Transforming rhinacanthin analogues from potent anticancer agents into potent antimalarial agents

Article abstract of DOI:10.1021/jm901545z

Twenty-six novel naphthoquinone aliphatic esters were synthesized by esterification of 1,4-naphthoquinone alcohols with various aliphatic acids. The 1,4-naphthoquinone alcohols were prepared from 1-hydroxy-2-naphthoic acid in nine steps with excellent yields. Twenty-four of the novel synthetic naphthoquinone esters showed significant antimalarial activity with IC ₅₀ values in the range of 0.03-16.63 μM. The length of the aliphatic chain and the presence of C-2′ substituents on the propyl chain affected the activity. Interestingly, compounds 31 and 37 showed very good antimalarial activity and were not toxic to normal Vero cells, and the PTI values of 31 (> 1990.38) and 37 (1825.94) are excellent. Both 31 and 37 showed potent inhibition against P. falciparum 3D7 cyt bc₁ and no inhibition on rat cyt bc₁. They showed IC₅₀ values in the nanomolar range, providing full inhibition of cyt bc₁ with one molecule inhibitor bound per cyt bc₁ monomer at the Q_o site.

Full text of DOI:10.1021/jm901545z

J. Med. Chem. 2010, 53, 1211–1221 1211
DOI: 10.1021/jm901545z
Transforming Rhinacanthin Analogues from Potent Anticancer Agents into Potent Antimalarial Agents
Ngampong Kongkathip,*,† Narathip Pradidphol,† Komkrit Hasitapan,† Ronald Grigg,‡ Wei-Chun Kao,§ Carola Hunte,§
Nicholas Fisher, Ashley J. Warman, Giancarlo A. Biagini, Palangpon Kongsaeree,^ Pitak Chuawong,† and
Boonsong Kongkathip*,†
†Natural Products and Organic Synthesis Research Unit (NPOS), Department of Chemistry and Center for Innovation in Chemistry, Faculty of
Science, Kasetsart University, Jatujak, Bangkok 10900, Thailand, ‡Molecular Innovation, Diversity and Automated Synthesis (MIDAS) Centre,
School of Chemistry, University of Leeds, Leeds LS2 9JT, U.K., §Institute of Membrane and Systems Biology, Astbury Center for Structural
Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K., Liverpool School of Tropical Medicine, Liverpool L3 5QA, U.K., and
^Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Received October 19, 2009
Twenty-six novel naphthoquinone aliphatic esters were synthesized by esterification of 1,4-naphtho-
quinone alcohols with various aliphatic acids. The 1,4-naphthoquinone alcohols were prepared from
1-hydroxy-2-naphthoic acid in nine steps with excellent yields. Twenty-four of the novel synthetic
naphthoquinone esters showed significant antimalarial activity with IC50 values in the range of
0.03-16.63 μM. The length of the aliphatic chain and the presence of C-20 substituents on the propyl
chain affected the activity. Interestingly, compounds 31 and 37 showed very good antimalarial activity
and were not toxic to normal Vero cells, and the PTI values of 31 (>1990.38) and 37 (1825.94) are
excellent. Both 31 and 37 showed potent inhibition against P. falciparum 3D7 cyt bc1 and no inhibition
on rat cyt bc1. They showed IC50 values in the nanomolar range, providing full inhibition of cyt bc1 with
one molecule inhibitor bound per cyt bc1 monomer at the Qo site.
Introduction
analogues might be a source of antimalarials. Our earlier
work on anticancer agents focused on C-(3)-aromatic esters of
rhinacanthins.1a We now report studies on C-(3)-aliphatic
esters that have led to potent antimalarial compounds.
In our previous papers1 we reported the syntheses of 1,2-
and 1,4-naphthoquinones such as rhinacanthin-M (1), -N
(2a), and -Q (2b) together with a substantial number of
C-(3)-analogues thereof. These compounds are natural pro-
ducts isolated from the roots and leaves of Rhincanthus
nasutus which are used in Thailand for the treatment of can-
cer.2 Several of the novel analogues1a exhibited potent antic-
ancer activity comparable to thatof adriamycin (doxorubicin)
(3) (Figure 1).
Quinones and, in particular, 1,4-naphthoquinones have
long been known to display antimalarial activity3 in addition
to a wide variety of other bioactivities.4 Pioneering work by
Wendel5 and by Fieser et al.6 on hydroxynaphthoquinones
formed the basis of the development of the antimalarial drug
atovaquone (4, Figure 2, Figure 3),7 while early clinical trials
on 4 demonstrated good initial control of malaria, and
currently 4 is used in combination with proguanil (5) under
the trade name Malarone.8 Atovaquone selectively inhibits
the cytochrome bc1 complex. The latter catalyzes electron
transfer from ubiquinol to cytochrome c and concomitantly
translocates protons across membranes.9,10 A further conse-
quence of inhibition of the cytochrome bc1 complex is the
indirect inhibition of pyrimidine biosynthesis.11
Chemistry
A total of 26 linear and branched aliphatic esters (Schemes 4
and 5 and Table 1) have been synthesized in our laboratory.
All of these were prepared by coupling of the appropriate C-(3)
aliphatic alcohol (10a-c) with linear and branched aliphatic
carboxylic acids 11 to afford esters 121 (Scheme 1). Naphtho-
quinone alcohol 10a was synthesized starting from the com-
mercially available 1-hydroxy-2-naphthoic acid in nine
sequential steps1a with excellent yields (Scheme 2). Naphtho-
quinone aliphatic esters (18-39) were synthesized from naph-
thoquinone alcohol 10a and various aliphatic acids by DCC/
DMAPa coupling in moderate to good yields (Table 1).
Naphthoquinone alcohol 10b could be obtained in good
yields via six steps1b by using 1-naphthol as starting material
(Scheme 3). Esterification of 10b with acetic acid, butyric acid,
and octanoic acid using CDI as a condensing agent afforded
naphthoquinone esters 42-44 in moderate yields (Scheme 4).
The cyclohexyl alcohol 10c was prepared from 22 by ring-
opening using 1% aqueous sodium hydroxide, and then
naphthoquinone ester 48 was subsequently obtained by cou-
pling 10c with octanoic acid using DCC in the presence of a
catalytic amount of DMAP (Scheme 5).
The structural similarities between rhinacanthin-C (6), -G
(7), -H (8), and -K (9)12 possessing C-(3)-aliphatic ester
side chains and atovaquone suggested that rhinacanthin
aAbbreviations: DCC, N,N0-dicyclohexylcarbodiimide; DMAP, 4-
dimethylaminopyridine; CDI, 1,10-carbonyldiimidazole; Qo site, quinol
oxidizing site of bc1 complex; Qi site, quinone reducing site of bc1
complex; DQH, decylubiquinol; YPD, yeast peptone dextrose; EDTA,
ethylenediaminetetraacetic acid.
*To whom correspondence should be address. For N.K.: phone,
þ6625625555, extension 2139; fax, þ6625793955; e-mail, fscinpk@
ku.ac.th. For B.K.: phone, þ6625625555, extension 2235; fax,
þ6625793955; e-mail, fscibsk@ku.ac.th.
r
2010 American Chemical Society
Published on Web 01/12/2010
pubs.acs.org/jmc

1212 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Kongkathip et al.
Figure 1. Structures of rhinacanthin-M (1), -N (2a), -Q (2b), and adriamycin (3).
Results and Discussion
The prepared 1,4-naphthoquinone aliphatic esters exhib-
ited significant antimalarial activity (Tables 2 and 3). Esters
with short chains (n=1, 2) showed less activity than those
with longer straight chains. Compounds 26 (n = 4), 29 (n=5),
30 (n=6), 31 (n=7), 33 (n=8), 34 (n=9), 35 (n=10), and 36
(n=11) showed quite strong antimalarial activity. Among the
compounds with linear aliphatic chain (n=1-11), compound
31 (n=7) showed the best activity and had no toxicity against
normal Vero cells corresponding to a favorably very high
potential therapeutic index (PTI > 1990). The length of the
aliphatic chain of this compound is the same as that of natural
rhinacanthin-C (6), -G (7), -H (8), and -K (9). The naphtho-
quinone aliphatic ester39(n=17) (IC50=0.133 μM) withquite
longer chain than n=11 showed similar activity as compound
31 (n = 7) (IC50=0.13 μM), whereas compounds 37 (n=13)
and 38 (n = 15) showed the best activity with favorable
IC50 values of 0.032 and 0.030 μM, respectively. In parti-
cular, compound 37 showed very high PTI (1826). It seems
that the optimum length of the aliphatic linear chain is n = 13
and 15.
Figure 2. Structures of atovaquone (4) and proguanil (5).
Antimalarial Activity
The synthetic 1,4-naphthoquinone aliphatic esters (18-39,
42-44, and 46) were evaluated for antimalarial activity using
dihydroartemisinin as reference (Tables 2 and 3). Plasmodium
falciparum (K1, multidrug resistant strain) was cultivated in
vitro according to Trager and Jensen.13a Quantitative assess-
ment of antimalarial activity in vitro was determined by
microculture radioisotope techniques based upon the meth-
ods described by Desjardins.13b
Cytotoxicity
Because most of the natural naphthoquinones contain an
R-methyl substituent on the aliphatic ester side chain, com-
pounds 20, 22, 27, and 32 with R-methyl substituents were
synthesized (as racemates) by the same method as mentioned
above. The test results showed that these naphthoquinone
esters showed stronger activity than the compounds contain-
Compounds 18-39 were subjected to cytotoxic evaluation
against the normal Vero cell lines employing the MTT colori-
metric method.14 The results are summarized in Table 2.
Activity of cyt bc1
With highly potent antimalarial activity and no cytotoxicity
against Vero cell lines, naphthoquinone esters 31 and 37 were
further tested for their inhibition of cyt bc1 of P. falciparum
3D7, yeast, rat, and purified yeast cyt bc1 by using decylubi-
quinol-linked cytochrome c reductase assay. The results are
shown in Tables 4 and 5.
ing linear chains of the same number of carbons (IC50
=
0.11-0.91 μM). Moreover, activity was enhanced 2-fold in
compound 25 with the S-configuration of the methyl sub-
stituent (IC50 = 0.11 μM) compared with that of the racemic
mixture 22 (IC50 = 0.22 μM).
While an R-methyl substituent enhanced the activity, com-
pounds 23 and 28 with β-methyl substituents showed reduced
activity. Compound23 showed about 2 times less activity than
22. An R-ethyl substituent, as in compound 24, also caused
reduced activity, compared with compound 22 (R-methyl
substituent). Compounds lacking gem-dimethyl substitution
in the propyl chain attached to the 1,4-naphthoquinone
Decylubiquinol/Cytochrome c Reductase Assay
Cytochrome bc1 activity15 was monitored spectrophotome-
trically in P. falciparum cell-free extracts and yeast mitochon-
drial membrane preparations by the steady-state decylubi-
quinol/cytochrome c reductase.
Figure 3. Structures of rhinacanthin-C (6), -G (7), -H (8), and -K (9).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1213
Scheme 1. Esterification of Naphthoquinone Alcohols 10a-c with Various Aliphatic Acids
Scheme 2. Synthesis of Naphthoquinone Alcohol 10aa
a Reagents and conditions: (i) MeI, K2CO3, acetone, reflux, 96%; (ii) LiAlH4, THF, 0 ꢀC to room temp, 94%; (iii) 1 M PBr3 in CH2Cl2, CH2Cl2, room
temp, 100%; (iv) methyl isobutyrate, LDA, THF, -78 ꢀC, 89%; (v) AlCl3, PhCl, reflux, 92%; (vi) LiAlH4, THF, 0 ꢀC to room temp, 88%; (vii) Fremy’s
salt, MeOH/DMF (3:1), 1 M NaOAc, room temp, 83%; (viii) DDQ, p-TsOH, PhH, reflux, 90%; (ix) 1% aq NaOH, reflux, 84%.
Scheme 3. Synthesis of Naphthoquinone Alcohol 10ba
a Reagents and conditions: (i) allyl bromide, K2CO3, reflux, 86%; (ii) 180 ꢀC, DMF, 87%; (iii) BH3 THF, then H2O2/NaOH, 79%; (iv) Fremy’s salt,
MeOH/DMF (3:1), 1 M NaOAc, room temp, 83%; (v) DDQ, p-TsOH, PhH, reflux, 93%; (vi) 1% aq NaOH, reflux, 89%.
3
nucleus, as in 42 and 43, were inactive, whereas 44 (IC50
=
agreement with previous data.16 IC50 values of 31 and 37
were determined as 840 and 770 nM, respectively, in deter-
gent environment. These inhibitors 31 and 37 apparently
have lower affinities than stigmatellin, explaining the 50
times higher IC50 values in detergent environment com-
pared to those in the membrane because binding of the
hydrophobic compounds competes with binding to deter-
gent micelles.
Inhibitor binding to Qo vs Qi site of cyt bc1 was distin-
guished by DQH induced redox difference spectra in the
presence of specific inhibitors. Stigmatellin binds specifically
to the Qo site while antimycin A inhibits cyt bc1 by occluding
the electron transfer from heme bH to the quinone substrate in
the Qi site.17 Both inhibitors (31 and 37) have IC50 values in
the nanomolar range, providing full inhibition of cyt bc1 with
one molecule inhibitor bound percyt bc1 monomer. Spectra of
DQH reduced inhibitor-cyt bc1 complex differ for Qo and Qi
site inhibitors at the absorbance for heme c1 at 553.6 nm
(Figure 4). Stigmatellin blocks electron transfer from the Qo
site to the iron sulfur cluster; thus, very limited heme c1 was
reduced (Figure 4, upper panel) by 45 μM DQH. However,
heme c1 was fully reduced in the presence of antimycin A
5.15 μM) and compound 46, having a spiro C-20 cyclohexyl
group (IC50 = 2.63 μM), showed only reduced activity. Thus
C-20 gem-dimethyl substitution of the propyl chain is favor-
able for good activity in this series of esters.
We have found that some of our compounds such as 31
and 37 showed very potent antimalarial activity but were
not toxic to Vero cells, so 31 and 37 were further tested in
vivo as well as the mechanism of their antimalarial activity.
As a known atovaquone, one of 1,4-naphthoquinone, has
been reported to inhibit the cyt bc1 of P. falciparum, this
inhibition may be a mechanism of our synthesized anti-
malarial naphthoquinones. So we investigated the inhibi-
tion mechanism of compounds 31 and 37 by decylubi-
quinol/cytochrome c reductase assay. Compounds 31 and
37 were determined for their cyt bc1 activity using stigma-
tellin as reference. Compound 31 inhibited P. falciparum
3D7, yeast, and rat with IC50 values of 5.4 ( 0.53, 16 ( 1.30,
and 3,000 ( 156 nM, respectively. Compound 37 also
showed inhibition with IC50 values of 8.0 ( 0.60, 79.6 (
3.41, and 2,495 ( 820 nM, respectively (Table 4). Our
test showed IC50 of stigmatellin as 9.1 nM (Table 5), in

1214 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Kongkathip et al.
(Figure 4, middle panel). Redox difference spectra quantifica-
tion (ε562-575nm) showed that one (0.9) of two b hemes were
reduced by DQH, indicating that one DQH was oxidized per
one cyt bc1 monomer as expected. The interaction site of
compounds 31 and 37 was determined by comparison with
these standards. Very limited heme c1 was reduced by
45 μM DQH in both 31 and 37 cyt bc1 complexes with
AU e 0.02 indicative for blocked electron transfer to the high
potential chain as observed for the stigmatellin-inhibited
complex, thus clearly revealing both 31 and 37 binding to
the Qo site (Figure 4, lower panel, solid and dotted lines).
Interestingly, less cytochrome b is reduced for the 31 and 37
probes (0.02 AU) compared to stigmatellin (0.05 AU), in-
dicating that these inhibitors may also bind to a certain extent
to the Qi site.
the structural differences between our naphthoquinone esters
and atovaquone, i.e., aliphatic ester moiety of our naphtho-
quinone versus a cyclohexyl unit of atovaquone, should
render different binding affinity toward cytochrome bc1 pro-
tein in P. falciparum. It is conceivable to anticipate that our
naphthoquinone esters would impart resistant in P. falcipar-
um. It is worth mentioning here that several antimalarial drug
resistance cases caused by mutations at other drug targets
have also been reported.24,25 To circumvent this problem, a
drug combination that acts on the principle of synergy might
avoid this problem as advocated by White.26 In the case of
our compounds we believe our ester 37 given in combination
with our reference drug dihydroartimisinin will be effective.
Dihydroartemisinin is the common metabolite of artemisinin
and its derivatives, artemether and artesunate. These are the
most active of all antimalarial drugs.26
The Qo site has been the target of both antimalarial drug
development and of agrochemical fungicides. Both applica-
tions have suffered from the development of resistance arising
from site mutations of the bc1 complex.18,19 Considerable
detailed evidence has accrued about the nature of these muta-
tions which will assist further drug development programs.
Very recently the Qo has also been proposed as the site of action
of the anticancer macrolides leucascandrolide A and neopen-
tolide20 and for the fungicidal crocacins A and D.21 Currently
there is no single marketed drug that is effective for the
treatment of multidrug resistant malarial parasites. However,
recent disclosures of the metabolism and pharmacokinetics of
GW844520 and other related 4-pyridone antimalarials indicate
that this class of compounds acts by selective inhibition of
mitochondrial electron transport at the cytochrome bc1 com-
plex involving the Qo site.22,23 In this case no significant cross-
resistance with atovaquone resistant strains of P. falciparum is
observed and the frequency at which resistant strains arise in
vitro is significantly lower than that displayed by atovaquone.
These observations indicate there are significant opportunities
for further exploration of the Qo site. Thus, it is of interest to see
whether the naphthoquinone ester system can be further
developed to produce a new antimalarial drug.
From our naphthoquinone basic structure, it would be said
that four functional parts showed important roles, which are
(i) 20-substituents of propyl chain, (ii) the chain length of
aliphatic chain, (iii) R-methyl substituent of aliphatic chain,
(iv) ester moiety. Synthesis of the modified antimalarial
naphthoquinones is being investigated by our group which
could be helpful in designing future antimalarial drugs caused
by mitochondria-containing parasite.
Conclusion
Twenty-six novel 1,4-naphthoquinone aliphatic esters were
synthesized, and twenty-four of them exhibited potent anti-
malarial activity,27 especially compounds 22, 25, 27, 31, 32, 37,
38, and 39 (IC50=0.03-0.22 μM). The R-methyl substituent
of the acid side chain affected the antimalarial activity.
Naphthoquinone aliphatic esters with R-methyl substituent of
the side chain were more active than those with β-methyl
substituent and the linear side chain with the same number of
carbon atoms. In addition, R-ethyl substituent showed less
activity than R-methyl substituent. C-20 Substituents of the
propyl side chain at the 3-position of the naphthoquinone
nucleus showed some effect on the activity. The C-20 dimethyl
group showed more potent activity than that with no substi-
tuent or with C-20 cyclohexyl group. Most of our naphthoqui-
nones showed no toxicity on normal Vero cell lines as well as
very good antimalarial activity. Compounds 31 and 37 showed
antimalarial activity with IC50 values of 0.13 and 0.032 μM,
respectively, whereas they showed no toxicity to Vero cell lines
(IC50 values of >258.75 and 58.43 μM, respectively), corre-
sponding to excellent PTI values of >1990 and 1826, respec-
tively. Compounds 31 and 37 showed potent inhibition against
P. falciparum 3D7 cyt bc1 with IC50 values of 5.4 ( 0.53 and
8.0 ( 0.60 nM, respectively, while they showed less inhi-
bition on yeast cyt bc1 (IC50=16 ( 1.30 and 79.6 ( 3.41 nM,
respectively) and no inhibition on rat cyt bc1 (IC50 = 3,000 (
156 and 2,495 ( 820 nM, respectively). Both 31 and 37 showed
IC50 values in the nanomolar range, providing full inhibition of
However, since our naphthoquinone esters affect cyt bc1
similar to atovaquone, it is our concern that the activity of
these newly synthesized naphthoquinone esters toward ato-
vaquone resistance P. falciparum might be different. And also
Scheme 4. Esterification of 10b with Aliphatic Carboxylic
Acids Using CDI as a Condensing Agent
Scheme 5. Synthesis of Cyclohexyl Alcohol 10c and Naphthoquinone Ester 46a
a Reagents and conditions: (i) 1% aq NaOH, reflux; (ii) octanoic acid, DCC, DMAP, CH2Cl2, room temp, 35% over two steps.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1215
Table 1. Esterification of 10a with Aliphatic Carboxylic Acids Using
DCC/DMAP as a Condensing Agent
data were obtained on the GCMS-QP-5050A. Nuclear magnetic
resonance spectra were recorded at 400 MHz on a Bruker
Advance DPX-400. Chemical shifts (δ) are given in parts per
million downfield from tetramethylsilane (TMS) as internal
standard. Coupling constants are given in hertz (Hz). The
following abbreviations are used: s=singlet, d=doublet, m=
multiplet, dd=doublet of doublet. Elemental analyses were per-
formed for compounds 18-39, 42-44, and 46 on an elemental
analyzer (EA), Perkin-Elmer PE2400 series II (Scientific and
Technological Research Equipment Centre, Chulalongkorn
University, Bangkok, Thailand), and the results are within
(0.4% of the theoretical values and indicate >95% purity;
values are presented in the Supporting Information. Column
chromatography was performed with flash silica gel (Merck
9385).
General Procedure for Synthesis of Naphthoquinone Aliphatic
Esters 18-39. A solution of naphthoquinone alcohol (0.2 mmol)
in dry dichloromethane (2 mL) was added to a stirred solution of
carboxylic acid (0.26 mmol) and 4-dimethylaminopyridine
(DMAP) (0.06 mmol) in dry dichloromethane (2 mL). The
reaction mixture was stirred at room temperature for 5 min
when a solution of 1,3-dicyclohexylcarbodiimide (DCC) (0.26
mmol) in dry dichloromethane (3 mL) was added. Stirring was
continued overnight at room temperature. The precipitate of
dicyclohexylurea was filtered off and the filtrate washed with
saturated ammonium chloride solution, water, dried (anhy-
drous Na2SO4), and filtered, and the filtrate was concentrated
in vacuo. The residue was purified by flash column chromatog-
raphy.
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Acetate (18). Flash column chromatography was
used, eluting with 5% ethyl acetate-hexane. The yellow oil was
obtained in 89% yield, and then it was crystallized with hexane-
dichloromethane to give yellow needles, mp 113.5-114.5 ꢀC. 1H
NMR (CDCl3, 400 MHz) δ: 0.92 (s, 6H, 2 ꢀ CH3), 1.95 (s, 3H,
COCH3), 2.6 1 (s, 2H, CH2Ar), 3.77 (s, 2H, OCH2), 7.40 (s, 1H,
OH), 7.62 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 7.69 (td, J =
7.6 Hz, J = 1.4 Hz, 1H, ArH), 8.02 (dd, J = 7.6 Hz, J = 1.4 Hz,
1H, ArH), 8.05 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Propionate (19). Flash column chromatography
was used, eluting with 2% ethyl acetate-hexane. The yellow oil
was obtained in 90%, and then it was crystallized with hexane-
dichloromethane to yield yellow needles, mp 89.0-90.0 ꢀC. 1H
NMR (CDCl3, 400 MHz) δ: 0.91 (s, 6H, 2 ꢀ CH3), 1.13 (t, J =
7.6 Hz, 3H, CH2-CH3), 2.31 (q, J = 7.6 Hz, 2H, CH2-CH3), 2.69
(s, 2H, CH2Ar), 3.86 (s, 2H, OCH2), 7.43 (s, 1H, OH), 7.70 (td,
J = 7.6 Hz, J = 1.2 Hz, 1H, ArH), 7.77 (td, J = 7.6 Hz, J = 1.2
Hz, 1H, ArH), 8.10 (dd, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH), 8.13
(dd, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Isobutyrate (20). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow oil
was obtained in 86%, and then it was crystallized with hexane-
1
dichloromethane to give yellow needles, mp 70.5-71.5 ꢀC. H
NMR (CDCl3, 400 MHz) δ: 0.99 (s, 6H, 2 ꢀ CH3), 1.17 (d, J =
7.0 Hz, 6H, (CH3)2-CH), 2.54 (sept, 1H, (CH3)2-CH), 2.69 (s,
2H, CH2Ar), 3.85 (s, 2H, OCH2), 7.44 (s, 1H, OH), 7.70 (td, J =
7.5 Hz, J = 1.2 Hz, 1H, ArH), 7.77 (td, J = 7.5 Hz, J = 1.2 Hz,
1H, ArH), 8.10 (dd, J = 7.5 Hz, J = 1.2 Hz, 1H, ArH), 8.13 (dd,
J=7.5 Hz, J=1.2 Hz, 1H, ArH).
cyt bc1 with one molecule inhibitor bound per cyt bc1 monomer.
Therefore, it is clear that our naphthoquinone esters 31 and 37
revealed their binding to the Qo site. It is not far-fetched to
think that inhibition of P. faciparum is an important attribute
of antimalarial activity; thus, it is of interest to see whether the
naphthoquinone ester system can be developed sufficiently to
produce a new antimalarial drug.
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Butyrate (21). Flash column chromatography was
used, eluting with 7% ethyl acetate-hexane. The yellow oil was
obtained in 87% yield, and then it was crystallized with hexane-
1
dichloromethane to give yellow needles, mp 66.0-67.0 ꢀC. H
Experimental Section
NMR (CDCl3, 400 MHz) δ: 0.85 (t, J = 7.4 Hz, 3H, CH3-CH2),
0.92 (s, 6H, 2 ꢀ CH3), 1.56 (m, 2H, CH3-CH2-CH2), 2.18 (t, J =
7.4 Hz, 2H, CH2-CH2-COO), 2.61 (s, 2H, CH2Ar), 3.79 (s, 2H,
OCH2), 7.37 (s, 1H, OH), 7.62 (td, J = 7.6 Hz, J = 1.4 Hz, 1H,
General Remarks. Melting points were determined on a Fisher
John apparatus and are uncorrected. The IR spectra were
recorded on an FTIR Perkin-Elmer system 2000. Mass spectral

1216 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Kongkathip et al.
Table 2. Antimalarial Activity and Cytotoxicity against Vero Cell Lines of Naphthoquinone Esters (18-39)
a IC50 = half-maximal inhibitory concentration. b Vero cell lines = African green monkey kidney cell. c The results are the mean of six replicate
determinations ( SD. d Used as antimalarial reference.
ArH), 7.69 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 8.02 (m, 1H,
ArH), 8.05 (m, 1H, ArH).
2 ꢀ CH3), 0.92 (s, 6H, 2 ꢀ CH3), 2.00 (m, 1H, CH(CH3)2),
2.08 (m, 2H, CH2C(O)OR), 2.61 (s, 2H, CH2Ar), 3.79 (s, 2H,
CH2OC(O)R), 7.37 (s, 1H, OH), 7.62 (td, J = 7.5 Hz, J = 1.4
Hz, 1H, ArH), 7.70 (td, J = 7.5 Hz, J = 1.4 Hz, 1H, ArH), 8.02
(m, 1H, ArH), 8.06 (m, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl 2-Ethylbutanoate (24). Flash column chromato-
graphy, eluting with 2% ethyl acetate-hexane, gave a yellow oil
(73%). 1H NMR (CDCl3, 400 MHz) δ: 0.80 (t, J = 7.4 Hz, 6H,
2 ꢀ CH3), 0.92 (s, 6H, 2 ꢀ CH3), 1.43 (m, 2H, CH2CH3), 1.53 (m,
2H, CH2CH3), 2.11 (m, 1H, CH2C(O)OR), 2.62 (s, 2H, CH2Ar),
3.81 (s, 2H, CH2OC(O)R), 7.38 (s, 1H, OH), 7.62 (td, J =
7.5 Hz, J=1.4 Hz, 1H, ArH), 7.69 (td, J=7.5 Hz, J=1.4 Hz, 1H,
ArH), 8.02 (m, 1H, ArH), 8.06 (m, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl 2-Methylbutanoate (22). Flash column chroma-
tography was used, eluting with 4% ethyl acetate-hexane. The
yellow gum was obtained in 81% yield. 1H NMR (CDCl3, 400
MHz) δ: 0.89 (t, J=7.2 Hz, 3H, CH3CH2), 0.99 (s, 6H, 2 ꢀ CH3),
1.14 (d, J=7.2 Hz, 3H, CH3CH(CO)), 1.46 (m, 1H, CH2CH3),
1.70 (m, 1H, CH2CH3), 2.35 (hextet, J = 6.8 Hz, 1H, CH3CH-
(CO)), 2.69 (s, 2H, CH2Ar), 3.87 (s, 2H, OCH2), 7.45 (s, 1H,
OH), 7.70 (td, J=7.5 Hz, J=1.2 Hz, 1H, ArH), 7.77 (td, J=
7.5 Hz, J = 1.2 Hz, 1H, ArH), 8.10 (ddd, J = 7.5 Hz, J = 1.2 Hz,
J=0.6 Hz, 1H, ArH), 8.13 (ddd, J=7.5 Hz, J=1.2 Hz, J=
0.6 Hz, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Isovalerate (23). Flash column chromatography,
eluting with 5% ethyl acetate-hexane, gave a yellow oil (82%).
1H NMR (CDCl3, 400 MHz) δ: 0.85 (d, J = 6.6 Hz, 6H,
(S)-3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-
dimethylpropyl 2-Methylbutanoate (25). Flash column chroma-
tography was used, eluting with 3% ethyl acetate-hexane.
The yellow gum was obtained in 76% yield. 1H NMR (CDCl3,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1217
Table 3. Antimalarial Activity of Naphthoquinone Esters (42-44
and 46)
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl-2-methylpentanoate (27). Flash column chroma-
tography was used, eluting with 3% ethyl acetate-hexane. The
yellow oil was obtained in 77% yield, and then it was crystallized
with hexane-dichloromethane to give yellow needles, mp 57.0-
58.0 ꢀC. 1H NMR (CDCl3, 400 MHz) δ: 0.88 (t, J=7.2 Hz, 3H,
CH3CH2CH2), 0.99 (s, 6H, 2 ꢀ CH3), 1.14 (d, J=7.2 Hz, 3H,
CH3CH(CO)), 1.24-1.42 (m, 3H, CH2CH2CH3), 1.65 (m, 1H,
CH2CH2CH3), 2.43 (hextet, J = 7.2 Hz, 1H, CH3CH(CO)), 2.69
(s, 2H, CH2Ar), 3.86 (s, 2H, OCH2), 7.45 (br s, 1H, OH), 7.70
(td, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH), 7.77 (td, J = 7.6 Hz, J =
1.2 Hz, 1H, ArH), 8.10 (dd, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH),
8.14 (dd, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl 3-Methylpentanoate (28). Flash column chroma-
tography was used, eluting with 3% ethyl acetate-hexane. The
yellow gum was obtained in 76% yield. 1H NMR (CDCl3, 400
MHz) δ: 0.79 (t, J = 7.4 Hz, 3H, CH3-CH2), 0.83 (d, J = 6.9 Hz,
3H, CH3-CH), 0.92 (s, 6H, 2 ꢀ CH3), 1.77 (m, 1H, CH), 2.00 (dd,
J=14.7 Hz, J=7.1 Hz, 1H, C(O)CH2CH), 2.19 (dd, J=14.7 Hz,
J = 7.1 Hz, 1H, C(O)CH2CH), 2.61 (s, 2H, CH2Ar), 3.79 (s, 2H,
CH2OC(O)R), 7.37 (s, 1H, OH), 7.62 (td, J=7.5 Hz, J=1.4 Hz,
1H, ArH), 7.69 (td, J = 7.5 Hz, J = 1.4 Hz, 1H, ArH), 8.02 (ddd,
J=7.5 Hz, J=1.4 Hz, J = 0.4 Hz, 1H, ArH), 8.06 (ddd, J=
7.5 Hz, J=1.4 Hz, J=0.4 Hz, 1H, ArH).
a IC50 = half-maximal inhibitory concentration. b The results are the
mean of six replicate determinations. c Used as antimalarial reference.
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Hexanoate (29). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow oil
was obtained in 74% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 54.5-
Table 4. Inhibition of P. falciparum 3D7, Yeast, and Rat cyt bc1 by 31
and 37
1
55.5 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.89 (t, J = 6.7 Hz,
3H, CH3CH2), 1.00 (s, 6H, 2 ꢀ CH3), 1.28 (m, 4H, (CH2)2CH3),
1.51 (quin, J = 7.5, 2H, COCH2CH2CH2), 2.27 (t, 2H, J = 7.5
Hz, COCH2CH2), 2.69 (s, 2H, CH2Ar), 3.87 (s, 2H, OCH2), 7.62
(br s, 1H, OH), 7.69 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 7.77
(td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 8.08 (dd, 1H, J = 7.6 Hz,
J=1.4 Hz, ArH), 8.12 (dd, 1H, J=7.6 Hz, J=1.4 Hz, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Heptanoate (30). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow oil
was obtained in 83% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 57.5-
a IC50 = half-maximal inhibitory concentration. IC50 values (presen-
ted as (SEM) were obtained from three separate assays and calculated
from four-parameter sigmoidal fits. b The cyt bc1 activity was monitored
spectrophotometrically as decylubiquinol-linked cytochrome c reduc-
tion assay.
1
58.5 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, J = 6.9 Hz,
3H, CH3CH2), 1.00 (s, 6H, 2 ꢀ CH3), 1.28 (m, 6H, (CH2)3CH2),
1.60 (quin, J = 7.4 Hz, 2H, COCH2CH2CH2), 2.28 (t, 2H, J =
7.4 Hz, COCH2CH2), 2.69 (s, 2H, CH2Ar), 3.87 (s, 2H, OCH2),
7.58 (br s, 1H, OH), 7.70 (td, J = 7.6 Hz, J = 1.1 Hz, 1H, ArH),
7.78 (td, J=7.6 Hz, J=1.1 Hz, 1H, ArH), 8.10 (dd, 1H, J=7.6
Hz, J=1.1 Hz, ArH), 8.13 (dd, 1H, J=7.6 Hz, J=1.1 Hz, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Octanoate (31). Flash column chromatography
was used, eluting with 5% ethyl acetate-hexane. The yellow oil
was obtained in 96% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 56.0-
Table 5. Inhibition of Activity of Purified Yeast cyt bc1 in Detergent by
Stigmatellin, 31, and 37
stigmatellin
31
37
a
IC50
9.1 nM
0.84 μM
0.77 μM
a IC50 = half-maximal inhibitory concentration. IC50 values were
calculated from logarithmic fits, and the mean value of two separate
assays is given.
400 MHz) δ: 0.89 (t, J=7.2 Hz, 3H, CH3CH2), 0.99 (s, 6H, 2 ꢀ
CH3), 1.14 (d, J = 7.2 Hz, 3H, CH3CH(CO)), 1.46 (m, 1H,
CH2CH3), 1.67 (m, 1H, CH2CH3), 2.35 (hextet, J=7.6 Hz, 1H,
CH3CH(CO)), 2.69 (s, 2H, CH2Ar), 3.87 (s, 2H, OCH2), 7.44 (s,
1H, OH), 7.70 (td, J=7.6Hz, J=1.6 Hz, 1H, ArH), 7.78 (td, J=7.6
Hz, J=1.6 Hz, 1H, ArH), 8.10 (m, 1H, ArH), 8.13 (m, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Pentanoate (26). Flash column chromatography
was used, eluting with % ethyl acetate-hexane. The yellow oil
was obtained in 45% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 51.0-
1
57.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.87 (t, J = 6.9 Hz,
3H, CH3CH2), 0.99 (s, 6H, 2 ꢀ CH3), 1.27 (br s, 8H, CH3-
(CH2)4CH2), 1.59 (m, 2H, COCH2CH2CH2), 2.26 (t, J = 7.6
Hz, 2H, COCH2CH2), 2.68 (s, 2H, CH2Ar), 3.85 (s, 2H, OCH2),
7.44 (s, 1H, OH), 7.69 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH),
7.76 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 8.09 (dd, J = 7.6 Hz,
J=1.4 Hz, 1H, ArH), 8.12 (dd, J=7.6 Hz, J=1.4 Hz, 1H, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl-2-methyl Octanoate (32). Flash column chroma-
tography was used, eluting with 4% ethyl acetate-hexane. The
yellow gum was obtained in 84% yield. 1H NMR (CDCl3, 400
MHz) δ: 0.86 (t, J = 6.8 Hz, 3H, CH3CH2CH2), 0.99 (s, 6H, 2 ꢀ
CH3), 1.13 (d, J = 7.0 Hz, 3H, CH3CH(CO)), 1.25 (m, 6H, CH2-
CH2CH2CH2CH3), 1.37 (m, 2H, CH3CH(CO)CH2CH2CH2),
1.64 (m, 2H, CH3CH(CO)CH2CH2CH2), 2.40 (hextet, J = 7.0
Hz, 1H, CH3CH(CO)CH2), 2.68 (s, 2H, CH2Ar), 3.85 (s, 2H,
1
52.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, J=7.3 Hz,
3H, CH3CH2), 0.99 (s, 6H, 2 ꢀ CH3), 1.32 (m, 2H, CH3-
CH2CH2), 1.58 (m, 2H, CH2CH2CH2), 2.27 (t, J=7.6 Hz, 2H,
COCH2CH2), 2.68 (s, 2H, CH2Ar), 3.85 (s, 2H, OCH2), 7.44
(s, 1H, OH), 7.69 (td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 7.67
(td, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH), 8.09 (dd, J = 7.6 Hz, J =
1.4 Hz, 1H, ArH), 8.13 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H, ArH).

1218 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Kongkathip et al.
Figure 4. Redox difference spectra of purified yeast cyt bc1 in the presence of inhibitors (stigmatellin (Stig), antimycin A (Ant A), 31, and 37).
Spectra were calculated as DQH-reduced cyt bc1 complex minus inhibitor-cyt bc1 complex. The cyt bc1 at 2 μM was used for each assay.
Vertical lines indicate the relevant absorption peaks for heme c1 (left) and heme b (right).
OCH2), 7.69 (td, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH), 7.76 (td, J =
7.6 Hz, J = 1.2 Hz, 1H, ArH), 8.09 (m, 1H, ArH), 8.12 (m, 1H,
ArH).
hexane-dichloromethane to give yellow needles, mp 68.0-
1
69.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.91 (t, 3H, J = 6.8
Hz, CH3CH2), 1.02 (s, 6H, 2 ꢀ CH3), 1.28 (m, 14H, (CH2)7), 1.61
(m, 2H, COCH2CH2), 2.29 (t, 2H, J = 7.6 Hz, COCH2CH2),
2.71 (s, 2H, CH2Ar), 3.88 (s, 2H, OCH2), 7.46 (s, 1H, OH), 7.73
(td, 1H, J = 7.6 Hz, J = 1.3 Hz, ArH), 7.80 (td, 1H, J = 7.6 Hz,
J = 1.3 Hz, ArH), 8.13 (dd, 1H, J = 7.6 Hz, J = 1.3 Hz, ArH),
8.16 (dd, 1H, J = 7.6 Hz, J = 1.3 Hz, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Nonanoate (33). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow oil
was obtained in 73% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 52.5-
1
53.5 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.90 (t, 3H, J = 6.7
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Tetradecanoate (37). Flash column chromatogra-
phy was used, eluting with 4% ethyl acetate-hexane. The yellow
solid was obtained in 80% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 79.0-
Hz, CH3CH2), 1.02 (s, 6H, 2 ꢀ CH3), 1.28 (m, 8H, (CH2)5CH2,
1.62 (m, 2H, COCH2CH2), 2.29(t, 2H, J = 7.5Hz, COCH2CH2),
2.71 (s, 2H, CH2Ar), 3.87(s, 2H, OCH2), 7.47 (s, 1H, OH), 7.73 (t,
1H, J = 7.5 Hz, ArH), 7.80 (t, 1H, J = 7.6 Hz, ArH), 8.03 (d, 1H,
J = 7.6 Hz, ArH), 8.06 (d, 1H, J=7.6 Hz, ArH).
1
80.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, 3H, J = 6.8
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Decanoate (34). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow oil
was obtained in 72% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 61.0-
Hz, CH3CH2), 0.99 (s, 6H, 2 ꢀ CH3), 1.25 (m, 20H, CH3-
(CH2)10CH2), 1.59 (m, 2H, COCH2CH2CH2), 2.26 (t, 2H, J =
7.6 Hz, CH2CH2CO), 2.68 (s, 2H, CH2Ar), 3.86 (s, 2H, OCH2),
7.44 (s, 1H, OH), 7.70 (td, 1H, J=7.6 Hz, J=1.2 Hz, ArH), 7.77
(td, 1H, J=7.6 Hz, J=1.2 Hz, ArH), 8.10 (dd, 1H, J=7.6 Hz, J=
1.2 Hz, ArH), 8.13 (dd, 1H, J=7.6 Hz, J=1.2 Hz, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Palmitate (38). Flash column chromatography
was used, eluting with 4% ethyl acetate-hexane. The yellow
solid was obtained in 77% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 76.0-
1
62.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.91 (t, 3H, J = 6.8
Hz, CH3CH2), 1.02 (s, 6H, 2 ꢀ CH3), 1.28 (m, 12H, (CH2)6CH2),
1.62 (m, 2H, COCH2CH2), 2.29 (t,2H, J = 7.6 Hz, COCH2CH2),
2.71 (s, 2H, CH2Ar), 3.88 (s, 2H, OCH 2), 7.46 (s, 1H, OH), 7.73
(dt, 1H, J = 7.6 Hz, J = 1.2 Hz, ArH), 7.80 (dt, 1H, J = 7.6 Hz,
J=1.2 Hz, ArH), 8.12 (d, 1H, J=7.6 Hz, ArH), 8.15 (d, 1H,
J=7.6 Hz, ArH).
1
77.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, 3H, J = 6.8
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Undecanoate (35). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow
solid was obtained in 83% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 64.5-
Hz, CH3CH2), 0.99 (s, 6H, 2 ꢀ CH3), 1.25 (br s, 24H, CH3-
(CH2)12CH2), 1.59 (m, 2H, CH2CH2CHCO), 2.26 (t, 2H, J =
7.6 Hz, CH2CH2CO), 2.68 (s, 2H, CH2Ar), 3.86 (s, 2H, OCH2),
7.45 (s, 1H, OH), 7.70 (td, 1H, J=7.6 Hz, J=1.2 Hz, ArH),
7.77 (td, 1H, J = 7.6 Hz, J = 1.2 Hz, ArH), 8.10 (dd, 1H, J =
7.6 Hz, J = 1.2 Hz, ArH), 8.13 (dd, 1H, J = 7.6 Hz, J = 1.2 Hz,
ArH).
1
65.5 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.90 (t, 3H, J = 6.8
Hz, CH3CH2), 1.01 (s, 6H, 2 ꢀ CH3), 1.28 (m, 12H, (CH2)7CH2),
1.62 (quin, 2H, COCH2CH2CH2), 2.29 (t, 2H, J = 7.6 Hz,
COCH2CH2), 2.71 (s, 2H, CH2Ar), 3.88 (s, 2H, OCH2), 7.46 (s,
1H, OH), 7.73 (td, 1H, J = 7.6 Hz, J = 1.1 Hz, ArH), 7.80 (td,
1H, J = 7.6 Hz, J = 1.1 Hz, ArH), 8.12 (dd, 1H, J = 7.6 Hz, J =
1.1 Hz, ArH), 8.15 (dd, 1H, J = 7.6 Hz, J = 1.1 Hz, ArH).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Dodecanoate (36). Flash column chromatography
was used, eluting with 3% ethyl acetate-hexane. The yellow
solid was obtained in 79% yield, and then it was crystallized with
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)-2,2-di-
methylpropyl Stearate (39). Flash column chromatography was
used, eluting with 4% ethyl acetate-hexane. The yellow solid
was obtained in 74% yield, and then it was crystallized with
hexane-dichloromethane to give yellow needles, mp 82.0-
1
83.0 ꢀC. H NMR (CDCl3, 400 MHz) δ: 0.88 (t, 3H, J=6.8
Hz, CH3CH2), 0.99 (s, 6H, 2 ꢀ CH3), 1.25 (br s, 28H, CH3-
(CH2)14CH2), 1.59 (m, 2H, CH2CH2CH2CO), 2.26 (t, 2H, J =
7.2 Hz, CH2CH2CO), 2.68 (s, 2H, CH2Ar), 3.86 (s, 2H, OCH2),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1219
7.44 (s, 1H, OH), 7.70 (td, 1H, J = 7.6 Hz, J = 1.2 Hz, ArH),
7.77 (td, 1H, J = 7.6 Hz, J = 1.2 Hz, ArH), 8.10 (dd, 1H, J = 7.6
Hz, J = 1.2 Hz, ArH), and 8.13 (dd, 1H, J = 7.6 Hz, J = 1.2 Hz,
ArH).
temperature for 16 h. The precipitate of dicyclohexylurea was
filtered off. The crude product was purified by flash column
chromatography, eluting with 3% ethyl acetate-hexane to
provide the desired product 46 (26.5 mg, 35% yield for two
1
General Procedure for Synthesis of Naphthoquinone Aliphatic
Esters 42-44. A solution of 1,10-carbonyldiimidazole (CDI)
(0.32 mmol) in dry THF (3 mL) was added dropwise to the
stirring and ice-cooled solution of carboxylic acid (0.32 mmol) in
dry THF (1 mL). After being stirred for 4 h at room tempera-
ture, the reaction mixture was placed in an ice-cooled bath and
to it was added dropwise a solution of 2-hydroxy-3-(3-hydro-
xypropyl)-1,4-naphthoquinone (0.22 mmol) in dry THF (1 mL)
over 5 min. After being stirred at room temperature for 18 h, the
reaction mixture was quenched with saturated ammonium chlo-
ride solution and extracted with dichloromethane (5 ꢀ 10 mL).
The combined organic layer was washed with saturated sodium
chloride solution (3 ꢀ 30 mL), dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography.
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)propyl
Acetate (42). Flash column chromatography was used, eluting
with 10% ethyl acetate-hexane. The yellow solid was obtained
in 32% yield, and then it was crystallized with hexane-
dichloromethane to give yellow needles, mp 104.0-106.0 ꢀC.
1H NMR, (400 MHz, CDCl3), δ: 8.15 (dd, 1H,ArH, J = 1.4, 7.5
Hz), 8.11 (dd, 1H, ArH, J = 1.4, 7.6 Hz), 7.79 (dt, 1H, ArH, J =
1.4, 7.5 Hz), 7.72 (dt, 1H, ArH, J = 1.4, 7.5 Hz), 7.40 (s, 1H,
ArOH), 4.13 (t, 2H, ArCH2CH2O, J = 6.5 Hz), 2.72 (t, 1H,
ArCH2, J = 7.5 Hz), 2.06 (s, 3H, O(CO)CH3), 1.95 (quint, 2H,
ArCH2CH2CH2O, J = 7.0 Hz).
steps) as a yellow gum and cyclized product 45 (16.6 mg). H
NMR (CDCl3, 400 MHz) δ: 0.81 (t, J = 3.4 Hz, 3H, CH3),
1.10-1.92 (m, 20H, CH2), 2.34 (t, J = 7.56 Hz, 2H, COCH2),
2.65 (s, 2H, ArCH2), 3.91 (s, 2H, OCH2), 7.37 (s, 1H, OH), 7.62
(dt, J = 7.5, 1.4 Hz, 1H, ArH), 7.68 (dt, J = 7.5, 1.4 Hz, 1H,
ArH), 8.01 (m, 1H, ArH), 8.04 (m, 1H, ArH).
FTIR, 13C NMR, MS, and elemental analysis data of com-
pounds 18-39, 42-44, and 46 are in Supporting Information.
Antimalarial Activity Assay. Plasmodium falciparum (K1,
multidrug resistant strain) was cultivated in vitro according to
Trager amd Jensen13a in RPMI 1640 medium containing 20 mM
HEPES (N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid),
32 mM NaHCO3, and 10% heat activated human serum with
3% erythrocytes and incubated at 37 ꢀC in an incubator with 3%
CO2. Cultures were diluted with fresh media and erythrocytes
everyday according to cell growth. Quantitative assessment of
antimalarial activity in vitro was determined by microculture
radioisotope techniques based upon the methods described by
Desjardins et al.13b Briefly, a mixture of 200 μL of 1.5%
erythrocytes with 1% parasitemia at the early ring stage was
pre-exposed to 25 μL of the medium containing a test sample
dissolved in 1% DMSO (0.1% final concentration) for 24 h,
employing the incubation conditions described above. Subse-
quently, 25 μL of [3H]hypoxanthine (Amersham) in culture
medium (0.5 μCi) was added to each well and plates were
incubated for an additional 24 h. Levels of incorporated radio-
actively labeled hypoxanthine indicating parasite growth were
determined using a TopCount microplate scintillation counter
(Packard). Inhibition concentration (IC50) represents the con-
centration that indicates 50% reduction in parasite growth. The
standard sample was dihydroartemisinin (DHA).
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)propyl
Butyrate (43). Flash column chromatography was used, eluting
with 10% ethyl acetate-hexane. The yellow solid was obtained
in 60% yield, and then it was crystallized with hexane-
1
dichloromethane to give yellow needles, mp 77.0-78.0 ꢀC. H
NMR, (400 MHz, CDCl3), δ: 0.95 (t, 3H, (CO)CH2CH2CH3,
J=7.5 Hz), 1.66 (sixt, 2H, (CO)CH2CH2CH3, J = 7.5 Hz), 1.93
(quint, 2H, ArCH2CH2CH2O, J = 6.5 Hz), 2.29 (t, 2H, O-
(CO)CH2CH2CH3, J = 7.5 Hz), 2.72 (t, 2H, ArCH2CH2CH2O,
J = 7.5 Hz), 4.14 (t, 2H, ArCH2CH2CH2O, J = 6.5 Hz), 7.39 (s,
1H, ArOH), 7.71 (dt, 1H, ArH, J = 1.4, 7.5 Hz), 7.78 (dt, 1H,
ArH, J = 1.4, 7.5 Hz), 8.11 (dd, 1H, ArH, J = 1.4, 7.5 Hz), 8.14
(dd, 1H,ArH, J = 1.4, 7.5 Hz).
Plasmodium falciparum (Strain 3D7) and Yeast (Saccharo-
myces cerevisiae) Culture. P. falciparum (strain 3D7) was
cultured in a 2% Oþ erythrocyte suspension as described in
Biagini’s method.28 S. cerevisiae was grown in YPD broth
at 28 ꢀC as described in Fisher’s method.29
Preparation of P. falciparum Cell-Free Extracts, Yeast Mito-
chondrial Membranes, and Rat Liver Microsomes. P. falciparum
was harvested from infected erythrocytes as described in
Biagini’s method.28 Parasite cell-free extracts were prepared
immediately prior to use by 20 s of sonication on ice. Yeast
mitochondrial membranes were prepared as described in
Fisher’s method.29 Adult male Wistar rats were obtained from
Charles River Laboratories (Margate, Kent, U.K.). Wistar rat
liver microsomes were prepared from male rats (125-170 g) as
described by Gill’s method.30
3-(1,4-Dihydro-2-hydroxy-1,4-dioxonaphthalen-3-yl)propyl
Octanoate (44). Flash column chromatography was used, elut-
ing with 10% ethyl acetate-hexane. The yellow solid was
obtained in 49% yield, and then it was crystallized with hexane-
1
dichloromethane to give yellow needles, mp 80.0-81.0 ꢀC. H
NMR, (400 MHz, CDCl3), δ: 0.89 (t, 3H, (CO)CH2(CH2)5CH3,
J = 6.9 Hz), 1.29-1.70 (m, 10H, (CO)CH2(CH2)5CH3), 1.93
(quint, 2H, ArCH2CH2CH2O, J=7.0 Hz), 2.30 (t, 2H, (CO)-
CH2(CH2)5CH3, J = 7.6 Hz), 2.72 (t, 2H, ArCH2CH2CH2O,
J = 7.5 Hz), 4.14 (t, 2H, ArCH2CH2CH2O, J = 6.5 Hz), 7.41 (s,
1H, ArOH), 7.72 (dt, 1H, ArH, J = 1.4, 7.6 Hz), 7.79 (dt, 1H,
ArH, J = 1.4, 7.6 Hz), 8.11 (dd, 1H, ArH, J = 1.2, 7.6 Hz), 8.14
(dd, 1H,ArH, J = 1.0, 7.6 Hz).
Decylubiquinol Preparation. Decylubiquinol (2,3-dimethoxy-
5-methyl-n-decyl-1,4-benzoquinol) was prepared by dithionite
reduction of decylubiquinone (Sigma) as described in Fisher’s
method.29
Decylubiquinol/Cytochrome c Reductase Assay. Cytochrome
bc1 activity was monitored spectrophotometrically in P. falci-
parum cell-free extracts and yeast mitochondrial membrane
preparations by the steady-state decylubiquinol/cytochrome c
reductase using ε550-542nm in a Cary 4000 UV-vis spectrophoto-
meter at 21 ꢀC. Assays were performed in 50 mM potassium phos-
phate buffer (pH 7.5) containing 2 mM EDTA, 10 mM KCN,
and 30 μM equine heart cytochrome c (Sigma) and the reactions
initiated by the addition of 50 μM decylubiquinol. The total
assay volume was 0.7 mL. Yeast and rat bc1 concentration were
assessed by the “dithionite reduced minus ferricyanide-oxi-
dized” difference spectra, using the extinction coefficient con-
(1-((3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl)-
cyclohexyl)methyl Octanoate (46). A solution of compound
45 (0.177 mmol) in 1% aqueous sodium hydroxide solution
(0.266 mmol) was refluxed for 1 h. After completion of the
reaction, the reaction mixture was cooled to room temperature
and neutralized with acetic acid to pH 7, then extracted with
dichloromethane (3 ꢀ 15 mL). After concentration, the inter-
mediate 10c was obtained together with a small amount of the
cyclized product 45 (monitoring by TLC). A solution of car-
boxylic acid (0.23 mmol), dicyclohexylcarbodiimide (DCC)
(0.23 mmol), and DMAP (0.053 mmol) was stirred in dry
CH2Cl2 (5 mL) at room temperature. After 3 h, a solution of
compound 45 and naphthoquinone alcohol intermediate 10c in
THF (2 mL) was added and then the mixture was stirred at room
stant ε562-575nm = 28.5 mM-1 cm-1 15a
and the concentrations
,
adjusted to 5 nM for the decylubiquinol/cytochrome c reductase
assay. The presence of contaminating hemozoin precludes the
use of spectrophotometric methods to directly determine cyt bc1

1220 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Kongkathip et al.
concentration in P. falciparum cell-free extracts. Therefore, total
protein concentration was first measured in the P. falciparum
cell-free extract by the Bradford method15b and then adjusted to
0.1 mg protein/mL for the decylubiquinol/cytochrome c reduc-
tase assay. Compounds 31 and 37 were prepared as 20 mM stock
solutions in DMSO and were added to the decylubiquinol/
cytochrome c reductase system without prior incubation. The
concentration of DMSO in the assay did not exceed 0.3% v/v.
IC50 values were calculated using four-parameter sigmoidal
curve fitting (Kaleidagraph, Synergy Software).
(3) Fotie, J. Quinones and Malaria. Anti-Infect. Agents Med. Chem.
2006, 5, 357–366.
(4) Verma, R .P. Anti-Cancer Activities of 1,4-Naphthoquinones: A
QSAR Study. Anti-Cancer Agents Med. Chem. 2006, 6, 489–499.
(5) Wendel, W. B. The Influence of Naphthoquinones upon the
Respiratory and Carbohydrate Metabolism of Malarial Parasites.
Fed. Proc. 1946, 5, 406–407.
(6) Fieser, L. F.; Berliner, E.; Bondhus, F. J.; Chang, F. C.; Dauben,
W. G.; Ettlinger, M. G.; Fawaz, G.; Fields, M.; Fieser, M.;
Heidelberger, C.; Heymann, H.; Seligman, A. M.; Vaughan, W.
R.; Wilson, A. G.; Wilson, E.; Wu, M. I.; Leffler, M. T.; Hamlin, K.
E.; Hathaway, R. J.; Matson, E. J.; Moore, E. E.; Moore, M. B.;
Rapala, R. T.; Zaugg, H. E. Naphthoquinones. I-XVII. J. Am.
Chem. Soc. 1948, 70, 3151–3244.
(7) Hudson, A. T. Atovaquone;A Novel Broad Spectrum Anti-
infective Drug. Parasitol. Today 1993, 9, 66–68 and references
therein .
(8) (a) Looareesuwan, S.; Chulay, J. D.; Canfield, C. J.; Hutchinson,
D. B. Malarone (Atovaquone and Proguanil Hydrochloride): A
Review of Its Clinical Development for Treatment of Malaria.
Malarone Clinical Trials Study Group. Am. J. Trop. Med. Hyg.
1999, 60, 533–541. (b) Baggish, A. L.; Hill, D. R. Antiparasitic Agent
Atovaquone. Antimicrob. Agents Chemother. 2002, 46, 1163–1173.
(9) Kessl, J. J.; Lange, B. B.; Merbitz-Zahradnik, T.; Zwicker, K.; Hill,
P.; Meunier, B.; Palsdottir, H.; Hunte, C.; Meshnick, S.; Trum-
power, B. L. Molecular Basis for Atovaquone Binding to the
Cytochrome bc1 Complex. J. Biol. Chem. 2003, 278, 31312–31318.
(10) Mather, M. W.; Darrouzet, E.; Valkova-Valchanova, M.; Cooley,
J. W.; McIntosh, M. T.; Daldal, F.; Vaidya, A. B. Uncovering the
Molecular Mode of Action of the Antimalarial Drug Atovaquone
Using a Bacterial System. J. Biol. Chem. 2005, 280, 27458–27465.
(11) (a) Hammond, D. J.; Burchell, J. R.; Pudney, M. Inhibition of
Pyrimidine Biosynthesis de Novo in Plasmodium falciparum by 2-(4-
t-Butylcyclohexyl)-3-hydroxy-1,4-naphthoqinone in Vitro. Mol.
Biochem. Parasitol. 1985, 14, 97–109. (b) Fry, M.; Pudney, M. Site of
Action of the Antimalarial hydroxynaphthoquinone, 2-[trans-4-(40-
Chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80).
Biochem. Pharmacol. 1992, 43, 1545–1553.
(12) Wu, T. S.; Hsu, H. C.; Wu, P. L.; Leu, Y. L.; Chan, Y. Y.; Chern, C.
Y.; Yeh, M. Y.; Tien, H. J. Naphthoquinone Esters from the Root
of Rhinacanthus nasutus. Chem. Pharm. Bull. 1998, 46, 413–418.
(13) (a) Trager, W.; Jensen, J. B. Human Malaria Parasites in Con-
tinuous Culture. Science 1976, 193, 673–675. (b) Desjardins,
R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Quantitative
Assessment of Antimalarial Activity in Vitro by a Semiautomated
Microdilution Technique. Antimicrob. Agents Chemother. 1979, 16,
710–718.
(14) Skehan, P.; Storeng, R.; Scudiero, D.; Monko, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screen-
ing. J. Natl. Cancer. Inst. 1990, 82, 1107–1112.
(15) (a) Vanneste, W. H. Molecular Proportion of the Fixed Cyto-
chrome Components of the Respiratory Chain of Keilin-Hartree
Particles and Beef Heart Mitochondria. Biochim. Biophys. Acta
1966, 113 (1), 175–178. (b) Bradford, M. M. A Rapid and Sensitive
Method for the Quantitation of Microgram Quantities of Protein
Utilizing the Principle of Protein-Dye Binding. Anal. Biochem.
1976, 72, 248–254.
(16) Wenz, T.; Covian, R.; Hellwig, P.; MacMillan, F.; Meunier, B.;
Trumpower, B. L.; Hunte, C. Mutational Analysis of Cytochrome
b at the Ubiquinol Oxidation Site of Yeast Complex III. J. Biol.
Chem. 2007, 282, 3977–3988.
(17) von Jagow, G.; Link, Th. A. Use of Specific Inhibitors on the
Mitochondrial bc1 Complex. Method. Enzymol. 1986, 126, 253–
271.
(18) Fisher, N.; Meunier, B. Molecular Basis of Resistance to Cyto-
chrome bc1 Inhibitors. FEMS Yeast Res. 2008, 8, 183–8192.
(19) Zhao, P.-L.; Wang, L.; Zu, X.-L.; Huang, X.; Zhau, C.-G.; Wu, J.-
W.; Yang, G.-F. Subnanomolar Inhibitor of Cytochrome bc1
Complex Designed by Optimizing Interaction with Conformation-
ally Flexible Residues. J. Am. Chem. Soc. 2009, DOI: 10.1021/
ja905756c.
(20) Altmann, K.-H.; Carreira, E. M. Unravelling a Molecular Target
of Macrolides. Nat. Chem. Biol. 2008, 4, 388–389.
(21) Crowley, P. J.; Berry, E. A.; Cromartie, T.; Daldal, F.; Godfrey, C.
R. A.; Lee, D.-W.; Philips, J. E.; Taylor, A.; Viner, R. The Role of
Molecular Modelling in the Design of Analogues of the Fungicidal
Natural Products Crocacins A and D. Bioorg. Med. Chem. 2008,
16, 10345–10355.
Protein Production and Purfication. Wild type yeast cells
(strain CKWT) were grown in YPGal medium (1% yeast
extract, 2% peptone, and 3% galactose) according to Wenz’s
method.16 Yeast mitochondrial membrane preparation and
cytochrome bc1 complex (cyt bc1) purification were described
in Wenz’s method.31 The purified cyt bc1 was stored in 50 mM
potassium phosphate buffer (pH 7.5, including 300 mM NaCl,
250 mM sucrose, and 0.05% UM) at -80 ꢀC. The cyt bc1 was
quantified by measuring the dithionite-reduced minus ferricya-
nite oxidized difference spectra at the R band of cyt bc1. The
extinction coefficient of b heme (ε562-575nm = 28.5 mM-1 cm-1
was used to calculate the cyt bc1 concentration.
,
The cyt bc1 Activity Assay. The reaction buffer for ubiqui-
nol-cytochrome c reductase activity of cyt bc1 contained 50
mM potassium phosphate buffer, pH 7.4, 0.05% UM, 1 mM
KCN, 0.2 mM EDTA, 250 mM sucrose, and 12 μM equine heart
cytochrome c. Purified cyt bc1 was diluted to 8 nM in the
reaction buffer. Stigmatellin and 31 and 37 inhibitors were
prepared as 40 and 20 mM stock solutions in DMSO, respec-
tively. Reactions were initiated by adding decylubiquinol
(DQH, final concentration of 50 μM) and monitored spectro-
scopically at 550 nm at room temperature. IC50 values were
determined with a concentration series of stigmatellin and 31
and 37 compounds. Inhibitors were added to the reaction
mixture prior to the start of the reaction.
Inhibitor Binding Assay. Cytochrome bc1 was diluted to 2 μM
in 50 mM potassium phosphate buffer containing 0.05% UM.
Inhibitors were added to cyt bc1 at a final concentration of 5 μM
31, 5 μM 37, 40 μM stigmatellin, and 40 μM antimycin A,
respectively. Compounds 31 and 37 contribute absorbance
between 500 and 600 nm; thus, 5 μM of both compounds was
used to avoid interference with the absorbance of heme R and β
bands. Redox difference spectra were recorded as DQH-reduced
cyt bc1-inhibitor complex minus protein-inhibitor complex.
Acknowledgment. This work was supported by the
Thailand Research Fund (TRF), Thailand. N.P. and K.H.
are Ph.D. students under the Royal Golden Jubilee Ph.D.
program, TRF. Financial support from the Center for
Innovation in Chemistry (PERCH-CIC) is also gratefully
acknowledged.
Supporting Information Available: FTIR, 13C NMR, MS, and
elemental analysis data for compounds 18-39, 42-44, and 46.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) (a) Kongkathip, N.; Luangkamin, S.; Kongkathip, B.; Sangma,
C.; Grigg, R.; Kongsaevee, P.; Prabpai, S.; Pradidphol, N.;
Piyaviriyagul, S.; Siripong, P. Synthesis of Novel Rhinacanthins
and Related Anticancer Naphthoquinone Esters. J. Med. Chem.
2004, 47, 4427–4438. (b) Kongkathip, N.; Kongkathip, B.; Siripong,
P.; Sangma, C.; Luangkamin, S.; Niyomdecha, M.; Pattanapa, S.;
Piyaviriyagul, S.; Kongsaeree, P. Potent Antitumor Activity of
Synthetic 1,2-Naphthoquinones and 1,4-Naphthoquinones. Bioorg.
Med. Chem. 2003, 11 (14), 3179–3191.
(2) Rojanapa, W.; Tepsuwan, A.; Siripong, P. Mutagenicity and
Antimutagenicity of Thai Medicinal Plants. Basic Life Sci. 1990,
52, 447–452.
(22) Xiang, H.; McSurdy-Freed, J.; Moorthy, G. S.; Hugger, E.;
Bambal, R.; Hau, C.; Ferrer, S.; Gargallo, D.; Davis, C. B.
Preclinical Drug Metabolism and Pharmacokinetic Evaluation of

Article
GW844520, a Novel Anti-Malarial Mitochondrial Electron Trans-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1221
(27) Kongkathip, N.; Kongkathip, B.; Hasitapan, K. Novel Antima-
larial 1,4-Naphthoquinone Aliphatic Ester Derivatives with Inhi-
bition of Plasmodium falciparum. Thailand Patent Application
0701003412, 2007.
(28) Biagini, G. A.; Fisher, N.; Berry, N.; Stocks, P. A.; Meunier, B.;
Williams, D. P.; Bonar-Law, R.; Bray, P. G.; Owen, A.; O’Neill, P.
M.; Ward, S. A. Acridinediones: Selective and Potent Inhibitors of
the Malaria Parasite Mitochondrial bc1 Complex. Mol. Pharmacol.
2008, 73, 1347–1355.
(29) Fisher, N.; Castleden, C. K.; Bourges, I.; Brasseur, G.; Dujardin,
G.; Meunier, B. Human Disease-Related Mutations in Cyto-
chrome b Studied in Yeast. J. Biol. Chem. 2004, 279, 12951–
12958.
port Inhibitor. J. Pharm. Sci. 2006, 95, 2657–2672.
(23) Yeates, C. L.; Batchelor, J. F.; Capon, E. C.; Cheeseman, N. J.;
Fry, M.; Hudson, A. T.; Pudney, M.; Trimming, H.; Woolven, J.;
Bueno, J. M.; Chicharro, J.; Fernandez, E.; Fiandor, J. M.;
Gargallo-Viola, D.; de las Heras, F. G.; Herreros, E.; Leon, M.
L. Synthesis and Structure-Activity Relationships of 4-Pyridones
as Potential Antimalarials. J. Med. Chem. 2008, 51, 2845–2852.
ꢀ
(24) Djimde, A.; Doumbo, O. K.; Cortese, J. F.; Kayentao, K.; Doum-
ꢀ
bo, S.; Diourte, Y.; Dicko, A.; Su, X.-Z.; Nomura, T.; Fidock, D.
A.; Wellems, T. E.; Plowe, C. V. A Molecular Marker for Chloro-
quine-Resistant Falciparum Malaria. N. Engl. J. Med. 2001, 344,
257–263.
(25) Cowman, A. F.; Morry, M. J.; Biggs, B. A.; Crosst, G. A. M.;
Foote, S. J. Amino Acid Changes Linked to Pyrimethamine
Resistance in the Dihydrofolate Reductase-Thymidylate
Synthase Gene of Plasmodium falciparum. Proc. Natl. Acad. Sci.
U.S.A. 1998, 85, 9109–9113.
(30) Gill, H. J.; Tingle, M. D.; Park, B. K. N-Hydroxylation of Dapsone
by Multiple Enzymes of Cytochrome P450: Implications for In-
hibition of Haemotoxicity. Br. J. Clin. Pharmacol. 1995, 40, 531–
538.
(31) Wenz, T.; Hellwig, P.; MacMillan, F.; Meunier, B.; Hunte, C.
Probing the Role of E272 in Quinol Oxidation of Mitochondrial
Complex III. Biochemistry 2006, 45, 9042–9052.
(26) White, N. J. Preventing Antimalarial Drug Resistance through
Combinations. Drug Resist. Updates 1998, 1, 3–9.