyl
sulfoxide (0.25 eq, 1.275 mL) and dichloromethane (1.275 mL).
The mixture was filtered, washed using methanol and dried at
RT to yield 45% of 2. After obtaining 2 (0.1 eq, 0.246 g) was
mixed with distilled water (2.5 eq, 4.2 mL) and ortho phosphoric
acid (1.9 eq, 0.89 g) and stirred at room temperature for 30 min.
The mixture was filtered and washed with distilled water. To the
crude product, methanol (0.75 eq, 0.325 mL) was added and
stirred at 5–101C for 1 h. Then acetone was added to the mixture
and stirred until PNDP particles were observed and filtered, and
washed with acetone again. The crude product solution was
mixed with 70% ethanol (3.5 eq, 5.84 mL) and ortho phosphoric
acid (0.5 eq, 0.23 g) and stirred at 60–651C for 1 h. The mixture
was allowed to cool to 10–201C, filtered and washed
with acetone, then dried under a vacuum at 501C to yield
95% of 3.
1
analysis. From the H-NMR spectrum of 5 it was observed that,
1
owing to strong H-13C coupling, the H-NMR signals of 5 were
split into two signals at 4.0–4.5 ppm.
[2H]-labeled pyronaridine tetraphosphate (7) was synthesized
by using paraformaldehyde-d2 (98%, Aldrich) by a microwave
irradiation technique. A mixture of paraformaldehyde-d2 (2.5eq,
0.36 g) and pyrrolidine (5 eq, 1.99 mL) in anhydrous ethanol (2.5 eq,
4.175 mL) was refluxed at 50–601C for 1 h, then 1 (0.25 eq, 0.419 g)
was added and the mixture was irradiated for 25 min so as to
reach a temperature of 1401C. The product yielded 45% of 6. After
obtaining 6 it was mixed with distilled water (2.5 eq, 4.2 mL) and
ortho phosphoric acid (1.9 eq, 0.89 g) and stirred at room
temperature for 30 min. The mixture was filtered and washed with
distilled water. To the crude product, methanol (0.75 eq, 0.325 mL)
was added and stirred at 5–101C for 1 h. Then acetone was added
J. Label Compd. Radiopharm 2009, 52 56–62
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S. H. Park et al.
Figure 4. HPLC chromatograms of synthesized compounds: (a) PNDP 3; (b) [13C]-PNDP 5; (c) [2H]-PNDP 7.
to the mixture until PNDP particles were observed and filtered,
and washed with acetone again. The crude product solution was
Experimental
Materials and methods
mixed with 70% ethanol (3.5 eq, 5.84 mL) and ortho phosphoric
acid (0.5 eq, 0.223 g) and stirred at 60–651C for 1 h. The mixture
was allowed to cool at 10–201C, filtered and washed with
acetone, then dried under a vacuum at 501C to yield 90% of 7.
In order to investigate the structural change upon a proton,
carbon exchange upon a [2H]-labeling, 1H and 13C-NMR analyses
were performed and it was found that there was no significant
structural difference between the labeled and unlabeled
compounds. It was observed from the NMR data of 7 that the
signal for the methyl group that disappeared at 4.22 ppm by a
[2H]-labeling effect was found to be absent in 3. The mass
spectrum of 3 was found to reveal a peak at m/z 518. The peak
of the 13C-pyronaridine ion of 5 was found at m/z 520.
[2H]-labeled pyronaridine ion of 7 was founded at m/z 522.
These results reveal that the isotope was labeled successfully to
pyronaridine and pyronaridine tetraphosphate.
2-Methoxy-7-chloro-10-(40-hydroxyanilino)-[b]-1, 5-naphthyri-
dine (PNC, 1) was obtained from Shinpoong Pharmaceutical,
Co., Ltd., Seoul, Korea. All other reagents were obtained from
Sigma Chemical Co., USA and were used as such without any
further purification.
All chromatographic separations were monitored by TLC
analyses and performed using glass plates precoated with
0.25 mm 230–400 mesh silica gel impregnated with a fluores-
cence indicator (254 nm). Solvent removal was accomplished
with an aspirator pressure using a rotary evaporator. Microwave
assisted organic synthesis was carried out using a microwave
reactor (Initatior 2.0, 400 W, Biotage, Sweden). H NMR and 13C
1
NMR spectra were recorded with a JNM-ECA500 (JEOL Ltd.)
operating at a 500 MHz frequency for 1H-NMR and at a 125 MHz
frequency for 13C-NMR. IR spectra were recorded on a VERTEX-
70 (BRUKER). MS data were obtained on a JMS-700 Mstation
(JEOL Ltd.). EA data were obtained from Flash EA 1112 Series (CE
Instruments).
The chemical purities of PNDP (3), [13C]- and [2H]-labeled
PNDP (5, 7) were determined by performing an HPLC (Agilent
1200 Series RRLC, Agilent technologies) using an optimaPAK C18
(5 mm; 4.6 Â 150 mm) column. HPLC solvents consisted of
potassium phosphate buffer: acetonitrile (9:1) (solvent A) and
potassium phosphate buffer: acetonitrile (2:8) (solvent B). One
liter of the buffer (pH 2) consisted of KH2PO4 (1.36 g) and
1-octanesulfonic acid sodium salt hydrate (2.163 g). HPLC
gradient: 0–17 min: a linear gradient to 70% A/30% B from
70% A/30% B; 17–25 min: a linear gradient to 60% A/40% B from
The chemical purity of 5 and 7 were determined by HPLC. The
retention times of PNDP (3), [13C]-PNDP (5) and [2H]-PNDP (7)
obtained from a UV–V is detector were found to be the same at
26.3 min as can be seen in Figure 4.
Stability test of [13C]-labeled PNDP and [2H]-labeled PNDP
[13C]- and [2H]-labeled PNDP (5, 7) were tested for their stability
by analyzing their compounds by HPLC at appropriate time
intervals, with 5 and 7 being stored at 41C for up to 3 months.
HPLC chromatograms taken before and after storage at 41C for
up to 3 months revealed a single peak of 499% chemical purity
with the same retention time indicating 5 and 7 to be highly
stable under standard storage conditions.
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J. Label Compd. Radiopharm 2009, 52 56–62

S. H. Park et al.
70% A/30% B; 25–30 min: a linear gradient to 60% A/40% B from Synthesis of pyronaridine tetraphosphate (PNDP, 3) from
pyronaridine (PND, 2)
60% A/40% B; 30–31 min: a linear gradient to 70% A/30% B from
60% A/40% B. The flow rate was maintained at 1 mL/min and the
column temperature was maintained at 401C.
Appropriate amounts of 2 (0.1 eq, 0.246 g), distilled water and
ortho phosphoric acid were mixed and stirred at RT for 30 min.
The molar composition of the mixture was: 0.1 PND: 1.9 ortho
phosphoric acid: 2.5 H2O. On completion of the reaction, the
mixture was filtered and washed with distilled water. Methanol
was (0.75 eq, 0.325 mL) added to the crude product and stirred
at 5–101C for 1 h. Then a sufficient amount of acetone was
added to the mixture until PNDP particles were observed,
filtered and washed with acetone again. The residue was added
to a mixture solution of 70% ethanol (3.5 eq, 5.84 mL) and ortho
phosphoric acid (0.5 eq, 0.223 g) and stirred at 60–651C for 1 h.
Then it was filtered and washed with acetone. The solid sample
was dried under a vacuum at 501C to yield 95% of 3. IR (KBr): n
3406, 2949, 1631, 1577, 1551, 1526, 1489, 1459, 1389, 1350,
Synthesis of pyronaridine (PND, 2) from 2-methoxy-7-
chloro-10-(40-hydroxyanilino)-[b]-1, 5-naphthyridine (PNC, 1)
by a classical method
A 250 mL round bottom flask containing a magnetic stir bar was
washed with double distilled water (DDW) under an ultrasonica-
tion. Then appropriate amounts of paraformaldehyde, pyrroli-
dine and anhydrous ethanol were added, in that order, and
refluxed at 50–601C for 1 h. 1 (1 eq, 1.676 g) was added to
complete the reaction mixture and stirred at 45–551C for
12–14 h. The final molar composition of any given synthesis
solution was: 1.00 PNC: 20 paraformaldehyde: 20 pyrrolidine: 10
ethanol. On completion of the reaction, distilled water (10 eq,
16.7 mL) was added to the mixture and stirred at 15–251C for
1 h, then filtered. The residue was dissolved in anhydrous
methanol (10 eq, 16.7 mL) and stirred at 5–101C for 2 h, then
filtered and washed with excessive methanol. The residue was
dissolved in a mixture of dimethyl sulfoxide (3 eq, 5.1 mL) and
dichloromethane (3 eq, 5.1 mL). The mixture was filtered,
thoroughly washed using methanol and finally dried to yield
40% of 2. IR (KBr): n 3307, 2965, 2872, 2806, 1621, 1571, 1519,
1493, 1465, 1403, 1377, 1305, 1271, 1094, 1068, 1000, 927, 867,
1
1285, 1091, 991 cmÀ1; H NMR (D2O):d 1.87 (s, 4H, CH2), 2.00 (s,
4H, CH2), 3.03 (s, 4H, CH2), 3.41 (s, 4H, CH2), 3.88 (s, 3H, OCH3),
4.32 (s, 4H, ArCH2), 7.10 (d, 1H, ArH), 7.38 (t, 2H, ArH), 7.42 (s,
12H, ArH), 7.73 (s, 1H, ArH), 8.02 (d, 1H, ArH); 13C NMR (D2O): d
22.4, 53.2, 54.0, 54.5, 111.3, 118.5, 122.1, 123.2, 124.9, 126.1,
127.4, 130.8, 131.3, 132.5, 132.7, 139.7, 140.7, 152.2, 154.5, 160.9;
MS m/z 518.4 [M1À(H3PO4)]. Calcd for C29H32ClN5O2: C 38.27, H
4.87, N 7.70. Found: C 38.04, H 4.86, N 7.39.
Synthesis of [13C]-labeled pyronaridine tetraphosphate
1
834, 808 cmÀ1; H NMR (CD3OD):d 1.81 (t, 8H, CH2), 2.61 (s, 8H,
CH2), 3.74 (s, 4H, ArCH2), 4.05 (s, 3H, OCH3), 7.01 (s, 2H, ArH), 7.27
(d, 1H, ArH), 7.64(d, 2H, ArH), 7.87 (s, 2H, ArH), 8.13 (d, 2H, ArH);
13C NMR (CD3OD): d 23.0, 53.1, 55.3, 119.5, 124.1, 124.7, 126.3,
127.0, 127.9, 135.2, 138.7, 141.9, 148.4, 154.6, 160.3; MS m/z
518.24 [M1]. Calcd for C29H32ClN5O2: C 67.23, H 6.23, N 13.52.
Found: C 66.89, H 6.33, N 13.27.
[13C]-labeled pyronaridine tetraphosphate (5) was synthesized
by using formaldehyde-13C (20%, Aldrich) by a microwave
irradiation technique. A 250 mL round bottom flask containing a
magnetic stir bar was washed with DDW under an ultrasonica-
tion. Then appropriate amounts of formaldehyde-13C, pyrroli-
dine and anhydrous ethanol were added, in that order, and
refluxed at 50–601C for 1 h. 1 (0.25 eq, 0.419 g) was added to
complete the reaction mixture and it was irradiated in a
microwave reactor for 25 min at 1401C. The final molar
composition of any given synthesis solution was; 1.00 PNC: 10
[13C]-formaldehyde: 20 pyrrolidin: 10 ethanol. On completion of
the reaction, distilled water (2.5 eq, 4.175 mL) was added to the
mixture and stirred at 15–251C for 1 h, then filtered. The residue
was dissolved in anhydrous methanol (2.5 eq, 4.175 mL) and
stirred at 5–101C for 2 h, then filtered and washed with excessive
methanol. The residue was dissolved in a mixture of dimethyl
sulfoxide (0.25 eq, 1.275 mL) and dichloromethane (0.75 eq,
1.275 mL). The mixture was filtered, thoroughly washed using
methanol and finally dried to yield 45% of 4. Appropriate
amounts of 4 (0.1 eq, 0.246 g), distilled water and ortho
phosphoric acid were mixed and stirred at RT for 30 min. The
molar composition of the mixture was: 0.1 PND: 1.9 ortho
phosphoric acid: 2.5 H2O. On completion of the reaction, the
mixture was filtered and washed with distilled water. Methanol
(0.75 eq, 0.325 mL) was added to the crude product and stirred
at 5–101C for 1 h. Then a sufficient amount of acetone was
added to the mixture until PNDP particles were observed,
filtered and washed with acetone again. The residue was added
to a mixture solution of 70% ethanol (3.5 eq, 5.84 ml) and ortho
phosphoric acid (0.5 eq, 0.223 g) and stirred at 60–651C for 1 h.
Then it was filtered and washed with acetone. The solid sample
Synthesis of pyronaridine (PND, 2) from 2-methoxy-7-
chloro-10-(40-hydroxyanilino)-[b]-1,5-naphthyridine (PNC, 1)
by a microwave irradiation technique
A 250 mL round bottom flask containing a magnetic stir bar was
washed with DDW under an ultrasonication. Then appropriate
amounts of paraformaldehyde, pyrrolidine and anhydrous ethanol
were added, in that order, and refluxed at 50–601C for 1h. 1
(0.25 eq, 0.419 g) was added to complete the reaction mixture and
it was irradiated in a microwave reactor for 25 min at 1401C. The
final molar composition of any given synthesis solution was: 1.00
PNC: 20 paraformaldehyde: 20 pyrrolidine: 10 ethanol. On
completion of the reaction, distilled water (2.5 eq, 4.175 mL) was
added to the mixture and stirred at 15–251C for 1 h, then filtered.
The residue was dissolved in anhydrous methanol (2.5eq,
4.175 mL) and stirred at 5–101C for 2 h, then filtered and washed
with excessive methanol. The residue was dissolved in a mixture of
dimethyl sulfoxide (0.25 eq, 1.275 mL) and dichloromethane
(0.25 eq, 1.275 mL). The mixture was filtered, thoroughly washed
using methanol and finally dried to yield 45% of 2. IR (KBr): n 3307,
2962, 2780, 1620, 1559, 1519, 1494, 1468, 1402, 1377, 1301, 1273,
1096, 1070, 1000, 929, 867, 836, 808 cmÀ1; 1H NMR (CD3OD):d 1.81
(t, 8H, CH2), 2.61 (s, 8H, CH2), 3.75 (s, 4H, ArCH2), 4.05 (s, 3H, OCH3),
7.01 (s, 2H, ArH), 7.28 (d, 1H, ArH), 7.65(d, 2H, ArH), 7.87 (s, 2H,
ArH), 8.13 (d, 2H, ArH); 13C NMR (CD3OD): d 23.0, 53.1, 55.3, 119.5,
124.1, 124.7, 126.3, 127.0, 128.1, 135.2, 138.7, 145.9, 148.4; MS m/z
518.24 [M1]. Calcd for C29H32ClN5O2: C 67.23, H 6.23, N 13.52.
Found: C 66.36, H 6.13, N 13.06.
was dried under
a
vacuum at 501C to yield
95% of 5. IR (KBr): n 3400, 2955, 1635, 1578, 1552, 1528,
1
1490, 1460, 1389, 1350, 1285, 1093, 991 cmÀ1; H NMR (D2O):d
J. Label Compd. Radiopharm 2009, 52 56–62
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

S. H. Park et al.
1.87 (s, 4H, CH2), 2.01 (s, 4H, CH2), 3.04 (s, 4H, CH2), 3.42 (s, 4H, reaction time and also increasing the yield of product formed.
CH2), 3.88 (s, 3H, OCH3), 4.18 (s, 4H, ArCH2), 4.47 (s, 4H, ArCH2), The labeled compounds thus synthesized had a chemical purity
7.11 (d, 1H, ArH), 7.35 (t, 2H, ArH), 7.43 (s, 2H, ArH), 7.74 (s, 1H, of 499% as determined by an HPLC and were also found to be
ArH), 8.03 (d, 1H, ArH); 13C NMR (D2O): d 22.4, 53.4, 54.0, 54.5, relatively stable up to 3 months when stored under standard
111.3, 118.5, 121.9, 122.1, 123.2, 124.9, 126.0, 127.3, 130.7, 131.2, conditions. They also revealed satisfactory 13C and 1H-NMR
132.5, 132.5, 139.6, 140.7, 152.2, 154.4, 160.8; MS m/z 520.4 [M1 spectrum and the labeled compounds showed different
À(H3PO4)]. Calcd for C29H32ClN5O2: C 38.27, H 4.87, N 7.70. molecular weights when compared with their corresponding
Found: C 32.86, H 4.52, N 6.39.
unlabeled reveled as determined by a mass spectroscopy. [13C]-
and [2H]-labeled PNDP can be used in preclinical studies to
determine the absorption, distribution and metabolism, as well
as excretion studies and pharmacokinetics of PNDP. As the
PNDP is labeled with [13C] and [2H] at a desired position they will
be highly stable inside the biological system and will facilitate
continuous detection of a drug and its subsequent metabolites.
Further, the use of PNDP labeled with stable isotopes, like [13C]
and [2H] is relatively safe because unlike unstable isotopes like
[14C]-, [3H]- and [131I]-labeled PNDP they do not pose the threat
of a radiation exposure. In addition to that, the advancements
made in the field of MS will facilitate in an easy detection of their
metabolites, while performing ADME and pharmacokinetic
studies using in vivo and in vitro systems.
Synthesis of [2H]-labeled pyronaridine tetraphosphate
[2H]-labeled pyronaridine tetraphosphate (7) was synthesized by
using paraformaldehyde-d2 (98%) by a microwave irradiation
technique. A 250 mL round bottom flask containing a magnetic
stir bar was washed with DDW under an ultrasonication. Then
appropriate amounts of paraformaldehyde-d2, pyrrolidine and
anhydrous ethanol were added, in that order and refluxed at
50–601C for 1 h. 1 (0.25 eq, 0.419 g) was added to complete the
reaction mixture and it was irradiated in a microwave reactor for
25 min at 1401C. The final molar composition of any given
synthesis solution was: 1.00 PNC: 10 [2H]-labeled paraformalde-
hyde: 20 pyrrolidine: 10 ethanol. On completion of the reaction,
distilled water (2.5 eq, 4.175 g) was added to the mixture and
stirred at 15–251C for 10 h, then filtered. The residue was
dissolved in anhydrous methanol (2.5 eq, 4.175 g) and stirred at
5–101C for 2 h, then filtered and washed with excessive
methanol. The residue was dissolved in a mixture of dimethyl
sulfoxide (0.75 eq, 1.275 ml) and dichloromethane (0.75 eq,
1.275 mL). The mixture was filtered, thoroughly washed using
methanol and finally dried to yield 45% of 6. Appropriate
amounts of 6 (0.1 eq, 0.246 g), distilled water and ortho
phosphoric acid were mixed and stirred at RT for 30 min. The
molar composition of the mixture was: 0.1 [2H]-PND: 1.9 ortho
phosphoric acid: 2.5 H2O. On completion of the reaction, the
mixture was filtered and washed with distilled water. Methanol
(0.75 eq, 0.325 mL) was added to the crude product and stirred
at 5–101C for 1 h. Then a sufficient amount of acetone was
added to the mixture until PNDP particles were observed,
filtered and washed with acetone again. The residue was added
to a mixture solution of 70% ethanol (3.5 eq, 5.84 mL) and ortho
phosphoric acid (0.5 eq, 0.223 g) and stirred at 60–651C for 1 h.
Then it was filtered and washed with acetone. The solid sample
was dried under a vacuum at 501C to yield 95% of 7. IR (KBr): n
3394, 3946, 1632, 1577, 1551, 1527, 1488, 1461, 1389, 1350,
Acknowledgement
This work was supported by the nuclear research and
development project from Korea Ministry of Education, Science
and Technology.
References
[1] H. M. Al Arishi, F. E. A. Ahmed, L. A. Al Bishi, Trans. R. Soc. Trop. Med.
Hyg. 2001, 95, 439–440.
[2] K. Yonemitsu, A. Koreeda, K. Kibayashi, P. Ng’walali, M. Mbonde,
J. Kitinya, S . Tsunenari, Leg. Med. 2005, 7, 113–116.
[3] P. G. Bray, S. Deed, E. Fox, M. Kalkanidis, M. Mungthin, L. W. Deady,
L. Tilley, Biochem. Pharmacol. 2005, 70, 1158–1166.
[4] D. Warhurst, Drug Resistance Updates 2001, 4, 141–144.
[5] C. P. Sanchez, W. D. Stein, M. Lanzer, Trends Parasitol. 2007, 23,
332–339.
[6] K. Kolaczinski, N. Durrani, S . Rahim, M. Rowland, Trans. R. Soc. Trop.
Med. Hyg. 2007, 101, 1081–1087.
[7] S. Gemma, G. Kukreja, G. Campiani, S. Butini, M. Bernetti, B. P. Joshi,
L. Savini, N. Basilico, D. Taramelli, V. Yardley, A. Bertamino,
E. Novellino, M. Persico, B. Catalanotti, C. Fattorusso, Bioorg. Med.
Chem. Lett. 2007, 17, 3535–3539.
[8] G. D. Li, Med. Hypotheses 2007, 68, 332–334.
[9] A. Radfar, A. Diez, J. M. Bautista, Free Radical Biol. Med. 2008, 44,
2034–2042.
[10] E. I. Elueze, S. L. Croft, D. C. Warhurst, J. Antimicrob. Chemother.
1996, 37, 511–518.
[11] S. D. Jayaraman, S. Ismail, N. K. Nair, V. Navaratnam, J. Chromatogr.
B 1997, 690, 253–257.
[12] C. P. Babalola, G. K. E. Scriba, A. Sowunmi, O. A. Alawode,
J. Chromatogr. B 2003, 795, 265–272.
1
1284, 1092, 990 cmÀ1; H NMR (D2O):d 1.87 (s, 4H, CH2), 2.00 (s,
4H, CH2), 3.04 (s, 4H, CH2), 3.42 (s, 4H, CH2), 3.87
(s, 3H, OCH3), 4.32 (s, 4H, ArCH2), 7.11 (d, 1H, ArH), 7.38 (t, 2H,
ArH), 7.42 (s, 2H, ArH), 7.72 (s, 1H, ArH), 8.04 (d, 1H, ArH); 13C NMR
(D2O): d 22.4, 30.2, 53.9, 54.5, 111.2, 118.5, 122.1, 123.2, 125.0,
126.0, 127.3, 130.7, 131.2, 132.4, 132.7, 139.5, 140.8, 152.1, 154.5,
160.8; MS m/z 522.5 [M1À(H3PO4)]. Calcd for C29H32ClN5O2:
C 38.27, H 4.87, N 7.70. Found: C 33.75, H 4.50, N 6.71.
[13] S. Ramanathan, S. Karupiah, N. K. Nair, P. L. Olliaro, V. Navaratnam,
W. H. Wernsdorfer, S. M. Mansor, J. Chromatogr. B 2005, 824, 45–50.
[14] C. Chang, L. H. Tang, C. Jantanavivat, Trans. R. Soc. Trop. Med. Hyg.
1992, 86, 7–10.
Conclusion
A method for synthesizing stable [13C]- and [2H]-labeled PNDP,
an antimalarial drug, using a microwave irradiation, has been
described in this study. The use of microwave irradiation
[15] L. Perreux, A. Loupy, Tetrahedron 2001, 57, 9199–9223.
¨
[16] P. Lidstrom, J. Tierney, B. Wathey, J. Westman, Tetrahedron 2001,
57, 9225–9283.
facilitated the use of high temperature thereby decreasing the [17] M. Larhed, A. Hallberg, Drug Discovery Today 2001, 6, 406–416.
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 56–62