Synthesis and antileishmanial activity of novel buparvaquone oxime derivatives

Article abstract of DOI:10.1016/j.bmc.2004.04.032

Novel oxime derivatives (2, 3 and 5) of buparvaquone (1) and O-methyl-buparvaquone (4) were synthesized and their in vitro activities against Leishmania donovani, the causative agent of visceral leishmaniasis (VL), were determined. Buparvaquone-oxime (2)

Full text of DOI:10.1016/j.bmc.2004.04.032

Bioorganic & Medicinal Chemistry 12 (2004) 3497–3502
Synthesis and antileishmanial activity of novel
buparvaquone oxime derivatives
a,
Antti Mantyla, Jarkko Rautio, Tapio Nevalainen, Jouko Vepsalainen,
a
a
b
*
€
€
Risto Juvonen, Howard Kendrick, Tracy Garnier, Simon L. Croft and Tomi Jarvinen
€
c
d
d
d
a
€
aDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
bDepartment of Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
cDepartment of Pharmacology and Toxicology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
dDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine,
Keppel Street, London, WC1E 7HT, UK
Received 16 January 2004; accepted 27 April 2004
Abstract—Novel oxime derivatives (2, 3 and 5) of buparvaquone (1) and O-methyl-buparvaquone (4) were synthesized and their in
vitro activities against Leishmania donovani, the causative agent of visceral leishmaniasis (VL), were determined. Buparvaquone-
oxime (2) was also studied as a bioreversible prodrug structure of buparvaquone (1). Buparvaquone-oxime (2) released bu-
parvaquone (1) in vitro when it was incubated with induced rat liver microsomes, which suggests that the oxime-structure is a useful
prodrug template for developing novel prodrugs of buparvaquone and other hydroxynaphthoquinones. Moreover, the formation of
NO2ꢀ, formed via oxidation of NO, was confirmed during the bioconversion. The release of NO from buparvaquone-oxime (2) may
provide an additional therapeutic effect in the treatment of leishmaniasis. Buparvaquone-oxime (2) and buparvaquone-O-methyl-
oxime (3) demonstrated moderate activity against amastigotes of the Leishmania species that causes VL. However, the studied
oximes (2, 3) most probably did not release buparvaquone (1) and NO during the present in vitro experiment. Further in vivo studies
are needed to verify the biological activity of buparvaquone-oximes in the treatment of leishmaniasis.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
toxicity, resistance and variable efficacy. Although new
drugs are on clinical trial,2 there remains an urgent need
for improved antileishmanials.
Leishmaniasis is a widespread parasitic disease that is
caused by protozoan parasites of the genus Leishmania
in tropical and subtropical areas in both the old and new
worlds.1 This disease occurs in two major forms; cuta-
neous (CL) and visceral leishmaniasis (VL). The para-
sitic protozoa Leishmania donovani is the causative agent
of VL, and species that cause CL are L. major, L. tropica
and L. aethiopica L. braziliensis, L. panamensis, L. am-
azonensis and L. mexicana.2 The most widely used
treatment for leishmaniasis over the past 50 years has
been pentavalent antimonial compounds.1;2 These drugs
are effective, but they have several disadvantages such as
the need for parenteral administration at high dosages,
long duration of therapy (several weeks), considerable
Some hydroxynaphthoquinones, for example bu-
parvaquone, have demonstrated activity against L.
donovani and some other Leishmania species, both in
vitro and in vivo as oral, subcutaneous and topical
administration, over 20 years ago.3–6 In our previous
paper, buparvaquone (1) the high activity against spe-
cies that cause CL was confirmed.7 The antiprotozoal
activity of hydroxynaphthoquinones has also been
demonstrated for other parasite species; for example,
Theilaria Parva,4 Eimeria tenella,4 Plasmodium falcipa-
rum,4 Trypanosoma cruzi,8 Trypanosoma bruce.8 The
efficacy of hydroxynaphthoquinones varies significantly
in different species, and the mechanism of action of
hydroxynaphthoquinones remains partly unclear. In
Plasmodium it has been proposed that these compounds
inhibit electron transfer at complex III of the mito-
chondrial respiratory chain.6;9 The other proposed mode
of action for hydroxynaphthoquinones involves the
Keywords: Buparvaquone; Leishmaniasis; Nitric oxide; Oxime; Pro-
drug.
* Corresponding author. Fax: +358-17-162-456; e-mail: antti.mantyla@
pp2.inet.fi
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.032

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A. M€antyl€a et al. / Bioorg. Med. Chem. 12 (2004) 3497–3502
ability of the drug to form free radicals during interac-
tion with the parasite’s respiratory chain.5;10 In the
presence of oxygen, the semiquinone radical reduces
oxygen to superoxide radicals.10 Besides the production
of superoxide (Oꢀ2 ), the generation of nitric oxide (NO)
by activated macrophages is an important mechanism to
eliminate intracellular Leishmania parasites.11–13 NO is
normally produced by inducing nitric oxide synthases
(iNOS) in the macrophage, which catalyzes the oxida-
O
N
O
H
RO
Scheme 2. The predominant Amphi conformer (E-isomer) of 2 (R ¼ H)
and 3 (R ¼ CH3).
tion of
sequent formation of
to NO biosynthesis by the oxidation of
studies have shown that various compounds such as
alkyl- and aryl-aldoximes, ketoximes, amidoximes and
guanidoximes containing a C@NOH structure can be
oxidized by liver microsomal cytochrome P450 with the
formation of nitrogen oxides, including NO, and the
L
-arginine by NADPH and O2, with the sub-
-citrulline and NO.14 In addition
-arginine, many
purified by flash chromatography, and in all cases the
products were obtained as mixtures of E- and Z-isomers
at the C@N double bonds. The pure geometrical isomers
were not isolated, as the literature suggests that the E-
and Z-forms are in a pH and temperature dependent
equilibrium.22–25 However, E- and Z-isomers of each
L
L
1
compound (2, 3 and 5) can be easily verified by the H
and 13C NMR chemical shifts from the downfield shift
of the C-8 proton for the major isomers of 2, 3 and 5,
due to the aromatic ring current.26 Experimental and
theoretical investigations on the structure of 2-hydroxy-
3-methyl-1,4-naphthoquinone show that it exists pre-
dominantly as the E-isomer (amphi conformer) by
forming a Nꢁ ꢁ ꢁH–O intramolecular hydrogen bond.27
Correspondingly, the same amphi conformer is sug-
gested for compounds 2 and 3 (Scheme 2). Steric factors
may explain the preference of the E-isomer for 5. The
HMBC NMR spectrum (the H–C long range correla-
tion) confirmed that the reaction of buparvaquone with
hydroxyl- and methoxyamine regio-selectively produced
the oximes only at the C-1 carbonyl, which has been
previously shown for the naphthoquinone lapachol
oxime.28 Hydrogen bonding is also a logical explanation
for the regio-selective reaction at the C-1 carbonyl
(Scheme 2).
corresponding compound containing
group.15–18
a
carbonyl
These findings led us to investigate various oxime
derivatives of buparvaquone as novel antileishmanials.
The present study describes the synthesis of bu-
parvaquone-oxime (2), buparvaquone-O-methyloxime
(3) and O-methyl-buparvaquone-oxime (5). The oxida-
tion of buparvaquone-oxime (2), which contains a
C@N–OH group, was studied in induced rat liver mi-
crosomes, to investigate the possible bioconversion of
this derivative (2) to the corresponding active metabo-
lite, buparvaquone (1), thus making the formation of
NO possible. Therefore, to further investigate the effects
of the oxime function on the activity of these com-
pounds were determined against the amastigote forms of
L. donovani in macrophages in vitro.
2. Results and discussion
2.1. Chemistry
2.2. Microsomal oxidation of buparvaquone oxime
Treatment of rats with known inducers of cytochrome
P450, such as phenobarbital (PB), dexamethasone
(DEX) and 3-methylcholanthrene (3-MC), results in
increased titers of cytochrome P450 in liver microsomes.
The ability of the buparvaquone-oxime (2) derivative,
containing a C@NOH group, to undergo a P450
dependent oxidative cleavage to buparvaquone (corre-
sponding C@O group) required the incubation of
buparvaquone-oxime (2) with rat liver microsomes in
The structures of buparvaquone-oxime (2), buparvaqu-
one-O-methyloxime (3) and O-methyl-buparvaquone-
oxime (5) are presented in Scheme 1. The compounds
were prepared according to the methods described by
Strenge and Rademacher.19;20 Buparvaquone was
treated with hydroxylamine hydrochloride or with O-
methylhydroxylamine hydrochloride to afford the oxi-
mes 2 and 3 in high yield. Compound 5 was synthesized
by the previous method using methylated buparvaquone
as a starting compound, which was prepared as
described earlier.21 The synthesized compounds were
ꢀ
Table 1. Formation of buparvaquone and NO2 during incubations of
buparvaquone-oxime (2) in the presence of NADPH (mean; n ¼ 2) and
rat liver microsomes (at 37 ꢁC) from untreated rats or from rats treated
with either 3-methylcholanthrene (3-MC), dexamethasone (DEX) or
phenobarbital (PB), or without microsomes (blank)
ꢀa
O
5
4
6
7
Microsomes
Formation of
buparvaquonea
Formation of NO2
3
2
OR
1
8
Without (blank)
Untreated
3-MC
Ndb
3.9
Ndb
Ndb
0.5
X
1: X = -O,
2: X = -NOH,
3: X = -NOCH3,
4: X = -O,
R = -H (Buparvaquone)
R = -H
R = -H
7.5
DEX
PB
11.8
10.6
1.8
0.1
R = -CH3
5: X = -NOH,
R = -CH3
a Results are expressed as nmol of product (5 mg of protein)ꢀ1 (24 h)ꢀ1
b Nd ¼ not detectable.
.
Scheme 1.

A. M€antyl€a et al. / Bioorg. Med. Chem. 12 (2004) 3497–3502
3499
the presence of NADPH (Table 1). Microsomes from
rats treated with the inducers had a higher ability to
oxidize buparvaquone-oxime (2) to buparvaquone than
the untreated microsomes. The metabolite of the oxi-
dation was verified to be buparvaquone (1) by HPLC
with an authentic buparvaquone standard. The forma-
tion of buparvaquone did not occur in the absence of
microsomes and, thus, the data suggests that the reac-
tion is catalyzed by microsomal oxidizing enzymes.
Also, the chemical degradation of buparvaquone-oxime
(2) to buparvquone was not observed during five days of
chemical stability determinations at pH 7.4 (data not
shown). Poor stability in aqueous solution does not limit
the use of the oxime structure in the case of
buparvaquone, in contrast to studies with other oxi-
L. donovani are shown in Table 3. The ED50 values for
amastigotes indicate that oximes 2, 3 and 5 had lower
activity than buparvaquone (1), whose high antileish-
manial activity against Leishmania spp. have been
shown in earlier studies.5–7 However, the antileishmanial
activity of 2 and 3 were comparable to the activity of
sodium stibogluconate (Sbv), the standard control
compound, whereas O-methyl-buparvaquone-oxime (5)
was almost inactive. It was likely that buparvaquone-
oxime (2), buparvaquone-O-methyloxime (3) and O-
methyl-buparvaquone-oxime (5) did not release
buparvaquone (1) and NO during the in vitro experi-
ments, and thus they demonstrated only moderate in
vitro activity against Leishmania. Thus, further in vivo
activity studies, which enable the oximes to release
buparvaquone (1) and NO via a P450 dependent oxi-
dative cleavage, are needed to verify the biological
activity of various buparvaquone-oximes against
Leishmania.
mes.29 Moreover, the formation of NO2 was observed
ꢀ
(Table 1) during the incubation with rat liver micro-
somes induced by the known inducers, as earlier
reported for other oxime structures.15;16;18 Thus, NO is
apparently released during the bioconversion of
buparvaquone-oxime (2) to buparvaquone (1). A major
limitation of these studies was the poor aqueous solu-
bility of buparvaquone-oxime (2) (Table 2) and
buparvaquone.7 Although 2% (v/v) ethanol was used as
a co-solvent, buparvaquone-oxime (2) was studied as a
suspension in the incubations. Due to an even lower
aqueous solubility of buparvaquone-O-methyloxime (3)
and O-methyl-buparvaquone-oxime (5) (Table 2), their
oxidation in microsomes were not investigated at all.
3. Conclusion
Novel buparvaquone-oxime (2), buparvaquone-O-
methyloxime (3) and O-methyl-buparvaquone-oxime (5)
were synthesized as novel antileishmanials. Compound 2
underwent an enzymatic oxidative cleavage to
buparvaquone and, thus, is a prodrug of buparvaquone
(1). In addition, the release of NO during bioconversion
may provide an additional therapeutic effect in the
treatment of leishmaniasis. Moreover, the present study
reports moderate in vitro activity for the studied com-
pounds (2 and 3) against intracellular L. donovani
amastigotes. The lower in vitro activity of the oxime
compounds compared to buparvaquone (1) is most
probably due to their slow conversion to buparvaquone
(1) during the in vitro experiment, and thus further in
vivo studies are needed to demonstrate the advantage of
the novel buparvaquone-oximes in the treatment of
leishmaniasis.
2.3. In vitro antileishmanial activity
The in vitro activity of buparvaquone-oxime (2), bu-
parvaquone-O-methyloxime (3) and O-methyl-bu-
parvaquone-oxime (5) against intracellular amastigotes
Table 2. Aqueous solubility of buparvaquone-oxime (2), buparvaqu-
one-O-methyloxime (3), O-methyl-buparvaquone-oxime (5) and
buparvaquone (BPQ) in 0.185 M borate buffer (pH 7.4) at room tem-
perature (mean SD; n ¼ 3)
Compound
Aqueous solubility (lg/mL)
BPQ
0.03 0.01a
5.42 0.46
<0.03
2
3
5
4. Experimental section
4.1. General procedures
Ndb
a From Ref. 7.
b Not determined.
1H and 13C NMR spectra were recorded on a Bruker
Avance DRX500 spectrometer (Bruker, Rheinstetter,
Germany) operating at 500.13 and 125.76 MHz,
respectively, using TMS as an internal reference for both
1H and 13C. Elemental analyses were carried out on a
ThermoQuest CE Instruments EA 110-CHNS-O ele-
mental analyser. Mass spectra were acquired by a LCQ
quadrupole ion trap mass spectrometer with an elec-
trospray ionisation source (Finnigan, San Jose, CA).
The samples were diluted with methanol (20 lg/mL) and
5 lL of samples were injected. TLC analyses were run on
Kieselgel 60 F254 plates (DC-Alufolien, Merck), and
column chromatography was performed with silica gel
60 (Merck) (0.063–0.200 mm). All solvents and reagents
were from commercial suppliers and used as received.
Table 3. The in vitro activity of buparvaquone-oxime (2), bu-
parvaquone-O-methyloxime (3), O-methyl-buparvaquone-oxime (5)
against L. donovani HU3 amastigotes
Compound
% Inhibition (lg/mL)
ED50
(lg/mL)
30
10
3
1
BPQ
––
––
––
––
––
––
––
––
0.14a
17.52
21.16
>30
2
3
5
86.9
86.1
3.6
12.2
1.9
0.0
0.0
––
11.4
0.0
39.7
Sbv (control)b 66.1
15.89
a From Ref. 7.
b Sodium stibogluconate (Pentostam).

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A. M€antyl€a et al. / Bioorg. Med. Chem. 12 (2004) 3497–3502
4.2. Chemistry
dropwise. The mixture was refluxed for 4 h and evapo-
rated to dryness under high vacuum. The crude product
was purified by column chromatography (petroleum
ether/ethyl acetate 10:1) affording 4 (Rf ¼ 0:42) as a
yellow oil (2.04 g, 6.0 mmol 98.7%): 1H NMR (CDCl3) d
0.81 (9H, s, –C(CH3)3), 0.82–1.10 (5H, m, CHC(CH3)3,
Hax), 1.49 (1H, br m, CH–CH2), 1.74 (4H, m, Heq), 2.50
(2H, d, J ¼ 7:3 Hz, CH2–CH), 4.11 (3H, s, OCH3), 7.69
(2H, m, H-6, H-7), 8.05 (2H, m, H-5, H-8).
4.2.1. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-hydroxy-
[1,4]naphthoquinone-1-oxime (2). To a solution of bu-
parvaquone (1) (1.00 g, 3.06 mmol) and hydroxylamine
hydrochloride (0.53 g, 5.66 mmol) in ethanol (100 mL)
was added sodium acetate (0.76 g, 9.19 mmol) in water
(2 mL) and the mixture was refluxed for 3 days. The
solvent was removed under reduced pressure, and a
crude product was purified by silica gel column chro-
matography (petroleum ether/ethyl acetate, 3:1) afford-
ing 2 (5:1 mixture of E/Z isomers) (Rf ¼ 0:35) as a
yellow solid (0.92 g, 2.69 mmol, 88.1%): mp (decom-
4.2.4. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-methoxy-
[1,4]naphthoquinone-1-oxime (5). Compound 5 was pre-
pared as a product of compound 2 from 4 (2.18 g,
6.4 mmol) to give a brown oil, which was purified by
column chromatography (petroleum ether/ethyl acetate
20:1) yielding 5 (Rf ¼ 0:25) as a viscose oil (10:1 mixture
of E/Z isomers) (0.3 g, 0.84 mmol, 13.1%). 1H NMR (d6-
DMSO) E-isomer d 0.79 (9H, s, –C(CH3)3), 0.82–1.00
(5H, m, CHC(CH3)3, Hax), 1.41 (1H, br m, CH–CH2),
1.69 (4H, m, Heq), 2.40 (2H, d, J ¼ 7:2 Hz, CH2–CH),
3.92 (3H, s, OCH3), 7.62 (1H, td, J ¼ 7:5, 1.2 Hz, H-6),
7.72 (1H, td, J ¼ 7:4, 1.5 Hz, H-7), 8.08 (1H, dd,
J ¼ 7:8, 1.5 Hz, H-5), 8.93 (1H, d, J ¼ 7:9 Hz, H-8),
13.47 (1H, br s, OH); Z-isomer d 3.76 (3H, s, OCH3),
7.55 (1H, td, J ¼ 7:3, 1.2 Hz, H-6), 7.65 (1H, td, J ¼ 7:3,
1.6 Hz, H-7), 7.95 (1H, dd, J ¼ 7:9, 1.4 Hz, H-5), 8.04
(1H, d, J ¼ 7:9 Hz, H-8) (other signals overlap with the
E-isomer). 13C NMR (d6-DMSO) E-isomer d 27.3, 27.6,
31.2, 32.4, 34.0, 37.6, 48.1, 62.0, 126.9, 127.0, 129.2,
130.1, 130.7, 131.2, 132.6, 141.6, 161.0, 185.7; Z-isomer
d 37.7, 48.1, 61.1, 123.4, 126.3, 129.4, 130.2, 131.3,
132.3, 133.1, 142.7, 156.4, 185.6 (other signals overlap
with the E-isomer). ESI-MS: 356.4 (M+1). Anal. Calcd
(C22H29NO3Æ0.5EtOAc) C: 75.16, H: 9.08, N: 3.51;
found C: 75.14, H: 7.37, N: 3.73.
1
posed). H NMR (d6-DMSO) E-isomer d 0.80 (9H, s,
–C(CH3)3), 0.82–1.01 (5H, m, CHC(CH3)3, Hax), 1.46
(1H, br m, CH–CH2), 1.69 (4H, m, Heq), 2.39 (2H,
d, J ¼ 7:2 Hz, CH2–CH), 7.61 (1H, td, J ¼ 7:5; 1:3 Hz,
H-6), 7.67 (1H, td, J ¼ 7:7, 1.6 Hz, H-7), 8.09 (1H, dd,
J ¼ 7:6, 1.6 Hz, H-5), 8.97 (1H, d, J ¼ 8:1 Hz, H-8), 9.61
(1H, br s, OH), 13.57 (1H, br s, OH); Z-isomer d 7.56
(1H, td, J ¼ 7:5, 1.4 Hz, H-6), 7.60 (1H, td, J ¼ 7:6,
1.6 Hz, H-7), 7.98 (1H, dd, J ¼ 7:6, 1.6 Hz, H-5), 8.08
(1H, d, J ¼ 7:8 Hz, H-8), (other signals overlap with the
E-isomer). 13C NMR (d6-DMSO) E-isomer d 26.8, 27.3,
29.9, 32.0, 33.3, 37.1, 47.5, 116.1, 125.8, 125.9, 129.2,
130.3, 130.4, 132.0, 139.7, 158.7, 183.3; Z-isomer d 29.3,
32.0, 33.3, 36.9, 47.5, 122.1, 125.2, 129.3, 130.7, 131.4,
(other signals overlap with the E-isomer). ESI-MS:
340.7 (M)1). Anal. Calcd (C21H27NO3Æ0.1H2O) C:
73.31, H: 7.97, N: 4.10; found C: 73.48, H: 7.99, N: 4.08.
4.2.2. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-hydroxy-
[1,4]naphthoquinone-1-(O-methyloxime) (3). To a solu-
tion of 1 (3.01 g, 9.22 mmol) and O-methyl-hydroxyl-
amine hydrochloride (1.95 g, 23.35 mmol) in ethanol
(200 mL) was added sodium acetate (2.28 g, 27.80 mmol)
in a mixture of water (5 mL) and refluxed for 5 days. The
solvent was removed under vacuum. 50 mL of water was
added, and the product was extracted with DCM
(3 · 75 mL). The combined organic layers were dried
with sodium sulfate and evaporated to give a yellow
solid as a 20:1 mixture of E/Z isomers (3.2 g, 9.00 mmol,
4.2.5. Preparation of hepatic microsomes. Wistar rats
(150–200 g) were treated ip for 4 days with either
phenobarbital (80 mg/kg/day in 0.9% NaCl solution), 3-
methylcholanthrene (20 mg/kg/day in olive oil), dexa-
methasone (50 mg/kg/day in olive oil) or 0.5 mL olive oil
(control rats).16 Microsomes were prepared as previ-
ously reported30 and stored at )80 ꢁC until use. Protein
concentrations were determined by using the Biorad
Protein Assay (BioRad, Hercules, USA), and cyto-
chrome P450 contents were determined as reported
earlier.31
1
97.6%): mp 150 ꢁC. H NMR (CDCl3) E-isomer d 0.80
(9H, s, –C(CH3)3), 0.82–1.10 (5H, m, CHC(CH3)3, Hax),
1.55 (1H, br m, CH–CH2), 1.75 (4H, m, Heq), 2.50 (2H,
d, J ¼ 7:3 Hz, CH2–CH), 4.28 (3H, s, OCH3), 7.57 (2H,
m, H-6, H-7), 7.66 (1H, s, OH), 8.26 (1H, m, H-5), 8.74
(1H, m, H-8); E-isomer: d 7.52 (2H, m, H-6, H-7), 8.12
(2H, m, H-5, H-8), 9.55 (1H, s, OH) (other signals
overlap with the E-isomer). 13C NMR (CDCl3, E-iso-
mer) d 27.3, 27.6, 30.5, 32.4, 33.8, 37.6, 48.1, 65.0, 118.3,
125.8, 127.1, 129.6, 131.2, 131.5, 132.0, 139.8, 155.9,
184.5. ESI-MS: 354.5 (M)1). Anal. Calcd (C22H29NO3)
C: 74.33, H: 8.22, N: 3.94; found C: 74.10, H: 8.26, N:
3.90.
4.2.6. Enzymatic incubations in microsomes. A stock
solution of buparvaquone-oxime (2) was prepared in
ethanol. 20 lL of the stock solution (final substrate
concentration of 0.95 mM), MgCl2 solution (5 mM),
liver microsomes (protein concentration of 4.4–6.3 mg/
mL) and 50 mM phosphate buffer pH 7.4 (total volume
1 mL) were incubated for 5 min at 37 ꢁC before adding
NADPH solution (final concentration of 4 mM). For the
blank sample, liver microsomes were replaced by the
same volume of water. Reactions were incubated in a
water bath at 37 ꢁC for 24 h, and then stopped either by
mixing 1 mL of ice-cold acetonitrile with the sample for
4.2.3. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-methoxy-
[1,4]naphthoquinone (4). Compound
1
(2.00 g,
6.10 mmol) and anhydrous potassium carbonate (3.50 g,
24.4 mmol) were suspended in 100 mL of dry acetone,
and dimethyl sulfate (2.3 mL, 24.4 mmol) was added

A. M€antyl€a et al. / Bioorg. Med. Chem. 12 (2004) 3497–3502
3501
€
€
HPLC analysis, or by heating the sample 5 min at 100 ꢁC
for NO2 assays. The samples were kept on ice, centri-
fuged for 10 min at 11,000 rpm and the supernatant was
analysed by HPLC.
Foundation of Orion Corporation to Antti Mantyla is
gratefully acknowledged. Tracy Garnier was supported
by The Sir Halley Stewart Trust, UK, Simon L. Croft
and Howard Kendrick received support from the
UNDP/World Bank/WHO Special Programme for re-
search in tropical diseases (TDR) and Jarkko Rautio by
the Academy of Finland. The authors thank Mrs. Helly
Rissanen and Mrs. Mia Reponen and Mr. Pekka Keski-
Rahkonen for their skilful technical assistance.
ꢀ
4.2.7. Determination of nitrite. For the determination of
nitrite (NO2ꢀ), enzymatic incubations in microsomes
were performed as described above, except nitrate
reductase (60 mU from Aspergillus niger) was added
after 23 h of incubation medium to reduce available
nitrate (NOꢀ3 ) to nitrite (NO2ꢀ). The incubation was
continued for 1 h after the addition of nitrate reductase.
Proteins were precipitated by heating, which also elim-
inated the bleaching effect of NADPH on the Griess
reaction. The samples were centrifuged for 15 min at
11,000 rpm. The concentration of nitrite in the super-
natants were determined by the Griess reaction, where
240 lL of sulfanilamide (1.8% m/V) solution in 1 M HCl
solution) and 160 lL of N-(1-naphthyl)ethylenediamine
dihydrochloride (0.3% m/V) solution in 1 M HCl solu-
tion were added to 800 lL of the supernatants before
measurement at an absorbance of 548 nm.32 For the
standard curve, solutions of known nitrite concentra-
tions (0.1–40 lM sodium nitrite solution) were prepared
into the mixture of 50 mM phosphate buffer pH 7.4 and
5 mM MgCl solution, and the absorbance was measured
after treatꢀment with Griess reagents. Quantitations of
total NO2 were obtained from the resulting standard
curve.
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Beckman System Gold Programmable Solvent Module
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