Synthesis of [13C6]primaquine

Article abstract of DOI:10.1002/jlcr.3039

In support of a program to identify toxic metabolites of the antimalarial, primaquine, its [¹³C₆] analog was prepared from [ ¹³C₆] anisole in seven steps. Copyright

Full text of DOI:10.1002/jlcr.3039

Research Article
Received 18 January 2013,
Accepted 26 January 2013
Published online 12 March 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3039
Synthesis of [13C6]primaquine
H. M. T. Bandara Herath,a James D. McChesney,c Larry A. Walker,a,b
and N. P. Dhammika Nanayakkaraa
*
In support of a program to identify toxic metabolites of the antimalarial, primaquine, its [13C6] analog was prepared from
[
13C6] anisole in seven steps.
Keywords: primaquine; carbon-13; malaria; 8-aminoquinoline
primaquine for use in hepatocyte, microsomal, or in vivo
metabolism studies.
Introduction
Compounds with isotopic labels (analogs) have been widely
utilized by pharmaceutical and biological scientists to study
biosynthesis, absorption, distribution, metabolism, and excretion
of those materials in animal and human studies.1 Historically, the
use of radiolabeled analogs was the method of choice because
detection and measurement of radioactivity was easily
accomplished. With the evolution of mass spectrometry (MS)
and especially the combined technology of HPLC (liquid
chromatography (LC)) and MS (LC/MS), the detection and
Experimental
General
[
13C6]Anisole and other reagents were purchased from Sigma-Aldrich (St
Louis, MO, USA). NMR spectra were recorded on a Varian-Mercury-plus-
400 or Varian Unity-Inova-600 spectrometer (Agilent Technologies, Palo
Alto, CA, USA) using CDCl3 and methanol-d4 unless otherwise stated.
MS data were obtained from an Agilent Series 1100 SL equipped with
quantitation of compounds labeled with stable isotopes (2H and an ESI source (Agilent Technologies, Palo Alto, CA, USA). Column
13C especially) in now easily and inexpensively accomplished.2
chromatography and preparative thin-layer chromatography (TLC) were
Also, the high sensitivity of modern MS instruments allows
detection of even trace quantities of metabolites without the
inconvenience and safety issues of handling radiolabeled materials.
carried out using Merck silica gel 60 (230–400 mesh) (Sigma-Aldrich
Corp., St Louis, MO, USA) and silica gel GF plates (20 ꢀ 20 cm, thickness
0.25 mm) (Analtech Inc., Newark, DE, USA), respectively.
Primaquine, an 8-aminoquinoline, is the only drug currently
Synthesis of [13C6]primaquine diphosphate
available for radical cure of relapsing malaria caused by
Plasmodium vivax.3 This drug is also used for the prophylaxis of
[13C6]4-Nitroanisole (3)
all types of malaria including P. falciparum4 that causes the most
A solution of HNO3 (70%, 40 mL) and NaNO2 (100 mg) was added to a stirred
solution of [13C6]anisole (1) (8g) in CHCl3 (80 mL) maintaining the temperature
20–25ꢁC. After 1 h, the reaction mixture was poured onto ice, and the organic
layer was separated, washed with water, dried, and evaporated. The resulting
products were chromatographed on silica gel and elution with hexanes/
EtOAc 99.8:02 yielded [13C6]4-nitroanisole (3) as a pale yellow solid. (6.4 g,
57%). m.p. 50–51ꢁC, IR (neat) nmax 1588, 1495, 1328, 1259, 1172, 1104, 1019,
843, 748 cmꢂ1, HRESIMS [M+Na]+ m/z 182.05244 (Calcd for [C[13]6C[12]
H7NO3 +Na]+ 182.0705). 1H-NMR (600 MHz, CDCl3) d ppm 3.88 (d, J=3.6Hz,
3H), 6.92 (d, J=166.8Hz, 2H), 8.16 (d, J= 169.2 Hz, 2H). 13C-NMR (150 MHz,
severe form and a majority of deaths from this disease, especially
among children in Africa.5 In combination with clindamycin, this
compound is also effective in prevention and treatment of
Pneumocystis pneumonia in AIDS patients.6 A major shortcoming
of this drug is its propensity to cause methemoglobinemia and
hemolysis, especially in individuals who are deficient in
glucose-6-phosphate dehydrogenase.7 Other 8-aminoquinolines
such as tafenoquine, which was developed as a safer alternative
to primaquine8 and sitamaquine, a drug candidate in clinical
trials for the treatment of visceral leishmaniasis, also have the CDCl3) d ppm 56.3 (s), 113.9 (tm, J = 65 Hz), 125.8 (tm, J = 67 Hz), 141.4
same drawback.9 There is strong evidence to indicate that
(td, J = 68, 8.2 Hz), 164.5 (td, J= 66, 8.2 Hz).
intraerythrocytic generation of reactive oxygen species by
redox-active metabolites of primaquine is the cause for
aNational Center for Natural Products Research, Research Institute of
Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS
this toxicity.10,11 Primaquine undergoes rapid metabolism in
humans and animal models, and the major metabolite,
carboxyprimaquine, accounts for most of the parent drug.12
Carboxyprimaquine is evidently not the metabolite responsible
for this toxicity.13 Toxic metabolites appear to be very reactive
and formed in small quantities. Hence, their identification in
complex metabolic mixtures remains a major challenge. Drug
candidates labeled with stable isotopes have been effectively
used to track metabolically derived compounds in complex
metabolic mixtures.1,2 Here, we describe the synthesis of [13C6]
38677, USA
bDepartment of Pharmacology, School of Pharmacy, The University of
Mississippi, University, MS 38677, USA
cIronstone Separations, Inc, 147 CR 245, Etta, MS 38627, USA
*Correspondence to: N. P. D. Nanayakkara, National Center for Natural Products
Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy,
The University of Mississippi, MS 38677, USA.
E-mail: dhammika@olemiss.edu
J. Label Compd. Radiopharm 2013, 56 341–343
Copyright © 2013 John Wiley & Sons, Ltd.

H. M. T. B. Herath et al.
[13C6]-p-Anisidine (4)
[13C6]6-Methoxy-8-(1-methyl-4-phthalimidobutylamino)quinoline (10)
Hydrazine hydrate (10 mL) was added to a stirred mixture of [13C6]- A mixture of [13C6]-8-amino-6-methoxyquinoline (8) (2.65 g) and 2-oxo-5-
4-nitroanisole (6.4 g) and Pd/carbon (10%, 800 mg) in ethanol (150 mL). phthalimido pentane (9) (4.49 g) in glacial acetic acid (15 mL) was treated
The reaction mixture was refluxed for 4 h and filtered through celite. with NaBH4 portion wise maintaining the temperature below 20ꢁC until
The filtrate was evaporated, and the product obtained was partitioned
reaction was complete as evidenced by TLC. The reaction mixture was
between ethyl acetate and water. The organic layer was washed, dried,
poured onto ice, basified with aqueous NaOH, and extracted with ethyl
and evaporated to afford [13C6]-p-anisidine (4) (5.1 g, 100%). m.p. 56–58ꢁC, acetate. The organic layer was washed with water, dried, and evaporated
IR (neat) nmax 3455, 3370, 1594, 1514, 1325, 1206, 1038, 842, 740 cmꢂ1
,
to afford a yellow gum. This gum was chromatographed over silica gel
HRESIMS [M+ H]+ m/z 130.0968 (Calcd for [C[13]6C[12]H9NO + H]+ and elution with ethyl acetate/hexanes 10:90 yielded a yellow solid that
130.1057). 1H-NMR (600MHz, CDCl3) d ppm 3.73 (d, J = 3.6Hz, 3H), 6.62 was crystallized from methanol to afford [13C6]-6-methoxy-8-(1-methyl-4-
(dm, J = 164 Hz, 2H), 6.74 (dm, J = 162 Hz, 2H). 13C-NMR (150 MHz, CDCl3) phthalimidobutylamino)quinoline (10) (3.6 g, 62%). m.p. 114–116ꢁC, IR
d ppm 55.7 (s), 114.7 (tm, J = 65 Hz), 116.4 (tm, J = 60 Hz), 140.0 (td, J = 62,
9.3 Hz), 164.5 (tdt, J = 66, 8.2, 1.5 Hz).
(neat) nmax 3393, 1762,1700, 1611, 1514, 1373, 1157, 1057, 815 cmꢂ1
,
HRESIMS: [M + H]+ m/z 396.2182 (Calcd for [C[13]6C[12]17H23N3O3 + H]+
396.2293). 1H-NMR (600 MHz, CDCl3) d ppm 1.27 (d, J = 6.0 Hz, 3H), 1.64
(m, 1H), 1.72–1.87 (m, 3H), 3.64 (heptet, J = 6.6 Hz, 1H), 3.71 (td, 6.6,
1.8 Hz, 2H), 3.85 (s, 3H), 5.98 (d, J = 7.6 Hz, 1H), 6.25 (dm, J = 156 Hz, 1H),
6.28 (dm, J = 161 Hz, 1H), 7.26 (sextet, J = 3.9 Hz, 1H), 7.66 (m, 2H), 7.78
(m, 2H), 7.87 (dd, J = 8.4, 1.2 Hz, 1H), 8.48 (m, 1H). 13C-NMR (150 MHz,
CDCl3) d ppm 20.5 (s), 25.4 (s), 33.9 (s), 37.9 (s), 47.7 (s), 55.2 (s), 91.6
(ddt, J = 71, 58, 3 Hz), 96.7 (tq, J = 70, 3 Hz), 121.8 (s), 123.1 (s), 129.8
(tm, J = 57 Hz), 134.7 (s), 135.3 (ddd, J = 64, 57, 3 Hz), 144.2 (s), 144.9
(tm, J = 68 Hz), 159.3 (tm, J = 70 Hz), 168.4 (s).
[13C6]4-Methoxy-2-nitroacetanilide (5)
A mixture of [13C6]-p-anisidine (5.1 g), acetic acid (20 mL), and acetic
anhydride (15 mL) was stirred at room temperature overnight. The
reaction mixture was cooled on an ice/salt bath and fuming nitric acid
(90%, 3.5 mL) was added dropwise maintaining the temperature below
10ꢁC. After the addition, stirring was continued for 2 h. The reaction mixture
was poured onto ice, and the product was filtered and dried (6.5 g, 72%). m.p.
116–118ꢁC, IR (neat) nmax 3377, 1699, 1507, 1326, 1213, 1026, 832 cmꢂ1
,
HRESIMS: [M + Na]+ m/z 239.0995 (Calcd for [C[13]6C[12]3H10N2O4 +Na]+
239.1005). 1H-NMR (600 MHz, CDCl3) d ppm 2.24 (s, 3H), 3.83 (t, J=2.0Hz,
3H), 7.21 (dm, J= 160 Hz, 1H), 7.64 (dm, J= 166 Hz, 1H), 8.61 (dm, J= 160 Hz,
1H), 13C-NMR (150 MHz, CDCl3) d ppm 25.4 (s), 55.9 (s), 108.4 (tt, J= 72,
2.2 Hz), 123.3 (tm, J=60Hz), 123.8 (tm, J= 63 Hz) 128.5 (tm, J= 72 Hz), 137.0
(tm, J= 71 Hz), 154.9 (tm, J= 64Hz).
[13C6]Primaquine diphosphate (11)
A
mixture of [
13C6]6-methoxy-8-(1-methyl-4-phthalimidobutylamino)
quinoline (10) (2.0 g) and hydrazine hydrate (1 mL) in ethanol (80 mL)
was refluxed for 4 h. The reaction mixture was cooled to room
temperature and filtered. The filtrate was evaporated, and the residue
was partitioned between ethyl acetate and aqueous NaOH (10%). The
organic layer was washed with water, dried, and evaporated. The
resulting gum was dissolved in ethanol (50 mL), treated with H3PO4
(2mL) in ethanol (8 mL), and left overnight under stirring. The product was
filtered and recrystallized from water/methanol to give [13C6]-primaquine
[13C6]4-Methoxy-2-nitroaniline hydrochloride (6)
A mixture of [13C6]4-methoxy-2-nitroacetanilide (5) (6.5 g) and HCl (37%,
10 mL) in ethanol (125 mL) was refluxed for 2 h. The reaction mixture was
evaporated to dryness to afford
[
13C6]4-methoxy-2-nitroaniline
hydrochloride (6) (5.6 g, 89%). m.p. 118–120ꢁC, IR (neat) nmax 3366,
2768, 2571, 1506, 1472, 1333, 1231, 1019, 803 cmꢂ1, HRESIMS: [M + H]+
m/z 175.0876 (Calcd for [C[13]6C[12]H9N2O3 + H]+ 175.1033). 1H-NMR
(600 MHz, CDCl3) d ppm 3.87 (t, J = 2.0 Hz, 3H), 7.21 (dm, 158 Hz, 1H),
7.43 (dm, 164 Hz, 1H), 7.65 (dm, 166 Hz, 1H). 13C-NMR (150 MHz, CDCl3)
d ppm 55.8(s), 108.5 (td, J = 70, 2.3 Hz), 123.6 (tm, J = 63 Hz) 124.2
(tm, J = 60 Hz), 128.4 (tm, J = 59 Hz), 137.1 (t, J = 75 Hz), 155.3 (tm, J = 64Hz).
[13C6]6-Methoxy-8-nitroquinoline (7)
A mixture of [13C6]4-methoxy-2-nitroaniline (6) (5.6 g), sulfomix14 (45 g),
water (15 mL), and glycerol (8 mL) was stirred at 130ꢁC for 4 h under
nitrogen. Additional glycerol (8 mL) was added, and the reaction was
continued for a further 2 h. The reaction mixture was poured onto ice,
basified with 10% aqueous NaOH, and left overnight. The precipitate
was filtered, dissolved in CH2Cl2, and passed through a silica gel plug.
The solvent was evaporated, and the residue was crystallized from
methanol to afford [13C6]6-methoxy-8-nitroquinoline (7) (3.4 g, 61%). m.p.
158–160ꢁC, IR (neat) nmax 1626, 1568, 1532, 1448, 1214, 1021, 835,
785 cmꢂ1, HRESIMS: [M + H]+ m/z 211.0995 (Calcd for [C[13]6C[12]
4H8N2O3 + H]+ 211.1033). 1H-NMR (600 MHz, CDCl3) d ppm 3.62 (s, 3H),
7.31 (brd, J = 162 Hz, 1H), 7.51 (m, 1H), 7.69 (brd, J = 162 Hz, 1H), 8.16
(d, J= 8.4 Hz, 1H), 8.83 (m, 1H). 13C-NMR (150 MHz, CDCl3) d ppm 56.1 (s),
109.5 (ddt, J= 72, 59, 4 Hz), 116.8 (tm, J= 69 Hz), 123.0 (s), 135.0 (ddd, J=74,
56, 2 Hz), 135.2 (s), 148.3 (tm, J= 73 Hz), 149.6 (s), 156.0 (tm, J=70Hz).
[13C6]8-Amino-6-methoxyquinoline (8)
A
mixture of [
13C6]-6-methoxy-8-nitroquinoline (3.4 g), Pd/C (10%,
700 mg) and hydrazine hydrate (3 mL) in ethanol (100 mL) was refluxed
for 2 h. The reaction mixture was filtered through celite, and the filtrate
was evaporated. The product obtained was partitioned between water
and ethyl acetate, and the organic layer was dried over Na2SO4 and
evaporated to give [
13C6]-8-amino-6-methoxyquinoline (8) (2.65 g,
95%) as a yellow gum. This was used in the next reaction without
further purification.
Scheme 1. Preparation of [13C6]primaquine.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 341–343

H. M. T. B. Herath et al.
diphosphate as orange crystals (11) (1.7 g, 74%). m.p. 203–205ꢁC, IR (neat)
n
Acknowledgements
max 1652, 1607, 1552, 1388, 1347, 1034, 937 cmꢂ1, HRESIMS: [M+ H]+ m/z
266.2096 (Calcd for [C[13]6C[12]9H21N3O + H]+ 266.2145). 1H-NMR
(600 MHz, D2O/CD3OD) d ppm 1.21 (brs, 3H), 1.69 (m, 4H), 2.93 (brs, 1H),
3.56 (brs, 1H), 3.79 (s, 3H), 6.48 (dm, J= 160Hz, 1H), 7.56 (d, J= 5.4Hz,
1H), 8.34 (s, 1H), 8.49 (s, 1H). 13C-NMR (150 MHz, CDCl3) d ppm 21.0 (s),
25.9 (s), 34.4 (s), 41.8 (s), 50.9 (s), 57.9 (s), 97.2 (m), 105.4 (tm, J =70Hz),
124.1 (s), 129.8 (t, J= 64Hz), 133.6 (t, J =59 Hz), 142.3 (t, J= 67 Hz), 144.9
(s), 162.2 (t, J =69Hz).
This study has received financial support from Bill & Melinda Gates
Foundation (Phase I Grand Challenges Explorations Award
No. OPP53288) and Army Medical Research & Material Command
Grant No. W81XWH-07-2-0095 and W81XWH-10-2-0059. The
NCNPR is also supported by USDA-ARS scientific cooperative
agreement No. 58-6408-2-0009.
Results and discussion
Conflict of Interest
Previously, in support of studies of primaquine metabolism,
our group prepared 5-deutero-, 7-deutero-, 6-methoxy-D3- and
10-deutero-, 40,40-dideutero-, analogs of primaquine.15,16 Because
of the exchangeability of deuteriums at the 5- and 7-positions
under acidic conditions,17 and the possibility of removal of both
the side chain and the methoxy group under metabolic
conditions,18 all of these analogs have serious limitations for
metabolic studies. Because all putative toxic metabolites of
primaquine contain the quinoline ring, 13C labeling of the
aromatic ring will afford a stable analog of six daltons higher
mass, which should be retained with metabolic changes incurred
in experimental systems. [13C6]-Primaquine diphosphate (11)
was prepared from commercially available [13C6]anisole (1)
(Scheme 1). Anisole was nitrated in a biphasic system in the
presence of sodium nitrite to yield a mixture of [13C6]-2- (2)
and [13C6]-4-nitroanisole (3). They were separated, and [13C6]-4-
nitroanisole was hydrogenated to yield [13C6]-p-anisidine (4).
[13C6]-p-Anisidine was acetylated and nitrated in situ to afford
[13C6]-4-methoxy-2-nitroacetanilide (5). Treatment of this
compound with HCl gave [13C6]-4-methoxy-2-nitroaniline (6)
that was converted to [13C6]-6-methoxy-8-nitroquinoline (7) by
the Skraup reaction. This product was hydrogenated, and the
resulting amine (8) was coupled to the side chain, 2-oxo-5-
phthalimido pentane (9), by reductive amination to give [13C6]-
6-methoxy-8-(1-methyl-4-phthalimidobutylamino)quinoline
(10). Deprotection by removal of the phthalimide group yielded
the free amine that was treated with H3PO4 and crystallized from
water/methanol to yield [13C6]-primaquine diphosphate (11).
The [13C6]-primaquine has proven to have great utility in
helping to track minor metabolites of primaquine in incubates
of human hepatocytes19 and should allow new insights into
the potential role of such metabolites in hematological or other
toxicities of primaquine.
The authors did not report any conflict of interest.
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Copyright © 2013 John Wiley & Sons, Ltd.
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