Correlation of antimalarial activity of artemisinin derivatives with binding affinity with ferroprotoporphyrin IX

Article abstract of DOI:10.1021/jm960767v

The antimalarial activity of a number of artemisinin derivatives, both newly synthesized and currently used as drugs, against Plasmodium falciparum in culture shows a correlation with their affinity of binding with ferroprotoporphyrin IX, as measured from the spectral change of the latter. The new C-16-functionalized artemisinin derivatives were obtained through a novel one-pot synthesis of artemisitene (2) from naturally abundant artemisinin (1), followed by Michael addition with nucleophiles. The correlation points to the biological significance of the interaction of these derivatives with ferroprotoporphyrin IX and may provide a basis for primary screening of peroxidic antimalarials of similar structures.

Full text of DOI:10.1021/jm960767v

©
Copyright 1997 by the American Chemical Society
Volume 40, Number 5
February 28, 1997
Articles
Cor r ela tion of An tim a la r ia l Activity of Ar tem isin in Der iva tives w ith Bin d in g
Affin ity w ith F er r op r otop or p h yr in IX
Sumpan Paitayatat,†,‡ Bongkoch Tarnchompoo,‡,§ Yodhathai Thebtaranonth, and Yongyuth Yuthavong*,†,§
‡
Departments of Biochemistry and Chemistry, Faculty of Science, Mahidol University, Rama 6 Road,
Bangkok 10400, Thailand, and National Science and Technology Development Agency, Rama 6 Road,
Bangkok 10400, Thailand
X
Received November 5, 1996
The antimalarial activity of a number of artemisinin derivatives, both newly synthesized and
currently used as drugs, against Plasmodium falciparum in culture shows a correlation with
their affinity of binding with ferroprotoporphyrin IX, as measured from the spectral change of
the latter. The new C-16-functionalized artemisinin derivatives were obtained through a novel
one-pot synthesis of artemisitene (2) from naturally abundant artemisinin (1), followed by
Michael addition with nucleophiles. The correlation points to the biological significance of the
interaction of these derivatives with ferroprotoporphyrin IX and may provide a basis for primary
screening of peroxidic antimalarials of similar structures.
The prevalence of malaria in many regions of the
world, together with the lack of vaccine and the emer-
gence of resistance to antimalarials in use, makes it
necessary to search for new antimalarials. Artemisinin
derivatives form a group of such new drugs currently
strongly with hemin (ferriprotoporphyrin IX) and its
ferrous form, ferroprotoporphyrin IX. In parasite me-
tabolism, hemin is left after digestion of hemoglobin by
the parasite. A potentially toxic compound, it is con-
verted to hemozoin (malarial pigment), which has been
1
under development and already in use in some
shown to be essentially â-hematin, a polymerized form
countries.2
-6
They share a common sesquiterpenoid
15
of heme devoid of immediate toxicity.
Some anti-
structure with an endoperoxide function which is es-
sential for the activity. Due to synergism with other
oxidant drugs and oxygen and reduction of activity by
agents which lower oxidative stress, we and others
proposed that the mechanism of their action involves
malarials, such as chloroquine, interfere with the poly-
merization, probably accounting for the generation of
toxicity to the parasite. The interaction of artemisinin
16
with hemin, which leads to adduct formation and
presumably interferes with the normal conversion of
hemin to hemozoin, could therefore form the basis of
its antimalarial action. Hemin or its immediate precur-
sor, ferroprotoporphyrin IX, could therefore be the
biological target of artemisinin. Alternatively, the
interaction could be similar to, and provide a model for,
the interaction of artemisinin with its real, as yet
unidentified target within the parasite. It is therefore
important to understand the mode of interaction of
artemisinin and its derivatives with hemin and ferro-
protoporphyrin IX and its relation with the antimalarial
action of the drugs. A correlation between a parameter
7
-11
exertion of oxidative stress on the parasite.
Al-
though the specific nature of the oxidative stress is as
yet unclear, the action probably involves desequestra-
tion of iron, as the drugs are strongly antagonistic with
such iron chelators as desferrioxamine, pyridoxal ben-
1
1
zoylhydrazone, and 1,2-dimethyl-3-hydroxypyrid-4-one.
Meshnick et al.
1
2-14
showed that artemisinin interacts
*
Address correspondence to this author.
Department of Biochemistry.
Department of Chemistry.
National Science and Technology Development Agency.
Abstract published in Advance ACS Abstracts, February 1, 1997.
†
‡
§
X
S0022-2623(96)00767-4 CCC: $14.00 © 1997 American Chemical Society

6
34 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Paitayatat et al.
of such interaction and the antimalarial activity would
provide strong evidence for its biological significance and
could furthermore give a convenient method for primary
screening of new artemisinin derivatives.
Sch em e 1
In this paper, we show that a number of artemisinin
derivatives, many of which are those with modifications
at C-16, interact strongly with ferroprotoporphyrin IX.
The dissociation constants for the interaction of the
artemisinin derivatives with ferroprotoporphyrin IX,
calculated from spectral change due to binding, show a
correlation with the antimalarial activity of the deriva-
tives. We also demonstrate that artemisitene, 2, can
be obtained from artemisinin, 1, by a one-pot selenoxide
elimination reaction. Compound 2 readily undergoes
a 1,4-addition reaction with nucleophiles; hence it serves
as a convenient precursor for the C-16-functionalized
artemisinin derivatives 3.
Ch em istr y
Artemisitene, 2, the oxidized form of artemisinin, 1,
is reported to be a minor constituent in an American
variant of Artemisia annua.17 The unique structural
feature of 2, bearing an exocyclic R,â-unsaturated ketone
moiety, chemically presents an opportunity for nucleo-
philic attack via a (Michael) 1,4-addition reaction. The
overall process, finally providing the Michael adduct,
e.g., 3, is equivalent to a straightforward derivatization
of the artemisinin molecule at the C-16 methyl carbon
(
3
see numbering in 1). Artemisinin derivatives such as
(and also 4 where Nu * H) would be highly attractive
Sch em e 2
for the structure-activity relationship study due to the
fact that they are structurally different from other
artemisinin derivatives which are now in use clinically,
e.g., artemether, arteether, and artesunate, 4b-d ,
respectively. The latter are products obtained from
derivatization of 1 at the C-10 carbonyl moiety and are
known to readily metabolize in vivo giving dihydro-
artemisinin, 4a .18
However, owing to the fact that artemisitene (2)
coexists with 1 in the plant in rather low and variable
quantities and also that its isolation and purification
1
7
from the crude extract are not straightforward, arte-
misitene has so far been underutilized.19 Obviously, a
semisynthesis of artemisitene from the cheap, naturally
abundant artemisinin would provide the solution to
overcome this drawback. Indeed, following a number
of unsuccessful attempts,20 artemisitene was recently
artemisitene (2) was obtained as the sole product in 73%
purified yield.
2
1
finally synthesized from artemisinin. Upon inspection
of the molecular model of 1 we concluded that the short
The origin of reaction regiospecificity, providing only
2 without any trace of its double-bond isomer, iso-
artemisitene 5, can be explained in terms of involving
the formation of lithium ester enolate 6 which stereo-
specifically reacts with phenylselenide bromide on the
â-face to provide 7 and finally 2 as shown in Scheme 2.
Artemisitene (2) readily reacts with sulfur, carbon,
and nitrogen nucleophiles to give the corresponding
Michael adducts 3 (3a -d in Scheme 1). In some cases
lithium anions derived from ferrocene derivatives were
deliberately employed as carbon nucleophiles (3b,c).
While only one (â-stereochemistry at C-9) isomer was
detected in the case of adduct 3a , isomeric mixtures
were obtained in the other cases.
and convenient method for conversion of 1 to 2 should
involve the selenoxide elimination reaction.2
2,23
This
mentioned approach has already been examined but
gave a negative result from which the starting material
and its C-9 stereoisomer, 9-epi-artemisinin, were ob-
2
0
tained. Therefore it was of importance to reinvestigate
the method of conversion of 1 to 2.
Here we report a straightforward, regiospecific, one-
pot, high-yield conversion of artemisinin (1) to arte-
misitene (2) involving selenoxide elimination and the
reaction of 2 with various nucleophiles in a Michael
addition fashion. Upon treatment of 1 with lithium
diisopropylamide (LDA) in tetrahydrofuran at -78 °C
followed by sequential additions of phenylselenenide
bromide2 and 30% aqueous hydrogen peroxide solution
followed by raising the reaction temperature to 0 °C,
Resu lts a n d Discu ssion
4
If the binding of artemisinin with ferroprotoporphyrin
IX is a biologically significant step, leading to killing of

Antimalarial Activity of Artemisinin
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 635
Ta ble 1. Dissociation Constants (Kd) and IC50 Values of
Artemisinin and Its Derivatives against P. falciparum in Vitro
from hemoglobin in the food vacuole after the parasite
has digested the globin component. Normally, it would
proceed to become ferriprotoporphyrin IX and subse-
quently hemozoin. Interaction with artemisinin or its
derivatives disrupts this normal process and may give
rise to potentially toxic agents. Meshnick et al. showed
that, after binding of artemisinin with hemin and
hemozoin, an adduct is formed with a molecular weight
4
compd
Kd (×10- M-1
)
IC50 (M)
-
-
-
-
9
8
8
9
1
2
3
3
3
3
4
4
4
4
4
0.24
0.26
0.34
0.14
0.25
0.20
0.21
0.27
0.32
0.38
0.05
5 × 10
3 × 10
2 × 10
1 × 10
a
b
-
-
-
-
-
-
-
9
c (isomer II)
2.1 × 10
3.2 × 10
1.1 × 10
4.1 × 10
2.4 × 10
6.2 × 10
3.9 × 10
8
d
a
b
c
d
e
9
12
almost equal to the sum of the two components.
9
However, the biological significance of such adduct
8
2
6,27
10
10
formation is unclear. Asawamahasakda et al.
showed
later that artemisinin and derivatives bind covalently
with parasite protein components, and the extent of this
binding reflects the antimalarial activities of the deriva-
tives. It was therefore postulated that artemisinin acts
in two steps: firstly, an artemisinin radical is formed
after interaction with suitable parasite component(s),
and secondly, the radical alkylates other essential
parasite component(s) thereby interfering with the
normal function of the component(s). Hemin or ferro-
protoporphyrin IX could, according to this scenario, play
a role in the generation of the artemisinin radical, but
although it is also alkylated by this radical, this alkyl-
ation was not considered vital for the killing of the
parasite. Our finding strengthens the role of ferro-
protoporphyrin IX in the initial interaction with arte-
misinin or its derivatives. It is presently unclear
whether this interaction leads to generation of arte-
misinin-derived free radicals or to the interruption of
the normal process of hemozoin formation, thereby
leaving the heme-artemisinin complex in the form
which causes damage to the parasite. Since the relation
between the binding of ferroprotoporphyrin IX and
artemisinin derivatives and the IC50 as shown in Figure
2 is an exponential one, the binding must lead to, or
reflect a process which leads to, amplification of the
parasite damage leading to killing.
d
F igu r e 2. Relation between dissociation constants (K ) and
antimalarial activity of artemisinin and its derivatives against
P. falciparum in vitro.
the parasite, or reflects an as yet unknown step leading
to such killing, the strength of binding may have
correlation with the killing effect of artemisinin deriva-
tives. The absorption peak of ferroprotoporphyrin IX
at 415 nm was strongly and immediately reduced by a
number of artemisinin and dihydroartemisinin deriva-
tives which have antimalarial activity (see Figure 1 in
the Supporting Information). In contrast, deoxyarte-
misinin, which is devoid of antimalarial activity, did not
give rise to a similar spectral change. From the
dependence of spectral change on the concentration of
the artemisinin derivatives, the dissociation constants
Although artemisinin is active against chloroquine-
resistant parasites and must therefore act differently
in the killing process, the interaction of artemisinin and
derivatives with heme draws some parallels with chlo-
roquine-hemin interaction, which presumably forms
16,28
the basis for its antimalarial action.
Another simi-
larity noted earlier is that a normally drug-sensitive
malaria parasite becomes resistant to both artemisinin
and chloroquine on infection of R-thalassemic erythro-
(
Kd) between these derivatives and ferroprotoporphyrin
IX can be calculated based on a simple binding model
Table 1). With the exception of artesunate (4d ), the
2
9
cytes. It is therefore possible that early events in the
interaction of the drug with the parasite are similar for
artemisinin and chloroquine.
(
values of Kd show a correlation with log IC50 (concentra-
tion of the drug required for killing 50% of Plasmodium
falciparum in culture) (Figure 2).
In our previous search for new artemisinin-based
derivatives with good antimalarial activity, we linked
various moieties to artemisinin or its dihydro derivative.
A similar spectral change has been shown to occur
with hemin (ferriprotoporphyrin IX) in the presence of
3
0
artesunate, albeit over a much longer period.25 We also
In an earlier approach, we linked the hydroxyl group
at C-10 of dihydroartemisinin to an alkyl chain which
is in turn linked to other functional groups. The ether
derivatives of dihydroartemisinin, which fall into the
same group of other artemisinin derivatives under
clinical development or already in clinical use, are
unstable in vivo, and all break down to dihydroarte-
misinin which is presumably the major active form of
confirm a similar interaction in our experimental sys-
tem, when the incubation was made in the absence of
the reducing agent dithionite (Figure 3 in the Support-
ing Information). The change can be observed after
several hours, instead of immediately as in the presence
of dithionite.
The correlation between antimalarial activity of ar-
temisinin derivatives and their dissociation constants
for the binding interaction with ferroprotoporphyrin IX
1
8,31,32
all these drugs.
This group of compounds would
not be expected to yield much information regarding the
requirement for interaction between artemisinin deriva-
tives with their biological targets. Nevertheless, they
(Figure 2) indicates that this interaction may be biologi-
cally significant. Ferroprotoporphyrin IX is released

6
36 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Paitayatat et al.
can still provide some structural information in binding
studies with isolated components.
solution was successively washed with saturated aqueous
sodium bicarbonate solution and water, then dried (MgSO
4
),
filtered, and evaporated to dryness. The crude product was
purified by silica gel column chromatography using a mixture
of hexane/ethyl acetate/methylene chloride (8:2:0.1) as eluant
to give, after crystallization from methylene chloride/hexane,
artemisitene (2) (409 mg, 73.21% yield).
The deviation shown by artesunate from the relation-
ship between the dissociation constants for ferroproto-
porphyrin IX binding and the antimalarial activity can
be explained from the fact that the active form of the
drug is probably dihydroartemisinin formed from the
decomposition of the hemisuccinate. The intact form
of the drug, if any exists in the test system, would be
less active as predicted from the relationship found from
other derivatives. This observation applies to all arte-
misinin derivatives which give rise to dihydroartemisi-
nin in vivo or in vitro. The relationship between the
biological activity of artemisinin derivatives and their
chemical property, such as interaction with ferroproto-
porphyrin IX, is obtained with more confidence by
studying stable derivatives, such as those based on
modification at C-16 as presented here.
2: white crystals; mp 163-164 °C (lit.17 mp 161-162 °C);
IR νmax (KBr) 3027, 2933, 1725, 1457, 1380, 1223, 1170, 1112,
-
1
+
1
3
034, 999, 880, 833 cm ; MS m/e (rel intensity) 248 (M -
1
2, 2), 230 (32), 220 (4), 205 (6), 190 (68), 93 (74), 55 (100); H
) δ 0.94 (d, J ) 5.6 Hz, 3H, H-15), 1.16 (m, 1H,
NMR (CDCl
3
H-8â), 1.38 (s, 3H, H-14), 1.33-1.53 (m, 4H, H-5a, H-6, H-5â,
H-7R), 1.67 (m, 1H, H-8R), 1.72 (m, 1H, H-7â), 1.91 (m, 1H,
H-5R), 2.00 (ddd, J ) 14.89, 4.67, 2.29 Hz, 1H, H-4â), 2.33 (m,
1
H, H-4R), 2.49 (dd, J ) 13.46, 4.52 Hz, 1H, H-8a), 5.60 (d, J
0.68 Hz, 1H, H-16), 5.93 (s, 1H, H-12), 6.47 (s, 1H, H-16);
)
1
3
C NMR (CDCl
7.55, 45.86, 49.93, 79.28, 93.36, 105.23, 130.19, 134.84,
62.61; [R] +125.97° (c 0.996, CHCl ). Anal. (C15 )C,H.
Nu cleop h ilic Ad d ition of Nu cleop h iles to 2. P r ep a r a -
3
) δ 19.68, 24.43, 25.26, 31.41, 33.50, 35.68,
3
1
D
3
20 5
H O
In conclusion, we have shown that a number of
artemisinin derivatives, many of which are those with
modifications at C-16, interact strongly with ferropro-
toporphyrin IX, causing a major immediate decrease in
the Soret band. The spectrum of hemin is also similarly
affected, albeit much more slowly. The dissociation
constants for the interaction of the artemisinin deriva-
tives with ferroprotoporphyrin IX, calculated from a
model of simple, reversible binding, show a correlation
with the antimalarial activity of the derivatives, as
assessed from P. falciparum in vitro culture. However,
artesunate shows a stronger antimalarial activity than
would be expected from the correlation with other
artemisinin derivatives. This correlation provides a
rationale for further investigation of the nature of the
interaction of artemisinin-derived compounds with fer-
roprotoporphyrin IX and its biological significance and
could form a basis for a convenient assay for the
screening of peroxidic antimalarials of similar structure
and mode of action. This would be very beneficial in
future drug development, considering the fact that no
such convenient assay exists at present.
tion of 3. Typical procedure for the preparation of 3a -c: To
a solution of lithium phenylthiolate (1.1 mmol), prepared in
situ from phenylthiol and n-butyllithium, in THF (1 mL) at
-
78 °C was added a solution of artemisitene (2) (280 mg, 1
mmol) in THF (2 mL), and the mixture was left stirring at
78 °C for 30 min. Saturated aqueous ammonium chloride
-
solution workup followed by extraction with methylene chlo-
ride, drying (MgSO ), and evaporation gave the crude product
4
which upon crystallization from ethyl acetate/hexane provided
the adduct 3a (280 mg, 71.8% yield) as a single (â, C-9) isomer.
3
a : white crystals, mp 99-100 °C; IR νmax (CHCl
3
) 2933,
2
877, 1728, 1584, 1440, 1379, 1106, 1010, 979, 878, 835, 692
-
1
+
+
cm ; MS m/e (rel intensity) 392 (M + 2, 4), 391 (M + 1,12),
+
3
90 (M , 53), 376 (0.2), 375 (0.9), 374 (1), 361 (0.6), 360 (0.1),
358 (0.2), 285 (16), 267 (7), 257 (3), 123 (100), 109 (51), 67 (28),
1
55 (68); H NMR (CDCl
(
1
(
3
) δ 0.99 (d, J ) 6 Hz, 1H, H-15), 1.20
m, 1H, H-8â), 1.35 (m, 1H, H-7R), 1.45 (s, 3H, H-14), 1.39-
.50 (m, 3H, H-8a, H-6, H-5â), 1.54-1.60 (m, 1H, H-7â), 1.66
ddd, J ) 12.96, 6.37, 3.16 Hz, 1H, H-8R), 1.90-1.98 (m, 1H,
H-5R), 2.06 (ddd, J ) 14.82, 4.41, 2.82 Hz, 1H, H-4â), 2.21
dd, J ) 13.53, 4.61 Hz, 1H, H-5a), 2.27 (ddd, J ) 11.65, 3.34,
(
0
.95 Hz, 1H, H-9), 2.39 (m, 1H, H-4R), 3.13 (dd, J ) 13.8, 11.67
Hz, 1H, H-16), 3.86 (dd, J ) 13.8, 3.34 Hz, 1H, H-16), 5.92 (s,
1H, H-12), 7.19 (dddd, J ) 7.23, 7.23, 1.40, 140 Hz, 1H, H-20),
7
1
4
1
.29 (m, 2H, Ar-H), 7.38 (m, 2H, Ar-H); 13C NMR (CDCl
3
) δ
9.78, 24.65, 25.40, 31.19, 33.78, 35.78, 37.50, 37.93, 41.07,
3.35, 50.27, 80.67, 94.00, 105.36, 126.43, 129.12, 129.35,
Exp er im en ta l Section
34.43, 170.49; [R]
D
3 26
+185.57° (c 0.499, CHCl ). Anal. (C21H -
Ch em istr y. NMR spectra were recorded on a Bruker DPX
SO
5
)C,H.
3
00 spectrometer. The IR spectra were determined on a
Anions of the ferrocene derivatives were prepared by treat-
ment of the corresponding ferrocenes with lithium diisopro-
pylamide.
3b: obtained as a mixture of two (C-17) stereoisomers in a
ratio of 1:1 (NMR analysis); yellow crystals; mp 139-140 °C;
J ASCO model A-302 or a Perkin-Elmer 2000 NIR FT Raman
spectrometer. UV spectroscopy was studied with a J ASCO
UVIDEC-650 spectrometer. Elemental analyses were carried
out on a Perkin-Elmer elemental analyzer 2400 CHN, and the
results are within 0.4% of the theoretical values. Mass spectra
were recorded on a J MS-DX 300 J EOL mass spectrometer.
Melting points were determined on an electrothermal melting
point apparatus. Artemisinin was purchased from the Na-
tional Center of Natural Science and Technology, Vietnam.
â-Arteether was obtained as a gift from Artecef BV, The
Netherlands.
Con ver sion of Ar tem isin in (1) to Ar tem isiten e (2). To
a solution of lithium diisopropylamide, LDA (2.2 mmol),
prepared by the conventional method from diisopropylamine
and n-butyllithium, in THF (5 mL) at -78 °C was slowly added
a solution of artemisinin (1) (564 mg, 2.0 mmol), and the
reaction mixture was left stirring at -78 °C for 10 min. A
solution of freshly distilled phenylselenenide bromide (519 mg,
86.05% yield; IR ν
max
3
(CHCl ) 3025, 2931, 2243, 1733, 1453,
-
1
1379, 1136, 1107, 1002, 882 cm ; MS m/e (rel intensity) 506
+
+
(M + 1, 26), 505 (M , 100), 489 (18), 463 (6), 445 (2), 389 (4),
1
250 (3), 237 (71), 224 (49), 165 (17), 121 (66); H NMR (CDCl )
3
δ 1.08 [m, 2H, H-8â (isomers I and II)], 1.27 [br s, 6H, H-15
(isomers I and II)], 1.41 [s, 6H, H-14 (isomers I and II)], 1.32-
1.49 [m, 9H, H-16 (isomer II), H-7R (isomers I and II), H-5â
(isomers I and II), H-6 (isomers I and II), H-5a (isomers I and
II)], 1.59-1.72 [m, 5H, H-5R (isomers I and II), H-8R (isomer
I), H-8a (isomers I and II)], 1.84-2.04 [m, 6H, H-16 (isomer
I), H-16 (isomer II), H-4â (isomers I and II), H-7â (isomers I
and II)], 2.22-2.56 [m, 6H, H-16 (isomer I), H-9 (isomers I
and II), H-4R (isomers I and II), H-8R (isomer II)], 2.63 [ddd,
J ) 13.97, 7.13, 7.13 Hz, 1H, H-16 (isomer I)], 3.92 [t, J )
7.88, 7.88 Hz, 1H, H-17 (isomer I)], 4.18-4.24 [m, 1H, H-17
(isomer II)], 4.19 [s, 5H, Ar-H (isomer II)], 4.22 [s, 5H, Ar-H
(isomer I)], 4.13-4.24 [m, 8H, Ar-H (isomers I and II)], 5.87
2
.2 mmol) in THF (2 mL) was introduced, and the solution
was again left stirring at -78 °C for 15 min, after which glacial
acetic acid (0.3 mL) was added followed by a solution of 30%
aqueous hydrogen peroxide (1.5 mL). The reaction tempera-
ture was raised to 0 °C, and the mixture was stirred for 30
min. Water (50 mL) was added and the organic material
extracted into methylene chloride. The methylene chloride
1
3
[s, 1H, H-12 (isomer I)], 5.90 [s, 1H, H-12 (isomer II)]; C NMR
(CDCl ) δ 19.68 (isomers I and II), 24.53 (isomers I and II),
3
25.27 (isomers I and II), 30.32 (isomer I), 30.58 (isomer II),
30.75 (isomers I and II), 32.13 (isomer II), 33.65 (isomer I),

Antimalarial Activity of Artemisinin
5.72 (isomer II), 37.34 (isomer I), 37.37 (isomer II), 39.77
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 637
3
1H, H-12 (isomer I)], 5.88 [s, 1H, H-12 (isomer II)], 6.89 [t, J
) 1.41 Hz, 1H, Ar-H (isomer I)], 6.91 [t, J ) 1.03 Hz, 1H, Ar-H
(isomer II)], 6.98 [s, 1H, Ar-H (isomer I)], 7.00 [s, 1H, Ar-H
(isomer II)], 7.43 [s, 1H, Ar-H (isomer I)], 7.46 [s, 1H, Ar-H
(isomer I), 41.56 (isomer II), 42.70 (isomers I and II), 43.17
(isomer I), 45.40 (isomer II), 50.17 (isomers I and II), 66.22
(isomers I and II), 66.46 (isomers I and II), 67.44 (isomers I
and II), 68.13 (isomers I and II), 68.35 (isomers I and II), 68.44
(isomer II)]; 13C NMR (CDCl
3
) δ 19.45 (isomer I), 19.49 (isomer
(
(
isomers I and II), 69.13 (isomer II), 69.26 (isomer I), 79.78
isomer II), 79.89 (isomer I), 82.48 (isomer I), 83.34 (isomer
II), 23.18 (isomer I), 24.40 (isomer II), 24.51 (isomer I), 24.82
(isomer I), 25.15 (isomer II), 30.80 (isomer II), 32.81 (isomer
II), 33.46 (isomer II), 35.54 (isomer II), 35.57 (isomer I), 37.13
(isomer I), 37.19 (isomer II), 39.43 (isomer II), 39.74 (isomer
I), 41.23 (isomer I), 43.44 (isomer I), 46.41 (isomer II), 49.44
(isomer II), 49.49 (isomer I), 50.00 (isomer I), 78.66 (isomer
I), 80.04 (isomer II), 93.82 (isomer I), 93.90 (isomer II), 105.45
(isomers I and II), 118.65 (isomers I and II), 129.83 (isomer
I), 130.04 (isomer II), 137.28 (isomer I), 137.70 (isomer II),
II), 93.71 (isomer I), 93.88 (isomer II), 105.31 (isomers I and
II), 120.29 (isomer I), 120.66 (isomer II), 170.68 (isomer I),
1
70.95 (isomer II); [R]
31NFeO )C,H.
c: obtained as two separable (R and â) C-9 stereoisomers
by PLC using silica gel/20% ethyl acetate in hexane) in a ratio
D
+44.97° (c 0.378, CHCl
3
). Anal.
(C
27
H
5
3
(
-
9
of 1:29, respectively (IC50) ) 2.1 × 10 M for isomer I and
-
9
IC50 ) 3.15 × 10 M for isomer II).
168.33 (isomer II), 168.49 (isomer I); [R]
D
+98.06° (c 0.21,
Isom er I: orange crystals; mp 155-156 °C; 3% yield; IR
CHCl ). Anal. (C18 )C,H.
3
24 2 5
H N O
ν
7
4
max (CHCl
3
) 3014, 2932, 1736, 1663, 1456, 1380, 1264, 830,
Compound 4e was obtained from the reduction of 3d with
sodium borohydride employing the known standard condi-
tions.33 NMR analysis (300 MHz) of the product revealed the
presence of two cis-isomers (C-9 and C-10).
-
1
+
61, 732 cm ; MS m/e (rel intensity) 508 (M , 100), 492 (20),
62 (8), 448 (3), 420 (3), 391 (3), 282 (8), 251 (2), 240 (8), 228
1
(69), 213 (21), 186 (35), 121 (77), 67 (13); H NMR (CDCl
3
) δ
0
1
1
1
1
.94 (d, J ) 5.42 Hz, 3H, H-15), 0.99-1.11 (m, 2H, H-7R, H-8â),
.38 (s, 3H, H-14), 1.28-1.47 (m, 3H, H-6, H-5a, H-5â), 1.65-
.70 (m, 2H, H-7â, H-16), 1.73-1.80 (m, 2H, H-8R, H-8a),
.92-2.02 (m, 2H, H-4â, H-5R), 2.10 (m, 1H, H-16), 2.35 (m,
H, H-4R), 2.80 (ddd, J ) 16.9, 8.83, 6.72 Hz, 1H, H-17), 3.16
4e: white crystals; mp 154-155 °C; IR νmax (KBr) 2938,
-
1
1653, 1519, 1457, 1377, 1284, 1129, 1095, 978, 846, 634 cm
;
+
+
MS m/e (rel intensity) 352 (M + 2, 0.5), 351 (M + 1, 0.7),
+
+
350 (M , 2), 332 (M - H
2
O, 36), 303 (56), 289 (9), 261 (44),
247 (13), 233 (24), 218 (34), 193 (19), 178 (50), 95 (27), 82 (100),
1
(ddd, J ) 16.9, 9.1, 5.19 Hz, 1H, H-17), 3.22 (ddd, J ) 8.78,
68 (97), 55 (44); H NMR (CDCl
3
+ CD OD) δ 0.81-0.91 [m,
3
8
1
5
2
6
.78, 4.16 Hz, 1H, H-9), 4.15 (s, 5H, Ar-H), 4.44 (t, J ) 1.76,
2H, H-8â (isomers I and II)], 0.86 [d, J ) 6.38 Hz, 3H, H-15
(isomer II)], 0.89 [d, J ) 6.38 Hz, 3H, H-15 (isomer I)], 1.21
[m, 2H, H-5a (isomers I and II)], 1.28-1.36 [m, 2H, H-6
(isomers I and II)], 1.33 [s, 3H, H-14 (isomer I)], 1.35 [s, 3H,
H-14 (isomer II)], 1.37 [m, 2H, H-7R (isomers I and II)], 1.52-
1.68 [m, 6H, H-5â (isomers I and II), H-8R (isomers I and II),
H-8a (isomers I and II)], 1.82 [m, 2H, H-7â (isomers I and II)],
1.95-2.12 [m, 4H, H-4â (isomers I and II), H-5R (isomers I
and II)], 2.29 [ddd, J ) 14.52, 14.52, 3.79 Hz, 2H, H-4R
(isomers I and II)], 2.69 [m, 1H, H-9 (isomer II)], 2.74 [m, 1H,
H-9 (isomer I)], 3.74 [m, 1H, H-16 (isomer II)], 3.78 [dd, J )
13.6, 5.95 Hz, 1H, H-16 (isomer I)], 4.10 [dd, J ) 13.6, 10.44
Hz, 1H, H-16 (isomer I)], 4.23 [dd, J ) 14.21, 5.03 Hz, 1H,
H-16 (isomer II)], 4.88 [d, J ) 3.34 Hz, 2H, H-10 (isomers I
and II)], 5.35 [s, 1H, H-12 (isomer II)], 5.56 (s, 1H, H-12 (isomer
I)], 6.87 [s, 1H, Ar-H (isomer II)], 6.89 [s, 1H, Ar-H (isomer
I)], 6.93 [s, 2H, Ar-H (isomers I and II)], 7.39 [s, 1H, Ar-H
.76 Hz, 2H, Ar-H), 4.74 (m, 1H, Ar-H), 4.78 (m, 1H, Ar-H),
1
3
.82 (s, 1H, H-12); C NMR (CDCl ) δ 19.68, 22.69, 23.50,
3
4.69, 25.04, 33.42, 35.75, 37.58, 38.31, 44.67, 49.79, 69.16,
9.20, 69.69, 72.12, 78.73, 79.18, 93.59, 105.25, 171.56, 203.77;
[
R]
Isom er II: red crystals; mp 120-121 °C; 87.05% yield; IR
) 3014, 2932, 1733, 1661, 1456, 1380, 1226, 1107,
D
+38.84° (c 0.515, CHCl
3 6
). Anal. (C27H32FeO )C,H.
ν
1
4
2
max (CHCl
3
-1
+
032, 831, 772, 737 cm ; MS m/e (rel intensity) 508 (M , 100),
92 (15), 474 (2), 448 (2), 420 (0.8), 391 (1), 282 (4), 251 (1),
1
40 (6), 228 (40), 213 (15), 186 (30), 121 (65), 67 (13); H NMR
(
1
1
3
CDCl ) δ 1.00 (d, 3H, J ) 6 Hz, H-15), 1.17 (m, 1H, H-8â),
.43 (s, 3H, H-14), 1.37-1.58 (m, 4H, H-6, H-5a, H-5â, H-7R),
.69-1.74 (m, 1H, H-8R), 1.76-1.82 (m, 1H, H-8a), 1.87-2.00
(
m, 2H, H-5R, H-7â), 2.04-2.11 (m, 1H, H-4â), 2.13-2.22 (m,
1
2
4
H, H-16), 2.29 (ddd, J ) 14.5, 7.74, 5.71 Hz, 1H, H-16), 2.33-
.46 (m, 2H, H-9, H-4R), 2.90 (m, 1H, H-17), 3.15 (m, 1H, H-17),
.22 (s, 5H, Ar-H), 4.50 (t, J ) 1.96, 1.96 Hz, 2H, Ar-H), 4.81
(isomer II)], 7.43 [s, 1H, Ar-H (isomer I)]; 13C NMR (CDCl
CD OD) δ 19.86 (isomer II), 20.04 (isomer I), 21.94 (isomer
+
3
(
dd, J ) 3.22, 1.89 Hz, 1H, Ar-H), 4.84 (dd, 1H, J ) 3.22, 1.89
3
1
3
Hz, Ar-H), 5.93 (s, 1H, H-12); C NMR (CDCl
3
) δ 19.75, 24.50,
5.37, 28.99, 31.21, 33.78, 35.79, 37.07, 37.37, 43.76, 44.47,
0.32, 69.15, 69.22, 69.69, 72.04, 78.81, 79.98, 93.79, 105.08,
71.59, 204.36; [R] +42.69° (c 0.56, CHCl ). Anal. (C27
)C,H.
II), 24.44 (isomer I), 24.85 (isomers I and II), 25.49 (isomer
II), 25.68 (isomer I), 33.47 (isomer II), 34.18 (isomer I), 36.11
(isomers I and II), 37.03 (isomers I and II), 37.57 (isomers I
and II), 40.98 (isomer II), 41.71 (isomer I), 44.16 (isomer II),
46.34 (isomer I), 51.12 (isomer II), 52.20 (isomer I), 79.61
(isomer II), 80.37 (isomer I), 87.79 (isomer I), 90.70 (isomer
II), 91.23 (isomer II), 91.81 (isomer I), 104.14 (isomer I), 104.40
(isomer II), 119.06 (isomers I and II), 128.67 (isomers I and
2
5
1
D
3
32
H -
FeO
6
Compound 3d was prepared by stirring a solution of
artemisitene (280 mg, 1 mmol) and imidazole (74.9 mg, 1.1
mmol) in THF (3 mL) at room temperature for 24 h. The
reaction mixture was evaporated to dryness and the crude
product recrystallized from ethyl acetate/hexane to yield 3d
as a mixture of two inseparable (R and â) isomers (269 mg,
II), 137.33 (isomers I and II); [R]
D
3
+ 154.55° (c 0.15, CHCl ).
Anal. (C18 )C,H.
26 2 5
H N O
Gen er al P r ocedu r es To Evalu ate th e In ter action Spec-
7
7.44% yield).
d : white crystals; mp 137-138 °C; IR νmax (CHCl
877, 1737, 1508, 1455, 1394, 1380, 1283, 1135, 1116, 1001,
72, 835 cm ; MS m/e (rel intensity) 348 (M , 20), 332 (0.6),
19 (15), 305 (9), 289 (4), 277 (9), 259 (16), 230 (12), 209 (5),
90 (26), 140 (15), 121 (23), 95 (30), 82 (75), 69 (100), 55 (45);
tr a of Dr u g-F er r op r otop or p h yr in IX. Hemin (0.4 mg,
-
4
3
3
) 2962,
6.13 × 10 mmol) was initially dissolved in 100 µL of 0.1 M
sodium hydroxide; then 29.9 mL of 1% SDS in PBS (pH 7.2)
was added. Various amounts of compounds (1-4e) were
dissolved in 120 µL of either MeOH or DMSO followed by
adding 1.08 mL of 1% SDS in PBS to obtain required
concentrations; 2-fold serial dilutions were then made. In a
1.5 mL cuvette was placed 0.5 mL of the hemin solution
together with 0.5 mL of the drugs. A few crystals of sodium
dithionite were added prior to measuring the spectra.
2
9
3
-
1
+
1
1
H NMR (CDCl ) δ 1.15 [m, 1H, H-8â (isomer I)], 1.35 [s, 3H,
3
H-14 (isomer I)], 1.38 [s, 3H, H-14 (isomer II)], 1.21-1.41 [m,
H, H-6 (isomers I and II), H-5â (isomers I and II), H-5a
8
(isomers I and II), H-8â (isomer II), H-8R (isomer II)], 1.46
[m, 1H, H-8a (isomer II)], 1.53 [m, 1H, H-8a (isomer I)], 1.70
[ddd, J ) 13.70, 6.78, 3.21 Hz, 2H, H-7â (isomers I and II)],
Biology. In Vitr o Assessm en t of An tim a la r ia l Activity.
The detailed protocol for in vitro antimalarial testing of drugs
against P. falciparum-infected red cells using a modification
1
2
2
1
5
1
.77 [ddd, J ) 13.19, 7.05, 3.56 Hz, 1H, H-8R (isomer I)], 1.85-
.03 [m, 4H, H-4â (isomers I and II), H-5R (isomers I and II)],
.26-2.38 [m, 2H, H-4R (isomers I and II)], 2.58 [ddd, J )
0.96, 5.00, 0.68 Hz, 1H, H-9 (isomer II)], 3.65 [ddd, J ) 9.79,
.44, 5.02 Hz, 1H, H-9 (isomer I)], 3.95 [dd, J ) 14.45, 9.7 Hz,
H, H-16 (isomer I)], 4.23 [dd, J ) 13.90, 10.96 Hz, 1H, H-16
3
of the [ H]hypoxanthine incorporation has been previously
published by Desjardins et al.34 Briefly, the drugs were
initially dissolved in dimethyl sulfoxide (DMSO) and diluted
with RPMI 1640 culture medium supplemented with 25 mM
3
Hepes buffer, 32 mM NaHCO , and 10% human plasma to the
required concentrations with a final concentration of 0.01%
(isomer II)], 4.51 [dd, J ) 13.90, 5.11 Hz, 1H, H-16 (isomer
II)], 4.56 [dd, J ) 14.45, 5.44 Hz, 1H, H-16 (isomer I)], 5.82 [s,
DMSO. A 200 µL portion of the 1.5% cell suspension with

6
38 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Paitayatat et al.
1
2
-2% parasitemia at early ring stage was pre-exposed to the
5 µL of the medium containing the drugs for 24 h and
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Goldberg, D. E.; Cerami, A.; Henderson, G. B. An Iron-
Carboxylate Bond Links the Heme Units of Malaria Pigment.
Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 325-329.
2 2 2
incubated at 37 °C with 5% O , 5% CO , and 90% N prior to
3
the addition of 25 µL of 0.5 µCi of [ H]hypoxanthine. After
(
16) Dorn, A.; Stoffel, R.; Matile, H.; Bubendorf, A.; Ridley, R. G.
Malarial Haemozoin/â-Haematin Supports Haem Polymerization
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3
[
H]hypoxanthine incorporation in each well was determined
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1
985, 441-442.
(
(
(
(
18) White, N. J . Clinical Pharmacokinetics and Pharmacodynamics
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Ack n ow led gm en t. This investigation received sup-
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and Biotechnology, Thailand (to Y.T. and B.T.), the
China Medical Board (to S.P.), the U.S. NIH ICIDR
Program (Grant No. U 01 AI 35827), and the U.S. AID
CDR Program (Project No. TA-MOU-C09-060) (to Y.Y.).
Senior Fellowship Award to Y.T. from the National
Science and Technology Development Agency (NSTDA)
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1
994, 88 (Suppl. 1), 41-43.
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(
(
(
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