Design, synthesis, and development of novel guaianolide-endoperoxides as potential antimalarial agents

Article abstract of DOI:10.1021/jm1006462

Design and synthesis of a guaianolide-endoperoxide (thaperoxide) 3 was pursued as a new antimalarial lead which was found to be noncytotoxic as compared to the natural product lead thapsigargin 2. Several analogues of 3 were successfully synthesized and found to be comparable to derivatives of artemisinin 1 in in vitro antimalarial assay. Among the synthesized compounds, 22 showed excellent in vitro potency against the cultured parasites (W2 IC ₅₀ = 13 nM) without apparent cytotoxicity. Furthermore, SAR trends in thaperoxide analogues are presented and explained with the help of docking studies in the homology model of PfSERCA(PfATP6).

Full text of DOI:10.1021/jm1006462

7864 J. Med. Chem. 2010, 53, 7864–7868
DOI: 10.1021/jm1006462
Design, Synthesis, and Development of Novel Guaianolide-Endoperoxides as
Potential Antimalarial Agents
Lingzhi Sun,† Falgun Shah,‡ Mohamed A. Helal,‡,^ Yunshan Wu,‡ Yakambram Pedduri,‡ Amar G. Chittiboyina,‡,
Jiri Gut,§ Philip J. Rosenthal,§ and Mitchell A. Avery*,†,‡,
†Department of Chemistry & Biochemistry, University of Mississippi, University, Mississippi 38677, ‡Department of Medicinal Chemistry, School
of Pharmacy, University of Mississippi, University, Mississippi 38677, §Department of Medicine, San Francisco General Hospital, University of
California, San Francisco, California 94143, and National Center for Natural Products Research; University of Mississippi, University,
Mississippi 38677. ^ Current Address: School of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.
Received May 28, 2010
Design and synthesis of a guaianolide-endoperoxide (thaperoxide) 3 was pursued as a new antimalarial
lead which was found to be noncytotoxic as compared to the natural product lead thapsigargin 2.
Several analogues of 3 were successfully synthesized and found to be comparable to derivatives of
artemisinin 1 in in vitro antimalarial assay. Among the synthesized compounds, 22 showed excellent in
vitro potency against the cultured parasites (W2 IC50 = 13 nM) without apparent cytotoxicity.
Furthermore, SAR trends in thaperoxide analogues are presented and explained with the help of
docking studies in the homology model of PfSERCA(PfATP6).
Introduction
Malaria remains a major cause of morbidity and mortality
worldwide, with about one million deaths annually.1 The
World Health Organization (WHO) estimates that half of
the world’s population is at risk of malaria, with the most
vulnerable populations including children under the age of
five and pregnant women in developing countries.2,3 At
present, with no available vaccine, the prevention and treat-
Figure 1. Structures of artemisinin, thapsigargin, and guaianolide-
endoperoxide (thaperoxide).
ment of malaria is increasingly difficult due to the growing
resistance of Plasmodium falciparum to most available anti-
malarial drugs.4,5 Currently, the sesquiterpene lactone arte-
misinin 1 and its derivatives (collectively called artemisinins)
are the only class of compounds consistently effective against
multidrug resistant strains of P. falciparum. Artemisinins are
now routinely recommended as first-line therapy for uncom-
plicated (as part of artemisinin-based combination therapy)6
and severe P. falciparum malaria. Interestingly, artemisinins
also display a wide variety of biological activities, including
cytotoxic, antibacterial, and antifungal activities.7-11 Exten-
sive efforts have been made so far for chemical modifications
of artemisinin to improve its therapeutic profile and to reveal
its mechanism of action.12-15 Considering the great therapeu-
tic importance of artemisinins, it is an appropriate strategy to
search for novel antimalarials with similar molecular scaffolds
to add to our antimalarial armamentarium. The natural
product sesquiterpene thapsigargin 2 caught our attention in
this regard as a potential lead compound for drug discovery.
Thapsigargin 2 (see Figure 1) belongs to the guaianolide
class of sesquiterpenes and was extensively studied as a highly
specific, potent irreversible inhibitor of SERCAa (sarco/en-
doplasmic reticulum Ca2þ-dependent ATPase) in the search
for promising new apoptotic agents.16-20 Interestingly, 2 also
showed moderate antimalarial activity with a newly proposed
mechanism of action inhibiting PfATP6, the P. falciparum
homologue of SERCA.21 During our synthetic studies on the
synthesis of 2, we designed a new guaianolide-endoperoxide 3
(named thaperoxide) as a precursor for its total synthesis
(Scheme 1). A closer observation of 3 indicates that it has
striking structural similarities with both 1 and 2. On the basis
of our computational model,22 we have found that 3 may
obviate the parasite cell permeability problem associated with
2, and also it can be a valuable probe to evaluate the newly
proposed mechanism of action of 1.21 Furthermore, we found
relatively few guaianolide-peroxides in the literature with the
unstable 1R,4R-peroxy-guaianolide scaffold.23-25 Our search
for guaianolides and guaianes revealed that some of them
have good antiparasitic activity.26 These observations includ-
ing our long-standing effortstounderstand themodeofaction
of 1 prompted us to develop an efficient synthetic strategy to 3
*To whom correspondence should be addressed. Phone: 662-915-
5879. Fax: 662-915-5638. E-mail: mavery@olemiss.edu.
a Abbreviations: SERCA, sarco/endoplasmic reticulum Ca2þ-depen-
dent ATPase; PfSERCA, P. falciparum homologue of SERCA; RCM,
ring closing metathesis; NaHMDS, sodium bis(trimethylsilyl)amide; Py,
pyridine; NOESY, nuclear Overhauser enhancement spectroscopy;
DBU, 1,8-diazabicycloundec-7-ene; DDQ, 2,3-dichloro-5,6-dicyano-
benzoquinone; PMB, p-methoxybenzyl; DIBAL-H, diisobutylalumi-
num hydride; RBC, red blood cell; SD, standard deviation; VERO,
monkey kidney fibroblasts.
r
pubs.acs.org/jmc
Published on Web 10/14/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7865
to evaluate its antimalarial activity and cytotoxicity, if pre-
sent. We planned to synthesize derivatives of 3 in order to
derive structure-activity relationships (SAR) of this series of
new compounds. As a preliminary study, we compared
obtained SAR results with our large pool of data generated
from derivatives of 127 and also performed docking studies
with the homology model PfSERCA.
zation. Accordingly, 8 was subjected to Dess-Martin period-
inane oxidation to yield ketone 5 in 84% yield. Our initial
efforts to homologate the C-7 position of 5 via enolization,
followed by treatment with various electrophiles, led to tedious
synthetic problems.30 After several attempts, a facile route was
then identified using ethyl pyruvate as a reactive electrophile
for aldol reaction. Thus 5 was subjected to kinetic enolization
using NaHMDS at -78 ꢀC, and subsequent treatment with
ethyl pyruvate gave 9 as a mixture of aldol products in 86%
yield. Without further separation, the aldol mixture 9 was then
subjected to dehydration using the Martin sulfurine, leading
to exclusive Z-olefin 10 as confirmed by a 2D NOESY
experiment.31 Subsequent olefin isomerization using DBU
led to the required conjugated diene 11 as a single diastereomer
as anticipated. The newly generated methyl stereo center of 11
was confirmed as 11R at a later stage. Further efforts to form
the lactone ring required a straightforward hydride reduction
of ketone in 11, followed by lactonization, to obtain 4. Sodium
borohydride mediated selective reduction of 11 produced a
corresponding secondary alcohol, which underwent in situ
lactonization, yielding lactone 4 in one pot with a 6R config-
uration. The obtained diene intermediate 4 was then subjected
to a final, crucial [4 þ 2] cycloaddition using singlet O2
(generated in situ in the presence of the photosensitizer methy-
lene blue) to furnish the single diastereomeric endo adduct 3 in
82% yield.32 The structure of 3 was unambiguously deduced
from single crystal X-ray diffraction studies of the correspond-
ing diol derivative 12,33 which was obtained by catalytic
hydrogenation of 3 without affecting the C7-C8 double bond.
Hence it was clearly evident from the X-ray studies that the
A-B-C rings are cis-fused and that the C11-methyl group is
in the β-configuration.
Chemistry
As shown in Scheme 1, we envisioned a stereoselective [4 þ 2]
cycloaddition of singlet oxygen on diene 4 for introduction of
the endoperoxide in 3. The synthesis of the key intermediate 4
was then anticipated from a bicyclic ketone 5 by homologa-
tion at C7 followed by lactonization, which in turn can be
obtained from bis-olefin 6 using ring closing metathesis
(RCM). A straightforward synthesis was planned for 6 from
the known compound 7.
Accordingly, the known ester 7 was obtained from
(S)-carvone28 and transformed into RCM precursor 6 in four
steps: (i) 3-OH protection, (ii) ester reduction, (iii) oxidation,
and (iv) homoallyl Grignard addition (Scheme 2). As predicted
by the Felkin-Ahn model, the addition of Grignard nucleophile
occurred from less hindered Si-face of corresponding aldehyde
intermediate to provide 6 exclusively.29 The proposed RCM of
6 cleanly furnished bicyclic intermediate 8 in the presence of
10 mol % of Grubbs’ II catalyst in dichloromethane in high
yield. As mentioned in the retrosynthetic analysis, introduction
of the lactone ring on the bicyclic system of 8 required C6-OH
oxidation for the C7-homologation by selective kinetic enoli-
Scheme 1. Retrosynthetic Analysis of Guaianolide-Endoperoxide
With the target compound 3 in hand, we evaluated it for
antimalarial activity against the chloroquine-resistant (W2)
P. falciparum strain. To our delight, the thaperoxide 3 showed
good antimalarial activity with at least 12-fold better activity
(See Table 1) than the corresponding guaianolide natural
product thapsigargin 2. The superior activity of thaperoxide
may be due to its greater parasite cell permeability than 2.
Additionally, unlike thapsigargin 2, thaperoxide 3 did not
show any cytotoxicity at 5 μg/mL concentration. Encouraged
by these results, we planned the synthesis of various analogues
of 3 based on our previous experience with sesquiterpene
Scheme 2. Stereoselective Synthesis of Thaperoxide 3a
a Reagents and conditions: (a) p-methoxybenzyl trichloroacetimidate, La(OTf)3, 0 ꢀC; (b) LAH, Et2O, rt; (c) DMP, DCM, 0 ꢀC to rt,
3-butenylmagnesium bromide, THF, -78 ꢀC; (d) Grubbs II catalyst, DCM, rt; (e) DMP, DCM, rt; (f) NaHMDS, ethyl pyruvate, THF, -78 ꢀC; (g)
Martin reagent, CHCl3, reflux; (h) DBU, DME, 0 ꢀC; (i) NaBH4, CeCl3 7H2O, MeOH, THF, 0 ꢀC; (j) methylene blue, O2, 0 ꢀC, DCM; (k) 10% Pd/C,
3
H2, CHCl3, rt. ORTEP representation of diol 12.

7866 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Sun et al.
lactones. In the first series, the seco analogues 13-18 were
synthesized from 3 (Scheme 3). We found LiBH4 to be a mild
reductant to open the lactone ring in 3 without affecting
endoperoxide functionality, furnishing diol 13 in a 94% yield.
Selective primary O-acetylation of 13 provided 15, which was
oxidized with the Dess-Martin reagent to give the ketone 17.
Meanwhile, PMB-deprotection of 13, 15, and 17, using DDQ,
provided the corresponding alcohols 14, 16, and 18, respec-
tively. In the second series of analogue synthesis, we pursued
partial reduction of the lactone in 3 to obtain lactol derivatives
and deoxo derivatives (Scheme 4).34 To synthesize the tetra-
cyclic analogues 21-24, the lactone 3 was converted to lactol
20 by treatment with DIBAL-H at -78 ꢀC. The ether deriva-
tives 21-22 were obtained directly by acid-catalyzed anome-
ric etherification, and deoxo analogues 23-24 were obtained
using triethylsilyl hydride promoted reduction of lactol 20.
Olefin-reduced congeners 28-31 were prepared from 25,
which was obtained by diimide reduction of 3.35 Thus lactone
reduction of 3 or 25 yielded lactol 20 or 28, followed by further
reduction with triethylsilyl hydride, furnished deoxoanalo-
gues 23, 24, or 31. Similar to lactol 20, lactol 28 was also
converted into corresponding ether derivatives 29-30. These
analogues were intended to mimic arteether and deoxoarte-
misinin compounds.
Table 1. Antimalarial Activity against W2 Clones of P. falciparum
compd no. IC50 (μM) ( SDb
compd no.
IC50 (μM) ( SDb
3
0.29 ( 0.01a
9.11 ( 0.21
NA
23
24
25
26
27
28
29
30
31
0.23 ( 0.09
0.57 ( 0.04
0.37 ( 0.00a
0.61 ( 0.03
0.27 ( 0.02
NA
13
14
15
16
17
18
19
20
21
22
Results and Discussions
NA
NA
The antimalarial activity of thaperoxide and its analogues
were determined against the chloroquine resistant W2
strain of P. falciparum (Table 1). Thaperoxide 3 displayed
submicromolar antimalarial activity and was 12-fold more
potent than thapsigargin 2.
NA
4.60 ( 0.10
1.05 ( 0.00
1.04 ( 0.01
0.62 ( 0.02a
0.013 ( 0.00a
3.40 ( 0.87
0.024 ( 0.02a
7.50 ( 0.04
0.005 ( 0.00
9.92 ( 0.10c
Modifications in the lactone ring had direct effects on the
activity and SAR, suggesting that the lactone moiety is
essential for antimalarial activity in this class of compounds.
Compounds lacking the lactone moiety, such as 13 and 18,
displayed a ∼31- and ∼16-fold decrease in the antimalarial
activity of the parent compound 3, whereas compounds
14-17 were inactive at 10 μM. Seco analogues of thaperoxide
showed only marginal antimalarial activity. Corresponding
artemisinin derivatives had variable antimalarial activity. A
few seco artemisinin analogues reported in the literature were
more potent than artemisinin, while most other derivatives
showed weak in vitro antimalarial potency. Other modifica-
tions in the lactone ring were performed based on the improved
potency and pharmacokinetic properties of artemether and
10-deoxyartemisinin derivatives.34,37,38 Among lactol deriva-
tives, acetal 22, with a β configuration at C-12, improved
antimalarial activity by ∼22 fold. Also, the β-isomer 22
was much superior in potency (∼48 fold) compared to the
1 (artemisinin)
2 (thapsigargin)
a These compounds did not show cytotoxicity against VERO
(monkey kidney fibroblasts) cells at the concentration greater than
5000 ng/mL.36 b SD = standard deviation. c Showed cytotoxicity against
VERO cells at 1200 ng/mL; NA = not active up to 10 μM.
Scheme 3. Synthesis of Thaperoxide Analogues without the
Lactone Moietya
a Reagents and conditions: (a) LiBH4, Et2O, rt; (b) DDQ, DCM,
H2O, rt; (c) Ac2O, Py, DMAP, DCM, 0 ꢀC to rt; (d) DMP, DCM, 0ꢀCtort.
Scheme 4. Synthesis of Acetal, Deoxo Derivatives of Thaperoxidea
a Reagents and conditions: (a) DIBAL-H, DCM, -78 ꢀC; (b) BF3 Et2O, benzene, EtOH, 45 ꢀC; (c) BF3 Et2O, Et3SiH, DCM, -78 ꢀC; (d)
3
3
KO2CNdNCO2K, HOAc, Py, MeOH, DCM, 0 ꢀC to rt; (e) DDQ, DCM, H2O, rt; (f) DMP, DCM, 0 ꢀC to rt.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7867
Figure 2. Docking pose of (a) 13, (b) 3, (c) 22 in the homology model of PfSERCA. Only side chain atoms of key residues of the active site are
shown for clarity. Figures were created using PyMOL1.1 (The PyMOL Molecular Graphics System, version 1.1, Schrodinger, LLC).
corresponding R-isomer 21. The same activity trend was observed
with C7-C8 hydrogenation congeners 29 and 30. The higher
potency of the β-isomer might be attributed to a favorable van
der Walls interaction at C-12 for 22 or 30 with corresponding
residues of the putative receptor (discussed below).
complementary shape of the sesquiterpene lactone to the
PfATP6binding pocket. Also, the lactone carbonyl was found
˚
to form a hydrogen bond (2.4 A) with the side chain amide of
Gln267. Furthermore, it was noted that the PMB group at the
C-3 position contributed to the binding of thaperoxide analo-
gues through hydrophobic interactions with Ile271, Val274,
Ile275, and Ile981 of the predicted PfATP6 active site. The
drastic reduction in the activity of 13 (seco analogue) com-
paredto 3 can result from the loss of the complementary shape
required for the proper hydrophobic interaction around
Phe264 placing R-hydroxy group on C-6 in the hydrophobic
environment (Figure 2a). The most active compound in this
series, 22, showed a similar binding mode to compound 3. The
improved activity of 22 might be attributed to additional
hydrophobic interactions between the ethoxy group on C-12
with Leu263 as well as the formation of a strong hydrogen
Modification in the cycloheptane ring, with complete satura-
tion of C7-C8, was expected to improve antimalarial activity by
obviating possible peroxide-diepoxidation rearrangement.39
However, saturation in the cycloheptane ring was found to
be unfavorable for antimalarial activity in the thaperoxide
series. Saturated analogues (25, 28-31) were less active than
the corresponding unsaturated analogues (3, 20-23). For
instance, saturated thaperoxide 25 was at least 1.3-fold less
potent than its unsaturated analogues. A similar trend was
observed for lactols 20 and 28, the latter was inactive at the
maximum tested concentration (10 μM). Saturated deoxo
analogue 31 was minimum 33-fold less potent than the corre-
sponding unsaturated deoxy derivative 23. However, to our
surprise, alcohol 26 in the C7-C8 saturated series was 1.7-fold
more potent than the unsaturated alcohol 19. Finally, the
effect of the C3-OPMB protecting group was observed, as the
C3-OH derivatives obtained by deprotection were inferior in
activity compared to compounds with the PMB group. For
example, compound 19 showed a 3.6-fold drop in potency
compared to that of 3 upon removal of the PMB group.
Similarly, the deprotected analogue of 25 (compound 26)
showed about half the potency of the parent compound.
We analyzed our SAR results based on interaction with the
proposed target PfATP6 by construction of a homology
model of PfATP6. This model was constructed using the
crystal structure of human SERCA (PDB CODE: 2AGV,
full details in Supporting Information). Docking of com-
pounds 3, 13, and 22 was carried out in the active site of
SERCA using the GOLD docking program (Figure 2).40 Top
ranked poses of these compounds are shown in Figure 2. In
the model, these compounds maintained the same orientation
of the tricyclic scaffold as seen in the X-ray structure of
thapsigargin in the complex with mammalian SERCA. The
key determinants of thaperoxide binding to PfATP6 involved
hydrophobic interactions with the active site residues of
SERCA, with the exposure of the peroxide bridge to the
solvent. Careful analysis of binding site interactions further
revealed three major structural features important for activity
within this series of compounds, namely the 5-7-5 tricyclic
system, the lactone ring, and the hydrophobic substituent at
the C-3 position. The tricyclic scaffold of compound 3
(Figure 2b) occupied the binding pocket around Phe264,
similar to the binding of thapsigargin in the crystal structure
of human SERCA. This interaction was facilitated by the
˚
bond with Gln267, which is within 2.0 A of 22 (see Figure 2c).
These observations showed that docking simulations were
able to support experimental results to some extent and
provided valuable insights about the structure-activity rela-
tionship of this novel series of peroxy analogues. Although
convincing, these results left certain ambiguities about
PfATP6 as a possible target for specifically artemisinin. We
noticed that no biochemical evidence of mobilization of
intracellular calcium intoP. falciparum resultantfrom binding
of artemisinin to PfATP6. Since the antagonistic relationship
between 1 and 2 is reported in an in vitro study supporting the
inhibition of pfATP6,21 we consider that the most active ana-
logues of thaperoxide (22 or 30, equipotent to artemisinin 1)
will provide more inputs in similar experiments. Further studies
are in progress in this regard and will be disclosed in due course.
Conclusion
In summary, we have designed and synthesized new guaia-
nolide-endoperoxides as potential antimalarial agents. In this
search, we identified thaperoxide (3)/analogues as simplified
analogues of thapsigargin 2 in terms of structural complexity
to reduce the synthetic challenge and cost. Most importantly,
installation of crucial peroxide pharmacophore on the back-
bone of guaianolide 3 improved its antimalarial potency and
precluded its cytotoxicity compared to 2. Various structural
features of 3, such as lactone, guaianolide tricyclic systems,
and peroxide moiety, are found to be critical to retain and to
improve its antimalarial activity. In addition, a homology
model of PfATP6revealed the importance of C3-hydrophobic
(OPMB) and C12-β substituents in 3 crucial for the potency in
this series. These detailed SAR studies provided important
information for future structural modifications. Further studies

7868 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Sun et al.
in this area are warranted and are currently underway in our
laboratory.
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Acknowledgment. L.S. is grateful to Dr. Fayang Qiu for
useful discussion and proofreading of manuscript. F.H.S. is
partially supported by grant no. 5P20RR021929 from the
National Center for Research Resources (NCRR) and from
NIH grant AI35707 to P.J.R. This investigation was con-
ducted in a facility constructed with support from research
facilities improvement program C06 RR-14503-01 from the
NIH NCRR. P.J.R is a Doris Duke Charitable Foundation
Distinguished Clinical Scientist.
Supporting Information Available: Synthesis methods and
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