Methylene Homologues of Artemisone: An Unexpected Structure–Activity Relationship and a Possible Implication for the Design of C10-Substituted Artemisinins

Article abstract of DOI:10.1002/cmdc.201600011

We sought to establish if methylene homologues of artemisone are biologically more active and more stable than artemisone. The analogy is drawn with the conversion of natural O- and N-glycosides into more stable C-glycosides that may possess enhanced biological activities and stabilities. Dihydroartemisinin was converted into 10β-cyano-10-deoxyartemisinin that was hydrolyzed to the α-primary amide. Reduction of the β-cyanide and the α-amide provided the respective methylamine epimers that upon treatment with divinyl sulfone gave the β- and α-methylene homologues, respectively, of artemisone. Surprisingly, the compounds were less active in vitro than artemisone against P. falciparum and displayed no appreciable activity against A549, HCT116, and MCF7 tumor cell lines. This loss in activity may be rationalized in terms of one model for the mechanism of action of artemisinins, namely the cofactor model, wherein the presence of a leaving group at C10 assists in driving hydride transfer from reduced flavin cofactors to the peroxide during perturbation of intracellular redox homeostasis by artemisinins. It is noted that the carba analogue of artemether is less active in vitro than the O-glycoside parent toward P. falciparum, although extrapolation of such activity differences to other artemisinins at this stage is not possible. However, literature data coupled with the leaving group rationale suggest that artemisinins bearing an amino group attached directly to C10 are optimal compounds.

Full text of DOI:10.1002/cmdc.201600011

DOI: 10.1002/cmdc.201600011
Full Papers
Methylene Homologues of Artemisone: An Unexpected
Structure–Activity Relationship and a Possible Implication
for the Design of C10-Substituted Artemisinins
[
b]
[b]
[b]
[b]
[c]
Yuet Wu, Ronald Wai Kung Wu, Kwan Wing Cheu, Ian D. Williams, Sanjeev Krishna,
[c]
[d]
[d]
[a, b]
Ksenija Slavic, Andrew M. Gravett, Wai M. Liu, Ho Ning Wong,
Richard K. Haynes*
and
[a, b]
We sought to establish if methylene homologues of artemi-
sone are biologically more active and more stable than artemi-
sone. The analogy is drawn with the conversion of natural O-
and N-glycosides into more stable C-glycosides that may pos-
sess enhanced biological activities and stabilities. Dihydroarte-
misinin was converted into 10b-cyano-10-deoxyartemisinin
that was hydrolyzed to the a-primary amide. Reduction of the
b-cyanide and the a-amide provided the respective methyla-
mine epimers that upon treatment with divinyl sulfone gave
the b- and a-methylene homologues, respectively, of artemi-
sone. Surprisingly, the compounds were less active in vitro
than artemisone against P. falciparum and displayed no appre-
ciable activity against A549, HCT116, and MCF7 tumor cell
lines. This loss in activity may be rationalized in terms of one
model for the mechanism of action of artemisinins, namely the
cofactor model, wherein the presence of a leaving group at
C10 assists in driving hydride transfer from reduced flavin co-
factors to the peroxide during perturbation of intracellular
redox homeostasis by artemisinins. It is noted that the carba
analogue of artemether is less active in vitro than the O-glyco-
side parent toward P. falciparum, although extrapolation of
such activity differences to other artemisinins at this stage is
not possible. However, literature data coupled with the leaving
group rationale suggest that artemisinins bearing an amino
group attached directly to C10 are optimal compounds. A brief
critique is made of proteomic studies purporting to demon-
strate the alkylation of intraparasitic proteins by alkyne- and
azide-tagged artemisinins and synthetic peroxides in relation
to mechanism of action.
Introduction
[
3]
Artemisinin (1) and its derivatives dihydroartemisinin (DHA, 2),
artemether (3), and artesunate (4) have high parasite kill rates
with broad-stage specificity, and they elicit faster clinical and
phase I metabolism and rearranges under physiological con-
[4]
ditions at pH 7.4 to peroxyhemiacetal 5, which is observed in
the plasma of patients treated with artesunate. Artesunate is
[5]
[1]
[6]
parasitological responses than any other antimalarial drug.
rapidly hydrolyzed to DHA in vivo, but likely because of the
However, all are thermally and chemically fragile, and as exem-
plified by DHA, the thermal instability engenders problems
protective effect of the ester group against first-pass metabo-
[7]
lism, it is a better source of DHA in plasma than DHA itself.
[
2]
during formulation and storage. DHA undergoes facile
The facile metabolism of artemether to DHA is reflected in the
detection of the latter compound in subjects administered
[8]
with artemether.
Artemisinins, especially DHA, elicit neurotoxicity in cell and
[a] Dr. H. N. Wong, Prof. R. K. Haynes
Centre of Excellence for Pharmaceutical Sciences, Faculty of Health Scien-
ces, North-West University, Potchefstroom 2520 (South Africa)
E-mail: richard.haynes@nwu.ac.za
[
9,10]
animal assays.
The neurotoxicity of DHA reaches the level
[9]
of activity that the drug displays in vitro against P. falciparum.
[11,12]
[b] Dr. Y. Wu, R. W. K. Wu, Dr. K. W. Cheu, Prof. I. D. Williams, Dr. H. N. Wong,
Although debate over the neurotoxicity of artemisinins
has
Prof. R. K. Haynes
subsided, the emerging resistance of the malaria parasite to
Department of Chemistry, The Hong Kong University of Science and Tech-
nology, Clear Water Bay, Kowloon, Hong Kong (P.R. China)
E-mail: haynes@ust.hk
chemotherapy involving current clinical artemisinins 2–4 in
[13]
Cambodia and other countries in south-east Asia now neces-
sitates administration of more protracted treatment regimens.
[
c] Prof. S. Krishna, Dr. K. Slavic
Centre for Infection, Division of Cellular and Molecular Medicine,
St. George’s Hospital, University of London, SW17 0RE (UK)
[12]
This mandates renewed vigilance of the neurotoxicity. Neu-
rotoxicity is also of concern wherein protracted treatment regi-
mens involving artemisinins are required for treatment of
[
d] Prof. A. M. Gravett, Dr. W. M. Liu
Department of Oncology, Division of Cellular and Molecular Medicine,
St. George’s Hospital, University of London,
Jenner Wing, London SW17 0RE (UK)
[14]
other diseases. Driven by the need to maintain an effective
antimalarial drug generation campaign that in view of the re-
sistance to the current clinical artemisinins has become a most
urgent task, a number of groups has prepared new derivatives
Supporting Information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600011.
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and analogues that do not have the shortcomings of the cur-
regimen of artemisone, corresponding to a total dose level of
one third of that of the comparator drug artesunate, followed
by a final dose of mefloquine was curative. The relatively
minor amounts of metabolites in plasma of the patients did
not include DHA. Both artemisone and its precursor artemiside
(8) display substantially better inhibition than either artemisi-
nin or artesunate in vitro against another apicomplexan organ-
[
15–17]
rent clinical artemisinins.
In the absence of an understand-
ing of the mechanism of action of artemisinin at the time we
commenced our own work in this area, we used a straightfor-
ward paradigm: because of neurotoxicity concerns, the deriva-
tive should not generate DHA in vivo and should not be too
[
18,19]
lipophilic.
This guided us in the preparation of C10 amino
[29]
derivatives such as compounds 6–8 with IC50 values in vitro
ranging from 0.25 to 0.63 nm against multidrug-resistant and
ism, namely Toxoplasma gondii.
Artemisinins possess anti-
[14,30]
cancer properties.
The efficacy of artemisone both alone
[
20,21]
sensitive strains of P. falciparum.
In contrast, values for ar-
and in combination with common anticancer agents was es-
tablished with in vitro assays. Artemisinin (1) and artemisone
(6) each caused dose-dependent decreases in cell number with
IC50 values significantly better in the latter. Combination stud-
ies showed that the antiproliferative effect of artemisone was
[
1b]
temether lie approximately between 2–5 nm. The develop-
ment candidate eventually chosen was artemisone 9. The
stop–go” criterion was its lack of neurotoxicity
[
22]
“
coupled
with acceptable physicochemical properties and an efficacy of
[31]
approximately 1 nm against multidrug-resistant and sensitive
enhanced by the addition of other drugs.
Artemisone is
[23]
strains of P. falciparum. The relatively low calculated (2.08)
among the most potent of all peroxides upon screening
against a panel of multidrug-sensitive and resistant tumor cell
and measured log P (2.49) values and acceptable water solubil-
À1
[32]
ity at pH 7.2 of 89 mgL mark artemisone as a relatively polar
lines. Thus, overall, and in particular because of non-neuro-
[
21]
compound. In comparison, artemether has a calculated log P
of 3.51, a measured log P of 3.98, and a water solubility at
toxicity, artemisone represents an attractive drug candidate for
treatment of malaria and other targets, especially those for
which protracted treatment regimens may be required.
However, one crucial aspect that was not satisfac-
À1 [21]
pH 7.2 of 117 mgL .
[2]
torily addressed is of stability. The thermal stability
of artemisone is similar to that of artesunate. Al-
though at pH 7.2 artemisone is hydrolytically stable
(
in contrast to artesunate), at pH 1.2, just like artesu-
[5]
[33]
nate, it is relatively rapidly hydrolyzed to DHA.
The hydrolysis likely is initiated by protonation of the
C10 nitrogen atom followed by cleavage to oxonium
ion 10 that adds water to generate DHA
[15]
(
Scheme 1). It is this relative instability at low pH
that needs to be addressed if a robust and equipo-
tent successor at least to artemisone is to emerge.
We sought to bypass this problem by preparing
Metabolism of artemisone to DHA does not take place, and
it is not an inducer of its own metabolism. Artemisone is more
potent than artesunate, especially against multidrug-resistant
amino derivatives with a nitrogen atom at C10 rendered less
basic by electron-withdrawing groups, as exemplified by polar-
substituted sulfamide 11 (with a calculated log P of 1.24) to be
described elsewhere. In complement with this approach, we
evaluated a second proposal for enhancing stability at low pH.
The C10ÀN1’ bond in artemisone (9) (Scheme 1) corresponds
to the N-glycoside linkage in N-glycosyl amines, wherein the
glycosyl moiety equates with the pyran ring of the artemisinin
nucleus. Attempts to develop carbohydrate-based therapeutic
agents have been complicated by the lability of N- and O-
acetal linkages between the carbohydrate and its bioconjugate
under weakly acidic conditions. A solution to this problem lies
in the replacement of the glycoside heteroatom with a carbon
[
24]
Plasmodium parasites, and is approximately 2–6 times more
potent in vitro than synthetic trioxolane development candi-
[
1,16,25,26]
dates.
Artemisone was the most efficient drug tested in
a murine model of cerebral malaria and prevented death even
[27]
when administered at late stages of cerebral pathogenesis.
Treatment of non-severe falciparum malaria in a primate
model with combinations of artemisone with other antimalarial
drugs was markedly superior to the same combinations con-
[
28]
taining the comparator drug artesunate. A phase IIa trial in-
volving oral treatment of malaria patients with a 2 day dose
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N
Scheme 1. S 1 decomposition pathway for artemisone proceeding via oxonium ion 10 under aqueous acidic conditions.
[38]
atom to provide unnatural C-glycosides that are resistant to
DHA (2) into the b-benzoate 16 by the Schmidt reaction.
degradation. Although there are conformational differences
The benzoate was treated with trimethylsilyl cyanide and SnCl4
as the catalyst to give solely the b-nitrile 17 in 83% yield after
direct crystallization (Scheme 2). This compound arises by an
[
34]
between natural and C-glycosides, the latter usually retain
[35]
the biological properties of the original carbohydrates. A rel-
evant example is provided by the glycolipid a-galactosylcera-
mide (KRN7000, 12) that exhibits potent antimalarial and anti-
tumor activities. The synthetic analogue 13, in which the exo-
cyclic heteroatom is replaced with a methylene group, is 1000-
fold more active than 12 against liver stages of Plasmodium
yoelii in a murine malaria model and 100-fold more potent in
S 1 reaction involving oxonium ion 10 (Scheme 1) formed
N
[15,38]
from the b-benzoate 16 and SnCl4.
The X-ray crystal struc-
ture (Figure S1, Supporting Information) indicates that the cya-
nide is axial on the chair pyranose ring. Although sterically
more hindered than the equatorial cyanide in the a epimer be-
[38]
cause of 1,3-diaxial interactions with the C8ÀC8a bond, the
axial cyanide is subject to anomeric stabilization involving s*–
[
36]
inhibiting metastasis in a murine melanoma model.
[15,38,39]
Thus, the insertion of a methylene group (ÀCH À) between
n orbital overlap, as in other derivatives of DHA.
2
the artemisinin nucleus and the thiomorpholine-S,S-dioxide
ring was planned. This will generate the methylene homo-
logues 14 and the epimer 15 of artemisone. In addition to en-
hancing stability, the methylene group should provide addi-
tional degrees of freedom to the molecule. If the thiomorpho-
line-S,S-dioxide is responsible for the enhanced efficacy of arte-
[
20]
misone, likely through enhancing permeability, or if it assists
in any interaction with proteins or receptors within the malaria
parasite or tumor cell, the homologues are expected to be
more active than artemisone. Such a transformation will not
[23]
adversely affect compound polarities: calculated log P values
for compounds 14 and 15 are each 1.7 (cf. artemisone 2.08).
Results and Discussion
Preparation of methylene artemisones
Scheme 2. Preparation of b-nitrile 17 and conversion into 10-a-amide 18
and 10-b-amide 19. Reagents and conditions: a) Me SiCN (3.0 equiv), SnCl
0.3 equiv), CH Cl
, À788C, 2 h, quench; direct crystallization from propan-2-
ol, 83%; b) KOH (3.5 equiv), tBuOH, 508C, 3.5 h, quant.; c) K CO (1.1 equiv),
3
4
The nitrile 17 was prepared in 76% yield from the a-acetate of
DHA and trimethylsilyl cyanide with TiCl as the catalyst in di-
(
2
2
4
2
3
[
37]
chloromethane.
We varied this procedure by converting
H
2
O
2
(30%, 15.0 equiv), THF, RT, 2 h then 408C, 30 min, 29% (Bz=benzoyl).
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[
43,44]
Because of the potential lability of the 1,2,4-trioxane unit
under the strongly acidic conditions normally required for the
hydrolysis of nitriles, hydrolysis in the presence of base was
nol
to give the crystalline a-homologue 14 of artemisone
in 40% yield from a-amide 19 and the crystalline b-homologue
15 in 45% yield from nitrile 17 (Scheme 4).
According to X-ray crystallography, the a epimer possesses
[
40]
carried out. The Hall method was adapted by heating a mix-
ture of the nitrile and potassium hydroxide in tert-butyl alcohol
at 508C, whereby the potassium hydroxide dissolved com-
pletely and the a-amide 18 was formed in quantitative yield
[20]
a chair pyranose ring, as is the case with artemisone, where-
as the b epimer has a slightly twisted half-chair pyranose ring
that attenuates the diaxial interaction between the C10 meth-
ylene substituent and the axial C8À8a bond (Figure 1). Notably,
the thiomorpholine-S,S-dioxide ring does not shield the perox-
ide bridge in either the a or b homologues; in both cases the
ring projects away from the trioxane nucleus. As outlined
below, the relative orientation of the thiomorpholine-S,S-diox-
ide ring in each of the compounds has no significant effect on
the biological activities of the homologues. Mechanistic con-
siderations aside, this is significant, as antimalarial activities of
artemisinins are sensitive to steric effects. Thus, for example, 9-
epiartemisinin with a C9 a-axial methyl group that flanks the
peroxide has relative activities ranging between 0.14 (D6) and
(
Scheme 2). At higher temperatures, decomposition took place.
[
41]
The other method involved the use of a mixture of aqueous
hydrogen peroxide and potassium carbonate in tetrahydrofur-
an (Scheme 2). The reaction provided the b-amide in 29%
yield. This fortuitous means of obtaining each amide epimer al-
lowed for the unambiguous assignment of stereochemistry to
1
each compound. In the H NMR spectrum of a-epimer 18,
J10,9 =11.2 Hz, indicative of a trans-diaxial arrangement of H10
and H9, and in b-epimer 19, J =6.2 Hz, indicative of a cis-
10,9
[
38]
axial-equatorial relationship. The structure of amide 18 was
confirmed by X-ray crystallography, wherein the trans-diaxial
relationship between H9 and H10 is apparent (Figure S2). The
evident relaxation of the anomeric effect for the carboxamide
substituent coupled with a greater 1,3-diaxial interaction in-
volving the axial amide with the C8ÀC8a bond in b-amide 19
that must be formed initially from nitrile 17 allows for base-
catalyzed equilibration to give equatorial epimer 18
[45]
0.67 (W2) of those of artemisinin.
Biological activities
Artemisone (9) and methylene homologues 14 and 15 were
screened in vitro against the chloroquine (CQ)-sensitive 3D7
strain of Plasmodium falciparum. Data are given in Table 1 and
dose–response curves are presented in Figure S3. It is empha-
sized that screening involving compounds 9, 14, and 15 was
carried out simultaneously in the one experimental set, that is,
differences in activities between artemisone and the homo-
logues are not artefactual. Thus, the homologues have compa-
rable activities, yet both are approximately an order of magni-
tude less active than artemisone (9).
(Scheme 3).
Although the peroxide restricts the type of reagents that
may be used for reduction of nitriles or amides to amines,
treatment of nitrile 17 and amide 18 with an excess of sodium
[
42]
borohydride–boron trifluoride in tetrahydrofuran
worked
well to provide crude aminomethyl artemisinin derivatives 20
and 21. The thiomorpholine-S,S-dioxide ring was then con-
structed around the primary amino groups in 20 and 21 by
treatment of the crude amines with divinyl sulfone in isopropa-
Scheme 3. Base-mediated conversion of axial amide 19 into equatorial amide 18.
Scheme 4. Conversion of nitrile 17 and amide 18 into methylene artemisone homologues. Reagents and conditions: a) NaBH
THF, reflux, 2 h; b) divinyl sulfone (2.0 equiv), isopropanol, reflux, 2 h; 14: 40% from amide 18, 15: 45% from nitrile 17.
4 3 2
(5.0 equiv), BF ·OEt (5.0 equiv),
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Figure 1. ORTEP plots obtained from X-ray crystallographic structural determination of artemisone (9), a-methylene artemisone (14) and b-methylene artemi-
sone (15).
transferring a hydride from NAD(P)H bound in a proximate site
Table 1. Activities in vitro of artemisone (9), a-methylene artemisone
14), and b-methylene artemisone (15) against P. falciparum CQ-sensitive
D7 strain.
[47,48]
in the C-terminal domain.
Thus, the FAD cofactor crucial
(
3
for reduction of disulfides to thiols by parasite flavin disulfide
reductases is cleanly and rapidly reduced by NAD(P)H-Fre to
[
a]
Compound
IC50 [nm]
the reduced conjugate FADH in aqueous buffer under physio-
2
artemisone (9)
a-methylene artemisone (14)
b-methylene artemisone (15)
1.09Æ0.105
8.85Æ1.68
8.50Æ1.59
logical conditions at pH 7.4. The reactions are conveniently fol-
lowed by UV spectroscopy. Treatment of the FADH generated
2
in this fashion with artemisinins such as artemisinin (1), arte-
mether (3), artesunate (4), and artemisone (9) results in rapid
[
[
a] Three independent experiments for each entry; evaluated with the
H]hypoxanthine incorporation assay.
3
oxidation of FADH to FAD and concomitant reduction of the
2
artemisinins to deoxyartemisinins. For artemisone (9), the path-
way is depicted in Scheme 5A, wherein hydride transfer results
in cleavage of the peroxide and expulsion of the thiomorpho-
line-S,S-dioxide ring; this leads to the ring-opened intermedi-
ate 22, which gives deoxydihydroartemisinin 23 and its epimer
Screening against A549 (lung adenocarcinoma epithelial),
HCT116 (colon), and MCF7 (breast) tumor cells was carried out.
Cell-counting experiments with either the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or trypan blue
assays indicated no observable effects for both compounds
against all cancer cell lines (Figure S4). Also, according to fluo-
rescence-activated cell sorting (FACS) analyses, there was no
clear effect for both homologues; probably there was a block-
[4,46]
24 as described in detail elsewhere.
The reduction also can
be effected by using an excess amount of the NADPH mimic
N-benzyl-1,4-dihydronicotinamide (BNAH) in the presence of
lumiflavine or riboflavin and artemisone in aqueous pH 7.4
buffer that allows for isolation and characterization of the re-
[4]
ade at the intermediate-grade G level of cancer cells. There
duction products from the artemisinin derivative. The reduc-
tion also works for totally synthetic antimalarial peroxides.
Thus, a trioxolane and a tetraoxane were readily reduced by
the NADPH-Fre-FAD system to the corresponding ketones; the
more active trioxolane was more rapidly reduced. Thereby, an
approximate correlation between the relative rates of oxidation
2
may be a growth inhibitory effect at concentrations <1 mm,
which may be at the level of G , but this is far from clear. In
2
notable contrast, artemisone displays IC50 values of 9.5Æ
0
.9 mm against HCT166 and 0.56Æ0.17 mm against MCF7 cell
lines and like artemisinins in general blocks at the G /G
1
2
[
31,32]
phase.
of FADH with the in vitro antimalarial activities of the peroxide
2
[46]
substrates can be drawn. Overall, it is argued that the rate of
intraparasitic redox perturbation should be considered as an
Mechanistic aspects
[49]
important determinant of in vitro antimalarial activities.
We have used the NAD(P)H:flavin oxidoreductase E. coli flavin
The Fre experiments were repeated here with artemisone (9)
reductase (Fre) as a mimic of flavin disulfide reductases that
and methylene artemisones 14 and 15 (Figure 2). FADH was
2
[
46]
maintain redox homeostasis in the malaria parasite. Fre does
not contain a flavin cofactor but reduces endogenous flavins
such as flavin adenine dinucleotide (FAD), flavin mononucleo-
tide, and riboflavin (RF) and exogenous oxidants such as meth-
ylene blue that bind to a receptor in the N-terminal domain by
first prepared by reduction of FAD (1 equiv) by NADH-Fre in
degassed pH 7.4 aqueous buffer solution. Then, each com-
pound (2 equiv each with respect to FAD in degassed MeCN)
was injected into the FADH solution. The relative reaction rate
2
was compared with that of the artemisone control experiment
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(
Figure 2). Although a faster reaction was observed with com-
pound 14, both homologues oxidized FADH more slowly than
2
artemisone. In principle, reduction of the methylene artemi-
sone, for example 14, to the corresponding deoxy compounds,
for example 26 (Scheme 5B), should have taken place under
these conditions. However, attempted isolation of pure prod-
ucts from the NADH-FAD-Fre reactions conducted as above
with each of the methylene artemisones or from the reaction
of methylene artemisone 14 (1 equiv) with BNAH (2 equiv) -RF
(
(
0.2 equiv) in degassed pH 7.4 MeCN-phosphate buffer solution
1:1) under an atmosphere of argon (cf. Scheme 5B) was not
successful. Apart from small amounts of unreacted methylene
artemisone 14, complex product mixtures were obtained, and
attempts to isolate discrete products from these mixtures were
not successful. However, direct analyses of the chromatogra-
phy fractions by mass spectrometry showed that the ring-
opened precursor 25 or equivalent (MS: m/z: calcd for
Figure 2. Oxidation of FADH
2
generated from NADH-Fre-FAD by artemisone
(
(
(
9), a-methylene artemisone (14), and b-methylene artemisone (15). FAD
230 nmol) was added to NADH (278 nmol)–Fre (0.1 nmol) in aqueous buffer
pH 7.4) under an atmosphere of argon and decay of FAD was followed by
monitoring the decrease in the absorbance at lmax =450 nm every 3 s until
the FAD absorbance no longer decreased. The FADH solution was treated
+
C H NO S : 418.2258; found: 418.2232) and deoxy product
20
36
6
2
+
2
6 (MS: m/z: calcd for C H NO S : 400.2152; found:
with a-methylene artemisone 14 (402 nmol, 2 equiv, in degassed MeCN) and
the increase in the absorbance at lmax =450 nm was monitored until con-
stant (brown line). The experiment was repeated with b-methylene artemi-
sone (15; 402 nmol, 2 equiv) (green line). The relative rates of oxidations
were compared with that of artemisone (9; 400 nmol, 2 equiv) (blue line)
and a blank experiment wherein degassed MeCN alone was added to the
20 34 5
400.2132) were present. Other products likely arising from de-
composition of ring-opened precursor 25 were also detected.
Full details are given in the Supporting Information and Fig-
ure S6.
2
FADH solution (purple line).
Scheme 5. A) Reduction of artemisone (9) by hydride transfer from FADH
2
(R=d-ribitol-5-diphosphate adenosine, BH=protic acid in cellular medium) to gen-
erate 22 as the ring-opened precursor of deoxydihydroartemisinin 23, epimer 24, and other products (for detailed discussions, see Refs. [4,47,50]). B) Pro-
posed reduction of methylene artemisone homologue 14 proceeding via intermediate 25; in this case, discrete products corresponding to deoxy product 26
could not be isolated, although these were identified in product mixtures by mass spectrometry.
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artemisinin in vitro against the CQ-resistant W2 and CQ-sensi-
Discussion
[52]
tive D6 strains, that is, it is some fivefold less active than ar-
[26]
The results of the efficacy studies are unexpected, and contrast
with the enhancement in antimalarial and antitumor activities
brought about by inserting methylene units into glycolipid 12
to generate analogue 13. In terms of absorption, the thiomor-
pholine S,S-dioxide group bestows acceptable physicochemical
tesunate.
However, such comparison of in vitro data be-
tween compound sets needs to be performed with care, as
such data should be gathered at the same time by using the
same assay methods for the respective compounds. An un-
equivocal answer to the C10 leaving group hypothesis can
only be provided by a systematic examination of compound
sets closely related by virtue of physicochemical properties, es-
pecially in relation to lipophilicity and spatial requirements,
with one subset bearing leaving groups at C10 and the other
bearing a carbon-linked or other “non-leaving” group at C10.
[
20,21]
properties on artemisone,
including an enhancement in
polarity. However, the group may have another less apparent
but important role in enhancing antimalarial activity. According
to one model put forward for the antimalarial mechanism of
[
4,46,49]
action of artemisinins, namely the cofactor model,
hy-
dride transfer to O1 of the peroxide bridge from FADH is cou-
2
pled with heterolytic cleavage of the peroxide and synchro-
nous unzipping with loss of the (protonated) C10 amino group
in providing ring-opened tricarbonyl compound 22, which un-
dergoes closure to the deoxydihydroartemisinin product 23
[
4]
and its epimer 24 (Scheme 5A). Significantly, analogous pro-
cesses occur for artemether (4) and artesunate (5), that is, no
products are obtained from this heterolytic process that retain
[
4]
the original group attached to C10. In the more specialized
cases of C10 aminoartemisinins, hydride transfer must be facili-
tated by protonation of the nitrogen atom, as illustrated for ar-
temisone in Scheme 5A. Thus, in terms of the cofactor model,
the feebler antimalarial activities of the methylene homologues
becomes apparent—there is no leaving group at C10, and
therefore, this process terminates with the formation of amino
intermediate 25, which transforms into deoxy product 26 and
the various other products arising from 25 by intramolecular
condensation or dehydration pathways.
[
15]
As we have pointed out elsewhere, the connectivity be-
tween the heteroatom attached to C10 and non-peroxidic
oxygen atoms with juxtaposed carbon atoms, as indicated in
Scheme 6, makes for an approximate “W” conformation that
must modulate electrophilicity of the peroxide. Further, hy-
dride transfer from FADH to O1 in artemisone (Scheme 5A) is
2
associated with synchronous ring opening to generate the
ring-opened reduction product 22. Thus, it is of interest to
speculate that the nature of the leaving group attached to C10
may become important; ideally, it will be one that will be acti-
vated, for example, by protonation in the biological medium at
essentially neutral pH. However, the dichotomy that leads to
At first sight, this implies that carba analogues of artemisi-
nins, that is, those bearing a carbon atom in place of the heter-
oatom attached to C10, may be intrinsically less active as anti-
malarial agents, even though the carba analogues will be more
lipophilic. Such an activity difference is more likely to be re-
vealed by comparison of in vitro data, for which differences
due to permeation, metabolism, and other pharmacokinetic as-
pects will be less important. The carba analogue 26 (calculated
log P 4.11) of artemether (3) (calcd log P 3.51) prepared from
an S 1 process, as illustrated in Scheme 1, under overtly acidic
N
conditions must be borne in mind; such can be obviated, for
example, by suitable formulations of the drug that resist gas-
tric pH. Be that as it may, it is apparent that artemisinin deriva-
tives bearing an amino group attached directly to C10 are ap-
preciably more active in vitro than their acetal counterparts
such as artemether (3), as exemplified by data for compounds
6–9 as discussed above.
[
50,51]
artemisinic acid,
is less active than artemether (3). Treat-
ment of CQ-resistant K1 and CQ-sensitive FC27 strains of P. fal-
ciparum with 5 nm artemether (3) and carba analogue 26 re-
[
51]
sulted in 100 and 45% inhibition respectively. Carba ana-
[
50]
logues of artesunate (4) are unknown, but compound 27 by
way of an approximate structural analogue is equipotent with
Scheme 6. Representation of hydride transfer from FADH
2
to O1 of artemisone (9) in synchrony with cleavage of the peroxide O1ÀO2 bond leading to the for-
mation of ring-opened product 22 (Scheme 5). The approximate W-conformation of N1’ÀC10ÀO11ÀC12 may allow transmission of inductive effects to the
peroxide and for subsequent concerted fragmentation triggered by the addition of hydride to O1.
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[
59]
caused by association of the alkyne with the heme iron. In
summary, it is premature to infer that the binding of the
alkyne- and azide-tagged artemisinins and trioxolanes to pro-
teins is mechanistically significant. Considerations of intrinsic
chemistry as experimentally verified under biologically realistic
conditions underscore the inability of artemisinins to undergo
Conclusions
Artemisone (9) exhibits potent antimalarial activities, and like
other compounds in its class, it provides a compelling starting
point for a drug-discovery effort based on this important class
of therapeutic agent. In commencing this work, we prepared
two artemisone homologues in which the thiomorpholine S,S-
dioxide group is attached through a methylene linker to C10
and which differ in stereochemistry at C10. These were de-
signed to have improved hydrolytic, thermal, and metabolic
stabilities and by analogy with unnatural C-glycosides vis-ꢁ-vis
their biologically active natural N-glycoside counterparts were
anticipated to possess enhanced biological activities. However,
in relation to biological activities, the converse turned out to
be true. The observations tend to suggest that the presence of
a leaving group at C10 of the artemisinin skeleton may influ-
ence the biological activity in a positive sense. This may be ra-
tionalized by our cofactor model, wherein in terms of compati-
bility with, and activation by proton transfer from, the biologi-
cal environment, an amino group at C10 appears to be ideal.
This in turn suggests that structurally more elaborate artemisi-
nins, for example, dimers or trimers attached at C10 through
carba-based linkers that display promising biological activi-
II
II
“activation” by heme-Fe or Fe and that further the putative
exquisitely oxygen-sensitive C radicals derived from reductive
cleavage of the peroxide cannot alkylate “important” biomole-
cules in what is a patently oxidizing and relatively oxygen-rich
[26]
environment within the intraerythrocytic parasite.
Experimental Section
General
Dihydroartemisinin was obtained from the Kunming Pharmaceuti-
cal Corporation, Kunming, China, or from Haphacen, Hanoi College
of Pharmacy, Vietnam. Other chemicals were purchased from com-
mercial sources and were used without further purification. Di-
chloromethane was distilled from calcium hydride. Diethyl ether
and tetrahydrofuran were dried with sodium and distilled from
sodium benzophenone ketyl prior to use. N,N-Dimethylformamide
was dried with calcium hydride, vacuum distilled, and stored over
type 4 ꢂ molecular sieves under an atmosphere of nitrogen. Ethyl
acetate and hexane for column chromatography were distilled
from calcium chloride. Triethylamine was dried with calcium hy-
[
53]
ties, may be rendered even more active by use of amino-
based linkers. Of course, the overall benefit may be ameliorat-
ed by the lower stability of the amino compounds in a biologi-
cal medium, and as a result, lower biological half-lives. Thereby,
the ultimate choice will rely on a cost–benefit analysis in rela-
tion to accessibility of the artemisinin derivative, its efficacy,
toxicity, and the specific nature of the target.
1
dride and stored over sodium hydroxide pellets. H NMR spectra
were recorded in CDCl with a Bruker-400 spectrometer, and
C NMR spectra were recorded in CDCl with a Bruker-300 spec-
3
13
3
trometer. Infrared spectra were recorded with a PerkinElmer Spec-
trum One spectrometer. Single-crystal X-ray structure measure-
ments were performed with a Bruker Smart-APEX CCD four-circle
diffractometer. All computations in the structure determination
and refinement were performed on Silicon Graphics Indy computer
by using programs of the Siemens SHELXTL PLUS (version 5) pack-
age. Melting points were recorded with a Leica Microscope Heat-
ing Stage 350 and are corrected. E. coli flavin reductase (Fre) was
Note added in proof: In relation to the mechanism, a referee
suggested that one should take cognizance of the reports pur-
porting to indicate that artemisinins and synthetic trioxolanes
tagged with alkyl chains bearing terminal alkyne or azide
groups evidently “alkylate” a considerable number of intrapara-
sitic protein targets, a process that is enhanced by heme
[46,47]
prepared as previously described
and was stored in phos-
[
54]
iron. What is apparent is that according to precedent for lip-
ophilic drugs, the technique confirms promiscuous binding to
proteins that are not necessarily associated with mechanism of
phate-buffered saline (PBS, pH 7.4) containing 10% glycerol at
À208C until use. UV/Vis spectra were recorded with a PerkinElmer
Lambda 900 UV/Vis/near-IR double beam spectrometer and Varian
Cary 50 UV spectrometer. Mass spectra were obtained with a GCT
Premier Mass Spectrometer by Waters Micromass operating in
[
55]
action. More importantly, the terminal alkyne or azide tag
very likely will sequester redox-active iron (either associated
chemical ionization (CI) mode, with CH or NH as the CI reagent
[
56]
4
3
with heme or labile) and, of course, copper through the for-
mation of alkyne or azide metal complexes, which thereby
would lead to an artificial heme or redox metal ion effect. In-
duced decomposition of the peroxide in the artemisinin or
synthetic trioxolane constrained by the proximate alkyne- or
azide-bound heme or redox metal in the intraparasitic environ-
ment will ensue. One has to wonder how the putative tagged
decomposition products—ketones, aldehydes, epoxides, and
gas.
Synthesis
1
0b-Cyano-10-deoxo-10-dihydroartemisinin (17): Tin(IV) chloride
(93.6 mL, 0.8 mmol, 0.4 equiv) in dichloromethane (1 mL) was
added to a stirred solution of 10b-dihydroartemisinyl benzoate
(16; 0.78 g, 2 mmol, 1 equiv) and trimethylsilyl cyanide (0.75 mL,
6 mmol, 3 equiv) in dichloromethane (5 mL) at À788C. The mixture
was stirred for 2 h and was then treated with hydrochloric acid
(2m, 3 mL). The mixture was extracted with dichloromethane (3ꢃ
[
26]
so on that do not possess antimalarial activities —arising
from redox metal ion catalyzed isomerization of the peroxide
[
57]
bridge in the artemisinin or synthetic peroxide may bind to
the proteins. In general, a terminal alkyne tag may be used
successfully as a surrogate for mapping behavior of endoge-
1
0 mL). The organic layers were combined, and the organic solu-
tion was washed with water (2ꢃ10 mL) and saturated aqueous
NaHCO solution (2ꢃ10 mL) and then dried (MgSO ). After filtra-
tion, the filtrate was concentrated under reduced pressure to leave
a crystalline residue. This was recrystallized directly from isopropa-
nol to give the product as fine colorless needles (0.97 g, 83%); mp:
3
4
[
58]
nous substrates such as alkenes that do not possess bioac-
tive peroxide groups. Further, terminal alkynes are potent
mechanism-based inactivators of cytochrome P450 enzymes
&
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1
1
6
3
2
62–1668C; H NMR: d=0.94–1.04 (m, 1H), 0.98 (d, J=6.4 Hz, 3H,
2 h. The mixture was cooled to ambient temperature and
quenched by the slow addition of water (20 mL). The mixture was
extracted with dichloromethane (3ꢃ30 mL). The organic extracts
were combined, washed with brine (20 mL), and dried (MgSO ).
4
After filtration and evaporation, crude amine 20 was used directly
-Me), 1.07 (d, J=7.6 Hz, 3H, 9-Me), 1.26–1.33 (m, 1H), 1.43 (s, 3H,
-Me), 1.46–1.54 (m, 2H), 1.60–1.65 (m, 1H), 1.74–1.86 (m, 2H),
.04–2.10 (m, 1H), 2.33–2.41 (m, 1H), 2.84–2.93 (m, 1H), 4.77 (d,
13
J=6 Hz, 1H, H-10), 5.53 ppm (s, 1H, H-12); C NMR: d=13.022,
2
6
6
1
1
0.16, 21.87, 24.44, 25.78, 28.91, 34.10, 35.97, 37.13, 44.44, 52.34,
5.75, 60.16, 90.34, 104.67, 117.91 ppm; IR (KBr): n˜ =491, 537, 596,
41, 713, 825, 841, 858, 884, 913, 937, 958, 981, 1015, 1044, 1053,
065, 1094, 1122, 1149, 1190, 1175, 1209, 1229, 1244, 1259, 1278,
without further purification; attempts at purification resulted in its
decomposition. Characteristic H NMR signals for compound 20
1
d=3.68–3.73 (m, 1H, H-10), 5.27 ppm (s, 1H, H-12). A solution con-
taining crude amine 20 (200 mg) and divinyl sulfone (0.1 mL,
1.0 mmol) in propan-2-ol (10 mL) was heated under reflux for 2 h.
The solvent was removed under vacuum. The residue was purified
by using column chromatography (hexane/ethyl acetate 2:1) to
afford a white crystalline product (145 mg, 34.9 mmol (40% from
amide 18). This was recrystallized from methanol to give 14 as col-
À1
306, 1334, 1384, 1458, 2879, 2943 cm ; MS: m/z: calcd for
+
+
C H NO ; 294.1705 [M+H] ; found: 294.1736. These data are in
agreement with reported values.
1
6
24
4
[37]
1
0a-Aminocarbonyl-10-deoxo-10-dihydroartemisinin (18): A mix-
1
ture consisting of nitrile 17 (0.293 g, 1 mmol), potassium hydroxide
0.196 g, 3.5 mmol, 3.5 equiv), and tert-butyl alcohol (10 mL) was
stirred at 508C in an oil bath for 3.5 h. Brine (10 mL) and water
5 mL) were then added, and the mixture was cooled to ambient
orless needles; mp: 135.2–135.98C; H NMR: d=0.80–0.82 (d, J=
(
6.8 Hz, 3H, 9-Me), 0.95–0.97 (d, J=6.4 Hz, 3H, 6-Me), 1.03–1.51 (m,
7H), 1.34 (s, 3H, 3-Me), 1.66–2.04 (m, 5H), 2.30–2.38 (td, J=3.6,
14.0 Hz, 1H), 2.58–2.61 (m, 1H), 2.62–2.67 (dd, J=5.2, 14.0 Hz, 1H,
(
temperature. The resulting solution was extracted with diethyl
ether (3ꢃ10 mL). The organic layers were combined and dried
-
CH -), 2.83–2.87 (dd, J=1.6, 14.0 Hz, 1H, -CH -), 3.06–3.09 (m, 6H),
2 2
3
.40–3.45 (m, 2H), 3.53–3.57 (m, 1H, H-10), 5.21 ppm (s, 1H, H-12);
1
3
(
MgSO ). After filtration, the filtrate was concentrated under re-
4
C NMR (300 MHz, CDCl ): d=14.03, 20.36, 21.57, 24.86, 26.06,
3
duced pressure to give product 18 as white plates (0.226 g, 99%);
2
8.88, 34.22, 36.24, 37.52, 45.79, 51.51, 51.85, 51.96, 57.48, 74.56,
2
0
1
mp: 75–818C; [a] = +154.03 (c=0.62 in CHCl ); H NMR: d=0.95
D
3
80.67, 91.65, 91.68, 104.21 ppm; IR (film): n˜ =824, 840, 858, 880,
03, 925, 943, 968, 993, 1012, 1022, 1043, 1063, 1088, 1099, 1123,
(
d, J=7.2 Hz, 3H, 6-Me), 0.97 (d, J=6 Hz, 3H, 9-Me), 1.03–1.10 (m,
9
1
1
1
H), 1.25–1.43 (m, 3H), 1.46 (s, 3H, 3-Me), 1.47–1.59 (m, 2H), 1.72–
.79 (m, 2H), 1.88–1.92 (m, 1H), 2.02–2.07 (m, 1H), 2.35–2.43 (m,
H), 2.48–2.53 (m, 1H), 3.91 (d, J=11.2 Hz, 1H, H-10), 5.32 (s, 1H,
1
2
190, 1218, 1226, 1242, 1272, 1302, 1330, 1376, 1452, 1631, 2842,
À1
+
870, 2917, 3448 cm ; MS: m/z: calcd for C H NO S : 416.2101
20
34
6
+
[M+H] ; found: 416.2120.
13
H-12), 5.36 (s, 1H, NH), 6.56 ppm (s, 1H, NH); C NMR: d=13.48,
1
1
0b-Aminomethylene-10-deoxo-10-dihydroartemisinin (21) and
0b-methyleneartemisone (15): BF ·OEt (0.62 mL, 5.0 mmol) was
20.18, 20.99, 24.71, 25.94, 31.22, 31.44, 33.91, 36.08, 37.36, 46.12,
51.63, 80.28, 91.39, 108.52, 173.20 ppm; IR (KBr): n˜ max =510, 563,
607, 697, 761, 929, 851, 878, 915, 941, 1043, 1060, 1087, 1134,
3
2
added slowly to a stirred mixture of 10b-cyano-10-deoxyartemisi-
nin (17; 293 mg, 1.0 mmol) and sodium borohydride (190 mg,
5.0 mmol) in dry tetrahydrofuran (10 mL) under an atmosphere of
nitrogen. The mixture was heated under reflux for 2 h and then
cooled to ambient temperature, which was followed by careful
quenching with water (20 mL). The mixture was extracted with di-
chloromethane (3ꢃ30 mL). The organic portion was combined,
1
2
3
196, 1228, 1245, 1280, 1381, 1453, 1612, 1637, 1663, 1693, 2872,
937, 3254, 3470, 3497, 3550 cm ; MS: m/z: calcd for C H NO :
5
12.1811; found: 312.1809 [M+H] .
À1
+
16
26
+
1
0b-Aminocarbonyl-10-deoxo-10-dihydroartemisinin (19): A mix-
ture consisting of nitrile 17 (293 mg, 1 mmol), tetrahydrofuran
5 mL), water (10 mL), potassium carbonate (152 mg, 1.1 mmol,
(
washed with brine (20 mL), and dried (MgSO ). After filtration and
4
1
1
.1 equiv) and hydrogen peroxide (2 mL, 15 mmol, 30% v/w,
5 equiv) was stirred for 2 h at room temperature. It was then
evaporation, milky crude amine 21 (167 mg) was obtained. Be-
cause this could not be adequately purified, it was used directly.
1
heated, with stirring, at 408C for 30 min. After cooling the reaction
mixture to ambient temperature, this was extracted with ethyl ace-
tate (3ꢃ5 mL). The organic layers were combined, and the organic
solution was washed with water (2ꢃ5 mL) and then dried over
MgSO . After filtration, the filtrate was concentrated by evapora-
tion under reduced pressure to leave a crystalline residue. This was
submitted to chromatography with ethyl acetate to give product
Characteristic signals for compound 21: H NMR (400 MHz, CDCl ):
3
d=4.43–4.78 (m, 1H, H-10), 5.32 ppm (s, 1H, H-12). A solution of
1
0b-aminomethylene-10-deoxyartemisinin (21; 167 mg, 0.4 mmol)
and divinyl sulfone (84 mL, 0.84 mmol) in isopropanol (6 mL) was
heated under reflux for 2 h. The solvent was removed under re-
duced pressure, and the residue was purified by column chroma-
tography (hexane/ethyl acetate 3:2 then 1:1) to afford a white crys-
talline product (123 mg, 0.30 mmol) in an overall yield of 45%
from nitrile 17. The homologue was recrystallized from methanol
4
20
1
9 as white plates (91 mg, 29%); mp: 157–1658C, [a] = +21.8
D
1
(c=0.55, CHCl ). H NMR: d=0.91–0.98 (m, 1H), 0.97 (d, J=6 Hz,
3
1
3
1
1
H, 9-Me), 1.10 (d, J=7.6 Hz, 3H, 6-Me), 1.41 (s, 3H, 3-Me), 1.12–
.45 (m, 4H), 1.65–1.85 (m, 3H), 1.92–2.32 (m, 2H), 2.31–2.39 (m,
H), 2.84–2.93 (m, 1H), 4.81 (d, J=6.2 Hz, 1H, H-10), 5.42 (s, 1H,
to afford colorless needles; mp: 102.1–102.68C; H NMR: d=0.87–
0
1
.88 (d, J=7.2 Hz, 3H, 9-Me), 0.96–0.98 (d, J=6.0 Hz, 3H, 6-Me),
.03–1.51 (m, 6H), 1.65 (s, 3H, 3-Me), 1.65–2.13 (m, 6H), 2.28–2.36
13
NH), 5.48 (s, 1H, H-12), 6.56 ppm (s, 1H, NH); C NMR: d=13.05,
(td, J=4.0, 13.6 Hz, 1H), 2.54–2.62 (m, 2H), 2.73–2.78 (dd, J=8.8,
2
7
9
3
0.25, 24.32, 25.04, 25.99, 29.97, 34.41, 36.57, 37.76, 43.97, 51.88,
3.89, 81.21, 90.52, 103.49, 174.55 ppm; IR (KBr): n˜ =600, 826, 884,
58, 1004, 1014, 1112, 1377, 1454, 1593, 1685, 2876, 2937, 3218,
1
5
2
3.6 Hz, 1H, -CH -), 3.06–3.22 (m, 8H), 4.56–4.60 (m, 1H, H-10),
2
13
.33 ppm (s, 1H, H-12); C NMR (300 MHz, CDCl ): d=12.08, 20.11,
3
4.83, 26.02, 30.21, 34.27, 36.58, 37.55, 43.72, 50.89, 51.52, 51.91,
À1
+
286, 3436, 3490 cm ; MS: m/z: calcd for C H NO : 312.1811
16
26
5
56.52, 71.83, 81.08, 90.01, 90.03, 102.93 ppm; IR (film): n˜ =824, 848,
+
[M+H] ; found: 312.1828.
8
1
77, 914, 936, 947, 971, 1016, 1043, 1055, 1091, 1125, 1189, 1231,
267, 1279, 1303, 1332, 1380, 1454, 1641, 2857, 2933, 3523,
1
1
0a-Aminomethylene-10-deoxo-10-dihydroartemisinin (20) and
0a-methylene artemisone (14): BF ·OEt (0.62 mL, 5.0 mmol) was
À1
+
+
3
595 cm ; MS: m/z: calcd for C H NO S ; 416.2107 [M+H] ;
20 34 6
3
2
found: 416.2098.
added slowly to a stirred mixture of 10a-aminocarbonyl-10-deoxy-
artemisinin (19; 311 mg, 1.0 mmol) and sodium borohydride
(
190 mg, 5.0 mmol) in dry tetrahydrofuran (10 mL) under an atmos-
phere of nitrogen. The mixture was then heated under reflux for
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UV experiments
ether (3 mL), and the organic layer was separated. The aqueous
layer was extracted with ethyl acetate (3ꢃ5 mL). The combined or-
ganic layer was washed with brine (2ꢃ5 mL) and then dried
Oxidation of FADH by artemisone (9) to FAD: The artemisone solu-
2
tion was prepared from artemisone (100.8 mg, 0.251 mmol) and
degassed MeCN (5 mL) to give a final concentration of 0.0502m.
The NADH solution was prepared from NADH (17.0 mg,
(
MgSO ). After filtration, the solution was concentrated under re-
4
duced pressure. The residue was submitted to chromatography
(
ethyl acetate/hexane/triethylamine 3:7:0.1) to give recovered 14
0
.0229 mmol) and degassed pH 7.4 aqueous buffer (1 mL) to give
+
(3.8 mg, 10%): MS: m/z: calcd for C H NO S : 416.2101; found:
20
34
6
a final calculated concentration of 0.0229m. The absorbance of
NADH measured at lmax =339 nm was 0.3968905 AU, and the ab-
sorption coefficient at lmax =339 nm was measured as
4
16.2101. Next was eluted a fraction (ꢀ3 mg) consisting of a com-
plex mixture that according to mass spectral analysis contained
products equivalent to ring-opened precursor 25 (MS: m/z: calcd
À1
À1
6
220m cm ; therefore, the actual concentration of the NADH so-
lution was 0.0116m. The FAD solution was prepared from FAD
8.4 mg, 0.0101 mmol) and degassed pH 7.4 aqueous buffer (2 mL)
+
for C H NO S : 418.2258; found: 418.2232) and deoxy product 26
20
36
6
+
(
MS: m/z: calcd for C H NO S : 400.2152; found: 400.2132). Prom-
inent peaks at m/z=388.2136 (MS: m/z: calcd for C H NO S :
88.2152) and 370.2020 (MS: m/z: calcd for C H NO S : 370.2047)
19 32 4
20 34 5
(
+
19
34
5
to give a final calculated concentration of 0.0051m. The absorb-
ance of FAD measured at lmax =450 nm was 0.5819063 AU and the
absorption coefficient at lmax =450 nm was 11300m cm ; there-
fore, the actual concentration of the FAD solution was 0.0047m.
The FAD reductase (Fre) was prepared in pH 7 buffer to give a final
concentration of 20 mm.
+
3
correlate with protonated molecular ions of products arising from
degradation of the initial deoxy products. Fragmentation peaks
correlating with b cleavage of the carbonyl side chain and the
methylene S,S-dioxo-4-thio-1-morpholine appendage in the pri-
mary products were also noted. The MS and details are given in
Figure S6a. A subsequent fraction isolated (ꢀ12 mg) was a mixture
À1
À1
The degassed pH 7.4 buffer (1900 mL) and the NADH solution
that according to mass spectral analysis contained the deoxy com-
(
1
24 mL, 0.278 mmol, ꢀ1.21 equiv) were added to a UV cuvette (d=
+
pound 25 (MS: m/z: calcd for C H NO S : 400.2152; found:
cm) at 228C and treated with the Fre solution (5 mL, 0.1 nmol). A
20 34
5
4
00.2162). Details are given in Figure S6b. However, attempts to
background was scanned from l=200 to 800 nm. The FAD solu-
tion (49 mL, 0.230 nmol, 1 equiv) was added with mixing. Scanning
was performed at lmax =450 nm at 3 s intervals until the concentra-
tion of the FAD no longer decreased. Then, the effect of immediate
repurify this sample for fuller characterization were not successful.
addition of the artemisone solution (8 mL, 0.402 mmol, ꢀ2 equiv) to Acknowledgements
the same cuvette on the rate of increase in the concentration of
the FAD was performed by monitoring the increase in absorption
Work at the Hong Kong University of Science and Technology
due to FAD at lmax =450 nm over 60 min. The results are presented
in Figure 2. The effect of adding the equivalent amount of de-
gassed MeCN (8 mL) containing no artemisone to the cuvette rep-
resents the blank experiment of Figure 2.
was performed in the Open Laboratory of Chemical Biology of
the Institute of Molecular Technology for Drug Discovery and Syn-
thesis with financial support from the Government of the HKSAR
University Grants Committee Areas of Excellence Fund (Project
Nos. AoE P/10-01/01-02-I and AOE/P-10/01-2-II) and the Universi-
ty Grants Council [Grant Nos. HKUST 6493/06M, and 600507].
Work at North-West University, South Africa, was performed
under financial support from North-West University and was
funded in part by the South African Medical Research Council
(MRC) with funds from National Treasury under its Economic
Competitiveness and Support Package.
Oxidation of FADH2 by a-methylene artemisone (14) to FAD: The
methylene artemisone solution was prepared from 14 (20.8 mg,
0
.0501 mmol) and degassed MeCN (1 mL) to give a final concentra-
tion of 0.0501m. The NADH, FAD, and Fre solutions were used as
above. The degassed pH 7.4 buffer (1900 mL) and the NADH solu-
tion (24 mL, 0.278 mmol, ꢀ1.21 equiv) were added to a UV cuvette
(d=1 cm) at 228C and treated with the Fre solution (5 mL,
0
.1 nmol). A background was scanned from l=200 to 800 nm. The
FAD solution (49 mL, 0.230 nmol, 1 equiv) was added with mixing.
Scanning was performed at lmax =450 nm at 3 s intervals until the
concentration of FAD no longer decreased. Then, the effect of
adding the solution of 14 (8 mL, 0.402 mmol, ꢀ2 equiv) to the cuv-
ette on the rate of increase in FAD was performed by monitoring
the increase in absorption due to FAD at lmax =450 nm over
Keywords: antimalarial activity
glycosides · peroxides · mechanisms
· antitumor agents · C-
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Received: January 7, 2016
Revised: May 13, 2016
Published online on && &&, 0000
&
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ÝÝ These are not the final page numbers!

FULL PAPERS
Managing malaria: Insertion of a meth-
ylene group between the S,S-dioxothio-
morpholine ring and the artemisinin nu-
cleus of artemisone generates homo-
logues that are surprisingly less effica-
cious than artemisone. This may be ex-
plained by hydride transfer from FADH2
in flavin disulfide reductases that regu-
late redox homeostasis to the peroxide,
activated by the S,S-dioxothiomorpho-
line leaving group in artemisone; such
activation is absent in the homologues.
Y. Wu, R. W. K. Wu, K. W. Cheu,
I. D. Williams, S. Krishna, K. Slavic,
A. M. Gravett, W. M. Liu, H. N. Wong,
R. K. Haynes*
&& – &&
Methylene Homologues of
Artemisone: An Unexpected
Structure–Activity Relationship and
a Possible Implication for the Design
of C10-Substituted Artemisinins
ChemMedChem 2016, 11, 1 – 13
www.chemmedchem.org
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These are not the final page numbers! ÞÞ